INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

1622-P 1624-P Hyperglycemia Mediated Accelerates Aging in Vascular Endothelial Unique Cellular Responses of Adult Blood-Derived Endothelial Pro- Cells genitor Cells and Mature Endothelial Cells to High Glucose ROKHSANA MORTUZA, SHALI CHEN, BIAO FENG, SUBRATA CHAKRABARTI, EMILY KEATS, ZIA A. KHAN, London, ON, Canada London, ON, Canada, Lindon, ON, Canada Diabetes leads to dysfunction of selected organ systems, and vascular Glucose-induced endothelial cell dysfunction is a main factor causing endothelial cell (EC) dysfunction and loss is the key initiating and perpetuating tissue damage in the eye, kidney, heart and nerve in Diabetes. We step in the development of secondary diabetic complications. A number hypothesized that hyperglycemia induced oxidative stress causes an of studies have investigated the effect of diabetes on non-vasculogenic accelerated aging process and that such process is mediated through precursor cells (also known as early endothelial progenitor cells). However, a alterations of silent information regulator proteins or sirtuins(SIRTs) through comparative study investigating the effects of high levels of glucose on the epigenetic mechanisms. proliferative/vasculogenic endothelial progenitor cells (EPCs; late EPCs) and We investigated glucose induced aging and alterations of SIRTs in two mature ECs is lacking. We herein determined the response of adult blood- types of endothelial cells, human umbilical vein endothelial cells(HUVECs) derived EPCs and mature ECs to high levels glucose. and human microvascular endothelial cells(HMECs). The cells were grown Mononuclear cells (MNCs) from normal adult blood were successfully and propagated in high glucose (25 mmol, HG) and low glucose(5 mmol, NG). differentiated into ECs and characterized through our established protocol. Cells were observed daily and images taken to analyze their rate of growth Mature ECs were obtained from neonatal foreskin. Cells were then subject to and confl uency. At each passage subculture, some cells were collected 5 mmol/L or 25 mmol/L glucose and cellular activity assays were performed. for analysis of SIRTs, excision repair cross complimenting 1 and 4(ERCC1, We further investigated the mechanisms of increased oxidative stress in ERCC4) by RT-PCR. Cells from each passage were stained for senescence these EC types. activated beta galactosidase(SA-βgal), a known biomarker of cellular aging. Blood-derived EPCs were characterized by fl ow cytometry, cell staining, Morphometric analyses were performed. and RT-PCR. The progenitor phenotype was confi rmed by our recently Both types of cells grew faster in NG compared to HG. When exposed to discovered endostatin response assay. Following characterization, the HG, both cell types showed evidence of early senescence as evidenced by cells were exposed to high levels of glucose for 6 and 12 days. Our results increased SA-βgal expression. The replicative capacities of these cells were show that short term exposure (6-day) to glucose increased endothelin-1 diminished and they developed an irregular and hypertrophic phenotype. while decreasing the endothelin receptors in both EPCs and mature ECs. Interestingly, we found that HMEC cells started showing such changes after Interestingly, long-term exposure of cells to glucose (12 days) increased passage 1 in HG(compared to passage 4 in NG), whereas such changes were endothelin receptors in mature ECs but not EPCs. These results suggest seen in HUVECs at passage 4 in HG(compared to passage 7 in NG). Almost that mature ECs are more susceptible to the adverse effect of high glucose. all HMECs in HG at passage 5(compared to only 1.5% in NG) and HUVECs Our results also show high glucose-induced E-selectin expression in EPCs in HG at passage 11(compared to 61.4% in NG) showed β-gal positivity. suggesting an angiogenic phenotype which was not evident in mature ECs. Quantitative RT-PCR analyses showed this aging process was associated Lastly, our data illustrate NADPH oxidase was increased in mature ECs but with decreased SIRT 1, 2, 5 expressions in both cell lines. ERCC1, ERCC4 remained unaltered in the EPCs. mRNA expression was found to be decreased in HMEC but increased in In conclusion, our results show differential response of adult blood EPCs HUVEC in earlier passages. Such changes were prevented by treatment with and mature ECs to high levels of glucose and evidence for the therapeutic resveratrol. potential of vasculogenic EPCs. Data from this study suggests that hyperglycemia leads to accelerated Supported by: Canadian Diabetes Association and the Lawson Health Research aging in the endothelial cells. Furthermore, such aging process is mediated Institute through SIRTs, ERCC1 and 4 and varies depending on the cell type.

1623-P INTEGRATED PHYSIOLOGY—INSULIN SECRETION Transcription Factor mafB and a Functional Link to E-cadherin IN VIVO Expression in β Cell Differentiation MARIKO TSUCHIYA, KEN TSUCHIYA, KOSAKU NITTA, ATSUSHI MAEDA, Tokyo, [See also: Presidents Posters 451-PP to 452-PP, page A125.] Japan It is essential to clarify the mechanism by which endocrine cells including β-cells differentiate or transform during development or regeneration in Guided Audio Tour: Insulin Secretion—In Vivo Physiology (Posters 1625-P the pancreas. First, we identifi ed a few cells in the ductal epithelium of to 1634-P), see page 13. the pancreas of newborn that stained positive for insulin and mafB and & 1625-P negative for E-cadherin, whereas almost all the surrounding duct cells Short-Term Administration of the Atypical Antipsychotic Olanzapine stained positive for E-cadherin, suggesting that there may be cross-talk Induces Meal-Specifi c Impairments in Glucose and Lipid Metabolism, between mafB and E-cadherin. The purpose of the present study was to Independent of Weight Gain or Psychiatric Disease elucidate the consequences of E-cadherin and mafB expression in relation KAREN L. TEFF, MICHAEL R. RICKELS, JOANNE GRUDZIAK, KARL RICKELS, Phila- to induction of ductal epithelial cell (β-cell progenitor) differentiation into delphia, PA endocrine cells by mafB. We used the technique to intravenously inject The atypical antipsychotics are associated with an increased incidence mice and transfect cultured cells with synthetic mafB-siRNA and analyzed of diabetes. The mechanisms underlying these metabolic defects are not the resulting alterations in gene and protein expression level by real-time understood, although it has been speculated that the initiating pathophysiology PCR or immunohistochemistry. To observe the precise mechanism of this is weight gain, secondary to centrally-mediated increases in appetite. Our process, we suppressed mafB gene expression to 40% of the control level objectives were to determine if olanzapine administration impairs insulin in AsPc1 cells, a cell line derived from a pancreatic carcinoma in which sensitivity and post-prandial responses to the physiologically-relevant stimulus maf expression has been confi rmed by real-time PCR. Suppression of mafB of a meal, independent of weight gain or psychiatric disease. Healthy male 2 Obesity expression by siRNA in AsPC1 cells resulted in upregulation of mRNA (1.4 subjects (BMI=69.8+2.6 Kg/m ) were admitted into the CTRC for 12-days.

POSTERS fold) and immunostaining intensity (1.4 fold) of E-cadherin. Expression of Activity was maintained for each subject at a level matched to their free α-catenin (1.2 fold), which is indispensable for cadherin-catenin-complex- living levels. On Days 1 and 3 in a randomized order, subjects underwent a 600

Integrated Physiology/ mediated cell adhesion, was also increased, and β-catenin was increased kcal mixed nutrient meal challenge (n=10) and a euglycemic, hyperinsulinemic by 3.5 fold and GSK3 was slightly suppressed (0.8 fold), suggesting that clamp (7/10 subjects). The meal was labeled with [1-13C] glucose to monitor mafB may regulate ductal epithelial progenitor differentiation by expression 2 glucose appearance and 6,6-[ H2]-glucose was infused to measure glucose of the cadherin-catenin complex in the WNT signaling pathway. In the in disposal and endogenous glucose production during both the meal challenge vivo study mafB-siRNA treatment in the mouse pancreas resulted in similar and the clamp. Subjects were then administered olanzapine (5 mg/d, Days 4-5; changes. Moreover, mafB suppression downregulated expression of γ-actin 10 mg/d, Days 6-12). On Days 10 and 12, the metabolic tests were repeated. by the results of microarray profi ling. Taken together, the fi ndings in this Weight remained stable throughout the study (69.8+2.6,pre:70.1+2.9 Kg/ study suggested that mafB, whose expression is modifi ed by other mafs or m2, post-olanzapine) and no increase in food intake was found. Olanzapine other genes, even in stress or metabolic conditions in the mature pancreas increased post-prandial glucose (area under the curve: pre, 4363+1981 vs may regulate the E-cadherin-catenin complex in the WNT signaling pathway, post, 6579+1838, P<0.02), insulin (6824+4162 vs 13676+6739, P<0.001) and resulting in cell adherence and endocrine cell differentiation. triglyceride (1962+3995 vs. 4200+3938, P<0.01) concentrations. Small but

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A442 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO signifi cant decreases in glucose disposal were found during the meal and the clamp (P<0.01) but no ef fects were obser ved on endogenous glucose production. These data demonstrate that short-term administration of olanzapine to normal subjects induces meal specifi c impairments in post-prandial glucose 1627-P and lipid metabolism, independent of weight gain or psychiatric disease. Hyperglycemic Clamp EvaluationWITHDRAWN of First and Second Phase Insulin The metabolic dysregulation likely contributes to the increased incidence of Secretion in Type 2 Diabetic Patients with BMI 24 - 34 Kg/m2 Submitted metabolic disease in patients treated with this and similar medications. to the Ileal interposition Associated with Sleeve Gastrectomy Supported by: NIH DK084383 (KLT). AUREO DEPAULA, STIVAL ALESSANDRO, ALFREDO HALPERN, SERGIO VENCIO, & Goiania, Brazil, São Paulo, Brazil 1626-P Previous studies had shown that laparoscopic ileal interposition associated Relation of Glucose Effectiveness to Insulin Resistance and Insulin with sleeve gastrectomy (II-SG) is a safe and effective operation for the Secretion: The Insulin Resistance Atherosclerosis Study (IRAS) treatment of type 2 diabetes (T2DM) in a non-morbidly obese population. In CARLOS LORENZO, MARIAN J. REWERS, ANDREW J. KARTER, ANTHONY J. this study we used the hyperglycemic clamp (HGC) to evaluate the fi rst and HANLEY, STEVEN M. HAFFNER, San Antonio, TX, Aurora, CO, Oakland, CA, Toronto, second phase insulin secretion, the gold standard for this purpose. ON, Canada A 3-hour HGC was performed consecutively in 18 T2DM patients in the Glucose effectiveness (S ), the ability of glucose to enhance its own G pre- and postoperative periods at a mean of 16 months, ranging 12-22. Mean cellular uptake and to suppress endogenous glucose production, is a risk age was 54.7 years, ranging 42 – 68. Mean diabetes duration was 11.3 years, factor for type 2 diabetes. Impaired glucose tolerance (IGT) and diabetes are ranging 4–25. Insulin therapy was been used by 61.1% of the patients. associated with lower S . To further examine the relation of S to glucose G G Postoperatively, mean BMI decreased from 30.9 Kg/m2 to 25.1 Kg/ tolerance status, we assessed the relation of S to insulin sensitivity and G m2. Insulin sensitivity evaluated at 140-180 min increased from a mean of insulin secretion in the Insulin Resistance Atherosclerosis Study. S , insulin G 73.5 mmol.min-1.nmol-1 to a mean of 130.1 mmol.min-1.nmol-1 following the sensitivity index (S ), and acute insulin response (AIR) were measured I operation. Acute c-peptide increment increased from a mean of 2540 pmol.l-1 in 1,265 participants aged 40 – 69 years by the frequently sampled to a mean of 3803 pmol.l-1, postoperatively. Acute c-peptide increment over intravenous glucose tolerance test. Individuals treated with glucose- acute glucose response increased from a mean of 32.1 pmol.mmol-1 to a lowering medications were excluded. S was 2.06 ± 0.03, 1.68 ± 0.04, and G mean of 52.2 pmol.mmol-1, postoperatively. The disposition index increased 1.51 ± 0.04 x 10-2 min-2 in individuals with normal glucose tolerance (NGT), from a mean of 14.2x104 to a mean of 24.6x104 postoperatively. Area under IGT, and diabetes, respectively (p <0.001). S had a U-shape relationship trend I the curve (AUC) of glucose from 10-180min decreased from 168.5 mmol.l-1 with S after adjusting for age sex, race/ethnicity, and research center. This G to 133.6 mmol.l-1. During the second phase, C-peptide levels increased from relationship was mostly positive in individuals with NGT (p <0.001) and trend 84.8x103 pmol.l-1 to 144x103 pmol.l-1 and when evaluated over the AUC of mostly negative in those with diabetes (p <0.001). trend glucose increased from 463 pmol.mmol-1 to 1119 pmol.mmol-1.

This Hyperglycemic Clamp study demonstrated improvement of fi rst and second phase insulin secretion after a mean of 16 months following the ileal interposition associated with sleeve gastrectomy in T2DM patients with AIR was directly related to S in all individuals (p <0.001) regardless of G trend BMI 24 – 34 kg/m2. glucose tolerance status. & 1628-P Early Improvement of Insulin Sensitivity but Not of Insulin Secretion in Severely Obese Type 2 Diabetic Patients after Gastric Bypass Surgery BERND SCHULTES, EVA SVEHLIKOVA, BARBARA ERNST, BRITTA WILMS, MARTIN THURNHEER, SAMRA ZAHIRAGIC, ALEXANDRU TUCA, BARBARA OBERMAYER- PIETSCH, OANA FREISINGER, THOMAS R. PIEBER, St. Gallen, Switzerland, Graz, Austria Gastric bypass surgery has been shown to resolve type 2 diabetes but the underlying mechanisms are incompletely understood. Here we report preliminary results of an ongoing study on changes in beta cell function and insulin sensitivity before and shortly after gastric bypass in 7 type 2 diabetic (DM) patients and 4 non-diabetic controls (C) with severe obesity. Before and 8 to 21 days after the surgery, patients were subjected to Obesity an oral glucose tolerance test (OGTT). Established models were used to POSTERS calculate various indices of glucose metabolism. During OGTT, glucagon-like

peptide 1 (GLP-1) and glucagon time profi les were measured. Integrated Physiology/ After surgery, a comparable decrease in body weight and BMI was achieved in DM and C. HbA1c, fasting glucose and 2h glucose response in the OGTT declined after surgery in DM (all p<0.05). None of the insulin secretion indices, e.g. early insulin response pointed to an improvement in insulin secretion which remained lower in DM compared to C. On the contrary, In summary, S declines and the relationship between S and insulin G G indices of insulin sensitivity signifi cantly increased in DM patients (e.g. sensitivity changes as the glucose tolerance deteriorates. Autoregulation of insulin sensitivity index: 0.003±0.02 vs. 0.03±0.02; p<0.01), who achieved glucose uptake tends to be preserved with signifi cant insulin resistance values almost comparable to those in C. The increase in plasma GLP-1 Supported by: NHLBI Grants: HL47887, HL47892, HL47902; GCRC Grants: M01RR431, and glucagon concentrations after the oral glucose load was signifi cantly M01RR01346 enhanced after surgery both in DM and C (all p<0.05).

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A443 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

Despite of a markedly enhanced GLP-1 response there was no improvement than those on PBO (p=0.002). PIO increased Si by 35% (p=0.014) whereas in insulin secretion but an unexpected increase in glucagon levels during the AIRg and GSIS were unchanged. GIP-SIS decreased by 33% in subjects on OGTT shortly after gastric bypass surgery. While gastric bypass surgery in PIO (p=0.013). Changes in GIP-SIS were negatively correlated with weight the short-term appears to leave beta cell function unaffected it obviously changes (p=0.032) and positively correlated with AIRg changes (p=0.002), exerts a strong stimulating effect on alpha cell function. Collectively, our but not with changes in Si (p=0.8). In summary we found that 12-weeks of data speak against a major contribution of incretin effects to the early treatment with PIO resulted in an overall decrease in GIP-SIS in subjects improvement in glucose metabolism after gastric bypass surgery which with well-controlled T2DM. This was not explained by increased peripheral appears to rely primarily on enhanced insulin sensitivity. insulin sensitivity, but was closely related to changes in weight and AIRg. DM C Thus, GIP-SIS mirrors overall β-cell function in this model. M/F Δ Δ Δ Δ Δ Δ pre-operative post-operative pre-operative post-operative Weight(kg) AIRg Si DI GSIS GIP-SIS (pM*min) (pM*min-1/10-5) (pM2/10-5) (pM/mM) (pM) GLP-1 AUC (pg/ml/min) 0.56±0.34 2.61±0.96 0.46±0.35 3.58±1.72 PIO 5/7 2.6±0.5 –2.3±6.4 0.77±0.30 0.40±0.38 –0.08±0.06 –13.6±5.2 Glucagon AUC (pg/ml/min) 11.27±3.10 52.20±16.19 10.11±4.22 53.36±12.14 PBO 7/5 –0.5±0.8 3.5±4.6 –0.37±0.45 –0.50±0.20 –0.08±0.04 –0.6±4.6 Supported by: EFSD/MSD Clinical Research Grant 2009 Supported by: Takeda; NIDDK

& 1629-P Impaired Insulinotropic Effect of Both GLP-1 and GIP Can Be Induced & 1631-P in Healthy Normal Glucose Tolerant Subjects Glucocorticoid-Induced Islet-Cell Dysfunction: Potential Role for KATRINE B. HANSEN, TINA VILSBØLL, JONATAN I. BAGGER, JENS J. HOLST, Altered Sympathovagal Balance? FILIP K. KNOP, Glostrup, Denmark, Gentofte, Denmark, Copenhagen, Denmark DANIEL H. VAN RAALTE, KELLY A.A. KWA, RENATE E. VAN GENUGTEN, JOHN M. The insulinotropic effect of the incretin hormones glucose-dependent KAREMAKER, MICHAELA DIAMANT, Amsterdam, The Netherlands insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is Glucocorticoids (GCs) induce insulin resistance, glucose intolerance and impaired in patients with type 2 diabetes. It remains unclear whether this diabetes. The effects of GCs on islet-cell function and the causal mechanisms impairment can be induced temporarily in healthy subjects by a short period remain more controversial. Here, we hypothesized that the GC prednisolone of glucose homeostatic dysregulation. (PRED) impairs islet-cell function and that alterations of autonomous nervous We aimed to investigate the insulinotropic effect of GIP and GLP-1 system (ANS) balance may contribute to these changes. compared to placebo (saline) before and after 12 days of glucose homeostatic Thirty-two healthy normoglycemic males (age: 21.0±2.1 years, BMI: -2 dysregulation in healthy subjects. 21.9±1.7 kg m ) were treated with either PRED 30 mg q.d. (PRED30), PRED The insulinotropic effect was measured using hyperglycemic clamps and 7.5 mg q.d. (PRED7.5) or placebo (PLB) for 14 days. At baseline and day 14 infusion of physiological doses of GIP, GLP-1 or saline in 10 healthy Caucasian of treatment, a combined hyperinsulinemic-euglycemic and hyperglycemic 2 clamp with additional arginine stimulation was done. Moreover, fasting males (age: 25±1 years (mean±SEM); BMI: 23±1kg/m ; HbA1c: 5.4±0.1%; no family history of diabetes) before and after intervention using high calorie glucagon levels were measured. Finally, ANS function was assessed by diet, sedentary lifestyle and administration of prednisolone (37.5 mg/day) measurement of heart rate variability and subsequent power spectral for 12 days. analysis. HFnorm was calculated as measure of parasympathetic activity, The intervention resulted in insulin resistance (IR) according to the LF/HF ratio was taken to represent the sympathovagal balance. homeostatic model assessment (1.2±0.2 vs 2.6±0.5, P=0.01) and glucose PRED dose-dependently decreased insulin sensitivity as compared to PLB: -1 -1 tolerance deteriorated as assessed by the area under curve (AUC) for mean differences: -2.1±0.8 mg kg min (P=0.021) for PRED7.5 and -1 -1 st plasma glucose during 75 g-OGTT (730±30 vs. 846±57mM×2h, P=0.021). The -4.5±0.7 mg kg min (P<0.001) for PRED30. PRED tended to increase 1 - and nd subjects compensated for the induced IR by signifi cantly increasing their 2 -phase glucose-stimulated C-peptide secretion (β=0.305; P=0.098) and post intervention insulin responses during saline infusion by 2.9±0.5 fold; (β=0.311; P=0.089) respectively; this effect was abolished when adjusted for signifi cantly (P=0.001) more than during GIP or GLP-1 infusions (1.78±0.3 and insulin sensitivity. C-peptide secretion following arginine stimulation on top 1.38±0.3, P=NS). of hyperglycemia (ASI-iAUCCP) was reduced both by PRED7.5: -2.7 (-5.2-0.3) These data show that impairment of the insulinotropic effect of both GIP nmol/L · min, P=0.05 and by PRED30: -3.0 (-7.6-0.2) nmol/L · min, P=0.02. and GLP-1 can be induced in healthy male subjects without risk factors for Fasting glucagon levels were dose-dependently increased: 2.2±0.9 type 2 diabetes. This fi nding indicates that the impaired insulinotropic effect pmol/L (P=0.04) for PRED7.5 and 4.3±1.2 pmol/L (P=0.003) for PRED30. PRED of the incretin hormones observed in type 2 diabetes is a consequence treatment tended to decrease HFnorm (β= -0.325; P=0.08). Overall, changes of insulin resistance and glucose intolerance rather than a primary event in Hfnorm inversely associated with fasting plasma glucose levels (β=-0.407; causing type 2 diabetes. P=0.03) and with fasting glucagon levels (β= -0.337; P=0.07). The change in Supported by: EFSD/Novartis Grant LF/HF ratio was negatively associated with ASI-iAUCCP (β= -0.365; P=0.05). In sum, PRED treatment dose-dependently impaired islet-cell function in healthy men. GC-induced alterations in sympathovagal balance may & 1630-P contribute to their effects on islet-cell function. Pioglitazone (PIO) Does Not Enhance Glucose-Dependent Insulino- Supported by: Dutch Top Institute Pharma T1-106 ADA-Funded Research tropic Peptide (GIP) Stimulated Insulin Secretion in Subjects with Well-Controlled T2DM WILLIAM G. THARP, OLGA SIDELEVA, DARIUSH ELAHI, COURTNEY LEDGER, & 1632-P RHONDA MAPLE, RICHARD PRATLEY, Burlington, VT, Baltimore, MD Deep Brain Stimulation in the Nucleus Acccumbens Alters Plasma Dysregulation of incretin action contributes to hyperglycemia in Type Insulin Concentrations in Rats 2 diabetes(T2DM). GIP stimulated insulin secretion(GIP-SIS) is markedly CHARLENE DIEPENBROEK, GEOFFREY VAN DER PLASSE, LESLIE EGGELS, ERIC blunted, possibly due to down-regulation of GIP receptor in β-cells by chronic FLIERS, ANDRIES KALSBEEK, MIREILLE J. SERLIE, SUSANNE E. LA FLEUR,

Obesity hyperglycemia. The promoter of GIP receptor contains a PPAR response Amsterdam, The Netherlands, Utrecht, The Netherlands POSTERS element. We hypothesized that treatment with PIO, a potent PPARγ agonist, The nucleus acccumbens (NAc), part of the reward centre in the brain, would improve GIP-SIS in subjects with T2DM. Twenty four subjects (12M/12F, has gained major interest because of its involvement in the development of

Integrated Physiology/ age 58.7+10.6, BMI 33.4+8.1) with well controlled T2DM (A1c<7%) on either obesity. Interestingly, viral tracing experiments revealed a neuroanatomical diet and exercise (D+E,n=9) or stable doses of metformin (MET,n=15) were connection between the NAc and the pancreas via the autonomic nervous recruited for a randomized, double-blind, placebo controlled study. Subjects system. We therefore hypothesize that the NAc could be involved in glucose underwent a frequently-sampled IVGTT followed by a graded glucose metabolism. To determine whether this anatomical connection is functional infusion and 30 min GIP infusion (2 pmol/kg/min) at baseline and after 12 in affecting insulin secretion, we applied deep brain stimulation of the NAc weeks of treatment with placebo (PBO) or PIO (45mg/day). The acute insulin and investigated the effects on levels of glucose and insulin. response to glucose (AIRg) and insulin sensitivity (Si) were derived from the Thirty male Wistar rats received bilateral stimulating electrodes in the Minimal Model, β-cell sensitivity to glucose (GSIS) was calculated as the NAc and a jugular vein catheter. Rats were subjected to 60 minutes of either slope of insulin to glucose during the glucose infusion and GIP-SIS as the 100 μA or 200 μA stimulation. Each animal served as its own control and was, increment in insulin secretion during the GIP infusion. GIP infusion increased controlled for body weight, randomly assigned to an experimental group. insulin secretion by 7-fold at baseline. Subjects on PIO gained more weight Blood samples were drawn prior to stimulation (t=-1), during stimulation

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A444 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

(t=5, t=10, t=15, t=30, t=60) and following cessation of stimulation (t=90 were increased in Atf6α-/- DO mice. They also exhibited mild liver steatosis, and t=120 minutes) to determine blood glucose and plasma insulin levels. To but serum triglyceride levels were lower with ATF6α defi ciency. Furthermore, exclude that the effects on glucose metabolism were stress-induced we also insulin-stimulated Akt and S6 phosphorylation was increased in skeletal measured plasma corticosterone levels. muscle from Atf6α-/- mice. Our data indicated that ATF6α contributes In the 200 μA stimulated animals, plasma insulin levels signifi cantly to both prevention and promotion of diabetes; it protects β cells from ER increased to 122% of base-line at onset of stimulation (t=5) compared to stress and suppresses gluconeogenic enzyme expressions in the liver, while sham stimulated animals whereas blood glucose values showed a slight (4%) it is involved in development of hypertriglycemia and insulin resistance in increase during the fi rst 15 min of stimulation. In contrast, stimulation at skeletal muscles. lower intensity (100 μA) did not affect either measure. Plasma corticosterone 1635-P levels were not signifi cantly changed as compared to the sham and 100 A μ β-Cell Overexpression of Selenoprotein S Protects Against Alloxan- conditions and thus can not explain changes in insulin secretion or the slight and Streptozotocin-Induced Diabetes but Not High Fat Diet-Induced glucose rise. Defects These data suggest a role for the NAc in the regulation of glucose TANYA SAMARASEKERA, KEN WALDER, NICOLE STUPKA, SHEREE MARTIN, metabolism and we hypothesize that the NAc controls the pancreas and NICOLE WONG, JENNY FAVALORO, JOSEPH PROIETTO, SOFIANOS ANDRIKO- thereby insulin secretion via the central nervous system. POULOS, Heidelberg Heights, Australia, Geelong, Australia Supported by: ZonMW The selenoprotein SEPS1, localized in the ER but also on the plasma membrane, was originally discovered in the type 2 diabetic model Psammomys & 1633-P obesus, where its expression was enhanced by fasting in the liver. A Synergism of Dopamine Agonist Plus GLP-1 Analog Therapy on subsequent study showed that SEPS1 overexpression in the pancreatic Improvement of Glucose Intolerance in Syrian Hamsters β-cell line MIN6 protected against oxidative stress-induced death. To further MICHAEL EZROKHI, SHUQIN LUO, YELENA TRUBITSYNA, ANTHONY H. CIN- examine the protective properties of SEPS1 we made two lines of transgenic COTTA, Tiverton, RI mice, line 16 and 20, in which SEPS1 overexpression was targeted to islet Previous studies have demonstrated that timed daily treatment of β-cells at high and low levels, respectively. Both transgenic lines showed bromocriptine, a dopamine D2 receptor agonist, to insulin resistant animals normal body weights and random plasma insulin levels. Interestingly, and humans improves postprandial insulin sensitivity. Such treatment SEPS1 high overexpressor (SHO) mice developed mild hyperglycaemia in reduces post-meal or post-glucose challenge levels of glucose while the non-fasted state and reduced islet insulin immunostaining. Glucose- simultaneously markedly reducing hyperinsulinemia. GLP-1 analogs such mediated insulin secretion (GSIS) was also lower in SHO mice compared as exendin-4, are known to enhance glucose – mediated insulin secretion with control mice. Unlike the SEPS1 low overexpressor (SLO) mice, the SHO and reduce glucagon secretion. Therefore, the possibility exists of a positive mice were protected against the in vivo diabetogenic effect of alloxan and interaction between such a postprandial insulin sensitizing mechanism streptozotocin (200 mg/kg i.p.). Following 200 mg/kg alloxan treatment with such an enhanced postprandial insulin secretory response to improve plasma insulin levels, islet insulin immunostaining and glucose-mediated glucose intolerance. insulin release were signifi cantly higher in SHO mice compared with control To test such a postulate, glucose intolerant insulin resistant Syrian mice. However, both SLO and SHO mice were susceptible to high fat diet- hamsters weighing 200 grams were treated with either vehicle (N=20) or induced β-cell dysfunction as evidenced by the development of glucose bromocriptine (4 mg/kg; N=20) for 2 weeks and then each group was further intolerance. Ex vivo islet studies showed that compared to control islets, SHO subdivided into 2 groups and treated with either vehicle or exendin-4 (4 µg/ islets have reduced intracellular ROS level and alloxan-induced cell-death. kg) just prior to being subjected to a glucose tolerance test (GTT) (3g/kg). qPCR revealed that SHO islets have upregulation of GPx1 and catalase, but Relative to the vehicle/vehicle (control), bromocriptine markedly reduced the reduced SOD1 and HO-1 genes. ER-stress related genes GRP78 and CHOP insulin area under the curve (AUC) by 39% (from 1311 ± 177 to 796 ± 119 were unchanged in SHO islets, but the splice variant XBP1(s) was increased ng*min/ml) (P=0.03) while reducing the glucose AUC by 21% (from 42576 ± 3889 compared to control islets. Western blot analysis showed that GRP78, to 33752 ± 2763 mg*min/dl) (P<0.09). Exendin-4, by contrast, had no effect on GRP94, and HO-1 proteins were present at normal levels, however PDX-1 and either the glucose or insulin AUC versus control. However, the combination α-tubulin protein levels were increased in the SHO islets compared to control of bromocriptine plus exendin-4 therapy resulted in a 60% reduction in the islets. Taken together, the data suggests that SEPS1 acts as an antioxidant glucose AUC relative to vehicle/vehicle controls (P=0.0002), but also in an and is effective in β-cells at a high level of expression specifi cally against increase in the plasma insulin levels relative to the bromocriptine therapy/ oxidative-stress insults. vehicle group (P<0.05) to those levels observed in vehicle/vehicle controls. These data indicate that 1) relative to controls, bromocriptine plus exendin-4 1636-P therapy synergizes to completely normalize glucose intolerance without Association of Short Chain Fatty Acids with Visceral Adiposity and raising plasma insulin levels and 2) bromocriptine therapy that improves Endogenous Glucose Production insulin resistance and reduces compensatory hyperinsulinemia also permits BRIAN T. LAYDEN, SUDHA K. YALAMANCHI, THOMAS M.S. WOLEVER, ANDREA the expression of a robust glucose-mediated insulin secretory response to DUNAIF, WILLIAM L. LOWE, Chicago, IL, Toronto, ON, Canada exendin-4 resulting in a synergistic effect on glucose intolerance. The composition of the gut fl ora has been linked to obesity, a major risk factor for type 2 diabetes. Understanding this association will & 1634-P provide new insight into obesity and type 2 diabetes. We hypothesized ATF6α Deletion Accelerates β Cell Loss but Improves Insulin Sensi- that serum concentrations of the short chain fatty acids (SCFA) acetate, tivity in Mice propionate and butyrate, major by-products of fermentation by gut fl ora, MASAHIRO USUI, HISAMITSU ISHIHARA, Sendai, Japan, Itabashi-ku, Japan are associated with body composition and glucose homeostasis. To explore Type 2 diabetes mellitus is caused by impaired insulin secretion and this hypothesis, eighteen obese women (7 with polycystic ovary syndrome increased insulin resistance. Recent studies reported that endoplasmic and 11 reproductively and metabolically normal control women) underwent reticulum (ER) stress is involved in both pancreatic β cell dysfunction and a maximal insulin-dose (400 mU/m2/min) euglycemic clamp study with stable peripheral insulin resistance. We hypothesized that activating transcription isotope measurement of endogenous glucose production. Body composition Obesity factor 6α (ATF6α), one of three ER stress sensor proteins, played a critical and fat distribution were assessed by computerized axial tomography (CAT) POSTERS role in development of diabetes mellitus. Here we investigated Atf6α null and dual photon x-ray absorptiometry. Fasting serum acetate levels were mice bred with genetic models of diabetes caused either by impaired insulin negatively associated (Pearson correlation coeffi cient 0.52, P=0.03) with Integrated Physiology/ secretion (Akita Ins2WT/C96Y mice) or by insulin resistance (Agouti yellow (Ay) CAT measures of visceral adiposity. Fasting serum acetate levels were obese mice). Atf6α-/- mice were also fed with a high fat diet to generate positively associated with post-absorptive endogenous glucose production diet-induced obese (DO) mice with insulin resistance. Hyperglycemia was (Pearson correlation coeffi cient 0.50, P=0.03). There were no other accelerated with further reduction in pancreatic insulin content in Atf6α- signifi cant associations between circulating SCFA levels and parameters of /-Ins2WT/C96Y mice. In Atf6α-/- Ay mice, glucose tolerance was impaired glucose homeostasis or body composition. These novel fi ndings suggest that with lower insulin secretion but interestingly they showed higher insulin SCFAs are related to visceral adiposity and play a role in the regulation of sensitivity. Atf6α-/- DO mice exhibited phenotypes similar to those of Atf6α- hepatic insulin action. These observations may explain, in part, how gut fl ora /- Ay mice. A compensatory increase in pancreatic insulin content was not contribute to metabolic regulation. observed in Atf6α-/- DO mice. Electron microscopic studies revealed swollen Supported by: Endocrine Fellows Foundation ER in Atf6α-/- DO β cells. In the liver, transcripts of gluconeogenic enzyme

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A445 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

1637-P pmol/min/g pancreas; Total-ISR 7.2±1.0 to 8.0±1.6 nmol/g pancreas, p=ns). DI Delayed Insulin Secretory Responses Are Seen in Patients with increased signifi cantly in EXE (1.0±0.1 to 2.2±0.3, p<0.01) not in SAL (1.5±0.2 Long Duration of Type 1 Diabetes and Residual Insulin* to 1.7±0.3, p=ns). No differences were found in glucagon suppression or JENNIFER SHERR, LINDA RINK, KEVAN HEROLD, New Haven, CT insulin/glucagon ratio between groups. (EXE 26.5±3.3 to 22.1±5.2 vs. SAL Calculation of insulin secretory rates (ISR) can provide quantitative and 25.0±5.5 to 20.2±2.4 mU/l×(ng/ml)-1). Continuous IV treatment with EXE qualitative information of insulin secretion during a mixed meal test (MMT). increased IS and DI whereas SAL treated NDB had a compensatory increase We previously reported that some patients with type 1 diabetes (T1D) have in ISR. In conclusion: EXE could act also as inducer of β-cells “rest”, possibly a delayed secretory response during a MMT compared to non-diabetic by improving IS. controls and that their decline in insulin secretion was less than patients Supported by: NIH R01-DKO80148 with T1D who showed an early secretion profi le (i.e. @< 45min). To determine if there is a relationship between disease duration and ISRs, data derived 1639-P from MMT were analyzed in 1) 20 subjects with T1D diagnosed < 1 year 2) Effect of Different Treatment Strategies on Postprandial Beta Cell 12 subjects with T1D 2-5 years duration, 3) 21 subjects with T1D >5 years, Function in Patients with Type 2 Diabetes Mellitus 4) 38 non-diabetic controls. All subjects with T1D <1 year had detectable THOMAS FORST, MARTIN LARBIG, STEFAN PSCHERER, BERNDT VON STRITZKY, c-peptide levels. 12% of subjects with T1D 2-5 years and 57% of T1D >5 ANDREAS PFÜTZNER, Mainz, Germany, Berlin, Germany, Traunstein, Germany years subjects had undetectable c-peptide levels and were excluded from Intact proinsulin is a marker of beta cell stress and CV risk. The aim of this further analysis. Chronobiological Series Analyzer (CSA) software was used cross-sectional study was to investigate the effect of different treatment to calculate ISR by deconvolution of C-peptide using a two-compartment strategies on postprandial release of intact proinsulin in patients with type model for hormone clearance. Parameters used accounted for the subject’s 2 diabetes mellitus (T2DM). age, sex, height, and diabetes status. Total ISR area under the curve (AUC) Subjects were well-controlled T2DM patients (A1C ≤ 7.1%) who were and time of peak insulin secretion was calculated for each subject. There treated with metformin and either insulin glargine (GLA, n=14), sulfonylurea was a decline in total insulin secretion with duration of T1D (p<0.0001 for (SU, n=17) or a DPP-4 inhibitor (DPP4, n=17) for 6-12 months and an age- all vs. D). The proportion of delayed responders was greater in pts with T1D matched non-diabetic control group (C, n=17). After an 8-hr fast, subjects vs. Non-Diabetic Controls (p<0.0001) and there was a trend for a higher received a standardized breakfast (27g protein, 15g fat, 48g carbohydrates). proportion of delayed responders with increasing duration of T1D (p=ns). We At baseline and for 5 hr postprandial, venous blood was drawn for conclude that there is an increase in delayed insulin secretory responses in measurement of intact proinsulin, insulin and blood glucose. Area under the individuals with T1D and that their decline in insulin secretion was less than concentration-time curve (AUC0-5h) was calculated by the trapezoidal rule. patients with T1D who showed an early secretion profi le (i.e. @< 45min) over Comparisons between treatments were made using t-tests. the fi rst 2 years of disease. Mean plasma concentration of intact proinsulin increased less over *Collaboration with DirecNet time with GLA or DPP4 than with SU; all increased more than in C. AUC0-5h T1D <1 year T1D 2-5 years T1D>5 years Non-diabetic values were lower (p=0.003) with GLA (4933 ± 2070 pmol/L) vs. SU (9003 N=20 N=10 N=12 controls ± 4348 pmol/L) as they were with DPP4 (4428 ± 2115 pmol/L; p=0.002). (A) (B) (C) N=38 AUC0-5h values were less in C (2466 ± 698 pmol/L) than in T2DM groups (all, (D) p≤0.002). Similar results were obtained for plasma insulin concentrations, Total Insulin Secretion AUC 33,844+3,652 14,796+3,455 9,520+2,465 45,836+2,038 while postprandial blood glucose concentrations were comparable between (pmol/240min) T2DM groups but greater than C (all, p<0.001). Mean+SEM Time of Peak ISR 98.3+15.0 108+19.1 113+16.2 29+1.1 (min) Mean+SEM % of Early Responders 35 30 20 97 (i.e. @<45 min) ADA-Funded Research

1638-P Effect of Continuous Exenatide Infusion on Insulin Action and Secretion in Partially Pancreatectomized Baboons FRANCESCA CASIRAGHI, ALBERTO OMAR CHAVEZ-VELAZQUEZ, ALBERTO DAVALLI, GREGORY AVEDIS ABRAHAMIAN, AMALIA GASTALDELLI, RAUL BASTARRACHEA, SUBHASH KAMATH, ROBERTA PETZ, PENGOU ZOU, PAUL HIGGINS, ZHI CHANG, ANTHONY COMUZZIE, GLENN HALFF, RALPH DEFRONZO, FRANCO FOLLI, San Antonio, TX, Milan, Italy, Pisa, Italy Type 2 diabetes (T2DM) onset is determined by β-cell failure to compensate for insulin resistance. The GLP-1 analogue, Exenatide (EXE) improves glycemic control in T2DM by enhancing insulin secretion. However, the effect of continuous chronic EXE infusion on α and β-cell function has been less studied. We examined the effect of a continuous IV infusion (via tether system) of: i) EXE (0.014 ug/kg.h, n=12) or ii) saline (SAL, n=12) on Despite comparable glycemic control with GLA, SU or DDP4 treatment, α and β-cell function in non-diabetic baboons (NDB). At baseline, baboons treatment with GLA or DPP4 resulted in relief of beta cell stress compared received a two-step Hyperglycemic (+125 and +225 mg/dl) Clamp followed with SU as refl ected by lower levels of intact proinsulin and insulin. Obesity by an IV Arginine bolus (0.15 g/kg) (HC+A). Immediately after HC+A baboons POSTERS Supported by: sanofi -aventis underwent a partial pancreatectomy (∼30% total mass) and started EXE or SAL infusion for 13 weeks. At the end of treatment, EXE infusion was 1640-P Integrated Physiology/ stopped for 72 h, HC+A was repeated and remnant pancreas was collected. Effect of Salsalate on Insulin Secretion and Clearance in Individuals Insulin sensitivity (IS) was measured as the glucose infusion rate per unit of with Impaired Glucose Metabolism insulin (M/I) during steady state conditions, and insulin secretory rate (ISR) JURAJ KOSKA, ALLISON B. GOLDFINE, PAUL R. CONLIN, DAWN C. SCHWENKE, was calculated by deconvoluting C-Peptide during the clamp. β-cell function FLORENCIA HALPERIN, CHRISTIAN MEYER, STEVEN E. SHOELSON, PETER D. (Disposition Index=DI) was calculated as ISR*M/I, α-cell function as % REAVEN, Phoenix, AZ, Boston, MA suppression of glucagon secretion from baseline. There were no signifi cant Impaired insulin secretion plays a key role in transition from impaired changes in food consumption, body weight and composition between the glucose metabolism (IFG and IGT) to type 2 diabetes. Previous smaller short-term studies have shown that the glucose-lowering effect of anti- EXE and SAL groups. M/I increased by ∼24% after EXE (p<0.06) and did not change after SAL. ISR increased signifi cantly in SAL group (Basal-ISR 2.0±0.3 infl ammatory doses of salicylates is accompanied by increased insulin [Pre] to 4.3±1.0 [Post] pmol/min/g pancreas, p<0.04, Total-ISR 5.6±0.4 to (I) levels. It remains uncertain however, whether this increase refl ects 7.7±1.1 nmol/g pancreas, p=0.08) but not in EXE (Basal-ISR 3.0±0.7 to 4.5±1.1 augmented β-cell function and/or changes in I clearance.

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A446 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

As part of a randomized, placebo-controlled study of salsalate (SAL) (Φd), to the rate of increase of glucose. The above basal insulin secretion and glucose metabolism in patients with IFG and IGT, we investigated is then linearly modulated by GLP-1 concentration, through parameter whether treatment with a high dose of SAL for 3 months would modulate (π), which represents the percent increase in secretion due to 1 pmol/l I and C-peptide (C) responses to a 75-g oral glucose tolerance test (OGTT), of circulating GLP-1, i.e. the GLP-1 induced potentiation. The model was and metabolic clearance of insulin (MCI) during both OGTT and euglycemic- tested on 22 subjects with Impaired Fasting Glucose (age=54.7±1.8 years, hyperinsulinemic clamp (EHC). Present analyses include data from 49 subjects BMI=32.9±1.2 kg/m2). Subjects were studied on 2 occasions with a standard (24 SAL/25 placebo) with follow-up OGTTs performed 2 and 3 months (Mo mixed meal. After a baseline meal study, subjects were randomized to 2 and Mo 3) after randomization and from 57 subjects (28 SAL/29 placebo) sitagliptin 100 mg or placebo taken daily over an 8-week treatment period. with follow-up EHC performed at study-end. Participants underwent a second meal study at the end of the treatment SAL reduced fasting glucose (Gluc) (11%, p=0.0001), did not change fasting I, period. Treatment with sitagliptin resulted in a slight decrease in total GLP- but reduced fasting C (31% Mo 2, p<0.0001; 19% Mo 3, p=0.003). Concurrently, 1 concentrations (AUC 7218±369 vs. 6759±308 pmol per 6 hr, p=6.2·10-2), the fasting MCI(=I/C) was decreased by SAL (29% Mo 2, p=0.0002; 27% Mo but did not alter fasting or postprandial glucose (AUC 3681±142 vs. 3543±84 3, p=0.002). Reductions in fasting Gluc accounted for 20% of the decline in mmol per 6 hr, p=1.1·10-2). C-peptide concentrations (AUC 956±78 vs. 954±67 fasting C. MCI was also delayed during the EHC (by 12%, p=0.05). The overall mmol per 6 hr, p=1.8·10-4) were also unchanged. The model described the Gluc, I or C responses (incremental area under the curve) during OGTTs were C-peptide data and provided a precise estimate of model parameters not signifi cantly changed by SAL. However, the acute I response from the including π (7.1±2.1 vs 8.1±1.8% per pmol/l, CV 24±4%). The oral GLP-1 model OGTT [insulinogenic index (IGI)=ΔI0-30min/ΔGluc0-30min] was higher at Mo 2 (57%, described here enables quantifi cation of GLP-1’s ability to potentiate insulin p=0.003) but less elevated at Mo 3 (18%, p=0.4). secretion from an oral test. In summary, treatment with SAL was associated with reduced I secretion Supported by: NIH DK-82396, DK-78646 in the fasting state, an increase in acute I response to a glucose load and a reduction in I clearance during both fasting and EHC. High therapeutic 1643-P doses of salicylates may help preserve insulin levels in individuals at risk for Fasting Glucose Does Not Mediate Hyperinsulinemic Compensation developing reduced β-cell function and type 2 diabetes. during Diet-Induced Insulin Resistance in Healthy Dogs Supported by: VA Merit Award MARILYN ADER, DARKO STEFANOVSKI, STELLA P. KIM, JOYCE M. RICHEY, VIORICA IONUT, JUSTIN DITTMANN, KARYN J. CATALANO, ISABEL R. HSU, 1641-P JENNY D. CHIU, ORISON WOOLCOTT, LISA N. HARRISON, DAN ZHENG, MAYA Effects of Alpha-Lipoic Acid on Lipid-Induced Insulin Resistance LOTTATI, CATHRYN M. KOLKA, SOPHIA YAE, HELEN LIU, ANA VALERIA B. and Beta-Cell Dysfunction in Obese and Overweight Non-Diabetic CASTRO, MORVARID KABIR, RICHARD N. BERGMAN, Los Angeles, CA Men Development of insulin resistance (IR) engenders a compensatory CHANGTING XIAO, ADRIA GIACCA, GARY F. LEWIS, Toronto, ON, Canada increase in plasma insulin in healthy subjects, and inadequate compensation Prolonged elevation of plasma free fatty acids (FFAs) induces insulin is considered a primary element in the pathogenesis of Type 2 diabetes. resistance and impairs pancreatic beta-cell adaptation to insulin resistance. The signal which heralds developing IR and initiates hyperinsulinemic The mechanisms whereby lipid induces these impairments are not fully compensation is unknown, but is widely assumed to be glucose. If true, defi ned, but may involve oxidative stress, infl ammation and endoplasmic development of IR must be accompanied by elevations in plasma glucose. reticulum stress. Alpha-lipoic acid (ALA), a commonly used health supplement We tested this supposition in the canine model of obesity. Normal, healthy with anti-oxidant, anti-infl ammatory and AMPK modulating properties, has male dogs (n=58) were fed a hypercaloric, fat-supplemented diet (+ 6 g/kg been shown to have some therapeutic value in type 2 diabetes and its per day) for 6 wks. Before and after dietary intervention, dogs underwent complications. Here we examined the effects of ALA on insulin sensitivity MRI to quantify total and regional (visceral, subcutaneous) adiposity and and beta-cell function in humans in the presence and absence of 24-h iv euglycemic hyperinsulinemic clamps to quantify total, peripheral, and lipid infusion administered to elevate plasma FFAs. Six male subjects (age hepatic insulin sensitivity. A subset of dogs (n=36) also underwent an = 46.6+/-3.3 years, BMI = 30.3+/-0.6 kg/m2) underwent 4 studies each, 4-6 insulin-modifi ed IVGTT to assess both sensitivity (using minimal model) and weeks apart, in random order: 1) SAL, 2 week oral placebo followed by acute insulin response to glucose (AIRg). Fat feeding caused modest, but 24-h iv infusion of saline; 2) IH, 2 week placebo followed by 24-h iv infusion signifi cant weight gain (28.8±0.4 to 31.2±0.5 kg; p<0.0001), but substantial of Intralipid plus heparin to raise plasma FFAs by approximately 2-fold; 3) increase in adiposity. Total adiposity expanded by 60±5%, refl ecting IH+ALA, 2 week ALA (1800 mg/day, divided into three equal doses) followed increases in both visceral (421±18 to 585±24 cm3) and subcutaneous (238±14 by 24-h infusion of Intralipid plus heparin; and 4) ALA, 2 week of ALA to 428±23 cm3) fat (p<0.0001 for all). We observed diet-induced IR in clamps followed by 24-h infusion of saline. Insulin secretion rates (ISR) and insulin (36.4±1.9 to 24.6±1.4 dl/min per kg per μU/ml, p<0.0001), at both peripheral sensitivity (SI) were assessed with a 2-h, 20-mmol/l hyperglycemic clamp and hepatic level (-84±35% and -85±31%, p<0.0001); IR was confi rmed by and a hyperinsulinemic-euglycemic clamp, respectively, after the above IVGTT (p<0.0001). Hyperinsulinemic compensation was observed in fasting 24-h infusions. ISR was not signifi cantly different between treatments. Lipid levels (10±1 to 13±1 μ/ml, p<0.0001) and increased AIRg (609±38 to 845±51 infusion impaired insulin sensitivity, with and without ALA pre-treatment. (μU/ml, p<0.0001). However, we observed no increase in fasting glucose Beta-cell function (disposition index) was similarly impaired by lipid infusion (pre-fat: 92.3±1.0, post-fat: 92.2±1.0 mg/dl, p=0.858). These data echo our in IH (IH= 63% of SAL, P<0.05) and IH+ALA (IH+ALA = 70% of SAL, P<0.05). prior studies in which diet-induced IR and hyperinsulinemia occurs in absence These results indicate that ALA, administered orally at this dose for 2 weeks, of any elevation in 24-hour glucose levels. In conclusion, large prospective does not protect against lipid-induced insulin resistance and beta-cell studies in insulin resistant dogs indicate that fasting glucose cannot be the dysfunction in overweight and obese humans. signal for hyperinsulinemic compensation. An alternative viable candidate mediating β-cell compensation could be nocturnally increased plasma free 1642-P fatty acids and/or leptin. ADA-Funded Research Estimation of GLP-1 Induced Potentiation of Insulin Secretion from an Oral Model 1644-P

FRANCESCO MICHELETTO, CHIARA DALLA MAN, ADRIAN VELLA, CLAUDIO Glucose Dependent Insulinotropic Peptide (GIP) Stimulates Insulin Obesity

COBELLI, Padua, Italy, Rochester, MN Secretion in Patients with Well-Controlled Type 2 Diabetes Mellitus: POSTERS Glucagon Like-Peptide-1 (GLP-1) is an important insulin secretagogue. Effects of Metformin and Gender

GLP-1-based therapies have been effective in improving glycemic control WILLIAM G. THARP, OLGA SIDELEVA, DARIUSH ELAHI, COURTNEY LEDGER, Integrated Physiology/ in type 2 diabetes. Recently we proposed a quantitative model of GLP- RHONDA MAPLE, RICHARD PRATLEY, Burlington, VT, Baltimore, MD 1 action on insulin secretion using data from a hyperglycemic clamp with Impaired incretin hormone signaling contributes to hyperglycemia in concomitant GLP-1 infusion. However, such methodology is not applicable T2DM. GIP stimulated insulin secretion (GIP-SIS) is markedly blunted in to studies utilizing an oral challenge. Hence we developed a model enabling patients with T2DM, but can be restored with intensive insulin therapy that quantitation of pancreatic response to GLP-1 secretion in response to a corrects hyperglycemia. Recently, metformin was shown to elevate GIP mixed meal. The model assumes that the above basal insulin secretion is receptor expression in murine islets and increase GIP-SIS in a β-cell line. made up of a static (SRs), and a dynamic (SRd) component. SRs is assumed We hypothesized that GIP-SIS would be enhanced in patients with T2DM equal to the provision of releasable insulin to β-cells, proportional, through treated with metformin. Subjects with well controlled T2DM (A1c<7%) on parameter (Φs), to glucose concentration. SRd represents the secretion of diet and exercise (D+E) or a stable dose of metformin (MET) were recruited insulin from the promptly releasable pool, proportional, through parameter and underwent a frequently-sampled IVGTT followed by a graded glucose

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A447 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

infusion and 30 min GIP infusion (2 pmol/kg/min). The acute insulin response to glucose (AIRg) and insulin sensitivity (Si) were derived from the Minimal Model, β-cell sensitivity to glucose (GSIS) was calculated as the slope of insulin to glucose during the glucose infusion, and GIP-SIS as the increment in insulin secretion above GSIS during the GIP infusion. Twenty-four subjects were studied (Table). There were no differences in A1C between treatments, but BMI was higher in the MET group than D+E (p=0.033) and higher in women than men (38.4±8.6 vs 28.4±3.4, p=0.001). AIRg, Si, and GIP-SIS did not differ by treatment, but GSIS was ∼3-fold higher in subjects on MET after controlling for AIRg and BMI (p=0.044, r2=0.70). GIP-SIS correlated with AIRg (p<0.001), but not GSIS. In multivariate analyses GIP-SIS was not associated with MET after controlling for AIRg, BMI, gender, and Si. In this model, gender was the only signifi cant predictor of GIP-SIS (p=0.023; r2=0.70). These data suggest that metformin enhances β-cell sensitivity to glucose without enhancing GIP-mediated incretin effects in people with well-controlled T2DM. The data also suggest that the incretin response to GIP may differ by gender, a potential effect that requires further exploration. 1647-P M/F BMI A1C AIRg(pM*min) Si(pM*min-1/10-5) GSIS(pM/mM) GIP-SIS(pM) Mammalian Target of Rapamycin Is Required for Insulin Secretion MET 7/8 35.5±9.3 6.3±0.5 30.6±10.5 2.35±0.38 0.48±0.13 40.8±10.6 in Pancreatic β-Cells JONGSUN PARK, SUNG HEE UM, JISOO PARK, DAVID D’ALESSIO, CHRISTOPHER B. D+E 5/4 30.0±4.6 6.6±0.4 21.6±14.4 2.95±0.71 0.16±0.04 30.5±7.5 NEWGARD, SARA C. KOZMA, Cincinnati, OH, Suwon, Republic of Korea, Durham, NC Supported by: Takeda Pharmaceuticals; NIDDK Defects in pancreatic β-cell function underlie the pathology of type 1 and 2 diabetes. In mammals, glucose homeostasis is maintained through insulin 1645-P mediated signaling events, including regulation of the insulin receptor, insulin Insulin Resistance Is Linked to AGEs Via SIRT1 Down-Regulation receptor substrate and phosphatidylinositol 3-kinase (PI3K). The importance WEIJING CAI, LI ZHU, XUE CHWITHDRAWNEN, GARY E. STRIKER, HELEN VLASSARA, New class 1 PI3K transduction pathway in β-cell sensing of nutrients, insulin York, NY secretion, and growth was revealed in studies in mice defi cient for specifi c Increased chronic infl ammation and oxidant stress (OS) contribute to signaling components. Consistent with these fi ndings, a key component in Insulin Resistance (IR). Elevated intracellular AGEs in human T2D may this pathway, the mammalian Target Of Rapamycin (mTOR), acts to integrate contribute to IR via a pro-infl ammatory mechanism which remains unclear. signals from nutrients, cellular energy status and insulin to control cell SIRT1, a de-acetylase and key regulator of infl ammatory and metabolic growth. The potential importance of mTOR Complex1 signaling in β cells is processes, is suppressed in diabetes, as is AGER1, an anti-AGE receptor. underscored by their exquisite sensitivity to both insulin and nutrients. We hypothesized that excessive AGEs in insulin-sensitive tissues neutralize To evaluate the impact of mTOR signaling in pancreatic β-cell function, AGER1, raising intracellular OS. It is known that OS suppresses SIRT1 and we generated mTOR fl oxed mice, in which the mTOR gene could be deleted + - promotes IR. in β-cells (mTORfl /fl RipCre mice and control mTORfl /fl RipCre mice). To control In vitro studies: Stimulation of differentiated 3T3-L1 adipocytes by for the effects of the Rip promoter, we also analyzed cohorts of wild type a chemically characterized AGE, methyl-glyoxal-BSA (MG) led to time- (C57BL/6) and Rip-cre transgenic mice. To determine whether in vivo effects dependent suppression of both AGER1 and SIRT1 expression. These of mTOR deletion were mediated by mTORC1 or mTORC2, we examined JNK-activation dependent changes were associated with: a. increased glucose-stimulated insulin secretion (GSIS) in the rat insulinoma INS-1- NF-kB p65 acetylation, b. decreased insulin receptor (InsR) and IRS-1 Tyr- derived β-cell line 832/3, transfected with the human pro-insulin gene and phosphorylation, c. increased IRS-1 and IRS-2 Ser-phosphorylation, and d. selected for their capacity to secrete insulin. In this case we used siRNAs to impaired insulin-stimulated 3H-glucose uptake. These effects were blocked deplete either raptor or rictor, specifi c molecular components of the mTORC1 by AGER1+ transduction and by anti-oxidants (N-acetyl-cystein, Apocynin), or mTORC2, respectively. but were restored after AGER1 mRNA silencing studies. In brief, the phenotype of the β-cell mTOR knockout mice is characterized In vivo studies: C57BL/6 mice were fed from birth an MG-rich diet (1mg/g) by glucose intolerance, loss of insulin secretion in response to glucose and (n=12). At 16 mos of age they had suppressed AGER1 and SIRT1 protein levels L-arginine as well as reduced islets number, which becomes more pronounced in fat, skeletal muscle and liver (by 60-80%, p<0.05), and high serum AGEs with age and diet. Like for mTOR, depletion of raptor, but not rictor, reduces (x2, p<0.04), compared to age-matched AGE-restricted mice. MG-fed mice strongly glucose induced insulin secretion in the 832/3 cell line. also had hyperinsulinemia (p<0.02), impaired glucose tolerance (by 37-50%, These results demonstrate that mTORC1-signaling controls pancreatic p<.03), high leptin (p<0.01) and low adiponectin (p<0.05), whereas AGE- islets development and regulates insulin secretion in β-cells. restricted mice remained normal. First two authors contributed equally to this work Conclusions: AGEs may promote IR, in part via OS-induced reduction Supported by: JDRF Grant (1-2009-293) of AGER1 and SIRT-1 in mice. AGE-restriction may protect against IR by maintaining SIRT-1 expression. 1648-P Supported by: AG-23188 and AG-09453 Overexpression of Glutathione Peroxidase 4 (GPX-4) Prevents Fat- Induced β-Cell Dysfunction In Vivo 1646-P KHAJAG KOULAJIAN, TEJAS DESAI, QITAO RAN, ADRIA GIACCA, Toronto, ON, Canada, San Antonio, TX WITHDRAWN We have previously shown that cytosolic superoxide plays a role in the decrease in β-cell secretory function induced by prolonged exposure to free

Obesity fatty acids (FFA) in vivo. However, since the antioxidants N-acetylcysteine

POSTERS (NAC) and taurine, which do not decrease superoxide, were effective in restoring β-cell function, other reactive oxygen species (ROS) are also

Integrated Physiology/ implicated. These ROS are likely Dichlorofl uorescein (DCF) positive, as DCF positive ROS were increased by oleate and normalized by NAC and taurine. Candidates are lipid peroxides which are detected by DCF and are known to cause impaired insulin secretion in vitro. To test the hypothesis that lipid peroxides play a causal role in fat-induced β-cell dysfunction in vivo, we used transgenic mice which overexpress the enzyme glutathione peroxidase 4 (GPX-4), a membrane enzyme which selectively decreases lipid peroxides. Chronically cannulated GPX-4 overexpressing mice (TG) and their littermate controls (WT) were intravenously infused with saline or oleate (0.3μmol/ min) for 48h, after which islets were isolated and incubated at 6.5 and 22 mM glucose. 48h FFA elevation impaired β-cell function in isolated islets of

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A448 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

WT mice compared to islets of saline infused WT mice. In contrast, islets of did not display any difference in glucose tolerance, b-cell Bmal1 female KO TG mice subjected to 48h elevation of FFA had no signifi cant impairment in displayed a signifi cant worsening. β-cell function compared to islets of saline infused mice (Insulin Release in These data indicate that female Bmal1 defi cient mice are protected from pmol/islet/h WT Saline: 0.27±0.03; WT Oleate: 0.14±0.03, p<0.01 vs. WT Saline; the metabolic derangements in glucose metabolism resulting from a disruption TG Oleate: 0.22±0.05, NS vs. WT Saline; TG Saline: 0.24±0.1, NS vs. WT Saline). of the circadian clock. Understanding the interplay of hormonal secretion and Thus overexpression of GPX-4 prevents the decrease in secretory capacity circadian clock will help in devising therapies to mitigate the increased risk of of the β-cell, suggesting that lipid peroxides play a causal role in fat-induced diabetes seen with circadian misalignment in women on shift work. β-cell dysfunction. 1651-P 1649-P Profound Impairment of β-Cell Morphology and Function in a Novel Predictive Model for Polycystic Ovary Syndrome Includes Risk Rat Model of Malnutrition Factors for Type 2 Diabetes and Obesity ROBERT H.J. BANDSMA, CHANGTING XIAO, CAMERON ACKERLEY, LINDA SZETO, MARKETA VANKOVA, JOSEF VCELAK, PETRA LUKASOVA, OLGA BRADNOVA, ADRIA GIACCA, GARY F. LEWIS, Groningen, The Netherlands, Toronto, ON, Canada MARTIN HILL, JANA VRBIKOVA, BELA BENDLOVA, Prague, Czech Republic Severe malnutrition is a major cause of global childhood mortality. It is Polycystic ovary syndrome (PCOS) is considered as a risk factor for type 2 associated with marked metabolic changes, including hypoalbuminemia, diabetes (T2D). It is often associated with obesity, beta-cell dysfunction or hepatic steatosis, oxidative stress and high mortality upon re-feeding. We insulin resistance but the genetic factors underlying the association of PCOS recently showed impaired glucose clearance related decreased insulin with T2D or obesity are unknown. secretion in malnourished children. There is a paucity of mechanistic studies The aim was to fi nd relevant parameters that determine the PCOS status examining the metabolic disturbances in this disorder, in part because a and are common for T2D and obesity. We evaluated simultaneously the well-characterized animal model is lacking. We created a novel rat model of beta and alpha-cell function by arginine secretion test, insulin sensitivity by post-weaning malnutrition and evaluated pancreatic β-cell function in vivo. euglycemic hyperinsulinemic clamp and both independently by oral glucose Rats were divided into 5 groups af ter weaning: 1. diet containing 4% protein tolerance test (OGTT) in 73 PCOS patients and 25 controls (BMI 26.9±5.4 (4%PROT), 2. diet containing 4% protein plus low dose lipopolysacharide (LPS, vs. 25.5±5.6 kg/m2; NS). The steroid, lipid spectra and anthropometric 2 mg/kg) biweekly two weeks into the diet (4%PROT+LPS), 3. 18% protein parameters were studied. The 34 SNPs of T2D and obesity candidate genes (18%PROT), 4. 18% protein plus LPS (18%PROT+LPS), and 5. normal chow were assessed. For simultaneous evaluation of relationships between PCOS diet (CONT). The 4% and 18% protein diets were otherwise matched and status and monitored parameters, a multivariate regression with reduction animals were pair-fed. After 4 weeks on the diets, a 17mmol/l hyperglycemic of dimensionality was used (Orthogonal Projections to Latent Structures- clamp was performed. OPLS; software SIMCA-P+ 12.0.0.0). Rats on the 4% protein diet, with and without LPS, had signifi cantly lower Using the OPLS method, we detected the positive relationships body weight, hypoalbuminemia and hepatic steatosis compared to the other between the PCOS status and parameters of insulin secretion (area under animals. During the clamp, glucose infusion rate was lower in 4%PROT+LPS curve of insulin - OGTT), body composition (WHR), steroids (total and (54±6 mg/kg/min), compared to 74±11, 88±6, 76±5 and 71±5 mg/kg/min in free testosterone, 17-hydroxyprogesterone) and family history of T2D. the 4%PROT, 18%PROT, 18%PROT+LPS and CONTR, respectively (P<0.05), The negative relationship was identifi ed between PCOS status and HDL- indicating impaired glucose homeostasis in the 4%PROT+LPS treated cholesterol. Positive relationships between PCOS status and major allele group. C-peptide concentration tended to be lower in 0.36±11 nmol/l in (recesive model) of Neurogenin 3 (Ngn3, rs4536103) and of minor allele 4%PROT+LPS vs 0.55±16 nmol/l in 4% and was signifi cantly lower (p<0.05) (dominant model) of Uncoupling protein 1 (UCP1, rs1800592) were observed. than C-peptide in 18%PROT and 18%PROT+LPS (1.03±0.28 and 0.75±0.09 In conclusion, PCOS status is primarily characterized by hyperandro- nmol/l, respectively) or CONTR (0.91±0.17 nmol/l). β-cells contained less genaemia but it shares common features with T2D and obesity such as dense insulin containing granules and showed mitochondrial abnormalities visceral fat accumulation, lower HDL-cholesterol and impaired insulin in 4%PROT and 4%PROT+LPS. secretion. However, hyperinsulinemia was related only to OGTT, not to We provide evidence that a low protein diet in young rats leads to a arginine test. Ngn3 variant plays a role in insulin secretion and UCP1 variant susceptibility for endotoxin induced impairment in glucose homeostasis, in energy dissipation and fat metabolism thus both genes could contribute related to decreased insulin secretion. The observation of β-cell dysfunction to the PCOS development. in malnutrition could lead to a re-evaluation of current treatment guidelines Supported by: IGA MHCR NS/9839-4, IGA MHCR NS/10209-3/2009 to improve survival. Supported by: Canadian Institute for Health Research 1650-P 1652-P Preserved B-Cell Function Protects Female Mice from Glucose Simultaneous Measurement of Insulin Sensitivity, Insulin Secretion Intolerance in the Absence of the Circadian Clock and the Disposition Index in Conscious Mice MI-SUN KIM, JEONGKYUNG LEE, RONYING LI, VICTORIA LIU, KE MA, DAVID D. LAURA C. ALONSO, YOSHIO WATANABE, DARKO STEFANOVSKI, EUHAN J. LEE, MOORE, VIJAY YECHOOR, Houston, TX SRIKANTH SINGAMSETTY, LIA C. ROMANO, BAOBO ZOU, ADOLFO GARCIA- Women on shift work who experience a disruption of their circadian OCANA, RICHARD N. BERGMAN, CHRISTOPHER P. O’DONNELL, Pittsburgh, PA, rhythm have elevated prevalence and incidence of diabetes. Dysregulation Los Angeles, CA of circadian rhythm results in disorders of glucose and lipid metabolism, Of the parameters that predict progression to diabetes in humans, fi rst- and insulin homeostasis at both physiological and molecular levels. While phase insulin secretion (AIRg), glucose effectiveness (Sg), insulin sensitivity the regulatory role of the circadian clock in islet function and glucose (Si), and the disposition index (DI), only Si can be reliably measured in metabolism is just being investigated, the molecular mechanism underlying conscious mice using currently available techniques. To measure DI, AIRg and the interactions of estrogen and progesterone and the circadian clock in the Sg, we developed the frequently sampled intravenous glucose tolerance test context of islet function and glucose metabolism remain entirely unknown. (FSIVGTT) for conscious mice. Adult male C57BL/6J, C57BL/6NTac, or Ob/Ob To explore this mechanism, we analyzed female mice defi cient in a key mice with arterial and venous catheters underwent FSIVGTT performed by

circadian regulator, Bmal1 (bHLH-PAS containing trascription factor). sampling arterial blood for glucose (and insulin) at (–10), (0), (1), (2), 3, (4), 5, Obesity

Bmal1 global knockout mice (global Bmal1 KO) are arrhythmic and display 6, (8), 10, (12), 14, (16), 18, (20), 25, (30), 40, 50, and (60) min after a 1 g/kg iv POSTERS a complete lack of circadian behavior in the absence of light entrainment. bolus of D50 at time 0.

We previously have shown that male Bmal1 KO mice display diabetes, Integrated Physiology/ hypoinsulinemia secondary to impaired glucose-stimulated insulin secretion (GSIS). In contrast to male mice, female Bmal1 KO mice on a normal diet are euglycemic with normal plasma insulin levels. However, female mice fed a high-fat diet display signifi cant glucose intolerance and signifi cantly impaired GSIS in vivo. Intriguingly, both female and male b-cell specifi c Bmal1 knockout mice (b-cell Bmal1 KO) display diabetes on normal diet, in contrast tothe global Bmal1 KO. Since global Bmal1 KO mice have abnormalities in estrogen and progesterone levels, to fully explore the interaction of estrogen and progesterone with the circadian clock in b-cells we analyzed ovarectomized (OVX) b-cell Bmal1 femal KO. In contrast to the wild type controls which

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A449 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

Conscious hyperinsulinemic euglycemic clamp was performed in 1654-P a separate set of Ob/Ob mice or lean controls. Si, AIRg, Sg and DI were The New Multiple Kglucose Analysis Program (MultiKgluc) Improves calculated by Minimal Model analysis of FSIVGTT data. Validating FSIVGTT the Effi ciency and Predictive Power for Overt Diabetes of the against the gold standard euglycemic clamp, the FSIVGTT-derived Si of lean Intravenous Glucose Tolerance Test (IVGTT) over Prior Methods for vs. Ob/Ob mice (10.6±.19 vs. 1.3±0.5 mU/ml/min, p=0.001) was equivalent to Assessment of Glucose Uptake Rate and Insulin Secretion/Action clamp glucose infusion rates (38.7±0.8 vs. 6.0±0.2 mg/kg/min, p<0.0001). BARBARA C. HANSEN, Tampa, FL AIRg was negatively correlated with AUC glucose (r2=0.63, p<0.0001), Both the euglycemic clamp and the minimal model approaches require suggesting that fi rst-phase insulin secretion does impact glucose disposal many samples obtained over two to three hours, and are costly and of minimal in rodents, a concept that has been disputed. Like obese humans, Ob/ob use in the usual clinical setting or in large scale studies. HOMA, QUICKI mice had increased AIRg (14265±726 vs. 9087±3.6, p<0.001), decreased and other single point approaches lack diabetes predictive validity within Sg (0.046±0.01 vs. 0.140±0.02 1/min, p<0.05), and decreased DI (1563±522 subjects. The present study was undertaken in order to improve diabetes vs. 7228±1227, p<0.01) relative to lean controls. These data will allow risk prediction by establishing a more reliable, effective, and effi cient application of mouse genetics to the determinants of AIRg, Sg and DI, and method of assessing glucose tolerance/insulin action and the interactions validate the conscious mouse as a model of glucose metabolism in normal of these with insulin secretion. Glucose uptake rates and insulin secretory and insulin resistant states. profi les following an intravenous glucose bolus were determined in 108 Supported by: HL063767 (COD), DK076562 (LCA), DK29867 and DK27619 (RNB). subjects maintained under uniform dietary and environmental conditions, with repeated measures in all subjects. Experiments had durations of 30 1653-P min to 3 hr, and were undertaken using nonhuman primates. Such primates The New GLP-1-Gastrin Dual Agonist ZP3022 Prevents Diabetes in are ideal subjects because the glucose tolerance and insulin secretory db/db Mice profi les of nonhuman primates are extraordinarily similar to those of KELD FOSGERAU, JAKOB L. TOLBORG, TORBEN ØSTERLUND, CHRISTIAN THORK- humans, with both species progressing naturally and spontaneously to the ILDSEN, CHRISTIAN GROENDAHL, TRINE S.R. NEERUP, Glostrup, Denmark most common form of type 2 diabetes. The MultiKgluc program determined Administration of a GLP-1 agonist in combination with Gastrin has been 8 log disappearance rates combined with the insulin secretion profi le per shown to increase beta-cell mass and improve glycemic control in diabetic experiment and produced valid and reliable estimates of progression of DM animals. Peptide ZP3022 is a novel and potent GLP-1-Gastrin dual agonist risk based only on samples obtained during the fi rst 30 minutes of an IVGTT. It being developed at Zealand Pharma A/S. further readily identifi ed any experimental deviations, thus assuring optimal In Vitro: Potency of peptides on the human GLP-1 (hGLP-1R) and Gastrin validation. Through sequential analyses, the data demonstrated the lack of (hCCKB-R) receptors was assayed in HEK293 cells stably expressing one of usefulness of samples obtained beyond 30 min, as all critical and predictive the receptors as cAMP and ERK phosphorylation formation, respectively. information was contained in the fi rst 30 min. Data further indicated that βcell secretory function should not be imputed to refl ect changes in insulin Table 1. Potency of peptides on the GLP-1 and the Gastrin receptors sensitivity. Within subject diabetes risk prediction is greatly enhanced by Substance hGLP-1R hCCKB-R this new, effi cient and cost effective analytical approach. (EC50±SD, nM) (EC50±SD, nM) Supported by: NIH NO1AG21012 and HHSN263200800022C Exendin-4 (n=6) 0.025±0.004 >1000 Liraglutide (n=2) 0.18±0.07 >1000 ZP3022 (n=2) 0.03±0.01 11.5±1.5 INTEGRATED PHYSIOLOGY—LIVER

In Vivo: 130 female db/db mice aged 6-7 weeks were used. Prevention: [See also: Presidents Posters 453-PP to 454-PP, page A126.] Animals were treated once daily for 93 days with vehicle or 100 nmol/kg of Exendin-4, Liraglutide or ZP3022. Treatment: Animals were administered vehicle for the fi rst 50 days before entering peptide treatment as described Guided Audio Tour: Liver—Animal Models (Posters 1655-P to 1662-P), above for 43 days. Drug holiday: Animals were treated with peptides for 50 see page 13. days as described above and then continued on vehicle for 43 days. Oral glucose tolerance tests (OGTTs) were performed at on-set (day 0) and days & 1655-P 50 (re-stratifi cation), 65, 78 and 91 in fasted mice. Figure 1 displays the Genetic Inactivation of Toll-Like Receptor 4 Alleviates Diet-Induced result from the repeated OGTTs as illustrated by the area under the curves. Nonalcoholic Steatohepatitis by Inhibiting Reactive Oxygen Species- We observed a signifi cant (p<0.001) improvement in glucose clearance by Evoked Activation of X-box Binding Protein-1 in ApoE-Defi cient Mice treatment with the GLP-1-Gastrin dual agonist compared to vehicle control DEWEI YE, FRANCOIS Y.L. LI., KAREN S.L. LAM, YU WANG, AIMIN XU, Hong Kong, regardless whether the treatment modality was prevention, treatment, China or drug holiday. This effect of the GLP-1-Gastrin dual agonist was most Nonalcoholic steatohepatitis (NASH) is the hepatic manifestation of pronounced. Accordingly, we observed in the drug holiday study that the metabolic syndrome characterized by steatosis and necroinfl ammation. effect of the GLP-1-Gastrin dual agonist was maintained weeks after the However, the precise mechanism underlying the progression from steatosis treatment was stopped, a fi nding we observed with the dual agonist ZP3022 to infl ammation remains poorly understood. Here we investigated the role of only and not for the two pure GLP-1 agonists. Toll like receptor 4 (TLR4), a key player in innate immunity and infl ammation, in the pathogenesis of NASH associated with obesity. We generated ApoE-/-/TLR4-/- mice and ApoE-/-/TLR wild type mice (ApoE-/-/TLR-WT) by cross-breeding the ApoE defi cient (ApoE-/-) strain with TLR4-defi cient mice, which were then fed a high fat high cholesterol (HFHC) diet for 12 weeks to induce obesity. The results showed that HFHC diet feeding induced typical symptoms of NASH in ApoE-/- mice, including elevated serum level

Obesity of Alanine aminotransferase and increased NASH score, and this change

POSTERS was accompanied by augmented expression of TLR4 in Kupffer cells. TLR4 inactivation protected against HFHC diet-induced liver injury and reduced -/- -/- -/- Integrated Physiology/ hepatic lipid contents in ApoE mice. Furthermore, ApoE /TLR4 displayed a signifi cant reduction in hepatic resident of macrophages and were less susceptible to diet-induced production of reactive oxygen species (ROS) and proinfl ammatory cytokines. In ApoE-/-/TLR4-/- mice on HFHC diet, the splicing of X-box binding protein-1 (XBP-1), a transcription factor involved in ER stress and infl ammation, was repressed in the liver tissue compared to those in ApoE-/-/TLR4-WT mice. Ex vivo experiments in primary rat Kupffer cells showed that treatment with antioxidants signifi cantly abrogated endotoxin- induced mRNA splicing of XBP-1 and attenuated production of cytokines. Furthermore, endotoxin-induced production of cytokines and activation of NF-κB were signifi cantly blunted by siRNA-mediated knocking down XBP-1

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A450 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER expression. These fi ndings suggest that TLR4 in Kupffer cells mediates the and mitochondrial β-oxidation of fatty acids, 2) PPARγ effects outside the progression from simple steatosis to infl ammation, partly by inducing the liver also impact hepatic β-oxidation, and 3) liver PPARγ may exert signifi cant production of ROS, thereby leading to the activation of XBP-1. effects upon skeletal muscle insulin sensitivity, even in the absence of Supported by: Hong Kong Research Grant Council (HKU 5/CRF/08 and HKU liver fat. We conclude that liver PPARγ may have important therapeutic 2/07C) implications for liver complications found in obesity and diabetes.

& 1656-P & 1658-P Liver-Specifi c Deletion of JAK2 Leads to Profound Fatty Liver but Rat Krueppel-Like Factor 10 (Klf-10) Gene Expression Is Regulated Suppression of Infl ammation and Protection Against Systemic Insulin by the Carbohydrate Response Element Binding Protein ChREBP in Resistance Primary Rat Hepatocytes RUBÉN GARCÍA, SALLY YU SHI, DIANA CHOI, ROBIN E. DUNCAN, SHUN YAN LU, KATSUMI IIZUKA, YUKIO HORIKAWA, REIKO TOMITA, TETSUYA SUWA, JUN STEPHANIE A. SCHROER, ERICA P. CAI, CHRISTINE TANG, ADRIA GIACCA, MARK TAKEDA, Gifu, Japan NAPLES, MARK J. DEKKER, KAY-UWE WAGNER, KHOSROW ADELI, RICHARD P. We previously reported that the carbohydrate response element binding BAZINET, MINNA WOO, Toronto, ON, Canada, Omaha, NE protein ChREBP plays an important role in the regulation of glucose and lipid Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic metabolism by regulating hepatic glycolytic and lipogenic gene expression. manifestation of the metabolic syndrome, a well-recognized precursor for We then established a mouse model infected by an adenovirus expressing many chronic diseases including type 2 diabetes, cardiovascular diseases dominant active ChREBP (daChREBP). Using the DNA microarray technique, and some cancers. NAFLD is a disease spectrum hypothesized to be we identifi ed the Krueppel-like factor 10 (Klf-10) as a candidate for one of induced by a two-step process: the fi rst hit, steatosis, sensitizes the liver the ChREBP target genes. Recently, Klf-10 has been identifi ed as a circadian to the infl ammatory damage induced by a second pathogenic insult, which transcriptional regulator that links the molecular clock to energy metabolism promotes steatohepatitis. The exact pathogenesis of NAFLD, however, is in the liver. Here we investigate whether ChREBP directly regulates Klf-10 not well understood. The Janus kinase-signal transducers and activators of gene expression in rat primary hepatocytes and the pancreatic beta cell transcription (JAK-STAT) pathway is involved in both infl ammatory signalling line INS-1E. Klf-10 mRNA is ubiquitously expressed and highly expressed and in lipid homeostasis in the liver and may contribute to the pathogenesis in muscles and the liver. The hepatic Klf-10 mRNA in ob/ob mice is about of NAFLD. To investigate the role of hepatic JAK2, an essential player in 2-fold higher than that in C57BL/6 mice. In rat primary hepatocytes and the JAK-STAT pathway, we generated mice with deletion of jak2 specifi cally INS-1E cells, glucose stimulation and overexpression of daChREBP induces in hepatocytes (L-JAK2 KO) using the cre-loxP system. L-JAK2 KO mice klf-10 mRNA in a dose-dependent manner. Consistent with these fi ndings, developed spontaneous hepatosteatosis early in life, similar to liver- overexpression of dominant negative Mlx inhibits glucose induction of liver specifi c growth hormone receptor- and STAT5-defi cient mice. However, in type pyruvate kinase (Lpk) and fatty acid synthase (Fasn) mRNA. A deletion contrast to these mice, L-JAK2 KO mice did not develop systemic insulin study of pGL3 vector with rat KLF-10 promoter and a CHIP assay against anti- resistance or glucose intolerance. Interestingly, the fatty acid composition ChREBP antibody demonstrated that the carbohydrate response element of liver triglycerides showed, in addition to increased palmitate, a persistent (ChoRE) is located between -200 and -100 bp in the rat Klf-10 gene promoter. predominance of the less toxic oleate, which was accompanied by reduced In addition, adenoviral overexpression of Klf-10 partly inhibits glucose hepatic infl ammation in L-JAK2 KO mice. Even when challenged with a induction of ChREBP target genes (Lpk and Fasn) in primary hepatocytes. In prolonged high fat diet, these mice were completely protected against conclusion, these fi ndings suggest that ChREBP directly regulates Klf-10 gene systemic infl ammation and insulin resistance, resulting in improved glucose expression at the transcription level and that Klf-10 also weakly contributes tolerance. Together, our fi ndings indicate a critical and unique role of hepatic to regulating ChREBP transactivities. Crosstalk between ChREBP and Klf-10 jak2 in the regulation of fatty acid metabolism and infl ammation, leading is involved in the regulation of the lipogenic pathway. to a complete halt in NAFLD progression and development of systemic insulin resistance in mice lacking JAK2 in hepatocytes. Targeting the JAK- STAT pathway may therefore provide a novel therapeutic strategy for the & 1659-P treatment of type 2 diabetes. Role of Sdf2l1, a Novel ER Stress-Related Protein, in the Regulation Supported by: CIHR; Ibercaja Foundation (Spain); BBDC-Novo Nordisk Student- of Hepatic Insulin Sensitivity ship TAKAYOSHI SASAKO, KOHJIRO UEKI, MITSURU OHSUGI, NAOTO KUBOTA, KAZUYUKI TOBE, TAKASHI KADOWAKI, Tokyo, Japan, Toyama, Japan & 1657-P Dys-regulation of dynamic metabolic changes in liver during the transition Liver-Specifi c Ablation of PPARγ Reduces Hepatic Mitochondrial between fasting and feeding leads to metabolic disorders. To further β-Oxidation and Lipid Accumulation, but Aggravates High Fat Diet- elucidate these changes, we compared global gene expressions in livers Induced Insulin Resistance of B6J mice in 24h-fasted and 6h-refed states by microarray analysis. ANISHA A. GUPTE, LAURIE J. MINZE, JESSICA R. WILES, MARTINEZ JESSICA, Interestingly several ER stress-related genes were up-regulated by ALAN R. COLLINS, CHRISTOPHER J. LYON, WILLA A. HSUEH, Houston, TX refeeding. Among them, we focused on Sdf2l1, a component of chaperone Fatty liver is thought to contribute to insulin resistance and diabetes. complexes, showing the second largest increase of all the genes. Indeed Although peroxisome proliferator-activated receptor-γ (PPARγ) activation hepatic expression of ER stress markers as well as Sdf2l1 was elevated can increase de novo lipogenesis in the liver during high fat diet (HFD), we prominently and transiently by refeeding in B6J mice. In order to explore the found that activation increases β-oxidation, improves liver mitochondrial role of Sdf2l1 in response to ER stress, we performed promoter assay and function and decreases liver fat accumulation (Gupte A, et al, Hepatology, ChIP assay, revealing that Sdf2l1 was regulated by Xbp1 and ATF6, despite 2010). However, very little is known about the role of liver-PPARγ and its absence of conventional ERSE in the promoter region. Since ER stress is effects, if any, upon liver metabolism. We thus generated mice with liver- thought to be a pivotal regulator of hepatic insulin sensitivity, to assess the specifi c PPARγ knockout (L-PPARγKO), and found that L-PPARγKO mice role of Sdf2l1 in this context, we knocked down hepatic Sdf2l1 expression fed obesogenic HFD for 3 months had 50% the liver fat of wild type (WT) by adenovirus-mediated RNA interference. Knocking down of Sdf2l1 in B6J littermates. However, L-PPARγKO had elevated fasting insulin vs. WT mice mice resulted in greater elevation of ER stress markers after refeeding, while and impaired insulin sensitivity when assayed by insulin tolerance test, it impaired glucose tolerance, attenuated insulin signaling after refeeding, Obesity but no differences in plasma triglycerides, phospholipids or free fatty acid and increased triglyceride contents. These prompted us to hypothesize that POSTERS levels. Insulin-stimulated Akt phosphorylation was suppressed in liver, fat dys-regulation of Sdf2l1 might contribute to insulin resistance in obesity. and skeletal muscle of WT mice on HFD vs. chow, but was increased in liver, Indeed in livers of db/db mice, a model of obesity and diabetes, Sdf2l1 was Integrated Physiology/ unchanged in fat, and reduced in skeletal muscle of HFD-fed L-PPARγKO mice down-regulated, accompanied by decreased binding of Xbp1, but not ATF6, vs. WT mice. Thus, L-PPARγKO mice have skeletal muscle insulin resistance, with the promoter region. By contrast, adenovirus-mediated restoration of but decreased liver fat and improved liver insulin sensitivity. Mitochondrial Sdf2l1 in livers of db/db mice improved glucose tolerance, insulin signaling function analyses revealed signifi cant suppression of β-oxidation in the after refeeding, and fatty liver. These data suggest that feeding induces ER livers of L-PPARγKO mice, but there was reduced expression of lipogenic stress in liver even under the physiological condition, while Sdf2l1 regulated genes (e.g. fatty acid synthase) corresponding with reduced hepatic fat by Xbp1 and ATF6 may switch off the ER stress response, maintaining accumulation, suggesting that liver-PPARγ directly regulates hepatic lipid hepatic insulin sensitivity after feeding. On the other hand, suppression of synthesis and metabolism. Surprisingly, the PPARγ agonist rosiglitazone hepatic Sdf2l1 expression in obesity may contribute to the development of restored β-oxidation in L-PPARγKO mice on HFD to that seen in WT mice. systemic insulin resistance, providing the possibility of Sdf2l1 to be a novel These data suggest: 1) liver PPARγ directly regulates hepatic lipid synthesis therapeutic target of obesity-induced diabetes.

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A451 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

resistant to high fat high sucrose (HFHS) diet-induced obesity and IR. We & 1660-P also saw an increase in basal hepatic ER stress in SKO mice (2-fold increase FoxO1 Regulates Sterol Regulatory Element Binding Protein-1c Gene in CHOP and BiP mRNA; p<0.05) and these stress markers were normalized in Expression DKO mice. Mechanistically, we hypothesize that this is due to the differential XIONG DENG, CHANDRAHASSA YELLATURU, WENWEI ZHANG, INSUG O-SULLIVAN, Ch metabolism of the two genotypes. We demonstrate that SKO mice are JAMES B. WILLIAMS, MARSHALL B. ELAM, EDWARDS A. PARK, RAJENDRA RAG- susceptible to high Ch diet (HCD)-induced hepatic ER stress (2.5-, 1.8-, 4.6- HOW, TERRY UNTERMAN, Memphis, TN, Chicago, IL fold increase in CHOP mRNA, SXBP1 mRNA, P-eIF2a) and this is abolished Induction of lipogenesis in response to insulin is critically dependent on the in DKO mice. Protection was also observed in DKO primary hepatocytes transcription factor, sterol regulatory element-binding protein-1c (SREBBP- treated with exogenous Ch dose-dependently. To further analyze the role 1c). FoxO1, a forkhead box class-O transcription factor, is an important of Ch in the regulation of hepatic ER stress, we examined the relationship target of insulin action, but its role in lipid metabolism has not been clearly between CHOP mRNA, tissue FC, and ER membrane FC in DKO, SKO, and WT defi ned. We conducted studies to examine the effects of FoxO proteins on mice. We observed that CHOP mRNA correlates poorly with tissue FC (R2 = SREBP-1c gene expression in the liver. Studies in transgenic mice show that 0.18), but very strongly with ER FC levels (R2 = 0.97). constitutively active FoxO1 (CA-FoxO1) suppresses expression of SREBP-1c mRNA in liver by ∼50%, while SREBP-1c expression is increased ∼2-fold in liver-specifi c FoxO knockout mice. Similarly, studies in primary hepatocytes show that CA-FoxO1 suppresses SREBP1-c expression and inhibits basal and insulin-stimulated SREBP-1c promoter activity, while siRNA knockdown of FoxO1 increases SREBP-1c mRNA and protein levels and enhances SREBP- 1c promoter activity. LXR, a nuclear receptor protein, plays an important role in promoting insulin-regulated SREBP-1c expression, and CA-FoxO1 signifi cantly blunts the effects of LXR and LXR agonist TO901317 on the activity of the SREBP-1c promoter and a luciferase reporter construct driven by 3 LXR-response elements. Chromatin immunoprecipitation assays reveal that CA-FoxO1 reduces the recruitment of LXR to the SREBP-1c promoter despite the absence of FoxO-binding sites. Together, these studies indicate that FoxO proteins can suppress the expression of SREBP-1c and that this effect may be exerted, at least in part, by inhibiting recruitment of and transactivation by LXR. Supported by: Department of Veterans Affairs Merit Review Program On chow diet, SKO mice have more and DKO mice have less ER FC than & 1661-P WT mice and on HCD, SKO mice accumulate excess ER FC, while DKO mice Investigation of the Role of Irs1 Ser302 in Insulin Sensitivity/Resist- do not. We conclude that DKO mice are protected from Ch-induced hepatic ance in Knock-In Mice ER stress by evading FC accumulation in the ER. This protection against ER KYLE D. COPPS, MORRIS F. WHITE, Boston, MA stress may also play a role in the observed insulin hypersensitivity in LCAT Hyperactivity of the nutrient-sensitive mTorc1→S6 kinase pathway impairs defi cient mice. insulin-stimulated Akt activity in conjunction with serine phosphorylation and degradation of the insulin receptor substrates, Irs1 and Irs2. In particular, serine 307 of Irs1 (mouse S302) is a major site of phosphorylation by S6K Guided Audio Tour: Liver—Clinical Studies and Physiology (Posters 1663-P in vitro, whereas phosphorylation of this site is decreased in tissue from to 1671-P), see page 11. insulin-sensitive S6k1 knockout mice. Thus, Ser302 phosphorylation might provide a link between abnormal physiologic states (potentially including & 1663-P nutrient excess) and insulin resistance. To test this hypothesis, knock-in mice Fasting and Postprandial Hyperglycemia in Prediabetes: Contribu- were made having a non-phosphorylatable alanine (A) substitution at S302. tion of Hepatic vs. Extrahepatic Insulin Action At age 4 months, male S302A homozygotes (A/A) of mixed 129xC57BL/6 RITA BASU, CRISTINA BAROSA, BARBARA J. NORBY, BETTYANN DICKE, JOHN background were signifi cantly glucose intolerant relative to similarly derived G. JONES, ANANDA BASU, ROBERT A. RIZZA, Rochester, MN, Coimbra, Portugal control knock-in mice (S/S) that expressed wild-type Irs1, but were also ∼10% The mechanism of fasting and postprandial hyperglycemia (IFG/IGT) more massive. By contrast, the A/A and S/S mice were indistinguishable remains poorly understood.To do so, 17 IFG/IGT and 12 age, sex and BMI by measurement of fasting blood glucose and insulin concentrations or matched subjects with normal fasting glucose and normal glucose tolerance sensitivity to IP-injected insulin. To more specifi cally assess the function of NFG/NGT underwent a four hour somatostatin clamp with replacement Irs, the A/A and S/S mice were intercrossed with mice bearing conditional glucagon and growth hormone. Insulin was infused (0.35mu/kgTBW/ (fl oxed) Irs1 and Irs2 alleles to generate males lacking Irs2 and retaining min) to maintain glucose in prediabetic range (∼6.1mM), deuterated water a single Irs1 A or S allele in the hepatocyte compartment. Comparison of ingested overnight and [1-13C] acetate infused to measure contribution these ‘genetically stressed’ mice (A/- or S/-) demonstrated the emergence of of transaldolase pathway (TA) to gluconeogenesis (GNG). Results were moderate glucose intolerance, but unaltered sensitivity to injected insulin, in compared using means of last forty minutes of basal and clamp. Fasting A/- mice between 4-6 months of age. The A/- mice were also slightly larger glucose (6.1±0.1vs.5.3±0.1mM), insulin (49.0±6.0vs.32.0±4.0pM) and on average than S/- mice at 6 months. Further experiments are in progress to c-peptide (1.1±0.1vs.0.76±0.05nM) levels were higher (p<0.02) whereas carefully evaluate the signaling capacity of the mutant S302A Irs1 protein, in glucagon (70.9±4.0vs.64.1±5.0pM) similar (p=ns) in IFG/IGT vs. NFG/NGT. the context of both normal and abnormal liver physiology caused by mTorc1 Clamp glucose, and all hormone concentrations were matched between activation. groups (p=ns). Fasting endogenous glucose production (EGP) and glucose disappearance (Rd) were higher (16.7±0.34vs.14.1±0.6 μmol/kg/min;p<0.001) Obesity

POSTERS & 1662-P in IFG/IGT vs.NFG/NGT. Clamp EGP remained higher (13.0±1.1vs.8.5±1.0 Lecithin:Cholesterol Acyltransferase (LCAT) Defi cient Mice Are μmol/kg/min;p=0.005) but Rd slightly lower (24.0±3.1vs.34.1±3.4 μmol/ kg/min;p=0.05) in IFG/IGT. Ratio of C3/C4glucose prior to and during

Integrated Physiology/ Resistant to Hepatic ER Stress and Insulin Resistance through Evasion of Cholesterol (Ch) Accumulation in the ER Membrane clamp were similar (p=ns) in both groups indicating equal TA exchange. LAUREN S. HAGER, LIXIN LI, LU LIU, GRAHAM MAGUIRE, MARK NAPLES, CHRIS Uncorrected GNG were similar prior to and during clamp (p=ns) while after BAKER, LILIA MAGOMEDOVA, CAROLYN L. CUMMINS, PHILIP W. CONNELLY, TA correction GNG rates were higher (p=0.02) in IFG/IGT before and during KHOSROW ADELI, DOMINIC S. NG, Toronto, ON, Canada clamp (5.7±0.4vs.4.9±0.2and 4.3±0.5vs.2.5±0.4 μmol/kg/min). In contrast LCAT mediates the esterifi cation of free cholesterol (FC) in lipoproteins uncorrected glycogenolysis (GGL) was higher in IFG/IGT before and during and its absence causes profound HDL defi ciency and hypertriglyceridemia. clamp (8.6±0.4 vs.6.9±0.5 and 6.1±0.3vs.3.5±0.4 μmol/kg/min;p=0.03) Although low HDL and high TG are typically seen in insulin resistance (IR), but the absolute rates and contribution following TA correction were our lab has reported that chow-fed LCAT null mice in the LDL Receptor more pronounced (11.0±0.5vs.9.1±0.4 and 8.7±0.4vs.6.0±0.3;p=0.01). We (LDLR) knockout background (DKO) are paradoxically more insulin sensitive conclude IFG/IGT have predominant defects in hepatic with subtle defects than LDLR single knockout (SKO) controls. We observed that DKO mice are in extra-hepatic insulin action. Overestimation of GNG in the absence of

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A452 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

TA correction can lead to loss of meaningful differences in GNG and GGL In conclusion, overweight/obese T2DM pts with NAFLD have a similar between study groups. Future studies are required to tease out differences degree of hepatic steatosis vs. non-DM, but NASH is more common and in isolated fasting hyperglycemia IFG/NGT vs. IFG/IGT. severe. Hepatic and ATIR play a key role, while hyperglycemia appears to Supported by: DK29953 have a minor role in the severity of NASH. Supported by: Burroughs Wellcome Fund ADA-Funded Research & 1664-P The Effect of Colesevelam Hydrochloride on Fasting and Postprandial & 1666-P Glucose Metabolism in Subjects with Type 2 Diabetes Role of Hepatic Insulin Resistance on Metabolic Factors and Liver GALINA SMUSHKIN, CHIARA DALLA MAN, MATHENI SATHANANTHAN, PAULA Histology in Obese Patients with NAFLD D. GIESLER, CLAUDIO COBELLI, ADRIAN VELLA, Rochester, MN, Padua, Italy CAROLINA ORTIZ-LOPEZ, ROMINA LOMONACO, MATTHEW PERRY, BEVERLY Lipid lowering therapy with a bile-acid sequestrant Colesevelam ORSAK, AMY WEBB, KENNETH CUSI, San Antonio, TX Hydrochloride in people with type 2 diabetes has been associated with The role of hepatic insulin resistance (HIRI) in nonalcoholic fatty liver a small but signifi cant decrease in HbA1c. However the mechanism of this disease (NAFLD) and steatohepatitis (NASH) is unclear. To better understand effect on glycemic control is unclear. To examine the effect of Colesevelam this relationship we studied 120 overweight/obese NAFLD pts (age=50±1, on fasting and postprandial glucose metabolism in type 2 diabetes we BMI=34.0±0.4 kg/m2) and 12 age/BMI-matched controls without NAFLD (no- studied 40 subjects using a double-blind, placebo-controlled, parallel-group NAFLD; MRS<5.4%). We measured: 1) liver fat by MRS; 2) adipose tissue IR design. Subjects were studied at baseline and after a 12 week treatment (Adipo-IR index [ATIR]= fasting plasma FFA x insulin [FPI]); 3) liver/muscle period using a labeled mixed meal consisting of 50g of bacon, 2 scrambled (Rd) insulin sensitivity (insulin clamp with 3H glucose) and hepatic IR (Hep- eggs and 75g of Jell-O labeled with [1-13C]-glucose. [6-3H] glucose was IR index [HIRI]= EGP x FPI) and 4) liver histology (biopsy; n=108). NAFLD infused intravenously to measure the systemic rate of meal appearance (mean LFAT=24±1%) vs. no-NAFLD pts had similar BP and hsCRP but worse 2 (Meal Ra). Infused [6,6- H2] glucose enabled measurement of endogenous ALT, TG/HDL, adiponectin and adipose tissue/liver/muscle IR (p<0.001). To glucose production (EGP) and glucose disappearance (Rd). Insulin sensitivity further explore the relationship between HIRI and metabolic/histological (Si) and β-cell responsivity indices (Φ) were estimated using the oral glucose parameters, we divided NAFLD pts in HIRI quartiles (Q). There was a ∼2- and C-peptide minimal models, respectively. The Disposition Index (DI) for fold increase in HIRI per Q (Q1:<8, more sensitive; Q4:>24, more IR). NAFLD each individual was subsequently calculated. Therapy with Colesevelam Q1 vs. no-NAFLD already had abnormal ALT/TG/HDL (p<0.05), dysfunctional was associated with a decrease in fasting (7.05±0.24 vs. 6.56±0.2mmol/l, adipose tissue (worse ATIR, %supprFFA, lower adiponectin) and muscle IR. p<0.01) and postprandial glucose concentrations (0.312±0.014 vs. A further worsening of these parameters occurred as hepatic IR increased 0.303±0.013mol per 6h, p<0.01). In contrast treatment with placebo did not from NAFLD Q1 to Q4 despite similar BMI and LFAT (all p<0.001,Table) and alter fasting (7.36±0.33 vs. 7.54±0.45mmol/l, p=0.37) or postprandial glucose so did liver histology (NASH activity score and fi brosis; both p<0.01]. HIRI concentrations (0.336±0.014 vs. 0.349±0.014mol per 6h, p=0.06). These correlated strongest with measures of adipose tissue IR (p<0.0001), much changes could not be explained by postprandial changes in insulin secretion less with clinical variables (p<0.05) but not with BMI or LFAT. and action as overall DI did not differ signifi cantly after treatment with In conclusion, there is a stepwise deterioration of metabolic and Colesevelam (318±56 vs. 324±55 10-14, dl/kg/min2 per mU/ml, respectively, histological variables as hepatic insulin sensitivity declines in overweight/ p=0.87). Although basal EGP decreased slightly after treatment (17.4±0.7 obese NAFLD pts. This is closely associated with measures of adipose tissue vs. 16.8±0.6mmol/kg/min), postprandial suppression did not differ (69.0±0.1 IR but not BMI or LFAT. vs. 68.0±0.1%). Peak and integrated Meal Ra as well as Rd did not differ No-NAFLD HIRI Q1 HIRI Q2 HIRI Q3 HIRI Q4 after treatment with Colesevelam. We conclude that the glucose-lowering effects of Colesevelam are attributable to direct effects on hepatic glucose Age(yrs) 50±3 51±2 51±2 51±2 48±2 production without signifi cant effect on meal induced insulin secretion or BMI (kg/m2) 32±1 33±1 33±1 34±1 35±1 gastrointestinal motility. LFAT % 2±3 24±2 21±2 24±2 29±2 Supported by: Daiichi-Sankyo ALT(IU/L) 34±12 54±8 51±8 61±7* 72±7*† TG (mg/dl) 87±35 131±23 153±24 171±21 271±22*† & 1665-P Insulin Resistance, but Not Hyperglycemia, Plays a Key Role in the HDL (mg/dl) 51±3 39±2* 39±2* 37±2* 34±2*† Severity of NAFLD in T2DM Patients ATIR (mmol/liter · µU/ml) 2±1 5±1 6±1* 7±1*† 14±1*† CAROLINA ORTIZ-LOPEZ, BEVERLY ORSAK, ROMINA LOMONACO, JOAN FINCH, Ins.supp.FFA (%) 71±7 66±4 49±5*† 48±4*† 39±4*† CELIA DARLAND, KENNETH CUSI, San Antonio, TX Rd (mg · KgLBM) 12±1 8±1* 7±1* 5±1*† 4±1*† The factors contributing to nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in pts with type 2 diabetes (T2DM) remain unclear. *p=0.05-0.01 (vs. no-NAFLD); †p<0.01 (vs. HIRI Q1) We examined the role of hyperglycemia and insulin resistance (IR) in 187 Supported by: Burroughs Wellcome Fund ADA-Funded Research NAFLD pts (age: 51±2; M/F: 66/34%; ethn: Hisp=60%/Cauc=28%/other=12%) with (n=96) or without (n=91 non-DM) T2DM. They were well-matched for BMI=34.9±0.6 vs. 34.2±0.6 kg/m2, total body fat (TBF by DXA)=33.6±0.9 & 1667-P vs. 33.8±0.9%, %obese=81% vs. 82% (all NS). We measured: 1) liver fat by α-Hydroxybutyrate and Linoleoyl-Glycerolphosphocholine as New MRS; 2) plasma glu/insulin/FFA during an OGTT; 3) adipose tissue IR (Adipo- Markers of Fatty Liver Disease IR index [ATIR]= fasting FFA x fasting plasma insulin [FPI]); 4) liver/muscle (Rd) AMALIA GASTALDELLI, ANDREA NATALI, WALTER GALL, CARLES LERIN, MARY insulin sensitivity (insulin clamp with 3H glucose) and hepatic IR (Hep-IR index ELIZABETH PATTI, JOHN PETRIE, BEVERLY BALKAU, CHRISTIAN ANDERWALD, [HIRI]= EGP x [FPI]) and 5) liver histology (biopsy; in 59 T2DM and 66 non-DM). MARTINE LAVILLE, RAFAEL SANCHEZ GABRIEL, ELE FERRANNINI, RISC While T2DM and non-DM were well matched for adiposity and had similar INVESTIGATORS, Pisa, Italy, Research Triangle Park, NC, Boston, MA, Glasgow, hepatic steatosis (MRS=24.7±1.2 vs. 22.4±1.3, NS), more T2DM developed United Kingdom, Villejuif, France, Vienna, Austria, Lyon, France, Madrid, Spain Increased levels of plasma α-hydroxybutyrate (α-HB) and decreased NASH (85% vs. 68%, p=0.04) and had worse infl ammation [p=0.007] and Obesity levels of plasma linoleoylglycerophosphocholine (L-GPC) have been recently fi brosis [p<0.05]. When examining the role of hyperglycemia on NASH in POSTERS T2DM (A1c=7.0±1.0%), there was no correlation between A1c and histology, described as markers of peripheral insulin resistance as measured by the euglycemic hyperinsulinemic clamp technique. Since both α-HB and L-GPC with only a trend for worse fi brosis if A1c>7.5%. In contrast, T2DM had more Integrated Physiology/ hepatic IR (HOMA, HIRI and suppression of EGP by low-dose insulin, p<0.05- are produced in the liver, with α-HB levels directly related to the rate of 0.001) with a strong correlation between HIRI and worse infl ammation and hepatic glutathione synthesis, the goal of this study was to test if α-HB and fi brosis (p=0.02-<0.01). T2DM had more dysfunctional adipose tissue (worse L-GPC are markers for fatty liver disease. In the RISC population (1,222 nondiabetic, normotensive subjects, age ATIR=10.1±0.8 vs. 6.0±0.8 mmol/liter · U/ml and supprFFA during OGTT 2 66±2% vs. 74±2% and low-dose insulin 43±3% vs. 58±3%; all p<0.001) and 44±8 years (mean±SD) [range 30-60], BMI 25.5±4.1 kg/m , [range 16.9-43.9]), on liver biopsies ATIR correlated closely with liver infl ammation (p=0.02), we measured circulating α-HB concentrations, liver enzymes (ALT, AST, but not fi brosis. In contrast, while Rd (muscle) was diminished by 24% in GGT), and plasma L-GPC levels. Fatty liver accumulation was assessed using T2DM (p<0.01) it did not correlate with histology. Biomarkers of systemic the recently validated fatty liver index, FLI. Hepatic insulin resistance (Hep- IR) was estimated in 380 subjects as the product of endogenous glucose infl ammation (IL-6, hsCRP, TNF-α and TGF-β) were similarly elevated in both 2 groups. production (by H2-glucose infusion) and fasting plasma insulin. To assess L-GPC levels directly in the liver, open liver biopsies were performed in the

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A453 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

fasting state during elective cholecystectomy from lean subjects without study was to delineate the impact of a selective increase in dietary fructose DM2 (n=8) and during gastric bypass from obese subjects with or without on net HGU (NHGU) and glycogen synthesis (GLY). Adult male dogs were fed DM2 (n=25). either a high-fructose/low-fat (17/26% of E; n=5) diet (FR) or a chow control (no After adjusting for center, sex, and age by multiple linear regression fructose/26% of E as fat; n=5) diet (CTR) for 4 wks. Dogs underwent hepatic/ analysis, α-HB was directly related to FLI (partial r = 0.21, p<0.0001), portal vein catheterization at 2wks and a hyperinsulinemic hyperglycemic whereas L-GPC was reciprocally related to FLI (partial r = -0.32, p<0.0001). clamp 2wks later. Somatostatin, basal intraportal (Po) glucagon, 4x basal Furthermore, a high α-HB and a low L-GPC both contributed to a high FLI Po insulin and peripheral glucose (to double the hepatic glucose load) were value independently of center, sex, and age. In the subgroup with tracer infused during experimental periods 1 (P1) and 2 (P2). Glucose was also infused measurements – and after adjustment for center, sex, age, and BMI – L-GPC Po (4.0 mg/kg/min) during P2 to activate a feeding signal. NHGU (mg/kg/min) was also reciprocally related to Hep-IR (partial r = -0.12, p=0.02). In support during P1 and P2 averaged 1.3±0.1 and 3.3±0.4 in CTR, and -0.8±0.2 (output) of these fi ndings, we further found that hepatic L-GPC were 5-fold lower in and 0.8±0.3 in FR (P<0.001 vs CTR), respectively. Diminished NHGU in FR the obese group compared to lean controls. was attributable to impaired suppression of endoRa (80±17% vs 57±17% in In conclusion, raised plasma concentrations of α-HB and L-GPC mark for CTR and FR, respectively) coupled with inadequate stimulation of HGU (mg/ the presence of fatty liver disease independently of sex and age. Low L-GPC kg/min; CTR: 1.9±0.2 [P1], 2.4±0.3 [P2]; FR: 0.5±0.2 [P1], 0.8±0.2 [P2], P<0.01 levels are also specifi cally associated with Hep-IR. Liver L-GPC levels were vs CTR). Terminal hepatic glycogen levels (mg/g) and direct GLY (mg/kg/min) also found to be signifi cantly lower in obese and T2D subjects. were 41±4 and 1.0±0.2 in CTR, and 29±3 and 0.4±0.0 in FR (P<0.01 vs CTR), Supported by: EU contract QLG1-CT-2001-01252, Astra Zeneca Sweden respectively. Blunted NHGU and GLY in FR were associated with 58±1% and 71±4% decreases (P<0.001 vs CTR) in hepatic glucokinase (HGK) protein & 1668-P content and activity, respectively, but there was no difference in relative Effect of Liver Fat Accumulation on Insulin Resistance and Severity HGK mRNA expression or liver triglyceride (LTG) levels between groups. In of NASH in Obese Patients with T2DM conclusion, a selective increase in dietary fructose is suffi cient to markedly ROMINA LOMONACO, BEVERLY ORSAK, CAROLINA ORTIZ-LOPEZ, JANET CHAN, reduce NHGU and GLY in association with a signifi cant post-transcriptional AMY WEBB, KENNETH CUSI, San Antonio, TX decline in HGK protein content in the absence of an elevation in LTGs. Thus, While hepatic steatosis appears to worsen insulin resistance (IR) and these data demonstrate the detrimental impact of high dietary fructose on cause an unfavorable metabolic profi le in obese type 2 diabetes mellitus hepatic glucose metabolism. (T2DM), it is unclear whether IR and nonalcoholic steatohepatitis (NASH) are directly proportional to the magnitude of liver fat accumulation. To & 1670-P this end we studied 104 pts with T2DM: age=55±1, gender (M/F)=66/34%, Effect of the Postprandial Increase in Glucagon Secretion on BMI=34.5±0.6 kg/m2, obese/overweight= 83/17%. We measured: 1) Hepatic Protein Metabolism LFAT=liver fat (MRS); 2) adipose tissue IR index(ATIR)= fasting FFA x fasting GUILLAUME KRAFT, KATIE C. COATE, BEN FARMER, DOMINIQUE DARDEVET, insulin [FPI]); 3)TBF=total body fat(DXA); 4)plasma glu/ins/FFA(OGTT); 5) MARTA S. SMITH, BAKULA TRIVEDI, KEN GRIMES, PATRICK DONAHUE, ALAN D. liver/muscle (Rd) insulin sensitivity (low/high-dose insulin clamp with 3H CHERRINGTON, MARY C. MOORE, Nashville, TN, St Genes Champanelle, France glucose); 6) hepatic IR index(HIRI)= EGP x FPI; 7) liver histology (biopsy; n=60). Glucagon (GGN) secretion is increased by amino acid ingestion and Mean LFAT was 22±1%, so we stratifi ed pts by the severity of LFAT as: no- GGN is known to increase net amino acid (AA) uptake by the liver, but NAFLD (LFAT<5%, controls), mild: 5-10%, moderate: >10-20%, severe: >20- the effects of lack of the normal postprandial rise in GGN on postprandial 30% or very severe: >30% LFAT. Liver fat was associated with a reduction protein synthesis are unknown. We assessed net hepatic AA uptake and in plasma adiponectin and increased insulin resistance in adipose tissue incorporation of [14C]Leucine in liver protein in 42h fasted dogs during a (ATIR, %ins supprFFA) and liver (HIRI) (Table) with no further deterioration study mimicking postprandial conditions: hyperinsulinemic (4X basal), beyond LFAT 10%. There was no relationship between increasing BMI and hyperglycemic (1.5X basal liver load), hyperaminoacidemic clamp (2X basal LFAT (Table). Histologically, an increase in LFAT in NAFLD pts from 5-10% vs. liver load) with both glucose and AA supplied portally. One group received any group LFAT>10% signifi cantly worsened liver infl ammation (p<0.03-0.01) basal GGN (BaGGN, n=5) and the other received 3x basal GGN (HiGGN, the and the NASH activity score (p=0.02-0.007), with no differences once LFAT physiological condition, n=6). was 20-30%. Net hepatic glucose uptake was increased in the BaGGN vs. HiGGN group In conclusion, in obese T2DM pts even a mild LFAT increase (5 to 10%) (2.29±0.21 and 1.35±0.25 mg/kg/min respectively, P<0.05). Hepatic load of severely alters metabolic parameters with no further worsening once LFAT AA was increased similarly in the 2 groups (1.27±0.06 and 1.35±0.07 × basal >10%. In contrast, histology continues to worsen with LFAT up to 20-30%, respectively for BaGGN and HiGGN; P=0.53). Total hepatic AA fractional but not beyond this point. extraction (FE) did not differ (23±1 and 26±1% respectively for BaGGN and No-LFAT Mild Moderate Severe Very Severe HiGGN, P=0.45), but the gluconeogenic AA hepatic FE was higher in the (<5%) (5-10%) (>10-20%) (>20-30%) (>30%) HiGGN group (23±3 and 32±2 % respectively with P<0.05). Elevation of GGN was associated with a decreased synthesis of both liver proteins and plasma Age (yrs) 60±3 59±2 54±2 55±1 54±1 secreted proteins (nmol leucine/mg protein/h : liver proteins = 6.3±0.7 and BMI (%) 33.1±2.1 35.5±1.6 34.8±1.3 35.0±1.1 35.0±1.1 2.8±0.5 for BaGGN and HiGGN, P<0.005; plasma secreted proteins: 8.0±0.8 A1c (%) 6.6±0.4 6.7±0.3 7.3±0.2 7.2±0.2 6.8±0.2 and 6.0±0.7 respectively for BaGGN and HiGGN, P=0.10). Molecular data Adiponectin (µg/ml) 11.1±2.1 8.1±1.4 9.6±1.1 7.8±1.0 9.8±1.0 are in accordance with that lower protein synthesis because GGN, through an activation of AMPK (1.0±0.20 and 1.33±0.05 relative phosphorylation ATIR (mmol/l · U/ml) 3±3 6±2 7±2 13±2*† 11±2*† of AMPK on Thr172, respectively for BaGGN and HiGGN, P=0.05), tends to % insulin supprFFA (%) 73±9 56±10 60±8 33±6*† 39±6*† increase phosphorylation of 4EBP1 (1.0±0.05 and 1.34±0.20, P=0.07). These HIRI(mg/kg-1 · min-1 · µU/ml) 5±2 27±9* 17±7* 26±5* 24±5* results suggest that the rise in GGN level during AA absorption stimulates AA hepatic uptake, especially gluconeogenic ones, but it also results *p<0.05-01 (no-NAFLD vs. NAFLD); †p<0.05-01 between mild vs. other in activation of AMPK, channeling AA toward gluconeogenesis and not NAFLD groups. primarily protein synthesis. Obesity

POSTERS Supported by: Burroughs Wellcome Fund ADA-Funded Research & 1671-P

Integrated Physiology/ & 1669-P Insulin Clearance Is a Major Determinant of Baseline Insulin Sensi- Increased Dietary Fructose Markedly Reduces Hepatic Glucose tivity in Healthy Dogs Uptake and Glycogen Synthesis MARILYN ADER, STELLA P. KIM, JOYCE M. RICHEY, VIORICA IONUT, DARKO KATIE C. COATE, GUILLAUME KRAFT, MARTA S. SMITH, BEN FARMER, JOSHUA STEFANOVSKI, KARYN J. CATALANO, KATRIN HUCKING, GREGG W. VAN CITTERS, D. ROOP, MASAKAZU SHIOTA, SUSAN VAUGHAN, SUSAN HAJIZADEH, ALAN D. ISABEL R. HSU, JENNY D. CHIU, ORISON WOOLCOTT, LISA N. HARRISON, DAN CHERRINGTON, Nashville, TN ZHENG, MAYA LOTTATI, CATHRYN M. KOLKA, JUSTIN DITTMANN, SOPHIA Impaired hepatic glucose uptake (HGU) contributes to postprandial YAE, HELEN LIU, ANA VALERIA B. CASTRO, MORVARID KABIR, RICHARD N. hyperglycemia (HG) and to the deterioration of HbA1c in diabetic individuals. BERGMAN, Los Angeles, CA We have shown previously that consumption of a high-fat/high-fructose Insulin resistance is a key risk factor for Type 2 diabetes and a major (52/17% of energy [E]) diet markedly impairs hepatic glucose disposition in the component of the metabolic syndrome. Insulin sensitivity (SI) can vary presence of hyperinsulinemia, HG and the portal glucose signal. The aim of this widely in healthy subjects, but decreased SI is most commonly associated

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A454 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER with increased body weight and/or adiposity. However, other blood- borne and metabolic factors can infl uence SI and be more substantial & 1673-P predictors of SI under baseline, non-stimulated conditions. We performed Glucose Lowering Effi cacy of LY2409021, a Small Molecule Gluca- a comprehensive assessment of metabolic function in healthy male dogs gon Receptor Antagonist, in ob/ob Mice and Streptozotocin-Treated (total n=90) to identify key determinants of insulin sensitivity. Whole- C57BL/6 Mice Expressing the Human Glucagon Receptor THOMAS B. FARB, WENZHEN MA, JAMES V. FICORILLI, ERIC D. HAWKINS, body SI (SIclamp), obtained from euglycemic clamp in all animals, was used as the primary outcome variable. SI was also measured independently by JOSEPH T. BROZINICK, KYLE W. SLOOP, REBECCA A. OWENS, MARTIN B. minimal model analysis of the IVGTT in a subset of animals (n=36). Total and BRENNER, PHILIP A. HIPSKIND, JULIE S. MOYERS, Indianapolis, IN regional (visceral, subcutaneous) adiposity were measured by MRI (n=90). Type 2 diabetes (T2DM) involves an imbalance in the relative actions of Glucose-stimulated insulin response was measured either by stepwise glucagon and insulin and is characterized by excessive glucagon-stimulated hyperglycemic clamp or acute insulin response from the IVGTT (n=86 and hepatic glucose production. LY2409021 was identifi ed as a potent and 36, respectively). Despite similar body weight (28.7±0.3 kg), baseline trunk selective antagonist that inhibits the human glucagon receptor (hGCGR) adiposity varied nearly 8-fold (172 to 1363 cm3), refl ecting a broad range with a Ki of 6.66 ± 0.64 nM and the mouse GCGR with a Ki of 75.3 ± 11.4 of both visceral and subcutaneous fat mass. Variability was also refl ected nM. Despite reduced mouse receptor affi nity, LY2409021 acutely lowers glucose in hyperglycemic ob/ob mice with an ED50 of 7.4 mg/kg. Once-daily in SIclamp (5.9 to 75.9 dl/min per kg per μU/ml). There was an expected dosing of LY2409021 at 50 mg/kg for 14 days in ob/ob mice signifi cantly negative association between fasting insulin and SIclamp (p<0.0001), but the relationship strengthened when considering the underlying physiologic decreased the plasma fructosamine levels from 335 ± 9.1 uM (day 1) to determinants of circulating insulin. In particular, insulin clearance was 271 ± 9.8 uM (day 14). Plasma glucagon levels were moderately increased, the dominant factor associated with baseline insulin sensitivity (r=0.51, along with a modest decrease in plasma insulin levels. Plasma free fatty p<0.0002), and this relationship was comparably strong with both clamp- acids, triglycerides, and cholesterol were unchanged. To examine glucose- and IVGTT-based variables. These data suggest that insulin clearance is a lowering effi cacy of the GCGR antagonist in a preclinical model using the major determinant of insulin sensitivity under baseline conditions, and may human receptor, we developed a C57BL/6 mouse line expressing the hGCGR be pivotal in understanding the mechanisms by which insulin resistance may in place of the mouse receptor and induced hyperglycemia by treatment develop. Changes in insulin clearance may result in hyperinsulinemia which, with streptozotocin. In these mice, LY2409021 reduced glucose with an in turn, may down-regulate insulin action in vivo. ADA-Funded Research ED50 of 1.39 mg/kg at 6 hours post-dose. Additionally, an ex vivo binding assay was established for receptor occupancy in liver tissue to address the extent of receptor blockade. Hepatic hGCGR occupancy reached 81.7% at 6 hours after treatment with 30 mg/kg of LY2409021. Thus, LY2409021 is a potent glucagon receptor antagonist that lowers glucose in murine models Guided Audio Tour: Liver—Preclinical Studies (Posters 1672-P to 1679-P), expressing either mouse or human GCGR. Despite longstanding knowledge see page 11. regarding the role of glucagon in T2DM pathophysiology, studies using small & 1672-P molecule hGCGR antagonists in patients with T2DM have yet to be reported. Metformin Inhibits Renal Gluconeogenesis in Zucker Diabetic Fatty LY2409021 was identifi ed as a novel molecule with suitable preclinical (ZDF) Rats: A Cellular Metabolomic Study with 13C-Lactate and properties - it is now being investigated as a new treatment approach for Carbon 13 NMR hyperglycemia in patients with type 2 diabetes. GABRIEL BAVEREL, BERNARD FERRIER, GUY MARTIN, AGNÈS CONJARD-DUPLANY, Lyon, France & 1674-P Metformin, one of the most widely used antidiabetic drugs, has been Glucagon-Like Peptide-1 Receptor (GLP-1R)-Mediated Improve- shown to decrease hyperglycemia in diabetic patients and in diabetic ments in Rodent Models of Nonalcoholic Steatohepatitis (NASH) animal models. This effect results in part from the inhibition of hepatic Occur Independently of Weight Loss gluconeogenesis. However, renal gluconeogenesis has also been recently JAMES L. TREVASKIS, PETER GRIFFIN, CARRIE WITTMER, BRENT A. NEUSCH- shown to be increased in diabetic subjects suggesting that this process may WANDER-TETRI, ELIZABETH M. BRUNT, CARRIE S. DOLMAN, MARY R. ERICKSON, signifi cantly contribute to systemic gluconeogenesis and, therefore, should DAVID G. PARKES, JONATHON D. ROTH, San Diego, CA, St. Louis, MO be inhibited. This is why the objective of this study was to investigate in vitro Pharmacotherapies for nonalcoholic fatty liver disease (NAFLD), and the effect of metformin on renal lactate gluconeogenesis by using a cellular its infl ammatory state NASH are lacking. In prior studies, GLP-1R agonists metabolomic approach. (exenatide or its analog AC3174) improved metabolic and histopathological For this, renal proximal tubules from fed ZDF rats, a good model of type markers of NAFLD/NASH in Lepob/Lepob mice maintained on chow or high 2 diabetes in which intrinsic renal gluconeogenesis has been shown to be trans-fat/high fructose/high cholesterol (HTF) diet. Our present studies augmented, were isolated with a collagenase method. Then, these tubules extend these earlier fi ndings in several ways. First, we assessed whether were incubated with 5 mM L-[1-13C]-, or L-[2-13C]- or L-[3-13C]lactate or 5 mM GLP-1 mediated improvements in HTF diet-induced NASH were solely due 13 L-lactate + 25 mM NaH CO3 as substrates. At the end of the incubation to AC3174-mediated weight loss. AC3174-treated mice (30 µg/kg/d for 28 period, substrate utilization and product formation were measured by both d) exhibited signifi cantly reduced body weight (by 8.3%), lower liver mass enzymatic and carbon 13 NMR methods. (by 14.2%), liver lipid (12.9%), plasma ALT and triglycerides compared to Metformin dose-dependently inhibited lactate gluconeogenesis (IC50 = vehicle controls. By contrast, a calorie-restricted weight-matched group 1.15 mM). The addition of 1 mM metformin also inhibited the removal of demonstrated modest non-signifi cant reductions in liver mass (9%) and liver 13 13 13 C-lactates, and the production of C-glucoses and CO2 without changing lipid (5.1%), and no difference in ALT and triglycerides, relative to vehicle the fi xation of 13C-bicarbonate. Combination of enzymatic and NMR controls. To confi rm the GLP-1R dependence of these improvements, effects measurements with a mathematical model of renal lactate metabolism of AC3174 infusion (30 µg/kg/d for 28 d) were compared in HTF maintained previously validated showed that 1 mM metformin caused an inhibition of wild-type (WT) and GLP-1R-defi cient (Glp1rKO) mice. AC3174 infusion to WT fl uxes through lactate dehydrogenase (by 27%), glucose-6-phosphatase mice elicited the expected reductions in weight (11.1%), liver mass (23%), liver (by 40%), pyruvate dehydrogenase (by 23%) and citrate synthase (by 21%). lipid (27%), ALT (55%), and triglycerides (29%) relative to vehicle, whereas By contrast, metformin did not alter fl uxes through pyruvate carboxylase treatment of Glp1rKO mice had no effect on these parameters. Finally, to Obesity POSTERS and phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine gain insight into micro- and macro steatotic improvements associated aminotransferase. with GLP1R agonism, detailed histopathological analyses was performed. It is concluded that renal lactate gluconeogenesis is very sensitive to the Maintaining Lepob/Lepob and C57BL6J mice on HTF diet for 8 or 12 weeks, Integrated Physiology/ inhibitory effect of metformin and that this effect does not result from the respectively, increased visible micro- and macrosteatosis in both strains of inhibition of fl uxes through all the key-gluconeogenic enzymes involved in mice. In Lepob/Lepob mice induction of fi brosis, coupled with ∼6-fold increase this pathway. in collagen-1 mRNA and protein was also evident. AC3174 treatment (30 µg/ kg/d for 28 days) reduced body weight (by 7.3% in Lepob/Lepob and 15.5% in WT mice vs. vehicle), body weight-adjusted liver mass (by 20% and 30%), and liver lipid (by 11% and 38%). Together, these data support the clinical evaluation of the utility of GLP-1R agonists for treatment of NAFLD/NASH.

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A455 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

& 1675-P & 1677-P Investigation of the Role of Hepatic Vagus Nerve on Hepatic Glucose Attenuation of Hepatic Steatosis Is Associated with Increased 2 Metabolism by Use of [6, 6- H2] Glucose Hepatic Phospholipid Eicosapentaenoic Acid (EPA) and Docosa- TAZU TAHARA, KUNPEI TOKUYAMA, NORIKAZU OGIHARA, MASATOSHI KIKU- hexaenoic Acid (DHA) in Diet-Induced Obese (DIO) Rats Fed a Canola/ CHI, Tokyo, Japan, Ibaraki, Japan, Saitama, Japan Flax Oil Blend It has been documented that portal glucose delivery creates an important DANIELLE P. HANKE, CARLA G. TAYLOR, PETER C. ZAHRADKA, Winnipeg, MB, signal to enhance liver glycogen deposition. The signaling pathway has been Canada established as a “portal signal”. We have identifi ed that hepatic glycogen Insulin resistance is the most important pathogenic factor in the etiology phosphorylase (GP) activity is a surrogate marker of portal signal, because of non-alcoholic fatty liver disease (NAFLD). NAFLD has been associated with GP activity was immediately reduced after glucose load from portal vein, but reduced omega-3 (n-3) fatty acid status. It has been proposed that n-3 fatty not from peripherally. To assess the role of hepatic vagus nerve on hepatic acids may prevent hepatic steatosis and progression to NAFLD by increasing glucose metabolism, glucose was infused into unrestrained conscious rats hepatic fatty acid oxidation via peroxisome proliferator activated receptor via either portal (Po) or peripheral (Pe) vein at a constant rate, and parameters a (PPARa) and decreasing hepatic fatty acid synthesis via sterol regulatory involved in hepatic glucose metabolism such as glucose production, glucose element-binding protein-1c (SREBP-1c). Furthermore, the effi cacy of the plant- output and glycogen deposition and GP activity were measured. Rats were based n-3 fatty acid, α-linolenic acid (ALA), as a dietary precursor of EPA and subjected to hepatic vagotomy (HV) or sham operation (SO) 7 days prior to DHA for modulating hepatic steatosis is unknown. Thus, the present study 2H the experiment. After 24-hour fasting, tracer amount of [6, 6- 2] glucose investigated hepatic steatosis, hepatic fatty acid composition and markers was infused for 180 minute into the peripheral vein for equilibration, and of fatty acid oxidation and synthesis in 6 week old Obese Prone Sprague kept infusion throughout the experiment. Then, glucose was infused portally Dawley rats fed high fat (55% energy) diets containing various fat types or peripherally (13.5 mg/ kg /min) for 120 minutes. Metabolic parameters including high oleic canola oil, canola oil, a canola/fl ax oil blend (C/F, 3:1), were determined by the mass isotopomer distribution analysis method. In saffl ower oil, soybean oil, or lard for 12 weeks. Upon completion of the study, SO, hepatic glucose output was signifi cantly reduced in Po than Pe after 30 the C/F and weight matched (WM) groups had the lowest percent liver lipid. minutes glucose infusion (3.13 and 6.05 mg / kg / min), while the reduction The C/F group had the highest amount of ALA in their diet and this resulted in glucose output observed in SO was disappeared in HV, and kept higher in the highest total n-3 and EPA in hepatic PL. The C/F group also had one of levels (16.7 and 23.2 mg / kg / min in Po and Pe). Hepatic glycogen deposition the highest DHA and lowest arachidonic acid (n-6) concentrations in hepatic was signifi cantly higher in Po than Pe after 120 minutes glucose infusion PL. Conversely, the type of fat or oil did not affect hepatic protein levels (57.1±2.1 vs 35.7±1.7μmol / g liver) in SO, whereas the glycogen deposition of PPARa and acyl-CoA oxidase or hepatic protein levels of SREBP-1c and in Po was reduced in HV compared with SO during portal glucose infusion acetyl-CoA carboxylase. In conclusion, our data indicates that the C/F diet, and was no siginifi cantly difference between Po and Pe (28.7±7.4vs 25.0±7.2 which was high in the plant-based n-3 ALA, can attenuate hepatic steatosis μmol / g liver). GP activity in SO was lower in Po than Pe after 120 minutes and favourably alter hepatic fatty acid concentrations of PL by increasing glucose infusion(1.04±0.21 vs 1.54±0.03μmol / g liver), but the difference EPA and DHA. However, the reduction in hepatic steatosis by the canola/fl ax in GP activity between Po and Pe was not observed in HV (0.91±0.18 and oil blend was not explained by the upregulation of PPARa and/or inhibition or 0.93±0.17 μmol / g liver). From the data we conclude that hepatic vagus suppression of SREBP-1c. nerve is important for the regulation of hepatic glucose production and Supported by: Canola Council of Canada glycogen deposition. & 1678-P & 1676-P Overexpression of SIRT1 in the Liver Deacetylates X-Box Binding Control of Blood Glucose in the Absence of Hepatic Glucose Pro- Protein 1 and Attenuates Hepatic Steatosis and Insulin Resistance duction Due to an Induction of Renal and Intestinal Gluconeo- in ob/ob Mice genesis by Gluca gon YU LI, KAZUTO NAKAMURA, WALSH KENNETH, MENGWEI ZANG, Boston, MA FABIENNE RAJAS, ELODIE MUTEL, AMANDINE GAUTIER-STEIN, AYA ABDUL- Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology WAHED, MARTA AMIGO, ANNE STEFANUTTI, GILLES MITHIEUX, Lyon, France of human type 2 diabetes (T2DM). Small molecular activators of SIRT1 were Since the pioneering work of Claude Bernard in the 19th Century, the shown to prevent diet-induced obesity and insulin resistance. Our previous scientifi c community has considered the liver to be the major source of studies have indicated that SIRT1 and resveratrol regulate hepatocyte lipid endogenous glucose production (EGP). We investigated the importance of the metabolism through activating AMPK. To extend these fi ndings, the goal of liver as a source of EGP, by using a mouse model of liver-specifi c deletion of the present study was to explore the mechanism of the therapeutic impact the glucose-6 phosphatase catalytic subunit gene (LG6pc-/- mice), encoding of NAD-dependent deacetylase SIRT1 on metabolic deterioration in T2DM. an essential enzyme for glucose production. We previously showed that an We demonstrated that adenovirus-mediated overexpression of SIRT1 in absence of hepatic glucose release had no major effect on the fasting plasma the liver of ob/ob mice attenuated hepatic steatosis, lowered hepatic glucose concentration. Instead, there was an early sequential induction of lipids contents, and ameliorated glucose tolerance and systemic insulin gluconeogenesis in the kidney and intestine, the alternative gluconeogenic resistance. Enhanced insulin sensitivity by SIRT1 was associated with organs. Here, we investigated molecular mechanisms underlying expression decreased ER stress markers such as GRP78 and CHOP. Importantly, X-box changes of gluconeogenic genes (Pck1 encoding phosphoenol carboxykinase binding protein 1(XBP1), a critical ER stress marker, was identifi ed as a direct and G6pc) in both kidney and intestine of LG6pc-/- mice. Male adult target of SIRT1, since co-immunoprecipitation showed the spliced form of B6.g6pclox/lox.SACreERT2/+ mice were treated with tamoxifen to obtain endogenous XBP1 physically interacted with SIRT1 in the mouse embryonic Lg6pc-/-. Blood was withdrawn for metabolic studies from 6h-fasted mice. fi broblasts (MEFs). Hepatic overexpression of SIRT1 increased its interaction Chip assays were performed with phospho-CREB antibody on G6pc and Pck1 with spliced XBP1 and resulted in the deacetylation of spliced XBP1 in ob/ promoters. At 6h of fasting, glucagon level was higher in LG6pc-/- mice than ob mouse livers, as compared to those of Ad-GFP-injected mice. Hepatic in control mice (538±9 pg/ml vs 280±19 pg/ml in control) while LG6pc-/- mice overexpression of SIRT1 also decreased SREBP-1c, key enzyme involved showed lower insulin levels than that of control mice. After confi rming the in lipid synthesis, in ob/ob mouse livers. In contrast, the promoter activity Obesity presence of glucagon receptors in the kidney and intestine, we showed that of SREBP-1c target, fatty acid synthase, was increased by SIRT1-/- MEFs, POSTERS P-CREB bound to the G6pc promoter in both organs. In contrast, P-CREB was compared with SIRT1+/+MEFs. Interestingly, serum levels of infl ammatory only bound to the Pck1 promoter in the intestine. Interestingly, we obtained marker monocyte chemotactic protein-1 (MCP-1) were reduced upon SIRT1 Integrated Physiology/ identical results in fed control mice in response of an IP injection of glucagon. treatment. Moreover, resveratrol decreased tunicamycin-induced NF-κB Complementary data showed that Pck1 expression was mainly induced in the promoter activity and mRNA levels of proinfl ammatory cytokine TNFα in kidneys by metabolic acidosis observed in LG6pc-/-. In conclusion, our study HepG2 cells. Taken together, our results 1) identify XBP1 as a novel target of provides a defi nitive quantitative estimate of the capacity of extrahepatic SIRT1 through the deacetylation regulation in the liver and in vitro; 2) reveal gluconeogenesis to sustain fasting EGP, regardless of the contribution of the that deacetylation of XBP1 by SIRT1 in the liver may serve as a therapeutic liver. Moreover, glucagon plays a central role to induce gluconeogenesis in strategy to attenuate obesity-induced ER stress, infl ammation, hepatic the kidney and intestine, as it is well known in the liver. steatosis, and insulin resistance. Supported by: Agence Nationale de la Recherche and Association Française des glycogénoses

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A456 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

1681-P & 1679-P Changes in Choline Metaboilism Increase Liver Insulin Sensitivity A-769662-Mediated AMPK Activation in the Liver Reverses Hepatic Independent of Adiponectin and SOGA Steatosis in Lipodystrophic Mice LESLIE FISCHER, LAURA JOSEPH, OMAR ABDELBAKY, RACHAEL COWHERD, MARC FORETZ, JOCELYNE LECLERC, BENOIT VIOLLET, Paris, France J. MCKEE ALDERMAN, MELISSA ASMAR, JONATHAN GUTIERREZ, ARLENE AMP-activated kinase (AMPK) is a key regulator of lipid metabolism BRIDGES, LEONARD COLLINS, JAMES SWENBERG, STEVEN ZEISEL, TERRY P. which increases fatty acid oxidation and decreases lipid synthesis. Leptin COMBS, Chapel Hill, NC and adiponectin, two adipokines secreted by adipose tissue, activate AMPK Several studies suggest that changes in choline metabolism can increase and stimulate lipid oxidation. Lipodystrophic syndrome is characterized by insulin sensitivity. We hypothesized that a decrease in the availability of loss of adipose tissue depots associated with low adipokine levels, hepatic choline elevates insulin sensitivity by increasing adiponectin and SOGA steatosis and severe insulin resistance. We hypothesized that fatty liver in (Suppressor Of Glucose by Autophagy), a target of adiponectin in the liver. lipodystrophy may be in part explained by an alteration in AMPK activity. (1) Phosphatidylethanolamine methyltransferase (PEMT) is the enzyme Inversely, pharmacological activation of AMPK in the liver may alleviate responsible for the synthesis of choline in the liver. Insulin sensitivity was hepatic steatosis associated with lipodystrophy. assessed by an oral glucose tolerance test using 2 mg/g glucose (n=4-6; We measured AMPK activity in liver from aP2-SREBP-1c mice, a p<0.05). Improved glucose tolerance was observed in PEMT homozygous lipodystrophy model transgenic mice. We found that AMPK activity was knockout (-/-) mice (n=5; p<0.05). This implies that decreased choline decreased by 50% and phosphorylation of acetyl-CoA carboxylase (ACC), availability may improve insulin sensitivity. (2) Seven female volunteers its downstream substrate, was reduced by 40%. Moreover, hepatic fatty consumed a choline-adequate (CA) diet for 10 days followed by a choline acid oxidation assessed by the plasma concentration of ketones bodies was defi cient (CD) diet for up to 42 days. At the end of the CA and the CD feeding decreased by 50% and hepatic triglyceride (TG) content was increased by regimen, we measured the rate of endogenous glucose production and the 5-fold. The small molecule A-769662 has been identifi ed as a potent and concentration of adiponectin and 28 kDa SOGA, a surrogate marker of SOGA direct activator for AMPK. Treatment of aP2-SREBP-1c mice with A-769662 activity in the liver. Fasting glucose production was measured using a stable for 1 week at a dose of 30 mg/kg b.i.d. increased phosphorylation of AMPK isotope labeled (2H) glucose injection. The CD regimen increased fasting and phospho-Ser79-ACC/ACC ratio in liver. Hepatic fatty acid oxidation was insulin sensitivity based on a decrease in the rate of glucose production completely restored. Importantly, hepatic TG and cholesterol content were (1.20 and 0.84 mg/kg/min) and the HOMA (125 and 145). Surprisingly, the decreased by 10-fold and 20%, respectively. elevation of insulin sensitivity occurred while circulating adiponectin (21.6 In conclusion, AMPK activity and fatty acid oxidation were decreased and 18.7 μg/ml) and 28 kDa SOGA (16.6 and 13.4) were reduced. (3) Primary in the liver of lipodystrophic mice whereas A-769662 treatment led to hepatocytes cultures in choline defi cient media reduced the stimulation of increased rates of fatty acid oxidation and abolished fatty liver. Our study proteolysis by nutrient deprivation. Choline defi ciency inhibited hepatocyte suggests that small molecule AMPK activators may have therapeutic secretion of valine, leucine and isoleucine, essential amino acids that cannot potential to reverse hepatic steatosis in patients with lipodystrophy or other be synthesized or metabolized by hepatocytes, indicating a decrease in metabolic diseases. proteolysis. These observations suggest that choline defi ciency has insulin mimetic effects. Furthermore, choline defi ciency can increase hepatic insulin 1680-P sensitivity independent of adiponectin and SOGA. De Novo Lipogenesis Induced Hepatic Steatosis and Insulin Resist- ance Involve ER Stress but Not Infl ammation in Contrast to Lipid 1682-P Oversupply Diabetes Is a Progression Factor for Liver Fibrosis and Induces LU-PING REN, STANLEY MH CHAN, XIAO-YI ZENG, ROSS D. LAYBUTT, TRISTAN Cirrhosis in a Mouse Diet-Induced Obesity Model of NASH J. ISELI, EDWARD W. KRAEGEN, GREGORY J. COONEY, NIGEL TURNER, JI-MING LISA LO, SUSAN V. MCLENNAN, JAMES G. KENCH, GEOFFREY W. MCCAUGHAN, YE, Sydney, Australia, Melbourne, Australia JAMES BONNER, PAUL F. WILLIAMS, MARK D. GORRELL, STEPHEN M. TWIGG, Mitochondrial dysfunction, infl ammation and endoplasmic reticulum (ER) Sydney, Australia stress have been implicated in hepatic steatosis and insulin resistance in Type 2 diabetes is an independent risk factor for progression of non- genetically obese and insulin resistant mice. The present study investigated alcoholic steatohepatitis (NASH) in man, however animal models to examine the role of mitochondrial dysfunction, infl ammation and ER stress in the this process have not been developed. We recently published that short term development of hepatic steatosis and insulin resistance due to hepatic 5 weeks duration of type 2 diabetes exacerbates liver fi brosis in a 15 weeks de novo lipogenesis from a high fructose diet (HFru), compared to that of mouse model of diet-induced obesity causing NASH. We now report that extrahepatic lipid oversupply from a high fat diet (HFat). Mice fed HFru or more severe pathology occurs when model duration is extended. HFat developed marked hepatic steatosis (triglyceride increased by 3.5 and Wild type C57BL/6 male mice (n=31) were fed a high fat diet (HFD: 2.4 fold, p<0.05) within 3 days, and at 7 day displayed glucose intolerance 45%kCal fat) or standard laboratory chow (CHOW: 12%kCal fat) ad libitum, (iAUC increased by 58% and 64%, p<0.05). Insulin-mediated phosphorylation for 15 weeks, when after randomisation some animals were made diabetic of Akt and GSK3β was suppressed to similar levels in both HFru and HFat (HFD+DM or CHOW+DM) with mini-dose STZ. After a further 22 weeks of mice (40-60%, p<0.05). However, neither HFruc nor HFat feeding altered diabetes animals were terminated. Liver histology was assessed and scored mitochondrial complexes or other key proteins PGC-1α, CTP-1, COX-1, using Blunt and Kleiner criteria and pro-fi brotic markers analysed by qRT- VDAC in the liver or the oxidation of plamitate. HFru feeding led to a 70% PCR. BGL, serum albumin and bilirubin were determined. increase in de novo lipogenesis and 3-8 fold greater expression of lipogenic enzymes ACC, FAS and SCD-1 (p<0.01). Associated with the elevated de Animal group BGL at Intra-acinar Fibrosis Score F2 F3 F4 Cirrhosis Present novo lipogenesis, both the PERK/XBP1 and eIF2 /IRE1 branches of the ER (n) Termination Infl am. Score F0-F1 (n) (n) (n) (n) α (mM) >1 (n) stress signaling pathway were signifi cantly activated. In contrast, HFat (n) feeding signifi cantly suppressed de novo lipogenesis (32%) and the protein expression of FAS and SCD-1 (45-80%) (p<0.05), with no signifi cant changes CHOW (5) 8.9±0.1 0 4 1 0 0 0 in either PERK/XBP1 or eIF2α/IRE1 ER stress signaling pathways. Among the CHOW+DM (5) 19.5±0.8* 1 4 0 1 0 0 investigated infl ammation markers, only JNK was found to be activated in Obesity HFD (9) 9.8±0.6 3 0 1 7 1 1 POSTERS HFat mice (p-JNK increased by 1.9 fold, p<0.01) but not in HFru mice. These HFD+DM (12) 21.6±3.1* 10* 0 4 2 6* 6* fi ndings indicate that the mechanism of initiating hepatic steatosis and insulin resistance by de novo lipogenesis is different from lipid supply. ER Data are mean±SEM, *P<0.05 vs CHOW. Integrated Physiology/ stress is closely associated with the development of hepatic steatosis and HFD+DM mice had signifi cantly increased intra-acinar infl ammation and insulin resistance induced by dietary fructose but not by dietary fat. JNK is the most severe stages of liver fi brosis. In addition 6/12 of the HFD+DM group unlikely to be a key mediator of hepatic insulin resistance linking ER stress showed changes of cirrhosis, determined by stage 4 fi brosis with disrupted and de novo lipogenesis. liver architecture. By qRT-PCR, hepatic pro-fi brotic markers including CTGF and TIMP-1, were increased in HFD+DM (by 6 and 38 fold respectively, each p<0.05 vs CHOW). The HFD+DM group also had liver dysfunction, with lower circulating albumin (CHOW:43.8±3.4 vs HFD+DM:29.2±2.5 g/L) and higher total bilirubin levels (CHOW:0.60±0.02 vs HFD+DM:1.13±0.28 g/L), each P<0.05. Early insulin treatment prevented fi brosis changes (not shown). This

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A457 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

novel long-term model indicates that diabetes contributes to progression 1685-P from NASH to more end-stage liver disease. Subsequent studies will further Effect of Rs7903146 TCF7L2 Gene Variant on Postprandial Glucose defi ne molecular mechanisms of this process. Supported by NHMRC of Metabolism in Subjects with Abnormal Glucose Regulation (AGR) Australia Project Grant #632822. GIUSEPPE DANIELE, AMALIA GASTALDELLI, ANDREA MARI, CRISTINA BIANCHI, Supported by: NHMRC of Australia Project Grant No. 632822 MELANIA MAGGINI, GIUSEPPE PENNO, ROBERTO MICCOLI, STEFANO DEL PRATO, Pisa, Italy, Padova, Italy 1683-P The pathogenetic contribution of common TCF7L2 gene variants to Differential Response of Mitochondrial Enzymes to Obesity and the risk of developing Type 2 diabetes remains far from being completely Excessive Dietary Fat Intake understood. This study was undertaken to evaluate the impact of this gene ELIZAVETA V. MENCHIKOVA, WAN HUANG, FREDERICO G.S. TOLEDO, ROBERT on postprandial glucose metabolism in subjects with AGR (i.e., IGT and/ M. O’DOHERTY, BRET H. GOODPASTER, VLADIMIR B. RITOV, Pittsburgh, PA or IFG). Fourteen subjects (age: 59±10 years; BMI 28±4 kg/m2) with risk- The current study was undertaken to compare the effect of obesity or conferring TCF7L2 genotypes (TT or CT at rs7903146) and 8 subjects (age excessive fat intake on specifi c activity (per mitochondria mass) of electron- 54±11; BMI 28±4) with wild-type genotype (CC) underwent a 4-hr meal- transport chain (NADH-oxidase) and TCA cycle (citrate synthase (CS) that tolerance test with [6,6-(3)H]glucose infusion and [U-13C]glucose ingestion provides entrance of acetyls into TCA cycle) enzymes in human skeletal muscle to study endogenous glucose production (EGP) and gut glucose absorption. and rat liver. Tissue cardiolipin (CL) content was measured as independent Plasma glucose (PG), insulin, C-peptide and glucagon concentrations were non-enzymatic marker of mitochondrial mass. We compared CL, NADH- measured. Mathematical modeling was used to quantify insulin secretory oxidase and CS activity in skeletal muscle biopsies from sedentary lean profi les. insulin sensitive (L, n=9, age 47±2, BMI 24±1) and sedentary obese insulin There was no difference between the 2 groups with respect to basal PG resistant (Ob, n=15, age 42±3, BMI 35±1) subjects. There is no difference (99±3 vs. 93±4 mg/dl), insulin (12±1 vs. 13±2 mU/l), C-peptide (3.4±0.1 vs. in CL content in skeletal muscle from L and Ob (74±9 and 76±6 μg/mU CK, 3.5±0.1 ng/ml), and EGP (13.6±2.2 vs. 18.1±3.1 μmol/kg/min). Only plasma respectively). The activity of NADH-oxidase was reduced signifi cantly in Ob glucagon was lower in the risk allele carriers (62±4 vs. 93±9 pg/ml; p=0.002). in comparison with L (5.63±1.59 and 2.45±0.23 U/mg CL (P=0.02) for L and Ob In response to meal ingestion there was no difference in PG and insulin subjects, respectively). However, the citrate synthase activity is signifi cantly secretion (insulin secretion rate, dose-response, and potentiation). Mean higher in obesity in comparison with lean group (35.8±3.4 and 54.5±5.3 U/mg EPG during the meal test did not differ between the 2 groups (4.4±1.1 vs. CL (P=0.02) for L and Ob, respectively). 7.1±2.1 μmol/kg/min). Total rate of appearance (Ra) tended to be lower in TT/ Analysis of liver mitochondria was performed on a rodent high-fat feeding CT (17.7±2.4 vs. 24.6±4.9 μmol/kg/min; p=0.37) mainly due to reduced oral model. Five weeks of high–fat feeding in rats (n=4) induces insulin resistance, Ra (13.2±1.8 vs. 19.9±3.8; p=0.16). In the whole population the latter was decreases specifi c NADH-oxidase activity (1.470±0.53 and 0.997±0.08 U/ positively correlated with mean PG following the meal ingestion (r=0.44; mg CL for control (C) and high fat-fed (HF) rats, respectively), signifi cantly p=0.07). Mean plasma glucagon remained constantly lower in TT/CT (97±11 increases citrate synthase activity (19.7±2.1 and 27.4±1.4 U/mg CL (P=0.023) vs. 60±4 pg/ml; p=0.018) accounting for slightly larger insulin:glucagon ratio for C and HF rats, respectively) and slightly decreases liver cardiolipin (1.09 vs. 0.85). content. We hypothesize that excessive dietary fat intake induces activation In conclusion, middle-aged individuals with AGR carrying the rs7903146 of CS (by induction of protein synthesis or by post-translational modifi cation TCF7L2 risk genotype do not have signifi cant abnormalities in glucose in liver and skeletal muscle) to utilize excessive acetyl-CoA produced in metabolism after the ingestion of a mixed meal. Moreover, they tend to have beta-oxidation pathway. lower levels of circulating glucagon and lower rate of appearance of oral These data highlight that mitochondrial TCA cycle and electron transport glucose into the systemic circulation chain enzymes respond differently to energy excess. Further investigation is Supported by: EFSD Merck grant needed to better understand the respective roles of specifi c mitochondrial function in obesity and insulin resistance. ADA-Funded Research 1686-P Exogenous Erythropoietin Attenuates Hepatic Dyslipidemia, Oxida- 1684-P tive Stress and Infl ammation in High-Fat Diet Mice Effect of Ecdysterone on Hepatic AMP Activated Protein Kinase in RAN MENG, YA-PING WANG, DA-LONG ZHU, Nanjing, China Mice Fed with a High-Fat Diet Erythropoietin(EPO), a well known hematopoietic cytokine, has been ZHONG Q. WANG, YONGMEI YU, XIAN H. ZHANG, DAVID RIBNICKY, WILLIAM T. demonstrated to attenuate insulin resistance in patients with end-stage renal CEFALU, Baton Rouge, LA, New Brunswick, NJ disease. However, the mechanism is still obscure, which was investigated Chronic nutrient overload leads to obesity and metabolic disorders, in the present study. Four-week-old male C57BL/6 mice were fed with high- including insulin resistance and type 2 diabetes. Ecdysterone (Ecdy), fat diet (HFD) for 12 weeks to induce insulin resistance (IR). Mice were then specifi cally20-hydroxyecdysone, is a steroid hormone from plants). Our arranged to get injections of recombinant human EPO (2000U/kg, EPO group) previous study showed that Ecdy may have anti-obesity and anti-diabetic or saline (HFD group) three times per week for fi ve weeks. Mice with EPO effects, but its molecular mechanisms remain largely unknown. It is well treatment exhibited signifi cantly slower weight gain compared to that of documented AMP activated protein kinase (AMPK) plays a key role in glucose HFD group without energy intake changes. Intraperitoneal glucose tolerance metabolism. In order to evaluate the effect of Ecdy on AMPK signaling, we tests, fasting insulin and glucose showed that EPO treatment signifi cantly examined hepatic AMPK pathway proteins in mice fed a low-fat diet (control) improved insulin sensitivity after EPO treatment. EPO treatment signifi cantly or high-fat diet (HFD) with or without low dose (25 mg/kg body weight, Ecdy reduced hepatic steatosis (H&E and red-oil O staining), decreased 45% L) or high-dose (50 mg/kg, Ecdy H) of Ecdy for 12 weeks. Body weight, fasting hepatic free fatty acids and down-regulated 40% sterol response element glucose and insulin concentrations were measured as well. At study end, binding protein 1c and 42% fatty acid synthase gene expression in the no differences in body weight, food intake or energy expenditure were liver (p<0.05 vs. HFD group). Meanwhile, EPO increased the activities of observed between HFD groups with or without Ecdy L, but the body weight total superoxide dismutase, Cu-Zn superoxide dismutase, glutathione and was signifi cantly lower in Ecdy H group relative to the HFD group (P<0.05). glutathione peroxidase (by 40%, 24%, 50%, 26%, respectively. p<0.05

Obesity Plasma glucose and insulin levels in Ecdy H group were also signifi cantly for each vs. HFD group), and decreased the activity of malondialdehyde

POSTERS lower than in the HFD group (P<0.01). Hepatic AMPK phos, AMPK α1 and by 25% (p<0.05 vs. HFD group) in the liver compared to HFD group. With AMPK α2 levels were signifi cantly higher in the HFD and the Ecdy L groups regard to the anti-infl ammatory function, the activity of nuclear factor κB

Integrated Physiology/ than in the LFD control (P<0.001, P<0.01 and P<0.05 respectively). AMPK α1 and gene expression of tumor necrosis factor-α, interleukin-6 and monocyte abundance was signifi cantly higher in the Ecdy H group than in LFD group chemotactic protein-1 were signifi cantly decreased in EPO group (15%, (P<0.05), but was signifi cantly lower than in the HFD group. AMPK α2 content 51%, 20%, 13%, respectively, p<0.05 for each vs. HFD group). Importantly, was modestly reduced in both Ecdy groups relative to the HFD group. These EPO receptor was detected in the liver. Taken together, our data indicate results suggest that high-fat diet altered hepatic AMPK signaling in mice that exogenous EPO could attenuate hepatic dyslipidemia, oxidative stress by increasing AMPK protein expression to compensate high-fat diet induce- and infl ammation induced by HFD. IR was ameliorated by EPO through its insulin resistant. Ecdy may partially attenuate the effects of high-fat diet multiple protective functions in HFD mice. Our fi ndings may result in novel on AMPK pathway with enhancing insulin sensitivity, especially at higher therapeutic strategies for IR and diabetes. dose. Supported by: Botanical Research Center Pilot Grant

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A458 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

1687-P at one month. Morphologically the KO livers are paler than WT livers with a Free Fatty Acid-Induced Hepatic Insulin Resistance Is Mediated by roughened surface and many small nodules. Microscopic examination of the Endoplasmic Reticulum Stress H&E stained liver sections showed that hepatocytes have varied morphology, CRISTINA DIRLEA, SANDRA PEREIRA, ADRIA GIACCA, Toronto, ON, Canada with occasional multiple nuclei, as well as enlarged nuclei and very small Insulin resistance is a common feature of obesity and is primarily caused condensed nuclei, indicating proliferation as well as apoptosis. Inactivation by an increased release of free fatty acids (FFA) and altered release of of SRp20 appears to inhibit hepatocyte maturation as livers exhibit several adipokines from the expanded adipose tissue. FFA-induced insulin resistance characteristics of fetal liver, including increased expression of proliferation has different mechanisms depending on the tissue type and although markers PCNA and Ki-67, increased alpha fetoprotein and the fetal isozyme previous work in our lab has shown activation of c-Jun-N-terminal kinase of pyruvate kinase (M-PK). On the other hand, the expression of albumin, (JNK) in the liver upon short-term lipid infusion, whether JNK is causal in Phenylalanine hydroxylase (PAH), gluconeogenic enzymes e.g., Glycogen FFA-induced insulin resistance is unknown. One potential mechanism of synthase-2 (Gys-2), PEPCK and G-6-Pase, and the adult isozyme of pyruvate JNK activation is as a result of endoplasmic reticulum (ER) stress. Therefore, kinase (L-PK) are signifi cantly reduced in SRp20KO liver. Moreover, FACS the present study investigates the causal roles of ER stress and JNK in analysis revealed signifi cant decrease of hepatocyte tetraploidy, which is FFA-induced insulin resistance. Wistar rats were infused intravenously an indicator of hepatocyte maturation. The livers also contain reduced for 7 hours with saline or Intralipid plus heparin to elevate plasma FFA levels of glycogen and the mice are prone to fasting-induced hypoglycemia. with or without an ER stress inhibitor (4-phenylbutyrate (4-PBA)) or a JNK The splicing pattern of the insulin receptor has also been changed in inhibitor (SP600125). Insulin-induced suppression of endogenous glucose SRp20KO liver. Collectively, our results establish that SRp20 is essential for production during hyperinsulinemic-euglycemic clamp was measured using morphological and functional differentiation of hepatocytes. tracer methods. 4-PBA co-infusion prevented FFA-induced hepatic insulin Supported by: VA Merit Grant resistance (P<0.05), whereas co-infusion of the JNK inhibitor only showed a tendency to prevent hepatic insulin resistance. We conclude that either the 1690-P JNK inhibitor was not effective in decreasing JNK activation in this model or Losartan Alone or Combined with Simvastatin Improved Visceral FFA-induced hepatic insulin resistance is prevented by the ER stress inhibitor Adipose Tissue and Infl ammation in Hypertensive Patients with Non- by mechanisms that are largely independent of JNK. Alcoholic Hepatic Steatosis ROBERTO FOGARI, AMEDEO MUGELLINI, PAMELA MAFFIOLI, GIUSEPPE DEROSA, 1688-P Pavia, Italy Glucotoxicity (GTX) Contributes to Progressive Reduction of Hepatic Aim of this study was to evaluate the effect of losartan (L) alone or Glucokinase (GK) in Zucker Diabetic Fatty Rats (ZDF) by Affecting a combined with simvastatin (S) on steatosis degree (SD), visceral adipose Post-Transcriptional Process tissue (VAT), insulin sensitivity, and Hs-CRP in hypertensive patients with KIICHIRO UETA, KUIKWON KIM, RICHARD L. PRINTZ, GREGORY A. MCCOY, non-alcoholic hepatic steatosis. TRACY P. TORRES, MASAKAZU SHIOTA, Nashville, TN After a 2 week placebo period, 150 hypertensive outpatients with non- We determined whether progressive reduction of GK expression during alcoholic hepatic steatosis were randomized to L 100mg/day or amlodipine development of diabetes in ZDF corresponds to changes in GKmRNA. (A) 10mg/day, for 6 months; subsequently S 20mg/day was added to both Expression levels were normalized to the average value of age-matched lean treatments for further 6 months. The patients performed an ultrasound littermates (ZCL). ZDF (6 h-fasted) were characterized by mild hyperglycemia examination to evaluate SD, [ultrasound grading (0-3)], and VAT diameter, (HG: 13±2 ZDF-E vs 7±1 ZCL; mM) and hyperinsulinemia (HI: 9±2 ZDF-E vs 1.3±0.2 [distance between the internal surface of the straight muscle of the abdomen ZCL; ng/mL) at an early (E; 10-11wk-old), HG (23±2 ZDF-M vs 7.8±0.4 ZCL-M) and the wall of the aorta], an euglycemic clamp [to evaluate glucose infusion and mild HI (2.8±0.3 ZDF-M vs 0.9±0.2 ZCL-M) at a middle (M; 14-15wk-old), rate (GIR)], and measurement of plasma Hs-CRP at baseline, after 6 and 12 and HG (31±1 ZDF-L vs 7.5±0.3 ZCL-L) and slight HI (1.6±0.2 ZDF-L vs 0.9±0.2 months of treatment. ZCL-L) at a late (L; 20-22wk-old) stage of diabetes. GKmRNA relative to ZCL Both drugs induced a signifi cant and similar SBP/DBP reduction. SD as well were 3.8±0.9-, 1.7±0.5- and 1.3±0.2-fold greater in ZDF-E, ZDF-M and ZDF-L, as VAT were reduced by L (-0.18, p<0.05 and -0.43, p<0.046, respectively), respectively. In contrast, GK levels were similar in ZDF-E (1.1± 0.1-fold), slightly but not by A. S addition signifi cantly amplifi ed the SD and VAT reduction in L lower in ZDF-M (0.8±0.1-fold) and signifi cantly lower in ZDF-L (0.3±0.1-fold). (-0.39, p<0.001 and -1.52, p<0.001, respectively) and also in A group, even if GKmRNA roughly correlated with plasma insulin (PI); whereas, GK inversely at a lesser degree (-0.18, p<0.04 and -0.32, p<0.05, respectively). L induced correlated with plasma glucose (PG). To further evaluate whether GTX affects a weak increase in GIR (+0.4 mg/min/kg, p=0.049), while A did not change GKmRNA and GK, ZDF-M were given a SGLT2 inhibitor, Canaglifl ozin (CA; 10 it. S addition signifi cantly increased GIR in both L (+1.3 mg/min/kg, p<0.01) mg/kg qd) or vehicle (V) orally for 6wks. In ZDF-V, PG (mM) increased from 20±3 and in A group (+0.5 mg/min/kg, p<0.05). Hs-CRP was signifi cantly reduced at 14wk to 27±2 at 20wk and PI (ng/ml) decreased from 2.5±0.2 to 1.5±0.2. by L (-0.4, p<0.05), but not by A (-0.2, ns). S addition ameliorated Hs-CRP While GKmRNA levels relative to ZCL were maintained (1.7±0.4 at 14wk and reduction in L (-0.6, p<0.041) and also in A group (-0.5, p< 0.05). The SD 1.4±0.3 at 20wk), GK levels were markedly reduced from 0.8±0.2 at 14wk to changes showed a signifi cant relationship with both GIR (r=-0.35, p<0.05), 0.3±0.1 at 20wk. In ZDF-CA, PG decreased within 1wk from 20±2 to 8±1 and and Hs-CRP (r=0.36, p<0.05) changes; also the VAT changes were correlated was maintained at near normal levels. PI was not signifi cantly different from to GIR (r=-0.37, p<0.05), and to Hs-CRP (r= 0.31, p<0.05) changes. that of ZDF-V. GKmRNA at 20wk (1.8±0.4) were similar with that of ZDF-V. L/S combination improved the hepatic SD and VAT diameter more than However, GK was maintained at 14wk levels (0.8±0.1). In response to gastric A/S combination. Although a possible explanation might be the greater infusion of a mixed meal (4.7 g glucose, 2.2 g protein and 0.6 g lipid per kg improvement in insulin resistance, it is possible the contribution of some body weight), the increment of PG (AUC mol × l-1 × min-1) and PI (AUC mol × l-1 other mechanism of the L/S combination, which yet needs to be clarifi ed. × min-1) were lower in ZDF-CA (3.9±0.5 and 589±55) than ZDF-V (5.9±0.6 and 750±50), which corresponded with sustained GK without an increment change 1691-P in GKmRNA. HG per se contributes to lower GK expression by affecting a post- PHF2 Demethylase Links ChREBP-Dependent Induction of Fatty transcriptional process. Acid Synthesis to the Development of Fatty Liver

JULIEN BRICAMBERT, JEAN GIRARD, CATHERINE POSTIC, RENAUD DENTIN, Obesity 1689-P Paris, France POSTERS Hepatocyte Maturation Is Arrested in Mouse Liver Lacking the Obesity and type 2 diabetes are associated with increased hepatic

Splicing Factor SRp20 lipogenesis. This results in fat accumulation in hepatocytes, a condition Integrated Physiology/ SUPRIYA SEN, NICHOLAS JG WEBSTER, San Diego, CA, La Jolla, CA known as hepatic steatosis, the most common cause of liver dysfunction SRp20 is a splicing factor and the smallest member of the highly conserved worldwide. Carbohydrate-responsive element–binding protein (ChREBP), a SR protein family. These proteins regulate both constitutive and alternative transcriptional activator of glycolytic and lipogenic genes, has emerged as a splicing in vivo and we have previously shown that SRp20 regulates splicing major player in the development of hepatic steatosis. However, the molecular of the insulin receptor gene in vitro. Recent studies have implicated SR mechanisms enhancing its transcriptional activity remain partially unknown. proteins in additional functions, including transcription, genomic stability, Posttranslational modifi cations of histone tails, including reversible mRNA export and translation. Here we used Cre-loxP-mediated tissue acetylation, methylation, phosphorylation and ubiquitination, modulate both specifi c recombination in mice to inactivate the SRp20 gene in liver as the chromatin structure and gene regulation. Although histone marks are altered whole body SRp20 knockout is embryonic lethal. The liver-specifi c SRp20 in response to cellular conditions the underlying mechanism by which this is knockout mice are smaller and have signifi cantly lower body and liver weight achieved is largely unknown.

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A459 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER

In this study, we identify the histone demethylase plant homeodomain EGP were reduced 18% (P<0.01) and 33%, respectively. Interestingly, PC (PHD) fi nger 2 (phf2) as an important regulator of both glycolytic and lipogenic ASO reduced epididymal fat mass 25% and hepatic triglyceride content gene expression acting as an upstream regulator of ChREBP activity. In 33% (both P<0.05). In ZDF rats, while PC ASO had no effect on body weight cultured mouse hepatocytes, using chromatin immunoprecipitation (ChIP) or adiposity, the effects on glucose persisted, with reductions in fasting analysis, we show that phf2 is recruited to the ChoRE containing region glucose [168±6.7 vs. 149±5.3 mg/dl (P<0.05)], basal EGP [9.3±0.9 vs. 6.9±0.4 of both glycolytic and lipogenic gene promoters (including LPK and FAS) mg/kg-min (P<0.05)] and clamped EGP [4.0±0.5 vs. 2.7±0.1 mg/kg-min in response to glucose and insulin stimulation. Then, phf2 specifi cally (P<0.05)]. In contrast, insulin–stimulated peripheral glucose disposal was demethylates di-methylated lysine 9 on histone H3 (H3K9Me2) allowing the unchanged in both models. Conclusion: ASO mediated inhibition of PC in vivo recruitment of ChREBP to these promoters to increase fatty acid synthesis. is a novel therapeutic approach that safely and effectively lowers plasma In the opposite, phf2 silencing results in increased H3K9Me2 methylation at glucose in a variety of rodent models by reducing hepatic glucose production these promoters, resulting in decreased of ChREBP occupancy and inhibition and surprisingly augmenting postprandial insulin concentrations. of fatty acid synthesis. Finally, in liver of fed diabetic db/db mice, high phf2 activity at these glycolytic and lipogenic gene promoters is associated with 1694-P decreased H3K9Me2 methylation and increased ChREBP transcriptional Reduced Hepatic Insulin Clearance as Measured by CEACAM-1 activity, correlating with the development of hepatic steatosis. Our fi ndings Expression Accounts for Hyperinsulinemia in Fat Fed Dogs suggest that inhibition of phf2 activity in liver may be benefi cial for treating MORVARID KABIR, MALINI S. IYER, DARKO STEFANOVSKI, ORISON WOOLCOTT, hepatic steatosis in state of obesity and type 2 diabetes. STELLA P. KIM, ISABEL R. HSU, KARYN J. CATALANO, JENNY D. CHIU, LISA N. Supported by: Agence Nationale de la recherhe (ANR-09-JCJC-0057-01) and by HARRISON, VIORICA IONUT, RICHARD N. BERGMAN, JOYCE M. RICHEY, Los the FRM Angeles, CA Hyperinsulinemic compensation (HC) whether by enhanced insulin 1692-P secretion or reduced insulin clearance is well established in our diet- Prenatal Protein Defi ciency Alters Circadian Rhythms in Core Clock induced insulin resistant canine model. However, the molecular correlates Oscillator Protein Expression in the Offspring underlying HC are not well understood. We undertook this study to elucidate ARMAND V. CENTANNI, DIANA C. ALBARADO, GREGORY M. SUTTON, Baton the molecular mechanisms associated with HC. Mild obesity and insulin Rouge, LA resistance concomitant with HC was induced in 9 male mongrel dogs by The mechanisms linking intrauterine growth retardation (IUGR) with feeding them high fat diet (HFD; 52% fat; n=9) for 22 weeks. The control adulthood obesity and diabetes are unknown. Previous studies performed group (C) was maintained on a standard diet (C; 33% fat; n=6). Liver and in our lab have demonstrated an altered circadian phenotype in 8 wk old pancreatic biopsies were collected from all animals for histology and gene male C57Bl/6J mice subjected to protein malnutrition (undernourished expression studies. We estimated beta cell function and proliferation by offspring, UO) in utero coupled with altered glucose homeostasis. Gene measuring gene expressions of pancreatic and duodenal homeobox1 (PDX1) expression of the nuclear receptor, Rev-erbα, a component of the circadian and insulin in pancreas. Additionally, we measured the gene that regulates clock mechanism in liver was dramatically reduced and out of phase in UO at hepatic insulin clearance, carcinoembryonic antigen-related cell adhesion 8 wk of age compared to control offspring (CO). Rev-erbα repressed genes molecule 1 (CEACAM1). Immunohistochemical measurements of total PDX1 involved in circadian regulation (Bmal1 and Per2) were increased in UO mice. and insulin showed no changes, between control and HFD groups. In addition, Surprisingly, protein expression of Rev-erbα in UO liver did not oscillate and nuclear/cytoplasmic PDX1 ratio, indicative of a beta cell defect remained was expressed at similar levels at circadian time points (CT) 1200 and 2400 unchanged. Consistent with immunohistology results, gene expressions of (noon and midnight) suggesting gene expression and protein translation are PDX1 (C: 1.16±0.32; HFD: 1.01±0.17) and insulin (C: 0.42± 0.12; HFD: 0.56± misaligned compared to CO. Phosphorylation of glycogen synthase kinase- 0.22) showed no signifi cant differences. In contrast, gene expression of 3β and protein kinase B, two second messenger signaling molecules that CEACAM1 in the liver was reduced by 70% in HFD group (C: 0.799±0.28; play a role both directly (GSK3β) and indirectly (PKB) in Rev-erbα regulation. HFD: 0.237±0.04; p<0.05), indicative of reduced hepatic insulin clearance. We now demonstrate that phosphorylation at the GSKβ site on Rev-erbα These data suggest that after 22 weeks of fat feeding, beta cell function is dramatically reduced in UO liver, suggesting altered transcription and does not contribute signifi cantly to HC, which is consistent with the in vivo regulation. These data suggest potentially cell surface signaling, perhaps data. Thus, the primary mechanism of HC induced by prolonged fat-feeding through insulin may play a role in impaired clock controlled processes in in a canine model is reduced hepatic insulin clearance. UO mice. We conclude that UO mice exhibit a metabolic disorder involving abnormal circadian patterns of gene and protein expression. Altered Rev- 1695-P erbα expression and function may be a key factor in metabolic dysregulation Regulation of SOGA (Suppressor of Glucose by Autophagy) in Diabetes associated with IUGR. ADA-Funded Research JENNIFER LAW, OMAR ABDELBAKY, LAURA JOSEPH, KEVIN FLURKEY, LALITHA KUNDURU, ELIZABETH ALDERMAN, RITA BASU, ROLAND TISCH, JOSEPH 1693-P GALANKO, JANET SNELL-BERGERON, DAVID MAAHS, DAVID HARRISON, ELIZA- Pyruvate Carboxylase, a Novel Therapeutic Target for Type 2 Diabetes BETH MAYER-DAVIS, TERRY COMBS, Chapel Hill, NC, Bar Harbor, ME, Rochester, NAOKI KUMASHIRO, SARA A. BEDDOW, IOANA FAT, SACHIN K. MAJUMDAR, MN, Aurora, CO FITSUM GUEBRE-EGZIABHER, JENNIFER L. CHRISTIANSON, PRASAD MANCHEM, Diabetes is linked to the elevation of gluconeogenesis. Suppressor of BRETT P. MONIA, SANJAY BHANOT, GERALD I. SHULMAN, VARMAN T. SAMUEL, glucose by autophagy (SOGA), a novel protein produced in the liver, inhibits New Haven, CT, Carlsbad, CA gluconeogenesis. Therefore, low SOGA can play a role in diabetes. To gain Fasting hyperglycemia in T2D is due to increased gluconeogenesis, a further insight about the role of SOGA in diabetes, we measured SOGA in process for which the enzymatic regulation is poorly understood. We recently patients with type I diabetes (T1D) using circulating levels of 28 kDa SOGA, a observed an association between fasting hyperglycemia and expression surrogate marker of SOGA expression in the liver. Our second objective was of pyruvate carboxylase (PC) but not PEPCK and G6Pase in several rodent to measure both 80 kDa liver SOGA and 25 kDa plasma SOGA in mice. SOGA models and thus, hypothesized that PC is an excellent therapeutic target for was measured by SDS-PAGE electrophoresis using species specifi c rabbit

Obesity T2D. To test this, we used antisense oligonucleotides (ASO’s) to decrease polyclonal antisera, enhanced chemiluminescence, digital image capturing

POSTERS PC expression in normal SD rats fed a regular chow, high-fat fed (HFF) SD and quantitative band assessment. (1) Plasma samples were obtained from rats and Zucker Diabetic Fatty (ZDF) rats. ASO’s specifi cally decrease target 85 patients with T1D (ages 6-18) during regular clinical visits. Circulating

Integrated Physiology/ expression in liver and adipose but not other tissues (e.g. pancreas). PC ASO levels of 28 kDa SOGA did not correlate with plasma glucose, HgA1c, time treatment decreased PC expression by ∼90%. In regular chow fed rats, this since insulin injection or age (n=10 per group; p>0.05). In a separate study, reduced plasma glucose concentrations in both the fasted [Cont: 109±1 vs. SOGA was measured in plasma samples obtained from adults during a PC: 103±1, (P<0.01)] and ad lib states [146±2 vs. 133±3 mg/dl (P<0.01)], without 3-stage euglycemic/ hyperinsulinemic clamp. By the end of the clamp, SOGA ketosis or lactic acidosis. Plasma insulin concentration was unchanged when was suppressed by 20% in healthy but not in patients with TID. Patients with fasting, but surprisingly, increased in the ad lib fed state [27±2 vs. 47±7 µU/ TID were insulin resistant based on the glucose infusion rate (n=10 per group; mL (P<0.01)]. In HFF rats, PC ASO reduced fasting plasma glucose [125 vs. p<0.05). (2) Prepubertal (3-4 weeks old) mice had 40-50% less SOGA in liver 108 mg/dl (P<0.01)], fasting insulin [12.9±1.5 vs. 8.5±1.2 µU/mL (P<0.05)] and and plasma than adult (3 months old) mice (n=5 per group; p<0.05). SOGA in HOMA-IR (4.0±0.5 vs. 2.3±0.4 mg/dl*µU/mL (P<0.01)]. The changes in insulin liver and plasma fell by 30% within 72 hours of the appearance of TID in NOD sensitivity were assessed with a euglycemic-hyperinsulinemic (4 mU/kg- mice (n=5 per group; p<0.05). Insulin resistant ob/ob mice showed a 30% min) clamp. Both basal endogenous glucose production (EGP) and clamped increase in liver and plasma SOGA after food intake was restricted by 50%

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A460 INTEGRATEDCATEGORY PHYSIOLOGY—LIVER for a period of 3-4 months (n=7 per group; p<0.05). In summary, the absence These data demonstrate that BH4 exerts glucose lowering effects through of any correlations between plasma glucose, HgA1c, time since insulin suppression of hepatic gluconeogenesis. injection and age in humans indicates the need to develop standardized We conclude that BH4 suppresses hepatic gluconeogenesis and activates RIA and ELISA methods or conveniently measuring SOGA in large sample AMPK and that intra-hepatocellular eNOS is required for these effects. sets. Hyperinsulinemia during euglycemic clamps reduced SOGA but not in Thus, BH4 has potential in a novel therapeutic approach to type 2 adults with TID. In mice, puberty stimulated liver and plasma SOGA however diabetes. TID and insulin resistance reduced SOGA. These observations support the hypothesis that low SOGA can play a role in diabetes. 1698-P The Effect of Cilostazol on Plasminogen Activator Inhibitor Type 1 1696-P Expression in Liver Sirt1 Expression Is Auto-Regulated through Expression of LKB1 and MI-KYUNG KIM, YUN A. JUNG, GWON SOO JUNG, HYE-SOON KIM, NAM- AMPK Via Negative Feedback KYEONG KIM, EON-JU JEON, HYUN-AE SEO, IN-KYU LEE, KEUN-GYU PARK, FAN LAN, JOSE M. CACICEDO, NEIL B. RUDERMAN, YASUO IDO, Boston, MA Daegu, Republic of Korea Both Sirt1 and AMPK play pivotal roles in cellular homeostasis and aging, Accumulating evidence suggests that plasminogen activator inhibitor however, relationship between Sirt1 and AMPK is not well understood. This (PAI)-1 plays an important role in the development of hepatic fi brosis via its study was aimed to elucidate this relationship. involvement in extracellular matrix remodeling. Cilostazol, a selective type Various cells including HepG2, human endothelial cells, keratinocytes, 3 phosphodiesterase inhibitor, is a potent antiplatelet agent currently used and 3T3L1 adipocytes were used. Lentivirus expressing shRNA was used to in clinical practice to treat patients with diabetic vascular complications. knockdown Sirt1. Lenti- or adenovirus expressing AMPK alpha 2 and LKB1 Our previous study showed that cilostazol inhibits vascular PAI-1 expression and their kinase-dead form were also created. through multiple mechanisms including transforming growth factor-b In HepG2 cells, lentivirus mediated Sirt1 knockdown caused an 80- (TGF-b) pathway. However, the effect of cilostzol on hepatic fi brosis 90% reduction in Sirt1 expression after 4-5 days, and decreased AMPK is unknown. Therefore, the aim of the present study was to determine phosphorylation due to LKB1 dysfunction. In contrast, in the cells with whether cilostazol prevents hepatic PAI-1 expression and fi brosis. We found chronic knockdown of Sirt1 after 2 passages, phosphorylation of AMPK that cilostazol effectively inhibits TGF-b-stimulated PAI-1, collagen and was unexpectedly increased. This increase was mediated by increased fi bronectin expression in liver cells. In addition, transient transfection study expression of AMPK alpha2 and decreased ATP due to lowered mitochondrial showed cilostazol inhibited TGF-b-stimulated and SMAD 3/4-stimulated Complex I expression. LKB1 protein abundance was also increased; however, PAI-1 promoter activity in a dose dependent manner. TGF-b increased phosphorylation of LKB1 substrate Mark1 protein and the phospho- to phosphorylation of Smad3, which is downstream of TGF-b, and nuclear total ratio of AMPK were actually decreased, suggesting persistence localization of phosphorylated Smad3. However, cilostzol inhibited nuclear of LKB1 dysfunction. In agreement with this, in Sirt1 knockdown 3T3L1 and cilostazol inhibited TGF-b-induced Smad3 phosphorylation and nuclear adipocytes, introduction of LKB1 K48R (mimicking LKB1 deacetylation form) localization of phosphorylated Smad3. In conclusion, cilostazol inhibited restored energy levels by increasing Complex I expression, and prevented TGF-b-stimulated PAI-1 expression and these data suggest it could be used senescence-associated infl ammation. We hypothesized that increased LKB1 to prevent hepatic fi brosis. and AMPK levels were signals that the cells try to restore Sirt1 levels by feedback mechanisms. To test this, we overexpressed AMPK alpha 2 or 1699-P LKB1 in various cells and found that increased expression of these proteins The Effect of Clusterin on Hepatic Lipogenesis dose-dependently increased Sirt1 levels and dominant negative forms HYE-YOUNG SEO, HAN-JONG KIM, JOON-YOUNG KIM, YOUNG-KEUN CHOI, JI- had opposite effects. In contrast, chemical activators of AMPK including HYUN KIM, KYUNG-SOOK LEE, CHAE-MYEONG HA, JUNG-GUK KIM, EON-JU AICAR, metformin, or A769662 had little or no effect on Sirt1 levels, and in JEON, HYUN-AE SEO, KEUN-GYU PARK, IN-KYU LEE, Daegu, Republic of Korea keratinocytes, AICAR in 16 hrs-incubation actually decreased Sirt1 levels. Fatty liver is common in obese subjects with insulin resistance. Sterol These results suggest thatLKB1 plays key roles for Sirt1’s actions to regulatory binding protein-1c (SREBP-1c) is a master regulator of lipogenic prevent cellular aging and to keepSirt1’s expression levels via negative gene expression in liver. Clusterin is an 80 kDa disulfi de-linked heterodimeric feedback. protein and plays a role in cholesterol transport from peripheral tissues to Chronic activation of AMPK without increasing its abundance on the other the liver. However, little is known about the effects of clusterin on hepatic hand has little effect on Sirt1 expression and in some cells may deplete its lipogenesis. Here, we examined whether clusterin regulates SREBP-1c expression. expression in liver. The effect of adenovirus-mediated overexpression of clusterin (Ad-Clu) on the expression of SREBP-1c stimulated by insulin or liver 1697-P X receptor (LXR) agonist in liver cell lines were determined by Northern and Tetrahydrobiopterin (BH4) Activates AMPK and Suppresses Hepatic Western blot analyse. The in vivo effects of Clusterin on hepatic lipogenesis Gluconeogenesis through ENOS-Dependent Pathway were verifi ed by tail vein injection of Ad-Clu into HFD fed mice. Transient YOSHIHITO FUJITA, ABULIZI ABUDUKADIER, AKIO OBARA, MASAYA HOSOKAWA, transfection study was performed to assess the effect of clusterin on SREBP- SHIMPEI FUJIMOTO, TORU FUKUSHIMA, YUICHI SATO, MASAHITO OGURA, 1c promoter activity. The effect of Clusterin on LXR-DNA binding activity was YASUHIKO NAKAMURA, NOBUYA INAGAKI, Kyoto, Japan examined by electrophoretic mobility shift assay. Ad-Clu inhibited insulin or Tetrahydrobiopterin (BH4) is an essential co-factor in endothelial nitric LXR agonist-stimulated SREBP-1c expression and its promoter activity. oxide synthase (eNOS) activity. We previously demonstrated that intra- hepatocellular eNOS is required for activation of AMP-activated protein kinase (AMPK) and suppression of hepatic gluconeogenesis by metformin in liver, which suggests that eNOS plays an important role in hepatic glucose metabolism. In this study, we investigated the effect of BH4 in suppression of hepatic gluconeogenesis using mouse hepatocytes.

In wild-type mouse hepatocytes, BH4 dose-dependently and time- Obesity dependently suppressed hepatic gluconeogenesis and activated AMPK. POSTERS BH4 had no effects on hepatic gluconeogenesis or AMPK activation in

eNOS-defi cient mouse hepatocytes. Sodium nitroprusside, a nitric oxide Integrated Physiology/ donor, suppressed hepatic gluconeogenesis and activated AMPK in both wild-type and eNOS-defi cient mouse hepatocytes. These fi ndings indicate that eNOS-dependent nitric oxide production enhanced by BH4 activates AMPK and suppresses gluconeogenesis in liver. To examine whether BH4 lowers blood glucose levels in vivo, 20mg/kg BH4 was injected intraperitoneally to streptozotocin diabetic wild-type mice fasted for 16 hours. Administration of BH4 signifi cantly lowered blood glucose levels and activated AMPK in liver tissues 2 hour after injection of BH4. By pyruvate tolerance test, BH4 injected intraperitoneally to wild-type mice signifi cantly decreased elevation of blood glucose levels compared with saline injection.

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A461 INTEGRATED PHYSIOLOGY—MACRONUTRIENTCATEGORY METABOLISM AND FOOD INTAKE

& 1701-P Metformin and Pioglitazone Altered Plasma Amino Acid Metabolites— Potential Biological Markers of Insulin Sensitivity AUDREY J. WEYMILLER, BRIAN IRVING, RICKEY E. CARTER, MAI PERSSON, LAURA TATPATI, SUMIT BHAGRA, MATTIAS SOOP, K. SREEKUMARAN NAIR, Rochester, MN, Austin, MN Insulin affects amino acid (AA) metabolism but AA may affect insulin action. To determine whether enhancing insulin sensitivity (SI) with insulin sensitizers alters AA and their metabolites, we randomized 25 participants with impaired fasting glucose or untreated T2DM (=Average [range]; In vivo model, Ad-Clu in liver of mice by tail vein injection inhibited Age=55 [27-71]; BMI=30.1 [24.9-35.9]; Fat%=46 [30.8-58.2]; FBS=129 [104- hepatic steatosis through inhibition of SREBP-1c expression. Additionally, 193]; Hgba1c=6.1[5-7.7]) in a double-blind design to receive placebo or Clusterin inhibited LXR-agonist-stimulated LXR-DNA binding to its response metformin+pioglitazone (met+pio). Before and following 12-weeks of either consensus element on SREBP-1c promoter. This study shows that Clusterin met+pio or placebo administration, SI was measured by hyperinsulinemic- inhibits hepatic lipogenesis in vitro and in vivo through downregulation of euglycemic clamp while replacing AA with infusion of 5% NephrAmine. In SREBP1c. The present study suggested that clusterin plays a critical role in samples collected prior to the infusions, plasma AA and their metabolite the regulating hepatic lipogenesis. concentrations were separated by ultra pressure liquid chromatography and detected by selected reaction monitoring on a triple Quadra pole mass spectrometer. Glucose Infusion Rate (μM/min/kg.FFM) to maintain similar INTEGRATED PHYSIOLOGY—MACRONUTRIENT glucose levels was higher (p<0.001) following met+pio (23.7±14.5 to 42.2±13.1, METABOLISM AND FOOD INTAKE p<0.001) than placebo (25.0±15.1 to 26.5±20.3; p=0.6). In comparison with the placebo, met+pio administration caused highly signifi cant changes [See also: Presidents Poster 455-PP, page A126.] in plasma concentrations (Δ met+pio vs. Δ placebo) of citrulline (-8.983 vs. 1.566, p<.0001), alpha-amino-adipic acid (-0.175 vs. -0.004, p=.024), ethanolamine (-1.488 vs. 0.217, p=.004), glutamic acid (-10.921 vs. 2.642, Guided Audio Tour: Macronutrients and Metabolism (Posters 1700-P to p=.001), glycine (29.687 vs. -0.524, p=.023), alpha-aminobutyric acid (3.178 1708-P), see page 13. vs. -1.431, p=.043), beta-aminoisobutyric acid (0.121 vs. -0.105, p=.021), arginine (-25.947 vs. -1.592, p=.039), and serine (13.033 vs. -1.299, p=.013). & 1700-P Thus improvement of insulin sensitivity determined by glucose infusion rate Infl uence of Age on the Brain Response to Eating in Adults Evaluated is associated with profound changes in 9 AA and metabolites indicating that Using Continuous Arterial Spin Labeling Functional Magnetic Resonance AA and metabolites are affected by the administration of insulin sensitizers. Imaging These metabolites of AA could, therefore, be used as potential biomarkers SARAH LEE, YEE S. CHEAH, YASHICA NATHAN, BULA WILSON, ANDREW of insulin sensitivity in humans. PERNET, MICHAEL J. BRAMMER, STEPHANIE A. AMIEL, FERNANDO O. ZELAYA, Supported by: NIH/NCRR CTSA Grant Number UL1 RR024150 and NIH RO1 DK London, United Kingdom 41973 Appetite and satiety dysregulation may result in overeating, obesity and Type 2 diabetes. Central control of appetite is an attractive target for weight & reduction and insulin sensitising therapies. Functional magnetic resonance 1702-P Effect of High-Protein and High Carbohydrate Diets on Insulin imaging (fMRI) using continuous arterial spin labeling (cASL), a non-invasive Resistance, Oxidative Stress, and Lipid Peroxidation in Obesee, method for quantifying regional cerebral blood fl ow (rCBF), has been used to Non-Diabetic, Premenopausal Women investigate the responses to a mixed meal ingestion, compared to water, in 2 groups of healthy volunteers with a signifi cant age difference. ABBAS E. KITABCHI, FRANKIE B. STENTZ, KRISTIN A. MCDANIEL, MERSCHON 11 healthy young adults (YA, 4 female): aged 19-33 yrs and 11 older adults HUTSON, JIM Y. WAN, FRANCES TYLVASKY, EBENEZER A. NYENWE, CHRISTOPHER (OA, 8 female) aged 38-52 yrs (p<0.01) had 4 cASL scans on a 1.5 Tesla MRI W. SANDS, Memphis, TN, Medicine, TN Various dietary interventions result in signifi cant weight loss and scanner at -14 (baseline), 0, +8 and +28 minute, where 0 was immediately reduction in cardiovascular risk factors, but advantages of these diets are not after consuming 50ml of water (fasted) or a 554kcal mixed meal (fed), in established. We hypothesized that high-carbohydrate (CHO) diet (HC) (55% random order. Imaging data were analysed using SPM5. CHO, 30% fat, 15% protein) results in greater triglycerides (TG), oxidative Global baseline CBF of grey matter showed no difference between 2 stress (DCF), and lipid peroxidation (MDA) than high-protein (HP) diet (30% groups (p=0.87). Regional, baseline rCBF was greater in YA than OA in Left- protein, 30% fat, and 40% CHO). To date, 13 (8HP, 5HC) non-diabetic, obese, insula, L-caudate, L-anterior/posterior cingulate (cluster level p<0.01) and pre-menopausal women were studied in a randomized prospective protocol. bilateral cingulate gyrus (p<0.01). The diets based on resting energy expenditures with 500 Kcal reductions With continued fasting, rCBF increased in Right-inferior parietal lobule were provided by pre-packaged food on a weekly basis. The data depict (p<0.01) and rCBF decrease in bilateral superior temporal gyrus (STG)(p<0.01) results at baseline and after 6 months of diet. in both groups; YA also had rCBF increase in R-dorsolateral prefrontal cortex (Brodmann area (BA) 9)(p<0.01). Parameters HP HP HP HC HC HC P** After feeding, rCBF increased in both groups in R-precentral gyrus measured Baseline P* 6 months Baseline P* 6 months (BA4)/bilateral medial frontal gyrus (BA6)(p<0.01). YA had greater rCBF in BMI (kg/m2) 42±7.0 0.01 38.8±7.3 37.5±4.9 0.02 35.3± 5.4 0.28 somatosensory cortex (BA2)(p<0.01); OA showed greater rCBF in bilateral % weight loss — 0.01 8.6±1.7 — 0.01 7.2 ±1.9 0.60 superior frontal gyrus (BA4/6) and L-precuneus (p<0.01). Both groups had a rCBF decrease in bilateral STG and culmen and L-insula. BP (sys/diast) 129/83±4/3 0.01 119/74±3/3 128/82 ± 3/3 0.01 120/75±3/3 0.70

Obesity In conclusion, food ingestion altered regional blood fl ow in brain regions TG (mg/dl) 102±14 0.01 85±10 102± 13 0.02 94±9 0.02

POSTERS known to be involved in appetite control, nutrient sensing and taste. The DCF (µM) 3.2±0.2 0.01 2.4±0.1 3.2± 0.2 0.04 2.9±0.1 0.02 preliminary study shows a dependence on age in rCBF response to food, MDA (µM) 1.1±0.08 0.02 0.7±0.05 1.1 ±0.09 0.04 0.9±0.7 0.04

Integrated Physiology/ which may contribute to the tendency to gain weight with increasing age and be involved in the greater risk of Type 2 diabetes in later life. HOMA-IR 3.6±0.9 0.01 2.7±0.4 3.5± 0.8 0.03 2.9 ±0.5 0.03 Supported by: Eli Lilly and Company; National Institute for Health Research %Adherence — — 91.8±5.1 — — 92.2 ±7.3 0.92 (NIHR) *Wilcoxon Sign Rank Test. ** Wilcoxon Rank Sum Test.

Conclusions: HP resulted in less insulin resistance (HOMA-IR), reduction in oxidative stress, lipid peroxidation, and TG than in HC diet with similar weight losses and dietary compliance in both diets. ADA-Funded Research

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A462 INTEGRATED PHYSIOLOGY—MACRONUTRIENTCATEGORY METABOLISM AND FOOD INTAKE

& 1703-P & 1705-P Meal-Induced Insulin Secretion Is Associated with Postprandial Glucose Transporter-1 in the Hypothalamic Glial Cells Regulates Triglyceride Clearance in Subjects Consuming Glucose, Fructose or Glucose Homeostasis High Fructose Corn Syrup-Sweetened Beverages CLAIR S. YANG, PATRICIA MIGHIU, MADHU CHARI, CAROL K.L. LAM, TONY K.T. ROEL G. VINK, KIMBER L. STANHOPE, ANDREW A. BREMER, VALENTINA MEDICI, LAM, Toronto, ON, Canada GUOXIA CHEN, TAK HOU FONG, VIVIEN LEE, ROSE MENORCA, NANCY L. KEIM, Circulating glucose inhibits glucose production in normal rats and PETER J. HAVEL, Davis, CA, Nashville, TN humans but this glucose effectiveness is disrupted in diabetes due partly It has been proposed that the adverse metabolic effects of chronic to glucotoxicity. The mechanisms responsible for the deleterious effect of consumption of sugar-sweetened beverages are a consequence of increased glucotoxicity remain unclear. We investigated whether glucotoxicity impairs meal-induced glucose and insulin excursions, i.e. dietary glycemic index. hypothalamic glucose effectiveness to regulate glucose homeostasis, and Our objective was to investigate the role of post-meal insulin peaks, which whether changes of glucose-transporter-1 (GLUT1) in the hypothalamic glial activate lipoprotein lipase (LPL), in postprandial triglyceride (TG) clearance in cells are responsible for the deleterious effects of glucotoxicity in rats in vivo. 48 adults (age: 18-40 years, BMI: 18-35 kg/m²) consuming glucose, fructose First, the ability of hypothalamic glucose infusion (defi ned as hypothalamic or high fructose corn syrup (HFCS) sweetened beverages. For 12 days glucose effectiveness; HGE) to elevate hypothalamic glucose levels and subjects resided at home and consumed their usual ad libitum diet along lower hepatic glucose production is disrupted in STZ-induced diabetes with with glucose, fructose or HFCS (16/group) sweetened beverages at 25% of hyperglycemia sustained for ∼24 hrs. Normalization of plasma glucose in total energy requirements. Subsequently, fasting ApoC3 and 23-h insulin and STZ-induced rats with i.v. phlorizin restored HGE. Next, HGE was evaluated TG levels were measured at the clinical research center while the subjects in normal rats induced with either whole body (via 24 hr i.v. glucose infusion) consumed the sweetened beverages with meals with an energy balanced or hypothalamic (via 24 hr hypothalamic glucose infusion) glucotoxicity, and diet (25% sugar-sweetened beverage, 30% complex carbohydrate, 30% HGE was disrupted in both glucotoxic models. Thus, glucotoxicity per se fat, 15% protein). Net TG clearance was calculated as the decrease of TG disrupts hypothalamic glucose effectiveness. Second, we discovered that 2 hours after the post-dinner TG peak. Consumption of glucose-sweetened the protein level of glial GLUT1 in the hypothalamus of STZ-induced diabetic beverages resulted in the largest glucose and insulin peaks and 23-h AUC. rats was reduced by ∼50% and that this reduction was fully reversed in STZ- These levels were lowest in subjects consuming fructose and intermediate induced rats that received i.v. phlorizin, suggesting glucotoxicity reduces in subjects consuming HFCS (effect of sugar P<0.001). There was a hypothalamic glial GLUT1. We generated adenovirus expressing GLUT1 signifi cant effect of sugar group on TG clearance, with increased clearance driven by a glial fribrillary acidic protein (GFAP) promoter (Ad-GFAP-GLUT1). after glucose (79 ± 7 mg/dL) and lower clearance after fructose (44 ± 7 mg/ Strikingly, direct injection of Ad-GFAP-GLUT1 (vs. Ad-GFAP-LacZ control) dL) and HFCS (47 ± 7 mg/dL) consumption. Furthermore, post-breakfast, into the hypothalamus of (A) STZ-induced rats with reduced hypothalamic -lunch, and -dinner insulin peaks were signifi cantly associated with post- glial GLUT1 acutely normalized plasma glucose levels, and (B) rats induced dinner TG clearance (all p< 0.005). Across sugar groups, mean insulin peaks with hypothalamic glucotoxicity restored HGE. Thus, over-expressing over 3 meals were correlated with TG clearance (r= 0.61; p<0.001). Another hypothalamic glial GLUT1 in two independent glucotoxic models restores potential contributor to TG clearance, ApoC3, which inhibits LPL, was not hypothalamic glucose effectiveness. Together, these data illustrate that associated with TG clearance (p= 0.87). These results indicate that the higher glucotoxicity impairs hypothalamic glucose effectiveness through changes insulin peaks resulting from glucose consumption enhance postprandial TG in hypothalamic glial GLUT1 and highlight the glucose regulatory role of clearance, while the reduced insulin peaks resulting from consumption of hypothalamic glial GLUT1. fructose-containing sugars result in lower postprandial TG clearance. Supported by: CIHR and BBDC

& 1704-P & 1706-P Metabolic Basis of “A-β+” Ketosis-Prone Diabetes (KPD) The Metabolomic Profi le of SRT2104, a Selective SIRT1 Activator JEAN W. HSU, SANJEET PATEL, FAROOK JAHOOR, IVONNE CORAZA, RAMAS- in High Fat Diet Fed Mice Reveals Signifi cant Improvements in WAMI NALINI, KEREM OZER, CHRISTIANE S. HAMPE, CHRISTOPHER B. NEW- Metabolic Function in Liver, Heart and Skeletal Muscle GARD, JAMES R. BAIN, ASHOK BALASUBRAMANYAM, Houston, TX, Seattle, VIPIN SURI, MEGHAN DAVIS, YONG QI, ELDEN O. LAINEZ, ANGELA M. COTE, WA, Durham, NC MARC O. JOHNSON, DAVID J. GAGNE, GEORGE P. VLASUK, JAMES L. ELLIS, “A-β+” Ketosis-Prone Diabetes (KPD), characterized by presentation with Cambridge, MA unprovoked diabetic ketoacidosis (DKA), male predominance, obesity and Cardiac and skeletal muscle are the major sites of energy generation r e v e r s i b l e b e t a c e l l d y s f u n c t i o n , i s a u n i q u e s y n d r o m e o f u n c l e a r p a t h o p h y s i o l o g y. and consumption with lipid oxidation serving as the major energy source A metabolomic comparison of A-β+ KPD patients to non-diabetic controls for cardiac muscle while skeletal muscle derives short term energy needs showed the former to have higher fasting plasma concentrations of the primarily from glucose substrates. Increases in myocellular lipids lead to acylcarnitine of betahydroxybutyrate, high concentrations of glutamate with insulin resistance, lipotoxicity and functional decline. In the current study, higher ornithine and lower citrulline, and diminished levels of branch-chain we used metabolomic profi ling to understand the mechanisms underlying amino acids and isovaleryl CoA-acyl carnitine. We hypothesize that A-β+ high fat diet (HFD)-induced lipotoxicity and alterations in carbohydrate and KPD patients have accelerated leucine-derived ketogenesis, with defects in lipid metabolism in C57/BL6 mice. We also utilized SRT2104, a specifi c small transferring nitrogen from glutamine/glutamate to the urea cycle and carbon molecule activator of the key metabolic regulator SIRT1 to assess the role of from glutamine/glutamate to the TCA cycle. We test these hypotheses SIRT1 in HFD-induced biochemical and physiological dysfunction. through stable isotope/mass spectrometry protocols in new onset A-β+ KPD HFD fed mice gained more weight, developed glucose intolerance and patients on stable insulin therapy compared to age-, BMI- and gender-matched hepatic steatosis. Administration of SRT2104 reversed HFD-induced non-diabetic controls, measuring fasting rates of lipolysis and fat oxidation; metabolic dysfunction and increased whole body carbohydrate and lipid acetyl CoA production, oxidation and its utilization for ketogenesis; and rate oxidation. Metabolomic analysis of liver, skeletal muscle and heart from of production of glutamine, its deamidation to glutamate, rate of transfer of HFD fed mice revealed considerable steatosis. Tissue acylcarnitines were its amide nitrogen to citrulline and oxidation of its carbon. Results from 8 KPD also elevated suggesting ineffi cient fatty acid oxidation. In the hearts from patients compared to 5 controls show: 1) Although entry rate of endogenous HFD fed mice, glycolysis appeared to stall at fructose-6-phosphate (F6P) and Obesity POSTERS leucine into the body’s free pool is not different, KPD patients have a higher glycolytic intermediates were channeled into the mannose-6-phosphate rate of leucine oxidation (55.2±1.7 vs. 47.9±3.1 µmol/kgFFM/h; P=0.05); 2) KPD pathway. HFD hearts also showed maltose accumulation indicating that have lower endogenous citrulline fl ux (5.6±0.5 vs. 7.5±0.7 µmol/kg/h; P<0.05), glycogen is being used as the glucose source even when plasma glucose Integrated Physiology/ with diminished rate of transfer of glutamine amide nitrogen to ornithine for levels were high. SRT2104 treatment reduced tissue acylcarnitines, de novo synthesis of citrulline; 3) KPD show a trend towards accelerated restored glycolytic fl ux as evidenced by reduced F6P and increased glutamine conversion to glutamate (Δ13C-glutamate enrichment 0.11±0.01 phosphoenolpyruvate, and corrected HFD-induced increase in fumarate and vs. 0.07±0.01 MPE/h; P=0.06), but this is not matched by increased oxidation malate levels suggesting that the activator restored fl ux through the Kreb’s of glutamate carbon; 4) there are no differences in lipolysis or fatty acid cycle. In addition, SRT2104 also reversed HFD-induced hepatic steatosis and oxidation. These kinetic data explain the metabolomic fi ndings, and suggest the increase in hepatic gluconeogenesis. that unique metabolic defects underlying the pathogenesis of A-β+ KPD could Our data provide important insights into the mechanisms underlying be increased leucine-derived ketogenesis and impaired anaplerotic entry of HFD-induced tissue dysfunction and also show the therapeutic potential of glutamate carbon into the TCA cycle. SIRT1 activation in cardiovascular abnormalities associated with metabolic Supported by: NIH-R21-DK082827 dysfunction.

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A463 INTEGRATED PHYSIOLOGY—MACRONUTRIENTCATEGORY METABOLISM AND FOOD INTAKE

1709-P & 1707-P Amino Acid Metabolites and Insulin Resistance in Human Inhibition of Delta-6 Desaturase Increases Phospholipid Linoleate BRIAN A. IRVING, AUDREY J. WEYMILLER, HUSNAIN SYED, HELEN KARAKELIDES, Content, Improves Cardiac Mitochondrial Function and Restores MATTIAS SOOP, RICKEY E. CARTER, K. SREEKUMARAN NAIR, Rochester, MN Glucose Tolerance in ob Mice Elevations in amino acids (AA), particularly branched chain AA (BCAA), MELISSA A. ROUTH, CHRISTOPHER M. MULLIGAN, CATHERINE H. LE, JULIANO have been reported to contribute to the development of insulin-resistance. SILVEIRA, GERRIT T. BOUMA, GENEVIEVE C. SPARAGNA, SIMONA ZARINI, We sought to determine the additive effects of AA, AA metabolites, and ROBERT C. MURPHY, ADAM J. CHICCO, Fort Collins, CO, Boulder, CO, Aurora, CO body composition on insulin sensitivity (SI). 24 non-diabetic lean (49+5 y, Epidemiological studies have linked a lower proportion of linoleic acid (LA, 22.8+0.4 kg/m2), 27 non-diabetic obese (49+4 y, 30.4+0.4 kg/m2), and 18:2n6) in serum phospholipids (PLs) to insulin resistance and cardiovascular 12 type 2 diabetic obese (57+4 y, 30.2+1.0 kg/m2) adults were studied. SI mortality in humans, but the mechanism and pathophysiological basis of was assessed using the steady-state glucose infusion rate (GIR, umol/ this phenomenon are unclear. A selective loss of LA also occurs in PLs from kgFFM/min) obtained during a standardized hyperinsulinemic (1.5 mU/ multiple tissues in obese/insulin-resistant (ob) vs. lean (C57Bl/6) mice, which kgFFM/min) euglycemic clamp. Body composition was determined using is paralleled by increases in long-chain PUFAs and an accumulation of their DEXA. Ultra-performance LC-MS/MS was used to quantify fasting AA eicosanoid derivatives. We hypothesized that this PL remodeling results and AA metabolites. Compared to the non-diabetic lean GIR (53+3) was from increased activity of delta-6 desaturase (D6D), the rate limiting enzyme progressively lower in both non-diabetic obese (40+3, p<0.05) and diabetic in long-chain PUFA biosynthesis that utilizes LA as a substrate, and explored obese adults (21+4, p<0.05). GIR was inversely correlated with the BCAA potential pathophysiological consequences of this phenomenon in ob mice. (valine: r=-0.56, leucine: r=-0.50, isoleucine: r-0.40, p<0.001) concentrations. qRT-PCR revealed a 1-2 fold induction of D6D mRNA in ob vs. lean mice Moreover, 20 of 35 AA metabolites showed signifi cant correlations with GIR. that paralleled increases in PL PUFA molar% ratios indicative of greater D6D As expected, GIR was also inversely correlated with %fat (r=-0.45, p<0.001). activity in heart, liver, serum and muscle (P < 0.01). Treatment of 4 mo old Values with signifi cant correlation coeffi cients were included into an initial male ob mice with the selective D6D inhibitor SC-26196 (SC, 100 mg/kg/d multiple regression model. Obeying the hierarchical principle, candidate for 4 wks) reversed D6D indices and increased the molar% of LA in tissue variables were eliminated in a backwards-stepwise fashion. The fi nal model PLs, and ameliorated 2 to 20-fold elevations in several pro-infl ammatory [GIR=age+sex+%fat+asparagine+serine+aspartic acid+sarcosine+beta- eicosanoid species present in the serum and hearts of untreated ob mice. SC alinine+alpha-aminoadipic acid+ornithine+tyorosine+valine] explained 67% treatment completely normalized the fatty acid composition of cardiolipin in of the variance in SI (p<0.001). In conclusion, the present data support that cardiac mitochondria, which was associated with improvements in the rate elevations in many AA including BCAA and AA metabolites are associated (state 3) and effi ciency (ADP/O ratio) of mitochondrial respiration (P < 0.05). with reduced SI. Moreover, the combination age, sex, percent body fat, along Interestingly, D6D inhibition also improved response to an acute glucose with a subset of AA metabolites explained ∼70% of the total variance in SI. challenge in ob mice (1 mg/g BW i.p.), indicated by lower peak (146% vs. Supported by: R01DK41973; UL1 RR024150 219% of fasting) and fi nal (85 vs. 180% fasting 2 hrs-post challenge) blood glucose levels vs. untreated ob mice (P < 0.01). These studies indicate that D6D plays a pivotal role in PL remodeling and eicosanoid production in ob 1710-P mice, and highlight the need for further investigation of PUFA metabolism Increased Nocturnal Lipid Oxidation in Young Men Who Had Low in the development of glucose intolerance and cardiac mitochondrial Birth Weight dysfunction associated with obesity/insulin resistance. CHARLOTTE BRØNS, SØREN LILLEØRE, CHRISTINE B. JENSEN, SØREN TOUBRO, Supported by: AHA Grant ALLAN VAAG, ARNE ASTRUP, Gentofte, Denmark, Bagsvaerd, Denmark, Copen- hagen, Denmark Low birth weight (LBW), a marker of an adverse fetal environment, is & 1708-P associated with increased risk of type 2 diabetes (T2D). Previous studies Improving Mitochondrial Fat Oxidation by Restoring Tissue Gluta- documented a range of early metabolic defects in young and healthy men thione Concentrations Increases Insulin Sensitivity, and Lowers born with LBW including abdominal obesity, mildly elevated fasting blood Hepatic Fat, Triglycerides and Body Weight in Aged Mice glucose levels, as well as liver and muscle insulin resistance. Altered RAJAGOPAL V. SEKHAR, DAN NGUYEN, SUSAN L. SAMSON, VASUMATHI T. energy expenditure (EE) and an increased lipid oxidation rate are common REDDY, Houston, TX features of the overt T2D phenotype, although the origin of these metabolic Introduction: We tested the hypotheses that glutathione defi ciency underlies dysfunctions as well as their exact role(s) in the development of T2D remains impaired mitochondrial fat oxidation in aging, and contributes to insulin resistance, controversial. Using respiratory chambers, we aimed to investigate whether elevated hepatic fat and obesity, and whether improving tissue glutathione young and healthy LBW men with a known increased risk of T2D exhibit concentrations with dietary precursors would restore these defects. altered 24-hours EE and/or fat oxidation rates even prior to the development Methods: 8 young (18w) and 16 old (76w) male C57BL/6 mice were of T2D, potentially representing primary metabolic defects due to altered studied. The old mice were matched for age, sex, weight, glucose tolerance developmental programming. Forty-six young, healthy and lean men and fat oxidation: one group (‘treated’) was pair fed to the other (‘untreated’) matched for age and BMI were included in the study. Twenty LBW subjects control group using an isocaloric-isonitrogenous diet for 6w (both groups had a birth weight below the 10th percentile for gestational age (2688 ± 269 ate identical daily calories and protein nitrogen - diet of the treated group g) and 26 control subjects had a normal birth weight (NBW) between the had higher content of glycine and cysteine (as n-acetylcysteine), precursor 50th and 90th percentile (3893 ± 207 g) (P < 0.0001). Subjects were fed a amino-acids for glutathione). standardized weight maintenance diet and 24-h EE, respiratory quotient (RQ), Outcome measures were liver and muscle glutathione, body weights and substrate oxidation rates were assessed using a respiratory chamber. and composition, fat oxidation, glucose and insulin tolerance, plasma No differences in overall average 24-h EE, RQ or substrate oxidation rates triglycerides, liver and renal profi les, and hepatic fat content. were observed between LBW and NBW subjects. Nevertheless, the LBW Results: Compared to young mice, untreated old mice had30-35% lower group exhibited signifi cantly lower mean adjusted RQ during sleep compared glutathione levels in the liver and muscle, 20% lower mitochondrial fatty- to NBW controls (0.81 ± 0.01 vs. 0.85 ± 0.01, P = 0.01), and with no difference acid oxidation, and signifi cantly higher hepatic fat, body weight, total body

Obesity in nocturnal EE between groups, the LBW subjects displayed a signifi cantly fat, and plasma triglycerides. POSTERS elevated rate of nocturnal fat oxidation compared with the NBW controls Compared to the untreated old control mice, the treated mice showed (2.55 ± 0.13 kJ/min vs. 2.09 ± 0.12 kJ/min, P = 0.02). signifi cantly improved liver and muscle glutathione concentrations by 20-

Integrated Physiology/ In conclusion, an increased rate of nocturnal lipid oxidation is an early 36%, and mitochondrial fatty acid oxidation by 22%, completely restored defect of metabolism in subjects born with LBW, potentially increasing the liver fat content, and improved body weight by 16%, triglycerides by 36%, and level of fasting glucose and risk of T2D via the induction of hepatic insulin insulin sensitivity by 66%, and no changes in expression of glutamylcystinyl resistance. ligase, an enzyme regulating glutathione synthesis. Supported by: Danish Diabetes Association, EFSD, EXGENESIS, Aase and Ejnar Conclusions: Glutathione defi ciency contributes to impaired mitochondrial fatty- Danielsen acid oxidation in aging, and predisposes to elevated hepatic fat, insulin resistance, dyslipidemia and obesity. Restoring glutathione concentrations reverses these defects. Using simple, safe and inexpensive amino-acid supplementation to boost glutathione levels may be developed as a novel nutritional approach to treat fatty liver disease, obesity and insulin resistance in aging. Supported by: Baylor Seed Fund

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A464 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE

1711-P INTEGRATED PHYSIOLOGY—MUSCLE The CB1 Inverse Agonist Rimonabant Prevents Obesity in Mice by Increasing Energy Expenditure Rather Than by Reducing Food Intake [See also: Presidents Posters 456-PP to 457-PP, page A126.] KATY JANE BROWN, HONG WANG, JENNIFER H. YOON, ROBERT H. ECKEL, Denver, CO The cannabinoid receptor CB1 in the CNS plays an important role in Guided Audio Tour: Muscle and Related Studies in Obesity Research regulating energy balance while peripheral CB1 receptors also appear to (Posters 1713-P to 1720-P), see page 15. modulate nutrient metabolism. Previously, treatment of ob/ob mice and diet- & 1713-P induced obese mice with the CB1 receptor inverse agonist rimonabant (RM) PUFA Acutely Decreases Muscle Triacylglycerol-Derived Fatty caused decreased food intake, weight loss, and increased muscle glucose Acid Uptake and Increases Postprandial Insulin Sensitivity uptake and oxygen consumption. We now tested the effect of RM on ANNEKE JANS, ELLEN KONINGS, ELLEN E. BLAAK, Maastricht, The Netherlands preventing diet-induced obesity in C57BL/6J mice. At 8-wks male mice were Increased fat storage in ‘nonadipose’ tissues such as skeletal muscle is ad libitum fed with either a 45% high fat (HF) diet or a 10% fat diet (HC), and a strong indicator of insulin resistance. There are indirect indications that also treated with RM (10 mg/kg/day) or saline by gastric gavage. Weight and dietary fat quality may modulate muscle lipid oxidation and accumulation, food intake were monitored daily and an IP glucose tolerance test (GTT) and resulting in a subsequent change in insulin sensitivity (S ). The study indirect calorimetry was completed after 7 wks of feeding and treatment. I objective was to examine acute effects of meals with various fatty acid (FA) Our data show that RM prevented weight gain of mice on both HF and HC compositions on skeletal muscle FA uptake and storage and postprandial S diets, making RM mice on both diets weigh similarly and less (10% , p<0.01) I ↓ in obese insulin-resistant subjects. In a single-blind randomized crossover than control mice on the HC diet. RM did not affect the average daily food study, 10 insulin-resistant men consumed 3 high-fat mixed meals (2.6 intake, but increased metabolic rate and physical activity by 40% (p<0.01) on MJ). The meals were high in saturated FA (SFA) (35.5en% SFA, 18.8en% both diets. Of interest, RM mice maintained the same level of whole body monounsaturated FA (MUFA) and 1.7en% polyunsaturated FA (PUFA)), high insulin sensitivity (by clamp), but showed a higher glycemic excursion by in MUFA (11.6en% SFA, 42.2en% MUFA and 4.6en% PUFA) or high in PUFA; IPGTT. RM mice also had markedly increased levels of FFA in fasting plasma 1:1 mix of n-3 and n-6 (14.4en% SFA, 11.9en% MUFA and 34.8en% PUFA). and increased LPL activity in skeletal muscle on both diets. Additionally, Fasting and postprandial skeletal muscle FA handling were examined by increased insulin stimulated glucose uptake (IGSG) was seen only in skeletal combining the forearm balance technique with specifi c isotope labeling of muscle in HF-fed RM mice and only in white adipose tissue in HC-fed RM endogenous and exogenous FA. [2H ]-palmitate was infused intravenously mice. These data suggest that the fuel partitioning among various tissues 2 to label endogenous triacylglycerol (TAG) and FFA in the circulation and the was modifi ed by RM treatment. Overall, our data suggest that RM prevents meals contained [U-13C]-palmitate to label chylomicron-TAG. Areas under the diet-induced weight gain mostly through increased physical activity and a curve (AUC) for glucose and insulin and the product of AUC x AUC x 10–6 negative energy balance. This effect could be attributable to the effect of gluc ins (PGI) were used as indices of S . The PGI after the SFA meal was signifi cantly RM on peripheral CB1 receptors in skeletal muscle and white adipose tissue, I higher as compared with the PUFA meal (p=0.027), with intermediate values but could also be mediated in part by the CNS effects of RM. for the MUFA meal. The total uptake of TAG-derived FA was signifi cantly Supported by: sanofi -aventis US lower in the early postprandial phase after the PUFA meal (AUC60-120, p<0.001) and tended to be correlated with PGI (r=0.602, p=0.086). The 1712-P MUFA meal resulted in higher chylomicron-TAG concentrations (p=0.021), The High Potassium Intake Improves High Fat Diet Induced Insulin but there were no differences in TAG extraction by the forearm muscle, Resistance in Male Wistar Rats possibly suggesting a lower clearance by other tissues like adipose tissue. HIROAKI SATOH, YOSHIYUKI SUGAYA, AKIHIRO KUDOH, SATORU YAMAZAKI, NORI- In conclusion, the reduced uptake of TAG-derived FA by forearm muscle TAKA MACHII, SATOKO WATANABE, HIROYUKI HIRAI, KOJI HASEGAWA, SHINICHI and the higher postprandial SI after a high PUFA meal may suggest that a NAKAJIMA, SANAE MIDORIKAWA, TSUYOSHI WATANABE, Fukushima, Japan reduced PUFA-induced muscle lipid accumulation might contribute to the A potassium-depleted diet has been known to lead to insulin resistance, higher postprandial SI. a resistance that was reversed when potassium was resupplied. Currently, Supported by: Dutch Diabetes Research Foundation no information under other circumstances; however, this mineral appears to have a close association with insulin resistance. & 1714-P In current studies, we investigated the effect of high potassium diet on TRB3 Regulates ER Stress-Induced Insulin Resistance in Skeletal insulin sensitivity in high fat (HF) fed male Wistar rats, whereby insulin Muscle Cells sensitivity was measured directly using the hyperinsulinemic-euglycemic HO-JIN KOH, MICHELLE M. JUNG, TARO TOYODA, DING AN, ROHIT N. KULKARNI, glucose clamp studies (at 25 mU/kg/min insulin infusion rate). Male Wistar MICHAEL F. HIRSHMAN, LAURIE J. GOODYEAR, Boston, MA rats were fed normal chow diet, or 60% HF diet containing with either 8% Although progress has been made in understanding the mechanisms of potassium-chloride or not, for 4 weeks. skeletal muscle insulin resistance, the role of altered signal transduction In control animals, in the basal state, HF feeding led to a ∼33% increase is not fully understood. Endoplasmic Reticulum (ER) stress has emerged in body weight, ∼30% increase in fasting insulin levels, no change in fasting as a signifi cant contributor to the development of insulin resistance in plasma glucose levels, and ∼15% increase in hepatic glucose output (HGO). multiple tissues. However, whether ER stress causes insulin resistance in During the clamp studies, the glucose infusion rate (Ginf) required to maintain muscle has not been explored, and the mechanism(s) underlying putative ER euglycemia, and the insulin stimulated glucose disposal rate (ISGDR), were stress-induced muscle insulin resistance is not known. TRB3 (tribbles 3) is a decreased by ∼15% and ∼19% in the HF diet fed control rats, respectively, pseudo-kinase that has been implicated in insulin resistance in the liver, and and the ability of insulin to suppress HGO was impaired by ∼58%, indicating ER stress has been reported to increase TRB3 in human hepatoma cell line an insulin resistant state. The HF containing potassium diet also led to a HepG2, although the two have not been linked mechanistically. To test the ∼33% increase in body weight. In the basal state, HF containing potassium hypothesis that TRB3 mediates ER stress-induced insulin resistance we used diet fed rats exhibited decreased insulin levels (∼20%) but no change in both adult skeletal muscle and C2C12 myoblasts. Overexpression of TRB3 in

fasting glucose levels compared to the HF fed controls. Furthermore, HF mouse tibialis anterior muscles by electroporation (4-fold) decreased insulin- Obesity containing potassium diet fed rats exhibited decreased plasma renin activity stimulated phosphorylation of IRS1 Y612 (22%; P<0.05) and Akt T308 (23%; POSTERS and angiotensin 2 levels but no signifi cant change in plasma aldosterone P=0.12). Mouse extensor digitorum longus (EDL) muscles were isolated

levels. In the clamp studies, the potassium fed animals exhibited increased and incubated with the ER stressors thapsigargin (THAP) and tunicamycin Integrated Physiology/ Ginf (∼30%) and ISGDR (∼35%) and decreased HGO (∼27%) compared to (TUNI). Both agents caused insulin resistance as determined by decreases the HF fed controls. Consistent with the clamp data, the insulin-stimulated in insulin-stimulated glucose uptake (23-25%; P<0.05). THAP and TUNI also phosphorylation of Akt was signifi cantly enhanced in liver and skeletal signifi cantly increased TRB3 mRNA (3.6- to 4.0-fold; P<0.001) and protein muscle. These results indicate that high potassium diet improves the insulin expression (>10-fold; P<0.01) in EDL. Consistent with adult skeletal muscle, resistance by decreasing plasma renin activity and angiotensin 2 levels. TRB3 overexpression in C2C12 myoblasts decreased insulin-stimulated In conclusion, potassium intake has benefi cial effects on whole body glucose uptake (40%; P<0.001) and IRS1 Y612 (67%; P<0.01) and Akt T308 (27%; insulin sensitivity in an insulin resistant state. P<0.05) phosphorylation. In C2C12 myoblasts, THAP and TUNI signifi cantly decreased insulin-stimulated glucose uptake (23-36%; P<0.001), IRS1 Y612 phosphorylation (30-46%; P<0.001), and Akt T308 phosphorylation (35-37%; P<0.01), and increased TRB3 expression. Knockdown of TRB3 (RNAi; 79%)

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A465 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE

prevented the THAP- and TUNI-induced decreases in insulin-stimulated We characterized the diastolic disturbances associated with chronic IRS1 Y612 phosphorylation and glucose uptake. These data suggest a novel pressure overload by AS alone or in the presence of DM. Diabetes exacerbates paradigm where ER stress enhances TRB3 expression leading to insulin the existing diastolic dysfunction of AS patients through extracellular matrix resistance in skeletal muscle. ADA-Funded Research (fi brosis and AGEs) and cardiomyocytes’ (increased Fpassive) alterations. This study highlights the need for earlier therapeutic interventions in order to & 1715-P prevent the dramatic structural myocardial changes and faster progression Effects of Muscle Specifi c Deletion of Carnitine Palmitoyltransfer- of diastolic dysfunction in diabetic AS patients. ase and Carnitine Acetyltransferase on Fuel Selection JINGYING ZHANG, ROBERT C. NOLAN, SARAH E. SEILER, TIMOTHY R. KOVES, & 1717-P INDU KHETERPAL, ROBERT D. STEVENS, OLGA R. ILKAYEVA, DEBORAH M. Curcumin Ameliorates Skeletal Muscle Atrophy in Type I Diabetic MUOIO, RANDALL L. MYNATT, Baton Rouge, LA, Durham, NC Mice Via Inhibiting the Protein Ubiquitination and Infl ammatory Mitochondrial fatty acid import and oxidation is initiated by carnitine Cyto kines palmitoyltransferase-I (CPTI). CPTI is located in the outer mitochondrial TAISUKE ONO, SHINTARO KINUGAWA, SHINGO TAKADA, TADASHI SUGA, membrane and catalyzes the formation of long chain acyl-carnitines from TSUNEAKI HONMA, ARATA FUKUSHIMA, MASASHIGE TAKAHASHI, MOCHAMAD carnitine and acyl-CoA. Short chain acyl-carnitines are formed via the ALI SOBIRIN, HIROYUKI TSUTSUI, Sapporo, Japan activity of carnitine acetyltransferase (CrAT) in the mitochondrial matrix. Skeletal muscle atrophy has been reported to be associated with an Even though CrAT’s role is not completely understood, it is thought to buffer increase in morbidity and mortality in patients with diabetes mellitus (DM), acetyl-CoA/CoA in the mitochondria. To understand the role of each enzyme especially in type 1 DM. It can be caused by an imbalance between protein in energy homeostasis, substrate utilization and insulin sensitivity; mice synthesis and degradation via mainly the activation of ubiquitin-proteasome were generated with muscle specifi c deletion of CPT-Im-/- and CrAT m-/-. CPT- due to chronic infl ammation. Curcumin, the active ingredient of turmeric, has Im-/- and CrAT m-/- mice have normal body weight, fat mass, fat free mass, various biological action including anti-infl ammatory properties. We thus energy expenditure and food intake. However, long chain fatty acid oxidation hypothesized that curcumin could ameliorate skeletal muscle atrophy in is greatly reduced in muscle homogenates and isolated mitochondria streptozotocin (STZ)-induced type 1 DM mice. from CPT-Im-/- mice. Given the popular hypothesis that impaired fatty acid C57BL/6J mice were injected intraperitoneally with 200mg/kg of STZ (DM) oxidation in skeletal muscle causes the accumulation of lipid intermediates or vehicle (Control). Each group of mice was randomly divided into 2 groups; leading to insulin resistance, we paradoxically observed improved GTT fed on the diet with or without curcumin (1.5g/kg/day) for 2 weeks. Thus, the and ITT in CPT-Im-/- mice relative to control mice. Metabolic chamber data experiment was performed in the following 4 groups of mice; Control (n=10), demonstrated that CPT-Im-/- mice oxidize more carbohydrate than control Control+Curcumin (n=10), DM (n=10), and DM+Curcumin (n=10). mice. Conversely, CrAT m-/- mice had impaired GTT and ITT relative to control Body weight, skeletal muscle weight, and cellular cross-sectional area mice. Metabolic chamber data revealed no difference in 24 hour RER of the skeletal muscle were signifi cantly decreased by 18%, 38% and between CrAT m-/- and control mice, but there is impaired switching from 42%, respectively, in DM compared with Control, which was signifi cantly fatty acid oxidation to carbohydrate oxidation during the transition from a attenuated in DM+Curcumin without affecting plasma glucose and insulin fasted to fed state. In vitro studies in muscle homogenates and isolated levels. There was no signifi cant difference in these parameters between mitochondria demonstrated that the addition of carnitine increased PDH Control and Control+Curcumin. Immunoblot analysis revealed that the activity and the complete oxidation of pyruvate in control mice but not CrAT ubiquitination of protein was increased by 27% in the skeletal muscle m-/- mice. In addition, the ability of pyruvate to suppress fatty acid oxidation from DM, which was inhibited in DM+Curcumin. Quantitative reverse was blunted in mitochondria from CrAT m-/- mice. Together our data indicate transcriptase-PCR analysis revealed that gene expression of muscle that, the loss of skeletal muscle CPTI results in reduced oxidization of long specifi c ubiquitin E3 ligase MuRF1 and MAFbx/Atrogin-1 was increased in chain fatty acids and stimulates glucose oxidation, whereas the loss of CrAT DM by 5.6- and 3.5- fold, respectively, and was inhibited in DM+Curcumin. in muscle predominately affects metabolic fl exibility. Moreover, gene expression of infl ammatory cytokines TNF-α and IL-1β Supported by: NIH ADA-Funded Research was also increased in the skeletal muscle from DM by 5.2- and 17.0-fold, respectively, and was inhibited in DM+Curcumin. & 1716-P Curcumin ameliorated skeletal muscle atrophy in DM mice via inhibiting Diabetes Mellitus Impact on Left Ventricular Myocardial Structure the protein ubiquitination and infl ammatory cytokines. Curcumin may be and Function in Aortic Stenosis before Valve Replacement benefi cial for the treatment of muscle atrophy in type 1 DM. INÊS FALCÃO-PIRES, NAZHA HAMDANI, CRISTINA GAVINA, JOLANDA VAN DER VELDEN, ATTILA BORBÉLY, HANS NIESSEN, GER STIENEN, ADELINO F. LEITE- & 1718-P MOREIRA, WALTER J. PAULUS, Porto, Portugal, Amsterdam, The Netherlands, Targeting Mitochondrial ROS Production in High-Fat Fed Mice Does Debrecen, Hungary, Amsterdam, Portugal Not Improve Skeletal Muscle Insulin Sensitivity or Mitochondrial Diabetes mellitus (DM) is an independent risk factor for progression of Function aortic valve stenosis (AS) and signifi cantly impacts longterm outcome after SABINA PAGLIALUNGA, BIANCA VAN BREE, PILAR VALDECANTOS, EMILIA valve replacement. High incidence of residual heart failure may account AMENGUAL-CLADERA, PATRICK SCHRAUWEN, JORIS HOEKS, Maastricht, The for this prognosis. We aimed to assess the impact of DM on diastolic (dys) Netherlands, Navarra, Spain, Palma de Mallorca, Spain function of AS patients. Reactive oxygen species (ROS) can reduce mitochondrial function by Patients with severe isolated AS (n=46) and AS plus type-II diabetes damaging mitochondrial proteins, lipids and DNA. Conditions of lipid (AS-DM+,n=16) with preserved left ventricular (LV) ejection fraction and oversupply such as high-fat (HF) feeding, type II diabetes, and obesity are no clinical or angiographic signs of coronary artery disease were studied. often associated with increased levels of ROS. Recent studies have identifi ed Doppler echocardiographic data was used to compare in vivo LV function. elevated ROS as a mechanism connecting skeletal muscle mitochondrial Biopsies were used to assess fi brosis, cardiomyocyte hypertrophy dysfunction to insulin resistance. Therefore, the aim of this study was to (MyD), advanced glycation endproducts (AGEs) and phosphorylation of investigate if mitochondrial ROS sequestering can circumvent mitochondrial

Obesity myofi lamentary proteins. Isolated and permeabilized cardiomyocytes were dysfunction upon HF feeding and to determine its impact on insulin sensitivity.

POSTERS used to measure active force (Factive), resting force (Fpassive) and myofi lament To this end, male mice were fed either a low-fat (LF) or HF diet for 16 weeks calcium sensitivity(pCa50). and treated with SkQ (in the drinking water), a mitochondrial-specifi c

Integrated Physiology/ In isolated AS, LV deceleration time and end-diastolic pressure were antioxidant used to target ROS production, or water as control (C) (n=7-10). augmented (239±19ms; 21.4±1.4mmHg, respectively) and the latter Glucose tolerance tests and insulin-stimulated signaling were determined signifi cantly correlated with increased fi brosis (12.9±1.1%; r=0.40,p=0.04) to assess (muscle) insulin sensitivity. Skeletal muscle mitochondria were + and MyD (22.9±0.5μm; r=0.60, p<0.001). In AS-DM patients, diastolic isolated to measure ROS by H2O2 production and to evaluate mitochondrial dysfunction (deceleration time and EDP) was exacerbated in comparison function by high-resolution respirometry. ROS production was elevated in with isolated AS (363±32ms, p=0.005; 28.2±3.7mmHg, p=0.04, HF-C mice (+37% vs. LF-C, P<0.05), but was not increased in HF-SkQ treated respectively), fi brosis, MyD and AGEs were further increased (25.6±1.3%, mice. Interestingly, the prevention of diet-induced increase in ROS in HF-SkQ p<0.001; 18.2±2.6μm, p<0.001; 31.4±6.1score/mm2, p=0.03, respectively). mice did not improve mitochondrial function. Pyruvate-supported respiration 2 2 Additionally, Fpassive (5.1±0.7kN/m vs 3.7±0.4kN/m ,p=0.002) and the ratio was reduced with HF feeding in control mice (-31% vs. LF-C, P<0.05) and between the phosphorylation of titin isoforms(P-N2BA/P-N2B) signifi cantly this decrease was not rescued with SkQ treatment. Both HF diet groups rose (0.61±0.10 vs 0.38±0.04,p=0.04). displayed impaired glucose tolerance compared to LF cohorts (area-under-

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A466 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE the curve analysis; LF-C: 2534±124, HF-C: 3371±186 mM*min, P<0.0001 and of uncoupling protein 2 and 3, and increased uncoupled mitochondrial LF-SkQ: 2510±139, HF-SkQ: 3099±108 mM*min, P<0.05; 1-way ANOVA). respiration compared with control muscles (p<0.05). The catalase expression However, there was no effect of SkQ treatment on glucose tolerance. Similar and activity in gastrocnemius muscle were also elevated. Therefore, these results were obtained for insulin signaling. Taken together, reducing ROS results indicate that hyperexpression of the constitutively active PPARβ/δ in at the level of the mitochondria in mice challenged with a HF diet did not skeletal muscle enhances insulin sensitivity under a HF feeding condition via improve mitochondrial function, whole body glucose tolerance or muscle facilitating fatty acid oxidation, accompanied with increased mitochondrial insulin sensitivity. These results question the causal role of ROS in the uncoupling and anti-oxidant defense, in skeletal muscle. development of muscle insulin resistance. Supported by: NILB1 (1R01HL085499 and 1R01HL084456)

& 1719-P Changes in Intramyocellular Lipid Accumulation and Insulin Sensi- Guided Audio Tour: The Muscle as a Target for Obesity Treatment and tivity after High-Fat Loading Are Associated with Transcriptional Research (Posters 1721-P to 1728-P), see page 15. Response in Human Skeletal Muscle SAORI KAKEHI, YOSHIFUMI TAMURA, YUKO SAKURAI, KAGEUMI TAKENO, & 1721-P MINAKO KAWAGUCHI, FUMIHIKO SATO, SHIN-ICHI IKEDA, MAENGKYU KIM, Membrane Localization of Unique Diacylglycerol Species Predict RISAKO YAMAMOTO, YUJI OGURA, NORIO SAGA, SHIZUO KATAMOTO, YOSHIO Insulin Resistance in Humans FUJTANI, TAKAHISA HIROSE, RYUZO KAWAMORI, HIROTAKA WATADA, Tokyo, BRYAN C. BERGMAN, DEVON M. HUNERDOSSE, ANNA KEREGE, MARY C. Japan, Chiba, Japan KOEHLER, LEIGH PERREAULT, Denver, CO, Aurora, CO The accumulation of intramyocellular lipid (IMCL) is regarded to be a We sought to evaluate if the cellular localization and molecular species of cause of insulin resistance. Thus, to elucidate the mechanism of the IMCL diacylglycerol (DAG) impacted insulin sensitivity and PKC activation in human accumulation will eventually contribute the full understanding of the skeletal muscle biopsies. Six healthy sedentary controls (Con: 2W, 4M, Age: pathophysiology of insulin resistance. One of the factors that increase 39.5±2.3 yrs, BMI: 33.3±1.4 kg/m2, Si:3.2±0.4 10-4/µU/ml), fi ve individuals IMCL level is dietary fat. However, the susceptibility for the increase of with type 2 diabetes (T2D: 0W, 5M, Age: 44±1.8 yrs, BMI: 30.1±2.3 kg/m2, Si: IMCL by dietary fat is highly variable. The aim of this study is to identify the 2.4±0.6 10-4/µU/ml), and ten endurance trained athletes (Ath: 2W, 8M, Age: molecular mechanism of the presence of the inter-individual variation. The 35.4±3.1 yrs, BMI:23.3±0.8 kg/m2, Si: 12.6±1.7 10-4/µU/ml) were studied. study subjects are 42 healthy men. They consumed high-fat diet for 3 days Insulin sensitivity was determined using an IVGTT, muscle biopsies taken following normal fat diet for 3 days. After each diet program, IMCL levels in after an overnight fast, fractionated using ultracentrifugation, and DAG the soleus were measured by 1H-MRS and peripheral insulin sensitivity (GIR) species measured using LC/MS/MS. Differences in DAG species between was evaluated by glucose infusion rate (GIR) of euglycemic-hyperinsulinemic groups were determined using ANOVA, corrected for multiple comparisons clamp. Among them, we picked up 2 groups of subjects: Low-responder (LR) using Bonferroni. Total muscle DAG concentration was higher in Con group, IMCL and GIR were changed within 10% from baseline after high- (13.3±1.0 pmol/ug protein) and T2D (15.2±1.0 pmol/ug protein) compared fat loading; High-responder (HR) group, IMCL was increased and GIR was to Ath (10.0±0.78 pmol/ug protein, p=0.002). The majority (76-86%) of DAG decreased after high-fat loading more than mean percent change of all study was localized in the membrane fraction for all groups, but this was lowest subjects. In each subject, information of gene expression profi le of muscle in the athletes (Con: 86.2±0.98% T2D:84.2±1.2% Ath: 75.9±2.7%, p=0.008). biopsy sample from vastus lateralis was obtained by a DNA microarray There were no differences in individual cytoplasmic DAG species between analysis (LR; n=5, HR; n=4). Gene Set Enrichment Analysis demonstrated no groups (p>0.12). Membrane DAG species C18:0/C20:4, Di-C16:0, and Di- signifi cant differences of the transcripts related to lipid metabolism between C18:0 were signifi cantly greater in T2D compared to the other groups. Only the groups before high-fat loading. However, signifi cant changes of gene total membrane DAG (R2=0.39,p=0.003) and Di-C18:0 (R2=0.35, p=0.004) expression in fatty acid transporter fundamental pathway (P=3.58E-05) and correlated to insulin sensitivity. There were signifi cant inverse relationships fatty acid oxidation fundamental pathway (P=0.0047) between before and (all p<0.003) between PKCe activation and cytoplasmic Di-14:0, Di-16:0, and after high-fat loading were observed in HR group, but not in LR group. In HR Di-18:0, suggesting cytoplasmic DAG localization may be protective against group, fatty acid transporter genes (FABP3, CD36) and fatty acid oxidation PKC activation. In the membrane, we found positive relationships between genes (CPT1a, ACSL, ACOX, ACAA, HADHB) are up-regulated after high-fat PKCe activation and C16:0/C18:1 (p=0.002), and C16:1/C18:1 (p=0.0005). loading. These data suggested that physiological response after high-fat These data indicate the majority of skeletal muscle DAG are localized in loading is closely associated with transcriptional response of genes related membranes, with DAG localized in the cytoplasm unrelated to insulin to lipid metabolism in skeletal muscle. resistance, and perhaps protective against PKC activation. Only certain membrane DAG species were related to PKC activation, and only DAG & 1720-P associated with membranes, particularly Di-C18:0, were negatively related Skeletal Muscle-Specifi c Activation of PPARβ/δ in Transgenic Mice to insulin sensitivity. Improves Insulin Sensitivity under High-Fat Feeding Condition by Facilitating Fatty Acid Oxidation and Uncoupling in Skeletal Muscle & 1722-P TEAYOUN KIM, JIAN LIU, PEIYONG WANG, DOUGLAS R. MOELLERING, MARIA Fyn Is a Muscle Mass Regulator and Controls Skeletal Muscle Macro - S. JOHNSON, TIMOTHY R. NAGY, LAN HE, QINGLIN YANG, Birmingham, AL autophagy by Activating STAT3/Bcl2 Signaling Previous studies have reported that skeletal muscle-specifi c over- EIJIRO YAMADA, CLAIRE C. BASTIE, JEFFREY E. PESSIN, Bronx, NY expression of Peroxisome Proliferator-activated Receptor β/δ (PPARβ/δ) Skeletal muscle represents 40-50% of the human body and in mammals, is in mice enhances muscle oxidative metabolism accompanied by increased one of the most important sites for the control of metabolism, body posture slow fi bers and prevent high fat (HF) diet-induced obesity. However, little is and physical activity. Muscle mass is defi ned by a balance between muscle known about the mechanisms underlying the benefi cial effects of skeletal protein synthesis and muscle protein breakdown. Recently autophagy, which muscle-specifi c PPARβ/δ activation on insulin sensitivity. To investigate is also implicated in diabetes and insulin resistance, was shown to regulate this, transgenic mice with skeletal muscle-specifi c hyperexpression of muscle mass. constitutively active PPARβ/δ (VP16-PPARβ/δ, SVPd) were subjected to either Previously we reported conventional Fyn null mice display increased Obesity chow or HF (60% fat kcal) diet for 22 weeks. After HF feeding, SVPd were energy expenditure and fatty acid oxidation due to increased AMPK activity. POSTERS resistant to weight-gain with markedly less fat mass compared with that We also demonstrated that Fyn negatively regulates LKB1. Two splicing

of controls (p<0.05). As results, the blood glucose level of SVPd during oral isoforms -T and -B of Fyn kinase are expressed throughout the body, but Integrated Physiology/ glucose tolerance test was substantially decreased compared with controls FynT is the predominant isoform expressed in skeletal muscle. Skeletal (p<0.05). Indirect calorimetry analysis revealed the total energy expenditure muscle specifi c FynT transgenic mice (HSA-FynT) displayed increased serum in SVPd was increased by 17% compared with controls (p<0.05) with similar triglyceride/free fatty acid levels. HSA-FynT mice also show a marked energy intakes. SVPd muscles showed signifi cantly increased mitochondrial reduction in white glycolytic muscle mass but the red oxidative muscle biogenesis and fat metabolism-related gene expression. SVPd muscle mass remained protected. Analyses of macroautophagy markers such as exhibited increased palmitate and oleate oxidation rates compared with the LC3-1,2 ratio, p62 expression, GFP-LC3 cleavage as well as autophagic controls (p<0.05). Fatty acid analysis on skeletal muscle samples using GC/FID fl ow studies have demonstrated a marked reduction of macroautophagy in (gas chromatograph/fl ame ionization detector) revealed long-chain fatty acids the skeletal muscle of HSA-FynT. Analyses of the macroautophagy signaling such as oleic, palmitic, and linoleic acids were substantially lower (p<0.05) pathways revealed a near complete absence of the Class III PI 3-kinase in SVPd than in control. SVPd muscles exhibited upregulated expression (Vps34) in the Beclin1/ATG14 complex due to increased expression of the

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A467 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE

inhibitory protein Bcl2. Phospho proteomic analysis and phospho-specifi c palmitate oxidation was 27% lower in red skeletal muscle from mTLR4 immunoblotting demonstrated an increased tyrosine phosphorylation of mice compared to wild-type (WT); no differences were observed in white STAT3, a known transcriptional activator of Bcl2 expression. Expression of muscle. Following 16 weeks of HF diet, fatty acid oxidation was signifi cantly a dominant-interfering STAT3 mutant (STAT3-Y705A) was a potent activator increased in WT mice (+17.8%, p<0.05) while this adaptation was not evident of skeletal muscle macroautophagy. Together these data demonstrate that in the mTLR4 mice (-5.2%). mTLR4 mice tended to gained more weight Fyn functions as a suppressor of macroautophagy through a STAT3 mediated (+17.2±1.2 vs. +15.1±0.9g, P=0.09) and became more glucose intolerant inhibition of the Vps34/Beclin1/ATG14 complex 1. (GTT AUC, 35.3±2.6 vs. 29.7±1.2, mmol, p<0.05) on HF diet compared to WT Supported by: NIH mice. Energy expenditure (kJ/h/kg FFM) was differentially affected by diet between mTLR4 and WT mice; no differences were observed in response to & 1723-P low fat diet (103.1±4.2 vs. 104.5±4.7) but values were signifi cantly lower in Macrophages Are Necessary for Exercise-Induced Enhancement of mTLR4 mice following HF diet (98.7±2.2 vs. 108.6±3.4, p<0.05). In conclusion, Insulin Sensitivity in Skeletal Muscle mTLR4 mice possess a heightened pro-infl ammatory milieu and blunted SHIN-ICHI IKEDA, YOSHIFUMI TAMURA, SAORI KAKEHI, YOSHIO FUJITANI, oxidative capacity in skeletal muscle, which are associated with impaired TAKAHISA HIROSE, RYUZO KAWAMORI, HIROTAKA WATADA, Tokyo, Japan adaptation to high fat feeding. These data suggest that increased TLR4 Type 2 diabetes and obesity are characterized by insulin resistance expression in skeletal muscle may play a role in the metabolic dysregulation in skeletal muscle. It has been well demonstrated that exercise increase associated with obese and diabetic states. insulin sensitivity in skeletal muscle. However, it remains still unclear Supported by: NIH-NIDDK (DK078765, MWH) ADA-Funded Research how a single bout exercise enhance subsequent insulin sensitivity. The understanding of this mechanism would eventually contribute to further development of treatment for type 2 diabetes. Recently, it has been reported & 1725-P that infl ammatory M1 macrophages (MAC) are associated with development Fibroblast Growth Factor 21 Protects Human Skeletal Muscle Myo- of insulin resistance and anti-infl ammatory M2 MAC have positive effect on tubes from Palmitate-Induced Insulin Resistance by Inhibition of insulin sensitivity in several insulin target organs, such as adipose tissue, Stress Kinase and NF-κB liver and skeletal muscle. We, therefore, hypothesized that MAC, especially MIN SUK LEE, EUN KYOUNG KIM, EUN SUK HA, SUNG-E. CHOI, TAE HO KIM, M2, are involved in the mechanisms of exercise-induced enhancement of SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, YUP KANG, KWAN-WOO LEE, insulin sensitivity in skeletal muscle. To test this hypothesis, we injected PBS- Suwon, Republic of Korea, Goyang, Republic of Korea containing liposome (PBS) or clodronate-containing liposome (CL), a MAC Insulin resistance is an important mechanism of type 2 diabetes mellitus. suppressor, to C57BL6J mice. Then, mice were subjected to a single bout Fibroblast growth factor 21 (FGF21) has been identifi ed as a potent metabolic of treadmill running (20m/min, 90 min). Twenty-four hour after exercise, we regulator with specifi c effects not only on glucose and lipid metabolism, but measured ex-vivo insulin-stimulated 2-deoxy glucose (DG) uptake in skeletal also as a regulator of energy balance, especially in adipocytes and the liver. muscle. We observed that a single bout exercise enhanced CD206 (M2 MAC However, it not yet clear whether FGF21 contributes to insulin resistance marker)-positive MAC accumulation and insulin-stimulated 2-DG uptake in in skeletal muscle cells. Palmitate causes insulin resistance in skeletal plantaris muscle in PBS group (Figure 1). However, CL treatment completely muscle cells through the activation of a chronic infl ammatory process. abolished MAC accumulation and enhanced insulin sensitivity in skeletal Here, we investigated the potential contribution of FGF21 to the insulin muscle (Figure 1). We also observed that basal AMPK phosphorylation and resistance induced by palmitate in skeletal muscle cells. First, confi rming insulin-induced phosphorylation state of Akt and AS160 were not changed the contribution of FGF21 to the palmitate-induced insulin resistance, we by exercise or CL treatment in plantaris muscle. From these results, we determined 2-NDBG uptake and levels of proteins related to insulin signaling conclude that MAC is involved in enhancement of insulin sensitivity in pathways (IRS-1, Akt) in human skeletal muscle myotubes (HSMM) exposed skeletal muscle after exercise, independent of Akt and AMPK activity. to palmitate (100, 200 μM) for 24 h and compared those in HSMM exposed to both palmitate and different doses of recombinant FGF21 (100, 200 ng/ mL). Second, to determine the mechanisms underlying the contribution of FGF21 to palmitate-induced insulin resistance, we compared levels of protein related to palmitate-induced insulin resistance (PKC-Θ, IKKβ, JNK, p38, IκBα, NF-κB) in HSMM exposed to palmitate and different doses of recombinant FGF21 (100, 200 ng/mL) for 24 h. Palmitate reduced the insulin- stimulated glucose uptake in HSMM. However, palmitate-reduced glucose uptake was restored by FGF21. Palmitate also inhibited phosphorylation of Akt, and thus impaired the insulin signaling pathway in HSMM, but FGF21 prevented the palmitate-inhibited phosphorylation of Akt. These results indicated that FGF21 prevented palmitate-induced insulin resistance in HSMM. Palmitate activated NF-κB in HSMM, and thus impaired the action of insulin and initiated chronic infl ammation in HSMM. However, FGF21 inhibited the palmitate-induced NF-κB activation in HSMM.The results of the present study suggest that FGF21 prevents palmitate-induced insulin resistance in HSMM by inhibiting activation of stress kinase and NF-κB.

& 1726-P Activation of Wnt/β-Catenin Signaling Improves Insulin Resistance & 1724-P in Skeletal Muscle Selective over Expression of Toll-Like Receptor 4 in Skeletal Muscle ELENI CHRISTODOULOU-VAFEIADOU, AURORE NAVIÈRE, MARYLINE FAVIER,

Obesity Causes Impaired Adaptation to High-Fat Feeding PASCAL MAIRE, ISABELLE GUILLET-DENIAU, Paris, France

POSTERS RYAN MCMILLAN, YARU WU, KEVIN VOELKER, JOHN KAVANAUGH, KRISTIN We reported that high glucose concentration up-regulated the lipogenic WAHLBERG, ANGELA ANDERSON, KIM HAYNIE, MORDECAI HARVEY, GABRIELLE factor SREBP-1c, leading to de novo lipogenesis, intramyocellular lipid

Integrated Physiology/ FUNDARO, ELIKA SHABROKH, MADLYN FRISARD, RANDY MYNATT, MATTHEW deposition and insulin resistance in rodent myoblasts. Furthermore, the HULVER, Blacksburg, VA, Baton Rouge, LA secreted protein Wnt10b and SREBP-1c showed inverse expression patterns Our laboratory has shown that toll-like receptor 4 (TLR4) is elevated in during myoblastic differentiation. Here we show that Wnt10b and SREBP- skeletal muscle of obese humans and its activation results in a partitioning 1c proteins are mutually exclusive in human cultured primary myoblasts of fatty acids toward storage at the expense of oxidative pathways. To better from healthy or obese and type 2 diabetic patients (OD). Moreover, insulin understand this phenomenon, we developed a mouse model, on C57Bl/6 had no effect on lipogenic genes transcription (FAS, ACC2, SREBP-1c) in background, with selective over expression of TLR4 in skeletal muscle myoblasts from OD patients, whereas myoblasts from healthy subjects (mTLR4). Mice were metabolically characterized on chow and a 45% high fat were highly sensitive to insulin. In addition, the selective GSK3 inhibitor (HF) diet. Skeletal muscle from mTLR4 mice displayed heightened activation 6-Bromo-indirubin-3’oxime (BIO), an activator of Wnt/β-catenin signaling, of pro-infl ammatory pathways as evidenced by increased protein levels decreased lipogenic proteins level and intramyocellular lipids in myoblasts of interleukin-6 and tumor necrosis factor-alpha. On a chow diet, fasting from OD patients, inducing a restored insulin sensitivity. Skeletal muscles

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A468 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE of genetically obese and diabetic (ob/ob) mice display a 50% size reduction serine/threonine phosphorylation of Akt/PKB in skeletal muscle and liver was and a dramatic lipid accumulation as compared to lean mice. To determine completely normalized by TZD treatment. PPARγ-sparing TZDs also increased whether activation of Wnt signaling could improve lipid deposition in vivo, the adipose tissue expression and plasma concentration of adiponectin. we performed a direct electrotransfection of Wnt10b cDNA or injected BIO Moreover, adipose tissue infl ammation and macrophage infi ltration were in Tibialis Anterior (TA) muscles of ob/ob mice. Both up-regulated Wnt10b strikingly suppressed by all TZDs. In conclusion, MSDC-0602 and MSDC- and down-regulated SREBP-1c proteins 21 days later, then inducing a 0160 are PPARγ-sparing TZDs that retain the ability to bind to mitochondrial drastic decrease in lipid deposition. Furthermore, mitochondrial Succinic membranes, exhibit potent anti-diabetic and anti-infl ammatory effects, and DeHydrogenase (SDH) staining showed that Wnt signaling increased may be useful for treatment of type 2 diabetes. oxidative fi bers number by 12% ± 3% in TA of ob/ob mice, while decreasing Supported by: STTR Grant (R41 DK084596) from NIDDK glycolytic fi bers of the same amount, the size and total number of fi bers remaining unchanged. Immunostaining of TA slices using antibodies raised 1729-P against the slow myosin MyhcI isoform and the fast MyhcIIA isoform A Mitochondrial Target of Pioglitazone Acutely Regulates Mito- (the most oxidative among fast isoforms) showed a 16% ± 4% increase in chondrial Respiratory Function oxidative fi bers number, the number of glycolytic fi bers being decreased AMANDA T. WHITE, SANDRA E. WILEY, WILLIAM G. MCDONALD, JERRY R. of the same amount. Our results show that activation of Wnt/β-catenin COLCA, ROLF F. KLETZIEN, ANNE N. MURPHY, La Jolla, CA, Kalamazoo, MI signaling in human and mouse skeletal muscle not only decreased lipid Thiazolidinediones (TZDs) such as pioglitazone (Pio) induce insulin deposition, but also induced a shift towards oxydative MyHC-expressing sensitization and mitochondrial biogenesis in skeletal muscle. Due to fi bers, leading to improved insulin sensitivity. reports of specifi c binding of Pio to mitochondria and rapid effects of Pio on Supported by: AFM metabolic signaling, we searched for a direct binding target in mitochondria using a photoaffi nity crosslinking derivative of Pio. We identifi ed a small & 1727-P novel protein, here termed Mitochondrial Target of Thiazolidinediones Mitochondrial Defi ciency of Aging Is Not Associated with Insulin (MTOT), that lacks domains of known function, resides in the inner Resistance or Higher Intramyocellular Lipid Storage mitochondrial membrane, and is expressed in a variety of tissues. Using FREDERICO G.S. TOLEDO, BRET H. GOODPASTER, PETER CHOMENTOWSKI III, Seahorse technology to monitor mitochondrial respiration, we found Pittsburgh, PA that low concentrations (100nM – 3µM) of Pio signifi cantly stimulated In young adults, insulin resistance has been associated with lower endogenous, state 4, and uncoupler-stimulated respiration after 48–120hr mitochondrial oxidative capacity in skeletal muscle. This association of treatment and as early as one hour post treatment in C2C12 mouse suggests a defi ciency in mitochondria that may promote intramyocellular myoblasts. MTOT over-expression suppressed rates of maximal respiration lipid (IMCL) deposition and insulin resistance. However, whether a defi ciency and led to fragmented morphology of mitochondria, although the stimulatory in mitochondria by itself is suffi cient to promote IMCL excess and insulin effect of Pio on endogenous, state 4, and uncoupler-stimulated respiration resistance in humans has not been established. Aging is associated with remained. However, in MTOT overexpressing cells, the stimulatory effects a decline in mitochondrial content and thus it is plausible that older adults of Pio were completely prevented or even reversed in the presence of with a decline in mitochondria should express higher IMCL accumulation and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), suggesting insulin resistance. To test this hypothesis, we studied the skeletal muscle involvement of MTOT in regulation of mitochondrial fatty acid oxidation. mitochondrial content of younger and older adults as it relates to IMCL Down-regulation of MTOT also modifi ed the effects of Pio on mitochondrial content and insulin sensitivity. Non-diabetic, middle-age (30-55 years-old) respiration, leading to an attenuated respiratory response to Pio. These data and older adults (>65) were divided into insulin-sensitive (IS) and resistant suggest that MTOT is necessary for the full response of cells to Pio-induced (IR) groups (Younger-IS, Younger-IR, Old-IS, and Old-IR). Euglycemic clamps respiratory stimulation, and imply that MTOT is a functional target of Pio at were used to measure insulin sensitivity defi ned as glucose disposal (mg/ mitochondria. Studies are ongoing to evaluate the involvement of this target kgFFM/min). Quantitative analysis of electron micrographs of skeletal in insulin sensitizing pharmacology. muscle biopsies was used to measure mitochondrial and IMCL content. In Supported by: R42DK081298 to RFK, ANM; T32 GM007752 to ATW the IR groups, glucose disposal was nearly half of the respective IS groups ADA-Funded Research (Younger: 5.8 vs 12.1; Old: 4.7 vs 10.7, P<0.01). There was no age effect on insulin sensitivity in both the IS and IR groups (P=ns). In younger adults, 1730-P insulin resistance was associated with 24% less mitochondrial content (2.95 A bla tion o f P DK1 in Vascular Endo t helial C ells De t erior a t e s S y s t emic vs. 3.90%, P<0.01). In contrast, mitochondrial content was similar between Insulin Sensitivity and Angiogenensis of Skeletal Muscles in STZ- the Old-IS and the Old-IR groups (2.45 vs. 2.52%, P=ns). The mitochondrial Induced Hyperglycemic Mice content in the Old-IS group was 37% lower than in the Younger-IS group KAZUHITO TAWARAMOTO, MITSURU HASHIRAMOTO, KO KOTANI, WATARU (P<0.01). Yet, despite this defi cit in mitochondria, IMCL content was similar OGAWA, HIDENORI HIRUKAWA, FUMINORI TATSUMI, SUMIKO HAMAMOTO, to the Younger-IS group (0.36% vs. 0.43%, P=ns). IMCL content in the IR MASASHI SHIMODA, YUKIKO KANDA, MASATO KASUGA, KOHEI KAKU, Kurashiki/ groups was higher than in the IS groups (P<0.05). These data reveal that Okayama, Japan, Kobe/Hyogo, Japan, Tokyo, Japan the association between insulin resistance and mitochondrial content is lost PI3K/ phosphoinositide-dependent protein kinase 1 (PDK1)/ Akt signal in with aging. More importantly, they show that the mitochondrial defi ciency of vascular endothelial cells (ECs) is the most important pathway in the process aging is by itself insuffi cient to lead to insulin resistance or increased IMCL of tissue angiogenesis. We previously reported that vascular ECs-specifi c accumulation. ADA-Funded Research PDK1 knockout (VEPDK1KO) leptin receptor defi cient mice deteriorated the systemic insulin sensitivity due to lowered vascularization in skeletal muscle. & 1728-P To further assess a role of PI3K signal in the systemic pathway of tissue Insulin Resistance in ob/ob Mice Is Ameliorated by Thiazolidinedi- angiogenesis under hyperglycemic condition, we generated streptozotocin ones That Do Not Activate PPARg (STZ)-treated hyperglycemia mice with (STZ-KO) or without (STZ-WT) BRIAN N. FINCK, ZHOUJI CHEN, WILLIAM MCDONALD, ROLF F. KLETZIEN, JERRY ablation of PDK1 in vascular ECs. PDK1 protein level in ECs was reduced by

R. COLCA, St. Louis, MO, Kalamazoo, MI 80-90 % in VEPDK1KO. The phospholylation of Akt at Thr308 stimulated with Obesity

The use of currently-approved thiazolidinediones (TZD) for treatment of insulin or vascular endothelial growth factor (VEGF) was reduced by 58% POSTERS insulin resistance and diabetes is limited by side effects that are mediated or 64%, respectively. Blood glucose level (STZ-WT:393±39.6 mg/dl, STZ- via ectopic binding and activation of the nuclear receptor PPARγ. To KO:404.8±40.2 mg/dl), plasma insulin level (STZ-WT:0.356±0.108 ng/ml, STZ- Integrated Physiology/ potentially circumvent these side effects, we have developed TZD analogs KO:0.306±0.037ng/ml), blood pressure (STZ-WT 118±9.4/69.2±9.1 mmHg, (MSDC-0602 and MSDC-0160) that are 100-fold less potent at activating STZ-KO:114±10.0/71.2±6.0 mmHg) and body-weight (STZ-WT 25.7±1.0 g, STZ- PPARγ compared to the high affi nity TZD, rosiglitazone. However, these KO24.3±1.7 g) were not different between two groups of mice. The ratio of compounds retain the ability to bind to mitochondrial membranes, which is IB4-lectin positive capillary/ one muscle fi ber in gastrocnemius muscle was a lesser known property of TZDs. We compared the anti-diabetic effi cacy decreased in STZ-KO (STZ-WT 0.85±0.11, STZ-KO 0.78±0.12, p<0.05, n=6). On of these “PPARγ-sparing” TZDs to rosiglitazone and pioglitazone in 6 week the other hand, plasma concentrations of soluble VCAM-1 and nitrate were old ob/ob mice. A four week course of all TZDs signifi cantly reduced fasting unaltered, VCAM-1 protein level was increased by 35% and eNOS protein plasma concentrations of glucose, insulin, and triglycerides. PPARγ-sparing level was reduced by 40% in gastrocnemius muscle of STZ-KO. Furthermore, TZDs also strongly diminished area under the curve in glucose tolerance the systemic insulin sensitivity measured by insulin-tolerance test (1mU/g and insulin tolerance tests. Consistent with these data, insulin-stimulated BW) was deteriorated in STZ-KO compared to STZ-WT (60 mins: STZ-WT

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A469 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE

28±6% of basal blood glucose, STZ-KO 42±7% of basal blood glucose, 1733-P p<0.05, n=6). The present results strongly suggest that the PI3K signal of Botanical Modulation of the Ubiquitin Proteasome System in Skeletal ECs is critical for angiogenesis in skeletal muscles under hyperglycemic Muscle status, deteriorating systemic insulin sensitivity. ZHONG Q. WANG, TONIYA ACHARYA, XIAN ZHANG, HEATHER KIRK-BALLARD, YONGMEI YU, GAIL KILROY, DAVID RIBNICKY, WILLIAM T. CEFALU, Z. ELIZABETH 1731-P FLOYD, Baton Rouge, LA, New Brunswick, NJ Adiponectin/AdipoR1 Regulate PGC-1α and Mitochondria by Ca2+ The insulin resistance associated with metabolic syndrome and type 2 and AMPK/SIRT1 in Skeletal Muscle, Leading to Amelioration of diabetes alters systemic glucose and protein metabolism. As the major site Obese Diabetes, like Exercise of glucose disposal and the primary reservoir of proteins, skeletal muscle MASATO IWABU, TOSHIMASA YAMAUCHI, MIKI O. IWABU, NAOTO KUBOTA, plays a central role in the development of insulin resistance. In insulin KOHJIRO UEKI, TAKASHI KADOWAKI, Tokyo, Japan resistance, the balance between skeletal muscle synthesis and degradation Pathophysiological mechanisms for mitochondrial dysfunction and insulin tilts toward protein degradation, a process that is regulated by the ubiquitin resistance seen in obese diabetes are not well clarifi ed. proteasome system. The elevated levels of protein degradation that occur We showed that muscle-specifi c disruption of AdipoR1 resulted in with insulin resistance are due to defects in skeletal muscle insulin signaling. decreased activation of AMP-activated protein kinase (AMPK)/SIRT1 by We have demonstrated that an ethanolic extract of Artemisia dracunculus L. adiponectin as well as decreased expression and increased acetylation of (Russian tarragon), termed PMI 5011, enhances insulin signaling in primary peroxisome proliferator activated receptor (PPAR)γ coactivator (PGC)-1α, human skeletal muscle culture and in rodent models of type 2 diabetes and decreased mitochondrial content and enzymes, decreased oxidative type I improves insulin action in vivo. These studies also demonstrate that PMI 5011 myofi bers, decreased oxidative stress-detoxifying enzymes and molecules increases the protein content of skeletal muscle. The purpose of this study is involved in fatty-acid oxidation, thereby leading to increased oxidative stress to determine if PMI 5011 regulates ubiquitin proteasome system activity in and increased tissue triglyceride content in skeletal muscle. Furthermore, skeletal muscle. Using C2C12 myotubes, we found that PMI 5011 enhances the muscle-specifi c AdipoR1 knockout mice (muscle-R1KO) exhibited increased effect of insulin on proteasome activity and ubiquitylation in skeletal muscle phosphorylation of p70 S6 kinase and JNK and also increased serine and that PMI 5011-mediated changes in ubiquitin proteasome system activity phosphorylation of IRS-1 as well as decreased GLUT4 expression and depend on phosphatidylinositol 3-kinase activity. In particular, PMI 5011 decreased Akt activation by insulin, which were associated with decreased affects the expression of the muscle-specifi c ubiquitin ligases Muscle Atrophy rates of glucose disposal. Importantly, all these alterations could result F-box protein (MAFbx/atrogin-1) and Muscle RING Finger-1 (MuRF-1), enzymes in insulin resistance and decreased exercise endurance. Moreover, obese that are required for skeletal muscle protein breakdown. In addition, PMI 5011 diabetic db/db mice exhibited almost all the same phenotypes in skeletal decreases the expression of MAFbx in vivo. These fi ndings indicate that PMI muscle observed in muscle-R1KO. Interestingly, AMPK activator AICAR 5011 may prevent skeletal muscle loss associated with insulin resistance and could only partially rescue the phenotypes of muscle-R1KO such as insulin type 2 diabetes via regulation of the ubiquitin proteasome system. Moreover, resistance and decreased mitochondrial content and function, whereas a role for PMI 5011 in regulating ubiquitin-dependent protein degradation exercise almost completely rescue their phenotypes. Interestingly, muscle- in skeletal muscle has broad implications for treatment of muscle loss in specifi c upregulation of AdipoR1 also could prevent against insulin resistance all catabolic diseases as the ubiquitin proteasome system is the common and mitochondrial dysfunction in obese diabetes. pathway responsible for determining skeletal muscle content. AdipoR1 appears to 1) regulate mitochondrial function and oxidative Supported by: NIH P50AT002776-01and COBRE P20-RR021945 stress in muscle as well as insulin sensitivity and exercise endurance, and 2) be required for adiponectin-induced PGC-1α expression and activation 1734-P via AMPK/SIRT1, and subsequent mitochondrial bioenergetics stimulated Cellular Mechanisms of Lipid-Induced Muscle Insulin Resistance with adiponectin in muscle. This study suggested that strategies to increase in Humans AdipoR1 in muscle may be logical approaches to providing a new treatment JULIA SZENDROEDI, TORU YOSHIMURA, ESTHER PHIELIX, MELISSA MARCUCCI, modality for mitochondrial dysfunction, insulin resistance and diabetes DONGYAN ZHANG, LARA THOMAS, SIMONE BAUDOT, CHRISTEL FUEHRER, linked to obesity. CHRISTIAN HERDER, PETER NOWOTNY, GERALD I. SHULMAN, MICHAEL RODEN, Düsseldorf, Germany, New Haven, CT 1732-P Insulin resistance in skeletal muscle plays a major role in the pathogenesis Angiotensin II Reduces Muscle Mitochondrial Content and Affects of type 2 diabetes but the underlying cellular mechanisms remain unknown. Glycemic Control Infl ammation associated with alterations in circulating adipocytokines MASANORI MITSUISHI, KAZUTOSHI MIYASHITA, AYAKO MURAKI, HIROSHI ITOH, and accumulation of intramyocellular lipid metabolites [ceramides, Tokyo, Japan diacylglycerols (DAGs)] have been implicated to cause insulin resistance Recent large-scale clinical trials have demonstrated that attenuation in animal studies but whether these mechanisms translate to humans is of angiotensin II (AngII) signaling by angiotensin II receptor blockers (ARB) unclear. We performed a comprehensive study to examine these potential prevents new onset of type 2 diabetes mellitus. In this context, we focused mechanisms in causing lipid-induced insulin resistance in man. We studied on whether AngII regulates mitochondrial content and function in the skeletal 16 healthy humans (11m/5f, 30±5 years, BMI: 23.5±2.2 kg/m2) and measured muscle, and subsequently affect glycemic control. C2C12 myotubular cells systemic levels of adipocytokines (n=6: TNFα, IL-6, sICAM, adiponectin, were treated with AngII for 48 hours. Mitochondrial mass estimated by means RBP-4, FGF-21), intramuscular DAG and ceramide content, expression and of fl uorescent staining decreased as a result of AngII treatment, in association activity of PKCs (b, Δ, θ), and mitochondrial function before and after a 4h with a signifi cant increase in mitochondrial ROS. Pharmacological blockade infusion of intralipid or glycerol. Following this, a hyperinsulinemic [40mU/ 2 2 of AT2R resulted in a reversal of the decrease in mitochondrial content, while (m .min)]-euglycemic clamp was performed combined with [6,6- H2]glucose, blockade of AT1R receptors did not affect it. Consistent results were obtained to assess muscle insulin responsiveness. Insulin sensitivity was markedly by genetic blockade of these receptors by small interference RNA. C57bl/6 lower during lipid infusion than during glycerol infusion (M-value: 3.7±0.6 mice were subjected to chronic infusion of AngII by means of an osmotic pump, vs. 10.9±1.4 [mg/(kg.min)], P<0.000001). Associated with this lipid-induced

Obesity combined with oral administration of the receptor blockers. The four groups insulin resistance, muscle DAG content increased by ∼110% (P<0.005) at

POSTERS were examined: control, AngII infusion, AngII infusion with AT1R blockade, 2.5h and remained elevated (∼40%, P<0.05) at 4h of lipid infusion. At 4h, or with AT2R blockade. Glucose levels after the glucose challenge test were PKCθ activity had increased by 64% (P<0.005). In contrast, there were

Integrated Physiology/ signifi cantly higher in the AngII-treated groups than controls, and the change no changes in the plasma concentrations of any of the adipocytokines or in glucose level was signifi cantly suppressed by either AT1R or AT2R blockade. muscle ceramide content despite the marked insulin resistance observed The mitochondrial content in quadriceps was reduced in the AngII group during the lipid infusion. Furthermore, activity of the other PKC isoforms, and this change was signifi cantly diminished by blockade of either AT1R or mitochondrial oxidative capacity and kinetics of respiratory chain complexes AT2R. Mitochondrial content expressed as mtDNA copy number signifi cantly I/II also remained unchanged during the lipid infusion. Conclusion: These correlated with muscle triglyceride and the index of glucose intolerance. data support the hypothesis that DAG-induced activation of PKCθ plays a Although oxygen consumption showed no signifi cant change, a decrease in fat central role in causing lipid-induced insulin resistance in human skeletal oxidation was observed in the Angiotensin II-infused group, and this change muscle. Furthermore, these data demonstrate that changes in circulating was reversed by blockade of either AT1R or AT2R. These fi ndings suggest that adipocytokines or alterations in muscle ceramide content are not associated the cardiovascular hormone, AngII, may have novel roles in the regulation of with acute lipid-induced muscle insulin resistance in humans. mitochondria and energy metabolism in skeletal muscle. Supported by: DFG SFB 575, EFSD Lilly

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1735-P 1737-P Cytokine Secretion from Diabetic Skeletal Muscle: Differential Direct Multi-Tissue Assessment of In Vivo Lipid Handling in Diabetic Regulation by LPS and Fatty Acids Rats Using Magnetic Resonance Spectroscopy THEODORE P. CIARALDI, YOUNG-SUN HONG, PAMELA TAUB, SUNDER R. MUDA- RICHARD AM JONKERS, LUC JC VAN LOON, KLAAS NICOLAY, JEANINE J. LIAR, ROBERT R. HENRY, La Jolla, CA, Seoul, Republic of Korea PROMPERS, Eindhoven, The Netherlands, Maastricht, The Netherlands Chronic low-grade infl ammation has been shown to contribute to Ectopic intracellular-lipid (ICL) accumulation is a hallmark of insulin the development of insulin resistance, mediated in part by increased resistance and type 2 diabetes (T2D), but the causes for this derangement macrophage infi ltration into adipose tissue and skeletal muscle. The possible remain unknown. We applied a 1H-[13C] magnetic resonance spectroscopy contribution of muscle cells to the production of cytokines and chemokines (MRS) method in a rat model of T2D to study in vivo lipid handling in insulin- was investigated using cultured human skeletal muscle cells (hSMC) from resistant liver and muscle at different stages in the pathogenesis of T2D. healthy subjects (H, n=4) and individuals with type 2 diabetes (T2D, n=7- Four groups of n=6 male Zucker diabetic fatty rats were used for this study: 10) Myotubes were treated with lipopolysaccharide (LPS, 100 ng/mL), obese, pre-diabetic fa/fa rats and lean, non-diabetic fa/+ littermates at the saturated (palmitate-Palm, 0.3 mM) and unsaturated (oleate,-Ol 0.3 mM) age of 6 weeks, and obese, diabetic fa/fa rats and lean, non-diabetic fa/+ fatty acids or pioglitazone (Pio, 10 μM) and the media was analyzed. Under littermates at the age of 12 weeks. MRS measurements were performed at control conditions over 24 hrs, T2D hSMC released considerably more IL-8 baseline and 4, 24 and 48 h after oral administration of 1.5 g [U-13C] Algal and GROα than H muscle cells, with smaller increases in IL-6 and MCP-1. In lipid mixture per kg body weight. Localized 1H-[13C] MRS was performed on T2D hSMC both LPS and Palm treatment signifi cantly stimulated the release a 6.3T Bruker MR system with voxels located in the liver or tibialis anterior of IL-6 and IL-15. Palm was more potent for these effects (ex. 557 ± 71% of muscle. Total ICL content and 13C-labeled ICL content were determined from 13 IL-6 secretion from untreated cells vs 166 ± 23% with LPS, p=0.0002), also the ICL-CH2 signal at 1.3 ppm. The absolute C enrichment was calculated signifi cantly stimulating TNFα secretion, along with a tendency to elevate by subtracting the natural abundance 13C-labeled ICL content determined at IL-8 release. Conversely, Ol treatment signifi cantly suppressed the release baseline from the 13C-labeled ICL content at 4, 24 and 48 h post 13C-labeled of IL-6, IL-8 and TNFα. Pio treatment was able to reduce the secretion of lipid administration. the same cytokines, though to a lesser extent than than Ol, except in the At baseline, total ICL content was higher in fa/fa rats compared with fa/+ case of GROα, where it was more effective (62 ± 13% of release in paired rats in both liver and muscle, and at both ages. Both in pre-diabetic and in control cells, p<0.05 vs 81 ± 21, p=ns). Pio treatment blunted LPS stimulation diabetic fa/fa rats, hepatic lipid uptake was increased compared with non- of IL-6 release. IL-8 and GROα act through a common receptor, CXCR2, which diabetic fa/+ rats, whereas the utilization of lipids in the liver did not seem was present in hSMC, suggesting that myotubes may be both a source and to be largely affected. Likewise, in muscle of diabetic fa/fa rats, lipid uptake target for this family of secreted factors. In summary: 1) Myotubes from T2D was higher than in muscle of fa/+ rats. In contrast, lipid uptake in muscle subjects display elevated release of a number of infl ammatory cytokines; 2) of younger, pre-diabetic fa/fa rats was lower than in controls. In muscle of Differential effects of saturated and unsaturated fatty acids on cytokine/ both pre-diabetic and diabetic fa/fa rats net lipid utilization appeared to be chemokine release mirror effects on myotube insulin sensitivity. We impaired compared with controls. conclude that T2D skeletal muscle cells through both intrinsic differences In conclusion, the novel application of 1H-[13C] MRS in combination in infl ammatory cytokine production and responses to extracellular factors, with 13C-labeled lipid administration allows for longitudinal multi-tissue can contribute directly to the development of insulin resistance, as well as assessment of in vivo lipid handling and will be instrumental to our augmenting macrophage infi ltration into muscle tissue. understanding of disturbed lipid handling in insulin resistant tissues. Supported by: VA Medical Research Service ADA-Funded Research 1738-P 1736-P Elevated Interstitial Free Fatty Acids Are Not Responsible for Development of Small Molecule AMPK Activators for Type 2 Diabetes Skeletal Muscle Insulin Resistance in the Fat-Fed Dog Model and Improvement of Muscle Endurance CATHRYN M. KOLKA, ANA VALERIA B. CASTRO, JOYCE RICHEY, ERLINDA L. YASUMICHI HITOSHI, HENRY NGUYEN, TIAN-QIANG SUN, YONCHU JENKINS, KIRKMAN, RICHARD N. BERGMAN, Los Angeles, CA VADIM MARKOVTSOV, IRA SMITH, WEI LI, TARIKERE GURURAJA, TAISEI KINO- Increased nocturnal plasma free fatty acids (FFA) accompany insulin SHITA, YINGWU LI, GERALD UY, ALISON PAN, KELLY MCCAUGHEY, GUILLER MO resistance induced by high fat feeding. It is possible that FFA are responsible GODINEZ, RANIEL ALCANTARA, STEPHANIE YUNG, RONGXIAN DING, DAVID for the insulin resistance, as elevated FFA due to infused intralipid inhibits CARROLL, DANE GOFF, SIMON SHAW, RAJINDER SINGH, TODD KINSELLA, insulin-mediated glucose uptake in skeletal muscle. FFA could infl uence DONALD G. PAYAN, San Francisco, CA muscle cells by moving from the plasma into the interstitial fl uid, where Regular exercise boosts endurance and cardiovascular health by promoting they could directly suppress insulin signaling. But, it is currently not known favorable structural and metabolic adaptations in skeletal muscle. During a whether elevated plasma FFA will result in elevated lipid at the surface bout of exercise, AMP-activated protein kinase (AMPK) activity increases, of the muscle cells. Measurements of FFA in the interstitial space using resulting in the upregulation of glucose uptake, β-oxidation of fatty acids, microdialysis have been problematic as FFAs can clog the pores of the increased glucose transporter 4 (GLUT4) expression, and mitochondrial microdialysis probe. As an alternative, we cannulate the lymph vessel to biogenesis. AMPK appears to be a central kinase for enhancement of provide interstitial fl uid. Anesthetized dogs were exposed to basal insulin muscle endurance as well as improvement of insulin sensitivity. As reported levels for 180 min prior to hyperinsulinemic clamp at 1mU/min/kg. Without previously, we have identifi ed a series of potent small molecule AMPK lipid infusion, FFA levels in artery, vein and interstitium declined during the activators using an adiponectin binding competition screen followed by a clamp experiments (0.27+0.06, 0.33+0.08 and 0.40+0.10mmol/L respectively, cellular AMPK activation assay. The lead series activate AMPK within a few to 0.09+0.03, 0.14+0.04 and 0.11+0.03 mmol/L respectively). A similar trend minutes in insulin target cells such as liver, muscle and fat cells in vitro, and occurred for glycerol and triglyceride. When lipid was infused, arterial and this is associated with reduced glycogen and triglyceride levels in liver cells, vein FFA levels increased to 5.94+1.33 and 5.43+0.91mmol/L respectively. decreased hepatic gluconeogenesis, inhibition of adipocyte lipolysis, as well Whole-body glucose utilization rate was signifi cantly impaired from as enhanced glucose transport and GLUT4 translocation in skeletal muscle 15.16+2.09 in control to 7.43+0.23mg/min/kg with lipid infusion (P<0.01).

cells. Interestingly activation of AMPK by the compounds is not AdipoR1 or Similarly, insulin-induced leg glucose uptake showed a trend to be impaired, Obesity

AdipoR2 dependent yet is dependent upon LKB1. In addition, mitochondria from 46.81+11.76 in control to 23.72+5.42mg/min with lipid infusion (P=0.11). POSTERS inhibition does not signifi cantly contribute to AMPK activation by the However, with lipid infusion, despite elevated FFA in artery and vein and

compounds, suggesting a novel mechanism of action. In db/db mice, the reduced glucose uptake, we observed no signifi cant increment in interstitial Integrated Physiology/ compounds activated AMPK in liver and muscle, enhanced GLUT4 expression FFA levels during the experiment (0.30+0.5mmol/L, P=0.722). Therefore, in muscle, improved glucose tolerance and lowered fasted blood glucose. we conclude that interstitial FFA are not elevated with acute lipid infusion, We tested whether the small molecules enhance running endurance in mice. and therefore cannot be responsible for the peripheral insulin resistance In our preliminary experiment, the compounds enhanced running time and observed during hyperlipidemia. distance. UCP3 protein level in gastrocnemius was also upregulated in the Supported by: NIH DK029867 and DK27619 ADA-Funded Research compound-dosed animals. Our results suggest that small molecule AMPK activators can be used for improvement of muscle endurance as well as treating type 2 diabetes.

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1739-P the presence of the IMCL accumulation. The understanding of this difference Failure of Angiotensin Converting Enzyme Inhibition To Reverse would eventually contribute to further understanding of pathophysiology of Insulin Resistance in Female Obese Zucker Rats insulin resistance. Here, we studied 37 non-athletes, non-obese healthy DINO PREMILOVAC, STEPHEN RATTIGAN, STEPHEN M. RICHARDS, MICHELLE A. men. The IMCL levels in tibialis anterior muscle were measured by 1H-MRS KESKE, Hobart, Australia and peripheral insulin sensitivity (GIR) was evaluated by euglycemic- Impairment in insulin-mediated microvascular perfusion in muscle con- hyperinsulinemic clamp (IIR=100mU/m2/min). Muscle biopsy sample was tributes to the development of insulin resistance and type 2 diabetes. obtained from vastus lateralis and gene expressionswere evaluated by RT- Angiotensin converting enzyme (ACE) inhibition has been reported to improve qPCR. After evaluation of IMCL and GIR, we included 17 from 37 subjects insulin sensitivity. Therefore, the aim of the current study was to determine whose IMCL levels were higher than mean value.Then, we compared gene whether ACE inhibition for 4 weeks improves insulin sensitivity by augmenting expression in skeletal muscle between subjects with upper (>11.0 mg/kg/ insulin-mediated microvascular perfusion in muscle. Female Obese Zucker min, n=6, HGIR group) and lower (<8.1mg/kg/min, n=6, LGIR group) tertiles (ZO) rats, at 10-12 weeks of age, were treated with quinapril (Sigma, 1mg/kg/ of GIR in the subgroup. Although, IMCL level was not signifi cantly different day provided in the drinking water) and compared to untreated ZO rats and between the groups, gene expression of fatty acid transporters, CD36 untreated age-matched lean Zucker (LZ) rats. Following 4 weeks of treatment, and FABP, are ∼2 times higher in HGIR group compared with LGIR group, overnight fasted anaesthetised rats were subjected to a hyperinsulinaemic while FATP gene expression level was decreased by 50% in HGIR group. euglycaemic clamp (20 mU/min/kg, 2hrs). Microvascular perfusion in muscle The expression levels of lipid oxidative genes (CPT1, HADHB, ASCL, ACC2, was assessed by metabolism of 1-methyl xanthine and muscle glucose uptake PDHa, ATGL) were 1.5∼4 times higher in HGIR group. While most of these by 14C-2-Deoxyglucose. After 4 weeks, untreated ZO rats had higher body gene expressions are regulated by PPARα and its co-activator PGC-1α weight (190±15 vs. 357±23 g, p<0.05), plasma glucose (6.8±0.3 vs. 12.4±2.3 whose activity is positively regulated by AdipoR1 signal cascade, PPARα mmol/L, p<0.05), plasma insulin (187±21 vs. 5689±1063 pmol, p<0.05), and and AdipoR1 expression levels were 2 times higher in HGIR group compared uterine horn fat mass (1.1±0.2 vs. 6.8±0.8 g, p<0.05) compared to untreated LZ with LGIR. On the other hand, the expression levels of lipid synthesis gene rats. Quinapril treatment of ZO rats for 4 weeks did not improve any of these (FAS) decreased by ∼50% in HGIR group, while other genes (GPAT, AGPAT2, parameters of insulin resistance. During the hyperinsulinaemic euglycaemic Lipin1, DGAT1) were comparable. In summary, our data suggest that higher clamp, untreated ZO rats displayed a 5.7 fold decrease in glucose infusion rate, expression level of PPARα and adiponectin signal might be a major cause of a 4.9 fold decrease in skeletal muscle glucose uptake, and a 2.2 fold decrease higher insulin sensitivity despite the presence of IMCL accumulation that is in microvascular perfusion compared to untreated LZ animals. Four weeks of typically observed in endurance athletes. quinapril treatment of ZO rats did not improve any of these parameters. The fi ndings of this study indicate that four weeks of quinapril treatment at 1mg/ 1742-P kg/day had no benefi cial effects on the progression of insulin resistance and High Salt Feeding Reduces Insulin Stimulated Glucose Disposal did not correct the microvascular dysfunction in muscle associated with insulin without Impairing Skeletal Muscle Microvascular Recruitment resistance in the female ZO rat. DINO PREMILOVAC, STEPHEN RATTIGAN, STEPHEN M. RICHARDS, MICHELLE A. KESKE, Hobart, Australia 1740-P Enhanced microvascular perfusion is important for nutrient exchange Flowmotion in Skeletal Muscle Is Associated with Increased Nutritive and contributes to insulin-mediated glucose disposal. We have previously Flow and Insulin Action demonstrated that acute nitric oxide (NO) synthase inhibition with NG- JOHN M. NEWMAN, RENEE M. DWYER, MICHELLE A. KESKE, STEPHEN M. nitro-L-arginine methyl ester (LN) blocks insulin-stimulated increases in RICHARDS, STEPHEN RATTIGAN, Hobart, Australia microvascular perfusion and reduces muscle glucose uptake (MGU) by Insulin action in skeletal muscle in vivo is infl uenced by the extent of ∼30%. Chronic high salt (HS) feeding induces insulin resistance; however, microvascular nutritive blood fl ow. whether this model has impaired insulin-stimulated microvascular perfusion Vasomotion is the rhythmic variation in blood vessel diameter which leads is unknown. To determine this, male Sprague Dawley rats, 3-4 weeks of age, to oscillations in blood fl ow distribution at the microvascular level to maintain were fed either a normal salt (NS, 0.5%) or HS (8%) diet. A second group of nutrient delivery (fl owmotion). The perfused rat hindlimb has been used HS rats were supplemented with LN (30mg/kg/min) in their drinking water. extensively to investigate muscle metabolism and alterations in the distribution After 4 weeks, overnight fasted anaesthetised rats were subjected to a of blood fl ow between nutritive and non-nutritive routes. This study was aimed hyperinsulinaemic euglycaemic clamp (10 mU/min/kg, 2hrs). All three groups at determining if the perfused rat hindlimb exhibits fl owmotion and whether had similar fasting plasma glucose and insulin levels. However, whole body this is infl uenced by vasoconstrictors that alter nutritive fl ow. Flowmotion in glucose uptake (GIR) and MGU, assessed by 14C-2-Deoxyglucose, were the constant-fl ow blood-perfused rat hindlimb muscle was monitored using a signifi cantly reduced in both HS and HSLN groups (table 1). Compared to laser Doppler fl owmetry (LDF) probe inserted into the tibialis anterior muscle NS, only the HSLN had elevated blood pressure (BP). Femoral arterial under basal conditions and during vasoconstriction with norepinephrine (NE) or vascular resistance (FVR), calculated as the BP/Femoral blood fl ow, was serotonin (5-HT). Wavelet analysis of the LDF signal was used to determine the signifi cantly increased by the HS diet, and even further exacerbated in the frequencies of fl owmotion in the range 0.009-0.3Hz. The two vasoconstrictors HSLN group. Insulin-stimulated microvascular perfusion in muscle, assessed increased perfusion pressure to the same extent, but had opposing effects on by metabolism of 1-methyl xanthine (1-MX), was not different between metabolism. the groups. These data suggest that in HS induced insulin resistant and Norepinephrine increased, while serotonin decreased oxygen and insulin- hypertensive states, insulin-mediated MGU is blunted while recruitment of mediated glucose uptake. Norepinephrine also increased the average LDF the microvasculature in skeletal muscle remains intact. signal strength by 38%, while serotonin decreased it by 47%. Flowmotion in the frequency range 0.06-0.3Hz was induced with the addition of NE, NS HS HSLN but not with 5-HT. These data suggest that despite the surgical isolation GIR (mg/min/kg) 11.9±0.7 9.5±0.4* 9.2±0.8* of the hindlimb muscles, there is the capacity to exhibit fl owmotion by a MGU (µmol/100g/min) 11.9±0.7 9.5±0.4* 9.2±0.8* vasoconstrictor that stimulates nutritive fl ow. BP (mmHg) 100±2 102±2 164±5# Flowmotion may be a physiological important regulator of microvascular FVR (mmHg.min/mL) 104±8 210±11# 338±45# Obesity nutritive fl ow that is important for insulin action in muscle.

POSTERS Supported by: NH&MRC and ARC, Australia Microvascular Perfusion 7.0±0.6 7.0±0.7 8.0±0.5 1741-P (nmol/min) Integrated Physiology/ Gene Expression Difference of Skeletal Muscle between Intramyo - Data expressed as mean±SE. *p<0.05 vs. NS, #p<0.05 vs. all other groups. cellular Accumulated Insulin Sensitive and Insulin Resistant Individuals n=6-9. MINAKO KAWAGUCHI, YOSHIFUMI TAMURA, KAGEUMI TAKENO, YUKO SAKURAI, FUMIHIKO SATO, SAORI KAKEHI, SHIN-ICHI IKEDA, MAENGKYU 1743-P KIM, RISAKO YAMAMOTO, YUJI OGURA, NORIO SAGA, SHIZUO KATAMOTO, Increased Cathepsin D Accumulation Contributes to Hyperglycemia- AKIO KANAZAWA, YOSHIO FUJITANI, TAKAHISA HIROSE, RYUZO KAWAMORI, Induced Cardiomyocyte Injury HIROTAKA WATADA, Tokyo, Japan SATORU KOBAYASHI, XIANMIN XU, MARIKO SASO-HAGIWARA, KAI CHEN, DEREK The accumulation of intramyocellular lipid (IMCL) is regarded to be a cause TIMM, TYLER JEPPERSON, QIANGRONG LIANG, Sioux Falls, SD of insulin resistance. On the other hand, increased accumulation of IMCL Although cardiovascular disease is the major cause of mortality and is also observed in insulin sensitive subjects (athlete’s paradox). It is still morbidity in diabetic patients, there are no effective preventive or thera- unknown why these insulin sensitive subjects are insulin sensitive despite peutic strategies available to ease this problem. Hyperglycemia is an

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A472 INTEGRATED CATEGORYPHYSIOLOGY—MUSCLE independent risk factor for diabetic heart failure. However, the underlying 1745-P mechanisms remain poorly understood. Previous studies suggest an Losartan Recruits Muscle Microvasculature and Prevents Lipid- association between diabetic cardiac injury and disturbed autophagy- Induced Metabolic Insulin Resistance lysosome pathway, but the specifi c changes and their signifi cance are not NASUI WANG, WEIDONG CHAI, EUGENE J. BARRETT, ZHENQI LIU, Charlottesville, fully characterized. Here, we show that the expression and distribution of VA Cathepsin D, an aspartyl protease normally restricted within lysosome, Patients with diabetes have increased plasma concentrations of free fatty were dramatically altered in cultured cardiomyocytes exposed to high acids (FFAs) which cause insulin resistance in multiple insulin responsive glucose. Specifi cally, Western blotting and RT-PCR showed that high glucose tissues, including the microvasculature. Microvascular insulin resistance (17 or 30 nmol/l) markedly increased the protein and mRNA expression levels contributes signifi cantly to metabolic insulin resistance seen in diabetes. of Cathepsin D in cardiomyocytes. Moreover, immunostaining analysis Blockade of angiotensin II type 1 receptor with losartan recruits muscle indicated that Cathepsin D lost its punctate distribution and was diffused in microvasculature and may improve insulin sensitivity. Whether losartan cardiomyocytes after high glucose treatment. To investigate the functional recruits muscle microvasculature and rescues insulin’s metabolic action in signifi cance of altered Cathepsin D in hyperglycemic toxicity, we treated the presence of high FFA concentrations is not known. cardiomyocytes with a low dose of the lysosome inhibitor Bafi lomycin A1 After an overnight fast, male Sprague-Dawley rats received a systemic (BFA, 0.2nmol/l). Remarkably, BFA inhibited Cathepsin D maturation and infusion of either saline or intralipid + heparin (3.3% and 30 U/ml) for 3 attenuated high glucose-induced myocyte injury, as shown by reduced hrs after an overnight fast, with a 3 mU/min/kg euglycemic insulin clamp cleavage of Poly (ADP-ribose) polymerase (PARP). Together, these fi ndings superimposed for the last 2 hrs. A third group received the same infusion suggest that increased expression and altered distribution of Cathepsin of intralipid + heparin and insulin, with a losartan injection (0.3 mg/kg, i.v. D may contribute to hyperglycemic cardiotoxicity. Consistently, we found bolus) 5 min before starting the insulin infusion. Muscle microvascular blood that Cathepsin D was also increased in the hearts of type 1 diabetic mice volume (MBV) and microvascular fl ow velocity (MFV) were measured using (Streptozotocin induced or OVE26 genetic model). Thus, future studies are contrast-enhanced ultrasound before and at 30, 60 and 120 min of insulin warranted to determine if Cathepsin D plays a similar role in diabetic cardiac infusion. Muscle microvascular blood fl ow (MBF) was calculated as the damage in vivo. product of MBV and MFV. Steady state whole body glucose disposal rates were calculated. Insulin infusion doubled muscle MBV at 30 min and this effect lasted for the entire 120 min (p<0.05) without affecting MFV, thereby leading to increased MBF. Lipid infusion abolished the insulin-mediated increase in muscle MBV and MBF, and lowered insulin-stimulated whole body glucose disposal (p<0.001). Losartan injection in the presence of lipid and insulin infusion resulted in 2-3-fold increase in MBV and MBF (p<0.05 for both), and rescued insulin’s actions to stimulate whole body glucose disposal (p=0.001). In conclusion, losartan potently recruits muscle microvasculature and attenuates metabolic insulin resistance induced by lipid infusion. This suggests that angiotensin II type 1 receptor blockade may improve muscle insulin sensitivity via microvascular recruitment and tissue delivery of ADA-Funded Research insulin. ADA-Funded Research

1746-P 1744-P Low Molecular Weight Adiponectin Does Not Acutely Modulate Increased Inducible Nitric Oxide Synthase Activity Is Responsible Muscle Hemodynamics or Metabolism In Vivo for Reduced Endothelin-1 Vasoconstrictor Responsiveness in Insulin CAROL T. BUSSEY, DINO PREMILOVAC, MICHELLE A. KESKE, STEPHEN RATTIGAN, Resistance STEPHEN M. RICHARDS, Hobart, Australia CAROL T. BUSSEY, MICHELLE A. KESKE, STEPHEN RATTIGAN, STEPHEN M. Adiponectin is an insulin sensitiser, with observed actions to stimulate RICHARDS, Hobart, Australia glucose uptake and fatty acid oxidation. Recent studies have shown that Increased levels of the potent vasoconstrictor endothelin-1 (ET-1) have adiponectin is also able to increase nitric oxide (NO) production by the been implicated in the development of insulin resistance, type 2 diabetes endothelium and relax pre-constricted isolated aortic rings, suggesting and hypertension, and particularly the endothelial dysfunction seen in that adiponectin may act as a vasodilator. We have prepared low these states. Endogenous ET-1 activity is consistently increased in these molecular weight adiponectin from bacteria and have previously shown pathologies, but opinion is divided as to whether ET-1 vasoconstrictor that this preparation opposes endothelin-1-mediated vasoconstriction. responsiveness is enhanced or impaired. Our study examined the sensitivity We hypothesised that adiponectin may be able to acutely improve insulin of skeletal muscle vasculature to ET-1 in rats made insulin resistant by high sensitivity in vivo, through both metabolic and hemodynamic mechanisms. fat feeding, using the isolated, pump-perfused hindlimb preparation. Male Adiponectin was infused before and during a euglycaemic hyperinsulinemic Sprague-Dawley rats were fed a high-fat diet for 4 weeks, resulting in an clamp in anaesthetised Sprague-Dawley rats. Adiponectin had no effect 81% increase in epididymal fat pad weight, accompanied by increased on insulin-mediated muscle glucose uptake or microvascular recruitment. fasting plasma insulin, but not glucose. Vasoconstriction by ET-1 (1 or 3nM) Similarly, mean arterial pressure and femoral blood fl ow were unaffected was reduced by the high-fat diet. Basal perfusion pressure was unaffected. by adiponectin infusion. There was also no difference in glucose infusion Treatment with the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L- rate, whole body glucose disposal or hepatic glucose production between an arginine methyl ester (L-NAME) signifi cantly increased ET-1 vasoconstriction acute infusion of adiponectin and vehicle. The present study indicates that in both normal and high-fat fed animals, reaching the same level, indicating the hemodynamic effects of adiponectin suggested by studies in cultured

increased NO bioavailability in insulin resistance. In the presence of 1400W, cells and isolated vessels may not be a major aspect of adiponectin action Obesity a specifi c inhibitor of the inducible NOS (iNOS) isoform, ET-1 reactivity in high- in vivo. Adiponectin may have more important roles in other processes, or POSTERS fat fed rats was restored to that from rats on normal chow. These fi ndings alternatively may be required chronically in order to modulate the vasculature

support the notion that high fat feeding increases muscle iNOS activity, and insulin sensitivity. Integrated Physiology/ counteracting ET-1 vasoconstriction. Alterations in the balance between constriction and NO-mediated dilation may account for differences observed 1747-P in ET-1 responsiveness in metabolic syndrome pathologies. Furthermore, Mitochondrial Function Does Not Deteriorate upon Short-Term Lipid the raised NO production in high fat fed rats may reduce responsiveness to Exposure in Young, Healthy Humans further NO-mediated dilation, and may explain the impairment of endothelial ESTHER PHIELIX, JULIA SZENDROEDI, GILLES SEQUARIS, PETER NOWOTNY, function seen in insulin resistant states. MICHAEL RODEN, Düsseldorf, Germany In sedentary subjects, fat accumulation in skeletal muscle (IMTG) tightly associates with muscular insulin resistance, a main risk factor for type 2 diabetes. Elevation of plasma free fatty acids (FFA) by lipid infusion, raises muscular fatty acid intermediates and induces insulin resistance and leads

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to IMTG accumulation. High FFA may compromise mitochondrial function, Single tibialis cranialis muscles (TCMs) of anaesthetised 7 week old termed mitochondrial lipotoxicity, which could contribute to insulin male rats were injected with 150μg of plasmid vector encoding AdipoR1 or resistance. Thus, we determined mitochondrial function during short-term AdipoR2 cDNAs, while empty vector was administered to the contralateral elevation of circulatory FFA. muscles as control. Immediately afterwards, each distal hindlimb was We included 12 young, healthy sedentary humans (8 males, 4 females; subjected to in vivo electrotransfer (IVE) using a pulse generator and calliper age: 29±2 yrs; BMI: 23.4±0.6 kg/m2) who underwent 4 hours of Intralipid electrodes. One week later, rats were euthanased and paired TCMs frozen (20%) infusion followed by an euglycemic-hyperinsulinemic clamp to assess for biochemical analysis and immunoblotting (n=8-12). insulin sensitivity. Detailed mitochondrial function, including ADP-coupled IVE of AdipoR1 and AdipoR2 cDNAs resulted in similar respective mean substrate oxidation on glutamate and succinate (state 3), FCCP-induced 556% (p<0.001) and 604% (p=0.015) increases in protein expression of maximal oxidative capacity (state u) as well as enzyme kinetics (Km and Vmax) each receptor in rat TCMs versus paired control muscles, in the presence of NADH and FADH2 dehydrogenase, was examined in permeabilized muscle of the same basal levels of adiponectin. Overexpression of either AdipoR fi bers using high-resolution respirometry. Muscle biopsies from the m.vastus caused increases in muscle glycogen content, with AdipoR2 IVE having the lateralis were taken before (baseline) and at 4 hours of intralipid infusion. greater effect (AdipoR1 17%, p=0.0011; AdipoR2 93% increase, p=0.0063). Elevation of plasma FFA (from 0.48±0.05 to 2.12±0.19 mmol/l, p<0.01) This was associated with increases in activating phosphorylation of Insulin resulted in an insulin-stimulated glucose uptake (ΔRd; clamp minus basal) Receptor substrate-1, Akt and Glycogen synthase kinase-3β, indicative of an of 3.7±0.6 ml×kg-1×min-1 but did not affect state 3 and state u respiration increase in fl ux through the PI3-kinase (insulin) signalling pathway. AMPK (state 3: 67±4 vs 62±5 pmol×mg-1×s-1; state u: 103±7 vs 100±11 pmol×mg-1 × phosphorylation was also increased by both manipulations, while APPL1 -1 s , p=n.s. for both vs. baseline). Also, enzyme kinetics of NADH and FADH2 expression was unaffected. dehydrogenases were not altered (Km NADH dehydrogenase: 1.3±0.4 vs Thus local overexpression of AdipoRs results in increased storage of 0.9± 0.3 mmol/l, Km FADH2 dehydrogenase: 2.9±0.5 vs 2.5±0.4 mmol/l, Vmax glycogen in muscle, probably mediated via increased PI3-kinase signalling. NADH dehydrogenase: 48±6 vs 46±4, Vmax FADH2 dehydrogenase: 63±5 vs These data imply that abundance of both AdipoRs is an important determinant 62±6, p=n.s. for all vs. baseline). No correlation was found between ΔRd and of adiponectin action in skeletal muscle. mitochondrial function. In conclusion, short-term increases in circulating FFA do not affect 1750-P mitochondrial function despite the development of insulin resistance. This Palmitate-Induced Perk Phosphorylation Precedes the Reduction supports the contention that muscular insulin resistance does not require in Glut4 Expression in L6 Myotubes mitochondrial dysfunction per se. PATRÍCIA EBERSBACH SILVA, ANA CLAUDIA POLETTO, MILANO FELIPE DOS SANTOS FERREIRA MARQUES, MARISTELA MITIKO OKAMOTO, GABRIEL 1748-P FORATO ANHÊ, UBIRATAN FABRES MACHADO, São Paulo, Brazil Mitochondrial Superoxide and Coenzyme Q in Insulin Defi cient High saturated fatty acids (SFA) levels lead to insulin resistance (IR) Rats: Increased Electron Leak in skeletal muscle by mechanisms not fully known. At the cellular level, JUDITH A. HERLEIN, BRIAN D. FINK, WILLIAM I. SIVITZ, Iowa City, IA insulin resistance is associated to a reduction in GLUT4 expression. Recent Mitochondrial superoxide is important in the pathogeneses of diabetes investigations suggested that endoplasmic reticulum (ER) stress plays and its complications. However, there is uncertainty regarding the intrinsic important role in the pathogenesis of diabetes and IR. The most immediate propensity of mitochondria to generate this radical. Studies to date suggest response to ER stress is translational attenuation signaled through the that superoxide production in by mitochondria of insulin sensitive target double stranded RNA-activated protein kinase-like ER kinase (PERK). The tissues of insulin-defi cient rodents is reduced or unchanged. Moreover, phosphorylation status of this protein is a key indicator of the presence little is known of the role of the Coenzyme Q (CoQ), whose semiquinone of ER stress. However, whether this translation attenuation is connected form reacts with molecular oxygen to generate superoxide. We measured with SFA-induced reduction in GLUT4 is not established. Therefore, this reactive oxygen species (ROS) production, respiratory parameters, and work aimed to verify the effects of palmitate on GLUT4 and endoplasmic CoQ content in mitochondria from gastrocnemius muscle of control and reticulum (ER) stress proteins in L6 muscle cells. GLUT4 expression and streptozotocin (STZ)-diabetic rats. CoQ content did not differ between PERK phosphorylation were analyzed in L6 myotubes treated with 0,75mM mitochondria isolated from vehicle- or STZ-treated animals. Under state of palmitate (DMEM+BSA1%) during different time intervals. GLUT4 and 4 or state 3 conditions, both respiration and ROS release were reduced in Phospho-PERK proteins expression were detected by Western Blotting-ECL. diabetic mitochondria fueled with succinate, glutamate plus malate, or with Data analysis was performed using ANOVA followed by Bonferroni post-test. all three substrates (continuous TCA cycle). However, H2O2 and superoxide The results showed a signifi cant reduction of the GLUT4 protein content since production were substantially increased in gastrocnemius mitochondria of the second hour of treatment with the fatty acid (control: 99.99±4.3, 2 hours: diabetic rats when expressed per unit oxygen consumed and, by inference, 66.37±7.2*, 4 hours: 65.03±8.0*, 6 hours: 67.16±8.8*, 8 hours: 60.91±11.3*, per unit electron transport. Based on substrate and inhibitor effects, the 10 hours: 54.33±11.5*, 12 hours: 58.88±5.5*, 16 hours: 72.22±10.7*, 20 hours: mechanism involved multiple electron transport sites. More limited results 63.78±9.1*, 24 hours: 63.62±7.6*, results expressed in arbitrary units, n=4 using heart mitochondria were similar. As expected, H2O2 release from to 8, *P<0.05). The statistics showed a signifi cant increase of Phospho- gastrocnemius mitochondria was less during state 3 respiration compared PERK protein content in the fi rst two hours of treatment with palmitate to state 4. However, this reduction was less for the diabetic compared to (control: 99.99±4.05, 2 hours: 191.80±36.09*, 4 hours: 124.80±18.85, 6 hours: control rats. We conclude that insulin defi cient diabetes does, in fact, alter 88.62±17.14, 12 hours: 94,91±23,33, results expressed in arbitrary units, n=4 electron transport properties causing excess electron leak to oxygen and to 8, *P<0.05). These data showed that palmitate impaired GLUT4 expression that mitochondrial superoxide production is best understood when viewed what is temporally preceded by an increase in phosphorylation of PERK. This in the context of electron transport activity. data reinforces the proposition that SFA-induced ER stress is associated with insulin resistance probably by reducing GLUT4 content. 1749-P Supported by: Fapesp 2010/09984-5 Overexpression of Adiponectin Receptors in Rat Skeletal Muscle

Obesity Causes Activation of the PI3-Kinase Pathway and Glycogen Accu- 1751-P

POSTERS mu la tion PPARγ Overexpression in Skeletal Muscle Protects Against High MARK E. CLEASBY, GREGORY J. COONEY, JONATHAN P. WHITEHEAD, EDWARD Fat Diet Induced Glucose Intolerance Via Improved beta-Cell

Integrated Physiology/ W. KRAEGEN, London, United Kingdom, Darlinghurst, Australia, South Brisbane, Function and Reduced Weight Gain Australia ANDREW W. NORRIS, SHANMING HU, JIANRONG YAO, ALEXANDER A. HOWE, Adiponectin is an adipokine whose secretion is reciprocally related to Iowa City, IA adiposity and insulin resistance. It has also been shown to exert a direct Peroxisome proliferator-activated receptor-gamma (PPARγ) is expressed insulin-sensitising effect on cultured myotubes and skeletal muscle strips, at low levels in skeletal muscle, where its metabolic role is important but believed to be mediated via membrane adiponectin receptors (AdipoRs) 1 incompletely understood. To test the hypothesis that increased skeletal and 2. Given the very high (μg/ml) plasma concentration of adiponectin, its muscle PPARγ activity is suffi cient to improve glycemia, we created actions are more likely to be determined by receptor abundance or post- muscle creatine kinase promoter-based transgenic mice (MCK-PPARγ). receptor events than by adipokine concentration. We aimed to determine These mice overexpressed PPARγ by 8±2 to 33±2 fold (p<0.005, n=7-9) in whether receptor expression determines the key metabolic endpoint of various muscle groups and exhibited a 49±32% increase (p<0.005, n=25- glycogen abundance in skeletal muscle and the mechanisms involved. 28) in muscle PPAR reporter activity in vivo. After 4 months high fat diet,

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A474 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

MCK-PPARγ were compared to related wildtype mice (CON). Weight gain we have investigated the cre/loxP-mediated, skeletal muscle-specifi c LXRα was 32% less in female MCK-PPARγ versus CON (p<0.05, n=5-14) despite knockout mouse line. After 8 weeks of high fat (HF, 60% kcal% fat) diet no difference in caloric intake. Fasting triglycerides were reduced 67±8% feeding, skeletal muscle-specifi c LXRα knockout mice (Sla-/-) showed a (p<0.01, n=5-14) in female MCK-PPARγ. By contrast in male mice, weight was similar increase in body weight relative to controls (transgenic mice with identical between genotypes. Glucose tolerance test area under the curve a skeletal actin promoter driven Cre expression, SKC). However, body (GTTAUC) was improved in female MCK-PPARγ by 34±6% (p<0.05, n=5-14) composition analysis revealed that fat mass was increased in Sla-/- relative versus CON. Male MCK-PPARγ also had improved glucose tolerance, with a to SKC mice (p < 0.05). Furthermore, the oral glucose tolerance test (1.5 g / kg 25±6% reduction in the 2-hour glucose level (p<0.05, n=16-21) versus CON. b.w.) revealed that Sla-/- mice exhibited a greater increase of blood glucose An insulin secretory response during glucose tolerance test, at 30 minutes content than SKC mice did (p < 0.05). In hyperinsulinemic euglycemic clamp versus baseline, occured in female MCK-PPARγ (p=0.01, n=12-14) despite the study using constant infusion of Humulin® (2.5 mU / kg / min) and 150 mg four months of high fat diet, whereas in CON an insulin secretory response / dL of euglycemia, the glucose infusion rate (GIR) was markedly lower in was absent. Importantly, insulin-stimulated 2-deoxyglucose uptake into Sla-/- mice than in SKC mice (p < 0.001). The skeletal muscle glucose uptake skeletal muscle during euglycemic hyperinsulinemic clamp was unimproved was also substantially decreased in Sla-/- relative to SKC mice (p < 0.01), in MCK-PPARγ versus CON (n=8-9). Similar effects occured in normal chow whereas liver and adipose glucose uptakes were not different between fed mice, with MCK-PPARγ exhibiting improvements versus CON in GTTAUC Sla-/- and SKC mice. While Sla-/- and SKC muscles showed a similar rate and glucose stimulated insulin secretion (18±2% in females p<0.005 and of complete palmitate oxidation, the Sla-/- muscle showed a substantially 49±13% in males p<0.05, respectively n=9-23). These results identify higher rate of incomplete palmitate oxidation than that of SKC muscle (p reduced obesity, reduced dyslipidemia, and improved insulin secretion < 0.05). These data provide the fi rst defi nitive evidence in intact animals as potential mediators by which muscle PPARγ activity improves glucose that LXRα in skeletal muscle plays an essential role in determining insulin tolerance. We speculate that these effects may occur via PPARγ action to sensitivity, potentially by regulating lipid metabolism of skeletal muscle. enhance lipid utilization in skeletal muscle. Supported by: NILBI (1R01HL085499 and 1R01HL084456) Supported by: R01-DK081548 (to A.W.N.)

1752-P INTEGRATED PHYSIOLOGY—OTHER HORMONES Role of Akt1/Akt2 in Cardiac and Skeletal Muscle KAZUMA KANEKO, KOHJIRO UEKI, TOSHIHIRO UMEHARA, MOTOHARU AWA- [See also: Presidents Posters 458-PP to 461-PP, page A127.] ZAWA, TAKAYOSHI SASAKO, MISTURU OHSUGI, MORRIS BIRNBAUM, TAKASHI KADOWAKI, Tokyo, Japan, Philadelphia, PA Recent studies have shown that insulin signaling in heart and skeletal Guided Audio Tour: Adipokines and Gut Hormones (Posters 1754-P to 1762-P), muscle (SKM) regulates growth and functions but seems less important for see page 13. glucose homeostasis. The exact role of Akt, however, in these tissues remains & 1754-P to be elucidated. Moreover, most of previous studies were conducted by Adiponectin Multimer Distribution and the Insulin-Like Growth using MCK-Cre mice to delete the target gene in muscle, resulting in lethality Factor (IGF)-Axis in Monogenic Insulin Resistance Caused by a within several weeks of age due to heart failure caused by the lack of insulin Heterozygous INSR Mutation signaling in heart. Therefore, the precise role of insulin signaling specifi cally KURT HØJLUND, HENNING BECK-NIELSEN, ALLAN FLYVBJERG, JAN FRYSTYK, in SKM is still unrevealed. Odense, Denmark, Aarhus, Denmark In this study, to gain more insight into the role of two major Akts, in heart Low levels of adiponectin, IGFBP-1, and IGFBP-2 correlates with several and SKM, we ablated Akt1 and Akt2 by crossing mice Akt1 fl oxed and Akt2 indices of insulin resistance and risk of type 2 diabetes. However, in insulin fl oxed mice with MCK-Cre or MLC1f-Cre mice, respectively, resulting in receptoropathies, plasma adiponectin is paradoxically increased and leptin both heart and SKM specifi c Akt1/Akt2 knockout (MCK-DKO) mice or SKM levels low despite severe insulin resistance, whereas the insulin-like growth specifi c Akt1/Akt2 knockout (MLC1f-DKO) mice. Moreover, to elucidate the factor (IGF)–axis is sparsely described. Here, we aimed to characterize the role of Akt in muscle of pathophysiological role for type 2 diabetes, we also multimeric distribution of adiponectin, total levels of adiponectin and leptin, investigated Akt related signaling in heart and SKM of db/db mice. and the IGF-axis in humans with a heterozygous INSR mutation (Arg1174Gln). Although each single knockout grew normally, MCK-DKO died within 5 Blood samples obtained in six Arg1174Gln carriers and 10 lean, healthy weeks after birth due to heart failure. The weight of heart and SKM were controls before and after an euglycemic-hyperinsulinemic clamp were signifi cantly decreased in MCK-DKO. Additionally, cardiac function analysis examined for plasma adiponectin and its multimers, leptin, total IGF-I, IGF-II, by echocardiography showed that ejection fraction was signifi cantly reduced free IGF-I, IGFBP-1 and IGFBP-2. in MCK-DKO. Despite the almost complete abolishment of Akt activity due to Despite 4-fold elevated fasting insulin levels and marked insulin Akt1/Akt2 deletion in heart and SKM, MCK-DKO showed normal glucose and resistance, the levels of total adiponectin, leptin, IGFBP-1 and IGFBP-2 were insulin tolerance at 3 weeks of age. On the other hand, MLC1f-DKO mice grew similar to those observed in lean controls, while total IGF-I, IGF-II and free normally but exhibited SKM atrophy and glucose intolerance in accordance IGF-I levels were increased in Arg1174Gln carriers. The relative fraction of with age. Quantitative RT-PCR analysis revealed genes regulating fatty acid HMW adiponectin was increased, whereas both the absolute concentration oxidation and oxidative phosphorylation were decreased signifi cantly in and fraction of LMW adiponectin were reduced in Arg1174Gln carriers. both heart of MCK-DKO and SKM of MLC1f-DKO. Moreover, these genes Interestingly, exogenous insulin failed to suppress total adiponectin in also decreased in both heart and SKM of db/db mice accompanied by the Arg1174Gln carriers, but reduced IGFBP-1 and increased IGFBP-2 to the same signifi cant reduction of Akt activity. extent as observed in controls. These data suggest that Akt1 and Akt2 play an important role for maintaining In the face of marked insulin resistance, carriers of a dominant-negative heart and SKM growth and function through regulating mitochondrial energy INSR mutation showed surprisingly normal levels of adiponectin, leptin, production and they may be a therapeutic target for type 2 diabetes itself IGFBP-1 and IGFBP-2, and a potential improved distribution of adiponectin and its complications, such as diabetic cardiomyopathy. multimers. This supports the hypothesis of an impaired ability of high fasting

insulin levels to suppress these markers of common insulin resistance in Obesity

1753-P insulin recepteropathies. However, the apparently normal acute regulation POSTERS Skeletal Muscle-Specifi c Liver X Receptor α Knockout Exacerbates of IGFBP1 and IGFB2 by insulin suggests that alternative mechanisms should

High-Fat Feeding Induced Insulin Resistance in Mice be considered. Integrated Physiology/ TEAYOUN KIM, JIAN LIU, OLGA ZHELYAVOBSKA, MARIA S. JOHNSON, TIMOTHY R. NAGY, YAN LI, QINGLIN YANG, Birmingham, AL & 1755-P Liver X Receptors (LXRs) belong to the nuclear receptor subfamily 1, group Adiponectin Actions Enhanced by Macrophage Adiponectin Recept- H, member 3 (LXRα) and member 4 (LXRβ). LXRα is highly expressed in liver, or Expression and moderately expressed in adipose tissue, macrophages, kidney, heart, NANLAN LUO, HONG CHUNG, W. TIMOTHY GARVEY, YUCHANG FU, Birmingham, AL and skeletal muscle, whereas LXRβ is ubiquitously expressed. These ligand- Adiponectin is one of several important, metabolically active cytokines activated transcription factors have been suggested as key regulators of secreted from adipose tissue. Epidemiologic studies have associated lipid, cholesterol, carbohydrate metabolism, and infl ammation. Despite low circulating levels of this adipokine with multiple metabolic disorders extensive previous studies on systemic LXRa knockout mouse line, the in including obesity, insulin resistance, type II diabetes, and cardiovascular vivo role of LXRa in skeletal muscle remains elusive. In the present study, disease. To investigate how adiponectin receptor enhances adiponectin

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A475 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

actions in vivo, we generated transgenic mice which specifi cally express the tub signaling pathways and its role in the regulation of food intake. Thus, gene coding for adiponectin receptor 1 (AdR1) in mouse macrophages using we investigated, herein, in vivo tub modulation/signaling and its effects the scavenger receptor A-I gene (SR-AI) enhancer/promoter. on food intake. Tub signaling was investigated by dissection of arcuate Analyzing this transgenic mouse model, we have determined that AdR1 (ARC), ventromedial/dorsomedial nuclei (MH), paraventricular (PVN), lateral expression in macrophages was associated with a signifi cantly decreased hypothalamus (LH). Tub pTYR was decreased after fasting and increased after cholesterol (45%, p<0.01) and triglyceride accumulation (34%, p<0.01) in refeeding in ARC, MH, PVN, LH. Acutely treatment with intracerebroventricular macrophages from transgenic mice when compared to that from the control, (ICV) insulin or leptin increased tub pTYR in a time/dose dependent manner wild-type mice. The total body weight, fat tissue and liver tissue masses in ARC, MH, PVN, LH from control mice. However, in DIO mice this effect were also lower in AdR1-TG mice compared to control mice. Mouse whole was blunted. PTP1B was highly associated with tub and treatment with ICV body dual-energy X-ray absorptiometry (DXA) and liver tissue section analyses PTP1B oligonucleotide antisense (ASO) increased tub pTYR in hypothalamic were conducted to further confi rm above data. To investigate systemic insulin nuclei from DIO mice, suggesting that PTP1B may be a regulator of tub pTYR. sensitivity in vivo, we performed glucose tolerance tests (GTT) and insulin Co-immunoprecipitation/tyrosine kinase assay in vivo showed that tub binds tolerance tests (ITT) in AdR1-TG mice and control wild-type (WT) animals. insulin receptor (IR) or JAK2 after ICV insulin or leptin respectively, suggesting Plasma glucose was consistently higher in wild-type animals during the GTT that tub is an IR and JAK2 substrate. We also observed that ICV insulin or when compared to levels in AdR1-TG mice fed either a normal diet or a high leptin induces the nuclear translocation of tub after 15min in ARC and after fat diet; plasma glucose levels were consistently lower in AdR1-TG mice fed 30min in MH, PVN, LH. Further, ASO tub treatment decreased by 30-40% either a normal diet or the high fat diet when compared to the wild-type (WT) the anorexigenic effects of leptin and insulin and in parallel, decreased control animals indicating signifi cantly improved insulin sensitivity in the AdR1 POMC levels, but not NPY, AgRP levels in ARC. Interestingly, this effect transgenic animals. We have also found that AdR1-TG mice have a decrease of was independently of STAT3, AMPK, Akt/Foxo1 pathways. In summary, our glucose production (71%, p<0.05) in its liver and an increase of glucose uptake data provide evidence that tub is a substrate of insulin receptor and JAK2 (23%, p<0.05) in its skeletal muscle compared to control mice. upon insulin and leptin stimulation, is downregulated in high fat feeding and These results indicated that macrophage adiponectin receptor 1 PTP1B induces tub dephosphorylation. In addition, insulin and leptin induce overexpression can systemically infl uence lipid and glucose metabolism in tub translocation to the nucleus, suggesting a possible role as a transcription metabolic active tissues by increasing adiponectin actions in these tissues, factor, and may modulate food intake by altering POMC expression in ARC. suggesting a novel role of macrophage adiponectin receptor in improving Supported by: FAPESP insulin sensitivity in vivo. ADA-Funded Research & 1758-P & 1756-P Possible Role of Leptin as a Stimulator for Nocturnal Lipolysis Adipokines and Insulin Secretion in Chronic Spinal Cord Injury STELLA P. KIM, ISABEL R. HSU, KARYN J. CATALANO, JENNY D. CHIU, PHILIP E. BRANDON HOLLIS, JENNIFER OLIVE, SHIRISH S. BARVE, LELILA GOBEJISHVILI, CRYER, RICHARD N. BERGMAN, Los Angeles, CA, St. Louis, MO SRI PRAKASH L. MOKSHAGUNDAM, Baton Rouge, LA, Louisville, KY Leptin is secreted by white adipose tissue and has been shown to induce With increasing life expectancy after spinal cord injury (SCI), type 2 lipolysis in adipocytes. It has been suggested that leptin may increase diabetes mellitus (T2DM) has been recognized as a major cause of morbidity sympathetic outfl ow to white adipose tissue, thus increasing fat mobilization. and mortality in these individuals. Reduction in lean body mass and increase It has also been shown that leptin receptors are expressed in adipocytes - in fat mass in SCI may contribute to the metabolic problems through changes suggesting that leptin may act directly, independent of central pathways, to in adipokines and cytokines. Systematic studies assessing the potential increase lipolysis via an autocrine effect. We have previously demonstrated relationship between adipokines and insulin secretion in SCI compared with that when insulin resistance and obesity are induced by a high fat diet in the appropriate age and BMI matched controls are limited. dog model, there is a signifi cant increase in the nocturnal levels of FFA despite Fourteen subjects with chronic SCI (mean duration 10.1years) and 14 unchanged fasting levels, emphasizing the importance of 24-hr measurements. age and BMI matched able-bodied controls were studied. There were no We sought to determine whether leptin may have a role in the increase in signifi cant difference in BMI, fat mass, and waist circumference between nocturnal FFA and whether the changes in leptin may be related to alterations SCI and control subjects. Fasting blood sample was drawn for measurement in plasma catecholamines over a 24-hr period in dogs fed a high fat diet for 6 of glucose, insulin, c-peptide, adiponectin, resistin, leptin, adipsin, visfatin, wks. Norepinephrine (NE) was measured in samples taken every hour beginning hsCRP, TNF-alpha, IL-6 and PAI-1. An oral glucose tolerance test (OGTT) was at 6AM until 6AM the next morning (n=8). There was no change plasma NE at performed for measurement of glucose and insulin levels. any time point over the 24-hr period (average NE wk 0: 260±36 vs wk6: 189±28 Insulin secretion was assessed by calculation of HOMA –B cell index, and pg/mL, p=NS). In a subset of these same animals (n=5), we were able to obtain early and total insulnogenic index. 24-hr leptin measurements. We found that there was a substantial increase Subjects with SCI had signifi cantly higher 2 hour glucose levels (125.15 + 45 in leptin levels that peaked at 3PM such that the 24-hr area under the curve vs 90.15 +20.54 mg/dl, p <0.05) and higher prevalence of glucose intolerance. was increased more than 2-fold (wk 0: 16.9±0.19 vs wk 6: 42.5±10.6 ng/mL·hr, Leptin (2983 +469 vs 1659+353 pg/ml, P <0.05) and visfatin (1091+339 VS p<0.05). This peak immediately preceded the nocturnal surge in FFA which 695+109 pg/ml, P <0.05) were signifi cantly higher in the SCI group. Total began at 6PM and continued through the night, peaking at 3AM. The increase insulinogenic index was lower in subjects with SCI (p=0.04). Leptin levels in in leptin, coupled with the unchanged plasma NE levels, suggests that leptin the SCI group signifi cantly correlated with fasting insulin (r=0.56, p=0.037), may be responsible for the increase in nocturnal FFA due to the direct action of c-peptide levels(r=0.811, p=0.001), HOMA B cell index (r=0.662, p=0.01), and leptin on adipocytes to increase lipolysis via an autocrine effect and not due to early Insulinogenic index (r=-.585, p=0.046). While visfatin correlated with an increase in sympathetic outfl ow to adipose tissue. fasting (r=.685, p=.007) and 2-hour glucose level(r=0.764, p=0.002), there was no relation to measures of insulin secretion. Despite absence of changes in measures of adiposity, signifi cant changes in adipokines were noted in SCI. Dysregulated adipokine secretion may contribute to the development of glucose intolerance in SCI. The mechanisms of altered leptin levels and its association with measures of insulin secretion Obesity

POSTERS in subjects with SCI needs further evaluation. Supported by: University of Louisville Multidisciplinary Grant Program Integrated Physiology/ & 1757-P Leptin and Insulin Induce Tub Translocation to the Nucleus and Modulate Food Intake and POMC Expression in Arcuate Nucleus PATRICIA O. PRADA, PAULA G.F. QUARESMA, ANDREA M. CARICILLI, ANDRESSA C. SANTOS, DIOZE GUADAGNINI, JOSEANE MORARI, EDUARDO R. ROPELLE, JOSE BARRETO C. CARVALHEIRA, LICIO A. VELLOSO, MARIO J. SAAD, Campinas, Brazil Mutation or knocking down tubby (tub) gene in mice induces maturity-onset obesity and insulin resistance, suggesting that tub could be an important regulator of energy balance. In cells and in adipose tissue insulin is able to induce tub tyrosine phosphorylation (pTYR). However, little is known about

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A476 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

& 1759-P & 1761-P Dissecting the Intestinal Cholescystokinin Signaling Pathway(s) Oxytocin and Brain Derived Neurotrophic Factor Each Mimic the That Regulate Glucose Production Effect of Leptin to Lower Food Intake but Not Blood Glucose in STZ- BRITTANY A. RASMUSSEN, DANNA M. BREEN, GRACE W.C. CHEUNG, ANDREA Induced Diabetic Rats KOKOROVIC, TONY K.T. LAM, Toronto, ON, Canada JAMES E. BLEVINS, MILES E. MATSEN, MICHAEL W. SCHWARTZ, GREGORY J. Diabetes and obesity is characterized by a disruption in glucose MORTON, Seattle, WA homeostasis due partly to an elevation of hepatic glucose production. To Recent evidence suggests that leptin signaling in the brain normalizes date, the mechanisms underlying the regulation of glucose production in blood glucose levels in a rat model of uncontrolled insulin-defi cient diabetes healthy and obese/diabetic settings remain to be elucidated. Duodenal lipid (uDM) via a mechanism independent of food intake inhibition. To identify metabolism is recently demonstrated to trigger a gut-brain-liver neuronal potential mediators of this leptin action, we investigated whether central axis to lower glucose production in rats in vivo. The gut derived hormone administration of either of two central targets of leptin action -- oxytocin cholecystokinin (CCK) mediates this gut lipid neuronal effect through the (OXY) and brain derived neurotrophic factor (BDNF) -- can mimic leptin’s anti- activation of CCK-A receptors. However, duodenal lipid-CCK activation fails diabetic effects. to lower glucose production in response to high-fat feeding, suggesting Following cannulation of the lateral ventricle, adult male Wistar rats duodenal CCK resistance. To begin locating the molecular signaling defect(s) received systemic injection of either vehicle or streptozotocin (STZ; 40mg/ of duodenal CCK-resistance, we fi rst discovered that high-fat feeding did kg/sc x2) to induce uDM. Four days later, rats received consecutive daily icv not alter duodenal CCK-A receptor expression, suggesting that duodenal injections of oxytocin (10ug/d), BDNF (5ug/d) or vehicle (sterile water) for CCK resistance occurs at the signaling level of the CCK-A receptors. We 7 days. To determine if oxytocin is required for the anti-diabetic effects of next investigated whether the activation of PKA, a downstream signaling leptin, a separate cohort of rats received an icv pre-treatment injection of the molecule of the CCK-A receptor, is suffi cient and necessary for duodenal CCK oxytocin receptor antagonist (OVT; 10ug/d) or its vehicle (0.9% saline), 1h prior to lower glucose production through a neuronal network in normal rats in to either icv leptin (4ug/d) or vehicle (PBS, pH 7.9) over the same time interval. vivo. First, intraduodenal infusion of the PKA activator, Sp-CAMPS, lowered While daily administration of oxytocin attenuated diabetic hyperphagia glucose production during the pancreatic (basal insulin) euglycemic clamps. (mean food intake (FI): 38.4±2.0 g for STZ-veh vs. 31.9±2.4 g for STZ-OXY; Co-infusion of Sp-CAMPS with the PKA inhibitor H89 intraduodenally P<0.05), it failed to lower blood glucose levels relative to STZ-veh-treated abolished the glucose production suppression effect. Second, intraduodenal rats (mean blood glucose: 383±16 mg/dl for STZ-veh vs. 372±14 mg/dl for administration of CCK-8 (the biologically active form of CCK) lowered glucose STZ-OXY; P<0.05). Moreover, icv pre-treatment with OVT failed to block production. Importantly, co-administration of CCK-8 with H89 abolished the either the anorexic or the anti-diabetic effects of leptin in uDM. ability of duodenal CCK-8 to lower glucose production. Third, the ability of Similarly, daily icv administration of BDNF completed blocked diabetic Sp-CAMPS to lower glucose production was abolished upon intraduodenal hyperphagia (mean FI: 42.1±3.6 g for STZ-veh vs. 23.1±1.9 g for STZ-BDNF; co-administration of the anesthetic tetracaine with Sp-CAMPS. In Summary, P<0.05) but only had a modest effect to lower blood glucose levels, and a these data together illustrate that duodenal PKA activation is suffi cient and follow-up study suggested that this glucose-lowering was due largely to necessary to lower glucose production via a neuronal network in normal reduced food intake. Taken together, these data suggest that while central rats. This set of preliminary data raises the possibility that high fat feeding administration of either oxytocin or BDNF can ameliorate diabetic hyperphagia, induces duodenal CCK resistance at the signaling level of duodenal PKA. neither is suffi cient to mimic the anti-diabetic effects of leptin. Supported by: CIHR We conclude that in uDM, leptin’s glucose-lowering effects occur independently of both oxytocin and BDNF signaling pathways. & 1760-P Gastrointestinal-Mediated Glucose Disposal in Vagotomized Subjects & 1762-P ASTRID PLAMBOECK, TINA VILSBØLL, CAROLYN DEACON, ANDRE WETTERGREN, Low Glucose Stimulates Ghrelin Release from Primary Cultures of SØREN MEISNER, CLAUS HOVENDAL, LARS BO SVENDSEN, FILIP KNOP, JENS Gastric Mucosal Cells HOLST, Hellerup, Denmark, Copenhagen, Denmark, Odense, Denmark PAUL K. PIPER, ICHIRO SAKATA, JEFFREY M. ZIGMAN, Dallas, TX After secretion, glucagon-like peptide-1 (GLP-1) is rapidly degraded by The peptide hormone ghrelin is released from a distinct group of the enzyme dipeptidyl peptidase-4 (DPP-4), resulting in less than 15% of gastrointestinal cells in response to caloric restriction and upon stress. Once the intact biologically active peptide reaching the systemic circulation. This released, ghrelin increases food intake and helps coordinate a response has led to the hypothesis that GLP-1 acts locally before being degraded. We to stress. Ghrelin secretion is particularly important during severe caloric aimed to evaluate the role of the vagus nerves for the effect of GLP-1 on restriction to prevent hypoglycemia. Yet, despite all that is known about gastrointestinal-mediated glucose disposal (GIGD). ghrelin action, little detail is known regarding ghrelin secretion. Here, we Eight truncally vagotomized (due to duodenal ulcer) subjects (age: 70±2 have used primary gastric mucosal cell cultures to investigate mechanisms years (mean±SEM); BMI: 24±1 kg/m2; fasting plasma glucose (FPG): 5.8±0.2 of ghrelin release, with an emphasis on the role of hypoglycemia. mM), 7 subjects previously treated for esophageal cancer with resection We assessed ghrelin release from primary cultures of gastric mucosal cells of the cardia including truncal vagotomy (age: 64±2 years; BMI: 23±1 kg/ exposed to various D-glucose concentrations, with or without the glucose m2; FPG: 5.4±0.1 mM) and 5 healthy control subjects (age: 68±2 years; BMI: antimetabolite 2-deoxyglucose (2-DG) or the sulfonylurea tolbutamide. We 25±1 kg/m2; FPG: 5.4±0.2 mM) were examined on three separate occasions: assessed ghrelin release following exposure to L-glucose, epinephrine and two 4h 50-g oral glucose tolerance tests (OGTTs) ± DPP-4 inhibition and an other potential secretagogues. We determined the expression profi le of isoglycemic intravenous (iv) glucose infusion (IIGI). various proteins involved in glucose metabolism for enriched populations of Isoglycemia during IIGIs was obtained using 24±2 g and 28±4 g of glucose ghrelin cells (isolated from ghrelin-hrGFP reporter mice) and for immortalized in patients with truncal vagotomy associated with surgery for duodenal ghrelin cell lines. ulcer and cardia resection, respectively (P=NS) and 17±2 g in healthy control Epinephrine and norephinephrine both stimulated ghrelin release from subjects (P<0.05), resulting in GIGD [100% x (glucoseOGTT - glucoseIIGI/ primary gastric mucosal cell cultures. Ghrelin secretion was negatively glucoseOGTT)] of 51±4 and 45±8% in the two vagotomized groups (NS) and correlated with D-glucose concentration but was unaffected by L-glucose. 67±4% in the healthy control subjects (P<0.05). Peak concentrations of 2-DG prevented the inhibitory effect of high D-glucose exposure on ghrelin Obesity POSTERS plasma glucose (PG) were similar in the two vagotomized groups (13.6 ±0.9 release, while tolbutamide exerted no direct effect on ghrelin secretion. vs. 13.3±0.8 mM, P=NS), and higher than in the control group (9.1±0.5 mM, GLUT4, GLUT5, glucokinase and sulfonylurea receptor (SUR1) mRNAs were P<0.003). There was no effect of DPP-4 inhibition on PG in any group. enriched within ghrelin cells. Integrated Physiology/ Vagotomized subjects have impaired glucose homeostasis, which may In conclusion, these experiments with 1° gastric mucosal cell cultures be due to accelerated gastric emptying. Interestingly, GIGD was diminished suggest that direct exposure of ghrelin cells to low ambient glucose stimulates in these subjects, suggesting that intact vagal innervation is important for ghrelin release, while high ambient glucose and glucose metabolism within maintenance of normal glucose homeostasis. ghrelin cells blocks ghrelin release. GLUT4, GLUT5 and glucokinase may be key Supported by: EFSD/Novo Nordisk Grant elements of the ghrelin cell machinery involved in reducing ghrelin release in high glucose conditions. The role of SUR1 in ghrelin release remains uncertain. These data suggest that ghrelin release may be enhanced directly by exposure of ghrelin cells to hypoglycemic conditions. Supported by: Endocrine Fellows Foundation

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A477 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

Guided Audio Tour: Incretin Hormone Biology (Posters 1763-P to 1772-P), see page 13. & 1765-P Glucose-Dependent Insulinotropic Polypeptide—A Bifunctional Blood 1763-P Glucose Stabilizer Chronic Administration of WITHDRAWNEzetimibe Increases Active Glucagon-Like MIKKEL CHRISTENSEN, LOUISE VEDTOFTE, JENS J. HOLST, TINA VILSBØLL, FILIP Peptide-1 and Prevents the Development of Type 2 Diabetes in Rats K. KNOP, Hellerup, Denmark, Copenhagen, Denmark SOO JIN YANG, JUNG MOOK CHOI, LISA KIM, WON JUN KIM, SE EUN PARK, Longstanding knowledge positions glucose-dependent insulinotropic EUN JUNG RHEE, CHEOL-YOUNG PARK, WON YOUNG LEE, KI WON OH, SUNG polypeptide (GIP) as an incretin hormone in healthy humans, but controversy WOO PARK, SUN WOO KIM, Seoul, Republic of Korea exists regarding the effects of GIP on glucagon secretion. We hypothesized Ezetimibe is a cholesterol-lowering agent targeting Niemann-Pick C1- that the glucagonotropic effect of GIP, like its insulinotropic effect, is like 1, an intestinal cholesterol transporter. Chronic administration of glucose-dependent. Therefore, we aimed to evaluate the effect of GIP on ezetimibe may ameliorate several metabolic disorders including hepatic plasma glucagon and insulin responses at three different glycemic levels. steatosis and insulin resistance. In this study, we investigated whether Ten healthy male subjects (age: 23±1 (mean±SEM) years; BMI: 22±1 chronic ezetimibe treatment prevents the development of type 2 diabetes 2 kg/m ; HbA1c: 5.5±0.1%) without family history of diabetes were studied on and alters levels of glucagon-like peptide-1 (GLP-1), an incretin hormone six separate days. Physiological doses of GIP or saline were administered involved in glucose homeostasis. Male LETO and OLETF rats at 12 weeks of intravenously (randomized and double-blinded) during 90 min of either -1 -1 age were treated with vehicle or ezetimibe (10 mg·kg · day ) for 20 weeks insulin-induced hypoglycemia, euglycemia or hyperglycemia (randomized). via stomach gavage. OLETF rats were diabetic with hyperglycemia and During hypoglycemia plasma glucose (PG) was gradually lowered from signifi cant decreases in pancreatic size and beta cell mass compared with mean fasting level of 5.0±0.1 mM (mean±SEM) to a plateau level of 2.8±0.1 lean controls. There was no difference in weight change and food intake mM. GIP infusion resulted in greater glucagon responses during the fi rst 30 between groups during the treatment period. However, chronic treatment of minutes compared to saline (area under curve: 76±17 vs. 28±16 pM×30 min, the OLETF rats with ezetimibe prevented the development of diabetes with P<0.008) and a small short-lasting (0-10 min) increment in plasma C-peptide reduced fasting serum glucose (7.2 ± 0.1 vs. 10.4 ± 0.2 mmol/l, P < 0.001) before PG was lowered. During euglycemia (mean PG: 5.0±0.1 mM) GIP and improved glucose control during oral glucose tolerance test (28504 ± infusion elicited larger glucagon (62±18 vs. -11±8 pM×90 min, P<0.005) and 1486 vs. 37512 ± 2052 area under the curve, P = 0.002) compared with OLETF C-peptide responses (9.7±2.1 vs. -3.8±1.0 pM×90 min, P<0.001) compared controls. Moreover, ezetimibe treatment rescued the reduced pancreatic to saline. During hyperglycemia (mean PG: 12.1±0.3 mM) comparable size and beta cell mass in OLETF rats. Consistent with the previous fi nding suppression of plasma glucagon (-461±81 vs. -371±50 pM×90 min, P=0.26) that diabetic patients showed high dipeptidyl peptidase-4 (DPP-4) activity was observed with GIP and saline infusions, whereas GIP infusion doubled and low levels of active GLP-1, DPP-4 activity was higher and active GLP- the C-peptide responses (255±25 vs. 117±13 nM×90 min, P<0.001). 1 tended to be lower in diabetic OLETF rats compared with lean controls. In conclusion, GIP has no effect on glucagon responses during Interestingly, ezetimibe signifi cantly decreased serum DPP-4 activity (OLETF hyperglycemia when it potentiates insulin secretion. In contrast, GIP ezetimibe 309.5 ± 7.3, OLETF control 478.2 ± 29.7 % LETO control; P < 0.001) increases glucagon levels during fasting and hypoglycemic conditions, and increased active GLP-1 (OLETF ezetimibe 14.8 ± 4.2, OLETF control 5.3 where it has little or no effect on insulin secretion. Thus, GIP seems to be a ± 0.3 pmol/l; P = 0.034) in OLETF rats. These fi ndings demonstrated that physiological bifunctional blood glucose stabilizer with diverging glucose- chronic administration of ezetimibe prevented the development of type 2 dependent effects on the two main pancreatic glucoregulatory hormones diabetes and increased active GLP-1 levels, suggesting possible involvement insulin and glucagon. of GLP-1 in the preventive effect of ezetimibe against type 2 diabetes.

& 1766-P & 1764-P Blocking GLP–1 Receptor Corrects Postprandial Hypoglycemia in Prohormone Convertase 2 Positive Enteroendocrine Cells Are More Hyperinsulinemic Hypoglycemia after Gastric Bypass Surgery for Abundant in Patients with Type 2 Diabetes—A Potential Source of Obesity Gut-Derived Glucagon MARZIEH SALEHI, LESLIE BAUM, RONALD L. PRIGEON, DAVID A. D’ALESSIO, FILIP K. KNOP, KRISTINE J. HARE, JENS PEDERSEN, JAKOB W. HENDEL, STEEN S. Cincinnati, OH, Baltimore, MD POULSEN, JENS J. HOLST, TINA VILSBØLL, Hellerup, Denmark, Copenhagen, Denmark, Severe hyperinsulinemic hypoglycemia occurring in patients several years Herlev, Denmark after gastric bypass surgery (GB) has been increasingly reported in recent In α cells, the precursor proglucagon (from the glucagon gene) is years. Affected patients have exaggerated insulin and glucagon like-peptide processed by prohormone convertase (PC)2 to glucagon, whereas entero- 1 (GLP-1) responses to meal ingestion compared to those who don’t have endocrine L cells utilize PC1 in the processing of proglucagon to the hypoglycemia after GB, raising the possibility that an enhanced GLP-1 action glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). Hyperglucagonemia contributes to abnormal glucose regulation. following oral glucose in type 2 diabetes mellitus (T2DM) is thought To determine the role of GLP-1 in postprandial glucose levels in this to arise as a consequence of dysfunctional α cells combined with β cell condition, 4 subjects suffering from neuroglycopenic hypoglycemia after GB insuffi ciency. However, in contrast to oral glucose, iv glucose does not elicit (H; age, 43±6 yrs; BMI, 36±2 kg/m2; 6.0±0.7 yrs post-op) and 3 asymptomatic hypersecretion of glucagon in T2DM. Therefore, we hypothesized that T2DM individuals with GB (A; age, 46±3 yrs; BMI, 35±6 kg/m2; 5.3±0.7 yrs post- patients possess the potential to release glucagon directly from the gut. op) were studied with a meal tolerance test on two days. On one day they 2 Ten male patients with T2DM (age: 51(41-62) years); BMI: 32(28-39) kg/m ; received the GLP-1 receptor antagonist, exendin-(9-39) [Ex-9] and on the HbA1c: 7.1(5.4-8.7)%) and 10 male healthy control subjects (age: 58(48-67) other a saline control. years; BMI: 31(26-36) kg/m2; HbA1c: 5.5(5.2-6.0)%) underwent a 4h meal test and a jejunoscopy (including jejunal biopsies) on two separate days. Patients with T2DM exhibited exaggerated postprandial plasma glucose excursions (379±76 (mean±SEM) vs. 77±33 mM×4h, P=0.001). Postprandial insulin (30±6 vs. 27±5 nM×4h, P=0.7) and C-peptide responses (175±25

Obesity vs. 188±24 nM×4h, P=0.7) were similar in the two groups, but patients POSTERS with T2DM exhibited higher postprandial glucagon responses (3.0±0.5 vs. 1.9±0.2 nM×4h, P=0.02). No differences in glucose-dependent insulinotropic

Integrated Physiology/ polypeptide (GIP), GLP-1, GLP-2 or peptide YY responses were observed. Similar numbers of endocrine cells (all stained for PC1) from jejunal biopsies were observed in the two groups; including GIP, GLP-1, and GLP-2 positive cells. Signifi cantly more PC2 positive cells were found among T2DM patients (70±8 vs. 44±4 cells/mm2, P=0.01). Similar levels of PC1 and PC2 gene GLP-1 blockade increased the AUCglucose and the time to reach nadir expression were observed in the two groups. glucose levels in both groups; eliminated hypoglycemia in all H subjects with Our results show that a high number of small intestinal endocrine cells in minimal effect on nadir glucose in A group. Ex-9 lowered insulin response to T2DM patients are equipped with PC2, which potentially - through processing of meal ingestion in both groups despite higher glucose levels. proglucagon to glucagon - contribute to the hyperglucagonemia of these patients; shifting the ‘pancreacentric’ view on type 2 diabetic hyperglucagonaemia towards a role for the gut in this pathophysiological trait.

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A478 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

prevented the drop of insulin levels in a dose-dependent manner. Effective Hypoglycemic-GB (H) Asymptomatic-GB (A) Statistical tests (p values) doses increased GLP-1 up to 2-fold and insulin up to 3-fold during the OGTT. Saline Ex-9 Saline Ex-9 H vs. A Ex-9 vs. Saline Interaction Administration of selective agonists at bile acid responsive receptors, Nadir glucose 2.3±0.5 4.1±0.4 3.9±0.3 4.2±0.2 0.19 0.00 0.01 the farnesoid-X receptor and TGR5, did not replicate the effects of ASBT (mmol/L) inhibition, suggesting an alternate mechanism. In summary, inhibition of Time to reach nadir 79±3 146±25 120±30 180±0 0.15 0.01 0.84 ASBT increases bile acids in the distal intestine, promotes GLP-1and insulin glucose (min) release and glucose-lowering, and may offer a new therapeutic strategy for type 2 diabetes mellitus. AUCglucose (mmol/L.min) 632±151 994±51 970±42 1056±73 0.16 0.03 0.12 AUCinsulin 59.4±14.7 30.6±3.6 40.1±16.2 28.6±11.9 0.40 0.11 0.44 (µmol/L.min) & 1769-P Novel Biological Action of the Dipeptidylpeptidase 4 Inhibitor, These fi ndings indicate that enhanced GLP-1 action contributes to Sitagliptin, as a GLP-1 Secretagogue postprandial hypoglycemia after GB and GLP-1 receptor antagonists are an GANESH V. SANGLE, LINA M. LAUFFER, ANTHONY GRIECO, PATRICIA L. attractive potential treatment for this condition. BRUBAKER, Toronto, ON, Canada Supported by: NIH-DK083554 Glucagon-like peptide-1 (GLP-1) is released from the intestinal L cell in response to nutrients and other secretagogues. GLP-1 has important anti- & 1767-P diabetic effects including enhancement of glucose-dependent insulin Glucose-Dependent Insulinotropic Polypeptide Suppresses the secretion. Sitagliptin prevents GLP-1 degradation by inhibiting dipeptidyl- Development of Atherosclerosis in Apolipoprotein E-Null Mice Via peptidase 4 (DPP4), and is used to lower HbA1c levels in patients with Its Own Receptors type 2 diabetes (T2D). Sitagliptin treatment for 4 wk increased bioactive MASAHARU NAGASHIMA, MICHISHIGE TERASAKI, KYOKO NOHTOMI, MASAKO GLP-1 levels by 80% and improved glycemic tolerance by 60% in neonatal- TOMOYASU, SHURI KANEYAMA, TAKUYA WATANABE, AKIRA MIYAZAKI, STZ rats, a T2D model. This effect was prevented by the GLP-1 antagonist, TSUTOMU HIRANO, Shinagawa, Japan, Hachiouji, Japan exendin-4(9-39NH2), confi rming GLP-1-dependence of the actions of Several reports have suggested glucagon-like peptide-1 has favorable sitagliptin. However, it is unclear if sitagliptin increases GLP-1 levels only effects on the suppression of atherosclerosis. Less is understood, however, via DPP4 inhibition or if it has actions on GLP-1 independent of DPP4. The on how atherosclerosis is affected by glucose-dependent insulinotropic effects of sitagliptin (0.1-2μM) directly on the L cell were thus tested using polypeptide(GIP). We report experimental evidence demonstrating that GIP the murine (GLUTag) and human (NCI-H716) L cell lines, as well as primary has direct anti-atherosclerotic properties. fetal rat intestinal L cells (FRIC). Sitagliptin increased total GLP-1 secretion in Starting from the age of 17 weeks, apolipoprotein (apo) E-null mice were the GLUTag and NCI-H716 cells, by up to 132 and 83%, resp. (P<0.01-0.001), placed on an atherogenic diet and administered daily subcutaneous infusions but did not change in GLP-1 secretion in FRIC cells. Treatment with the of vehicle (saline), GIP(1-42) (25 nmol/kg/day), GIP(3-42) (25nmol/kg/day), structurally-unrelated DPP4 inhibitor, MK0626 (1-50µM) did not affect GLP-1 (Pro3)GIP (25nmol/kg/day), or GIP(1-42) plus (Pro3)GIP for 4 weeks. The secretion in any of the cells. infusions did not signifi cantly affect body weight, food intake, plasma lipids, Sitagliptin and MK0626 inhibited DPP4 enzymatic activity in NCI-H716 or glucose. GIP(1-42) suppressed the atherosclerotic lesions in the whole cells, by 69 and 62%, resp. (P<0.05), but had no effect in GLUTag and aorta by 53%, suppressed the plaque volume in the aortic valve by 38%, FRIC cells, indicating that the effect of sitagliptin on GLP-1 secretion was and suppressed macrophage infi ltration by 48%, compared with the vehicle independent of protease inhibition. Unexpectedly, sitagliptin increased the controls. In contrast, GIP(3-42) had no effects on these atherosclerotic phosphorylation of ERK1/2 in GLUTag and NCI-H716 cells, by 352% and 22%, changes, and the co-infusion of (Pro3)GIP abolished the anti-atherosclerotic resp. after 5 min; however, MEK-1/2 inhibition (PD98059/U0126) had no effect of GIP(1-42). The infusion of (Pro3)GIP alone had no effect on the effect on sitagliptin-induced GLP-1 secretion in these cells (n=3-6). Sitagliptin development of atherosclerosis. did not induce changes in Akt phosphorylation (n=3). Taken together, these An oxidized LDL-induced foam cell formation was detected in the peritoneal fi ndings demonstrate that sitagliptin increases GLP-1 levels not only via macrophages obtained from apo E-null mice with various infusions. GIP(1- DPP4 inhibition, but also through novel, direct actions to stimulate GLP-1 42) infusion suppressed the foam cell formation by 30%, whereas GIP(3-42) secretion by the intestinal L cell via a MEK-independent, ERK1/2-dependent infusion had no suppressive effect whatsoever. Meanwhile, the co-infusion pathway (supported by an IISP grant from Merck). with (Pro3)GIP nullifi ed the suppressive effect of GIP(1-42). Supported by: Merck IISP grant; Canadian Diabetes Association GIP receptor mRNA was detected in human and murine macrophages, vascular smooth muscle cells, and endothelial cells by RT-PCR. GIP receptors & 1770-P were also histochemically stained in the vascular endothelium, media, and Non-Invasive SPECT Imaging of Pancreatic Islets Targeting Glucagon- plaque of aorta in the apo E-null mice. Like Peptide-1 Receptors GIP (1nM) in vitro suppressed the proliferation of smooth muscle cells HIROYUKI FUJIMOTO, KENTARO TOYODA, HIROYUKI KIMURA, YUU OGAWA, ERI determined by BrdU staining, the gene expressions of ICAM-1 and MCP-1 in MUKAI, ZHUANG XIAOTONG, HIDEKAZU KAWASHIMA, HIROKAZU MATSUDA, endothelial cells, and macrophage foam cell formation. KONOMU HIRAO, HIDEO SAJI, NOBUYA INAGAKI, Kyoto, Japan, Osaka, Japan These results suggest that GIP has direct anti-atherosclerotic effects on The volume of pancreatic β-cells is known to decrease during diabetes the cells mainly responsible for atherogenesis via its own receptors. development and progression. A technique for measurement of β-cell volume in vivo is required, but a non-invasive method to detect pancreatic β-cells & 1768-P has not been developed. We previously reported the possibility of the probe Antidiabetic Effect of Inhibitors of Apical Sodium-Dependent Bile targeting glucagon-like peptide-1 receptors (GLP-1R). The aim of this study Acid Transport (ASBT) is whether the mass of pancreatic β-cells can be imaged non-invasively XIAOZHOU YAO, JUDI A. MCNULTY, DONALD I. ANDERSON, YAPING LIU, by single-photon emission computed tomography (SPECT) targeting GLP- CHRISTOPHER C. NYSTROM, DALLAS K. CROOM, SEAN A. ROSS, DEEPAK K. 1R, using 123I-labeled exendin(9-39) (Ex(9-39)). For SPECT imaging, lysine

RAJPAL, KIMBERLY D. HAMLET, JON L. COLLINS, CHARI D. SMITH, ANDREW A. residue at 12 or 27 or N-terminus of Ex(9-39) was iodobenzoylated. For Obesity

YOUNG, LIHONG CHEN, Research Triangle Park, NC evaluation of their receptor specifi city, binding assay in vitro was preformed POSTERS Bile acids are increasingly recognized as metabolic modulators. We using dispersed cells of islets isolated from 6-week-old male ddY mice. For 125 evaluated the effects on glucose homeostasis of a potent ASBT inhibitor assessment of in vivo selectivity, each IB-Ex(9-39) was labeled with [ I] Integrated Physiology/ (264W94), which blocks intestinal absorption of bile acids and increases ([125I]IB-Ex(9-39)), and biodistribution study was performed by intravenous fecal bile acid concentration in Zucker Diabetic Fatty (ZDF) rats. Drug was administration of them to 6-week-old male ddY mice. At appropriate time orally administered twice daily for two weeks in doses of 0.001, 0,01, 0,1, 1 points after administration, selected organs and blood were harvested and 10 mg/kg (n=8/dose group). Non-fasting blood glucose, HbA1c, insulin, and their weight and radioactivities were measured. In a blocking study, plasma total glucagon-like peptide 1 (tGLP-1) and bile acid concentrations excess non-radioactive exendin(9-39) was administere. Furthermore, and fecal bile acids were measured weekly. An oral glucose tolerance most promising IB-Ex(9-39) was labeled with [123I] ([123I]IB-Ex(9-39)), and test (OGTT) was performed at the end of the study. Oral administration of systematically administered and its retention in pancreas was imaged by 264W94 dose-dependently decreased plasma bile acids, and increased fecal SPECT. The results of binding assay analysis showed competitive inhibition of bile acid and non-fasting plasma tGLP-1 throughout the course of the study. specifi c [123I]IB-Ex(9-39) binding by non-radioactive Ex(9-39). Biodistribution Treatment with 264W94 signifi cantly decreased HbA1c and glucose, and study showed that radioactivities of pancreas were 17 to 45 %dose/g.

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A479 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

Pre-administration of excess non-radioactive exendin(9-39) signifi cantly Of note, plasma Ex levels were approx. 8-fold higher in GLP-1R KO mice blocked the radioactivity of pancreas after [125I]IB-Ex(9-39) injection. SPECT (450±139 pg/ml) vs. WT (55±20 pg/ml) (p<0.02). was performed 30 min after [123I]IB-Ex(9-39) administration to mice, which These data support the concept that Ex requires a functional GLP-1R to revealed the remarkable accumulation of radioactivity in pancreas. These exert its chronic effects in mice. The differential plasma levels of Ex in WT results indicated that Ex(9-39) could serve as a useful probe for non-invasive vs. KO mice suggest that the GLP-1R may play an important role in the plasma SPECT imaging of pancreatic β-cells. Grant Acknowledgement: The Program clearance of Ex, via either a direct receptor-mediated effect, or an indirect for Promotion of Fundamental Studies in Health Sciences of the National effect on renal clearance. The apparent lack of effects of Ex in GLP-1R KO Institute of Biomedical Innovation (NIBIO). mice indicates that novel GLP-1 receptors may not substantially contribute to the chronic metabolic actions of GLP-1R agonists. & 1771-P Basal Plasma Glucose, Insulin and Glucose-Dependent Insulino- 1773-P tropic Polypeptide Constitute Important Determinants of Fasting Adipocyte Expression of the Glucose-Dependent Insulinotropic Hyper glucagonemia in Type 2 Diabetes Polypeptide Receptor (GIPR) Involves Gene Regulation by PPARγ JONATAN I. BAGGER, FILIP K. KNOP, MAI-BRITT T. NIELSEN, JENS J. HOLST, and Histone Acetylation TINA VILSBØLL, Hellerup, Denmark, Hvidovre, Denmark, Copenhagen, Denmark SU-JIN KIM, CUILAN NIAN, CHRISTOPHER H.S. MCINTOSH, Vancouver, BC, Canada Fasting hyperglucagonemia in patients with type 2 diabetes mellitus Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal (T2DM) contribute to the exaggerated fasting plasma glucose (FPG) levels of hormone that exerts insulinotropic and growth and survival effects on these patients. However, the mechanisms underlying elevated basal plasma pancreatic β-cells. Additionally, there is increasing evidence supporting an glucagon levels are poorly understood. We aimed to clarify the relationship important role for GIP in the regulation of adipocyte metabolism. In earlier between basal glucagon levels and several metabolic parameters including studies, both peroxisome proliferator-activated receptor (PPAR)α and PPARγ the gut incretin hormones glucose-dependent insulinotropic polypeptide have been implicated in the regulation of GIPR mRNA expression in β-cells. (GIP) and glucagon-like peptide-1 (GLP-1). In the curent study we examined the molecular mechanisms involved in the Blood from patients with T2DM (N=104, 27% women; age: 57±1 years regulation of GIP receptor (GIPR) expression in 3T3-L1 adipocytes. (mean±SEM); body mass index (BMI): 30±1 kg/m2; FPG: 10.8±0.3 mmol/L; GIP acted synergistically with insulin to increase neutral lipid accumulation HbA1c: 8.0±0.2%; diabetes duration: 53±6 months) and healthy control during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. subjects (N=71, 27% women; age: 56±1 years; BMI: 28±1 kg/m2; FPG: Both GIPR protein and mRNA expression increased during 3T3-L1 cell 5.6±0.1 mmol/L (P<0.001 vs. T2DM); HbA1c: 5.6±0.1% (P<0.001 vs. T2DM)) differentiation and this was associated with up-regulation of nuclear levels was sampled in the fasting state. Multiple linear regression analysis with of sterol response element binding protein 1c (SREBP-1c) and PPARγ, as well basal plasma glucagon as the dependent parameter and BMI, age, diabetes as acetylation of histone H3/H4. The PPARγ receptor agonists LY171883 and duration, insulin resistance according to the homeostatic assessment model rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, (HOMAIR), HbA1c, FPG and basal insulin, C-peptide, GIP and GLP-1 plasma whereas the antagonist, GW9662, ablated expression. Additionally, both concentrations as independent parameters was performed. PPARγ and acetylated histone H3/H4 were shown to bind to a region of the Patients with T2DM had higher fasting levels of plasma glucagon (12.9±0.5 GIPR promoter containing the peroxisome proliferator response element vs. 8.5±0.5 pmol/L, P<0.001), insulin (57±4 vs. 43±4 pmol/L, P=0.013), (PPRE). Knockdown of PPARγ in differentiated 3T3-L1 adipocytes using C-peptide (874±44 vs. 660±40 pmol/L, P=0.001) and GIP (15.5±1.7 vs. 11.1±0.9 RNA interference (RNAi) reduced GIPR expression, supporting a functional pmol/L, P=0.03). HOMAIR was most pronounced among T2DM patients regulatory role. Additionally, unlike the signifi cantly reduced β-cell GIPR (3.7±2.5 vs. 1.6±1.4, P<0.0001). The included independent parameters among expression in rodent models of T2DM, both GIPR and nuclear PPARγ levels T2DM patients explained 42% (r2 =0.42, P<0.0001) of the variation in their in adipose tissue of the VDF Zucker were found to be selectively elevated in fasting plasma glucagon levels. Interestingly, the only signifi cant contributors epididymal, retroperitoneal and mesenteric adipose tissue of obese VDF rats, were FPG (P=0.002), fasting plasma insulin (P=0.004) and fasting plasma compared to lean littermate controls. Taken together, the studies show that GIP (P=0.031), which all positively related to fasting hyperglucagonemia in GIP and insulin act in a synergistic manner on 3T3-L1 cell development and patients with T2DM. None of these parameters predicted fasting glucagon adipocyte GIPR expression is up-regulated through a mechanism involving levels in healthy subjects. interactions between PPARγ and a GIPR promoter region containing an Our data suggest that elevated fasting plasma GIP levels, in addition acetylated histone region. to hyperglycemia and hyperinsulinemia, may play a role in the fasting Supported by: Canadian Diabetes Association and Canadian Institutes for Health hyperglucagonemia observed in patients with T2DM. Research 1774-P & 1772-P Apelin Expression in Human Adipocytes Is Associated with Insulin Long-Term Metabolic Benefi ts of Exenatide in Mice Are Mediated Sensitivity Solely Via the Known Glucagon-Like Peptide 1 Receptor (GLP-1R) PATRICK YUE, FAHIM A. ABBASI, ALICIA C. DENG, TRACEY L. MCLAUGHLIN, KRYSTYNA TATARKIEWICZ, EMMANUEL SABLAN, CLARA POLIZZI, DAVID GERALD M. REAVEN, PHILIP S. TSAO, Stanford, CA PARKES, San Diego, CA Apelin, a recently discovered peptide hormone, has been shown to The GLP-1R is expressed in multiple tissues and its activation results in improve systemic insulin sensitivity in animals. Early studies have reported metabolic benefi ts including enhanced insulin secretion, slowing of gastric that apelin’s impact on insulin sensitivity is mediated by its actions in emptying, decreased food intake, and improved hepatic steatosis. There skeletal muscle. However, because apelin is also secreted by adipocytes, is limited and inconclusive knowledge if these effects are solely GLP-1R- it is possible that adipose tissue might have a role in regulating insulin- mediated with chronic exposure. Therefore, we performed a 3-month study glucose homeostasis. Human subjects were recruited and classifi ed as with supramaximal doses of exenatide (Ex) in mice lacking a functional GLP- insulin resistant (IR) or insulin sensitive (IS) based on steady-state plasma 1R (GLP-1R KO). glucose (SSPG) determination during an insulin suppression test using a GLP-1R KO and wild-type (WT) control mice were fed a high fat diet (58% prespecifi ed cutoff of 180 mg/dL. Peri-umbilical subcutaneous adipose

Obesity fat). Ex (30 nmol/kg/d) was infused SC for 12 wks. HbA1c, plasma glucose, biopsy specimens were obtained from IR and IS human subjects matched POSTERS insulin, amylase (A), lipase (L), ALT, AST, body weight (BW), and food intake for body mass index (BMI). Samples underwent mRNA isolation and cDNA (FI) were measured. At study end, oral glucose tolerance (OGTT) and gastric preparation, and then assessed for apelin mRNA expression by quantitative

Integrated Physiology/ emptying (GE) were assessed. Terminal organ weights, hepatic lipid content real time PCR. Additionally, serum levels of glucose, insulin, adiponectin, and (HLC), and plasma Ex levels were measured. apelin were measured from blood samples derived from each subject. No Ex produced no signifi cant effects on any endpoint in GLP-1R KO mice at statistically signifi cant differences in fasting glucose, gender distribution, or study end. In contrast, in WT mice Ex decreased vehicle (veh)-corrected BW BMI were observed between groups; however, IR subjects were signifi cantly (40±2%), cumulative FI (maximally to 70±3% at wk 2), and glucose (by 25±5 older, had higher insulin levels, and lower adiponectin levels. Adipose mg/dl). Ex reduced insulin (ng/ml) (0.5±0.2) vs. veh (1.8±0.5), OGTT glucose tissue apelin mRNA expression was 1.6-fold elevated in IR subjects (p < AUC0-2h (mg·h/dl) (642±70) vs. veh (972±93), ALT (U/l) (20±4) vs. veh (47±10), 0.01), and correlated strongly with SSPG. Bivariate analysis to reexamine and HLC (g/g) (0.059±0.004) vs. veh (0.107±0.010); (p<0.05). Ex increased apelin mRNA expression and SSPG as continuous values demonstrated a pancreatic (0.0097±0.0004) and renal (0.0101±0.0002) weight (g/g BW) vs. signifi cant relationship between the two (Spearman’s rho = 0.48; P <0.001). respective veh (0.0058±0.0005 and 0.0081±0.0005). Ex did not signifi cantly Finally, multiple regression analysis revealed that the association between change HbA1c or GE, and importantly had no effect on A, L, or liver weight. adipocyte apelin expression and SSPG was independent of age, BMI, gender,

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A480 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES insulin, glucose, adiponectin, and plasma apelin (p = 0.011). In conclusion, 1777-P apelin expression in adipose tissue is associated with insulin sensitivity as cGMP-Dependent Protein Kinase Cascades Regulate Muscle Mito- assessed by SSPG. This association is independent of plasma apelin levels, chondria and Prevent Obesity and Diabetes and suggests that apelin’s impact on systemic insulin sensitivity may be KAZUTOSHI MIYASHITA, KAZUWA NAKAO, HIROSHI ITOH, Tokyo, Japan, Kyoto, infl uenced by its effects in adipose tissue. Japan Supported by: NIH: 1K08DK080463 (to PY), 5R01DK071333 (to PST), 5R01DK071309 Natriuretic peptides (NP) have been characterized as vascular hormones (to GMR) which regulate vascular tone via cyclic GMP-dependent protein kinase (cGK). 1775-P Recent clinical studies have shown that plasma NP levels were lower in B-Type Natriuretic Peptide Affects the Response to Intravenous persons with the metabolic syndrome. Our study was aimed to elucidate the Glucose in a Placebo-Controlled Cross-Over Study in Healthy Volun- roles for NP/cGK cascades in energy metabolism. We used BNP transgenic teers (BNP-Tg) and cGK-Tg mice and analyzed the metabolic consequences of MICHAEL RESL, BIRGIT HEINISCH, GREISA VILA, MICHAELA RIEDL, EVELYNE chronic activation of NP/cGK cascades in vivo. We also examined the effect WOHLSCHLAEGER-KRENN, GIOVANNI PACINI, MARTIN CLODI, ANTON LUGER, of NP in cultured myocytes. BNP-Tg mice fed on high-fat diet were protected Vienna, Austria against diet-induced obesity and insulin resistance; and cGK-Tg mice had B-type natriuretic peptide (BNP) is a hormone secreted from the heart reduced body weight even on standard chow with giant mitochondria in response to volume load and serves clinically to help reduce the cardiac densely packed in the skeletal muscle. Both mice showed an increase in work load. BNP is as well a reliable biomarker in the diagnosis of cardiac muscle mitochondrial content and fat oxidation through up-regulation of dysfunction and heart failure. As patients with heart failure present an PGC-1a and PPARd. The functional NP-receptors, GCA and GCB, were down- increased risk for developing diabetes, we aimed to investigate the role regulated by feeding high-fat diet; while GCA+/- mice showed increases in of BNP on parameters of glucose metabolism in a placebo-controlled body weight and glucose intolerance when fed on high-fat diet. NP directly crossover study performed in 10 healthy volunteers (25±1 years; BMI 23±1 increased the expression of PGC-1a and PPARd and mitochondrial content in kg/m2; fasting glucose 83± mg/dl). Participants received intravenously either cultured myocytes. The fi ndings together suggest that NP/cGK cascades can placebo or 3 pmol/kg/min BNP-32 for 4h. One hour after beginning the BNP/ promote muscle mitochondrial biogenesis and fat oxidation, as to prevent placebo infusion, a 3h intravenous glucose tolerance test (0.33 g/kgglucose obesity and glucose intolerance. Thus, the vascular hormone, NP, would + 0.03 U/kg insulin at 20 min) was started. Plasma glucose, insulin and contribute to effi cient coordination of oxygen supply and utilization. C-peptide were frequently measured for minimal model analysis. Supported by: Japanese Ministry of Education, Culture, Sports, Science, and BNP increased the glucose distribution volume (13±1 %BW vs. 11±1, Technology P<0.002), leading to an overall reduction of glucose concentration (P<0.001) 1778-P especially during the initial 20 min of the test (P=0.001), accompanied by a Characterization of a Potent Glucokinase Activator with Diminished reduction of the initial C-peptide levels (4.3±0.4 ng/ml vs. 4.9±0.3, P=0.015). Risk of Hypoglycemia On the other hand, BNP had no specifi c impact on beta cell function (129±17 NICOLE BARUCCI, ALAN S. ROBERTSON, PATRICIA BOURASSA, LEVENIA vs. 124±11 pmolCP/mmolG), insulin clearance (8.7±1.1 vs. 8.3±0.7 ml/min/kg) BAKER, DAWN MATHER, DAVID G. PERREGAUX, JUDITH L. TREADWAY, JUSTIN and insulin sensitivity (10±1vs. 9±2 104min-1/(µU/ml)), all P>0.6. WALTON, KAREN ATKINSON, JOHN HELLEMBAEK, BEIJING TAN, JEFFREY A. Intravenous administration of BNP increases glucose distribution volume PFEFFERKORN, CARINE BOUSTANY, THERESA D’AQUILA, JOHN LITCHFIELD, lowering plasma glucose concentrations after a glucose load without ROBERT J. AIELLO, Groton, CT, New Haven, CT affecting beta cell function and insulin sensitivity. These results speak for Several glucokinase activators have been characterized in the clinic the concept that BNP improves diabetes in patients with heart failure, and but have been discontinued from drug development due to the risk of open otherwise new questions regarding BNP-induced positive effects on hypoglycemia. GK-1 is a novel systemic glucokinase activator designed to glucose metabolism. reduce the risk of hypoglycemia while maintaining glucose lowering effi cacy. Supported by: ÖNB Project Nr 13583 To characterize GK-1’s in vivo effi cacy, an acute oral glucose tolerance test 1776-P was performed in Sprague-Dawley rats. GK-1 dose dependently reduced the Catestatin (Human Chromogranin A352-372) Exerts Both the Insulin glucose excursion curve compared to vehicle control (∼27% AUC reduction Sensitizing and Anti-Hypertensive Effect in db/db Mice at 100mg/kg dose). Additionally, GK-1 did not lower fasting plasma glucose J I AY U R R . G AY EN, A R S H I J H A , S T EFA N O G EN T I L E, G AU TA M K . B A N DYO PA D H YAY, levels beyond the threshold of 65 mg/dL. A tissue penetration study was SUSHIL K. MAHATA, La Jolla, CA performed in rats to characterize pharmacokinetic and tissue distribution Chromogranin A (human CHGA/ mouse Chga), a 48 kDa acidic secretory properties of GK-1 in plasma, pancreas, liver, CSF and brain. Unbound proprotein, gives rise to several bioactive peptides including the cate- tissue GK-1 exposure levels in pancreas and liver were in equilibrium with cholamine release-inhibitory and anti-hypertensive peptide catestatin (CST: plasma; however brain and CSF exposure levels were 5 to 10 fold lower than human CHGA352-372). Although CHGA is overexpressed in hypertensive plasma indicating an impaired CNS penetration. Finally, we addressed the subjects, CST is diminished in hypertensive subjects. Consistent with human effect of GK-1 on counter-regulatory response (CRR) biomarkers during the fi ndings, targeted ablation of the Chga gene resulted in high blood pressure, steady state phase of a hypoglycemic clamp in which blood glucose levels higher plasma catecholamines and reactive oxygen species and decreased were maintained at 45-50mg/dL for the duration of the clamp. The glucose nitric oxide. CST replacement in Chga-KO mice normalizes both blood infusion rate increased by 2 mg/kg/min in rats treated with GK-1 at the dose pressure and plasma catecholamines, pointing to catecholamine regulation of 100 mg/kg versus vehicle control. Baseline-corrected plasma levels of of blood pressure in this genetic model. Besides the anti-adrenergic and anti- glucose, insulin, C-peptide, corticosterone, epinephrine and norepinephrine hypertensive effects, CST caused improved glucose tolerance in high fat were similar between groups at steady-state. Interestingly, plasma glucagon diet (HFD) fed obese & insulin resistant mice, which prompted us to validate levels appeared to be lower in the GK-1 treated rats versus vehicle, however this unexpected fi ndings in db/db mice, which is a model for diabetes and this was not statistically signifi cant. In conclusion, GK-1 was found to be a hypertension. We sought to establish that CST could control both glucose “brain-impermeant” glucokinase activator with glucose lowering capabilities level and blood pressure. CST was infused (5 µg/gm body wt/day) into db/ and a diminished risk of interference with the counter-regulatory response

db mice for two weeks via osmotic minipumps and then subjected to GTT. during hypoglycemia. Obesity

Consistent with HFD-diet fed mice, CST caused a 25% drop in fasting plasma POSTERS glucose level in db/db mice. Suppression of glycogenolysis by CST might have 1779-P

contributed to its insulin-sensitizing effect in db/db mice. CST infusion for 2 Circadian Control of Energy Balance by Synaptotagmin 4-Directed Integrated Physiology/ weeks resulted in ∼20% decrease in both SBP and DBP without affecting the Hypothalamic Oxytocin Release heart rate. After 4 weeks of CST infusion, the SBP and DBP were decreased GUO ZHANG, DONGSHENG CAI, Bronx, NY even further (by ∼35%) without changing the heart rate. These fi ndings Energy homeostasis and circadian rhythms are controlled primarily by indicate that CST controls blood pressure in db/db mice by improving hypothalamic arcuate nucleus and suprachiasmatic nucleus, respectively. vascular resistance, which is at least in part due to changes in NPY level. Recent research has revealed that circadian rhythmic abnormality Intriguingly, CST did not suppress the expression of gluconeogenic genes underlies obesity development; however, it remains unknown how these such as glucose-6 phosphatase or phosphoenolpyruvate-carboxy kinase. two pathological processes are mechanistically linked. By targeting the These novel fi ndings of CST may have far reaching impact in regulation of paraventricular nucleus – a hypothalamic region anatomically innervated insulin resistance and blood pressure. by both arcuate nucleus and suprachiasmatic nucleus, this study aimed to Supported by: VA Merit Award identify a neuropeptide program that might integrate the circadian and the

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A481 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

metabolic controls of appetite/body weight. We found that oxytocin release generates Ang (1-7) by degrading Ang I. Ang (1-7) mediates its vasodilatation displayed distinct circadian rhythmicity in normal mice, with hypothalamic action through a G protein-coupled receptor (Mas). In our previous studies and circulating levels of oxytocin gradually increased during the daytime and we demonstrated an age-dependent increase of blood pressure in db/db drastically declined during the nighttime. The circadian release of oxytocin type 2 diabetic mice. The goal of this study was to evaluate whether there is was found to underlie the temporal patterns of appetite in a negative an age-dependent changes in aortic ACE, ACE2 and NEP protein expression manner in normal chow-fed mice. In contrast, chronic high-fat diet (HFD) in db/db diabetic mice. Plasma ACE and ACE2 activities were measured. feeding impaired oxytocin release predominantly during the daytime, leading Western blot analysis demonstrated a signifi cant decrease in aortal ACE2 to vanished circadian rhythms of oxytocin release. This change underlied the and NEP protein expression in 12 wk hypertensive db/db mice compared to prominent promotion of appetite by HFD during the daytime rather than during controls (p<0.001). However, kidney NEP protein expression was not altered in the nighttime. To explore molecular mediators, we examined the relationship 8wk and 12wk db/db mice. In addition there was no difference in Mas receptor between oxytocin and synaptotagmin 4 (Syt4), suggested by our recent work protein expression in the kidney of 12 wk db/db mice compared to control. There showing that Syt4 is an exocytotic inhibitor of oxytocin release (Zhang et al, was a signifi cant increase in plasma Ang II and ACE activity in 8wk and 12 wk Neuron, in press). Data revealed that Syt4 localization in oxytocin vesicles db/db mice (p<0.001). In conclusion, alteration in the balance between ACE/ was >10-fold less active during the daytime than that during the nighttime. Ang II and Ang (1-7) forming enzymes (ACE2 and NEP) could contribute to the Consistently, HFD feeding elevated Syt4 localization in oxytocin vesicles development of hypertension and renal dysfunction in db/db diabetic mice. predominantly during the daytime, leading to the circadian arrhythmicity of Supported by: AHA oxytocin release. Physiological studies using Syt4 knockout mice revealed that Syt4 inhibition prevented HFD-induced circadian arrhythmicity in oxytocin 1782-P release and appetite, which accounted for the anti-obesity phenotype of Development of Non-Invasive PET Probe for Quantifying Pancreatic Syt4 knockout mice. In conclusion, Syt4-directed oxytocin release represents β-Cell Mass Using Fluorine-18-Labeled Exendin-4 a hypothalamic neuropeptide program that mediates circadian regulation/ KENTARO TOYODA, HIROYUKI KIMURA, HIROYUKI FUJIMOTO, ZHUANG dysregulation of energy and body weight balance. XIAOTONG, ERI MUKAI, YUU OGAWA, KONOMU HIRAO, HIROKAZU MATSUDA, Supported by: NIH ADA-Funded Research HIDEKAZU KAWASHIMA, MASASHI UEDA, TAKASHI TEMMA, HIDEO SAJI, NOBUYA INAGAKI, Kyoto, Japan, Osaka, Japan The volume of pancreatic β-cells is known to decrease during diabetes 1780-P development and progression. A technique for measurement of β-cell Circulating Plasma Pigment Epithelium-Derived Factor (PEDF) Levels volume in vivo is required, but a non-invasive method to detect pancreatic Are Associated with Insulin Resistance in Women with Estrogen β-cells has not been developed. We previously reported the possibility of the Defi ciency probe targeting glucagon-like peptide-1 receptors (GLP-1R). MICHELE M.A. YUEN, WING SUN CHOW, CHEN CHENG, TING CHUNG PUN, The aim of this study is whether the mass of pancreatic β-cells can be LAW RENCE S.C. LAW, ANNETTE W.K. TSO, AIMIN XU, KAREN S.L. LAM, Hong quantifi ed non-invasively by positron emission tomography (PET) targeting Kong, China GLP-1R, using fl uorine-18-labeled exendin-4 ([18F]exendin-4). Pigment epithelium-derived factor (PEDF) is implicated in murine insulin For developing PET imaging probe, the lysine residue of exendin-4 at 12 resistance and metabolic dysfunctions. Women have lower serum PEDF levels was labeled with [18F]. For evaluating receptor specifi city, binding assay in and 17β-estradiol inhibits the transcription of PEDF in human ovarian epithelial vitro was performed using dispersed cells of islets isolated from 6-week-old cells. To investigate whether estradiol regulates PEDF in vivo, we studied the male ddY mice. For assessment of in vivo selectivity, biodistribution study changes in PEDF levels in 21 pre-menopausal women (age = 49.6 ± 3.5 years) was performed by intravenous administration of [18F]exendin-4 to 6-week- following bilateral oophorectomy for benign gynaecological conditions. old male ddY mice. At appropriate time points after administration, selected Body mass index (BMI), waist circumference (WC), percentage body fat, organs and blood were harvested and their weight and radioactivities and plasma PEDF (ng/ml) and estradiol (pmol/l) levels were measured before were measured. Furthermore, [18F]exendin-4 (3MBq) was systematically and after oophorectomy. The changes (Δ) in PEDF, estradiol, homeostatic administered and its retention in pancreas was imaged by PET using model assessment of insulin resistance (HOMA-IR) and quantitative insulin- microPET system (Explore Vista, GE). sensitivity check index (QUICKI) were analyzed with repeated measure The results of binding assay analysis showed competitive inhibition ANOVA. The mean duration between pre- and post-operative assessment of specifi c [18F]exendin-4 binding by non-radioactive exendin(9-39) and was 4.0±0.7 months. BMI was marginally reduced in the post-operative its IC50 was 4.4 nmol. Biodistribution study showed that radioactivity of 2 2 period (25.2 ± 4.0 kg/m pre-op vs. 24.8 ± 4.3 kg/m post-op; p = 0.048). There pancreas was 20.4 %dose/g and highest at 60 min. The ratio of radioactivity were no signifi cant changes in WC or percentage body fat The marked post- in pancreas to that in blood, liver, intestine, and kidney were 30.9, 35.9, operative reduction in plasma estradiol levels (225 [109-418] pmol/l pre-op 8.0, 2.4, respectively. MicroPET was performed 60 min after [18F]exendin-4 vs. 32 [19-50] pmol/l post-op; p < 0.001) was associated with increases in administration to mice, which revealed the remarkable accumulation of plasma PEDF levels (7.7 ± 1.6 ng/ml pre-op vs. 8.8 ± 1.5 ng/ml post-op; p = radioactivity of [18F]exendin-4 in pancreas. 0.001). An inverse relationship was found between Δestradiol and ΔPEDF (r = These results indicated that exendin-4 could serve as a useful probe for -0.497; p = 0.022). Insulin resistance increased post-operatively, as indicated non-invasive PET imaging for the evaluation of pancreatic β-cell mass. by HOMA-IR (1.06 [0.73-1.93] pre-op vs. 1.63 [1.06-2.26] post-op; p = 0.002) Supported by: The Program for Promotion of Fundamental Studies in Health and QUICKI (0.38 ± 0.04 pre-op vs. 0.36 ± 0.03 post-op; p = 0.006). ΔHOMA- Sciences of the National Institute of Biomedical Innovation (NIBIO) IR and ΔQUICKI became insignifi cant (p = 0.101 and 0.141 respectively) after adjusting for ΔPEDF. In conclusion, the reduction in plasma estradiol levels following oophorectomy was signifi cantly associated with increases in 1783-P plasma PEDF levels and insulin resistance. Our data suggest that estradiol is Differential Effect of Sham Feeding on Meal Glucose Tolerance in a physiological regulator of PEDF expression in humans and that an increase Hypoglycemic vs. Asymptomatic Individuals after Gastric Bypass in circulating PEDF contributes to the increase in insulin resistance in these Surgery women with estrogen defi ciency following bilateral oophorectomy. MARZIEH SALEHI, AMALIA GASTALDELLI, RONALD L. PRIGEON, DAVID A.

Obesity D’ALESSIO, Cincinnati, OH, Pisa, Italy, Baltimore, MD

POSTERS Gastric bypass surgery (GB) induces diabetes remission independent of weight 1781-P loss. While the underlying mechanism for this effect is not known, nutrient- Decreased Aortic Angiotensin Converting Enzyme 2 (ACE 2) and Integrated Physiology/ stimulated β- and α-cell secretion is greater after GB compared to non-operated Neprilysin (NEP) Protein Expression in db/db Diabetic Mice subjects. Also, hyperinsulinemic hypoglycemia, a rare late-complication of HARSHITA CHODAVARAPU, NARGES KABLAN, RENDONG QUAN, KHALID GB is associated with an even more rapid and pronounced β-cell response to ELASED, Dayton, OH meal ingestion. The enhanced postprandial islet responses after GB raise the Cardiovascular disease is a long term complication of diabetes, which possibility that increased vagal activation, which controls early-phase islet remains a leading cause of morbidity and mortality. There is evidence of function, plays a central role in glucose metabolism after GB. activation of renin angiotensin system (RAS) in diabetic animals and humans. To evaluate the contribution of vagal activation by oral sensory stimulation Emerging data shows that the vasoconstrictor actions of Angiotensin II (Ang to glucose regulation, 4 patients with neuroglycopenic hypoglycemia after II) may be opposed by formation of the vasodilator, Ang (1-7). ACE2 recently GB (H) and 4 asymptomatic subjects with GB (A) (age, 43±6 vs. 47±3 yrs; BMI, was identifi ed as a homologue of ACE that preferentially forms Ang-(1-7) from 36±2 vs. 35±5 kg/m2; 4-6 vs. 4-7 yrs post-op in H vs. A, respectively) were Ang II. Neutral endopeptidase (NEP), a cell surface metallopeptidase also studied with a meal tolerance test (Ensure plus, 350kcal) on two days, one

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A482 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES control and one preceded by a sham feeding (chewing and expectorating alterations in ER stress and the UPR. Isolated rat neonatal cardiomyocytes peanut butter sandwich, 255kcal). Areas under the curves (AUC) for glucose were treated with 5mmol/L glucose (LG), 33mmol/L glucose (HG), or 33mmol/L and insulin response to meal ingestion over the premeal values were glucose+30nmol/L Ex4 (HGEx4) for 48hr. Cell death was analyzed using compared using two-way repeated ANOVA. trypan blue and TUNEL. HGEx4 attenuated HG-induced cell death (Trypan H subjects had lower nadir glucose, lower AUCglucose, and higher AUCinsulin Blue: LG=8.3±1.3, HG=24.0±1.0, HGEx4=13.5±1.5%); (TUNEL: LG=9.0±1.0, values compared to A group during control studies. Sham feeding had no effect HG=21.0±0.2, HGEx4=12.0±1.4%). Cardiomyocytes were treated with LG, on the glucose and insulin responses in the H subjects. However, in A subjects HG, or HGEx4 for 1, 6, and 12 hr and analyzed for expression of ER stress and sham feeding caused a signifi cant decrease in AUCglucose(p<0.05), and an increase UPR markers. Data here are presented as fold induction at 12hr compared to in meal stimulated AUCinsulin to the level of the H subjects (p<0.05). baseline (LG at 0 hr) and controlled to β-actin. HG increased transcript levels of the ER stress marker Glucose Regulated Protein 78 (GRP78), but HGEx4 reduced GRP78 to levels observed with LG (LG=3.2±0.2, HG=4.0±0.1; HGEx4=3.1±0.4). HGEx4 did not reduce IRE1α activation induced by HG. Increased expression of X-box Binding Protein (XBP)-1 by HG was attenuated by HGEx4 to levels comparable to those in response to LG (LG=1.4±0.1, HG=3.4±0.1, HgEx4=1.6±0.2). XBP-1 regulates the pro-apoptotic C/EBP HOmologous Protein (CHOP). HGEx4 reduced CHOP levels induced by HG (LG=5.0±0.7, HG=6.7±0.5, HGEx4=3.8±0.1). The Growth Arrest and DNA Damage-inducible 34 (GADD34) protein was quickly (1h) induced by HG. Both LG and HGEx4 resulted in a mild but delayed (12h) increase in GADD34. In sum Ex4 attenuates hyperglycemia- induced cardiomyocyte apoptosis. This is associated with a restoration of ER stress and UPR markers to expression patterns observed in low glucose conditions. This shows the potential therapeutic use of GLP1R agonists in the These fi ndings indicate that the differences in insulin secretion and meal treatment of diabetic heart disease. glucose tolerance between H and A subjects are at least partly accounted for by neural responses elicited by sham feeding. 1786-P Supported by: NIH-DK083554 (MS) Exendin-4 Increased Bone Mineral Density through Osteocyte in 1784-P Normal and Type 2 Diabetic Rat Exenatide Improves Pancreatic Beta Cell Function in a Canine Model JU-YOUNG KIM, SEONG-KYU LEE, KYUNG-JIN JO, JI-HYUN KIM, DONG-MEE of Obese Mild Type 2 Diabetes LIM, KANG -WOO LEE, KEUN-YOUNG PARK, GWANPYO KOH, BYUNG-JOON KIM, VIORICA IONUT, HUIWEN LIU, HASMIK MKRTCHYAN, DARKO STEFANOVSKI, Daejeon, Republic of Korea, Jeju, Republic of Korea ORISON WOOLCOTT, ANA VALERIA B. CASTRO, JOSIANE L. BROUSSARD, Long-term hyperglycemic condition can infl uence to bone mineral density MORVARID KABIR, MALINI IYER, RICHARD N. BERGMAN, Los Angeles, CA in type 2 diabetes. However, the relationship between osteoporosis and type Exenatide (EX) is a GLP-1 receptor agonist used in the therapy of type 2 2 diabetes is much complicated and is still debated. Glucagon-like peptide-1 diabetes. It has been shown that EX treatment results in improved glycemic (GLP-1) receptor has shown some role in the control of bone resorption in control and weight loss, but the mechanism of EX action on glucose knockout mouse study. But, the effect of GLP-1 on bone formation and the homeostasis is not completely understood. mechanism of this phenomenon remain unclear. Sclerostin, secreted from Obese insulin resistant mild type 2 diabetic dogs on a high fat hypercaloric osteocyte which may be controlled osteoblast in bone, is a potent inhibitor of diet were treated for 12 weeks with either EX (sc, 10 mcg bid; n=6) or placebo bone formation. In the present study, we have explored the effect of treatment (PB; n= 4). Body weight (BW), beta cell function and insulin sensitivity were with exendin-4 (Ex-4; 10 nmol/kg), GLP-1 receptor agonist, twice daily for 3 measured before, during and after the treatment. weeks on bone turnover factors and bone mineral density (BMD) in OLETF and As expected, with fat feeding, BW increased in the PB group (+ 0.48±0.05 SD rat. Expression of sclerostin in serum was measured by ELISA. Also, we kg) but not in the EX group (-0.32±0.09 kg). Consequently, insulin sensitivity investigated whether Ex-4 reduces sclerostin levels associated with bone was maintained, and even increased in the EX group (5.03 ± 1.1 to 5.8± 0.9 formation during normal or high-glucose (30 mmol) conditions in MLO-Y4 cells, mU/l-1*min-1) but worsened with PB (5.2±0.8 to 3.3 ±0.3 mU/l-1*min-1). osteocyte-like cell lines. Expression of sclerostin was determined by real-time EX effect on beta cell function was measured by three methods: a) RT-PCR and western blot. Ex-4 was reduced sclerostin expression during intravenous glucose tolerance test and calculation of the disposition index normal or high-glucose conditions in MLO-Y4 cells. Also, we found that Ex-4 (DI); b) stepped hyperglycemic clamp; and c) static incubation with glucose of increases BMD (fi gure A) by reduced sclerostin (fi gure B) in OLETF and SD rat. pancreatic islets isolated from EX and PB animals. EX improved islet function: We show that Ex-4 can interact with the sclerostin to promote bone formation. hyperglycemic clamp revealed an increased ability of beta cells to respond These fi ndings suggest that Ex-4 exerts osteogenic effects in normal and to prolonged hyperglycemia in the EX group (wk 12 AUC 22±9 nmol/l*min type 2 diabetes models, and might be useful as a pharmacological agent for vs. 19±7 nmol/l*min at wk 0), but not in the PB group (AUC 10±2 nmol/l*min improving the defi cient bone formation associated with type 2 diabetes. at wk 12 vs. 10±3 nmol/l*min at wk 0). Beta cell’s ability to compensate for changes in insulin sensitivity, the disposition index (DI), improved in the EX group, but declined in the PB (EX: 982±174 at wk 0 vs. 1189±211 at wk 12; PB: 917±285 at wk 0 vs. 669±127 at wk 12; p<0.001). Importantly, 12 weeks of EX treatment resulted in beta cell changes that were maintained ex vivo: static incubation with glucose of islets isolated from EX treated animals resulted in a higher insulin response than that of islets from the PB group (AUC EX 714±288 pmol/l*min vs. PB 449 ±167 pmol/l*min, p<0.05). Our data indicates that EX treatment improves in vivo beta cell function

and results in changes in the beta cell that are maintained beyond the acute Obesity

effect of the drug. POSTERS Supported by: Amylin Pharmaceuticals, Inc.

1785-P Integrated Physiology/ Exendin-4 (Ex4) Attenuates Hyperglycemia-Induced Cardiomyocyte Apoptosis in Association with Alterations in Endoplasmic Reticulum (ER) Stress and the Unfolded Protein Response (UPR) CRAIG W. YOUNCE, JULIO E. AYALA, Orlando, FL Diabetic heart disease is often associated with hyperglycemia-induced cardiomyopathy and cardiomyocyte apoptosis. Hyperglycemia-induced apoptosis is attributed to ER stress and the UPR. Activation of the glucagon- like peptide-1 receptor (GLP1R) improves cardiac function in ischemic heart failure models. We tested the hypothesis that GLP1R activation with Ex4 attenuates hyperglycemic-induced cardiomyoycte death in association with

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1787-P 1789-P Implication of Corticotropin-Releasing-Hormone on INS-1 and Pan- Involvement of Hypothalamic AMPK and Histamine on Olanzapine- creatic Islets In Vitro Induced Glucose Intolerance in Mice BARBARA LUDWIG, JANINE SCHMID, CHRISTIAN ZIEGLER, MONIKA EHRHART- MEGUMI ASATO, YOKO ISHIKAWA, HIROKO IKEDA, ATSUKO KAMEI, KENJI BORNSTEIN, STEFAN R. BORNSTEIN, Dresden, Germany ONODERA, JUNZO KAMEI, Tokyo, Japan, Kanagawa, Japan Hormonal factors, including corticotropin-releasing-hormone (CRH) and Atypical antipsychotic drugs are well known to produce metabolic glucocorticoids (GC) regulate the activity of the hypothalamic-pituitary- disturbance. Treatment with some atypical antipsychotic drugs such as adrenal (HPA) axis in response to stress. This axis is kept in balance by clozapine or olanzapine induces the impairment of glucose metabolism, the negative feedback effects of GC on the CRH synthesis and secretion which increases the risk for developing metabolic side-effects, including in the hypothalamus. Recently CRH has been identifi ed to promote β-cell weight gain, dyslipidemia, insulin resistance and hyperglycemia. We have proliferation and potentiates insulin secretion in a glucose dependent already reported that central administration of olanzapine produces glucose manner. On the other hand, GCs are referred to as diabetogenic hormones intolerance. Recent study showed that clozapine activated adenosine due to the induction of gluconeogenesis, implication on development of 5’-monophosphate-activated protein kinase (AMPK) through histamine H1 insulin resistance, effects on adipocytes and the functionally insulin- receptors in the hypothalamus. AMPK is known to sense the energy status antagonizing effects. Therefore, an imbalance of the CRH/GC system may of cells and to regulate fuel availability. In central nervous system, AMPK lead to metabolic dysfunction and diabetes. modulates feeding and systemic energy homeostasis. Thus, it is possible GC access to intracellular receptors is regulated by two isoforms that atypical antipsychotic drugs such as clozapine and olanzapine induces of 11β-hydroxysteroid dehydrogenase (11β-HSD) which catalyse the glucose intolerance by activating AMPK in the hypothalamus. Therefore, the interconversion of physiologically active GC to its inactive metabolite present study was designed to investigate the involvement of histamine and (11β-HSD-2) and vice versa (11β-HSD-1). AMPK on olanzapine-induced glucose intolerance. In the glucose CII test, In the present study we analyzed the mechanism of CRH mediated both intraperitoneally (i.p.) or intracerebroventricually (i.c.v.) administration β-cell regulation and asked if pancreatic islets respond directly to CRH by of olanzapine dose-dependently induced glucose intolerance as compared interference with 11β-HSD activity and therefore GC activity. with the vehicle-treated group. The glucose intolerance induced by i.c.v. In in vitro studies we could demonstrate that CRH and its receptor are treatment with olanzapine was attenuated by i.p. pretreatment with expressed on mRNA and protein levels in INS-1 cells, rat and human islets. histamine synthesis inhibitor, α-fl uoromethylhistidine (FMH). The glucose We also found expression of both 11β-HSD isoforms in rat and human intolerance induced by i.c.v. treatment with olanzapine was also attenuated islets. CRH exposure of islets signifi cantly decreased mRNA levels of by i.c.v. pretreatment with an AMPK inhibitor, compound C. In addition, both 11β-HSD-isoforms as measured by quantitative RT-PCR. The specifi c i.c.v. treatment with an AMPK activator, 5-aminoimidazole-4-carboxamide iso-enzyme activity was analyzed by measuring the production of active ribonucleoside (AICAR) also produced the same changes in serum glucose GC. Following CRH treatment, active GC levels were signifi cantly reduced levels as olanzapine. In the western blot test, olanzapine increased the indicating an overbalance in favour of 11β-HSD-2 activity. Stimulation with phosphorylation of AMPK in the hypothalamus, though total amount of CRH resulted in signifi cantly increased insulin secretion. Moreover, CRH- AMPK was not affected. These results indicate that both histamine and receptor activation caused a signifi cant increase of cell proliferation and AMPK in the hypothalamus are involved in the olanzapine-induced glucose reduced cell apoptosis. intolerance. Furthermore, the present study suggests that olanzapine might We suggest that CRH may not only be of signifi cance within the activate hypothalamic AMPK via histaminergic system. endocrine stress system for triggering and sustaining obesity and metabolic dysfunction, but may also play a direct role for glucose homoeostasis and 1790-P the regulation of β-cell mass. Low Estradiol Concentrations in Males with Hypogonadotrophic Hypogonadism and Type 2 Diabetes 1788-P SANDEEP DHINDSA, RICHARD FURLANETTO, MEHUL VORA, HUSAM GHANIM, Improved Glycemic Control Enhances the Incretin Effect in Patients PARESH DANDONA, Buffalo, NY, Chantilly, VA with Type 2 Diabetes One-third of men with type 2 diabetes have hypogonadotrophic hypo- ZHIBO AN, SADIA ALI, COLLEEN ROGGE, FAY HAILES, CATHY BAILEY, BRENDA gonadism(HH). It has been suggested that HH in these men may be due to WENSTRUP, BRIANNE REEDY, MARZIEH SALEHI, DAVID A. D’ALESSIO, Cincinnati, OH an increase in plasma estradiol(E2) concentrations secondary to an increase The incretin effect is impaired in type 2 diabetes, and diabetic subjects have in aromatase activity in the adipose tissue which leads to the suppression diminished responses to incretins. However, it is not clear whether the defects of hypothalamo-hypophyseal-gonadal(HHG) axis. We investigated the are specifi c for incretin stimulated insulin secretion or simply another aspect of hypothesis that plasma E2 concentrations are signifi cantly greater in type 2 generalized β-cell dysfunction. Correction of chronic hyperglycemia improves diabetic males with HH as compared to those without HH. Plasma estradiol, incretin action in animals. Further, normalization of hyperglycemia in diabetic testosterone(T), LH and sex hormone binding globulin(SHBG) concentrations patients improves the potentiation of glucose-stimulated insulin secretion were measured in fasting blood samples of 236 men with type 2 diabetes by GLP-1 and GIP. The aim of this study was to determine whether glycemic (mean age: 56±12; range:23-83 years; mean BMI: 35±7;range:17-59kg/ control specifi cally improves the incretin effect in humans. Six type 2 diabetic m2) attending a tertiary diabetes referral center. Total T was measured by subjects with moderate to poor control (age 55±3; BMI 34±2) were studied liquid chromatography tandem mass spectrometry(LC-MS/MS). In 196 men, twice using a glucose clamp-OGTT protocol before and after 8 weeks of long- total E2 was measured by an immunoassay. Free E2 and T concentrations acting insulin treatment titrated to a fasting glucose target of 6.0 mM, causing were calculated using total E2, T, albumin and SHBG. In 99 men, total E2 Hg A1C to reduce from 8.2±0.2 to 6.8±0.2 %. Following an overnight fast and was measured by the more specifi c LC-MS/MS assay. Free E2 and free T basal blood draws (-20-0 min), glucose was given intravenously (IV) to raise concentrations in these men were measured by tracer equilibrium dialysis. blood glucose by ∼6.5 mM. At 90 min 75 g of glucose was given orally, and HH was defi ned as free T<3.5 ng/dl along with LH<10 IU/L. E2 concentrations the IV glucose infusion was adjusted to maintain the blood glucose constant. between men with and without HH were compared after adjusting for The incretin effect was calculated by comparing plasma insulin levels before age and BMI differences. The calculated free E2 concentration in men

Obesity and after the oral glucose load. In the basal period, P1 (0-90 min) and P2 (90- with HH was lower than that in eugonadal men (median 0.047[0.035-

POSTERS 270 min), the blood glucose levels were 8.6±0.6, 15.1±0.8, and 16.3±1.3 mM 0.068] vs. 0.063[0.046-0.077] ng/dl, p<0.001). Directly measured free E2 at visit 1; and 5.9±0.6, 13.2±0.8, and 14.3±1.0 mM at visit 2, respectively. concentrations were also lower in men with HH (median 0.023[0.008-0.049]

Integrated Physiology/ The incremental insulin response to IV glucose (P1) increased from 0.10±0.06 vs 0.045[0.027-0.069] ng/dl, p=0.01). In multiple regression analysis, free (visit 1) to 0.16±0.08 nM (visit 2). The response to oral glucose ingestion at E2 was independently and directly related to free T(β=0.24, p=0.001) but fi xed hyperglycemia (P2) increased from 0.42±0.30 (visit 1) to 0.90±0.38 nM not to BMI(β=0.10, p=0.17) or age(β=-0.07, p=0.37). Since total and free (visit 2). After 8 weeks of treatment, the average increase of the incremental E2 concentrations are signifi cantly lower in HH males, the syndrome of HH insulin response to oral glucose was 8 fold greater than the increase to IV cannot be due to an excess of E2. The diminished E2concentration appears to glucose (p=0.2), with 5 out of 6 subjects having a 4 fold or greater increase. be a function of its precursor, T. The pathogenesis of HH in type 2 diabetes Our data support the hypothesis that intensifi ed insulin treatment to improve needs to be investigated further. ADA-Funded Research glycemic control leads to a disproportionate improvement of insulin secretion in response to oral, compared to isoglycemic IV, glucose stimulation in patients with type 2 diabetes. Supported by: VA Merit Award to D.D.

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1791-P 1793-P Mechanism Underlying Metformin-Induced Secretion of Glucagon- Overexpression of Fetuin-A in LDL-R Defi cient Mice Induces Insulin Like Peptide-1 from the Intestinal L-Cell Resistance, Dyslipidemia and Increased Atherosclerosis ANDREW J. MULHERIN, AMY H. OH, HELENA KIM, ANTHONY GRIECO, LINA M. MICHAEL LEHRKE, KATJA PIOTROWSKI, CHIRAZ EL-AOUNI, MELANIE BECKER, LAUFFER, PATRICIA L. BRUBAKER, Toronto, ON, Canada SUSANNE HELBIG, KLAUS G. PARHOFER, ULI C. BROEDL, PETER BOECKSTEGER, The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted by the CORINNA LEBHERZ, Aachen, Germany, Munich, Germany intestinal L-cell in response to both nutrient and neural stimulation, leading Fetuin A is a potent mineralization inhibitor and association studies in to enhancement of glucose-dependent insulin secretion. GLP-1 is therefore patients with end stage renal disease have been shown, that there exists a most attractive therapeutic approach for the treatment of Type 2 Diabetes a negative correlation between serum fetuin A levels and cardiovascular Mellitus. The anti-diabetic drug, metformin, has previously been shown events. Recently reports are increasing that apart from its impact on to increase circulating levels of GLP-1, although its mechanism of action mineralization fetuin is an inhibitor of the insulin receptor cascade creating is currently unknown. To elucidate this mechanism, GLP-1 secretion was insulin resistance and its well know effects on atherosclerosis development. measured in murine GLUTag, human NCI-H716, and rat FRIC L-cell cultures In this study we wanted to elucidate if there is a causal relationship between treated with metformin (5-2000µM) or AICAR (100-1000µM), activators of fetuin A and atherosclerosis development and furthermore if its effects on AMPK. Neither metformin nor AICAR directly stimulated GLP-1 secretion, glucose homoeostasis or mineralization are predominating. despite a 1.7±0.2-fold increase in AMPK phosphorylation (P<0.01, n=8). Adult Hepatic overexpression of Fetuin A was performed using adeno-asso- Wistar rats treated with metformin (300mg/kg po) and AICAR (250mg/kg ciated viral vectors. sc) showed an increase in plasma total GLP-1 over a 2h period, peaking at LDLreceptor defi cient mice were used since they develop atherosclerotic 37±9pg/ml and 29±9pg/ml (P<0.001) respectively, compared to basal (7±1 lesions on a high fat diet in a reproducible manner. Mice were studied over a pg/ml). Plasma activity of the GLP-1 degrading enzyme dipeptidylpeptidase- period of 16 weeks. During this time repeated measurements of body weight, IV was not affected by metformin treatment, further indicating metformin’s serum lipids, glucose and insulin levels were performed as well as glucose activity as a GLP-1 secretagogue. Pre-treatment with the non-specifi c and insulin tolerance tests. At day of sacrifi ce the aorta was harvested to muscarinic antagonist atropine (1mg/kg iv) decreased the area-under- determine atherosclerotic lesion size. Liver and adipose tissue was taken for curve (AUC) of metformin-induced GLP-1 secretion by 55±11% (P<0.05, further analysis. n=9). Pre-treatment with the M3 muscarinic receptor antagonist 4-DAMP Fetuin A overexpression led to a signifi cant increase in body weight (500µg/kg iv) also decreased the GLP-1 AUC by 48±8% (P<0.05), whereas compared to control animals without changes in food intake. They the antagonists pirenzepine (M1) and gallamine (M2) had no effect (n=6- furthermore developed a pro-atherogenic lipid profi le with a signifi cant 9). Furthermore, chronic subdiaphragmatic vagotomy decreased basal increase in total and non-HDL cholesterol. Glucose and insulin sensitivity secretion (7±1pg/ml vs 13±1pg/ml in sham rats, P<0.001, n=8), but did not were signifi cantly decreased in mice overexpressing fetuin A and fasting alter the GLP-1 response to metformin treatment. In contrast, pre-treatment glucose was increased. These metabolic changes fi nally led into an signifi cant with the gastrin-releasing peptide (GRP) antagonist, RC-3095 (100µg/kg sc), augmentation in atherosclerotic plaque area in fetuin A treated animals. reduced the GLP-1 response to metformin by 55±6% (P<0.01, n=6) at t=30 Hepatic overexpression of fetuin A induces a pro-atherogenic phenotype min. In summary, metformin increases plasma GLP-1 secretion in vivo but not with insulin resistance, weight gain and dyslipidemia. Further studies are in vitro, independent of DPP-IV inhibition, through a mechanism involving the underway to elucidate the underlying pathways and to determine the parasympathetic nervous system and GRP, but not the vagus nerve. morphology of the atherosclerotic plaques. Supported by: Canadian Diabetes Association 1794-P 1792-P Pigment Epithelium-Derived Factor (PEDF) Is Associated with BMI Osteocalcin and Residual β-Cell Function in Type 1 Diabetes and Vitamin D in Adolescents and Young Adults with and without ANNA RITA MAURIZI, NICOLA NAPOLI, ROCKY STROLLO, ANGELO LAURIA, Diabetes SILVIA MANFRINI, ANN SCHWATZ, PAOLO POZZILLI, Rome, Italy, San Francisco, CA REBECCA J. BROWN, HUSSAM J. ALAMRI, GABRIELLA AITCHESON, MARY The skeleton is an important target of diabetes complications. The WALTER, KRISTINA I. ROTHER, Bethesda, MD relationship between type 1 diabetes (T1D) and bone metabolism appears to Pigment epithelium-derived factor (PEDF) is elevated in adults with type be complex and the mechanisms leading to bone loss in TD1 remain unclear. 1 diabetes (T1D) with microvascular complications and in type 2 diabetes It is reported that not only poor glycemic control has negative effects on (T2D). Little is known about PEDF in young patients with diabetes, prior to bone metabolism, but also the rate of bone turnover may in turn regulate the development of complications. glucose homeostasis. We measured fasting serum PEDF in 48 healthy subjects, 11 patients with Aim of the present study was the evaluation of the relationship between T1D, and 11 patients with T2D, ages 12-25 years, and studied its correlation markers of bone formation [osteocalcin (OC), bone alkaline phosphatase with metabolic risk factors. Subjects with diabetes had no micro- or (bALP)], bone mineral density (BMD) and body composition] and metabolic macrovascular complications. Serial PEDF after a 75g glucose load was control in T1D patients. A total of 45 patients affected by T1D aged 18- measured in 12 subjects. 50 years (29 males 16 females) and 37 age and gender-matched healthy subjects underwent assessment of biochemical bone formation markers (OC and bALP), BMD and measurement of body composition using dual energy X-ray absorptiometry. In T1D patients Hb1Ac, fasting C-peptide and daily insulin requirement (I.R.) were also evaluated. A two-sides paired T-test was used to assess differences between patients with T1D and controls. Pearson’s correlation coeffi cients were used for assessing the association between two continuous variables. No differences were observed in OC and bALP levels between the two groups. T1D patients and controls showed

also similar fat mass and BMD at all sites. In T1D patients fat distribution, Obesity

expressed as abdominal fat/total fat ratio, was positively correlated with PEDF levels were highest in T2D and lowest in T1D (Figure A, Unadjusted POSTERS HbA1c levels (r=0.5, p=0.032) and with I.R. (r=0.4, p=0.02). Finally, a positive PEDF, p= 0.0015). PEDF was positively correlated with BMI, C-peptide, HOMA-

correlation between osteocalcin and fasting C-peptide was observed in IR, leptin, C reactive protein, and systolic blood pressure, and negatively Integrated Physiology/ T1D patients (r=0.5, p<0.001). In conclusion, patients affected by T1D show correlated with adiponectin and Vitamin D. On multivariate analysis, BMI minor differences in body composition and no difference in BMD compared (p=<0.0001, β=182.3) and Vitamin D (p=0.014, β=-86.5) remained signifi cant with normal subjects. The novel fi nding of a positive correlation between predictors of PEDF. After BMI and Vitamin D adjustment, diagnosis was no osteocalcin and residual C-peptide observed in T1D is consistent with longer a signifi cant predictor of PEDF (Figure A, Adjusted PEDF, p=0.21). experimental models, suggesting a direct effect of this osteoblast-derived In subjects who underwent serial testing, PEDF levels showed a gradual protein on β-cell function. decrease after the glucose load, (Figure B, *p<0.05 relative to baseline). In young patients with diabetes, PEDF was indistinguishable from healthy controls after adjusting for covariates; thus increases in PEDF appear to be related to vascular complications. The positive correlation between PEDF and BMI has been observed in older cohorts, and is likely due to secretion of

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A485 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

PEDF by adipocytes. The negative correlation between Vitamin D and PEDF is novel, and may be related to inverse correlations of Vitamin D and PEDF with metabolic risk factors. Further prospective studies are needed to determine the chronology and clinical importance of PEDF changes in diabetes.

1795-P Potential Gastrointestinal Mechanisms for the Anti-Diabetic Effect of Metformin LIHONG CHEN, CHARI D. SMITH, YAPING LIU, MAGGIE S. MCINTYRE, DANA P. DANGER, JUDI A. MCNULTY, TYMISSHA D. JACKSON, SEAN A. ROSS, JEAN- LOUIS D. KLEIN, JAMES M. WAY, CHRISTOPHER C. NYSTROM, DALLAS K. CROOM, DONALD I. ANDERSON, ALAN J. CUNNINGHAM, ANDREW A. YOUNG, Research Triangle Park, NC Metformin is a fi rst-line oral treatment for type 2 diabetes mellitus. Its many reported mechanisms of action include increasing the sensitivity of liver, muscle, fat, and other tissues to insulin. Despite ample evidence for the effi cacy of orally-administered metformin, there are no clinical reports of an antidiabetic effect of parenterally-administered metformin. Its action may involve gastrointestinal (GI) targets where concentrations are high at effective doses (up to 2500 mg in humans). We evaluated its effects on the GI mechanisms, sodium-glucose co-transport (SGLT1) and apical sodium- 1797-P dependent bile transport (ASBT), each of which exhibits antidiabetic effect Serum Fibroblast Growth Factor 21 Is Associated with the Fasting in animal models. Uptake of labeled glucose and labeled taurocholate into Plasma Glucose, γ-Glutamyltransferase, and Presence of Diabetic cells respectively expressing human SGLT1 and human ASBT was assessed Retinopathy in Type 2 Diabetes in vitro at different concentrations of metformin. It could dose-dependently EUN KYOUNG KIM, MIN SUK LEE, JIYEONG KWAK, YONG JUN CHOI, EUN SUK and fully inhibit SGLT1 and ASBT, respectively, and was effective on those HA, SUNG-E. CHOI, SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, YUP KANG, tranporters at concentrations estimated to prevail within the GI lumen at KWAN-WOO LEE, Suwon, Republic of Korea effective antidiabetic doses. Acute oral administration of metformin at Fibroblast growth factor (FGF) 21, a hormone secreted primarily by the doses that chronically correct diabetes in Zucker Diabetic Fatty (ZDF) rats liver, has benefi cial effects on glucose and lipid metabolism in animal (300mg/kg b.i.d.) signifi cantly decreased post-challenge glucose excursions models. However, its role in the pathogenesis of type 2 diabetes in humans in both ZDF and normal rats, consistent with an effect on glucose transport. remains to be defi ned. This study quantifi ed the circulating plasma FGF-21 Acute in vivo studies indicated that metformin could dose-dependently levels and examined their relationship with the fasting plasma glucose inhibit ileal bile salt reuptake, and increase fecal concentrations up to 3-fold. level, insulin resistance, and metabolic biomarkers in subjects with varying The chronic antidiabetic effects of metformin, manifest as dose-dependent degrees of obesity and glucose tolerance. The serum FGF21 levels were reductions in non-fasting glucose and HbA1c in ZDF rats, were associated determined by enzyme-linked immunosorbent assays in 58 normal glucose with an enhanced GLP-1 response in nutrient challenges. In summary, the tolerance (NGT), 22 impaired glucose tolerance (IGT), and 143 type 2 diabetic glucose-lowering effi cacy of metformin in preclinical diabetic models could subjects, and their associations with parameters of adiposity, glucose, and reside, at least partly, in the direct and/or indirect consequences of its lipid profi les and levels of liver injury markers were studied. In the type 2 luminal effects to inhibit SGLT1 and/or ASBT. diabetic subgroup, the association with serum FGF21 and the presence of diabetes complications was investigated. The serum FGF21 levels increased 1796-P progressively from 168.3 ± 138.1 to 199.1 ± 175.0 to 216.8 ± 228.9 pg/ml in NGT, IGT, and type 2 diabetic subjects, respectively. The FGF21 levels Prolonged Initial Glucagon Response in Patients with Type 2 Dia- correlated positively with the fasting plasma glucose (r = 0.187, p = 0.028) betes Following a Mixed Meal and -glutamyltransferase (r = 0.346, p = 0.001) levels in type 2 diabetics. MARJAN ALSSEMA, JOSINA M. RIJKELIJKHUIZEN, ELISABETH M. EEKHOFF, γ The serum FGF21 levels were signifi cantly higher in type 2 diabetics with LEEN M. ‘T HART, GIEL NIJPELS, JACQUELINE M. DEKKER, Amsterdam, The Nether- diabetic retinopathy (371.1 ± 515.6 vs. 199.5 ± 193.2 pg/ml; p = 0.002) and lands, Leiden, The Netherlands carotid artery plaques (260.2 ± 283.0 vs. 157.0 ± 118.2 pg/ml; p = 0.015) than As a pancreatic hormone, glucagon promotes the conversion of liver in those without either condition. The FGF21 levels correlated positively with glycogen into glucose when plasma glucose levels decrease. Inappropriate the urine albumin-creatinine ratio (r = 0.244, p = 0.011) in type 2 diabetics. suppression of glucagon after oral glucose has been described for type 2 The serum FGF21 levels are signifi cantly associated with the fasting plasma diabetes patients. The effect of a physiological mixed meal stimulus on glucose, -glutamyltransferase, and presence of diabetic retinopathy in type glucagon profi les as compared to oral glucose are largely unknown. γ 2 diabetes. In a population-based study, 21 persons with and 182 without type 2 diabetes received a standardized mixed meal (75 g carbohydrates, 50 g fat and 24 g proteins) and an oral glucose tolerance test (75 g glucose) on 1798-P separate occasions. Glucagon profi les up to t=120 minutes (oral glucose Serum Levels of Adipocyte Fatty Acid-Binding Protein Are Associated tolerance test) and t=240 minutes (mixed meal test) were measured by with the Severity of Coronary Artery Disease in Chinese Women radioimmunoassay, after extraction. Time-dependent glucagon profi les MI ZHOU, YUQIAN BAO, ZHIGANG LU, HUATING LI, YE WANG, MEIFANG GAO, were analysed by linear mixed models. MENG WEI, WEIPING JIA, Shanghai, China Persons with diabetes had higher mean levels of glucagon in the fasting Background: Adipocyte fatty acid-binding protein (A-FABP) has been state (13.9 pmol/l versus 9.8 pmol/l, p<0.001) than persons without. Glucagon described as a novel adipokine, playing an important role in the development

Obesity levels were suppressed following the oral glucose load, with stronger of metabolic syndrome, type 2 diabetes and atherosclerosis. In this study,

POSTERS suppression at t=120 after glucose intake in type 2 diabetes as compared we investigated the relationship between serum levels of A-FABP and the to persons without diabetes (p-value interaction=0.06). After the mixed presence and severity of coronary artery disease (CAD) in Chinese subjects.

Integrated Physiology/ meal, levels of glucagon initially increased and than stabilized (no diabetes) Methods: Circulating A-FABP level was determined by enzyme-linked or decreased (diabetes). The initial rise in glucagon at t=30 was stronger in immunosorbent assay in 341 Chinese subjects (221 men, 120 women) who patients with diabetes as was the later decrease in glucagon levels at t=180 underwent coronary angiography. According to the number of diseased (p-value for interaction<0.01). vessels, CAD patients were further divided into 1-vessel and multi-vessel Glucagon levels decrease after oral glucose, but increase after a mixed disease groups. The extent and severity of CAD was assessed by assigning meal. Following a mixed meal, the initial increase in glucagon is stronger points to each lesion, and the coronary atherosclerosis index (CAI) was and prolonged in patients with diabetes, which may contribute to glucose defi ned as the sum of these scores. intolerance in type 2 diabetes patients. Results: Of the 341 subjects, 94 were without CAD (46 men, 48 women), Supported by: Unrestricted Investigator Initiated grant from Merck & Co. Inc while 247 suffered from CAD (175 men, 72 women). A-FABP levels in patients with CAD were signifi cantly higher compared with non-CAD subjects (P=0.029 in men; P=0.031 in women).

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A486 INTEGRATED PHYSIOLOGY—OTHERCATEGORY HORMONES

metformin (N=16) or combination of sitagliptin and metformin (N=15) for one week. Serum concentrations of total and active ghrelin were determined in all subjects immediately before and 2 hours after meal challenge. Same testes were repeated among patients with diabetes after receiving drug therapy for a week. Results demonstrated that postprandial active ghrelin was more signifi cantly suppressed in patients with diabetes than in non- diabetic controls. Maximum suppression was seen after patients were put on treatment and after meal challenge. Active ghrelin was more signifi cantly decreased than total ghrelin in diabetic patients (by 36%, p<0.001). In patients taking sitagliptin, total ghrelin (means±SEM) fell from 382±60 pg/ ml at baseline to 345±58 pg/ml (p>0.05) whereas active ghrelin decreased from 140±26 pg/ml to 85±12 pg/ml (p<0.01). A similar drop was observed in all medication groups. There was no statistical difference in ghrelin levels In multiple logistic regression analysis, serum A-FABP was an independent between the three treatment groups. Active ghrelin concentration correlated risk factor for CAD in women (OR=12.078, P=0.031). Serum A-FABP increased negatively with BMI in diabetic patients (P<0.05). In conclusion: sitagliptin signifi cantly in multi-vessel diseased patients than in non-CAD subjects and metformin either alone or in combination suppressed active ghrelin (P=0.011 in men, P=0.004 in women), and showed a positive correlation more signifi cantly in patients with diabetes when compared to healthy with CAI (r=0.134, P=0.032) after adjustment for age and gender. In addition, controls. Postprandial ghrelin suppression was strongest after medication amino terminal pro-brain natriuretic peptide (NT-proBNP) was demonstrated and meal challenge. The result of this study warrants further investigation of to be positively and independently correlated with A-FABP in all subjects the signifi cance of ghrelin suppression in patients with diabetes. (β=0.075, P=0.014). Conclusions: Serum A-FABP is closely associated with the presence and severity of CAD in Chinese women. A-FABP levels are also associated with 1801-P the circulating levels of NT-proBNP. Synergism by Individual Macronutrients Explains the Marked Incretin Supported by: Chinese National 973 Project (2007CB914702) and Islet Hormone Response to Mixed Meal—Exploration of a Novel Experimental Tool, the Oral Meal Tolerance Test in Mice 1799-P LINDA HANSSON, JENNY VIKMAN, GIOVANNI PACINI, BO AHRÉN, Lund, Sweden, SIRT1-Mediated Regulation of FGF21 Expression in Lean Mice Probed Padova, Italy with Small Molecule Activators and Genetic SIRT1 Deletion The glucagon-like peptide-1 (GLP-1) and insulin responses to oral glucose ANGELA M. COTE, MARC O. JOHNSON, KRISTINE STEARNS, MEGHAN L. DAVIS, have been well characterised. Fatty acids and proteins also stimulate GLP- DAVID J. GAGNE, MARIE YEAGER, JAMES L. ELLIS, VIPIN SURI, GEORGE P. 1 and insulin secretion, however, the relative contribution of the macro- VLASUK, Cambridge, MA nutrients to the GLP-1 and insulin response to mixed meal is not known. The endocrine hormone Fibroblast Growth Factor 21 (FGF21) has broad We therefore explored a novel tool - the oral meal tolerance test (MTT) in metabolic actions including weight loss, increased insulin sensitivity mice - to evaluate the differential macronutrient impact on GLP-1 and insulin and reduction of triglycerides in rodent models of obesity and metabolic responses to mixed meal. dysfunction. FGF21 is induced in mouse liver by prolonged fasting, a Anesthetized C57BL/6J mice were orally administered either a mixed ketogenic diet, and by agonists of peroxisome proliferator activated meal consisting of glucose, whey protein and peanut oil with total caloric receptor alpha (PPARa). The protein deacetylase SIRT1 also modulates FGF21 load 0.285 kcal or single macronutrients in the same quantity as in the mixed expression as hepatocytes defi cient in SIRT1 show reduced PPARa induced meal, i.e., 0.17 kcal glucose, 0.055 kcal whey protein or 0.055 kcal peanut oil. FGF21 expression while modest overexpression of SIRT1 increases FGF21 For comparison, a test with glucose alone at same caloric load as mixed meal expression in hepatocytes. However, the mechanism of SIRT1-mediated (0.285 kcal) was given. Glucose, insulin and intact GLP-1 were measured in regulation of FGF21 secretion in mice has not been fully explored. blood collected before and at specifi c time points after oral challenge. To further understand the role of SIRT1 in mice, we analyzed the effect of The early (15 min) insulin response to MTT was substantially higher genetic and pharmacological manipulation of SIRT1 activity on the induction (2.5±0.3 nM) than the response to the single macronutrients (887±140 pM of FGF21 by fasting as well as by two specifi c ligands of PPARa. A 24 hour fast for glucose, 118±45 pM for whey protein and 46±27 pM for peanut oil), in increased FGF21 expression in liver as well as serum FGF21 levels by 8-10 fold. fact signifi cantly higher than the added responses for the three individual Oral administration of a specifi c small molecule SIRT1 activator during fasting macronutrients (P<0.001). The early insulin response to MTT was higher also further enhanced FGF21 serum levels by about two-fold. Administration of when compared to the matched caloric load with glucose alone (1.5±0.3 nM; PPARa ligands WY14643 or GW7647 to ad lib fed mice also resulted in a dose P=0.02). Furthermore, whereas MTT signifi cantly increased plasma GLP-1, dependent increase in hepatic FGF21 expression as well as serum FGF21 levels no such increase was observed by the individual macronutrients. of up to 60 fold over vehicle treated ad lib fed mice. Concomitant administration This novel approach to explore incretin and insulin secretion in mice of a sub-maximal dose of PPARa agonists WY14643 or GW7647 and a SIRT1 revealed that the marked early insulin response to mixed meal emanates from activator to ad lib fed mice signifi cantly increased serum FGF21 levels beyond a synergistic, rather than an additive effect of the macronutrients and this is those induced by the PPARa agonist alone. most likely caused by increased GLP-1 secretion. Hence, superior to glucose, We also analyzed the effects of fasting or PPARa agonists on FGF-21 the MTT offers a physiological tool for exploration of incretin and islet hormone induction in SIRT1 conditional knockout mice. In contrast to the positive secretion in studies on integrative metabolism and drug development. action of SIRT1 activator on FGF-21 induction, genetic deletion of SIRT1 signifi cantly muted induction of FGF21 expression in liver as well as serum 1802-P FGF-21 levels. Taken together these observations suggest a key role for System Level Analysis of the Use of Alpha Cell Inhibitors (ACI) To Repair SIRT1 in the regulation of FGF-21 levels. In addition, these observations Defective Glucagon Counterregulation (GCR) in Insulin Defi ciency suggest that regulation of FGF-21 levels may contribute to the metabolic LEON S. FARHY, ANTHONY L. MCCALL, Charlottesville, VA GCR is a key protection against hypoglycemia impaired in diabetes. Our benefi ts of genetic or pharmacological SIRT1 activation. Obesity

recent studies suggest that partial glucagon inhibition may improve GCR. POSTERS 1800-P Here, we continue the analysis of the use of ACI to repair defective GCR using our mathematical model of the pancreatic network in which α-cells

Sitagliptin Suppresses Ghrelin Hormone in Patients with Diabetes Integrated Physiology/ BERHANE SEYOUM, ALEMU FITE, ABDUL B. ABOU-SAMRA, Detroit, MI are suppressed by glucose, β-cells, and auto-feedback. We have shown that Ghrelin is an appetite-stimulating hormone mainly produced by the stomach. this construct precisely reconstructs the GCR control mechanism and its Circulating levels of ghrelin concentration increase in fasting states and fall impairment in diabetes. following meal. Sitagliptin is an orally available new class of anti-diabetic We estimated the improvement in defective GCR caused by constant drug that inhibits dipeptidyl peptidase-4 (DPP-4) enzyme leading to 2-3 fold infusions of ACI depending on the extent to which they inhibit the feedback- increase in the serum concentration of endogenous glucagon-like peptide-1 independent (basal) and feedback-regulated (pulsatile) glucagon (FIG and FRG, (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This study respectively). To this end, we tabulated the predicted hypoglycemia-stimulated was performed to determine the effects of sitagliptin on circulating levels glucagon release assuming graded levels of FIG and FRG inhibition by ACI. of ghrelin. Control subjects (N=15) and 46 diabetic patients were recruited Restoring GCR requires >40% reduction of FIG, but <80% suppression of FRG. for the study. The diabetic patients were treated with sitagliptin (N=15), 40% reduction in both FIG and FRG restores the GCR response to hypoglycemia,

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A487 OBESITY—ANIMALCATEGORY

but glucagon remains inappropriately elevated during eu- and hyperglycemia. glucose load were measure in 97 patients with NGT, 73 patients with IGT and Normalization of glucagon requires reduction of FIG accompanied by only minor 48 patients with T2DM. Correlation coeffi cients between aldosterone and FRG suppression. Table 1 summarizes the impact of ACI on the fold increase in HOMA-IR were studied according to Pearson analysis. Levels of serum and glucagon in response to hypoglycemia assuming 0% to 100% reduction in FIG urine aldosterone and PRA were compared in these three groups of patients. (by rows) and FRG (by columns). Values >7 (bold) indicate restoration to levels The infl uential factors of insulin resistance were screened by logistic regression similar to the normal pancreas (vs. 1.6 in insulin defi ciency). analysis.The correlation coeffi cient index of U-ald and fasting serum insulin FIG↓/FRG→ 0% 20% 40% 60% 80% 100% concentration was 0.211 (P<0.01), whereas the correlation coeffi cient index of serum aldosterone concentration and Log-HOMA-IR was 0.133 (P =0.05). 0% 1.6 1.5 1.4 1.4 1.3 1 The infl uential factors of insulin resistance were BMI and U-ald according 20% 4.3 2.8 2 1.6 1.5 1 to the Logistic regression analysis, and the OR values were 8.497 (95%CI: 40% 8.6 7.5 6 3 1.9 1 3.403-21.219, P<0.001) and 3.480 (95%CI: 1.016-11.966, P=0.047) respectively. 60% 13.3 11.6 9.8 7.7 3.1 1 The patients with T2DM got the highest level of U-ald and PRA compared to the patients with NGT and IGT. The IGT group got the lowest of level of PRA. 80% 18.9 17.3 15.6 13.1 7.2 1 (3) The correlation coeffi cient index of U-ald and BMI was 0.185 (P<0.01). The 100% 28.8 28.8 28.8 27.2 28.3 patients with higher BMI got higher U-ald than the patients with lower BMI. In conclusion, repair of defective GCR is possible by ACI that suppress The whole day urine aldosterone excretion is the independent risk factor of preferentially the basal rather than the pulsatile glucagon release. Since insulin resistance and closely related with BMI. The patients with T2DM got deconvolution of hormone time series can distinguish between these two the highest level of U-ald and PRA. The study indicated the association of secretory components our results suggest a hypothesis to test experimentally RAAS system, esp.aldosterone, and insulin resistance. that ACI with mostly basal suppression may be used as a protection against hypoglycemia in insulin defi ciency. OBESITY—ANIMAL Supported by: NIDDK grant R01 DK082805 1803-P [See also: Presidents Posters 462-PP to 465-PP, page A128.] The Relationships of Pancreatic Polypeptide with Aging, Obesity and Diabetes ZHIKE CHEN, ZHUO LIU, CHEE W. CHIA, OLGA D. CARLSON, JOSEPHINE M. EGAN, Guided Audio Tour: Animal Models for the Study of Obesity (Posters 1805-P Baltimore, MD to 1812-P), see page 11. Insulin, glucagon, pancreatic polypeptide (PP), somatostatin and ghrelin & 1805-P are secreted from fi ve distinct cells in islets of Langerhans. While the Loss of Hypothalamic Autophagic Activity Mediates the Central regulation of insulin secretion and its dysregulation in type 2 diabetes Induction of Obesity have been extensively investigated, the other four hormones are often over QINGYUAN MENG, DONGSHENG CAI, Bronx, NY shadowed. Plasma glucagon levels are elevated in type 2 diabetes and are Autophagy is an important player for maintaining cellular homeostasis. implicated in the pathogenesis of the elevated fasting glucose levels. PP Recent research has suggested that defective hepatic autophagy contributes has been largely ignored. We hypothesized that PP secretion might also be to obesity-associated insulin de-sensitization, and upregulation of auto- dysregulated in type 2 diabetes and insulin resistance states. The purpose phagic activity in the liver counteracts peripheral insulin resistance. Our of this study was to investigate the relationships between PP plasma levels research investigated whether hypothalamic autophagy was involved in with aging, obesity and diabetes in the fasting state and after oral glucose the neural control of feeding and body weight balance. First, we performed tolerance test (OGTT). immunostaining of a key autophagic mediator, autophagy-related gene-7 Using data collected from the Baltimore Longitudinal Study of Aging (ATG-7), and found that ATG-7 was abundantly expressed in the mediobasal (BLSA), we found that in non-diabetic healthy subjects the plasma PP levels hypothalamus (MBH). Detailed examination revealed that ATG-7 was present were signifi cantly elevated in aged subjects (80-90 years old) compared with mainly in neurons but barely detectable in glial cells (such as astrocytes) of young subjects (30-40 years old) in the fasting state (50.2±7.6 vs. 30.6±5.0 this region. Then, we explored if changes in hypothalamic autophagic activity pM, p<0.05). The 2 hours post-OGTT area under curve (AUC) of plasma PP might underlie the development of obesity under high-fat diet (HFD) feeding levels was signifi cantly elevated in aged subjects compared with young condition. Compared to normal chow feeding, chronic HFD feeding signifi cantly subjects (5972.0±628.0 vs. 3877.4±508.4, p<0.05). Most interestingly, we decreased hypothalamic ATG-7 mRNA levels and the ratio of LC3-II to LC3-I found that AUC of plasma PP levels was negatively correlated with BMI mRNA expression. Subsequently, we knocked down ATG-7 gene in the MBH (range 18 to 45) in non-diabetic healthy subjects with beta = -0.3, p = 0.017 of normal chow-fed mice via intra-MBH injections of ATG-7 shRNA or control after adjusting for age. We also found that in type 2 diabetic subjects the lentiviruses. Despite the normal chow feeding which continued following viral plasma PP levels were signifi cantly elevated compared with non-diabetic injections, mice with MBH-directed ATG-7 knockdown developed a moderate subjects in both the fasting state (47.7±6.3 vs. 28.8±3.2 pM, p<0.05) and 2 overweight/obesity phenotype (weight gain: control 0.29±0.08 vs. ATG-7 hours post-OGTT (61.6±9.7 vs. 27.9±3.3 pM, p<0.01). The 2 hours post-OGTT shRNA 0.61±0.12 g/week, P<0.05). The obesity phenotype of these mice was AUC of plasma PP levels was elevated in diabetic subjects compared with verifi ed by MRI scanning which confi rmed an increase in fat mass but not lean non-diabetic subjects (7032.5±1029.2 vs. 4123.2±419.0, p<0.01). mass. The obesity-prone phenotype was attributed to overeating (average We conclude that the plasma PP levels increased as a factor of aging and food intake: control 4.17±0.14 vs. ATG-7 shRNA 4.79±0.20 g/day, P<0.05) and the diabetic state, but not as a result of obesity. Further investigation of the impaired energy expenditure (O2 consumption: control 4.4±0.18 vs. ATG-7 function of PP may contribute to better understanding of the pathogenesis shRNA 3.69±0.10 L/kg/h, P<0.05). Interestingly, ablation of JNK1 using JNK1 of diabetes and even aging. knockout mice prevented the obesogenic effect of delivering ATG-7 shRNA Supported by: NIH Intramural Research Program of the NIA lentiviruses in the MBH. This observation suggested that JNK1 represents an upstream molecule that inhibits hypothalamic autophay to affect body weight 1804-P balance. In conclusion, loss of autophagic capacity in the hypothalamus directs

Obesity energy and body weight imbalance. The Serum Aldosterone Concentration and Plasma Renin Activity in POSTERS Supported by: NIH Populations with Different Glucose Tolerance Status & 1806-P SHAO LING ZHANG, GUO SHU YIN, MU CHAO WU, MING TONG XU, FENG LI, LI

Integrated Physiology/ Bariatric Surgery Regulates Tissue Specifi c Infl ammation in High YAN, HUA CHENG, Guangzhou, China, Shantou, China Fat Diet-Induced Obese (DIO) Mice The relationship between aldosterone and glucose metabolism is poorly QIANG GAO, LIAN LI MA, WENWEI YAN, PHILLIP E. WILLIAMS, DAVID H. understood. The aims of this study was to evaluate the relationship of serum WASSERMAN, NAJI N. ABUMRAD, DENG PING YIN, Nashville, TN aldosterone with insulin resistance by comparing serum aldosterone level Visceral fat deposition induces chronic low grade infl ammatory reactions, in different glucose tolerant status.In this study, 233 patients with normal causing insulin resistance. However, recent results show that surgical glucose tolerance (NGT), 109 patients with impaired of glucose tolerance removal of visceral fat fails to reverse metabolic disease, and intrahepatic (IGT) and 112 patients with T2DM were recruited. Serum aldosterone fat, not visceral fat, is linked with metabolic complications of obesity. We concentration, urine aldosterone excretion for 24 hours (U-ald) and plasma have developed mouse bariatric surgery models, including gastric banding, renin activity (PRA) were measured in all of the participants. Fasting plasma sleeve gastrectomy (SG), Roux-en-Y gastric bypass (RYGB) and biliopancreatic glucose concentration and plasma glucose concentration after glucose load, diversion (BPD). The results showed that SG and RYGB represent reliable fasting serum insulin concentration and serum insulin concentration after 75g

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A488 OBESITY—ANIMALCATEGORY restrictive and GI bypass models, respectively. By using SG and RYGB mouse elicits anorectic effects at doses comparable to GLP-1. Some actions of OXM, models, we tested whether the effects of bariatric surgery in the treatment for example inhibition of gastric acid secretion and stimulation of heart rate, of obesity and insulin resistance are associated with the regulation of are independent of GLP1R. Because glucagon decreases food intake in rodents infl ammation in visceral adipose tissue (VAT) and liver in DIO mice. SG and humans, we examined whether the chronic effect of OXM on food intake induced weight loss, reduced fat mass and improved glucose tolerance in and body weight is distinct from that obtained with an equipotent GLP1R- glucose tolerance tests within 4 wks, but the effects were not completely selective agonist in mice and investigated whether any such differential sustained. However, RYGB resulted in a sustained prevention of weight gain effects were mediated by GCGR activation. We identifi ed an equipotent and improved liver steatosis and insulin resistance through 12 wks. Both RYGB GLP1R-selective peptide agonist that differs from OXM by only one residue and SG inhibited CD11b macrophages and Th1 (CD3+/IFNg+) subsets in liver by (Gln3→Glu, OXMQ3E), but has no signifi cant GCGR agonist activity in vitro. -60% and -42%, respectively, as well as in VAT (-85% and -64%, respectively) First we demonstrated that chronic treatment of diet-induced obese mice and enhanced Th2 (CD3+/IL-10+) subsets in liver (+100%), but not in VAT at 7 with OXM impacts superior body weight loss compared to similar treatment days post-surgery. RYGB, but not SG, suppressed phosphorylation of IRS- with equimolar doses of OXMQ3E. Next, a chronic pair-feeding study revealed 1/Ser307, inhibited (-47%) M1 (CD11c) macrophage polarization, promoted that the superior weight loss effi cacy of OXM was due to increased energy (+78.5%) M2 (CD206) macrophage polarization and enhanced (+100%) CD3+/ expenditure and inhibition of food intake. Finally, co-administration of OXM FoxP3+ regulatory T cells in the liver. RYGB, but not SG, reduced circulating and a glucagon receptor antagonist abolished the incremental body weight insulin (-85%), leptin (-45%) and IL-6 (-64%) by one week post-surgery. Our lowering effect of OXM compared to OXMQ3E. Our data provide new insights study suggests that SG induces weight loss and improves glucose tolerance in into the mechanism of action of OXM and reveals that activation of GCGR is the short term; however, RYGB persistently improves insulin resistance. Early involved in the chronic body weight-lowering action of OXM. positive effects of RYGB and SG in the improvements of insulin resistance appear to be modulated by the inhibition of infl ammation in liver and VAT. & 1809-P RYGB specifi cally inhibits infl ammatory M1 macrophages, enhances M2 Catestatin (Human Chromogranin A352-372) May Improve Obesity macrophages and promotes regulatory T cell subsets in the liver. through Lipid Mobilization Supported by: NIH grants to D.W. and N.A., JDRF grant to D.Y. JIAYUR R. GAYEN, HOWON LEE, SUSHIL K. MAHATA, GAUTAM K. BANDYO- PADHYAY, La Jolla, CA, San Diego, CA & 1807-P Catestatin (CST), a proteolytically cleaved peptide from the proprotein Depletion of α/β T Cells Attenuates Obesity-Associated Infl amma- Chromogranin A (human CHGA/mouse Chga), is secreted from endocrine tion and Metabolic Abnormalities and neuroendocrine glands. By virtue of its potent antagonism to nicotine- ILVIRA M. KHAN, XIAOYUAN PERRARD, JERRY PERRARD, AMIR MANSOORI, C. evoked catecholamine actions, CST has been proposed to act through WAYNE SMITH, HUAIZHU WU, CHRISTIE M. BALLANTYNE, Houston, TX the modulation of the nicotinic-acetylcholine receptor (nAChR) and the Recent investigations have linked obesity with low-grade chronic adrenergic receptors (ADR). In the present investigation, we found that infl ammation in adipose tissue (AT), which causes AT dysfunction, thus CST stimulated secretion of adiponectin from 3T3-L1 cells and completely contributing to obesity associated metabolic abnormalities. Diet-induced blocked adiponectin secretion induced by isoproterenolol (ADRB agonist). obesity is correlated with accumulation of “classically activated” (M1) While activation of ADRA by phenylephrine was ineffective in inducing macrophages in AT, which secret proinfl ammatory cytokines and impair adiponectin secretion it potentiated CST-induced adiponectin secretion by insulin signaling and lipid storage in adipocytes. In addition, obesity alters T 4 fold, implicating a synergistic interaction between CST and ADRA. Lack cell numbers in AT. However, the role of T cells in metabolic complications of CST in Chga knockout (Chga-KO) mice resulted in an increase of adipose of obesity is less defi ned. Using mouse model of diet-induced obesity, we tissue mass, which was decreased after CST infusion, pointing to a role of found that both αβ and γδ T cells reside in perigonadal AT of lean mice. CST in the regulation of fat deposition and obesity. CST administration to However, only αβ T cells were signifi cantly increased in AT of obese mice Chga-KO mice (5 mg/g body weight/day for 10 days) though reduced fat mass, (25.05 + 4.38%) compared with lean controls (8.89 + 3.60%, n=7/group, enhanced glycerol and NEFA (non-esterifi ed fatty acid) release (both by 50% P<0.0001). Therefore, we studied the role of αβT cells in AT infl ammation compared to saline treated control), it decreased molar ratio of the NEFA to and metabolic dysfunctions using TCRβ-null mice defi cient in α/β T cells, glycerol in the released products. Whether oxidation or re-esterifi cation of with C57BL/6J as WT controls. Obesity was induced by high fat diet (HFD, NEFA played a role in low NEFA to Glycerol ratio in the released products 21% fat) feeding with mice on normal diet (5% fat) as lean controls. TCRβ- was evaluated by injecting 1-14C-palmitate into Chga-KO mice and analyzing null mice gained similar amount of body weight (43.92 + 3.77g) and fat mass its incorporation and oxidation. CST augmented fatty acid oxidation (by 4-5 (43.45 + 6.60% fat) as WT (46.91 + 4.2g body weight and 45.03 + 2.98% fold) in both tissues and moderately stimulated (by 2-fold) incorporation into fat). Nevertheless, we observed lower fasting glucose levels and improved liver lipids without stimulating incorporation into adipose tissue lipids. CST- glucose and insulin tolerance in obese TCRβ-null mice compared to obese stimulated adipokine production from adipocytes might have promoted fatty WT. Compared to obese WT, obese TCRβ-null mice had reduced levels acid oxidation through activation of a G-protein linked AMPK pathway. Gene of IFN-γ, a Th1 cytokine, and decreased M1 macrophage contents with expression studies revealed CST upregulation of genes that facilitated fatty concomitant decreases in levels of cytokines (TNF-α) and chemokines (MCP- oxidation such as, PPARalpha, ACOX, UCP2 and CPT1, and downregulation of 1, RANTES) in AT, indicating that HFD-induced AT infl ammation was blunted lipogenic genes, SREBP1c and FAS. We conclude that CST plays a crucial role in the absence of α/β T cells. Obese TCRβ-null mice also had signifi cant in the regulation of obesity through its infl uence on lipid mobilization. reduction in triglyceride content in skeletal muscle (109.8 + 17.12 mg/g tissue Supported by: VA Merit Grant in TCRβ-null mice vs 130.9 + 29.69 in WT, n=13/group, P<0.05). In conclusion, αβ T cells, which are increased in AT with obesity, play an essential role in & 1810-P obesity-associated AT infl ammation and metabolic complications. Adipose Tissue-Specifi c Suppression of c-JUN Terminal Kinase Supported by: R01DK078847 to C.M.B. R01HL098839 to H.W. Attenuates Obesity-Related Metabolic Disorders in a Genetic Mouse Model of Obesity and Type 2 Diabetes CHENG CHEN, XINMEI ZHANG, RACHEL L.C. WONG, SOOKJA CHUNG, AIMIN & 1808-P XU, KAREN S.L. LAM, Hong Kong, China The Glucagon Receptor Is Involved in Mediating the Body Weight Mice with genetic or diet-induced obesity have markedly increased Obesity Lowering Effects of Oxyntomodulin activity of c-Jun NH2-terminal kinase (JNK), a key regulator of infl ammatory POSTERS JENNIFER KOSINSKI, JAMES HUBERT, PAUL CARRINGTON, JAMES MU, responses, in their liver, muscle and adipose tissue (AT). To determine the ELISABETTA BIANCHI, ANTONELLO PESSI, RANABIR SINHAROY, DONALD MARSH, role of JNK in adipose tissue infl ammation and insulin sensitivity, we have Integrated Physiology/ ALESSANDRO POCAI, Rahway, NJ, Pomezia, Rome, Italy, Rome, Pomezia, Italy previously generated a transgenic mouse model that expresses a dominant Oxyntomodulin (OXM) is a peptide secreted postprandially from the L-cells negative form of JNK (dn-JNK) under the transcriptional control of the ap2 of the gut that has a weak affi nity for both the GLP-1 receptor (GLP1R) and gene promoter, leading to the selective inactivation of JNK1 and JNK2 in the glucagon receptor (GCGR). Peripheral administration of OXM in humans AT. In this study, we investigated whether selective AT JNK inactivation and rodents causes weight loss reducing food intake and increasing energy could ameliorate the development of metabolic dysfunctions in db/db mice, expenditure. Because the acute anorectic effects of OXM are abolished a mouse model of genetic obesity and type 2 diabetes. We cross-bred the in GLP1R-defi cient mice or by co-administration of the GLP1R antagonist ap2-(dn)-JNK(+/-) mice with C57 BKS db/db(+/-) mice to obtain db/db(-/-)/ exendin(9–39), it has been suggested that OXM modulates energy intake ap2-dn-JNK(-/-) mice as controls, and db/db mice with AT overexpression solely through weak GLP1R agonism. OXM shows 10- to 100-fold reduced of dn-JNK: db/db(-/-)/ap2-dn-JNK(+/-). The db/db(-/-)/ap2-dn-JNK(+/-) potency compared with the cognate ligands GLP-1 and glucagon. However, it transgenic mice had reduced body weight gain from week 5 of age (p<0.05

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A489 OBESITY—ANIMALCATEGORY

versus controls, despite similar intake), which may be attributed in part to and 129S1/SvImJ (129/Jax) and a closely related strain of 129 mice, 129S6/ an increased locomotor activity (p<0.05 versus controls during dark cycle) SvEvTac (129/Tac) from Taconic Farms. Upon high fat feeding, B6 mice became and greater energy expenditure and lipid utilization, as refl ected by a higher obese and insulin-resistant, while 129/Tac mice became obese but retained oxygen consumption rate (VO2) and lower respiratory exchange ratio (RER, normal insulin sensitivity and 129/Jax mice were protected against both VCO2/VO2), compared to controls. The transgenic mice were less insulin obesity and insulin resistance. To study the composition of gut microbiota, we resistant and had lower glucose levels during intraperitoneal GTT (p<0.05 performed quantitative real-time PCR analysis of bacterial DNA of the feces of versus controls). Furthermore, JNK inactivation in the db/db mice led to the 3 strains of mice. This revealed profound differences in the relative amount reduced adipocyte size in both white and brown AT, a lesser degree of of bacterial species with 129/Tac mice having higher levels of Bacteroidetes AT macrophage infi ltration and hepatic steatosis, and reduced circulating than 129/Jax mice, and both strains of 129 having much higher levels of levels of A-FABP (p<0.01 versus controls), in keeping with the JNK-mediated Lactobacillus species than the B6 mice. To “normalize” of the environmental regulation of A-FABP expression. In conclusion, the selective suppression factors, infl uencing gut microbiota, we established our own inbred colonies of of JNK activation in AT alone was suffi cient to attenuate the development the B6, 129/Jax and 129/Tac mice. This produced a profound remodeling of the of obesity-induced metabolic disturbances, even in the context of a genetic gut microbiota of all three mice lines when compared to the original. Moreover, predisposition to obesity due to leptin resistance. following reprogramming, the 129/Tac mice lost their susceptibility to diet- Supported by: Hong Kong Research Council (GRF 767208M) induced obesity and became very similar to the obesity resistant 129/Jax mice. These changes in obesity susceptibility in 129/Tac mice were associated & 1811-P with a major increase in Bacteriodes species and a decrease in Firmicutes. Inactivation of the Fto Gene in the Adult Mouse Thus, the genetic background and the original gut microbiome are important FIONA MCMURRAY, CHRIS D. CHURCH, FRANCES M. ASHCROFT, ROGER D. COX, infl uences in the susceptibility of mice to diet-induced obesity, which can be Harwell, United Kingdom, Oxford, United Kingdom reprogrammed by exposing mice to new environmental factors. In 2007 a genome wide association study, originally for type 2 diabetes, linked SNPs in intron 1 of FTO with an increased body mass index. The Guided Audio Tour: Animal Models in Obesity Research (Posters 1813-P to 1820-P), see page 11. individuals with the ‘risk’ allele are on average 3 kg heavier than those with the ‘protective’ allele. Mouse models have been identifi ed and generated & 1813-P including a conditional knockout allele, which is lean when globally Increased GLP-1 Secretion in VSG Is Not Due to Rapid Gastric Empty ing expressed, and a conditional overexpression allele of FTO, which has DARLEEN A. SANDOVAL, ADAM CHAMBERS, JOYCE SORRELL, ALFOR LEWIS, increased body weight when globally expressed. The results from these and BRIANNE REEDY, MARGARET STEFATER, HILARY WILSON-PEREZ, STEVE C. other studies suggest that the SNPs in FTO lead to weight gain by increasing WOODS, DAVID D’ALESSIO, RANDY J. SEELEY, Cincinnati, OH the activity and/or expression levels of FTO. Vertical sleeve gastrectomy (VSG), a surgery where 80% of the stomach We are utilising the conditional knockout to globally inactivate Fto in the along the greater curvature is removed, causes sustained weight loss in adult mouse. This has been achieved by crossing the fl oxed Fto knockout human and rodent models and improves glucose tolerance similar to roux-en-Y allele to a tamoxifen inducible ubiquitous Cre line. Male mice were either gastric bypass (RYGB). In both weight loss surgeries there is a substantial not treated, receive tamoxifen (200mg/kg) or an equivalent amount of increase in circulating levels of the incretin, glucagon like peptide-1 (GLP-1). vehicle at 6 weeks by oral gavage for 5 days. Three weeks after tamoxifen The increase in GLP-1 with RYGB has been attributed to rapid passage of administration recombination of the fl oxed Fto allele was observed together nutrients through the gastro-jejunostomy into the L-cell rich part of the gut. with signifi cantly decreased Fto mRNA levels, and decreased FTO protein in However, with VSG there is no intestinal manipulation and the mechanism liver and brain. for increased GLP-1 secretion is unknown. Previous studies in humans have The effect of inactivating FTO in the adult causes signifi cantly reduced demonstrated that rapid delivery of glucose to the upper gut stimulates weight gain following tamoxifen treatment (P=0.0217, 6-12 weeks repeated release of GLP-1. To test whether this mechanism might be operative in measures 2 way ANOVA). This is statistically signifi cant from 8 weeks when VSG, dietary-induced obese male Long Evans rats were implanted with a compared to vehicle controls (P=0.035, pair wise t-test). duodenal catheter immediately after VSG (n=12) or sham (n=12) surgery. At least one month after surgeries, 16h fasted rats were either infused with 2.5ml of 25% dextrose at 0.4 ml/min or, one week later, were gavaged with the same volume and concentration of dextrose. Peak glucose levels after the gavage were comparable, but glycemia was signifi cantly lower in VSG vs. sham animals at 45 and 60 minutes, consistent with increased glucose clearance. Similarly, after the dextrose infusion at 0.4ml/min, glucose levels were signifi cantly lower at all time points in VSG vs. sham. Interestingly, active GLP-1 levels were signifi cantly higher in VSG vs. sham animals after both the gavage and duodenal infusion. Since SGLT1 has been suggested to be related to GLP-1, duodenal SGLT1 expression was determined in separate groups of ad lib fed rats with VSG or sham surgery and was found to be signifi cantly greater in VSG vs. sham animals. These data suggest that that differences in SGLT1 expression but not potential acute differences in gastric emptying play a role in increased GLP-1 secretion after VSG. Supported by: NIH NIDDK (DK075365) and Ethicon Endo Surgery The aim was to test whether loss of FTO in the adult would have an effect on body weight and composition, and to overcome postnatal growth & 1814-P retardation and decreased survival seen previously. Inactivation of FTO in ASP1941, a Novel SGLT2-Selective Inhibitor, Exerts Anti-Obesity the adult caused reduced weight gain, and this ongoing experiment will yield Effects by Increasing Fatty Acid Oxidation in Diet-Induced Obesity more data about the phenotype. We have demonstrated so far that FTO Rats Obesity TOSHIYUKI TAKASU, YUKA HAYASHIZAKI, KEISUKE MITSUOKA, MASANORI

POSTERS could be a pharmacological target for the treatment of common obesity. Supported by: MRC and Wellcome Trust YOKONO, RUMI KIHARA, SOUSUKE MIYOSHI, SHIGERU YOSHIDA, EIJI KURO- SAKI, Ibaraki, Japan Integrated Physiology/ & 1812-P ASP1941 is a novel, selective sodium-dependent glucose co-transporter 2 Environmental Remodeling of the Gut Microbiome Can Erase Differ- (SGLT2) inhibitor that is currently under clinical development for the treatment ences between Inbred Mice in Susceptibility to Diet-Induced Obesity of patients with type 2 diabetes mellitus. In this study, the anti-obesity effects OLIVIER BEZY, GRAHAM SMITH, MICHAEL ROURK, C.RONALD KAHN, Boston, MA of ASP1941 were investigated in diet-induced obesity (DIO) rats treated for 4 Alterations in gut microbiota by dietary and environmentally acquired weeks with ASP1941 (1–10 mg/kg once daily). Plasma and urine parameters, bacteria, such as increased Firmicutes and decreased Bacteroidetes, have and indirect calorimetry were measured. Furthermore, body composition and been implicated in the development of obesity and altered energy balance in abdominal fat mass (visceral and subcutaneous) were analyzed using dual- mice and humans. In the present study, we have assessed the importance of energy X-ray absorptiometry and computed tomography, respectively. genetic background and environmental factors on gut microbiome composition ASP1941 dose-dependently suppressed an increase in body weight (70.9 and its infl uence on differences in susceptibility to diet-induced obesity. To do ± 3.9 g vs 109.3 ± 5.1 g in vehicle group) and epididymal fat mass (10.6 ± 0.9 so, we studied two common mice lines from Jax laboratories, C57BL/6J (B6) g vs 13.4 ± 0.6 g in vehicle group), and statistic signifi cance was observed in

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A490 OBESITY—ANIMALCATEGORY the ASP1941 10 mg/kg group (P<0.05). ASP1941 treatment dose-dependently Caspase-1-/- animals were protected against the development of high fat increased fasting plasma non-esterized fatty acid (1.54 ± 0.12 eEq/L vs 1.04 ± diet induced obesity. Despite a similar caloric intake, HFD-feeding of wild- 0.10 eEq/L in vehicle group) and 3-hydroxy butyrate (2273 ± 397 μmol/L type mice led to higher plasma insulin, leptin and resistin levels as compared vs 899 ± 92 μmol/L in vehicle group). Indirect calorimetry showed that the to caspase-1-/- animals. Importantly, caspase-1-/- animals were resistant to respiratory ratio was signifi cantly lowered by ASP1941 (0.87 ± 0.01 vs 0.77 ± 0.01 the development of insulin resistance. Protection against obesity was not in vehicle group; P<0.001). ASP1941 decreased heat production rate (HPR) caused by a decrease in intestinal TG uptake in caspase-1-/- animals, as from glucose (6.5 ± 2.4 cal/min vs 27.6 ± 2.7 cal/min in vehicle group), but assessed by gavage of glycerol tri[3H]oleate-labeled olive oil after Triton increased HPR from fat (35.6 ± 0.3 cal/min vs 18.2 ± 1.5 in vehicle group). WR1339 injection. In contrast, the VLDL-TG production was decreased by Although lean body mass was not affected signifi cantly by ASP1941, both -49% (P<0.01) in the absence of caspase-1 together with an impairment in visceral fat mass and subcutaneous fat mass were signifi cantly less than VLDL-TG clearance. Finally, metabolic cage studies revealed an enhancement those of vehicle group in the ASP1941 10 mg/kg group (visceral 74.8% of in total energy expenditure in the absence of caspase-1. vehicle and subcutaneous 78% of vehicle; P<0.05 in both cases). In conclusion, absence of caspase-1 protects against the development In conclusion, these results show that ASP1941 promoted the utilization of diet-induced obesity that may partly be mediated through effects on of fatty acids instead of glucose as an energy source, thereby reducing both energy expenditure and a reduced fl ux of triglycerides from the liver towards visceral and subcutaneous fat mass, and without affecting lean body mass adipose tissue. We envision that inhibition of caspase-1 may be a useful in DIO rats. therapeutic strategy for treatment of obesity. & 1815-P Supported by: Dutch Diabetes Research Foundation Disturbed Diurnal Rhythm and High Fat Diet Additively Deteriorate Insulin Sensitivity & 1817-P CLAUDIA P. COOMANS, SJOERD A A. VAN DEN BERG, THIJS HOUBEN, JO- Interaction between Infl ammatory, Adiposity Negative Feedback, HANNES A. ROMIJN, KO WILLEMS VAN DIJK, NIENKE R. BIERMASZ, JOHANNA and Satiety Signals at the Onset of High-Fat Feeding H. MEIJER, Leiden, The Netherlands, Amsterdam, The Netherlands KELLY A. ROGERS, RICHARD L. PRINTZ, KEVIN D. NISWENDER, Nashville, TN Disturbances of circadian rhythm are associated with increased incidence A number of biochemical mechanisms relating the development of of obesity and type 2 diabetes. By disturbing circadian rhythm using constant neuronal insulin and leptin resistance with accompanying weight and fat light, we examined the causal relation between disturbed rhythm and insulin mass gain have been reported after prolonged high-fat (HF) feeding (8- sensitivity. 16wks), yet fat mass accrues rapidly in rodents (11% gain by day 2). We Mice were subjected to 12-h/12-h light/dark cycle (LD) or continuous hypothesized that neuronal insulin and leptin resistance are primary to the light (LL) for 5 weeks during chow and high fat feeding (45 energy% lard). development of diet-induced obesity (DIO), will therefore occur early, and Behavioral activity was monitored using infrared motion detectors. For 4 may result from infl ammatory signaling via activation of Inhibitor of kappa B days, energy metabolism, food intake and activity were studied by indirect kinase β (IKKβ). Hypothalamic insulin and leptin sensitivity were assessed calorimetry. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic relative to low-fat (LF) fed controls by intracerebroventricular (icv) injection clamp at the start of the active phase and at the start of the resting phase. of insulin (10mU) or leptin (3µg) in Long-Evans rats fed a HF diet for 2 and Body composition was assessed by DEXA scan. 7 days. We observed that hypothalamic insulin and leptin resistance were In chow-fed mice, LL disturbed behavioral circadian rhythmicity as mechanistically and temporally uncoupled at the onset of HF DIO with leptin evidenced by periodogram analysis (rhythm strength -58%,P<0.01). The resistance associated with IKKβ activation, detected at day 2 of HF diet, circadian pattern in feeding and activity as observed in LD mice was lost and prior to insulin resistance (day 7). However, IKKβ activation and leptin in LL mice. Total energy intake and activity were not different between LL sensitivity were restored to control levels at day 7. Central inhibition of IKK and LD mice, resulting in similar body weights and composition at week 5. (PS-1145, 10μg icv) had no effect on food intake in LF fed rats, but reduced LD mice showed circadian variation of insulin sensitivity, with lowest insulin 24h food intake in HF fed rats at day 2 (80±7 vs 22±6 kcal, p<0.001), but sensitivity in the resting phase compared to the active phase (glucose not day 7. As hypothalamic insulin and leptin action are thought to sensitize infusion rate (GIR) -45%,P<0.01). This diurnal rhythm of insulin sensitivity hindbrain to the satiety factor cholecystokinin (CCK), we assessed the was lost in LL mice, resulting in signifi cantly decreased insulin sensitivity ability of intraperitoneal CCK (0.5µg) to modulate HF food intake. CCK failed compared to LD mice in the active phase. to reduce 30min food intake in the presence of leptin resistance and IKKβ In high-fat fed mice, circadian rhythmicity was disturbed by LL (rhythm activation (day 2 of HF diet), but was effective despite insulin resistance strength -49%,P<0.01), without affecting body weight. High-fat diet induced (day 7 of HF diet). Pretreatment with IKK inhibitor (3μg icv) on day 2 also insulin resistance evidenced by lower GIR compared to chow diet (GIR restored sensitivity to CCK (85% vs 7% reduction in food intake) and the -59%,P<0.01) in LD mice. In LL mice, insulin resistance induced by high fat ability of icv leptin to activate Stat3. These data suggest that in early DIO 1) feeding was further aggravated compared to LD mice in the active phase. hypothalamic leptin resistance may be the primary determinant of increased In conclusion, continuous light disrupts the normal rhythm present in adiposity upon exposure to HF diet, 2) IKKβ inhibition normalizes HF diet energy metabolism and insulin sensitivity. High-fat diet aggravates insulin induced food intake, and 3) a functional interaction exists between IKKβ, resistance induced by continuous light. These data indicate an additive leptin action and the food intake lowering effects of CCK. effect of high fat diet and disturbed diurnal rhythms in the development of Supported by: DK085712 insulin resistance. The data are important to humans suffering from rhythm disturbances, such as elderly and shift workers. & 1818-P & 1816-P Macrophage, Its Signifi cance in Regulating Energy Homeostasis Absence of Caspase-1 Protects Against Diet-Induced Obesity and BONGGI LEE, LIPING QIAO, BRICE KINNEY, JUSTIN LEVAUGHN, CHEUNG WAI, Insulin Resistance: Identifi cation of Underlying Mechanisms ROBERT MAK, ESTA STERNECK, JIANHUA SHAO, San Diego, CA, Lexington, KY, RINKE STIENSTRA, JANNA A. VAN DIEPEN, CEES J. TACK, PATRICK C.N. Frederick, MD RENSEN, LEO A.B. JOOSTEN, MIHAI G. NETEA, THIRUMALA-DEVI KANNEGANTI, Macrophages present in almost all tissues and are important in tissue Nijmegen, The Netherlands, Leiden, The Netherlands, Memphis, TN development. Obesity associates with increased macrophage infi ltration in The world-wide epidemic of obesity has accelerated the number of white adipose and other tissues. However, whether residential macrophages Obesity individuals diagnosed with type 2 diabetes. Obesity is characterized by directly regulate energy metabolism, particularly under the physiological POSTERS an enlargement of adipose tissue mass that promotes the development of conditions, are largely unknown. CCAAT/enhancer-binding proteins (C/ a chronic low grade infl ammation. Obesity-induced infl ammation leads to EBPs) are highly expressed in macrophages and play an important role in Integrated Physiology/ enhanced production of numerous pro-infl ammatory mediators including IL- macrophage activation. Our study showed that the expression of C/EBPβ 18 and IL-1β that may cause insulin resistance. In order to become active, is remarkably elevated in peritoneal macrophages from diet-induced obese both IL-18 and IL-1β are processed by the cysteine protease caspase-1. C57BL/6 mice. We created macrophage-specifi c C/EBPβ knockout (MβKO) Previously, we have shown that caspase-1 is activated in adipose tissue mice using Cre-Loxp approach to study the role of C/EBPβ in macrophage of obese animals. Therefore, the aim of this study was to test whether activation and the regulatory effects of intact macrophage on energy caspase-1 contributes to the development of obesity and insulin resistance metabolism. C/EBPβ protein was barely detected in macrophages from and to identify possible underlying mechanisms. MβKO mice, with no signifi cant compensational increase of C/EBPα. Similar Both wild-type and caspase-1-/- animals were fed a high fat diet to induce to systemic C/EBPβ knockout mice, body size and fat mass of MβKO mice obesity. Metabolic cage studies, hyperinsulinemic-euglycemic clamps and were signifi cantly smaller than wild type (WT) mice. However, percent mechanistic studies on triglyceride (TG) metabolism were performed. of body fat of MβKO mice was signifi cantly higher than WT mice, which

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A491 OBESITY—ANIMALCATEGORY

indicates that MβKO mice have increased adiposity. Signifi cantly elevated tolerance tests (area under curve) up to 8 wks after surgery (compared serum leptin levels were observed in MβKO mice, despite the fact that with pair-fed sham-treated mice, 993±189 vs 1856±169 at 1 wk, p<0.001; their hematological parameters, serum insulin and glucagon were within 937±154 vs 1927±269 at 2 wks, p<0.01; 1564±224 vs 2108±186 at 4 wks, p< normal ranges. Leptin-suppressed food intake and body weight reduction 0.05; and 1348±137 vs 2074±243 at 8 wks, p<0.01). In older diabetic BKS-db in MβKO mice were signifi cantly less than WT mice, which further suggest mice, RYGB did not reverse diabetes (blood glucose levels, ≥400mg/dl), and that knocking out C/EBPβ in macrophages impairs leptin sensitivity in mice. immunohistochemical examination showed a reduction of insulin+ cells in The expression of key lipogenic genes was signifi cantly increased in livers pancreatic islets. Similarly, RYGB did not reverse STZ-induced diabetes in and white fats of MβKO mice. On the other hand, the levels of F4/80 and MIP-Luc mice (blood glucose > 400mg/dl, n=6) that had reduced β-cell mass other macrophage markers were remarkably decreased in white fat, liver evidenced by reduced islet bioluminescence. These results indicate that and skeletal muscle of chow or high fat diet-fed MβKO mice. Using bone RYGB effectively prevents the progression to diabetes if performed prior to marrow-derived macrophages, we found the expression of alternative onset of severe β-cell damage by a mechanism independent of reduction of activated macrophage markers were signifi cantly decreased in C/EBPβ body weight or fat mass. defi cient macrophages at basal and after IL-4 induction. These results Supported by: NIH grant R01 DK050277 and DK059637 to D.W., R01 DK070860 to indicate that 1) C/EBPβ plays an important role in macrophage activation N.A., JDRF grant 1-2008-159 to D.Y. and tissue infi ltration; 2) residential macrophages may actively regulate energy metabolism. 1821-P Supported by: NIDDK ADA-Funded Research AICAR Reverses the Effects of Adiponectin Defi ciency Related Kidney Disease & 1819-P ANNA V. MATHEW, ANNE-EMILEE DECLEVES, SHINICHI OKADA, MARILYN Sterol Regulatory Element Binding Protein-1c (SREBP-1c) Is Differ- FARQUHAR, KUMAR SHARMA, La Jolla, CA ent ly Regulated by TNF-Related Apoptosis Inducing Ligand (TRAIL) Obesity related morbidity including kidney disease is on the rise. in High-Fat Diet Induced Obese Mice Adiponectin is secreted by adipocytes and is negatively correlated with SO-YOUNG PARK, MI-KYOUNG PARK, YING HAN, SU KYUNG PARK, DUK KYU obesity and albuminuria. In our studies we have identifi ed that adiponectin KIM, HYE-JEONG LEE, Busan, Republic of Korea knockout mice are associated with proteinuria TRAIL is a member of TNF family of cytokines, which exist either as type II We planned to further characterize the effects of adiponectin defi ciency membrane or as a soluble protein. Although the well-characterized activity of and AMPK stimulation in young adiponectin KO mice. We studied kidney TRAIL is represented by its anti-cancer activity, little is known regarding the sections of adiponectin knockout mice and adiponectin heterozygote mice effects of TRAIL on metabolic pathways. To address this point, we studied using electron microscopy. Adiponectin knockout mice at one and two in vivo effects of TRAIL in high-fat diet induced obese mice. SREBP-1c is a month had foot process effacement and the heterozygotes did not reveal transcription factor which is synthesized as a precursor in the membranes of any visible changes. The degree of effacement was quantifi ed using ratios the endoplasmic reticulum and which requires post-translational modifi cation of cumulative foot process basal plate width maintaining contact with the to yield its transcriptionally active nuclear form. SREBP-1c induces the glomerular basement membrane to fi xed length of the glomerular basement expression of a family of genes involved in glucose utilization and fatty membrane. acid synthesis. C57BL/6 Mice were classifi ed into two groups and fed with ZO1 immunofl uorescence staining of adiponectin knockout mice kidney the normal (ND) and high-fat diet (HFD) for up to 22 weeks. All mice of the at one and two month of age reveal diffuse and diminished ZO1 staining high-fat diet group developed obesity and type 2 diabetes. Seven days after of glomeruli when compared to age matched controls which demonstrate a adenoviral-mediated hTRAIL (ad.hTRAIL) and control GFP (ad.GFP) delivery more linear staining pattern. The staining pattern is linear in heterozygous to each groups, mice were killed and samples were collected and analyzed. mice and in KO mice treated with AICAR. The expression of ad.hTRAIL was determined by western blotting with liver We designed an experiment with 3 groups of 2 month old mice (n=10 tissue. Overexpression of ad.hTRAIL signifi cantly decreased accumulation of each); Wild type mice (WT-S) treated with 0.2 normal saline i.p. for 7 days; hepatic lipid and plasma lipid profi les in HFD mice. The expression of hepatic Adiponectin KO mice (KO-S) treated with 0.2 normal saline i.p. for 7 days and SREBP-1c was increased by ad.hTRAIL in ND mice, compared with ad.GFP. Adiponectin KO mice (KO-A) treated with 0.5 gm/gm body weight of AICAR The expression of SREBP-1c was increased in HFD mice, compared with i.p. for 7 days. We found that the kidney and heart of the KO-S group weighed ND mice as expected by high lipogenic activity. However, the expression of signifi cantly higher in when compared to the WT-S group; this difference SREBP-1c was decreased by ad.hTRAIL in HFD mice, compared with ad.GFP normalized with AICAR treatment. We also measured the concentration of in HFD mice. The target genes of SREBP-1c, fatty acid synthase, acetyl CoA phosphorylated AMPK (PAMPK) in kidney tissue of the 3 groups and found carboxylase, and stearoyl CoA desaturase 1 were altered signifi cantly by the PAMPK (p=0.011) and PAMPK/protein ratio (p=0.012) was decreased in the changes of the expression of SREBP-1c. Taken together, TRAIL alters the KO-S when compared to the WT-S; this recovers with AICAR treatment. the expression of lipogenic genes through SREBP-1c and the expression These studies help us understand the effects of adiponectin in disease of SREBP-1c is differently regulated by TRAIL in response to high-fat diet. states with the adiponectin knockout model and delineate the pathways These fi ndings suggest that TRAIL might have a novel function for lipid that connect adiponectin, AMPK and ZO1 localization in podocytes. These metabolism via SREBP-1c related mechanism, modulating lipogenesis in pathways might open novel targets of therapy in ameliorating these disease lipid-rich environment and improving hepatic steatosis. processes. Supported by: U01, ADA (K.S.) & 1820-P Roux-en-Y Gastric Bypass Prevents the Progression to Diabetes in 1822-P Obese Mice Antisense Reduction of FGFR4 Expression: A Novel Therapeutic DENG-PING YIN, QIANG GAO, LIAN-LI MA, PHILLIP E. WILLIAMS, OWEN MC- Approach for Obesity GUINNESS, ALVIN C. POWERS, DAVID H. WASSERMAN, NAJI N. ABUMRAD, XING XIAN YU, PRASAD MANCHEM, LYNNETTA M. WATTS, JUAN RAMIREZ, Nashville, TN KAUSHIK CHAKRAVARTY, BRETT P. MONIA, SANJAY BHANOT, MICHAEL L. MC-

Obesity Obesity is associated with insulin resistance and increased risk of CALEB, Carlsbad, CA

POSTERS developing type 2 diabetes (T2D). C57BLKS-db/db (BKS-db) mice, a model of We recently reported that antisense reduction of FGFR4 expression insulin resistance, develop diabetes with aging. We tested whether Roux- lowered adiposity and improved insulin sensitivity and that it also

Integrated Physiology/ en-Y gastric bypass (RYGB), an effective therapy for morbid obesity and T2D augmented appetite suppressant-induced anti-obesity effect in rodents (Yu in humans, can prevent disease progression in young pre-diabetic (6-7 wks et al, Diabetes, Suppl 1, A83, 2009 & A474, 2010). To expand these fi ndings, old, n=6) and/or reverse diabetes in older diabetic BKS-db mice (14-15 wks we evaluated the anti-obesity effect of FGFR4 inhibition in DIO mice with old, n=4), as well as in wild-type STZ-induced diabetic mice (FVB) expressing caloric restriction (CR). Specifi cally, DIO C57BL/J mice fed a 58% fat diet ad luciferase under the control of the mouse insulin promoter (MIP-Luc, n=6). lib were treated with saline, a control (CTL) ASO or FGFR4 ASO (R4 ASO) at All BKS-db mice that underwent RYGB were compared to pair-fed BKS- 25 mg/kg twice weekly. After 2 wk of treatment (Rx), they were switched to db mice that underwent a sham surgery (n=4/group). In the young BKS-db CR by providing 95% of the amount of food consumed daily by R4 ASO group mice, RYGB and pair-fed sham-treated groups experienced similar weight during the fi rst 2 wk of Rx. The Rxs were continued for another 4 or 6 wk. In loss and gain and changes in fat mass throughout the fi rst eight wks. RYGB both studies, R4 ASO reduced liver FGFR4 mRNA levels by 75-80% whereas in young BKS-db mice maintained normoglycemia (blood glucose levels < CTL ASO had no effect. CR signifi cantly decreased both BW and body fat 200mg/dl) and improved glucose tolerance during intraperitoneal glucose content (BF) of saline-treated group with CR (saline-CR) as compared to

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A492 OBESITY—ANIMALCATEGORY saline-treated group fed ad lib. CTL ASO did not change BW or BF versus energy) induced impaired glucose tolerance when analyzed by the intra- saline-CR. However, R4 ASO further lowered BW by 7-11% and BF by 23- peritoneal glucose tolerance test in mice. COMT-inhibitor treatment for last 25%. In addition, R4 ASO reduced epididymal fat pad wt by 16-22% and 1 week displayed the exacerbation of glucose tolerance defects and insulin peri-renal fat pad wt by 29-33% without effect on lean mass. As expected, resistance in mice with a high-fat diet. Such impaired glucose tolerance CR signifi cantly reduced whole body VO2. In contrast, R4 ASO prevented the was associated with the hepatosteatosis, decreased liver glycogen level decline in VO2. In separate studies, R4 ASO Rx of lean C57BL/J and CD-1 associated with HIF-1α and macrophage accumulation in epididymal fat. A mice for 3 mo resulted in > 75% reduction in liver FGFR4 mRNA and was well signifi cant angiogenesis in the mesenteric fat was also observed in high- tolerated without any overt side effects, indicating lack of mechanism based fat fed mice treated with COMT-inhibitor. These abnormal adiposity and toxicity with chronic reduction of FGFR4. To further explore mechanism of glucose tolerance defect with the abnormal angiogenesis in COMT-inhibitor action, the effect of R4 ASO Rx on FGF15 levels was determined. Four-wk treated mice were ameliorated by the 2-ME treatment. 2-ME suppressed Rx of DIO mice reduced liver FGFR4 mRNA by > 75%, increased ileum FGF15 HIF-1α and macrophage accumulation in the epididymal fat. These results mRNA by > 5-fold and plasma FGF15 protein levels by > 2-fold. These rodent indicated that 2-ME could be the potential target drug for the treatment of fi ndings were confi rmed in Cynomolgus monkeys, where Rx with R4 ASO for 3 metabolic defects via inhibition of abnormal adipogenesis, angiogenesis and mo reduced liver FGFR4 mRNA by 70% and caused > 5-fold increase of ileum infl ammation especially in low COMT genotype population. FGF19 mRNA and 2-fold increase of plasma FGF19 protein levels without any Supported by: Kanae Foundation (K.K.) overt toxicities. These data provide further support that peripheral inhibition of FGFR4 with antisense drugs is an attractive therapeutic approach for obesity. 1825-P Cathepsin K, L Expression in Adipocytes Is Upregulated by Palmitate 1823-P Via TNFα and IL-6 Caloric Restriction Reverses Elevated ER Stress and Oxidative JEONG SEON YOO, YOUNGMI LEE, EUN HAE LEE, JIWOON KIM, SHINYOUNG Stress in Liver of ob/ob Mice LEE, JANG-HAN JUNG, JI SUN NAM, SHIN AE KANG, TAE-WOONG NOH, MIN ATSUYUKI TSUTSUMI, HIROYUKI MOTOSHIMA, SHUJI KAWASAKI, SATOKO HO CHO, JONG SUK PARK, KYUNG WOOK KIM, JAE-WOO KIM, CHUL WOO AHN, HANATANI, MOTOYUKI IGATA, TATSUYA KONDO, TAKESHI MATSUMURA, KAKU BONG SOO CHA, EUN JIG LEE, SUNG KIL LIM, KYUNG RAE KIM, HYUN CHUL LEE, TSURUZOE, TAKESHI NISHIKAWA, EIICHI ARAKI, Kumamoto, Japan Seoul, Republic of Korea Endoplasmic reticulum (ER) stress and oxidative stress have been Aim: Cathepsin family is lysosomal cysteine protease. It is recently proposedto play a crucial role in the development of insulin resistance reported that cathepsin K, L, S control adipogenesis and relate to acute and diabetes in obesity. Although caloric restriction (CR) improves various coronary syndrome. However, it is not clear whether cathepsins expression obesity-related disorders, the effects of CR on ER stress and oxidative stress can be affected by saturated fatty acid which has a critical role in metabolic in obesity have not been elucidated. disease. We examined the hypothesis that palmitate upregulates cathepsin To investigate how CR affects ER stress, oxidative stress and insulin K, L, S expression in obese adipose tissue and infi ltrating macrophages have signaling in obesity, a leptin-defi cient ob/ob mice under CR (ob-CR) or ad crucial role in this process via infl ammatory cytokines. libitum (AL)–feeding (ob-AL) for four weeks were compared on daily food- Methods and Results: 3T3-L1 cells were fully differentiated to mature intake, body weight (BW), glucose metabolism, ER stress, oxidative stress adipocytes for 8 to 10 days and treated with palmitate, lipopolysaccharide and insulin signaling. To reduce BW to the level of lean littermates fed AL (LPS) which is ligand of TLR4 (toll-like receptor 4). Real-time PCR revealed that (lean-AL), the ob-CR were given reduced chow (2.0 g/day) to a level which not cathepsin S but cathepsin K, L expression is upregulated by palmitate as previously reported to extend lifespan of ob/ob mice. Glucose and insulin dose- and time-dependent manner. But there was no additional effect when tolerance, markers for ER stress and oxidative stress (protein nitrotyrosine), we use palmitate-treated RAW264.7 cells’ media instead of direct treatment and insulin signaling were investigated in these animals. to 3T3-L1 cells. Cathepsin K, L is upregulated after treatment of TNFa or IL-6 CR reduced BW in ob-CR, resulted in comparable BWs between ob-CR like palmitate. Six-week-old C3H/HeN mice with normal TLR4 and C3H/HeJ and lean-AL after 2 weeks, both of which became smaller than ob-AL in BW. mice with TLR4 gene’s mutation got LPS injection ip (1 mg/kg) or were fed The ob-CR showed improved glucose tolerance and hepatic insulin action with high fat diet (60% fat of total calories) for 13 weeks and sacrifi ce to compared with ob-AL. Signifi cant increases in ER stress markers (such as harvest epididymal fat. Cathepsin K, L, S expression increased in both kinds phosphorylation of PERK and eIF2α, and expression of ATF4 and GRP78 of mice fed high fat diet compared with chow diet. But we cannot fi nd similar mRNA) and in protein nitrotyrosine were observed in liver and epididymal pattern at mice which got LPS ip injection. fat from ob-AL compared with those from lean-AL. CR signifi cantly reduced Conclusions: We concluded that cathepsin K, L expression in fat tissue are all of these markers in ob/ob mice. CR also signifi cantly reduced both serine- upregulated by saturated fatty acid via infl ammatory cytokines, not TLR4, phosphorylation of IRS-1 and JNK phosphorylation in liver of ob/ob mice. thereby might have a critical role in developing acute coronary syndrome Reduction in ER stress by CR tended to be stronger than that obtained by of metabolic syndrome and there are no additional effects of infi ltrating treatment with 4-phenyl butyric acid, which is known to reduce ER stress macrophages. in vivo. In conclusion, four weeks of CR effectively reduced ER stress and oxidative 1826-P stress, and improved insulin action via suppression of JNK-mediated IRS-1 Comparison of Genetic Factors Controlling Abdominal Fat Distribu- phosphorylation in liver of ob/ob mice. tion and Glucose Metabolism in A/J and SM/J Mice MISATO KOBAYASHI, TAMIO OHNO, MASAKO KUGA, MASAHIKO NISHIMURA, 1824-P ATSUSHI MURAI, FUMIHIKO HORIO, Nagoya, Japan Catechol-o-Methyltransferase Defi ciency Is Associated with Abnorm- Obesity is a major risk factor for insulin resistance, type 2 diabetes, al Adiposity and Glucose Intolerance: Implication for a New Mecha- dys lipidemia, cardiovascular disease, fatty liver and stroke. However, nism of Metabolic Syndrome each abdominal fat depot, such as mesenteric or epididymal, differently MEGUMI KANASAKI, KEIZO KANASAKI, DAISUKE KOYA, Uchinada, Japan contributes to the development of insulin resistance. Previously, we mapped a major quantitative trait locus (QTL, T2dm2sa) for impaired glucose Catechol-o-methyltransferase (COMT), an enzyme linked with several Obesity neuronal/psychological diseases, metabolizes a group of catechols including tolerance on chromosome (Chr.) 2 and revealed that the chromosomal region POSTERS catecholamines and catechol estrogens. 2-methoxyestradiol, one of the near T2dm2sa on Chr. 2 had affective genes on not only glucose tolerance, but also the accumulation of body fat, using SM.A-T2dm2sa congenic metabolite of catechol estrogens via COMT, exerts as an anti-angiogenesis Integrated Physiology/ and a vascular protective factor via inhibition of hypoxia-inducible factor mice. SM.A-T2dm2sa mice possess the A/J-allele T2dm2sa region in SM/J (HIF)-1α. Several single-nucleotide polymorphisms (SNPs) in the human mice. To identify the genetic regions that contribute to fat accumulation in COMT gene have been observed and some of which may result in the epididymal and mesenteric fat depots and to examine whether or not the depletion of catalytic activity, such as the well-known functional Val158Met genetic regions that affect glucose metabolism and body fat distribution isoform. These COMT SNPs have been associated with hypertension, are coincident, we performed QTL analyses using (A/J×SM.A-T2dm2sa)F2 preeclampsia, obesity, increased fat mass and obesity in diabetes. However, intercross mice. SM.A- T2dm2sa mice shows lower glucose tolerance and the contribution of COMT suppression in the onset of metabolic diseases higher epididymal fat weight compared with A/J mice. has not been determined. Using COMT-inhibitor (Ro 41-0960), here we Signifi cant QTLs for mesenteric fat were detected on Chrs. 5 (Mfatq3sa, show that COMT-inhibitor accelerate adiposity with the impaired glucose LOD: 3.5), 7 (Mfatq1sa, LOD: 7.7) and 16 (Mfatq2sa, LOD: 5.0). Signifi cant tolerance in mice fed a high-fat diet. A 2-week high-fat diet (60% fat in total QTLs for epididymal fat were detected on Chrs. 10 (Efatq2sa, LOD: 3.8) and

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A493 OBESITY—ANIMALCATEGORY

12 (Efatq1sa, LOD: 4.5). Thus, QTLs for mesenteric and epididymal fat depot mice with B6 background over 8 generations and made the Kl/+ mice with were mapped on the different chromosomal regions. The A/J allele on Chrs. hyperadiponectinemia. These mice were mated with each other to generate 7, 10 and 16 contributed to increase mesenteric or epididymal fat weights, Kl/Kl mice with hyperadiponectinemia. We measured the bodyweights oppositely the SM/J allele on Chrs. 5 and 12 contributed to increase these of these mice until they died naturally. Moreover, the levels of serum fat weights. This suggests that the fat accumulations in individual fat depots adiponectin (measured by ELISA) and the expressions of Klotho protein in are controlled by distinct genomic regions. In addition, the affected allele several organs (measured by Western blotting) in Kl/Kl mice overexpressing exists in both of the parental strains. adiponectin gene were compared with those of control Kl/Kl mice at the Signifi cant QTLs for impaired glucose tolerance and blood glucose age of 7 week. concentration have been detected on Chrs. 3, 6, 11 and 18. Comparison of Results: 1. We succeeded in producing Kl/Kl mice which overexpress a these QTLs for abdominal fat distribution with those for glucose metabolism human adiponectin gene in the liver. The serum levels of external human revealed that the genetic factors controlling fat accumulation in adipose adiponectin in these mice were extremely high (95.4±58.1μg/ml). tissue do not coincide with those controlling glucose tolerance and blood 2. The serum levels of internal mouse adiponectin in Kl/Kl mice with glucose concentration in (A/J×SM.A-T2dm2sa) F2 mice. hyperadiponectinemia were signifi cantly higher than those of control Kl/Kl mice (56.7±8.9 vs. 34.6±3.9μg/ml, p<0.001). 1827-P 3. Although their body weights were almost the same, Kl/Kl mice with Effect of Sucrose vs. Fructose-and-Glucose on the Body Mass of the hyperadiponectinemia did not signifi cantly prolong their average life span Rat compared with control Kl/Kl mice (61.8±9.1 vs. 59.7±17.0 days). GRZEGORZ WYSTRYCHOWSKI, WLADYSLAW GRZESZCZAK, EWA OBUCHO- 4. The expression levels of Klotho protein in the kidney, which is WICZ, ANTONI WYSTRYCHOWSKI, Zabrze, Poland, Katowice, Poland one of the major Klotho-protein producing organs, in Kl/Kl mice with U.S. boost in obesity coincided with high-fructose corn syrup (HFCS, hyperadiponectinemia did not show signifi cantly higher levels compared to mixture of 55% fructose (F) and 42-45% glucose (G)) domination as sweetener those of control Kl/Kl mice. in soft drink industry. Obesogenic effect of HFCS has been claimed with Conclusion: Adiponectin did not affect the expression of Klotho protein little scientifi c evidence and attributed to syrup-specifi c compounds like and the shortened life span of Kl/Kl mice. dicarbonyls or higher F content as compared with sucrose (S). We aimed to 1829-P verify it by assessing impact of S vs. HFCS-like mixture of F&G on weight and Effects of Ectopic Lipid Accumulation in Liver and Muscle on Mito- basic metabolic markers in rat model. chondrial Function 24 M 6-w.o. S-W rats were assigned to 9 weeks of unlimited drinking of GILLES SÉQUARIS, JULIA SZENDROEDI, JÖRG KOTZKA, ESTHER PHIELIX, BIRGIT 5% S solution (8 rats, S group), 5% solution of 55% F & 45% G (8 rats, F&G) KNEBEL, HANS-JOACHIM PARTKE, DIRK MÜLLER-WIELAND, MICHAEL RODEN, or tap water (8 rats, W). Chow was provided ad libitum. Metabolic markers at Düsseldorf, Germany, Hamburg, Germany study completion, weight changes, and fl uid, chow and energy intakes were Ectopic lipid accumulation in liver and muscle has been associated with compared between groups. abnormal mitochondrial function and insulin resistance. However, it is unclear F&G and W gained more weight than S (table). There was trend for fl uid, whether hepatic lipid accumulation directly affects mitochondrial function. chow, and corresponding caloric intakes to be greater in F&G than in S. To address this question, we examined two transgenic mouse strains which Total energy intake in W tended to be higher than in S, while lower than in develop non-alcoholic fatty liver disease (NAFLD) due to liver- (alb-SREBP- F&G. Serum cholesterol was higher in F&G and S. Triglycerides tended to be 1c, n=7) and adipose tissue- (aP2-SREBP-1c, n=8) specifi c overexpression increased in F&G. Uric acid and G levels did not differ (not shown). of the lipogenic transcription factor sterol regulatory-element binding S F&G W protein-1c (SREBP-1c), and matched control mice (con, n=10). Alb-SREBP-1c mice exhibit a primary moderate NAFLD due to liver-specifi c stimulation of Baseline weight [g] 184±19 175±7 174±15 lipid metabolism, whereas aP2-SREBP-1c mice develop excessive NAFLD Weight gain/day [g] 3.9±0.4 4.5±0.4** 4.7±0.6## secondary to lipid overfl ow from adipose tissue. Animals were fed regular Fluid intake/day [ml] 80.5±9.2 89.2±15.0* 51.1±4.2##$$ chow and sacrifi ced at the age of 18 weeks. Mitochondrial (mt) content Chow intake/day [g] 21.1±1.1 22.9±1.0* 27.5±1.0##$$ (mtDNA:nuclearDNA ratio) was assessed from quantitative PCR. Mt function was measured by high-resolution respirometry (Oroboros, Austria) Energy from fl uid/day [kJ] 65.2±7.5 69.4±11.7* 0##$$ in permeabilized liver and soleus muscles. AP2-SREBP-1c mice had 37% Energy from chow/day [kJ] 253.7±13.1 274.7±12.3* 330.6±11.7##$$ higher liver weight (aP2-SREBP-1c: 1.55±0.11 vs. alb-SREBP-1c: 1.04±0.10, Total energy intake/day [kJ] 318.9±5.7 344.1±23.9* 330.6±11.7#$ con: 1.13±0.17 [g]; p<0.001) and 66% higher hepatic state 3 respiration Serum cholesterol [mmol/l] 1.17±0.17 1.23±0.15 0.93±0.13##$$ (aP2-SREBP-1c: 53±14 vs. alb-SREBP-1c: 33±8 and con: 32±9 [pmol.s-1.mg- 1]; p<0.005) Mt content was 10% lower in liver (aP2-SREBP-1c: 1.46±0.13; Serum triglycerides [mmol/l] 0.72±0.25 0.99±0.39 0.66±0.24$ ∼ alb-SREBP-1c: 1.63±0.08; con: 1.57±0.08; P<0.05) and soleus (aP2-SREBP-1c: *P <0.1, **P <0.05 F&G vs. S; #P <0.1, ##P <0.05 W vs. S; $P <0.1, $$P <0.05 1.55±0.11, alb-SREBP-1c: 1.63±0.11; con: 1.73±0.09; P<0.005). Mitochondrial W vs. F&G; Mann-Whitney U-test function in soleus muscle was comparable in all groups. Unrestricted intake of liquid F&G results in greater increase in rat weight Conclusion: The lower mitochondrial content of liver and soleus muscle as compared with S. As ingestion of sweetened fl uid tends to be higher and suggests an early abnormality in these models. In lipid-overfl ow induced compensatory decrease in solid food intake lower in animals on F&G than on NAFLD, liver but not skeletal muscle, exhibits greater oxidative capacity, S, lesser satiety with F&G (and consequently HFCS) seems likely and may be possibly to compensate for the excessive lipid availability. responsible for weight gain. Both F&G and S appear to raise cholesterolemia in rats. Larger study shall verify these results. 1830-P Effects of Fenofi brate and Pioglitazone on the ATGL Level in High 1828-P Fat Diet Rats Effects of Adiponectin on the Life Span of Klotho Mice: From the LI WEI, CHAOYU ZHU, JUNXI LU, WEIPING JIA, Shanghai, China Obesity Generation of Klotho Mice Overexpressing Adiponectin Gene Triglyceride (TG) accumulation in tissues is positively associated with POSTERS SHUICHI OTABE, TOSHIHIKO HASHINAGA, XIAOHONG YUAN, TOMOKA FUKU- insulin resistance. Adipose triglyceride lipase (ATGL) is an important TG TANI, TSUYOSHI OHKI, SATOMI KAKINO, NOBUHIKO WADA, YAYOI KURITA, hydrolase in adipose tissue, liver and skeletal muscle of rodents. PPAR Integrated Physiology/ HITOMI NAKAYAMA, YUJI TAJIRI, KENTARO YAMADA, Kurume, Japan agonists can signifi cantly decrease TG accumulation or enhance insulin Aim: We previously reported that adiponectin had a life-extending sensitivity. This study aims to evaluate the effects of PPAR-alpha and PPAR- effect in mammals. Meanwhile, a presenile mouse strain using insertional gamma agonists on the ATGL expression of adipose tissue and skeletal mutagenesis of the Klotho gene was developed; mice which are homozygous muscle in high fat diet rats. for the Klotho mutant (Kl/Kl mice) have reduced Klotho gene expression and Eight-week-old SD rats were randomized into four groups: normal diet have exhibited rather short life span and several other signs of aging. Hence, (NC), high fat diet (HFD), high fat diet and treated with fenofi brate (HFD-FT), to investigate whether adiponectin can restore the life span of Kl/Kl mice, high fat diet and treated with pioglitazone (HFD-PT). Western blot was used we overexpressed human adiponectin gene in Kl/Kl mice. Then, we observed to assess protein level of ATGL in adipose tissue and skeletal muscle. TG in the phenotypes of Kl/Kl mice with hyperadiponectinemia. skeletal muscle was also measured. Methods: As Kl/Kl mice are infertile, we crossed fertile Klotho mutant After eight weeks of high fat diet, SD rats showed characters of obesity, mice (Kl/+ mice) with previously reported human adiponectin transgenic hyperglycemia and hyperlipidemia. Lipid profi les and insulin sensitivity

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A494 OBESITY—ANIMALCATEGORY were improved by fenofi brate and pioglitazone. High fat diet induced TG obese rats, which was partly improved by fenofi brate (EDV increased 43.3%, accumulation in skeletal muscle was also decreased 20% by fenofi brate P<0.01). Correlation analysis showed ACR had a positive correlation with and 24% by pioglitazone. ATGL protein expression was respectively reduced FFAs, PF/BW and VF/BW (r=0.751, 0.783, 0.712, respectively, P<0.01) and 40% and 18% in adipose tissue and skeletal muscle of HFD rats and was had a negative correlation with EDV(r=-0.781, P<0.01). We conclude that increased after pioglitazone treatment in these tissues. ATGL was increased elevated circulating FFAs level may cause increased MAU in obese rats by in skeletal muscle but has no change in adipose tissue by fenofi brate. impairment of EDV and increased MAU of obese rats could be improved by Fenofi brate and pioglitazone treatment improved insulin sensitivity and reducing circulating FFAs level. increased ATGL protein and coincide with decreased TG content in skeletal Supported by: National Nature Science Foundation of China 81070675 muscle. These fi ndings suggest that fenofi brate and pioglitazone ameliorate insulin resistance through decreased ectopic TG content mediated by 1833-P increased ATGL in mainly tissues. Hyperleptinemia Induces Leptin Resistance and Further Worsening Supported by: National Natural Science Foundation of China Project (30670989) Obesity SATOKO SENDA, ATSUSHI INOUE, RYO SUZUKI, NOZOMU KAMEI, TAKU 1831-P WATANABE, ARSHAD MAHMOOD, AKIKO TAKIKAWA, AMINUDDIN AMIN, Fenofi brate Involves in CCK-Induced Reduction of Food Intake in SHIHO FUJISAKA, YUKIKO KOSHIMIZU, MANABU ISHIKI, ISAO USUI, TAKASHI Rats KADOWAKI, KAZUYUKI TOBE, Toyama, Japan, Tokyo, Japan YING HAN, MI-KYOUNG PARK, SO-YOUNG PARK, SU KYUNG PARK, DUK KYU Leptin resistance is the cause of obesity. It is known that insulin receptor KIM, HYE-JEONG LEE, Busan, Republic of Korea substrate(IRS)-2-PI3 kinase pathway is important for leptin signal as well Fenofi brate has been reported to decrease food intake independent of as JAK-STAT3 pathway in the hypothalamus. IRS-2-/- mice are the model leptin in rodents. We hypothesized that fenofi brate might decrease food of obesity due to leptin resistance associated with hyperleptinemia intake via cholecystokinin (CCK) dependent pathway. Otsuka Long-Evans genetically because of severely reduced leptin-stimulated PI3 kinase Tokushima fatty (OLETF) rats, which genetically lack CCK receptor as a result activity in the hypothalamus. To know the mechanism how leptin resistance of mutation, were divided into OLETF-fenofi brate group (n=5) and OLETF- causes obesity, we investigated the effect of calorie restriction and β3- control group (n=5). OLETF-fenofi brate group was fed with standard rat chow adrenergic receptor(AR) agonist treatment on leptin resistance in IRS- and fenofi brate (30mg/kg/day) and OLETF-control group was only fed with 2-/-mice. Three weeks of calorie restriction reduced the body weight and standard rat chow for the same period as control. Long-Evans Tokushima serum leptin concentration and restored leptin sensitivity in IRS-2-/- mice. Otsuka (LETO) rats which have normal CCK receptor, as control groups to Furthermore, calorie restriction signifi cantly restored the leptin-induced OLETF rats, were also divided into LETO-fenofi brate group (30mg/kg/day) phosphorylation of signal transducers and activators of transcription 3 (n=5) and LETO-control group (n=5). After 11 weeks of fenofi brate treatment, (STAT3), and neuropeptide(pro-opiomelanocortin: POMC and neuropeptide food intakes of the fenofi brate group were signifi cantly decreased than those Y: NPY) gene expression in the hypothalami of IRS-2-/- mice. In addition, of the control group in the LETO rats (P< 0.05), but there was no signifi cant calorie restriction reduced the expression of SOCS3 in the hypothalami difference in the OLETF rats. The levels of blood β-ketone, plasma leptin, of IRS-2-/- mice. In contrast, two weeks of treatment with β3AR agonist and fasting blood sugar (FBS) did not show signifi cant difference between reduced the serum leptin concentration without affecting adiposity. The the fenofi brate and control groups neither in the LETO rats nor in the OLETF β3AR agonist treatment also restored leptin sensitivity. Furthermore, the rats. To investigate the expression of CCK by fenofi brate, the protein level leptin-stimulated phosphorylation of STAT3, and POMC and NPY gene of CCK were analyzed with small intestinal tissue of the rats. Fenofi brate expression were restored in the hypothamami of these mice. In addition, the treatment groups showed that the protein levels of PPARa and CCK were expression of SOCS3 in the hypothalami was reduced in these mice. These signifi cantly increased in the small intestine than control groups both in the results suggest that the amelioration of hyperleptinemia is important for the LETO and OLETF rats. To examine the expression of CCK by fenofi brate, the restoration of leptin sensitivity. Hyperleptinemia caused leptin resistance Caco-2 cells, intestinal epithelial cell line, were cultured in the presence of by reducing leptin-induced phosphorylation of STAT3 due to the increased fenofi brate for 24 hours. Fenofi brate treatment signifi cantly increased the SOCS3 gene expression. protein expressions of PPARα and CCK in the Caco-2 cells. In conclusion, fenofi brate treatment decreased food intake in the LETO rats which have 1834-P normal CCK receptor, but not in the OLETF rats which lack CCK receptor. The Increased Infl ammation in a Rhesus Monkey Model of Dietary possible anorexigenic mechanism by fenofi brate might be up-regulation of Fructose-Induced Metabolic Syndrome CCK in the small intestine. ANDREW A. BREMER, KIMBER L. STANHOPE, JAMES L. GRAHAM, BETHANY P. CUMMINGS, PETER J. HAVEL, Nashville, TN, Davis, CA 1832-P Previous studies from our laboratory have demonstrated that chronic High Free Fatty Acids Level Related with Microalbuminuria in Obese consumption of a high-fructose diet induces insulin resistance and dyslipid- Rats emia in rhesus monkeys. However, the effect of a high-fructose diet on XIAO DONG SUN, YE RONG YU, LI NA HAN, BEN WANG, Chengdu, China infl ammation in nonhuman primates is not known. Therefore, we evaluated It is known that obesity is associated with microalbuminuria (MAU), which the effects of fructose on indices of infl ammation in a rhesus monkey model is not only an early manifestation of kidney damage, but also an independent of diet-induced metabolic syndrome. risk factor for ischemic cardiovascular disease. High free fatty acids (FFAs) For this study, a total of 29 adult male rhesus monkeys wereevaluated for level is a common feature of obesity and can cause impaired endothelium- one year. In addition to a standard ad libitum diet (31% energy as protein, 11% dependent vasodilatation (EDV). We hypothesized that increased release energy as fat, and 58% energy as carbohydrate), each animal was provided of FFAs from excessive visceral fat accumulation is related to increased 500 ml/day of a fruit-fl avored 20% fructose-sweetened beverage. Total urine albumin excretion and reduced circulating FFAs level by fenofi brate and percent body fat were determined by DEXA and glucose tolerance and has renal protective effects in MAU by improving endothelial dysfunction insulin sensitivity were assessed with intravenous glucose tolerance tests. in diet-induced obese rats. Male Wistar rats 4 weeks in age were Fasting glucose, insulin, total cholesterol, HDL-cholesterol, LDL-cholesterol,

randomly divided into three groups. The rats in the control group, obesity triglyceride, apolipoprotein (Apo)-B, Apo-A1, Apo-C3, adiponectin (total and Obesity group and fenofi brate group were fed with normal diet, high-fat diet, and HMW isoforms), and leptin were also measured. To evaluate circulating POSTERS high-fat diet plus fenofi brate (100mg/kg/d), respectively. At the end of 24 infl ammatory markers, fasting monocyte chemotactic protein-1 (MCP-1) and

weeks, body weight increased 32.8% in obese rats compared to control C-reactive protein (CRP) concentrations were measured. In the monkeys Integrated Physiology/ group. The serum FFAs and TG levels increased signifi cantly in obese rats. that developed insulin resistance and dyslipidemia (but not diabetes), one Fenofi brate intervention decreased serum FFAs and TG levels by 43.4% and year of fructose consumption increased MCP-1 concentrations by 25% 48% respectively, accompanied by reduced ratio of perirenal fat to body (from 232±20 pg/ml to 314±27 pg/ml, P<0.05) and CRP concentrations by weight (PF/BW) and visceral fat to body weight(VF/BW). Urinary albumin/ 60% (from 1.63±0.26 mg/L to 2.60±0.26 mg/L, P<0.001). In the 4 monkeys creatinine ratio (ACR) increased in obese rats (56.09±22.64 VS 19.52±7.30 that developed insulin resistance, dyslipidemia, and frank diabetes, MCP-1 mg/g, P<0.01), which was decreased 62.6% after fenofi brate intervention. concentrations increased by 35% (from 340±141 pg/ml to 451±172 pg/ml, Endothelial function was determined by comparing the vasorelaxation P<0.001). response of thoracic aorta segment to acetylcholine (to determine EDV), These results demonstrate that infl ammation as assessed by increases with that of sodium nitroprusside (to determine endothelium-independent of circulating MCP-1 and CRP is increased with insulin resistance and vasodilatation) in organ bath. Severe EDV impairment was observed in dyslipidemia in this nonhuman primate model, paralleling what has been

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A495 OBESITY—ANIMALCATEGORY

reported in humans. Thus, fructose-fed rhesus monkeys represent a novel in regulating growth and metabolism. While growth defects were overcome nonhuman primate model useful for investigating the pathophysiology, in het mice, the elevated adiposity persisted, which suggests even small prevention, and treatment of the metabolic and infl ammatory changes that reductions in CNS Rictor function may result in obesity. lead to an increased risk for type 2 diabetes and atherosclerotic cardio- Supported by: DK085712 vascular disease. Supported by: NIH Grants R21 AT250099 and R21 AT003645 1837-P ADA-Funded Research Obesity/Diabetes-Prone Atypical Antipsychotics Elicit a Rapid Metabolic Toxicity in Mice Treated with CPT1 Inhibitors 1835-P RONALDO PAOLO L. PANGANIBAN, SELENA A. DAVIS, VANCE L. ALBAUGH, Lack of Inteferon-γ Results in Reduced Body Weight and Better CHRISTOPHER J. LYNCH, Hershey, PA Glucose Tolerance Atypical antipsychotics (AAP) frequently elicit serious metabolic side NICOLE WONG, BARBARA C. FAM, GITTA R. CEMPAKO, GREGORY R. STEINBERG, effects including obesity and diabetes. Paradoxically, olanzapine lowers THOMAS T.W. KAY, JOSEPH PROIETTO, SOFIANOS ANDRIKOPOULOS, Heidelberg plasma FFA by impairing lipolysis and elevating rates of lipid oxidation (∼2- Heights, Australia, Hamilton, ON, Canada, Fitzroy, Australia fold). Dark cycle RER lowering in mice corresponds to the propensities of AAPs Obesity is a chronic low-grade infl ammatory disease that is caused by to cause metabolic side effects. Recent studies suggest that increased FFA increased energy intake and reduced energy expenditure. Studies using oxidation in diabetes is detrimental and that experimental therapeutics aimed animal models with deletion of infl ammatory cytokines have produced at further increasing fat oxidation should be abandoned in favor of efforts confl icting results with some showing increased weight gain and others to increase glucose utilization. Therefore, we hypothesized that preventing showing no effect or even reduced body weights. Clearly more work is AAP-stimulated fat oxidation would improve glycemia and normalize FFAs by necessary to understand the role of cytokines on body weight control. switching fuel utilization back to carbohydrate. To test this, male C57BL/6 Interferon-gamma (IFNγ) is a cytokine derived from T-cells that plays an mice were treated with tetradecylglycidic acid (TDGA) or etomoxir using important role in the innate and adaptive immune response particularly to dosing schemes that completely inhibit carnitine palmitoyltransferase I viral infections. Emerging evidence suggests that increases in the T-cell (CPT1). Mice were subsequently injected with 2-10 mg/kg of olanzapine, population in adipose tissue may contribute to obesity and the associated clozapine or vehicle (∼9PM). CPT1 inhibitors alone had little effect on metabolic syndrome. Recent studies have shown that IFNγ was increased calorimetry parameters and, as expected, clozapine and olanzapine lowered with obesity in both human and rodent models while defi ciency of IFNγ RER to ∼0.7, but had little effect on VO2. On the other hand, complete CPT1 resulted in better glucose tolerance in mice. However, how IFNγ affects inhibition led to rapid lowering of body temperature and energy expenditure, glucose tolerance is not known. The aim of this study was to assess the most animals died within hours. Even with incomplete CPT1 inhibition effects of IFNγ on both body weight and glucose metabolism using a global olanzapine still lowered RER, however VO2 dropped by ∼2/3 or more within -/- knockout mouse model. To do this, male IFNγ and wild-type C57BL/6 mice minutes of administration. The time course for recovery of VO2 was slower were monitored for 20 weeks for body weight, food intake and physical than expected given the half-life of these drugs in mice, but this is consistent activity on a standard rodent chow diet (3.3 kcal/g). At the end of the study, with slower overall metabolic rates after AAP treatment; some still died. IPGTT, ITT and hyperinsulinaemic/euglycaemic clamps were performed. As opposed to directly making conditions favorable for lipid oxidation, the Expression levels of arcuate nucleus NPY, AgRP and POMC mRNA as well converse effects of AAPs on glucose and FFA metabolism likely result from as circulating leptin levels were also determined. The results show that the block of an early step in glucose metabolism. The metabolic switching IFNγ-/- mice had reduced weight gain associated with decreased food intake appears to be required to survive. If new CPT1 inhibitors are developed for and increased physical activity. NPY and AgRP mRNA expression was clinical applications, their use in those with obesity or diabetes associated reduced, while POMC mRNA expression was increased as were plasma with atypical antipsychotic treatment should be carefully monitored. leptin levels. This led to improved glucose tolerance and insulin sensitivity Supported by: NIH (DK084428) and the Pennsylvania DOH using Tobacco due to greater suppression of endogenous glucose output, associated with Settlement funds decreased hepatic glucose-6-phosphatase activity. We conclude that global deletion of IFNγ in mice resulted in reduced body weight associated with 1838-P negative energy balance, glucose tolerance and improved hepatic insulin Obesity-Induced Hypoxia Regulates the Polarization of Adipose sensitivity. Our fi ndings demonstrate that IFNγ plays a critical role in the Tissue Macrophages regulation of body weight and subsequently glucose metabolism. SHIHO FUJISAKA, ISAO USUI, MASASHI IKUTANI, YUKIKO KANATANI, SATOKO SENDA, AKIKO TAKIKAWA, AMINUDDIN AMINUDDIN, HIKARI SUZUKI, MANA- 1836-P BU ISHIKI, MINORU IWATA, YOSHINORI NAGAI, KIYOSHI TAKATSU, KAZUYUKI Neuronal Rictor Deletion Leads to Obesity, Growth Defects and TOBE, Toyama, Japan Glucose Intolerance Adipose tissue macrophages (ATMs) consist of at least two different HEIDI E. KOCALIS, RICHARD L. PRINTZ, KEVIN D. NISWENDER, Nashville, TN subtypes i.e., classically activated M1 ATMs and alternatively activated Insulin has pleiotropic effects in the central nervous system (CNS), and M2 ATMs. We have recently reported that M1 and M2 ATMs are clearly neuronal resistance to insulin is implicated in the pathogenesis of obesity separated by fl ow cytometry using CD11c and CD206 as M1 and M2 markers, and dysglycemia. Akt is a down-stream mediator of insulin action on growth respectively. The M1 and M2 ATMs from epidydimal adipose tissue of high and metabolism. Akt is activated via phosphorylation at serine473 by the fat-fed mice exhibited very much different gene expression patterns. For rapamycin-insensitive companion of TOR (Rictor) containing complex, example, pro-infl ammatory cytokines, such as TNFα and IL-6, were mainly mTORC2, in addition to PDK1 phosphorylation at threonine308. The role of expressed in M1 ATMs, whereas, IL-10, an anti-infl ammatory cytokine, mTORC2 signaling targets in CNS regulation of energy homeostasis is not was highly expressed in M2 ATMs. The purpose of this study is to clarify well established. Given the role of Akt in energy and glucose homeostasis, the relationship between the characters of ATMs and hypoxia in adipose and the role of mTORC2 in Akt activation, we hypothesized that neuronal tissue. To assess the hypoxic conditions in adipose tissue, mice were Rictor gene deletion would lead to obesity and glucose intolerance. intraperitoneally injected with 60mg/kg pimonidazole, a chemical probe for

Obesity Cre-Lox P mediated deletion in neurons (Nestin-Cre) lead to gene dose hypoxia, 30 minutes prior to sacrifi ce. Immunoblotting with anti-pimonidazole

POSTERS dependant reductions in Rictor mRNA, and signifi cantly reduced serine473 antibody revealed that the hypoxic signals in epididymal adipose tissue phosphorylation in null mice. Null mice displayed a profound growth defect were enhanced in high fat-fed mice. Flow cytometry analysis demonstrated

Integrated Physiology/ that resolved by 12 weeks of age, after which, body weight and length were that the uptake of pimonidazole in M1 ATMs was stronger than that of M2 similar to controls. After 20 weeks of chow diet feeding, het mice gained ATMs. In the stromal vascular fraction of epididymal adipose tissues, the 68% (11.1±0.6g, P<0.05) and null mice gained 100% (13.2±0.6g, P<0.05) more expression of hypoxia-related genes such as HIF1α, VEGF, GPX1, EglN2, VHL body weight than controls (6.6±0.6g). Similarly, fat mass gain was 150% and PDK1 were dramatically upregulated by high fat feeding. Furthermore, higher in both het (3.0±0.2g, P<0.05) and null mice (3.0±0.2g, P<0.05) than the expressions of the hypoxia-related genes were higher in M1 ATMs than controls (1.2±0.2g). Lean mass gain was similar between control (5.1±0.3g) those in M2 ATMs. To know the effect of hypoxia on macrophage polarization, and het mice (4.5±0.4g), while null mice gained more lean mass (8.8±0.6, we cultured bone marrow-derived macrophages in hypoxic chamber with P<0.05). Glucose tolerance (area under the curve) was measured by IPGTT 1%O2 condition for 24 hours. In these cells, the expression of CD11c, an M1 in only het and control mice, but was found to be impaired in het mice vs. marker of ATMs was increased. These result indicated that obesity-induced controls (het; 5119±391 vs. control 3799±278, P<0.05) despite similar four- hypoxia may have some roles in M1 polarization of ATMs. hour fasting glucose levels. In conclusion, neuronal Rictor plays a critical role

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A496 OBESITY—ANIMALCATEGORY

1839-P 1841-P Omega-3 Fatty Acids Protect Against Glucose Intolerance and Prevention of Obesity and Insulin Resistance by Overexpressing Attenuate Ceramide Accumulation with High Fat Diet HMGA1 in Adipose Tissue IAN LANZA, AGNIESZKA ZABIELSKI, PIOTR ZABIELSKI, DANIEL JAKAITIS, ALTAMIRA ARCE, SYLVIE FRANCKHAUSER, MIQUEL GARCÍA, TURA FERRÉ, IVET BUSHRA ALI, K. SREEKUMARAN NAIR, Rochester, MN ELIAS, CARLES ROCA, PEDRO J. OTAEGUI, ANNA PUJOL, FÀTIMA BOSCH, EFRÉN Lipotoxicity is implicated as a cause of insulin resistance. Accumulation RIU, Bellaterra, Spain of lipid metabolites (e.g., ceramides) may interfere with insulin signaling in Obesity represents one of the major health risks in modern societies. muscle. Dietary omega-3 fatty acids (n3PUFA) are reported to protect against Adipocyte functionality is lost during obesity and has been related to impaired insulin resistance with high-fat diets. We determined the impact of n3PUFA transcriptional regulation of the key factors that control adipogenesis. on the content and composition of lipid metabolites in skeletal muscle. Mice Among them, the high mobility group A1 (HMGA1) proteins may play an were fed for 10wks with normal fat diet (NF, 10% fat), high fat diet (HF, 60% important role in adipogenesis. Nevertheless, the mechanisms by which fat), or high fat diet + fi sh oil (HF+N3, 60% fat with 3.4% kcals from n3PUFA). HMGA1 proteins may affect adipose tissue functionality in vivo are still Oral glucose tolerance tests (OGTT) were performed at baseline and 10wks. unknown. To examine the effects of adipose tissue HMGA1 expression in the Muscle tissue was homogenized and separated into sarcoplasmic, myofi brillar, development of obesity and insulin resistance, we generated transgenic mice and mitochondrial fractions. Ceramides and long chain acyl coA (LCACoA) overexpressing HMGA1 specifi cally in adipose tissue (aP2-HMGA1). HMGA1 were measured by mass spectrometry using internal standards for individual expression in fat tissues was markedly increased compared with control species. OGTT revealed signifi cant reductions in glucose tolerance in HF animals. Intriguingly, these transgenic mice showed decreased epididymal but not HF+N3 or NFD. Total ceramide was increased in HF (15.69±0.41 ng/ WAT and BAT weight and lower triglyceride content than control mice. mg tissue) and HF+N3 (14.38±0.16) compared to NFD (13.05±0.43). Closer This was parallel to decreased levels of serum free-fatty acid, triglyceride examination of individual ceramide species revealed that n3-PUFAs completely and glycerol. However, these transgenic mice were normoglycemic and prevented HF-induced increases in medium chain saturated (C18) ceramides normoinsulinemic and no differences in glucose tolerance and insulin (NF: 7.38±0.31, HF: 9.41±0.27, HF+N3: 8.08±0.17 ng/mg tissue), but signifi cantly sensitivity were observed between groups. In contrast, when fed a high- increased levels of long chain monounsaturated (C24:1) ceramides ceramides fat diet (HFD), body weight gain was lower in transgenic than in control (NF: 2.74±0.06, HF: 2.45±0.06, HF+N3: 2.76±0.04 ng/mg tissue). Regression mice. This was parallel to lower WAT and BAT weight. However, HFD fed analyses revealed that C18 ceramide content is a strong predictor (R2=0.57, transgenic mice showed increased liver weight that was related to increased P<0.0001) of glucose intolerance. In contrast, other long-chain ceramides (C20, intra-hepatic triglyceride content. Nevertheless, transgenic mice under a C24, C24:1) were poor predictors (R2= 0.03, 0.04, 0.002, respectively). Similarly, HFD showed increased glucose tolerance and whole body insulin sensitivity. we found that LCACoA, the precursor to ceramide, was increased in HF, but These transgenic mice also showed decreased levels of serum free fatty signifi cantly attenuated with n3-PUFA. These results support a hypothesis acid, triglyceride and glycerol compared to controls. The overexpression of that n3-PUFAs protect against HF-induced glucose intolerance by altering the HMGA1 in adipose tissue of transgenic mice led to a marked decrease in the content and composition of ceramides in skeletal muscle. Lower LCACoA levels expression of genes involved in fatty acid metabolism and in mitochondrial n3-PUFA diet indicates that either more circulating lipid is oxidized by skeletal biogenesis and function, suggesting mitochondrial dysfunction of the muscle or that more lipid is directed away from skeletal muscle toward storage adipose tissue in these transgenic mice. Currently, we are further analyzing in adipose tissue. the mechanisms by which HMGA1-mediated adipose tissue dysfunction Supported by: KL2-RR-084151 (I.R. Lanza) in transgenic mice protects them against high-fat diet-induced insulin resistance and obesity. 1840-P Supported by: Grants from the MICINN of Spain (SAF2008-03083) and EU (MIRG- Physiological Anandamide Levels Potentiate Glucose-Stimulated CT-2007-20) Insulin Secretion in Isolated Canine Pancreatic Islets ORISON O. WOOLCOTT, JOYCE M. RICHEY, ROBERT H. CHOW, MORVARID 1842-P KABIR, MALINI IYER, ERLINDA L. KIRKMAN, DARKO STEFANOVSKI, MAYA Proinfl ammatory Cytokines Inhibit Ventromedial Hypothalamus LOTTATI, L. NICOLE HARRISON, VIORICA IONUT, DAN ZHENG, ISABEL R. HSU, Glucose Sensing STELLA P. KIM, KARYN J. CATALANO, JENNY D. CHIU, RICHARD N. BERGMAN, REEMA VAZIRANI, SRINIDHI DESHPANDE, VANESSA ROUTH, Newark, NJ Los Angeles, CA Obesity is a major risk factor for type 2 diabetes mellitus. The two disorders Anandamide (AEA) is an endogenous cannabinoid ligand and known to often present concurrently and are accompanied by hyperleptinemia and stimulate insulin secretion. Interestingly, AEA plasma levels are elevated hyperinsulinemia. Interestingly, proinfl ammatory cytokines such as tumor in obesity. Therefore we hypothesize that AEA may contribute to the well- necrosis factor-α (TNFα) and interleukin-1β (IL1β) are also elevated. This characterized insulin hypersecretion in response to insulin resistance in obesity. observation has led to the hypothesis that obesity is an infl ammatory Dog pancreatic islets were isolated at sacrifi ce. Insulin secretion, disease. While the functions of insulin and leptin in energy homeostasis have expressed as stimulation index (Sindex, ratio of glucose-stimulated insulin been studied extensively, less is known about the roles of proinfl ammatory secretion [GSIS, insulin secreted during 1h-stimulation with glucose 15 cytokines. The ventromedial hypothalamus (VMH) plays a key role in the mmol/L] over basal insulin secretion [BIS, 1h-unstimulated period with regulation of energy homeostasis and is a site of leptin and insulin action. glucose 3 mmol/L]) was assessed in batches of 15 islets during static Proinfl ammatory cytokines may also act in the VMH. The VMH contains incubation in presence or absence of AEA. All experiments were done in 3-6 glucose inhibited (GI) neurons that are believed to play an important role in replicates. Supraphysiological concentrations of AEA (10 μmol/L) stimulated energy balance. GI neurons increase their electrical activity as extracellular BIS two-fold (p=0.018) in lean animals (n=7, BW=28.8 ± 1.0 kg). In another glucose levels decrease. The mechanism by which this occurs involves group maintained on a high fat diet for 22 weeks (n=5, BW=34.2 ± 1.6 kg), the metabolism of glucose which increases the ATP:AMP ratio. Glucose AEA stimulated BIS four-fold (p=0.043). Similarly, AEA 10 μmol/L potentiated defi cit increases AMP, resulting in the phosphorylation and activation of GSIS in lean and HFD animals (by three- [p=0.018] and four-fold [p=0.043], AMP-activated kinase (AMPK). AMPK closes a chloride channel, resulting respectively). Cannabinoid 1 receptor (CB1R) antagonist rimonabant inhibited in depolarization and increased action potential frequency. We have

AEA effects on GSIS but not BIS, in both groups. Together, these results previously shown that leptin inhibits AMPK and blunts the response of VMH Obesity

suggest that supraphysiological AEA effects on GSIS are mediated through GI neurons to decreased glucose. Since leptin is also classifi ed as a cytokine, POSTERS CB1R. The presence of CB1R and CB2R in isolated islets was confi rmed by we hypothesize that proinfl ammatory cytokines act similarly. In support of

real-time PCR. In addition, physiological AEA concentrations (10 nmol/L) this, we found that a proinfl ammatory cytokine cocktail (40ng/mL TNFα and Integrated Physiology/ potentiated GSIS (Sindex: 8.26 ± 1.72 vs. 6.69 ± 1.83; p=0.043) in another 10ng/mL IL1β) inhibits VMH AMPK phosphorylation in response to decreased group of lean dogs (n=5, BW=27.2 ± 0.1 kg). Rimonabant (100 nmol/L) and glucose from 2.5 to 0.3mM (40% decrease in AMPK Thr172 phosphorylation; the transient receptor potential vanilloid 1 channel (TRPV1) antagonist p<0.05) in adult mice. Furthermore, using membrane potential dye imaging, iodoresiniferatoxin (100 nmol/L) but not CB2R antagonist AM 630 (100 we have found that IL1β (10ng/mL) inhibits the response of VMH GI neurons nmol/L) showed a trend to inhibit AEA effects on GSIS (p=0.138). Taken to decreased glucose from 2.5 to 0.7mM (% cells responding=9.8% control together, these results suggest that AEA effects on GSIS, at physiological vs 6.1% IL1β; p<0.05). These data suggest that proinfl ammatory cytokines concentrations, might be mediated partially through CB1R and TRPV1. may play a role in energy homeostasis by regulating the glucose sensitivity Our results are consistent with the notion that elevated anandamide of VMH GI neurons. plasma concentrations in obese patients might be suffi cient to contribute to Supported by: R56 DK55619, R21 CA139063 the compensatory hyperinsulinemia in response to insulin resistance.

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A497 OBESITY—ANIMALCATEGORY

1843-P 1845-P Pyruvate Dehydrogenase Kinase 2 (PDK2) Defi ciency Reduces Reduction of Adipose 11β-Hydroxysteroid Dehydrogenase Type 1 Fasting Blood Glucose Levels and Fat Accumulation in the Liver of and Hexose-6-Phosphate Dehydrogenase Expression Ameliorates Mice Fed a High Fat Diet Obesity and Insulin Resistance in Type 2 Diabetic db/db Mice YOUNGHOON GO, NAM HO JEOUNG, JIYUN JEONG, HYEON-JI KANG, KEUN- WEI WANG, JUAN ORTEGA, RENE PORCHE, KABIRULLAH LUTFY, THEODORE GYU PARK, EON-JU JEON, CHANG JOO OH, AE-KYUNG MIN, JEONG-KOOK KIM, FRIEDMAN, YANJUN LIU, Los Angeles, CA, Pomona, CA SUN JOO LEE, HYUN-AE SEO, ROBERT A. HARRIS, IN-KYU LEE, Daegu, Republic Prereceptor activation of glucocorticoids via the NADPH-dependent of Korea, Indianapolis, IN 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has been identifi ed Regulation of the activity of the pyruvate dehydrogenase complex as an important risk factor in metabolic syndrome. Hexose-6-phosphate (PDC) is critical for disposal of excess glucose, fuel selection by tissues, dehydrogenase (H6PDH) mediates intracellular NADPH availability to mouse and conservation of substrates for glucose synthesis. A dominant role for 11β-HSD1 and thus ensures 11β-HSD1 driving tissue GC production linked to PDK4 in down regulation of PDC activity is suggested by the remarkable up the development of type 2 diabetes and obesity. regulation of PDK4 during fasting. This hypothesis is supported by reduced Berberine (BBR) is an anti-infl ammatory drug and has recently shown to fasting levels of blood glucose levels and three carbon gluconeogenic improve obesity and insulin resistance. Here, we evaluated the potential substrates in PDK4 knockout (PDK4-/-) mice. Relative to PDK4, less evidence benefi cial effects of BBR in type 2 diabetic mice by examining the possible has existed for an important role for PDK2 in glucose homeostasis. Although infl uence of BBR on the expression of GR, 11β-HSD1, and H6PDH in vivo ubiquitously expressed, PDK2 is only modestly increased by fasting, and PDK2 and in vitro in adipocytes. Treatment of db/db mice with BBR for 4 weeks defi ciency has no effect upon fasting blood glucose levels in chow fed mice. markedly reduced adipose 11β-HSD1 activity and GR mRNA levels and However, in studies with mice fed a high fat diet for 16 weeks (12-13 mice reduced weight gain, hyperglycemia, and insulin resistance. The reduction per group), PDK2 defi ciency reduced body weight (WT vs PDK2-/-, 49.8±0.8 vs of adipose 11β-HSD1 was accompanied by BBR-induced inhibition of 42.8±1.2 g, Mean±SE, P<0.001), lowered fasting blood glucose levels (WT vs H6PDH mRNA expression in the adipose tissues. Incubation of 3T3-L1 PDK2-/-, 180±9 vs 153±8 mg/dL, P<0.05) improved glucose tolerance (WT vs adipocytes with increasing concentrations of BBR led to a dose-dependent PDK2-/-, 31600±1900 vs 21100±1100 mg/dL x min area under curve, P<0.001), down-regulation of 11β-HSD1 mRNA levels with corresponding reduction and reduced liver fat relative to wild type C57BL/6J mice. Sterol-regulatory- of H6PDH expression. These fi ndings suggest that BBR exerts some of element-binding protein-1c (SREBP1c), acetyl-CoA carboxylase (ACC), and its benefi cial effects, at least in part, by inhibiting adipose 11β-HSD1 and fatty acid synthase (FAS) were signifi cantly reduced in the liver of PDK2-/- mice H6PDH expression. compared with wild type mice. Interestingly, epididymal fat weight/body Supported by: NIDDK SC1DK087655 weight increased in PDK2-/- mice compared to wild type mice (WT vs PDK2-/-, 3.93±0.39 vs 5.59±0.25%, P<0.01). GLUT4 and hexokinase II were increased in adipose tissue of PDK2-/- mice, suggesting increased glucose metabolism in the epididymal fat of these mice. Furthermore, the infl ammatory cytokine 1846-P tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 Regulation of Secretion and Expression of Adiponectin by Insulin (MCP-1) were signifi cantly decreased while adiponectin was increased in and β-Agonists in Rat -/- epididymal fat tissue of PDK2 mice, suggesting improved adipose tissue LI CONG, GANG LI, NA YIN, QIANG LI, ALLAN ZHAO, Harbin, China, Pittsburgh, PA function. These results suggest an important role for PDK2 in regulating lipid Adiponectin plays an important role in regulating insulin sensitivity and metabolism in the liver and adipose tissue. metabolism of carbohydrate and lipid. It has proved that concentrations of adiponectin are decreased in obesity suggesting that altered adiponectin 1844-P homeostasis may play a role in the development of obesity-related insulin Reduced Sex Hormone-Binding Globulin Levels with Obesity in resistance. Current study is to determine the regulation of secretion and Male Monkeys expression of adiponectin in vivo. Male Wistar rats were fasted for 18 hr and MARJAN KAREGAR, DUSHAN GHOORAY, BARBARA HANSEN, STEPHEN J. allowed to refeed with/without a β3-adrenergic receptor agonist infused WINTERS, Louisville, KY, Tampa, FL into refeeding rats. Insulin clamp was used for measuring the effect of Sex hormone binding globulin (SHBG) transports testosterone and euglycemic-hyperinsulinemic clamp and hyperglycemic-hyperinsulinemic other steroids in the blood, and regulates the clearance and production clamp on adiponectin secretion and expression. Plasma adiponectin levels of testosterone in men. The level of SHBG is infl uenced by a variety of were determined by radioimmunoassay. Real-time PCR was used for hormones as well as nutritional and genetic factors. SHBG levels decline detecting the expression of adiponectin genes in rat primary adipocytes. with increasing obesity, and low SHBG levels predict the development of Refeeding of 18-hr fasted rats increased plasma adiponectin concentration the metabolic syndrome (MetS) and type 2 diabetes (T2DM). While there is (∼2 fold) and adipose tissue adiponectin expression (∼3 fold). A 3-hr a strong body of evidence linking hyperinsulinemia to low SHBG in obesity, euglycemic-hyperinsulinemic clamp elevated the plasma concentration of other mechanisms involving hepatic fat and hepatic nuclear factor-4α have adiponectin by 1.6±0.3 fold and increased adiponectin mRNA expression also been proposed. SHBG is present in the plasma of human and non- in adipose tissue by 3.1±0.9 fold compared to saline-infused animals. human primates, but since it is undetectable in adult rodent plasma, its Hyperglycemic-hyperinsulinemic clamps increased plasma adiponectin molecular regulation has not received intense study. Because of its strong and adiponectin mRNA levels that were similar to those observed with association with the MetS and T2DM, we propose that study of SHBG in euglycemic clamps. BRL37344, a specifi c β3-agonist completely blocked the obese non-human primates will be informative. In this study, we measured stimulatory effects of refeeding on the plasma adiponectin, but did not alter the impact of obesity on SHBG levels in the monkey using a solid phase adiponectin mRNA levels in adipose tissue. In conclusion, the secretion and assay based on SHBG binding to concanavalin A-Sepharose. Blood samples expression of adiponectin are acutely regulated by nutritional status in vivo. were obtained after an overnight fast using ketamine anesthesia from 32 Moreover, insulin and β-agonists act directly at the adipocyte to regulate the male rhesus monkeys (Macaca mulatta) age 10.3-36 yrs (18.6±1.0) weighing secretion and expression of adiponectin in vivo. 8.95-24 kg (16.0±0.67). Mean (±SEM) serum testosterone was 90.1±14.6 ng/ Supported by: National Natural Science Foundation of China (NSFC, No.30700281)

Obesity dl, and SHBG was 51.5±4.82 nmol/l. There was highly signifi cant (p<0.01)

POSTERS inverse relationship between body weight and SHBG (r=-0.46). On the other hand, testosterone was not signifi cantly related to body weight (r=-0.1),

Integrated Physiology/ and neither SHBG (r=-0.07) nor testosterone (r=-0.006) was related to age. Therefore, the monkey is an excellent model to examine the role of SHBG in 1847-P obesity and T2DM. Low testosterone levels in these animals and the lack of Role of Kinin B2 Receptor in Mitochondrial Activity and Diet Induced the expected inverse relationship to body weight may be due to age or the Obesity Resistance fasting conditions and anesthesia used for blood sampling. FELIPE C.G. REIS, FABIANA L.T. MOTTA, SANDRO M. HIRABARA, MILENE S. OR- Supported by: NIA N01AG31012 and NIA HHSN2532008002C MANJI, KELY S. DE PICOLI, EDSON L. SANTOS, CHARLLES H.M. CASTRO, CLÉLIA R.A. BERTONCINI, BEATRIZ H. KIYOMOTO, RUI CURI, RONALDO CARVALHO ARAUJO, JOÃO BOSCO PESQUERO, São Paulo, Brazil, Dourados, Brazil Kinins mediate pathophysiological processes related to hypertension, pain and infl ammation through the activation of two G protein-coupled receptors, named B1 (B1r) and B2 (B2r). These peptides are related to glucose homeostasis

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A498 OBESITY—ANIMALCATEGORY however their effects on energy balance were elucidated by our group recently 1849-P showing that kinin B1r participates in the regulation of energy balance via a The Contribution of Enhanced Myocardial mTOR/S6K1 Activation mechanism that could involve the modulation of leptin sensitivity. Now, using to Impaired Insulin Signaling, Oxidative Stress and Diastolic B2 knockout mice and in vivo techniques, we present here evidence of a novel Dysfunction in Obese db/db Mice role for kinin B2r in the regulation of mitochondrial activity and adiposity. VINCENT G. DEMARCO, JAVAD HABIBI, LAKSHMI PULAKAT, ADAM T. WHALEY- Kinin B2r defi ciency in mice (B2-/-) resulted in less fat content, higher CONNELL, LIXIN MA, MELVIN R. HAYDEN, ROGER D. TILMON, C.B. KRUEGER, NA- glucose uptake in isolated muscle (C), increased lean mass and robust THAN REHMER, MONA GARRO, MING YANG, JAMES R. SOWERS, Columbia, MO protection against high fat diet (HFD)-induced weight gain (A). Under HFD Obesity, the metabolic syndrome and heart failure (HF) are epidemics in B2-/- exhibited improved lipid oxidation and increased energy expenditure the United States. Obesity related HF is characterized by decreased insulin (B). Surprisingly, B2r defi ciency led to an augment in mitochondrial activity metabolic signaling and diastolic dysfunction. We have investigated the role (E), fatty acid β-oxidation related gene expression (D) and superoxide of mTOR/S6K1 activation in impairment of insulin metabolic signaling and dismutase activity in muscle (F). Finally, B2r mRNA levels were signifi cantly diastolic relaxation in young db/db mice. The initial diastolic fi lling rate, as lower in 3T3-422A differentiated adipocytes (G) and during the early days of measured by high resolution MRI, was decreased in 12 week hyperglycemic C2C12 myoblast differentiation (H). Together, our data suggest that kinin B2 db/db mice compared to age matched controls. There were contemporaneous receptors participate in the regulation of energy balance via a mechanism increases in reactive oxygen species (ROS), increased small abnormal that may involve the modulation of mitochondrial activity in muscle fi bers. mitochondria, lipid accumulation and interstitial fi brosis in db/db heart. There Supported by: FAPESP, CNPq, CAPES was increased Thr 389 phosphorylation (P)/activation of S6K1, enhanced association of S6K1 with IRS-1 and Ser307 P of IRS-1. This was associated with decreased total IRS-1 and a tendency (not signifi cantly) for decreased P/activation of Akt. This is consistent with fi ndings in other tissues where S6K1 P has been associated with Ser P and associated ubiquinization/ 1848-P degradation of IRS-1. A mitochondrial source of increased ROS is likely as Saturated and Unsaturated Fat-Induces Tissue-Specifi c Changes the number of mitochondria was increased and NADPH oxidase activity was in Critical Nodes of Insulin Resistance in Diet-Induced Obese (DIO) not increased in db/db heart. In summary, young db/db mice manifested Rats diastolic dysfunction possibly due to decreased insulin metabolic signaling, SURESH K. MOHANKUMAR, PETER ZAHRADKA, DANIELLE P. HANKE, LINDA increased mitochondrial generation of ROS, and interstitial fi brosis. SIEMENS, CARLA TAYLOR, Winnipeg, MB, Canada Supported by: NIH R01-HL-63904 and VA Merit (JRS), and VA Career Development The liver and peripheral tissues including skeletal muscle and adipose Award (AWC) play a pivotal role in the pathogenesis of insulin resistance (IR) induced by high fat diets (HFD). However, the originating nodes of IR in these tissues 1850-P remain poorly understood. The present study therefore investigated the The Inhibition of Cannabionoid Receptor CB1 Increases GLUT4 tissue-specifi c changes of protein markers of IR in response to isocaloric Expression in Adipocytes and Adipose Tissue HFD of either saturated fat (SF) or unsaturated fat (USF) in obese-prone DANIELA T. FURUYA, ANA C. POLETTO, HELAYNE S. FREITAS, UBIRATAN F. rats. Fasting serum analysis indicated that rats fed HFD for 12 weeks were MACHADO, São Paulo, Brazil insulin resistant with marked elevation in the fasting glucose (245±22 Evidences have suggested that the endocannabinoid system is overactive mg/dL), insulin (45±5 µU/ml) and HOMA (27±5). Comparative analysis of in obesity, resulting in enhanced endocannabinoid levels in both circulation gastrocnemius muscle (GM), liver (L) and epididymal adipose (EA) tissues and visceral adipose tissue. The cannabinoid CB1 receptor is expressed indicated that the degree of relative phosphorylation/activation of the in the adipose tissue besides the brain. Few studies in vitro suggest that key nodes of IR was greater in some tissues versus others: IRS1-s636/639, CB1 activation increases glucose uptake in adipocytes. The objective of the GM>L=EA; IRS1-s307, GM>L=EA; AKT-s473, GM=EA>L; JNK, L>GM=EA; ERK, present study was to investigate the CB1 receptor modulation on glucose EA>GM=L, although there were no changes in the respective protein levels. transporter GLUT4 expression and the related mechanisms in adipocytes and These results suggest that activation of these critical nodes in response adipose tissue of obese mice. For this, 3T3-L1 adipocytes were incubated to high fat is tissue-specifi c. Next, we determined whether SF and USF in the presence of a selective agonist of CB1 receptor (1 mM ACEA) or a differentially infl uence the critical nodes of IR. The HOMA value of SF and selective antagonist of CB1 receptor (0.1, 0.5 or 1 mM AM251) or both. After USF fed rats (37±4 and 19±2) indicated that SF fed rats were more insulin 2, 4 or 24 hours, cells were harvest to evaluate GLUT4 mRNA (Real Time resistant than USF. However, there were no differences between SF and USF PCR) and protein (Western blotting), and NFkappaB activation specifi cally in the activation of the above-mentioned IR nodes. Interestingly, the animals in the promoter of SLC2A4 gene (EMSA) which encodes GLUT4 protein. fed USF had active tyrosine phosphorylation of AKT-t308, a positive regulator Additionally, insulin sensitivity in vivo and GLUT4 protein from adipose tissue of insulin signaling, in GM and EA (64% and 32% increase versus SF) despite were assessed in monosodium glutamate–induced obese mice untreated or no differences in caloric intake. These results paralleled the reductions of treated with AM251 (0.01 mg/kg body weight). Acute and chronic incubation fasting glucose, insulin and HOMA in USF fed rats (-27, -31 and -48% versus with AM 251greatly increased GLUT4 protein content [0.1, 0.5 or 1 µM AM251 SF group), suggesting that USF sustains metabolic functions of GM and EA for 4 hours treatment, 309%, 313% or 357% vs C, respectively, P<0,001; and, tissues. In summary, these data establish IRS1/AKT in muscle, JNK in liver for 24 hours treatment, 155% (P<0,01), 169% (P<0,01) or 204% (P<0,001), and AKT/ERK in adipose tissue as key players in the pathogenesis of IR in respectively]. Moreover, 2 hour-treatment of AM251 increased GLUT4 mRNA response to HFD and USF delays the progression of IR via AKT. [0.5 or 1 µM AM251, 50% (P<0.01) or 79% (P<0.001), respectively]. Supported by: Canola Council of Canada AM251 was able to increased NF-kappaB activation in adipocytes only after 24 hour incubation. In obese mice, the forementionated concentration of AM251 reversed insulin resistance, but did not have any effect on GLUT4 mRNA and protein expression. In conclusion, the present data shows that the blockade of CB1 receptor markedly increases GLUT4 expression

in adipocytes, and that NFkappaB seems to be involved in the chronic Obesity

modulation of GLUT4 expression. POSTERS Supported by: FAPESP (08/09194-4 and 07/50554-1) Integrated Physiology/ 1851-P The Novel SGLT2 Inhibitor BI 10773 Prevents Pioglitazone-Induced Weight Gain and Further Improves Glycemic Control in Dietary- Induced Obese Rats ROLF GREMPLER, LEO THOMAS, ACHIM SAUER, MICHAEL MARK, PETER EICKELMANN, STEVEN VICKERS, SHARON C. CHEETHAM, THOMAS KLEIN, ROBERT B. JONES, Biberach an der Riss, Germany, Nottingham, United Kingdom The novel potent and selective SGLT2 inhibitor BI 10773 is in late stage clinical development for the treatment of type 2 diabetes. In addition to its anti-diabetic properties, BI 10773 causes a reduction of body weight and

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A499 OBESITY—HUMANCATEGORY

body fat content in dietary-induced obese rats (DIO rats). PPARg agonists, sought to determine the biological effects of a TrkB agonist and identify its like Pioglitazone (Pio), are effi cacious drugs for the treatment of type 2 mechanism of action. diabetes. However, a common side effect of this drug class is weight gain. Diet-induced obese (DIO) mice were injected with a potent and highly Here, we investigated whether BI 10773 could attenuate Pio-induced selective mouse monoclonal TrkB agonist and the effects on body weight weight gain of DIO rats after 4 weeks treatment with 10mg/kg BI 10773, and food intake were determined. We observed that TrkB activation induced 10mg/kg Pio and the combination of both (n=10 per group). Pio signifi cantly robust and reversible weight loss accompanied by a concomitant reduction in increased body weight (+6.8 %), whereas BI 10773 led to weight loss (-3.1%) food intake. To gain further insight into the hypophagic effect of the agonist, compared to control animals. Animals that received BI 10773 in combination we studied circadian meal patterns and observed that TrkB agonism reduced with Pio had no signifi cant increase in body weight (+1.6% vs. vehicle). the amount of food ingested per meal but did not affect meal frequency. As DIO rats are insulin-resistant and hyperinsulinemic we investigated In addition, pair-feeding experiments showed that body weight was mostly the effect of the combination on metabolic parameters. Both compounds regulated by food intake. Given the high selectivity of the blood-brain-barrier, signifi cantly reduced fed plasma insulin levels (vehicle: 5.27 ± 0.7; BI 10773: it is unknown whether the effects of this TrkB agonist on food intake are 3.53 ± 0.4; Pio: 2.27 ± 0.3 ng/ml) which was further reduced (p=0.054) by the mediated through activation of TrkB receptors in the central nervous system combination (BI 10773 + Pio: 1.55 ± 0.1 ng/ml). Furthermore, the combination (CNS) or periphery. To address this, we peripherally injected fl uorescently of BI 10773 + Pio reduced plasma triglyceride levels, whereas either labeled TrkB agonist and detected fl uorescence in the medium eminence and compound alone had no effect. As expected, BI 10773 and BI 10773 + Pio the arcuate nucleus of the hypothalamus as well as the dorsal vagal complex markedly increased urinary glucose excretion. of the hindbrain. Collectively, our studies show that TrkB agonism decreases After termination of the study carcass analysis was performed to assess body weight mainly by increasing satiety through direct activation of the body composition. The fat content was 147.4 + 7.8 g in the vehicle group brain satiety centers. compared to 140.7 + 7.1 g (BI 10773). Pio increased body fat content to 1854-P 170 + 6.8 g but the gain in body fat was prevented by combination treatment Weight Control Diminishes Progression of Insulin Resistance Due (BI 10773 + Pio: 149.6 + 7.2 g). to Estrogen-Deprived Condition Our data show that BI 10773 prevented the Pio-induced body weight gain MUJALIN PRASANNARONG, KANOKWAN VICHAIWONG, VITOON SAENGSIRI- and the associated gain of body fat after 4 week treatment of DIO rats. SUWAN, Bangkok, Thailand Furthermore, BI 10773 improved hyperinsulinemia of DIO rats alone and in Prolonged estrogen deprivation in ovariectomized rats resulted in the combination with Pio. Our data support the concept of a combination of BI phenotypic features of the metabolic syndrome including increased visceral 10773 with Pio for the treatment of type 2 diabetes. fat accumulation, dyslipidemia, impaired glucose tolerance, and insulin resistance of skeletal muscle. Because these animals are also hyperphagic, 1852-P the development of metabolic defects under estrogen-deprived condition Tissue-Specifi c Dysregulation of Hexose-6-Phosphate Dehydrogen- could be confounded by excessive calorie consumption. To determine the role ase and Glucose-6-Phosphate Transporter Expression in a Mouse of calorie consumption in the development of the phenotypic characteristics Model of Type 2 Diabetes of the metabolic syndrome in animal under prolonged estrogen deprivation, YANJUN LIU, YUICHI NAKAGAWA, YING WANG, WEI WANG, JUAN ORTEGA, adult female Sprague-Dawley rats were randomly assigned to sham- THEODORE C. FRIEDMAN, Los Angeles, CA, Hamamatsu, Japan operated (SHAM), ovariectomized (OVX), pair-fed ovariectomized (OVX+PF), Tissue-specifi c amplifi cation of glucocorticoid action through 11β-hydroxy- or body weight-controlled ovariectomized (OVX+WC) groups. After a 12- steroid dehydrogenase type 1 (11β-HSD1) affects the develop ment of the week experimental period, whole-body glucose tolerance, skeletal muscle metabolic syndrome. insulin action, intra-abdominal fat content and serum lipid profi le were Hexose-6-phosphate dehydrogenase (H6PDH) mediates intracellular evaluated. When compared to OVX group, both pair-feeding and weight NADPH availability for 11β-HSD1 and depends on the glucose-6-phosphate control paradigms signifi cantly reduced intra-abdominal fat accumulation by transporter (G6PT). To assess whether the tissue-specifi c alterations of 32% and 44% (p<0.05). Serum ratio of low-density lipoprotein cholesterol/ H6PDH and G6PT expression could contribute to local glucocorticoid action total cholesterol decreased by 7% and 10% (p<0.05) and high-density and insulin resistance in type 2 diabetes and obesity, we characterized the lipoprotein cholesterol/total cholesterol increased by 10% and 31% (p<0.05). role of H6PDH and G6PT in pre-receptor metabolism of glucocorticoids by However, glucose intolerance and insulin resistance of skeletal muscle examining the production of the hepatic 11β-HSD1-H6PDH-G6PT system in glucose transport observed in OVX animals were effectively corrected only a mouse model of type 2. in those underwent weight control paradigm. Compared with OVX group, the We observed that increased production of hepatic H6PDH in diabetic glucose-insulin index decreased by 0.67-fold and insulin action on skeletal db/db mice was paralleled by upregulation of hepatic G6PT production and muscle glucose transport rate elevated by 1.86–fold in OVX+WC group elevated circulating levels of corticosterone. In contrast, pharmacological (p<0.05). In spite of the amount of calorie consumption matched to SHAM inhibition of H6PDH and G6PT expression decreased NADPH production group, OVX+PF animals still exhibited whole-body and skeletal muscle accompanied by reduction of 11β-HSD1 in the liver and subcutaneous fat, insulin resistance. These results underscore the role of estrogen deprivation and attenuated the phenotype of type 2 diabetes. Incubation of mouse in the development of metabolic disorders. Moreover, we have shown that a hepatocytes with corticosterone enhanced G6PT and H6PDH production greater extent of calorie manipulation effectively prevents the development with corresponding activation of 11β-HSD1 and PEPCK. Knockdown of of the phenotypic features characterizing the metabolic syndrome occurring H6PDH by siRNA attenuated the corticosterone-induced H6PDH production under prolonged estrogen-deprived condition. in these intact cells. Addition of the G6PT inhibitor to primary hepatocytes Supported by: Offi ce of Higher Education Commission, Thailand suppressed H6PDH production. These fi ndings suggest that increased hepatic and adipose H6PDH and G6PT expression may contribute to 11β-HSD1 upregulation of local glucocorticoid action that may be related to OBESITY—HUMAN the development of type 2 diabetes. Supported by: NIDDK SC1DK087655 [See also: Presidents Posters 466-PP to 468-PP, page A129.] Obesity

POSTERS 1853-P TrkB Agonist Induces Body Weight Loss by Activating Satiety Centers Guided Audio Tour: Pharmacologic Agents for Obesity (Posters 1855-P to

Integrated Physiology/ in the Brain 1862-P), see page 11. TIFFANY GARESKI, SARAH WILL, DAVID KUBASIAK, THADDEUS UNGER, & 1855-P KIMBERLY COUGHLAN, GUO FENG, YING SUN, XIANGPING LI, ARIFUL QADRI, Dipeptidyl Peptidase 4 Is a Novel Adipokine Potentially Linking DARRELL PANZA, SEUNG HAHM, JULI JONES, JANET PAULSEN, RUTH GIMENO, Obesity to the Metabolic Syndrome MYLENE PERREAULT, Cambridge, MA HENRIKE SELL, SUSANNE FAMULLA, LAMERS DANIELA, SONJA HARTWIG, STEFAN Brain-derived neurotrophin factor (BDNF) and its receptor, tropomyosin- LEHR, JOHANNES RUIGE, MARGRIET OUWENS, MIKAEL RYDEN, PETER ARNER, related kinase B (TrkB), have emerged as critical regulators of central neural JÜRGEN ECKEL, Düsseldorf, Germany, Ghent, Belgium, Stockholm, Sweden circuits involved in energy homeostasis. Obesity is the hallmark of the metabolic syndrome and frequently associates The potential therapeutic application of TrkB activation has been with the development of type 2 diabetes and cardiovascular disease. Enlarge- demonstrated in several rodent models in which BDNF administration ment of adipose tissue leads to dysregulation of adipokine secretion induced weight loss mainly through appetite suppression. In this study, we representing a potential critical pathogenic link between obesity, insulin

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A500 OBESITY—HUMANCATEGORY resistance and type 2 diabetes. By comprehensive proteomic profi ling of we evaluated the difference between subjects for cancer surveillance (CS) the human adipocyte secretome we identifi ed DPP4 as a novel adipokine. and subjects for medical check-up (MC), and determined the impact of life This study assessed the association of the adipokine DPP4 to the metabolic style and other parameters on aBAT. We analyzed 31,088 subjects who syndrome. performed 18F-fl uorodeoxyglucose (FDG) PET/CT scans from July 2006 to June Fully differentiated adipocytes exhibit a substantially higher release of 2010 at Shanghai and examined the characteristics of subjects with aBAT. DPP4, when compared to preadipocytes or macrophages. DPP4 release We found that aBAT predominantly exists from anterior neck to the thorax. from adipocytes is increased after treatment with insulin and TNFα. Direct Active BAT was seen in 410 of the 31,088 subjects (1.32%), which is lower addition of DPP4 to adipocytes impairs insulin signaling at the level of than previous fi nding (5.37%). Among CS patients, aBAT was found in 146 insulin-stimulated Akt phoshorylation. A signifi cantly higher level of DPP4 of 14,389 (1.01%), while it was 264 of 16,699 (1.58%, p<0.05) in MC group. protein expression was seen in visceral as compared to subcutaneous fat of The prevalence of aBAT was higher in women (2.36% [273 of 11570]) than in obese patients. DPP4 expression in lean subjects was signifi cantly lower in men (0.70% [137 of 19518], P<0.0001). In both groups, the prevalence of aBAT both depots compared to the obese patients and with no regional difference. is higher in women than men (1.59% vs 0.61%, 3.16% vs 0.77%, P<0.0001, DPP4 serum concentrations signifi cantly correlated with adipocyte size, respectively). In univariate analyses of CS group, aBAT was most frequently serum leptin and various clinical parameters related to the metabolic detected in women (P=0.0005), patients in the bottom third for age (<39.7 syndrome as BMI, fasting insulin and hs-CRP. Using adipose tissue explants years, P=0.0077), the least obese patients (P<0.0001), and those who had from lean and obese subjects, we observed a 2-fold increase in DPP4 release never smoked, less drinking, moderate exercise and no fatty liver (P<0.0001, in obesity that strongly correlated with adipocyte volume and parameters of P=0.0015, P=0.0309 and P=0.0003, respectively). The univariate analyses the metabolic syndrome. DPP4 release from adipose tissue was decreased of MC group showed the same results except for exercise (P=0.1937). In to the lean level after weight reduction of the obese patients induced by age- and sex- adjusted multivariate analyses, BMI, smoking and exercise in bariatric surgery. Both circulating DPP4 and DPP4 released from adipose the CS group remained signifi cant. BMI, smoking and thyroid disease in MC tissue strongly correlated positively with an increasing risk score for the group remained signifi cant. In conclusion, in Chinese adults, the detectable metabolic syndrome. aBAT correlates inversely with BMI, smoking, exercise and thyroid diseases, DPP4 is a novel adipokine which may be linked to adipocyte hypertrophy suggesting a central role in adult metabolism and possibly a contributing and adipose tissue infl ammation. As DPP4 release strongly correlates with role to the increasing rate of metabolic syndrome among East Asians. adipocyte size, adipose tissue could represent an important source of DPP4 Supported by: Grants 30900502 and 81070680 from NSFC. in obesity. We therefore suggest that DPP4 may be critical in linking adipose tissue and the metabolic syndrome. & 1858-P Differential Effects of Gastric Bypass and Banding on Postprandial & 1856-P Glucose and Insulin Secretion in Diabetes and Healthy Obese Lipid Accumulation Products Index as a Better Predictor for Insulin MIRJAM A. LIPS, MARJOLEIN A. WIJNGAARDEN, GERRIT H. DE GROOT, Resistance and Glucose Intolerance Than BMI in Young Obese Women EDO AARTS, IGNACE M.C. JANSSEN, BERT VAN RAMSHORST, BART A. VAN with Polycystic Ovary Syndrome WAGENSVELD, DINGEMAN J. SWANK, FRANCOIS VAN DIELEN, KO WILLEMS JEE-YOUNG OH, HYE JIN LEE, YOUNG SUN HONG, YEON-AH SUNG, Seoul, VAN DIJK, HANNO PIJL, Leiden, The Netherlands, Beverwijk, The Netherlands, Republic of Korea Arnhem, The Netherlands, Nieuwegein, The Netherlands, Amsterdam, The Nether- Elevated waist circumference and elevated triglycerides has been lands, Gouda, The Netherlands, Eindhoven, The Netherlands considered as a surrogate marker of cardiovascular risk. BMI may not to Bariatric surgery alters glucose homeostasis in obese patients with and be the best marker for obesity-related disease. Lipid accumulation product without type 2 diabetes (T2DM) within weeks. The effects on postprandial (LAP) index, an ordinal scale combining waist circumference and triglycerides glucose metabolism and insulin secretion seem to depend on type of surgery concentration, was suggested as a better marker for cardiovascular disease and on preoperative glucose homeostasis. This study aimed to quantify than BMI. postprandial glucose and insulin levels before and after laparoscopic We examined whether the LAP index is associated with cardiovascular adjustable gastric banding (LAGB) or roux-en-y-gastric bypass (RYGB) in risk factors and a better predictive marker than BMI in obese women with normal glucose tolerant (NGT) subjects and subjects with T2DM. polycystic ovary syndrome (PCOS). We recruited 223 obese (BMI ³23kg/m2) We studied 3 groups patients at baseline and 3 weeks after surgery; a women with PCOS and 230 age-, and BMI-matched regular cycling control NGT group (n=11; age 46,3±6,3y, BMI 43,1±3,0kg/m2) undergoing LAGB, a women by voluntary participation for the prevalence study. PCOS was NGT group (n=16, age 48,6±6,5y, BMI 44,2±3,3kg/m2) undergoing RYGB diagnosed by using NICHD, and LAP index was calculated using the formula and a diabetic group (n=15, age 51,3±7,3y, BMI 43,5±4,2kg/m2) undergoing [waist circumference(cm)-58] x TG(mmol/L). Glucose intolerance was defi ned RYGB. A healthy control group (n=12, age 49±6,1y, BMI 21,7±1,6kg/m2) was as IFG, IGT or diabetes, and insulin resistance as HOMA value greater than also studied. 90 percentile of obese controls. We performed a meal tolerance test and measured glucose and insulin Mean age and BMI were 24±4 yr and 25.6±2.2 kg/m2 in PCOS and 24±4 yr for 3 hours. A paired Student’s t-test was used to compare groups before and 25.2±1.8 kg/m2 in controls. LAP index was signifi cantly higher in women and after surgery. with PCOS than controls (32.2±21.9 vs. 23.6±15.6, P<0.001). In women with In NGT subjects, in response to LAGB, glucose decreased slightly PCOS, LAP index was more strongly correlated with postload 2-hr glucose, (p=NS), while insulin levels were approximately 30% lower (90,9mU/l vs. fasting and 2-hr insulin, and HOMA-IR than BMI, in contrast, BMI was 64,2mU/l, p=NS). In contrast, in NGT subjects after RYGB, peak glucose more strongly correlated with body fat mass than LAP index. By logistic levels increased from 6,9±1,1 to 8,2±1,6mmol/l (p<0,05) in the face of a regression, LAP index was signifi cantly associated with glucose intolerance higher insulin peak (58,7mU/l vs. 141,6mU/l, p<0,05), and an increased area (OR 2.6, 95% CI 1.4∼5.1), and insulin resistance (OR 2.9 95% CI 1.3∼6.5) but under the insulin curve (AUC) (p<0,05). In T2DM patients, RYGB ameliorated BMI did not show any signifi cant association. These associations were not glucose metabolism: peak glucose (12,6 vs.10,9mmol/l, p<0,05) and AUC observed in control subjects. glucose (1899 vs.1479mmol/L/3h, p<0,05) decreased, while peak insulin In conclusion, LAP index could be a better predictable marker for increased (52,3 vs. 86,3mU/l, p<0,05), and AUC insulin did not change. recognizing the insulin resistance and glucose intolerance in young obese These data suggest that in NGT subjects, LAGB enhances insulin sensitivity Obesity

women with PCOS. within 3 weeks. In contrast, RYGB appears to adversely affect glucose POSTERS metabolism in these patients, whereas it ameliorates glucose metabolism in T2DM patients. The results support observations in rodents indicating 1857-P Integrated Physiology/ The Prevalence and Related Factors of Active Brown Adipose that nutrient entry in the proximal gut activates neuroendocrine systems Tissue in Chinese Adults WITHDRAWN regulating glucose metabolism in NGT subjects. Remarkably, bypass of the ZHAOYUN ZHANG, AARON M. CYPESS, QING MIAO, HONGYING YE, CHONG WEE same gut segment ameliorates glucose tolerance in T2DM patients. LIEW, QIONGYUE ZHANG, RUIDAN XUE, QIQUN TANG, YIHUI GUAN, YIMING LI, Shanghai, China, Boston, MA Previous studies have shown active brown adipose tissue (aBAT) is present in adults and may play important roles in regulating energy homeostasis. Very little is known about the prevalence of aBAT in Chinese adults. Almost all human cohort studies were done in patients for cancer surveillance and thus may not represent healthy subjects. Due to the large amount in our cohort,

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A501 OBESITY—HUMANCATEGORY

and was also strongly enriched among genes correlated with % fat mass in & 1859-P adipose (-logB-Hp-value=3.2), which includes thioredoxin reductase-1 as the Liraglutide Provides Weight Maintenance and Additional Weight strongest correlated gene (ρ=0.65, p<0.0001). Interaction network analysis Loss after Low Calorie Diet-Induced Weight Loss in Obese Subjects within genes correlated with TXNRD1 expression indicated a signifi cant without Diabetes: The SCALE™ Maintenance Study network that includes genes involved in thioredoxin mediated oxidative THOMAS A. WADDEN, PRISCILLA HOLLANDER, SAMUEL KLEIN, KEVIN NIS- stress defense mechanism (TXNRD1, TXN and TXNIP) and angiogenesis WENDER, VINCENT WOO, PAULA HALE, TU DUYEN LE THI, LOUIS J. ARONNE, (ANG and RNH1). Pro and anti-angiogenic factors were negatively and Philadelphia, PA, Dallas, TX, St. Louis, MO, Nashville, TN, Winnipeg, MB, Canada, positively correlated respectively with obesity. Nine SNPs were associated Princeton, NJ, Copenhagen, Denmark, New York, NY with expression of 10 local (±500kb) transcripts. SNP rs6861681 was the Weight loss is diffi cult to achieve and sustain by lifestyle therapy alone. strongest cis-eQTL for CPEB4 (P=2.93x10-10). Liraglutide, a once-daily human glucagon-like peptide-1 analog approved Conclusions. Our study suggests a novel interaction of up-regulated TXN- for the treatment of T2DM, induced dose-dependent wt loss in obese TXNRD1 system mediated oxidative stress defense and down-regulated subjects without diabetes in a phase 2 study, with highest effi cacy shown angiogenesis pathway as an adaptive response in obese non-diabetic by a 3mg/d dose. The present randomized, placebo-controlled trial tested subjects. A subset of obesity associated SNPs regulates expression of the ability of liraglutide (3mg/d) to maintain diet-induced wt loss. Subjects transcripts as cis-eQTLs. who lost >5% body wt after a 4–12 week run-in (RI) with a low-calorie diet (1200–1400kcal/d) and exercise counseling were randomized to receive liraglutide or placebo plus a 500kcal/d defi cit diet and exercise regimen for & 1861-P 56 weeks. Of 511 subjects entering RI, 422 were randomized 1:1 to the 2 Increased VLDL-TG Secretion Precedes Impaired Control of Endo- arms. The mean RI wt loss for all randomized subjects was 6.0% (6.3kg). genous Glucose Production in Obese, Normoglycaemic Men Over the next 56 weeks, additional wt loss occurred post-randomization (PR) LARS P. SØRENSEN, ESBEN SØNDERGAARD, BIRGITTE NELLEMANN, JENS S. (6.1% vs 0.05%, liraglutide vs placebo, p<0.0001)(Table). A larger fraction of CHRISTIANSEN, LARS C. GORMSEN, SØREN NIELSEN, Aarhus, Denmark liraglutide than placebo subjects maintained RI wt loss, or lost ≥5% additional The purpose of the study was to assess basal and insulin-mediated VLDL- wt. Completion rates, serious adverse events (AEs) and withdrawals due to TG kinetics and the relationship between VLDL-TG secretion and hepatic AE were similar for each group. Nausea was reported by more subjects on insulin resistance assessed by endogenous glucose production (EGP) in liraglutide vs placebo, particularly during titration; 64% of liraglutide cases upper-body obese (UBO) and lean men. 2 were mild and most declined by 4–6 weeks. Psychiatric AEs were reported Twelve normoglycaemic UBO (waist-hip ratio>0.9, BMI>30 kg/m ) and 12 2 by a similar percent of liraglutide (11.3%) and placebo (12.4%) subjects. In lean (BMI 20-25 kg/m ) age-matched men were included. Ex vivo-labeled 14 3 sum, liraglutide 3mg/d not only maintained diet-induced weight loss, it [1- C]VLDL-TG and [3- H]glucose tracers were infused postabsorptively and achieved additional clinically-relevant weight loss in a majority of subjects during a hyperinsulinemic euglycaemic clamp to determine VLDL-TG kinetics while demonstrating safety and overall tolerability similar to placebo. and EGP. Body composition was determined by DXA- and CT-scanning. Energy expenditure and substrate oxidation rates were measured by indirect Full analysis set Liraglutide Placebo calorimetry. N=207 N=206 In the basal period VLDL-TG secretion rates were increased in the UBO Additional Δwt(PR),% (kg)a –6.1c(–5.7)c –0.05(0.16) compared with lean men (1.25±0.34 vs. 0.86±0.34 µmol/kg FFM/min, Maintained RI wt loss, %b 81c 49 p=0.011) whereas there was no difference in clearance rates (150±56 vs. Lost ≥5% body wt(PR), %b 51c 22 162±77 ml/min, p=0.931) resulting in greater VLDL-TG concentrations (0.74±0.40 vs. 0.38±0.20 mmol/l, p=0.011). Insulin-mediated suppression of VLDL-TG secretion was impaired in UBO men (-36±18 vs. -54±10%, Safety analysis set N=212 N=210 p=0.008), and whereas clearance rates decreased in UBO men there was Subjects with AEs, n(%) no signifi cant change in lean men (-17±18 vs. 7±20%, p=0.007) resulting Total 194(91.5) 186(88.6) in impaired reduction of VLDL-TG concentrations in UBO men (-22±20 vs. Serious 9(4.2) 5(2.4) -56±11%, p<0.001). Withdrawals, n(%) Corrected for FFM, EGP was reduced in UBO men in the basal period Total 59(27.8) 69(32.9) (2.0±0.2 vs. 2.3±0.2 mg/kg FFM/min, p<0.001) whereas there was no From AEs 18(8.5) 18(8.6) difference during hyperinsulinemia (0.43±0.25 vs. 0.32±0.45 mg/kg FFM/ Nausea, % 47.6 17.1 min, p=0.139). Insulin-mediated suppression of EGP was comparable (-79±12 Vomiting, % 17 2 vs. -87±18%, p=0.250). Insulin mediated glucose disposal (GIR and Rd) were aANCOVA, blogistic regression, cp<0.0001 signifi cantly reduced in UBO compared with lean men. Conclusions: 1) Basal VLDL-TG secretion rates are increased in normoglycaemic but insulin resistant UBO men resulting in hypertri glyceri- demia. 2) Insulin-mediated suppression of EGP is preserved in UBO men & 1860-P whereas suppression of VLDL-TG secretion is impaired. Inability to suppress An Integrative Genomics Approach Identifi es Activation of Thio re- hepatic VLDL-TG secretion under conditions of hyperinsulinemia appears to doxin/Thioredoxin Reductase-1 Mediated Oxidative Stress De fense be an early pathological fi nding in male upper body obesity. Path way and Inhibition of Angiogenesis in Obese Non-Diabetic Human Subjects SWAPAN K. DAS, NEERAJ K. SHARMA, SANDRA J. HASSTEDT, ASHIS K. MON- & 1862-P DAL, LIJUN MA, KURT A. LANGBERG, STEVEN C. ELBEIN, Winston-Salem, NC, Naltrexone SR/Bupropion SR Therapy Reduced Body Weight and Salt Lake City, UT HbA1c in Overweight/Obese Women with Type 2 Diabetes Context. Obesity is a complex disease that involves both genetic and PRISCILLA HOLLANDER, JUDY LOPER, LAURENCE MIGNON, COLLEEN BURNS, environmental perturbations to gene networks in adipose tissue and is

Obesity RAUL HARRIS-COLLAZO, DENNIS KIM, Dallas, TX, Columbus, OH, La Jolla, CA proposed as a trigger for metabolic sequelae at the systems level. POSTERS The effects of 32 mg/day naltrexone sustained-release (SR)/360 mg/day Objective. We hypothesized that expression of adipose tissue transcripts bupropion SR combination therapy (NB32) were evaluated in COR-Diabetes, in gene networks for adaptive response would correlate with % fat mass in

Integrated Physiology/ a Phase 3, double-blind, placebo-controlled, 56-week study in overweight/ healthy non-diabetic subjects to maintain metabolic equilibrium and would obese subjects with type 2 diabetes (T2DM; baseline HbA1c 7-10% using overlap with genes modulated in response to elevated fatty acid. oral (or no) anti-diabetes medications). Randomization was 2:1 to NB32 or Design and Patients. Genome wide transcript profi les were determined in placebo. As approximately 80% of the prescriptions for obesity therapies are sc. adipose of 136 nondiabetics and in palmitate induced cell culture models fi lled by women, we conducted a subgroup analysis of women in this study. based on HapMap TLs. Genotype information and gene expression data Women represented half of the subjects enrolled in COR-Diabetes (N=285, in nondiabetic subjects was integrated to characterize the function of 41 56% of study population). Baseline characteristics were similar between obesity associated GWAS SNPs. groups: 73% Caucasian, mean age 53 years, BMI 36.5kg/m2, weight 97kg, Results. Genes in infl ammation-immune response, ER-stress, and cell- and HbA1c 7.8-7.9%. Completion rates were 45.6% for NB32 and 54.4% for extracellular matrix interaction were signifi cantly correlated with % fat mass. placebo. Week 56 weight loss was greater (p<0.001) with NB32 (-5.4±0.5%, The NRF2-mediated oxidative stress response pathway emerged as the most N=144) vs. placebo (-2.2±0.6%, N=84; modifi ed ITT-LOCF population: subjects enriched pathway among genes differentially expressed by palmitate in TLs

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A502 OBESITY—HUMANCATEGORY with ≥1 post-baseline weight on study drug). More NB32-treated women (vs. subjects, 31 had NFG, 33 had IFG, and 18 had type 2 diabetes. PHLPP-1 protein placebo) achieved weight loss of ≥5% (46.5 vs. 23.8%; p<0.001) and ≥10% expression was 2-fold higher in the three obese groups compared with non- (22.2 vs. 7.1%; p<0.01). NB32-treated women experienced HbA1c reductions obese (P=0.004). No differences were observed among obese subjects with of 0.5±0.1 vs. 0.0±0.1% with placebo (p=0.001), and a higher percentage NFG, IFG or type 2 diabetes. PHLPP-1 abundance was positively correlated achieved HbA1c <7%: 45.7 vs. 23.9% (p=0.002). While NB32-treated women with basal Akt Ser473 phosphorylation (r=-0.48; P=0.001), BMI (r=0.44; experienced greater improvements (p<0.05) vs. placebo in HDL-C (+3.31 vs. P<0.0001),circulating insulin levels (r=0.35; P<0.0001), and HOMA (r=0.38; -0.35 mg/dL) and triglycerides (-13.4 vs. +5.8%) there were no differences P<0.0001). Similarly, PHLPP-1 abundance was 2-fold higher in the skeletal between groups with respect to LDL-C, blood glucose, and insulin changes. muscle of 12 obese compared with 8 non-obese subjects (P<0.0001). Insulin The most frequent adverse events were nausea, vomiting, and diarrhea. In treatment of hepatoma HepG2 cells resulted in a dose and time-dependent conclusion, in overweight/obese women with type 2 diabetes, NB32 led to up-regulation of PHLPP-1. A 3 fold overexpression of PHLPP-1 in HepG2 cells clinically meaningful weight loss, which was similar to that observed in the resulted in impairment in insulin signalling, with a 70% reduction of Akt- men, and to reductions in HbA1c. Ser473 phosphorylation, 60% reduction in GSK-3α/β phosphorylation and a 65% reduction of glycogen synthesis, as compared with cell transfected with empty vector (all p<0.01). A 3 fold overexpression of PHLPP-1 in L6 myocytes caused a 50% reduction insulin stimulated Akt-Ser473 phosphorylation Guided Audio Tour: Interventional Treatment of Obesity (Posters 1863-P and 80% reduction in glucose transport (all p<0.01). Increased abundance to 1870-P), see page 15. of PHLPP-1, whose expression is regulated by insulin, may represent a new & 1863-P early molecular defect in insulin resistant states such as obesity. Variation in the Obesity Risk Gene FTO Determines the Fasting and Postprandial Cerebral Processing of Food Stimuli & 1865-P MARTIN HENI, RALF VEIT, CAROLINE KETTERER, STEPHANIE KULLMANN, Omentin Levels Are Signifi cantly Decreased in Patients with Morbid MARTINA GUTHOFF, HARALD STAIGER, FAUSTO MACHICAO, HANS-ULRICH Obesity and Increase after Bariatric Surgery HÄRING, HUBERT PREISSL, ANDREAS FRITSCHE, Tübingen, Germany FLORIAN HOELLERL, JOHANNA MARIA BRIX, GERIT HOLGER SCHERNTHANER, Variation in the FTO locus is the strongest genetic determinant of body HANS PETER KOPP, EVA GULZ, GUNTRAM SCHERNTHANER, Vienna, Austria weight. Besides the expression of this gene in the periphery, it is strongly Patients with morbid obesity have a high risk for cardiovascular (CV) expressed in the brain including the hypothalamus, a brain region involved morbidity, mortality and diabetes. The only effective method to reduce in food intake and metabolism. In humans there is fi rst evidence that FTO weight and the associated risks is bariatric surgery. Omentin, a novel infl uences food intake and nutrient specifi c food preference. We recently adipokine, – preferentially produced by visceral adipose tissue - was found demonstrated in humans that variation in this gene is associated with to be associated with insulin resistance and obesity. Thus it was of interest resistance to cerebral insulin action. In this study, we investigated dif ferences to evaluate Omentin levels in patients with morbid obesity before and after in brains’ processing of food-related stimuli between FTO genotypes in the weight loss induced by bariatric surgery. fasting state and during an oral glucose tolerance test. We included 81 morbid obese patients (MO, 69 women, BMI 47±7 kg/m²) We studied 24 healthy subjects genotyped for FTO SNP rs8050136, the and 39 healthy controls (BMI 26±3 kg/m²). All patients were investigated frequency of the obesity-risk A-allele was 38%. Participants were examined before and one year after gastric bypass surgery. Apart from weight and at three time points (0, 30 and 120 minutes) after ingestion of 75g glucose CV risk-markers, an oral glucose tolerance test (oGTT) was performed and solution or water (both 300 ml) on two days in a randomized order. Functional insulin resistance (IR) was calculated by using HOMA. Omentin levels were magnetic resonance imaging (fMRI) during visual food picture presentation determined in serum samples by an ELISA. was performed and blood oxygenation dependent-level (BOLD) was Omentin levels were signifi cantly decreased in MO vs healthy controls: measured at the three time points. 461±153 vs 619±196 ng/ml; p<0.001. After weight loss induced by bariatric We detected signifi cant differences in the cerebral processing of surgery Omentin levels signifi cantly increased (461± 153 vs 515±177 ng/ml; food stimuli between FTO genotypes in regions important for food intake p= 0.003). behaviour and energy balance. Differences were detected by functional Delta Omentin levels (pre minus post) were signifi cantly associated magnetic resonance imaging (fMRI) before and after ingesting glucose: with fasting plasma glucose (p=0.032), one hour (p=0.002) and two hours In the fasting state, food pictures elicited signifi cantly increased activity (p=0.007) plasma glucose levels in the OGTT. in risk allele carriers in the left fusiform area (p=0.01), a region important This is the fi rst study demonstrating (a) decreased Omentin levels in for object recognition and hedonic processing of food pictures. They also patients with MO compared to healthy controls and (b) a signifi cant increase showed lower activity in the hypothalamus, the brain’s key regulator of food of Omentin levels after dramatic weight loss induced by bariatric surgery. intake and metabolism 30 minutes after glucose load (p=0.02). Another area Since Omentin was shown to be associated with insulin sensitivity and processing food-related stimuli, the left orbitofrontal cortex showed higher glucose metabolism, the observed signifi cant increase in Omentin after activity 120 minutes post-load (p=0.03). dramatic weight loss might help to understand the benefi cial effects of For FTO’s obesity risk allele carriers, we detected alterations in food gastric bypass surgery. stimulated activity in three brain areas important for the regulation of food intake. This might help to understand how genetic variation infl uences body weight. & 1866-P Naltrexone SR/Bupropion SR Therapy in Overweight/Obese Subjects & 1864-P with Type 2 Diabetes Mellitus: Effects on Glycemic Control after Increased Expression of the Akt-Specifi c Phosphatase PH Domain Accounting for the Infl uence of Rescue Medications for Hyper- Leucine-Rich Repeat ProteinWITHDRAWN Phosphatase 1 (PHLPP-1) in Obese glycemia Subjects Is Associated with Insulin-Resistance HAROLD BAYS, SHERWYN SCHWARTZ, FRANK GREENWAY, BRENDA CLAPPER, FRANCESCO ANDREOZZI, CRISTINA PROCOPIO, ANNALISA GRECO, GAIA WHEDY WANG, DENNIS KIM, EDUARDO DUNAYEVICH, Louisville, KY, San CHIARA MANNINO, CLAUDIA MIELE, GREGORY ALEXANDER RACITI, CLAUDIA Antonio, TX, Baton Rouge, LA, La Jolla, CA IADICICCO, FRANCESCO BEGUINOT, ANTONIO E. PONTIROLI, MARTA L. HRIBAL, Obesity and adiposity promote type 2 diabetes mellitus (T2DM) and Obesity FRANCO FOLLI, GIORGIO SESTI, Catanzaro, Italy, Naples, Italy, Milan, Italy, San metabolic comorbidities. COR-Diabetes was a Phase 3, double-blind, placebo POSTERS Antonio, TX (PBO)-controlled, 56-week study evaluating naltrexone sustained-release To determine the contribution to peripheral insulin resistance of a recently (SR) 32 mg /bupropion SR 360 mg per day combination therapy (NB32) in Integrated Physiology/ identifi ed PH domain leucine-rich repeat protein phosphatase-1(PHLPP-1), overweight/obese subjects with T2DM (baseline HbA1c 7-10%) treated with which dephosphorylates Akt at Ser473 causing inhibition of its activity, we oral or no anti-T2DM drugs. Randomization was 2:1 (NB32:PBO). Completion measured its abundance in adipose tissue and skeletal muscle from obese rates were 52% for NB32 and 59% for PBO. Modifi ed ITT-LOCF population subjects. We also overexpressed PHLPP-1 in HepG2 and L6 cells to study included subjects with ≥1 post-baseline weight on study drug (NB32 N=265, its effect on insulin signaling. Subcutaneous adipose tissue samples were PBO N=159). Baseline characteristics: 54% female, 80% Caucasian, mean 2 obtained from 82 subjects with morbid obesity and 10 nonobese subjects age 54y, BMI 37kg/m , weight 106kg, and HbA1c 8.0%. Compared to with NFG. Quantifi cation of PHLPP-1 in human tissues was performed by PBO, NB32 resulted in greater weight loss (-5.0% vs -1.8%; p<0.001) and immunoblotting. The functional consequences of recombinant PHLPP-1 greater achievement of ≥5% weight loss (44.5% vs 18.9%; p<0.001). Fewer overexpression on insulin signalling and metabolic effectors in hepatoma NB32-treated subjects required rescue medications for unacceptable HepG2 cells and L6 myocytes, were also investigated. Of the 82 obese hyperglycemia during the study (22.3% vs 35.2%; p<0.01). To minimize

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A503 OBESITY—HUMANCATEGORY

the confounding effect of differential use of rescue medications, the last hsCRP (-33.7 vs. -3.7%), insulin (-11.5 vs. +3.3%), and glucose (-3.07 vs. -1.70 available glycemic measure prior to initiation of rescue medication was mg/dL). Larger improvements in weight-related quality of life measures carried forward in the following post hoc analyses for glycemic parameters: were observed with NB32 vs. PBO (p<0.001). Blood pressure was generally Compared to PBO, NB32 improved HbA1c (-0.6% vs 0.1%; p<0.001), fasting unchanged in all groups; pulse was ∼1 bpm higher with NB than PBO. NB32- glucose (-11.2% vs 2.0%; p=0.001), and HOMA-IR (-19.0% vs -5.7%; p<0.05); treated women reported greater reductions vs. PBO-treated women (p<0.05) fasting insulin was numerically reduced for NB32 (-12.8% vs -6.8%; p=0.20). in frequency of food cravings, diffi culty resisting food cravings, and diffi culty The most frequent adverse events were nausea (42.3% NB32 vs 7.1% PBO), controlling eating. The most frequent adverse events with NB were nausea, constipation (17.7% vs 7.1%), and vomiting (18.3% vs 3.6%). Nausea was constipation, headache, dry mouth, and insomnia. NB32 was generally mostly mild/moderate in severity, transient, and occurred early in the study. well-tolerated, led to clinically meaningful, sustained weight loss and In overweight/obese subjects with T2DM, NB32 produced improvements improvements in metabolic parameters, quality of life and control of eating, in body weight and glycemic control compared to PBO. These results were and may be of particular value in the treatment of obesity in women. associated with signifi cant reductions in rescue therapy use for unacceptable hyperglycemia, which, when accounted for, resulted in more prominent & 1869-P improvements in glycemic parameters. A New Body Adiposity Index (BAI) Which Applies for Both Sexes and Two Ethnic Groups & 1867-P RICHARD N. BERGMAN, DARKO STEFANOVSKI, THOMAS A. BUCHANAN, ANNE Association between Cathepsin K and Energy Homeostasis in Post- E. SUMNER, JAMES C. REYNOLDS, NANCY G. SEBRING, ANNY H. XIANG, menopausal Women RICHARD W. WATANABE, Los Angeles, CA, Bethesda, MD, Pasadena, CA BARBARA BUDAY, PETER F. PACH, BOTOND L. NAGY, MARTA VITAI, LÁSZLÓ Obesity is a growing problem in the United States and throughout the KORÁNYI, Balatonfüred, Hungary, Veszprém, Hungary world. Background: Cathepsin K (CatK) is an osteolytic enzyme involved in bone It is a risk factor for Type 2 diabetes, as well as cardiovascular disease, resorption and currently a pharmateutical industry target for treatment hypertension and cancer. It is important clinically to estimate percent body of osteoporosis.It has an established role in infl ammation and several fat of individual patients. The body mass index, or BMI, has been used to malignancies and is a newly identifi ed factor important for adipogenesis. assess body fat for almost 200 years. Defi ciency or selective inhibition of CatK activity reduces preadipocyte BMI is known to be of limited accuracy, and is different for males and differentiation and body weight, serum insulin and glucose levels in diet females with similar percent body adiposity. induced obese mice. Here we introduce a new index of adiposity, the Body Adiposity Index Patients and Methods: We investigated serum CatK levels in 55 post- (BAI). We used a Mexican-American population from the “BetaGene” study, menopausal women with normal (NGT, n=21) and impaired glucose tolerance to develop the BAI. Percent body fat, as measured by the DEXA, was used as (IFG, IGT, drug naive type 2 DM, n=34). OGTT, IVGTT and hyperinsulinemic a “gold standard” for validation. Hip circumference (R=0.602) and height (R=- euglycemic clamp were done to assess glucose tolerance, insulin secretion 0.524) were strongly correlated with percent body fat and therefore chosen and insulin sensitivity (whole body and muscle glucose uptake, M and Mm, as principal anthropometric measures upon which we based BAI. We defi ned mg/min/kg). Adipocytokines, serum lipids, markers of bone turn over were the novel parameter as (BAI= ([hip circumference]/([height]1.5)–18). Among also measured. the Beta-Gene participants (n=1733), the BAI was able to estimate percent Results: CatK levels did not differ signifi cantly between NGT and IFG/ body fat for adult men and women without numerical correction (R=0.79, IGT/2DM subjects (9.85±8.57 vs.11.04±9.35 pmol/l, p=0.61). CatK showed see Figure). signifi cant (p<0.05) negative bivariate correlation with BMI (r=-0.32), body The BAI measure was validated in the “TARA” study of African Americans. fat percent (BFP, r=-0.28), OGTT glucose area under the curve (AUC) (r=-0.26), Correlation between DEXA-derived percent adiposity and the BAI was OGTT insulin AUC, serum leptin (r=-0.31), clamp M (r=0.27), Mm (r=0.34). R=0.85 for TARA with a concordance of C_b=0.95. BAI provides an accurate It showed no correlation with osteocalcin (OCN) and osteoprotegerin but estimate of percent adiposity in men and women, Mexican-American and strong negative correlation (r=-0.46) with RANKL (receptor activator for African-American, and appears to work in Caucasian Americans. BAI is nuclear factor κB ligand), i.e. members of the “OCN/insulin axis”, linking measured without weighing, which may render it useful in settings where bone turn over to glucose/insulin metabolism. These associations were measuring accurate body weight is problematic. We have defi ned a new independent of glucose homeostasis, i.e. stayed signifi cant when assessed parameter, the BAI, which can be calculated from hip circumference and separately in the NGT and IFG/IGT/2DM group. Correlations with OGTT height only. It can be used in the clinical setting to predict percent adiposity insulin levels and RANKL stayed signifi cant but disappeared with M and Mm even in remote locations with very limited access to reliable scales. The BAI after adjustment with BMI and BFP. estimates percent adiposity directly. Conclusion: Our results suggest a different role of CatK in the energy homeostasis of humans compared to mice. Further investigations are needed to elucidate the metabolic role of CatK in humans.

& 1868-P Effects of Naltrexone SR/Bupropion SR on Weight and Obesity- Related Health Risks in Overweight/Obese Women from the COR-II Trial DOMENICA RUBINO, CAROLINE APOVIAN, LAURENCE MIGNON, COLLEEN BURNS, RAUL HARRIS-COLLAZO, DENNIS KIM, Arlington, VA, Boston, MA, La Jolla, CA The effects of 32mg/day naltrexone sustained-release (SR)/360mg/day bupropion SR combination therapy (NB32) were evaluated in overweight/ obese subjects in COR-II, a Phase 3, double-blind, placebo(PBO)-controlled,

Obesity 56-week study. Randomization was 2:1 to NB32 or PBO. As approximately

POSTERS 80% of the prescriptions for obesity therapies are fi lled by women, we conducted a subgroup analysis on women in this study. Of 1496 participants,

Integrated Physiology/ 1267 were women, with these baseline characteristics: 82% Caucasian, mean age 44 years, BMI 36.1 kg/m2, weight 97 kg, waist circumference 107 cm, HDL-C 53.8 mg/dL, LDL-C 120.7 mg/dL, triglycerides 123.7 mg/dL, hsCRP 6.6 mg/L, insulin 14.1 µU/mL, and glucose 93.9 mg/dL. Completion rates were 52% for NB32 and 53% for PBO. Week 56 weight loss was greater (p<0.001) with NB32 (-6.6%) vs.PBO (-1.4%) (modifi ed ITT-LOCF population: ≥1 post- baseline weight on study drug). More NB32-treated women achieved ≥5% (51.2 vs. 17.9%), ≥10% (29.5 vs. 6.8%), and ≥15% (14.5 vs. 2.9%) weight loss (all p<0.001). NB32-treated women also experienced greater improvements (p<0.05) vs. PBO in waist circumference (-6.9 vs. -2.2 cm), HDL-C (+4.07 vs. -1.29 mg/dL), LDL-C (-6.93 vs. -3.08 mg/dL), triglycerides (-10.0 vs. -2.7%),

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A504 OBESITY—HUMANCATEGORY

& 1870-P & 1872-P Relationship between Total Body Fat and Body Fat Distribution and Relationship between Changes in Plasma Adiponectin Concentra- Left Ventricular Diastolic Dysfunction in Men with the Metabolic tion and Insulin Sensitivity after Extended-Release Niacin Therapy Syndrome GEMMA FRATERRIGO, ELISA FABBRINI, STEVE O’RAHILLY, SAMUEL KLEIN, St. ANNIE MORIN, JEAN-PIERRE DESPRÉS, MARIE ARSENAULT, MARIE-KRISTELLE Louis, MO, Cambridge, United Kingdom ROSS, JEAN BERGERON, ANGELO TREMBLAY, NATHALIE ALMERAS, PAUL POIRIER, Obesity is associated with insulin resistance and dyslipidemia, which are Quebec, QC, Canada important risk factors for cardiovascular disease. Extended-release (ER) Left ventricular diastolic dysfunction (LVDD) has been observed in niacin is a nicotinic acid formulation used to treat dyslipidemia in obese subjects with metabolic syndrome. We examined the relationship between subjects. Nicotinic acid binds to the GPR109A receptor in adipose tissue, several indices of adiposity (waist circumference, waist-to-hip ratio, waist- which stimulates adiponectin secretion. Although increased adiponectin to-height ratio, body mass index, body fat estimated by bioimpedance, and secretion should improve insulin sensitivity, treatment with nicotinic acid visceral adiposity assessed with computed tomography) and LVDD in men derivatives often causes insulin resistance. The purpose of this study was with metabolic syndrome without heart disease. A total of 76 men between to test the hypothesis that ER niacin-induced changes in plasma adiponectin 30-65 years (age 48.5 yrs) evaluated by echocardiography for the presence concentration are associated with an amelioration of ER niacin’s adverse of LVDD, using doppler mitral infl ow with/without Valsalva maneuver, effect on insulin action. A euglycemic-hyperinsulinemic clamp procedure, in 2 pulmonary venous fl ow and tissue doppler imaging, were included. LVDD conjunction with [6,6- H2]glucose tracer infusion, was used to assess skeletal was graded according to guidelines. All had normal left ventricular ejection muscle insulin sensitivity, before and after 16 wks of ER niacin therapy (2000 fraction. Twenty-one subjects were classifi ed as normal, whereas 55 were mg/d) in 9 obese subjects (age: 42±14 y; BMI: 35.1±3.9 kg/m2; means ± classifi ed as abnormal in terms of LVDD, including 17 grade I, 33 grade 2, SD) who had normal oral glucose tolerance. In addition, body composition and 5 as unclassifi ed. Groups were comparable regarding adiposity indices. was assessed by using DXA. ER niacin therapy did not affect body weight Visceral adipose tissue volume was also similar between groups: normal (99.1±12.6 kg before and 100±13.1 kg after) or % body fat (37.8±7.5 % before diastolic function (mean volume 1778±480 cm3) vs. LVDD (1938±460 cm3); and 37±7.5 % after). However, ER niacin therapy caused a 22% reduction in grade I (1924±531 cm3) vs. grade II (1923±439 cm3). However, subcutaneous skeletal muscle glucose disposal during insulin infusion (183±6.7% increase adipose tissue volume was different between groups; 2121±828 cm3 for the during insulin before treatment and 142±78% increase during insulin after group with normal function vs. 1698±499 cm3 for LVDD (p=0.04). There was treatment). The decline in glucose disposal was inversely correlated with an no signifi cant difference between grades I and II. Ratio of visceral adipose increase in serum adiponectin concentration (r=0.665, p=0.05). tissue volume on total adipose tissue volume was also statistically different between normal function (0.47±0.11) and LVDD (0.53±0.07) (p=0.01). Again, no signifi cant difference was found between grades I and II. Thus, after adjustment for age, no adiposity indices explained the presence or the severity of LVDD. Furthermore, there was no difference regarding fasting glucose, insulinemia, C-peptide, insulin resistance, lipid profi le, C-reactive protein and adiponectin between groups. In conclusion, with age taken into consideration, we did not observed any signifi cant relationship between indices of adipose tissue distribution and LVDD in our sample of asymptomatic men with features of the metabolic syndrome.

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& 1871-P Reduction in the Expression of β-Amyloid Precursor Protein (APP) Following Roux-en-Y Gastric Bypass Surgery (RYGB) and Weight Loss PARESH DANDONA, HUSAM GHANIM, SCOTT MONTE, CHANG LING SIA, These results demonstrate that ER niacin causes skeletal muscle insulin JEROME SCHENTAG, SANDEEP DHINDSA, JOSEPH CARUANA, Buffalo, NY resistance, independent of changes in body weight or body fat. However, Obesity and type 2 diabetes are associated with an increase in the ER niacin also increases plasma adiponectin concentration, which blunts ER incidence and prevalence of Alzheimer’s disease (AD). We have recently niacin’s adverse effect on insulin action. demonstrated that peripheral blood mononuclear cells (MNC) express APP, the precursor of β-amyloid, which forms the pathognomonic plaques in the & 1873-P brain. We hypothesized that APP expression diminishes after the marked Short-Term Overfeeding in Lean Men Decreases Insulin Sensitivity caloric restriction and weight loss associated with RYGB. Fifteen type 2 and Decreases Parasympathetic Activity as Measured by Heart diabetic patients with morbid obesity underwent RYGB following which Rate Variability their caloric intake diminished and they lost 38.5 ±2.9 Kg in weight over 6 NICOLETTE M. LAMMERS, JOHN M. KAREMAKER, BEREND E. WESTERHOF, months. BMI fell from 54.4±3.1 to 40.5± 2.9 Kg/m2. There was a signifi cant MYRTE BRANDS, MARIETTE T. ACKERMANS, ERIC FLIERS, SUSANNE E. LA fall in plasma concentrations of glucose (from 148±8 to 101±4mg/dl), insulin FLEUR, MIREILLE J. SERLIE, Amsterdam, The Netherlands (from 18.5±2.2 to 8.6±1.0µU/ml) and HOMA-IR (from 7.1±1.1 to 2.1±0.3).). The The autonomic nervous system is involved in body weight regulation and expression of APP mRNA fell by 31±9% and that of protein fell by 22±12%. In insulin sensitivity. It has been suggested that obesity and its metabolic addition, there was a reduction in other AD related genes including presinilin consequences are in part explained by changes in the activity of the 2 (PN2) which fell by 27±10%, ADAM-9 which fell by 35±12% and GSK-3β parasympathetic and sympathetic nervous system. We performed this which fell by 28±6% (P<0.01 for all). PN2 mediates the conversion of APP study to determine the infl uence of a hypercaloric diet (HD) on the autonomic Obesity POSTERS into β-amyloid while GSK-3β hyperphosphorylates tau protein to form the nervous system in non-obese subjects. neurofi brillary tangles in the brains of patients with AD. These changes We submitted healthy lean men to a HD to reach a 5% increase in body occurred in parallel with reductions in other pro-infl ammatory mediators weight over a period of 5 weeks. We measured plasma glucose and insulin, Integrated Physiology/ including CRP (from 10.7±1.6 to 5.8±1.0mg/L, P<0.001). Thus, the reversal 24hrs excretion of catecholamines and resting energy expenditure (REE) of the pro-infl ammatory state of obesity is associated with a concomitant after an overnight fast before and after the HD. Heart rate (HR) and blood reduction in the expression of APP and other AD related genes in MNC. pressure were recorded continuously for 10 minutes in the supine and 10 We conclude that obesity and caloric intake modulate the expression of minutes in the standing position. Barorefl ex sensitivity was assessed with APP in MNC. If indeed, this effect also occurs in the brain, this may have spectral analysis of the heat rate and blood pressure variability. implications for the pathogenesis and the treatment of AD. It is relevant that We studied 9 lean men (age 37 [27-43] years and BMI 23.6 [20.6-25.6] cognitive function has been shown to improve with weight loss following kg/m2). They gained 7 [5.1-7.6] % of their initial body weight. Plasma insulin bariatric surgery. increased after the HD (insulin before 27 ± 38.6 vs. after 48 ± 30.8 pmol/L, p = Supported by: NIH ADA-Funded Research 0.012). HOMA-IR increased signifi cantly after the HD. REE and catecholamine excretion did not change. HR increased in the supine position (HR supine

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before 63 ± 8 vs. after 69 ± 8 bpm, p = 0.049). Low frequency gain (LF-gain), least-squares (LS) mean percent weight loss at Week 56 was signifi cantly a measure for barorefl ex sensitivity, decreased after the HD (LF-gain before greater for both doses of PHEN/TPM CR compared with PBO (ITT-LOCF; 13.0 ± 9.7 vs. after 9.5 ± 6.1 msec/mmHg, p = 0.017). HF variability (HFvar), P<0.0001): 2.3%, 8.2%, and 10.5% for PBO, 7.5/46, and 15/92, respectively. primarily determined by respiratory vagal functions, decreased after the HD PHEN/TPM CR was generally well tolerated; most common adverse events (HFvar before 496 ± 795 vs. after 354 ± 519 msec², p = 0,050). LF/HF ratio, an were constipation, dry mouth, and paraesthesia. Weight loss associated indicator of sympathovagal balance, did not change after the HD. with PHEN/TPM CR led to resolution of the MetS diagnosis in nearly half of Our results indicate that a hypercaloric diet induces insulin resistance in subjects, and resulted in improvements in the individual components of the lean healthy men and has signifi cant effects on cardiovascular autonomic MetS trait cluster. indices which point to changes towards sympathetic predominance/vagal Table. LS Mean Change in MetS Components at Week 56 (MetS Sample withdrawal. This suggests that functional changes in the autonomic nervous ITT-LOCF) system are part of the adaptive response to a hypercaloric milieu and may in PBO 7.5/46 15/92 part mediate the changes in glucose metabolism. WC change (cm) -3.7 -8.4* -10.1*‡ & 1874-P SBP change (mm Hg) -2.8 -5.5† -6.7* Naltrexone SR/Bupropion SR Combination Therapy Improves Control DBP change (mm Hg) -3.2 -3.8 -4.3† of Eating and Reduces Food Cravings in Overweight and Obese Subjects FG change (mg/dL) -1.8 -4.6† -6.6* JAMES O. HILL, HOLLY WYATT, SONJA K. BILLES, COLLEEN BURNS, RAUL TG % change (%) -3.9 -16.6* -17.9* HARRIS-COLLAZO, EDUARDO DUNAYEVICH, JOHN BLUNDELL, Denver, CO, La Jolla, CA, Leeds, United Kingdom HDL-C % change (%) 4.0 8.8† 10.3* The effects of naltrexone/bupropion combination therapy (NB) on obesity *P<0.0001 vs PBO; †P<0.05 vs PBO; ‡P<0.05 vs 7.5/46 and eating behavior (Control of Eating Questionnaire: CoEQ, twenty 100mm Supported by: Vivus, Inc. visual analog scales) were evaluated in obese/overweight individuals in the COR-II study at baseline and Weeks 8, 16, 28, and 56. This Phase 3, double-blind, placebo-controlled, 56-week study, randomized 1496 subjects & 1876-P in a 2:1 ratio to NB32 (32mg naltrexone sustained-release [SR]/360mg Impact of Maximum BMI Against Development of Retinopathy and bupropion SR) or placebo. The completion rate was 54% in each group. Increase of an Albumin to Creatinine Ratio (ACR): The Report of Baseline characteristics for the modifi ed ITT-LOCF population (subjects with Longitudinal National Survey of Early-Onset Type 2 Diabetes ≥1 post-baseline weight on study drug) were: 84% female, 85% Caucasian, YASUKO UCHIGATA, NAOKO TAJIMA, RIMEI NISHIMURA, NARIHIKO YOSHIOKA, mean±SD age 44±11y, weight 100±16kg, and BMI 36±4kg/m2. Week 56 TATSUHIKO URAKAMI, NOBUYUKI KIKUCHI, AKIRA TAKEDA, TAISUKE OKADA, weight loss was greater (p<0.001) with NB32 (-6.4%) vs. placebo (-1.2%) EIICHI ARAKI, YASUO OHASHI, YASUHIKO IWAMOTO, Tokyo, Japan, Sapporo, and more NB32-treated vs. placebo-treated subjects achieved weight loss Japan, Yokohama, Japan, Tottori, Japan, Kochi, Japan, Kumamoto, Japan ≥5% (50% vs. 17%) and ≥10% (28% vs. 6%). NB32-treated subjects reported Early-onset type 2 diabetes can be detected early by school urinalysis reduced diffi culty in control of eating at Week 56 (CoEQ item 19; p<0.01) and, screening in Japan. However, patients tend to stop treatment, thus in a post hoc analysis, at all other time points (p<0.01). Post hoc analyses becoming susceptible to severe diabetic complications from their 30s. We showed differences (p<0.05) at Week 8 between NB32 and placebo in other started a nationwide prospective study on the development of diabetic CoEQ items indicating reduced desire for sweet and non-sweet tasty foods, complications, in which type 2 diabetes patients diagnosed at the age ≤ reduced cravings for chocolate and other sweet foods, decreased hunger, 19 years and <30 years of age at the end of 2006 were recruited (except reduced frequency and strength of food cravings, decreased diffi culty MODY, GAD-Ab positive patients, and insulin-treated patients in one resisting food cravings, decreased eating in response to cravings, and month after the diagnosis).The aim was to identify clinical parameters for increased ability to resist craved foods. Many remained improved at Week prognostic factors against new development of retinopathy or ACR ≥300 28 and some at Week 56. There was a trend for NB32 to be associated with mg/gCr as endpoints. Cox’s proportional hazards model was used. The numerical improvements in mood-related items compared to placebo. Blood subjects for retinopathy endpoint were 417 (male/female,202/215; clinical pressure was generally unchanged in all groups; pulse was approximately characteristics at enrollment were;19.5±4.6 years old; BMI,30.2±3.5 kg/ 2 2 1 bpm higher with NB than placebo. The most common adverse events m (maximum BMI,30.7±4.0 kg/m); systolic/diastolic blood pressures, were nausea, constipation, headache, insomnia and dry mouth. Compared 122.2±17/71.7±13 mmHg; A1C,7.9±2.5%; total cholesterol,190.1±41 mg/dl; to placebo, NB32 was generally associated with greater control of eating, and ACR,56.1±292 mg/gCr). After a follow-up period of 2.5±0.9 years, 28 reduced food cravings, and greater control over the response to food subjects (6.7%) reached the endpoint. Maximum BMI (p=0.020, 95%CI: 1.01 cravings. These results suggest that improved control of eating may be one to 1.17) and diabetes duration (p=0.013, 95%CI: 1.03 to 1.25) were identifi ed mechanism for weight loss with NB32. as prognostic factors. The subjects for nephropathy endpoint were 445 Supported by: Orexigen Therapeutics (male/female, 211/234; clinical characteristics at enrollment were;19.9±4.7 years old; BMI,29.9±3.5 kg/m2 (maximum BMI, 30.7±4.0 kg/m2); systolic/ diastolic blood pressures,122.5±17 /71.8±12 mmHg; A1C,8.2±2.5%; total & 1875-P cholesterol,190.2±40 mg/dl; and ACR,25.2±45.0 mg/gCr). After a follow-up Weight Loss with Controlled-Release Phentermine/Topiramate period of 2.1±1.1 years, 13 subjects (2.9%) reached the endpoint. Maximum (PHEN/TPM CR) Reverses Metabolic Syndrome (MetS) and Improves BMI (p=0.035, 95%CI: 1.01 to 1.16) was identifi ed as a prognostic factor. In Associated Traits conclusion, apart from the parameters of glucose metabolism, lipid profi le, W. TIMOTHY GARVEY, ROBERT R. HENRY, CRAIG A. PETERSON, CHARLES H. and blood pressure, maximum BMI was identifi ed as a prognostic factor for BOWDEN, Birmingham, AL, San Diego, CA, Mountain View, CA the development of background retinopathy and an ACR ≥300 mg/gCr. The MetS is a risk factor for type 2 diabetes and is exacerbated by obesity. independent importance of BMI was confi rmed in care of early-onset type When ≥3 of these 5 criteria are met, MetS can be diagnosed: waist 2 diabetes. circumference (WC) ≥102 cm in men, ≥88 cm in women; triglycerides (TG) Supported by: Manpei Suzuki Diabetes Foundation

Obesity ≥150 mg/dL; high-density lipoprotein cholesterol (HDL-C) <40 mg/dL in men, POSTERS <50 mg/dL in women; systolic blood pressure (SBP) ≥130 & diastolic BP (DBP) ≥85 mm Hg; and fasting glucose (FG) ≥100 mg/dL. A 56-week, Phase 3 trial & 1877-P Naltrexone SR/Bupropion SR Combination Therapy Reduced Weight Integrated Physiology/ (CONQUER) evaluated PHEN/TPM CR for weight loss in overweight/obese subjects randomized to receive placebo (PBO), PHEN 7.5 mg/TPM CR 46 and Improved Weight-Related Quality of Life and Physical Health in mg (7.5/46), or PHEN 15 mg/TPM CR 92 mg (15/92); all subjects received Overweight and Obese Subjects lifestyle intervention. In subanalysis, at baseline, 59.8% (n=1460) of subjects RONETTE L. KOLOTKIN, CHRISTOPHER STILL, HOLLY MAIER, COLLEEN BURNS, met criteria for MetS. By Week 56, 43.7% and 51.1% of subjects treated with RAUL HARRIS-COLLAZO, EDUARDO DUNAYEVICH, LOUIS J. ARONNE, Durham, 7.5/46 and 15/92, respectively, had resolution of their MetS compared with NC, Danville, PA, La Jolla, CA, New York, NY only 28.9% of subjects receiving PBO (P<0.0001). In subjects without MetS The effects of naltrexone sustained-release (SR)/bupropion SR combina- at baseline (n=983), progression to MetS was 23.3% on PBO and 14.5% and tion therapy (NB) on weight loss, weight-related quality of life (using the 16.3% on 7.5/46 and 15/92, respectively (P<0.02). For subjects with MetS at Impact of Weight on Quality of Life-Lite [IWQOL-Lite] questionnaire) and baseline, treatment with PHEN/TPM CR resulted in greater improvements physical and mental health (using the SF-36 Health Survey) were evaluated in the individual components of MetS when compared with PBO (Table) and in overweight/obese individuals in the COR-II study, a Phase 3, double-blind,

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A506 OBESITY—HUMANCATEGORY placebo-controlled 56-week study. Subjects (N=1496) were randomized 2:1 to but the sleeve gastrectomy groups in those studies have all been 80 or less NB32 (32mg naltrexone SR/360mg bupropion SR) or placebo. The completion patients. This large study compares the laparoscopic gastric bypass (LGB) rate was 54% in each group. Baseline characteristics for the modifi ed ITT- and the laparoscopic sleeve gastrectomy (LSG) performed by the same two LOCF population (subjects with ≥1 post-baseline weight on study drug) were: surgeons in a community practice. We conducted a retrospective chart 84% female, 85% Caucasian, mean±SD age 44±11y, weight 100±16kg, BMI review to compare the complications associated with the LGB and the LSG 36±4kg/m2, IWQOL-Lite Total score 73±17 (moderate impairment), IWQOL- operatively and during 6 weeks post-operatively. Weight loss induced by Lite subscales: Physical Function 70.4±20.4, Self-Esteem 56.2±25.6, Sexual the two surgeries and the percentage of insured patients versus private Life 75.5±26.3, Public Distress 86.6±18.0, Work 87.1±17.1, and SF-36: pay patients was compared. There were 835 LGB and 766 LSG patients. The Physical Component 50.2±7.1 and Mental Component 54.4±7.3 (a score of 50 prevalence of complications was greater with the LGB both operatively (55.4% is equivalent to average health). Week 56 weight loss was greater (p<0.001) vs. 4.0%, p<0.001) and during the fi rst 6 weeks post-operatively (15.0% vs. with NB32 (-6.4%) vs. placebo (-1.2%). NB32-treated subjects reported 4.3%, p<0.001). The specifi c complications with a higher prevalence included greater improvements vs. placebo in IWQOL-Lite Total score (p<0.001) and gastrointestinal bleeding (3.0% vs. 0.1%, p<0.001), small bowel obstruction in all subscales except Work (p<0.01 for subscales). In post hoc analyses, (1.1% vs. 0%, p<0.025) and cardiopulmonary complications (2.3% vs. 0.3%, more NB32-treated vs. placebo-treated subjects experienced clinically p<0.001). LGB patients lost signifi cantly more weight than LSG patients at meaningful improvements in IWQOL-Lite Total score (53% vs. 33%; p<0.001). both 1 and 2 years (-36.4% vs. -29.0% and -42.7% vs. -30.3%, respectively, NB32-treated subjects experienced greater improvements vs. placebo in the p<0.001). Insurance covered 90% of LGB patients and only 8.2% of LSG SF-36 Physical Component Summary score (p<0.001), as refl ected by greater patients. Based on this largest yet reported series, we conclude that the improvements vs. placebo in the Physical Functioning, Role Physical, General LSG is a safer operation, but it results in less weight loss compared to the Health, and Vitality components (all p<0.01). There was no treatment LGB. However, LSG weight loss reported in this study is similar to published difference in the SF-36 Mental Component. Blood pressure was generally LGB weight loss. Insurance coverage for LSG is much less common than the unchanged in all groups; pulse was approximately 1 bpm higher with NB more established LGB. than placebo. The most common adverse events were nausea, constipation, Supported by: Ethicon headache, dry mouth and insomnia. NB was generally well-tolerated, and led to reduced weight and improved weight-related quality of life and physical 1880-P health. Angiogenin Increases after Bariatric Surgery in Morbid Obesity Supported by: Orexigen Therapeutics JOHANNA MARIA BRIX, FLORIAN HOELLERL, HANS PETER KOPP, GERIT HOLGER SCHERNTHANER, EVA KRATZ, GUNTRAM SCHERNTHANER, Vienna, Austria & 1878-P Angiogenin – a 14,124-Da soluble protein - is an important angiogenic Randomized, Placebo-Controlled Trial of Lorcaserin for Weight growth factor that has a great infl uence on the process of creation of new Loss in Patients with Type 2 Diabetes vessels. In addition, angiogenin induces nitric oxide synthesis in endothelial PATRICK M. O’NEIL, MEREDITH C. FIDLER, MATILDE SANCHEZ, NEIL J. WEISS- cells. Since patients with morbid obesity (MO) have an increased risk for MAN, STEVEN R. SMITH, CHRISTEN M. ANDERSON, WILLIAM R. SHANAHAN, CVD and information about angiogenesis is lacking in these high risk patients Charleston, SC, San Diego, CA, Washington, DC, Winter Park, FL we were interested in angiogenin levels in patients with MO compared to Lorcaserin (Lor) is a selective 5HT2C agonist that has been shown to induce healthy lean controls (CO), as well as before and after bariatric surgery weight loss in non-diabetic obese and overweight patients (pts). BLOOM- (BS). DM was a 52 week (wk), randomized, double-blind, placebo-controlled study We included 80 patients (mean age: 39± 11 years; mean BMI: 45.8 ± 6.9 in pts with type 2 diabetes inadequately controlled with oral agents (HbA1C kg/m²) with MO in comparison with 40 CO (mean age: 47± 8 years; mean 7-10%), 18 - 65 years-old, with a BMI between 27 and 45 kg/m2. Pts were BMI: 25.3 ± 5.7 kg/m²). All patients were investigated before and 2 years initially randomized 1:1:1 to Lor 10 mg BID (L), 10 mg QD, and placebo (P). after BS. Apart from weight and CV risk-markers (blood pressure, lipids), Enrollment in the 10 mg QD group was halted prematurely to accelerate trial a glucose tolerance test (75g), renal and infl ammation parameters were completion, and results for this group (n=95) are not presented. The primary assessed. Angiogenin levels were assessed by a commercial ELISA. endpoints, at wk 52 in hierarchical order, were: proportion of patients with Patients with MO had signifi cant lower angiogenin levels than CO: 548 ± ≥5% weight loss; absolute change in body weight and proportion of patients 163ng/ml vs 624±123 ng/ml; p= 0.04. In the MO patients angiogenin levels with ≥10% weight loss. At baseline (BL), mean BMI was 36 kg/m2, 46% were increased signifi cantly two years after bariatric surgery: 548 ± 163ng/ml male and the mean age was 53 years. HbA1C was 8.1%, systolic and diastolic vs 612±164 ng/ml; p<0.001. In the correlation analysis Δangiogenin levels blood pressure (BP) were 127 and 78 mmHg, respectively; and LDL, HDL and were associated with ΔTriglycerides (R= 0.361; p=0.009) and Δ2hour total cholesterol, and triglycerides were 95 mg/dL, 45 mg/dL, 173 mg/dL postprandial Insulin (R= 0.404; p= 0.024) In a multivariate model Δ2hour and 169 mg/dL, respectively. Most pts were White (62%), Black (20%) or postprandial Insulin (Beta= 0.4070; p=0.021) remained as a single predictor Hispanic (13%). 66% of 256 L and 62% of 253 P pts completed the trial. At for Δangiogenin. wk 52 (MITT-LOCF), 37.5% and 16.1% of pts receiving L and P, respectively, Patients with MO have signifi cantly lower angiogenin levels compared achieved ≥5% weight loss (p<0.001), and 16.3 and 4.4% achieved ≥10% with healthy controls, but angiogenin levels increase signifi cantly after weight loss (p<0.001), respectively. LS Mean weight loss in the L group weight loss. Since angiogenin can induce angiogenesis and helps formatting was 4.5% vs. 1.5% in the P group (p<0.001). Signifi cant improvements (LS new blood vessels, our results might help to understand the benefi cial mean ±SE) over P were also seen for HbA1C (-0.93 ±0.06% vs. -0.44 ±0.06%, effects of bariatric surgery regarding CV morbidity and mortality. p<0.001), fasting glucose (-27.4 ±2.5 mg/dL vs. -11.9 ±2.5 mg/dL, p<0.001) and HOMA-IR (-0.54 ±0.11 vs. -0.23 ±0.11, p=0.02). BP did not change signifi cantly 1881-P in either group. L was associated with greater incidence of headache (14.5% Associations of Polymorphisms in Several Common Obesity- L vs. 7.1% P) and fatigue (7.4% L vs. 4.0% P). Symptomatic “hypoglycemia” Susceptibility Genes with Body-Mass Index and Other Anthro- events were reported by 7.4% and 6.3% of L and P pts, respectively. SAEs pometric Characteristics were infrequent and similarly distributed. Discontinuations for adverse PETRA LUKASOVA, MARKETA VANKOVA, JOSEF VCELAK, OLGA BRADNOVA, events were 8.6% for L and 4.3% for P pts. Lor over a one-year period HANA KVASNICKOVA, BELA BENDLOVA, Prague, Czech Republic Obesity produced statistically signifi cant weight loss and improved glycemic control Large-scale genome-wide association studies have identifi ed several POSTERS in obese and overweight pts with type 2 diabetes. genetic obesity-related loci with different effect sizes. We aimed to

assess the effects of 10 obesity-susceptibility variants rs9939609 in FTO; Integrated Physiology/ 1879-P rs12970134 in MC4R; rs1800592 in UCP1; rs7561317 in TMEM18; rs29941 A Retrospective Chart Review of Laparoscopic Sleeve Gastrectomy in KCTD15; rs6232 in PCSK1; rs7498665 in SH2B1; rs10913469 in SEC16B; and Laparoscopic Gastric Bypass Patients in a Private Surgical rs4923461 and rs925946 in BDNF genes on body-mass index, waist circum- Practice fer ence and body composition. Our study group contained 597 women (18- BROOKE E. BAYHAM, DRAKE BELLANGER, FRANK L. GREENWAY, WILLIAM 50 years; BMI 16-47 kg/m2) without diagnosis of diabetes mellitus type 2. JOHNSON, Baton Rouge, LA, Gonzales, LA The strongest association with BMI revealed rs9939609 (FTO; p=0.009), Gastric bypass is the standard operation for obesity surgery in the than rs29941 (KCTD15; p=0.029) and rs7561317 (TMEM18; p=0.041) and United States. It results in approximately 35% weight loss at 1-2 years with marginally infl uenced rs925946 (BDNF; p=0.067) and rs7498665 (SH2B1; maintenance of a 30% weight loss for over a decade. Sleeve gastrectomy is p=0.071). Risk alleles of rs7561317 (TMEM18; p=0.014) and rs7498665 a newer obesity surgery that has similar weight loss in comparative studies, (SH2B1; p=0.047) were associated with higher waist circumference. The

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A507 OBESITY—HUMANCATEGORY

rs29941 (KCTD15; p=0.006) and rs7561317 (TMEM18; p=0.043) had effect those completing the study so far(n=34) BMI fell from 49 ± 7 to 34 ± 4kg/m2, also on waist-hip ratio. The carriers of minor alleles G in homozygote forms and weight from 137 ± 22 to 96 ± 15 kg. Median HBA1c fell from 7.8 (IQR 7.2, in variants rs4923461 (BDNF; p=0.036) and rs1800592 (UCP1; p=0.033) had 9.1) to 5.9 (IQR 5.4, 6.4). increased muscle mass in body composition, furthermore, GG homozygotes Bariatric surgery provides early clinical benefi ts to a patient group already in rs1800592 had decreased fat mass (p=0.037) and also increased muscle- participating in fully-funded proactive chronic disease management for fat ratio (p=0.026). In summary, we confi rmed associations of majority of the diabetes. Referral for surgery appears to be lower for Pacifi c and Asian 10 obesity-susceptibility variants with several obesity-related parameters; peoples. the most complex infl uence on anthropometric features was identifi ed in variant rs7561317 in TMEM18 gene. 1884-P Supported by: IGA MHCR NS/9839-4 and NS10209-3/2009 Basal Endogenous Glucose Production and Insulin Levels Are Reduced 2 Weeks after Bariatric Surgery with No Effect on Hepatic 1882-P and Peripheral Insulin Sensitivity Associations of Waist Circumference and Body Mass Index with BARBARA A. DE WEIJER, EDO AARTS, IGNACE M. JANSSEN, ARNOLD VAN DE Fasting Blood Glucose among Overweight African American Parish- LAAR, KARIN KAASJAGER, MARIETTE T. ACKERMANS, ERIC FLIERS, MIREILLE ioners in a Southern Semi-Urban Community J. SERLIE, Amsterdam, The Netherlands, Arnhem, The Netherlands RICHARD W. SATTIN, LOVORIA B. WILLIAMS, JAMES K. DIAS, THOMAS JOSHUA, Bariatric surgery has very early metabolic effects on glucose metabolism LUCY N. MARION, Augusta, GA before the occurrence of clinically signifi cant weight loss. This suggests an African-Americans (AAs) are at high risk for type 2 diabetes (T2D) partly acute effect of the surgery itself, i.e. bypassing the nutrient fl ow from the because rates of obesity are about twice as high as that in the overall US proximal gastrointestinal tract. We hypothesized that Roux-en Y gastric population. Although body mass index (BMI) has been considered an accurate bypass surgery (RYGB) ameliorates glucose metabolism by increasing clinical measure of body fat, central adiposity, approximated by waist hepatic and peripheral insulin sensitivity. circumference (WC), has been found to be a reliable predictor of metabolic We studied glucose metabolism and lipolysis in the basal state and during disturbances, morbidity and mortality. Our objective is to determine, in a a hyperinsulinemic euglycemic clamp using stable isotopes two weeks cohort of overweight AAs participating in the Fit Body and Soul Study (FB&S), before and two weeks after RYGB. the statistical relationships between WC, BMI, demographic characteristics, We included 12 pre-menopausal Caucasian women (median age 38 [26- and fasting blood glucose (FBG). FB&S is a single-blinded, cluster randomized, 49] yrs, median BMI 45 [39.2-53.9] kg/m2) scheduled for RYGB. Two weeks community trial developed to test the effectiveness of a faith-based after RYGB median weight loss was 5.5kg [2-14 kg]. adaptation to the Diabetes Prevention Program. AA church parishioners, in Basal insulin and glucose levels decreased after surgery (insulin pre- a semi-urban Southern US community aged 20 to 64 years, with a BMI of surgery 73 [21-122] vs post-surgery 48 [15-120] pmol/L, p = 0.0025 and 25 kg/m2 or greater and without a self-reported history of diabetes, were glucose pre-surgery 5.5 [4.4 – 6.9] vs post-surgery 4.8 [3.9 – 5.7] mmol/L, eligible to participate. Baseline demographic and measurement data from p = 0.0058 resp.). Endogenous glucose production (EGP) was lower after the participants in the fi rst ten of 20 churches available were collected, surgery (before 13.5 [11.3- 15.9] vs after 11.4 [10.3 – 14.2] μmol/kgFFM/ including age, gender, BMI, WC, and FBG. Standard descriptive statistics min, p = 0.0078). Insulin levels were lower during the clamp after surgery, were calculated and multiple linear regression and correlation analyses suggesting enhanced clearance. Hepatic and peripheral insulin sensitivity were conducted to model FBG. From September 2009 through August 2010, (both corrected for insulin) did not change after surgery. FFA were increased 393 AAs were consented. Eighty-four percent were female; the average after surgery in the basal state (0.76 [0.67–1.13] vs 0.94 [0.73-1.33] mmol/L, age of those consented was 46.8 years (95% confi dence interval (CI): [45.6, p = 0.0049) and during the fi rst step of the clamp (0.13 [0.02 – 0.29] vs 0.34 47.8]); the average BMI was 35.4 kg/m2 (95% CI: [34.7, 36.2]); the average [0.06 – 0.49] mmol/L, p = 0.0010). Lipolysis expressed per REE tended to WC was 107.7 cm (95% CI: [106.1, 109.4]); the average FBG was 93.0 mg/ increase in the basal state (259 [182 - 426] vs 257 [167 - 384] µmol/kcal, p = dl (95% CI: [91.4, 94.5]). The partial correlation of FBG with BMI, adjusted 0.0674), and was higher during hyperinsulinemia (91 [70 - 298] vs 132 [100 - for age and gender, was 0.189 (p-value < 0.001). The partial correlation of 403] µmol/kcal, p = 0.0020). FBG with WC, adjusted for age and gender, was 0.192 (p-value < 0.001). Our Within 2 weeks, bariatric surgery reduces basal EGP, insulin and glucose fi ndings suggest that both BMI and WC are important contextual variables levels but has no acute benefi cial effect on hepatic or peripheral insulin and provide similar results for the study of T2D in AAs. sensitivity. The latter may be explained by higher rates of lipolysis and Supported by: NIH grant DK082401 exposure to FFA induced by the hypocaloric state.

1883-P 1885-P Bariatric Surgery in a Multi-EthnicWITHDRAWN Type 2 Diabetes Population CREW (Calcium REduces Weight) Study: Impact of Calcium Supple- BRANDON J. ORR-WALKER, MARIE WHITE, IRENE ZENG, Auckland, New Zealand mentation on Weight, Adiposity, Glycemia and Hypertension This study is to determine the effect of adjunctive support compared with LAKSHMI KANT SHANKHDHAR, KSHITIJ SHANKHDHAR, UMA SHANKHDHAR, standard care in a morbidly obese, diabetic cohort on body mass index (BMI) SMITA SHANKHDHAR, Lucknow, India undergoing publicly-funded bariatric surgery with secondary endpoints of Aims: CREW study aimed to observe impact of Calcium supplementation glycemic control (HbA1c), blood pressure, lipids, pulmonary function and (CaS) on wt, waist circumference (WC), body fat% (BFP), BP, glycemia in T2 quality of life. As the study has not yet been completed, unblinded results diabetic (2D) & nondiabetic (ND) women. of recruitment and clinical response for completers to 1 year post surgery Methods: 120 drug naïve hypertensive obese female patients, both ND are presented. All subjects have type 2 diabetes on hypoglycaemic agents & 2D, were divided into 2 control (NDCo&2DCo) & 2CaS receiving (NDCa and are enrolled in a diabetes chronic care management program (CCM) and 2DCa) subgroups, with age (ND=44.6±9.86, 2D=50.85±7.37yrs), Wt which provides free quarterly review in primary care, in addition to the usual (ND=94.7±9.07, 2D=89.0±8.07Kg), BMI (ND=37.91±4.41, 2D=36.75±3.37Kg/ funded laboratory, pharmaceutical and hospital services. M2), WC (ND=102±6.44, 2D=100.9±4.08Cm) & BFP (ND=41.0±5.26, 2D= Entry to the study was for those enrolled for at least one year in CCM 39.96±3.0%). They were advised 300 lesser calories, 30min brisk walk for 3

Obesity with a BMI ≥35, 7.0≤ HBA1c ≤10.0 on hypoglycaemic medication, serum months along with education.

POSTERS creatinine ≤150 (≤ 120 for women) and urinary albumin creatinine ratio ≤ Results: NDshowed more reduction than 2D in Wt (NDCo=3.6 vs 200 mg/mmol. Additional surgical criteria limiting surgery to non-smokers 2DCo=1.13%, NDCa=5.78 vs 2DCa=5.23%), WC (NDCo=1.91 vs 2DCo=0.59%,

Integrated Physiology/ aged 20-60, with a weight of ≤160kg were applied resulting in a potential NDCa=1.76 vs 2DCa= 1.58%) & BFP (NDCo=4.87 vs 2DCo=1.63%, NDCa=5.36 recruitment population of 1924 patients from the total 7367 enrolled. (27% vs 2DCa=3.33%). CaS reduced SBP more (2DCa=8.2 Vs 2DCo=3.03%) than Maori, 55% Pacifi c, 11% European, 7% Asian). Informed consent for both DBP (2DCa=2.92 vs 2DCo=1.26%) in diabetics. Glycemia improved more with surgery and the study was required for inclusion. CaS:2DCa (FBG=19.08, PPBG=25.9, A1c=8.8%) vs 2DCo (FBG=7.5, PPBG=8.8, Of those referred to the study team by their family practitioner, 113 were A1c= 4.9%) not enrolled( 63 did not meet the study criteria, 9 had other contraindications, Conclusions: CaS plays positive role in reduction of adiposity, glycemia 34 declined surgery, 2 declined study involvement but had surgery, others 5), & BP. and 9 withdrew after randomisation. Overall Pacifi c and Asian peoples are underrepresented by referral, resulting in lower inclusion in the study. Rates of non-enrolment were similar. Enrolees had a mean age of 48 years, and 64% were female, 33% Maori, 30% Pacifi c, 35% European and 1% Asian. Of

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A508 OBESITY—HUMANCATEGORY

Observations: adipokines levels and HOMA-IR. The transcriptional expressions of PPAR-γ NDCo (n=30) NDCa (n=30) and RBP4 in SAT were negatively correlated with serum total cholesterol, LDL-cholesterol (P<0.05). This study indicated that SAT is inadequately Baseline Mean Final Mean p value Baseline Mean Final Mean p value differentiated and negatively associated with serum cholesterol levels in Wt (Kg) 96.10±7.76 92.70±8.42 0.004 93.30±10.45 87.90±11.96 0.0003 overweight condition, which might be one cause to explain that SAT plays the protective role in the development of obesity-related complications. BMI (Kg/M2) 38.31±4.28 36.74±4.62 0.006 37.51±4.28 35.28±4.06 0.0004 WC (Cm) 99.00±4.00 97.10±5.13 0.003 102±6.61 100.50±7.26 0.0014 1887-P BFP (%) 41.01±4.70 38.98±4.90 0.002 40.99±6.02 38.64±5.42 0.00003 Diabetes Prevention and Normalization of Fasting Glucose in SBP (Hg mm) 132.80±5.67 127.60±4.19 0.003 131.00±7.37 126±.0.00 0.0263 Subjects with Prediabetes Using Controlled-Release Phentermine/ Topiramate (PHEN/TPM CR) in a 2-Year Weight Loss Intervention DBP (Hg mm) 80.60±1.34 78.80±2.14 0.0148 81.00±3.16 78.80±3.15 0.0646 DONNA H. RYAN, W. TIMOTHY GARVEY, BARBARA TROUPIN, WESLEY W. DAY, Baton Rouge, LA, Birmingham, AL, Mountain View, CA 2DCo (n=30) 2DCa (n=30) Weight loss of 5%-10% in patients with prediabetes may prevent pro- Wt (Kg) 88.30±8.52 87.30±8.80 0.0236 89.70±8.00 85.00±8.11 0.000003 gression to type 2 diabetes mellitus (T2DM). In the 56-week CONQUER study, BMI (Kg/M2) 36.93±3.19 36.49±3.07 0.0250 36.58±3.70 34.65±3.49 0.000003 once-daily, PHEN/TPM CR led to signifi cant weight loss, improvements in glycemic parameters, and reduced overall rates of progression to T2DM. WC (Cm) 101.00±2.86 100.40±2.45 0.0025 100.80±5.20 99.20±5.15 0.00002 SEQUEL, a 52-week extension in subjects completing CONQUER, maintained BFP (%) 40.32±2.16 39.66±2.08 0.0002 39.61±3.74 38.11±3.40 0.00000 the original blinding and randomization (placebo [PBO], PHEN 7.5 mg/TPM CR SBP (Hg mm) 132.40±6.31 128.20±3.45 0.0358 133.80±10.47 122.40±4.50 0.0014 46 mg [7.5/46], and PHEN 15 mg/TPM CR 92 mg [15/92]) and evaluated the DBP (Hg mm) 78.80±3.15 77.80±4.15 0.1231 82.00±4.32 79.60±0.84 0.052 long-term effects of PHEN/TPM CR when added to lifestyle interventions for weight loss. This pre-specifi ed subanalysis evaluated weight loss and FBG (mg%) 121.20±20.24 112.00±8.76 0.0588 131.00±18.01 106.00±7.45 0.0028 changes in glycemic status in subjects who had prediabetes at CONQUER PPBG (mg%) 153.60±21.64 139.00±16.94 0.0276 165.60±27.93 122.70±10.01 0.0006 baseline Week 0, n=316), defi ned as impaired fasting glucose (≥100 and A1c (%) 7.5±0.22 7.1±0.18 0.0002 7.67±0.43 6.99±0.20 0.00006 ≤125 mg/dL) or impaired glucose tolerance (oral glucose tolerance test ≥140 and ≤199 mg/dL). After a total of 108 weeks of treatment, least-squares (LS) mean percent weight loss was signifi cantly greater with both doses of 1886-P PHEN/TPM CR compared with PBO (P<0.0001; ITT-LOCF): 2.2%, 11.1%, and Depot-Specifi c Expression of Adipogenic Genes in Subcutaneous 12.7% for PBO, 7.5/46, and 15/92, respectively. Dose-related improvement and Omental Adipose Tissues of Women and Their Relationship with in fasting insulin (µIU/mL) at week 108 was observed with PHEN/TPM CR Dyslipidemia when compared with PBO: LS mean change of -2.8, -5.0, and -5.5 for PBO, HONGYUN LU, XIAOFENG LI, PANWEI MU, JIANG WEI, LONGYI ZENG, Guangzhou, 7.5/46, and 15/92, respectively (P=0.0314 vs PBO for 15/92). More subjects China treated with PHEN/TPM CR achieved normoglycemia than those receiving Depot differences in adipose tissue function may have implications PBO; progression to T2DM was more common with PBO (Table). PHEN/TPM for insulin resistance (IR), the adipogenesis inability of the subcutaneous CR was well tolerated over 108 weeks; most common adverse events were adipose tissue (SAT) may cause ectopic fat deposition and IR, but the constipation, dry mouth, and paraesthesia. molecular mechanism remains unclear. This study aimed to measure gene Table. Percentage of Subjects With Change in Prediabetes Status at Week expression involved in adipocytes differentiation and adipokines secretion 108 (ITT-LOCF) in human SAT and omental adipose tissue (OAT), and then investigate their PBO (n=103) 7.5/46 (n=83) 15/92 (n=130) relationship with obesity and IR. We examined the expression of candidate genes from 29 lean and 12 overweight non-menopausal women. Serum Achievement of 33.0 37.4 54.6* leptin, adiponectin, retinol-binding protein 4(RBP4) and fasting insulin (FINS) normoglycemia (%) levels were measured by using ELISA. IR was evaluated by the HOMA model Progression to T2DM (%) 5.8 6.0 0.8* index (HOMA-IR). We found that serum concentrations of leptin and RBP4 *P=0.0004 vs PBO were elevated and adiponectin decreased in overweight patients compared to lean controls (P<0.01). For all subjects, the transcriptional expressions of In subjects with prediabetes enrolled in a weight loss study, PHEN/TPM PPAR-γ, leptin and β-catenin were signifi cantly higher in SAT than OAT, while CR was associated with long-term improvements in fasting insulin and led RBP4, CEBP-α and adiponectin expression were signifi cantly lower in SAT to reductions in the rate of progression to T2DM. than OAT. Supported by: Vivus, Inc.

1888-P Distinct Exhaled Gas Profi les in Obese, T1DM, and T2DM during Glucose and Insulin Fluctuations STACY R. OLIVER, MATT K. CARLSON, JOANNE CHEUNG, ALEXANDRA R. LY, REBECCA L. FLORES, DONALD R. BLAKE, PIETRO R. GALASSETTI, Irvine, CA Exhaled breath analysis is increasingly considered an alternative testing methodology in obesity (OW), type 1 and type 2 diabetes (TIDM, T2DM). While we have previously measured plasma glucose, insulin, and lipids through breath analysis in healthy and T1DM subjects, accuracy was still inadequate for clinical applications; possibly due in part to lack of identifi cation of exhaled gas patterns, specifi c to each dysmetabolic condition. We therefore Obesity attempted to identify group-specifi c exhaled gas patterns in 22 healthy (29±1 POSTERS yrs; 10F), 23 T1DM (23±2 yrs; 12F), 6 OW (40±4 yrs; 6F), 10 T2DM (44±3 yrs; 5F) subjects, during 4 hours of controlled glycemic and insulinemic fl uctuations Integrated Physiology/ (hyperglycemia of ∼220 mg/dL HG, euglycemic hyperinsulinemia, INS). Gas concentrations in matched breath/room air samples collected at 12 time- points were determined by gas chromatography/mass spectrometry. Of >100 tested gases, acetone, MeONO2, EtONO2, CH3Br, i-butane, isoprene, C2Cl4 had especially distinct profi les in OW, T1DM and T2DM. OW had lowest acetone and MeONO2, greatest EtONO2 and CH3Br uptake throughout the study, and a signifi cant increase of C2Cl4 in HG and INS. T1DM had OAT in overweight patients was characterized with higher transcriptional the highest acetone levels, and intermediate MeONO2, EtONO2, CH3Br, expressions of PPAR-γ, C/EBP-α, leptin, RBP4 and lower level of adiponectin, i-butane, isoprene and C2Cl4 concentrations. T2DM had the highest isoprene but these expression diversity wasn’t direct associated with serum and i-butane levels in all test conditions. Our data support the concept that

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different metabolic conditions can induce signifi cantly divergent exhaled gas aggressive weight loss intervention being more likely to facilitate remission profi les. Integrations of this information into condition-specifi c predictive of disease. Larger and longer-term studies are required. models (for instance use of different gases in predictive algorithms for Supported by: Allergan glycemia in obese, T1DM and T2DM) is likely to considerably improve accuracy of breath based measurements of plasma variables, accelerating 1890-P the development of portable, clinically usable breath testing devices. Ectopic Fat Accumulation and Metabolic Syndrome (MSYN) Are Asso- ciated with Reduced Subcutaneous Adipose Tissue (SAT) Lipid Storage in Obese Postmenopausal Women (OPW) MONICA C. SERRA, ALICE S. RYAN, RONALD L. PRIGEON, ANDREW P. GOLDBERG, Baltimore, MD Impaired expandability of SAT and the accumulation of fat in visceral adipose tissue (VAT) and muscle (Low Density Lean Tissue [LDLT]) may lead to metabolic dysfunction in obesity. We hypothesize that a decreased ability to store triglycerides (TG) in SAT, due to low adipose lipoprotein lipase activity (LPL), results in lipid overfl ow into VAT and LDLT stores, insulin resistance (IR [measured by HOMA-IR]) and MSYN. To test this hypothesis we measured body composition (DXA, CT), MSYN variables, fat cell weight (FCW) and LPL in abdominal and gluteal adipose biopsies in 125 Caucasian overweight and OPW (25-44 kg/m2; age 45-77 yrs) grouped into lowest and highest quintiles of VAT/total abdominal fat (VAT/[VAT+SAT]; mean±SD: 0.17±0.02 vs. 0.37±0.05). OPW in the highest quintile had a greater prevalence of impaired glucose tolerance (IGT; 67% vs. 20%, P<0.01) and MSYN (68% vs. 28%, P<0.01) compared to the lowest. Despite similar obesity (lowest versus highest quartile: BMI: 31±4 vs. 33±5 kg/m2; body fat: 47±5 vs. 46±5%), the lowest quintile had higher SAT (P<0.005) and 12% lower abdominal FCW than the highest quintile, which was associated with a trend toward higher abdominal LPL (P=0.06) and higher gluteal LPL (P<0.01). FCW in SAT correlated with abdominal LPL in the lowest (r=0.76, P<0.0001) and highest quintile (r=0.42, P<0.05) (Table: *P<0.05, **P<0.01, †P=0.06). Lowest Quintile Highest Quintile Supported by: NIH 1UL1RR031985, K24 DK085223 ADA-Funded Research (N=25) (N=25) VAT (cm2) 100±23 228±79** SAT (cm2) 498±107 389±134** LDLT (cm2) 14±4 21±5** 1889-P 2h OGTT (mmol/L) 6.4±2 8±2** Early Aggressive Weight Loss Efforts Using Adjustable Gastric Band- HOMA-IR 2.4±1.4 4.2±1.7** ing Leads to Improvement or “Remission” of Type 2 Diabetes Mellitus TED OKERSON, MICHAEL OEFELEIN, SUNIL BHOYRUL, PAMELA BARNETT, JOHN Abdominal Fat Cell Weight (µg lipid/cell) 0.68±0.21 0.76±0.19 B. DIXON, APEX, Irvine, CA, La Jolla, CA, Melbourne, Australia Gluteal Fat Cell Weight (µg lipid/cell) 0.72±0.20 0.73±0.19 Bariatric surgery (malabsorptive or restrictive techniques) has been Abdominal LPL Activity (nmol/min/106cells)1 4.0±2.1 2.7±1.9† established as an effective treatment to reduce weight in severely obese Gluteal LPL Activity (nmol/min/106cells)1 5.5±5.8 2.9±3.3** patients. This study reports the 1 year “remission” (elimination of hypo- glycemic medication) and/or improvement (reduction in hypoglycemic 1Adjusted by FCW medication) of type 2 diabetes mellitus (T2D) after laparoscopic placement The lower LPL and SAT and trend for larger abdominal FCW suggest OPW of an adjustable gastric band (AGB) as documented by T2D medication with the highest VAT/(VAT+SAT) have less capacity than the lowest quintile reduction/discontinuation, and the accompanying change in BMI and co- to store TG in SAT, despite similar adiposity. Thus, the inability to store morbidities. The APEX study is an ongoing 5-year, prospective, multi-center, excess TG in SAT appears to result in ectopic lipid accumulation in VAT and open-label, observational study to assess weight reduction, co-morbidities muscle, IR and MSYN. and quality of life after implantation of the LAP-BAND AP® gastric band, a restrictive weight loss technique. This is an interim analysis of subjects who reported daily medical therapy for T2D before AGB and who have completed 1891-P the 1 year post-operative scheduled visit. At baseline (BL), 94 out of 436 Ef fect of Duodenal-Jejunal Bypass Surgery on Glucose Homeostasis subjects (22%) reported T2D requiring daily medical therapy; data from 64 and β-Cell Function in Overweight and Obese Subjects with Type 2 subjects contained suffi cient information to assess outcome at 48 weeks. Diabetes (T2D) Overall, 86% had remission and/or improvement in T2D, with remission more ELISA FABBRINI, BRUCE PATTERSON, CARLOS SCHIAVON, JOSE CORREA, JOAO likely to occur in patients treated earlier after the diagnosis of T2D: SALLES, BERNARDO WAJCHEMBERG, SAMUEL KLEIN, RICARDO COHEN, St. Louis, MO, São Paulo, Brazil Remission Improvement Stable Worse The purpose of the present study was to test the hypothesis that bypass of %(n) 34 (22) 52 (33) 13 (8) 2 (1) the upper gastrointestinal tract has therapeutic effects on glucose homeostasis Mean Duration T2D(mo) 63 76 90 1 and β-cell function in patients with T2D. A 2-h OGTT was performed in 35 Obesity overweight-obese adults with T2D (BMI:27.0±4.0 kg/m2) before and 6, 9, and POSTERS BL BMI 46 44 52 47 12 months after duodenal-jejunal bypass (DJB) surgery, which involves creating Δ BMI -8.9 -7.6 -8.1 -2.9 a duodenojejunostomy without gastric restriction or exclusion. HOMA-IR, the Integrated Physiology/ Δ Wt (lb) -55 -48 -52 -18 incremental increase in the area under the curve above baseline (iAUC) for % Wt Δ -19 -21 -15 -6 glucose (GLU), insulin (INS), and C-peptide (C-PEP) after GLU ingestion, and selected indices of β-cell function (c-peptide secretory response [C-PEP Baseline BMI, reductions in BMI and % change in weight were not iAUC÷GLU iAUC], and insulinogenic index [INS iAUC÷GLU iAUC during 120 min statistically different among the groups, although numbers were small. OGTT]) were determined. A scoring system based on the number and dose As in patients with T2D, resolution or improvement also occurred in other of medications taken was used to assess the use of diabetes medications. pre-existing co-morbidities measured: hypertension (78%), hyperlipidemia We conclude that DJB surgery improves glycemic control, manifested by a (57%), depression (71%), obstructive sleep apnea (69%) and GERD (93%). decrease in HbA1C with less diabetes medications and improved oral glucose These data suggest that a minimally-invasive restrictive gastric banding tolerance. Our data suggest the mechanism responsible for this therapeutic procedure in obese patients with T2D results in clinically meaningful weight effect is an increase in β-cell response and moderate weight loss. These loss, as well as a reduction in T2D medication requirements, with an earlier fi ndings have important implications in understanding the factors involved

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A510 OBESITY—HUMANCATEGORY in the pathogenesis of T2D, and suggest that altering the intestinal site of with obesity. We enforced VLCD treatments (600-800 kcal/ day) more than delivery of ingested nutrients has therapeutic effects. minimum 1week for high obesity 18 patients, and then we measured urinary Before surgery 6 M 9 M 12 M 8-OHDG and 8EPG, and serum IL-6, IL-18, TNF-α, VCAM-1, L-selectin, MCP-1, ICAM-1 and adiponectin before and after VLCD. BMI and circumference at Weight (kg) 79.4±11.9 74.7±10.9* 75.5±11.2* 75.9±12.4* navel decreased after VLCD (34.1kg/m2 from 36.3kg/m2, and 105.6cm from HOMA-IR 5.2±3.1 5.7±3.2 5.6±2.6 6.4±3.3 112.7cm, respectively, p<0.01). Friedewald LDL cholesterol and triglyceride Medication score 1.40±0.55 0.89±0.65* 1.04±0.73* 1.05±0.76* decreased signifi cantly aftrer VLCD, too(93.8mg/dl from 116.8mg/dl, and HbA1c (%) 9.3±1.7 7.0±1.0* 7.1±1.2* 7.4±1.7* 85.7mg/dl from 110.9mg/dl, respectively p<0.01). On oxidative stress markers and cytokines, urinary 8-OHDG increased in 19.6ng/ml from 11.8ng/ GLU iAUC (mg/dLx120min) 16300±4600 13700±4400* 12500±3900* 11700±4000* ml (p<0.05). Serum IL-18 increased in 295.6pg/ml from 229.1pg/ml (p<0.05), C-PEP iAUC (µg/mLx120min) 158±62 306±66* 376±112* 363±90* VCAM-1 increased in 791.3ng/ml from 662.1ng/ml (p<0.01) and L-selectin INS iAUC (µU/mLx120min) 882±360 1560±700* 1540±620* 163±610* increased in 922.1ng/ml from 857.4ng/ml (p<0.05) after VLCD. On the other C-PEP secretory response 0.011±0.008 0.026±0.019* 0.035±0.021* 0.035±0.014* hand, serum IL-6 decreased in 4.55pg/ml from 5.78pg/ml (p<0.05), and high- (c-PEP iAUC/GLU iAUC) molecular weight adiponectin showed a tendency to increase after VLCD (1.62μg/ml from 0.88μg/ml, p=0.052). Urinary 8EPG, serum TNF-α, MCP- Insulinogenic index 0.06±0.04 0.13±0.09* 0.16±0.13* 0.15±0.07* 1 and ICAM-1 did not show a meaningful change after VLCD treatment. (INS iAUC/GLU iAUC) Although serum IL-6 decreased, the oxidative stress marker and plural Values are means±SD. cytokines such as urinary 8-OHDG, serum IL-18, VCAM-1 and L-selectin Value different from before surgery: * p<0.05 increased. These fi ndings suggested that sudden weight decrease by the VLCD treatment in patients with obesity might give vessels damage not only 1892-P from oxidative stress markers, but also from cytokines. Effect of High-Protein and High-Carbohydrate Weight Reduction Diets on Bone Density and Body Composition in Obese Non-Diabetic, 1894-P Pre-Menopausal Women Effect of Weight Gain and Weight Loss on Insulin Sensitivity and FRANCES A. TYLAVSKY, ABBAS KITABCHI, FRANKIE STENTZ, KRISTIN MCDANIEL, Insulin Secretion in Adults Who Maintain Normal Glucose Tolerance EBENEZER A. NYENWE, JIM WAN, CHRISTOPHER SANDS, Memphis, TN during a 5-Year Period Caloric defi cits and protein intake have been implicated in the maintenance of KRISTINE FÆRCH, DORTE VISTISEN, TORBEN JØRGENSEN, DANIEL R. WITTE, the human skeleton. With weight reduction as a prime approach for treatment of Gentofte, Denmark, Glostrup, Denmark type 2 diabetes, we examined the effect of 2 hypo-caloric diets containing high- To improve prevention of type 2 diabetes it is important to know how carbohydrate (CHO) HC (55% CHO, 30% fat, 15% protein (PRO)) or high protein (HP) glucose homeostasis is regulated in healthy individuals. We studied whether (30% protein, 30% fat, and 40% CHO) on skeletal health and body composition. insulin sensitivity and insulin secretion changed in response to weight gain and The HP and HC diet provided 1684 and 1725 mg of calcium respectively. To date, weight loss in individuals maintaining normal glucose tolerance (NGT) during 13 (8 HP, 5 HC) non-diabetic, obese, pre-menopausal women have completed a a 5-year period. From the Inter99 study we included 2,822 participants with 6 month randomized trial. The diets based on resting energy expenditures with NGT both at baseline and after 5 years. All were classifi ed as having either 500 Kcal reductions per subject were provided by pre-packaged ready to eat food increased (progressors) or decreased (regressors) their BMI during the 5-year items. Dual energy x-ray absorptiometery of the whole body were performed at period. Progressors (n=1,787) and regressors (n=1,035) were further divided randomization and at the conclusion of the trial to estimate changes in fat, lean into tertiles of BMI increase or decrease. From oral glucose tolerance tests, at and bone density(BMD). Protein intake was 1.15 g/kg on HP and 0.61g/kg on HC. baseline and 5 years, we calculated two estimates of insulin sensitivity: Gutt The table shows that there were similar losses of whole body (WB), lean and fat index, refl ecting mainly peripheral insulin sensitivity and AUCglu30xAUCins30, mass regardless of diet assignment. However there appears to be a differential refl ecting mainly hepatic insulin sensitivity. We also estimated absolute early loss of BMD between the 2 diets. insulin secretion as well as two estimates of insulin secretion relative to insulin sensitivity: ‘peripheral’ and ‘hepatic’ disposition indices. Large weight gain and HP HC large weight loss was associated with signifi cantly altered ‘hepatic’ disposition Parameter Baseline 6 months P* Baseline 6 months P* P** index, whereas medium weight loss was associated with improved ‘peripheral’ BMI (kg/m2) 42.0 ± 7.0 38.8 ±7.3 .0078 37.5 ± 4.9 35.3 ±5.4 .0625 .2844 disposition index. After adjusting for age, sex, intervention group, and baseline variables of insulin sensitivity or secretion there was no difference in the % weight loss 7.7 ± 4.7 6.0 ±4.3 .4351 change of ‘peripheral’ disposition index between those in the three weight gain 2 BMD (g/cm ) 1.17 ± .07 1.17 ±.07 .8438 1.12 ± .05 1.14 ±.06 .1250 .0505 or weight loss groups (P≥0.144), but those with a large weight gain reduced FAT Mass (kg) 53.1 ± 12.2 47.7 ±12.4 .0078 47.8 ± 13.5 44.7±14.0 .0625 .2844 their ‘hepatic’ disposition index more than those with a small weight gain Lean Mass (kg) 57.3± 9.1 53.8 ±9.2 .0078 54.6 ± 8.7 52.4±8.9 .0625 .5237 (P=0.010). These data suggest that small weight gain or small weight loss is not associated with changes of insulin sensitivity and/or secretion. % Adherence 91.8±5.1 92.2 ± 7.3 .9153 * Wilcoxon Signed-Rank Test (baseline to 6 months) ** Wilcoxon Rank Sum Test (difference between HP and HC over 6 months) In a short term hypo-caloric feeding study with adequate calcium intake, there were no defi cits in BMD for those on HP but a marginal increase in BMD was seen for those on HC. Whether these differences in BMD between the 2 diets are maintained requires long term requires longer term follow-up. ADA-Funded Research Obesity

1893-P POSTERS Effect of Very Low Calorie Diet (VLCD) on Oxidative Stress Markers and Cytokines in Patients with Obesity Integrated Physiology/ SHUKI USUI, YASUSHI TOUJOU, KAORU ISO, NAOKI HIROI, EIICHI MURAKAMI, KOUJI KUBOKI, GEN YOSHINO, Tokyo, Japan With diabetes and obese patients, we found increase of oxidative stress markers and cytokines. It is thought that they play an important role for the onset and development of a complication such as arteriosclerosis. On the other hand, very-low-calorie-diet (VLCD), supplying the energy and nutrient of the necessary least amount, can achieve a weight reduction effect equal to starvation therapy. However, the changes of oxidative stress markers and cytokines by VLCD are not clarifi ed enough. The aim of this study was to clarify the infl uence to oxidative stress markers and cytokines by VLCD in patients

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A511 OBESITY—HUMANCATEGORY

1895-P had 5-year follow-up data available. Patient’s current T2DM status was Effects of a Three-Month Combined Exercise Program on Fibroblast determined by biochemical analyses and review of medications. Remission Growth Factor 21 and Fetuin-A Levels and Arterial Stiffness in Obese was defi ned as fasting glucose <120 off diabetic medications. Forty one Women T2DM patients who underwent bariatric surgery had 5 year data available HYE JIN YOO, HO CHEOL HONG, HAE YOON CHOI, MYONG JIN CHO, YOON JUNG (59% follow-up rate, 32% male, mean [±SE] age 49.3±1.6 yrs). Mean duration KIM, JOO HYUNG KIM, CHAI RYOUNG EUN, SAE JEONG YANG, HEE YOUNG KIM, of T2DM was 97.6 months (range 3-468). 29 patients underwent RYGB, 12 had JI A. SEO, NAN HEE KIM, SIN GON KIM, SEI HYUN BAIK, DONG SEOP CHOI, KYUNG gastric restriction. Results are presented in Table 1. At mean follow-up of 72 MOOK CHOI, Seoul, Republic of Korea months (range 65-83), T2DM remained resolved or improved in 84%. Insulin We examined the relationship between brachial-ankle pulse wave velocity and oral hypoglycemic medications were stopped or reduced, respectively, (baPWV) refl ecting arterial stiffness and the levels of novel hepatokines in all these patients. In those whose T2DM status was unchanged at 5 fi broblast growth factor 21 (FGF21) and fetuin-A. In addition, we evaluated the years (66% restrictive procedures), all had regained weight in that interval. effect of a three-month combined aerobic and resistance exercise program on Bariatric surgery can induce a signifi cant and sustainable improvement in FGF21 and fetuin-A levels as well as arterial stiffness in obese women. T2DM. Lasting remission appears to be related to the duration and severity Forty non-diabetic, obese women (BMI = 27.6 ± 2.4 kg/m2) were included of diabetes preoperatively, and the extent and durability of weight loss. in the study and were compared before and after a three-month exercise These data further support bariatric surgery as an effective treatment for program, which was composed of 45 minutes of aerobic exercise at an obesity and T2DM, which should be considered early in the course of the intensity of 60–75 % of the age-predicted maximum heart rate and 20 disease. minutes of resistance training fi ve times a week. Table 1. Outcomes 5 years postop At baseline, baPWV levels were signifi cantly correlated with age, BMI, systolic blood pressure, high-density lipoprotein cholesterol, fasting glucose, Remission Improvement No change and serum FGF21 levels. In a multiple stepwise regression analysis, baPWV T2DM 42% 42% 16% levels were independently associated with age, BMI, SBP, FGF21, and fetuin-A Hypertension 14% 44% 42% levels (R2 = 0.744). After the exercise program, BMI, waist circumference, Dyslipidemia 46% 23% 31% SBP, DBP, and triglyceride levels were signifi cantly decreased. Moreover, baPWV values were signifi cantly improved (P < 0.001) while FGF21 levels Duration of T2DM (months) 43.0±14 207±70 236±116 declined signifi cantly (P = 0.043). However, fetuin-A levels were not changed BMI, kg/m2 signifi cantly (P = 0.202). Pre 49.3±2.0 49.4±3.7 44.4±4.6 A three-month combined exercise program signifi cantly decreased the 5yr 36.2±2.1 40.6±3.9 41.0±2.7 arterial stiffness and FGF21 levels in obese Korean women. HbA1C % Pre 7.2±0.5 8.0±0.4 8.6±0.2 5yr 6.1±0.2 7.2±0.6 7.6±0.6 Fasting glucose (mg/dL) Pre 122.3±5.4 134.9±20.4 164.7±40.9 5yr 111.3±8.3 122.1±9.3 160.3±66 Blood pressure (mmHg) Pre 141/79 130/74 139/72 5yr 135/73 122/75 137/75 Total cholesterol (mg/dL) Pre 183±12.3 169.7±14.3 164.5±21.5 5yr 181.3±16.5 166.1±13.0 190.7±25.7

1897-P Genome-Wide Association for Abdominal Fat Deposition Reveals Novel Loci CAROLINE S. FOX, L. ADRIENNE CUPPLES, CHARLES C. WHITE, TAMARA B. HARRIS, INGRID BORECKI, YONGMEI LIU, ON BEHALF OF THE VATGEN CONSORTIUM, Framingham, MA, Boston, MA, Bethesda, MD, St Louis, MO, Wake Forest, NC Background: Body fat distribution is associated with metabolic risk above and beyond total adiposity. In order to uncover novel loci for body fat distribution independent of body mass index, we performed genome-wide association of visceral fat among participants of European ancestry. Methods: Subcutaneous and visceral fat were measured using standard methods in AGES (n=3172), Family Heart Study (n=2659), the Framingham Heart Study (n=3158), and the Health ABC study (n=1568). Genotyping was performed using standard arrays and imputed to ∼2.5 million Hapmap SNPs. Each study performed a genome-wide association analysis of SAT, VAT, and VAT/SAT ratio in the overall sample and in women and men separately. A weighted z-score meta-analysis was conducted across all 4 studies. Results: Overall, 5559 women and 4997 men were available for analysis. For the VAT/SAT ratio, our top p-value was rs11118316 near the LYPLAL1 gene Obesity (p=3.1*10E-09). For SAT, the best SNP was in the FTO gene (p=5.9*10-08). POSTERS 1896-P Five-Year Follow-Up of Type 2 Diabetic Patients Who Underwent Bari- We uncovered no genome-wide signifi cant fi ndings for VAT. Given the known differences in body fat distribution between women and men, we performed

Integrated Physiology/ atric Surgery HELEN M. HENEGHAN, FADY MOUSTARAH, SHAI MERON-ELDAR, SANGEETA sex-specifi c analyses. Our top fi nding was on chromosome 2p11 near TNHSL2 KASHYAP, JOHN KIRWAN, BIPAN CHAND, STACY BRETHAUER, TOMAZ ROGULA, for VAT in women (p=1.6*10-08). We also interrogated the 14 recently- MATTHEW KROH, LAURENCE KENNEDY, PHILP R. SCHAUER, Cleveland, OH published loci for body fat distribution for waist-hip-ratio adjusted for In addition to weight loss, bariatric surgery has powerful metabolic BMI. We observed no signifi cant associations with VAT (all p-values>0.05), effects, including resolution of obesity-related comorbidities such as type whereas associations were detected for VAT/SAT ratio with LYPLAL1, GRB14, 2 diabetes mellitus (T2DM). However, the durability of these benefi ts is and ADAMTS9 (p-value range 0.0016-0.03); these same SNPs were also largely unknown. The aim of this study was to determine 5-year outcomes associated with SAT (p<0.01). of morbidly obese diabetic patients who underwent bariatric surgery, and to Conclusions: Genome-wide association for visceral and subcutaneous fat identify factors associated with durable diabetes remission. We identifi ed revealed several loci of interest. Replication efforts are ongoing to confi rm all T2DM patients who underwent bariatric surgery at our institution, and these fi ndings.

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1898-P 1900-P Heritability of the Risk Factors Characteristic for the Metabolic Impact of Obesity on Quality of Life, Work Productivity and Resource Syndrome: A Twin Study Use among Type 2 Diabetics GYÖRGY JERMENDY, LEVENTE LITTVAY, RITA STEINBACH, ÁDÁM JERMENDY, JAN-SAMUEL WAGNER, MARCO DIBONAVENTURA, KELLY BELL, MICHAEL JÁNOS OSZTOVITS, Budapest, Hungary POLLACK, JOHN GRAHAM, New York, NY, Plainsboro, NJ, Wilmington, DE Both genetic and environmental factors play role in the pathogenesis of the This study assessed the association of body mass index (BMI) with metabolic syndrome and obesity. The precise magnitude of genetic infl uence health-related quality of life (HRQoL), work productivity, and resource use on the components of the metabolic syndrome is poorly described. among type 2 diabetes patients. A total of 8,255 patients who reported In our study, a total of 101 (63 monozygotic [MZ] and 38 dizygotic [DZ]) being diagnosed with type 2 diabetes and provided weight and height adult twin pairs (n=202; mean age: 43.3±15.8 years) were investigated. information to the 2009 National Health and Wellness Survey, a cross- Past medical history was recorded and physical examination was carried sectional survey representative of the US population, were included. Obese out in each subject. Fasting venous blood samples were used for measuring (BMI>30; n=5324), overweight (BMI: 25.0-29.9; n=2140), and normal weight laboratory parameters. Intraclass correlations were evaluated and patients (18.5-24.9; n=791) were compared, controlling for demographic heritability model analyses (A-C-E model) were performed. All parameters and health characteristics. HRQoL (SF-12), work productivity within the were corrected for age, gender and BMI values. past 7-days (WPAI), and healthcare resource use within the past 6-months The intraclass correlation for waist circumference was higher in MZ were assessed and evaluated. The sample was mostly male (57.5%) with a (r=0.744; p<0.0001) than in DZ twin pairs (r=0.065; p=0.794) and the heritability mean age of 58.5 (SD=12.4). Compared to normal patients, obese patients was estimated as high as 70.7% (95% CI: 42.6 – 89.3%). Very similar results were signifi cantly (p<.05) more likely to report lower household income, were found regarding weight (MZ: r=0.880 p<0.0001; DZ: r=0.437; p=0.019; exercise less, have at least one cardiovascular comorbidity, and to use heritability 88.1%, 95% CI 58.4 – 93.5%). The intraclass correlation for insulin. After controlling for demographic and health characteristics, obese systolic blood pressure was higher in MZ (r=0.577 p<0.0001) than in DZ twin patients reported signifi cantly lower mean physical (38.2 vs. 42.8, p<.0001) pairs (r=0.019 p=0.944) and the heritability was estimated as 57.7 % (95 % SF-12 component summary scores, more time missed from work (5.4% vs. CI 27.9 – 74.9%). As for diastolic blood pressure, the heritability was 56.8% 3.0%, p=.0345), impairment while at work (20.9% vs. 16.9%, p=.0235), and (95% CI 23.3 - 73.4%). Shared environmental factors predominated in HDL- impairment of activities in daily living (39.3% vs. 31.3%, p<.0001) than cholesterol and blood glucose values (54.7% and 59.2%, respectively) while normal weight patients. The mean number of physician visits in the past 6 unique environmental factor had the highest proportion of total phenotypic months (7.0 vs. 6.1, p=.0010) was higher among obese patients than normal variance in serum triglycerides values (57.8%). weight patients. Signifi cant differences were not observed between normal Our data indicate that among components of the metabolic syndrome and overweight individuals. Obesity was independently and signifi cantly genetic factors have substantial infl uence on waist circumference and associated with lower levels of physical HRQoL, lower work productivity, blood pressure while shared and unique environmental factors predominate greater impairment in activities of daily living, and greater healthcare alterations of HDL-cholesterol, fasting blood glucose and serum triglycerides resource use. Obesity-related impairments were clinically meaningful for values in healthy adult subjects. The different heritability of the individual physical HRQoL and amounted to a mean 2 hours 15 minutes per week in risk factors challenges the original unifying concept of the metabolic work productivity losses. These fi ndings suggest, in addition to glycemic syndrome. control, effective management of weight may improve health outcomes and Supported by: sanofi -aventis, Hungarian Diabetes Ass., Hungarian Society of productivity among type 2 diabetes patients. Hypertension Supported by: BMS; Conducted by Kantar Health

1899-P 1901-P IFG Development Risk Is Related to Age and Insulin Resistance and In Humans, Olanzapine Acutely Elicits Some but Not All of the Same Not Body Weight in the Morbidly Obese Endocrine-Metabolic Changes That Precede Increased Adiposity in MACIEJ PAWLOWSKI, MALGORZATA GILEWSKA, JERZY LOBA, LESZEK CZU- Rodent Models PRY N IAK, Lodz, Poland VANCE L. ALBAUGH, RAVI SINGAREDDY, DAVID MAUGER, CHRISTOPHER J. LYNCH, Obesity is an independent risk factor of glucose intolerance. Impaired Hershey, PA fasting glucose (IFG) is a relatively new clinical entity of glucose metabolism In humans and rodent models, olanzapine and other atypical antipsychotics disturbances. The purpose of our study was to identify risk factors for the cause unexplained metabolic side effects including diabetes mellitus and development of IFG in the morbidly obese but otherwise healthy subjects. obesity. Surprisingly, circulating FFAs also appear depressed after chronic The study group comprised 235 non-smoking normotensive obese patients treatment in humans and animals. In animals, chronically elicited effects are (83 males and 152 females, mean age [±SD] 44.2±14.1 years, body weight preceded by rapid changes in glucose tolerance, insulin sensitivity and leptin. 110.7±20.9 kg, BMI 43±2.6 kg/m2). In all subjects body weight, height, WHR, In an animal model, Olanzapine rapidly lowered FFAs by impairing lypolysis blood pressure, fasting plasma glucose (FPG), insulin, triglycerides, total and increasing peripheral lipid oxidation. In a double blind, randomized, cholesterol, LDL and HDL-cholesterol, HbA1c, liver function tests (LFTs) and placebo-controlled crossover study (Clinicaltrials.gov: NCT00741026) we thyroid-stimulating hormone (TSH) concentration were measured. Lipid and sought to examine the clinical relevance of acute actions of Olanzapine in insulin resistance indices (HOMA, QUICKI) were calculated. The subjects animal models. Male (8) and female (7) volunteers were recruited [18-30 were subdivided into two groups according to their fasting plasma glucose: years old, BMI 18.5-25]; subjects with psychiatric or medical disorder that normal glucose tolerance (NGT, FPG<100 mg/dl, n=157) and IFG (n=78). BMI might impact measures were excluded. One male subject did not complete and WHR were similar in NGT and IFG groups (40.2±6.5 vs 41.0±5.2 kg/m2; the study. Placebo or Olanzapine (10 mg/day) was provided for three days 0.92±0.08 vs 0.94±0.08, respectively). IFG subjects were signifi cantly older prior to a fasting oral glucose tolerance test (oGTT) and other blood draws. (46.7±10.9 vs 40.6±14.2 years, p<0.05), had higher fasting plasma glucose The main outcomes examined were oGTT, FFA and fasting leptin along (108.2±5.7 vs 87.82±7.9 mg/dl, p<0.05), insulin (23.85±13.2 vs 15.15±8.6 with triglycerides, total cholesterol, and high- and low-density lipoprotein mU/L, p<0.05), HbA1c (5.99±0.44 vs 5.39±0.22 %, p<0.05), HOMA index cholesterol, heart rate, blood pressure, body weight and BMI. Olanzapine

(6.24±3.4 vs 3.36±1.9, p< 0,05) and lower QUICKI index (0.49±0.06 vs increased glucose Area Under the Curve (AUC) by 42%. In contrast to Obesity

0.58±0.14, p<0,05) than NGT subjects. Plasma total and LDL-cholesterol rodents where fasting plasma leptin concentrations were acutely reduced, POSTERS levels in both groups were similar. IFG subjects had signifi cantly higher fasting plasma leptin in humans was elevated 24%. Similar to animal

plasma triglycerides (2.14±0.91 vs 1.67±0.64 mmol/l, p<0.05),HDL-cholesterol models and consistent with changes observed after chronic administration Integrated Physiology/ (1.19±0.26 vs 1.37±0.31 mmol/l, p<0.05) and LDL/HDL, TC/HDL, TG/HDL in humans, FFA were depressed by 32%. Triglycerides were elevated 22%, ratios than the individuals with NGT. Moreover, the IFG group had higher whereas HDL declined 11% after Olanzapine (P<0.02). Other measures were systolic (137.18±17.0 vs 128.23±14.0 mmHg, p<0.05) and diastolic (85.43±12.9 unchanged. In summary, Olanzapine acutely exerts some but not all of the vs 78. 77±10.9 mmHg, p<0.05) blood pressure. There were no differences in rapid endocrine/metabolic alterations observed in rodent models of atypical LFTs or TSH between the groups. In conclusion, in morbidly obese subjects antipsychotic drug side effects that precede increased adiposity and food the IFG risk is related to age and insulin resistance rather than to the degree intake. The acute effects of olanzapine on glucose tolerance and FFA may be or type of obesity. Moreover, morbidly obese subjects with IFG are at greater rapidly elicited in humans as in rodent models. cardiovascular risk. Supported by: NIH (DK084428) and the Pennsylvania DOH using Tobacco Supported by: Medical University of Lodz Settlement funds

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1902-P 1904-P Intentional Weight Loss Is Associated with Increased Total and Lean Mass Is Inversely Associated with Insulin Sensitivity in Non- High Molecular Weight Adiponectin in Men and Women with Type Diabetic Overweight and Obese Black Women 2 Diabetes in Look AHEAD (Action for Health in Diabetes); Findings SHANNON M. JENKINS, KRISTEN J. PEPIN, BRITTANY O. AICHER, GLORIA on Fitness and Adiposity Change ZALOS, BERNARD V. MILLER III, KONG Y. CHEN, ANNE E. SUMNER, RICHARD O. L. MARIA BELALCAZAR, STEVEN M. HAFFNER, WEI LANG, RON C. HOOGEVEEN, CANNON III, Bethesda, MD KATHERINE M. DONADIO, DAWN C. SCHWENKE, F. XAVIER PI-SUNYER, RUSSELL The prevalence of obesity is higher in blacks than whites. In addition, P. TRACY, ANDREA M. KRISKA, CHRISTIE M. BALLANTYNE, Galveston, TX, Houston, impairment in insulin-mediated glucose uptake tends to be higher in blacks than TX, Winston-Salem, NC, Phoenix, AZ, New York, NY, Burlington, VT, Pittsburgh, PA whites. As skeletal muscle is a major site of insulin-mediated glucose uptake, we Adiponectin and particularly high molecular weight adiponectin (HMW-A) reasoned that race differences in lean mass glucose uptake may exist. levels are low in people with type 2 diabetes. Little is known about the One hundred twenty-two non-diabetic women [age 45±11 (mean±SD) differential effects of weight loss and increased fi tness on total and HMW-A years, range 22-67 years; BMI 34±6 kg/m2, range 25-58 kg/m2; 62% in obese diabetic persons. In a subset of 1,397 Look AHEAD participants (with black, 38% white] were enrolled. Lean mass and truncal fat (% total fat) characteristics similar to the whole sample) we evaluated the effects of a were measured by dual-energy x-ray absorptiometry. Since lean mass is 1-year intensive lifestyle intervention for weight loss (ILI) and of usual care correlated with height (r=0.41, P<0.01), an index of lean mass (LMI) was (Diabetes Support and Educations [DSE]) on total and HMW-A levels using a derived by adjusting to the square of the height. Lean mass glucose uptake quantitative ELISA and investigated the independent contribution of adiposity was estimated using the minimal model. The insulin sensitivity index (SI) and fi tness changes on their levels. Look AHEAD is a randomized trial evaluating was log-transformed to achieve normality, with lower values consistent whether ILI will reduce cardiovascular events/mortality when compared to with reduced lean mass insulin sensitivity. DSE in obese type 2 diabetic persons. Subjects (mean age 57.2 years; BMI LMI was greater in blacks than whites (18±2 versus 17±2 kg/m2, P<0.01); 36.3 kg/m2; submaximal fi tness 5.2 METS) had baseline total and HMW-A truncal fat was similar between races (47±6 versus 46±6%, P=0.35). LMI levels (median [interquartile range]) of 4.1 (3.0, 5.8) and 1.6 (1.0, 2.7) in men was positively associated with truncal fat for blacks (r=0.45, P<0.01) and and 5.1 (3.8, 7.5) and 2.2 (1.4, 3.5) µg/mL in women, respectively. At 1-year, ILI marginally so for whites (r=0.29, P=0.05). In blacks, LMI was inversely participants lost 8% of baseline weight and increased fi tness by 21% (vs 0.6% correlated with SI, but not for whites (Figure). By multiple regression analysis and 6% in DSE, respectively). ILI increased baseline total adiponectin by 24% with age, truncal fat, LMI, and race entered as covariates, truncal fat was 2 in men and 6% in women and HMW-A by 36% and 15% in men and women, the best predictor of SI (β= -0.02, R =0.38, P<0.01), followed by LMI (β= -0.03, respectively (p<0.0001 for all, ILI vs. DSE). Regression analyses (adjusting for R2=0.09, P<0.01) and race (β= 0.10, R2=0.03, P<0.01). Analyzing blacks and baseline adipokine level, randomization arm, demographics, pertinent medical whites separately, LMI remained an independent predictor of SI in blacks (β= history and medication use) showed that weight loss was associated with -0.03, R2=0.08, P<0.01), but not in whites. increased total and HMW-A in men (p<0.0001 for both) and in women (p<0.05 Increased lean mass is an independent predictor of diminished insulin for both), whereas improved fi tness was associated with increased total and sensitivity in overweight and obese black, but not in white women, and may HMW-A in women (p≤ 0.005 for both) but not in men (p=0.18 and 0.6 for total contribute to the greater burden of conditions related to insulin resistance. and HMW-A, respectively). We conclude that ILI differentially raises total and HMW-A levels in obese persons with type 2 diabetes, with greater increases in men and in the HMW-A oligomer. Weight loss in men and weight loss and improved fi tness in women were independent predictors of increased total and HMW-A levels at 1-year. Supported by: HL090514, HL090514S3

1903-P Is the Waist-to-Stature Ratio a Better Predictor Than Other Anthro- pometric Indicators To Predict Future Diabetes Risk? A Meta-Analysis SATORU KODAMA, CHIKA HORIKAWA, YORIKO HEIANZA, AYUMI SUGAWARA, 1905-P YOKO IBE, YOKO YACHI, MIHO MAKI, MIAO SHU, KAZUMI SAITO, HIROHITO SONE, Lipoxygenase Expression in Obese Diabetic Human Adipose Tissue Ibaraki, Japan and Changes with Bariatric Surgery Epidemiological studies have indicated that various obesity indicators are ANCA D. DOBRIAN, JOSHUA J. BROTMAN, QIAN MA, DAVID C. LIEB, JERRY L. associated with the development of type 2 diabetes. Recently, the waist-to- NADLER, Norfolk, VA stature ratio (WSR) was suggested to be useful to predict future diabetes Central obesity is associated with insulin resistance (IR), type 2 diabetes risk. However, it has not been established whether the WSR is more strongly (T2DM) and cardiovascular disease (CVD). A chronic infl ammatory state in associated with diabetes risk than other anthropometric indicators. Our aim omental (OM) fat is thought to participate in the pathology of T2DM. The of the meta-analysis is to quantify the relationship between anthropometric 12- and 15-lipoxygenases (ALOX) generate infl ammatory lipids linked to IR indicators and diabetes risk, focusing on the comparison of the WSR with and CVD in animal models. ALOX are expressed in human adipose tissue (AT), other anthropometric indicators. An electronic literature search was however their role in IR and infl ammation in human obesity is not completely conducted using MEDLINE (1950 to Nov. 23, 2010) and EMBASE (1974 to Nov. understood. The aim of this study was to investigate the expression of different 23, 2010) for prospective studies that investigated the relationship between ALOX isoforms in subcutaneous (SC) and OM human AT in obesity and T2DM obesity and diabetes risk. Studies were included that 1) examined at least and following bariatric surgery. Expression of the pro-infl ammatory cytokines one of the following anthropometric indicators in addition to WSR, which IL-12, IL-6 and IFNγ were also examined by real-time PCR. Data were analyzed were body mass index (BMI), waist circumference (WC), and waist-to-hip by paired and unpaired Student’s t test. Paired biopsies of SC and OM adipose ratio (WHR), and 2) that made it possible to estimate relative risk (RR) of tissues were collected from 21 obese subjects (7 T2DM, 14 controls). BMI was diabetes for a 1 standard deviation (SD) increase in these obesity indicators. similar between the 2 groups, while HbA1C% was signifi cantly higher in the

Obesity Pooled RRs for incremental increases, expressed as RRWSR, RRBMI, RRWC, T2DM group. Paired biopsies were also collected from 8 T2DM subjects at the

POSTERS and RRWHR, and the differences among these associations were calculated time of bariatric surgery and again after a median of 12.5 months. ALOX12, 15a with a multivariate random-effects model that considers both within- and and 15b were expressed solely by the stromal vascular cells in AT. ALOX12,

Integrated Physiology/ between-study correlations. Twelve eligible studies were identifi ed and all 15a, 15b, IL-6 and IL-12a were signifi cantly higher in OM vs. SC by 1.3-2.1-fold gave information on the RR of diabetes for a 1 SD increase in WSR, BMI, (p<0.05) in both T2DM and obese controls. In the OM tissue, ALOX12, IL-12a and WC, and WHR. The pooled RRs [95% confi dence interval] for these obesity IFNγ were all signifi cantly higher in T2DM vs. controls by 1.2-1.8-fold (p<0.05). indicators were 1.56 [1.44 to 1.68] (RRWSR), 1.51 [1.41 to 1.62] (RRBMI), 1.57 No differences in expression of any of the genes in SC tissue were found [1.46 to 1.70] (RRWC), and 1.46 [1.36 to 1.58] (RRWHR). WSR had a stronger between T2DM and controls. None of the tested genes correlated with BMI association with diabetes risk than BMI (P=0.03) and WHR (P=0.04). There but ALOX12 showed a signifi cant positive correlation (r2=0.573, p<0.05) with was no signifi cant difference in the strength of association with diabetes HbA1c levels in the OM depot. Following bariatric surgery ALOX12 expression risk between WSR and WC (P=0.73). It is concluded that WSR is slightly was signifi cantly decreased by 1.6-fold (p<0.05) in OM but not in SC. These new superior to BMI or WHR for predicting future diabetes risk. However, results suggest that ALOX12 is an important infl ammatory marker correlated measuring stature in addition to WC does not appear to provide additional with IR in human AT. Furthermore, ALOX12 may be an attractive therapeutic information for detecting subjects at high risk for diabetes. target to reduce infl ammation in central obesity.

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1906-P 47%, 43% and 40% at 2, 2.5 and 3 yrs respectively. Participants who maintained Long-Term Weight Loss and Improvement in Glycemic Parameters their weight loss within 5 lbs were successful in retaining the achieved A1c with Low-Dose, Controlled-Release Phentermine/Topiramate (PHEN/ target. The signifi cant improvement in BP, lipid profi le and microalbuminuria seen TPM CR) at 12 wks and 1 yr disappeared on longer-term follow up. ROBERT F. KUSHNER, W. TIMOTHY GARVEY, BARBARA TROUPIN, WESLEY W. Conclusion: Following the initial 12 wks of intensive non-surgical diabetes DAY, Chicago, IL, Birmingham, AL, Mountain View, CA weight management in routine practice, participants may be able to maintain Obesity is an epidemic linked to chronic comorbidities, including type 2 ∼7% weight loss for 3 yrs on their own. However, some of the metabolic diabetes mellitus (T2DM). Previously, weight loss associated with PHEN/ improvements seen with initial weight loss did not last long-term except in TPM CR demonstrated improvements in glycemic parameters through 56 those who maintained their exit weight within 5 lbs. weeks in the CONQUER study. SEQUEL, an extension of CONQUER, was conducted to evaluate long-term (108 weeks) effects of PHEN/TPM CR on 1908-P weight loss and glycemic parameters related to T2DM. This double-blind Lorcaserin, a Selective 5-HT2C Agonist, Evaluated as a Weight Loss extension maintained the original blinded treatment groups for an additional Agent in Obese and Overweight Patients with Type 2 Diabetes 52 weeks in subjects completing 56 weeks of CONQUER: placebo (PBO; Treated with Sulfonylureas or Metformin n=227), PHEN 7.5 mg/TPM CR 46 mg (7.5/46; n=153), or PHEN 15 mg/TPM MEREDITH C. FIDLER, RONALD SHAZER, MATILDE SANCHEZ, SCOTT STUBBE, CR 92 mg (15/92; n=295). All subjects with T2DM were managed to the ADA CHRISTEN M. ANDERSON, WILLIAM R. SHANAHAN, San Diego, CA standards of care. In the overall ITT-LOCF sample, least-squares (LS) mean Lorcaserin (Lor) is a selective 5HT2C agonist that has been shown to percent weight loss was signifi cantly greater with both doses of PHEN/TPM induce weight loss in non-diabetic obese and overweight patients in two CR compared with PBO (P<0.0001) at Week 108: 1.8%, 9.3%, and 10.5% previous phase 3 trials. BLOOM-DM was a 52-wk, randomized, double-blind, for PBO, 7.5/46, and 15/92, respectively. Similarly, subjects with T2DM at placebo-controlled study in patients (pts) with type 2 diabetes inadequately baseline (n=145) experienced signifi cantly greater weight loss with both controlled with oral agents (HbA1C 7-10%), 18 to 65 years-old, and with a doses of PHEN/TPM CR than with PBO (P≤0.0003): LS mean percent weight BMI between 27 and 45 kg/m2; 256 pts took Lor 10 mg BID (L), 252 pts took loss was 2.0%, 9.0%, and 9.0% for PBO (n=55), 7.5/46 (n=26), and 15/92 (n=64). placebo (P). At baseline (BL), 49.6% took metformin (M) without sulfonylurea Treatment with PHEN/TPM CR was also associated with improvements in (SFU) and 50.4% took SFU with or without M (analyzed as SFU subgroup); several glycemic parameters compared with PBO (Table). Subjects receiving 41.9% took both. Wk 52 results were analyzed to determine whether the 15/92 were more likely to undergo a reduction in concomitant antidiabetic effi cacy or safety of Lor was affected by baseline antidiabetic medication. medications compared to subjects receiving PBO who were more likely to At BL, mean BMI was 36.0 kg/m2, mean age was 52.7 years and 54% of pts require an increase. PHEN/TPM CR was well tolerated over 108 weeks with were female. The trial was completed by 66.0% of L and 62.1% of P. Using an overall completion rate of 84% on study drug. The most common adverse MITT/LOCF analysis, 37.5% of pts on L and 16.1% of pts on P (p<0.001) lost events were similar to those experienced at Week 56 of CONQUER, with ≥5% of baseline body weight. Analyses by baseline antidiabetic medications lower incidence rates at 2 years. The sustained weight loss demonstrated presented in the table show that effi cacy was not affected by treatment with PHEN/TPM CR led to long-term improvements in several glycemic with M or SFU. Changes in lipids and blood pressure were comparable parameters over 2 years. in patients in the M and SFU subgroups. More pts on M alone than SFU Table. LS Mean Change in Glycemic Parameters at Week 108 (Overall receiving L achieved HbA1C levels <7% (SFU 42.2%, M 58.2%), due in part to Sample; ITT-LOCF) lower mean BL values in pts receiving M (SFU 8.3%, M 7.8%). Symptomatic Change at Week 108 hypoglycemia occurred more frequently in L than P treated pts, and more frequently with SFU (L 28.6%, P 19.2%) than M (L 9.6%, P 2.4%); no severe Baseline PBO 7.5/46 15/92 hypoglycemia occurred. Weight loss was greater in L than P pts with type HbA1c (%) 6.0 0.2 0.0* 0.0* 2 diabetes whether treated with M or SFU. SFU predictably increased the Fasting glucose (mg/dL) 109.1 3.7 0.1 -1.2* frequency of hypoglycemia as compared to M in both treatment groups. Fasting insulin (µIU/mL) 17.4 -2.6 -5.3* -5.2* L P *P≤0.005 vs PBO SFU (n=126) M (n=125) SFU (n=125) M (n=123) Supported by: Vivus, Inc. % achieving ≥5% weight loss 31.7 43.2 16.0 16.3 Change from Baseline 1907-P Body weight (kg) -4.2 -5.7 -2.0 -1.7 Long-Term Weight Reduction Is Achievable in Clinical Practice HbA1C (%) -1.0 -0.9 -0.4 -0.5 after Non-Surgical Diabetes Weight Management Program OSAMA HAMDY, AMR MORSI, NUHA ELSAYED, ANN GOEBEL-FABBRI, GILLIAN Fasting plasma glucose (mg/dL) -34.2 -23.8 -9.7 -13.0 ARATHUZIK, JACQUELINE SHAHAR, AMANDA KIRPITCH, MICHAEL SEE, JOHN HOMA-IR -0.57 -0.47 -0.28 -0.13 ZREBIEC, PAMELA NEEDLE, ANDREA PENNEY, ALI NABOUSH, JO-ANNE Fasting insulin (µIU/mL) -2.9 -2.4 -0.6 -3.3 RIZZOTTO, MARTIN J. ABRAHAMSON, Boston, MA Weight Achievement and Intensive Treatment (Why WAIT) is an intensive 12-wk multidisciplinary diabetes weight management program designed 1909-P for clinical practice. Participants in this program were followed for 3 yrs to Lower Serum Creatinine Is Related to Prevalence of Glucose Ab- evaluate its long-term outcomes. Anthropometric, blood pressure (BP) and normality among Obese Korean Men metabolic parameters were measured after 12 wks and during routine clinic BO GWANG CHOI, JI HYUN KANG, YOUNG BAE LIM, MI RA KIM, YUN KYOUNG visits. JEON, SANG SOO KIM, BO HYUN KIM, YONG KI KIM, IN JU KIM, Busan, Republic We enrolled 141 (91f/50m) obese patients with diabetes (127 type 2 and of Korea 14 type 1) with mean age of 53.3±10.3 yrs (28-81 yrs), diabetes duration Serum creatinine is possibly related to skeletal muscle mass, which is of 9.5±9.2 yrs, weight of 241.2±42.2 lbs, BMI of 38.6±5.3 kg/m2, A1c of one of the target tissues for insulin. The aim of this study is to evaluate Obesity 7.5±1.3%. Six patients dropped out. Enrollment was sequential over time the association between serum creatinine level and the components of POSTERS (10-15/group) - 135 patients completed 2 yrs of follow up, 105 completed metabolic syndrome (MS) among adult Korean. We used data from 2,154 2.5 yrs and 70 patients completed 3 yrs. The program included: change in Korean men (aged ≥30 years) with serum creatinine level ≤1.2 mg/dL, based Integrated Physiology/ diabetes medications to enhance weight loss with reduction or elimination on the national data from representative sample of the forth Korea National of weight promoting drugs, a prescribed lower calorie diet using customized Health and Nutrition Examination Survey (KNHANES IV-2) in 2008. Fasting menus of natural food and meal replacements (∼40% carbohydrates and 20- glucose, HOMA-IR, HDL cholesterol and systolic blood pressure (SBP) 30% protein), prescribed strength and cardiovascular exercise and weekly were signifi cantly different among different creatinine categories in whole didactic & behavioral support sessions. At program end, participants were population. There were no signifi cant differences of the prevalence in the instructed to follow the same plan on their own. components of MS and HOMA-IR among different creatinine categories in After 12 wks, average weight loss was -24.1±10.7 lbs (-9.9%, p<0.001), and the normal and underweight men. Among obese men, the fasting glucose, average A1c was 6.6±0.93 (p<0.001). After 2, 2.5 and 3 yrs, weight remained HOMA-IR and SBP in the lowest creatinine category (≤0.80mg/dL) were lower at -18.2 (-7.2%), -17.2 (-6.9) and -17.2 (-7.0%) respectively (p<0.001). Around the highest and the lowest category of creatinine was associated with an 82% achieved target A1c of <7% at 12 wks, but this percentage was down to increased risk of glucose abnormality, but not other components of MS.

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The multiple adjusted odds ratio for those who had the lowest creatinine In contrast to normal-weight insulin-resistant people, MHO individuals level (≤0.80mg/dL) was 1.785 (95% CI 1.145– 2.781) compared with those show decreased HF-risk in a 6-year follow-up study. who had the highest creatinine level (1.01–1.20). In conclusion, lower serum creatinine was associated with an increased risk of glucose abnormality, but 1912-P not other components of MS among obese Korean men. Pathogenic Seipin Mutant Suppresses Adipogenesis Via ER Stress- Associated Infl ammatory Response 1910-P WENJIE QIU, KENNETH WEE, SHIGEKI SUGII, WEIPING HAN, Singapore, Singapore Metabolic Syndrome Is Associated with Impaired Pulmonary Func- Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is an autosomal tion in a Restrictive, but Not Obstructive, Pattern, in Japanese Men recessive disease characterized by the near complete absence of adipose TAKEHIDE OGIHARA, NORIKO CHIKATSU, TAKESHI NAWA, RYOICHI NAMBA, tissue from birth or early infancy, and development of metabolic syndrome in YUSUKE YAMAMOTO, MICHIKO MURAOSA, SHINJI HIRAI, YUJI OKA, Hitachi, Japan adulthood, such as early onset diabetes mellitus, severe insulin resistance, Metabolic syndrome (MS) has been shown to be associated with impaired hypertriglyceridemia and fatty liver. The mechanism whereby pathogenic pulmonary function, especially chronic obstructive pulmonary disease (COPD). mutations in BSCL2/seipin gene cause lipodystrophy is unknown. In this Systemic infl ammation is a possible mechanism underlying the association study, we investigated the role of a pathogenic missense mutant seipin- between these disorders. In this study, we investigated the effect of MS on A212P in the regulation of adipocyte differentiation. We established two pulmonary function by analyzing data from 9,573 men aged 22 to 89 years. stable 3T3-L1 cell lines expressing wild type seipin (3T3-wt) or seipin- Based on the diagnostic criteria for MS in Japan, current/former-smokers A212P (3T3-mut). When treated with the standard adipogenic cocktail, and those who never smoked were each divided into MS and non-MS groups. 3T3-wt cells exhibited proper time course of differentiation into mature Then we examined the relationship between MS and measures of pulmonary adipocytes, indistinguishable from non-transfected 3T3-L1 cells. In contrast, function (including percentage predicted forced vital capacity; %FVC, adipogenesis was signifi cantly inhibited in 3T3-mut cells, possibly due percentage predicted forced expiratory volume in 1 s; %FEV1, and FEV1- to- to reduced PPARγ expression, as the defective adipogenesis could be FVC ratio). Next, we investigated the correlation between visceral fat area partially rescued by either treatment with pioglitazone, a PPARγ agonist, or and each measure of pulmonary function. Lastly, we analyzed the infl uence of overexpression of PPARγ. Gene expression profi ling by microarray analysis MS diagnostic criteria on each measure of pulmonary function using stepwise revealed that the inhibition of adipogenesis was associated with activation regression analysis. Non-smokers and smokers with MS showed signifi cantly of infl ammatory pathways and ER-stress response. These results suggest lower %FVC and %FEV1 than those without MS, respectively. However, that the pathogenic missense mutant seipin-A212P may induce ER stress- FEV1- to-FVC ratio was signifi cantly higher in subjects with MS than in those associated infl ammation to inhibit adipogenesis. without MS. Among smokers and non-smokers, visceral fat area showed weak Supported by: Agency for Science Technology and Research negative correlations with %FVC and %FEV1. In the regression equation with %FVC as a dependent variable, waist circumference, blood pressure, lipids 1913-P and fasting blood glucose were signifi cant negative explanatory variables. Patterns of Weight Changes over the First Year of an Intensive However, in that with FEV1- to-FVC ratio, lipids and fasting blood glucose were Weight Loss Intervention Are Associated with Longer Term Weight positive explanatory variables. Thus, indicators of impaired pulmonary function Maintenance and Changes in Cardiovascular Disease Risk Factors: associated with MS are %FVC and %FEV1, suggesting that MS is associated Results from the Action for Health in Diabetes (Look AHEAD) with impaired pulmonary function in a restrictive, but not obstructive, pattern. MARK A. ESPELAND, REBECCA H. NEIBERG, GEORGE A. BRAY, LUCY FAULCON- As airfl ow limitation for the diagnosis of COPD is judged by FEV1- to-FVC ratio, BRIDGE, KAREN C. JOHNSON, ABBAS E. KITABCHI, DALANE W. KITZMAN, AMY there is a risk of missing early COPD among smokers with MS. Therefore, D. OTTO, RENA WING, Winston-Salem, NC, Baton Rouge, LA, Philadelphia, PA, smokers with MS should be advised early of the need for smoking cessation. Memphis, TN, Pittsburgh, PA, Providence, RI Weight loss patterns during the fi rst year of an intensive lifestyle inter- 1911-P vention may predict longer term weight loss maintenance and risk factor Metabolically Healthy Obese Individuals Have Decreased Heart changes. Principal components analysis was used to identify two major weight Failure Risk Compared to Normal-Weight People in a Six-Year Medi- loss patterns among 2,438 individuals, aged 45-75 years with type 2 diabetes ter ranean Study mellitus, who were enrolled intervention of Look AHEAD: a randomized clinical VASILLIS VOULGARIS, CHRISTINA VOULGARI, NICHOLAS TENTOLOURIS, PAUL trial to test whether an intensive lifestyle intervention designed to promote and POIRIER, Athens, Greece, Quebec, QC, Canada maintain weight loss reduces the risk of major cardiovascular disease events. Heart failure (HF) is one of the leading causes of mortality and its prevalence The fi rst pattern corresponded to the accumulated weight loss over time (i.e. continues to rise, despite the decline in cardiovascular death rates. Among the area transcribed by the weight loss curve). The second described, for a given the risk factors identifi ed as consistently associated with its development amount of weight loss during the fi rst year, whether this occurred relatively are Obesity and Metabolic Syndrome (MetS). “Metabolically Healthy Obese” quickly and then stabilized or whether it occurred more gradually and continued. (MHO) people, despite their body fat, display a favorable metabolic profi le The characteristics and 4-year changes in weight and risk factors (glycosolated characterized by high levels of insulin-sensitivity, and a benefi cial blood- hemoglobin – HbA1c; systolic blood pressure, HDL-cholesterol, LDL-cholesterol, pressure, glycemic, lipids, and infl ammation profi le. It remains controversial and triglycerides) of participants grouped according to their expressions of whether this healthier metabolic profi le is translated into a lower cardiovascular these patterns were contrasted. Individuals who, during the fi rst year of the risk compared with normal-weight individuals with MetS. intervention, achieved a greater accumulated weight loss and/or a more A total of 550 individuals without diabetes or baseline macrovascular gradual and sustained weight loss had better 4-year weight loss maintenance, complications were studied. Participants were classifi ed by presence independent of a number of characteristics traditionally linked to weight loss (n=271) or absence (n=279) of MetS and by BMI (BMI; <25 Kg/m2=normal- success (p<0.001). Compared to other participants, those with the greatest weight, n=177; 25-29.9 Kg/m2=overweight, n=234; ≥30 Kg/m2=obese, accumulation of weight loss during the fi rst year had better 4-year levels of HbA1c n=139). MetS was diagnosed with the NCEP-ATP-III criteria. Left ventricular and systolic blood pressure, even after adjustment for 4-year weight losses. A functional capacity, myocardial structure and performance were assessed greater accumulation of weight loss and a more gradual and sustained pattern

Obesity echocardiographically. of weight loss in response to an intensive lifestyle intervention are associated

POSTERS Six-year HF incidence was compared by obesity and metabolic status. with better long-term maintenance. Greater accumulated weight loss over the Obesity status or BMI were not associated with increased HF risk (HR 0.88, fi rst year is associated with better longer term risk factor profi les, independent

Integrated Physiology/ 95%CI 0.41-1.32). Presence of MetS conferred 2.5-fold higher HF risk (HR of longer-term weight loss maintenance. 2.5, 95%CI 1.68-3.40). Overweight/obese individuals without MetS had the lowest 6-year HF risk (HR 1.12, 95%CI 0.35-1.33) compared to normal- 1914-P weight people with MetS (HR 2.33, 95%CI 1.25, 4.36, P=0.007). From the PGC1-α Gly482Ser Polymorphism Predicts Improved Metabolic, individual components of MetS, impaired fasting glucose (HR 1.5, 95% Infl ammatory and Vascular Outcomes Following Gastric Bypass 1.23-1.92), hypertension (HR 1.1, 95% 1.03-1.47), dyslipidemia (HR 0.48, 95% Surgery 0.39-0.58) and central-obesity (HR 1.33, 95% 1.17-1.64) were all associated SYLKA R. GELONEZE, BRUNO GELONEZE, JOSEANI MORARI, MARCELO M. LIMA, with increased HF risk. Physical inactivity (<7.5 MET-h/week), smoking and JOSÉ R. MATTOS-SOUSA, JOSÉ C. PAREJA, ELINTON A. CHAIM, LÍCIO A. VELLOSO, factors commonly associated with MetS like insulin-resistance, low-grade Campinas, Brazil infl ammation (high-sensitivity C-reactive protein, microalbuminuria) were Bariatric surgery has a positive impact on cardiovascular risk. PGC1α is independently associated with HF incidence. a co-activator of PPARγ involved in the regulation of metabolic functions.

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A516 OBESITY—HUMANCATEGORY

There is an association of the Gly482Ser polymorphism with type 2 min) postmenopausal (45-76 yr) women. We measured insulin sensitivity (M) diabetes and obesity. No previous study has evaluated the association of and metabolic fl exibility via indirect calorimetry after an overnight fast and this polymorphism with the outcomes of bariatric surgery. We tested the 2 hrs into an 80 mU.m-2.min-1 hyperinsulinemic-euglycemic clamp. Women hypothesis that Gly482Ser polymorphism could predict clinical, laboratorial underwent a VO2max test, DXA and CT scans and a subset (n=62) did a 1 mg and structural atherosclerotic marker [carotid intima-media thickness dexamethasone suppression test of plasma cortisol (DST). Plasma cortisol (C-IMT)] outomes. Patients (n=55) were submitted to Roux-en-Y gastric levels decreased signifi cantly after DST (11.4 ± 0.6 vs. 5.0 ± 0.4 ng/ml, bypass (RYGB) and evaluated for anthropometric, C-IMT, metabolic and P<0.0001), a 54% suppression. Metabolic fl exibility was not associated with infl ammatory parameters at baseline, 1, 6 and 12 mo after RYGB. The VO2max, total body fat, visceral fat, or subcutaneous abdominal fat but was polymerase chain reaction- restriction fragment length polymorphism associated with muscle attenuation (r=-0.32, P<0.05). Metabolic fl exibility was performed. Serum IL-6, adiponectin, leptin, insulin and MCP-1 were correlated with glucose120 min of OGTT (r=-0.25, P<0.05), fasting insulin (r=-0.26, determined. An euglycemic-hyperinsulinemic clamp was performed. C-IMT P<0.01), and insulin sensitivity (r=0.45, P<0.0001), and % cortisol suppression was measured 1.0 cm distal to the bulbus over a length of 1.0 cm of carotid (r=0.27, P<0.05). We conclude that metabolic fl exibility is associated with arteries. At baseline, all parameters were similar between groups. At 1-year ectopic lipid deposition in muscle, insulin sensitivity and suppression of after surgery, BMI dropped from 45 to 28 in the Gly/Gly group, and from 44 to cortisol by dexamethasone in obese, sedentary postmenopausal women. 28 in Gly/Ser+Ser/Ser group. There was a signifi cant reduction in the waist/ hip ratio only in the Gly/Ser+Ser/Ser group. Lipid profi le improved after 12 1917-P mo similarly in both groups. Fasting blood glucose dropped signifi cantly only Prevalence of Newly Diagnosed Diabetes and Cardiovascular Risk in the Gly/Ser+Ser/Ser. Insulin, HOMA-IR and clamp results, and adipokines Factors (CVRFs) in a Young,WITHDRAWN Asymptomatic, and Physically Active (leptin, adiponectin) presented signifi cant changes similarly in both groups. Population with a Normal and Elevated Body Mass Index (BMI) Both Gly/Gly and Gly/Ser+Ser/Ser groups presented reductions of us-CRP, GLENN K. LEE, HUI WEN SIM, YANNI TAN, THAZIN MA, KIAN HWA LEW, MCP-1 and IL-6. Signifi cant inter-group differences were detected for us-CRP, ENG CHUN TAN, LI CHING LEE, CHEE FANG SUM, HEAN YEE ONG, Singapore, blood leukocyte counts and IL-6. Both Gly/Gly and Gly/Ser+Ser/Ser patients Singapore, Georgetown, Malaysia presented signifi cant reductions at 12 mo (0.20 mm, and 0.27 mm, respectively, Background: Obesity is associated with signifi cant morbidity and mortality. p<0.05 between groups). The presence of the Gly482Ser polymorphism While BMI is a crude surrogate marker of body fat, it remains a simple and predicts metabolic and infl ammatory outcomes, ultimately leading to a more inexpensive technique to quantify obesity. In a low-risk population, we pronounced reduction in a structural marker of atherosclerosis, in patients aim to determine the association of BMI with CVRFs and newly diagnosed undergoing RYGB. The evaluation of this polymorphism may contribute to diabetes. refi ne the selection of candidates for surgery. Methods: We studied 3026 subjects referred for routine employment health screening. Patients with pre-existing diabetes mellitus and/or vascular 1915-P disease were excluded. Each subject had anthropometric measurements and Plasma Insulin and Infl ammatory Markers Prior to Weight Loss Can CVRF parameters taken. Predict Dietary Responders Results: The mean age was 38.9±5.4 years, 89.9% were males. The LING CHUN KONG, FROOGH HAJDUCH, PIERRE HENRI WUILLEMIN, JEAN prevalence of newly diagnosed diabetes was 2.4%. The majority of subjects PHILLIPPE BASTARD, SORAYA FELLAHI, DOMINIQUE BONNEFONT-ROUSSELOT, were low risk using the Framingham Risk Score (84.5%). The mean BMI was RANDA BITTAR, ARNAUD BASDEVANT, JEAN DANIEL ZUCKER, JOËL DORÉ, 25.2±4.0kg/m². A positive correlation was seen between BMI and prevalence KARINE CLÉMENT, SALWA RIZKALLA, Paris, France, Jouy-en-Josas, France of CVRFs (p<0.001 for all). At a normal BMI of 20-22kg/m², only 42.0% and The ability to identify obese subjects who will lose weight in response to 51.3% had normal blood pressure and LDL levels, and at a BMI of <20kg/ energy restriction is an important strategy in obesity clinical care. m², the prevalence of newly-diagnosed diabetes was naught. Prevalence of Fifty obese/overweight subjects were submitted to an energy restricted raised LDL levels plateaus beyond BMIs of 24kg/m². Patients with BMI >20kg/ high protein diet for 6 weeks followed by a 6 weeks-maintenance diet. Based m² have increased odds of pre- and hypertension (OR 4.19, 95%CI 3.14–5.59), on their trajectories of weight loss during the study, three subjects clusters abnormal fasting glucose (2.05, 1.03–4.06), total cholesterol (2.70, 1.95–3.74), were identifi ed. Cluster A (n=17) and Cluster B (n=15) lost more weight during LDL (3.91, 2.73–5.59), and HDL (4.90, 2.00–12.04) (p<0.05 for all). the diet period, however during the stabilization phase cluster A continued to Conclusions: A signifi cant proportion of our subjects with a normal (Asian) loose weight, whereas cluster B remained stable. Cluster C (n=17) lost less BMI of <23kg/m² were found to have elevated CVRFs on routine screening. and rapidly regained weight during the stabilization. At baseline, subjects in The step-wise rise and additive nature of these CVRFs and her consistent cluster C had the highest plasma insulin (P=0.01), IL-6 (P=0.05) and adipose correlation with a rising BMI of above 20kg/m² suggest that an ideal tissue infl ammatory marker (HAM56, P=0.03), and the lowest plasma free BMI conferring optimal levels of measurable CVRFs including a very low fatty acid excursions after a glucose charge (FFA AUC, P=0.03). Gut microbiota prevalence of diabetes may perhaps be a below-average value of 18-20kg/ was profi led from faecal samples by qPCR in all the subjects. Intriguingly m² for our population. subjects in cluster C had the highest level of Lactobacillus/Leuconostoc/ Pediococcus group before diet. During the dietary program subjects in cluster C consumed more starchy foods, less protein and raw vegetables. Spearman correlations revealed positive relationship between weight regain after diet and HOMA-IR (P=0.0002, rs=0.5), infl ammatory markers (leucocytes numbers: P=0.05, rs=0.27; Neutrophils: P=0.05, rs=0.28; IL-6: P=0.002, rs=0.43) as well as the number of Lactobacillus/Leuconostoc/Pediococcus group (p=0.005, rs=0.4) at baseline. Bayesian network (BN) was performed for prediction of the 3 clusters by using the data prior to the weight-loss dietary program. According to the learnt structure of BN, the levels of 4 biomarkers (plasma insulin, IL-6, leucocytes numbers and adipose tissue HAM56) at baseline were suffi cient to characterize the distribution of the 3 clusters. The prediction of clusters Obesity

was 75.5% (37 among 49 subjects). We concluded that individual responses to POSTERS hypocaloric high protein dietary program could be predicted by plasma insulin, IL-6, leucocytes numbers and adipose tissue HAM56 levels prior to diet. Integrated Physiology/ Supported by: French National Agency for Research

1916-P Predictors of Metabolic Flexibility in Obese Postmenopausal Women ALICE S. RYAN, SHAWNA L. MCMILLIN, ANDREW GOLDBERG, Baltimore, MD Obesity is characterized by metabolic infl exibility, a condition described as an inability to switch from fat oxidation during fasting to carbohydrate oxidation during hyperinsulinemia. Cortisol plays a role in fat accumulation, insulin resistance and glucose metabolism. We sought to examine predictors of metabolic fl exibility in 110 obese (37-59% fat), sedentary (19 ± 0.5 ml/kg/

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A517 OBESITY—HUMANCATEGORY

1918-P of food motivation in response to visual food cues. Ten healthy subjects Prevalence-Adjusted Cost of Comorbidities in Overweight/Obese (mean age 31±8, A1c 5.4±0.4) underwent a 2mU/kg.min hyperinsulinemic Patients with a Body Mass Index 25-34.9 vs ≥35 euglycemic clamp (goal=plasma glucose ∼90mg/dl) while viewing high fat, DIANA I. BRIXNER, MORGAN BRON, BRANDON BELLOWS, XIANGYANG YE, low fat food and non-food pictures, 2 hours after receiving a standardized VENKATESH HARIKRISHNAN, GARY M. ODERDA, Salt Lake City, UT, Deerfi eld, IL meal. Liking and wanting ratings were collected for each picture. Hunger Obesity is associated with an increased prevalence of comorbidities. ratings were collected at baseline, 60, 85min and at 2 hrs. Initially plasma This study looked at prevalence and cost of comorbidities in 2 categories of glucose rose from 83±9 mg/dL at baseline to 98±11 mg/dL after 10mins. overweight/obese patients based on BMI. During the interval between 10 and 60 min plasma glucose showed more Prevalence of 25 comorbidities in patients with BMI 25-34.9 and ≥35 was variability, although mean levels decreased slightly to 93±7mg/dL (it calculated and ranked based on data from the GE Centricity EMR research decreased by 14+/-4 mg/dL in 6 subjects and increased by 11+/-5 mg/dL in database. Patients whose BMI was within NHLBI guideline categories for 4 subjects). Subsequently, plasma glucose stabilized at 95±9mg/dL until the overweight (25-29.9), class I obesity (30-34.9) and class II or III obesity (≥35) end of the clamp. Hunger ratings, after subjects viewed food and non-food between 3/1/05 and 6/30/09 were included. BMI index date was defi ned as images, signifi cantly increased from 1.7±0.7 at baseline to 4.0±0.8 at 60min date of either only BMI on record, or latest BMI for patients with 2 or more (p=0.01) and 5.2±2.9 at 2 hrs. (p=0.004). BMI readings. Prevalence of comorbidities was based on presence of ICD- Hunger levels at the end of the clamp were associated with glucose at 9 code within 2 y prior to BMI index date. Annual comorbidity-associated 10min (r=0.816,p=0.004) and 60min (r=-0.702,p=0.02) and the drop in glucose costs were based on an ICD-9 code for overweight or obesity and patient between 10 and 60mins (r=0.848,p=0.002). The changes in glucose were not activity for 1 y post comorbidity index date in Thomson Reuters MedStat associated with signifi cant increases in plasma glucagon, cortisol, FFA, or MarketScan database. Prevalence-adjusted cost of comorbidities was ghrelin from baseline levels. In addition, leptin levels increased (p<0.02). determined by multiplying annual comorbidity-associated cost per patient Furthermore, there were no signifi cant correlations between any hormone from MarketScan by comorbidity prevalence rate ratio from 2 BMI groups level and hunger rating. Mean liking and wanting scores did not change of the EMR database. signifi cantly during the clamp. Our data suggest that small decrements in The GE EMR database identifi ed 109,885 patients with BMI ≥25 and 2 y EMR plasma glucose within the normal range provoke increased hunger ratings activity before BMI index date. The 3 comorbidities with highest prevalence (BMI when individuals are exposed to visual food cues, which in turn may facilitate 25-34.9, ≥35) were hyperlipidemia (14.6%, 16.3%), hypertension (10.2%, 17.7%), food consumption among vulnerable individuals. and back pain (7.5%, 8.3%). Annual comorbidity-associated cost per overweight/ obese patient was $1136 for hyperlipidemia, $1511 for hypertension, and $2081 1921-P for back pain. Prevalence-adjusted per patient cost increased from BMI 25-34.9 Subjects with Type 2 Diabetes Meeting the Metabolic Syndrome to BMI ≥35 in hyperlipidemia ($166 to $185), hypertension ($154 to $268), and Criteria Have High Levels of Visceral Adipose Tissue and Liver Fat back pain ($156 to $173). As an example, a plan with 1,000 members with BMI JESSICA D. SMITH, ANNE-LAURE BOREL, JULIE-ANNE NAZARE, STEVEN M. 25-34.9 would have hyperlipidemia-associated cost of $166,000 vs $185,000 for HAFFNER, BEVERLEY BALKAU, ROBERT ROSS, NATALIE ALMÉRAS, JEAN-PIERRE a plan with 1,000 members with BMI ≥35. DESPRÉS, INSPIRE ME IAA INVESTIGATORS, Quebec, QC, Canada, Houston, TX, This study confi rmed the incremental impact of weight on prevalence and Villejuif, France, Kingston, ON, Canada cost of selected comorbidities in overweight or obese patients. Management The added value of a clinical diagnosis of the metabolic syndrome or therapies that can lower patients’ BMI have the potential to decrease (MetS) remains debated. The present analysis examined the relevance comorbidity rates and may lower costs for health plans. of the MetS in explaining heterogeneity of cardiovascular disease risk in type 2 diabetes (T2D). In order to evaluate the effects of MetS versus T2D 1919-P we defi ned subjects from the INternational Study of Prediction of Intra- Short-Term Evolution of the Insulin Resistance Depending on the Abdominal Adiposity and its Relationships with CardioMEtabolic Risk/ Type of Bariatric Surgery Intra-Abdominal Adiposity (INSPIRE ME IAA) into four groups: those without LOURDES GARRIDO-SANCHEZ, MORA MURRI, JOSE RIVAS-BECERRA, DIEGO Met S or T2D (MetS-T2D-), those with Met S only (MetS+T2D-), those with FERNANDEZ-GARCIA, JUAN ALCAIDE, EDUARDO GARCIA-FUENTES, FRANCISCO T2D only (MetS-T2D+) and those with both (MetS+T2D+). We evaluated TINAHONES, Tarragona, Spain, Malaga, Spain several cardiometabolic risk factors, including visceral adipose tissue Obesity is very often accompanied by other diseases, the most common being (VAT), abdominal subcutaneous adipose tissue (SAT) and liver attenuation type 2 diabetes mellitus and cardiovascular complications. Bariatric surgery by computed tomography. We performed multivariate analyses of the is almost the only effective strategy for treating morbidly obese patients. cardiometabolic risk factors, including both T2D and MetS in the model, The aims of this study was evaluate the metabolic changes that occur in the and adjusting for age, BMI, and ethnicity for men and women separately. early stage after two types of bariatric surgery, biliopancreatic diversión of Subjects (N=4055) were recruited from 29 countries in Asia, Europe, Latin Scopinaro (BPD) and Sleeve gastrectomy (SG), in morbidly obese persons. The America and North America by their physician and were between the ages study was undertaken in 31 morbidly obese persons (7 men and 24 women) 15 of 39 to 71 for men and 44 to 71 for women. VAT was signifi cantly higher in days before and 15, 30, 45 y 90 days after bariatric surgery (times 0, 1, 2, 3, and subjects with the MetS in those both with and without T2D. For men, the 4, respectively). There is a signifi cant improvement in anthropometric variables presence of T2D was not associated with a further increase in VAT (model as result of the two types of bariatric surgery, without signifi cant differences p<0.0001, MetS p<0.0001, T2D p=0.7), whereas MetS+T2D+ women had the between both types of interventions. In patients undergoing BPD, serum highest level of VAT (model p<0.0001, MetS p<0.0001, T2D p=0.0001). The glucose, cholesterol, triglycerides, HDL-cholesterol and FFA were signifi cantly lowest liver attenuation, indicating the highest liver fat content, was also reduced. The changes that occur in these biochemical variables following the observed among MetS+T2D+ men (model p<0.0001, MetS p<0.0001, T2D SG were not signifi cant. Insulin resistance decreased signifi cantly over the p<0.0001) and women (model p<0.0001, MetS p<0.0001, T2D p<0.0001). CRP 90 days after surgery, showing the highest decrease at 15 days. Meanwhile, was increased in subjects both with and without T2D by the presence of in patients undergoing SG, insulin resistance worsened at 15 days and later MetS. CRP did not differ between MetS+T2D- and MetS+T2D+ groups in men diminished. In conclusion, this study shows that the surgical technique that (model p<0.0001, MetS p=0.0003, T2D p=0.5) and women (model p<0.0001, MetS p<0.0001, T2D p=0.9). We conclude that clinical assessment of MetS

Obesity excludes the duodenum (BPD) has immediate postoperative changes in the

POSTERS degree of insulin resistance in morbidly obese patients compared to those allows the identifi cation of individuals with high levels of VAT and liver fat, techniques that do not exclude (SG). irrespective of the absence/presence of T2D. Supported by: sanofi -aventis

Integrated Physiology/ Supported by: JCI-2009-04086, CP04/00133, PS09/01060, PS09/00997

1920-P 1922-P Small Decrements in Glucose Increase Hunger in the Presence of The Association of the rs1049353 Polymorphism of the CNR1 Gene Visual Food Cues with Hypoadiponectinemia in Subjects with Abdominal Obesity RENATA BELFORT DE AGUIAR, SARITA NAIK, DONGJU SEO, RAJITA SINHA, ILIE-ROBERT DINU, SIMONA GEORGIANA POPA, MARIA MOTA, EUGEN MOTA, ROBERT SHERWIN, New Haven, CT CAMELIA STANCIULESCU, MIHAI IOANA, Craiova, Romania Hypoglycemia is associated with increased hunger, triggering increased The endocannabinoid system (ECS) is involved in energy balance and food consumption. The mechanism for how circulating glucose infl uence appetite stimulation. CNR1 is the gene encoding type 1 cannabinoid food-seeking behavior is unclear. In this study, we evaluated the effects of receptor, implicated in the metabolic functions of ECS. Adiponectin is the small changes in glucose within the physiological range on measurements most abundant adipose-specifi c adipocytokine with key metabolic functions.

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A518 OBESITY—HUMANCATEGORY

Several studies indicated that obese subjects, particularly those with 1924-P visceral fat accumulation, have reduced plasma levels of adiponectin. Other The Lipid Accumulation Product Is Associated with Impaired results indicate that rs1049353 (1359G/A) polymorphism of the CNR1 gene is Glucose Tolerance in PCOS Women associated with increased abdominal circumference (AC). ELISABETH WEHR, THOMAS R.WITHDRAWN PIEBER, BARBARA OBERMAYER-PIETSCH, Graz, The aim of our study was to investigate the association between the Austria rs1049353 polymorphism and low adiponectin levels in subjects with Women with polycystic ovary syndrome (PCOS) frequently suffer from abdominal obesity (AO). metabolic disturbances, in particular from insulin resistance and impaired The study included 377 subjects divided in: 219 subjects with AO glucose tolerance (IGT). Lipid accumulation product (LAP) is an emerging (AC≥102cm in men; AC≥88cm in women) and 158 subjects without AO. The cardiovascular risk factor, which is calculated from waist circumference groups were divided in 2 subgroups: one with low adiponectin levels (<5,6 (WC) and triglyceride levels. The aim of this study was to investigate the μg/ml for men; <7,1 μg/ml for women) and another with normal adiponectin association of LAP with IGT in women with PCOS and in control women. levels. The LAP was calculated as (WC [cm] – 58) x (triglycerides [mmol/l]) in Fasting plasma adiponectin levels were determined using ELISA 392 PCOS and 67 BMI-matched control women within the same age range. immunoassay and the genotyping of the rs1049353 polymorphism was Metabolic, endocrine, and anthropometric measurements and oral glucose made using PCR technique. De Finetti’s program was used for the statistical tolerance tests were performed. analysis. PCOS women had signifi cantly higher LAP levels than control women in In the group with subjects with AO: There was no deviation from Hardy- age-adjusted analyses (22.2 [10.9-46.2] and 16.3 [9.4-34.9], respectively, Weinberg equilibrium of genotypes distribution in both subgroups. The p=0.001). In PCOS and control women, age, BMI, blood pressure, fasting and difference of the allelic frequency indicated that the G-allele seems to confer stimulated glucose, fasting and stimulated insulin, free testosterone and FAI risk for hypoadiponectinemia (OR=2.295, CI=1.518-3.468) and the A-mutant progressively increased whereas SHBG decreased across LAP quartiles. LAP allele seems to be protective (OR=0.436, CI=0.288-0.659). The presence of was positively correlated with HOMA-IR (r=0.556, p<0.001). In PCOS women the GG genotype (OR=3.919, p<0.001) and GA genotype (OR=2.294, p=0.003) and control women, ROC curve analysis revealed that the best cut-off confers risk for hypoadiponectinemia. To increase the test sensitivity value for LAP to defi ne the presence of IGT was 44.1 and 40.8, respectively of χ2, the Cochran-Armitage test was made. The corrected values were (sensitivity 79.5% and specifi city 80.5%, AUC 0.86; and sensitivity 83.3 % ORcorrectedG=2.012 and ORcorrectedA=0.493 (p<0.001). and specifi city 90.5%, AUC 0.86, respectively). In PCOS women and control In subjects without AO: The G allele did not associate with hypo- women, ROC curve analyses for BMI (0.77 and 0.64, respectively) and adiponectinemia. WC (0.80 and 0.71, respectively) to defi ne IGT revealed lower AUCs. ORs In conclusion, this study indicates that in subjects with AO, the presence for IGT for PCOS women in the highest LAP, BMI, and WHR quartile were of G allele of the rs1049353 polymorphism in CNR1 gene is associated with 41.81 (5.52-316.54), 10.24 (2.94-35.63), and 18.45 (4.19-81.30), respectively, hypoadiponectinemia. when compared to PCOS women in the lowest LAP, BMI, and WHR quartile, Supported by: National University Research Council - MEDC-ANCS Grant, PN respectively. II - ID 711

1923-P The Attitudes of Patients with Type 2 Diabetes and a BMI of 30-40 kg/m2 Regarding the Safety and Effi cacy of Bariatric Surgery MARION L. VETTER, SCOTT RITTER, SHERI VOLGER, JACQUELINE C. SPITZER, KAREN HOFFER, ANTHONY N. FABRICATORE, NOEL N. WILLIAMS, THOMAS A. WADDEN, DAVID B. SARWER, Philadelphia, PA A FDA advisory panel recently recommended approval of an adjustable gastric band for weight reduction in select patients with a body mass index (BMI) as low as 30 kg/m2. Given the lower indication for the device and the growing interest in bariatric surgery as a potential treatment for type 2 diabetes, the feasibility of randomized trials to compare the effi cacy of the various surgical procedures and nonsurgical lifestyle modifi cation should be explored. We evaluated the attitudes of type 2 diabetic individuals with a BMI of 30-40 kg/m2 regarding the safety and effi cacy of gastric bypass and gastric banding, as well as their willingness to participate in a randomized bariatric surgery trial. Using a 40-item questionnaire developed specifi cally for the study, we anonymously surveyed 513 individuals via U.S. mail who were identifi ed from the University of Pennsylvania’s Integrated Clinical and Research Database (PICARD). We asked about attitudes towards current LAP is an easily obtainable and cheap marker associated with IGT in PCOS diabetes treatments, the perceived safety, effi cacy, and acceptability of and control women. bariatric surgery as a therapy for diabetes and obesity, and willingness to be randomized to a surgical procedure. One-hundred and thirty of 513 1925-P (25%) individuals responded. Respondents had a median (interquartile Variants within DDX60L and Intergenic to CD83 and RNF182 Are range) age of 58 (51,63) years and BMI of 32.9 (30.9,35.2) kg/m2. Roughly Associated with BMI in Pima Indians half (66/130) were female. Overall, 64/128 (50%) respondents had negative KE HUANG, JANEL MACK, ROBERT HANSON, WILLIAM C. KNOWLER, CLIFTON views of bariatric surgery, with 46/126 (37%) reporting that it was unsafe. BOGARDUS, LESLIE BAIER, Phoenix, AZ Only 34/123 (28%) considered bariatric surgery to be an effective treatment

A genome-wide association study (GWAS) was previously done in 1149 Obesity

for type 2 diabetes, whereas the vast majority (105/123, 85%) believed that Pima Indians using the Affymetrix 1-million SNP array to identify obesity POSTERS diet and exercise improved glycemic control. Less than 20% of respondents genes. Among the signals undergoing follow-up are SNPs on chr 4 at 169.3 were willing to be randomized to a surgical procedure for the treatment of

Mb and chr 6 at 14.4 Mb that were associated with BMI (e.g. rs17612333, Integrated Physiology/ diabetes (19/118, 16%) or obesity (20/114, 18%). In conclusion, despite the p=1.7x 10-6, and rs6459353, p=5.1x 10-3). SNPs were further genotyped in an potential increased eligibility for adjustable gastric banding, respondents expanded, population-based sample of 3501 full-heritage Pima Indians and had fairly negative views about bariatric surgery. the associations with BMI remained signifi cant (p= 4.0 x 10-5 for rs17612333 Concerns about safety and effi cacy among patients with a BMI of 30-40 and p= 6.6 x 10-4 for rs6459353, adjusted for age, sex, and birth-year and kg/m2 must be addressed to enhance the feasibility of randomized trials of accounting for family membership). Analysis of these 2 SNPs in an additional bariatric surgery. sample of 3723 mixed heritage Native Americans showed associations with Supported by: RCI-DK086132 BMI of p= 0.1 for rs17612333 and p= 2.6x 10-3 for rs6459353 (P adjusted for age, sex, birth-year and family membership, and estimates of admixture) and combining all individuals (n=7224) provided stronger evidence for association (p= 2.2x 10-5 for rs17612333, p= 5.6x 10-6 for rs6459353). rs17612333 maps

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A519 OBESITY—HUMANCATEGORY

within an intron of DDX60L which encodes a ATP-dependent helicase binding with RNA. CD83 and RNF182 are the only 2 genes within ±500kb of rs6459353. CD83 is a cell-surface glycoprotein that regulates immunity response, and RNF182 is a brain-enriched E3 ubiquitin ligase that may participate in controlling the neurotransmitter release mechanism. To explore potential associations between these 3 genes and BMI, the putative promoter regions and all exons and exon-intron boundaries of these genes were sequenced in 24 Pima Indians. 4 novel SNPs were identifi ed (2 in DDX60L, 1 in CD83 and 1 in RNF182); one novel SNP within DDX60L predicts a 1323F/I substitution and the other 3 SNPs are in untranslated regions. These SNPs, in addition to 53 tag SNPs from the HapMap (CHB) are currently being genotyped in our samples.

1926-P Vitamin D Insuffi ciency, a Risk Factor for Prediabetes and Diabetes Type 2 in Abdominal Obesity Women TATIANA KARONOVA, ELENA MICHEEVA, DANY AZZI, ELENA KONOPLYANNI- KOVA, IDELYA MAMINA, ELENA KRASILNIKOVA, EUGENIYA PATRAKEEVA, ELENA GRINEVA, EUGENIY SHLYAKHTO, St. Petersburg, Russia Some studies suggest that serum 25(OH) vitamin D concentration is inversely associated with insulin resistance and the prevalence of diabetes type 2. However due to the uncertainty of these associations, we examined the relationship between serum concentration of 25(OH)D and glucose as well as insulin levels, HOMA-IR, and ISI (0,120) in abdominal obesity women. We examined 162 women 40 to 52 years old (mean age 46.9±5.5) with abdominal obesity (according to IDF criteria, 2005). The mean waist circumference was 96.5±1.7 cm, body mass index (BMI) was 31.3±0.4 kg/m2, waist-to-hip ratio (WHR) - 0.9±0.004. Serum 25(OH)D and insulin levels were determined by ELISA, plasma glucose levels - by standard biochemistry. All patients underwent standard oral glucose-tolerance test (75 g of glucose). Insulin resistance was evaluated by HOMA-IR and insulin sensitivity by ISI (0, 120). Glucose level was 6.1±0.2 mMol/L, insulin level 14.9±0.9 (mIU/ml), HOMA- IR - 3.2±0.1, ISI (0, 120) – 7.52±0.39. Serum 25(OH)D level was from 19.4 to 134.1 nMol/L (mean 55.6±2.9). Vitamin D insuffi ciency and defi ciency was revealed in 83.6% of patients, only 16.4% women had normal 25(OH)D level. Glucose intolerance was revealed in 44 women (25.9%), diabetes type 2 was diagnosed in 25 women (16.1%). Correlation analysis showed inversed correlations between serum 25 (OH)D level and fasting glucose, insulin levels (r=0.14, p=0.05; r=0.20, p=0.008 respectively) and ISI (0,120) (r=0.33, p=0.001). We found that HOMA-IR was related to BMI value (r=0.5, p=0.001), VDR and vitamin D level associated variants infl uence metabolic and but not with 25(OH)D levels (r=-0.14, p=0.08). endocrine parameters including 25(OH)D levels in PCOS women. Our results showed that women with abdominal obesity have higher tendency for developing vitamin D defi ciency and this later is correlated with high fasting plasma glucose, serum insulin levels and decreased tissues 1928-P insulin sensitivity. Hence vitamin D insuffi ciency might possibly be a risk Weight Loss and Improvements in Markers of Metabolic Risk in factor for pre diabetes and diabetes type 2. Overweight and Obese Subjects Completing 56 Weeks of Treatment with Naltrexone SR/Bupropion SR KEN FUJIOKA, SONJA K. BILLES, COLLEEN BURNS, RAUL HARRIS-COLLAZO, 1927-P DENNIS D. KIM, EDUARDO DUNAYEVICH, FRANK L. GREENWAY, La Jolla, CA, Vitamin D-Associated Polymorphisms Are Related to Insulin Resist- Baton Rouge, LA ance and Vitamin D Defi ciency in Polycystic Ovary Syndrome The naltrexone/bupropion combination (NB) produces signifi cant weight ELISABETH WEHR, THOMAS R.WITHDRAWN PIEBER, BARBARA OBERMAYER-PIETSCH, Graz, loss in obese/overweight individuals. The effi cacy and safety of NB was Austria evaluated in a subgroup analysis of subjects completing COR-I, a Phase 3, Women with polycystic ovary syndrome (PCOS) frequently suffer from double-blind, placebo-controlled, 56-week study. Subjects (N=1742) were insulin resistance, which might be related to vitamin D metabolism. We randomized 1:1:1 to NB32 (32mg naltrexone sustained-release [SR]/360mg aimed to investigate the association of polymorphisms in the vitamin D bupropion SR), NB16 (16mg naltrexone SR/360mg bupropion SR) or placebo. receptor (VDR) gene and vitamin D level associated genes with metabolic Baseline characteristics of completers (NB32 N=296, NB16 N=284, placebo and endocrine parameters in PCOS and control women. N=290) were similar to the randomized population: age 46 y, 82% female, Metabolic, endocrine, and anthropometric measurements and oral 76% Caucasian, weight 100 kg, BMI 36 kg/m2, waist circumference 110 cm, glucose tolerance tests were done in 545 PCOS and 145 control women. HDL-C 52.0 mg/dL, LDL-C 121.3 mg/dL, triglycerides 130.3 mg/dL, hs-CRP Genotyping of VDR (Cdx-2, Bsm-I, Fok-I, Apa-I, and Taq-I), GC, DHCR7, and 6.02 mg/L. Week 56 weight loss was greater (p<0.001) with NB32 (-8.1%) Obesity CYP2R1 polymorphisms was performed. and NB16 (-6.7%) vs. placebo (-1.8%). More (p<0.001) NB32- and NB16- POSTERS 25-hydroxyvitamin D (25[OH]D) levels correlated negative with insulin treated vs. placebo-treated completers achieved weight loss ≥5% (62% and resistance and positive with insulin sensitivity (p<0.05 for all) in PCOS 55% vs. 23%), ≥10% (34% and 30% vs. 11%) and ≥15% (17% and 14% vs. 3%). Integrated Physiology/ women. In PCOS women, the VDR Cdx-2 “AA” genotype was associated with NB32 and NB16 were associated with improvements vs. placebo (p<0.05) in lower fasting insulin (p=0.039) and HOMA-IR (p=0.041) and higher QUICKI waist circumference (-6.9 cm and -5.3 cm vs. -2.7 cm), HDL-C (+3.5 mg/dL and (p=0.012) and MATSUDA-index (p=0.003). The VDR Apa-I “AA” genotype +3.6 mg/dL vs. -0.1 mg/dL), triglycerides (-13.3% and -8.8% vs. -2.8%), insulin was associated with lower testosterone (p=0.028) levels. In PCOS women, sensitivity (HOMA-IR; -22.9% and -15.8% vs. -5.6%), fasting insulin (-19.6% 170 women (31.2%) presented with 25(OH)D levels <20 ng/ml. PCOS women and -12.2% vs. -4.0%), fasting glucose (-3.9 mg/dL and -3.1 mg/dL vs. -1.4 carrying the GC “GG” genotype and the DHCR7 “GG” genotype had a higher mg/dL), and hs-CRP (-34.0% and -33.1% vs. -18.9%). NB-treated completers risk for 25(OH)D levels <20 ng/ml (OR 2.53 [1.27-5.06], p=0.009, and OR 2.66 also reported greater improvements in weight-related quality of life and [1.08-6.55], p=0.033, respectively) when compared to PCOS women carrying control of eating. Systolic and diastolic blood pressures were reduced the GC “TT” genotype and DHCR “TT” genotype in multivariate analyses. from baseline in all treatment groups, though placebo was associated with We observed no association of genetic variations and PCOS susceptibility. larger reductions than NB. The most common adverse events (NB>placebo) were nausea, constipation, headache, dry mouth, and nasopharyngitis. In

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A520 ISLET BIOLOGY—APOPTOSISCATEGORY subjects completing 56 weeks of treatment, NB was generally well tolerated comparable levels with fresh islets. Improved proIAPP processing in exenatide- and associated with dose-dependent sustained weight loss and numerous treated islets was associated with reduced hIAPP proto(fi brils) detected by improvements in weight-related risk factors, including lipids and glycemic electron microscopy and TUNEL-positive β-cells (human: -Ex: 15.2±2.4% vs +Ex: parameters, as well as improved quality of life and control of eating. 7.6±1.7%; mouse: -Ex: 12.3±2.7 vs +Ex: 5.8±2.3, P<0.05). These data suggest Supported by: Orexigen Therapeutics that treatment with GLP-1 analogues such as exenatide may enhance proIAPP processing and reduce amyloid formation in T2D patients and pre-transplant ISLET BIOLOGY—APOPTOSIS culture of human islets.

[See also: Presidents Posters 469-PP to 470-PP, page A130.] & 1931-P Mammalian Sterile 20-Like Kinase 1 (MST1) Mediates Pancreatic β-Cell Apoptosis Guided Audio Tour: Apoptosis (Posters 1929-P to 1937-P), see page 11. AMIN ARDESTANI, FEDERICO PARONI, JULIE KERR-CONTE, KATHRIN MAEDLER, Bremen, Germany, Lille, France & 1929-P Apoptotic cell death is the hallmark of the loss of insulin producing b-cells Impact of Vildagliptin on Glucose Tolerance, and beta Cell Mass in and a fundamental cause of diabetes. Loss of function, of survival signals Insulin Receptor Substrate-2-Knockout Mice and activation of pro-apoptotic mediators are characteristic for diabetic KOICHIRO SATO, AKINOBU NAKAMURA, JUN SHIRAKAWA, TOMONORI MURA- β-cells. In search for a common pro-apoptotic pathway, we investigated OKA, YU TOGASHI, KAZUKI ORIME, YASUO TERAUCHI, Yokohama, Japan whether the serine/threonine kinase Mammalian sterile 20-like kinase 1 Loss of beta-cell mass underlies the development of diabetes and insulin (MST1), triggers β-cell death in diabetes. receptor substrate-2 (Irs2) is critically required for beta cell growth and Exposure of isolated human islets and INS1-cells to a diabetic milieu survival. GLP-1 reportedly increased islet proliferation and reduced apoptosis (cytokines IL-1β/INF-γ, high glucose or thapsigargin) signifi cantly increased of beta-cells in rodents. In this study, we explored the chronic effect of MST1 activity (phosphorylation & cleavage) and Akt phosphorylation was dipeptidyl peptidase-4 (DPP-4) inhibitor vildagliptin on glucose tolerance acutely increased, but dramatically decreased after 48h and 72h. Increased and beta-cell mass in Irs2-knockout (Irs2-/-) mice fed a high-fat diet for 20 MST1 activity and its downstream effectors p38 and JNK correlated with weeks. Wild type (WT) mice and Irs2-/- mice of the same genetic background increased b-cell apoptosis demonstrated by increased caspase-3- and PARP- were fed standard-chow until 8 weeks of age, and then given free access cleavage. Blocking MST1 by overexpression of dominant negative protein to high-fat diet for 20 weeks. Half of the animals in each genotype were (DN-MST1) inhibited b-cell apoptosis and potentiated insulin-induced AKT given vildagliptin in the drinking water (0.3 mg/ml) and others were given phosphorylation, whereas MST1 overexpression was suffi cient to induce tap water without vildagliptin. Whereas there were no differences in body and potentiate b-apoptosis and abolished AKT phosphorylation. Activation of weight or food intake, vildagliptin signifi cantly reduced fasting blood glucose the PI3K-Akt pathway through exogenously added insulin or GLP1 abrogated in Irs2-/- mice. In both genotypes of mice, vildagliptin signifi cantly decreased glucose-, cytokine- and thapsigargin-induced MST1 activation and β-cell apoptosis, whereas suppression of PI3-K/Akt signaling (by LY294002, AIP-2 or AUC0-120min blood glucose and signifi cantly increased the insulin response to glucose during oral glucose tolerance test (OGTT). To evaluate the chronic siRNA to Akt), induced MST1 activity and b-cell apoptosis. Our results indicate effect rather than the direct effect, we also performed OGTT one day after that MST1 and Akt negatively regulate each other and constitute a stress- sensitive survival pathway. Under acute stress conditions, Akt promoted discontinuation of vildagliptin administration. AUC0-120min blood glucose was still signifi cantly decreased and the insulin response to glucose during OGTT cell survival by inhibiting MST1, but prolonged stress decreased Akt, which was signifi cantly increased in Irs2-/- mice treated with vildagliptin compared allowed pro-apoptotic MST1 signaling. MST1 overexpression caused PDX1 with those without vildagliptin. Histochemical analysis of pancreatic islets shuttling from the nucleus to the cytosol and its subsequent degradation, revealed that treatment with vildagliptin signifi cantly increased beta-cell which occurred by direct MST1-induced phosphorylation of PDX1. Importantly, mass (p<0.05) and proportion of beta-cells to islet cells (p<0.01) in Irs2-/- mice. MST-1 was highly activated in islets isolated from patients with T2DM, from Vildagliptin decreased the proportion of TUNEL-positive beta-cells (p<0.05), diabetic db/db mice and high fat/high sucrose fed mice. but failed to increase the BrdU incorporation in Irs2-/- mice. These results Our results show that MST1 plays an important role in triggering b-cell suggest that vildagliptin improved glucose tolerance and increased beta-cell death via suppressing survival signals and PDX1 functionality. Its inhibition mass by reducing apoptotic beta-cells in Irs2-/- mice and that vildagliptin is provides a new strategy to restore b-cell survival. able to reduce apoptotic beta-cells in Irs2-independent manner. & 1932-P & 1930-P Identifi cation of microRNA Expression Profi le Induced by Palmitate Exenatide Treatment Improves Processing of Pro-Islet Amyloid in Human Pancreatic Islets Poly peptide in Human and Mouse Islets during Culture YUNXIA ZHU, HONGDONG WANG, XIAO HAN, Nanjing, China HAO CHEN, TIMOTHY J. KIEFFER, C. BRUCE VERCHERE, ZILIANG AO, MADELEINE Among all the pancreaticβ-cell dysfunction related factors in T2DM, FFA SPECK, DAVID THOMPSON, GARTH L. WARNOCK, LUCY MARZBAN, Vancouver, seems to play a key role in inadequate insulin secretion. Several proteins BC, Canada were involved in the pathology of FFA-induced β-cells impairment, such Islet amyloid is a pathologic characteristic of type 2 diabetes (T2D) that also as pdx1, neurod1, sur2, kir6.1, oncut2, vamp2, et al. Since the discovery forms in islets during culture and post-transplant. Islet amyloid is comprised of microRNAs (miRNAs), an entirely new line of thoughts has emerged as mainly of human islet amyloid polypeptide (hIAPP; amylin), a β-cell peptide regard to the relationship between miRNAs and disease proteins. Moreover, that is produced by cleavage at the C- and N-termini of its precursor proIAPP. a particular link seems present between miRNA and palmitate-induced Elevated synthesis, fi brillogenic sequence, and impaired processing of proIAPP β-cells failure. To investigate the possible involvement of miRNAs in due to β-cell dysfunction have been implicated in hIAPP aggregation. Exenatide palmitate-induced human islets impairment, the isolated adult human islets is a long acting analogue of glucagon-like peptide-1 (GLP-1) that is used for were picked up and incubated for 48 hours in the presence of 0.5 mmol/l treatment of T2D and has been shown to enhance β-cell mass and function. palmitate. The GeneChip® miRNA Array (Affi metrix) was used to identify the We tested the effect of exenatide on restoring impaired proIAPP processing expression profi le and 289 miRNAs were detected. 11 miRNAs were highly in islet β-cells. Freshly isolated human or hIAPP-expressing transgenic mouse expressed in untreated human islets. Especially, 21 miRNAs up to two fold islets were cultured in CMRL or Hams-F10 (5 or 10 mmol/l glucose), respectively, altered were determined in palmitate treated group. Among those miRNAs, without or with exenatide (10 nM, 7 d). (Pro)IAPP forms were detected in 9 miRNAs were up-regulated and 12 miRNAs were down-regulated. For islet lysates by Western blot followed by immunoblot using an antibody that further functional analysis, we used miRNA target prediction algorithm recognizes all (pro)IAPP forms or antibodies that we generated specifi c for N- named miRanda and Targetscan to search possible regulation target of and C-termini of human proIAPP. Mature IAPP was the major form detected in these miRNAs. Connected with pancreatic β-cell proliferation and insulin fresh human and mouse islets with low levels of immature proIAPP also present. secretion associate proteins, miR-377 may involve in β-cell failure via down- Islet culture caused impaired proIAPP processing manifest as elevated proIAPP regulating the protein level of puma, bcl-xL, kir 6.1, sur2, et al. The reported and its intermediates and reduced mature IAPP compared to fresh islets. The miR-146a was also signifi cant increased in treated group, demonstrating major intermediate form present in cultured human and hIAPP-expressing mouse POSTERS

that miR-146a played a main role in human islets induced by palmitate as Islet Biology/ islets was N-terminally unprocessed proIAPP, suggesting that mainly cleavage at well. Our results may provide other insights into the causes of β-cell failure Insulin Secretion N-terminus was impaired. Exenatide-treated islets had markedly lower proIAPP implicating the development of type 2 diabetes. and its intermediates and higher mature IAPP than untreated cultured islets and Supported by: National Basic Research Program of China (2011CB504003)

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