INSULIN ACTION—ADIPOCYTECATEGORY BIOLOGY

1692-P 1694-P Stress in Beta Cells Obtained with Laser Capture Microdissection Biopsy-Proven Insulitis of Clinical Islet Transplantation Is Not from Cadaver Pancreases of Brain Dead Donors Reversed by Steroid Therapy AREF EBRAHIMI, MIN-HO JUNG, JONATHAN M. DREYFUSS, HUI PAN, DENNIS ANNA LAM, BEHRUZ RAHIMI, SHARLEEN IMES, KIM SOLEZ, JAMES SHAPIRO, C. SGROI, SUSAN BONNER-WEIR, GORDON C. WEIR, Boston, MA PETER A. SENIOR, Edmonton, AB, Canada Brain death of pancreas donors is thought to lead to the expression of Gradual decline in islet function remains a challenge in clinical islet infl ammatory, stress and apoptotic pathways in isolated islets resulting in transplantation (CIT), but acute graft loss is relatively uncommon. Here we poor clinical outcomes. To test this hypothesis we obtained cadaveric pan- describe a case of acute decline in graft function with histology suggesting creases from brain dead pancreatic donors (n=7, mean age 5011) and normal an immune mechanism. A 49 year old female (BMI 24.4 kg/m2, insulin 0.3 pancreatic tissue obtained at surgery done for pancreatic neoplasms (n=7, U/kg) with type 1 diabetes for 37 years underwent two CIT (6071 and 6827 age 699). Frozen sections were subjected to laser capture microdissection islet equivalents/kg) following alemtuzumab induction with tacrolimus (TAC, to obtain beta-cell rich islet tissue, from which extracted RNA was analyzed mean 8 ug/L) and mycophenolate mofetil for maintenance. Initial engraft- with Affymetrix arrays. Gene expression of the two groups was evaluated ment was reasonable (β2 score of 12 at 1 week), but β2 score gradually with principle component analysis (PCA), and differential expression anal- declined (Figure 1) rising to 19 after the second CIT. Insulin independence ysis was performed for genes and pathways. The pathways expressed at was achieved only briefl y following each CIT. There was an acute decline the highest signifi cance included Glycolysis, Unfolded Protein Response, in β2 score after day 120 (Figure 1) prompting liver biopsy. Three collections MTORC1 Signaling, and Pancreatic Beta Cells. Pathways of Apoptosis were of insulin staining cells heavily infi ltrated with mononuclear infl ammatory not differentially expressed. cells and eosinophils were seen. TAC was increased (mean 14 ug/L) and A striking fi nding was the unfolded protein response (UPR), which is a pro- prednisone 50 mg/day started, but graft function did not recover. Pre-CIT tective component of the endoplasmic reticulum (ER) stress response that anti-insulin antibodies were positive and anti-GAD negative, while panel if severe can cause apoptosis. An important group of protective chaperone reactive antibodies were 0% pre- and post-CIT. This is the fi rst report of genes were upregulated in the cadaver donors (HSP90B1, HSPA5, PDIA6, biopsy proven insulitis post-CIT, which may be due to acute rejection and/ DnaJB9 and DnaJC3). In addition the protective genes of ER-associated pro- or recurrent autoimmunity. Unfortunately, this did not reverse with steroid tein degradation (ERAD) proteins (DERL 1, 2, 3) were upregulated. In con- therapy. Further study of allo- and auto-immunity in acute CIT failure may trast the proapoptotic “executioner” genes CHOP and JNK were not upregu- allow targeted immune therapy. lated. Other evidence of stress included upregulation of genes associated Figure 1. Post-CIT Graft Function as Measured by BETA-2 Score, a Clinical with NFkB activation and TNF pathways, including TNFRSF1A, IL17RB and Composite Score (Fasting C-peptide, Fasting Blood Glucose, Insulin Dose, IL13RA1. Other upregulated genes of interest included REG1B, LDHA and Tis- and HbA1C) after Two Transplants. sue Factor. In conclusion, while there was little evidence of active apoptosis in beta cells from cadaver donors, stress markers were found that could represent vulnerability resulting from brain death and/or the trauma of organ preser- vation. This could account for some of the early beta cell death found with islet transplantation. Supported By: National Institutes of Health

1693-P Abnormal Responses to Oral and Intravenous Glucose Stimula- tion in Intestinal Transplant Recipients With or Without Pancreas Allograft DAHAE LEE, LAURENS CEULEMANS, DANIEL JACOBS-TULLENEERS-THEVISSEN, BART KEYMEULEN, ILSE WEETS, DIETHARD MONBALIU, JACQUES PIRENNE, CHANTAL MATHIEU, PIETER GILLARD, Leuven, Belgium, Jette, Belgium Technique of liver-small intestinal transplantation (LITx) often includes a partial or whole pancreas allograft to preserve bile duct continuity. Aim of the study was to measure responses to oral and intravenous (IV) glucose stimulation in LITx recipients in comparison to recipients of isolated small intestinal transplant (ITx) and nondiabetic controls. Plasma glucose, C-peptide and insulin were measured during 180-minute oral glucose tolerance test (OGTT) and 160-minute hyperglycemic clamp (HG clamp) in nondiabetic LITx (n = 3) and ITx (n = 3) recipients under immuno- INSULIN ACTION—ADIPOCYTE BIOLOGY suppression with azathioprine, tacrolimus and low-dose corticosteroids. Ten

POSTERS healthy volunteers served as controls. Insulin Action/ All but 1 LITx and ITx recipients showed abnormal glucose profi le after Moderated Poster Discussion: Feel the Burn—Factors that Activate OGTT despite higher basal C-peptide. 2/3 ITx recipients showed higher glu-

Molecular Metabolism Brown Fat (Posters: 1695-P to 1701-P), see page 14. cose peak during OGTT and 45% lower functional beta cell mass (FBM) than controls. In 1 LITx recipient with partial pancreas allograft, OGTT resulted in & 1695-P delayed but higher glucose peak in parallel with high C-peptide and insulin Differential Roles of Fox0 Proteins in White and Brown Adipose levels, leading to rapid decrease in glucose and severe hypoglycemia. Both Tissue LITx recipients with partial pancreas allograft had FBM above percentile 85 ERICA HOMAN, BRIAN T. O’NEILL, MASAJI SAKAGUCHI, CHRISTIE PENNIMAN, of controls. One LITx recipient with whole pancreas allograft showed very DOMENICO ACCILI, C. RONALD KAHN, Boston, MA, New York, NY high baseline C-peptide, due to systemic venous drainage of recipient pan- Insulin and IGF-1 are essential for mediating normal adipocyte differen- creas and high insulin resistance (HOMA-R=3.60). Glucose peak during OGTT tiation and metabolism. In insulin/IGF responsive tissues, insulin and IGF-1 was higher and delayed but without late hypoglycemia. During all phases induce phosphorylation of the Forkhead Box O family of transcription factors of the HG clamp, C-peptide and insulin were higher with FBM calculated at (Fox0s) via action of Akt maintaining their cytoplasmic localization, reversing 150% of controls. the activation of multiple Fox0-responsive genes involved in maintenance of Intestinal transplant recipients show abnormal responses to both oral energy homeostasis, glucose metabolism, cell cycle arrest, and cell death. and IV glucose stimulation. Several factors may explain these observations: We explored whether Fox0 activation controls the decrease in white and intestinal denervation, loss of normal anatomy, loss of FBM and insulin resis- brown fat mass, glucose intolerance, and decrease in brown fat activation tance. Analysis of these complex cases can help to understand regulation of observed in mice with fat-specifi c knockout of both the insulin and IGF-1 glucose metabolism in normal and posttransplant conditions. receptors (FIGIRKO mice). To this end, we generated mice with fat-specifi c knockout of the insulin and IGF-1 receptors, as well as knock-out of the three Fox0s expressed in fat [Fox01, Fox03, and Fox04] (F-Quint KO) and compared these with control and FIGIRKO mice. As previously observed, FIGIRKO mice

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A440 INSULIN ACTION—ADIPOCYTECATEGORY BIOLOGY exhibited a marked loss of all white and brown fat depots, marked hypergly- & 1698-P cemia, insulin resistance, fatty liver and inability to maintain body tempera- Raptor Defi ciency Promotes Browning of White Adipose Tissue via ture during a cold challenge. By contrast, the F-Quint KO mice had similar glu- Adiponectin-dependent Mechanisms cose tolerance to control animals, had normalized brown fat mass and were XIAOFENG DING, YAN LUO, XIN YANG, XIAOQING WANG, XIANYUN ZHENG, able to maintain body temperature during cold exposure. However, like the XUEXIAN YANG, MEILIAN LIU, Albuquerque, NM, Changsha, China FIGIRKO mice, the F-Quint KO mice failed to respond to insulin stimulation, Beige adipocytes burn lipid by dissipating energy in the form of heat and had markedly decreased subcutaneous fat pad weights, absence of perigo- offer a new way to battle obesity and its related disorders. However, what nadal fat pads, and hepatosteatosis. Thus, we demonstrate differential intracellular signaling pathways drive the browning effect remains largely regulation of Fox0 proteins in adipose tissue such that suppression of Fox0s unknown. Here, we show that inhibition of mammalian target of rapamycin by insulin/IGF-1 action is critical for brown fat differentiation and systemic complex 1 (mTORC1) by adipose specifi c-deletion of raptor (raptor fKO), a glucose metabolism, but not critical for the regulation of white adipose tis- key component of mTORC1 promoted browning of inguinal WAT (iWAT) and sue mass and systemic insulin sensitivity. enhanced basal and cold-induced energy expenditure, concurrently with an Supported By: National Institutes of Health (5T32DK007260-39) increase in population of beige cells in iWAT. However, disrupting raptor had no signifi cant effect on CL316,243-induced UCP1 expression and oxygen & 1696-P consumption in the primary brown adipocytes. On the other hand, raptor defi - Regulatory Control of Adipose Mass: Insights from a Novel Model ciency led to marked downregulation of expression and circulating level of of Lipodystrophy adiponectin and upregulation of type 2 infl ammatory cytokines such as IL-4 MASAJI SAKAGUCHI, SHIHO FUJISAKA, WEIKANG CAI, MASAHIRO KONISHI, and IL-13. Similar to raptor fKO mice, adiponectin-defi cient mice displayed BRIAN T. O’NEILL, HIROKAZU TAKAHASHI, C. RONALD KAHN, Boston, MA, increased basal and cold-induced energy expenditure, elevated browning Toyama, Japan effect and upregulation of IL-4/13 pathway. The effects of raptor defi ciency White and brown adipocytes are important for regulation of energy stor- on browning effect and thermogenesis were diminished by administration of age and expenditure and, under normal conditions, remain relatively stable, adiponectin receptor agonist adipoRon in vivo. Our study identifi ed mTORC1 with very low turnover rates. Insulin and IGF1 signaling regulate development signaling and adiponectin as the key regulators of recruitment and activation and function of both brown and white adipose tissue. To better understand of beige adipocytes. these hormones and factors regulating adipose tissue mass in adult animals, Supported By: American Diabetes Association (1-13-JF-37 to M.L.); American we created inducible fat-specifi c knockouts of IR, IGF1R or both using fl oxed Heart Association mice and mice carrying a tamoxifen-inducible Cre-ERT2 transgene on the adiponectin promoter. Within 3 days after tamoxifen treatment, mice with & 1699-P KO of IR or both IR and IGF1R displayed a >70% decrease in WAT and BAT NFIA Controls the Brown Fat Gene Program by Co-Localizing with mass, associated with marked decreases of serum adiponectin and leptin, PPARgamma at Cell Type-Specifi c Enhancers and a metabolic syndrome including severe hyperglycemia, hyperinsuline- YUTA HIRAIKE, HIRONORI WAKI, JING YU, MASAHIRO NAKAMURA, KANA mia, β-cell proliferation and cold intolerance. However, within 10 to 30 days MIYAKE, KEN SUZUKI, RYO NAKAKI, WEI SUN, TOMOHISA AOYAMA, YUSUKE these phenotypes disappeared, and knockout mice displayed virtually com- HIROTA, GAKU NAGANO, HARUYA OHNO, KENJI OKI, MASAYASU YONEDA, plete recovery of WAT and BAT mass. Based on FACS analysis of the stro- SHUICHI TSUTSUMI, HIROYUKI ABURATANI, TOSHIMASA YAMAUCHI, TAKASHI movascular fraction and lineage-tracing with a Rosa26-Tomato/GFP (mTmG) KADOWAKI, Tokyo, Japan, Hiroshima, Japan reporter, this recovery was due to proliferation of preadipocytes and regen- Brown fat dissipates energy in the form of heat, and is a promising target eration of new adipocytes. Leptin administered by subcutaneous pump infu- for treatment of obesity. However, global landscape of brown fat develop- sion during this 30 day period completely prevented development of hyperg- ment is not entirely understood. Here we performed FAIRE-seq on murine lycemia and fatty liver, but had no signifi cant effect on loss of WAT and BAT brown and white fat tissues and found that the binding motif for Nuclear fac- following IR/IGFR knockout. In vivo leptin also suppressed the increase in tor I (NFI) transcription factor is enriched within brown-fat-specifi c open chro- preadipocyte proliferation and recovery of fat mass, as monitored by FACS matin regions. Of the four isoforms of NFI family, NFIA is highly expressed analysis and mTmG lineage tracing. In vitro, on the other hand, leptin had no in brown fat compared to white fat or muscle. Introduction of NFIA into effect on proliferation of brown or white preadipocytes and only minimal myoblasts results in lipid accumulation, activation of the brown-fat-specifi c effects on their differentiation. Thus, in this new model, development of lip- gene program and suppression of muscle genes. Conversely, knockdown of odystrophy provokes a powerful stimulus for the regeneration of both white NFIA in brown adipocytes suppressed the brown-fat-specifi c genes. NFIA and brown adipose tissue. The factors that regulate regeneration of adipose selectively co-localize with PPARgamma at the brown-fat-specifi c enhanc- mass are distinct from leptin, but are, at least in part, regulated by leptin or ers, and co-localization of NFIA facilitates binding of PPARgamma, leading its effects on metabolism. to increased chromatin accessibility and active transcription. Brown fat of Supported By: Japan Society for the Promotion of Science NFIA knockout mouse neonates show impaired expression of brown fat genes and reciprocal elevation of muscle genes. Finally, human perirenal & 1697-P brown fat of patients with pheochromocytoma show concurrent increase Fat-specifi c Rheb Ablation Promotes Beige Fat Development and in NFIA and UCP1 expression. Collectively, these results indicate that NFIA

Thermogenesis is a novel key transcription factor that co-localizes with PPARgamma and POSTERS WEN MENG, XIUCI LIANG, HONGZHI CHEN, SIJIA HE, FANG HU, FENG LIU, Chang - activates the brown-fat-specifi c gene program. Insulin Action/

sha, China, San Antonio, TX Molecular Metabolism Beiging of white adipose tissue (WAT) has potential anti-obesity and & 1700-P antidiabetes effects, yet its underlying mechanisms remain elusive. We have Regulation of Brown Fat Activation and Development by Insulin’s recently demonstrated that fat-specifi c knockout of Grb10 in mice upregu- Specifi c Actions on the Endothelium lates mTORC1 signaling, promotes thermogenesis, and increases the expres- KYOUNGMIN PARK, QIAN LI, CHRISTIAN RASK-MADSEN, ERNESTO MADD- sion of thermogenic and lipolytic genes. To directly elucidate role of mTORC1 ALONI, MOGHER KHAMAISI, MATTHEW D. LYNES, ALISON BURKART, MANOJ in beige fat development, we generated mice in which the expression of GUPTA, YU-HUA TSENG, GEORGE L. KING, Boston, MA the mTOR upstream activator, Rheb, is disrupted in adipose tissue. Adipose- Dysfunctional white and brown adipose tissues (WAT and BAT) can con- specifi c Rheb ablation induces WAT beiging, enhances energy expenditure tribute to insulin resistance and diabetes. Since angiogenesis in fat plays and cold tolerance, increases resistance to high-fat diet-induced obesity, an important role in their function, we explored the possibility that enhanc- and improves global insulin sensitivity in vivo. Protein kinase A (PKA) activ- ing insulin actions in the endothelium can increase energy expenditure, fKO ity and UCP1 expression are higher in subcutaneous WAT of Rheb mice decrease WAT and improve insulin sensitivity by enhancing BAT activation compared to control mice. On the other hand, Rheb overexpression inhibited and development. Insulin’s actions on endothelium were enhanced by selec- PKA activity and UCP1 expression in adipocytes. Our results identify adipose tively overexpressing insulin receptor subtract 1 in endothelial cells to gen- Rheb as a key regulator of WAT beiging and reveal a potential mechanism erate the ECIRS1 transgenic mice which exhibited enhanced activation of underlying the crosstalk between the mTORC1 and PKA signaling pathways Akt/eNOS pathway and NO production by >5 fold. When fed with high fat in adipocytes. diet (HFD, 60% fat), ECIRS1 mice gained 12% less weight, had 15% reduc- Supported By: National Basic Research Program of China (2014CB910501 to tion in WAT mass and increased BAT by 2.3 fold vs. wild type mice (Wt). F.L.); National Natural Science Foundation of China (81130015 to F.L.), (31471131 ECIRS1 mice attained lower blood glucose levels in glucose tolerance test. to F.H.)

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A441 INSULIN ACTION—ADIPOCYTECATEGORY BIOLOGY

The anti-obese action, and improved insulin sensitivity in ECIRS1 mice were reduced by miRNA494 inhibitor for 0.8- and 0.6- fold, respectively. More- prevented by a general inhibitor of eNOS activity (L-NAME). CLAMS analysis over, expression of UCP-1 and miRNA-494 were strongly stimulated by iso- showed that ECIRS1 mice had higher rates of respiratory exchange rate by proterenol, beta adrenergic agonist, 20- and 13-fold, respectively. Finally, we 46% ±11% (p<0.05), but without differences in motor behaviors and food tested the expression of UCP-1 and miRNA-494 in the adipose tissue from intake. The increase in energy expenditure and decrease in WAT suggested 3 hours of cold exposed mice, and found that these expression were signifi - an improvement of mitochondrial activity in BAT from ECIRS1 mice. Bioen- cantly increased compare to the mice in room temperature. ergetic analysis of isolated mitochondria from BAT of ECIRS1 mice showed Our result suggests that mitochondrial biogenesis enhanced by miRNA- higher oxygen consumption rate by 23% ±11% (p<0.05), which were pre- 494 under stimulation of ADRB3 maybe a potential mechanism of browning vented by L-NAME. The size and vascularity of BAT were also increased by process. 43% ± 13% and 37% ±16% respectively (p<0.05) which were also inhibited by L-NAME. The distribution of mitochondrial size in BAT, measured by elec- 1703-P tron microscopy, exhibited increases in size by 63% ± 14% and 34% ±10% Effect of Weight Loss on Adipose Senescent Cells in Obesity on RD and HFD respectively, vs. Wt mice. These fi ndings are the fi rst to dem- ANA ESPINOSA DEYCAZA, BARBARA (GISELLA) CARRANZA-LEON, ESBEN SON- onstrate that enhancing insulin action specifi cally to the endothelium can DERGAARD, DEBRA HARTENECK, MARIA MORGAN-BATHKE, DANAE A. DELI- regulate the development and activation of BAT leading to an improvement VANIS, MICHAEL D. JENSEN, Rochester, MN, Nashville, TN of systemic insulin sensitivity in diet-induced obesity. Senescent preadipocytes cannot replicate or differentiate into mature Supported By: National Institute of Diabetes and Digestive and Kidney Diseases adipocytes. Senescent cells are more prevalent in adipose tissue (AT) of obe- sity and may play a role in AT dysfunction. Because weight loss improves AT 1701-P dysfunction, we studied whether weight loss reduces the number of senes- WITHDRAWN cent cells in AT. Seventeen overweight/obese participants (4 men) underwent body com- position studies (DXA) and femoral-abdominal subcutaneous AT biopsies, for senescent cell determination, before and after weight loss. Senescent cells in AT were identifi ed by senescence associated β-galactosidase staining and expressed as proportion of total number of nucleated cells (DAPI). Adipose dysfunction was estimated using AT insulin resistance (Adipo-IR) calculated as fasting palmitate concentration x fasting insulin concentration (N=14). We used a comprehensive lifestyle intervention weight loss program. Median age was 39 years (range: 23-55) and BMI 33.1 kg/m2 (range: 29.6- 36.5). At baseline, the percentage of AT senescent cells averaged 2.8% and 4.4% in the abdominal subcutaneous and femoral fat, respectively (p=0.001, abdomen vs. femoral). There was a positive correlation between % body fat and both abdominal AT senescent cells (rs= 0.58, p=0.02) and femoral AT senescent cells (rs= 0.49, p=0.052). The median loss of 11% of their initial body weight (IQR 7.4, 13.5%) resulted in a median Adipo-IR reduction of -1.3 mmol/l X pmol/l (IQR -0.6, -3.4) (p=0.007), but no signifi cant change in senes- cent cells proportion in the abdominal or femoral fat depots. The median change in senescent cells was 0.48 (IQR -0.38, 1.9) (p= 0.12) in the abdominal AT and 1 (IQR -0.5, 1.84) (p= 0.22) in the femoral AT. Despite a positive relationship between senescent cells in AT and adipos- ity, the proportion of senescent cells in AT is not affected by weight loss. The improvement in AT insulin sensitivity after weight loss without reductions in senescent cells suggests that senescence might not play a role in obesity- associated AT dysfunction. Supported By: National Institutes of Health

1704-P 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Mediates Insulin Resistance through JNK Activation in Adipocytes CHAO ZHENG, GUANG LIANG, Wenzhou, China Inducing insulin resistance is the major side effect of glucocorticoids. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that

POSTERS 1702-P catalyzes the conversion of the inactive glucocorticoid to active form. how- Insulin Action/ Adipose Mitochondrial Biogenesis in Browning and a Potential Role ever, the molecular mechanisms by which glucocorticoid induce insulin resis-

Molecular Metabolism of miRNA-494 tance remain limited. JNK plays an important role in insulin resistance. We MENGISTU LEMECHA SHUKARE, KATSUTARO MORINO, TAKESHI IMAMURA, hypothesize that JNK may mediate 11β-HSD1-induced insulin resistance. HIROTAKA IWASAKI, NATSUKO OHASHI, HIROTAKA YAMAMOTO, SATOSHI UGI, Our results found that JNK was activated in adipose tissue of HFD mice and HIROSHI MAEGAWA, Otsu, Japan in cultured adipocytes after glucocorticoids stimulation or overexpressing Cold exposure causes browning in adipose tissue. Mitochondrial bio- 11β-HSD1. Inhibition of 11β-HSD1 blocked the activation of JNK in adipose genesis is activated by various stimuli including beta-3 adrenergic recep- tissue of HFD mice as well as in cultured adipocytes stimulated with gluco- tor (ADRB3). However, molecular mechanisms underlying this phenomenon corticoids or overexpressing 11β-HSD1. Furthermore, prednisone stimulation especially by micro-RNA remains unknown. In this study, we tested the mito- or 11β-HSD1 overexpression signifi cantly impaired insulin signal pathway, chondrial biogenesis during adipogenesis and browning in 3T3L1 adipocyte. while these effects were reversed by JNK inhibitor C66 or dominant nega- Protein expression of mitochondrial transcription factor A, succinate tive JNK, respectively. Finally, oral administration with either PF00195715 or dehydrogenase, pyruvate dehydrogenase, and adenine nucleotide translo- C66 in obese mice remarkably mitigated insulin resistance. Taken together, cator proteins were increased during differentiation of 3T3L1 from day 0 to glucocorticoids and 11β-HSD1 mediate insulin resistance through JNK acti- day 8. These proteins were further increased during browning stimulation vation in adipocytes. Our fi ndings suggest that inhibition of JNK represents for 6 days with triiodothyronine, 3-isobutyl-1-methylxanthine and rosigli- a valid strategy for treating insulin resistance induced by glucocorticoid tazone. We also observed robust change in the expression of miRNA-494 excess and abuse. which we have been reported the role in mitochondrial biogenesis in skel- Supported By: National Natural Science Foundation of China (81200630 to etal muscle. Uncoupling protein 1 (UCP-1), peroxisome proliferator-activated C.Z.), (81500291 to G.L.); Natural Science Fund Committee of Zhejiang Province receptor gamma, coactivator 1 alpha, and ADRB3 mRNAs were increased (12H07001 to C.Z.); Wenzhou Science and Technology Bureau (H20150001 to C.Z.) 1.2-, 1.2-, 1.3-fold, respectively by miRNA-494 overexpression compared to empty vector in adipocytes. Furthermore, mRNA of UCP-1 and ADRB3 were

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A442 INSULIN ACTION—ADIPOCYTECATEGORY BIOLOGY

1705-P 1707-P A Human Mutation in PI 3-Kinase Creates Tissue Specifi c Insulin Increased Adipokine Production in the Chow-Fed Adipocyte-specifi c Resistance OSMR Knockout Mouse MARIE H. SOLHEIM, JONATHON N. WINNAY, JASON K. KIM, ANDERS MOLVEN, CARRIE M. ELKS, JENNIFER L. BAILEY, HARDY HANG, RANDALL L. MYNATT, PAL R. NJOLSTAD, C. RONALD KAHN, Boston, MA, Worcester, MA, Bergen, Nor- JACQUELINE M. STEPHENS, Baton Rouge, LA way Oncostatin M (OSM), an IL-6 family cytokine produced by adipose tissue Phosphatidylinositol 3-kinase (PI3K) is a central component of insulin macrophages, is highly up-regulated in obesity. Acting through its cognate signaling in control of glucose metabolism, cell growth/differentiation, and receptor (OSMR), OSM signifi cantly up-regulates pro-infl ammatory and pro- apoptosis. We have previously identifi ed a heterozygous missense mutation fi brotic genes in cultured adipocytes, and OSMR knockdown prevents these (Arg649Trp) in the p85 regulatory subunit of PI3K (PIK3R1) in patients with changes. We hypothesized that adipocyte-specifi c OSMR knockdown would a familial syndrome characterized by insulin resistance, partial lipodystro- have similarly benefi cial effects in vivo. Adiponectin-Cre mice were crossed phy and short stature. We have generated knock-in mice with this mutation with fl oxed OSMR mice to create mice lacking OSMR in mature adipocytes to investigate the mutation’s role in insulin action and signaling. Similar to (OSMR FKO). Chow-fed male OSMR FKO mice and littermate controls were the patients, mutant mice showed a reduction in body weight and length, examined at 7-8 months of age. No signifi cant differences in body weight or partial lipodystrophy and systemic insulin resistance. This was associated 4-hour fasting blood glucose were detected between genotypes. Akt phos- with a reduced capacity of insulin and other growth factors to activate phorylation in response to an acute insulin bolus was blunted in epididymal the PI 3-kinase pathway and its downstream targets such as Akt in vivo in adipose tissue of OSMR FKO mice, suggesting decreased adipose tissue insu- liver, muscle and fat, as well as in vitro in in hepatocytes and immortalized lin sensitivity. When assessed by protein array, epididymal fat of OSMR FKO brown preadipocytes derived from these mice. Euglycemic-hyperinsuline- mice also revealed signifi cant increases in several insulin resistance- and mic clamp studies revealed marked insulin resistance in the knock-in mice infl ammation-associated adipokines: IGF binding protein 3 (increased 3.5- with a 58% reduction in the glucose infusion rate (46.4±6.6 vs. 73.3±3.0 fold), lipocalin 2 (increased 1.5-fold), C-reactive protein (increased 2-fold), mg/kg/min, p<0.01) and an associated decrease in the rate of glucose turn- and resistin (increased 2.5-fold). Collectively, these data suggest a possible over 51.8±3.4 mg/kg/min vs. 63.8±2.0 mg/kg/min, p<0.05) when compared requirement for intact adipocyte OSM signaling in maintenance of adipose to controls. In addition, hepatic glucose production was suppressed during tissue homeostasis and insulin sensitivity. Current efforts are focused on the clamp by only 76% in mutant mice compared to the 100% suppression in elucidating the molecular mechanisms responsible for altered adipose tissue controls (p<0.01). Surprisingly, no difference was observed in glucose uptake homeostasis in the OSMR FKO mouse model. in skeletal muscle and brown adipose tissue between control and mutant Supported By: National Institutes of Health (P20 GM103528) mice, whereas white adipose tissue glucose uptake was marked reduced (70.8±12.2 vs. 32.6±4.2 nmol/g/min) (p<0.01). Thus, despite ubiquitous 1708-P expression of the mutant allele, the mutation uncovers differences in insulin CB1 Antagonist Increased Natriuretic Peptide Receptors that Pro- signaling and metabolic phenotype among insulin sensitive tissues with a mote Fat Browning in the Subcutaneous and Visceral Fat in the Fat- clear reduction in adipose tissue glucose uptake and a relative failure of Fed Dog insulin to suppress gluconeogenesis, creating a novel form of tissue-specifi c MALINI S. IYER, RICHARD N. BERGMAN, JOYCE M. RICHEY, ISAAC ASARE insulin resistance. BEDIAKO, ORISON O. WOOLCOTT, STELLA P. KIM, CATHRYN KOLKA, DEBORAH J. CLEGG, MORVARID KABIR, Los Angeles, CA 1706-P We have recently demonstrated that CB-1 receptor antagonism increased Regulation of Filamin A Cleavage Prevents Adipocyte Lipid Accumu- expression of genes involved in browning of adipose tissues, specifi cally in la tion the subcutaneous (SC) and visceral (VIS) depots. The mechanism (s) by which JYOTI RANJAN, SAIE MOGRE, KALYANI V. GUNTUR, STEPHANE GESTA, VIVEK the CB1 antagonist Rimonabant (RIM) promotes adipocyte browning is K. VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN R. NARAIN, Framing- unknown. Natriuretic peptides (NPs) are a group of peptide-hormones mainly ham, MA secreted from the heart which promote browning of the white adipose tis- FlnA was identifi ed as a potential target for obesity from the Berg Inter- sues. The current study examines the longitudinal changes of NPs receptors rogative Biology® platform using an adipocyte model generated by modu- (NPR1, 2, 3) expression in the SC and VIS depots by CB1 antagonist RIM. lation of glucose and lipids in vitro. Filamin A (FlnA) is a 280 kDa scaffold- Conscious dogs were fed a high fat diet (HFD, 52% fat) for 6 weeks followed ing protein with reported function in regulating lipid droplet formation and by a continued 16 weeks of fat feeding with either HFD + placebo (PL) (n=9) in insulin signaling. We have previously demonstrated that reducing FlnA or HFD + RIM (1.25 mg/kg per day; n=11). Biopsies from SC and VIS depots expression in human adipocytes leads to enhanced lipid accumulation and were obtained for gene expression: before HFD (Pre-fat), after 6 weeks of basal lipid mobilization. FlnA expression profi le in intra-abdominal white fat (HFD) and 16 weeks of HFD +/- RIM. RIM increased NPR1 expression adipose tissue of high-fat diet mice indicated an increase in FlnA cleavage. in SC depot by 2.5 fold (P<0.05) and in VIS depot by 5 fold (P<0.001) com- In several cell types, FlnA cleavage is known to be regulated by calcium- pared to HFD groups. Similarly, RIM increased NPR2 by 4 times in the SC and dependent cysteine protease, calpain. As expected, treatment of immortal- VIS depots (P<0.001). There is a tendency for NPR3 to be increased by CB1 ized human adipocytes with calpain resulted in FlnA cleavage, while treat- antagonist only in the VIS depot by 2 times (P=0.052). Our data suggests that

ment with calpastatin (a specifi c calpain inhibitor) reduced it. Furthermore, one of the mechanisms by which the CB1-R antagonist increases browning POSTERS increased FlnA cleavage induced by calpain was associated with increased of adipose tissue is through upregulation of the key factors, NPs. Increasing Insulin Action/ triglyceride accumulation in adipocytes subjected to conditions recapitulat- of the browning process in the SC and VIS depots is an important mechanism Molecular Metabolism ing pathology of obesity. Interestingly, treatment with calpastatin, as well by which the CB1 antagonist regulates energy homeostasis. as other calpain inhibitors (acetyl calpastatin, PD150606 and ALLN peptide) Supported By: Sanofi prevented triglyceride accumulation induced by simulated conditions. This effect was also associated with increased adiponectin expression, secre- 1709-P tion, and a reduction in basal lipolysis. Characteristic changes observed in Effect of Low Carbohydrate Weight Loss Diet on Adipose Cell Size response to calpastatin treatment could be prevented by knocking down TRACEY MCLAUGHLIN, LI-FEN LIU, ERIN AVERY, WEN-JUN SHEN, CORAAL expression of FlnA, suggestive of the requirement of FlnA for calpastatin to COHEN, FREDERIC KRAEMER, SAMUEL CUSHMAN, CHRISTOPHER D. GARDNER, mediate these changes. Altogether, these data demonstrate that inhibition Stanford, CA, Bethesda, MD of FlnA cleavage by calpain inhibitors could effectively prevent increased Larger adipose cell size is associated with risk for insulin resistance and adiposity. Therefore in adipose tissue, increasing FlnA levels by prevention type 2 diabetes. Diets high in carbohydrate (CHO) are insulinogenic and may of its cleavage could serve as an effective therapeutic strategy for treat- promote fat storage in adipocytes, thus contributing to cell enlargement. ment of obesity and associated metabolic disease. The goal of the current study was to test the hypothesis that weight loss via CHO vs. fat restriction would lower ambient insulin concentrations and reduce adipose cell size. To test this, 90 healthy obese participants were randomly assigned to low CHO or low fat diet for 6 mos. Intake of CHO or fat was limited to 20g/d for 8 weeks and then gradually increased. Abdominal adipose tissue biopsies were performed at baseline and 6 mos in diet-com- pliant participants who lost ≥5% initial body weight. Adipose cell size distri- bution was measured via Beckman Coulter Multisizer III (A), yielding adipose

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A443 INSULIN ACTION—ADIPOCYTECATEGORY BIOLOGY

cell size (peak diameter), and distribution (nadir separates small vs. large cell to control. Triglyceride accumulation, as well as basal and isoproterenol populations). Ambient insulin and FFA concentrations were measured dur- stimulated lipolytic activity, were signifi cantly increased in shFlnA com- ing diet-congruent 4-hr meal tolerance tests (MTT). 30 subjects completed. pared to shCont adipocytes. However, these changes were independent of Mean age, BMI, and female sex were 40 yrs, 33 kg/m2, and 50%female. an improved adipocyte differentiation, as expression of adipogenic markers Subjects lost 9±3 kg. The low CHO diet decreased insulin but not FFA concen- (PPARg, Cebpa, FABP4 and GLUT4) was not affected. In addition, increased trations, whereas the low fat diet consistently decreased FFA (B). Adipose adiponectin expression and secretion was noted in shFlnA adipocytes, cell size decreased signifi cantly on the low CHO but not the low fat diet (C). In although insulin stimulated Akt phosphorylation was not changed. Interest- conclusion, a low CHO vs. low fat hypocaloric diet reduces adipose cell size ingly, when adipocytes were subjected to a high nutrient environment in but yields greater circulating FFA. vitro, consisting of high levels of glucose, lipid and insulin, cells with reduced Figure. FlnA expression exhibited higher triglyceride storage capacity than control. Altogether, the data suggests that FlnA is involved in limiting triglyceride storage capacity of human adipocytes. Therefore, methods to increase FlnA expression in adipose tissue represent a potential strategy to limit adipose tissue expansion. In many cell types, proteases have been involved in regu- lating intracellular levels of FlnA and might therefore represent potential alternative targets to mitigate adipose tissue expansion.

1712-P Characterization of Infl ammation during the Development of Obe- sity in Cats EMILY C. GRAFF, HAN FANG, ANNIE MAGUIRE, OLGA C. NORRIS, ALISON R. Supported By: National Institutes of Health EMMERT, ROBERT L. JUDD, Auburn, AL, Columbia, MO In humans, obesity is characterized by systemic and adipose tissue infl am- 1710-P mation that contribute to secondary disease processes, such as type 2 dia- Protective Role of High Adiponectin Levels on Metabolic Functions betes mellitus (T2DM) and atherosclerosis. Cats are a naturally occurring in a Mouse Model of PCOS model of human obesity and T2DM. However, obese cats do not develop BELEN CHANCLON GARCIA, ANNA BENRICK, YANLING WU, LAILA HADI, STE- atherosclerosis and studies suggest that this may be due to a unique immune PHEN FRANKS, ELISABET STENER-VICTORIN, INGRID WERNSTEDT ASTER- response during the development of feline obesity. Therefore, the goal of HOLM, Gothenburg, Sweden, London, United Kingdom this study was to investigate systemic and adipose tissue infl ammation dur- Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects ing the development of obesity and insulin resistance in cats. Adipose tis- 5-10% of women in their reproductive age. PCOS is often related to meta- sue, whole blood, serum and MRI data were collected from twelve, lean, bolic disturbances, such as obesity and insulin resistance. Serum adiponec- male neutered, cats at baseline and following 18 months of ad libitum chow tin and adipocyte size are the strongest factors linked to decreased insu- diet. Changes in insulin sensitivity, lipid metabolism, adipokine profi les, sys- lin sensitivity in PCOS. Therefore, we hypothesize that adiponectin has a temic and adipose tissue infl ammation, and distribution of fat mass were protective effect on the development of metabolic dysfunction in this PCOS evaluated. The cats had a signifi cant increase in total fat mass, with pref- model. To address this we have investigated the metabolic function in mice erential expansion of subcutaneous adipose depots. Increased adiposity in over-expressing adiponectin in the adipose tissue (tg) and in knockout (ko) cats resulted in decreased insulin sensitivity, altered lipid metabolism and mice with or without dihydrotestosterone (DHT)-induced PCOS. DHT-pellets changes in adipokine profi les. During the development of obesity, the mean were implanted subcutaneously in pre-pubertal female mice to induce PCOS adipocyte area increased 138% in abdominal and 143% in subcutaneous adi- while controls received placebo-pellets. Adiponectin tg, ko and wt mice pocytes. No changes in circulating leukocytes were noted. However, serum were divided into placebo and DHT groups. Insulin/glucose tolerance, and concentrations of MCP-1 and TNF-α were signifi cantly increased. Crown- body composition measurements were performed between 14-16 weeks of like structures (CSL) were rare to absent in all adipose tissue samples and age. Wt-DHT mice displayed reduced serum adiponectin levels and became numbers of CLS were independent of time (baseline vs. endpoint) and loca- insulin resistant compared to controls, while tg-DHT mice were protected tion (subcutaneous vs. abdominal). In conclusion, during the development of against this effect of DHT. Ko-DHT mice developed more severe insulin obesity and insulin resistance, cats have altered lipid metabolism, adipocyte resistance than wt-DHT animals. Both wt-DHT and ko-DHT groups displayed hypertrophy, and changes in adipokines and circulating cytokines consistent impaired glucose tolerance compared to placebo but there was no signifi - with what is described in humans. However, unlike humans, cats preferen- cant difference between groups. Preliminary data shows that wt-DHT ani- tially expand subcutaneous adipose tissue depots rather than intra-abdom- mals have larger adipocytes than controls, and that tg-DHT mice are pro- inal and they do not develop classic adipose tissue infl ammation, character- tected against this effect. Gene expression analysis of the gonadal adipose ized by the development of CLS. depot shows decreased levels of genes involved in metabolic pathways like AdipoR2, IRS1, PPARγ and ChREBP in both wt-DHT and ko-DHT, which was 1713-P

POSTERS not observed in tg-DHT mice. Moreover, pancreas of the tg-DHT mice show KBRPL2001, a Novel GPR120 Agonist, Improves Insulin Sensitivity Insulin Action/ increased mRNA levels of insulin receptor, Glut2 and IGF1R, genes related and Glucose Tolerance and Decreases Hepatic Steatosis in Rodent

Molecular Metabolism to beta cell viability. There were small differences in body weight and body Models of Type 2 Diabetes composition between genotypes. We conclude that adiponectin have a pro- RAGHURAM ANUPINDI, RAJIV SHARMA, RAGHIB HUSAIN, Kalyani, India tective role on metabolic functions in this PCOS mouse model. GPR120 (FFAR4), a member of rhodopsin-like family of GPCRs, is widely expressed in many tissues and its activation improves many aspects of meta- 1711-P bolic homeostasis. KBRPL2001 is a selective GPR120 agonist (hEC50: 51 nM) Manipulation of Filamin A Expression in Adipocytes as a Potential that showed improvement in these metabolic parameters. KBRPL2001 treat- Modality to Limit Adipose Tissue Expansion ment displayed increase in GLUT-4 localization at the plasma membrane of SYAMALA AKELLA, ISHITA DEB MAJUMDAR, KALYANI V. GUNTUR, STEPHANE stimulated cells when compared with LA treated 3T3 adipocytes. In STC1 GESTA, VIVEK K. VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN R. cells, treatment with KBRPL2001 exhibited activation of MAPK pathway, as NARAIN, Framingham, MA determined by increased phosphorylation of Erk at 3 and 10uM treatment. FlnA was identifi ed as a major modulator of obesity phenotype using the Chronic administration of KBRPL2001 in diet-induced obese (DIO, 60% fat) Berg Interrogative Biology® platform from an in vitro obesity model contain- mice (30 mg/kg, p.o., b.i.d., 7 wk) caused a signifi cant reduction in the AUC ing adipocytes as one of the cellular model. Filamin A (FlnA) is a 280 kDa of blood glucose levels in OGTT (20%, p<0.001), reduction in fasting blood scaffolding protein that crosslinks actin to form fi lamentous network in cyto- glucose (192 to 148 mg/dL) and plasma insulin (2.3 to 1.2 ng/mL), demonstrat- plasm. Although it is reported to play a role in lipid droplet formation and ing its potential in improving insulin sensitivity. The treatment resulted in in insulin signaling, the function of FlnA in regulating adipocyte biology is signifi cant reduction (p<0.05) in body weight gain compared to DIO vehicle. still poorly understood. To determine the role of FlnA in adipocyte functions, In epididymal fat pads of DIO mice treated with KBRPL2001, decrease in immortalized human preadipocyte cell lines stably expressing either a con- pro-infl ammatory Adipose Tissue Macrophages (ATMs) and increase in anti- trol (shCont) or FlnA shRNA (shFlnA) were generated. FlnA protein expres- infl ammatory ATMs was observed. Similarly, increased presence of anti- sion was reduced by 50 to 60% in shFLNA expressing adipocytes compared infl ammatory macrophages was seen in liver tissue of compound treated

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A444 INSULIN ACTION—CELLULARCATEGORY AND MOLECULAR METABOLISM animals, indicating its potential to reduce infl ammation associated with insu- altogether, the relatively mild and muscle type specifi c phenotypes of both lin resistance. KBRPL treatment also showed signifi cant reduction in plasma the β- and γ-actin KO mice indicate that mature skeletal muscle does not triglycerides (p<0.001) and a corresponding, non-signifi cant decrease in liver rely on cortical remodeling of either β- or γ-actin for glucose transport to the triglycerides, indicating its potential in addressing hepatic steatosis/NAFLD. extent suggested by cell culture studies, although compensation between The preclinical data suggest that KBRPL2001, a novel GPR120 agonist, can be the two actin isoforms cannot be excluded. a potential therapy for type 2 diabetes and metabolic disorders. Supported By: Danish Diabetes Academy; Novo Nordisk Foundation

1716-P INSULIN ACTION—CELLULAR AND MOLECULAR Lipoprotein Lipase Is a Key Regulator of Energy Metabolism in the METABOLISM Brain KIMBERLEY D. BRUCE, ALENA RODRIGUEZ, SACHI GORKHALI, ANDREW LIBBY, 1714-P ROBERT H. ECKEL, Aurora, CO Vascular Endothelial Cell-specifi c PDK1 Knockout Mice Present Lipoprotein Lipase (LPL) is a key regulator of lipid metabolism. Abnormal Insulin Resistance and Deterioration of Mitochondrial Biogenesis LPL activity and expression has been implicated in the pathogenesis of a in Muscle under STZ-induced Hyperglycemia plethora of metabolic diseases. While the nutritional and molecular regula- ATSUSHI OBATA, KAZUHITO TAWARAMOTO, SEIZO OKAUCHI, TOMOHIKO tion of LPL in peripheral tissues is fairly well established, the role and regu- KIMURA, HIDENORI HIRUKAWA, MASASHI SHIMODA, KENJI KOHARA, AKIHITO lation of LPL in the brain is much less clear. Nonetheless, LPL is abundantly TANABE, TOMOE KINOSHITA, SHINJI KAMEI, TOMOATSU MUNE, KOHEI KAKU, expressed in several regions of the brain, and a reduction in neuronal LPL HIDEAKI KANETO, Kurashiki, Japan expression is thought to be involved in hypothalamic lipid sensing, hyper- The phosphatidylinositol 3-kinase signaling pathway in vascular endothe- phagia and the development of obesity (Wang et al. 2011. Cell Metab). To lial cells is important for systemic angiogenesis and glucose metabolism. We gain novel insights into the function of LPL in the brain, immortalized hypo- previously reported that vascular endothelial cell-specifi c PDK1 knockout thalamic neurons were genetically manipulated to either over, or under mice (VE-PDK1-KO mice) (Tie2 promoter-Cre/ PDK1fl ox/fl ox mice) presented the express LPL (Libby et al., 2015. BBRC). Using these cells we explored the improvement of insulin resistance and glucose tolerance (Mol Endocrinol. effect of nutritional cues, and the dynamics of candidate molecular factors 26, 95-109, 2012). In this study, we showed that VE-PDK1-KO mice presented thought to be involved in LPL inhibition (Angptl4), and lipoprotein receptor insulin resistance under STZ-induced hyperglycemia. This opposed pheno- lipid uptake (VLDLR, and ApoER2). Interestingly, LPL activity and expres- type suggests some uncovered pivotal roles of PDK1 in endothelial cells. We sion was lowest at high glucose conditions (20 mM). We also found that investigated the mechanism which induces insulin resistance in VE-PDK1-KO Angptl4 expression was profoundly increased in cells with reduced expres- mice under STZ-induced hyperglycemia. STZ (150 µg/g) was intraperitoneally sion of LPL (p < 0.001 vs. WT), consistent with a lack of Angptl4-mediated injected to VE-PDK1-KO and control fl ox/fl ox mice at 6 weeks of age. Ani- LPL inhibition. VLDLR gene expression was increased when LPL was knocked mals whose random fed glucose level was over 300 mg/dl were selected as down (p < 0.01 vs. WT), suggesting that VLDLR expression may be compen- diabetic mice. At 12 weeks of age, capillary blood volume in skeletal muscle satorily increased following reduced LPL-mediated lipid uptake. In contrast, was signifi cantly lower in VE-PDK1-KO mice than control mice accompanied ApoER2 expression was markedly reduced when LPL was knocked down with decrease of eNOS level. The protein level of p-Akt in skeletal muscle (p < 0.01 vs. WT), implying that ApoER2 may be involved in LPL-dependent was also decreased in VE-PDK1-KO mice, suggesting that insulin resistance lipid uptake. Taken together our fi ndings show for the fi rst time that neuronal was induced in skeletal muscle. Immunohistological analysis revealed the LPL is responsive to hypothalamic glucose concentrations, and is intricately decrease of endothelial cell number in skeletal muscle. TUNEL-positive involved in neuronal lipoprotein receptor-mediated lipid uptake. endothelial cells were increased in skeletal muscle in VE-PDK1-KO mice. Supported By: National Institutes of Health Furthermore, mRNA level of PGC1α was reduced. The downstream mRNA levels of PGC1α which regulate mitochondrial biogenesis such as Tfam, 1717-P NRF1 and NRF2 were also signifi cantly decreased in VE-PDK1-KO mice. In A Novel Factor POlDIP2 Is Suppressed in Livers of Type 2 Diabetic fact, mRNA levels of Cpt1β and ACADM were reduced in skeletal muscle Mice and Dysregulates Hepatic Cholesterol Homeostasis in VE-PDK1-KO mice. In conclusion, VE-PDK1-KO mice present insulin resis- KEYANG CHEN, CHENGWEI YANG, TAO LI, QUNAN WANG, KEVIN JON WIL- tance due to decreased endothelial cell number and capillary blood volume LIAMS, Hefei, China, Philadelphia, PA and deterioration of mitochondrial biogenesis in muscle under STZ-induced The NADPH oxidase-4 (NOX4) has emerged as a major metabolic regulator hyperglycemia. that fails to function normally in type 2 diabetic liver (ATVB 2012;32:1236- 1245). The molecular basis for its dysfunction has not been established. 1715-P Polymerase-δ interacting protein-2 (POLDIP2) was identifi ed as a NOX4 The Role of β- and γ-actin in Insulin-stimulated Glucose Transport partner for strengthening focal adhesions in vascular smooth muscle cells AGNETE B. MADSEN, JONAS R. KNUDSEN, YELIZ ANGIN, LYKKE SYLOW, ERIK A. (Circ Res 2009;105:249-259). We hypothesized a role for POLDIP2 in diabetes RICHTER, THOMAS E. JENSEN, Copenhagen, Denmark and lipid metabolism. Insulin-stimulated glucose uptake into skeletal muscle accounts for the Here, we found that hepatic POLDIP2 protein levels are substantially decreased in hyperphagic, obese, T2DM KKAy mice compared with nondi- POSTERS

majority of whole body insulin-stimulated glucose disposal, but the under- Insulin Action/ lying basic molecular mechanisms governing this process remain unclear. abetic KK littermate controls. Moreover, hepatic cholesterol content was y Accumulating evidence in cell culture and rodents suggest that the cortical doubled in KKA vs. KK mice, and it signifi cantly and inversely correlated Molecular Metabolism actin cytoskeleton plays a signifi cant role in the insulin-stimulated translo- with Poldip2 mRNA levels in liver. To identify mechanisms of POLDIP2 dys- cation of glucose transporter 4 (GLUT4) to the plasma membrane. Based on regulation in T2DM, we found that high levels of glucose (25mmol/L) - alone this it was hypothesized that muscle-specifi c knockout (KO) of either β- or or with insulin (10nM), leptin (8nM), or AGEs (200ug/ml) - sharply suppress γ-actin in adult mice would cause pronounced alterations in substrate utili- POLDIP2 protein expression in cultured rat hepatocytes. Treatment of cul- zation and glucose tolerance in vivo and reduce insulin-stimulated glucose tured hepatocytes with high glucose plus insulin doubled cellular cholesterol transport ex vivo. No genotype differences were observed in in vivo mea- content, similar to our fi ndings in vivo. Importantly, siRNA-mediated knock- surements including body composition, fasting insulin and glucose levels, down of POLDIP2 in cultured hepatocytes signifi cantly decreased insulin- and indirect calorimetry, except for a decreased glucose tolerance in the induced production of regulatory hydrogen peroxide, suggesting that POL- γ-actin KO mice (19%, p=0.024). Ex vivo, maximal insulin-stimulated 2-deoxy- DIP2 is required for healthy NOX4 activity. glucose transport was lower in soleus muscle in both β- and γ-actin KO mice To address POLDIP2 function in vivo, we used adenoviral particles to y (40%, p=0.001 and 33%, p=0.002 in β- and γ-actin KO mice, respectively). restore hepatic levels of POLDIP2 protein to normal in T2DM KKA mice. This In the β-actin KO mice no genotype differences were observed in any of intervention increased hepatic hydrogen peroxide production, decreased the measured protein expressions and phosphorylations in either muscle, hepatic cholesterol content by nearly 50% to reach levels seen in livers of nor was glucose transport affected in EDL from neither the β- or γ-actin KO nondiabetic KK littermates, and lowered plasma LDL cholesterol levels. mice. However, the γ-actin KO mice showed reduced insulin-stimulated Akt Taken together, our fi ndings demonstrated that a novel factor, POLDIP2, serine 473 and p70S6 kinase threonine 389 phosphorylation, although not regulates cholesterol homeostasis in liver, is defi cient in T2DM, and may signifi cant when related to total protein expression. These data imply that therefore contribute to metabolic dysregulation in states of overnutrition. γ-actin may be more important for glucose transport into skeletal muscle, Supported By: American Diabetes Association (1-13-BS-209 to K.J.W.); National due to the more pronounced phenotype of γ-actin vs. β-actin KO. However, Natural Science Foundation of China

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A445 INSULIN ACTION—CELLULARCATEGORY AND MOLECULAR METABOLISM

1718-P cally induced hypophosphatemia due to ablation of the renal sodium phos- Obesity Increased Mitochondrial DNA Damage and DAMPs which phate co-transporter NaPi2a, have reduced spontaneous activity and forced Regulate Infl ammatory Signaling and Insulin Resistance exercise capacity. We examined the potential role of muscle mitochondrial LARYSA YUZEFOVYCH, VIKTOR PASTUKH, MYKHAILO RUCHKO, MICHELE ATP synthetic rate (VATP) in this process using 31P magnetic resonance SCHULER, GLENN WILSON, JON SIMMONS, WILLIAM RICHARDS, MARK spectroscopy (MRS) saturation transfer (31P-ST) in vivo. Basal and insulin- GILLESPIE, LYUDMILA RACHEK, Mobile, AL stimulated VATP were reduced in mice on low phosphate diet or in NaPi2a Mitochondrial DNA (mtDNA) damage has been implicated in the devel- knockout mice (NaPi2a-/-) using this non-invasive technique. Likewise, VATP opment of insulin resistance (IR), since mtDNA is highly specialized and was reduced in a patient with hypophosphatemia due to a mutation in the encodes for proteins essential for energy metabolism and, also, mtDNA dam- gene encoding the renal phosphate transporter NAPI2C. Restoration of nor- age heightens mitochondrial oxidative stress, which is very critical for IR. mophosphatemia normalized VATP in NaPi2a-/- mice and the patient with Recently it has been shown that cells damaged by mechanical or infectious a mutation in the gene encoding the renal phosphate transporter NAPI2C injury release pro-infl ammatory mtDNA Damage Associated Molecular Pat- Using L6 and RC13 rodent myocytes and isolated muscle mitochondria, we terns (DAMPs) into the circulation (c-mtDNA). In this study, we demonstrate showed that VATP is directly related to cellular and mitochondrial phosphate in obese type 2 diabetes (T2D) patients and mice fed a high fat diet (HFD) uptake. Therefore, decreased muscle mitochondrial ATP synthetic rate may that elevated c-mtDNA correlate closely with IR. HFD-fed mice defi cient in in part explain the muscle weakness seen in hypophosphatemia and may the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), displayed serve as a non-invasive marker for hypophosphatemic myopathy. skeletal muscle mtDNA damage, elevated c-mtDNA, and an IR phenotype. Supported By: Austrian Science Fund Importantly, OGG1-defi cient mice reconstituted with human OGG1 (hOGG1) targeted specifi cally to mitochondria were protected against mtDNA dam- 1721-P age and elevations in c-mtDNA fragments and have reduced obesity, both Transgenic Mouse Model of Breast Cancer Develops Insulin Resis- systemic and tissue infl ammation and IR. Importantly, administration of exog- tance in Skeletal Muscle enous mtDNA DAMPs induced infl ammatory signaling, hyperglycemia and IR HEE JOON KANG, HYE-LIM NOH, SUCHAORN SAENGNIPANTHKUL, JOSE in vivo, providing a direct causative role for mtDNA DAMPs in the develop- MERCADO-MATOS, RANDALL H. FRIEDLINE, JONG HUN KIM, TAEKYOON KIM, ment of IR. Furthermore, exogenous mtDNA fragments induced TLR9-medi- KUNIKAZU INASHIMA, BORAM HAN, ALYCIA QUICK, XIAODI HU, KAREN KELLY, ated NF-κB activation, increased mitochondrial oxidative stress, reduced KI WON LEE, LESLIE M. SHAW, JASON K. KIM, Worcester, MA, Seoul, Republic mitochondrial function and suppressed insulin-mediated glucose uptake in of Korea vitro. Collectively, these fi ndings show that mtDNA damage with attendant Epidemiologic evidence suggests a potential link between cancer and dia- mitochondrial dysfunction and release of pro-infl ammatory mtDNA DAMPs betes that share multiple risk factors. Here we examined glucose metabo- contribute to the obesity-related IR phenotype and point to the prospect of lism in a transgenic mouse model of breast cancer expressing an oncogene, developing new diagnostic and treatment strategies focused on assessment polyoma middle T antigen driven by the Mouse Mammary Tumor Virus pro- and protection of mtDNA integrity. moter (MMTV-PyMT). A hyperinsulinemic-euglycemic clamp was performed Supported By: American Diabetes Association (7-13-BS-139-BR to L.R.) in female MMTV-PyMT mice with palpable tumors at the mammary region and control mice at 8~9 weeks of age (n=9~12/group). Despite similar body 1719-P weights, MMTV-PyMT mice developed insulin resistance with a signifi cant decrease in whole body glucose turnover (Figure 1; *P<0.05). This was largely WITHDRAWN due to a 20% decrease in insulin-stimulated glucose uptake in skeletal mus- cle (Figure 2). Insulin resistance was selective to muscle in MMTV-PyMT mice that showed normal glucose metabolism in white and brown fat as well as liver. MMTV-PyMT mice showed signifi cant increases in plasma IL-6, G-CSF, and MCP-1 levels (Figure 3), whereas adipokine levels did not differ between groups. Overall, these results indicate that tumor-bearing MMTV- PyMT mice develop insulin resistance in skeletal muscle. Also, our fi ndings suggest a novel paradigm in which cytokines and chemokines derived from the tumor microenvironment affect systemic glucose metabolism thereby providing a direct link between cancer and insulin resistance. Figure. POSTERS Insulin Action/ Molecular Metabolism

1720-P Regulation of Basal and Insulin-stimulated Rates of Muscle ATP Synthesis by Plasma Phosphate Assessed by 31P NMR Spectros- Supported By: National Institutes of Health (R01-DK080756, U24-DK093000, copy R24-DK090963) DOMINIK PESTA, DIMITRIOS TSIRIGOTIS, DOUGLAS E. BEFROY, DANIEL CABAL- LERO, MICHAEL JURCZAK, GARY W. CLINE, SYLVIE DUFOUR, ANDREAS L. BIRKENFELD, DOUGLAS ROTHMAN, THOMAS CARPENTER, CARL INSOGNA, KITT F. PETERSEN, CLEMENS BERGWITZ, GERALD I. SHULMAN, New Haven, CT, Toronto, ON, Canada, Dresden, Germany Hypophosphatemia in intensive care patients leads to muscle weak- ness resulting in respiratory and heart failure. Similarly, mice with geneti-

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A446 INSULIN ACTION—CELLULARCATEGORY AND MOLECULAR METABOLISM

1722-P 1724-P Structural and Functional Analysis of the Diabetes Risk Factor Insulin Regulates Glucose Transporter 4 Traffi cking in Diabetic Brain ZnT8/SLC30A8 Zinc Transporter Protein RAHUL AGRAWAL, ADRIANA VIEIRA-DE-ABREU, SIMON J. FISHER, Salt Lake MARK J. DANIELS, MACIEJ JAGIELNICKI, MARK YEAGER, Charlottesville, VA City, UT The human zinc transporter ZnT8 (SLC30A8), expressed predominantly in Evidence indicates that the brain may be insulin sensitive, but the mecha- pancreatic β-cells, is key in maintaining the concentration of blood glucose nism by which insulin may regulate glucose transport in critical brain regions through its role in insulin storage, maturation, and secretion. This transporter remains uncharacterized, especially in diabetes. The objective of the current is associated with type 2 diabetes through a risk allele that encodes a major study was to test the hypothesis that systemic insulin crosses the blood non-synonymous SNP at Arg325, and also with type 1 diabetes as a major brain barrier and acts both chronically and acutely in regulating glucose autoantigen. Interestingly, non-functional mutants reduce diabetes risk, transporter 4 (GLUT4) content and translocation in the hypothalamus (HYP) suggesting a therapeutic function for compounds that inhibit ZnT8 activity. and hippocampus (HIPP). C57Bl6 male mice (3 months old) were injected with This ~40 kDa protein is known to form homodimers in β-cells, where it streptozotocin (200 mg/kg; DIAB) or vehicle (nondiabetic controls; CON). regulates transport of Zn2+ into insulin secretory granules. Despite its role After 2 weeks of hyperglycemia (glucose > 300 mg/dl), DIAB mice were in diabetes and its concomitant potential as a drug target, little is known injected intraperitoneally with saline or insulin (5U/mouse; DIAB+INS) and about the structure or mechanism of ZnT8. The only structural information sacrifi ced after 40 min. Systemic insulin administration acutely stimulated available is from studies on the homologous bacterial transporter YiiP. Our brain insulin signaling as noted by increased Akt phosphorylation by 40 and goal is to provide insight into the function of ZnT8 as a Zn2+/H+ antiporter by 32% in HYP and HIPP regions. As compared to nondiabetic controls, chroni- structural and functional studies. cally insulin-defi cient DIAB mice demonstrated 55% and 40% decreased We successfully purifi ed ZnT8 constructs from a Pichia pastoris overex- plasma membrane (PM) GLUT4 content in HYP and HIPP, respectively. Acute pression system. With negative-stain electron microscopy (EM) and RELION systemic insulin administration increased relative PM GLUT4 content 3-fold single particle image analysis, the molecular boundary of an EM density map (in HYP) and 2-fold (in HIPP) in DIAB mice, indicating that peripheral insulin at 20Å resolution was suffi ciently well defi ned that we could dock our homol- rapidly stimulates brain GLUT4 recruitment to the PM. These results indi- ogy model of ZnT8. Our V-shaped structure is consistent with a homodimer cate that the chronic diabetes-induced down-regulation of PM GLUT4 can be and the bipartite appearance of each monomer is interpreted as the 22 kD reversed by acute insulin therapy. alpha-helical TM bundle and the 10 kD C-terminal domain. Figure. Insulin Acutely Restores Low PM GLUT4 Content in Hypothalamus Complementary to our structural analyses, we have developed an in vivo of Diabetic Mice. functional assay in our P. pastoris expression system and found that ZnT8 enhances Zn2+ effl ux. We also purifi ed ZnT8 constructs from insect cells and reconstituted the proteins into proteoliposomes. These vesicles showed pH- dependent uptake of Zn2+ consistent with a Zn2+/H+ antiport. The results of this research will be a starting point for drug design in targeting diabetes and its resulting complications. Supported By: American Diabetes Association (7-13-BS-038 to M.J.D.)

1723-P Epithelial Sodium Channel Inhibition with Amiloride Prevents Vas- cular Stiffening in Response to a Western Diet in Female Mice ANNAYYA AROOR, VINCENT G. DEMARCO, GUANGHONG JIA, JAVAD HABIBI, MONA GARRO, ZHE SUN, LUIS MARTINEZ-LEMUS, GERALD MEININGER, IRIS Supported By: National Institutes of Health JAFFE, JAMES R. SOWERS, Columbia, MO, Boston, MA Over-nutrition/obesity predisposes individuals, particularly women, to 1725-P arterial stiffening, an independent predictor of future adverse cardiovascu- Zinc Finger Protein 407 Overexpression Improves Glucose Homeo- lar events. We have recently developed a clinically relevant murine model stasis in Mice fed a high fat and high fructose diet (western diet, WD) which causes vas- ALYSSA CHARRIER, Cleveland, OH cular stiffness. In this model very low dose administration of the mineralo- Peroxisome proliferator-activated receptor gamma (PPARg) controls insulin corticoid (MR) antagonist spironolactone prevented development vascular sensitivity by regulating the expression of genes involved in glucose homeo- stiffness in females on a WD. One of the mechanisms by which MR acti- stasis, adipogenesis, and lipid metabolism. We recently discovered that zinc vation promotes endothelial stiffness is through increased expression and fi nger protein 407 (Zfp407) defi ciency in cultured adipocytes decreased the activation of epithelial sodium channel (ENaC) in endothelial cells (EC). In expression of PPARg target genes, including glucose transporter 4 (Glut4), this study, we tested whether amiloride, an inhibitor of ENaC, decreases thereby reducing insulin-stimulated glucose uptake. Co-overexpression of aortic stiffness in vivo and ex vivo aortic explants in WD fed female mice. Zfp407 and PPARg enhanced the expression of a luciferase reporter con- Four week-old C57BL6/J female mice were fed a WD with excess fat (46%) struct utilizing a canonical PPARg DNA binding site demonstrating a syn- and fructose (17.5%) with or without amiloride (1mg/kg/day) for 16 weeks. ergistic effect of Zfp407 on PPARg target gene expression. Therefore, we POSTERS Insulin Action/ Compared to mice fed a control diet (CD), aortic stiffness, determined by hypothesized that Zfp407 overexpression would increase PPARγ activity in vivo PWV, was signifi cantly increased in females on a WD and this cor- and improve glucose homeostasis in vivo, thus representing a novel thera- Molecular Metabolism responded to WD-induced increase in EC stiffness, measured ex vivo by peutic approach for treating type 2 diabetes. We generated a new trans- atomic force microscopy. These increases in stiffness were prevented by genic mouse strain (ZFP-TG) that specifi cally overexpressed Zfp407 in skel- administration of a very low dose of the ENaC inhibitor, amiloride. Moreover, etal muscle (19-fold) and heart (3-fold). Transcriptome analysis by RNA-Seq incubation of aortic explants ex vivo with 1 µM amiloride, a dose which it identifi ed 1,300 differentially expressed genes in the muscle of ZFP-TG mice, is more specifi c for ENaC, resulted in decrease in aortic stiffness in aorta among which PPARγ target genes were signifi cantly enriched. PPARγ mRNA from WD fed female mice. Moreover, in another study ENaC expression was and protein levels did not differ between ZFP-TG and control mice, suggest- also increased in WD fed mice which was decreased in EC specifi c MR KO ing that Zfp407 post-translationally regulates PPARγ activity. Among PPARγ mice with concomitant decrease in endothelial stiffness. Taken together, target genes, Glut4 mRNA and protein levels were increased in heart and these fi ndings support the notion that a WD promotes ECMR activation of muscle. The increase in Glut4 and other transcriptional effects of Zfp407 ENaC in ECs and associated vascular stiffness which is a marker/predictor overexpression together decreased total body weight and lowered plasma of cardiovascular disease. glucose levels relative to control littermates. Additionally, ZFP-TG male mice Supported By: National Institutes of Health had decreased plasma insulin levels and the HOMA-IR score was decreased in both male and female ZFP-TG mice compared to WT controls. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating meta- bolic disease. Supported By: American Diabetes Association (1-16-PDF-018)

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A447 INSULIN ACTION—GLUCOSE TRANSPORTCATEGORY AND INSULIN RESISTANCE IN VITRO

1726-P nant cortical actin cytoskeleton component. Furthermore, actin remodeling Interrogating the Signaling Mechanisms of GPR119: a Novel Target is abolished in the insulin-resistant state. In mature skeletal muscle insulin- for Type 2 Diabetes Mellitus (T2DM) and Obesity stimulated glucose uptake is blunted by actin depolymerizing agents, indi- PRAMISHA ADHIKARI, EMMANUEL STURCHLER, PATRICIA MCDONALD, Jupiter, cating that this could be a conserved function. However, further evidence is FL needed to support this hypothesis. Here we imaged isolated individual skel- GPR119 has emerged as a promising new drug target for the treatment etal muscle fi bers expressing GFP-tagged βcyto-actin and used Fluorescence of T2DM and obesity. This GPCR is expressed on intestinal L cells and pan- Recovery After Photobleaching (FRAP) to evaluate whether insulin induced creatic β-cells and as such, provides a unique opportunity to target a single any changes in the mobility of βcyto-actin, as suggested in cell culture and receptor promoting insulin secretion, β-cell proliferation, and secretion of heart muscle. We found that βcyto-actin co-localized with the Z-disks in a stri- gut-derived hormones (incretins) that induce postprandial insulin secre- ated pattern throughout the fi ber. In addition, βcyto-actin was located around tion. Collectively, these responses contribute to maintain normoglycaemia. the nuclei. After insulin treatment, we detected neither changes in the distri- Agonist activation of GPR119 leads to an increase in intracellular cAMP bution of βcyto-actin in live fi bers nor changes in the pattern of F-actin in fi xed fi bers. When FRAP was measured in the well resolved regions surrounding via coupling to Gαs-protein. G-protein signaling is terminated by β-arrestin recruitment, however, it is now appreciated that β-arrestins can also act as the nuclei, GFP βcyto-actin exhibited limited dynamics that were not signifi - signal transducers. Recently, the concept of biased signaling or functional cantly changed by insulin. We verifi ed, by immunoblotting, that insulin did selectivity, whereby, a GPCR preferentially activates G protein signaling over affect the muscle fi bers. Thus, our data do not support the hypothesis that β-arrestin signaling, or vice versa, has expanded the range of ligand activ- the insulin-induced changes in actin dynamics observed in muscle cells are ity to target GPCRs. Such ligands, termed “biased” ligands, have recently conserved into mature skeletal muscle. gained increasing attention due to their potential to provide therapeutic ben- Supported By: National Institute of Arthritis and Musculoskeletal and Skin efi t over existing therapies. However, whether pathway-selective GPR119 Diseases targeting can provide therapeutic advantages is yet to be determined. The aim of this study is to interrogate the contribution of G-protein and β-arrestin 1729-P signaling in mediating the physiological functions of GPR119. Ideally, GPR119 Both Enhancers and Potent Inhibitors Were Identifi ed in a Screen biased ligands would facilitate such studies, however as no such ligands for Small Molecule Modulators of Acute Insulin Action exist, we have taken a genetic approach. Recombinant cell lines have been CYNTHIA CORLEY MASTICK, PAUL DUFFIELD BREWER, IRINA ROMENSKAIA, generated that stably express mutant GPR119 that can selectively activate Reno, NV Gαs or β-arrestin mediated signaling. Our results demonstrate that modifi ca- Using a 1° assay for acute insulin action that is sensitive, responsive, and tions of the carboxy-terminal tail of GPR119 generates a Gαs biased signaling highly reproducible, we identifi ed 12 novel enhancers and 26 novel inhibi- receptor, activation of which results in an attenuation of both MAPK phos- tors of acute insulin action in a pilot screen 1120 drugs. Using our 2° kinetics phorylation and desensitization as compared to wildtype receptor. We are assays to characterize these compounds, novel mechanisms for enhancing currently investigating the physiological consequences of selectively engag- and inhibiting acute insulin action were discovered. In addition, drugs that ing Gs at the GPR119 receptor. may have adverse effects on glucose homeostasis and be contraindicated in diabetes were identifi ed. Enhancers include 6 cardiac glycosides related to 1727-P ouabain. All are Na+/K+-ATPase inhibitors. Ouabain accelerates both endo- The Relevance of Insulin Signaling in the Regulation of Pluri- cytosis and exocytosis of Glut4 (1.5- and 3.3-fold). These compounds increase potency cell surface Glut4 by inhibiting sorting into the highly regulated storage ves- MANOJ K. GUPTA, DARIO F. DE JESUS, ROHIT N. KULKARNI, Boston, MA icles (GSVs). Inhibitors include 11 “antibiotics.” Five affect protein synthesis, Self-renewal of mouse induced pluripotent stem cells (miPSCs) is achieved but are structurally and mechanistically distinct. A requirement for protein by the modulation of dedicated transcription circuits that eliminate differen- synthesis for acute insulin action has not been described. Hits also include tiation-inducing signals. Among these circuits the relevance of insulin recep- 10 phenothiazines and dibenzazepines. These drugs cause acute insulin resis- tor (IR)-mediated signaling in regulating the identity of miPSCs is not fully tance (hyperinsulinemia/normoglycemia) in humans, through an unknown understood. Here we report the relevance of insulin receptor (IR) dependent mechanism. Four different phenotypes were observed for the inhibitors, indi- and independent signaling pathways in the maintenance of pluripotency. We cating distinct mechanisms of action. Membrane traffi cking: the nutraceu- derived IR Knock-Out (IRKO) miPSCs from E14.5 mouse embryonic fi broblasts tical resveratrol and the natural product piperlongumine decreased surface (MEFs) of global IRKO mice using a cocktail of four reprogramming factors, Glut4 to the same extent and as rapidly as the PI3-kinase inhibitor LY294002 namely Oct4, Sox2, Klf4 and cMyc. iPSCs were maintained in a 2- inhibitor (LYi; t½=5 min). LYi inhibits exocytosis. Protein synthesis: the natural product (2i) feeder-free system. All clones stained positive for alkaline-phosphatase licorine had the same slow kinetics and effi cacy as puromycin (t½=30 min). and formed teratomas containing the three lineages. Gene expression analy- Signal transduction: propranolol and the natural product parthenolide had sis revealed an upregulation of several genes associated with pluripotency kinetics similar to an Akt inhibitor (t½=10 min). Endosomal pH: the phenothi- including Klf4, Lin28a, Tbx3 and cMyc (p<0.05, n=3 clones/group) in IRKO azine maprotiline and the anti-malarial chloroquine had very fast transition iPSCs as compared to controls (C). Oct4 and Nanog protein levels were 4-fold kinetics (t½=3 min). These amines inhibit endocytic acidifi cation. (p<0.05, n=3) and 3-fold (p<0.01, n=3) increased in IRKO iPSCs as compared Supported By: American Diabetes Association (1-12-BS-132 to C.C.M.)

POSTERS to C respectively and were confi rmed by immunofl uorescence staining. Basal Insulin Action/ signaling analysis demonstrated downregulation (p<0.05, n=3) of phospho- 1730-P STAT3, p-mTOR and p-ERK, despite an increase (p<0.05, n=3) in total protein Molecular Metabolism N-WASP-Cortactin Signaling Promotes Skeletal Muscle GLUT4 levels of mTOR and ERK in IRKO iPSCs. Furthermore, stimulation of phospho- Vesicle Translocation ERK by leukemia inhibitory factor (LIF) was decreased by 3-fold (p<0.05, n=3) RAGADEEPTHI TUNDUGURU, JEFFREY S. ELMENDORF, DEBBIE C. THURMOND, in IRKO iPSCs as compared to C. Therefore, IRKO iPSCs provide a unique Indianapolis, IN, Duarte, CA opportunity to explore the signifi cance of insulin receptor signaling in the Insulin maintains glucose homeostasis by mobilizing GLUT4 vesicles from maintenance of pluripotency and its crosstalk with key pluripotency related intracellular compartments to the plasma membrane (PM) of muscle and signaling pathways. adipose cells, facilitating glucose uptake into these cells. Defects in GLUT4 translocation are associated with peripheral insulin resistance, pre-clinical diabetes and eventual progression to type 2 diabetes. Recruitment of GLUT4 INSULIN ACTION—GLUCOSE TRANSPORT AND to the PM of skeletal muscle cells requires fi lamentous (F)-actin remodeling. INSULIN RESISTANCE IN VITRO Recent in vitro data implicates the neural Wiskott-Aldrich syndrome protein (N-WASP) in insulin-dependent cortical F-actin rearrangement. However, 1728-P the mechanism of action of N-WASP in regulating this cortical actin net- work, and any relatedness to skeletal muscle function, is unexplored. Here Insulin-induced βcyto-actin Dynamics in Live Skeletal Muscle JONAS R. KNUDSEN, KRISTIEN J. ZAAL, EVELYN RALSTON, THOMAS E. JENSEN, we show that inactivation of N-WASP by its specifi c inhibitor, Wiskosta- Copenhagen, Denmark, Bethesda, MD tin, fully abrogates the insulin-stimulated increase in GLUT4 translocation Prediabetic insulin-resistance impedes insulin-stimulated translocation of to the plasma membrane in skeletal muscle cells. Toward interrogating the the glucose transporter 4 (GLUT4) to the surface membrane in skeletal mus- underlying mechanism, interactions between N-WASP and Cortactin were cle. In muscle cell culture, accumulating evidence suggests that this translo- assessed, given that Cortactin is an actin binding protein and implicated in actin remodeling in clonal muscle cells. Indeed, a ~1.5 fold increased bind- cation requires increased dynamics and remodeling of βcyto-actin, the domi-

For author disclosure information, see page A696. & Moderated Poster Discussion ADA-Supported Research

A448 INSULIN ACTION—GLUCOSE TRANSPORTCATEGORY AND INSULIN RESISTANCE IN VITRO ing of Cortactin to N-WASP with insulin stimulation was detected using whereby increased HBP activity increases Sp1 transcriptional activation of a mouse skeletal muscle lysates as well as L6-mycGLUT4 cell lysates. In sum, cholesterolgenic program thereby elevating PM cholesterol and compromis- these results suggest that N-WASP facilitates Cortactin-mediated F-actin ing cytoskeletal structure essential for insulin action. remodeling for insulin-stimulated GLUT4 vesicle translocation to the PM of Supported By: American Diabetes Association (1-15-BS-053 to J.S.E.); Eli Lilly skeletal muscle cells. Additional studies delineating the signaling elements and Company surrounding N-WASP-Cortactin, with the ultimate goal of identifying thera- peutic targets in this pathway, are currently underway. 1733-P Supported By: American Diabetes Association (1-15-BS-053 to J.S.E.); National Carnitine Acetyltransferase: A New Player in Skeletal Muscle Insu- Institutes of Health; JDRF lin Resistance? SOFIA BERG, NILS FÆRGEMAN, MICHAEL GASTER, Odense, Denmark 1731-P Carnitine acetyltransferase (CRAT) defi ciency has previously been shown The Regulation of Glucose Transport Is Altered during Diabetes- to result in muscle insulin resistance due to accumulation of long-chained induced Atrial Fibrillation acylcarnitines. Myotybes established from type 2 diabetes mellitus subjects ZAHRA MARIA, ALLISON CAMPELO, BRENDA SMITH, BENJAMIN SCHERLAG, express primary insulin resistance. The aim of this study was to examine VERONIQUE A. LACOMBE, Stillwater, OK, Oklahoma City, OK whether myotubes established from obese persons with and without type 2 Diabetes and obesity have been identifi ed as major risk factors for atrial diabetes mellitus (T2DM), and lean controls express differences in CRAT and fi brillation (AF). However, whether a metabolic substrate underlies AF is in acylcarnitine species precultured under physiological conditions. Primary unknown. Glucose transport into the cell via Glucose Transporters (GLUTs) myotubes obtained from obese persons with or without T2DM and lean con- is the rate-limiting step of glucose utilization. Although GLUT4 is the major trols (N=10 in each group) were established at normophysiological condition isoform in the heart, GLUT8 has recently emerged as a novel cardiac iso- and harvested for LC-MS-based profi ling of acylcarnitines. The level of CRAT form. However, its role in the heart is not well known. We hypothesized mRNA and protein levels were determined by quantitative PCR (qPCR) and that GLUT-4 and -8 translocation to the atrial cell surface will be impaired Western Blotting. Our results show that the protein and mRNA levels of during type 2 diabetes (T2Dx)-induced AF. AF was induced by transesopha- CRAT are unchanged in obese with and without T2DM persons compared geal atrial pacing in healthy and long-term high-fat-diet (HFD)-induced T2Dx to lean controls. We measured 14 different acylcarnitine species and show rodents. Expression of GLUTs and key proteins involved in the insulin signal- that the myotube levels of palmitoylcarnitine (C16) and octadecanoylcarni- ing pathway was measured by Western blot in cardiac myocytes. Active cell tine (C18) were slightly reduced in T2DM patients. Moreover, the total level surface GLUT content was measured using the state-of-the-art biotinylated or the levels of the other individual acylcarnitine species were unaltered photolabeled assay in the perfused heart. After 6 months on a HFD, mice between the three groups. The present results indicate that CRAT is not were obese and hyperglycemic, and developed insulin resistance compared important for primary insulin resistance. Long-chain acylcarnitines do not to mice on a control diet. T2Dx animals showed an increased susceptibil- accumulate in obese persons with and without T2DM under physiological ity and propensity for AF. In the T2Dx atria, active cell surface and total conditions, suggesting that the main factor that causes primary insulin resis- GLUT4 content was down-regulated (by 66% and 40%, respectively, P<0.05) tance in T2DM persons still remains unclear. under basal condition. Long-term HFD-induced T2Dx resulted in impairment in Akt and AS160 phosphorylation, which was signifi cantly correlated with 1734-P GLUT4 protein content in the atria. These data suggest an impairment of the insulin signaling pathway, which was further confi rmed by altered traf- WITHDRAWN fi cking of both GLUT-4 and -8 to the cell surface upon insulin stimulation in the T2Dx atria. In conclusion, our data suggest that: 1.) T2Dx increases the vulnerability to AF; 2.) GLUT-4 and -8 traffi cking is altered in the T2Dx atria due to impairments in the insulin signaling pathway. Therefore, alterations in atrial glucose transport may induce perturbations in energy production and could provide a metabolic substrate for atrial fi brillation during diabetes and obesity. Supported By: Harold Hamm Diabetes Center

1732-P Evidence for a Cholesterolgenic Response as a Basis of Insulin Resistance in Mice Fed a High-Fat Diet JEFFREY S. ELMENDORF, LIXUAN TACKETT, BRENT A. PENQUE, NOLAN J. HOFF- MAN, WHITNEY J. SEALLS, JOSEPH T. BROZINICK, Indianapolis, IN, Stony Brook, NY, Sydney, Australia Clonal cell studies demonstrate that excess hexosamine biosynthesis pathway (HBP) activity increases O-linked N-acetylglucosamine modifi cation POSTERS of the transcription factor Sp1, leading to transcriptional activation of HMG- Insulin Action/

CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthe- Molecular Metabolism sis. This HBP-induced cholesterolgenic transcriptional response increases cholesterol in the plasma membrane (PM), while reducing cortical fi lamen- tous actin (F-actin) that is essential for insulin-stimulated GLUT4-mediated glucose transport in 3T3-L1 adipocytes and L6 skeletal muscle myotubes. To gain in vivo understanding of cholesterol-associated insulin resistance, 4-wk old male C57BL/6J mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates, to better mimic the average human diet in Western societies. At 8 wks, both glucose and insulin tolerance were impaired in HF- fed mice. Consistent with these data, fed insulin levels were signifi cantly increased by HF-feeding compared to LF-fed mice. Mixed hindlimb skeletal 1735-P muscle from these HF-fed mice showed a 34% increase in PM cholesterol Receptor-mediated Glucose Regulation in Stem Cell-derived compared to LF-fed mice. In line with cell culture fi ndings, demonstrating Hepato cytes, Cardiomyocytes, and Skeletal Myoblasts increased PM cholesterol causes a loss of both F-actin and insulin-stimu- DAVID MANN, NATSUYO AOYAMA, COBY CARLSON, MIKE HANCOCK, BLAKE lated glucose transport; both were decreased by 21% and 26%, respectively ANSON, Madison, WI in muscle from HF-fed mice compared to LF-fed mice. Epididymal fat pads Glucose homeostasis is tightly regulated in vivo as dysregulation results also displayed a HF-feeding induced accumulation of PM cholesterol, as in tissue damage to multiple organ systems, including hepatic, cardiac and well as O-GlcNAc modifi cation of Sp1 and higher binding affi nity of Sp1 to skeletal muscle tissues. Owing to functional limitations, there is a dearth the promoter region of HMGR. Together, these data suggest a mechanism of in vitro models to study normal and diseased tissues. Many primary tis-

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A696.

A449 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

sue isolates and immortalized cell lines alter their responsiveness to stimuli insulin signaling. Therefore we studied the role of CBP/p300 interacting in culture over time and become insulin resistant. Induced pluripotent cells transactivator-2 (CITED2), a negative regulator of HIF activity, in endothelial (iPSCs) and the differentiated cell types derived therefrom afford a limit- cell insulin signaling. We generated an animal model with endothelial spe- less supply of donor consistent tissue for glucose regulation research and cifi c loss of Cited2 using Cre-Lox recombination. In isolated primary endothe- therapeutic discovery. lial cells, loss of Cited2 enhanced insulin stimulated Akt phosphorylation at The liver is the major metabolic regulatory organ potentiating serum glu- Ser473 by 3.1±0.8 fold while ERK1/2 phosphorylation remained unchanged. cose levels. In hepatocytes, expression of glucokinase is regulated by insulin Consistent with this observation, biological functions of insulin were potenti- and glucagon. Here we show in iPSC-derived hepatocytes the cell signaling ated by Cited2 deletion. Cited2 knockout enhanced the effect of insulin on events triggered by these hormones, as well as the ability to monitor gluco- endothelial cell proliferation by 72±9%, and potentiated the ability of insulin neogenesis. to increase Vegfa mRNA by 42±9% and phosphorylation of endothelial nitric Skeletal muscle is a peripheral target for insulin action and thus is an oxide synthase by 2.1±0.3 fold. Cited2 deletion did not affect phosphorylation important cell type in diabetes research for studying glucose transport. Here of the insulin receptor at Tyr1345 or insulin receptor substrate (IRS)-1 expres- we show that iPSC-derived skeletal myoblasts can be differentiated to elon- sion. However, loss of Cited2 resulted in a 66±9% increase in IRS-2 mRNA gated myogenin-positive myotubes capable of glucose uptake in response to and a 3.6 fold increase in IRS-2 protein. Therefore, loss of Cited2 enhances insulin in a dose-dependent manner. Importantly, this signal increase can be endothelial cell insulin signaling via derepression of IRS-2 expression. Con- modulated by the GLUT4-specifi c inhibitor, Indinavir. sistent with a role for CITED2 as a novel mediator of endothelial insulin resis- Cardiomyocytes preferentially consume fatty acids for ATP production. tance, Cited2 mRNA was increased 10.2±0.3 fold (n=2) in endothelial cells However, under particular circumstances, glucose uptake is increased to freshly isolated from mice with diet-induced obesity vs. lean controls. Impor- optimize energy production. We report here an assay to measure intracel- tantly, our fi ndings extend to human disease as CITED2 was elevated 3.8 fold lular glucose levels as demonstrated by an increase in response to insulin in mammary artery from 5 patients with type 2 diabetes vs. 4 nondiabetic and other related molecules. This assay not only enables metabolic studies controls. These data identify CITED2 as a novel modulator of endothelial insu- in human iPSC-derived CMs, but it sets the stage for comparison of disease- lin signaling that is dysregulated in patients with type 2 diabetes. Inhibition specifi c samples to wild-type/normal control cells. of CITED2 is a potential approach to selectively enhance IRS-2/Akt signaling in endothelial cells and prevent vascular complications. 1736-P Supported By: Danish Diabetes Academy; National Institutes of Health An In Vitro Contraction Model that Replicates Exercise-mediated (R21CA185196); Diabetic Complications Consortium (U24 DK076169); Diabetes Effects on Glucose Metabolism in C2C12 Skeletal Muscle Cells also Research Wellness Foundation Protects against Lipid-induced Insulin Resistance STEPHAN NIEUWOUDT, ANNY MULYA, CIARÁN E. FEALY, ELIZABETH E. MAR- & 1738-P TELLI, SATHYAMANGLA V. NAGA PRASAD, JOHN P. KIRWAN, Cleveland, OH, Diabetes Remission Induced by the Central Action of Fibroblast Kent, OH Growth Factor 1: Role of Insulin In vitro models to replicate the metabolic effects of exercise and probe JENNIFER M. ROJAS, JARRAD M. SCARLETT, MILES E. MATSEN, KARL J. KAI- the cellular and molecular mechanisms that mediate skeletal muscle insulin YALA, GREGORY J. MORTON, MICHAEL W. SCHWARTZ, Seattle, WA resistance are beginning to emerge. Herein we describe an in vitro contrac- In rodent models of type 2 diabetes, hyperglycemia is transiently ame- tion model that allows isolation of the specifi c effects of exercise on skeletal liorated by either systemic or intracerebroventricular (icv) administration muscle glucose metabolism that are typically seen in vivo. We validated the of fi broblast growth factor (FGF)-19 or FGF-21. Based on the relatively long model by probing the effects of contraction on insulin-stimulated glucose duration (up to 42 h) of the antidiabetic action of FGF1 following systemic uptake and lipid-induced insulin resistance. The model utilizes a customized administration, we investigated its glucose-lowering potential following icv electrical pulse stimulation (EPS) system to provide the excitation-contrac- injection. As reported elsewhere at this meeting, we found that a single icv tion stimulus to muscle cells. Following 16 hours of stimulation at 1 Hz (1.5 injection of recombinant murine FGF1 (mFGF1) at a dose (3 µg) 10-fold below V/mm), fully differentiated C2C12 myotubes were challenged with 0.5 mM that needed for systemic effi cacy induces weight-loss independent diabe- palmitate (saturated fatty acid) for 4 hours. EPS increased the basal glucose tes remission in both ob/ob (on C57Bl6J background) and db/db mice with uptake to the same level as insulin stimulation alone (1 µM, 30 minutes) in moderate hyperglycemia. Specifi cally, blood glucose (BG) values declined non-EPS control cells (P=0.70). The palmitate challenge signifi cantly sup- from ~300 to ɖ 200 mg/dl for up to 4 wk in db/db and 18 wk in ob/ob mice pressed insulin-stimulated glucose uptake (P<0.05), a key effect of skele- following icv FGF1 (icv Veh vs. FGF1, P<0.0001 by linear mixed model analy- tal muscle insulin resistance. Most importantly, EPS effectively protected ses). However, icv mFGF1 was ineffective in mice with severe, uncontrolled against lipid-induced insulin resistance (P<0.05). Western blotting shows hyperglycemia (BG >300 mg/dl, including db/db, wild-type C57BL6J (WT) that the protective effect on the insulin signaling pathway is signifi cant for receiving a high dose of the pancreatic β-cell toxin streptozotocin, and ob/ the Threonine 308 phosphorylation residue site of Akt/PKB. Insulin receptor ob crossed onto the diabetogenic BTBR background). To test the hypothesis substrate-1 (IRS-1) associated phosphoinositide 3-kinase (PI3K) basal activ- that glucose lowering elicited by icv FGF1 requires an intact insulin signal, ity, upstream of Akt/PKB signaling, was affected by contraction as well, we administered the high-affi nity insulin receptor (IR) antagonist S961 to with whole cell insulin stimulated PI3Kα activity matching glucose uptake diet-induced obese WT mice as a continuous subcutaneous infusion at a

POSTERS results. These data suggest that in vitro EPS of skeletal muscle cells pro- dose (29 nmol/wk) designed to achieve hyperglycemia (~300 mg/dl) com-

Insulin Action/ vides an experimental model that reproduces the effects of exercise that are parable to that observed in moderately diabetic ob/ob mice that respond observed in vivo, and show that the protection against lipid-induced insulin robustly to icv FGF1. Although transient anorexia induced by icv mFGF1 was Molecular Metabolism resistance is mediated through intrinsic regulation of insulin signaling. not altered by systemic IR blockade, the antidiabetic effect of FGF1 was Supported By: National Institutes of Health completely blocked. We conclude that 1) diabetes remission can be induced by the action of brain FGF1, and 2) an intact insulin signal is required for this effect. INSULIN ACTION—SIGNAL TRANSDUCTION, Supported By: National Institute of Diabetes and Digestive and Kidney Diseases INSULIN, AND OTHER HORMONES (DK083042, DK090320, DK101997 to M.W.S.), (DK089056 to G.J.M.), DK007247, DK103375; Nutrition Obesity Research Center (DK035816); University of Washing- ton Diabetes Research Center (DK017047) Moderated Poster Discussion: Advances in FGF-21 and Insulin Action (Posters: 1737-P to 1743-P), see page 15. & 1739-P A Novel Mechanism for Fibroblast Growth Factor-21 to Regulate & 1737-P Hepatic Insulin Sensitivity via Inhibiting Mammalian Target of The Transcriptional Coregulator CITED2 Impairs Vascular Endothe- Rapamycin Complex 1 lial Cell Insulin Signaling by Suppressing IRS-2 QI GONG, ZHIMIN HU, FEIFEI ZHANG, XIN CHEN, HAOYANG JIANG, JING GAO, SAM M. LOCKHART, XUANCHUN WANG, DITTE SORENSEN, SALLY DUN- XUQING CHEN, YAMEI HAN, QINGNING LIANG, LEI SHI, EUGENE CHIN, YU WOODIE, LARS M. RASMUSSEN, CHRISTIAN RASK-MADSEN, Boston, MA, Syd- WANG, HUI XIAO, FEIFAN GUO, YONG LIU, MENGWEI ZANG, AIMIN XU, YU LI, ney, Australia , Odense, Denmark Shanghai, China, Hong Kong, China, Boston, MA Insulin signaling in endothelial cells regulates leukocyte adhesion, angio- The hepatokine FGF-21 has emerged as a novel metabolic regulator that genesis and vascular permeability. Hypoxia-inducible factor (HIF) regulates has potential to treat diabetes and obesity. We recently demonstrated that

For author disclosure information, see page A696. & Moderated Poster Discussion ADA-Supported Research

A450 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

SIRT1 and RARβ are key upstream regulators of FGF-21 to regulate hepatic potency (IC50 ~ 10 pM). Glucagon-stimulated maximal glucose output was lipid metabolism. Although pharmacological and physiological studies have dose-dependently suppressed by insulin, indicating functional integration of demonstrated benefi cial functions of FGF-21 in the liver, the downstream insulin and glucagon signaling. Under modeled conditions, human iPS-derived signaling pathways mediating these activities remain largely unknown. We hepatocytes expressed multiple markers of mature hepatocytes (TAT, TDO2, showed that administration of FGF-21 inhibited mTOR/S6K1 activity and HPX, albumin), including modulators of glucose metabolism (G6PC, PEPCK, increased phosphorylation of Akt and GSK3β to improve hepatic insulin FBP), insulin signaling (IRS1, pI3K, AKT, mTOR) and lipid metabolism (PGC1α, sensitivity and systemic glucose homeostasis in HFHS diet-fed mice. Strik- CPT1a, LIPC). Cultured human iPS-derived hepatocytes exhibited morpho- ingly, these effects were abrogated by hepatic knockdown of FGF-21’s core- logic features reminiscent of human liver, including cobblestone epithelial ceptor βKlotho using adenovirus-mediated short-hairpin RNA. FGF-21 defi - morphology, glycogen storage, and capacity for intracellular lipid accumu- ciency potentiated hepatic mTORC1 activity and resulted in attenuated Akt lation, dependent on extracellular substrate availability. Development of phosphorylation in acute insulin-treated mice. Importantly, we showed that iPS-derived hepatocellular culture models will enable new opportunities for insulin- and nutrient-stimulated activation of mTORC1 activity was inhibited advancing mechanistic understanding of human hepatocyte physiologic and by FGF-21 overexpression in human HepG2 cells, leading to increased phos- pathophysiologic states. phorylation of Akt. FGF-21 was suffi cient to increase glycogen synthesis in primary mouse hepatocytes. Moreover, hepatic overexpression of mTORC1’s & 1742-P downstream effector S6K1 abolished FGF-21’s augmentation of insulin sen- Insulin Uptake by the Brain Endothelial Cell (BEC) Is Receptor- sitivity in HFHS diet-fed mice. Our fi ndings indicate that FGF-21-mediated dependent and Blunted by High-Fat-Diet Feeding inhibition of mTORC1 may represent a molecular mechanism by which phar- SARAH M. GRAY, KEVIN W. AYLOR, EUGENE J. BARRETT, Charlottesville, VA macologic and genetic manipulation of FGF-21 ameliorate hepatic insulin Insulin access to the brain may be critical for appetite regulation, metabo- resistance, hyperglycemia, and type 2 diabetes. lism, and cognition. Prior work suggests insulin crosses the blood-brain bar- Supported By: National Natural Science Foundation of China (81270930, rier (BBB) to reach brain interstitial fl uid where it can act on neurons. Little is 31471129) known about the transit process in the BEC and if insulin resistance affects it. We tested whether BECs have an insulin receptor (IR)-mediated vesicular & 1740-P transport system and if high-fat diet (HFD)-induced insulin resistance affects Intracellular and Extracellular Domain-Dependent Effects of Insu- this. We fed rats HFD or normal chow (ND) for 4 weeks before isolating and lin and IGF-1 Receptors culturing BECs. We examined BEC insulin uptake using radiolabeled insulin WEIKANG CAI, MASAJI SAKAGUCHI, ANDRE KLEINRIDDERS, BRIAN T. O’NEILL, (125I-ins), insulin signaling, and mRNA and protein expression. 125I-ins uptake JONATHON N. WINNAY, JEREMIE BOUCHER, C. RONALD KAHN, Boston, MA was decreased in BECs from HFD rats compared to ND (p<0.01). 10 nM insu- The insulin receptor (IR) and IGF-1 receptor (IGF1R) are highly homologous, lin increased p-Akt (Ser473) and p-eNOS (Ser1177) similarly in BECs from but mediate distinct cellular and physiological functions. To defi ne how the HFD and ND rats. Insulin did not increase ERK (Thr202/Tyr204), Src (Tyr416), different domains of these receptors contribute to their unique signaling and or caveolin-1 (Tyr14) phosphorylation in either group. IR-β mRNA and IR-β functions, we created preadipocyte cell lines in which both endogenous IR protein were not signifi cantly different between HFD and ND. We then and IGF1R had been genetically deleted and then reconstituted them with used cell-surface biotinylation and western blotting to test whether insu- normal IR, IGF1R, or two chimeric receptors: one with the IR extracellular lin affected IR-β endocytosis. Plasma membrane sheets were isolated from domain (ECD) fused to intracellular domain (ICD) of the IGF1R [IR/IGF1R] and rat microvascular BECs treated with 10 nM insulin or control. Compared to the other with the ECD of the IGF1R fused to the ICD of the IR [IGF1R/IR]. control, insulin treatment decreased plasma membrane-bound IR-β indicat- Both cells expressing receptors with the ICD of the IGF1R (i.e., IGF1R and ing receptor-mediated endocytosis. Blocking PI3-kinase or MEK pathways IR/IGF1R) showed higher mitogenic activity, while cells expressing recep- decreased insulin signaling in whole-cell lysates, but did not prevent IR-β tors with the ICD of the IR (i.e., IR and IGF1R/IR) had more robust glycolytic endocytosis. This is consistent with our previous fi ndings that blocking these responses to ligand stimulation. These correlated with increased activation pathways did not decrease 125I-ins uptake. In conclusion, insulin promotes of Shc, Gab-1, ERK1/2 and p70-S6K1 pathways in cells expressing recep- BEC insulin uptake by stimulating IR-β endocytosis. HFD feeding reduced tors with IGF1R ICD, while cells expressing receptors with IR ICD displayed 125I-ins uptake despite preserved signaling to Akt and eNOS. These fi ndings higher phosphorylation on IRS1. Receptors with IGF1R ICD were also more underscore the need to unravel the precise mechanisms regulating insulin potent in regulating gene expression in pathways involved in proliferation uptake in BECs. and cell surface protein expression, whereas receptors with IR ICD were Supported By: National Institutes of Health; American Heart Association more potent in regulating genes involved in metabolic pathways, especially glucose metabolism. Surprisingly, some differences in intracellular signaling & 1743-P and gene expression regulations also correlated with the unique extracellu- Quantitative Proteomics of Mouse Intestinal Mucosa Lacking Insu- lar domains of the two receptors. Changing amino acid residue 961 adjacent lin Receptors in the intracellular NPxY motif from leucine (present in the IR) to phenylala- STINA JENSEN, SARAH WHEELER, HENNING HVID, ERWIN SCHOOF, THOMAS nine (found in the IGF1R) resulted in changing the signaling and gene expres- KISLINGER, BO F. HANSEN, ERICA NISHIMURA, GRITH S. OLSEN, PATRICIA L. sion pattern for IR-like to IGF1R like. These studies demonstrate how domain BRUBAKER, Toronto, ON, Canada, Måløv, Denmark structural differences between IR and IGF1R result in differential regulation Mice with targeted deletion of the insulin receptor (IR) in the intestinal POSTERS of signaling, gene expression, and cellular functions between IR and IGF1R. epithelium (IE-irKO) have been used to study the role of the IR in the gastroin- Insulin Action/

testinal tract (GIT). In general, IE-irKO mice do not show a strong metabolic- Molecular Metabolism & 1741-P or intestinal-physiological phenotype. Hence, we used an unbiased Mass Modeling Integrated Insulin and Glucagon Signaling in Human iPS Spectrometry approach to quantify proteins in jejunal and colonic mucosa Derived Hepatocytes from IE-irKO and the two control mice: IE-irKOfl / fl and VIL-Cre. Mice were fed WILLIAM C. ROELL, SIMONE GUPTA, JIANNONG DAI, MELISSA K. THOMAS, a chow- or high-fat-sucrose western diet (WD) for 12 wk. By proteomic analy- Indianapolis, IN sis, 7931 proteins were identifi ed. Principle component analysis (PCA) clearly Mechanisms by which integration of hepatic insulin and glucagon signal- separated proteins from chow- and WD-fed mice. Moreover, the PCA indi- ing is dysregulated in type 2 diabetes are incompletely understood, in part cated a distinct differential protein expression in the colonic mucosa of WD- due to limitations in translating rodent hepatic physiology to humans and to fed IE-irKO mice compared to WD-controls. A greater number of signifi cantly variable fi delity of disease modeling from cadaveric tissues. Human induced different proteins were found in IE-irKO as compared to control mice when pluripotent stem cell (iPS) differentiated models provide a unique opportunity animals were fed the WD instead of chow. Paneth cell products, including to address gaps in understanding hepatic physiology and pathophysiology. Lysozyme, CRIS1C-2/3, Angiogenin-4, and Interlectin-1a, were signifi cantly By modeling culture conditions for hepatocytes differentiated from human reduced in IE-irKO jejunal mucosa, as were both Glucose-dependent Insuli- iPS cells, we explored their capabilities to recapitulate physiologic counter- notropic Peptide (GIP) and Neurotensin. Mucin-2 was decreased in both jeju- regulation of hepatic glucose production. In this model, glucagon stimulated num and colon of IE-irKO mice. A reduction of the SLC amino acid transport- hepatic glucose production with an approximate potency of EC50 ~ 5 nM. ers and members of the ATPase family was also found in the colonic mucosa Specifi city of this regulation was demonstrated by suppression of glucagon- of WD-fed IE-irKO mice. Finally, Apolipoprotein A-I and II were increased in stimulated glucose production with glucagon receptor antagonist co-admin- WD-fed IE-irKO colon and jejunum, respectively. Tissue resistance studied istration. Treatment of human iPS derived hepatocytes with insulin resulted by Ussing Chamber and in vivo permeability to FITC-dextran (4 kDa) did not in physiologic, dose-dependent suppression of glucose output with marked demonstrate any alterations in intestinal barrier integrity in IE-irKO. Together

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A451 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

the data demonstrate altered protein expression in the intestinal mucosa of activation. For its activation, the Akt is translocated from cytoplasm to cell IE-irKO mice which was enhanced when the animals were challenged with membrane. This process is through Lys63-linked ubiquitination by TRAF6, an a WD. In jejunum, Paneth-, Goblet- and Enteroendocrine cell products were ubiquitin E3 ligase. However, it remains unclear where the interaction occurs. reduced, suggesting a role for the IR in the regulation of both host protective Here, we show that TRAF6 through its RING fi nger domain interacts with Akt, functions and gut hormone expression in the GIT. leading to Akt ubiquitination, which is essential for Akt activation upon insulin stimulation. Absence of TRAF6 or its RING fi nger domain resulted in impaired 1744-P the Akt ubiquitination thereby prevented it from activation. These results Effect of Metformin on Phosphorylation Profi le of Insulin Resistant suggest that TRAF6 induces Akt ubiquitination by interaction of its RING fi n- Primary Human Skeletal Muscle Cells ger domain with Akt, which regulates Akt activity in insulin signaling. ABDULLAH MALLISHO, MICHAEL CARUSO, NISHIT SHAH, BERHANE SEYOUM, Supported By: Auburn University at Montgomery (to G.T.); Alabama Agricultural YUE QI, DIVYASRI DAMACHARLA, DANJUN MA, XIANGMIN ZHANG, ZHENG- Experimental Station (to R.J.) PING YI, Detroit, MI Metformin is the principle biguanides and the fi rst line drug therapy used 1747-P for treatment of type 2 diabetes. However, the mechanism of action of Met- Higher Postprandial GLP-1 Responses Are Associated with formin is still not fully understood. The mainstay of action has been for a Improved Insulin Action in Liver and Muscle but Not in Adipose Tis- long time attributed to its hepatic effect on decreasing hepatic glucose pro- sue in Morbidly Obese Nondiabetic Subjects duction. However, Metformin has also been shown to improve insulin sen- PIM W. GILIJAMSE, KASPER W. TER HORST, MARIETTE T. ACKERMANS, sitivity in skeletal muscle. Phosphatases and kinases are proteins required JOHANNES A. ROMIJN, MAX NIEUWDORP, MIREILLE J. SERLIE, Amsterdam, for dephosphorylation and phosphorylation of proteins, respectively, in cells Netherlands during various signaling pathways. These phosphorylation events carry the Glucagon-like peptide 1 (GLP-1) is an incretin which stimulates insulin potential to be used as drug targets. In the present work, we studied the secretion and improves insulin sensitivity. It remains to be established to effect of Metformin on phosphorylation changes of proteins from primary what extent meal-induced GLP-1 responses contribute to insulin-mediated cell culture of human skeletal muscle tissue from obese/overweight insulin glucose and lipolysis fl uxes in humans. We aimed to study these metabolic resistant participants using phosphoproteomics. We identifi ed 2930 phos- fl uxes in relation to the postprandial GLP-1 response.We studied 29 mor- phorylation sites in 1085 proteins. Of particular interest, Metformin treat- bidly obese nondiabetic subjects (18 ş, 11 š; age 43 ± 2 y; BMI 43 ± 1 kg/m2). ment signifi cantly changed phosphorylation levels of 23 sites, including 6 The GLP-1 response was assessed during an oral liquid mixed meal test (50 g phosphorylation sites in 4 phosphatase subunits and 17 phosphorylation carbohydrates, 40 g protein, 67 g fat) and basal and insulin-mediated glucose sites in 9 kinases and kinase subunits. These results provide new informa- and lipolysis fl uxes were measured using a two-step euglycemic hyperinsu- 2 2 tion on the mechanism of action of Metformin in human skeletal muscle cells linemic clamp with infusion of [6,6- H2]glucose and [1,1,2,3,3- H5]glycerol. Liver derived from overweight/obese participants and can potentially be use to fat content was assessed by magnetic resonance spectroscopy. Following the better understand how Metformin increase skeletal muscle insulin sensitiv- mixed meal, plasma GLP-1 levels increased 7 ± 1-fold after 30 min and did not ity in vivo in humans. return to baseline within 4 h. There was a negative correlation between the GLP-1 and insulin AUCs (r = -0.45, p = 0.015), indicating that a larger GLP-1 1745-P response was associated with lower insulin secretion following the mixed meal. Glargine and Detemir Do Not Increase Tumour Incidence or Multi- The postprandial GLP-1 responses correlated strongly with hepatic (r = 0.59, plicity Compared with Unmodifi ed Insulin in a Carcinogen-induced p < 0.01) and peripheral insulin sensitivity (r = 0.53, p < 0.01), but not with Rat Model of Breast Cancer adipose tissue insulin sensitivity (r = -0.20, p = 0.33) or basal endogenous YUSAKU MORI, EUNHYOUNG KO, STUART C. WIBER, GEORGE I. FANTUS, ALAN glucose production (r = -0.18, p = 0.41). Finally, postprandial GLP-1 responses MEDLINE, ADRIA GIACCA, Toronto, ON, Canada did not correlate with gender, age, BMI, waist circumference, or liver fat (p > Glargine and determir are commonly used insulin analogues for basal insu- 0.05), indicating that differences in baseline characteristics did not infl uence lin replacement. Concern raised by several studies that glargine increases the fi ndings. Postprandial GLP-1 responses correlated positively with insulin the risk for all-type cancer was not confi rmed by subsequent studies. How- action in liver and skeletal muscle but not in adipose tissue in obese subjects. ever, whether glargine increases the risk for breast cancer is not fully clari- The inverse correlation between postprandial GLP-1 and postprandial insulin fi ed. This is of particular importance now, with two new glargine prepara- suggests that the relationship between GLP-1 levels and overall metabolic tions, biosimilar and centrated glargine, approved. To gain insight into this health is stronger than the incretin effect. issue from preclinical studies, four wk old female Sprague-Dawley rats were Supported By: European Union started on a high fat diet to induce insulin resistance (hyperinsulinemic clamp onfi rmed) and given the carcinogen N-Methyl-N-nitrosourea (50mg/kg) 1 wk 1748-P later. At 9 wk of age, the rats were randomly assigned to 4 groups: vehicle, Curcumin Increases the Expression of Carbohydrate Responsive NPH (unmodifi ed human insulin), glargine, and detemir (n=30/group). After Element-binding Protein in Male Mouse Hepatocytes via Stimulat- 6 weeks of treatment (15 U/kg/day, 5 days/wk), mammary tumours were ing p21 Activated Protein Kinase counted and extracted. No insulin increased mammary tumour incidence. KEJING ZENG, LILI TIAN, ADAM SIREK, WEIJUAN SHAO, LING LIU, YU-TING

POSTERS However, tumour multiplicity (number of tumours per rat or per tumour- ALEX CHIANG, JONATHAN CHERNOFF, DOMINIC NG, JIANPING WENG, TIANRU Insulin Action/ bearing rat) was increased with NPH or glargine (p<0.05, respectively) and JIN, Guangzhou, China, Toronto, ON, Canada, Philadelphia, PA

Molecular Metabolism almost increased with detemir (p=0.2) with no difference among insulins. In response to hyperglycemia, carbohydrate-responsive element-binding Mammary tumours (all carcinomas) expressed higher levels of insulin recep- protein (ChREBP) facilitates hepatic lipogenesis in order to reduce the load tor (IR) and insulin-like growth factor-1 receptor (IGF-1R) than non-tumorous of plasma glucose. We found previously that insulin can stimulate ChREBP mammary gland. Compared to MCF-7 human breast cancer cells, IR was expression via inactivating a transcriptional repressor, the POU homeodo- higher in mammary tumours while IGF-1R expression was much higher in main protein Oct-1. Here we demonstrated that the effect of insulin in atten- MCF-7. Glargine and detemir increased Akt phosphorylation in tumours, but uating the chromosomal Oct-1 occupation on ChREBP promoter and stimulat- Erk 1/2 phosphorylation was not changed by any insulin. In summary, in this ing ChREBP expression is mediated by an Akt-independent ERK activation. model of estrogen-dependent breast cancer in insulin resistant rats, insulin Interestingly, the curry dietary compound curcumin was shown to stimulate glargine and detemir did not increase tumor incidence and multiplicity to a ChREBP expression in mouse hepatocytes, and short-term curcumin gavage greater extent than unmodifi ed human insulin. increased hepatic expression of ChREBP. We then demonstrated that cur- cumin can also stimulate ERK phosphorylation and Oct-1 nuclear exclusion 1746-P in hepatocytes in the presence of Akt inhibition. Furthermore, like insulin, The RING Finger Domain of TRAF6 Interacts with Akt for Its Ubiquit- curcumin treatment was shown to stimulate Thr423 phosphorylation of p21- ination and Activation on Insulin Stimulation activated protein kinase (Pak-1). The inhibition of Pak-1 or genetic knockout GEETHA THANGIAH, CHEN ZHENG, VISHAL KOTHARI, ANDREA CARTER, JAKE of Pak-1 attenuated the effect of curcumin on stimulating ChREBP expres- SUSTARICH, RAMESH JEGANATHAN, Montgomery, AL, Auburn, AL sion and reducing Oct-1 levels. Together, our observations indicated a novel Akt, a serine/threonine kinase, also known as protein kinase B, is respon- signaling cascade, Pak-1/ERK/Oct1, for curcumin in exerting its glucose low- sible for glucose metabolism upon insulin signaling. Binding of insulin to its ing effect via promoting hepatic lipogenesis. receptor on cell membrane activates several downstream signaling com- Supported By: Canadian Institutes of Health Research ponents in sequence, which eventually leads to Akt phosphorylation and

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A452 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

1749-P diabetes (T2D) in humans. Consequently, IDE has awaken a great level of HKDC1, a Novel Fifth Hexokinase, Is Involved in Whole-Body Glu- interest as a therapeutic target in the treatment of T2D patients. However, cose Metabolism the physiological role of IDE on the regulation of glucose homeostasis, and CAROLINA M. PUSEC, EMILY D. SMITH, ANTON E. LUDVIK, WILLIAM L. LOWE, its potential therapeutic benefi t, remains largely unknown. To shed light on BRIAN T. LAYDEN, Chicago, IL the role of IDE in the regulation of glucose metabolism in T2D patients, we Hexokinases are the fi rst enzyme in the glycolysis pathway, catalyzing analyzed IDE levels in cadaveric human pancreata from T2D patients that the phosphorylation of glucose to yield glucose-6-phosphate (G-6-P) as its underwent the classical treatments used for the control of diabetes, i.e., product. Four well-characterized hexokinase isoforms exist. Recently, Irwin oral hypoglycemic agents (OHAs) or insulin therapy. In addition, we mea- et al. suggested a novel hexokinase-like gene, called Hexokinase Domain sured IDE levels in pancreata of a pre-clinical mouse model of diabetes (db/ Containing 1 (HKDC1) as another hexokinase. Interestingly, through a GWAS db mouse). we identifi ed and reported a strong genetic association between variants Islets histomorphometry showed that the ratio beta-/alpha-cells was in the HKDC1 gene and 2 hour plasma glucose levels in women that were reduced by 50% in T2D patients vs. control subjects. In parallel, the percent- 28 weeks pregnant. We also reported that HKDC1 is a fi fth hexokinase age of alpha-cells was two-fold higher in T2D patients than in control sub- validated by hexokinase assays. However, these data showed that, despite jects. Furthermore, T2D patients showed a two-fold increase in IDE positive high sequence homology to hexokinase 1, the enzymatic activity of HKDC1 cells in comparation with control subjects. Similar results were found in db/ is more similar to glucokinase. To explore the in vivo role of HKDC1, global db mice. Finally, isolated rodent islets treated with insulin exhibited upregu- HKDC1 mice (HKDC1tg/tg) were generated. Homozygous knockout was an lation (~50%) of IDE levels. embryonic lethal mutation. We therefore used heterozygous HKDC1tg/twt In conclusion, we demonstrated that IDE expression is upregulated in mice and found impaired glucose tolerance with aging, but no other overt human pancreata of insulin treated T2D patients. Of note, the vast major- metabolic phenotypes. To circumvent the in utero lethality, we have now ity of IDE-positive cells were alpha-cells, suggesting that insulin therapy generated adult HKDC1 global knock mice via a cross of HKDC1fl / fl with a through upregulation of IDE levels may represent a new cellular mechanism tamoxifen-inducible Beta-Actin Cre mouse line. Thus far, we have observed to counteract glucagon production in alpha-cells, contributing to improved that these mice have elevated insulin in the post-absorptive state, while overall glucose homeostasis in T2D patients. maintaining normal body mass, fasting blood glucose and insulin levels, and Supported By: Ministerio de Economia y Competitividad post-absorptive glucose levels after tamoxifen treatment. We are currently examining the role of HKDC1 in this model through additional extensive phe- 1752-P notyping studies. Altogether, our data indicate that HKDC1 is a novel fi fth Gut Microbiome Produced N-Formyl Peptides Act through the hexokinase that contributes to whole-body glucose metabolism. Receptor FPR1 to Modulate Insulin Secretion and Metabolism Supported By: National Institute of Diabetes and Digestive and Kidney Dis- JOSHUA WOLLAM, ANDREW M. JOHNSON, YONGJIANG XU, MOHIT JAIN, JER- eases; National Institutes of Health ROLD M. OLEFSKY, La Jolla, CA In western societies, the increasing prevalence of obesity has been 1750-P accompanied by a dramatic increase in type 2 diabetes. Diet-induced obe- Unraveling the Mechanism behind the Benefi cial Effects of Omega-3 sity is associated with systemic infl ammation, which is recognized as a con- Fatty Acids: The FFAR4-Beta Arrestin-2 Interaction tributor to insulin resistance and diabetes. However, the mechanisms by EVELYN WALENTA, VIVIAN A. PASCHOAL, DA YOUNG OH, La Jolla, CA which the diet infl uences infl ammation remain poorly understood. A key link Recent studies demonstrated that the free fatty acid receptor 4 (FFAR4, between diet and metabolism is the microbiome of the gastrointestinal (GI) formerly GPR120) is the control point for the robust effects of omega-3 tract, producing metabolites that infl uence host physiology. Interestingly, the fatty acids (ω-3 FAs) to mediate anti-infl ammatory and insulin sensitizing composition of commensal microbiota and associated metabolites changes actions. This has been an important discovery in our understanding of the dramatically in obesity. Among these, N-formyl peptides are bacterial meta- interconnections between obesity, infl ammation, and insulin resistance. The bolic byproducts and infl ammatory chemokines that activate the G-protein detailed mechanism by which fi sh oil supplementation to dietary regimens coupled N-formyl peptide receptor 1 (FPR1). We have found that levels of exerts benefi cial effects is of high interest. Not only will the discovery of this the N-formyl peptide fMLF are elevated in systemic circulation of wild-type mechanism help to improve the administration of fi sh oil in a better targeted (WT) obese mice fed a high fat diet (HFD) compared to lean mice. Knockout way, this will also help to identify and develop ligands for FFAR4, which is a of the FPR1 receptor (KO) improves glucose tolerance and insulin secretion promising new drug target. in HFD conditions, dependent upon the enteroendocrine-produced hormone It has been shown that the ability of beta arrestin-2 (ARRB2) to physi- glucagon-like peptide-1 (GLP-1), which promotes insulin secretion. Further- cally associate with FFAR4 is likely a key mechanism in the anti-infl amma- more, treatment of WT HFD mice with an FPR1 antagonist improves glucose tory pathway induced by ω-3 FAs. Investigating downstream, we analyze tolerance and increases plasma GLP-1 and insulin levels. Obese HFD KO mice the for the FFAR4-ARRB2 interaction required phosphorylation of FFAR4. We also display a dramatically altered microbiome composition compared to WT identify a single phosphorylation site (T242) to be necessary for the anti- littermates, as well as reduced circulating fMLF levels, suggesting feedback infl ammatory effects of FFAR4 activation by ω-3 FAs as well as a new FFAR4 mechanisms exist between the intestinal microbiome and FPR1 signaling to specifi c agonist, CpdA. Furthermore, we compare the metabolic phenotypes infl uence metabolism. Overall, the altered microbiome in obesity modulates

of ARRB2 whole body knockout mice to FFAR4 whole body knockout mice. metabolic status through production of N-formyl peptides and activation POSTERS We can show that ARRB2 is necessary for the positive effects of ω-3 FAs on of FPR1 in the GI tract, leading to suppressed GLP-1 production and insu- Insulin Action/ reduced lipid accumulation as well as expression of infl ammatory markers. lin secretion. FPR1 antagonists may prove to be valuable therapeutic treat- Molecular Metabolism Consequently, the ARRB2 knockout mice do not display the improved insulin ments providing protection against systemic infl ammation, hyperglycemia, sensitivity of wild type mice when fed an ω-3 FA rich diet or the new FFAR4 GI disorders, metabolic disease and insulin resistance. specifi c agonist, CpdA. This is refl ected in impaired hepatic glucose produc- Supported By: National Institutes of Health tion suppression and muscle glucose disposal. Taken together, we show that the positive effects of ω-3 FAs or CpdA can 1753-P only operate through the FFAR4-ARRB2 pathway, and that ARRB2 is required Brain Insulin Signaling Is Increased in Insulin-Resistant States and for the benefi cial effects of FFAR4 activation by ω-3 FAs or its specifi c ago- May Abet Alzheimer’s Disease Development nist on lipid accumulation, infl ammation, and insulin sensitivity. MINI P. SAJAN, ROBERT IVEY, MARGARET FARESE-HIGGS, CSILLA ARI, SHIJIE Supported By: Larry L. Hillblom Foundation; American Heart Association SONG, MICHAEL LEITGES, URSULA BRAUN, BARBARA C. HANSEN, ROBERT FARESE, Tampa, FL, Oslo, Norway, Gulfport, FL 1751-P Increased coexistence of Alzheimer’s disease (AD) and type 2 diabetes Insulin Degrading Enzyme Is Upregulated in Type 2 Diabetes Patients mellitus (T2DM) suggests that insulin resistance abets AD development, but Pancreatic Alpha Cells mechanisms are obscure. Nevertheless, it is commonly assumed that the CRISTINA M. FERNADEZ-DIAZ, LUIS ESCOBAR-CURBELO, JOSE F. LOPEZ- brain itself is insulin-resistant (IR) in states of systemic insulin resistance, ACOSTA, JULIAN SANZ-ORTEGA, GERMAN PERDOMO, IRENE COZAR-CASTEL- and insulin treatment (Rx) for AD is in trials. Here, we examined insulin LANO, Valladolid, Spain, Madrid, Spain, Burgos, Spain signaling in brains of IR high-fat-fed mice, ob/ob mice, mice with impaired Insulin Degrading Enzyme (IDE) is an endopeptidase with a broad spec- glucose transport owing to muscle-specifi c aPKC-λ knockout (MλKO), and trum of activity against proteins such as insulin, glucagon and beta-amyloid. monkeys with long-standing obesity/T2DM. In normal mice, 15-min insulin Interestingly, the IDE gene has been associated with the risk to suffer type 2 Rx increased brain Akt and aPKC activity. In all IR models, however, basal

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A696.

A453 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

Akt and aPKC activities were maximally increased by hyperinsulinemia as (9-36), the inactive metabolite of GLP-1 (7-36) lacking the two N-terminal they were unaffected by insulin Rx. Moreover, Akt hyperactivation in all IR amino acids, by turning it into a ligand able to activate the GLP-1R in almost models led to hyperphosphorylation of Akt substrates, GSK3β, mTOR, Fox01, the same extent as GLP-1 (7-36) does. We designed small molecule PAMs, Fox03a, and Fox04. And, with decreased Fox0 activity, PGC-1α levels were which highly activate the GLP-1R in the presence of inactive GLP-1 (9-36) decreased in all IR models. Akt hyperactivation was also confi rmed in indi- metabolite. Using a recombinant cell line overexpressing the GLP-1R, we can vidual neurons of anterocortical and hippocampal regions that house impor- show that the receptor is activated almost as effi ciently as with the active tant cognition/memory centers. Most notably, Aβ1-40/42 peptide levels were GLP-1 (7-36). In the presence of inactive GLP-1 (9-36) metabolite, our PAMs increased by insulin in normal mice and maximally increased basally in all IR trigger glucose-stimulated insulin secretion in rodent as well as in human models. And, and, in long-standing IR in obese/T2D monkeys, increases in pancreatic islets. Oral glucose tolerance tests in diabetic mice models show Aβ1-40/42 peptide levels were accompanied by diminished amyloid precursor that glucose levels are robustly reduced and insulin levels are increased fol- protein levels, indicating continued conversion to Aβ1-40/42. Tau-PO4 was also lowing administration of PAM and exogenous GLP-1 (9-36). Furthermore, increased in ob/ob mice and T2D monkeys. Importantly, with correction of a moderate reduction in blood glucose level is observed by exploiting the hyperinsulinemia by liver-specifi c inhibition of aPKC and improved systemic endogenous GLP-1 inactive metabolite levels only. insulin resistance, brain insulin signaling normalized. As Fox0s and PGC-1α We conclude that the development of highly potent PAMs with good phar- are essential for memory and maintenance of neuronal structure, function macokinetic properties could be an attractive alternative to GLP-1 peptide and regeneration and, as Aβ1-40/42 and Tau-PO4 may accumulate in interneu- mimetics. ronal plaques and intraneuronal tangles, the aforesaid aberrations may link hyperinsulinemia states obesity and T2DM to AD. 1756-P Supported By: National Institutes of Health; U.S. Department of Veterans Affairs Association between Low Body Mass Index (BMI) and Insulin Sen- sitivity 1754-P AKANKASHA GOYAL, ANNEKA WICKRAMANAYAKE, SHANKAR VISWANA- PKCε and CDKs Regulate Insulin Signaling in Response to a 3-Day THAN, RIDDHI DASGUPTA, MERCY INBAKUMARI, CHAITHANYA MURTHY High-Fat Diet KOCHER LAKOTA, PRAVEEN GANGADHARA, NIHAL THOMAS, MEREDITH HAWK- BRANDON M. GASSAWAY, MAX C. PETERSEN, HANS R. AERNI, YULIA V. INS, New York, NY, Bronx, NY, Vellore, India SUROVTSEVA, KARL W. BARBER, JOSHUA B. SHEETZ, SVETLANA ROGULINA, A few reports have suggested a J-shaped relationship between BMI and JANIE MERKEL, VARMAN T. SAMUEL, GERALD I. SHULMAN, JESSE RINEHART, insulin sensitivity, with progressive insulin resistance as BMI falls below West Haven, CT, New Haven, CT 18.5 kg/m2, suggesting a unique phenotype of “low BMI diabetes.” We there- Insulin resistance is the main driving force behind type 2 diabetes mellitus fore studied insulin sensitivity using gold-standard methodologies across a (T2DM). In the liver, diacylglycerol (DAG) is a key mediator of lipid-induced spectrum of BMI ranging from 14 to 27 kg/m2 in subjects with and without hepatic insulin resistance by activating protein kinase C epsilon (PKCε), diabetes. Euglycemic (90 mg/dl) hyperinsulinemic (80 mU/ m2/min) clamp which in turn directly inhibits the insulin receptor kinase resulting in hepatic studies were performed in n=35 ethnically homogeneous Indian males with insulin resistance. Knockdown of PKCε in liver protects rats from lipid- non-ketogenic diabetes mellitus (DM) (avg age 36.5 y; HbA1c 10.5%; range: induced hepatic insulin resistance in vivo by increasing InsR kinase activity. % body fat by DXA 7.1-31.8) compared with n=16 matched nondiabetic (ND) However, PKCε might not be the best therapeutic target for T2D since this subjects (avg: age 33.4 y; HbA1c 5.3%; range: % body fat 10.6-26.5), using kinase controls other important signaling networks unrelated to insulin sig- 6, 6-deuterated glucose to quantify glucose uptake. Therapeutic regimens naling. We hypothesize that DAG-activated PKCε would act on a network of were intensifi ed for two weeks to eliminate glucose toxicity in DM subjects. proteins that would be comprised of known and novel members of the insu- Plasma levels of fat-derived proteins including adiponectin were measured lin signaling pathway. This PKCε network is currently unknown and would by chemiluminescence. A direct linear correlation was found between BMI potentially contain new candidates for antidiabetic therapeutics. Here we and insulin resistance in both groups, with an estimated average 0.48 unit used quantitative phosphoproteomics combined with a systems-biology decrease in glucose uptake per one unit increase in BMI (p<0.0001, adjust- inspired functional screen to identify and characterize a novel hepatic PKCε ing for diabetes status and adiponectin). Additionally, an average 2.85 unit network. We quantifi ed changes in protein phosphorylation in insulin-sen- decrease in glucose uptake was estimated in the DM subjects compared to sitive liver, lipid-induced insulin resistant liver, and lipid-loaded liver made ND (p=0.0001, adjusting for BMI and adiponectin). These studies are novel insulin sensitive by knockdown of PKCε and uncovered over 550 changes in in their documentation of improved insulin sensitivity at lower BMIs in both the phosphoproteome. We observed that Cyclin Dependent Kinases (CDKs) DM and ND subjects, with a progressive linear decrease in glucose uptake were the primary divers of changes in the phosphoproteome in response with increasing BMI. Across this BMI range, DM subjects were more insulin to the HFD. We also determined which of the observed phosphoproteins resistant than ND subjects of comparable BMI and fat mass, underscoring were direct PKCε or CDK substrates by employing a novel kinase substrate the importance of “glucose toxicity.” When controlling for BMI, changes in expression platform. Finally, we investigated our novel PKCε and CDK net- adiponectin at low BMI’s had no signifi cant effect on glucose uptake. These works with a high-content siRNA screen in cultured hepatocytes, where we are the fi rst studies to examine insulin resistance in DM subjects with low examined the role of our liver phosphoproteins as novel inhibitors or activa- BMI using sophisticated methodologies and promote a shift in our under- tors of canonical insulin signaling. The results of these experiments have standing of the phenotype of low BMI diabetes.

POSTERS identifi ed a more expanded network of proteins involved in insulin signaling, Insulin Action/ which includes PKCε and CDK substrates that may direct new therapeutic 1757-P approaches for T2DM.

Molecular Metabolism Insulin Regulation of TLR4 Signaling in Human Leukocytes Supported By: National Institutes of Health; National Science Foundation; How- LOUIS F. AMOROSA, ZHIYONG ZHANG, MARIE MACOR, SUSSETE COYLE, LEON- ard Hughes Medical Institute ARD LEE, BEATRICE HAIMOVICH, New Brunswick, NJ Human peripheral blood leukocytes (PBL) are a readily available model 1755-P for studying infl ammatory cellular signaling. We used these cells to investi- GLP-1R Oral Positive Allosteric Modulators (PAM): Another Approach gate insulin signaling in patients with chronic and acute insulin resistance. to Tackle T2DM Our recent studies demonstrated that MMP9 expression in PBL is involved KRISTIN BREITSCHOPF, ELISABETH DEFOBA, MATTHIAS LOEHN, HANS MAT- in the cleavage of AMPKinase, in vivo and in vitro, following stimulation TER, MARÍA MÉNDEZ PÉREZ, MICHAEL PODESCHWA, NILS RACKELMANN, with lipopolysaccharides, a TLR4 ligand. MMP is also expressed in PBL of JENS RIEDEL, PAVEL SAFAR, Frankfurt, Germany, Tucson, AZ free living type 2 diabetic patients and patients who develop acute insulin The Glucagon-like peptide 1 (GLP-1) hormone is secreted from intestinal resistance associated with cardiopulmonary bypass surgery. These stud- L-cells after food intake and plays a crucial role in blood glucose regula- ies link MMP expression and AMP Kinase degradation with a cascade of tion, and in the control of appetite and body weight. Activation of the cor- transcription factor phosphorylations associated with clinical insulin resis- responding GLP-1 receptor (GLP-1R) triggers glucose-dependent insulin tance. (Zhang Z, et al, J Immunol, 2015). In a subset of these patients with secretion and leads to suppression of glucagon secretion in the pancreas. type 2 diabetes, we have identifi ed additional differences in signaling inter- For the treatment of type 2 diabetes several pharmaceutical approaches, mediates including AKT phosphorylated on Ser473. Insulin, but not LPS, like GLP-1 mimetics (e.g., liraglutide (Victoza) or lixisenatide (Lyxumia)) and induced mTORC2-dependent phosphorylation of AKT on Ser473 and Fox01/ DPP-4 inhibitors (e.g., sitagliptin (Januvia)), are directed to enhance the acti- O3 on Thr24/25. Furthermore, insulin and AS1842856, a Fox01 inhibitor, sup- vation of the GLP-1R. We are following a new approach aiming at positive pressed the expression of LPS-induced signaling intermediates in a dose- allosteric modulators (PAMs). These PAMs modulate the activity of GLP-1 and time-dependent manner. We conclude that insulin is a homeostatic

For author disclosure information, see page A696. & Moderated Poster Discussion ADA-Supported Research

A454 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES regulator of PBL response to TLR4 ligands through Fox01 phosphorylation. 1760-P The infl ammatory signaling signature expressed in PBL of type 2 diabetic Insulin Sensitivity and Clearance Determine Time to Post-Clamp patients suggests TLR4 dominance and lack or defective insulin signaling. Glycemic Stabilization in Nondiabetic Subjects The model demonstrates that the mechanism PBL employ to react to infl am- IBIYE OWEI, NIDHI JAIN, NKIRU UMEKWU, DAVID JONES, SAMUEL DAGOGO- matory stimuli, is associated with impaired transduction of the insulin signal JACK, Memphis, TN as found chronically in PBL of patients with type 2 diabetes and acutely in Measurement of insulin sensitivity with hyperinsulinemic euglycemic nondiabetic patients following cardiopulmonary bypass. clamp involves constant infusion of insulin along with variable dextrose (D20) in overnight fasted subjects. There is individual variation in the time 1758-P to post-clamp glycemic stabilization, the determinants of which are unclear. Plasma SerpinB1 Levels Are Strongly Correlated with Circulating Here, we tested the hypothesis that post-clamp glycemic stabilization ANGPTL8 Levels in Patients with Type 2 Diabetes is dependent on insulin clearance and insulin sensitivity. We studied 220 SHINSUKE TOKUMOTO, AKIHIRO HAMASAKI, YUKIKO KAWASAKI, SACHIKO healthy subjects (110 black {B} and 110 white {W}). Each subject underwent HONJO, YOSHIYUKI HAMAMOTO, Osaka, Japan anthropometry, oral glucose tolerance test (OGTT), and measurement of Background and Aims: SerpinB1, a protease inhibitor secreted by the plasma insulin and c-peptide levels. Insulin sensitivity was assessed using liver, has recently been described as a potent stimulator that increases beta the hyperinsulinemic clamp (ISI) and HOMA-IR. Insulin clearance was calcu- cell proliferation in mice and humans. While we previously reported that lated using the molar ratio of fasting C-peptide and insulin concentrations. ANGPTL8 correlated with insulin secretion capacity, the pathophysiological At the end of the clamp, insulin infusion was stopped and dextrose infusion role of SerpinB1 in patients with type 2 diabetes mellitus (T2DM) remains continued. Plasma glucose (PG) was determined every 10 min, with gradual poorly understood. The aim of the study was to evaluate the relationship weaning of D20 infusion. Glucose stabilization time (GST) was defi ned as the between plasma SerpinB1 levels and other biomarkers including ANGPTL8. interval between stopping insulin infusion and D20 infusion. The mean (±SD) Materials and Methods: Overnight fasting plasma samples from 8 healthy age of our cohort was 46.3±9.96 yr and BMI was 30.7±8.43kg/m2. The fi nal subjects and 29 T2DM patients were collected. HbA1c, fasting plasma glu- PG (mg/dl) at discharge was 124.2±26.9 for the entire cohort, 123.9±25.8 cose, total cholesterol, triacylglycerol, and creatinine clearance (CrCl) cal- (men), 124.3±27.4 (women), 122.1±26.2 (B) and 126.1±27.4 (W). There were no culated by 24-hour urine collection were measured in all T2DM patients. ethnic or gender differences in the fi nal PG level. The mean GST (min) was Plasma levels of serpinB1 (Cusabio, Catalogue No.CSB-EL021065HU) and 72.2±31.4 for all subjects, 77.7±34.8 (men), 69.7±29.6 (women), 73.3±33.2 (B) ANGPTL8 (Eiaab, Catalogue No.E11644h) were determined by enzyme-linked and 71.1±29.8 (W). The GST did not differ signifi cantly by race, gender or age. immunosorbent assay according to the manufacturer’s protocol. Correla- Using linear regression models, the signifi cant predictors of post-clamp GST tions were evaluated by Spearman’s rank test. P values <0.05 were consid- were 2 hr OGTT PG (p=0.0002), insulin clearance (p=0.02), ISI (p=0.03) and ered statistically signifi cant. HOMA-IR (p=0.01). We conclude that glucose stabilization following hyper- Results: Plasma serpinB1 levels were signifi cantly higher in T2DM insulinemic euglycemic clamp was not related to gender or ethnicity, but patients (0.37 ± 0.20 ng/ml) than in healthy subjects (0.21 ± 0.16 ng/ml; p < was signifi cantly delayed by lower glucose tolerance, insulin resistance, and 0.05). In T2DM patients, plasma serpinB1 levels showed a signifi cant posi- decreased insulin clearance. tive association with ANGPTL8 (r = 0.40 p = 0.02), and, both serpinB1 and Supported By: American Diabetes Association (7-07-MN-13 to S.D-J.); R01 ANGPTL8 levels signifi cantly correlated only with age (serpinB1: r = 0.42 DK067269, R01 DK067269-04S1 p = 0.02; ANGPTL8: r = 0.46 p = 0.01), duration of diabetes (serpinB1: r = 0.53 p = 0.003; ANGPTL8: r = 0.45 p = 0.01), and CrCl (serpinB1: r = −0.39 p = 0.03; 1761-P ANGPTL8: r = −0.45 p = 0.01). Characterization of the Cardiac Function of Human Insulin and the Conclusion: We showed that serpinB1 levels were higher in T2DM patients Long-Acting Insulin Analogs M1-glargine and Degludec in Adult Rat than in healthy subjects, suggesting that serpinB1 plays a role in regulation Ventricular Cardiomyocytes of beta cell mass in human. Since both serpinB1 and ANGPTL8 levels corre- PAULUS WOHLFART, THORSTEN M. HARTMANN, NINA WRONKOWITZ, JUER- lated with each other and with the same parameters, both proteins could be GEN ECKEL, NORBERT TENNAGELS, Frankfurt, Germany, Düsseldorf, Germany similarly regulated in T2DM patients. An important aspect for the long-term use of insulin analogues are car- diovascular effi cacy and safety data. Previously, we have shown that under 1759-P steady state conditions, insulin glargine (IGla) and its active metabolite M1 Model of the Glucose-Insulin-Glucagon Dynamics after Subcutane- (IGlaM1) as well as insulin degludec (IDeg) all elicit a comparable signaling ous Administration of a Glucagon Rescue Bolus in Healthy Humans and contractility response in different cardiomyocyte cell models Indeed, SABRINA L. WENDT, JAN K. MOELLER, AHMAD HAIDAR, BRITTA V. BYSTED, under steady state conditions all insulins displayed a similar AKT phospho- CARSTEN B. KNUDSEN, HENRIK MADSEN, JOHN B. JOERGENSEN, Glostrup, rylation and contractility of electrically paced ARVM after 15 min. However, Denmark, Kongens Lyngby, Denmark, Montreal, QC, Canada for IDeg a delayed onset of action could be observed. In the current study, In healthy individuals, insulin and glucagon work in a complex fashion to we investigated the early response of human insulin (HI), GlaM1 and IDeg in maintain blood glucose levels within a narrow range. This regulation is dis- adult rat cardiomyocytes (ARVM). In the early phase (1-5 min), only HI and torted in patients with diabetes. The hepatic glucose response due to an IGlaM1 were able to stimulate fractional shortening of total cardiomyocytes elevated glucagon level depends on the current insulin concentration and length in a comparable dose-dependent manner (1, 10, and 100 nM), with a POSTERS thus endogenous glucose production (EGP) can not be modelled without maximal shortening at 100 nM of 8.8 ± 1.1% and 7.3 ± 0.3%, respectively. Insulin Action/ In contrast, IDeg displayed no clear dose-response up to 1000 nM and a knowledge of the concentration of both hormones in plasma. Furthermore, Molecular Metabolism literature suggests an upper limit to EGP irrespective of glucagon levels. We maximum increase of only 3.6 ± 0.3% at 1000 nM. The difference might build a simulation model of the glucose-insulin-glucagon dynamics in man be explained by a different binding behavior of IDeg towards the cell mem- including saturation effect of EGP. Ten healthy subjects received a 1 mg sub- brane embedded insulin receptor (IR). The IC50 values observed in radioac- cutaneous (SC) glucagon bolus (GlucaGen®). Plasma samples were collected tive insulin displacement assays differs between detergent solubilized (S-IR) until 300 minutes post dose and analyzed for glucagon, insulin, and glucose and membrane embedded IR (M-IR) preparations (IC50 values [nM] S-IR vs. concentrations. All observations were used to fi t a physiological model of M-IR: 0.4/ 0.6 (HI), 0.7/ 0.9 (GlaM1), 2.3/ 18.2 (IDeg)), which could result in a the glucose-insulin-glucagon dynamics using the Hovorka model with a different association kinetic. In summary, the data demonstrated that IDeg, novel multiplicative description of the effects of insulin and of glucagon on and IGlaM1 show a different onset of action in AVRM most probably due to EGP. Bayesian estimation by Maximum a Posteriori using prior knowledge a different IR kinetic. However, under steady-state-conditions, a similar effi - reported in literature was used to estimate the model parameters for each cacy can be observed. Whether those differences might translate to in vivo subject. Profi le likelihood plots were used to investigate parameter identifi - conditions needs further investigation. ability. Unidentifi able parameters were fi xed at their prior mean values. The Supported By: Sanofi new model enables simulations of the glucose-insulin-glucagon dynamics in humans at both low and high glucagon concentrations (180-8000 pg/mL) and physiologic insulin concentrations (1.2-81.9 mIU/L). The model can be used for simulation of glucagon bolus strategies for treatment of hypoglycemia and for in silico simulation of dual-hormone artifi cial pancreas algorithms. Supported By: Zealand Pharma A/S

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A455 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

1762-P 1764-P Impaired Cardiomyocyte Autophagic Flux in Ischemic/Reperfused Circulating Apolipoprotein J Is a Novel Marker of Insulin Resist- Diabetic Heart: Critical Role of Hypoadiponectinemia ance YAJING WANG, BING LIANG, WAYNE LAU, BERNARD LOPEZ, THEODORE CHRIS- JI A. SEO, MIN-CHEOL KANG, SANG SOO KIM, SOO HYUN HONG, MICHELLE TOPHER, XINLIANG MA, Philadelphia, PA CHUNG, THEODORE P. CIARALDI, ROBERT R. HENRY, YOUNG-BUM KIM, Ansan, Autophagy is an important regulator of myocardial ischemia/reperfusion Republic of Korea, Boston, MA, La Jolla, CA, Del Mar, CA (MI/R) injury. However, whether and how autophagy is altered in I/R diabetic Insulin resistance is a major causal factor for developing type 2 diabetes. heart remains unknown. Adiponectin (APN) is a cardioprotective molecule. Apolipoprotein J (ApoJ, also called clusterin), a secreted sulfated glycopro- APN knockout (APNKO) markedly increases MI/R injury. However, whether tein, has been implicated in altered pathophysiologic states such as cardio- and how hypoadiponectinemia may alter cardiac autophagy in I/R diabetic vascular diseases, Alzheimer’s Disease, and cancer. However, the metabolic heart is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes function of ApoJ in glucose homeostasis remains unclear. This study sought (HFD), and APNKO mice, we demonstrated that autophagosome formation to determine whether serum ApoJ levels correlate with insulin resistance and was signifi cantly inhibited (P<0.01 vs. NC) and autophagosome clearance was if they change after an intervention that improves insulin sensitivity. Serum virtually abolished (P<0.01 vs. NC) in HFD heart subjected to MI/R. APNKO ApoJ, insulin resistance status using HOMA-IR and QUICKI were measured largely reproduced the phenotypic alterations occurring in HF-D heart, with in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosigli- both autophagosome formation and clearance inhibited after MI/R. Treat- tazone or Metformin treatment for 4 months on serum ApoJ levels were also ment of HFD and APKNO mice with AdipoRon (an APN receptor agonist) evaluated in T2D subjects. ApoJ protein was measured by immunoblotting. stimulated autophagosome formation, increased autophagosome clear- Fasting serum ApoJ levels were elevated in humans with type 2 diabetes ance, reduced infarct size, and improved cardiac function (P<0.01). Mecha- (T2D vs. ND; 141.3±9.3 vs. 100±9.1 AU, p=0.002). In ND subjects, insulin- nistically, AdipoRon caused signifi cant phosphorylation of AMPK, BeclinT119, resistant subjects had higher levels of serum ApoJ than insulin-sensitive and VPS34S164, and enhanced expression of lysosome protein LAMP2 both in subjects. Circulating ApoJ levels strongly correlated with fasting insulin, vivo and in isolated adult cardiomyocytes. Pharmacologic (Compound C) or HOMA-IR, QUICKI, and body mass index. Multiple regression analysis indi- genetic (dominant negative AMPKα2 overexpression) AMPK inhibition abol- cated that ApoJ levels were a signifi cant independent association factor for ished AdipoRon-induced BeclinT119/VPS34S164 phosphorylation and autopha- HOMA-IR even after adjustment for age, sex, and body mass index. Rosigli- gosome formation. However, AdipoRon-induced LAMP2 expression and tazone treatment of subjects with T2D resulted in a reduction in serum ApoJ autophagosome clearance were not affected by AMPK inhibition (P>0.05 levels (before vs. after treatment; 100.0±20.6 vs. 67.6±7.9 AU, p<0.001) but vs. vehicle or WT). Collectively, these results demonstrate for the fi rst time Metformin had no effect on ApoJ levels. In conclusion, serum ApoJ levels that hypoadiponectinemia impairs autophagic fl ux, contributing to enhanced are closely correlated with the magnitude of insulin resistance regardless of MI/R injury in diabetic individuals. Restoring APN signaling systems by APN obesity and decreases after insulin-sensitizer treatment in type 2 diabetes. receptor agonist activates APMK-mediated autophagosome formation and Thus, circulating ApoJ could be a novel marker of insulin resistance. AMPK-independent autophagosome clearance, representing a novel inter- Supported By: American Diabetes Association (7-12-BS-094 to Y-B.K.); U.S. vention against MI/R injury in diabetic conditions. Department of Veterans Affairs Supported By: American Diabetes Association (1-14-BS-218 to Y.W.) 1765-P 1763-P Uterus Remains Sensitive to Insulin Induced AKT Activation due Electrical Stimulation of Peripheral Sympathetic Nerve Fascicle to Impaired Inhibition through IRS-1 in a Lean, Hyperinsulinemic Enhances Non-Insulin-mediated Glucose Uptake in Rats Transgenic Mouse DAISUKE SATO, RIKU YAMAGUCHI, HIROYUKI SASAKI, MASATAKA KUSUNOKI, CLARE A. FLANNERY, CAITLIN RADFORD, FARRAH SALEH, DEREK LEROITH, ZHONGGANG FENG, TAKAO NAKAMURA, Yamagata, Japan, Nagoya, Japan, EMILY J. GALLAGHER, HUGH S. TAYLOR, New Haven, CT, New York, NY Yonezawa, Japan Type 2 diabetes is an independent risk factor for developing endometrial Regarding the possibility that the sympathetic nervous system could adenocarcinoma. We hypothesized that hyperinsulinemia promotes abnor- regulate peripheral glucose uptake, we reported that electrical stimulation mal endometrial proliferation, and sought to examine uterine insulin sensi- of peripheral sympathetic nerve fascicle (microstimulation), eliciting small tivity in vivo. We previously found endometrial hyperplasia occurring in the muscle contraction, induced transient blood glucose (BG) reduction within lean, euglycemic, but hyperinsulinemic MKR transgenic mouse, despite a 30 s of start of the microstimulation while plasma insulin (PI) changed little 60% reduction in mitogenic insulin receptor (IR-A) mRNA, relative to WT. in rats. In the present study, we evaluated the effects of microstimulation To determine uterine insulin sensitivity, we semi-quantifi ed phosphory- with no muscle contraction, and assessed the glucose uptake caused by the lated and total signaling proteins using western blot and densitometry from stimulation. extracted uteri of MKR (n=9) and WT (n=5) mice at 15 weeks of age. Each We microneurographically detected peripheral sympathetic nerve signal group was analyzed in the proliferative and secretory phases (PP, SP) by to locate the tip of microelectrode in a sympathetic fascicle in the rat sciatic 2-tailed t-test after log transformation. nerve, and then electrically stimulated the fascicle via the microelectrode We found Akt-ser473 phosphorylation was increased by 188% in PP (n=7). Based on our preceding study, the stimulation intensity was set at (p=0.02) and 244% in SP (p=0.05) of MKR, whereas p-MAPK-thr202/tyr204

POSTERS 0.05-0.10 V lower than muscle contraction threshold to enhance sympathetic was similar between MKR and WT. In all MKR, phosphorylation of inhibitory Insulin Action/ nerve activity. BG and PI levels were measured throughout the microstimula- IRS-1 site ser636/639 was absent in both phases, while phosphorylated in tion. Glucose uptake was assessed as glucose infusion rate (GIR) measured WT. P-IRS-1-ser318 was reduced by 66% in SP, vs. WT (p<0.05). Mean PTEN Molecular Metabolism before, during, and after the microstimulation with the euglycemic clamp were similar. method in additional rats (n=9). Here we isolated the endometrial effect of excess insulin from obesity As a result, the microstimulation transiently reduced BG (from 68 ± 4 to and unopposed estrogen in an in vivo model. Hyperinsulinemia did not induce 64 ± 3 mg/dL, p<0.01, mean ± SD) 30 s after start of the microstimulation resistance to insulin signaling in the uterus, as reported in muscle and adi- while PI changed little (from 2.0 ± 0.7 to 1.7 ± 0.7 ng/mL) as previously seen pose. Despite a protective reduction in mitogenic IR-A, the endometrium in the microstimulation accompanying muscle contraction. The microstimu- remains sensitive to insulin induced AKT activation due to impaired inhi- lation signifi cantly increased GIR from 12.9 ± 1.5 to 15.2 ± 1.5 mg/kg/min bition through IRS-1. In this setting, PTEN remains a protective barrier to (p<0.01). After the termination of the microstimulation, GIR was remained unopposed AKT activation. In women with type 2 diabetes, endogenous higher (14.7 ± 1.8 mg/kg/min) than that observed before the stimulation hyperinsulinemia may promote abnormal cellular proliferation, leaving (p<0.05) although GIR seemed to slightly decrease. the endometrium susceptible to unregulated growth if PTEN is disrupted These results indicate that the transient BG reduction induced with the through mutagenesis. Mutations of PTEN, rendering it inactive or defi cient, microstimulation might be attributed to enhancement of non-insulin-medi- are commonly found in endometrial adenocarcinoma. ated glucose uptake, and that the effect could remain even after terminating Supported By: Eunice Kennedy Shriver National Institute of Child Health and the microstimulation. Human Development (K08HD071010) Supported By: Japan Society for the Promotion of Science

For author disclosure information, see page A696. & Moderated Poster Discussion ADA-Supported Research

A456 INSULIN ACTION—SIGNAL TRANSDUCTION,CATEGORY INSULIN, AND OTHER HORMONES

1766-P exposing hepatocytes to sFRP4 impaired the insulin-mediated phosphory- Genetic Ablation of Interleukin-10 Receptor in Skeletal Muscle lation of Akt-Ser473 by 27% and its substrates glycogen synthase kinase Affects Glucose Metabolism in Diet-induced Obese Mice 3β-Ser9 (GSK3β-Ser9) by 27%, and Forkhead box proteins O1/3a (Fox01/3a)- SEZIN DAGDEVIREN, HYE-LIM NOH, HEE JOON KANG, SUCHAORN SAENGNI- Thr24/32 by 34% (all p<0.05). Although sFRP4 did not affect insulin-mediated PANTHKUL, RANDALL H. FRIEDLINE, XIAODI HU, KUNIKAZU INASHIMA, CAIT- phosphorylation of Akt-Ser473 and GSK3β-Ser9 in hSkMC, insulin-mediated LYN C. KEARNS, MARILIA L. LOUBATO, CECILIA P. UONG, JASON K. KIM, Worces- phosphorylation of Fox01/3a-Thr24/32 was reduced by 30% (p<0.05). In con- ter, MA clusion, increased expression of sFRP4 associates with insulin resistance, Skeletal muscle insulin resistance is a major characteristic of obesity and which may result from defects in the phosphorylation of the Forkhead tran- type 2 diabetes and is causally associated with infl ammation. We have pre- scription factors. viously shown that interleukin-10 (IL-10) improves muscle glucose metabo- Supported By: German Center for Diabetes Research lism partly by attenuating local infl ammation in obese mice. To further exam- ine the effects of IL-10, we generated mice with muscle specifi c deletion 1768-P of IL-10 receptor (MCK-IL10r KO) that developed without obvious anomaly Proteotoxicity Constrains the Structure of a Globular Protein: Insulin and showed normal glucose metabolism on chow diet. MCK-IL10r KO and as a Case Study wild-type (WT) mice were fed a high-fat diet (HFD) for 6 wks (n=6~11) and 16 NISCHAY K. REGE, FARAMARZ ISMAIL-BEIGI, NELSON B. PHILLIPS, JONATHAN wks (n=5~7), and insulin sensitivity was assessed using hyperinsulinemic- WHITTAKER, MICHAEL A. WEISS, Cleveland, OH euglycemic clamp in awake mice. Both groups of mice became obese after 6 Insulin is a peptide hormone that initiates the uptake of glucose from the or 16 wks of HFD (Figure 1). After 6 wks of HFD, basal glucose levels tended blood by peripheral tissues. The absolute or relative lack of insulin is the to be higher in MCK-IL10r KO mice (211±12 vs. 176±12 in WT mice; P=0.08). cause of type 1 or type 2 diabetes mellitus, respectively. Insulin’s evolution MCK-IL10r KO mice became more insulin resistant than WT mice after 6 and shows interplay between optimizing its ability to bind to its target, insulin 16 wks of HFD with signifi cant decreases in whole body glucose turnover receptor (IR), and maintaining a tertiary structure that confers resistance in these mice (Figure 2; P<0.05). This was mostly due to ~25% reductions in to proteotoxic aggregation: although insulin forms amyloid fi brils in phar- skeletal muscle glucose metabolism in HFD-fed MCK-IL10r KO mice as com- macological formulations, insulin derived amyloids are not seen in human pared to HFD-fed WT mice (Figures 3 and 4). These results indicate that IL-10 pathophysiology. Among insulin’s highly conserved structural motifs is a directly affects skeletal muscle insulin resistance, and our fi ndings identify type I β-turn spanning residues 20-23 of its B chain. We hypothesize that this a novel role of IL-10 receptor-mediated signaling in the regulation of muscle structural motif is conserved as a result of its role in protecting against amy- glucose metabolism. loid fi bril formation rather than its necessity for IR engagement. Our results Figure. indicate that although elimination of this turn through mutation of GlyB23 attenuates insulin’s biological activity, the substitution of PheB24 for glycine restores activity through the creation of a putative non-canonical turn. How- ever, these double mutant analogs show increased susceptibility to amyloid formation compared to wild type insulin. Double mutants also show dimin- ished ability to assemble into oligomers that protect the molecule from non- native aggregation. These results underscore potential proteotoxicity’s role as a driving force for protein evolution and its contribution to amyloid-based diseases and diseases of protein misfolding. Supported By: National Institutes of Health; National Institute of Diabetes and Digestive and Kidney Diseases

1769-P

WITHDRAWN

Supported By: National Institutes of Health (R01-DK080756, U24-DK093000, R24-DK090963)

1767-P Increased Expression of sFRP4 Associates with Insulin Resistance POSTERS Insulin Action/ in Liver and Skeletal Muscle

TINA HÖRBELT, DEIKE HESSE-WILTING, MARLIES BEKAERT, MAREN CARSTENSEN- Molecular Metabolism KIRBERG, FREDERIQUE VAN DE VELDE, YVES VAN NIEUWENHOVE, BRUNO LAPAUW, ANNETTE SCHÜRMANN, CHRISTIAN HERDER, MICHAEL RODEN, D. MARGRIET OUWENS, Düsseldorf, Germany, Potsdam, Germany, Ghent, Belgium Secreted frizzled-related protein 4 (sFRP4) is a regulator of the activity of the Wnt signaling pathway, which is released by multiple tissues including adipose tissue. Emerging evidence links increases in circulating sFRP4 levels to the development of β-cell dysfunction. Yet, it is currently unclear whether changes in the expression of sFRP4 in adipose tissue affect insulin sensitiv- ity in peripheral tissues, such as the liver and skeletal muscle. Therefore, we examined whether the expression of sFRP4 is changed in visceral adipose tissue (VAT) biopsies obtained from a cohort of morbidly obese men with (n=27) and without type 2 diabetes (n=39), and normal-weight men (n=15) undergoing abdominal surgery. Furthermore, we analyzed whether recom- binant sFRP4 affects insulin signaling in primary murine hepatocytes and primary human skeletal muscle cells (hSkMC). Levels of sFRP4 mRNA were 3.2-fold increased in VAT from morbidly obese men, independent of type 2 diabetes, vs. normal weight men (p<0.001), and associated positively with BMI (r=0.58, p<0.001) and HOMA-IR (r=0.38, p<0.001). A negative associa- tion was found with circulating adiponectin levels (r=-0.42, p<0.001). In vitro,

ADA-Supported Research & Moderated Poster Discussion For author disclosure information, see page A696.

A457 INTEGRATED PHYSIOLOGY—INSULINCATEGORY SECRETION IN VIVO

1770-P & 1772-P DNA Repair Enzyme OGG1 Regulates Obesity and Insulin Sensitiv- Association of a Type 1 Diabetes Genetic Risk Score with C-Peptide ity with Age and Age of Diagnosis in Type 1 Diabetes LARYSA YUZEFOVYCH, MYKHAILO RUCHKO, MICHELE SCHULER, LYUDMILA DELNAZ ROSHANDEL, RICHARD A. ORAM, ANDREW T. HATTERSLEY, MICHAEL RACHEK, Mobile, AL N. WEEDON, ROSE GUBITOSI-KLUG, SHELLEY B. BULL, ANDREW D. PATERSON, Oxidative stress and mitochondrial dysfunction in skeletal muscle are DCCT/EDIC RESEARCH GROUP, Toronto, ON, Canada, Exeter, United Kingdom, important factors leading to insulin resistance (IR) during the aging process Cleveland, OH but the underlying mechanisms are still unknown. Among the potential tar- A proportion of people with type 1 diabetes (T1D) have detectable C-pep- gets is mitochondrial DNA (mtDNA), since mtDNA is highly specialized and tide years after diagnosis: they maintain better glycemic control and experi- encodes for proteins essential for energy metabolism and, also, damage to ence lower risk for diabetic complications. Multiple loci have been identifi ed mtDNA heightens mitochondrial ROS (mtROS) production that is very criti- for risk of T1D, but it is unknown whether they or a T1D genetic risk score cal for IR. Despite extensive age-related studies on mtDNA mutations, until (GRS) are associated with C-peptide. recently, the integrity of mtDNA and repair mechanisms have received little The study population included 1303 Caucasian subjects from the Diabetes attention in diabetes research. We hypothesized that progressive oxidative Control and Complications Trial (DCCT), recruited in 2 cohorts: primary and mtDNA damage triggers development of age-associated oxidative stress in secondary with 1-5, and 1-15 yrs of diabetes, respectively. We calculated skeletal muscle, and thus, potentiates development of IR with age. To vali- a weighted T1D GRS of 30 SNPs of which 5 were in HLA. T1D GRS, a DR3/ date this hypothesis, we performed studies in vivo using DNA repair enzyme, DR4 GRS (2 SNPs) and individual SNPs (coded additively) were tested for 8-oxoguanine (8-OxoG) DNA glycosylase (Ogg1) knockout and overexpressing association with stimulated C-peptide after a standard meal measured at models: 1) Ogg1 -/- (KO); 2) Tg MTS-hOGG1 (Tg, mice overexpressing human DCCT baseline. Separate analyses of primary cohort (N = 651), secondary OGG1 (hOGG1, subunit 1-α) in mitochondria; 3) wild type (WT, ogg1 +/+). We cohort with duration 1-5 yrs (N = 135), and secondary cohort with duration evaluated obesity and IR phenotypes, oxidative mtDNA damage, oxidative 5-15 yrs (N = 517) were combined through meta-analysis using Tobit models stress and expression of several mitochondrial proteins in skeletal muscle for c-peptide adjusting for sex, age at diagnosis and duration. Similarly, T1D from young (~5 month old) and aging (~15 month old) males from all three OGG1 GRS, DR3/DR4 GRS and individual SNPs were tested for association with groups. Our results showed that mitochondrial hOGG1 overexpression amelio- age of diagnosis using linear regression. rated age-associated obesity and IR phenotypes and protected against oxi- T1D GRS and DR3/DR4 GRS were not associated with stimulated C-pep- dative mtDNA damage and oxidative stress. Additionally, protein content for tide. The risk alleles (T) of SNPs in HLA-A24 region (rs1264813, β (SE) = -0.3 transcriptional coactivator peroxisome proliferator activated receptor alpha (0.1), p = 3E-5) and Insulin gene (rs689, β (SE) = -0.2 (0.1), p = 0.01) were (PGC-1α) and mitochondrial protein porin was signifi cantly increased in skel- associated with lower stimulated C-peptide. T1D GRS (β (SE) = -0.8 (0.1), p = etal muscle from aging Tg mice. Collectively, these proof-of-concept results 3E-8), DR3/DR4 GRS (β (SE) = -0.5 (0.2), p = 6E-4) and the risk allele (T) of provide fi rst direct evidence that oxidative mtDNA damage triggers oxidative the non-HLA SNP rs5753037 (HORMAD2, β (SE) = -1.0 (0.3), p = 4E-4) were stress and development of age-associated IR in mice, thus making mtDNA all negatively associated with age of diagnosis, and exceeded Bonferroni damage and repair a novel target for treatment of IR and type 2 diabetes. correction (< 0.002). At all loci, there was no signifi cant heterogeneity of the effect among the 3 subgroups. We identifi ed risk alleles of 2 T1D susceptibility loci, HLA-A24 and INS, INTEGRATED PHYSIOLOGY—INSULIN SECRETION associated with lower C-peptide levels. T1D GRS, DR3/DR4 GRS and IN VIVO rs5753037 were associated with age of diagnosis. Supported By: National Institutes of Health (U01 DK094157, U01 DK094176, DP3 DK104438) Moderated Poster Discussion: Insulin Secretion in Animals and Humans (Posters: 1771-P to 1777-P), see page 14. & 1773-P Racial Differences in Early GIP Contribute to Postprandial Hyperin- & 1771-P sulinemia in Black Women: The Federal Women Study Insulin Receptor Knockout Using Two Beta-Cell-Specifi c Cre Mouse PAOLA C. ALDANA, AMBER COURVILLE, MARY WALTER, MICHELLE T. DUONG, Lines Transiently Improves Glucose Homeostasis BRIANNA A. BINGHAM, LILIAN S. MABUNDO, MADIA RICKS, JOON HA, ARTHUR SØS SKOVSØ, LYNDA ELGHAZI, DEREK A. DIONNE, MELISSA M. PAGE, HONG S. SHERMAN, ANNE E. SUMNER, STEPHANIE T. CHUNG, Bethesda, MD LI, DARIA HUTCHINSON, XIAOKE HU, FARNAZ TAGHIZADEH, ERNESTO BERNAL- Black women are more hyperinsulinemic than whites after exposure to MIZRACHI, JAMES D. JOHNSON, Vancouver, BC, Canada, Miami, FL intravenous glucose but the post-meal insulin response by race is unclear. Insulin signaling is important for glucose homeostasis and is disrupted in Since incretins mediate 50% of postprandial insulin secretion, we hypothe- type 2 diabetes. The insulin receptor is highly expressed in beta cells, where sized that black women would have higher insulin, active glucagon-like pep- its function remains to be fully established. Previous studies on mice gener- tide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) compared ated to delete insulin receptors (InsR f/f) from beta-cells reported impaired glu- to whites during a 2 h mixed meal test (MMT). To quantify insulin and incretin cose tolerance, reduced insulin secretion, and reduced beta-cell mass. How- response, early (0-30 min) and total (0-120 min) area under the curves were ever, the so-called BIRKO model employed Cre recombinase under the control calculated for the MMT (52% carbohydrate, 15% protein, 33% fat) in 36 fed- of a short fragment of the RIP, which led to deletion of the insulin receptor in erally employed women without diabetes (17 African-American, 6 African both beta-cells and the brain. We generated two new mouse models to re- immigrants, 13 whites; age 43±9 (mean±SD), range 25- 62 y; BMI 30.8±5.8, address the function of insulin receptors specifi cally in beta-cells. First, we range 20.5-45.3 kg/m2). Glucose tolerance status was previously determined crossed InsR f/f mice with a mouse line expressing Cre within the endogenous by OGTT and percent (%) body fat by DXA. Analyses of covariance were Ins1 gene locus (Ins1 Cre), as our group and others have shown little or no Ins1 used to examine insulin and incretin response by race accounting for glucose gene expression in the brain. At 25 weeks of age, female InsR f/f:Ins1 Cre mice and % fat. Glucose tolerance status did not differ by race. During the MMT, exhibited improved glucose tolerance relative to InsR wt/wt:Ins1 Cre littermate glucose concentrations did not differ by race (P≥0.50). After adjusting for % f/f Cre Obesity controls. Interestingly, InsR :Ins1 mice were also signifi cantly heavier fat, early insulin response was 50% higher in black vs. whites (insulin0-30min

POSTERS from 7-16 weeks of age. Second, we employed the tamoxifen-inducible Ins1- 1100±770 vs. 719±371, P=0.01) and total insulin response was 20% higher ERT Cre transgenic mouse model, which has been shown to have virtually no (insulin0-120min 8012±5645 vs. 6598±3305 µU·min/mL, P=0.10) but this did not f/f ERT Integrated Physiology/ recombination in the brain. Similarly, InsR :Ins1-Cre mice had signifi cantly reach signifi cance. Postprandial early and total GIP was higher in blacks vs. improved glucose tolerance 4 weeks after tamoxifen injection relative to both whites (GIP0-30min: 3468±3053 vs.1778±1003, GIP0-120min: 54159±22792 vs. InsR wt/wt:Ins1-CreERT and InsR f/f littermate controls, although this reversed to 39849±15582 pg·min/mL, both P<0.03). There were no differences by race impaired glucose homeostasis later in life. There were no signifi cant dif- in GLP-1 or C-peptide concentrations. Early GIP (P<0.001) and % fat (P<0.001) ferences in body weight in the tamoxifen-inducible model, suggesting that mediated 46% of the racial differences in early insulin response. Greater effects of beta-cell InsR deletion were restricted to developing and/or young early GIP was related to black race (P=0.03), % fat (P=0.04), and glucose0-30min mice. Collectively, these data are consistent with the concept that insulin (P<0.01). Overall, racial differences in postprandial insulin response were inhibits its own secretion, at least initially. Together, these data provide new identifi ed during a meal and may be partially explained by higher early GIP, information about the function of beta-cell insulin receptors and will provide not GLP-1, in black women relative to whites. more insight into the pathogenesis of type 2 diabetes. Supported By: National Institute of Diabetes and Digestive and Kidney Diseases Supported By: Canadian Institutes of Health Research

For author disclosure information, see page A696. & Moderated Poster Discussion ADA-Supported Research

A458