COMPLICATIONS—HYPOGLYCEMIA

COMPLICATIONS—HYPOGLYCEMIA AMP-activated protein kinase (AMPK) activity may be integral to cellular glucose-sensing. AMPK activity is suppressed after RH and the α2 catalytic [See also: Presidents Poster 379-PP, page A105.] subunit of AMPK is required for KATP-dependent GS in some hypothalamic neurons. Here we show that mouse hypothalamic GT1-7 cells (i) exhibit GS behavior in electrophysiological experiments, (ii) are hyperpolarized by Guided Audio Tour: Hypoglycemia, Glucose Variability, and Vascular Effects the SUR-1-selective KATP channel opener, NN414, (iii) are hyperpolarized (Posters 478-P to 485-P), see page 15. by the GK inhibitor, alloxan. Moreover, lowering the extracellular glucose concentration from 2.5 mM induced K opening in a concentration-

ATP POSTERS & 478-P dependent manner, which was reversed by the KATP blocker, tolbutamide Complications The Effect of Glucose Variability on the Rate of Hypoglycemia Events or the GK activator, GKA50. Finally, lentiviral delivery of shRNA directed Acute and Chronic YONGMING QU, SCOTT JACOBER, QIANYI ZHANG, LINDA WOLKA, J. HANS to α2AMPK reduced GT1-7 cell α2AMPK protein levels, which resulted in DEVRIES, Indianapolis, IN, Amsterdam, The Netherlands the loss of GS behavior with exposure of cells to 0.5 mM glucose no longer The goal of diabetes treatment is to control blood glucose (BG) while causing cell hyperpolarization. Inhibition of UCP2 with genipin mimicked the limiting hypoglycemia. It has been hypothesized that high glucose variability phenotype of α2AMPK knockdown cells, suggesting that UCP2 activity is (GV) leads to more frequent hypoglycemia. Our aim was to evaluate the required for GS. In conclusion, the GT1-7 cell line closely mimics classical effect of GV on the rate of hypoglycemia. GS behavior in so far as electrical activity is regulated by glucose and the This post-hoc analysis included T2D patient data from three 24-week glucose signal can be modulated through manipulation of GK and SUR- insulin trials including patients on twice-daily (BID) insulin lispro mixtures 1 KATP channels. GT1-7 cells may thus provide a unique model cell line for (n=805), daily (QD) insulin glargine (GL, n=1,019), insulin lispro protamine investigating GS mechanisms. Furthermore, suppression of α2AMPK activity suspension (ILPS, n=353) (QD or BID), and insulin detemir (DE, n=166) (QD or in these cells blocks GS behavior. These data suggest that suppression BID), all with continuation of prestudy oral antihyperglycemic medications. of hypothalamic α2AMPK activity inhibits normal GS behavior and may At baseline, mean (±SD) age was 56.9±9.7 years, duration of diabetes was contribute to defective hypoglycemia detection. 9.5±6.1 years, A1C was 8.9±1.1%, and 51.9% were male. We studied the Supported by: JDRF, Anonymous Trust relationship between hypoglycemia rate (between 12 and 24 weeks) and the following 12 variables regarding intra-day and inter-day GV and mean BG: & 480-P daily mean BG, fasting BG (FBG), A1C, standard deviation (SD) and coeffi cient Acute Effects of Hypoglycemia and Hyperglycemia on Pro-Athero- of variation (CV) of intra-day BG, intra-day minimum BG, intra-day difference thrombotic Risk in Non-sDiabetic Humans between minimum and maximum BG, average inter-day SD and CV, Mean NINO GOGITIDZE JOY, JENNIFER PERKINS, ANTOINETTE RICHARDSON, MAKA Amplitude of Glycemic Excursions (MAGE), Mean Absolute Glucose change HEDRINGTON, LISA YOUNK, IAN DAVIS, DONNA TATE, STEPHEN N. DAVIS, Balti- (MAG), and Average Daily Risk Range (ADRR), all derived from 7-point self- more, MD, Chapel Hill, NC, Nashville, TN monitored BG profi les taken at 24 weeks. A Generalized Boosted Model The comparative effects of acute moderate hyperglycemia and hypo- (GBM) was used to evaluate the relative importance of these variables and glycemia on vascular biologic mechanisms have not been determined. To select variables with p-value <0.05. investigate this question 45 individuals (21M/24F, 38±3yrs, BMI 29±2kgm2, The Figure shows the relative infl uence on the hypoglycemia rate (%) and HbA1C 5.2±0.2%) participated in four single blind glucose clamp studies. the p-values. The intra-day BG CV, FBG, intra-day minimum BG and inter-day Protocols P1 and P2 consisted of 4hrs euinsulinemic/hyperglycemia (200mg/ BG CV were signifi cantly correlated with the rate of hypoglycemia events. dl) or hyperinsulinemic/hyperglycemia respectively (both with pancreatic We conclude that intra-day and inter-day GV are related to hypoglycemia clamp). P3 and P4 consisted of 2hr hyperinsulinemic/euglycemic (89mg/ events even after adjusting for mean BG and A1C. dL) or hyperinsulinemic/hypoglycemic (51mg/dL) clamps respectively. Two D doppler ultrasound was used to determine brachial artery endothelial function. All end of clamp measurements were performed at the same time of day. Insulin levels during P1 were 26±3 μU/mL. Insulin levels during all hyperinsulinemia protocols (P2, P3, P4) were similar (139±13μU/mL). Results from fi nal 30 minutes of glucose clamps are shown in table 1. Table 1. Δ Responses from Baseline to fi nal 30 minutes of clamps. VCAM-1 PAI-1 P-selectin NO-mediated Vasodilation (ng/ml) (ng/ml) (pg/ml) (Mean Max % change) P1 (euins/hypergly) 20.8+14.2≠ -0.3±1.6≠ 16.7±7.4≠ -5.2± 1.3≠ P2 (hyperins/hypergly) -61.3±36.3 -12.0±4.7 -7.8±10.8 0.3 ± 1.2 P3 (hyperins/eugly) -88.9±30.6 -1.5±1.4 -13.1±10.3 0.9 ± 0.8 P4 (hyperins/hypogly) 86.6±24.6* 8.3±3.5* 42.1±9.6* -3.9 ± 1.0≠ * p<0.05 Signifi cantly different from all protocols ≠ p<0.05 Signifi cantly different from protocols P2 and P3 In summary, acute hypoglycemia had a greater effect on reducing Supported by: Eli Lilly and Company fi brinolytic balance ( ↑PAI-1), increasing adhesion molecules (VCAM-1) and platelet activation (P-Selectin) as compared to hyperglycemia. However, & 479-P hypoglycemia had similar adverse effects on endothelial function as Hypothalamic Glucose-Excited Neurons Require α2AMPK for compared to hyperglycemia. Hyperinsulinemia protected the vasculature Glucose Sensing against hyperglycemia but not hypoglycemia. CRAIG BEALL, LEE HAMILTON, JENNIFER GALLAGHER, FIONA ASHFORD, KATH- In conclusion, moderate hypoglycemia caused equivalently impaired RYN WRIGHT, MARC SOUTAR, MIKE L. ASHFORD, RORY J. MCCRIMMON, endothelial function, but signifi cantly increased pro-thrombotic and pro- Dundee, United Kingdom infl ammatory effects as compared to hyperglycemia. Thus the combined Hypothalamic glucose-excited (GE) neurons are postulated to contribute effects of hypoglycemia appear to result in a greater pro-atherothrombotic to whole body glucose homeostasis and participate in the detection risk as compared to acute hyperglycemia. of hypoglycemia. However, the glucose sensing (GS) mechanism is Supported by: NIH incompletely understood and may be defective in type 1 diabetes where frequent hypoglycemia occurs (recurrent hypoglycemia (RH)). Classical and probably defi ning features of glucose-sensing cells are the presence of glucokinase (GK) and the sulphonylurea receptor 1 (SUR1)-containing ATP- + sensitive K channel (KATP). Recent work has also shown that hypothalamic

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A133 COMPLICATIONS—HYPOGLYCEMIA

origin of the astrocytic response. These data suggest that NST astrocytes are & 481-P intrinsically glucosensitive and are capable of actively signaling in response Effects of Hypoglycemia on Endothelial Function and Athero throm- to low glucose availability. This active signaling may play a pivotal role in the botic Balance in Type 2 Diabetes (T2DM) central detection of hypoglycemia and the instigation of the CRR. NINO GOGITIDZE JOY, MAIA MIKELADZE, MAKA S. HEDRINGTON, LISA YOUNK, Supported by: NIH Grants: NS52142, DK56373, NS55866, and NS60664 LINDSAY PULLIAM, IAN DAVIS, DONNA B. TATE, STEPHEN N. DAVIS, Baltimore, MD Recent randomized clinical trials have reported an association between & 483-P hypoglycemia and cardiovascular adverse events. The physiologic mecha-

POSTERS Acute Insulin-Induced Hypoglycemia Induces a Rapid Increase in

Complications nisms responsible for these fi ndings have not been extensively studied. Hypothalamic Synaptic Plasticity Acute and Chronic Therefore, the aim of this study was to test the hypothesis that 2 hrs of RAIMUND I. HERZOG, TUKIET T. LAM, ROBERT S. SHERWIN, New Haven, CT moderate hypoglycemia acutely impairs vascular biologic mechanisms in Intensive insulin therapy increases the risk of hypoglycemia, which leads T2DM. to brain energy defi cit and potentially signifi cant brain injury. Eleven T2DM (2M/9F) (45±4 yrs, BMI 38±3kg/m2, HbA1c 8±1%) participated How this acute metabolic challenge alters proteins and their activation in single 2 day studies. Day 1 consisted of a 2 hour hyperinsulinemic/ state within the hypothalamus, a brain region that plays a key integrative euglycemic (eugly) clamp (89±1mg/dL) and day 2 a 2hr hyperinsulinemic/ role in the response to hypoglycemia has received little attention. Such hypoglycemic (hypo) clamp (58±0.3mg/dL). Insulin levels during clamps were data might contribute to a better understanding of the mechanisms involved similar (126±2 U/mL). Two D doppler ultrasound was used to determine μ in limiting brain injury. We analyzed the hypothalamic phospho- and non- endothelial function. phospho proteome of the hypothalamus of non-diabetic rats undergoing an End of clamp (fi nal 30 minutes) responses of ICAM-1, VCAM-1, P-Selectin, acute 2 hr episode of insulin-induced (10mU/kg i.p.) hypoglycemia (plasma PAI-1 (Plasminogen activator inhibitor 1) and VEGF (Vascular endothelial glucose ∼45mg/dl). Mass spectrometry based phospho-proteome analysis growth factor) were all increased (p<0.05) during hypo as compared to eugly. allows the simultaneous observation of a vast number of phosphorylation Nitric Oxide (NO) and non-NO mediated vasodilation were both impaired events on different proteins at once. Following protein extraction and (p<0.05) during hypo. Glu-C trypsin enzymatic digestion the resulting peptide samples were See results in Table 1. enriched with TiO2 for phospho-peptides. These were analyzed using four Table 1: Δ Responses from baseline to fi nal 30 minutes of clamps injections on a nano-UPLC LTQ-Orbitrap tandem mass spectrometry system. Eugly Hypo Proteins were identifi ed using the MASCOT search algorithm and relative ICAM-1 (ng/ml) -9+8 13+4* expression ratios determined via a label free approach using the Progenesis LC-MS software suite. Protein hits that reached a statistically signifi cant VCAM-1 (ng/ml) -25+37 100+32* ANOVA of p<0.05 were considered for further GO-category analysis. P-Selectin (pg/ml) -6+37 25+9* Among proteins signifi cantly down regulated following hypoglycemia, those PAI-1 (ng/ml) -11.5+3 -2.7+1.5* belonging to the categories GO:0031110 (negative regulation of microtubule polymerization or depolymerization) and GO:0051494 (negative regulation VEGF (pg/ml) -5+9 15+9≠ of cytoskeleton organization) were overrepresented. Conversely, phospho NO-mediated vasodilation 1.3+0.5 -3+0.9* proteins upregulated with acute hypoglycemia included those belonging to non NO-mediated vasodilation 1.9+1 -3.1+1.3* GO:0051128 (regulation of cellular component organization), GO:0043005 * p <0.05 signifi cantly different from euglycemia. (neuron projection) and GO:0051726 (regulation of cell cycle). We conclude ≠p=0.05 different to euglycemia that acute hypoglycemia increases plasticity in the rat hypothalamus In summary, moderate hypo increased vascular adhesion molecules (ICAM- consistent with neuronal and synaptic re-organization in response to the 1, VCAM-1), markers of angiogenesis (VEGF), platelet activation (P-Selectin) metabolic challenge. This rapid adaptation may maximize the functional and decreased fi brinolytic balance ( ↑PAI-1) in T2DM. Additionally, hypo capacity of the hypothalamus to limit brain injury. acutely impaired endothelial function via both NO and non NO mechanisms. Supported by: Yale DERC Pilot and YCCI Grant KL2 RR-024138 We conclude that acute hypoglycemia can result in a spectrum of deleterious vascular effects including impaired endothelial function and activation of & 484-P prothrombotic, pro-infl ammatory and pro-atherogenic mechanisms in T2DM. Recurrent Hypoglycemia Selectively Reduces Adrenal Cate chola- Supported by: National Institute of Health mine Release Evoked by Muscarinic Acetylcholine Receptors BRANLY O. ORBAN, VANESSA H. ROUTH, BARRY E. LEVIN, JOSHUA R. BERLIN, & 482-P Newark, NJ Astrocytes in the Nucleus of the Solitary Tract Actively Signal during A serious complication in the treatment of diabetes is reduced release Low Glucose Availability of counter-regulatory (CR) hormones, such as epinephrine (Epi), in response DAVID H. MCDOUGAL, GERLINDA E. HERMANN, RICHARD C. ROGERS, Baton to iatrogenic hypoglycemia. Recurrent hypoglycemia alone blunts the Epi Rouge, LA response to insulin-induced hypoglycemia (Hypo). Adaptations in brain Hypoglycemia triggers a series of physiological adjustments collectively regions regulating sympathetic outfl ow contribute to reduced CR Epi known as the counter-regulatory response (CRR) to return serum glucose to release. Peripheral sympathetic nerve function has also been studied, but euglycemic levels. The CRR of insulin-dependent diabetics becomes aberrant whether recurrent hypoglycemia directly affects adrenal medulla function and potentially life-threatening after their fi rst exposure to hypoglycemia. is unknown. In this study, Hypo was induced by sc insulin (1U/kg) in 8-week Understanding this impaired refl ex is hindered by not knowing the cellular and old male Sprague Dawley rats once a day for 3 days (Ins). Control rats (Con) molecular basis of hypoglycemic detection in the normal condition. Recent received sc saline injections. On day 4, Hypo was induced in both groups work suggests that brainstem astrocytes may be important chemodetectors while measuring plasma catecholamine (Cat) levels. On day 5, left adrenal for autonomic control and they have also been shown to contribute to the glands were isolated and perfused to determine Cat release rate evoked initiation of the CRR. Therefore, these experiments were designed to see by acetylcholine (Ach) as well as by muscarinic and nicotinic Ach receptor if brainstem astrocytes actively signal during conditions of low glucose agonists, pilocarpine (Pilo) and nicotine (Nic), respectively. Release rates availability. In vitro, confocal live cell imaging of acute brainstem slices from were also measured in adrenal glands isolated from rats not exposed to adult Long Evans rats was used to quantify cellular activation of astrocytes Hypo (Naïve). Cat content was measured in the right adrenal glands. and neurons in the nucleus of the solitary tract (NST) evoked by these During day 4 Hypo, the time course of glucose decline and minimum challenges. Changes in intracellular calcium levels refl ected the activation of plasma glucose levels in Con and Ins groups were similar. However, the pre-labeled astrocytes and neurons. Slices were exposed to a 10min interval peak Epi response was signifi cantly reduced in the Ins group. After isolating of low glucose or glucoprivic challenge [0.5mM glucose, 1.0mM glucose adrenal glands on day 5, maximum Cat release rate evoked by Ach (10µM or 5mM 2-deoxyglucose (2-DG)]. These conditions evoked four distinct to 30mM), Ach concentration producing half-maximal release rate, release response patterns in both NST astrocytes and neurons. The most prevalent rate evoked by 1 mM Nic and Cat content were similar in Con, Ins and response pattern in both cell types was an increase in intracellular [Ca2+] Naïve groups. However, Cat release rate in response to 10mM Pilo was (i.e., activation). Activated astrocytes had signifi cantly larger responses and signifi cantly reduced in the Ins, but not the Con group when compared to the shorter latencies to respond relative to the NST neurons. Additionally, the Naïve group (p<0.05). Our results show that recurrent hypoglycemia did not astrocytic activation induced by low extracellular glucose was maintained change glucose levels during Hypo, though CR Epi release was blunted, as even in the presence of 1μM TTX, eliminating the possibility of a neuronal previously reported. We also observed a selective reduction in muscarinic

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A134 COMPLICATIONS—HYPOGLYCEMIA

Ach receptor mediated adrenal Cat release rate. These results suggest provide a therapeutic opportunity to sustain catecholamine biosynthetic that recurrent Hypo could impair intrinsic adrenal medullary Epi release in levels commensurate with release. addition to blunting central stimulation of sympathetic outfl ow. 487-P & 485-P Early Alzheimer-Type Dementia and Type 2 Diabetes: Analysis Using Hypothalamic S-nitrosylation Contributes to the Impairment of the Voxel-Based Specifi c Regional Analysis System for Alzheimer’s Counter-Regulatory Response Following Recurrent Hypoglycemia Disease (VSRAD)

XAVIER FIORAMONTI, ADAM DEAK, SRINIDHI DESHPANDE, CORINNE LELOUP, MAMI YOSHIDA, AKIO SAEKI, YASUKI FUKUDA, TAKESHI KUZUYA, NAKAAKI POSTERS LUC PENICAUD, ANNIE BEUVE, VANESSA ROUTH, Newark, NJ, Dijon, France OHSAWA, SHIGERU YOSHIDA, SHIGEAKI SATO, TOSHIAKI HNAFUSA, Ibaraki, Complications Intensive insulin therapy improves diabetic complications but increases Japan, Ohtu, Japan, Takatuki, Japan Acute and Chronic hypoglycemia risk. Recurrent hypoglycemia (RH) impairs the counter- [Purpose] Diabetes is known to be a risk factor for Alzheimer’s disease regulatory response (CRR) which restores euglycemia. The CRR requires (AD). Hyperglycemia itself and insulin resistance is suggested to be the ventromedial hypothalamus (VMH) nitric oxide (NO) production. However, underlying mechanism. Voxel-based specifi c regional analysis system for when NO is produced in the presence of reactive oxygen species (ROS) Alzheimer’s disease (VSRAD) is the software of automatic image processing protein S-nitrosylation occurs and desensitizes NO signaling. Hypoglycemia and statistical analysis of the degree of parahippocampal cortex atrophy and increases VMH ROS levels (149 ± 22%, n=7). We hypothesize that during is widely used for early diagnosis of AD in Japan. The purpose of this study hypoglycemia, ROS increases VMH S-nitrosylation and impairs the CRR. In is to assess the factor related with the development of early AD in patients support of this, we found that 3 consecutive daily episodes of RH increase with diabetes using VSRAD. VMH S-nitrosylation. We then determined whether decreased ROS production [Subjects and Methods] We studied 181 out-patients with type 2 diabetes prevents the impaired CRR after RH by treating rats with the antioxidant over 60 years old (Age 69.0±9.3 years, male 87/female 94, HbA1c 6.0±1.2 N-acetyl cysteine (NAC) in their drinking water (5 g/l) for 9 days before and %). The patients were divided into four groups according to the degree of during RH. After RH, glucose levels fell further and glucagon production was atrophy with VSRAD, 0-1: little, 1-2: moderate, 2-3: considerable, 3-: marked inhibited in response to insulin-hypoglycemia (glucose nadir RH: 39.3 ± 1.1 atrophy. BMI, insulin resistance, glucose/lipid metabolism, and diabetic mg/dl; controls: 53.5 ± 1.8 mg/dl - glucagon nadir RH: 115 ± 13 ng/l; control: complications were also assessed in the patients. 244 ± 25 ng/l; n=13; p<0.05). Interestingly, after NAC pre-treatment there [Results] The numbers of patients in each group were 0-1:81, 1-2:60, 2-3:19, were no signifi cant differences in glucose nadir or glucagon levels between 3-:21. Compared with patients with little atrophy, patients in the groups with animals exposed to RH and saline controls (glucose nadir RH + NAC: 39.6 ± the atrophy degree of 1 or higher, showed signifi cantly older age (71.8±8.2 vs 3 mg/dl; controls + NAC: 35 ± 3 mg/dl - glucagon nadir RH + NAC: 288 ± 21 64.9±8.8 years old ,p=0.012), elevated BMI (25.5±3.6 vs 23.6±3.6,p=0.022), ng/l; controls + NAC: 333 ± 21 ng/l; n=8; p>0.05). Thus, NAC pretreatment higher 2-hour-postprandial plasma glucose (221±69 vs 179±46mg/dl, prevented the impaired CRR. NAC pre-treatment also prevented increased p=0.0061), and increased HOMA-R (3.2±4.0 vs 1.25±1.25,p=0.0037). No VMH S-nitrosylation after RH. Finally, we performed a hyperinsulinemic/ signifi cant difference was seen between the two groups in the duration hypoglycemic after third ventricular injection of the nitrosylating agent of diabetes, HbA1c, fasting plasma glucose, blood pressure, LDL, HDL, S-nitro-L-cysteine (CSNO, 0.5 mM, 5μl, 0.5 μl/min). The glucose infusion rate triglyceride, creatinin, and uric acid level. Patients in the groups of more necessary to maintain the hypoglycemic plateau during the last 30 minutes severe atrophy had signifi cantly increased incidence of hypoglycemia (more of the clamp was higher in CSNO injected animals (control: 9.21 ± 0.5 mg/kg/ than three times / month) and higher prevalence of visceral obesity (p<0.05). min vs CSNO: 11.02 ± 0.4 mg/kg/min; p<0.05). Thus, S-nitrosylation impairs No signifi cant relation was seen between the atrophy and the presence the CRR. Our study suggests that NAC prevention of VMH S-nitrosylation of diabetic retinopathy / nephropathy / neuropathy, cerebrovascular may be useful in preventing the impaired CRR after RH. disease, coronary artery disease, arteriosclerosis obliterans, hypertension, Supported by: DK81358 (VHR), JDRF (XF) dyslipidemia, and the smoking. [Conclusion] Hypoglycemia and insulin resistance were suggested to be 486-P related with the development of early AD in patients with diabetes. A Molecular Explanation of Hypoglycemia-Associated Autonomic Failure (HAAF): Downregulation of Adrenal Medullary Epinephrine 488-P (Epi) Biosynthesis and Release EphA5 Receptors in the Ventromedial Hypothalamus (VMH) Modu- NECLA KIRTOK, BISTRA NANKOVA, OWEN CHAN, EDMUND F. LA GAMMA, late Insulin and Glucagon Secretion Valhalla, NY, New Haven, CT BARBARA SZEPIETOWSKA, WANLING ZHU, ADAM HORBLITT, ROBERT S. Even in healthy adults, recurrent hypoglycemia (RH) alone can cause SHERWIN, New Haven, CT counter regulatory (CR) failure and hypoglycemia-unawareness. We Interactions between EphA5 receptors and ephrinA5 ligands in brain hypothesize that progressive loss of the Epi response in HAAF arises modulate neuron/glia interactions and synaptic plasticity, whereas in from a depletion of the releasable pool of adrenal Epi due to biosynthesis pancreatic islets activation of EphA5 receptors by ephrinA5 ligands suppress lagging behind release. Despite the undisputed role of hypoglycemia insulin secretion. We have previously reported that local activation of VMH per se, the mechanisms underlying HAAF remain to be fully elucidated. EphA5 receptors by ephrinA5-Fc exogenous ligand increases glucagon We tested the hypothesis that loss of the sympathoadrenal response in responses to hypoglycemia in normal rats. To examine whether VMH EphA5 HAAF may stem from down regulation of the rate limiting enzyme in Epi receptor expression might alter glucagon and insulin responses to changes biosynthesis, tyrosine hydroxylase (TH), in the adrenal gland. To address in glucose, we microinjected into the VMH of male Sprague-Dawley rats this question, we induced HAAF in normal Sprague-Dawley rats using adeno-associated virus containing a shRNA (or control sequence) to diminish once or twice daily intraperitoneal injections of insulin (2U/kg) or saline VMH EphA5 receptors. Subsequently, animals (5-8 per group) underwent (control animals) for 3 consecutive days. Then, following an overnight fast, hyperinsulinemic (20mU/kg/min)-hypoglycemic (50mg/dl) or hyperglycemic the animals were subjected to a hyperinsulinemic-hypoglycemic glucose (220 mg/dl) clamp studies. Down-regulation of VMH EphA5 receptors clamp on day 4 to evaluate CR responses. Five hours after the start of the increased the glucose infusion rate required to maintain hypoglycemia and insulin infusion, the animals were sacrifi ced and adrenal medullae were suppressed both glucagon [213 ± 54 pg/ml vs. 322 ± 34 in controls, p< 0.01] collected for quantifi cation of TH mRNA levels using northern blots. This and epinephrine release [2347 ± 633 pg/ml vs. 4567 ± 909 in controls, p< 0.05] was used as a surrogate marker of catecholamine biosynthetic capacity. during hypoglycemia. Furthermore, knockdown of VMH EphA5 receptors Our data shows that single daily RH via low dose insulin injection does increased glucose-stimulated insulin secretion 2-fold (p<0.01) as well the not induce HAAF. This is in contrast to current models of HAAF that use glucose infusion rate required during the hyperglycemic clamp by 50%. A excessively high doses of insulin (10U/kg) where even one injection per day modest, but insignifi cant increase in body weight was seen 6 weeks after for 3d can produce HAAF. This suggests that the dose of insulin may have knockdown of VMH EphA5 (p>0.10). We conclude that expression of EphA5 an important role in HAAF. On the other hand, twice daily injections of low receptors in the VMH, and in turn local neuron/glia interactions within this dose insulin can lead to signifi cant impairments in both the glucagon and Epi critical glucose sensing brain region may infl uence pancreatic islet function responses. More importantly, we identifi ed the novel fi nding that loss of the during changes in systemic glucose levels. These data imply that a neural Epi response in this latter group was associated with suppressed induction circuit linking the VMH and the islet may modulate glucose homeostasis. of adrenomedullary TH mRNA. We speculate that initial stimulation of the Supported by: JDRF adrenals induces, while more frequent stimulation suppresses the induction of TH mRNA. This suggests that attenuation of signal-transduction may

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A135 COMPLICATIONS—HYPOGLYCEMIA

489-P Evidence That Islet Sympathetic Nerves (ISN) and Islet Beta Cells Are Destroyed Via Different Mechanisms in Type 1 Diabetes (T1D) QI MEI, DARYL HACKNEY, HO CHANG, KARIN BORNFELDT, GERALD J. TABORSKY, JR., Seattle, WA Activation of ISN and suppression of islet beta cells both contribute to the glucagon response to insulin-induced hypoglycemia. In autoimmune

POSTERS T1D, most ISN and islet beta cells are lost, coincident with an early, marked

Complications impairment of this glucagon response. To better understand the cause of this Acute and Chronic glucagon impairment, we sought to determine if the mechanism underlying ISN loss is different from that causing beta cell loss. We used a transgenic mouse model of T1D in which a viral glycoprotein (GP) of the lymphocytic choriomeningitis virus (LCMV) is driven by the rat insulin promoter (RIP); therefore this viral GP is expressed solely in islet beta cells. Systemic LCMV injection caused a marked loss of both islet beta cells (glucose = 523 ± 40 mg/dl) and ISN (no LCMV = 104 ±17 µm2 /islet vs LCMV = 19 ± 4 µm2 /islet; Δ = -82%; p < 0.002; both n=3). This beta cell loss is caused by GP-specifi c T-lymphocytes. Since ISN do not express this viral GP, the loss of ISN must be due to a different mechanism. It is known that binding of brain derived neurotrophic factor (BDNF) to the 491-P p75 neurotrophin receptor (p75NTR) prunes redundant sympathetic axons Multiple Daily Insulin (MDI) and Continuous Subcutaneous Insulin during target innervation. Since activated B lymphocytes release BDNF and Infusion (CSII) Are Associated with Lower Risk of Death among since they invade the T1D islet, we hypothesized that, in T1D, the p75NTR on Patients Reporting Severe Hypoglycemia ISN is activated and causes the loss of ISN. ROZALINA GRUBINA, NILAY D. SHAH, HOLLY K. VAN HOUTEN, ROBERT A. To test this hypothesis we crossed p75NTR knockout mice with RIP-GP WERMERS, STEVEN A. SMITH, Rochester, MN mice and then injected them with LCMV. ISN area (62 ± µm2/islet; n=3) was Most patients with diabetes ultimately require insulin therapy, yet this 3-fold greater in mice lacking p75NTR than in controls (19 ± 4 µm2/islet; n=3; causes signifi cant anxiety, particularly among patients with history of p<0.001). Thus, knocking out p75NTR in RIP-GP mice signifi cantly preserves severe hypoglycemia (SH). The ADA recommends modifying treatment ISN, despite equivalent diabetes (523 ± 45 mg/dl), again suggesting a goals and regimens if SH occurs. Multiple daily insulin (MDI) and continuous different mechanism. subcutaneous insulin infusion (CSII) improve glycemic control and lower risk We conclude that the mechanism causing the loss of ISN in T1D is distinct, of microvascular complications, but are associated with higher risk of SH. at least in part, from that causing the loss of islet beta cells. Further, p75NTR Whether patients with history of SH should avoid MDI-CSII is unknown. might be a therapeutic target for protecting ISN and thereby mitigating the All patients (n=1130) seen in a specialty diabetes clinic between August impairment of the glucagon response to insulin-induced hypoglycemia in 2005 and July 2006 were questioned by their healthcare providers (n=31) T1D. about presence of SH during the preceding 6 months. Surviving patients Supported by: NIH/NIDDK and VA were reassessed by postal survey in quarter 1 of 2010, querying their glycemic control: oral agent(s), provider-prescribed insulin (PPI), bolus/basal/ 490-P sliding scale MDI or CSII, and/or non-insulin injectables. Information about Lack of Association between Residual Insulin Production and Gluca- non-surviving patients was obtained from the medical record. Demographics gon Response to Hypoglycemia in Youth with Short Duration of Type were compared using the two sample t-test, and risk of death is expressed 1 Diabetes as risk ratio [RR; 95% confi dence interval). JENNIFER SHERR, WILLIAM TAMBORLANE, EVA TSALIKIAN, LARRY FOX, BRUCE At enrollment, 79 of 1130 patients (7%) reported SH; of these, 62 (78.5%) BUCKINGHAM, STUART WEINZIMER, NEIL WHITE, ANA MARIA ARBELAEZ, were alive at time of reassessment. Of the 79 patients, 4 (5%) used oral DONGYUAN ZING, KATRINA RUEDY, CRAIG KOLLMAN, ROY BECK, DIRECNET, agent(s), 16 (20%) PPI, 32 (41%) MDI-CSII, and 1 (3%) non-insulin injectables. New Haven, CT, Iowa City, IA, Jacksonville, FL, Stanford, CA, St. Louis, MO, Tampa, FL Survivors were younger than those who had died (54 ± 16 vs. 66 ± 13 years; Glucagon plays a key role in the counterregulation of hypoglycemia. The p=0.01), but there was no difference in duration of diabetes (26 ± 13 vs. 23 ± natural history of impaired glucagon responses to hypoglycemia early in the 12 years; p=0.29). While baseline HbA1c was identical in those using and not course of type 1 diabetes (T1D) and whether such impairments are related to using MDI-CSII (7.7 ± 4.0 vs. 7.6 ± 2%), MDI-CSII was associated with 20% loss of residual β-cell function have not been established. To examine these lower risk of death (RR 0.2; 0.09-0.5). The follow-up survey was completed questions, the Diabetes Research in Children Network (DirecNet) studied by 32 survivors (52%) and 12 (38%) reported continued SH. There was no 20 T1D adolescents and children (age 9-18 yrs, A1c 6.8+0.9%) with T1D<52 difference in mode of diabetes management compared to those without SH. wks (6-13 wks: n=3; 14-26 wks: n=7; 27-39 wks: n=4; 40-52 wks: n= 6). Each In this cohort of ambulatory patients reporting SH, MDI-CSII was subject underwent a mixed-meal tolerance test (MMTT) to assess β-cell asso ciated with lower risk of death. While further research is needed function and a hyperinsulinemic (2mU/kg/min) euglycemic (glucose ∼95mg/ to understand this relationship, MDI and CSII appear to be reasonable dL) hypoglycemic clamp (nadir glucose ∼55mg/dL) to assess glucagon treatment options in patients with SH. responses (defi ned as an increase in plasma glucagon levels >16 pg/mL from baseline). Peak stimulated c-peptide levels were >0.2 nmol/L (range 492-P 0.12-1.96) in all but 1 subject (17yrs, T1D 49 wks duration), whereas only 11 Prediction and Prevention of Inpatient Hypoglycemia (55%) subjects had retained plasma glucagon responses to hypoglycemia. STEPHEN SCHAFERS, MICHAEL ELLIOTT, RICHARD REICHLEY, JULIE SILVERSTEIN, Moreover, the change in plasma glucagon levels with hypoglycemia did not JANET MCGILL, GARRY S. TOBIN, St. Louis, MO correlate with the peak c-peptide (Figure) or the incremental c-peptide AUC Inpatient hypoglycemia is associated with serious morbidity and may (Spearman rho=0.0, 95%CI= (-0.44, +0.44)) during the MMTT or with the time cause death. Hypoglycemia has been reported in up to 20% of hospitalized from diagnosis (Spearman rho=+0.10(-0.36, +0.52)). These data demonstrate diabetic patients. Severe hypoglycemia (SH, glucose < 40 mg/dl) is the most that many youth with T1D lose their ability to mount plasma glucagon common medication error “never event” based on Medicare guidelines. responses to hypoglycemia during the fi rst year of diabetes. In addition, This study was undertaken to identify risk factors for SH, to apply that although the sample size is small, these preliminary observations do not knowledge to the development of a prediction algorithm, and to institute a support the hypothesis that the loss of glucagon responses to hypoglycemia prevention program at a tertiary medical center. We analyzed all SH events is closely linked to loss of residual -cell function. β for patient characteristics, medication prescribing and administration errors, and other predisposing factors for 6 months in 2009. One-hundred seventy- two adjudicated patients with SH events were then used as a validated population to develop a computer generated algorithm to predict SH. This algorithm was tested on a subsequent population of inpatients found to have any glucose <90 mg/dl during their hospital stay (N=9,997 unique patients) corresponding to the same time period. The population was assessed at 2

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A136 COMPLICATIONS—HYPOGLYCEMIA levels of hypoglycemia < 60 mg/dl (N=955) and < 40mg/dl (N=232). Chi-square time to blood glucose ≥70 mg/dL (3.9 mmol/L) was measured as the primary analyses and t-tests were run to identify bi-variate associations. Signifi cant endpoint. Recovery to ≥65 mg/dL (3.6 mmol/L) and counterregulatory variables were entered into a partition analysis to identify interactions. hormone levels were also assessed. Logistic regression was performed to calculate parameters related to the The mean increases in recovery time to 70 mg/dL [90% CI] for the 200- odds of hypoglycemia below each cut point. ROC curve analysis was used to mg and 1000-mg doses were 12.2 min [-1.9, 26.3] and 25.1 min [11.0, 39.3] determine sensitivity and specifi city at various cut points. The cut points that compared to placebo. Mean recovery times [range] to 70 mg/dL by treatment resulted in 50% sensitivity for each hypoglycemia level were determined. were 33.3 [23-46], 45.0 [29-79], and 59.1 [37-78] min for placebo, 200-mg These were tested against the adjudicated patients with SH. Variables and 1000-mg MK-0893, respectively. Mean recovery times [range] to 65 POSTERS related to blood glucose < 60mg/dl were basal, prandial, and adjustment mg/dL were 29.6 [18-43], 38.9 [24-74], and 51.6 [33-67] min, respectively. Complications scale insulin dose, weight, creatinine clearance, and sulfonylurea use. Glucagon levels were increased by MK-0893 prior to clamp initiation, Acute and Chronic The AUC value for this equation was 0.692 and the Nagelkerke r-square and both glucagon and epinephrine levels increased substantially during was 0.134. The 50% sensitivity cut point correctly identifi ed 70% of the hypoglycemia. MK-0893 treatment was generally well tolerated. adjudicated patients. The same analysis for < 40mg/dl gave an AUC value of These data indicate that MK-0893 slows recovery from hypoglycemia 0.697 and the Nagelkerke r-square was 0.095. The 50% sensitivity cut point in normal subjects in a dose-dependent fashion, but the 200-mg dose correctly identifi ed 55% of the adjudicated cases. This algorithm will be (∼60% blockade) produced little delay despite glucose-lowering effi cacy used to develop an alert system aimed at predicting and preventing serious demonstrated at that level of functional receptor blockade in a separate hypoglycemia at our institution. study in patients with T2DM (ADA abstract submitted). These results suggest that competitive GRAs may not delay recovery from hypoglycemia 493-P to a clinically important degree at doses that can improve glycemic control. Predictors of Outpatient Visits for Hypoglycemia in Type 2 Diabetes Patients on Oral Antidiabetic Agents 495-P JASON C. SIMEONE, BRIAN J. QUILLIAM, Kingston, RI Recovery from Hypoglycemic Clamp in Type 2 Diabetes (T2DM) To identify predictors of hypoglycemia-related emergency department Patients during β-Adrenergic Blockade Plus Glucagon Receptor (ED) and outpatient visits in enrollees with Type 2 diabetes. Blockade with MK-0893 We used the 2004-2008 MarketScan® database to conduct a nested MATTHEW D. TROYER, MARCUS HOMPESCH, THOMAS JAX, BARNALI PRA- case-control analysis. Enrollees included in the cohort were ≥18 years of age MANIK, FANG LIU, LINDA MORROW, KHIN WIN, TOM REYNDERS, CHENGCHENG with type 2 diabetes, and were taking an oral antidiabetic agent at cohort LIU, SAMUEL ENGEL, TIM HEISE, ELSA MALIWAT, WILLIAM S. DENNEY, DAVID E. entry. We required > 12 months of continuous enrollment in a non-capitated KELLEY, JOHN A. WAGNER, S. AUBREY STOCH, Whitehouse Station, NJ, San Diego, plan and excluded persons with type 1 or gestational diabetes. A total of CA, Neuss, Germany, Chula Vista, CA, Brussels, Belgium 11,375 cases and 68,247 controls were selected and 6:1 matched using Glucagon receptor antagonists (GRAs) are drug candidates that may incidence density sampling. We used a conditional logistic regression model improve glycemic control in T2DM, but GRAs may also delay recovery to estimate adjusted odds ratios (AORs) and corresponding 95% confi dence from hypoglycemia, particularly with concomitant β2-receptor blockade. intervals for potential predictors. We previously studied the effect of monotherapy with MK-0893, a Cases were more likely than controls to have diabetic complications and potent, competitive GRA, and observed a modest delay in recovery from other comorbid conditions, and to be utilizing more antidiabetic or other hypoglycemia in healthy subjects. To better understand the effect of medications prior to the index date. Males had lower odds of hypoglycemia β-adrenergic blockade combined with a GRA in T2DM patients, we examined (AOR: 0.83; 95% CI: 0.80-0.87). Enrollees aged 50-59 had the lowest odds of the effect of combining the β1/β2-blocker propranolol with a dose of MK- hypoglycemia, compared to enrollees aged 18-29 (AOR: 0.49; 95% CI: 0.40- 0893 that provides near-maximal glucagon receptor blockade. 0.61). Enrollees in the Northeast (relative to South) had the lowest odds of In this randomized, double-blind study, 22 patients (age 41-59 years, hypoglycemia (AOR: 0.85; 95% CI: 0.78-0.93). Presence of a macrovascular 16 male, washed off oral antihyperglycemic therapy) were titrated onto a (AOR: 1.78; 95% CI: 1.68-1.89) or a microvascular complication (AOR: 2.80; stable dose of oral propranolol (80 mg t.i.d.). Patients then received a single 95% CI: 2.64-2.97) increased the odds of hypoglycemia. With no antidiabetic dose of 1000-mg MK-0893 or placebo in a 2-period crossover and underwent drug therapy as the referent, insulin monotherapy was associated with the a hypoglycemic clamp in each period with blood glucose = 50 mg/dL (2.8 highest odds of hypoglycemia (AOR: 2.28; 95% CI: 2.01-2.59). Monotherapy mmol/L) for 30 minutes. After discontinuation of each clamp, recovery with sulfonylureas (AOR: 1.16; 95% CI: 1.06-1.26) or meglitinides (AOR: time to blood glucose levels ≥65 mg/dL (3.6 mmol/L) was assessed as the 1.40; 95% CI: 1.02-1.93) had increased odds of hypoglycemia. Metformin primary endpoint. Mean recovery time [95% CI] was delayed with MK-0893 monotherapy (AOR: 0.77; 95% CI: 0.72-0.83) and thiazolidinedione + propranolol vs. placebo + propranolol treatment to 103 [88, 118] vs. 71 monotherapy (AOR: 0.87; 95% CI: 0.78-0.96) had lower odds of hypoglycemia. [55, 88] minutes. The estimated mean difference [90% CI] in recovery time DPP-4 and alpha-glucosidase inhibitors were not predictive. between the two treatments was 32 [15, 49] minutes and did not meet the Medications should be carefully chosen for patients at high risk of prespecifi ed clinical importance boundary of 60 minutes (p = .005). MK-0893 hypoglycemia, particularly those with micro- or macrovascular complications treatment was associated with signifi cantly increased plasma glucagon and or those taking insulin, meglitinides, or sulfonylureas. cortisol during recovery. Geometric mean ratios (MK-0893 + propranolol/ Supported by: Takeda Pharmaceuticals, Inc. placebo + propranolol) [95% CI] for hormone AUC0-180min were: glucagon, 3.76 [1.52, 9.27]; cortisol, 1.86 [1.07, 3.25]; epinephrine, 1.34 [0.53, 3.40]; growth 494-P hormone, 1.93 [0.88, 4.27]. Recovery from Hypoglycemia in Healthy Subjects Is Preserved These results indicate that a high dose, competitive GRA plus β-blockade Despite Glucagon Receptor Blockade by MK-0893 delays recovery from hypoglycemia, but may delay recovery less than in MATTHEW D. TROYER, MARCUS HOMPESCH, BARNALI PRAMANIK, WEI prior studies in which glucagon secretion was inhibited. Recovery may have ZHENG, KHIN WIN, STEPHANIE DUNBAR, SUSIE LI, MARCELLA RUDDY, JOHN been facilitated by a compensatory counterregulatory hormone response, AMATRUDA, KEITH KAUFMAN, JOHN WAGNER, S. AUBREY STOCH, Whitehouse including larger increases in glucagon levels. Station, NJ, Chula Vista, CA Glucagon receptor antagonists (GRAs) are drug candidates that may improve glycemic control in Type 2 diabetes (T2DM) but may also delay recovery from hypoglycemia. To better understand this risk, we studied the effect of MK-0893, a potent, competitive GRA, on recovery from hypoglycemia in healthy subjects. This was a double-blind, placebo-controlled, crossover glucose clamp study in 13 healthy males 18-37 years old. MK-0893 doses were chosen to provide clinically effi cacious levels of functional glucagon receptor blockade. Subjects were randomized to a sequence of 2 of 3 treatments: single-dose placebo, 200-mg (∼60% blockade) or 1000-mg MK-0893 (∼90% blockade), separated by a 3-week washout. After each treatment, a hypoglycemic clamp was initiated and blood glucose was maintained at 50 mg/dL (2.8 mmol/L) for 30 minutes (min). Upon discontinuation of the clamp, recovery

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A137 COMPLICATIONS—HYPOGLYCEMIA

496-P 498-P Reduced Rates of Severe Hypoglycemia in Children and Adolescents Response to Induced Hypoglycemia in Type 1 Diabetes: Insulin De- with Type 1 Diabetes over the Decade 2000-2009 gludec Elicits an Enhanced Counter-Regulatory Hormone Response SUSAN M. O’CONNELL, MATTHEW N. COOPER, MAX K. BULSARA, ELIZABETH Compared to Insulin Glargine A. DAVIS, TIMOTHY W. JONES, Perth, Australia, Fremantle, Australia THOMAS PIEBER, STEFAN KORSATKO, GERD KÖHLER, SIGRID DELLER, GERLIES We have previously reported increased rates of hypoglycemia associated BOCK, SAMRA ZAHIRAGIC, JULIA MADER, CARSTEN ROEPSTORFF, SØREN with improved glycemic control in a large population-based sample of RASMUSSEN, HANNE HAAHR, SIMON HELLER, Graz, Austria, Soeborg, Denmark,

POSTERS children with T1D, however therapeutic improvements may have altered Sheffi eld, United Kingdom

Complications hypoglycemia incidence. In this study, rates of severe hypoglycemia (SH) Hypoglycemia remains a major side-effect of intensifi ed insulin therapy Acute and Chronic (an event leading to loss of consciousness or seizure) in a population based and intact hormonal counter-regulation has an essential role in promoting sample of childhood onset T1D were examined and clinical associations of glucose recovery from low glucose levels. In this double-blind, two-period, these events investigated. cross-over trial we compared counter-regulatory hormone responses during Data were extracted from the Western Australia Childhood Diabetes hypoglycemia induced by insulin glargine (IGlar) or ultra-long-acting insulin Database. Clinical data, including hypoglycaemia history is prospectively degludec (IDeg; formerly named SIBA). Subjects with type 1 diabetes (n=28, recorded at each 3 monthly visit. Data from 1,683 patients with T1D from age 41±12 yrs, diabetes duration 21±10 yrs, A1C 7.8±0.6%) were randomized 2000-2009 inclusive (age at diagnosis 10.5 ± 4.2 years [mean ± SD], range to IDeg or IGlar once daily for 5 days. At midnight on Day 5, each subject 1–18) were analysed. Rates of SH were investigated with respect to A1c, received a dose that was 3-times their individual daily requirement, and treatment type, duration of diabetes, age and gender using interaction terms euglycemia was maintained at 5.5 mmol/l by a variable iv glucose infusion fi tted within a negative binomial regression model. until 7 AM. Plasma glucose (PG) was then allowed to decline to 3.5 mmol/l In total 7,378 patient-years of data and 780 severe events were recorded. (maintained for 30 min) and thereafter to a nadir of 2.5 mmol/l (maintained for The rate of SH/100 patient years in 2000 was 14.3, peaking at 17.3 in 15 min). After nadir, PG was raised to 3.9 mmol/l and maintained at this level 2001. From 2004 there was a sharp decline, with the lowest rate of 5.8 for 2 h, then raised to 5.5 mmol/l. Hormonal counter-regulation was assessed in 2006. There was a negligible decline in HbA1c levels of 0.07% per year at baseline and at PG levels from 4.5–2.5 mmol/l. Rate of PG decline and (p = 0.03). Using multivariate analysis optimal recommended A1c (<7%) was mean PG at nadir were similar between groups. There was a signifi cantly signifi cantly associated with higher risk of SH (IRR=3.0, p<0.001; CI 1.9- greater growth hormone and cortisol response during hypoglycemia (from 4.7) relative to A1c>10%. Compared to previous fi ndings, children <6 years PG 4 mmol/l to PG nadir) with IDeg compared to IGlar (AUChormone [ng*min/ were no longer at increased risk of SH. From 2000 to 2009 the proportion ml]: 498 vs. 204 (treatment ratio (TR): 2.44 [1.30; 4.60] p<0.01); 10834 vs. of patients using pump (1% vs 26%) and injections ≥ 4/day (8% vs 25%) 8808 (TR: 1.23 [1.01; 1.50] p<0.05)) and a trend towards a higher epinephrine increased. Using interaction models, from age >13 years the estimate for response which failed to reach statistical signifi cance (AUC [pg*min/ml]: SH events for the prospective 12 month period was 64% lower for patients 6636 vs. 4726 (TR: 1.40 [0.96; 2.04] p=0.07)). Norepinephrine and glucagon using pump therapy compared to injections (p <0.001). responses were similar for both groups. At PG nadir, glucose infusion rates There has been a decrease in rates of SH over the past decade despite in both groups were comparable, whereas during recovery, less glucose static glycemic control. More intensive blood glucose monitoring, analog was required to alleviate hypoglycemia with IDeg (AUCGIR[mg/kg]: 201 vs. insulins, more widespread use of pump therapy are likely to have contributed 285 (TR: 0.71 [0.53; 0.93] p<0.02)). In summary, hypoglycemia induced by to the change, but their relative importance cannot be determined. IDeg is associated with a signifi cantly greater counter-regulatory hormone response raising the possibility that enhanced counter-regulation with IDeg 497-P may contribute to the observed reduced risk in hypoglycemia. Reduction of Hypoglycemia Burden Following Three Months of Continuous Glucose Monitoring in Patients with Impaired Aware- 499-P ness of Hypoglycemia Safety and Effi cacy of Insulin Aspart and Soluble Human Insulin in PRATIK CHOUDHARY, PETROS THOMAKOS, BULA WILSON, ANDREW PERNET, Elderly Type 2 Diabetes Patients: Results from UpGrade Observa- DAVID HOPKINS, STEPHANIE A. AMIEL, London, United Kingdom tional Study Introduction: Impaired awareness of hypoglycaemia (IAH) affects 25-30% THE UPGRADE ITALIAN STUDY GROUP, Rome, Italy of patients with type 1 diabetes and increases risk of severe hypoglycaemia Insulin therapy is associated with increased concern of major hypos 6-fold. Meticulous hypoglycemia avoidance can restore awareness, but is in elderly type 2 diabetes patients, due to the higher rate of concomitant diffi cult to achieve. Availability of continuous glucose monitoring (CGM) disease and polytherapy, and to the lack of hypos awareness. In this abstract with hypoglycaemia alarms may assist in restoration of awareness. We data from a subanalysis of the UpGrade 26-week observational study on hypothesized that a short intensive intervention with CGM may restore insulin aspart (IAsp) versus soluble human insulin (SHI) in Italian elderly hypoglycemia awareness and provide lasting reduction in hypoglycemia (>70 years) patients is provided. Safety and effectiveness were assessed burden. in routine clinical practice in 3 visits (baseline, 13-week and 26-week) Methods: 20 subjects with hypoglycaemia unawareness were randomized performed in patients attending outpatient diabetes clinics. to 10 weeks of optimized care support with CGM (CGM+) or without (CGM- Overall, 1385 subjects (out of 4185 enrolled in the UpGrade study) were ). We measured HbA1c, Clarke score, Gold score and acquired at least 4 included in this subanalysis, treated with >2 daily boluses of rapid-acting days’ blind CGM to assess glycemic control, hypoglycemia awareness insulin. Approximately 71% were treated with IAsp. No oral agents were and hypoglycemia burden was assessed over 4 weeks at baseline and on allowed except metformin. completion of the 10 week study period using Ryan scores and blind CGM. Mean age was 76.9 y, diabetes duration 19.7 y, BMI 28.9 kg/m2, males Results: Mean HbA1c reduced similarly in both groups [0.23% vs. were 45%. Despite the old age, 97% were treated with 3-4 daily injections, 0.22%; CGM+ vs. CGM-; p=0.9] from 7.6%; with no signifi cant change in with a higher prevalence of basal-bolus in the IAsp than in the SHI group Gold scores [5.5 vs 5.8; CGM+ vs CGM-]. Five of 11 subjects randomized to (57% vs. 41%). Mean total insulin daily dose was 0.60 U/kg in IAsp-group, CGM+ fi rst reported any improvement in Gold score (the others remaining and 0.54 U/kg in SHI-group. HbA1c decreased from 61.9 to 58.3 (-3.6) mmol/ stable vs. 2/9 randomized to CGM-. 7/11 of CGM+ had an improvement in mol in IAsp-group, and from 59.0 to 57.6 (-1.4) mmol/mol in SHI-group. No Ryan score compared with 3/9 CGM-. Hypoglycemia burden (Ryan score) clinically signifi cant change was observed in therapy and dose after 26 fell signifi cantly after 10 weeks of CGM [266.5 vs. 614.3; CGM+ vs CGM-; weeks, thus confi rming a lack of therapy intensifi cation also in the elderly. p=0.03], with comparable but not statistically signifi cant reduction in time Multivariate analysis for hypos showed signifi cant risk reduction with use of spent below 4mmol/l on blind CGM[-16% vs -7%; CGM+ vs CGM-; p=0.32]. IAsp (for major HR=0.70, p=0.0478; for minor HR=0.69, p=0.0013). Strong positive There was also a similar reduction in capillary glucose readings < 2.5 mmol/l correlation was found for major hypos with baseline HbA1c class >64 mmol/mol [-2.25 vs. -1.27 readings / month CGM+ vs CGM-; p=0.6]. (HR=2.49, p=0.002). As in recent literature fi ndings, this could be explained with Conclusion: These data suggest that a short duration of continuous higher glycaemic instability in patients with poorer control. A higher body weight CGM may be able to reduce hypoglycaemia burden, without deteriorating was inversely related to major hypos risk (HR=0.84, p<0.0001). overall glucose control, in those with persistent impaired awareness of Overall, in elderly type 2 diabetics >70 years, IAsp was associated with hypoglycaemia. a signifi cantly lower risk of both major and minor hypos vs. SHI. As in the Supported by: DiabetesUK overall population, IAsp also allowed a higher reduction of HbA1c over 26 weeks. Supported by: Novo Nordisk - Italy

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A138 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

500-P COMPLICATIONS—MACROVASCULAR— Self Report of Severe Hypoglycemia Is Associated with Increased ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND Risk of Death in Diabetic Patients HUMAN DIABETES ROZALINA GRUBINA, NILAY D. SHAH, HOLLY K. VAN HOUTEN, ROBERT A. WERMERS, STEVEN A. SMITH, Rochester, MN [See also: Presidents Posters 380-PP to 383-PP, page A105.] Hypoglycemia is a cause of signifi cant morbidity in patients with diabetes mellitus. Recent post-hoc analyses of the ACCORD and ADVANCE trials

have found a potential correlation between documented hypoglycemia Guided Audio Tour: Atherogenesis and the Arterial Wall in Human Diabetes POSTERS and increased risk of microvascular and macrovascular complications, and (Posters 502-P to 509-P), see page 13. Complications Acute and Chronic mortality. Whether self-report of hypoglycemia among ambulatory patients & 502-P is associated with increased mortality, however, is unknown. Paracrine Dysfunction in Diabetic Endothelial Progenitor Cells All patients seen in a specialty diabetes clinic between August 2005 YAGNA P. JARAJAPU, MOHAN K. RAIZADA, STEPHEN H. BARTELMEZ, MARIA B. and July 2006 were questioned about the frequency of mild or severe GRANT, Gainesville, FL, San Francisco, CA hypoglycemia over the preceding 6 months. A total of 1130 patients with Bone marrow-derived endothelial progenitor cells (EPCs) participate in established diabetes mellitus were seen by 31 providers, including physicians, vascular repair and promote vascular growth by releasing proangiogenic midlevel providers, nurse educators, and dieticians. Mild hypoglycemia factors. We and others have shown that the vasoreparative function of EPCs was defi ned as presence of symptoms (dizziness, blurry vision, confusion, is impaired in diabetes (DM). We hypothesized that diabetic EPC dysfunction and sweating) that could be managed without external assistance, while is associated with defective paracrine signaling. Circulating Lin-CD45midCD34+ severe hypoglycemia required assistance from others. Patients’ clinical cells from DM (n=35) and nondiabetic (ND, n=27) individuals were studied. status was reassessed in November 2010, after mean 5 years of follow-up. EPCs were plated in basal medium (Stemspan) for 18-20 hours and the cell- Demographics were compared using the two sample t-test, and risk of death free conditioned-medium (CM) was concentrated and analyzed for 28 different is expressed as odds ratio (OR, 95% Confi dence Interval). cytokines and growth factors. The effects of CM on migration (modifi ed At enrollment, 641 of 1130 (57%) patients reported 1 episode of ≥ Boyden chamber assay) and proliferation (CFSE cell proliferation assay) of hypoglycemia, and 79 (7%) reported severe hypoglycemia. Those with ND-CD34+ cells was assessed. Paracrine effects of cells on vasculature severe hypoglycemia were more likely to have longer duration of diabetes were assessed in pressure-mounted rat posterior cerebral arteries (RPCAs). (26 ± 12 years vs. 16 ± 12 years; p<0.001) and slightly higher HbA1c (7.6% ± The effect of CM was evaluated in wire-mounted mouse mesenteric arteries 1.5 vs. 7.2% ± 1.4; p=0.07) compared to those with no or mild hypoglycemia. (MMAs) as was generation of cGMP by CM. TNFa, MCP1, IL-1, IL-6, IL-8 and Importantly, report of severe hypoglycemia was associated with higher decorin levels were higher and stem cell factor, hepatocyte growth factor and 5-year mortality rate (21.5% vs 12.8%; p=0.0361). thrombopoietin levels were lower in CM from DM- compared to ND-cells. ND- Self-report of severe hypoglycemia in this ambulatory specialty clinic CM induced migratory response in ND-EPCs whereas DM-CM had no effect. population was associated with longer duration of diabetes, slightly higher ND-CM enhanced proliferation of EPCs whereas DM-CM did not. Intraluminal HbA1c levels, and increased mortality during a 5-year follow-up period. ND-cells signifi cantly attenuated pressure (70 mmHg)-induced constriction in Further research is needed to determine whether severe hypoglycemia is RPCAs while DM-cells were ineffective. In the presence of ND-CM, relaxation a cause or a clinical marker of increased mortality. Nonetheless, physicians to bradykinin was increased and the potency of phenylephrine-induced should routinely question patients about presence of hypoglycemia, and constriction was decreased in MMAs whereas in the presence of DM-CM educate them about its risk factors, symptoms, treatment, and prevention. relaxation to bradykinin was not affected but the sensitivity to phenylephrine- constriction was increased. This was associated with decreased cGMP 501-P generation in MMAs by DM-CM compared to ND-CM. These results support Severe Hypoglycemic Episodes in STAR 3: Baseline and Behavioral that the vasoreparative dysfunction of diabetic EPCs is due to 1) an altered Predictors profi le of paracrine factors, 2) an intrinsic defect in their migration and YOGISH C. KUDVA, MARK DANIELS, JENNIFER GREEN, JOHN B. WELSH, FRAN- proliferation and 3) ineffective interaction with vessels. CINE R. KAUFMAN, QINGQING YANG, SCOTT W. LEE, JOHN SHIN, Rochester, Supported by: EY 007739; EY012601; DK 090730 MN, Orange, CA, Durham, NC, Northridge, CA STAR 3, a multi-center, randomized, controlled trial, showed a signifi cant & 503-P reduction in A1C with sensor-augmented pump therapy (SAP) compared to Use of P53 Silenced Endothelial Progenitor Stem Cells (hEPC) in multiple daily insulin injections (MDI) in 485 subjects (329 adults and 156 Diabetic Peripheral Vascular Disease children), without increasing severe hypoglycemia (SH). At 1 year, the rate SABYASACHI SEN, MARY YOUNG, XIAOLING HILL, JOSEPH JERRY, Springfi eld, MA of SH in the SAP group did not differ signifi cantly from that in the MDI group Literature shows that peripheral blood derived EPCs can be matured to adult (13.31 vs. 13.48 cases per 100 person-yr, respectively, P=0.58). In the SAP EC, however senescence of EPCs in high glucose (HG) is known to occur. This group, 21 subjects (17 adults and 4 children) experienced 32 episodes of SH may lead to reduction in the number of EPCs leading to poor wound healing in and 223 did not. Of the 32 episodes, 1 subject had 5 episodes, 1 subject had diabetes. This senescence is secondary to p53 activation. We cultured hEPCs 3 episodes, 5 subjects had 2 episodes, and 14 subjects had 1 episode. In the and exposed them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to MDI group, 17 subjects (13 adults and 4 children) experienced 27 episodes of 360mg%) glucose and assessed cell survival by FACS analysis using propidium SH and 224 did not. Of the 27 episodes, 1 subject had 7 episodes, 1 subject iodide (PI) stain. There was signifi cant cell death of EPCs noted in HG within had 3 episodes, 2 subjects had 2 episodes, and 13 subjects had 1 episode. 48hrs. Such quick onset of cell death in HG is evident in EPCs, compared to Those with SH and those without SH were similar with respect to age, human umbilical vein endothelial cells (HUVEC), which are mature endothelial BMI, and baseline A1C, but differed with respect to duration of diabetes cells (EC). As senescence of EPCs in HG has been linked to p53 activation, we (22.8±13.4 with SH vs. 14.7±12.0 years without SH, p<0.0001). Within the obtained mouse peripheral blood derived EPCs from mouse p53 KO and WT SAP group, subjects with and without SH differed with respect to mean animals and observed that p53 KO EPCs are more resistant to death compared amplitude of glycemic excursions (MAGE). MAGE was 95.3±21.9 mg/dl in to p53 WT in HG. We cultured EPCs from p53KO mouse peripheral blood which SAP subjects with SH versus 84.1±20.2 mg/dl in SAP subjects without SH evolved into mature mouse EC (MEC) without senescence. MEC retained all (P=0.02 over 1 year). A1C at 1 year was 7.7±0.9% in the SAP group with SH endothelial properties such as cobblestone appearance, tube-formation on compared to 7.5±0.8% in the group without SH (P=0.08). Pump and glucose matrigel and several EC gene expression such as pecam-1 (also known as data from the 1- and 7-day intervals prior to each SH event were examined CD-31), vwf (von-Willebrand’s factor), kdr (also known as vascular endothelial to identify predictors of SH. Overall insulin delivery patterns, total daily growth factor receptor 2, VEGF-2), e-nos (endothelial nitric oxide synthase) doses of insulin, and mean glucose levels were similar over the week of were maintained at similar level to WT EPC. We subsequently used Lenti- the SH event. There was a trend toward higher MAGE values on the day shRNA to silence p53 in human EPCs and noted better survival and maturity prior to the SH event compared to the day of the SH event (P=0.08). SH towards adult human EC with no loss of function comparable to HUVEC for episodes occurred in the minority of STAR 3 subjects and these were not over 8wks. These fi ndings illustrate that EPCs are more susceptible to death signifi cantly associated with most baseline and pump characteristics, other in HG and EPCs with P53 silenced survive better in high glucose. Preliminary than duration of diabetes. Most subjects with SH experienced only one such results from transplantation of P53 silenced EPCs in hyperglycemic mice episode. Modifi able risk factors that could be targeted to reduce SH include with ischemic wound demonstrated better healing compared to WT-EPC reducing glycemic variability. transplanted group indicating therapeutic benefi t in transplanting P53 silenced Supported by: Medtronic, Inc. EPC to prevent peripheral vascular disease in a setting of diabetes. Supported by: Collaborative Biomedical Research Fund

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& 504-P Toll-Like Receptor 4 (TLR4) and Vascular Infl ammation Following & 506-P Weight Loss in Patients with Metabolic Syndrome Arterial Stiffness in Youth and Young Adults with and without Type PADMA SATHYANARAYANA, SADEKA SHAHANI, VADIM SHERMAN, SOMA- 1 Diabetes: The SEARCH CVD Study SEKHAR KONDURU, MANDEEP BAJAJ, Houston, TX R. PAUL WADWA, ELAINE M. URBINA, JENNIFER TALTON, RALPH D’AGOSTINO, Toll-like receptor 4 (TLR4) expression is increased in obesity and type JR., MAMTA JAISWAL, AMY S. SHAH, SANTICA MARCOVINA, RICHARD F. 2 diabetes and TLR4 may be a mediator of vascular infl ammation and HAMMAN, STEPHEN R. DANIELS, LAWRENCE M. DOLAN, DANA DABELEA, Aurora, CO, Cincinnati, OH, Winston-Salem, NC, Seattle, WA

POSTERS insulin resistance. We examined the effect of weight loss on mononuclear

Complications cell TLR4 protein and vascular infl ammation following bariatric surgery Recent data suggest sub-clinical vascular damage, including increased Acute and Chronic in morbidly obese non-diabetic humans with metabolic syndrome. Six arterial stiffness (AS) may be identifi ed in youth with type 1 diabetes subjects (age=38±4 y, BMI=48.5±4.9 kg/m2, body weight=135.1±13.8 Kg) (T1D) well before clinical signs of vascular disease are evident. Therefore, underwent a 75 gram Oral Glucose Tolerance Test (OGTT) and peripheral we examined AS in 237 youth with T1D (age 19.5±2.8 years, T1D duration blood mononuclear cell isolation before and 6 months after bariatric surgery. 10.1±3.9 years) compared to 125 non-diabetic control subjects (age 19.2±3.0 Following bariatric surgery, body weight (delta= 35.0 kg, p< 0.05), fasting years) who participated in the SEARCH CVD study in Colorado and Ohio. AS plasma glucose (104±5 to 84±2 mg/dL, P<0.05), fasting plasma insulin (23±2 measures included carotid-femoral vascular segment pulse wave velocity to 12±1 μU/ml, P<0.05) decreased and insulin sensitivity index (Matsuda (PWV), augmentation index adjusted to a heart rate of 75 beats/ minute Index) increased (1.4±0.2 to 2.5±0.5, p<0.05). Fasting plasma free fatty acids (AI75) using the SphygmoCor Vx (AtCor Medical, Lisle, IL), and brachial (FFA) did not change signifi cantly (0.69 ± 0.05 to 0.63 ± 0.05 mmol/L) but distensibility (brachD) using the DynaPulse 2000 (PulseMetric, San Diego, plasma FFA concentrations during the OGTT were signifi cantly decreased CA). Body mass index (BMI), blood pressure (BP), fasting lipids and A1C were (0.45 ± 0.04 to 0.37 ± 0.02 mmol/L, p<0.05) following weight loss. Plasma measured. General linear models were used to explore group differences adiponectin was increased (6.5 ± 0.5 to 11.6 ± 0.9 μg/mL, p<0.01) following (T1D vs. controls) in measures of AS, after adjustment for covariates. After weight loss. Mononuclear cell TLR4 protein was reduced (p<0.05) and was adjusting for age, sex and race, T1D status is associated with higher PWV associated with a reduction (p<0.05) in mononuclear cell NFkappaB (p65) but not with AI75 or brachD (Table). In multivariate models, BMI and systolic following weight loss. Conclusions: These results suggest that weight BP are associated with worse AS (higher PWV and lower brachD (p<0.05)). loss following bariatric surgery is associated with a signifi cant reduction In models limited to only T1D subjects, higher A1C is associated with higher in mononuclear cell TLR4 and vascular infl ammation (NFkappaB) in patients PWV (p<0.01). with metabolic syndrome. ADA-Funded Research T1D Controls p-value brachD, %Δ/mmHg* 6.4±0.3 6.7±0.3 0.06 & 505-P AI75, %, adjusted for height* 1.5±2.7 0.3±2.6 0.32 Different Effects of Type 2 Diabetes Mellitus and Essential Hyper- PWV, meters/second* 6.0±0.2 5.8±0.2 0.04 tension on Aortic, Carotid and Peripheral Vascular Stiffness GIUSEPPE PENNO, GIUSEPPE A. DANIELE, LAURA PUCCI, DANIELA LUCCHESI, * Mean ± standard error adjusted for age, sex and race ROSAMARIA BRUNO, LORENZO GHIADONI, ELEONORA RUSSO, CRISTINA In conclusion, T1D adversely affects arterial stiffness as measured by BIANCHI, MONIA GAROFOLO, STEFANO TADDEI, ROBERTO MICCOLI, STEFANO carotid-femoral PWV by late adolescence. BMI, blood pressure and glycemic DEL PRATO, Pisa, Italy control appear to be modifi able risk factors for vascular disease in youth Type 2 diabetes (T2D) and hypertension (HYP) both accelerate vascular with T1D. aging. Arterial stiffness increases with age and in presence of CV risk Supported by: NIH R01 DK078542 factors. Stiffening of large arteries with advancing age and risk factors exposure meanly involves the elastic aorta and carotid. We evaluated the impact of T2D, HYP, and their combination on aortic, carotid and & 507-P peripheral arteries stiffening. 114 subjects were enrolled: 18 normotensive Does Intensive Multifactorial Treatment of Screen-Detected (NT), 37 hypertensive (HT), 20 T2D normotensive (DMNT), and 39 T2D Diabetes Affect Aortic Stiffness? ADDITION-Denmark hypertensive (DMHT). Applanation tonometry was used to measure aortic NANNA B. JOHANSEN, DORTE VISTISEN, SIGNE SS RASMUSSEN, NIELS WIIN- (carotid to femoral, aPWV) and peripheral (carotid to radial, pPWV) pulse BERG, MORTEN CHARLES, KNUT BORCH-JOHNSEN, TORSTEN LAURITZEN, wave velocity. Common carotid intima-media thickness (IMT) and carotid ANNELLI SANDBÆK, DANIEL R. WITTE, Gentofte, Denmark, Copenhagen, Denmark, diameter (CD) were obtained by B-mode ultrasound image (Carotid Studio). Frederiksberg, Denmark, Århus, Denmark, Odense, Denmark Common carotid stiffness (CCS) was determined from stroke change in Aortic stiffness is an independent risk factor for cardiovascular disease lumen area and local pulse pressure by applanation tonometry. HYP groups (CVD). In microalbuminuric type 2 diabetes patients, intensive multifactorial (HT and DMHT) have similar BP and lipids; T2D (DMNT and DMHT) have treatment of type 2 diabetes reduces the risk of CVD by more than 50%. The similar HbA1c and lipids. pPWV was similar in all groups. aPWV signifi cantly aim of this study is to examine the effect of intensive multifactorial treatment increased from NT (7.2±1.0) to HT (8.1±1.4) and DMNT (8.2±0.8), with the on aortic stiffness in a population with screen-detected diabetes. highest values in DMHT (10.6±1.9 m/s; p<0.001). CCS had a similar behaviour As part of a population based screening and intervention study in general (NT 6.0±0.7, DMNT 6.5±1.2, HT 6.6±1.2, DMHT 7.3±1.2 m/s; p<0.01). HYP practice, 1533 Danes aged 40-69 years were diagnosed with screen- carried a higher risk of having increased (above the median) aPWV (OR: detected diabetes. The general practitioners were randomized to provide 6.9; 5-95%CI: 1.9-24.8), CCS (OR: 2.8; 1.1-7.4), and CD (OR: 3.9; 1.4-10.6) either intensive multifactorial or conventional treatment. At 5 year follow- regardless of age and diabetes, while the differences were not signifi cant up, a random subsample of 427 patients had measurements of aortic for pPWV and IMT. T2D carried a higher risk of having increased (above the stiffness by carotid-femoral pulse wave velocity (PWV). The effect of the median) aPWV (OR: 9.6; 3.3-27.2) and IMT (OR:2.7; 1.1-6.6) regardless of age intervention on PWV was analyzed by mixed effect models adjusted for and hypertension, while the differences were not signifi cant for pPWV, CD clustering (all patients within a practice were randomized to either intensive and CCS. Both T2D and HYP are associated with increased aPWV, and their or conventional treatment), mean blood pressure and heart rate at follow-up combination induces an even greater aortic stiffening. HYP is characterized to account for its direct functional effects on PWV. We additionally adjusted by vascular stiffening at both the aortic and carotid level. In contrast, T2D for age and sex. is associated only with increased aPWV. The two conditions also differ for From the study sample at follow-up, mean age was 65.7 years (SD: 9.8), carotid remodeling characteristics, since HYP determines carotid dilation 260 patients (61%) were men, mean PWV was 9.54 m/s (SD: 2.12), and 270 while T2D is associated with carotid wall thickening. patients (63%) were in the intervention group. PWV was 0.43 m/s lower in patients from the intervention group than patients from the conventional treatment group (Table 1). Intensive multifactorial treatment of screen-detected diabetes in general practice has a benefi cial effect on aortic stiffness. Extrapolating our results based on estimates from cohorts with baseline PWV, the effect observed in our study would correspond to a 2% lower risk of CVD and a 3% lower risk of all cause mortality over a period of 10 years.

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A140 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

p<0.001). Patients with PN (n=36) had also higher OPG levels than patients Table 1. Intervention versus conventional treatment without PN (17.09±5.78 vs 14.94±3.94 pmol/l, p<0.011). OPG levels were Pulse wave velocity (m/s) 95% CI p-value signifi cantly associated with ABI (r=-0.309, p<0.001), VPT (r=0.370, p<0.001) and NDS (r=0.324, p<0.001). The association between OPG concentrations Model 1 -0.44 (-0.86 ;-0.01) 0.05 and ABI remained signifi cant after adjustment for age, diabetes duration, Model 2 -0.43 (-0.79 ;-0.06) 0.02 sex and presence of PN (r=-0.220, p=0.010). The associations between OPG levels and VPT, NDS remained signifi cant after adjustment for age, diabetes Model 1: adjusted for clustering (general practice), mean blood pressure duration, sex and presence of PAD (r=0.236, p=0.005 and r=0.241, p=0.004, POSTERS and heart rate at time of measurement respectively). Complications

Model 2: model 1 + adjustment for age and sex Serum OPG levels are increased in diabetic patients with either PAD or PN Acute and Chronic and are associated independently with the severity of these complications. & 508-P Guided Audio Tour: Heart Disease in Human Diabetes (Posters 510-P Tumor Necrosis Factor alpha and Interleukin-10 Genetic Polymorph- to 517-P), see page 11. isms and Susceptibility to Peripheral Artery Diseases Associated with Type 2 Diabetes Mellitus and Hypertension in Brazilian Patients & 510-P ETHEL SPICHLER, DAYSE SILVA, ELIZEU FAGUNDES DE CARVALHO, ROMULO Atherogenic Dyslipidemia Is Associated with an Increased Risk of VIANNA, MARILIA PANICO, ELAINE TAVARES, JOSE ADLER PEREIRA, RENATA Silent Coronary Stenoses in Patients with Type 2 Diabetes ADLER PEREIRA, ANNE SPICHLER, DAVID SPICHLER, Rio de Janeiro, Brazil, San BERNARD CHANU, MINH TUAN NGUYEN, ISABELA BANU, SABRINA SCHIHEB, Diego, CA EMMANUEL COSSON, PAUL VALENSI, Bondy, France Polymorphisms of Tumor Necrosis Factor alpha (TNF-α) and Interleukine-10 The ACCORD-Lipid trial has recently provided additional evidence for the role (IL-10) cytokines may have critical functional effects, contributing to the of atherogenic dyslipidemia (low HDL-cholesterol and high triglycerides (TG)) in magnitude of innate immune response toward Peripheral Artery Diseases cardiovascular events in patients with type 2 diabetes (T2D). The aim was to (PAD) in association with Diabetes Mellitus (DM) and Hypertension. To evaluate the risk of silent myocardial ischemia (SMI) and coronary stenoses (CS) determine whether single nucleotide polymorphisms G308A, G238A and associated with atherogenic dyslipidemia in asymptomatic T2D patients. G863T of TNF-α and G1082A, C819T and C592T of IL-10 have an association Among the 852 diabetic patients who have been prospectively screened with PAD in patients with DM and Hypertension. Mutation detection and for SMI since 1991 in our department, 410 had T2D and LDL-cholesterol <130 genotyping of both cytokines were performed on DNA from 102 patients with mg/dl. SMI was assessed on stress myocardial scintiscan and SC on coronary PAD aging ≥ 30 years and with Ankle-Brachial Index ≤ 0.9 and population angiography performed in those with SMI. Atherogenic dyslipidemia was controls. This model was analyzed for demographic and clinical variables. defi ned by HDL-C ≤34 mg/dl and TG ≥200 mg/dl. SMI was found in 134 of the Whole gene sequencing of TNF-α and IL-10 coding regions were carried out 410 patients considered in the current analysis, and 47 had CS. CS+ patients using regular PCR and specifi c primers. Statistical analysis was conducted had higher TG (p=0.01). Atherogenic dyslipidemia was present in 28 patients using SPSS, ABIGeneScan and Genotyper software, and signifi cance level (with LDL-C very similar as in those without atherogenic dyslipidemia) was < 0.05. and was more prevalent in CS+ patients (19.1% vs 5.2%; p=0.002). After PAD patients enrolled and controls showed: age (mean 70.9 ±10.2 years), adjustment on lipid lowering treatments, atherogenic dyslipidemia was and 59.1% female.(<0.05). More than half patients self-reported smoking associated with a higher risk of SMI (OR 2.13 [0.95-4.76]; p=0.06) and CS (OR status and 9.1% of stroke. Abnormal systolic and diastolic pressure, as well 4.83 [1.99-11.74]; p<0.001). Among the 192 patients with LDL-C <100 mg/dl, as fasting plasma glucose, LDL-Cholesterol, renal function, and homocysteine 70 were SMI+ and 23 CS+. These CS+ patients had lower HDL-C (p=0.004) were signifi cant in comparison with controls. G238A and G863T TNF and higher TG levels (p=0.10) with a higher rate of atherogenic dyslipidemia heterozygous frequencies in (PAD and controls) showed: (7.1 and 19 %), (21.7% vs 5.7%, p=0.02), and after adjustment on lipid lowering treatment (33.3 and 25.5%) respectively, while 2% for homozygous only in the control atherogenic dyslipidemia was associated with a higher risk of CS (OR 5.17 group. G308A homozygous were found only in the control group. Regarding [1.52-17.61]; p=0.009). IL-10 gene, G1082A homozygous were found only in PAD (3.5%). Genotyping These data suggest that in patients at LDL-C goal, atherogenic dyslipidemia C592T homozygous was identifi ed only in control group (35%) however, contributes to the residual risk of silent coronary stenoses. C819T showed similar frequencies. There were signifi cant differences in the allelic distribution of TNF α -863, -308 and IL-10 -592 (<0.05) polymorphisms between populations. Our data provide a role for TNF-α -863, -308 and IL- & 511-P 10 -592, in PAD associated to DM and hypertension suggesting a probable C ar diovascular Risk Predic tion Is Improved by Adding A sy mp tomatic contribution for genetic susceptibility. Coronary Status to Routine Risk Assessment in Type 2 Diabetic Supported by: FAPERJ Patients EMMANUEL COSSON, MINH TUAN NGUYEN, BERNARD CHANU, ISABELA & 509-P BANU, SABRINA CHIHEB, KARIM TAKBOU, PAUL VALENSI, Bondy, France Serum Osteoprotegerin Levels Correlate with Severity of Peripheral The aim of the study was to evaluate whether silent myocardial ischemia Arterial Disease and Peripheral Neuropathy in Patients with Type (SMI) and silent coronary artery disease (CAD) provide signifi cant additional 2 Diabetes value to routine cardiovascular risk assessment in type 2 diabetic patients IOANNA ELEFTHERIADOU, PINELOPI GRIGOROPOULOU, VASILIKI ARGIANA, without cardiac history or symptom. ALEXANDER KOKKINOS, STAVROS LIATIS, DESPOINA PERREA, NICHOLAS We followed up for a fi rst cardiovascular event (cardiac death, acute KATSILAMBROS, NICHOLAS TENTOLOURIS, Athens, Greece coronary syndrome, heart failure, stroke, amputation, peripheral or Increased OPG levels have been found in diabetic patients with micro- secondary coronary revascularization) 688 (322 men, 59±8 years) out of and macrovascular complications. Recent studies have shown that serum 731 consecutive type 2 diabetic patients with ≥1 additional risk factor who osteoprotegerin (OPG) concentrations correlate with severity of peripheral had been prospectively screened between 1992 and 2006 for SMI by stress arterial disease (PAD) in patients without type 2 diabetes mellitus (T2DM). myocardial scintigraphy and for silent CAD by coronary angiography. However, no data exist on the association between serum OPG levels and SMI was found in 207 (30.1%) patients and CAD in 76 of those with SMI. A severity of PAD and peripheral neuropathy (PN) in patients with T2DM. The total of 98 patients had a fi rst cardiovascular event during a 5.4±3.5 [range: aim of this study was to look for potential association between serum OPG 0.1-19.2] year period. Both SMI and CAD predicted signifi cantly cardiovascular levels and severity of PAD and PN in patients with T2DM. events. Cox regression analysis considering parameters which predicted A total of 74 patients (148 feet) with T2DM were recruited (mean age cardiovascular events in univariate analyses but not SMI and CAD (“routine 67.8±9.0 years, duration of diabetes 15.3±10.9 years). Serum OPG levels assessment”) showed that macroproteinuria (hazard ratio 3.33 [1.74-6.35], were measured using ELISA. PAD was diagnosed by means of ankle-brachial p<0.001), duration of follow-up after year 2000 <10% of the overall follow- index (ABI≤0.9). Patients with ABI values >1.3 were excluded from further up (HR 3.74 [2.10-6.66], p<0.001) and peripheral/carotid occlusive arterial analysis (8 feet). Diagnosis of PN was based on neuropathy disability score disease (PCOAD: HR 4.33 [2.15-8.71], p<0.001) independently predicted (NDS) and vibration perception threshold (VPT). events. SMI (HR 1.76 [1.00-3.12], p=0.05) or CAD (2.28 [1.24-4.57], p<0.01) Patients with PAD (n=27) had signifi cantly higher serum OPG levels in added into the model were also independently associated with events. SMI comparison with those without PAD (17.97±4.88 vs 14.84±4.83 pmol/l, added to prediction of an event at 5-year of follow-up above and beyond

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A141 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

routine risk prediction (c statistic with or without SMI 0.788 [0.720-0.855] case, the ECG was classifi ed as either likely AMI or likely non-AMI by the and 0.705 [0.616-0.794]; Hosmer-Lemeshow χ2 5.13, p=0.643 and 1.34, cardiologists or the CA. “Gold standard” was the fi nal clinical diagnosis. p=0.932, respectively). Statistical analysis was McNemar’s test with continuity correction. To conclude, SMI and silent CAD are predictive for cardiovascular events Results: The 155 DM2 patients were 50% male, mean age 56.8 ± 12.0 in type 2 diabetic patients independently of routine assessment, especially yrs; 44 pts had a fi nal clinical diagnosis of AMI (17 ST elevation MI [STEMI], represented by PCOAD, macroproteinuria and more intensive treatment 27 non-ST elevation MI [NSTEMI]) and 111 had no AMI. Diagnostic results after year 2000. in Table: POSTERS Complications & 512-P Acute and Chronic Evaluation of Clinical Predictors of Vulnerable Coronary Plaque Detected by CT Angiography in Asymptomatic Patients with Type 2 Diabetes (T2DM) KAZUYA FUJIHARA, HIROAKI SUZUKI, AKIRA SATO, YORIKO HEIANZA, SATORU KODAMA, KAZUMI SAITO, KAZUTO KOBAYASI, AKIMITSU TAKAHASHI, SHIGERU YATOU, NOBUHIRO YAMADA, HIROHITO SONE, HITOSHI SHIMANO, Mito, Japan, Tsukuba, Japan This study aimed to elucidate the clinical predictors of vulnerable coronary plaque by 64-slice CTA in asymptomatic patients with T2DM. From April 2009 to November 2010, 73 Japanese T2DM patients who had a maximum carotid intima-media thickness (IMT) ≥1.1 mm, electrocardiogram (ECG) abnormality, or positive exercise ECG test results underwent 64-slice Conclusions: Relative to standard 12L ECG read by cardiologists, CA CTA. Luminal narrowing of ≥50% on CTA was defi ned as indicating CAD. showed signifi cant gains in sensitivity for ACS diagnosis in DM2 patients, Vulnerable coronary plaque was considered present in patients with CAD without loss in specifi city. Sensitivity gains were particularly high in patients with positive vessel remodeling and low-attenuation plaque (<50 HU). exhibiting NSTEMI, the most common form of AMI in DM2. Quantitative Vulnerable carotid plaque was defi ned as echolucent plaque on carotid 3D analysis of ECGs in DM2 patients with suspected AMI may improve B-mode ultrasound. diagnostic accuracy relative to the standard 12-lead ECG. Logistic regression analyses were performed to identify variables related to vulnerable coronary plaque. Forty-three patients had coronary & 514-P artery stenosis, 21 of which had vulnerable coronary plaque. Hypertension Survival Following Acute Myocardial Infarction in South Asians (p=0.033), duration of diabetes (p=0.037), and Max IMT (p=0.002) were and White Europeans in the UK independent predictors of coronary artery stenosis. LDL-C/HDL-C (L/H) NITIN N. GHOLAP, RAJ L. MEHTA, MELANIE J. DAVIES, IAIN SQUIRE, KAMLESH (p=0.008) and vulnerable carotid plaque (p=0.014) were independent KHUNTI, Leicester, United Kingdom predictors of vulnerable coronary plaque. Max IMT ≥1.7 mm predicted Some UK studies have reported higher case-fatality rates following acute coronary artery stenosis with sensitivity of 88.4%, specifi city of 58.6%, myocardial infarction (AMI) in British South Asian (SA) driven mainly by positive predictive value (PPV) of 76.0%, and negative predictive value (NPV) higher rates of diabetes mellitus (DM) compared to white European (WE) of 77.3%. The combination of Max IMT ≥1.7 and L/H ≥2.5 predicted the patients. Following AMI, both antecedent DM and admission hyperglycaemia presence of vulnerable coronary plaque with sensitivity of 76.2%, specifi city regardless of DM predicts adverse outcomes. We compared survival rates of 78.0%, PPV of 59.3%, and NPV of 89.0%. post AMI in relation to abnormal glucose metabolism in SAs and WEs drawn These data suggest that measurement of carotid max IMT, echolucency from a multi-ethnic UK population. and the L/H ratio may improve selection of patients with T2DM for CTA. We conducted a retrospective cohort study of 4111 (SAs 18%) consecutive Sensitivity, specifi city, PPV and NPV for vulnerable coronary plaque patients with AMI admitted between October 2002 and September 2008. Cox regression models were constructed to identify determinants of 30- Sensitivity Specifi city PP NPV days and 1-year mortality, entering ethnicity, random admission blood (%) (%) (%) (%) glucose and antecedent DM individually and together along with other Max IMT carotid artery ≥1.7 100.0 43.1 42.0 100.0 relevant variables. Vulnerable carotid plaque 66.7 66.7 45.2 82.9 SAs were younger (62.0 vs. 67.3 years, p < 0.05) and more likely to have L/H ≥2.5 76.2 58.8 43.2 58.7 hypertension (55.2% vs. 49.4%, p = 0.004) or DM (39.7% vs. 16.1%, p < 0.05) at presentation compared to WEs. Thirty day and 1-year mortality were L/H 4.0 78.6 90.2 54.5 75.4 ≥ 10.0% and 15.2% in SAs compared to respectively 9.9 % and 16.7 % in WEs. Max IMT carotid artery ≥1.7 and L/H ≥2.5 76.2 78.0 59.3 89.0 On multivariate analysis (including antecedent DM but excluding admission Max IMT carotid artery ≥1.7 or L/H ≥4.0 100.0 39.2 40.3 100.0 blood glucose) the association of SA ethnicity with mortality was signifi cant Vulnerable carotid plaque or L/H ≥4.0 81.0 62.7 47.2 88.9 (HR 1.48, CI 1.03, 2.13). However on inclusion of admission blood glucose in the model, this association of ethnicity with mortality became non- signifi cant (HR 1.31, CI 0.86, 1.99). Conversely each unit (mom/L) increase in admission blood glucose was associated with 7% increase in mortality (HR 1.07, CI 1.04, 1.10), after adjusting for all covariates. Furthermore exclusion & 513-P of ethnicity and antecedent DM from the model did not alter the predictive Quantitative 3-Dimensional Electrocardiography Substantially WITHDRAWN value of admission blood glucose (HR 1.08, CI 1.05, 1.10). Similar associations Improves Diagnosis of Acute Myocardial Infarction in Type II Diabetic were observed for one year mortality. Patients Despite higher prevalence of DM in SAs, their mortality post AMI was IHOR GUSSAK, DHANUNJAYA LAKKIREDDY, SUBBA VANGA, SUDHA BOMMANA, similar to WEs. Furthermore, admission hyperglycaemia more so than BRIAN J. WENZEL, GORAN SIMIC, BOSKO BOJOVIC, SAMUEL GEORGE, DORIN antecedent DM was an important predictor of increased mortality post PANESCU, Santa Clara, CA, Kansas City, KS, Kansas City, MO AMI. To improve survival, active management of admission hyperglycaemia Background: Acute myocardial infarction (AMI) diagnosis in type II should be considered in patients with AMI, regardless of DM or ethnicity. diabetes (DM2) patients is diffi cult and ECG fi ndings are often inconclusive. Supported by: CLARHC Project We developed computer algorithms (CA) to process standard 12-lead ECG input data for quantitative 3-dimensional analysis, which analyzes an array of over 100 3D-based AMI diagnostic markers, which we hypothesized may & 515-P improve AMI diagnostic accuracy in DM2 patients. Myocardial Metabolic Flexibility in Complicated Type 2 Diabetes Methods: We identifi ed 155 consecutive DM2 patients age >25 yrs with JACQUELINE T. JONKER, NATHANJA TJEERDEMA, MIEKE T.J. BUS, HILDO J. chest discomfort or shortness of breath who were evaluated at an urban LAMB, AAN V. KHARAGJITSINGH, ALBERT DE ROOS, JOHANNES A. ROMIJN, emergency department (130 pts) or the cardiac cath lab (25 pts) for possible JOHANNES W.A. SMIT, Leiden, The Netherlands, Hague, The Netherlands AMI. The fi rst digital 12-lead ECG for each patient, obtained within 30 min A very low calorie diet induces an increase in plasma levels of non- of presentation, was evaluated by 2 blinded expert cardiologists. The same esterifi ed fatt y acids (NEFA). This is associated with an increase in myocardial fi le was then analyzed by the CA for quantitative 3D ECG analysis. In each triglyceride (TG) content and a decrease in diastolic cardiac function in

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A142 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES healthy subjects and patients with uncomplicated type 2 diabetes mellitus (DM2). Whether this fl exibility of myocardial TG content is also present in DM2 patients with cardiovascular complications is yet unknown. Therefore we aimed to assess whether myocardial TG accumulation and cardiac function are amendable to dietary intervention by a 3-day very low calorie (VLCD) diet in DM2 patients with cardiovascular complications. Fourteen patients with DM2 were included (age: 57±3 years, BMI 28.9± 1.1 kg/m2, HbA1c: 7.7 ± 0.4%). Inclusion criteria were: ischemia on single POSTERS photon emission computed tomography and/ or >70% occlusion at computed Complications tomography coronary angiogram. Left ventricular (LV) function, myocardial Acute and Chronic TG content and hepatic TG content were determined using magnetic Supported by: NIH DK080756 ADA-Funded Research resonance (MR) imaging and proton MR spectroscopy before and after a 3-day VLCD (471 kcal/day). We calculated diastolic deceleration of the early & 517-P fi lling phase (E deceleration) and the ratio between early and atrial fi lling Lung Fibrosis as a New Possible Diabetic Complication Is Pre- phase (E/A ratio) as measures for LV diastolic function. dominantly Induced by Angiotensin II We found plasma levels of NEFA were 0.63 ± 0.08 mmol/l at baseline JUNLING YANG, YI TAN, LINING MIAO, LU CAI, Changchun, China, Louisville, KY and increased to 1.22 ± 0.08 mmol/l after the VLCD (P<0.05). Myocardial TG Diabetic patients have a high risk of pulmonary dysfunction, particularly content increased from 0.66 ±0.08% at baseline to 0.89 ±0.08% after the with restrictive dysfunction, suggesting a fi brotic process. Clinical VLCD (P<0.05). This was associated with a decrease in LV diastolic function histological examination also showed the existence of lung fi brosis in (E/A ratio: 1.03±0.06 (baseline); 0.92±0.06 (VLCD), P<0.05). Plasma glucose diabetic patients compared to non-diabetic patients. The present study levels and hepatic TG content did not signifi cantly change. aimed at defi ning whether and how diabetes causes lung fi brosis. Lung In conclusion, a short-term very low calorie diet in patients with type samples from streptozotocin-induced type 1 diabetic mice, spontaneously 2 diabetes mellitus with cardiovascular complications increases plasma developed type 1 diabetic OVE26 mice and their age-matched controls, were non-esterifi ed fatty acids and is associated with increased myocardial investigated with histopathological and biochemical analysis. In diabetes, TG accumulation and a decrease in diastolic cardiac function. This fi nding histological examination with Sirius-red staining and H & E staining showed indicates that patients with DM2 and cardiovascular complications are still signifi cant fi brosis, along with massive infl ammatory cell infi ltration. Lung susceptible to dietary interventions, thereby opening perspectives to dietary fi brosis and infl ammation were confi rmed by real time PCR and Western interventions aimed at improving cardiac function blotting assays for the increased fi bronectin, connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 and tumor necrosis factor-α & 516-P mRNA and protein expressions. Diabetes also signifi cantly increased NADPH Diet-Induced Obesity and Weight Loss Following Low-Fat Diet Exert oxidase (NOX) expression and protein nitration, along with up-regulation Reciprocal Effects on Cardiac Remodeling and Diastolic Function in of angiotensin II (Ang II) and its receptor expression. In vitro exposure of Mice lung fi broblasts to Ang II increased CTGF expression in a dose- and time- YOSHIHIRO AZUMA, DAE YOUNG JUNG, UMBER SHAFIQ, JASON K. KIM, Wor- dependent manner, which could be abolished by inhibition of superoxide, cester, MA NO, and peroxynitrite accumulation. Furthermore, chronic infusion of Ang Cardiac remodeling and ventricular hypertrophy are major characteristics II to normal mice at a subpressor dose induced diabetes-like lung fi brosis, in obese and diabetic subjects. In this study, we examined the effects of diet- and Ang II receptor AT1 blocker (losartan) abolished the lung fi brotic and induced obesity on cardiac parameters including diastolic function. C57BL/6 infl ammatory responses in diabetic mice. These results suggest that lung mice at 5 months of age were fed a high-fat diet (HFD) for 2 months, and fi brosis is able to be induced directly by diabetes and Ang II plays a critical echocardiography was performed before and after HFD (n=12). Age-matched role in diabetic lung fi brosis via NOX activation-mediated nitrosative control mice were fed chow diet (n=6) and showed no changes in any of the damage. ADA-Funded Research cardiac parameters. HFD increased body weight by ∼50% (Fig. 1; *P<0.05), and caused a ∼20% increase in left ventricular posterior wall thickness (LVPW; Fig. 2) with a signifi cant reduction in LV internal dimension, suggesting Guided Audio Tour: Cardiovascular Disease Risk Factors in Human Diabetes ventricular hypertrophy. LV mass and cardiac output were not altered after (Posters 518-P to 525-P), see page 11. HFD (Fig. 3), but diastolic function (E’; early ventricular relaxation) measured using tissue Doppler imaging was reduced by ∼15% following HFD (Fig. 4). This resulted in a signifi cant decrease in E’/A’ (atrial contraction) in HFD- & 518-P fed mice. Our second study examined the effects of weight loss on cardiac Effects of Rosuvastatin on Levels of Serum YKL-40 and Urine Albumin parameters of obese mice. C57BL/6 mice were fed HFD for 6 months to Excretion in Type 2 Diabetic Patients with Hypercholesterolemia induce obesity, and were switched to a low-fat diet (LFD) for 2 months (n=6). YOSHIFUMI SUZUKI, TAKAMICHI YAMANE, YUKIE SAKUMA, RIE IWAI, MASAKO LFD caused a 20% reduction in weight loss in obese mice (Fig. 1). Weight TAGUI, SHOJI YOSHIDA, NAOTAKE HASHIMOTO, Asahi, Japan, Yachiyo, Japan YKL-40, secreted from macrophages in atherosclerotic plaques and loss resulted in ∼15% decreases in LVPW (Fig. 2) and interventricular septum wall thickness, and a 30% reduction in LV mass (Fig. 3). Diastolic function vascular smooth muscle cells, is associated with infl ammation and (E’) tended to improve following LFD (Fig. 4). Thus, these data demonstrate endothelial dysfunction. Increasing levels of albuminuria are closely linked that obesity-induced cardiac remodeling and LV hypertrophy are improved by to an increased risk of cardiovascular disease. In our preceding study, serum weight loss in mice. Importantly, our fi ndings of reduced E’ after a short-term YKL-40 levels were strongly correlated with urine albumin excretion, and HFD and improved E’ following weight loss indicate an important role of a multiple logistic regression analysis identifi ed YKL-40 as a signifi cant diastolic abnormality during early cardiac remodeling in obese mice. independent predictor of microalbuminuria in type 2 diabetic patients with normo- or microalbuminuria. Recently lipid-lowering therapy with statins has been reported to show the regression of atherosclerotic plaques, suggesting that statins exert a variety of several “pleiotropic actions”. The aim of the study is to assess the effects of therapy with rosuvastatin(Ros) on markers for endothelial function including YKL-40 and urine albumin excretion in patients with type 2 diabetes. A total of 96 patients with normo- or microalbuminuria, and with LDL-C≥120mg/dL were treated with Ros 2.5 or 5mg for 24 weeks. Ros therapy signifi cantly improved lipid parameters (LDL-C: 141±15 vs. 89±24mg/dL, p<0.0001, HDL-C: 52±11 vs. 54±12mg/ dL, p=0.016). Serum YKL-40 levels were signifi cantly decreased by Ros (132.0±81.5 vs. 110.9±70.7ng/mL, p<0.0001). The decrease in YKL-40 induced by Ros was correlated with the reduction in LDL-C (r=0.269, p=0.0027) and the increase in HDL-C (r=-0.255, p=0.013). Urine albumin-to-creatinine ratio was also reduced (52.7±70.6 vs. 44.3±56.3 mg/gCre, p=0.017). In addition, Ros was evidently reduced serum levels of infl ammatory markers such as hs-CRP (133±166 vs. 88±107μg/dL, p=0.0002) and IL-6 (1.9±1.3 vs. 1.7±1.0

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A143 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

pg/mL, p=0.0052). In conclusion, Ros improved levels of serum YKL-40 to fi brin (KD 299±115 and 79±25 nM for diabetic and control plasminogen, and urine albumin excretion, associated with endothelial function, and of respectively; p<0.05). Poor glycaemic control was associated with impaired infl ammatory markers in addition to lipid-lowering effects. These fi ndings plasmin activity, partly corrected by improving glycaemia. Plasminogen may in part explain the stability and regression of atherosclerotic plaques glycation at baseline and post intervention was (34.6±1.5 and 28.1±2.4 induced by statins. Such evidence could suggest a new therapeutic use µmol/L, respectively; p<0.05) compared with 5.7±3.9 µmol/L for controls of statins in the prevention of development of nephropathy and vascular (p<0.01). MS of diabetes plasminogen showed increased fructosyl-lysine complications in patients with type 2 diabetes. product with preferential glycation of residues lysine 107 and 557 in vitro, providing a mechanistic explanation for altered plasminogen-fi brin POSTERS Complications & 519-P interaction and decreased plasmin protease activity, respectively. Acute and Chronic Similar Circulating Concentrations of Adhesion Molecules between We demonstrate that glycation alters plasminogen-fi brin interactions Type 1 Diabetes and LADA Patients: Results from the Action LADA and affects plasmin generation and protein activity. These changes can be Study modulated by improving glycaemic control, identifying a previously unknown MINH-NGUYET PHAM, MOHAMMED HAWA, MICHAEL RODEN, GUNTRAM link between plasma glucose levels, fi brinolytic potential and cardiovascular SCHERNTHANER, PAOLO POZZILLI, RAFFELLA BUZZETTI, WERNER SCHERBAUM, risk in subjects with diabetes. JOCHEN SEISSLER, HUBERT KOLB, STEVEN HUNTER, RICHARD DAVID LESLIE, Supported by: Diabetes UK NANETTE CATHRIN SCHLOOT, ACTION LADA STUDY GROUP, Düsseldorf, Germany, London, United Kingdom, Vienna, Austria, Rome, Italy, Belfast, United & 521-P Kingdom Infl uence of a Short-Term Change in Glucose and Cholesterol Con- Systemic concentrations of adhesion molecules are associated with centra tions on Aspirin Esterase Activity in Patients with Type 2 increased cardiovascular complications. We compared patients with type Diabetes Mellitus 2 diabetes known to be at high risk for cardiovascular disease with patients ALEJANDRO GUGLIUCCI, KAZUHIKO KOTANI, SHUN ISHIBASHI, RUSSELL CACCA- who have type 1 diabetes or LADA (Latent Autoimmune Diabetes in Adults) VELLO, MICHIAKI MIYAMOTO, Vallejo, CA, Tochigi, Japan and hypothesized that these immune mediators are elevated in patients While aspirin is one of the widely-used drugs for the prevention of with type 2 diabetes. atherothrombosis in patients with diabetes mellitus (DM), further research We analyzed serum concentrations of sE-Selectin, sICAM-1 and sVCAM-1 regarding aspirin metabolism are still required. Although few studies have by multiplex beads based technology in 90 type 1 diabetes (age 45.3±10.1 investigated the intrinsic mechanisms of aspirin resistance, the alteration yrs, F/M=28/62), 61 LADA (51.8±10.2 yrs, F/M=35/26 yrs), 465 type 2 of serum aspirin esterase (AE) activity, which participates in aspirin diabetes (54.9±9.1 yrs, F/M=202/263) and 41 healthy subjects (47.2±9.4 yrs, pharmacokinetics, may account for part of the mechanisms. Some studies F/M=25/16). Diabetes duration in all patients obtained from the Action LADA have shown increased activity of AE in diabetic patients but there has cohort was maximum 5 years. Multivariate regression analysis was used to been controversy. The relationship between AE and DM remains yet to compare cytokines of four groups adjusted for sex, age, BMI, blood pressure be fully established and deserves further exploration. Since these studies and diabetes duration. were performed with a cross-sectional study design, prospective and Increased concentrations of adhesion molecules were detected in patients intervention studies are also required. In addition, it would be of interest with type 2 diabetes (p < 0.02). Type 1 diabetes, LADA and control subjects to know whether glycemic control can enhance aspirin effectiveness The were similar in their immune mediator levels. Signifi cantly increased levels aim of the present study was to investigate the infl uence of a short-term of adhesion molecules in type 2 diabetes compared to type 1 diabetes, change of plasma glucose and serum lipid concentrations on serum aspirin LADA and healthy subjects persisted in spite of adjusting for age, sex, BMI, esterase (AE) activity, which can contribute to aspirin effectiveness and its blood pressure and diabetes duration (p < 0.04). Diastolic and systolic blood resistance, in DM patients. The glucose, lipid and AE levels were measured pressure were positively associated with sE-Selectin adjusted for age, sex in the pre- and post-phase of a 2-week intervention program on glycemic and BMI in patients (p < 0.045). Higher BMI and increased concentrations of control, based on the diet therapy with an increased physical activity, adhesion molecules were positively associated in all groups (p < 0.0001). among patients with type 2 DM (n = 19, mean age = 57.4 years, hemoglobin Overall, our results show that metabolic confounders affect systemic A1c = 8.5%). During the period that produced improvement of metabolic concentrations of adhesion molecules that associate with increase measures, age- and sex-adjusted linear regression analyses revealed that cardiovascular risk. However, even after adjustment for these confounders changes in AE were signifi cantly and positively correlated with those of total type 2 diabetes had higher concentration of these markers suggestive of cholesterol (correlation coeffi cient = 0.521, P < 0.05) and glucose (0.469, P a diabetes type related effect. LADA and type 1 diabetes were similar, < 0.05), respectively. These data suggest that improvement of glucose despite their different pathogenic process leading to slower deterioration and cholesterol measures may contribute to aspirin effectiveness in DM of β-cell function in LADA and faster deterioration of β-cell function in type patients. The fi ndings warrant more studies in this area. 1 diabetes. & 522-P & 520-P Postprandial Hyperglycemia and/or Hyperinsulinemia Is Associated Fructosamine-Modifi ed Plasminogen in Diabetes Reversibly Inhibits with Cardiovascular Risk Factors in Healthy Male Subjects Fibrinolysis by Reducing Plasminogen-Fibrin Interactions and Alter- YOSHINORI MIYAZAKI, MAKOTO FURUGEN, HIROSHI AKASAKA, SHIGEYUKI ing Plasmin Activity SAITOH, TETSUJI MIURA, Muroran, Japan, Sapporo, Japan RAMZI A. AJJAN, TOBY GAMLEN, KRISTINA F. STANDEVEN, SALIHAH MUGHAL, Using the measurements in medical check-up with 75g-OGTT, we KATHARINA HESS, FLADIA PHOENIX, PAUL J. THORNALLEY, M.M. ANWAR, N. investigated associations between cardiovascular risk factors and plasma RABBANI, HELEN PHILIPPOU, PETER J. GRANT, Leeds, United Kingdom, Aachen, levels of glucose (PG) and insulin (PI) in healthy male subjects (n=620) who Germany, Warwick, United Kingdom were not taking any medications known to affect glucose tolerance, blood Fibrinolysis is dependent on plasminogen/fi brin interactions and inhibition pressure (BP) or plasma lipids. According to 2003 ADA criteria, subjects of this process is associated with atherothrombotic events in diabetes. We were classifi ed as NFG/NGT (N, n=499), isolated IFG (iIFG, n=63), isolated investigated the effects of plasminogen glycation on plasmin generation, IGT (iIGT, n=29), and combined IFG and IGT (IFG/IGT, n=29). Insulin action was protein activity, interaction with fi brin and fi brinolysis using plasma samples evaluated by HOMA-IR (an index of hepatic insulin resistance) and Matsuda and purifi ed protein from type 1 diabetes (T1DM) subjects before and index (an index of whole body insulin sensitivity) {=10,000/ [(FPGxFPI) after improving glycaemic control. Plasminogen activity was measured by x(meanPGxmeanPI)]1/2} calculated from the OGTT. Age and BMI were similar chromogenic, turbidimetric and conversion assays, binding to fi brin using in all 4 groups. HOMA-IR was higher in iIFG (2.38) and IFG/IGT (2.58) compared plasmon resonance and glycation by GlyPro test/mass spectrometry (MS). with N (1.79) (p<0.05). Matsuda index was lower in iIFG (6.74), iIGT (6.26) and HbA1c reduction by 0.9% in 27 subjects was associated with decreased IFG/IGT (5.01) compared with N (9.94) (p<0.05). Mean BP (MBP) was higher plasma clot lysis from 516±16 to 400±16 sec (p<0.001), with no change in iIGT (101mmHg) versus N (92mmHg) (p<0.01). Plasma triglyceride (TG) level seen in clots made from purifi ed fi brinogen, indicating the difference with and TG/HDL ratio were higher in IFG/IGT versus N (173 vs. 128mg/dl and 3.99 plasma clots is fi brinogen independent. Diabetes-purifi ed plasminogen vs. 2.78, both p<0.01), although HDL, LDL and LDL/HDL ratio were similar in demonstrated reduced fi brinolytic potential compared with control protein the groups. In backward stepwise multiple regression analysis using age, (723±16 and 317±0.4 sec, respectively; p<0.01), which was partly restored by BMI, PG and PI at 0, 60, 120 min during the OGTT as independent variables, improving glycaemia (450±8 sec, p<0.05 compared with baseline). Diabetes increases in BMI, PG120 and PI60 were independently related to increases altered both fi brin-dependent plasmin generation and plasminogen binding in MBP (p<0.0001) and TG/HDL ratio (p<0.0001). Increases in BMI and PI60

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A144 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES were independently related to an increase in LDL/HDL ratio (p<0.0001). 42.36% of the cohort reached adequate control of blood glucose Matsuda index inversely correlated to MBP (r=-0.19, p<0.001), TG/HDL ratio (HbA1C<6.5%), blood pressure (< 130/80 mmHg), or blood lipid (LDL-C<2.5 (r=-0.15, p<0.001) and LDL/HDL ratio (r=-0.15, p<0.01), although HOMA-IR mmol/L), respectively while only 4.22% reached all three targets. There positively correlated to only MBP (r=0.13, p<0.01) but not to TG/HDL ratio or was a statistically signifi cant association of CVD risk factors with micro- LDL/HDL ratio. These results indicate that postprandial hyperglycemia and/ and macro-vascular complications (p<0.001) except peripheral vascular or hyperinsulinemia due to the impairments of whole body insulin sensitivity disease, and hypertension was a stronger indicator (Figure 1).While 40% in addition to obesity are closely related to risk factors of atherosclerotic T2D patients reached adequate blood glucose control, only 4.22% also macrovascular disease in pre-diabetic male subjects. reached blood pressure and lipid control. Without signifi cant effort,rapid POSTERS

rise of CVD incidence would result in signifi cant social and economic burden. Complications & 523-P A longitudinal study to determine the clinical outcomes of better control of Acute and Chronic Genetic Variability in Adapter Proteins with PH Domain, PTB Domain CVD risk factors is thus necessary. and Leucine Zipper Motif 1/2 (APPL1/2) Is Associated with the Risk of Coronary Artery Disease in Type 2 Diabetics in a Chinese Population XIAOWEI MA, SHAN DING, XIAODAN MA, NAN GU, XIAOHUI GUO, LINONG JI, Beijing, China Adaptor proteins containing PH domain, PTB domain, and leucine zipper motif 1 and 2 (APPL1/2) play a key role in cell proliferation as homodimers or heterodimers in many tissues. APPL1 or 2 as an adaptor for adiponectin receptors mediates the signaling pathway of adiponectin which acts as an anti- atherosclerotic adipokine. This study aimed to investigate whether or not genetic variations in the APPL1/2 genes mediating these effects affect the risk of coronary artery disease (CAD) in Chinese type 2 diabetic patients.7 haplotype-tagging single nucleotide polymorphisms (SNPs) were selected from CHB HapMap database (Phase II) and total 203 CAD-positive cases and 106 CAD-negative controls with type 2 diabetes were genotyped for the Supported by: Merck 7 tag-SNPs by the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) assay.The minor allele G of rs4640525 at APPL1 & 525-P locus was protective from CAD in type 2 diabetic patients, with the carriers Time-Varying Effects of Blood Pressure on Cardiovascular Disease— of genotype CC at higher risk of CAD compared to non-carriers (OR =2.830, A Simulation Study 95%CI 1.285-6.230, p = 0.010; OR’ =4.992, 95%CI =1.758-14.173, p’ = 0.003, KERENAFTALI KLEIN, KAMLESH KHUNTI, SANJOY PAUL, Brisbane, Australia, after adjustment for the other known CAD risk factors); the homozygotes of Leicester, United Kingdom AA at rs11112412 in APPL2 gene had higher risk of CAD compared to those The blood pressure (BP) ranges associated with vascular events in Type of GG (adjusted OR’ =5.697, 95%CI 1.006-32.257, p’ = 0.049).We concluded 2 diabetes patients is not clear. Our objective was to determine the time- that genetic variation(s) in APPL 1/2 is associated with CAD risk in type 2 varying effects of high and low BP on macro-vascular events (MVE) and all diabetes in the Chinese population. cause mortality (ACM) in a virtual diabetes population who were thought to be under intensive or standard glucose and BP control therapy. Table. 1 The association of the SNPs in AMPKα2 gene with the risk of CAD Using the distributions of baseline age, weight, HbA1c and longitudinal BP SNP Genotype CAD (n) Non-CAD(n) OR p OR’ p’ measurements over 5 years, observed in the BP arm of ADVANCE study, 5 appl1 rs4650525 CC 191 90 2.830 0.010 4.992 0.003 sets of 5-years followup data on 10000 patients were simulated. Mean (SD) age of patients was 66 (6) years, weight 78 (17) kg, HbA1c 7.5 (1.6) %, SBP CG+GG 12 60 1 1 / DBP 137 (21) / 78 (10) mmHg at baseline. Time to event data for MVE and APPL2 rs11112412 AA 15 53 1.325 0.583 5.697 0.049 ACM were simulated using reported event rates for two arms of the study. AG 80 44 0.964 0.884 1.086 0.798 The simulated event rates were 8 and 7% on average respectively for MVE and ACM in the standard treatment group. The event rates and distributions GG 100 6 1 1 of risk factors over time were consistent in all simulated data sets. ‘p: adjusted for age, sex, BMI, duration of diabetes mellitus, lipid profi le, Adjusting for baseline age, weight and treatment, 10 mmHg rise in SBP hypertension and smoking status and DBP increased the risk of MVE by 24% (CI of HR: 1.09 – 1.29) and ACM Association of SNPs at APPL1/2 loci with CAD in Type 2 Diabetics by 17% (CI of HR: 1.10 – 1.26) respectively. Compared to SBP range of 105 – Supported by: National 973 Project (2006CB503903,2006CB503908) 129 mmHg, subjects with SBP between 130 – 139 mmHg had 46% and 13% higher risk for MVE and ACM respectively. Compared to normal DBP (70 – 79 & 524-P mmHg), subjects with DBP < 70 mmHg indicated possible increased risk of Nationwide Assessment of Cardiovascular Risk Control in Type 2 MVE (CI of HR: 0.85 – 1.26) and ACM (CI of HR: 0.67 – 1.03). The risks were Diabetics in China-CCMR-3B Study explored for various categories of BP levels compared to normal BP (Table). This novel simulation approach provides robust estimates of time-varying LINONG JI, DAYI HU, CHANGYU PAN, GUANGWEI LI, CHANGSHENG MA, GUANG effects of low and high BP on CV risks on a large virtual patient population NING, XUEWANG LI, YONG HUO, XINGWU RAN, JIANPING WONG, QIUHE JI, with characteristics very close to a recent mega trial, and emphasises the BENLI SU, RENMING HU, HAN LEI, QIFU LI, CHUANMING HAO, DANYI ZHANG, urgency for treatment of hypertension in diabetes patients. CCMR-3B, Beijing, China, Shanghai, China, Cheng Du, China, Guangzhou, China, Xi An, China, Da Lian, China, Chongqing, China, Berwyn, PA Diabetes prevalence has grown rapidly in China. It potentiates signifi cant BP, mmHg Macrovascular Event All Cause Mortality burden of cardiovascular disease (CVD). China Cardiometabolic Registries HR 95% CI HR 95% CI (CCMR), the largest clinical outcomes assessment program, has been SBP 105-129 Reference designed to establish systemic evaluation on disease progression and infl uencing factors. As part of CCMR, the Nationwide Assessment of CVD SBP < 105 0.98 0.67 - 1.45 0.92 0.60 - 1.41 Risk Factors: Blood Pressure, Blood Lipid, and Blood Glucose, in Chinese SBP 130-139 1.46 1.16 - 1.82 1.13 0.87 - 1.47 Patients with Type 2 Diabetes (T2D) (CCMR-3B) was conducted to assess SBP ≥ 140 1.58 1.30 - 1.91 1.58 1.29 - 1.95 the level of CVD risk control and its impact on incidence of diabetic DBP 70-79 Reference complications. CCMR-3B is targeting to enroll 25,000 diabetic patients across all major regions of China from all three tiers of hospitals. T2D DBP < 70 1.04 0.85 - 1.26 0.83 0.67 - 1.03 patients with diagnosis for 6 months or more are enrolled from out-patient DBP ≥ 80 1.24 1.04 - 1.47 1.11 0.92 - 1.34 clinics by endocrinologists, cardiologists, and nephrologists simultaneously to ensure broad and representative patient population. A total of 5099 patients was included in this interim analysis. Among them were 41.89% male, 47.72% age 65 years or older, 32.52% smokers, and 55.38% BMI > 24 Kg/m2. The analysis showed that 39.38%, 31.65%,

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A145 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

526-P 528-P 8-Hydroxy-2-Deoxyguanosine Prevents Plaque Formation in a Altered Topoisomerase Activity Mediates Hyperglycemic Induced Partial Ligated ApoE Knockout Mouse, an Acute Model of Athero- Myocardial MitochondrialWITHDRAWN DNA (mtDNA) Damage sclerosis STEVEN HICKS, BRIAN PITEO, JAMIE MATHEW, SUSHMA MEDIKAYLA, NAZAR JOO YOUNG HUH, HANJOONG JO, MYUNG-HEE CHUNG, HUNJOO HA, Seoul, LABINSKYY, JOHN G. EDWARDS, Valhalla, NY Republic of Korea Diabetic cardiomyopathy initially presents with diastolic dysfunction, Atherosclerosis is a progressive disease characterized by the accumulation but evolves into decompensated eccentric hypertrophy. Mitochondrial

POSTERS of lipids and fi brous elements in the large arteries. Abnormal proliferation and dysfunction has a signifi cant role in the development and complications

Complications migration of vascular smooth muscle cells (VSMCs) along with accumulation of diabetic cardiomyopathy. Less clear are the mechanisms that lead Acute and Chronic of extracellular matrix (ECM) are important pathogenic mechanisms in lesion to failure since the mitochondria have defense mechanisms to manage development. Infl ammation and oxidative stress are known to be related in this diabetic-induced oxidant stress. This suggests that there may be limits process, but an effective therapeutic modality remains elusive. 8-Hydroxy- to adaptability or that the chronic hyperglycemia ultimately produces an 2-deoxyguanosine (oh8dG), which is known as a marker of oxidative stress, accumulation of errors with which the mitochondria are unable to cope. We has been recently reported to possess anti-oxidative and anti-infl ammatory have examined the impact of diabetes on myocardial mitochondria using effect. In this study, we investigated the effects of oh8dG on advanced lesion two models of chronically elevated glucose; in culture using the cardiac development in a partial carotid ligation model, which is a newly developed derived H9c2 cell line and neonatal cardiomyocytes and in vivo using the and more physiologically relevant model of disturbed fl ow. Partially ligated Goto-Kakizaki (GK) diabetic rat. 13 days of elevated glucose in H9c2 cells apoE knockout mice fed with high fat diet developed an advanced lesion in or neonatal cardiomyocytes resulted in signifi cant (p,.05) mitochondrial 2 weeks and oh8dG treatment signifi cantly reduced plaque formation along dysfunction that was accompanied by increased apoptosis as a latter with reduced superoxide formation, monocyte/macrophage infi ltration, and event. Chronically elevated glucose signifi cantly increased mtDNA damage ECM accumulation. Further studies performed with cultured VSMCs in vitro globally and across the region encoding for the three subunits of cytochrome showed that oh8dG inhibited FBS- or PDGF-induced proliferation. Also, rat oxidase (COX). Using the temporal temperature gradient electrophoresis aortic ring assay showed that oh8dG dramatically decreased PDGF-induced (TTGE) method to analyze left ventricle (LV) mtDNA mutations, we VSMC proliferation and migration. Angiotensin II-induced increase in rac1 observed a number of basepair substitutions or insertions in the 3’ end of activity was ameliorated by oh8dG pretreatment in VSMCs. This indicates a COX3 from GK LV, but not from Wistar LV controls. Concomitant with this possible involvement of rac1 in anti-infl ammatory and anti-oxidative effect was a signifi cant reduction in LV cytochrome oxidase activity. Signifi cant of oh8dG. Our results have shown for the fi rst time that oh8dG treatment increases in mitochondrial-dependent DNA cleavage were observed from suppresses plaque formation in vivo and VSMC activation in vitro possibly hyperglycemic cells. Immunoprecipitation of mitochondrial extracts with an through inhibition of rac1. This points to a novel mechanism of action of mtTOP1 antibody signifi cantly decreased DNA cleavage indicating at least oh8dG to reduce oxidative stress and infl ammation, and emphasize a new a partial role for mtTOP1. These results point towards a signifi cant role for therapeutic avenue to benefi t atherosclerosis. mitochondrial topoisomerases in mediating diabetic-induced myocardial Supported by: 800-20080663, 800-20090208; J.Y.H. supported by Brain Korea 21 mitochondrial DNA damage. The accumulation of mtDNA mutations and concomitant mitochondrial dysfunction may be the underlying pathology for 527-P many complications of diabetes, including vascular dysfunction and heart Accelerated Diabetic Complications in Japanese Type 1 Diabetes failure. with Nonalcoholic Fatty Liver Disease Supported by: Supported in part by NIH HL43023 and HD065551 CHIHIRO YONEDA, HIROYUKI MATSUURA, JUN OGINO, TAKENORI HARUKI, YOSHIFUMI SUZUKI, NAOTAKE HASHIMOTO, Yachiyo, Japan, Asahi, Japan 529-P Nonalcoholic fatty liver disease (NAFLD) is a signifi cant health problem and A Positive Relationship of Glycemic Levels with the Development there have been many reports of the association between type 2 diabetes, of Diabetic Cardiomyopathy and Serum CTGF Level in the Patients but the signifi cance of NAFLD in type 1 diabetes has not been fully discussed. with Type 2 Diabetes Therefore, we studied the association of NAFLD in Japanese type 1 diabetes YUZHI YANG, LIJUAN XU, KUN FENG, Harbin, China and attempted to clarify the phenotype of type 1 diabetes with NAFLD. The The objective of this study was to investigate the relationship of glycemic mean patient age was 56.8 and 46.8 years old with (N=15) and without levels with diabetic cardiomyopathy (DC) and serum connective tissue NAFLD (N=42) There was a signifi cant difference in the present body mass growth factor (CTFG) in type 2 diabetic patients. index (BMI) (P<0.0001) and in the maximal BMI in the past (P<0.05) between Fifty type 2 diabetic patients (25 with and 25 without DC) with HbA1c the two groups. Patients with NAFLD had a longer duration of diabetes more than 7% and 30 age-matched health adults were included for (P<0.05) and higher HbA1c (P=0.06). The patients with NAFLD had signifi cant monitoring the glucose levels continuously for three days by Continuous higher AST, ALT and g-GTP concentration and these data were coincident Glucose Monitoring System (CGMS), based on which the standard deviation with the presence of NAFLD diagnosed by ultrasonography. Moreover, of mean blood glucose (SDBG), the mean amplitude of glycemic excursions the dyslipidemia in triglyceride and HDL-cholesterol concentrations, and (MAGE), the largest amplitude of glycemic excursions (LAGE), the area under hypertension occurred more frequently in the NAFLD group; although a the curve (AUC), and the absolute mean of daily differences (MODD) were signifi cantly greater number of patients were treated using antihypertensive calculated. Blood serum CTGF levels were measured by ELISA assay, and its agents or HMG-CoA reductase inhibitors. Patients with NAFLD had a association with other general biochemical measurements were analyzed stronger family history of diabetes (P<0.05), and the prevalence of the fi rst among the groups. degree of family history was increased signifi cantly compared with patients Results showed: (1) CTGF levels were signifi cantly higher in DC teams without NAFLD (P<0.05). The treated basal total and per kg insulin doses and (982.78±263.56 ng/l) than that inT2DM without DC teams (435.45±172.31 shows that the patients with NAFLD required a higher dose of insulin (P<0.01, ng/l) or in healthy subjects (296.86±153.21 ng/l, F=76.25, P<0.05); (2) 2hPG, P<0.05) despite having higher HbA1c concentrations. The total insulin dose SDBG, MAGE,LAGE, MODD and AUC levels in the diabetic patients with DC was higher in NAFLD patients (P<0.05). Notably, urinary albumin excretion were remarkably higher than that of the patients without DC or the healthy (mg/g×creatinine) and the prevalence of carotid plaques were signifi cantly group (F=24.34, 31.67, 39.48, 30.75, and 24.69, respectively, P<0.05); (3) The higher in patients with NAFLD (P<0.05, P<0.05) compared with patients serum CTGF levels were positively correlated with 2hPG, 24h-ALB, SDBG, without NAFLD. Logistic analysis of the association with NAFLD showed LAGE, MAGE, MODD, and AUC levels (r=0.723, 0.695, 0.613, 0.524, 0.634, that basal insulin dose and BMI were independent risk factors for NAFLD 0.561, and 0.537, respectively, P< 0.05). (P<0.05). Urinary albumin excretion rate was associated with age, systolic These results suggest that glycemic increase may be related to the blood pressure and the presence of NAFLD (P<0.05). Our results suggest that development of DC by increasing profi brotic mediator CTGF that induce the presence of NAFLD in Japanese type 1 diabetes may be associated with myocardial fi brosis. Therefore, the increase in serum CTGF level may b a the progression of diabetic nephropathy and macroangiopathy. very important index in diagnosis and prognosis of DC.

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530-P associated with weight, gender, MVD, PAD and angina pectoris. Thus BNDF Asymmetric Dimethylarginine (ADMA) Does Not Affect Endothelial might be protective from macrovascular complications, which has to be Function in Subjects with Alterations of Glucose Regulation investigated in a case-control trial. DANIELA LUCCHESI, LAURA PUCCI, ROSAMARIA BRUNO, LORENZO GHIADONI, ELEONORA RUSSO, GIUSEPPE A. DANIELE, MONIA GAROFOLO, STEFANO TADDEI, 532-P ROBERTO MICCOLI, GIUSEPPE PENNO, MARKUS C. STUHLINGER, STEFANO DEL Clinical Outcomes Following Cardiac Rehabilitation in Diabetic vs. PRATO, Pisa, Italy, Innsbruck, Austria Non-Diabetic Patients—A Need for More Comprehensive Diabetic

ADMA has been related to endothelial dysfunction (ED) and athero- Cardiac Rehab POSTERS sclerosis. Increased ADMA has been described in type 1 and type 2 diabetic HORMOZ KIANFAR, SEEMA PATEL, HARMONY LEIGHTON, BHARATHI REDDY, Complications subjects with nephropathy or CVD. Studies assessing ADMA in people JOHN NICHOLSON, Flushing, NY Acute and Chronic with uncomplicated diabetes report confl icting results. ADMA, SDMA Background: Diabetes is a major risk factor for cardiac morbidity and (symmetrical dimethylarginine) and L-arginine (L-arg; HPLC), brachial artery mortality. fl ow-mediated dilation (FMD) and nitrate endothelium-independent dilation Cardiac rehabilitation (CR) has been shown to be benefi cial in both (GTN) were evaluated in 26 subjects with normal glucose tolerance (NGT), diabetic and non-diabetic patients, with signifi cant improvement in exercise 34 with pre-diabetes (IFG or IGT, preDM) and 18 with newly diagnosed capacity. However, due to increased macrovascular disease in diabetics, we type 2 diabetes (newT2DM; OGTT). Groups were similar for gender, hypothesize that diabetic patients will have worse long term clinical outcomes smoking, BMI, waist, dBP, total and LDL-C, apoA1 and apoB, fi brinogen, as compared to those without diabetes after completion of the CR. fasting insulin, eGFR and cystatin C. Age, fasting and post-load glucose, Methods: We performed an observational cohort study of 178 (54 diabetic glucose area under the OGTT curve (AUCgluc), HbA1c (6.0±0.4, 6.5±0.6 vs and 124 non-diabetic) patients who completed a minimum of 24 weeks 5.5±0.4%, p<0.0001) and triglycerides were higher in preDM and newT2DM; of CR from January 2008 to December 2009. We defi ned major adverse HDL-C was lower. In pre-DM, sBP (127±13) was in between NGT (118±15) cardiac events (MACE) as death, MI, stroke, coronary revascularizations and and newT2DM (137±15 mmHg, p=0.0005). GTN decreased (D% 9.9±3.4, hospitalization for heart failure or chest pain. A telephone questionnaire was 8.8±3.3 and 7.4±3.9; p=0.081) with signifi cant differences between NGT and used to determine events during a 16-month average follow-up period. newT2DM (p=0.025). FMD was lower in newT2DM (D% 4.4±3.3) and preDM Results: Indications for CR were similar between the two groups, with the (D% 6.0±2.8) than in NGT (D% 7.9±3.6, p=0.0017). L-arg was similar in NGT exception of diabetics undergoing more coronary bypass grafting (50% vs. (97.5±20.0 µmol/l) and preDM (97.1±20.6), but lower in newT2DM (81.2±18.9, 39 %). Baseline exercise capacity was similar in both groups and standard p=0.015). ADMA progressively reduced from NGT (1.33±0.96 µmol/l) to cardiac rehabilitation yielded similar improvements in exercise capacity. preDM (1.02±0.79, p=0.14 vs NGT) and newT2DM (0.72±0.53, p=0.017 vs During the follow up period post CR, MACE occurred at a signifi cantly higher NGT). SDMA was similar in NGT (1.78±0.74 µmol/l) and preDM (1.56±1.02), rate in diabetic patients as compared to non-diabetic patients (48% vs. 32% but lower in newT2DM (0.97±0.35, p=0.002 vs NGT, p=0.02 vs preDM). No p <0.05). Diabetic patients had higher rates of hospitalizations (20% vs. association was observed between ADMA (or SDMA) and eGFR or cystatin 10%), MI (3.7% vs. 1.6%) and revascularizations (24% vs.11%) as compared C. No correlation between ADMA and FMD (r=0.14, p=0.23). By multiple to non-diabetic patients. regression, only AUCgluc (p=0.002) and sBP (p=0.047) were inversely Conclusion: Despite similar improvements in exercise capacity with CR, related to FMD. AUCgluc, inversely, (p=0.024) and ADMA (0.044) correlated diabetic patients had worse long term clinical outcomes as compared to with GTN. In conclusion, uncomplicated newT2DM and pre-DM have lower patients without diabetes. Our results stress the need to identify diabetic ADMA, in presence of impaired FMD. ADMA was not related to endothelial patients undergoing CR and target them for aggressive risk reduction function. In these subjects with early abnormalities of glucose regulation, management. CR programs for diabetics should not be limited to exercise ED seems not a result of eNOS inhibition by ADMA. training, but also focus on more intensive weight management, lipid lowering, glycemic control and diabetic education in the hopes of reducing 531-P future major cardiac events. Brain Derived Neurotrophic Factor Levels in Patients with Diabetes— Protection from Macrovascular Disease? 533-P DAVID LORANT, MARTIN URSLI, CLEMENS HOEBAUS, JOHANNA-MARIA BRIX, Determinants of the Variability in the Recovery Rate of Platelet Cyclo- RENATE KOPPENSTEINER, GUNTRAM SCHERNTHANER, GERIT-HOLGER SCHERN- oxygenase Activity during Chronic Therapy with Low-Dose Aspirin THANER, Vienna, Austria in Type 2 Diabetes Diabetes patients have impaired regenerative functions. Likewise, DARIO PITOCCO, BIANCA ROCCA, FRANCESCA SANTILLI, LUCIANA MUCCI, vascular, pancreatic and brain stem cells were found to be reduced in animal GIOVANNA PETRUCCI, ESTER VITACOLONNA, STEFANO LATTANZIO, DOMENICO models of diabetes. We investigated the levels and associations of Stem MATTOSCIO, FRANCESCO ZACCARDI, ROSSELLA LIANI, NATALE VAZZANA, Cell Growth Factors in Patients with Diabetes. ADRIANA DEL PONTE, ELISABETTA FERRANTE, FRANCESCA MARTINI, PAOLA We measured Brain Derived Neurotrophic Factor (BDNF), Vascular RIZZO, GIOVANNI GHIRLANDA, GIOVANNI DAVÌ, CARLO PATRONO, Rome, Italy, Endothelial Growth Factor (VEGF), Angiogenin (ANG), and Interleukin-18 Chieti, Italy (IL18) in 213 consecutive patients with Diabetes mellitus (HbA1c 8.0±1.6 Aspirin is currently recommended for cardiovascular prevention in type 2 rel.%, Age 53.0±15.0 years, 49.3% female, Diabetes Duration 15±11 years) by diabetes (T2DM). However, primary prevention trials failed to substantiate ELISA. 79% were treated with Insulin. All patients underwent standardized its effi cacy in T2DM and incomplete platelet inhibition has been reported. interviews and blood tests. Patients were compared with 90 age-and gender Hyperglycemia has been often hypothesized to contribute to such an matched controls (CO). incomplete inhibition. We tested whether a faster recovery of platelet COX- Patients with Diabetes had higher IL18 levels (301 (214,409) vs 271 1 activity and/or a suboptimal 24-hour glycemic control would account for (219,372); p=0.031) and lower ANG levels (291 (216,401) vs 349 (259,615); an incomplete thromboxane (TX) A2 inhibition by low-dose aspirin during p=0.004) compared to CO. Among patients with diabetes the Stem Cell the 24-hour dosing interval. One-hundred T2DM patients on chronic low- Growth Factors showed different associates (univariate regression, beta in dose aspirin (100 mg daily) were studied. Serum TXB2, an index of platelet brackets): VEGF was signifi cantly related to BMI (0.156), hypertension (0.202), COX-1 activity, was measured every 3 hours, between 12 and 24 hours macrovascular disease (MVD) (0.204), stroke (0.163), peripheral arterial after a witnessed aspirin administration, to characterize the kinetics of disease (PAD) (0.202), and mediasclerosis (0.160). Angiogenin was related platelet COX-1 recovery (phase 1); 46 out of 100 patients underwent 24- to Age (0.250), BMI (0.252), Systolic Blood Pressure (SBP) (0.193), weight hour continuous glucose monitoring (CGMS) as well. Thirty-three patients (0.243), HDL (0.249), triglyceride (0.248), MVD (0.335), hypertension (0.310), with the steepest COX-1 recovery slopes were subsequently randomized hyperlipidemia (0.230), PAD (0.306), Angina Pectoris (0.168), Myocardial to receive aspirin 100 mg daily, 200 mg daily or 100 mg twice daily for 28 Infarction (0.240), and Stroke (0.197). Contrariwise, BDNF was indirectly days (phase 2). On day 29, COX-1 recovery was reassessed over the 12 to 24 related to weight (-0.151), gender (-0.205), MVD (-0.167), PAD (-0.149), and hour dosing interval. COX-1 activity displayed linear kinetics with large inter- Angina Pectoris (-0.178). IL18 was related to Age (0.162), HbA1c (0.203), BMI individual variability in recovery slope. Multiple linear regression showed (0.192), weight (0.216), HDL (-0.312), triglyceride (0.271), gender (0.144), MVD that mean platelet volume, higher BMI quartiles and age predicted serum (0.146), and Hypertension (0.250). TXB2 recovery slope, independently of other variables. None of the CGMS In Summary, only IL18 was associated with HbA1c. VEGF and ANG variability indices, (mean 24h glycemic value, standard deviation, MAGE showed positive associations with MVD, PAD and stroke. In addition, VEGF and CONGA1-5) as well as HbA1c and fasting glucose correlated with serum was associated with mediasclerosis. Contrariwise BDNF was negatively TXB2 recovery slopes. A twice-daily aspirin regimen, but not a doubling of

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A147 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

the dose, completely corrected the abnormal recovery slope of platelet COX- group from 60 to 120 minutes was signifi cantly increased in comparison with 1 activity. We conclude that inter-individual variability in the recovery rate of that of the controls, while there was no signifi cant difference between OGTT platelet COX-1 activity during the aspirin dosing interval most likely refl ects group and controls abnormal megakaryopoiesis associated with T2DM while is not infl uenced Those results suggest that the differences of dietary carbohydrate content by 24-hr glucose control or other glycemic indices; inadequate TX inhibition induce differences in attenuation of endothelial function in the postprandial can be overcome by a twice daily regimen. state.

POSTERS 534-P 536-P Complications Diabetes Effects on Progression of the Ankle Brachial Index Effect of Aging on Progression of Atherosclerosis in Type 2 Diabetes Acute and Chronic NKETI I. FORBANG, YIHUA LIAO, MARY MCDERMOTT, MICHAEL CRIQUI, La Jolla, Patients CA, Chicago, IL KENTARO WATANABE, TATSUYA SUZUKI, MOTOSHI OUCHI, KAZUNARI SUZUKI, Diabetes mellitus is a risk factor for peripheral arterial disease (PAD) KENICHI SEKIMIZU, HIROSHI NAKANO, KENZO OBA, Tokyo, Japan and medial arterial calcifi cation (MAC). The ankle brachial index (ABI) is The aim of our study was to evaluate the effect of aging on progression abnormally low in PAD, but high in the presence of MAC in the leg arteries. of atherosclerosis in type 2 diabetes patients by using ultrasound variables We studied associations of diabetes mellitus with change in the ABI over of the carotid arteries. time. Our study subjects were 68 Japanese patients treated for type 2 diabetes Over four years, 551 men and women participants with and without PAD in our division. They were divided into three groups by age: a middle- were identifi ed from Chicago-area medical centers and followed prospectively aged group (less than 65 years), an elderly group (65 to 74 years), and an with annual ABI measurements. Co-morbid illness was determined at advanced-age group (75 years or older). The ultrasound variables were the baseline from medical records, patient self-report, laboratory values, and average intima-media thickness (IMT), the average pulsatility index (PI), and a primary care physician questionnaire. Participants who underwent lower the sum of the plaque scores (PLQS) of the right and left carotid arteries. extremity revascularization during follow-up were censored at the time of We measured these variables again an average of 1.5±0.8 years after the revascularization. Prevalent PAD at baseline was defi ned as ABI < .90. fi rst measurements, and calculated the annual rates of change of IMT, PLQS, Adjusting for ABI in the previous year, annual average ABI changes were and PI. We evaluated the differences of the effects of aging on ultrasound -.011 (95% CI: -.02, -.002) in diabetics with PAD, -.013 (95% CI: -.02, -.007) variables between the three age groups. in non-diabetics with PAD, -.001 (95% CI: -.013, .01) in diabetics without The change in IMT in the elderly group, and the PI of the advanced-age PAD, and .008 (95% CI: .001, .014) in non-diabetics without PAD. Mean (SD) group were signifi cantly higher than those of the middle-aged group, while ABI at baseline were .68 (.13), .67 (.14), 1.08 (.11), and 1.09 (.09) respectively. there were no signifi cant inter-group differences in the changes of PLQS. Compared to non-diabetics without PAD, annual ABI changes were Furthermore, there were signifi cant correlations between BMI and the signifi cantly different in diabetics with PAD and non-diabetics with PAD change of PI in the middle-aged group, the LDL/HDL ratio and the change of (p-value <.05 for both). After adjustment for age, gender, ethnicity, current IMT in the advanced-age group, and the pressure pulse and the change of smoking, hypertension, HDL cholesterol, CRP, Ddimer, and the number PI the in the advanced-age group, while there was no signifi cant correlation of cardiovascular diseases (CVD) reported at baseline, diabetes (Beta=- among the other clinical characteristics and ultrasound variables. Also, .03, 95% CI: -.07, 0.1) was not independently associated with ABI change. there were non-signifi cant, correlations between age and both the change Signifi cant predictors of ABI decline were; higher baseline ABI (Beta=- of IMT and PI in all subjects, and diastolic blood pressure and the change of .18, 95% CI: -.26, -.11), pack years of smoking (Beta=-.001, 95% CI: -.002, IMT in the elderly group. In linear-regression analysis adjusted for clinical -0.0004), total cholesterol (Beta=-.0008, 95% CI: -.0013, -.0003), and lower characteristics, age was signifi cantly associated with the change of IMT and body mass index (BMI, kg/m2) (Beta=.005, 95% CI: .001, .008). Associations with that of PI in all subjects. However, there was no signifi cant association were similar for limbs with higher ABI. between age and change of PLQS. In this cohort of patients, greater ABI decline was associated with higher These results suggest that the effect of aging on progression of plaque baseline ABI, pack-years smoking, total cholesterol, and lower BMI. Diabetes formation was weaker than that on progression of thickening of intima- did not affect ABI change, possibly due to antagonistic effects of promoting media thickness, or on increase of vascular stiffness. both atherosclerosis and arterial stiffening. 537-P 535-P Effect of Aliskiren on QT Dispersion in Diabetic and Non-Diabetic Differences in Dietary Carbohydrate Content Induce Differences in Hypertensive Patients Postprandial Attenuation of Endothelial Function ROBERTO FOGARI, AMEDEO MUGELLINI, ANNALISA ZOPPI, GIUSEPPE DEROSA, KAZUNARI SUZUKI, KENTARO WATANABE, MOTOSHI OUCHI, KENICHI SEKIMIZU, Pavia, Italy TATSUYA SUZUKI, HIROSHI NAKANO, KENZO OBA, Tokyo, Japan Aim of this study was to compare the effect of aliskiren on QT dispersion The aim of our study was to evaluate the differences in attenuation of in hypertensive patients with or without diabetes After a 2 week placebo endothelial function in the postprandial state induced by differences in period 98 mild hypertensive patients with well controlled type 2 diabetes dietary carbohydrate content. (HbA1c < 7%) and 102 mild hypertensive patients without diabetes were The study subjects were 19 healthy Japanese men. The subjects were treated with aliskiren 300 mg for 12 weeks. At the end of the placebo period divided into three groups: the 75gOGTT (OGTT) group (300 kcal: containing and of treatment period BP was evaluated and an ECG was recorded using a 75 g carbohydrates; n=7), the CalorieMateTM (CM) intake group (400 kcal: paper speed of 50 mm/s. The QT intervals were measured manually in all the containing 40.7 g carbohydrates; n=5), and the controls in an overnight- 12 leads in blinded fashion and 3 consecutive QT intervals were measured fasting condition (n=7). Brachial artery fl ow-mediated dilation (FMD) of and averaged for each lead. The QT dispersion was corrected for the heart the OGTT or the CM group was measured at 0 minute (FMD0), 60 minutes rate (QTc). (FMD60), and 120 minutes (FMD20) after loading. The FMDs of the controls The treatment induced a signifi cant and similar SBP/DBP reduction in were measured for 3 consecutive hours in the fasting state from morning. both groups of patients (p<0.001 vs baseline). In hypertensive patients with The plasma glucose and immunoreactive insulin (IRI) levels of the 75gOGTT type 2 diabetes aliskiren induced a signifi cant reduction in QTc dispersion and CM groups were measured at 0, 30, 60, and 120 minutes after loading. (-10.6±22.6 ms, p=0.03) and in QTmax (-12.1±22.1 ms, p=0.02); the QTc FMD60 and FMD120 of the OGTT group were signifi cantly lower than that dispersion change did not correlate with BP change. In hypertensive patients of FMD0, while there were no signifi cant differences between the FMD0, without diabetes aliskiren induced a smaller and non signifi cant reduction in FMD60, FMD120 of CM groups or controls. On the other hand, the FMD60’s of QTc dispersion (-4.8±19.6 ms, p=0.09) and in QTmax (-3.9±19.6 ms, p=0.11). the OGTT and CM groups were signifi cantly lower than those of the controls, Aliskiren reduces QT dispersion in hypertensive diabetic patients and this but no signifi cant differences between three groups in FMD0, or FMD120. effect is not related to the BP lowering. It suggests that aliskiren has the Concerning correlations between FMD and both plasma glucose and IRI potential to reduce severe arrhytmic complications in this type of patients. in the OGTT and CM groups, there were signifi cant correlations between FMD120 and the plasma glucose at 60 minutes after loading, but none between the other FMDs and both plasma glucose and IRI. Also, the change of FMD in the OGTT group from 0 to 60 minutes was signifi cantly decreased against that of controls, while there was no signifi cant difference between the CM group and the controls. In contrast, the change of FMD in the CM

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A148 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

538-P baseline in the ACG compared with the STG: from 42 pg/mL (IQR 12-97) to Effect of Structured Blood Glucose Monitoring Versus Usual Care 49 pg/mL (IQR 11-118) (P=0.0122) vs. 39 pg/mL (IQR 12-104) to 42 pg/mL (IQR on Levels of hs-CRP—A Marker of Cardiovascular Risk—In Poorly 17-84) (P=0.9263), with no between-group difference (P=0.1732). In subgroup Controlled Type 2 Diabetes Patients analysis, NT-pro BNP remained stable in all patients with HbA1c decreases OLIVER SCHNELL, ILDIKO AMANN-ZALAN, ZHIHONG JELSOVSKY, CHRISTOPHER >0.5 %; patients with less HbA1c improvement experienced a worsening of PARKIN, BETTINA PETERSEN, MATTHIAS SCHWEITZER, LAWRENCE FISHER, their NT-pro BNP levels (P<0.003). In patients with >100 pg/mL NT-pro BNP WILLIAM POLONSKY, Munich, Germany, Mannheim, Germany, Tampa, FL, (N= 105) – an indicator of future cardiovascular risk – ACG patients (N=48) remained stable while STG patients (N=57) levels decreased signifi cantly

Indianapolis, IN, San Francisco, CA, San Diego, CA POSTERS

Hs-CRP is a sensitive biomarker of cardiovascular (CV) risk. In the Structured (P=0.0159). More STG patients reached levels <100 pg/mL compared with Complications Testing Program (STeP) study, the use of structured self-monitoring of blood ACG patients: 81% vs. 71%; P<0.04). Our approach to structured SMBG Acute and Chronic glucose (SMBG) was recently shown to improve glycemic control in type 2 contributed to signifi cant reductions in CV risk in patients with non-insulin diabetes patients compared to non-specifi ed SMBG. We assessed the effect treated T2DM. Reductions in NT-pro BNP were even more profound in of the structured SMBG intervention on hs-CRP levels in 483 patients with patients with elevated risk indicated by NT-pro BNP >100 pg/mL. non-insulin treated type 2 diabetes patients. Data for this evaluation were derived from the STeP study, a 12-month, cluster-randomized clinical trial 540-P that assessed the use of structured SMBG on glycemic control compared to Effects of Cyanidin-3-Glucoside on Glycated LDL-Induced Production usual care. Baseline characteristics were comparable among the 2 groups. of Reactive Oxygen Species and NADPH Oxidase Activation in Vascu- In the study, Hs-CRP measurements were taken at baseline and months 3, lar Endothelial Cells 6, 9 and 12 during the study, using the Hitachi system (Roche Diagnostics, XUEPING XIE, RUOZHI ZHAO, GARRY SHEN, Winnipeg, MB, Canada Rotkreuz, Switzerland). Mean (SD) baseline value: Control=6.9 (9.1) mg/L, Cardiovascular complications are major cause of death in diabetic patients. Experimental=6.6 (14.1) mg/L. At months 3, 6, 9 and 12, experimental Oxidative stress in blood circulation or vasculature has been considered subjects showed signifi cantly lower mean (SE) change in hs-CRP levels as an important etiological factor for cardiovascular disease. Levels of (mg/L) compared to controls: -1.7(0.4) vs. -0.1(0.5), P=0.008, -0.0(0.5) vs. glycated low density lipoprotein (glyLDL), a type of advanced glycation end 0.0(0.5), P<0.002; -2.0(0.5) vs. -0.5(0.5), P<0.02, -1.8(0.5) vs. -0.2(0.5), P<0.02), products, are commonly increased in diabetic patients. Our previous studies respectively. Experimental subjects with hs-CRP levels >3.0 showed even demonstrated that glycated low density lipoprotein (glyLDL) increased the greater improvement at months 3, 6, 9 and 12 compared to control subjects: production of reactive oxygen species (ROS), activated NADPH oxidase -3.1(0.7) vs. -1.1(0.7), P=0.04; -3.9(0.7) vs. -1.0(0.7), P<0.01); -3.9(0.8) vs. (NOX) and suppressed mitochondrial electron transport chain (mETC) -1.4(0.7), P<0.02; -3.2(0.8) vs. -1.0(0.7), P<0.04), respectively. As evidenced complex enzyme activities in vascular endothelial cells (EC). Cyanidin-3- by hs-CRP measurements, our collaborative approach to structured SMBG glucoside (C3G), a type of anthocyanin abundant in dark-skin berries or red contributed to signifi cant reductions in cardiovascular risk in patients with wine, reduced oxidative stress and insulin resistance in diabetic animals. The non-insulin treated type 2 diabetes. Reductions in hs-CRP were even more present study examined the effect of C3G on redox status, NOX activation profound in those patients with higher hs-CRP levels at entry to the study. and mitochondrial enzyme activity in porcine aortic EC (PAEC) with or without glyLDL treatment. C3G alone reduced H2DCF-DA fl uorescence in PAEC. Co- treatment of C3G normalized glyLDL-induced impairment of redox status in EC. GlyLDL increased NOX activity in EC and the abundance of NOX2 or p22phox in EC. C3G inhibited glyLDL-induced upregulation of NOX activity and the abundance of NOX2 and p22phox in EC. C3G prevented glyLDL- induced inhibition on mETC Complex III enzyme activity in EC. The fi ndings of the present study suggest that C3G may prevent diabetes-associated LDL- induced oxidative stress through the protection of mETC and NOX activities in cultured aortic EC, which potentially attenuate vascular injury in diabetic condition. Supported by: CDA, HSFC, CIHR

541-P Effects of Salsalate on Cardiovascular Risk Factors in Impaired Glu- cose Tolerance FLORENCIA HALPERIN, ALLISON B. GOLDFINE, PAUL R. CONLIN, JURAJ KOSKA, DAWN SCHWENKE, STEVEN SHOELSON, PETER D. REAVEN, Boston, MA, Phoenix, AZ 539-P Chronic infl ammation may play a role in the pathogenesis of type 2 Effect of Structured Blood Glucose Monitoring Versus Usual Care diabetes (T2D) and atherosclerosis. Targeting infl ammation with salsalate on Levels of NT-Pro BNP—A Marker of Cardiovascular Risk—In (SAL), which inhibits NF-κB activity, has been shown to improve glycemia Poorly Controlled Type 2 Diabetes Patients in T2D and overweight individuals. To study the effects of SAL on risk OLIVER SCHNELL, ILDIKO AMANN-ZALAN, ZHIHONG JELSOVSKY, CHRISTOPHER factors for cardiovascular disease (CVD), 70 predominantly male veterans PARKIN, BETTINA PETERSEN, MATTHIAS SCHWEITZER, LAWRENCE FISHER, with abnormal glucose tolerance (IFG/IGT) enrolled in a randomized double- WILLIAM POLONSKY, Munich, Germany, Mannheim, Germany, Tampa, FL, Indiana- blinded study of SAL 4 g/day or placebo (P) for 12 weeks were evaluated for polis, IN, San Francisco, CA, San Diego, CA changes in CVD risk markers. Baseline characteristics were similar between NT-pro BNP is a sensitive biomarker of cardiovascular (CV) risk. In groups (age 58±8 yrs, BMI 33±7 kg/m2, A1c 6.0±0.3%, systolic/diastolic blood the Structured Testing Program (STeP) study, the use of structured self- pressure 132±13/82±9 mm Hg, total cholesterol 170±31 mg/dl, LDL 103±25 monitoring of blood glucose (SMBG) was shown to improve glycemic control mg/dl, HDL 37±9 mg/dl, TG 150±56 mg/dl). SAL treatment signifi cantly in type 2 diabetes patients compared to non-specifi ed SMBG. We assessed lowered TG (ANCOVA p=0.009; Δ TG in SAL vs P -34 vs 7 mg/dl at week the effect of the structured SMBG intervention on NT-proBNP levels in 483 12). There was no overall effect of treatment on HDL (ANCOVA p=0.2), but patients with non-insulin treated type 2 diabetes patients (T2DM). Data within the SAL group HDL increased (Δ HDL 12 weeks vs baseline 3 mg/dl, were derived from STeP study, a 12-month, cluster-randomized clinical trial p<0.01). Increased calculated LDL was also observed (ANCOVA p=0.02, Δ that assessed use of structured SMBG (STG) on glycemic control compared LDL in SAL vs P 8 vs -7 mg/dl at week 12). There was no change in weight, to usual care (ACG). Baseline characteristics were comparable. NT-pro BNP blood pressure, total cholesterol, LDL/HDL ratio or CRP, and no change in measurements were taken at baseline, month 6 and month 12, using the endothelium-mediated vasodilation by peripheral arterial tonometry (Endo- Elecsys 2010 system (Roche Diagnostics, Rotkreuz, Switzerland). Median PAT, Itamar Medical, Israel). Use of lipid-lowering or antihypertensive agents NT-pro BNP levels at baseline were 32 pg/mL (IQR 10-95) for the STG and 36 did not change or differ signifi cantly between groups. In sum, in this group pg/mL (IQR 10-91) for the ACG. NT-pro BNP level tended to be stable over the of patients at higher risk for T2D and CVD, SAL treatment led to modest course of the study in the STG (P=0.3067), while levels in the ACG increased improvement in TG, an increase in calculated LDL (which could be explained signifi cantly (P=0.0292), with no between-group difference (P= 0.2542). Per by lower TG) with no change in LDL/HDL, and no adverse effects on other protocol analysis showed a more pronounced change in NT-pro BNP from CVD risk factors. In the context of concern about the cardiovascular safety

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of diabetes medications, salsalate may be a novel therapy for patients serum amyroid A protein (SAA) for infl mmation. The value was compensated with abnormal glucose tolerance with a neutral effect on cardiovascular with each ApoA-I level and compared with those in plasma levels. risk. Further study is needed to elucidate the effects of longer-term SAL ApoA-I levels in HDL fractions(1.70±0.43 μg/dl) were correlated well with treatment on glycemia, lipids and CVD outcomes. those in plasma (114±17.5mg/dl). SAA in HDL(16.1±14.2ng/ml) was similar with Supported by: The VA Clinical Sciences Research Program plasma levels (16.6±14.3ng/ml). PON-1 activities in HDL(18.2±4.57×104U/l/ min) were 5.7 times higher than those in plasma levels (3.20±1.52×104U/l/ 542-P min). However, CML levels in HDL were below sensitivity, although those in plasma were 0.26±0.09 mg/ml. POSTERS Exenatide Effects on Cardiovascular Risk Biomarkers in Patients Complications with Type 1 Diabetes Higher levels of PON-1 activity and undetectable CML levels in HDL Acute and Chronic MAY ALATTAR, REBECCA J. BROWN, KRISTINA I. ROTHER, TAMMY NGUYEN, revealed strong anti-glucoxidative nature of HDL even in diabetic patients. Bethesda, MD These fi ndings demonstrated that the ability of HDL to inhibit glycation Exenatide (Byetta™) therapy has been shown to improve blood glucose and lipid oxidation could refl ect changes in specifi c enzyme and protein control and cardiovascular disease (CVD) risk biomarkers in patients with components in HDL particles. type 2 diabetes (T2DM) as it induced weight loss, decreased HbA1C, total cholesterol (TC), triglycerides (TG), and LDL, and increased HDL. It also 544-P increased adiponectin (ADPN), a polypeptide with anti-infl ammatory and Genetic Variability in AMPKα2 Gene Is Associated with Risk of cardioprotective properties after prolonged therapy (12 months). An increase Coronary Artery Disease in Chinese Type 2 Diabetics in ADPN may be a useful marker for decreased coronary artery disease risk. RUIFEN DENG, XIAOWEI MA, SHAN DING, XIAODAN MA, NAN GU, XIAOHUI We hypothesized that exenatide therapy in individuals with type 1 GUO, JIANPING LI, LINONG JI, Beijing, China diabetes (T1DM) would result in improved CVD profi les similar to the effects AMP-activated protein kinase (AMPK) is a sensor of fuel supply that is seen in patients with T2DM. composed of α, β and γ subunits, of which AMPKα2 is highly expressed in the 14 subjects with long-standing T1DM (mean duration 20.2±11.4 years) myocardial muscle, skeletal muscle and liver. AMPK can inhibit the growth participated in a cross-over study (6 months on and 6 months off exenatide). of the vascular smooth muscle and the apoptosis induced by hyperglycemia Plasma samples were collected at baseline, and on and off exenatide. through mediating adiponectin-evoked AMPK activation and thus anti- Statistical analysis using ANOVA fi xed effect model was applied to atherosclerosis. To study whether or not the genetic variability in AMPKα2 determine changes in body weight, HbA1C, fasting and postprandial blood gene affects the risk of cardiovascular disease, we selected 5 haplotype- glucose, daily insulin requirements, plasma ADPN, TC, TG, LDL and HDL on tagging single nucleotide polymorphisms (SNPs) (rs1418442, rs2143754, and off exenatide. rs2796534, rs11206887, rs2143749) at AMPKα2 locus according to the CHB 2 Variable On exenatide Off exenatide P value database from HapMap phase II (r <0.8 and MAF≥0.05), and genotyped 326 unrelated Han subjects with type 2 diabetes (180 CAD-positive and 146 HbA1c (%) 6.6±0.6 6.8 ±0.6 0.3878 CAD-negative individuals) using the polymerase chain reaction - restriction Fasting plasma glucose (mg/dl) 142.5 ± 4.42 132.2 ± 3.45 <0.0001 fragment length polymorphism (PCR-RFLP) assay. For each SNP, the odds Postprandial plasma glucose (mg/dl) 142.5 ± 4.42 135.5 ± 4.43 0.0005 ratios (ORs) were estimated for CAD comparing with the CAD-negative Insulin requirement (unit/kg/day) 0.48 ± 0.11 0.55 ±0.13 0.0062 individuals by logistic regression analysis (SPSS version 17.0). The carriers of genotype GG at rs11206887 had a higher risk compared to non-carriers Total cholesterol (mg/dl) 149.5±32.9 151.7 ±34.0 0.8667 (OR=2.553, 95%CI =1.235-5.278, p = 0.011) for CAD. After adjusted for sex, Triglycerides (mg/dl) 70.1±35.5 59.6 ±19.6 0.3411 age, BMI, smoking status and diabetic duration, the difference was still LDL (mg/dl) 80.3±25.7 82.4 ±27.3 0.8351 statistically signifi cant (OR’=2.542, 95%CI =1.184-5.476, p’ = 0.017). Besides, HDL(mg/dl) 60.4±18.0 64.4 ±17.9 0.5530 GG homozygotes at rs2143749 were found at the increased risk compared to CC/CG carriers with statistical boundary (OR=1.68, 95%CI =1.029-2.741, p Adiponectin (mcg/l) 696.30±786.5 683.79 ±1045.5 0.9717 = 0.038)(Table1). Our fi ndings suggested that genetic variability at AMPKα2 In conclusion, exenatide therapy in patients with T1DM resulted in locus may be associated with the risk of CAD in type 2 diabetes in the signifi cant weight loss, decreasedpostprandial blood glucose excursions Chinese population. and a decrease in the daily insulin requirements; however, therapy for 6 SNP CAD+ n(%) CAD- n(%) OR (95%CI) p OR’ (95%CI) p’ months had no effect on CVD risk markers. We conclude that if lipid levels are optimal at the start of therapy, exenatide is unlikely to infl uence lipid rs11206887 values any further. A longer duration of therapy might be required to observe GG 31 17.2 11(7.5) 2.553(1.235-5.278) 0.011 2.542(1.184-5.476) 0.017 an effect on ADPN similar to what has been shown in patients with T2DM. AG/AA 149(82.8) 135(92.5) 1 1 rs2143749 GG 62 34.8 35(24.1) 1.68 (1.029-2.741) 0.038 1.566(0.934-2.626) 0.089 543-P CG/CC 116(65.2) 110(75.9) 1 1 Functional Characterization of HDL in Patients with Type 2 Diabetes ’Adjusted for sex, age, BMI, smoking status and diabetic duration. Using Simple Separation Method of Plasma HDL Fraction JUTARO TANABE, HIROSHI MURAKAMI, NAOKI TAMASAWA, MIKI YAMASHITA, Supported by: National 973 Project (2006CB503903, 2006CB503908) KOTA MATSUKI, HIROSHI MURAKAMI, JUN MATSUI, TOSHIHIRO SUDA, Hirosaki, Japan Low HDL-C is characteristic in patients with type 2 diabetes and metabolic syndrome associated with hypertriglyceridemia and small, dense LDL. 545-P Besides the ability to promote the effl ux of cholesterol from cells, HDL also HDL-Dysfunction (Paraoxonase- PON1) Plays a Role in Carotid has antioxidant, anti-infl ammatory, and antithrombotic properties. Intima Media Thickness (CIMT) in Non-Diabetic Postmenopausal We developed a simple method to separate HDL fraction from human African American Women Than in White American Women plasma, by which several properties of HDL function were possible to be TRUDY R. GAILLARD, MICHAEL R. GO, SAMPATH PARTHASARATHY, KWAME evaluated simultaneously from plasma in type 2 diabetic patients. OSEI, Columbus, OH Subjects were 15 patients with t ype 2 diabetes mellitus (8 males, 7 females, Introduction: CIMT is greater in African Americans (AA) than white age 61.8 ±4.7 y.o.) who were admitted to our department. Laboratory data Americans (WA). This occurs despite higher HDL-C and lower triglycerides in were HbA1c 8.5±1.3%, TC 171.8±18.5mg/dl, LDL-C 101.8±14.1 mg/dl, HDL-C AA than whites. We have recently demonstrated that HDL is dysfunctional 40.5±8.7mg/dl and TG 147.8±48.5mg/dl. Fasting plasma sample was applied as assessed by paraoxonase (PON1). However, the association of PON1 to polyacrylamide gel electrophoresis (PAGE); Lipophor, Jokoh, Japan, then and CIMT in postmenopausal AA women (AAW) remains controversial. sharp HDL fraction was recovered using Phoreto-yield protein collection Therefore, we examined the association between CIMT and PON1 in AAW device (Jokoh, Japan) equipped with cut-off MW 15,000. In this procedure, and White American women (WAW). native HDL fraction was obtained within 1 hour. Calculated HDL recovery was Subjects and Methods: We studied AAW(n-21) and WAW(n=21), mean age- 50.9% and CV% was under 10%. We analyzed several functional markers as 57.2±3.6, yrs, BMI-30.3±6.3 kg/m2, SBP-120.7±15.5 and DBP-74.3±7.4mmHg. follows in native HDL fraction simultaneously: (1) carboxymethyl-lysin(CML) Fasting blood samples were obtained for PON1, oxidized LDL (ox-LDL), lipids/ for glucoxidation, (2) paraoxonase-1 (PON-1) activity for anti-oxidation (3) lipoproteins, apolipoprotein A1 (apoA1) and B (apoB) and hsCRP. Standard

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A150 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

OGTT was performed. Insulin resistance was calculated by HOMA-IR lower HDL (p=0.018) compared to those with hsCRP<3 mg/mL. Recipients and body composition by DEXA. CIMT was performed in a subset of our with type 2 and/or post-transplant diabetes had a higher hsCRP, than type population, (WAW, n=8) and (AAW, n=12) by B mode ultrasonography. 1 diabetes and non-diabetes (p=0.003). Those with hsCRP >3 mg/L were Results: AAW were more obese (BMI- 32.8±6.5 vs 27.8±5.0 kg/m2, p= more likely to report a fever or infection in the past month but there was no 0.007 and % body fat- 47.6±7.6 vs 42.6±8.6%, p=0.05) vs WAW. There were difference based on recent hospitalization or history of chronic infectious no differences in glucose tolerance, blood pressure or HOMA-IR. We found no disease (CMV, Hepatitis B, C, EBV, HTLV). Many factors were not associated differences in total cholesterol, HDL-C, LDL-C and apoB. However, we found with hsCRP: age, years of dialysis, BP, HbA1C, total or LDL-C, eGFR, urine alb/ lower triglycerides (62.2±23.4 vs 88.3±46.5mg/dl, p=0.02) and higher apoA1 creat, PTH, homocysteine, gender, graft type, smoking status, statin use, POSTERS

(185.2±29.7 vs 159.7±46mg/dl, p=0.03) in AAW. PON1 was lower (0.97±0.35 aspirin use, or race/ethnicity (p>0.05). However, sirolimus (61% vs. 39%; Complications vs 2.09±0.29ng/ml, p=0.0001) and hsCRP (3.96±3.7 vs 2.2±2.5mg/L, p=0.07) p<0.0001) and tacrolimus (74.4% vs. 59.4%; p=0.0053) use were more common Acute and Chronic and ox-LDL (8.2±2.5 vs. 4.5±1.2U/L, p=0.0001) were signifi cantly higher in our in those with hsCRP >3 mg/L. hsCRP was also signifi cantly associated with AAW vs WAW. CIMT (0.69±0.10 vs 0.61±0.02mm, p=0.04) was signifi cantly CVD progression as expressed by CIMT in a general linear regression model higher in AAW than WAW. PON1 negatively correlated with CIMT in our (p=0.01). Conclusions: In a representative KTX cohort, hsCRP is signifi cantly population (r=-0.42, p=0.06). After adjusting for race, a 1 unit increase in elevated in half, even long after KTX, and was positively correlated with PON1 was associated with a 0.0038mm decrease in CIMT. CVD progression after KTX. CVD risk factors most closely associated with Conclusion: We speculate that the higher CIMT seen in AAW can be hsCRP were BMI, features of insulin resistance (e.g., elevated triglycerides attributed in part to the HDL dysfunction (PON1) with the resultant HDL- and HOMA-IR), and type 2 or post-transplant diabetes. Whether hsCRP is associated antiinfl ammatory and antioxidant properties. Further studies, also an independent CVD risk factor is yet to be determined. with larger sample sizes are warranted to confi rm these fi ndings. Supported by: NIH R01 34-5226-2002 (Clinicaltrials.gov: NCT00374595) Supported by: Award # UL1RR025755, National Center for Research Resources, NIH 548-P Hyperglycemia after Coronary Artery Bypass Grafting and Its 546-P Effects on Hospital Outcomes High Prevalence of Atherosclerotic Plaque Detected by the Carotid DAOUD DAOUD, SUSAN BAIMBRIDGE, CHARLES BAIMBRIDGE, GLENN R. and Lower Extremity Artery Ultrasonography in Hospitalized Chinese CUNNINGHAM, Houston, TX Type 2 Diabetic Patients We investigated the relationship between postoperative hyperglycemia LIANXI LI, FANG LIU, YUQIAN BAO, WEIPING JIA, Shanghai, China and outcomes (OCs) in patients who underwent isolated coronary artery Aims: Our aim was to evaluate the prevalence of atherosclerosis detected bypass grafting (CABG). by the carotid and lower extremity artery ultrasonography and the risk We conducted a retrospective study of 1504 consecutive patients who had factors associated with atherosclerosis in hospitalized Chinese type 2 isolated CABG at St Luke’s Episcopal Hospital between 1/1/07 and 12/31/09. diabetic patients. Patients were stratifi ed into 5 groups based on their preoperative risk of Methods: A number of 709 hospitalized Chinese type 2 diabetic patients mortality (MR), using the Society of Thoracic Surgeons’ criteria. Average were prospectively assessed.Atherosclerosis was defi ned as the presence glucose levels for the fi rst 2 days after surgery (ave.2d.g) were determined of either carotid or lower extremity arterial atherosclerotic plaque in any and correlated with relevant OCs: ICU time (ICUT), post-op length of stay of the above-mentioned artery segments. Body measurements including (LOS), ventilation time (VT) and overall complications (Comp). The ave.2d.g height, weight, waist circumference and hip circumference, resting heart values within each group were divided into 4 quartiles (QTs). Continuous rate and blood pressure, fasting and 2-h postprandial blood measures were OCs were considered cases if values were greater than the median. Logistic investigated. Diabetic retinopathy was assessed by fundus photographs. regression analysis was used to determine if the OCs were related to Diabetic nephropathy was assessed by 24-h albumin excretion rate. Both glycemic control. carotid and lower extremity arterial atherosclerosis were measured by The number of patients in each MR group 1 through 5 (low to high) Doppler ultrasound. The prevalence of atherosclerosis was calculated, and was 761, 344, 139, 80 and 180, respectively. Consistent and signifi cant the risk factors associated with atherosclerosis were evaluated using binary correlations between OCs and glycemic control were confi ned to Group 1. logistic regression. The Mean±SD glucose levels for each QT were 131.7±7.3, 147.3±4.0, 163±5.6 Results: (1) The prevalence rate of atherosclerosis was 79.4% in and 199.1±25.7 mg/dL. Glycemic control was not correlated with red blood hospitalized Chinese patients diagnosed with type 2 diabetes, 81.2% in male cell transfusion, septicemia, deep sternal wound infection or mortality. patients, and 77.6% in female patients. There was no signifi cant difference Table 1. Odds Ratios (ORs) for OCs vs. 2 Day Average Glucose Levels in the prevalence of atherosclerosis in patients between the sexes. (2) Atherosclerotic patients were older and had longer duration of diabetes, Outcome median 2nd vs. 1st QT 3rd vs. 1st QT 4th vs. 1st QT +10 mg/dL Increase higher systolic blood pressure, neutrophil percentages, common carotid OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI) and lower extremity artery IMT. In addition, the prevalence of hypertension, p value p value p value p value cardiovascular and cerebrovascular diseases were higher in type 2 diabetes ICUT 32.63 1.31 (0.87-1.96) 1.63 (1.09-2.46)0.02 1.86 (1.23-2.79) 0.003 1.07 (1.02-1.13)0.007 with atherosclerosis. (3) Atherosclerosis was signifi cantly associated with (hours) 0.19 duration of diabetes, older age, systolic blood pressure, and low extremity LOS 7 1.36 (0.85-2.17) 1.70 (1.07-2.69)0.02 2.39 (1.52-3.78)0.001 1.09 (1.04-1.15)0.001 artery IMT based on logistic regression analysis. (days) 0.20 Conclusions: The prevalence of atherosclerosis was high in Chinese in- VT 8.87 1.81 (1.20-2.72)0.004 1.72 (1.14-2.59)0.01 2.32 (1.53-3.50)0.001 1.09 (1.03-1.15)0.002 patients with type 2 diabetes, especially in elderly patients. Combination of (hours) carotid and lower extremity ultrasound examination resulted in a signifi cant Comp 280¹ 1.74 (1.13-2.67) 1.63 (1.06-2.52)0.03 1.82 (1.18-2.80) 0.006 1.06 (1.01-1.12) 0.03 improvement in the detection of type 2 diabetes with atherosclerosis. N(%) (36.8) 0.01 547-P ¹Number of cases (%) hsCRP Is Associated with Cardiovascular Disease Progression after Hyperglycemia in the fi rst two days after CABG appears to affect adversely Transplant multiple postoperative outcomes. MEGHAN GAULE, ELIZABETH LYDEN, FANG YU, TERICA HUDSON, JENNIFER Supported by: Internal funds LARSEN, Omaha, NE Introduction: Cardiovascular disease (CVD) remains the greatest cause 549-P of death after kidney transplant (KTX) and infl ammation is one of the Hyperhomocysteinemia Exacerbated Hyperglycemia-Induced Endo- nontraditional risk factors associated with CVD. Methods: In recipients thelial Dysfunction by Modulation of Cyclooxygenase-Calpain >6mo after KTX with a GFR>30 ml/min, we evaluated the association of Signaling highly sensitive CRP (hsCRP) with other CVD risk factors and CVD progression ZHONGJIAN CHENG, XIAOHUA JIANG, STANISLAV SIDOROV, PU FANG, MEGHAN as analyzed by carotid intima media thickness (CIMT). Results: The study PANSURIA, ROSARIO SCALIA, DOMENICO PRATICO, WILLIAM DURANTE, population (n=342;192 M/150 F) with mean age of 52+1y was 5.9+0.3y since XIAOFENG YANG, HONG WANG, Philadelphia, PA, Columbia, MO KTX (0.5-33.8y). 50% had diabetes. Of this cohort, 50.3% had an elevated Accumulative evidence indicates that elevated plasma homocysteine hsCRP (>3mg/mL), and this group was closer to transplant (p=0.0003), had a (Hcy) level is a stronger risk factor of cardiovascular diseases in diabetes. higher BMI (p<0.0001), HOMA-IR (p=0.003), and triglycerides (p=0.0023), and We examined the effect of hyperhomocysteinemia (HHcy) on endothelial

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A151 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

function in hyperglycemic mice. Moderate HHcy and severe HHcy (plasma 551-P Hcy: 30 and 256 μM, respectively) was developed by feeding a high Infl uence of Diastole Duration on Subendocardial Myocardial Via- methionine diet (2%) for 8 weeks in 8-week old male cystathionine beta- bility and Role of Cardiac Autonomic Dysfunction in Type 2 Diabetic synthase (CBS+/+ and CBS-/+) mice. Hyperglycemia (HG) was generated by and Obese Patients streptozotocin (40 mg/kg for 5 consecutive days, i.p.) started at the time QINDA CHEN, YAHYA JABER, HUBERT DABIRE, SABRINA CHIHEB, ISABELA of dietary intervention. Severe HHcy impaired endothelium-dependent BANU, MINH TUAN NGUYEN, EMMANUEL COSSON, PAUL VALENSI, Shanghai, vascular relaxation to acetylcholine (ACh) in the absence and presence China, Bondy, France, Maisons-Alfort, France of HG in CBS-/+ mice (98% to 77% and 83% to 63%, respectively).

POSTERS Cardiac autonomic dysfunction (CAD) characterized by reduced vagal

Complications Endothelium-independent vascular relaxation to sodium nitroprusside activity and relative sympathetic overdrive might shorten diastole duration Acute and Chronic was not changed by HHcy. Vascular relaxation to ACh was completely (DD) and thus impair coronary perfusion. The aim was to study the relation abolished by L-NAME (nitric oxide (NO) synthase inhibitor) and normalized between DD and subendocardial myocardial viability (SEVR) and the by indomethacin (cyclooxygenase (COX) inhibitor) in all mouse groups. infl uence of CAD in patients with type 2 diabetes (T2D) or obesity. COX-1 inhibitor SC560 and COX-2 inhibitors celecoxib or NS398 improved We included 607 T2D (58.5±10.8 yrs) and 185 non diabetic obese patients vascular relaxation to ACh in HHcy-HG mice. Calpain inhibitory therapy (40.5±13.5 yrs, BMI 39.1±6.4 kg/m2), all free of cardiac history. Using using a calpain inhibitor MDL28170 (1 mg/kg for 2 weeks, i.p.) completely applanation tonometry (SphygmoCor®), we measured heart rate (HR), DD, rescued severe HHcy-impaired vascular relaxation to ACh both in the aortic and radial blood pressures and stiffness (amplifi cation index AIx) absence and presence of HG. Furthermore, preincubation with calpain and pulse wave velocity. The percentage of DD (DD% = (DD/duration of inhibitors (MDL28170, ALLM and calpeptin) normalized vascular response heart period) x100) and SEVR (areas under diastolic/systolic aortic pressure to ACh in HHcy-HG mice. Calpain activity was increased in cultured mouse curves) were calculated. primary aortic endothelial cells from HHcy and HHcy-hyperglycemic mice CAD was assessed in 482 of the patients (349 T2Ds and 133obeses) using (125% and 261%, respectively). Finally, L-Hcy (500 µM, 48h) increased standard tests (deep-breathing, lying-to-standing, Valsalva) which mostly calpain activity in cultured human aortic endothelial cells (HAECs) and that depend on vagal control. CAD was found in 237 T2Ds, and among the obese is potentiated by D-glucose (44 mM, 273% and 743%, respectively). SC560 patients, CAD was absent in 74 (G0), moderate (G1: 1 abnormal test) in 43, and celecoxib markedly reversed HHcy-induced calpain activation in and confi rmed or severe (G2: 2 or 3 abnormal tests) in 16 patients. HAECs, suggesting that activation of calpain is secondary from activation In T2D and obese patients, DD% correlated negatively with HR (r=-0.826 of COX by HHcy and/or HG. Conclusions: HHcy exacerbates HG-induced and r=-0.638, p<0.0001, respectively) and was lower in CAD+ than in CAD- endothelial dysfunction (ED). Modulation of COX-calpain signaling may patients (p<0.01 and 0.05, respectively; p=0.002 and p<0.0001 after HR play a critical role in HHcy-induced ED in diabetes. adjustment at 75 bpm). In obese patients, DD% was already lower in G1 vs G0 (p=0.05), independently of age. In T2D and obese patients, SEVR correlated 550-P strongly with DD% (r=0.927 and r=0.536, p<0.0001). SEVR correlated only in Infl uence of Cigarette Smoking on QTc Interval Duration in Patients T2Ds weakly with aortic AIx adjusted at HR 75 bpm (r=-0.371, p<0.0001) and with Type 2 Diabetes Mellitus neither with pulse pressures nor pulse wave velocity. PETROS THOMAKOS, STAVROS LIATIS, STAVROULA KALOPITA, IOANNIS VLAHO- To conclude, in T2D and obese patients, both HR acceleration and reduced DIMITRIS, CHRYSSOULA STATHI, NICHOLAS KATSILAMBROS, KONSTANTINOS vagal activity contribute to diastole shortening, which play a more important MAKRILAKIS, Athens, Greece role than artery stiffness in the impairment of subendocardial myocardial Prolongation of the QT interval corrected for heart rate (QTc) is associated viability and may thus compromise coronary prognosis. with a lowered ventricular fi brillation threshold and an increased incidence of sudden cardiac death. Both type 2 diabetes (T2D) and cigarette smoking 552-P are well identifi ed risk factors for cardiovascular disease. The mechanism by Is There a Diagnostic or Mechanistic Role for Epicardial Adipose which smoking may lead to cardiovascular events in T2D patients has not Tissue in Diabetes Related Coronary Disease? been adequately investigated. YING Q. LIU, JENCIA WONG, FEI JUN, LYNDA MOLYNEAUX, DAVID CELERMAJER, Aim of the present study was to assess the relationship between smoking ZHANG RONG XU, DENNIS K. YUE, Beijing, China, Sydney, Australia and QTc interval (measured by continuous ECG monitoring) in T2D patients. Epicardial Adipose Tissue (EAT), considered an extension of the visceral fat A total of 72 T2D patients (36 chronic smokers, 36 non-smokers), age- and depot, correlates well with the Metabolic Syndrome and has recently been sex-matched, on oral antidiabetic agents, underwent continuous 24-hour found to be increased in diabetic patients. It has been suggested that EAT ECG Holter monitoring. QTc interval during the day and night was separately may be useful as a new independent marker for coronary artery disease (CAD) analyzed. in diabetes. Furthermore, EAT may release pro-atherosclerotic cytokines and Smokers (21 men/15 women) and non-smokers (20 men/16 women) excess local EAT may determine the regional distribution of CAD. We therefore were not different regarding age [mean±SD]: [54.9±9.0 vs. 56.7±8.2 years examine EAT volume and distribution in 189 consecutive Chinese type 2 (p=0.362)], diabetes duration [5.5±4.6 vs. 5.3±4.3 years (p=0.83)], HbA1c diabetes patients stratifi ed according to presenting features with 90 (47.6%) [7.3±1.2 vs. 6.9±0.9% (p=0.09)] and systolic blood pressure [125.1±14.5 vs. asymptomatic, 62 (32.8%) symptomatic with chest pain and 37 (19.6%) with 128.2±15.4 mmHg (p=0.38)], but smokers had a lower BMI [29.8±5.1 vs. known CAD. All patients underwent dual source 64 slice CT angiography with 32.6±5.2 kg/m2 (p=0.02)]. measurements taken of EAT volume, coronary calcium score (CCS), a marker Smokers showed increased QTc duration during the 24 hours [439.25±26.95 of CAD and Gensini score, a measure of the extent and severity of CAD. The vs. 425.91±23.28 ms (p=0.03)] as well as during day [439.14±24.31 vs. relationship between regional EAT volume and atherosclerotic burden (< or 427.86±24 ms (p=0.053)] and night period [440.91±32.3 vs. 426.55±25.34 ≥ 50% stenosis) in the corresponding arterial bed was also evaluated. The ms (p=0.046)]. After adjusting for BMI and HbA1c (using linear regression mean ± SD for age was 60.6 ± 11.4 years and 56.6% were male. There was analysis), smoking was independently and signifi cantly associated with a signifi cant but weak correlation between logEAT volume and Gensini score increased QTc during the 24 hours (standardized β=0.27, p=0.03), as well as (r=0.2 p=0.008) and CCS (r=0.2, p=0.007). There was a statistically signifi cant during the day (β=0.25, p=0.046) and night period (β=0.27, p=0.037). difference in CCS between the three groups but no difference in EAT volume Cigarette smoking is associated with prolongation of the QTc interval between the groups [Table]. In the multivariate analysis of Gensini score as in T2D patients and this effect may be implicated with an increased the dependent variable, EAT volume was not a signifi cant predictor of CAD. cardiovascular risk in this population. There was also no increase in the EAT volume at sites of ≥ 50% stenosis. We conclude that EAT is not a clinically useful marker of CAD and that CCS is a better measure. There is also no evidence that regional EAT volume is associated with local coronary stenosis. n=189 Asymptomatic for CAD Symptomatic for CAD Past CAD p-value n=90 n=62 n=37 Total Calcium Score 5.0 [0.1-156.4] 84.4 [1.6-437.6] 74.3 [0.1-28.3] 0.004

Total Gensini Score 9 [3-20] 14 [4-35] 15 [4-39] 0.054

EAT Volume (cm3) 151.8 [119.8-179.3] 148.8 [121.5-196.6] 148.0 [129.3-224.0] 0.5

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A152 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

553-P Peripheral Endothelial Function and Silent Coronary Artery Disease in Type 2 Diabetic Patients MINH TUAN NGUYEN, ISABELLE PHAM, ALAIN NITENBERG, PAUL VALENSI, EMMANUEL COSSON, Bondy, France Endothelial dysfunction is early involved in atherosclerosis. The aim of the study was to assess if peripheral endothelial dysfunction was

associated with silent myocardial ischemia (SMI) and the presence of occult POSTERS signifi cant coronary artery disease (CAD) in type 2 diabetic patients (T2D) Complications (NCT00685984). Acute and Chronic The humeral diameter was measured by ultrasound examination before and one minute after ischemic hyperaemia. Endothelium-dependent fl ow- mediated dilation (FMD: change in diameter in %) was calculated in 30 control subjects ≤ 40 years (19 males, body mass index (BMI) 22±2 kg/m², 25±6 years), 30 overweighed or obese subjects without diabetes (3 males, BMI 34±4 kg/m², 40±13 years) and 116 asymptomatic patients with T2D for 15±7 years (70 males, BMI 31±5 kg/m², 62±8 years). All the T2D patients had additional risk factors and fulfi lled the criteria of the French Alfediam- SFC guidelines for SMI screening. They were prospectively screened for SMI defi ned as an abnormal stress myocardial scintigraphy and/or stress echo- 555-P cardiography. A coronary angiography was performed in those with SMI. Postprandial Serum Responses of Glucose, Insulin and Triglycerides FMD was 4.5±3.3; 4.6±6.8 and 1.2±3.9% in control, overweighed or obese Are Not Associated with Carotid Intima-Media Thickness in Non- subjects and T2D patients (p<0.0001), respectively. SMI was found in 58 Diabetic Adults with Overweight T2D patients, and 19 of them had CAD. FMD was impaired in the patients KARIN B. GAST, RENÉE DE MUTSERT, EELCO J. DE KONING, WOUTER J. JU- with SMI (SMI+: 0.6±4.2% / IMS-: 2.0±3.5%, p<0.05), and further impaired KEMA, FRITS R. ROSENDAAL, KLAUS F. RABE, TON J. RABELINK, JOHANNES and in those with CAD (CAD+:-0.8±4.5% / CAD-: 1.8±6.7%, p<0.01). In A. ROMIJN, JAN W. SMIT, SASKIA MIDDELDORP, Leiden, The Netherlands, multivariate analyses taking into account SMI (model 1) or CAD (model 2) and Amsterdam, The Netherlands the potential confounding factors (age, gender, HbA1c, nephropathy, cardiac A frequent intake of meals and snacks results in a postprandial state the autonomic neuropathy, smoking, BMI, LDL-cholesterol, lipid treatment, majority of the day. As post-challenge elevations of glucose and lipid levels systolic blood pressure, renin-angiotensin system blocker), only SMI (model are associated with an increased risk of cardiovascular disease, our objective 1: p<0.01) or CAD (model 2: p<0.001) were independently associated with was to investigate the association between serum responses of glucose, FMD impairment. insulin and triglycerides after a mixed meal and carotid Intima-Media These results show for the fi rst time that the peripheral endothelial thickness (cIMT) in non-diabetic persons with overweight.In this cross- dysfunction observed in high risk T2D patients is independently associated sectional analysis of the baseline of the NEO (Netherlands Epidemiology of Obesity) study, a cohort of persons of 45-65 years with a Body Mass Index with the presence of SMI and silent CAD. 2 FMD measurement might be used as a non invasive method in screening (BMI) ≥27 kg/m , glucose, insulin and triglycerides concentrations were for SMI and CAD. measured in fasting blood samples, 30 and 150 min after a liquid mixed meal (600 kcal, 75g carbohydrates, 24g proteins, 23g fat). CIMT was measured by ultrasound in both common carotid arteries. We performed linear 554-P regression analyses of fasting, postprandial serum levels and area under Postprandial Endothelial Function Does Not Differ by Race: An the curves with cIMT, adjusting for age, sex, ethnicity, education and waist Insulin Resistance Paradox circumference. After exclusion of participants with known diabetes (n=111), RANGANATH MUNIYAPPA, VANDANA SACHDEV, STANISLAV SIDENKO, MADIA this analysis included 1211 participants with a mean (SD) age of 56 (6) years, RICKS, AMBER B. COURVILLE, ANNE E. SUMNER, Bethesda, MD mean BMI of 30.8 (3.7) kg/m2, 6% CVD. Mean cIMT was 0.665 (0.086) mm. Insulin resistance is typically associated with postprandial hyperlipidemia Postprandial responses of glucose (fi gure 1), insulin and triglycerides differed and endothelial dysfunction. As black women are more insulin-resistant between sexes. Overall, there were no associations between postprandial than white women, it is assumed that black women have worse endothelial responses of glucose, insulin or triglycerides and cIMT. Subgroup analyses function. However, no studies have been performed comparing endothelial showed a weak association between the glucose response at 30 min and function in black and white women. We determined postprandial (PP) cIMT (β:0.004, 95%CI:-0.001,0.009) in women. In conclusion, cIMT in a large endothelial function following a high-fat (HF) breakfast (40% fat, 40% cohort of non-diabetic persons with overweight was in the normal range and CHO, 20% protein) in 35 women (17 black, 18 white) who were age and there were no associations between fasting or postprandial responses of 2 BMI-matched (age: 37 ± 11y; BMI: 30 ± 6 kg/m ). All women consumed glucose, insulin and triglycerides. weight-maintenance diets with a macronutrient distribution of 33% fat, 52% CHO, 15% protein for 7-days prior to the assessment of endothelial function. Endothelial function, defi ned by percent change in brachial artery fl ow- mediated dilation (FMD), was measured at 0, 2, 4 and 6h following the HF meal. Race differences were not signifi cant in baseline FMD, total body fat, abdominal visceral fat, or fasting levels of glucose, insulin, A1C, total cholesterol, LDL-cholesterol, serum estradiol, or hsCRP. Although black women were more insulin-resistant (SI: 3.7 ± 1.5 v 5.2 ± 2.6, p < 0.05), both fasting triglyceride (TG: 48 ± 18 vs. 97 ± 49 mg/dL, P<0.01) and PP-TG 2 levels (AUC0-4h: 154 ± 52 vs. 307 ± 159 mg/dL.min.10 , P<0.01) were lower in black than white women. Bivariate analysis revealed that 4hr-PP-FMD was positively related to PP-TG-AUC (r= 0.5) in black women, but negatively related in white women (r=-0.6). However, PP endothelial function was not different between the groups (P>0.1 for group x time interactions) (Figure). These data suggest than insulin resistance does not appear to be a key determinant of postprandial endothelial function.

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A153 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

556-P are suffi ciently adequate for detecting asymptomatic CHD in Japanese Relation between Aorta Stiffness and Advanced Diastolic Function subjects with type 2 diabetes. Indices in Patients with Diabetes Mellitus Type 1—Assessment with MRI and Speckle Tracking Strain Analysis 558-P LINDA D. VAN SCHINKEL, DOMINIQUE AUGER, SASKIA G.C. VAN ELDEREN, NINA SDF-1β Protects Cardiac Cells from Palmitate-Induced Nitrosative AJMONE, VICTORIA DELGADO, ARNOLD C.T.A. NG, JOHANNES W.A. SMIT, JEROEN Stress-Mediated ER Stress and Cell Death through Activation of J. BAX, ALBERT DE ROOS, JOS J.M. WESTENBERG, Leiden, The Netherlands AMPK-Mediated IL-6 Excretion

POSTERS Increased pulse wave velocity (PWV) is a marker for aortic stiffness YUGUANG ZHAO, YI TAN, WEI LI, LU CAI, Changchun, China, Louisville, KY Complications and is associated with cardiovascular disease. PWV can be accurately Elevated saturated free fatty acids including palmitate (Pal) often occur Acute and Chronic assessed with MRI. Whether an increased MRI PWV translates in early in the patients with obesity and diabetes, and are also primary trigger for diastolic dysfunction in patients with diabetes mellitus type 1(DM1) remains cardiac cell death, but its mechanisms remain largely unknown. Stromal unexplored. The purpose of this study is to evaluate the correlation between cell-derived factor-1beta (SDF-1β) was cardiac protective, but whether it PWV and advanced diastolic function parameters in patients with DM1. also protects the heart from lipotoxicity remains unknown. Using H9c2 Normal PWV age-relation was determined in 25 healthy volunteers, age cardiac cell line, we studied the apoptotic effect of Pal and its possible 18-65 years with a 1.5T MRI scanner and is represented by the formula: protection by SDF-1β. Exposure of H9c2 cells to Pal at 62.5 nM for 16 h PWV=A×AGE+B, with A±SE=0.03±0.01m/s/year and B±SE=3.70±0.46m/s. causes a signifi cant apoptotic effect (DNA fragmentation and cleaved Next, PWV was assessed in 41 DM1 patients. Subjects were divided into caspase-3). Pal also induced signifi cant increases in 3-nitrotyrosine at 3 – 3 groups. Group 1 (normal PWV) consisted of patients with normal PWV 6 h and endoplasmic reticulum (ER) stress [GRP78, CHOP and caspase-12] values up to 1×SE, group 2 (moderately increased PWV) had values up to at 6 – 9 h after treatment. Inhibition of NADPH oxidase (NOX)-activation 2×SE and group 3 (high PWV) had a PWV>2×SE. Then patients underwent or scavenging superoxide and peroxynitrite with their specifi c inhibitors echocardiography for assessment of conventional diastolic function indices abolished Pal-induced ER stress and cell death. Inhibition of ER stress (e.g. isovolumic relaxation time (IVRT), transmitral early (E) and atrial (A) with 4PBA and TUDCA prevents cell death. It suggests that Pal-induced peak fi lling velocities, E/A-ratio). Tissue Doppler imaging was performed to cell death is mediated by ER stress-associated cell death pathway determine mean mitral annulus velocity (E’). Moreover, longitudinal speckle activated by NOX-activation associated nitrosative stress. Pretreatment tracking strain analysis was performed to derive advanced diastolic indices with SDF-1β abolishes Pal-induced nitrosative damage, ER stress and [peak transmitral E-wave to strain rate during the isovolumic relaxation cell death, and induces AMPK-mediated IL-6 production. AMPK activator period (SRIVR)] and left atria l(LA) systolic strain. Next the peak E/SRIVR signifi cantly prevents Pal-induced cardiac apoptosis and increases IL-6 ratio was calculated. while AMPK inhibito abolishes SDF-1β’s cardiac protection. Direct addition Of 41 DM1 patients (age 50±9years), 19 were in group 1 (PWV of recombinant human IL-6 to cell cultures offers a signifi cant prevent of 5.72±0.78m/s), 9 in group 2 (PWV 6.37±0.58m/s) and 13 in group 3 (PWV Pal-induced ER-stress and cell death. These results suggest that cardiac 9.87±2.06m/s). After correction for age, gender and mean arterial pressure, apoptotic effect of Pal is mediated by NOX activation-triggered nitrosative aortic PWV was signifi cantly correlated with SRIVR (p=0.000,β=-0.71), E/ damage and ER stress cell death pathway. SDF-1β prevents Pal apoptotic SRIVR (p=0.002,β=0.61) and LA strain (p=0.014,β=-0.47). effect via activating AMPK-mediated IL-6. The fi nding that SDF-1β protects In patients with DM1, increased PWV is correlated with advanced indices cardiac cell death from fatty acid opens a new road for the research on for diastolic dysfunction. Increased MRI PWV may therefore be a marker of cardiac protection by SDF-1β that provides cardiac protection independent early left ventricular diastolic dysfunction. of stem cell mobilization. ADA-Funded Research Supported by: The Netherlands Heart Foundation (Project UL 2009-4548) 559-P 557-P Secretory Products from Epicardial Adipose Tissue from Patients Screening Method for Asymptomatic Coronary Heart Disease in with Type 2 Diabetes Affect MiRNA Expression in Cardiomyocytes Japanese Subjects with Type 2 Diabetes MARCEL BLUMENSATT, SABRINA GREULICH, DANIELLA HERZFELD, BUJAR YUKIKO KAWASAKI, YOSHIYUKI HAMAMOTO, SACHIKO HONJO, KANAKO MORI, MAXHERA, ARTUR LICHTENBERG, JUERGEN ECKEL, MARGRIET OUWENS, HIROKI IKEDA, YOSHIHARU WADA, KAZUHIRO NOMURA, YORIHIRO IWASAKI, Düsseldorf, Germany HIROYUKI KOSHIYAMA, Osaka, Japan Epicardial adipose tissue (EAT) is a visceral fat depot around the heart, We investigated the usefulness of screening methods using the treadmill which is not separated by a fascia. Therefore, factors secreted from EAT can tolerance test (TTT) as the fi rst line test or the risk-guided approach including directly affect the function of the myocardium. We have recently found that the 1998 ADA guideline for the detection of asymptomatic coronary heart the secretory profi le of EAT is altered in patients with type 2 diabetes (T2D). disease (CHD) in Japanese patients with type 2 diabetes. Subjects included Furthermore, conditioned media (CM) generated from EAT from patients with consecutive inpatients with type 2 diabetes (n= 481). They were checked T2D (CM-EAT-T2D) induce cardiomyocyte dysfunction as illustrated by the with electrocardiogram (ECG) at rest and TTT, and those with abnormal TTT induction of insulin resistance and reductions in sarcomere shortening and fi ndings were investigated with stress myocardial perfusion scintigraphy cytosolic Ca2+-fl uxes. Here, we examined whether the detrimental effects (MPS), and those with abnormal MPS fi ndings were examined with coronary induced by CM-EAT can be ascribed to alterations in miRNA expression in angiography (CAG). The number of patients who met the criteria of ADA cardiomyocytes. Therefore, primary adult rat cardiomyocytes were incubated guideline, or those of patients with cardiac autonomic neuropathy (CAN) or with CM generated from EAT-biopsies collected from patients without (ND) maximal carotid artery intima-media thickness (max IMT) of ≥1.1mm were and with T2D undergoing open heart surgery. Alterations in expression levels examined. CAN was defi ned as the coeffi cients of variation of R-R intervals of 343 rat miRNA species (miRBase V13.0) were quantifi ed by real-time PCR. of ECG less than 2%. A total of 69 had positive TTT, among whom, a total Adult rat cardiomyocytes were found to express 164 miRNA species. Of of 22 had positive MPS. A total of 14 subjects received CAG, and eight of these, let7c and the miRNA-143/145 cluster were selectively up-regulated by them (only 1.7% of the total subject) were fi nally indicated to have CHD. The CM-EAT-T2D compared to CM-EAT-ND. In addition, miRNA-26a, miRNA-26b, patients who met the criteria of ADA were 53.6% of TTT-positive patients, miRNA-425, miRNA-191 and miRNA-218 were signifi cantly down-regulated 45.5% of MPS-positive patients, and 62.5% of CAG-positive patients, by CM-EAT-T2D versus CM-EAT-ND. respectively. The patients with CAN and/or increased max IMT were Levels of these miRNA species were not affected by CM from subcutaneous 46.0/75.4%, 55.0/77.3% and 57.1/87.5% of patients with positive TTT, MPS and pericardial adipose tissue from the same patients. Predicted target genes and CAG, respectively. for these miRNAs include the key regulator of myocardial Ca2+-metabolism, The unexpectedly low rate of asymptomatic CHD in our screening SERCA2a, which is downregulated by CM-EAT-T2D in cardiomyocytes. In suggested that considerable subjects with asymptomatic CHD had escaped line with this, preliminary validation experiments suggest that silencing of the present screening method; in fact, among those with negative results, let7c increases the abundance of SERCA2a. Collectively, these data suggest three patients developed CHD during the follow-up. Furthermore, it was that alterations in miRNA expression in cardiomyocytes induced by factors suggested that the risk-guided approach with the ADA guideline or that secreted from epicardial adipose tissue could contribute to the pathogenesis incorporating CAN and/or increased max IMT with risk factors would have of diabetes-related heart disease. overlooked some subjects with CHD, even those who could be detected in the present screening. These results suggest that neither the screening methods using TTT as the fi rst line test nor the risk-guided approach including the ADA guideline

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A154 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

560-P SPA is reduced after even short-term improvement of glycemic control and Soluble CD36 (sCD36) in Maturity Onset Diabetes of the Young adiponectin inhibits directly SPA. (MODY) Subjects MA P. KYITHAR, SIOBHAN BACON, AASE HANDBERG, MARIA M. BYRNE, Dublin, 562-P Ireland, Aarhus, Denmark The Effects of the Metabolic Syndrome on Early Subclinical Diastolic MODY subjects are lean, insulin sensitive and usually have normal lipid Dysfunction Is Primarily Driven by the Inclusion in the Defi nition of profi le. Other than the long duration of hyperglycemia, MODY patients have Hyperglycemia

minimal additional cardiovascular risk factors. However, studies have shown ALOIS SALLER, PAOLA BIGOLIN, MARINO BRUSEGHIN, MARCO ARBOIT, POSTERS that hepatocyte nuclear factor (HNF) 1A-MODY subjects have an increased ISABELLA BARZON, Padua, Italy, Padova, Italy Complications risk of cardiovascular morbidity. CD36, a transmembrane glycoprotein, has The purpose of this study was to evaluate the presumed importance of Acute and Chronic been associated with infl ammation, atherosclerosis and platelet activation, hyperglycaemia (HG) as the basic mechanism underlying early subclinical and increased plasma sCD36 levels have been correlated with plaque diastolic dysfunction (DD) by recent more sensitive transthoracic instability. This study evaluated sCD36 levels in different forms of MODY echocardiographic (TTE) techniques in patients (pts) with Metabolic and to compare this to type 2 diabetes (T2DM) & HNF1A-mutation negative Syndrome (MeS). MeS ATPIII criteria were assessed in 154 adults with controls with normal glucose tolerance (NGT). normal systolic left ventricular (LV) function. As controls served 45 subjects Plasma sCD36 levels were measured by ELISA in 32 HNF1A-, 11 glucokinase which presented less than 2 risk factor components except impaired fasting (GCK)-, 9 HNF4A-MODY, 19 T2DM & 10 NGT subjects. HbA1c, fasting insulin glucose (CS-IFG) and 22 type II diabetes pts without criteria for MeS (DM- & C-peptide, oral glucose insulin sensitivity index (OGIS) & area-under the MeS). TTE was used to assess LV structure (LV mass) and DD: by colour curve (AUC) insulin were measured from 2-hour 75-g oral glucose tolerance M-mode TTE and fl ow propagation velocity (pv) in order to calculate E/pv test. Subjects were BMI-matched. sCD36 levels (mean ± standard error of ratio and colour-guide pulsed wave tissue Doppler from the apex to calculate means) in HNF1A subjects were signifi cantly lower compared to NGT controls E/E’ ratio. All patients had normal global systolic function (ejection fraction (0.71±0.07 vs. 1.15±0.16 arbitrary units,P=0.02). No signifi cant difference 67.4±6%, mean±S.D.; 51-69%, min-max). 114 pts (84%) presented left was seen in sCD36 levels among HNF1A, GCK & HNF4A subjects. sCD36 ventricular hypertrophy (LVH) and 45 pts (31%) had DD. Logistic regression levels in MODY groups did not differ when compared with BMI-matched analysis model showed that only HG was signifi cantly associated with both T2DM subjects. In GCK-MODY, a signifi cant inverse correlation was seen DD parameters (E/E’ 4.7 O.R. 95% CI. (0.99-22.14), p=0.05; E/pv 13.2 O.R. between sCD36 and HbA1c (r=-0.63,P=0.04) & OGIS (r=-0.82,P=0.004). A 95% CI. (1.6-109.2), p=0.017). Subgroup analysis: The prevalence of LV DD positive correlation was found between sCD36 & fasting insulin in NGT derived by E/E’ and E/pv in the MeS group with IFG ranged from 34.4%- subjects (r= 0.75,P=0.02). sCD36 levels did not correlate with age, diabetes 41.3%, in MeS without IFG from 10%-5.3%, in CS-IFG from 6.7%-4.4% and duration, BMI, fasting insulin & C-peptide, OGIS & AUC insulin in HNF1A, DM-MeS from 22%-22.7 (p<0.001 for all). The prevalence of LV DD derived HNF4A and T2DM subjects. by E/E’ and E/pv in the MeS group with LVH ranged from 31.1%-37%, in the MeS without LVH from 30.8%-30.8%, in CS-IFG pts with LVH from 9.1%- 9.1% and in CS-IFG without LVH from 4.3%-0% (p<0.01 for all). In according to previous studies MeS is associated with DD independent of LVH. The effect of the MeS on DD is primarily driven by the inclusion in the ATP III defi nition of HG, itself a well established, potent cardiovascular risk factor.

563-P The Effects of Uninephrectomy and Accompanied ACEI Treatment on Myocardial Gene Expression HEUNG MAN LEE, YI SUI, HAILU ZHAO, JENNIFER M.T. SIU, RICHARD K.W. CHOY, XIAODAN FAN, WING-YEE SO, JULIANA C.N. CHAN, RONALD C.W. MA, Hong Kong, China Our data suggest that HNF1A mutations may play a role in regulation of Type 2 Diabetes is commonly associated with renal and cardiovascular sCD36 levels and sCD36 may be a measure of insulin sensitivity in GCK- complications. Patients with diabetic kidney disease are at risk of progression MODY subjects. to end stage renal disease, and have increased risk of heart failure and cardiovascular events. The mechanism linking kidney disease with increased 561-P cardiovascular risk is not clear. We utilized the uninephrectomized rat Spontaneous Platelet Aggregation Evaluated by Laser Light Scatter model (UNX rats) to study cardiac gene expression profi le under conditions in Patients with Type 2 Diabetes: Effects of Short-Term Improved mimicking diabetic kidney disease. Six months after removal of kidney, UNX Glycemic Control and Adiponectin rats developed multiple abnormalities including reduced kidney function, KENJI HARA, YOSHIMASA ASO, KYOKO OMORI, TOMOKO TERASAWA, RIKA pancreatic islet β-cell dysfunction, glucose intolerance and dyslipidemia. In NARUSE, KOHOZO TAKEBAYASHI, TOSHIHIKO INUKAI, Koshigaya, Japan UNX rats, there is evidence of myocardial damage. Using the Agilent Rat 44K Spontaneous platelet aggregation (SPA) is enhanced in patients with type whole genome microarray that covers over 20,000 genes, we identifi ed a 2 diabetes. Adiponectin may inhibit platelet aggregation. The aims of the predominant trend of activated expression of ribosomal proteins, suggesting present study were to identify factors associated with in vitro SPA measured an increased protein synthesis capacity in the heart of UNX rats. Moreover, by a laser light scattering method, and to investigate the effects of short-term activated expression in genes involved in ribosome biogenesis and protein glycemic control and adiponectin on SPA. We investigated SPA in 20 healthy translation were also observed. This trend of increased protein translational control subjects and 82 patients with type 2 diabetes. Next, we evaluated capacity was confi rmed by qRT-PCR to show increased expression of the changes of SPA and serum high molecular weight (HMW) adiponectin after 28S ribosomal RNA. Supporting the increased translational capacity in 2 weeks of improved glycemic control in 20 hospitalized diabetic patients. the heart of UNX rats, chaperone proteins which are important for protein Using washed platelets from 10 subjects, in vitro SPA was measured over 15 folding also showed increased expression in the microarray results and min in the absence or presence of recombinant adiponectin (20μg/ml). SPA validated by qRT-PCR. Although ACEI treatment signifi cantly improved renal was defi ned as formation of small aggregates (SA) under constant stirring dysfunction, insulin secretion and glucose tolerance in the UNX rats, the in the absence of any agonists. SPA was increased in diabetic patients increased expression of ribosomal proteins and chaperone proteins in the compared with control subjects. In diabetic patients, SPA was positively UNX heart was not altered by ACEI treatment. Translational activation has correlated with plasma fi brinogen, fasting plasma glucose, glycated albumin, been observed in myocardial hypertrophy. Increased ribosome biogenesis in and high-sensitivity C-reactive protein. Step-wise multivariate analysis the hearts of UNX rats suggested UNX rats may be a useful model to study showed plasma fi brinogen to be the strongest independent determinant the pathogenesis of cardiac dysfunction in diabetic kidney disease. of SPA. SPA decreased signifi cantly after 2 weeks of glycemic control in diabetic patients. There was a signifi cant negative correlation between changes of SPA and those of HMW adiponectin during treatment. The in vitro study showed that adiponectin inhibited the spontaneous aggregation of washed platelets. In conclusion, hyperfi brinogenemia and hyperglycemia are independently associated with SPA in patients with type 2 diabetes.

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A155 COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND HUMAN DIABETES

564-P 566-P The Relationship of Serum Infl ammatory Factors, Insulin Resistance Tissue Inhibitors of Metalloproteinase 1 and 2 and Matrix Metallo- and Sub-Clinical Vascular Disease in Type 2 Diabetic Patients proteinase-9 in Prediabetes XIAO-ZHEN JIANG, QUAN JIANG, XIAO-HUI ZHAO, YU-FENG ZOU, Shanghai, JUNPING CHEN, DONGXU FU, JULIE A. STONER, MINGYUAN WU, TIMOTHY J. China LYONS, Oklahoma City, OK Object: Chronic low-grade infl ammation in subjects with type 2 diabetes Vascular complications in Type 2 Diabetes may be initiated years earlier is associated with health complications including insulin resistance in the prediabetic stage. Matrix metalloproteinases (MMPs) and their tissue

POSTERS and cardiovascular diseases. However,there were little study about the inhibitors (TIMPs) are implicated in vascular function and remodeling. We

Complications relationship of serum infl ammatory factors and sub-clinical vascular hypothesize that an imbalance between MMPs and TIMPs is present in Acute and Chronic disease or insulin resistance. We investigated the association of serum prediabetes, associated with cardiovascular dysfunction and an increase of C-reactive protei(CRP), interleukin-6(IL-6), adiponectin (APN), leptin (LEP), tissue advanced glycation end-products (AGE). To test this hypothesis, we tumor necrosis factor-α (TNF-α), matrix metallo -proteinases(MMP-9), measured serum levels of MMP-9, TIMP-1, and TIMP-2 with ELISA assays tissue inhibitor of metalloproteinase-1 (TIMP-1), insulin resistance and sub- in 89 subjects, who were classifi ed by 2h 75g oral glucose tolerance tests clinical vascular disease in type 2 diabetic patients in this study. Methods: as prediabetic (n=35, 29 females) or normal glucose tolerant (NGT, n=54, Serum levels of CRP, IL-6, APN, LEP, TNF-α, MMP-9 and TIMP-1 of 199 T2DM 43 females). Cardiovascular function was assessed by pulse wave analysis patients without clinical macroangiopathy were tested. We measured their (PWA), and skin AGE content by an investigational device - SCOUT which carotid intima-media thickness (IMT) by ultrasound, and calculated HOMA- scans and analyzes skin AGE-related autofl uorescence to provide a ‘SCOUT IR of each patient. If the IMT was greater than 0.9mm, the patients were score’. Means were analyzed with a two-sample t-test and Pearson’s partial assigned to sub-clinical vascular disease group (group A), the rest were in correlation coeffi cients were used for age- and body mass index-adjusted non-clinical vascular disease group (group B). Results: CRP (3.19±1.33 vs analyses for females, while non-parametric alternatives were used for males 2.55±1.05), IL-6 (3.5±0.93 vs 2.79±0. 72), MMP-9 (72.01±17.91 vs 54.83±17.20), due to small sample size. Serum TIMP-1 levels were signifi cantly higher in TIMP-1 (63.09±8.11 vs 43.38±7.78), LEP (0.85±0.44 vs 0.62±0.36) and HOMA- prediabetic than NGT females and males (p<0.0001 and 0.026 respectively). IR (6.78±2.98 vs 4.93±2.93) were higher but APN (8.83±3.99 vs 9.79±4.47) Among female only, prediabetes was associated with a decreased MMP- was lower in group A than in group B (P<0.05). There were no difference 9/TIMP-1 ratio (p=0.0049); TIMP-1 was associated positively with SCOUT in TNF-α (P>0.05) between two groups. In group A, APN was negatively score (p=0.044) and negatively with mean arterial pressure (p=0.048); and correlated to HOMA-IR and IMT (P<0.05). CRP, LEP, MMP-9 and TIMP-1 was MMP-9/TIMP-1 ratio was positively associated with diastolic blood pressure positively correlated to HOMA-IR and IMT (P<0.05), TNF-α was positively (p=0.035). Among males, MMP-9 and MMP-9/TIMP-1 ratio were positively correlated to IMT (P<0.05), but TNF-α was not signifi cantly correlated to correlated with diastolic blood pressure (p=0.010 and 0.023 respectively), HOMA-IR (P>0.05). HOMA-IR was positively associated with C-IMT (P<0.05). while TIMP-2 was positively correlated with estimated stroke volume Conclusions: Levels of serum APN ,CRP, IL-6,LEP, MMP-9, TIMP-1 signifi cantly index (p=0.031) and negatively with pulse rate (p=0.043). In conclusion, the changed at the stage of sub-clinical vascular disease, which suggested us to elevation of TIMP-1 in prediabetes and its positive association with AGEs inhance the prevention therapy to slow down the process of macrovascular in females suggests that AGE formation may reduce extracellular matrix complications when they occured. (EM) degradation. Together with the correlations of TIMP-1 with PWA Supported by: Health Bureau of Shanghai Pudong New Area measures, the data suggest that impaired EM remodeling may contribute to cardiovascular dysfunction, especially diastolic dysfunction, in prediabetes. 565-P Supported by: Talley Research Award Thrombosis Risk Assessment of Diabetes by Dielectric Spectroscopy ISAO UCHIMURA, YOSHIHITO HAYASHI, MAKOTO KAIBARA, Tokyo, Japan, 567-P Saitama, Japan Type 2 Diabetes and the Progression of Visualized Atherosclerosis The measurements of whole blood coagulability and platelet aggregability to Clinical Cardiovascular Events are very important to reduce the risk of thrombosis or to avoid unexpected CHRISTOPH H. SAELY, PHILIPP REIN, ALEXANDER VONBANK, KURT HUBER, bleeding in diabetic patients treated with antiplatelet or anticoagulant HEINZ DREXEL, Feldkirch, Austria therapy. The dielectric spectroscopy (DS) of whole blood from healthy We aimed at prospectively evaluating to what extent pre-existing volunteers showed dielectric relaxation in the specifi c frequency range (Anal. coronary artery disease (CAD) accounts for the increased long-term vascular Chem. 82: 9769, 2010.). And the blood coagulability measured by DS showed event risk of patients with type 2 diabetes (T2DM). We hypothesized that the signifi cant correlation with the data by free oscillation rheometer(Ti). baseline CAD among patients with T2DM may account substantially for their In this study, we measured the coagulation of whole blood obtained from increased cardiovascular risk. diabetes (n=28) with DS. Blood was taken using 3.8% tri-sodium citrate Over 8 years we recorded vascular events in 750 consecutive patients solution. DS measurements at 37C in 1kHz,10KHz,756KHz and10MHz were whose baseline CAD state was verifi ed angiographically. performed by an impedance analyzer (Agilent 4294A). The capacitor-type The prevalence rates of CAD (87.8% vs. 80.4%; p=0.029) and of signifi cant sample holder has 2.0 mm space between two titanium electrodes. After coronary stenoses ≥50% (69.5% vs. 58.4%; p=0.010) as well as the extent the addition of 250 mM CaCl2 solution to blood, measurements were started. of CAD, defi ned as the number of signifi cant coronary stenoses (1.7±1.6 vs. The dielectric spectra were recorded up to 60 min and were normalized by 1.4±1.5; p=0.014) were higher in patients with T2DM (n=164) than in non- the spectrum at the time zero(e/et=0). With holder rotation the normalized diabetic subjects. During follow-up, T2DM strongly predicted vascular events spectra (NS) of whole blood from diabetes showed elevated peak levels (n=257) independently from the presence and extent of baseline CAD (hazard from early phase of coagulation in both 756kHz and 10MHz. The time at ratio (HR) 1.36 [1.03-1.81]; p=0.032); conversely, the presence and extent of peak level of the NS in 10MHz showed positive correlation with Ti(r=0.541). baseline CAD predicted vascular events independently from T2DM (HRs 3.29 Peak levels in 10MHz showed signifi cant correlation with BMI (r=0.49), [1.93–5.64]; p<0.001 and 1.37 [1.23–1.53]; p<0.001, respectively). The overall FPG (r=0.554), LDL-C (r=0.395) and Hct (r=0.522). Without holder rotation risk increase conferred by T2DM was driven by the extremely high 53.3% NS showed elevated peak levels in 1kHz and 10kHz from diabetes who event rate of patients with both T2DM and signifi cant CAD at baseline; showed acceleration of blood sedimentation rate. The blood from diabetics individuals with T2DM who did not have signifi cant CAD at baseline showed treated with aspirin showed the delay of time of MAX coagulation with a signifi cantly lower event rate (22.0%; p<0.001). NS. Dielectric responses in low frequency suggested red cell aggregation We conclude that T2DM and angiographically visualized coronary athero- during coagulation process, and those in middle or high frequency also sclerosis are mutually independent predictors of vascular events. The overall suggested a formation of stable fi brin net. Therefore, the present study risk increase conferred by T2DM is driven by accelerated progression of pre- strongly suggested that DS is simple and accurate method for measuring existing atherosclerosis to clinical cardiovascular events. blood coagulability and will provide the risk assessment and the safety to diabetics taking antiplatelet or anticoagulant.

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A156 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

568-P sulfate-containing proteoglycan. Radiotracer studies in rats showed that Vascular Smooth Muscle Dysfunction Is Differently Associated MGmin-LDL had similar fractional clearance rate in plasma to unmodifi ed with Vascular Complications and Risk Factors from Endothelial Dys- LDL but increased partitioning onto the aortal wall. Mass spectrometry function in Type 2 Diabetic Patients peptide mapping identifi ed an arginine “hot spot” site of modifi cation by NAOYA KAWANO, MASANORI EMOTO, YUKO YAMAZAKI, HIROMI URATA, methylglyoxal in apolipoprotein B100 in MGmin-LDL. A computed structural SHOKO TSUCHIKURA, KOKA MOTOYAMA, TOMOAKI MORIOKA, KATSUHITO MORI, model predicted that MGmin-LDL was structurally distorted, increasing SHINYA FUKUMOTO, TETSUO SHOJI, YASUHISA OKUNO, YOSHIKI NISHIZAWA, the surface exposure of a proteoglycan binding domain. We conclude that methylglyoxal modifi cation of LDL forms small, dense LDL with increased

MASAAKI INABA, Osaka, Japan POSTERS

Atherosclerosis and arteriosclerosis are mainly caused by dysfunction atherogenicity, providing a new route to atherogenic LDL which likely Complications of components of artery, vascular endothelial cell, smooth muscle cell, contributes to the escalation of risk of CVD in diabetes. Acute and Chronic and extracellular matrix. Endothelial dysfunction is well established as a Supported by: The British Heart Foundation predictive surrogate marker of cardiovascular event. But, little is known regarding clinical implications of vascular smooth muscle dysfunction on & 570-P cardiovascular disease and microangiopathy. In the present study, we aimed Biological Signifi cance of Charge-Based Heterogeneity in Diabetic to clarify the association of arterial function with micro-/macroangiopathy VLDL Subfractions and conventional cardiovascular risk factors in 190 type 2 diabetic patients CHU-HUANG CHEN, JONATHAN LU, DAVID A. ENGLER, LIANG-YIN KE, RAMAZAN (T2DM: age, 64±11 (SD) years old; duration of diabetes, 12±10 years). Flow- YILMAZ, RICHARD A.F. DIXON, SHU-HUA CHEN, CHAO-YUH YANG, Houston, TX, mediated dilatation (FMD) as an endothelial function and nitoroglycerin- Kaohsiung, Taiwan, Taichung, Taiwan mediated dilatation (NMD) as vascular smooth muscle function were Plasma very low-density lipoprotein (VLDL) in patients with type 2 assessed by a novel ultrasound equipment, UNEXEF18G (Unex Co. Ltd., diabetes mellitus (DM) damages vascular endothelial cells (ECs), but Japan). the mechanisms are not well understood. Comprising heterogeneous In all T2DM, FMD was 6.7±3.9%, ranging from 0.7 to 19.2%, and NMD particles, VLDL’s harmful components have not been identifi ed. Thus, we 13.1±6.6%, 0.5 to 28.9%. There were no signifi cant differences in FMD chromatographically resolved VLDL from 9 type 2 DM patients (fasting between in T2DM with and without micro- or macroangiopathy. However, glucose 9.4±1.2 mmol/L; VLDL 1.20±0.32 mmol/L) and 9 nondiabetic NMD in T2DM with micro- and macroangiopathy was signifi cantly lower individuals (fasting glucose 5.2±0.5 mmol/L; VLDL 0.48±0.05 mmol/L) into than those without both anigopathy. 5 increasingly electronegative subfractions (V1-V5) to study the in-vitro Although both FMD and NMD were lower in accordance with the characteristics of each. V5 composed 11±4% (0.14±0.05 mmol/L) of diabetic progression of CKD stage (FMD, stage1 9.4±5.0, stage2 6.6±3.7, stage3 and 2±1% (0.02±0.01 mmol/L) of nondiabetic VLDL (P<0.01). In cultured 6.3±3.4, stage4/5 5.3±3.9, p=0.005; NMD, stage1 17.9±6.2, stage2 14.1±6.5, human aortic ECs, V5 (0.05 mmol/L) induced apoptosis in 40% of cells in stage3 11.3±6.1, stage4/5 7.8±3.2, p<0.0001), the decrease of NMD was 24 hours, whereas V1 had a negligible effect. V5 was not effective at <0.03 greater than that of FMD. On multiple regression analysis, age (β=-0.186, mmol/L but killed nearly all cells at >0.1 mmol/L. To assess receptor-binding p=0.030), smoking (β=-0.174, p=0.018), systolic blood pressure (β=-0.166, affi nities, V5 and V1 were labeled with different fl uorescent dyes and added p=0.031), and HbA1c (β=-0.156, p=0.031) were found to be a signifi cant simultaneously to EC cultures. At 15 minutes, cells were fi lled exclusively independent contributor to FMD (R2=0.159, P<0.0001) and, age (β=-0.269, with V5 particles. When added separately, only V5-treated cells produced p=0.002) and eGFR (β=0.262, p=0.002) to NMD (R2=0.283, P<0.0001). In reactive oxidative substances. Quantitative mass spectrometry revealed that conclusion, NMD is differently associated with vascular complications and V5 was associated with apo(a) and contained 1.5-fold more apolipoprotein risk factors from FMD in type 2 diabetic patients. (apo)E than V1. Both apo(a) and apoE are natural ligands for VLDL receptors (VLDLR). Receptor-associated protein (RAP) prevents ligand association with VLDLR and LDL receptor. Lectin-like oxidized LDL receptor-1 (LOX-1) COMPLICATIONS—MACROVASCULAR— is a receptor for modifi ed LDL. V5-induced EC apoptosis was signifi cantly CELLULAR MECHANISMS OF ATHEROGENESIS attenuated by RAP but not LOX-1 neutralizing antibodies, suggesting that IN DIABETES V5 was endocytosed by receptors that recognize apoE but not apoB, such as VLDLR. V5-derived neutral lipids induced apoptosis in more than 70% of [See also: Presidents Poster 384-PP, page A106.] cells; V5 phospholipids induced apoptosis in 20%. However, V1 lipids had no effect. Thus, V5, whose affi nity to VLDLR is probably promoted by its increased association with apoE and apo(a), exerts EC toxicity through its Guided Audio Tour: Mechanisms of Atherosclerosis in Diabetes—Animal various lipid components, suggesting that the pathogenicity of diabetic VLDL and Cellular Models (Posters 569-P to 576-P), see page 13. is attributable in part to its high content of V5. & 569-P Supported by: NIH Grant HL-63364 ADA-Funded Research Conversion of Low Density Lipoprotein to the Small, Dense Pro- Atherogenic Form by Glycation with Methylglyoxal & 571-P NAILA RABBANI, LISA GODFREY, MINGZHAN XUE, PAUL J. THORNALLEY, Severe Hypercholesterolemia Induces a Unique Subpopulation of Coventry, United Kingdom Macrophage Foam Cells in ApoE -/- Mice Cardiovascular disease (CVD) is a major cause of premature mortality in KOKA MOTOYAMA, JINGJING TANG, IVAN GOMEZ, MASANORI EMOTO, diabetes. Increased retention of low density lipoprotein (LDL) in arterial walls MASAAKI INABA, ELAINE W. RAINES, Osaka, Japan, Seattle, WA is a key mediator of arterial atherosclerosis. Low density lipoprotein (LDL) Macrophages play a key role in the development of infl ammatory lesions of modifi ed by methylglyoxal is a quantitatively important form of advanced atherosclerosis. Recently, there has been increasing realization that macrophage glycation endproduct (AGE)-modifi ed LDL in human subjects, increasing subsets respond differently to specifi c stimuli, in particular dependent upon Th1 4-fold in patients with diabetes. The aim of this study was to investigate vs. Th2 immune stimuli. Foam cells are a hallmark of developing lesions, whose structural and functional changes of LDL when it is modifi ed to physiological abundance increases with the extent of hypercholesterolemia, but it is unclear extent by methylglyoxal. how the immune response is altered by foam cell formation. Human LDL was isolated from peripheral venous plasma and glycated To begin to address this question, we have compared the characteristics by methylglyoxal in vitro to minimal extent (MGmin-LDL). Particle size was of peritoneal macrophages from ApoE-/- mice fed a high fat diet (plasma determined by electron microscopy and atherogenicity by binding to arterial cholesterol > 1500 mg/dl) with ApoE-/- mice fed a normal chow diet (plasma proteoglycan and vortex mixing-stimulated aggregation in vitro, and binding cholesterol 502 mg/dl). Consistent with previous studies, ApoE-/- mice on to rat aorta in vivo. a chow diet display a Th1 immune response, but those on a high fat diet MGmin-LDL contained 1.6 molar equivalents of modifi cation - mostly show Th2 dominance. To clarify macrophage characteristics with different hydroimidazolone AGE. MGmin-LDL had decreased particle size (nm, mean diets, macrophages were collected from the peritoneal cavity following ± SD): MGmin-LDL 26.0 ± 1.7 and LDL 29.1 ± 1.8; P<0.001 (n = 70). It also had administration of the sterile irritant thioglycollate, and fl ow cytometry was 2-fold increased affi nity for arterial proteoglycan – biglycan, aggrecan and used to evaluate macrophage lipid content simultaneously with evaluation perlecan. It also had increased tendency to aggregate in vitro. Glycation of of different cell surface and intracellular markers. LDL by glucose was without similar effect. Cell culture studies showed that Distinct large and small subpopulations are detected in thioglycollate- MGmin-LDL normal binding to the LDL receptor, did not bind the scavenger elicited peritoneal macrophages based on their size and granularity. Elevated receptor and had increased binding affi nity for cell surface heparin numbers of the small cell population are seen in ApoE-/- mice fed a high

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A157 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

fat diet, and these small cells have an increased proportion of foam cells. explained 12% of the variance in circulating sRAGE, a level similar to that This small macrophage subpopulation is further characterized by elevated previously reported by us for known non–genetic determinants. We note levels of CX3CR1 and CCR7, but decreased levels of Ly6C, which suggests that both these SNPs show ethnic variation in a direction that is consistent anti-infl ammatory features. Based on intracellular markers, this small with the previously reported much lower total sRAGE in those of African macrophage subpopulation displays increased levels of SOCS-3 and the Th2 Caribbean origin versus whites. In conclusion we report a novel tagging marker Arginase I, but decreased levels of SOCS-1 and the Th1 marker iNOS. variant rs2071800 in the RAGE-HLA-DQA2 region independently associated However, pro-infl ammatory markers NFkB (p65), CD80 and CCR2 were also with sRAGE levels and confi rm the known association of sRAGE levels with elevated in this small macrophage subpopulation. These fi ndings suggest rs2070600 in the RAGE gene. Variation in the frequencies of these tagging POSTERS

Complications the possibility that severe hypercholesterolemia in ApoE-/- mice induces a markers, as seen in the HAPMAP samples may explain the ethnic differences

Acute and Chronic Th2-prone macrophage foam cell subpopulation, but also promotes a distinct in circulating sRAGE levels. phenotype in response to the complex micro-environment in vivo. Supported by: Grant from Pfi zer, Inc.

& 572-P & 574-P Tight Control of Type 1 Diabetes May Be Cholesterologenic Diabetes Mellitus Accelerates Cartilaginous Metaplasia and Calci- MAY-YUN WANG, SUZANNE STROWIG, SARA K. MCCORKLE, XINXIN YU, fi cation in Atherosclerotic Vessels of LDLr Mutant Mice GREGORY O. CLARK, PHILIP RASKIN, ROGER H. UNGER, Dallas, TX NGOC B. NGUYEN, VEENA NAIK, MEI Y. SPEER, Seattle, WA In normal islets, the juxtaposed α-cells are the fi rst targets of secreted Vascular calcifi cation is highly prevalent in patients with type II diabetes insulin. Since the paracrine level of insulin is >100 times normal peripheral mellitus (T2D) and is associated with a high risk of cardiovascular morbidity vein levels, to normalize hyperglucagonemia and hyperglycemia in type 1 and mortality in this population. Although it has long been considered a diabetes (T1DM) must require maintenance of near-paracrine levels of degenerative process leading to passive deposition of calcium-phosphate hyperinsulinemia in the general circulation. To test this, we assayed plasma salts in necrotic tissue, a growing number of studies have evidenced that insulin levels in 10 well-controlled T1DM patients before and after insulin vascular calcifi cation is a cell-mediated, highly regulated process that injection and a mixed meal (Boost). Mean insulins were 44±28 before and recapitulates bone formation and remodeling. To understand the relation 115±87 ng/ml 1 h after a preprandial bolus. Age-matched nondiabetics of T2D and vascular calcifi cation as well as the mechanisms that regulate given the same meal averaged 10±4 and 38±22 ng/ml at equivalent times. the development of bony and cartilaginous elements in T2D vessels, To determine if iatrogenic hyperinsulinemia contributes to atherogenesis we attempted to develop a T2D mouse model with features of human in T1DM, 12 T1DM (NOD) mice were optimally controlled with insulin (0.2 cardiovasculopathy. We found that low density lipoprotein receptor mutant units bid). Glucose levels averaged 130±71 mg/dL 3h after insulin; plasma (LDLr-/-) mice fed with diabetogenic/procalcifi c diet developed obesity, insulin averaged 3.9±1.1 before and 14.3±4.5 ng/ml 3h after insulin, which hyperglycemia, hyperlipidemia, atherosclerosis, and vascular cartilaginous was 5 and 15 times the levels of 0.8±0.25 and 1±0.32 ng/ml in nondiabetic metaplasia and calcifi cation, similar to those observed in human T2D vessels. littermates at the same times of day. On high dose insulin hepatic expression Importantly, T2D accelerated the development of vascular cartilaginous of cholesterologenic transcription factors SREBP-2 and SREBP-1a, and their metaplasia and calcifi cation when compared with studies of non-T2D LDLr- targets, HMG-CoA reductase and PCSK9, rose from low levels in untreated /- mice fed with procalcifi c diet. By 18 weeks under the diets, incidence of diabetes to nondiabetic levels. However, while they remained low on cartilaginous metaplasia in T2D vessels increased by 3 folds (100% in T2D both low dose insulin monotherapy (0.02U bid) and leptin monotherapy, vs 33% in non-T2D) and of calcifi cation by 15 folds (75% in T2D vs 5% in non hyperglycemia was uncontrolled (588±17 mg/dL) on low insulin monotherapy T2D). Genetic fate mapping of smooth muscle (SM) cells in LDLr-/- mice via whereas stable normal glycemia (80±13 mg/dL) with normal HgA1c was SM22-Cre recombinase and Rosa26 Cre reporter transgenic alleles revealed maintained with leptin monotherapy. Our results imply that leptin might that cells of osteochondrogenic properties were largely derived from cells help reduce both hyperglycemia-induced microvascular complications and of SM origin. Finally, an increased expression of receptor for advanced macrovascular complications of T1DM. Because the prevalence of coronary glycation end products was found in T2D LDLr-/- vessels, co-localizing with artery disease in middle-aged T1DM patients is so high and is not well early osteochondrogenic markers and cells of chondrocyte morphology. correlated with hyperglycemia, it seems prudent to explore further the role These results are the fi rst to elucidate a role of T2D in vascular cartilaginous of iatrogenic hyperinsulinemia as a contributing factor. metaplasia and calcifi cation. Studies on the mechanisms involved in this life- threatening complication are still ongoing. & 573-P Supported by: NIH K01 DK075665, New Investigator Award administered by NIH Genome Wide Scan of Soluble RAGE Levels in a Diabetic Population P30 DK017047 Reveals a Novel Associated Variant in RAGE-HLA-DQA2 Region HELEN COLHOUN, HARSHAL DESHMUKH, JOHN BETTERIDGE, AUROBINDO & 575-P CHATTERJEE, PAUL DURRINGTON, JOHN FULLER, CRAIG HYDE, SHONA LIVING- Hyperglycemia Induced 26S Proteasome Activation Is an Early STONE, PAUL MCKEIGUE, ANDREW NEIL, VALENTINE CHARLTON-MENYS, Event in Streptozotocin-Treated Mice WARREN BAO, DAVID DEMICCO, GREGORY PRESTON, KATHRYN TAN, GRAHAM HONG-TAO LIU, SHUJIE YU, JIAN XU, Oklahoma City, OK HITMAN, Dundee, United Kingdom, London, United Kingdom, Groton, MA, The ubiquitin-proteasome system, a major route for degradation of Manchester, United Kingdom, Edinburgh, United Kingdom, Oxford, United Kingdom, cellular proteins, has been implicated in certain cancers, neurodegenerative New York, NY, Hong Kong, China disorders, and cardiovascular diseases. Although we have previously We previously reported that higher levels of total soluble receptor for shown that 26S proteasome activation might cause endothelial dysfunction advanced glycation endproducts (sRAGE) predicts coronary heart disease in a mouse model of diabetes, evidence of 26S proteasome activation in in the Collaborative Atorvastatin Diabetes Study (CARDS). The extent to diabetes in vivo is lacking. Here we report that 26S proteasome activation which genetics infl uences sRAGE levels needs to be determined in order for induced by acute hyperglycemia is an early event in the streptozotocin (STZ) sRAGE to be used as a biomarker in the development of RAGE inhibitors. treated mice. To assess proteasome functionality in vivo, we employed mice To identify loci associated with sRAGE levels in samples from CARDS that expressed a reporter UbG76V-Green fl orescence protein (GFP) (Jackson participants (N=594), a genome wide scan using the Perlegen 6 SNP set (n Lab). The reporter was designed with GFP fused to a mutant ubiquitin =525,625 SNPs) was carried out followed by imputation of loci not directly moiety which allows quantifi cation of 26S proteasome activity in vivo with typed using IMPUTE 2 and HAPMAP II data. sRAGE was measured using the GFP fl uorescence and protein levels. The mice were rendered diabetic by R&D Systems Quantikine Immunoassay (assay coeffi cient of variation 4.4%). STZ injection (STZ: 50 mg/kg/d, sham: sodium citrate; i.p. 5d. n=3/group, We tested for an association of both directly typed and imputed SNPS with age: 10 wks). Acute hyperglycemia was defi ned as a fasting (4 hrs) blood sRAGE using multiple linear regression implemented in SNPTest adjusted glucose level of >300 mg/dL 1 week after the fi nal dose of STZ. Compared for age, sex and BMI. Conditional haplotype-based testing implemented in to sham mice, acute hyperglycemic mice presented a signifi cant decrease in PLINK was used to test for the independence of associations found. Results: both GFP fl orescence and protein levels in several tissues including aortic 12 SNPS reached genome wide signifi cance level of p<10-8 all of which were artery, brain, fat, heart, kidney, liver, lung, pancreas, skeletal muscle, and in the RAGE locus or +/- 150 kb upstream or downstream of it. These markers spleen. The GFP reduction correlated with increased 26S proteasome were tagged by two missense mutations rs2070600 (P =1.5x10-11 beta= activity (chymotrypsin-like) in the same tissue preparations. Further, the -6.8(±0.9)) in the RAGE gene region on chromosome 6 and rs2071800 in the increased 26S proteasome activity was also detected in age matched wild HLA-DQA2 region (P=1.2 x10-8 beta=-4.4(±0.7)) that were associated with type mice (C57BL/6J) that received the same STZ regimen (STZ vs. sham; sRAGE independently of each other. Together variation at these two loci n=5/group). Importantly, the impaired endothelial function, as measured

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A158 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES by acetylcholine-induced vasorelaxation, could be effectively mitigated 578-P by pharmacological (MG132: 5 mg/kg/d, i.p. 2d; sham: DMSO/saline. n=5/ Comparative Effects of Insulin Glargine and NPH Insulin on Nitric group) or genetic (siRNA knockdown of PA700, a regulatory complex in 26S Oxide Bioavailability and Nitroxidative Stress in Aortic and Glomer- proteasome, i.v. 7d. n=5/group) inhibition of the 26S proteasome in these ular Endothelium from Diabetic Rats mice. These data suggest that acute hyperglycemia functionally activates R. PRESTON MASON, ROBERT F. JACOB, LU-LIN JIANG, ADAM M. JACOBY, the 26S proteasome, which may serve as an initial step in the pathogenesis RUSLAN KUBANT, TADEUSZ MALINSKI, Boston, MA, Beverly, MA, Athens, OH of diabetic vascular dysfunction, a hallmark of atherosclerosis. Endothelial cell (EC) dysfunction in diabetes is characterized by reduced Supported by: SDG (AHA) and CoBRE (NIH) –

nitric oxide (NO) and increased peroxynitrite (ONOO ) levels, an imbalance POSTERS

causally related to atherothrombotic disease. In this study, we hypothesized Complications & 576-P that EC dysfunction in diabetic rats is reversed to a greater extent with Acute and Chronic Ceramide-Induced Vascular Dysfunction Results from PP2A Asso- a long-acting insulin analog (insulin glargine) versus an intermediate- ciation with ENOS acting analog (NPH insulin) due to enhanced glycemic control. To test this QUAN-JIANG ZHANG, NICHOLAS B. DEETER, ALEXANDREA NICHOLS, MICHOLE hypothesis, male WKY rats were treated with a nicotinamide-streptozotocin DEESING, CHRIS KOWALSKI, WILLIAM L. HOLLAND, SCOTT A. SUMMERS, E. DALE regimen (65 mg/kg/day) to induce diabetes. After confi rming hyperglycemia, ABEL, J. DAVID SYMONS, Salt Lake City, UT, Dallas, TX, Singapore, Singapore rats were treated with insulin glargine (4 U/kg/day), NPH insulin (2 U/kg, We have previously reported that de novo ceramide biosynthesis twice a day) or vehicle for 4 weeks. At the end of treatment, aortic and decreases endothelium-dependent vasorelaxation by impairing endothelial glomerular ECs were assayed ex vivo for nitric oxide (NO) and peroxynitrite nitric oxide (NO) synthase (eNOS) phosphorylation at serine 1177 (p-eNOS) (ONOO–) release using amperometric approaches. Changes in EC function in obese mice and in isolated arteries. To explore potential mechanisms, were correlated with fasting glucose levels. After 4 weeks, both insulin bovine aortic endothelial cells (BAECs) were incubated for 180-min ± 500 analogs signifi cantly lowered glucose levels as compared to control levels uM palmitate (pal) ± the ceramide synthesis inhibitor myriocin (myr, 10 (483 ± 49 mg/dL, p<0.001); however, a greater effect was observed with uM). Basal or stimulated p-eNOS was assessed in cells treated with or insulin glargine versus NPH insulin (126 ± 8 and 182 ± 22 mg/dL, respectively; without 100 nM insulin (ins) for the fi nal 10-min. Myr prevented pal-induced p<0.001). Insulin glargine also enhanced NO release in aortic and glomerular ceramide accumulation (p<0.05; n=6), and reversed pal-induced reductions ECs by 82% and 112% (p<0.001), respectively, as compared to NPH insulin, (p<0.05) in basal and ins-stimulated p-eNOS, eNOS dimer formation, and NO which increased aortic and glomerular NO release by 47% and 69% (p<0.001), production (n=10-20). Pal-induced, ceramide-mediated reductions in p-eNOS respectively. In glomerular ECs, insulin glargine and NPH insulin reduced were neither secondary to defective kinase signaling to eNOS, increased ONOO– levels by 50% and 32% (p<0.001), resulting in a four-fold and two- fold increase in the NO/ONOO– ratio, respectively; both agents had similar O2•¯ production, nor peroxynitrite accumulation (n=12-60). Pal-induced reductions (p<0.05) in basal and ins-stimulated p-eNOS in BAECS were effects on ONOO– and ratios of NO/ONOO– release in aortic ECs. These data negated both by the PP2A inhibitor okadaic acid (4 nM; n= 6-40), and following demonstrate that insulin treatment, in addition to lowering blood glucose gene silencing of PP2A using siRNA (n=6). eNOS was immunoprecipitated levels, enhanced NO release and reduced oxidative stress in arterial and from BAECs treated ± pal ± myr. Pal led to PP2A association with eNOS renal ECs in diabetic animals. These effects were signifi cantly greater with (p<0.05), which was prevented by myr (n=9-11). To determine if similar insulin glargine, as compared to NPH insulin, suggesting an improved effect mechanisms were present in vivo, mice with ablation of one allele of of long-acting insulin analogs on glycemic control and eNOS function. dihydroceramide desaturase (des1 +/-) and wild type littermates (des1 +/+) consumed high-fat (HF) or standard chow for 12 weeks. HF feeding induced 579-P vascular ceramide accumulation and arterial dysfunction (p<0.05) in des1 DHA Increases HO-1 Expression and Antioxidative Activity through +/+ but not in des1 +/- mice (n=9 per group). HF diet lowered (p<0.05) arterial Activation of Nrf2 in Vascular Endothelial Cells p-eNOS to a greater extent in des1 +/+ versus des1 +/- mice. (p<0.05) and ATSUSHI ISHIKADO, YOSHIHIKO NISHIO, KATSUTARO MORINO, ATSUSHI increased PP2A association with eNOS in vessels from des1 +/+ but not MUKOSE, SATOSHI UGI, HAJIME KONDO, YOKO SONO, TAKETOSHI MAKINO, des1 +/- mice (n=3-4 per group). Thus, ceramide-mediated impairment in ATSUNORI KASHIWAGI, HIROSHI MAEGAWA, Otsu, Japan, Takatsuski, Japan eNOS phosphorylation and vascular dysfunction might be mediated by PP2A N-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to reduce association with eNOS. cardiovascular events independently of the classical risk factors for Supported by: NIHR15HL091493 ADA-Funded Research atherosclerosis, suggesting the direct effects on vascular tissues. In this study, we have tested the hypothesis that n-3 PUFAs increase antioxidative 577-P activity through the activation of Nrf2, a master regulatory transcriptional Arachidonic 12/15-Lipoxygenase Is Involved in the Development of factor, in vascular tissues. Diabetic Cardiomyopathy First of all, we fed C57BL6 or Nrf2 (-/-) mice (n=6-10) with fi sh oil diet (FD) for MASAYA SAKAMOTO, HIROSHI SUZUKI, YOUSUKE KAYAMA, YOSUKE KAYAMA, 3 weeks. FD signifi cantly increased mRNA or protein expression of HO-1, an HIYOUYUKI IUCHI, TOHRU MINAMINO, KATSUYOSHI TOJO, KAZUNORI Nrf2 target gene, by 2.1 or 1.5 fold in thoracic aorta, while the effect of FD was UTSUNOMIYA, Tokyo, Japan, Chiba, Japan not observed in the Nrf2 (-/-) mice. Secondly, human umbilical vein endothelial Adverse effects of hyperglycemia on endothelial function, interstitial fi brosis cells were stimulated with docosahexaenoic (DHA), eicosapentaenoic (EPA), and cardiomyocytes are known as pathogenesis of diabetic cardiomyopathy. arachidonic (ARA) or linoleic acid (LA). DHA and ARA (25-100 μM) dose- But precise mechanism underlying diabetic cardiomyopathy is still unknown. dependently increased HO-1 mRNA (DHA 100 μM; 46.3 fold, ARA 100 μM; To investigate the molecular mechanisms of diabetic cardiomyopathy, we 40.2 fold) and protein expressions, but EPA and LA showed less effects. DHA created a diabetic cardiomyopahty model (streptozotocin (STZ) injection and ARA (100 μM) also increased the intranuclear expression (8.4 and 8.5 rat) and performed microarray analysis. Echocardiography showed that STZ fold) and DNA binding (4.5 and 3.3 fold) of Nrf2, while EPA and LA had no injection rat developed diastolic and systolic dysfunction and cardiac fi brosis effects. The siRNA of Nrf2 signifi cantly reduced the DHA or ARA-induced was increased in STZ injection rat. Among the genes analysis, we found HO-1 mRNA or protein expression. Furthermore, pretreatment with DHA (75 that arachidonic 12/15-lipoxygenase (12/15-LOX) was upregulated in the μM) prevented the tert-butyl hydroperoxide-induced LDH release, cell death heart of STZ rat. Northern blotting and ELISA analysis demonstrated that or ROS production. These effects of DHA were canceled by HO-1 inhibitor or mRNA level of 12/15-LOX and production of 12/15-HETE, a major metabolite the siRNA of Nrf2. To consider the biological mechanisms of DHA and ARA of 12/15-LOX, were also found upregulated in the heart of STZ rat compared on Nrf2 activation, cells were also treated with the inhibitors or siRNAs of to those of control rat heart. To investigate the role of 12/15-lipoxygenase enzymes such as cyclooxygenases and lipoxygenases or nuclear receptor in the cardiomyopathy, we then created STZ injection mice (WT-STZ) and PPARα. However, they did not affect HO-1 mRNA expression induced by compared to those of 12/15-LOX KO (KO-STZ) mice. Interestingly, disruption DHA or ARA. In contrast, N-acetyl cysteine markedly reduced the DHA or of 12/15-LOX improved diastolic and systolic dysfunction and signifi cantly ARA-induced HO-1 mRNA expression, suggesting the role of intracellular reduced cardiac infl ammation and the expression of reactive oxygen species. ROS for Nrf2 activation by DHA and ARA. Our results suggest that cardiac 12/15-LOX is involved in the development Thus, we have found that DHA shows preventive effects against oxidative of diabetic cardiomyopathy and that inhibition of 12/15-LOX could be a novel stress through the Nrf2 activation in vascular endothelial cells, which may treatment for this condition. explain the mechanism of the cardioprotective effects of DHA.

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A159 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

580-P 582-P Direct Demonstration of the Importance of Insulin Receptor in the Even Mild Hyperglycemia Disturbs Vascular Homeostasis in Humans Angiogenic Action of Bone Marrow Derived Mesenchymal Stem BREGTJE A. LEMKES, SARAH E. SIEGELAAR, MAX NIEUWDORP, WIM KULIK, Cells JOOST C. MEIJERS, JOOST B. HOEKSTRA, FRITS HOLLEMAN, Amsterdam, The KOJI MIZUTANI, GREGORY S. WHITE, IN-KYUNG JEONG, HYUNJIN NOH, AKIRA Netherlands MIMA, QIAN LI, CHRISTIAN RASK-MADSEN, DAVID J. MOONEY, GEORGE L. Hyperglycemia induces oxidative stress, damage to the endothelial KING, Boston, MA, Cambridge, MA glycocalyx and a prothrombotic shift in coagulation and fi brinolysis.

POSTERS Insulin’s direct actions to decrease ischemia and accelerate wound The aim of this study was to determine at which level of glycemia these

Complications healing have been discussed, yet this topic remains controversial. The changes occur. In 11 healthy young males we performed a stepwise normo- Acute and Chronic positive effect of insulin on perfusion has been attributed to increasing the insulinemic hyperglycemic clamp at plasma glucose (PG) levels of 6, 8 and expression of angiogenic cytokines such as vascular endothelial growth 10 mmol/l for two hours each (fi g A). Octreotide was infused to suppress factor (VEGF). We have shown that insulin and/or bone marrow derived endogenous insulin release. Measurements were performed at baseline, mesenchymal stem cells (MSC) embedded in the hindlimb skeletal muscle after a 1-hr octreotide run-in, every 30 minutes during the clamp and at 24- of mice with surgically-induced ischemia can increase the expression of hrs. Oxidative stress was assessed by determining malondialdehyde (MDA) angiogenic cytokines and improve perfusion. To directly demonstrate the using tandem mass spectrometry; coagulation activation by von Willebrand positive effect of insulin and MSC on perfusion after ischemia, we implanted factor (vWf, Elisa) and prothrombin fragment 1+2 (F1+2, Elisa); fi brinolysis by insulin scaffold with MSC isolated from a vascular endothelial cell insulin plasmin-alpha2-antiplasmin complexes (PAP, Elisa) and d-dimer (BCS-XP); and receptors (IR) knockout (VENIRKO) mouse. MSC were isolated and cultured glycocalyx turnover by hyaluronic acid (HA) and hyaluronidase (both Elisa). from bone marrow of VENIRKO or control mice (C57BL). In MSC from VENIRKO MDA showed a gradual increase highly correlating with PG (ρ=0.82, mice, IR-β expression was 65% less than in control MSC. Activation of Akt p<0.001, fi g B). F1+2 signifi cantly increased at 6 mmol/l, maintaining and Erk by insulin (10nM) was signifi cantly decreased in VENIRKO MSC as the same level at all PG concentrations (fi g C) and both PAP and d-dimer compared with control MSC by 39% and 31% (p<0.01). Insulin increased increased signifi cantly at 8 mmol/l, indicating coagulation activation mRNA expression of VEGF and FGF and secreted VEGF in supernatants in followed by fi brinolyis (fi g D&E). vWf showed a small, but signifi cant control MSC by 159%, 154% and 139%, respectively. In contrast, insulin decrease at all PG levels (max 9%, p=0.01). No difference in HA levels was did not increase expression of VEGF/FGF in VENIRKO MSC. Perfusion was detected, however hyaluronidase showed a gradual decrease, signifi cant at quantitated in ischemic hindlimbs of C57BL mice using blank scaffold, insulin 10 mmol/l (fi g F), indicating increased hyaluronidase substrate binding. There scaffold, control MSC, control MSC/insulin, VENIRKO MSC and VENIRKO was no indication of a cumulative effect of PG on any of the parameters. In MSC/insulin. Even at 1 week post surgery, laser Doppler scan demonstrated conclusion, oxidative stress as well as coagulation activation already start that insulin scaffolds enhanced perfusion recovery as compared with blank at near normal PG levels (6-8 mmol/l), while endothelial glycocalyx changes scaffolds. After 4 weeks, perfusion of insulin scaffold and control MSC/ occur at 10 mmol/l. insulin scaffold was signifi cantly higher than blank by 143% and 146% (p<0.05). Control MSC recovered perfusion more effectively than VENIRKO MSC. Moreover, control MSC/insulin scaffold group demonstrated better recovery than VENIRKO MSC/insulin scaffold group. Those results clearly demonstrate that insulin receptors on MSC can enhance angiogenesis and perfusion in response to insulin. Methods that improve insulin actions on MSC may lead to novel therapies that substantially improve diabetic wound healing.

581-P Effects of ER Stress Induced by Tunicamycin on the Activity of Endothelial Nitric Oxide Synthase JI HEE YU, KEE-HO SONG, CHANG HEE JUNG, WOO JE LEE, JOONG-YEOL PARK, Seoul, Republic of Korea Metabolic diseases including obesity and diabetes are causally related to the development of atherosclerosis, which is known as an infl ammatory disease. Endoplasmic reticulum (ER) stress has been known to link between metabolic stress and infl ammation. However, the effect of ER stress on the eNOS activity in vascular endothelial cells, which has an important role in regulating endothelial function, is currently unclear. In this study, we investigated the effect of ER stress on eNOS activity and the effects of various drugs on vascular ER stress. Human aortic endothelial cells (HAECs) were used in whole experiments. Tunicamycin (Tm) was used to induce ER stress. We measured the phosphorylation of eNOS and examined whether PKC is involved in this pathway using GFX, a PKC inhibitor. To identify the specifi c isoform of PKC involved in ER stress-induced eNOS inactivation, we also measured cytosolic and membranous forms of PKC βII and PKCδ. Treatment of Tm decreased the phosphorylation of eNOS at the site of Ser1177 but increased it at Thr495 site. Tm-induced changes in eNOS phosphorylation were reversed with GFX pretreatment. Tm induced translocation of PKCδ but not PKC βII from the cytosolic to membrane fraction. ALA and rosiglitazone, but not telmisartan decreased ER stress and ER stress-induced eNOS inactivation. In conclusion, Tm-induced ER stress inactivates eNOS by activating PKCδ. ER stress-induced eNOS inactivation 583-P might lead to decrease in NO bioavailability, endothelial dysfunction and Induced Pluripotent Stem Cells Inhibit Apoptosis and Fibrosis in eventually atherosclerosis. ALA and rosiglitazone might be used to reverse Streptozotocin-Induced Diabetic Cardiomyopathy in Rats ER stress-induced endothelial dysfunction. SARAH NEEL, DINENDER SINGLA, Orlando, FL This is never tested before if modifi ed ES, iPS cells, or factors released from these cells can inhibit apoptosis and fi brosis in the streptozotocine (STZ) induced cardiomyopathy. Therefore, we determined the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a pro- pancreatic endodermal transcription factor, induced pluripotent stem (iPS) cells generated from H9c2 cardiomyoblasts, or their respective conditioned media (CM) on diabetic cardiomyopathy. Experimental diabetes was induced

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A160 COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES in rats (8-10 weeks old) by intraperitoneal (IP) STZ injections (65 mg/kg body In a random sample of 104 participants from the 24-week L2T3 trial weight for 2 consecutive days). Animals were divided into six groups; control, (NCT00405418)in which 973 insulin-naïve patients with type 2 diabetes treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, inadequately controlled on oral agents were randomized to insulins glargine STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, cells (1 X 106/ or detemir, we measured these parameters at baseline and at the end of the mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 study at a central laboratory. Data are expressed as medians (interquartile consecutive days. Animals were sacrifi ced and hearts were harvested at day range) and tested non-parametrically; multivariate analysis was performed 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess by linear regression. apoptosis and fi brosis. M-mode echocardiography fractional shortening was Baseline 24 Weeks P-value POSTERS used to assess cardiac function. Animals transplanted with ES cells, iPS cells, Complications or both CMs showed a signifi cant (p<0.05) reduction in interstitial fi brosis (Ptf1a Coagulation Acute and Chronic cells: 0.03 ± 0.01, Ptf1a CM: 0.05 ± 0.01, iPS cells: 0.01 ± 0.00, and iPS CM: 0.03 Antithrombin (%) 106 (99-114) 104 (97-113) 0.059 ± 0.01 vs. STZ: 0.09 ± 0.02 mm2, p<0.05), and apoptosis (TUNEL staining; Ptf1a Factor VIII (%) 71 (55-84) 65 (52-84) 0.095 cells: 0.35 ± 0.02%, and iPS cells: 0.39 ± 0.04%, vs. STZ: 1.17 ± 0.21%, p<0.05; Von Willebrand factor (%) 110 (84-146) 112 (83-141) 0.583 CASPASE 3 activity; Ptf1a cells: 0.13 ± 0.02, Ptf1a CM: 0.14 ± 0.01, iPS cells: 0.13 ± 0.01, and iPS CM: 0.14 ± 0.01 vs. STZ:0.22 ± 0.01, p < 0.05) compared with Prothrombin fragment 1+2 (pmol/l) 165 (136-218) 163 (135-194) 0.039 STZ group. Echocardiography showed a signifi cant improvement in fractional Fibrinolysis shortening in cell and media transplanted groups (Ptf1a cells: 39.37 ±0.90%, Plasmin-antiplasmin complex (µg/l) 369 (306-466) 416 (343-515) <0.001 Ptf1a CM: 35.95 ± 1.16%, iPS cells: 40.29 ± 1.41%, and iPS CM: 36.13 ±1.29% Plasminogen activator inhibitor-1 (ng/ml) 100 (64-159) 87 (56-139) 0.007 vs. STZ: 29.19 ± 0.80%, p<0.001) compared with STZ. In conclusion, our data suggest that ES cells, iPS cells, and/or CMs inhibit apoptosis, reduce fi brosis, Clot lysis time (min) 71 (60-85) 65 (58-77) 0.001 and improve cardiac function in STZ-treated diabetic rats. During the trial HbA1c dropped from 8.8±0.9% to 7.2±0.9% (mean±sd) in Supported by: NIH this cohort. Markers of coagulation or glycobiology (data not shown) were not affected. However, signifi cant changes were found in the markers of 584-P fi brinolysis. In multivariate analysis, endpoint HbA1c was the most important Infl uence of Endogenous Glucose-Dependent Insulinotrophic Poly- predictor of these improvements. In conclusion, initiation of basal insulin peptide on Atherosclerosis therapy ameliorates the hypofi brinolytic state in type 2 diabetes, without YASUNORI TAKATA, JUNICHI FUNADA, YUJI MATSUMOTO, GO HIASA, RYOICHI affecting general markers of coagulation or glycobiology. KAWAMURA, WATARU NISHIDA, HIROSHI ONUMA, HARUHIKO OSAWA, Toon, Supported by: sanofi -aventis Japan, Saijyo, Japan, Ozu, Japan Recent animal studies have reported the benefi cial effect of the 586-P interrupting of glucose-dependent insulinotrophic polypeptide (GIP) receptor Insulin Treatment Reduces Atherosclerotic Intimal Lesions and (GIPR) signaling in metabolic disorders. We have recently reported that Matrix Metalloproteinase-9 Expression in Diabetic Apolipoprotein circulating GIP levels were associated with the prevalence of cardiovascular E-Defi cient Mice disease (CVD). In contrast, emerging studies have demonstrated the cardio- CORINNE A. SCHUYLER, NGA N. TA, YANCHUN LI, MARIA F. LOPES-VIRELLA, protective effect of glucagon like peptide-1 (GLP-1) receptor agonists. To YAN HUANG, Charleston, SC clarify the infl uence of endogenous incretin levels on cardiovascular system, Patients with diabetes mellitus have increased mortality and morbidity of we elucidated the interaction between fasting GIP and GLP-1 in 122 subjects, cardiovascular diseases as compared with nondiabetic patients. Although and we performed in vitro molecular analysis. the clinical studies have shown that effective glycemic control with insulin Simple regression analysis demonstrated that the fasting GIP was inversely treatment in patients with type 1 diabetes is associated with reduced correlated with fasting GLP-1 (r=-0.21, p=0.04). Multivariate regression analysis cardiovascular events, the underlying mechanisms have not been well revealed that the GIP/GLP-1 ratio was associated with known cardiovascular understood. In the present study, we employed apolipoprotein E-defi cient risk factors such as visceral fat area (β=0.28, p<0.01), fasting insulin (β=0.36, (apoE-/-) mice as a model of atherosclerosis and induced diabetes in mice p<0.001), HOMA-IR (β=0.30, p<0.001), total cholesterol (β=0.34, p<0.001), with streptozotocin injection. We treated diabetic apoE-/- mice with apolipoproteinB-48 (β=0.37, p<0.001), remnant-like particle cholesterol insulin for 20 weeks and studied the effect of insulin treatment on intimal (β=0.28, p<0.01), leptin (β=0.24, p<0.01), pulse wave velocity (β=0.19, p<0.01) lesion size and matrix metalloproteinase (MMP)-9 expression known to be and carotid intima-media thickness (β=0.28, p=0.02) after adjustment for age, involved in plaque destabilization. Results showed that insulin treatment, gender and fasting plasma glucose. which effectively reduced plasma glucose level by 76% in diabetic mice, To further clarify the mechanism of the link between GIP and athero- attenuated diabetes-increased intimal lesion size by 60%. Insulin treatment sclerosis, we analyzed the expression of GIPR in human carotid athero- also signifi cantly inhibited diabetes-increased MMP-9 expression, but sclerotic lesion. RT-PCR and Western blotting revealed that GIPR was had no effect on tissue inhibitor of metalloproteinase (TIMP)-1 expression present in human atherosclerotic lesion, especially in macrophages. To in atherosclerotic plaques, indicating that insulin treatment may inhibit identify the direct effect of GIP on macrophage, we performed in vitro matrix-degrading activity in atherosclerotic lesions by decreasing MMP- analysis. QRTPCR analysis demonstrated that GIP stimulation (100 nM for 9/TIMP-1 ratio. Furthermore, we observed that insulin treatment did not 24 hours) up-regulated the gene expression of interleukin 6, chemokine (C-C reduce diabetes-increased macrophage content, but inhibited expression motif) receptor (CCR) 2, CCR5 and scavenger receptor type A in human THP-1 of interleukin-6 (IL-6), a stimulator for MMP-9 expression. In vitro studies macrophages (all p<0.05 vs. control by unpaired t-test). showed that IL-6-stimulated MMP-9 secretion from mononuclear cells Given our fi ndings and previous reports, it is possible that the imbalance cultured in normal glucose (5 mM)-containing medium was 40% of that from between circulating GIP and GLP-1 may contribute to metabolic disorders mononuclear cells cultured in high glucose (25 mM)-containing medium. and atherosclerosis, resulting in the development of CVD. Taken together, this study has shown for the fi rst time that insulin treatment in diabetic apoE-/- mice attenuates diabetes-increased intimal lesion and 585-P reduces the expression of MMP-9 and IL-6, indicating that glycemic control Initiation of Insulin Therapy Ameliorates Fibrinolysis in Type 2 by insulin treatment leads to stabilization of atherosclerotic plaques. Diabetes Supported by: VA Merit Review Grant and NIH DE016353 BREGTJE A. LEMKES, J. HANS DEVRIES, MAX NIEUWDORP, RICARDO DE GROOTH, JOOST B. HOEKSTRA, JOOST C. MEIJERS, FRITS HOLLEMAN, Amsterdam, The Netherlands, Gouda, The Netherlands The prothrombotic state found in type 2 diabetes pairs hypercoagulability with impaired fi brinolysis. Both may be related to hyperglycemia-induced damage to the endothelial glycocalyx. This layer of proteoglycans and glycosaminoglycans protecting the vascular endothelium is involved in regulation of hemostasis. Our aim was to determine the effect of improved glycemic control by the initiation of insulin therapy on circulating components of the endothelial glycocalyx and markers of coagulation and fi brinolysis.

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A161 COMPLICATIONS—NEPHROPATHY

587-P 589-P Mechanisms of a Proatherogenic Crosstalk between Human Peri- Spatial Population Dynamics of Mouse Endothelial Progenitor Cells vascular Adipose Tissue and Smooth Muscle Cells in Diabetes RAPHAELA SCHLICH, DANIELA LAMERS, SABRINA GREULICH, BUJAR MAX HERA, HIDEHITO SAITO, YASUHIKO YAMAMOTO, SEIICHI MUNESUE, TAKUO WATA- ARTUR LICHTENBERG, HENRIKE SELL, JUERGEN ECKEL, Düsseldorf, Germany NABE, HIROSHI YAMAMOTO, Kanazawa, Japan In the context of obesity, perivascular adipose tissue (PAT) might be an Recent evidence suggests that circulating endothelial progenitor cells activator of vessel wall dysfunction. In previous studies we demonstrated (EPC), derived from bone marrow (BM), maintain endothelial integrity and

POSTERS that adipocyte-conditioned media (CM) induced proliferation of human vascular homeostasis. Reduced EPC number and function in diabetes may

Complications smooth muscle cells (SMC). CM combined with oleic acid (CMOA) be responsible for the development of vascular complications. However, the Acute and Chronic enhanced proliferation in a synergistic way via iNOS expression, NO and cell surface markers for EPC and distribution of the classifi ed heterogeneous proinfl ammatory signaling. The aim of this study was to identify mechanisms population are not fully characterized. To identify EPC subclasses that are responsible for the synergistic effect of CMOA on SMC proliferation. affected by diabetes, we comprehensively analyzed by multicolor fl ow The proliferative synergism of CMOA was accompanied by signifi cantly cytometry a total of 32 repertoires of the cell surface markers for mouse increased lipid accumulation. CM and CMOA increased the expression of EPC, including Lin, CD31, CD34, Flk1, Sca1, and cKit. The cell number of each CD36 while FATP4 remained unaltered. CD36 silencing decreased OA- and repertoire was counted in peripheral blood (PB), BM and spleen from multiple CMOA-induced proliferation. As the synergistic effect of CMOA could not be low dose streptozotocin (STZ)-induced diabetic C57BL/6J (STZ-diabetes) completely abrogated by CD36 silencing it can be speculated that increased mice, db/db mice, and the age-matched non-diabetic controls. Cell numbers of lipid storage can explain the effect of OA but not the synergism of CMOA. 10 subclasses previously defi ned as mouse EPC were signifi cantly decreased Analysis of adipokine concentration in various CM revealed that VEGF content in PB from STZ-diabetes mice and these were reversed by insulin treatment. signifi cantly correlated with proliferation. Recombinant VEGF mimicked the Among them, PI-Lin-CD31+CD34-Flk1-Sca1+cKit- EPC cluster with the highest proliferative effect of CM. CMOA induced VEGF release from SMC and number was also decreased in BM, but paradoxically increased in spleen, increased VEGFR1 expression indicating an auto/paracrine contribution to both from STZ-diabetes and db/db mice. Insulin treatment reversed the proliferation. VEGF blocking completely prevented the proliferative effect decrease both in PB and BM, and the increase in spleen. Total EPC number of CM and OA while the effect of CMOA could be reduced. Analysis of VEGF was found to be about 10-fold higher in spleen than in PB. The other clusters release by adipose tissue explants revealed a signifi cant 2.5-fold increased of spleen EPC were signifi cantly decreased in both STZ-diabetes and db/ secretion from PAT of diabetics compared to controls or subcutaneous fat. db mice, and this was reversible by insulin treatment. We thus revealed Combining CD36 silencing and VEGF blocking, CMOA-induced proliferation for the fi rst time the spatial changes of mouse EPC numbers in diabetes. could be further decreased but not to control levels. CD36 silencing affected Especially, PI-Lin-CD31+CD34-Flk1-Sca1+cKit- EPC is assumed to be damaged neither VEGF release nor VEGFR1 expression by SMC. This indicates that in the production from BM and in the mobilization from spleen reservoir VEGF and CD36 act independently and are not suffi cient to explain the CMOA in diabetes, maybe resulting in low circulating EPC. These results suggest synergism. that circulating EPC supply can be impaired in several steps by diabetes, In this study we identifi ed VEGF as an important factor of adipocyte- contributing to the diabetic vascular dysfunctions. induced proliferation of SMC whose release is increased specifi cally in PAT of diabetic patients. The release of VEGF combined with elevated circulating levels of free fatty acids and increased CD36 expression could be crucial COMPLICATIONS—NEPHROPATHY elements in a proatherogenic crosstalk between PAT and the vessel wall. [See also: Presidents Posters 385-PP to 387-PP, page A106.] 588-P Rho/Rho-Kinase Mediates Thrombin-Induced MCP-1 Induction in Vascular Endothelial Cells Guided Audio Tour: Potential Mechanisms Underlying Pathogenesis of Dia- DAIJI KAWANAMI, KEIICHIRO MATOBA, SHO ISHIZAWA, YASUSHI KANAZAWA, betic Nephropathy (Posters 590-P to 596-P), see page 15. TAMOTSU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan & 590-P Thrombin is a pro-infl ammatory molecule that has been implicated as Toll-Like Recepotor 4-Mediated, Hyperinsulinemia-Independent an indcuer of atherosclerosis in patients with diabetes. Thrombin has Progression of Diabetic Nephropathy by Hyperlipidemia been shown to increase expression of chemokine including monocyte TAKASHIGE KUWABARA, KIYOSHI MORI, MASASHI MUKOYAMA, MASATO chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the KASAHARA, HIDEKI YOKOI, YOKO SAITO, HIROTAKA IMAMAKI, TOMOKO development of atherosclerosis. However, precise mechanism underlying KAWANISHI, AKIRA ISHII, KENICHI KOGA, KEITA PIERRE MORI, YUKIKO KATO, MCP-1 induction by thrombin still remains unknown. Small G protein RhoA, AKIRA SUGAWARA, KAZUWA NAKAO, Kyoto, Japan, Osaka, Japan and its effector Rho-kinase has been implicated in the pathogenesis of Hyperlipidemia is an independent risk factor for the progression of diabetic atheosclerosis but its role in regulation of MCP-1 is not fully understood. nephropathy, and nephropathy induced by hyperlipidemia is reported to be In this study, we aimed to elucidate the role of Rho/Rho-kinase in thrombin- caused, at least in part, through activation of fatty acid synthesis and lipid mediated MCP-1 induction. We stimulated human umbilical vein endothelial accumulation in the renal tubules. In the present study, we investigated cells (HUVEC) with thrombin and then examined MCP-1 mRNA expression. whether, in the absence of metabolic syndrome (obesity and insulin Real-time PCR analysis revealed that thrombin induced MCP-1 mRNA resistance), concomitant occurrence of diabetes and hyperlipidemia causes expression in endothelial cells. To understand the role of Rho/Rho-kinase in exacerbation of renal lesions. We treated toll-like receptor 4 knockout this induction, we examined whether Y-27632, a specifi c Rho-kinase inhibitor, (TLR4 KO) and wild-type (WT) mice with streptozotocin (STZ) and/or 45% inhibits thrombin-mediated MCP-1 induction. Y-27632 potently inhibited high fat diet (HFD). As a signal transduction pathway, we focused upon MCP-1 induction, demonstrating that Rho/Rho-kinase signaling pathway TLR4 and one of its endogenous ligands, myeloid-related protein 8 (MRP8 mediates MCP-1 induction by thrombin. We next used a variety of MAPK or S100A8), since glomerular mRNA expression of both MRP8 and TLR4 was inhibitors to elucidate signaling pathways that is involved in MCP-1 induction upregulated in our microarray analysis of diabetic nephropathy models, and by thrombin. We observed that SB203580, a specifi c p38MAPK inhibitor, was further enhanced by HFD. Treatment with HFD alone had little effects attenuated MCP-1 induction. In addition, Western blot analysis revealed upon kidneys of WT and KO mice. In insulin-defi cient WT STZ mice, addition that thromin induced p38MAPK phosphorylation. Furthermore, we found of HFD aggravated diabetic nephropathy, as indicated by marked increase that Y-27632 attenuates thrombin-mediated p38MAPK phosphorylation, in albuminuria, glomerular mesangial expansion, infi ltration of macrophages indicating that Rho/Rho-kinase mediates MCP-1 induction via p38MAPK and upregulation of pro-infl ammatory and pro-fi brotic genes in glomeruli and signaling pathway. Finally, we performed chemotaxis assay utilizing Boyden renal interstitium, and decrease in glomerular podocin expression. Despite chamber to examine the biological signifi cance of Rho-kinase inhibition similar degrees of metabolic abnormalities in blood samples and renal lipid in thrombin-mediated MCP-1 induction. Y-27632 signifi cantly decreased accumulation to WT mice, additional effects of HFD upon renal lesions were the chemotactic activity of thrombin-stimulated HUVEC supernatant on almost completely abolished in KO STZ mice. MRP8 protein in the kidney monocytes. These fi ndings demonstrate that Rho/Rho-kinase signaling was predominantly expressed by macrophages and, in cultured mouse bone pathway plays a critical role in thrombin-mediated MCP-1 expression and marrow-derived macrophages, fatty acid amplifi ed MRP8 gene expression function. We conclude that Rho/Rho-kinase is an important target in the only under high-glucose conditions. Furthermore, phosphorylation of renal development of new therapeutic strategy for atherosclerosis. IRF3 in WT STZ mice was enhanced by HFD, suggesting the activation of TLR4-downstream, TRIF-dependent pathway. In conclusion, synergistic

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A162 COMPLICATIONS—NEPHROPATHY exacerbation of microvascular injury by diabetes and hyperlipidemia does not necessarily require obesity and hyperinsulinemia, and is associated with & 593-P activation of MRP8/TLR4/TRIF-dependent pathway. The Role of Ets-1 for the Upregulation of NAD(P)H Oxidase NOX4 in Diabetic Renal Tissue & 591-P MASAKAZU FUJII, TOYOSHI INOGUCHI, YASUTAKA MAEDA, NORIYUKI SONODA, KUNIHISA KOBAYASHI, RYOICHI TAKAYANAGI, Fukuoka, Japan P2X Defi ciency Attenuates Renal Disease Induced by High-Fat 7 We reported markedly lower prevalence of nephropathy in diabetic Diet patients with Gilbert syndrome, a congenital hyperbilirubinemia (JAMA

ANNA SOLINI, STEFANO MENINI, CHIARA ROSSI, CARLO RICCI, ELEONORA POSTERS

2007; 298(12):1398-400), suggesting a benefi cial effect of bilirubin(BIL) Complications SANTINI, CLAUDIA BLASETTI FANTAUZZI, ANTONIO SALVATI, CARLA IACOBINI, on diabetic(DM) nephropathy. To directly examine this, we showed that Acute and Chronic GIUSEPPE PUGLIESE, Pisa, Italy, Rome, Italy administration of BIL and biliverdin(BVD) protected against oxidative stress Renal disease associated with type 2 diabetes and the metabolic and renal damage in db/db mice via downregulating renal NAD(P)H oxidase syndrome is characterized by a distinct infl ammatory phenotype. The NOX4 (Kidney Int 2010;78(9):905-919). However, the detailed regulation purinergic system plays a relevant role in the infl ammatory and immune mechanism for NOX4 expression has not been elucidated yet. In this study, response to injurious stimuli, particularly through the P2X receptor, which is 7 we show the role of transcriptional factor Ets-1 on the regulation of NOX4 expressed in resident and non-resident kidney cells, such as mesangial cells expression in cultured normal human mesangial cells(NHMCs) and DM renal and macrophages. In addition to stimulating the release of pro-infl ammatory tissues and thus show that Ets-1 may be a novel therapeutic target molecule cytokines, it has been shown to promote mesangial cell apoptosis and matrix for DM nephropathy. production, these effects being opposed by the P2Y receptors, and to be First we investigated the effect of Ets-1 overexpression on NOX4 upregulated in glomeruli from animal models of diabetes, hypertension and expression in the NHMCs. The levels of mRNA / protein for NOX4 were glomerulonephritis. This study aimed at verifying the hypothesis that P2X 7 signifi cantly increased from 48 hours after transfected with the plasmid promotes the development of lipid-induced renal disease by inducing renal containing Ets-1 gene. Next, we performed the transfection using siRNA tissue infl ammation and consequent dysregulated glomerular remodeling. To directed against Ets-1. After knockdown of Ets-1 by siRNA, the expression of this end, P2X knockout (KO) and coeval wild type (WT) mice were fed a high- 7 NOX4 mRNA / protein was signifi cantly decreased. fat diet (HFD, 60% saturated fat) or a normal-fat diet (NFD, 4% saturated Furthermore, we evaluated the mRNA / protein revels for Ets-1 and NOX4 fat) for 4 months. Body weights were signifi cantly higher in KO vs. WT mice in kidneys from db/db mice. The mRNA / protein revels for Ets-1 / NOX4 were and increased in both genotypes upon HFD. HFD-induced glomerular lesions signifi cantly increased in db/db compared with control db/+. Administration were attenuated in KO vs. WT mice. This was confi rmed by morphometric of BVD completely normalized Ets-1 / NOX4 expression, oxidative stress analysis, showing signifi cantly lower values (P<0.001) in KO vs. WT mice of markers (8-hydroxy-2’-deoxyguanosine and dihydroethiduim staining), mean glomerular area (3,165±183 vs. 3,453±212 µm2), mean (605.3±60.3 vs. albuminuria and renal mesangial expansion in parallel. In the NHMCs, 780.8±80.8 µm2), and fractional (19.2±2.0 vs. 22.7±2.6 %) mesangial area. angiotensin II (AngII) stimulated Ets-1 / NOX4 expression. BIL / BVD inhibited This was associated with signifi cantly reduced glomerular expression of the AngII-induced Ets-1 / NOX4 expression in parallel, but other antioxidants extracellular matrix proteins fi bronectin and collagen IV, the macrophage NAC / LA did not. marker F4/80 and the advanced lipoxidation endproducts 4-hydroxy-2- α In conclusion, Ets-1 plays a role in the regulation of NOX4 expression in nonenal-adducts as well as with decreased kidney mRNA levels of collagen the NHMCs. In DM kidneys, upregulation of NOX4 may be at least in part IV, F4/80 and the pro-infl ammatory cytokine monocyte chemoattractant due to increased levels of Ets-1. The benefi cial effect of BIL / BVD on DM protein-1. Phosphorylation of ERK1/2-mitogen-activated protein kinase and nephropathy may be mediated by downregulation of Ets-1 / NOX4. Ets-1 may phosphoinositide-3-kinase were up-regulated upon HFD in WT and KO mice, be a novel therapeutic target molecule for DM nephropathy. respectively. These data show that P2X7 plays an important role in HFD- induced renal disease by modulating glomerular cell apoptosis and matrix accumulation and renal tissue infl ammation. & 594-P Supported by: Research Foundation of the Italian Society of Diabetology Diabetes in a Novel Mouse Model of Mitochondrial Dysfunction— (Fo.Ri.SID) ADA-Funded Research Testing the “Unifying Hypothesis” ANDRZEJ S. JANUSZEWSKI, BEN MA, YUAN ZHANG, RACHEL BLAKE, IAN & 592-P TROUNCE, CARL A. PINKERT, DARREN J. KELLY, ALICIA J. JENKINS, Melbourne, Australia, Auburn, AL Palmitate-Induced Mitochondrial Dysfunction and Apoptosis in Our novel xenomitochondrial mice (MM) have an introduced, interspecifi c Mouse Podocyte 145 amino acid substitutions in mtDNA-encoded oxidative phosphorylation SHANHUA XU, RANJAN DAS, XIANGLAN QUAN, CHOON HEE CHUNG, EUN- subunits, promoting its mild dysfunction. We previously demonstrated YOUNG LEE, SUN JU CHOI, YUP KANG, KYU-SANG PARK, Wonju, Republic of increased tissue AGEs in association with renal fi brosis and systolic cardiac Korea, Cheonan, Republic of Korea, Suwon, Republic of Korea dysfunction in diabetic (DM) MM. Aim of the project was to determine if tissue Podocytes play a major role in glomerular fi ltration barrier, but also oxidative stress is increased in DM MM and is related to DM complications. participate in pathogenic process of diabetic nephropathy. Diabetic patients Four groups of (n=12) 24-week old mice were studied: 1) wild-type (WT) Non- have elevated plasma levels of saturated free fatty acid (FFA); however, the DM; 2) MM Non-DM; 3) WT streptozotocin (STZ)-induced DM and iv MM pathophysiologic effects of palmitate, the predominant circulating FFA, have STZ-DM. DM duration was 16 weeks. Post-sacrifi ce heart, kidney and skin not been clearly elucidated in podocyte. Here, we investigated the effect were frozen until analysis. AGEs (CML, pentosidine) were measured by GC/ of palmitate on reactive oxygen species (ROS) production, mitochondrial MS and HPLC respectively. Paraffi n-embedded kidney sections were stained function, and cell death in mouse podocyte. Palmitate conjugated with fatty for nitrotyrosine (NT, refl ecting oxidative stress), and kidney and heart – acid-free bovine serum albumin (BSA) was administered to conditionally with Picro Sirius Red (fi brosis) and with PAS (glomerulosclerosis). Staining immortalized mouse podocytes. Incubation with palmitate decreased cell intensity was determined using Analytical Imaging Station software (6.0, viability in a time (6∼48hrs) and dose (50∼300mM) dependent manner. Ontario, Canada). DAPI staining and immunoassay for cytoplasmic histone-associated DNA HbA1c was increased in DM vs. non-DM mice (WT: 6.7±0.4 vs. 4.4±0.4%; fragments showed a marked increase in apoptotic cell death by palmitate. p=0.001 and MM: 6.8±0.4 vs. 4.0±0.3%; p<0.0001) and was similar in WT- Palmitate elevated the ROS production in cytoplasm as well as mitochondria DM and MM-DM. There was renal hypertrophy, increased renal NT and and depolarized the mitochondrial membrane potential, which were fi brosis in MM-DM animals vs. WT DM. Renal NT correlated with renal and measured by fl uorescence spectrophotometer and microscopic imaging cardiac fi brosis and with skin AGEs. Skin AGEs also correlated with renal system. Especially, palmitate-treated podocytes elicited a depolarizing dysfunction (Table). response to oligomycin implying a functional impairment of mitochondria. Morphodynamic changes of mitochondria to fragmentation by palmitate treatment were observed. Palmitate upregulated ER stress proteins such as GRP78/Bip, spliced Xbp1 and CHOP in podocyte. These data suggest that palmitate as a major saturated FFA leads to mitochondrial dysfunction and ER stress resulting in apoptosis of podocyte, which may participate in the pathogenesis of glomerular complications in diabetes.

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A163 COMPLICATIONS—NEPHROPATHY

Mitochondrial electron transport chain dysfunction in the DM MM is A total of 2,268 glomerular and 1,709 tubular proteins were identifi ed, of associated with increased oxidative stress (NT), AGEs, tissue fi brosis and which 54 and 27, respectively, were altered (defi ned as 1.5x fold change and dysfunction. q* ≤ 0.05) by administration of VEGF antibody. Proteins altered by α-VEGF WT WT-DM MM MM-DM P P antibody were analyzed using Ingenuity Pathway Analysis. GO functions non-DM non-DM MM-DM vs. WT-DM vs. were primarily related to alterations in fatty acid metabolism in glomeruli MM non-DM MM-DM (BH p-value < 10^-8, Fisher exact test, q* ≤ 0.05) and cellular development in Kidney weight (mg/g) 13.7±0.8 12.4±1.0 14.0±0.6 15.8±0.8 0.01 0.002 tubules (BH p value < 10^-3, Fisher exact test, q* ≤ 0.05). Network analysis of the glomerular and tubular altered proteomes revealed several novel POSTERS Oxidative stress (%) 4.5±0.7 18.0±3.1 14.9±2.4 30.1±4.5 0.001 0.009 Complications potential VEGF regulatory networks including metabolism, activity and Acute and Chronic Kidney fi brosis (%) 0.8±0.3 2.0±0.4 0.4±0.3 3.2±0.3 0.0002 0.002 storage of retinoids and HNF4-α. Also, it provided additional evidence of a Heart fi brosis (%) 1.3±0.3 1.5±0.3 1.8±0.4 2.5±0.3 0.048 link between the PPAR’s and VEGF activity. These results demonstrate that systems biology strategies that explore effects of blocking VEGF activation Correlations Nitrotyrosine on the diabetic renal cortical proteome can provide novel insights into the Heart fi brosis R=0.42; p=0.01 pathophysiology of diabetic microvascular complications. Kidney fi brosis R=0.37; p=0.02 Skin CML R=0.34; p=0.04 Skin Pentosidine R=0.34; p=0.048 Guided Audio Tour: Clinical Aspects of Diabetic Nephropathy (Posters 597-P to 603-P), see page 15. Skin CML Skin pentosidine Kidney fi brosis R=0.75; p<0.00001 R=0.51; p=0.003 & 597-P Urinary Albumin/Creatinine R=0.37; p=0.02 Non-Alcoholic Fatty Liver Disease (NAFLD) Is Associated with an Increased Risk for Diabetic Nephropathy MARIKO HIGA, KAORU YAMASHITA, RYO IGA, AYUMI YOSHIFUJI, EIKO YOSH- & 595-P IDA, TAKAMASA ICHIJO, HIROMI OUCHI, TOSHIKI INOKUCHI, Yokohama, Japan, Characterization of microRNAs Derived from the Plasmacytoma Tokyo, Japan Variant Translocation 1 Gene (PVT1) as Mediator of Diabetic Kidney [AIM] There is now growing evidence that non-alcoholic fatty liver disease Disease (NAFLD) may be linked to an increased risk of developing cardiovascular M. LUCRECIA ALVAREZ, JEFF KIEFER, JOHANNA K. DISTEFANO, Phoenix, AZ disease and chronic kidney disease. The aim of this study was to assess Diabetic nephropathy (DN) is the most common disorder leading to end whether NAFLD, as diagnosed by ultrasound, the most widely used imaging stage renal disease (ESRD). Previously, we observed association between test for detecting hepatic steatosis, is associated with an increased risk of variants in PVT1 and ESRD attributed to type 1 or 2 diabetes. We also diabetic nephropathy in type 2 diabetic patients. [METHOD] The subjects of found that PVT1 is upregulated by high glucose (HG), and PVT1 knockdown this study were 60 type 2 diabetic patients (age 63.7±9.0 years, 30 males signifi cantly reduced mRNA and protein levels of major extracellular matrix and 30 females). Patients with macroalbuminuria were excluded. NAFLD was (ECM) components and key regulators of ECM, including transforming diagnosed by liver ultrasound fi ndings. Urinary albumin (UAE) was measured growth factor beta 1 (TGFB1). However, the mechanism by which PVT1 from an early morning urine sample as the albumin-to-creatinine ratio and the exerts these effects remains unknown. PVT1 does not encode a protein, level of kidney function was defi ned by estimated GFR (eGFR) using MDRD. The but does contain 6 microRNAs (miRNAs), which may mediate PVT1 effects following parameters were investigated; HbA1c, liver enzymes, lipid levels, on ECM. The goal of this study was to characterize PVT1-derived miRNAs serum leptin, ferritin and unsaturated fatty acid (PUFA) levels. [RESULTS] in mesangial cells (MC). We fi rst evaluated the effect of glucose on PVT1- Among these 60 patients, 32 patients had NAFLD (FL group) and 28 patients derived miRNA expression by culturing MC under normal glucose (NG) did not have NAFLD (non FL group). No signifi cant differences in age, duration and HG conditions for 8 days and quantifying miRNA expression by qPCR. of diabetes, smoking, or kind of antihypertensive agent used were found Levels of all PVT1-derived transcripts expressed in MC (miR-1205, miR- between the 2 groups. BMI, waist circumference, and AST and leptin levels 1207-3p, miR-1207-5p, miR-1208) increased 2.5-3.5-fold in response to HG. in the FL group were signifi cantly higher than those in non FL group. UAE was We also determined that miR-1207-5p is the major PVT1-derived miRNA 12.0±11.7 mg/gCr in the non FL group and 32.7±40.3 mg/gCr in the FL group, expressed in MC, and the presence of a Smad binding element in the mature which was signifi cantly higher than those in the non FL group (p<0.05), while sequence of this miRNA suggested that it may be regulated by the TGFB- there were no signifi cant differences in blood pressure, HbA1c, lipid, ferritin, Smad signaling pathway. Thus, we treated MC with 0, 2, 5 or 10 ng/ml of PUFA levels or eGFR between the 2 groups. Log UAE showed signifi cant TGFB for 24 h and measured miR-1207-5p and PVT1 expression by qPCR. We positive correlations with waist circumference (r=0.64, p<0.01) and systolic found that miR-1207-5p, but not PVT1 itself, was upregulated by TGFB1, and blood pressure (r=0.49, p<0.001), respectively. [CONCLUSION] Our fi ndings increased up to 7 times in a dose-dependent manner. Targets of miR-1207-5p suggest that NAFLD is associated with an increased prevalence of diabetic include stromal antigen 1 (STAG1) and syndecan-1 (SDC1), which antagonize nephropathy. This signifi cant association might point to a link between the TGFB signaling, and we investigated miRNA effects on these genes using pathogenesis of NFLD and diabetic nephropathy, suggesting that abdominal luciferase reporters containing the STAG1 and SDC1 3’-untranslated regions obesity and low-grade infl ammation might be key features in the development (UTRs) cotransfected with miR-1207-5p mimics in HEK293 cells. We found of both hepatic steatosis and microalbuminuria in type 2 diabetic patients. that miR-1207-5p mimic signifi cantly inhibited 3’UTR luciferase activity of both genes. A decreased expression of STAG1 and SDC1 mediated by miR- & 598-P 1207-5p might favor a more sustained and intense TGFB signaling in MC Albuminuria Does Not Predict All-Cause Mortality in Patients with under HG. These results provide new insight into mechanisms underlying the Type 2 Diabetes and Low Glomerular Filtration Rate development of DN. ADA-Funded Research SALVATORE DE COSMO, OLGA LAMACCHIA, ELEONORA MORINI, STEFANIA FARIELLO, ANTONIO PALENA, MARIA ROSARIA SORRENTINO, ANNA RAUSEO, & 596-P SABINA PINNELLI, VINCENZO TRISCHITTA, MAURO CIGNARELLI, San Giovanni Renal Proteomic Effects of a Neutralizing VEGF Antibody on Diabetic Rotondo, Foggia, Italy, Rome, Italy Nephropathy Albuminuria predicts mortality in type 2 diabetes (T2D). Whether glomerular JONATHAN M. STARKEY, K.C. RHAO, YINGXIN ZHAO, WANDA S. LEJEUNE, MAS- fi ltration rate (GFR) modulates this effect is a matter of uncertainty which ARU MIYAGI, TIMOTHY S. KERN, RONALD G. TILTON, Galveston, TX, Cleveland, OH was investigated in this study. Two cohorts of T2D patients were recruited To better understand the role of VEGF in the pathophysiology of diabetic in Apulia (Italy) for genetic studies on incident mortality: one in the Gargano nephropathy, we quantifi ed changes in the glomerular and cortical tubular region, at “CSS” Hospital in San Giovanni Rotondo from November 1th 2000 proteomes of type 1 diabetic rats with or without treatment with a to September 30th 2005 (“Gargano Mortality Study”, GMS, n=1,028) and one neutralizing VEGF antibody using 18O isotope labeling and 2D-LC-MS/MS at University Hospital of Foggia from January 7th 2002 to September 30th to map dysregulated protein pathways and networks through bioinformatic 2008 (“Foggia Mortality Study, FMS, n=1,146). Information on vital status analysis. Male Sprague-Dawley rats (n=10) were administered 50 mg/kg (by direct contact or by queries to the registry offi ce of cities of residence) STZ IP and the experimental arm (n = 5) were treated with VEGF antibody was obtained from March 1th to November 30th 2010 in 1,885 study patients: bi-weekly IP following confi rmation of hyperglycemia. Data were obtained n=838 from GMS and n=1047 from FMS. Of these, 1,762 (866M/896F, using a Thermo-Finnigan LTQ LC-MS/MS. Data analysis utilized MASCOT age=63±11 yrs, duration of diabetes, DD=12±10 yrs, A1c levels=8.8±2.0%) for peptide identifi cation and in-house software for ratio assignment.

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A164 COMPLICATIONS—NEPHROPATHY had both GFR (from serum creatinine by MDRD formula) and albuminuria development of DN. Therefore, we examined 30 type 2 diabetic patients [22 (albumin/creatinine ratio, ACR) measurements; 349 had low-GFR (<60ml/ men, age 49 ± 9 (mean ± SD) years, known diabetes duration 12 ± 7 years, 22 min/1.73m2), 496 micro-albuminuria (Mi-A: ACR>2.5/3.5 mg/mmol in F/M) normoalbuminuria (NA), and 8 microalbuminuria (MA)] without any defi nable and 144 macro-albuminuria (Ma-A: ACR>30 mg/mmol). During a follow-up renal disease other than DN on renal biopsy. Light and electron microscopic of 61+33 months, 266 deaths occurred. In the pooled analysis of both GMS morphometric analyses provided quantitative glomerular structural changes. and FMS, after adjusting for sex, DD, A1c, smoking habit and study sample, Patients were followed up for 6.2 ± 3.5 years, and GFR measurements were the risk of death was higher in patients with Mi-A (HR=1.51; 95%C.I. 1.14- taken at every 6 months using plasma clearance of iohexol. The GFR value 1.99, p=0.004) and Ma-A (HR=2.60; 1.81-3.74, p<0.0001), as compared to at biopsy correlated with the fi ltration surface per glomerulus [Sv(PGBM/ POSTERS those with normal ACR. Similar results were obtained when GMS and FMS glom)], but not with the mesangial fractional volume or other morphometric Complications were analyzed separately (p of study sample-by-albuminuria status=0.36). parameters. Sv(PGBM/glom) value correlated with the yearly GFR decrease Acute and Chronic Prediction of death by Mi-A and Ma-A was strongly signifi cant in patients following the renal biopsy, but not with the GFR change for the longer period. with normal GFR (n=385, HR=1.60, 1.12-2.28, p=0.009; n=77, HR=2.55, 1.48- The GFR values showed a close negative correlation with the subsequent 4.40; p=0.001) while barely detectable and not signifi cant, in those with low- yearly changes in GFR. GFR showed statistically signifi cant linear decrease GFR (n=111, HR=1.02, 0.64-1.62, p=0.92; n=67, HR=1.43, 0.85-2.39, p=0.17). A in 9 of the 30 patients, slopes of regression lines were almost zero in 11 signifi cant albuminuria-by-GFR states interaction in modulating risk of death patients, GFR increased and decreased in parabolic fashion in 2 patients, was observed (p=0.032). In our samples, albuminuria is not associated with and statistically signifi cant regression curves/lines were not available in all-cause mortality in T2D patients with low-GFR, thus questioning its role the remaining 8 patients. Nine of the 11 patients who showed statistically as a risk factor in this subset of patients. signifi cant decline of renal function were without appreciable worsening of albuminuria, while only 2 patients developed persistent proteinuria during & 599-P the observation period. Conclusions: In renal biopsy-proven NA and MA type Diabetic Retinopathy and Microalbuminuria Could Determine Renal 2 diabetic patients, GHF is closely associated with an increased glomerular Function Progress in Type 2 Diabetic Patients fi ltration surface. Elevated GFR would predict subsequent GFR decline AKIKO TSUCHIYA, TATSUMI MORIYA, SONOMI WAKAKURA, JUNRO OGAWA, without worsening of albuminuria. AKINORI HAYASHI, SHINICHIRI OKIZAKI, MASAYOSHI SHICHIRI, Sagamihara, Japan & 601-P According to the albuminuria (UAE) or diabetic retinopathy (DR) grades, Impact of Glucose Level on Estimated Glomerular Filtration Rate in when renal functional decline occurs remains unclear. To clarify factors Diabetic Patients affecting glomerular fi ltration rate (GFR) and UAE during extensive, long- HAIBING CHEN, LEI ZHANG, YUQIAN BAO, WEIPING JIA, Shanghai, China term follow-up, we examined 32 type 2 diabetic patients by percutaneous Objective: To investigate the infl uence of blood glucose level on the renal biopsy (23 men, age 49±10 yrs, known duration 13±8 yrs [mean±SD], estimated glomerular fi ltration rate (eGFR) by Cockcroft-Gault (CG) and 20 patients had DR, 17 normoalbuminuria [NA], 15 microalbumiuria [MA]) Modifi cation of Diet in Renal Disease (MDRD) formula in diabetic patients, divided into 4 groups according to the presence or absence of DR and MA: and to explore the differences between the CG and MDRD formula to NA(R-), NA without DR; NA(R+), NA with DR; MA(R-), MA without DR; and estimated GFR in different levels of blood glucose in the diagnosis of MA(R+), MA with DR. Electron microscopic morphometric analyses gave moderate renal insuffi ciency. Methods: In 1210 diabetic patients (650 quantitative glomerular structural changes including mesangial fractional males and 560 females), we searched for an association between HbA1c 99m volume [Vv(Mes/glom)]. Light microscopy tissues were categorized as: CI, and isotopically measured GFR (iGFR) ( Tc-DTPA) and eGFRCG and normal/near normal renal structure; CII, typical diabetic glomerulopathy; CIII, eGFRMDRD equation. The patients were divided into normal GFR group atypical injury patterns. Patients were followed up for 6.2 ± 3.5 years, and (n=589, iGFR≥90ml·min-1per1.73m2), gently GFR group (n=470, 60≤iGFR<90 annual GFR and UAE measurements were performed. At the renal biopsy: ml·min-1per1.73m2), moderate and sever GFR group (n=151,30≤iGFR<60 Vv(Mes/glom) correlated with UAE in patients with DR (r=0.46, p<0.05), but ml·min-1per1.73m2). According to the quintile HbA1c level (7.1, 10.5%), the not in patients without DR. Group comparisons: All of the MA(R+) patients patients were divided into four groups. Patients whose HbA1c<7.1% were were categorized as CII with signifi cantly different histological patterns defi ned as well controlled group, those HbA1c≥10.5% were defi ned as poorly among all 4 groups. Vv(Mes/glom) was higher in MA(R+) and NA(R+) than controlled group. Results: HbA1c was signifi cantly correlated with the iGFR, in NA(R-). Follow-up: GFRs were lower in MA(R+) and GFR decline /yr was eGFRCG, eGFRMDRD. eGFRMDRD seemed to signifi cantly overestimate GFR both faster in MA(R+) than in NA(R-) and NA(R+). Stepwise regression analysis in all GFR subgroup. However, eGFRCG seemed to signifi cantly underestimate showed that the categorization using both DR and MA affected GFR decline GFR in well controlled group. Bland-Altman analysis indicated that the bias of (F=6.26, p<0.05). eGFRMDRD with iGFR in poorly controlled group were higher than those in well NA(R-) NA(R+) MA(R-) MA(R+) controlled group. Compared with the poorly controlled group, the 15% and 30% accuracies of eGFR in the well controlled group were signifi cantly GFR at baseline 113±22 118±29 136±29 131±55 MDRD higher. The bias of eGFRCG with iGFR were signicantly higher than those of GFR at follow-up 113±9 115±29 123±32 81±42 eGFRMDRD with iGFR. The areas under the ROC curves were signifi cantly Vv(Mes/glom) 0.20±0.03 0.26±0.08 0.23±0.04 0.29±0.06 higher in the eGFRMDRD than in the eGFRCG in poorly controlled group, the CI/CII/CIII 2/0/4 2/5/4 1/1/4 0/7/0 diagnostic accuracies were better for the MDRD, except for the diagnosis of moderate renal failure in well-controlled patients. Conclusions: eGFR Conclusions : In type 2 diabetes: 1) Renal structural-functional relationships with MDRD and CG formula in diabetic patients may lead to discrepancies become more obvious under the presence of DR, 2) Renal function in NA with the real GFR value. eGFRMDRD overestimates the real GFR value. The patients might be preserved regardless of the DR, 3) MA(R+) but not MA(R-) MDRD equation was more accurate and robust in poorly controlled diabetic showed typical diabetic glomerulosclerosis and GFR decline. Therefore, DR patients with moderate renal insuffi ciency. and albuminuria should be considered to determine whether or not renal function progresses in type 2 diabetic patients. & 602-P HbA1c Variability and Risk of Development of Microalbuminuria & 600-P in Japanese Patients with Type 2 Diabetes: A Pilot Study, Tsukuba Glomerular Hyperfi ltration and Increased Glomerular Filtration Kawai Diabetes Registry 3 Surface Are Associated with Renal Function Decline in Normo- and AYUMI SUGAWARA, KOICHI KAWAI, SHINOBU MOTOHASHI, KAZUYA FUJI- Microalbuminuric Type 2 Diabetic Patients WARA, REIKO HIRASAWA, KAYANO IGAWA, KAZUTO KOBAYASHI, KAZUMI TATSUMI MORIYA, AKIKO TSUCHIYA, SONOMI WAKAKURA, JUNRO OGAWA, SAITO, KATSUYA YAMAZAKI, HIROHITO SONE, Mito, Japan, Tsukuba, Japan AKINORI HAYASHI, SHIN-ICHIRO OKIZAKI, MASAYOSHI SHICHIRI, Sagamihara, Although glycemic instability has been considered as a potential risk Japan factor for development of microvascular complications in diabetes, clinical Recently, albuminuria and glomerular fi ltration rate (GFR) were indicated evidence has been inconsistent. In addition, although HbA1c variability to be independent predictors of renal or cardiovascular outcomes in the has been reported to be predictive of microvascular complications in type general population or and among type 2 diabetic patients. However, little 1 diabetes, whether it applies to type 2 diabetes is unknown. Therefore, is known about whether or not glomerular hyperfi ltration (GHF) at the early we assessed the effect of HbA1c variability on the risk of microalbuminuria stages of diabetic nephropathy (DN) in type 2 diabetic patients is related to in type 2 diabetes. Study subjects were 233 normoalbuminuric (fi rst and renal structural changes and if it can be used to predict the further functional second sample urinary albumin-to-creatinine ratios <30 mg/g) patients (162

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A165 COMPLICATIONS—NEPHROPATHY

males, 71 females) with type 2 diabetes who fi rst visited the Kawai Clinic 604-P (typical diabetes clinic in a suburb of Tokyo) in 2000-2002 and who were A Role of Change in Renal Expression of Sphingosine-1-Phosphate followed for at least 3 years. The observation period was from the fi rst clinic Receptor 2 and Sphingosine Kinase in the Pathogenesis of Diabetic visit to the date of development of microalbuminuria (ACR ≥30 mg/g in at Nephropathy least 2 of 3 consecutive urine samples) or to their last ACR measurement. SHO ISHIZAWA, JUNKO TAKAHASHI-FUJIGASAKI, YASUSHI KANAZAWA, HbAlc was determined at least quarterly and ACR every 6 months during KEIICHIROU MATOBA, DAIJI KAWANAMI, TOMOKO ITOU, JUN KINOSHITA, the observation period. The standardized SDs of HbA1c (SD-HbA1c) were TAMOTSU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan compared with the risk of development of microalbuminuria with adjustments

POSTERS More recently, we have reported that activation of Rho/ROCK pathway

Complications for age, sex, disease duration, blood pressure, total cholesterol, smoking plays a role in the pathogenesis of diabetic nephropathy causing excess Acute and Chronic status, and baseline HbA1c. The mean±SD age of the subjects at baseline production of extracellular matrix and oxidative stress; however, the was 54.2±10.2 years. At study entry, 181 patients used antidiabetic agents mechanism by which Rho/ROCK pathway is upregulated in diabetic kidney (172, oral hypoglycemic; 8, insulin) and 38 patients used antihypertensive still remains unknown. Sphingosine-1-phosphate (S1P) is a bioactive agents. During the mean follow-up period of 7.4±2.5 years, 89 patients sphingolipid produced via phosphorylation of sphingosine by sphingosine developed microalbuminuria. Cox proportional modeling showed that SD- kinase (SPHK) and binds S1P receptor (S1PR)-2 which leads to potent HbA1c (0.70±0.34) was a predictor of microalbuminuria (HR, 95%CI 2.69; activation of Rho/ROCK pathway. We found that treatment of cultured renal 1.08-6.70, p=0.03) independent of blood pressure (1.01; 1.00-1.03, 0.13), fi rst epithelial cells with S1P induces activation of Rho/ROCK pathway through ACR (1.11; 1.08-1.15, 0.00) or the other parameters stated above. Baseline S1PR-2, which results in decrease in expression of E-cadherin and increase HbA1c was not an independent factor for development of microalbuminuria. in α-SMA-positive cells. These data suggest that S1P may cause epithelial- In conclusion, our result suggests that HbA1c variability contributes to mesenchymal transition (EMT) of renal tubules activating Rho/ROCK development of microvascular complications in type 2 diabetes. We will pathway. Hence, we hypothesize that S1P synthesis may be accelerated in conduct a further study of a larger sample to confi rm these results. diabetic kidney, which is responsible for activation of Rho/ROCK pathway. To elucidate contribution of S1P to diabetic nephropathy, this study aimed to & 603-P investigate change in expression of SPHK and S1PR-2 in kidney of diabetic Circulating Markers of Protein Glycation, Oxidation and Nitration in and non-diabetic mice. Twelve week-old-diabetic mice (db/db) and control Type 2 Diabetes and Effect of Decline in Renal Function (db/m) mice were used. By real-time RT-PCR, expression of SPHK-1 mRNA GEORGE JERUMS, SIANNA PANAGIOTOPOULOS, RICHARD J. MACISAAC, in the whole kidney of db/db mice was signifi cantly higher than that in db/m DENNIS K. YUE, GREG R. FULCHER, MATTHEW A. ROBERTS, JAMES R. LARKIN, mice, indicating that SIP is excessively increased in the diabetic kidney ANTONYSUNIL ADAIKALAKOTESWARI, NAILA RABBANI, PAUL J. THORNALLEY, tissue. No signifi cant change in expression of S1PR-2 mRNA was detected Heidelberg, Australia, Sydney, Australia, Coventry, United Kingdom between db/db and db/m mice. To characterize distribution of S1P receptor, Impairment of renal function in diabetes has been associated with raised immunohistochemical examination was performed for S1PR-2. S1PR-2 was serum levels of advanced glycation endproducts (AGEs) and, conversely, expressed mainly in tubules. Above all, expression of S1PR-2 was apparently hyperfi ltration has been associated with low circulating levels of AGEs. high in distal tubules compared to other tubules. In conclusion, together with Proteins are modifi ed by glycation, oxidation and nitration in vivo. Serum our previous in vitro study, it could be postulated that S1P synthesis may contains glycation, oxidation and nitration adducts in protein and related be accelerated in the kidney tissue of diabetes, which results in activation free adducts (glycated, oxidized and nitrated amino acids) – the latter formed of Rho/ROCK pathway through S1PR-2. Activation of Rho/ROCK pathway in by proteolysis of damaged proteins. The aim of this study was to explore the renal tubular cells may induce EMT of renal tubules leading to interstitial relationship of circulating markers of protein damage to glomerular fi ltration fi brosis of diabetic kidney. rate (GFR) in type 2 diabetes. Study participants had type 2 diabetes and 5 categories of estimated 605-P GFR: 15-30, 31-60, 61-90, 91-120 and > 120 ml/min/1.73 m2 (n = 10-15 per Activation of Peroxisome Proliferator-Activated Receptor δ Inhibits category). Fasting serum samples were obtained prior to a routine clinic Streptozotocin-Induced Diabetic Nephropathy through Anti-Infl am- visit and stored at -20°C until assay. Fourteen glycation, oxidation and ma tory Mechanisms in Mice nitration adducts were quantifi ed by stable isotopic dilution analysis liquid HIROMI TACHIBANA, DAUSUKE OGAWA, YUICHI MATSUSHITA, NORIKO YAMA- chromatography-tandem mass spectrometry - free adducts by analysis of a MOTO, NORIKO TOYOTA, CHIKAGE SATO, JUN WADA, KENICHI SHIKATA, 12 kD ultrafi ltrate and protein adducts after exhaustive enzymatic hydrolysis HIROFUMI MAKINO, Okayama, Japan of serum protein. Study group means are compared. Activation of the nuclear hormone receptor peroxisome proliferator- Overall, free adducts were more sensitive to decline in GFR than protein activated receptor δ (PPARδ) has been shown to improve insulin resistance, adducts. For AGE free adducts, methylglyoxal-derived hydroimidazolone adiposity, and plasma HDL levels. Several studies have reported that (MG-H1) increased from 234 to 2611 nmol/L, Nε-carboxymethyl-lysine activation of PPARδ is atheroprotective; however, the role of PPARδ in renal (CML) from 60 to 277 nmol/L and pentosidine from 0.15 to 2.04 nmol/L with function remains unclear. Here, we report the renoprotective effects of PPARδ decline of GFR (P<0.001). For oxidation adducts, dityrosine increased from activation in a model of streptozotocin-induced diabetic nephropathy. Eight- 0.89 to 6.36 nmol/L and N-formylkynurenine (NFK) from 5.4 to 12.2 nmol/L week-old male C57BL/6 mice were divided into three groups: (1) non-diabetic whereas methionine sulfoxide (MetSO) decreased from 6.2 to 3.4 µmol/L. control mice, (2) diabetic mice, and (3) diabetic mice treated with the PPARδ The nitration free adduct, 3-nitrotyrosine (3-NT), increased from 4.5 to 21.8 agonist GW0742 (1 mg/kg/day). GW0742 was administered by gavage for 8 nmol/L. In serum protein, AGEs showed modest increases, dityrosine, NFK weeks after inducing diabetes. GW0742 decreased urinary albumin excretion and 3-NT were unchanged whereas MetSO increased with decline in GFR. without altering blood glucose levels. Macrophage infi ltration, mesangial These results are consistent with modest increases in glycation and matrix accumulation, and type IV collagen deposition were substantially methionine oxidation of protein in the circulation and marked decreases attenuated by GW0742. The gene expression of infl ammatory mediators in in clearance of most glycation, oxidation and nitration free adducts with the kidney cortex, such as monocyte chemoattractant protein (MCP-1) and decline in GFR in type 2 diabetes. osteopontin (OPN), was also suppressed. In vitro studies demonstrated that PPARδ activation increased the expression of anti-infl ammatory corepressor B cell lymphoma-6, which subsequently suppressed MCP-1 and OPN expression. These fi ndings uncover a previously unrecognized mechanism for the renoprotective effects of PPARδ agonists and support the concept that PPARδ agonists may offer a novel therapeutic approach for the treatment of diabetic nephropathy.

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A166 COMPLICATIONS—NEPHROPATHY

CCR2 antagonism was investigated in two models of type 2 diabetes: diet-induced obese mice and db/db mice. Glycemic control and adiponectin production were assessed in fasted mice. Adipose macrophages were quantifi ed by fractionation of epididymal fat pads followed by fl ow cytometry to identify macrophages. Albuminuria was determined in db/db mice housed in metabolic caging overnight. Treatment with an orally active CCR2 antagonist signifi cantly improved multiple metabolic/renal parameters in obese, diabetic mice, including POSTERS

hyperglycemia, insulin sensitivity, and serum adiponectin and, in db/db Complications

mice, albuminuria and serum markers of renal function. The metabolic Acute and Chronic improvements correlated with a signifi cant reduction in adipose tissue macrophage numbers. These data implicate CCR2-driven processes in the pathology of type 2 diabetes and associated co-morbidities such as diabetic nephropathy. CCR2 Antagonist Effect in Diabetic Mice (mean ± SD) Normal Vehicle-Treated CCR2 Antagonist Treated Lean Mice Obese Mice Obese Mice Fasting Blood Glucose (mg/dL) 105 ± 5 205 ± 9 140 ± 9* Adipose Macrophages (cells/g) 34000 ± 9300 65000 ± 8500 27000 ± 6500* Urinary Albumin (ug/day) 110 ± 27 1058 ± 381 347 ± 152* * p < 0.005 vs vehicle

607-P Changes in Skin Microcirculation in Patients with Diabetic Nephro- pathy: Infl uence of Growth and Infl ammatory Markers PRZEMYSLAW MIARKA, MALGORZATA WALUS-MIARKA, MARCIN KRZAN- OWSKI, MARZENA DUBIEL, MALGORZATA STOMPOR, TOMASZ GRODZICKI, WLADYSLAW SULOWICZ, MACIEJ MALECKI, BARBARA IDZIOR-WALUS, Krakow, Poland Introduction: Periphery skin microcirculation dysfunction is a common element of hemodynamic irregularities in diabetic patients. Abnormalities of microcirculation are the basis of microvascular complications in diabetes and are present in many organs. The aim of the study was to evaluate the association between certain growth factors and infl ammatory markers and skin microcirculation assessment parameters in patients with type 2 diabetes. Material and methods: The study group included 70 patients with type 2 diabetes and diabetic nephropathy divided into 4 groups according to GFR values: group 1 - GFR>90 ml/min/1.73m2, n=19 group 2 - GFR 60-89 ml/ min/1.73m2, n=13; group 3 - GFR 30-59 ml/min/1.73m2, n = 22 and group 4 -GFR 15-29 ml/min/1.73m2, n=16). Forearm skin microcirculatory blood fl ow was measured using laser doppler fl owmetry. Mean blood fl ow in basal conditions -MFb, the dynamic parameters such as mean blood fl ow in 44 degrees Centigrade -MF44 and peak fl ow in reactive hyperemia –PF were evaluated. In each patient growth and infl ammatory markers: TGF β, PDGF-BB, VEGF, IL-6, TNF α RII (using ELISA), hsCRP and fi brinogen were measured. Results: In the whole group of patients signifi cant associations between PF and TNF α RII (r=0.44, p<0.0001) and TGF β (r= -0.27, p<0.05) and between MF-44 and TNF α RII (r=0.27, p<0.035) were observed. At the earliest stage of CKD (group 1) signifi cant correlations between PF and infl ammations markers hsCRP (r= -0.565, p=0.02) and fi brinogen (r= -0.836, p<0.0001) were found. In patients with more advanced CK (group 2) an association between PF and fi brinogen (r= -0.642, p<0.05) and MF-44 and TNF α RII (r=0.65, p<0.05) were found, while in group 3 only correlation between PF and TNF α RII (r=0.478, p<0.05) and PDGF-BB (r=-0.629, p=0.005) were observed. In whole group of patients PF correlated signifi cantly with creatinine (r=0.6, p<0.0001), while TNF α RII with GFR (r=-0.7, p <0.00001). 606-P The results of this study indicate that in patients with diabetic nephropathy CCR2 Antagonism Improves Renal Function and Hyperglycemia in skin microcirculation parameters are associated with renal function as well Preclinical Models of Type 2 Diabetes as with infl ammation markers and at later stages with growth factor levels. TIMOTHY SULLIVAN, ZHENHUA MIAO, ROBERT BERAHOVICH, NIKY ZHAO, TREVOR CHARVAT, JAY P. POWERS, JUAN JAEN, THOMAS SCHALL, Mountain 608-P View, CA Clusterin Inhibits Angiotensin II Induced Renal Fibrosis The role of infl ammation in the diabetic syndrome continues to be MI-KYUNG KIM, GWON-SOO JUNG, YUN-A JUNG, NAM-KYEONG KIM, HYE- elucidated, especially the impact that infi ltrating monocytes can have SOON KIM, HYUN-AE SEO, EON-JU JEON, IN-KYU LEE, KEUN-GYU PARK, Daegu, on multiple tissues involved in the disease. Increased adiposity leads to Republic of Korea recruitment of infl ammatory myeloid cells into adipose tissue and production Progression of diabetic nephropathy is characterized by fi brosis of of factors known to impair insulin sensitivity (i.e. TNFα, IL-6, and MCP-1). renal glomerulus and tubulointerstitial region. Angiotensin (Ang) II is Also, myeloid cell recruitment into diabetic kidneys has been associated a major effect molecule in the development of diabetic nephropathy with poor renal outcome. The current studies were undertaken to address through proinfl ammatory and profi brotic effects and glomerular capillary the ability of interventional CCR2 antagonism to improve metabolic and hypertension. Clusterin is implicated in several physiological processes, but renal function in obese, diabetic mice.

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A167 COMPLICATIONS—NEPHROPATHY

the effect of clusterin on kidney is unknown. Here, we examined whether MT expression in human renal proximal tubular epithelial cells (HRPTECs). clusterin prevents Ang II induced renal fi brosis in vitro and in vivo. First, Hypoxia (1% O2) increased MT mRNA levels in HRPTECs. Whereas, 2.5 adenovirus-mediated overexpression of clusterin inhibited Ang II-stimulated ng/ml TGF-β1 decreased MT mRNA levels down to ∼50 % of the control PAI-1, type I collagen, and fi bronectin expression in cultured rat mesangial in normoxia. Immunofl uorescence staining showed that hypoxia induced cells and renal tubular epithelial cells. Transient transfection study showed strong fl uorescence intensity of MT signals in HRTPECs, while a weaker that clusterin inhibited Ang II-stimulated PAI-1 promoter activity in a dose immunoreactivity was observed by TGF-β1. In the immunohistochemical dependent manner. Infusion of Ang II using osmotic minipump markedly analysis using the kidneys of 17 weeks old ZDF rats, MT expression increased the expression of PAI-1, type I collagen, and fi bronectin. However, was upregulated in the tubules of juxtamedullary region, which also POSTERS

Complications up-regulation of clusterin expression in the kidney by adenovirus expressing demonstrated strong intensity of HIF-1α, hypoxic probe pimonidazole, and Acute and Chronic clusterin inhibited Ang II-induced renal fi brosis. Kidneys of clusterin -/- mice oxidative stress marker 8-hydroxy-2’-deoxyguanosine, compared with lean increased renal fi brosis compared to those of wild type mice. Moreover, control rats. Intriguingly, MT was faintly expressed in severe fi brotic kidneys clusterin -/- mice exhibited more markedly increased tubulointerstitial in 39 weeks old ZDF rats. damage and fi brosis after Ang II infusion compared to wild type mice. Taken In conclusion, our data, for the fi rst time, suggest that DN may be together, clusterin inhibits Ang II induced renal fi brosis, and it could be a associated with differential expresssion of renal tubular MT in response to therapeutic target for renal fi brosis. hypoxia and profi brotic TGF-β1, and that MT may serve as a potential target for the therapeutic remedy for DN. 609-P Diabetic Nephropathy in the KK.Cg-Ay/J Mouse Model of Type 2 611-P Diabetes Effect of Early Intensive Insulin Therapy on MKK3/6-p38 Pathway in STEPHEN P. O’BRIEN, LUCY PHILLIPS, MANDY M. SMITH, HONG LING, YVES Renal Cortex of Diabetic Rats SABBAGH, STEFAN WAWERSIK, STEVEN R. LEDBETTER, CYNTHIA M. ARBEENY, DONGHONG FANG, HONGYU GUAN, LIEHUA LIU, XUESI WAN, YANBING LI, Framingham, MA Guangzhou, China Diabetes is the major cause of chronic and end-stage renal disease, EDIC trial reported the phenomenon “metabolic memory” for the fi rst and identifying animal models that are representative of human diabetic time. However, little is known about the underlying mechanisms. It has been nephropathy (DN) is of great importance in developing effective treatments. showed that p38 mitogen-activated protein kinase (p38) was involved in the The KK.Cg-Ay/J (KK-Ay) model is an obese model of type 2 diabetes with development of diabetic nephropathy (DN) and can be activated by oxidative renal impairment. Our goal was to further characterize the progression of stress, which has been thought to be a potential factor in “metabolic renal disease in this model and to correlate serum, urine and renal pathology memory”. P38 has also been found to participate in transforming growth fi ndings with those seen in human DN. In this study, female KK-Ay mice factor β1 (TGF-β1) signaling cascades, a key factor that was involved in the and control KK.Cg-a/a were studied at 8 to 24 weeks of age. To maximize pathogenesis of DN. Therefore this study examined whether p38 pathway phenotypic differences, KK-Ay mice were fed a diet containing 24% fat while was involved in “metabolic memory”. We also examined the activity of KK.Cg-a/a were fed a 12% fat diet. During this study, KK-Ay mice developed TGF-β1 and the p38 pathway members, MAPK kinase 3/6 (MKK 3/6), cAMP- progressive albuminuria and glomerulosclerosis compared to control animals. responsive element binding protein (CREB, immediate downstream of p38) Relative to controls, 24 week old KK-Ay mice had elevated HbA1c, insulin, and and MAPK phosphatase-1 (MKP-1). leptin levels. Concurrently, 24 week old KK-Ay mice were albuminuric but were High-fat diet and low dose streptozotocin (STZ) induced diabetic rats not azotemic or hypertensive. Total kidney and liver weights were increased, were divided randomly to diabetic group (DM group, no insulin treatment, consistent with renal hypertrophy and hepatic steatosis. Histological n=8), early intensive insulin therapy group (EI group, n=9) and late insulin assessment of renal tissue indicated progressive glomerular hypertrophy therapy group (LI group, n=7). Non-diabetic SD rats served as controls (NC with segmental to global expansion of the mesangial matrix, and glomerular group, n=8). Three days after STZ injection, once induction of diabetes hypercellularity is consistent with membranoproliferative glomerulopathy. was confi rmed, NPH (6-8 U per day) was used in EI group to maintain good Increases in glomerular size, cell number, and nuclear size were found, while glycemic control (GC, nonfasting blood glucose≤8 mmol/L) for the entire 16 total podocyte number, was unchanged. Tubular lesions were multifocal and weeks. In LI group, GC was induced after 8 weeks of poor glycemic control mild, and consisted of dilation, intra-tubular protein casts, and epithelial cell (nonfasting blood glucose≥16.7 mmol/L). Real time PCR revealed that as degeneration, necrosis and regeneration. In this model, we demonstrate compared with NC group, levels of CREB and MKP-1 mRNA in renal cortex novel fi ndings where urine nephrin levels increase progressively over the of DM group increased to 3.2±0.42 and 3.1±0.75 folds respectively (P<0.05), time, indicating progressive podocyte injury. This elevatation in urinary in EI group remained normal (P>0.05), and in LI group increased to 2.6±0.56 nephrin and albuminuria correlate to glomerular pathology. These data and 3.3±0.93 folds respectively (P<0.05). Expression of phosphorylated suggest that KK-Ay develop progressive glomerulopathy and that podocyte (active) p38 (P-p38), P-MKK3/6, P-CREB and MKP-1 protein showed a similar injury is an early event. The model has characteristics of early stage renal pattern. However, levels of TGF-β1 mRNA were similar in NC, EI and LI group disease, and it is possible that aging or a secondary insult could drive disease (P>0.05). to overt renal insuffi ciency characteristic of late stage human disease. Our results suggested that increased renal cortical p38 activity in diabetic Supported by: Genzyme Corporation rats was attenuated by early intensive insulin treatment but not late insulin therapy. The MKK3/6-p38 pathway may play an important role in “metabolic 610-P memory”, but not TGF-β1 signaling cascades. Dysregulation of Metallothionein in a Rat Model of Type 2 Diabetic Nephropathy: Divergent Roles of Hypoxia and TGF-β1 612-P MANAMI KOBAYASHI, YUMI TAKIYAMA, YUKIHIRO FUJITA, JUN HONJO, YASU- Effects of Single Doses of Pioglitazone and Rosiglitazone on Sodium TAKA TAKEDA, HIROYA KITSUNAI, HIDEMITSU SAKAGAMI, YUICHI MAKINO, Excretion in Healthy Volunteers MASAKAZU HANEDA, Asahikawa, Japan CHRISTELLE FOUCHER, DOMINIQUE CRIMET, ELIZABETH ALLEN, DMITRI KAZEI, Oxidative stress is thought to play an important role in the onset and EDU ZONDAG, CLAIRE NEE, TROY ZUMBRUNNEN, Daix, France, London, United progression of diabetic nephropathy (DN). Continued hyperglycemia results Kingdom, Weesp, The Netherlands, Marietta, GA in excessive production of reactive oxygen species (ROS) in diabetic kidney, Thiazolidinediones (TZDs) are potent glucose lowering agents known to leading to cellular dysfunction accompanied with subjugated antioxidant cause fl uid retention in susceptible subgroups of patients. The mechanism mechanisms. On the other hand, chronic hypoxia and TGF-β1 contribute to is multifactorial and may involve vasodilatation and increased renal sodium the development of renal fi brosis in DN. For investigation of whether other reabsorption. We hypothesized that single doses of TZDs could induce change key factors contribute to the pathogenic mechanism of DN, we analyzed in sodium reabsorption. In an exploratory, open-label, randomized, placebo- microarray data form microdissected juxtamedullary proximal tubules in a controlled, 3-way cross-over study, 12 healthy males received single doses rat model of type 2 diabetes, Zucker Diabetic Fatty (ZDF) rats. A total of of pioglitazone 45mg (Actos®), rosiglitazone 8mg (Avandia®) and placebo 27,342 transcripts were analyzed, among them, 47 were induced over (Pl) separated by a 7-day washout. During each study period dietary sodium twofold change in the diabetic kidney of 17 weeks old ZDF rat. One of them intake was standardized (5g of salt/day). Urine collections were performed which were upregulated in diabetic renal tubules was metallothionein(MT)-1. over 24h before dosing and fractionated collections on dosing days as follows: MT is a cysteine-rich protein with low molecular weight, and act as at 0h concomitant to dosing, every 30min from 0-6h, every 3h from 6-12h and an antioxdant against the toxicity of metals, ischemia, and ROS. To then 12-24h after dosing. After 1L water load 1h before dosing, water balance investigate the pathological role of MT, we fi rst studied the regulation of was maintained by replacing fl uid loss (voided urine volume and metabolic

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A168 COMPLICATIONS—NEPHROPATHY loss) with drinking water every 30min from 0-6h, then ad libitum. Sodium eNOS mRNA and synergistically ameliorated the kidney damage. The excretion (Naexc) was calculated in each urine collection. Cumulative Naexc kidney dysfunction and pathological changes were signifi cantly associated was calculated on 0-24h and area under the curve at 4-6h (AUCNaexc 4-6h). with cholesterol, with markers of cholesterol absorption (campesterol and At 4-6h post-dose, AUCNaexc 4-6h was signifi cantly lower with pioglitazone cholestanol), and with 8-OHdG. Multiple regression analysis revealed that (LSmean [95%CI]: 7.39 [6.10, 8.67] mmol*h vs. 9.21 [7.93, 10.50] mmol*h the markers of cholesterol absorption were independently associated with with Pl, p=0.027). In addition, signifi cant differences in Naexc were observed the kidney damage in the ezetimibe-treated rats. These results suggest that at 4-4.5h (p<0.05) and 5.5-6 h (p<0.01) intervals with pioglitazone. A trend ezetimibe confers renoprotective effects by inhibiting excess cholesterol for a similar but weaker effect was seen with rosiglitazone (AUC : absorption, which in turn reduces oxidative stress, and that pitavastatin

Naexc 4-6h POSTERS

8.26 [6.97, 9.54] mmol*h, p=0.227 vs. Pl). Cumulative Naexc over 24h was additively ameliorates kidney damage by increasing NO production via Complications also lower with pioglitazone: 60.5 [50.2, 70.8] mmol vs. 68.0 [57.7, 78.3] mechanisms independent of cholesterol reduction. Acute and Chronic mmol with Pl, 68.5 [58.2, 78.8] mmol with rosiglitazone (NS). Treatments were well tolerated. This study was able to detect differences in sodium 615-P excretion at 4-6 h post dose following single doses of pioglitazone 45mg and Gender Disparities in Diabetic Nephropathy rosiglitazone 8mg compared to placebo. MARGARET K. YU, COURTNEY REES, BESSIE A. YOUNG, Seattle, WA Diabetes confers an increased cardiovascular risk to women compared to 613-P men. There is increasing recognition of gender disparities in diabetes with Enhanced Angiotensin Converting Enzyme 2 Inhibits Angiotensin II- respect to traditional cardiovascular risk factors. Chronic kidney disease Induced Collagen Production by Regulating AT1 Receptor- Phospho- (CKD) is an established risk factor for cardiovascular disease, but the effect inositide3- Kinase-Akt Pathway of gender on diabetic nephropathy is unclear. We investigated whether there LE BU, XIAOYUN CHENG, JUNJIE ZOU, XIANG GAO, YI BAO, WEI TANG, SHEN were gender disparities in cardiovascular risk factors and CKD in a cohort of QU, ZHIMIN LIU, Shanghai, China diabetic patients from a managed care setting. Recent reports show a protective role of angiotensin-converting enzyme The Pathways Study is a prospective cohort of ambulatory, diabetic 2(ACE2) against glomerular diseases, especially the accumulation of patients from a large managed care population in Seattle, WA. Baseline extracellular matrix proteins. However, the mechanism regulating this effect characteristics were analyzed for gender differences in cardiovascular risk appears to be complex and poorly understood. Our aim was to investigate factors, including CKD. whether ACE2 blockades the profi brotic effects of Ang II in mouse mesangial 3968 patients met inclusion criteria. The prevalence of hypertension was cell line, MES-13. We demonstrate at least four noteworthy results. First, greater in women than men (44.97% vs 41.04%, p=0.008). Baseline LDL was Ang II induced the mRNA synthesis of neither type III nor type IV, but type signifi cantly higher in women (p=0.000); their mean LDL was 115.9 mg/dl I collagens in mesangial cells. Second, Ang II, via AT-1R, increased collagen (95% CI 114.0-117.8) compared to 107.8 mg/dl (95% CI 106.2-109.4) for men. synthesis through PI3K-Akt signaling pathway. Third, the activation of PI3K/ Women were more likely to have an LDL >130 (OR 1.55, 95% CI 1.32-1.81,

Akt from Ang II/AT-1R was Gα5-cAMP -dependent but PKA-independent and p=0.000), less likely to have their cholesterol checked (OR 0.73, 95% CI 0.65- involved TGFβRI. Finally, ACE2 gene transfer suppressed the expression of 0.83, p=0.000), and less likely than men to be prescribed a statin (26.48% pAkt induced by angiotensin (Ang) II, accompanied by a decreased level of vs 35.36%, p=0.000). There was no difference in HbA1c by gender. Overall, collagen type I in cells. Based on these data, we propose that Ang II binding fewer women had CKD stage 5 at baseline (43.75% vs 56.25%, p=0.000). to AT-1R triggered sequential activation of adenylyl cyclase and TGFβRI When stratifi ed by race, this difference was only signifi cant in Caucasians and led to activation of the PI3K signaling pathway. Phosphorylation of the (p=0.000); amongst Blacks and Asians the trend was towards more women downstream signaling molecule Akt resulted in an increase in type I collagen with CKD stage 5 at baseline, although this was not signifi cant. synthesis. ACE2 gene transfer signifi cantly prevented collagen synthesis In diabetics, there are gender disparities in cardiovascular risk factors as and phosphorylation of Akt stimulated by Ang II. In summary, the present well as in diabetic nephropathy. There appears to be a dissociation between study defi nes a regulatory role for the ACE2 gene in the Ang II/AT-1R-PI3K/ the gender effect on traditional cardiovascular risk factors (which are more Akt pathway and a protective effect of ACE2 against collagen deposition in prevalent in women) and in diabetic nephropathy, which warrants further mesangial cells. The benefi cial effect of ACE2 over expression in mesangial investigation. cells suggested that the ACE2 gene might be a novel therapeutic target for glomerular diseases, such as diabetic nephropathy. Elucidating further 616-P details in this signaling pathway should allow us to better understand the HDL Cholesterol as a Predictive Factor for Associated Micro- regulatory roles of ACE2 in collagen metabolism and promote an ACE2 albuminuria in Type 2 Diabetes Mellitus Patients treatment for glomerular diseases. OVIDIU BRADESCU, ROSEMARIE URDEA, CRISTIAN SERAFINCEANU, Bucharest, Supported by: National Natural Science Foundation of China (81001572) Romania The impact of various clinical and biologic factors on the occurence of 614-P microalbuminuria in type 2 diabetes patients was subject of many research Ezetimibe Prevents Kidney Damage in 5/6 Nephrectomized Rats Fed approaches. Nevertheless, the relation between HDL cholesterol and High-Cholesterol by Suppression of Cholesterol Absorption albumin excretion rate (AER) was less intensively investigated. YUSAKU MORI, TSUTOMU HIRANO, Shinagawa, Japan We have reviewed medical records of 251 type 2 diabetic patients In this study we attempted to elucidate the relationship between free of renal failure (116 men and 135 women) mean age 70,4±10,0 years, cholesterol absorption and kidney damage by investigating the reno- diabetes duration 27,4±8,2 years, BMI 26,4±4,5 Kg/m², HbA1c 9,1±1,8%, HDL protective effect of ezetimibe, an inhibitor of cholesterol absorption, in cholesterol 40,9±8,9 mg/dl, total cholesterol 204,0±49,1 mg/dl, triglycerides 5/6 nephrecto mized rats. Nephrectomized or sham-operated rats were 164,4±71,8 mg/dl, serum creatinine 0,9±0,2 mg/dl, creatinine clearance fed 1% cholesterol with ezetimibe (10 mg/kg/day) for 8 weeks. The sham- 88,0±14,9 ml/min/1,72m², serum urea 45,0±17,7 mg/dl, uric acid 5,4±1,2 operated rats fed the high-cholesterol exhibited hypercholesterolemia, mg/dl, AER 45,4±44,1 mg/24 hours and erythrocyte sedimentation rate proteinuria, and glomerulosclerosis with macrophage infi ltration in the (ESR) 31,7±26,4 mm/1h. We divided the subjects in two groups: one with kidney, and ezetimibe attenuated all of these changes. The nephrectomized normoalbuminuria (AER < 30 mg/24 hours) and one with microalbuminuria rats exhibited hypercholesterolemia, decreased creatinine clearance, (30 mg/24 hours ≤ AER < 300 mg/24 hours). We have performed logistic proteinuria, and increased urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG). regression to assess the factors which might be predictive for associating They also exhibited glomerulosclerosis with macrophage infi ltration microalbuminuria. As men and women were matched for age, HbA1c, and interstitial fi brosis, and down-regulation of endothelial-nitric oxide creatinine clearance and AER, we also computed the binary regression synthase (eNOS) mRNA in the remnant kidney. The high-cholesterol diet separately for male and female patients. increased serum cholesterol further and worsened the kidney damage The logistic regression model was signifi cant (Omnibus test = 0,012, with increased 8-OHdG. Ezetimibe attenuated the hypercholesterolemia by Hosmer and Lemeshow test = 0,401). Among all the factors included in the inhibiting cholesterol absorption, and it signifi cantly ameliorated the kidney model, HDL cholesterol and diabetes duration resulted to be signifi cant for dysfunction and pathological changes. All of these benefi cial effects of associating microalbuminuria (Exp(b) = 0,939, 95% confi dence interval = ezetimibe were associated with reduced 8-OHdG. 0,906-0,972, p < 0,001 and Exp(b) = 1,037, 95% confi dence interval = 1,001- Pitavastatin treatment (3 mg/kg/day) did not reduce cholesterol levels 1,074, p = 0,046 respectively). This prediction remained signifi cant for HDL or 8-OHdG, but it suppressed the kidney damage with up-regulated eNOS cholesterol when analysed separately by sexes (p = 0,042 in men, p = 0,002 mRNA. The combination of ezetimibe and pitavastatin remarkably increased in women).

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A169 COMPLICATIONS—NEPHROPATHY

HDL cholesterol along with duration of diabetes are signifi cantly related to 619-P microalbuminuria in type 2 diabetic patients. HDL cholesterol is associated Long Duration Complications-Free Diabetes Is Characterized by with microalbuminuria irrespective of sex in type 2 diabetes mellitus, but the Increased Monocyte Toll like Receptor Expression: Is This a Pro- relationship is more powerful in women. tective Phenotype? JENCIA WONG, WENSHENG BAO, DANQING MIN, BELINDA BROOKS, ANA 617-P CHARLTON, BRIAN HARRISBERG, DENNIS K. YUE, SUSAN V. MCLENNAN, Camper- High Glucose Increases Mitochondrial Superoxide Generation by down, Australia, Sydney, Australia

POSTERS Modulating RBP4 Receptor in Human Renal Proximal Tubular Cells Diabetes duration is one of the strongest determinants for the development Complications SHYI JANG SHIN, CHAO HUNG CHEN, Kaohsiung, Taiwan of diabetic complications. Those with long term diabetes but free from Acute and Chronic Our previous study showed that RBP4 was signifi cantly elevated in type 2 complications therefore represent a unique phenotype in which to explore diabetic patients with albuminuria and high apo-RBP4/holo-RBP4 can induce protective molecular mechanisms. Toll Like Receptors (TLR) expressed by apoptosis through the activation of STRA6/JNK, p38MAPK phosphorylation mononuclear cells play a critical role in the activation of the innate immune in HEK cells. In this study, we aimed to investigate the infl uence of high system, an important etiological factor in diabetes and its complications. glucose concentration on the expression of STRA6/CRBP/RAR, and, in Moreover, increased monocyte TLR2 and TLR4 in new onset type 2 diabetes contrast, the effect of holo-RBP4 addition on high glucose-activated reactive (T2DM) has been reported. Here we explore the hypothesis that differential oxygen generation in HEK cells. Our results showed that high glucose monocyte behaviour in regards to the expression of TLR2 and TLR4 may strongly enhanced the expression of STRA6, and decreased ATP/ADP ratio, modify susceptibility to long term diabetes complications risk. ATP synthase expression, mitochondrial cytochrome c level and increased Patients with T2DM (age62.2±11.5yrs) of duration over 10 years superoxide generation (MitoSOX red staining). All of the changes stimulated (18.2±9.5yrs) i) without diabetic complications (C-ve n=25), ii) with by high glucose can be reversed by the addition of holo-RBP4. Furthermore, microvascular complications (C+ve n=24) and iii) non diabetic controls (n=24, STRA6 siRNA didn’t change the infl uence of holo-RBP4 addition on ATP/ age 50.7±9.9yrs) were studied. Monocytes were isolated by density gradient ADP ratio, ATP synthase expression, mitochondrial cytochrome c level and the ability of lipopolysaccharide (LPS:-1ng/ml for 4hours) to stimulate and superoxide generation in HEK cells cultured in normal glucose levels. TLR2 and TLR4 mRNA was determined by qRT-PCR. Results at basal (0LPS) However, STRA6 siRNA can inhibit the rescue by holo-RBP4 addition on high and as change from basal after LPS stimulation (+LPS) are expressed as glucose-suppressed ATP/ADP ratio, ATP synthase expression, mitochondrial mean±SEM. cytochrome c level and high glucose-stimulated superoxide generation mRNA Controls Diabetes C-ve Diabetes C+ve in HEK cells. These results implicate that high glucose may increase TLR2 (0 LPS) 1.3±0.3 3.7±0.3* 1.8±0.8 mitochondrial superoxide generation by modulating RBP4 receptor in human proximal tubular cells. TLR4 (0 LPS) 1.6±1.7 10.9±1.4* 1.7±1.5† TLR2(+LPS) 147.1±24.1 138.2±16.2 50.8±17.8*† 618-P TLR4 (+LPS) 58.4±15.3 43.8±8.7 11.4±9.1*† Hyperglycemia Induced Inactivation of VEGF by PKC-δ/SHP-1: A * p<0.01 vs controls, † p<0.01 vs diabetes C-ve by ANOVA. Cause of Podocyte Apoptosis in Diabetic Nephropathy AKIRA MIMA, MUNEHIRO KITADA, PEDRO GERALDES, QIAN LI, WEIER QI, KOJI C-ve monocytes were associated with increased monocyte TLR2 and MIZUTANI, CHRISTIAN RASK-MADSEN, GEORGE L. KING, Boston, MA TLR4 mRNA at baseline and also maintained a normal response to LPS. In Hyperglycemia induced podocyte apoptosis is likely a critical step in the contrast, in C+ve cells the response to LPS was attenuated signifi cantly. There pathogenesis of diabetic nephropathy. We propose that hyperglycemia can was no correlation between TLR expression and BMI, HbA1c or diabetes cause podocyte apoptosis by inhibiting the actions of podocyte survival duration. These data suggest that C-ve monocytes maintain their “activation factors, such as inactivating the cellular effects of vascular endothelial potential” and such long term robust monocyte innate immune response cell growth factor (VEGF). Recently, we reported that hyperglycemia can may be protective against the development of diabetic complications. These activate the protein kinase C (PKC)-δ/ Src homology-2 domain-containing data add to accumulating evidence suggesting that differential monocyte phosphatase-1 (SHP-1) retinal pathway leading to the deactivation of biology may underlie the propensity to develop diabetic complications. platelet-derived growth factor (PDGF) and subsequent pericyte apoptosis and retinopathy (Nat. Med. 2009). To establish that PKC-δ/SHP-1 activation can 620-P inactivate VEGF in vivo, we studied changes in diabetic Sprague-Dawley rats N-Terminal ProBNP(NT-ProBNP) Is a Predictor of the Progression and PKC-δ null mice which were treated with streptozotocin. Hyperglycemia’s of CKD as Well as CVD in Type 2 Diabetes inhibitory effects on VEGF actions in cultured mouse podocytes were also KUMIKO HAMANO, MASANORI TAKAHASHI, AIKO OOKA, ASAMI TANAKA, assessed utilizing adenoviral vectors expressing dominant-negative (DN) RIEKO KOMI, Kawasaki, Japan, Kamakura, Japan PKC-δ and small interfering RNA (siRNA) of SHP-1. High glucose levels (HG) NT-proBNP is postulated as a diagnostic and prognostic biomarker of (25mM) increased PKC-δ, p38 MAPK and SHP-1 expressions by 2.6 ± 0.3- heart failure and cardiovascular mortality in type 2 diabetes. We recently fold, 1.9 ± 0.1-fold and 1.3 ± 0.1-fold, respectively, consistent with increased reported that NT-proBNP could be a marker of silent myocardial ischemia apoptosis of the podocytes (1.8-± 0.2-fold). Most of the HG’s apoptotic in type 2 diabetes. It is known that NT-proBNP is elevated in CKD refl ecting effects and inhibitory effects on VEGF actions can be neutralized by DN reduced glomerular fi ltration. An additional mechanism might be present PKC-δ and siRNA of SHP-1, independent of NF-kB activation. Similarly, when considering the concept of cardiorenal continuum. We tested whether chronic diabetes (3 months of duration) in both streptozotocin-treated mice NT-proBNP could predict progression of CKD. We consecutively recruited 157 and rats increased PKC-δ, p38 MAPK and SHP-1 activation via inhibition of patients (age 62.7±0.9, HbA1c 8.8±0.2,Cr 0.90±0.02mg/dl). Serum NT-proBNP VEGF signaling and accelerated podocyte apoptosis in the renal glomeruli. measurement and echocardiogram were performed and subjects were All of these renal cortex abnormalities were signifi cantly decreased in followed up for 5 years. Primary endpoints were defi ned as 1) 30% decrease diabetic PKC-δ null mice compared to diabetic controls. of eGFR due to low baseline Cr and 2) CVD event and death. NT-proBNP was Our results demonstrate that hyperglycemia activates PKC-δ and p38 correlated with left ventricular mass index and Cr. NT-proBNP levels were MAPK-mediated activation of SHP-1. These changes result in VEGF-A increased according to CKD stages (median 21, 66, 154, 480 pg/ml, stage 1, receptor-2 deactivation to cause podocyte apoptosis and initiate diabetic 2, 3, 4 respectively). During the follow-up period, twenty patients reached nephropathy in vivo. For the fi rst time, these fi ndings identify activation of the renal endpoint and had signifi cantly higher baseline NT-proBNP (207 the tyrosine phosphatase SHP-1 as a target of hyperglycemia, resulting in vs.82, p<0.05). Patients were stratifi ed by NT-proBNP concentrations above the inhibition of endogenous protective factors and consequent podocyte and below the optimal cut-off points by ROC analysis. Survival curves using apoptosis. the Kaplan Meier calculation demonstrated signifi cantly higher proportions of subjects with NT-proBNP above 196 had progressive decline of eGFR (Log rank test: p=0.0192). Fifty-three (group 1) had history of CVD at baseline. Eighteen patients (group 2) had new incidences of CVD or death. Mean levels of NT-proBNP were 189, 121 and 49 for group 1, 2 and 3 (those without CVD events), respectively and the difference between groups was signifi cant (p<0.05). Subjects with NT-proBNP level above 60 had signifi cantly higher risks of CVD and death than those below 60 (p=0.0125) by Kaplan Meier analysis. In conclusion, both cardiac and renal functions are refl ected by NT-

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A170 COMPLICATIONS—NEPHROPATHY proBNP and it also predicted not only future CVD event or death as well as CKD progression. NT-proBNP might be one of key molecules linking kidney Exp(b) 95% confi dence interval p value with CVD and eventual mortality. It is possible to stratify diabetic patients Total cholesterol 1,010 1,000 - 1,020 0,047 at high risk by simple measurement of NT-proBNP. HDL cholesterol 0,943 0,899 - 0,989 0,016 Hemoglobin 0,665 0,468 - 0,945 0,023 621-P ESR 0,981 0,966 - 0,997 0,020 Plasma SSAO Activity Is Increased in Type 1 Diabetes and Its

Vascular Complications but Does Not Correlate with HbA1c HDL cholesterol, total cholesterol, hemoglobin and ESR are signifi cantly POSTERS ANDRZEJ S. JANUSZEWSKI, NICHOLAS J. MASON, KEVIN G. ROWLEY, CONNIE associated with microalbuminuria in insulin treated type 2 diabetic Complications S. KARSCHIMKUS, JAMES D. BEST, DAVID N. O’NEAL, ALICIA J. JENKINS, patients. Acute and Chronic Melbourne, Australia Semicarbazide-sensitive amine oxidase (SSAO) which catalyses amines 623-P into aldehydes with release of ammonia and hydrogen peroxide, is implicated Rac1 Activation by High Glucose Is Mediated by ELMO1 in Kidney in: apoptosis, atherogenesis, cell adhesion, leukocyte traffi cking and glucose Cells transport. SSAO activity contributes to vascular dysfunction and oxidative STEFANIA COTTA-DONE, SERDAR TUNCALI, JULIANNA ROSS, NHAN L. TRAN, stress in diabetes (DM). JOHANNA K. DISTEFANO, Phoenix, AZ Aim of this project was to quantify plasma SSAO activity in Type 1 diabetic Diabetic nephropathy is the major cause of end-stage renal disease in (T1DM) and non-diabetic subjects, including its relationships with renal and developed countries and is associated with substantial morbidity and vascular function, complications, oxidative stress and glycemia. mortality in individuals with diabetes. Genetic determinants are among the T1DM subjects, n=94, aged (mean±SD) 40±14 years, DM duration 20±13 factors that contribute to the development of diabetic nephropathy, and we years, including 34 with microvascular complications (CX[+]) were compared and others showed that variants in the gene encoding the engulfment and with 96 healthy, non-DM subjects (CON), aged 40±12 years. Vascular cell motility 1 protein (ELMO1) are associated with diabetic kidney disease function measures were: blood pressure, small (SAE) and large (LAE) artery across multiple populations and independent of type of diabetes. elasticity. Biochemical measures included fasting glucose, HbA1c, renal ELMO1 is known to bind with Dock180, a bipartite guanine nucleotide function (cystatin C, urinary albumin/creatinine, GFR by Cockcroft-Gault exchange factor (GEF) specifi c for Rac1, which mediates actin cytoskeleton formula), homocysteine (Hcy), vascular infl ammation (sVCAM-1, sICAM-1, dynamics and lamellipodia formation. ELMO1 is also known to increase s-eSelectin). Plasma SSAO activity was quantifi ed by HPLC. expression of extracellular matrix proteins in kidney cells. However, SSAO activity (mean±SD) was increased in T1DM vs. CON (1023±266 vs. the mechanisms by which disruption of ELMO1 function mediates 742±193 mU/L; p<0.00001) and was also higher in CX[+]DM vs. CX[-]DM renal dysfunction remain unknown. To begin to delineate the molecular (1127±245 vs. 982±269 mU/L; p=0.01). In DM SSAO activity correlated with mechanisms by which ELMO1 may contribute to the development of diabetic GFR (r=-0.44; p=0.0001), cystatin C (r=0.47; p=0.0001), sVCAM-1 (r=0.41; nephropathy, we investigated the relationship between ELMO1 and Rac1 p=0.0001), sICAM-1 (r=0.33; p=0.002), Hcy (r=0.53; p=0.0001) levels and SAE using human embryonic kidney cells (HEK-293). We fi rst demonstrated that (r=-0.23; p=0.03). In CON, but not in DM, SSAO activity correlated with HbA1c Rac1 is activated under conditions of high glucose (25.5 mM), but not normal (r=0.26; p=0.02). Factor analysis revealed that renal dysfunction (cystatin C, glucose (5.5 mM) or osmolarity control (25.5 mM mannitol) in these cells. GFR), Hcy and sVCAM-1 are major determinants of SSAO in T1DM. We also determined that inhibition of endogenous ELMO1 expression SSAO activity is increased in T1DM vs. non-diabetic subjects and in T1DM by siRNA oligonucleotides signifi cantly impeded high glucose-mediated subjects with vs. without complications. In T1DM SSAO activity is related to Rac1 activation compared to non-specifi c control siRNA. We are currently renal function, Hcy and vascular infl ammation, but not to HbA1c. exploring molecular mechanisms by which ELMO1 mediates high glucose- induced Rac1 activation. Together, these fi ndings show that Rac1 activation 622-P is sensitive to high glucose concentration in kidney cells, but that depletion Predictive Factors for Associating Microalbuminuria in Insulin of ELMO1 abrogates this effect, and provide the fi rst evidence supporting Treated Type 2 Diabetes Patients a mechanism by which ELMO1 may mediate detrimental effects of high ROSEMARIE URDEA, OVIDIU BRADESCU, CRISTIAN SERAFINCEANU, Bucharest, glucose on kidney cells. Romania Microalbuminuria (MA) is a well-known risk factor for cardiovascular 624-P disease in type 2 diabetes patients. The impact of various clinical and biologic Regression and Progression of Estimate GFR and Urine Albumin to factors on the occurence of nephropathy in these patients was subject of Creatinine Ratio in Type 2 Diabetic Patients: A Three-Year Follow- many research approaches. However, there are few studies regarding the Up Study at Diabetes Clinics in Japan link between modifi ed HDL cholesterol and MA. YUKO WATANABE, HITOMI FUJII, TAKAICHI MIYAKAWA, Tama, Japan We have reviewed medical records of 160 type 2 diabetic patients We conducted a 3-year observational cohort study to reveal changes of without renal failure (64 men and 96 women) mean age 69,5±9,8 years, estimated glomerular fi ltration rate (eGFR) and urine albumin to creatinine diabetes duration 26,8±7,4 years, BMI 26,1±4,3 Kg/m², HbA1c 9,3±1,9%, HDL ratio (ACR). A total of 259 consecutive adult type 2 diabetic patients seen at cholesterol 41,3±9,1 mg/dl, total cholesterol 204,4±51,6 mg/dl, triglycerides four diabetes clinics in the Tokyo Metropolitan Area were enrolled in 2007. 164,9±66,6 mg/dl, serum creatinine 0,9±0,2 mg/dl, creatinine clearance Eligible patients had serum creatinine and spot urine ACR measured at least 88,2±14,4 ml/min/1,72m², serum urea 46,3±19,5 mg/dl, uric acid 5,3±1,2 mg/ two times by immunoturbidimetric assay in each year, and an eGFR was dl, AER 39,4±35,8 mg/24 hours, hemoglobin 12,6±2,3 g/dl and erythrocyte calculated by the MDRD Study equation with the Japanese coeffi cient. We sedimentation rate (ESR) 34,5±29,3 mm/h. These patients received excluded patients with kidney failure. Patients had a mean age (SD), 61.3 insulin in monotherapy or combined therapy for 6,8±5,6 years: 0,55±0,21 (10.7); female, 40%; mean BMI, 24.8 kg/m2 (4.1); mean HbA1C, 7.5% (1.3); units per body weight. We divided the subjects into two groups: one with eGFR 77.3 ml/min/1.72m2 (20.5); and ACR 100.8 mg/g creatinine (344.8). 77% normoalbuminuria (< 30 mg/24 hours) and one with MA (between 30 and 299 of patients were on oral antidiabetic agents and/or insulin; 34% of patients mg/24 hours). We have performed logistic regression to assess the factors were on ARBs. After 3 years, patients who achieved ADA treatment goals which might explain the MA. The factors included in the regression model were 41% for HbA1C, 54% for SBP, and 81% for DBP, respectively. As for were age, diabetes duration, insulin treatment duration, insulin amount LDL-cholesterol, 57% of patients achieved our goal of 120mg/dl. Progression per body weight, BMI, HbA1c, HDL and total cholesterol, serum creatinine, was determined if eGFR decreased or ACR increased more than one stratum serum urea, creatinine clearance, uric acid and ESR. after 3 years (for eGFR strata: eGFR > 90; eGFR 60 ∼ 89; and eGFR 30 ∼ 59 The logistic regression model was signifi cant (Omnibus test = 0,006, and for ACR starata: ACR < 30; ACR 30 ∼ 299; and ACR > 300), respectively; Hosmer and Lemeshow test = 0,883). Among all the factors included in regression was determined vice versa. Patients whose eGFR progressed the model, HDL and total cholesterol, hemoglobin and ESR resulted to be (N = 43) showed a mean eGFR decrease of 22.5, had higher eGFR at start signifi cant for association with microalbuminuria (see table 1). irrespective of age and sex (P = 0.001), and were on ARB more frequent (P = 0.04). Patients whose eGFR regressed (N = 24) showed a mean eGFR increase of 14.3. Patients whose ACR regressed (N = 22) showed a mean ACR decrease of 353 despite longer duration of diabetes and high ACR level at start, whereas patients whose ACR progressed (N = 36) showed a mean ACR increase of 374. Progression of ACR level correlated to high prevalence

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A171 COMPLICATIONS—NEPHROPATHY

of macroangiopathy (P = 0.007). In this cohort, 70% of patients remained at the same eGFR or ACR stratum after 3 years. Conclusions: In “real world” GFR LOSS ABSENT MODERATE RAPID clinical practice, 80% of patients were able to maintain or even improve their (<2%/yr) (2-4%/yr) (>4%/yr) eGFR and/or ACR by treating diabetes and its consequences vigorously. timepoint baseline 2 year baseline 2 year baseline 2 year plasma marker 625-P TNF free [pg/ml] 5.0 4.7 5.0 5.5 5.8 5.5 Relationship between Postprandial Hyperglycemia and Renal Tubu- TNF total [pg/ml] 7.1 6.1 7.8 7.6 9.5 8.6 POSTERS lar Function in Patients without Diabetes Complications MOTOSHI OUCHI, KAZUNARI SUZUKI, KENICHI SEKIMIZU, KENTARO WATANABE, TNFR1 [ng/ml] 1.4 1.3 1.3 1.3 1.7 1.8 Acute and Chronic TATSUYA SUZUKI, HIROSHI NAKANO, KENZO OBA, Tokyo, Japan TNFR2 [ng/ml] 2.3 2.2 2.4 2.3 2.8 3.0 The aim of our study was to evaluate whether postprandial hyperglycemia Markers correlated between timepoints signifi cantly (r >0.5, p value <0.01 increased the risk of progression of renal tubular dysfunction by using serum for each one). TNF receptors, but not TNF, were characterized by extremely 1,5-anhydroglucitol (1,5-AG) as a marker of the postprandial glucose level, low intraindividual variation over time (<11%). In addition, high baseline and urinary N-acetyl-β-D-glucosaminidase (NAG) index as a marker of renal concentrations of either TNF free or TNF total did not result in the increase tubular function. of TNFRs over time. As study subjects, we recruited 735 patients who were treated in our In conclusion, plasma levels of TNF receptors do not increase with renal outpatient department and maintained an HbA1c of less than 7.4%. Patients function loss in subjects with type 1 diabetes, but, as shown in the former who were pregnant had HBV/HCV infection, liver cirrhosis, chronic kidney study, their elevation precede kidney dysfunction. They seem not to be disease (serum creatinine 106.1μmol/Lor over), proteinuria, or renal infl uenced by TNF levels and have a very low intra-individual variation. These glycosuria, or were taking herbal medicine or steroid hormone therapy, or features suggest that TNFRs are not only predictors, but also risk factors of had a past history of gastrectomy, were excluded. We divided the subjects early GFR loss in Type 1 Diabetes. ADA-Funded Research into the following two groups: those with diabetes,(A1c of less than 7.4%, 32 to 93 years of age), and those without diabetes (A1c of less than 6.2%, 22 to 95 years of age). Furthermore, we divided them into three equal-sized 627-P groups (tertiles) according to the serum 1,5-AG levels (low, medium and high) Serum Resistin and Kidney Function: A Family Based Study in Non in each group. We investigated the relationship between serum 1,5-AG and Diabetic Untreated Whites with Normal Glomerular Filtration Rate the urinary NAG index in subjects with or without diabetes. CLAUDIA MENZAGHI, LUCIA SALVEMINI, GRAZIA FINI, DAVIDE MANGIACOTTI, In tertiles divided according to the serum 1,5-AG levels of the subjects MADDALENA GIORELLI, CONCETTA DE BONIS, SALVATORE DE COSMO, with diabetes, the FPG and A1c of the low 1,5-AG tertile were signifi cantly VINCENZO TRISCHITTA, San Giovanni Rotondo, Italy higher than those of both middle and high 1,5-AG tertiles, while there was no High serum resistin levels have been associated with kidney dysfunction. signifi cant inter-tertile difference in eGFR and serum creatinine. In contrast, Most of these studies have been performed in samples including in subjects without diabetes, the eGFR and serum creatinine of the low individuals with severe kidney impairment and/or hypertension, diabetes, 1,5-AG tertile were signifi cantly higher than those of both middle and high coronary artery disease. Thus, the observed association might have been 1,5-AG tertiles, while there was no signifi cant difference in FPG and A1c confounded by these disorders and related treatments. Our aim was to among the tertiles. The urinary NAG-index levels of the low tertile group study the relationship between serum resistin (ng/ml by ELISA), albumin/ with and without diabetes were signifi cantly higher than those of the middle creatinine ratio (ACR, mg/mmol) and glomerular fi ltration rate (GFR, ml/min, and high tertile groups.There were correlations between serum 1,5-AG and as assessed by the reciprocal of serum cystatin C) in a family-based sample the urinary NAG-index of subjects with and without diabetes, but those of 671 (256M/415F; age 39.8±14.2 yrs), non diabetic, untreated Whites with correlations were not signifi cant. normal GFR (≥60 ml/min). A linear mixed effects model and bivariate analyses These results suggest that postprandial hyperglycemia may be a risk were used to evaluate the phenotypic and genetic relations between serum factor for renal tubular dysfunction in non-diabetic subjects as in diabetes. resistin levels and both ACR and GFR. All analyses were adjusted for sex, age, age2, systolic blood pressure, smoking habits and physical exercise. 626-P Resistin levels were slightly positively associated with ACR ( ±SE=0.055±0.023, p=0.017) and strongly inversely related to GFR levels Repeated Measurements of TNF Markers in Subjects with Type 1 β ( ±SE=-0.011±0.0024, p=4.7-6). Of note, 7% of GFR variance was accounted Diabetes and Early Renal Impairment β for by resistin levels. In addition, a genetic correlations ( g=-0.20±0.096; MONIKA A. NIEWCZAS, TOMOHITO GOHDA, WILLIAM H. WALKER, ADAM M. ρ p=0.032) was observed between resistin and GFR. When the resistin gene SMILES, LINDA H. FICOCIELLO, JAMES H. WARRAM, TANYA N. MAYADAS, (RETN) SNP rs3745367, previously associated with serum resistin in the ANDRZEJ S. KROLEWSKI, Boston, MA same setting (p=0.04), was included into the model, such genetic correlation We have already shown that soluble Tumor Necrosis Factor receptors was completely abolished ( g=-0.07±0.10; p=0.51). (TNFRs) are strong predictors of early glomerular fi ltration rate (GFR) loss ρ The association between resistin and ACR was maintained when GFR in type 1 diabetes. Aim of this study was to evaluate a variation over time was included into the model ( ±SE=0.049±0.024, p=0.041); likewise, both of TNF markers in plasma in subjects with type 1 diabetes in the context of β the phenotypic ( ±SE=-0.0096±0.0024, p=6.3-5) and the genetic ( g=- early nephropathy course. Our study group consisted of 77 subjects with β ρ 0.19±0.096; p=0.04) associations between resistin and GFR were maintained type 1 diabetes, no proteinuria and preserved renal function at baseline, after adjusting for ACR. followed then for 10 years. Plasma concentrations of TNF total, TNF free, Serum resistin levels are independently associated with ACR and GFR. TNFR1 and TNFR2 were measured in each individual in two timepoints, at Serum resistin and GFR share also some common genetic background, baseline and two year later. Subjects with annual GFR decrease A: <2%, B: mostly represented by the RETN locus. Our data, obtained in the absence of 2-4%, C: >4% were considered to have A: an absent, B: a moderate and C: a confounding effects of diabetes, kidney impairment and related treatments, rapid GFR loss, respectively. strongly suggest that resistin plays a role in modulating kidney function. Moderate GFR loss occurred in 33 subjects (43%) and rapid loss of renal function in 25 subjects (32%). Plasma levels of each of the four markers were comparable between baseline and follow-up in the entire group. Moreover, 628-P they did not differ in any of GFR outcome related strata. Mean levels of TNF The Role of Glucosepane and Other AGEs in Microvascular and markers in two timepoints and in regards to renal outcome are presented Neuropathic Complications in Type 1 Diabetes in the table. VINCENT M. MONNIER, DAVID R. SELL, CHRISTOPHER M. STRAUCH, WANJIE SUN, SAUL M. GENUTH, THE DCCT RESEARCH GROUP, Cleveland, OH, Rockville, MD Prior to DCCT closeout (1993), two 4 mm skin biopsies (Diabetes 48:870- o 80, 1999) were obtained and stored under N2 at -80 C in anticipation that novel markers of complication risk would later become available. The tissue was enzymatically proteolyzed and several novel collagen-linked advanced glycation endproducts (AGEs) were determined by LC/MS in subjects with intensive (n= 122, INT) or conventional (n=94, CON) treatment, and 43 non- diabetic (ND) controls. Glucosepane (GSPNE), a recently described AGE and major protein cross-link in human extracellular matrix, stood out among six

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A172 COMPLICATIONS—NEUROPATHY

AGE markers. Mean (± SD) age adjusted GSPNE values in nmoles/mg collagen comprised of Mediterranean diet and structured exercise on these measures were 1.64 (0.40) in ND, 2.41 (0.69) in INT and 2.76 (0.70) in CON (p<0.0001 for in subjects with MS. trend and P <0.0001 for INT versus CON). GSPNE was signifi cantly (p<0.0001) We utilized left ventricle (LV) positron emission tomography with [11C] correlated with age (r=0.46), duration of type 1 diabetes (T1D) (r=0.51), meta-hydroxyephedrine ([11C]HED), quantitative heart rate variability fructose-lysine (r=0.48), and mean DCCT A1C up to the biopsy (r=0.61). GSPNE (HRV) testing, and highly sensitive mass-spectrometry to measure plasma was associated with having had experienced a signifi cantly higher rate of nitrotyrosine to explore relationship between CAN and oxidative stress in sustained 3 step progression of retinopathy in DCCT (odds ratio (OR) / unit 25 subjects with MS and 25 healthy controls (mean age 46±11 vs. 43±3 years increase: 2.5, p=0.0027), sustained ≥ 3 microaneurysms (MA) in those with no respectively). Adherence to the intervention was monitored weekly with the POSTERS baseline retinopathy (OR: 4.8, p<0.0001), AER>40 mg/24 hr (OR: 5.3, P<.0001), Mediterranean Diet Score, weekly exercise logs and VO2. Complications and confi rmed clinical neuropathy (CCN) (OR: 3.4, p=0.0150). After adjustment At baseline MS subjects presented with changes in HRV suggesting Acute and Chronic for mean DCCT A1C, GSPNE remained signifi cant for >3 MA (p=0.0252) and sympathetic stimulation, abnormal LV [11C]HED retention (0.044±0.007 AER (p=0.0006). All complications were strongly associated with A1c, but this vs.0.075± 0.01 ml/min/ml, p<0.0001) and elevated plasma nitrotyrosine association was either completely lost (AER) or reduced (> 3 step retinopathy (42.5±10.1 vs. 5.07±1.3 mmol/mol tyrosine, p< 0.001) compared to healthy progression and > 3 MA) when adjusted for GSPNE. Conclusions: The fact controls. Plasma nitrotyrosine decreased signifi cantly (27 ± 11 mmol/mol that Glucosepane is robustly associated with nephropathic and retinopathic tyrosine, p< 0.05 vs. baseline) and LV [11C]HED retention defi cits normalized outcomes in spite of adjustment for A1c, and conversely the association of in 17 MS subjects who completed the 24-week lifestyle intervention. these outcomes with A1c was nullifi ed or weakened by adjustment for GSPNE, These data support that MS subjects present with increased oxidative stress suggests that GSPNE is a likely mediator in the pathogenetic pathway from and defi cits of HRV and LV [11C]HED suggestive of CAN. Preliminary data obtained hyperglycemia to microvascular complications. Moreover, GSPNE levels are in subjects with MS who adhered and completed the lifestyle intervention ameliorable by INT treatment of T1D. demonstrate reduction in these defi cits, suggesting the salutary effects of the Supported by: JDRF intervention in this high-risk population. ADA-Funded Research

& 631-P COMPLICATIONS—NEUROPATHY Is Vitamin D Defi ciency a Risk Factor for Diabetic Peripheral Neuro- pathy? [See also: Presidents Posters 388-PP to 389-PP, page A107.] NABILA ABDELLA, DIA SHIHAB, KHALID AL-JARALLAH, HISHAM AL MOHAMMEDI, OLUSEGUN MOJIMINIYI, Kuwait, Kuwait Recent reports suggest high prevalence of vitamin D defi ciency in various Guided Audio Tour: New Ideas on the Diagnosis and Treatment of Diabetic populations. Despite reports linking Vitamin D defi ciency with increased Nerve Disease (Posters 629-P to 636-P), see page 11. risk of diabetes mellitus (Types 1 and 2) and the complications, there is limited data on patients with diabetic peripheral neuropathy (DPN). The & 629-P main aim of this study was to evaluate the prevalence and associations Early Detection of Nerve Damage by Skin Biopsy Compared with of Vitamin D defi ciency in 210 (67 Males, 143 Females) patients with Type Quantitative Small and Large Fiber Function Tests in Recently 2 diabetes with and without DPN. We measured fasting glucose, lipid Diagnosed Type 2 Diabetic Patients profi le and assessed diabetic control with HbA1c. Vitamin D status was DAN ZIEGLER, IRIS ZIEGLER, MARIA JEZIORSKA, RAYAZ A. MALIK, KLAUS- determined by measuring 25-dihydroxyvitamin D (25-OH D) levels. Presence WERNER SCHULTE, CLAUDIA SOMMER, MICHAEL RODEN, GDC STUDY GROUP, or absence of coronary heart disease (CHD) was determined and early- Düsseldorf, Germany, Manchester, United Kingdom, Würzburg, Germany morning urine microalbumin:creatinine ratio was measured to establish It is unclear whether small and large nerve fi ber dysfunction and pathology the degree of microalbuminuria. All patients were assessed clinically using develop sequentially or in parallel in type 2 diabetic subjects. We assessed neuropathy symptom score (NSS), neuropathy disability score (NDS) and by peripheral nerve morphology and function in 51 recently diagnosed type neurophysiology using nerve conduction study (NCS). 87 patients had DPN 2 diabetic patients (age: 55.1±11.1 (mean±SD) years, diabetes duration: and these patients had signifi cantly longer duration of diabetes and HbA1c 18.2±14.1 months, BMI: 31.4±5.8 kg/m², systolic/diastolic blood pressure: but age, gender, incidence of retinopathy and CHD were not signifi cantly 131±16.7/73.6±8.5 mmHg, triglycerides: 188±120 mg/dl, cholesterol: LDL: different from those without DPN. Vitamin D was signifi cantly lower in those 125±41.9 mg/dl, HDL: 46.6±11.7 mg/dl, HbA1c: 6.6±1.4%) and 43 healthy with DPN (36.9 (39.9) nmol/L) compared to those without (58.32 (58.9) nmol/L), subjects of similar age by intraepidermal nerve fi ber density (IENFD) in 3-mm p= 0.001. 81.5% of patients with DPN had vitamin D defi ciency as compared punch biopsies from the distal leg using immunohistochemistry with PGP to 60.4% of those without. Vitamin D showed signifi cant correlations 9.5 antibody, motor and sensory nerve conduction velocity (MNCV, SNCV), with Total cholesterol (p = 0.008), LDL-Cholesterol (p = 0.04) and urine sensory nerve action potential (SNAP) amplitudes, and quantitative sensory microalbumin:creatinine ratio (p=0.009). Binary logistic regression analysis testing (QST) using vibration perception thresholds (VPT) and thermal showed that DPN was signifi cantly associated with Vitamin D defi ciency perception thresholds (TPT). Age- or height-dependent limits of normal (Odds ratio = 3.47; p = 0.043) after inclusion of potential confounders such th th were defi ned at the 95 or 5 percentiles, respectively. In the lower limbs, as duration of diabetes, HbA1C and urine microalbumin:creatinine ratio. We peroneal MNCV and malleolar VPT were the most frequently abnormal tests conclude that low vitamin D is an independent risk factor for DPN. Further in 43.1% each, followed by IENFD in 39.2%, sural SNCV in 35.3%, sural SNAP studies are required to confi rm if Vitamin D supplementation could prevent in 32.0%, cold TPT in 27.5%, and warm TPT in 7.8% of the diabetic subjects. or delay the onset of DPN. Three or more abnormal tests out of 7 were observed in 45.1% of the patients, while 62.7% showed both at least 1 abnormal small and large nerve fi ber test. IENFD correlated signifi cantly with sural SNCV (r=0.289; p=0.04), but & 632-P not with the other tests studied. In conclusion, both small and large nerve GLP-1, Insulin, or Thiazolidinediones Afford Neuroprotection in fi bers are frequently affected in recently diagnosed, well controlled, type 2 Diabetic Encephalopathy through Different Anti-Apoptotic Pathways diabetic patients, suggesting an early parallel involvement of the various MASAHIRO OKOCHI, NAOTSUKA OKAYAMA, RYOSUKE KIMURA, GOJI SHIGEKI, fi ber types. Skin biopsy detects small fi ber neuropathy independent of QST YASUHIRO TAMURA, KAORU ASADA, KOHEI HATTORI, AKITOMO GOTO, YASU- and more frequently than thermal perception thresholds. TAKA KAMIYA, TSUNEO OHNO, TAKASHI JOH, Inazawa, Japan, Nagoya, Japan Patients with long-standing diabetes commonly develop cognitive & 630-P impairment, defi ned as diabetic encephalopathy, which is involved in oxidative Cardiovascular Autonomic Neuropathy, Oxidative Stress and Life- stress-induced neuronal apoptosis as well as the impairment of brain insulin style Intervention in Subjects with Metabolic Syndrome action. However, the treatment strategy has not been elucidated. GLP-1, RODICA POP-BUSUI, DAVID RAFFEL, MELVYN RUBENFIRE, ANURADHA GIRI, insulin or thiazolidinediones have been shown to exert neuroprotective JAEMAN BYUN, KATHLEEN RYAN, MORTON BROWN, SUBRAMANIAM PENN- actions in Alzheimer’s disease. GLP-1 receptors are widely expressed in ATHUR, Ann Arbor, MI neurons throughout the brain, and GLP-1 readily crosses the blood-brain Cardiovascular autonomic neuropathy (CAN) strongly associates with barrier. Additonally, intranasal insulin administration has been reported to increased cardiovascular disease (CVD) mortality. Presence of metabolic prevent cognitive impairment in patients with Alzheimer’s disease. Hence, syndrome (MS) also associates with increased CVD risk. the present study was conducted to determine the effects of GLP-1, insulin, We explored the relationship between components of the MS, CAN and thiazolidinediones on oxidative stress-induced neuronal apoptosis in and oxidative stress, and the effects of an intensive lifestyle intervention diabetic condition. Human brain endothelial cells and rat pheochromocytoma

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A173 COMPLICATIONS—NEUROPATHY

cells, both of which are used commonly as neuronal cell models, were PGP9.5 and NADPH diaphorase staining disclosed signifi cant reduction of exposed to 25mM glucose and/or 1mM MG with/without with 0.1nM GLP-1, myenteric neuronal area, intramural axonal fi bers and the number of nNOS 100 nM insulin, thiazolidinediones (10 µM troglitazone, 10 µM pioglitazone), positive myenteric neuronal cells in diabetic rats. Similarly, expressions or 30 µM GW9662 (a PPARγ antagonist). Oxidative stress triggered PTP of both PKB/AKT and pAKT in the intestinal tissues of diabetic rats were opening, cytochrome c release, BcL-2 downregulation, caspase-9 and -3 suppressed. There were also reduced mRNA expressions of fi broblast activation, and cell apoptosis. GLP-1 or insulin protected against oxidative growth factor (FGF), vascular endothelial growth factor (VEGF), insulin-like stress-induced apoptosis through PI3K/Akt/mTOR/Nrf2/GCL/redox signaling growth factor (IGF)-I, nNOS and CHAT in the intestine of diabetic rats. ASC- pathway. Inhibitors of PI3K, Akt, and mTOR abrogated the cytoprotective Tx improved all these abnormalities. There was no signifi cant infl uence of POSTERS

Complications effect of GLP-1 or insulin. GLP-1 also induced EGFR phosphorylation, and Tx on the mRNA expressions of these factors, pAKT and AKT expression

Acute and Chronic inhibitors of EGFR, EGFR ligands abrogated the GLP-1 cytoprotective effect, and neuropathology of intestinal wall in normal control rats. BrdU-positive indicating the different neuroprotective pathway of GLP-1. On the other hand, transplanted cells were sparsely located in transplanted site of animals. The thiazolidinediones protected against oxidative stress-induced apoptosis in results demonstrated that topical application of autologous ASC into the gut association with the upregulation of Bcl-2 protein, consequently led to the wall improved diabetic gastroenteropathy in rats. Future clinical application inhibition of caspase-9 and -3 activation, those of which were inhibited by of ASC-Tx may be warranted for hitherto untreatable condition of diabetic GW9662, indicating the involvement of PPARγ. In conclusion, GLP-1, insulin gastroenteropathy. or thiazolidinediones afford neuroprotection through enhancing different anti-apoptotic signaling pathway, and represent a promising treatment & 635-P modality for diabetic encephalopathy. Autonomic Function Testing and Cardiac MIBG in Type 1 Diabetes Mellitus & 633-P TRIANTAFILLOS DIDANGELOS, EFSTRATIOS MORALIDIS, FOTIOS ILIADIS, ALEX- Diabetes Reduces the Sterol Sensor SCAP in the Brain Altering ANDRA NTEMKA, ANNA GOTZAMANI-PSARRAKOU, APOSTOLOS HATZITOLIOS, Brain Cholesterol Metabolism and Brain Function Thessaloniki, Greece RYO SUZUKI, HEATHER FERRIS, C. RONALD KAHN, Boston, MA This study compares autonomic function tests (AFT) with cardiac Diabetes mellitus is associated with a variety of cerebral complications, sympathetic innervation imaging with 123I metaiodobenzylguanidine (MIBG) including cognitive dysfunction, depression, and increased risk of Alzheimer’s in patients with type 1 diabetes mellitus (T1DM). disease. The brain is the most cholesterol-rich organ in the body, most of Forty-nine patients (29 male, 20 female), aged 36±10 years (range 19-62), which comes from in situ synthesis. Recently we showed that in multiple with a duration of T1DM 19±6 years (range 7-31), without known diabetic mouse models of diabetes, there is a global down-regulation of cholesterol complications and receiving only insulin were enrolled prospectively. synthetic genes and their major transcriptional regulator SREBP-2 in the brain, Participants were evaluated clinically for autonomic dysfunction with the leading to a broad reduction of cholesterol synthesis. We now show in the mean circular resultant (MCR), Valsalva maneuver (Vals), postural index brain of streptozotocin (STZ)-induced diabetic mice there is a 50% decrease (PI) and orthostatic hypotension (OH) assessment. Tests were interpreted in levels of the sterol sensor protein SCAP, which serves as a binding partner as normal-abnormal according to age and the number of abnormal tests in regulation of SREBP processing. This is present in both the cerebral was also considered. Within one month patients underwent cardiac MIBG cortex and hypothalamus. To defi ne the effect of SCAP defi ciency in brain, imaging and the ratio of the heart to upper mediastinum count density (H/M) we generated brain-specifi c SCAP knockout mice by crossing nestin-Cre at 4 hours post-injection was calculated (abnormal if H/M<1.80). transgenic mice with Scap fl oxed mice. Homozygous disruption of Scap gene There were 28 cases with at least 1 abnormal AFT and 37 with abnormal in the brain caused perinatal lethality with microcephaly. To better mimic MIBG studies. the effect of the 50% reduction of SCAP observed in diabetes, we created Among patients with normal MCR, Vals, PI and OH tests 70%, 71%, 73% mice with haploinsuffi ciency of the Scap gene in the brain. These mice were and 68%, respectively, had abnormal MIBG studies. Among patients with an viable and had a ∼30% reduction in brain cholesterol synthesis measured abnormal MCR, 21% had a normal H/M, but all other abnormal AFT values 3 using H2O, similar to that observed in STZ-diabetic mice. The brain-Scap were invariably associated with H/M abnormality. heterozygotes also exhibited growth-retardation with a 63% reduction in In patients with only one abnormal AFT, 21% had a normal MIBG study, circulating IGF-I levels (P<0.01). Food intake was mildly increased correlating whereas among those with no clinical evidence of autonomic neuropathy with upregulation of Agrp in the hypothalamus. Stress-related hormones, 71% had an abnormal H/M. All patients with no less than 2 abnormal AFT including corticosterone and norepinephrine, were normal at rest, however, also had an abnormal MIBG study. a mild stress (isolation in an unfamiliar paper box) caused more than 2-fold A marginally signifi cant correlation (r=-0.281, p=0.050) was found between increases in the hormone levels in the heterozygotes compared to control the number of abnormal AFT and the H/M value. (P<0.05). Neurological function testing, including novel object recognition These results reinforce the concept of cardiac MIBG as a more sensitive test and open fi eld test, also revealed impairment in cognitive and behavioral technique for the assessment of autonomic neuropathy in T1DM patients function. Thus, reduction of SCAP and consequent suppression of cholesterol in comparison to clinical evaluation. It is shown also that the criterion of synthesis in the brain may play an important role in the cerebral dysfunction “at least 2 abnormal autonomic function tests” correctly identifi es T1DM observed in diabetic states. patients with cardiac sympathetic system impairment in MIBG studies.

& 634-P & 636-P Amelioration of Experimental Diabetic Gastroenteropathy in Dia- Accelerated Intraepidermal Nerve Fiber Density (IENFD) Decline betic Rats Autologously Transplanted with Adipose-Tissue-Derived Precedes Progression to Neuropathic Symptoms in Patients with Mesenchymal Stem Cells Diabetes HIROKI MIZUKAMI, KAZUNORI TAKAHASHI, KAZUHIRO SUGIMOTO, MASAYUKI J. ROBINSON SINGLETON, CHARLES LATNER, COLLIN J. ARSENAULT, MICHAEL BABA, SOROKU YAGIHASHI, Hirosaki, Japan, Aomori, Japan T. PORZIO, A. GORDON SMITH, Salt Lake City, UT Gastroenteropathy is a serious complication of diabetes leading to Once established, diabetic neuropathy has proven resistant to medical diffi culty of blood glucose control and poor prognosis. There is no effective therapy. Presymptomatic detection would permit targeted early preventive treatment for this serious disorder. Stem cell therapy is now a promising treatment that might prove more effective. The Cutaneous Measures of approach for reparative therapy. We applied adipose-tissue derived mesen- Neuropathy in Diabetes (CMND) natural history cohort study compares chymal stem cells (ASC) to diabetic gastroenteropathy in diabetic rats. peripheral nerve measures for their ability to predict progression to Streptozotocin-induced diabetic rats with 16 wk-duration were transplanted symptomatic diabetic neuropathy in a cohort of 210 type II diabetes subjects. with autologous ASC (x106) labeled with BrdU, retrieved from subcutaneous Neuropathy designation requires symptoms or exam signs plus presence of fat prior to diabetes onset, into the serosa of terminal ileum. After 4 wk two confi rmatory abnormalities; NCS quantitative sensory testing, QSART observation period, intestinal passage, mRNA expressions of growth factors or IENFD. Annually, a blinded Investigator interviews each CMND subject and neuronal nitric oxide synthase (nNOS), choline acetyltransferase (CHAT), to determine if symmetrical, length dependent numbness or paresthesias the number of nNOS-positive neuronal cells and neuropathology of intestinal are present. T-test statistics compare measures of peripheral nerve function walls were examined. Intestinal passage estimated 30 minutes after and metabolic indices in the cohort progressing to neuropathy symptoms gavage of 10% methylen blue was accelerated in diabetic rats (38.9±4.1 with indices for subjects without neuropathy, or with baseline neuropathy. vs. 72.8±8.4cm, p<0.01) and ASC transplantation (Tx) improved the passage Change in nerve measures over the fi rst year was also compared between (52.9±5.1cm, p<0.05 vs. diabetes). Neuronal distribution as revealed by groups.

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A174 COMPLICATIONS—NEUROPATHY

140 CMND subjects have been followed for three years. Of 84 subjects patients receiving 25mg, 1–50mg and 2–75mg) and placebo intravenously without baseline neuropathy, 17 (20%) developed neuropathy symptoms for 3 weeks. The treatment with the same drug orally in the previously over the follow-up period. Mean baseline values for most nerve function established doses was followed in ambulatory setting for 3 months. Pain tests were signifi cantly worse for progressors than for the non-neuropathy relief (SFMPQ-VAS), quality of life improvement (EuroQol EQ-5D) and cohort, and similar to those for the neuropathy cohort. In the fi rst year mean occurrence of adverse events were assessed weekly during hospitalization IENFD at proximal and distal thigh and ankle decreased signifi cantly, and and monthly during the period of ambulatory follow-up. cold detection threshold (CDT) percentile increased for progressors, but not A ³50% reduction in VAS was noted in 7 (44%) patients on α-lipoic acid for other cohorts. One year NCS, QSART, and vibration QST measures did and 6 (38%) patients on amitriptyline (P=0.19). A comparable reduction in POSTERS not progress signifi cantly for any group. Incident neuropathy subjects had pain intensity was obtained with both drugs from the fi rst week on (ΔVAS Complications signifi cantly higher mean baseline triglycerides and lower HDL than both -13±13mm vs. -13±14mm, P=0.94, respectively). The improvement in quality of Acute and Chronic the non-neuropathy (p<0.005) and neuropathy cohorts (p<0.05). Baseline life was signifi cantly higher in patients treated with amitriptyline after 1 and age, BMI and HgbA1c did not differ signifi cantly between groups. IENFD 3 weeks of the observation when compared to ALA (respectively: ΔEQ1week and CDT are dynamic small fi ber measures that may allow presymptomatic 9±13 vs. -1±10, ΔEQ3week 28±21 vs. 12±20, P<0.05). The adverse events were diabetic patients at high neuropathy risk to be recognized as targets for early reported only with amitriptyline, dry mouth being the most common. preventive therapy. In conclusion, amitriptyline and α-lipoic acid are similarly effective in Supported by: NIH RO1 DK064814, NIH NCRR UL1RR025764 the treatment of painful diabetic polyneuropathy. Amitriptyline has greater benefi cial impact on the quality of life, but is associated with a higher risk 637-P of adverse effects. A Rapid, Low-Cost, Point-of-Care Test for Diabetic Peripheral Neuropathy 639-P BONNIEJEAN BOETTCHER, BRIAN TRACEY, XUAN KONG, MIKE WILLIAMS, SHAI Antinociceptive Effect of Bumetanide in a Rat Model of Diabetic N. GOZANI, Waltham, MA Neuropathy Annual screening for diabetic peripheral neuropathy (DPN) is recommended FILIPE FERNANDES-CONTI, AMANDA ARAUJO, FRANCISCO J. ALVAREZ- for early detection of this microvascular complication and to track its LEEFMANS, MAURICIO DI FULVIO, São Paulo, Brazil, Dayton, OH progression. The most widely utilized screening method is the 5.07/10-g Neuropathy is the most common complication and the major source of monofi lament. Nerve conduction studies (NCS) are the most accurate and morbidity associated with diabetes. It affects ∼50% of diabetic patients reproducible test for DPN. However, they are not used for screening due to with long-standing disease. Pain is present in ∼35% of all diabetic their expense, limited availability, and complexity. In response, we developed patients. Clinically, painful diabetic neuropathy (PDN) is characterized a fast, low-cost ($10-15), point-of-care sural nerve conduction velocity (CV) by hyperalgesia and allodynia. Currently, there are no treatment options test. As a measure of nerve conduction, we hypothesized that the test would to relieve the pain completely, making PDN difficult to manage. The have a strong association with HbA1c. targets of PDN are the primary sensory neurons (PSN), the cells that Fifteen consecutive subjects (8 female) with diabetes mellitus (DM; 3 Type convey somatosensory information to the spinal cord and brainstem. I, 12 Type II) who responded to study recruitment notices were evaluated. GABA depolarizes the central terminals of PSNs via activation of Cl– Ages ranged from 33 to 76 yrs and disease durations from 1 to 24 yrs. Sural channels coupled to GABAA receptors. GABA-mediated depolarization of CVs were measured bilaterally. In addition to the most recent HbA1c (range PSNs determines presynaptic inhibition, a pivotal mechanism in gating 4.7-10.3), the following variables were modeled: age, height, diabetes type the flow of nociceptive information into the spinal cord. Various types and duration. The relationship between HbA1c and CV was evaluated by of peripheral injury appear to increase depolarization of PSNs to a level multivariate regression with the fi nal model retaining variables at p<0.1. sufficient to initiate action potentials in their intraspinal terminals. These Complete tests were performed in 29 sural nerves; excessive signal action potentials, known as dorsal root reflexes (DRRs), play a key role in variability prevented evaluation of 1 nerve. The average data collection the development of hyperalgesia and allodynia. Depolarization and DRRs time was 10±5 (range 2-16) seconds per nerve. 25 nerves had a detectable of PSNs are made possible by the Na+, K+, 2Cl– cotransporter NKCC1 that – response (>1.5 μV) from which a CV could be determined (range 23.3-48.8 maintains [Cl ]i above electrochemical equilibrium in these cells. The meter/sec). The remaining 4 nerves were undetectable and no CV was resulting outward Cl– gradient and its modulation are thought to play a reported. The regression analysis demonstrated a statistically signifi cant fundamental role in the control of information flow in nociceptive primary (p=0.001) and clinically meaningful 2.1 meter/sec decrease in CV for every afferents. We investigated the effect of bumetanide, an inhibitor of 1% increase in HbA1c. Age, height, and diabetes duration were also NKCC1 on the thermal nociceptive threshold (TNT) of diabetic and normal associated with CV. rats. TNT of diabetic rats was significantly lower (p<0.001) than that In previous studies performed using traditional NCS techniques, sural CV in normal rats (45.6±0.1°C and 47.4±0.1°C, respectively). Bumetanide decreased by about 1-3 meter/sec for every 1% increase in HbA1c. In the administration (i.p.) at non-diuretic doses increased TNT of hyperalgesic present study, point-of-care sural CV exhibited a similar association to HbA1c. diabetic rats to that of normal animals. The results show that bumetanide However, unlike time consuming traditional techniques, this test required has antinociceptive effects in rats with PDN. This suggests that NKCC1 only 30-60 seconds per nerve including preparation. Rapid, low-cost, point- in PSNs may be a potential pharmacological target for the treatment of of-care sural CV testing has the potential to improve DPN screening. PDN. ADA-Funded Research

638-P 640-P Amitriptyline Versus α-Lipoic Acid in the Treatment of Painful Assessing Severity of Peripheral Neuropathy through Quick Measure- Diabetic Polyneuropathy ment of Sudomotor Function ANNA PSUREK, MARTA WRÓBEL, ALEKSANDRA SZYMBORSKA-KAJANEK, JEAN-HENRI CALVET, HENRI GIN, ROSELYNE BAUDOIN, CHRISTELLE RAFFAITIN, GRZEG ORZ WYSTRYCHOWSKI, DOMINIKA ROKICKA, WLADYSLAW GRZESZ- VINCENT RIGALLEAU, CONCEPTION GONZALEZ, Paris, France, Bordeaux, France CZAK, KRZYSZTOF STROJEK, Zabrze, Poland Sweat dysfunction leading to abnormal skin conditions including dryness, Painful diabetic polyneuropathy is a serious health problem. There is a fi ssures and blisters could increase diabetic foot risk. A consensus statement need of a medication that provides meaningful pain relief, improves quality of American Diabetes Association has suggested that sudomotor function of life and is well tolerated. Nowadays amitriptyline is a gold standard should be included in diagnostic test for the detection of neuropathies in in treatment of polyneuropathy. The results of recent studies suggest diabetes but lack of trained personal have restricted the widespread of study effectiveness of α-lipoic acid (ALA) in pain reduction in patients suffering of sudomotor function. The aim of this study was to evaluate SUDOSCAN™ from diabetic polyneuropathy. The aim of this study was to compare the a new quick, non-invasive, quantitative method to measure sudomotor effi cacy and safety of ALA and amitriptyline in alleviating pain associated dysfunction as a screening tool of diabetic peripheral neuropathy. with diabetic polyneuropathy. This is the fi rst head-to-head study comparing 142 diabetic patients (age 62 ± 18 years, diabetes duration 13 ± 14 years, both agents. HbA1c 8.9 ± 2.5%) had monofi lament test, were measured for vibration In this randomized, single-blind, clinical trial 32 patients with diabetic perception threshold (VPT) using a biothesometer and had retinopathy polyneuropathy who had a pain score of at least 40mm in a 0-100mm VAS status as indicator of microvascular lesions. Sudomotor dysfunction was scale, were assigned to sequential treatment with 600mg α-lipoic acid once assessed by measuring electrochemical sweat conductance (ESC, µS). daily intravenously and placebo orally for 3 weeks or to treatment with Patients place their hands and feet (area with highest sweat gland density) amitriptyline orally, at minimal effective doses in the 25-75 mg range (13 on large electrodes where a low voltage is applied. ESC results from

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A175 COMPLICATIONS—NEUROPATHY

chloride movements and electrochemical reaction in response to electric cholesterolemia. However, no data exist regarding the effect of treatment stimulation. with statins on BRS in patients with T2DM. In the present prospective study As diabetic peripheral nerve function is disturbed in nerve length- we examined the effect of treatment with atorvastatin for 1 year on BRS in dependant manner, patients were classifi ed in three groups according to the subjects with T2DM. results of their feet ESC: <40µS high sweat dysfunction, 40-60µS moderate Thirty-six patients with T2DM and LDL cholesterol levels >100 mg/dl were sweat dysfunction, > 60 µS no sweat dysfunction. Data for biothesometer, treated with 10 mg atorvastatin daily and a low-fat diet for 1 year. monofi lament and retinopathy status are presented in the table below. According to their BMI, there were 16 were obese (33.79±3.47 kg/m2) and Results are expressed as median (iqr), comparisons were done using 20 normal weight (26.87± 3.03 kg/m2) subjects. Participants in the obese and POSTERS

Complications Kruskal-Wallis test: normal weight groups were comparable in terms of gender, age, diabetes

Acute and Chronic duration, LDL cholesterol and HbA1c. BRS was assessed by the spontaneous Feet ESC < 40µS Feet ESC 40-60 µS Feet ESC > 60 µS p ® (n=33) (n=43) (n=66) method, using the BaroCor system. Serum lipids, HbA1c and BRS were evaluated at baseline and 3, 6 and 12 months after intervention. Age (yrs) 63 (11) 65 (14) 58 (25) 0.02 Total cholesterol and LDL cholesterol concentrations were reduced BMI (kg/m2) 31 (8) 32 (9) 27 (11) 0.0005 signifi cantly at the examined time intervals in comparison with baseline after

HbA1C (%) 8.2 (2) 9.7 (2) 8.9 (2) NS treatment with atorvastatin (p<0.001). No signifi cant change was observed Biothesometer (V) 28 (14) 25 (25) 11 (17) <0.0001* in BMI, blood pressure and HbA1c. When all patients were evaluated as one group, BRS showed a signifi cant increase (20.25% versus baseline, p=0.005) No monofi lament perception (%) 70 69 29 <0.0001* after 12 months of therapy. When analysis was repeated according to Retinopathy (%) 61 42 14 <0.0001* obesity status, in the normal weight participants BRS was increased as early * remained signifi cant after adjustment for age and BMI. as 3 months after intervention (by 14.58%, p=0.03) and further improvement was demonstrated at 6 months (by 38.59%, p=0.04) which maintained at 12 ™ Foot sweating status using SUDOSCAN a simple, quick and quantitative months (by 38.57%, p=0.014). On the contrary, no signifi cant change in BRS method may be used as a screening tool of diabetic peripheral neuropathy was observed in the obese participants. for early prevention of foot skin lesion and foot ulceration. Long-term treatment with atorvastatin therapy improves BRS in normal weight but not in obese subjects with T2DM. 641-P Autonomic Nervous System and Renal Function Assessment in Type 643-P 1 Diabetes Mellitus Does the Combination of Cardiovascular Autonomic Neuropathy and TRIANTAFILLOS DIDANGELOS, EFSTRATIOS MORALIDIS, FOTIOS ILIADIS, Corrected QT Interval Improve the Prediction of Total and Coronary ALEXANDRA NTEMKA, CHARALAMPOS MARGARITIDIS, ANNA GOTZAMANI- Artery Disease Mortality in Type 1 Diabetes? PSARRAKOU, DIMITRIOS GREKAS, Thessaloniki, Greece GEORGIA PAMBIANCO, TINA COSTACOU, TREVOR J. ORCHARD, Pittsburgh, PA This study investigates if there is an association between glomerular It has recently been reported that the combination of reduced heart rate fi ltration rate (GFR) measurement, as index of renal function, and autonomic variation and QT corrected interval strongly predicts mortality in type 1 function assessment in patients with type 1 diabetes mellitus (T1DM). diabetes (T1D). To confi rm this fi nding, total and coronary artery disease (CAD) Forty-six patients (28 male, 18 female), aged 36±10 years (range 19-62), mortality were examined using 20 year follow up data from the Pittsburgh with a duration of T1DM 19±6 years (range 7-31), without known diabetic Epidemiology of Diabetes Complications (EDC) study of childhood onset complications and receiving only insulin were enrolled prospectively. T1D (mean baseline age 28 and diabetes duration 19 years). Cardiovascular Participants were evaluated clinically for autonomic dysfunction with the autonomic neuropathy (CAN) was determined by the heart rate response to mean circular resultant (MCR), the Valsalva maneuver (Vals), postural index deep breathing (E:I ratio) and examined both continuously and dichotomized (PI) and orthostatic hypotension (OH) assessment. The number of abnormal as >=1.1 (normal) and <1.1. QT interval was corrected using Bazett’s formula clinical tests was also considered. Within one month patients underwent (QTc=QT/sqrt RR) and dichotomized at the standard cutpoint of <=416 msec 51 glomerular fi ltration rate measurement with Cr-EDTA (expressed in ml/ (normal) and >416 msec (abnormal). CAN and QTc were subsequently combined 2 123 min/1.73m ) and cardiac I metaiodobenzylguanidine (MIBG) imaging [with using these dichotomized variables (CAN+QTc). Mortality was classifi ed by the ratio of the heart to upper mediastinum count density (H/M) at 4 hours death certifi cate and/or hospital/autopsy/next of kin interview records when post-injection calculated]. appropriate. Analyses allowed for diabetes duration, HbA1c, albumin excretion The values of examined variables were as follows [mean±1SD (range)]: GFR rate (AER, 2 of 3 timed urines), HDL and nonHDL cholesterol, and hypertension. 98±17 (61-137), MCR 40±29 (5-108), Vals 1.55±0.30 (1.11-2.29), PI 1.33±0.17 Results from Cox proportional hazard models are presented univariately (1.03-1.81), OH 5±9 (0-30) and H/M 1.65±0.20 (1.30-2.34). (model 1) and multivariably with forward selection (model 2). CAN, QT, and GFR correlated signifi cantly with age (r=-0.377, p=0.011), the duration of CAN+QT were forced into the multivariable models. diabetes (r=-0.514, p=0.000), Vals (r=0.377, p=0.010), OH (r=-0.448, p=0.002) and the number of abnormal clinical tests (r=-0.366, p=0.012). MCR, PI and Total Mortality CAD Mortality the H/M showed no signifi cant correlation with GFR. There was also no Model 1 Model 2 Model 1 Model 2 signifi cant difference in GFR between male and female patients. (n=97 cases/507) (n=92 cases/492) (n=47 cases/507) (n=46 cases/492) In multiple backward regression analysis (in which variables were entered HR(CI) HR(CI) HR(CI) HR(CI) if they were signifi cant in univariate analysis) only the duration of the disease CAN(dichotomized) 5.51(3.6-8.2) 1.57(.96-2.6) 5.57(3.1-9.9) 1.21(.61-2.4) was found to be an independent predictor of GFR. In T1DM patients GFR is associated with clinically evaluated autonomic QT(dichotomized) 1.58(1.1-2.3) 1.21(.79-1.8) 1.67(.94-2.9) 1.21 (.67-2.2) function (which predominantly address the parasympathetic system) but not CAN+QT(dichotomized) 5.02(3.3-7.6) 1.66(1.03-2.7) 4.77(2.6-8.7) 1.25 (.64-2.5) with cardiac MIBG measurements (which refl ect sympathetic dysfunction). Multivariably, CAN in combination with QTc interval only modestly However, once the duration of T1DM is taken into account it is likely that this improves the prediction of total mortality compared to either component association is no more signifi cant. alone and neither predicts CAD specifi c mortality. These results further support earlier observation that the CAN and QT association with mortality 642-P largely refl ect confounding, particularly by renal disease. Different Effect of Atorvastatin on Barorefl ex Sensitivity in Obese and Normal Weight Patients with Type 2 Diabetes 644-P PINELOPI GRIGOROPOULOU, IOANNA ELEFTHERIADOU, CHRISTOS ZOUPAS, Enhanced Rho/ Rho - Kinase Pathway in Diabetic Rat s A f fec t s Schwann ALEXANDROS KOKKINOS, DESPOINA PERREA, IOANNA BALLA, VASILIKI ARGI- Cell Adherens Junctions and Induces Peripheral Nerve Defi cits ANNA, NICHOLAS KATSILAMBROS, NICHOLAS TENTOLOURIS, Athens, Greece YASUSHI KANAZAWA, JUNKO TAKAHASHI-FUJIGASAKI, SHO ISHIZAWA, Barorefl ex sensitivity (BRS) is an index of arterial barorefl ex function NAOKO TAKABAYASHI, KUMIKO ISHIBASHI, KEIICHIRO MATOBA, DAIJI KAWA- and an index of autonomic nervous system activity. Decreased BRS is NAMI, TAMOTSU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan associated with electrical instability of the myocardium, impaired blood Rho-kinase is a serine-threonine kinase, a downstream effector of a pressure regulation and increased risk for cardiovascular events. Impaired small GTPase protein Rho. Rho/Rho-kinase pathway participates in various BRS has been described in obesity and in patients with type 2 diabetes biological events, such as cell adhesion. We previously reported that Rho mellitus (T2DM). Statins improve BRS in non-diabetic subjects with hyper- and Rho-kinase activity was enhanced in sciatic nerves of the diabetic rats,

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A176 COMPLICATIONS—NEUROPATHY and a selective Rho-kinase inhibitor, fasudil, improved both motor nerve 646-P conduction velocity and sensory defi cit in diabetic rats, yet the underlining High Prevalence of Sleep Apnea in Type 1 Diabetes Mellitus: The mechanisms remain unknown. We hypothesized that the upregulation Role of Cardiovascular Autonomic Neuropathy of Rho/Rho-kinase pathway might alter expression and distribution of CAROLINA CASTRO PORTO-SILVA, LUIZ CLEMENTE ROLIM, DALVA POYARES, E-cadherin and β-catenin in sciatic nerves of diabetic rats. E-cadherin JOAO ROBERTO SA, SERGIO TUFIK, ADEMIR BAPTISTA SILVA, SERGIO ATALA is a Rho/Rho-kinase-related adhesion molecule, distributed at adherens DIB, São Paulo, Brazil junctions (AJs) in Schwann cells to maintain proper structure and function of Recent studies have correlated cardiovascular autonomic neuropathy peripheral nerves. E-cadherin function at the AJs requires association with

(CAN) to sleep-disordered breathing (SDB) in diabetic patients. We POSTERS cytoplasmic partners called catenins. evaluated the sleep pattern of 20 type 1 diabetes mellitus (T1DM) with and Complications Sprague–Dawley rats were rendered diabetic with streptozotocin, and without CAN and 22 healthy individuals, matched by age, sex and BMI. All Acute and Chronic divided in the 3 groups, normal control rats, diabetic rats, and diabetic rats subjects were submitted to the Epworth Somnolence Score (ESS) and to a treated with a Rho-kinase inhibitor, fasudil. After 4 weeks, immunoblot polysomnography according to the American Sleep Disorders Association. and immunostainning for E-cadherin and β-catenin were performed, using CAN was assessed by the Ewing protocol. The statistical analysis was resected sciatic nerves. A slight decrease in expression of E-cadherin and made by Pearson chi-square for categorical variables and t-tests for β-catenin was observed in diabetic rats. Histologically, both proteins were continuous variables. A p-value <0.05 was considered signifi cant. After the distributed mainly at nodes of Ranvier and Schmidt-Lanterman incisures (S- cardiovascular test, the T1DM patients were divided in two groups: 9 that L), which contain AJs in myelin sheath formed by Schwann cells. To analyze had CAN(CAN+) and 11 that didn’t have (CAN-). The duration of T1DM was alteration in distribution of the proteins semi-quantitatively, the distribution of not statistically different between the groups as well as the mean A1c. ESS both proteins at S-L was scored on digital photographs by blinded examiners. showed signifi cant difference between T1DM patients and the control group, The distribution of E-cadherin in the diabetic rats signifi cantly became unclear, as well as within the diabetic groups. The results of the polysomnography which was restored by administration of fasudil. However, the distribution of showed that the sleep effi ciency was severely impaired in CAN+ patients. β-catenin did not alter signifi cantly. Our results indicated that upregulation Sixty-seven percent of CAN+ presented more than 5 obstructive apneas/ of Rho/Rho-kinase might dislocate E-cadherin in the Schwann cell AJs, and hypopneas per hour, in contrast to 25% of the CAN- and 4,5% of the control this could be one of the pathological mechanisms for the nerve defi cits in the group. diabetic rats.Other catenins, such as α, γ, and p120 catenins might be involved in the disturbance of Schwann cell AJs. ADA-Funded Research Table 1. Clinical and polysomnographic aspects of the groups Control(C) CAN (-) T1DM CAN (+) T1DM p 645-P Age (yrs) 23.2 ±3.9 27.0±8.2 32.7 ±6.8 C vs CAN- vs CAN+; NS GLP-1 Represents a Promising Treatment Modality for Glycemic Mean A1c (%) NA 7.5±1.7 9.0±2.3 C vs CAN- vs CAN +; NS Fluctuation-Induced Diabetic Encephalopathy Duration of disease (yrs) NA 16.7±7.8 14.8±5.3 NS MASAHIRO OKOCHI, NAOTSUKA OKAYAMA, RYOSUKE KIMURA, GOJI SHIGEKI, YASUHIRO TAMURA, KAORU ASADA, KOHEI HATTORI, AKITOMO GOTO, YASU- ESS>9 (% of patients) 18 45.4 55.5 C vs CAN- vs CAN+; p<0.05 TAKA KAMIYA, TSUNEO OHNO, TAKASHI JOH, Inazawa, Japan, Nagoya, Japan Sleep effi ciency (%) 85.1±9.4 88.4±5.5 80.2±10.7 CAN- vs CAN+; p=0.04 Diabetic encephalopathy, characterized by cognitive impairment in Microarousal index (events/h) 7.0± 5.0 7.7±4.1 12.4±5.1 CAN- vs CAN+; p=0.03 diabetic patients, involves oxidative stress-induced neuronal apoptosis, REM (%) 15.9 ±27.0 17.3±2.8 17.5±8.0 NS followed by hippocampal atrophy. Recent evidence indicates postprandial plasma glucose excursion is associated with cognitive impairment. GLP-1 SWS (%) 20.3±7.2 22.6±6.8 18.9±9.6 NS readily enters the brain, prevents neuronal cell apoptosis, and improves OSAHI (events/h) 2.1±2.3 2.9±1.7 6.3±4.6 CAN- vs CAN+; p=0.03 cognitive impairment in Alzheimer’s disease. Therefore, we investigated This pilot study shown that T1DM have more SDB than healthy age and the protective action of GLP-1 against glycemic fl uctuation-induced diabetic BMI-matched individuals and there is certainly an association between CAN encephalopathy in vitro and in vivo. We fi rst compared cognitive impairment and SBD in T1DM. Therefore T1DM, specially with CAN, should be screened and hippocampal atrophy on MMSE and MRI between 300 type 2 diabetic to SBD and its potential complications. patients and 300 nondiabetic control subjects, and determined the important factors affecting hippocampal atrophy. Second, neuronal cells were exposed for 1 week in media containing different glucose concentrations: 5mM, 25 647-P mM, or a daily alternating 5 or 25 mM glucose plus 500 µM MG with or Hyperglycemia Alters the Pro- and Anti-Infl ammatory Cytokine Levels without 0.1 nM GLP-1, and cell apoptosis, cellular redox, G6PD activity, in the Amygdala of Diabetic Rats apoptosis signaling pathway were examined. In vivo, glycemic fl uctuation CARLOS A. GARCIA, SRI VIDYA DEVI BOYANA, REJEANA M. STEPHENS, Kings- and poor glycemic control were associated with hippocampal atrophy. ville, TX In vitro, chronic hyperglycemia exacerbated MG-induced apoptosis that Diabetes affects the functioning of the central nervous system (CNS). corresponded to exaggerated redox imbalance, impaired G6PD activity, and Pathophysiological abnormalities including cognitive impairments are well enhanced expression of Apaf-1. Glycemic fl uctuation further enhanced MG- documented, particularly in those with poor glycemic control. The amygdala, induced apoptosis in association with greater redox imbalance and G6PD an important emotion regulating region, and the hypothalamus, which links impairment and elevated Apaf-1 expression. GLP-1 attenuated glycemic the CNS to the endocrine system, may be impacted by hyperglycemia. Early fl uctuation-induced neuronal cell apoptosis. GLP-1 induced phosphorylation events, prior to clinically signifi cant CNS changes, may include alterations of EGFR, Akt, mTOR, p70S6K, the upregulation of GCL, and nuclear in pro- and anti-infl ammatory cytokine levels. The purpose of this study translocation of Nrf2. Inhibitors of EGFR, EGFR ligands, a metalloproteinase, is to investigate the effects of hyperglycemia on the production of the PI3K, Akt and mTOR abrogated the GLP-1 cytoprotective effect. These results pro-infl ammatory cytokine Tumor Necrosis Factor–alpha (TNF-α) and the suggest that glycemic fl uctuation exacerbates diabetic encephalopathy than production of the anti-infl ammatory cytokine Interleukin 10 (IL-10) in the sustained chronic hyperglycemia through enhancing neuronal cell apoptosis amygdala and hypothalamus of streptozotocin (STZ)-induced diabetic Long via aggravated redox imbalance, G6PD impairment, and elevated Apaf-1 Evans rats. Age and sex-matched rats were separated into two groups (n=8), expression. GLP-1 treatment affords neuroprotection in glycemic fl uctuation- control and diabetic (STZ:60 mg/kg dissolved in 0.5ml 0.05M citrate buffer, induced diabetic encephalopathy through activating EGFR/PI3K/Akt/mTOR/ pH 4.2). One month after the onset of diabetes, both groups were sacrifi ced Nrf2/GCL/redox signaling pathway. and brains dissected. The amygdala and hypothalamic tissues were collected for biochemical analysis. The content of TNF-α and IL-10 was measured by enzyme-linked immunosorbent assays (ELISAs) in brain tissue homogenates. In the diabetic amygdala, the pro-infl ammatory TNF-α content increased from 1870.5 pmoles/mg protein to 2190.39 pmoles/mg protein while the anti-infl ammatory IL-10 content signifi cantly decreased (p<0.01) from 702.97 pmoles/mg protein to 115.68 pmoles/mg protein. The hypothalamus TNF-α content remained approximately the same in the two groups (control=7042.81 pmoles/mg protein, diabetic= 6988.13 pmoles/mg protein). This data provides evidence that hyperglycemia selectively increases the levels of TNF-α in the amygdala which is coupled with a signifi cant decrease in the levels of the anti-infl ammatory cytokine IL-10 while having no affect

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A177 COMPLICATIONS—NEUROPATHY

in the hypothalamus. The results suggest: i) a novel cytokine involvement of early infl ammation in the amygdala, ii) a TNF–mediated apoptotic pathway Repeat Test Kappa P in a longer period of hyperglycemia, and iii) an alteration of neuronal function RR (N=42) R<15 RR≥15 0.7083 <0.0001 in the diabetic CNS. Initial Test RR<15 15(36) 3(7) Supported by: D06RH09012 (HRSA) RR≥15 3(7) 21(50) 648-P VR (N=27) VR≤1.5 VR>1.5 0.8466 <0.0001 VR≤1.5 10(37) 1(4) POSTERS Improvement of Diabetic Neuropathy Applying TAT-Mediated En- Complications hanced Delivery of Metallothionein and SOD VR>1.5 1(4) 15(56) Acute and Chronic YONGSOO PARK, DONGSOO MIN, LEEJIN PARK, HYUNOK KIM, Seoul, Republic ΔBP (N=25) ΔBP OH+ ΔBP OH- 0.3363 =0.0847 of Korea ΔBP OH+ 1(4) 2(8) D i a b e t i c n e u r o p a t h y ( D N ) m i g h t b e r e s u l t e d f r o m o x i d a t i v e s t r e s s i n n e u r o n a l tissues due to glucotoxicity, hypoxia, and advanced glycation endproduct ΔBP OH- 1(4) 21(84) (AGE) accumulation. Therefore, antioxidant treatment may improve neuronal Very good test-retest categorical agreement was found for R-R (86%) and repair in patients with DN. The biological properties of metallothionein VR (93%). OH occurred infrequently (4 subjects demonstrated OH+ on either (MT) and superoxide dismutase (SOD) are related to their free-radical test and 1 demonstrated OH+ on both). Still, 84% of subjects had no OH (OH-) scavenging abilities, but their access into the biologic membrane is limited. on both the initial and the repeat test. These fi ndings demonstrate good We investigated whether Tat-MT (and Tat-SOD) fusion protein applying a intra-subject reproducibility of CAN tests performed in EDIC. new protein transduction technology may transduce into the PC12 cells, Supported by: NIH, NIDDK surrogate to peripheral nerve cells and protect them from various forms of oxidative damage. Tat-MT and Tat-SOD were successfully delivered into and 650-P persisted in the PC12 cells. The intracellular activities of MT and SOD were Loss of Sural Responses Estimated by a Rapid and Easily Administered found to increase in line with the amount of protein delivered into the cells. Technique Identifi es Greater Neuropathy Severity and Risk in a Tat-MT and Tat-SOD in combination signifi cantly inhibited hyperglycemia, General Diabetes Population hypoxia and AGE-induced apoptotic and infl ammation signaling in PC12 cells. SHAI N. GOZANI, AARON I. VINIK, Waltham, MA, Norfolk, VA We then examined in vivo transduction of Tat-MT and Tat-SOD in OLETF Abnormalities of sural nerve conduction (NC) are sensitive indicators rats. A single intraperitoneal injection of Tat-MT and Tat-SOD resulted in of diabetic peripheral neuropathy (DPN). The sural sensory nerve action the delivery of these antioxidants to the neuronal tissues. Increased radical potential (SNAP) amplitude is often dichotomized as detectable or scavenging activity, decreased apoptosis of mitochondrial and ER pathway undetectable. A detectable sural has been used as a marker for less severe by inhibiting redundant NFkB, AMPK and MAPK signaling and, therefore, DPN in clinical trials. More severe neuropathy is a predictor of foot ulcers improvement in remyelination and nerve conduction were found in rat sciatic and risk of falling and fractures. In this study we evaluated whether the nerves after the treatment of Tat-MT and Tat-SOD. Tat-MT and Tat-SOD in dichotomous sural response identifi es this at risk population in a general combination signifi cantly repressed three different ROS-mediated injuries diabetes population using a rapid, easily administered quantitative test. (hyperglycemia, hypoxia and AGE) and resultant neuronal damage in PC12 We analyzed 10,000 consecutive point-of-care NC studies uploaded to the cells through inhibiting the activation of redundant NFkB, AMPK, MAPK and NC-stat® database. The studies were performed in US based endocrinology ER stress signaling pathways and its downstream infl ammatory mediators. and primary care clinics for evaluation of DPN. Each study included data Antioxidant combination treatment coupled with adequate delivery system from at least 1 median, peroneal, and sural nerve. The studies were grouped may facilitate improvement in repairing mechanisms in patients with DN. according to the dichotomous sural response (detectable SNAP > 2 μV), with group differences evaluated by the two-group t-test. 649-P 1872 (18.7%) studies had an undetectable sural response. All electro- Intra-Subject Reproducibility of Cardiac Autonomic Neuropathy physiological parameters exhibited statistically signifi cant differences Tests in the Diabetes Control and Complications Trial/Epidemiology between the two cohorts. Patients with detectable surals had motor of Diabetes Interventions and Complications Study (DCCT/EDIC) amplitudes that were 0.6–1.1 mV larger than those with undetectable CATHERINE L. MARTIN, BARBARA H. BRAFFETT, RODICA POP-BUSUI, EVA L. surals. Prior studies have demonstrated that amplitude differences of this FELDMAN, JAMES W. ALBERS, CATHERINE SOMMER, PHILLIP A. LOW, PATRICIA magnitude are clinically meaningful in DPN. A. CLEARY, WILLIAM H. HERMAN, DIABETES CONTROL AND COMPLICATIONS The results of this study support use of this easily administrated evaluation TRIAL/EPIDEMIOLOGY OF DIABETES INTERVENTIONS AND COMPLICATIONS of the sural response as an indicator of neuropathy severity in patients with (DCCT/EDIC), Ann Arbor, MI, Rockville, MD, Rochester, MN diabetes. Patients with an undetectable sural had substantially lower motor DCCT participants were invited to join the EDIC study in 1994: 96% agreed amplitudes suggesting greater axon loss and more severe neuropathy. to participate. Cardiac autonomic neuropathy (CAN) tests were performed in These patients are candidates for more aggressive management of their either year 13 or 14 of EDIC follow-up and again in EDIC year 16 or 17, at which neuropathy and its consequent risk. time we evaluated the same-day, test-retest, intra-subject reproducibility of Detectable Undetectable Diff. EDIC CAN tests. Subjects across the 28 EDIC sites were randomly selected to repeat one Peroneal n component of the CAN test battery; heart rate variability to paced breathing Latency (µsec) 3.9 (0.7) 4.2 (0.8) +0.3, p<0.001 (R-R), Valsalva ratio (VR) or postural blood pressure change (ΔBP). Results Amplitude (mV) 3.0 (1.7) 1.9 (1.5) -1.1, p<0.001 were categorized using thresholds used to defi ne the presence of CAN F-wave Latency (msec) 55.0 (6.5) 58.7 (7.1) +3.7, p<0.001 in DCCT/EDIC; an R-R value of < 15, a VR < 1.5, and for ΔBP, orthostatic hypotension (OH+) defi ned as a fall in diastolic blood pressure of > 10 mmHg Median n. at any time during 10 minutes of standing. The ANS 2000 (Hokanson Inc, Motor Amplitude (mV) 4.7 (1.9) 4.0 (1.9) -0.7, p<0.001 Bellevue WA) was used for all CAN tests. Kappa statistic was used to assess Sensory Amplitude (µV) 20.7 (14.6) 13.0 (11.6) -7.7, p<0.001 categorical agreement between the initial and the repeat test. In all, 175 subjects were identifi ed for repeat R-R (80), VR (56) and ΔBP (49) testing. The identifi ed subjects were representative of the EDIC cohort in age (49.8±7.0 years), gender (51% male), diabetes duration (28.5 ±5.0 years), and DCCT group (47% Intensive). Preliminary fi ndings comparing initial and repeat test results are shown.

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A178 COMPLICATIONS—NEUROPATHY

651-P 653-P Low-Dose Insulin Deactivates p44/42 MAPK and Ameliorates Neuroprotective Effect of Insulin-Like Growth Factor I on Cardio- Peripheral Sensory Nerve Dysfunction in Rats with Streptozotocin- vagal Function in Type 1 Diabetes Induced Diabetes VICTOR M. HERRERA, LEONELO E. BAUTISTA, MARI PALTA, Madison, WI KAZUHIRO SUGIMOTO, MASAYUKI BABA, SOROKU YAGIHASHI, MINORU YASU- The purpose of this analysis was to test the hypothesis that besides its JIMA, Hirosaki, Japan, Aomori, Japan well-known effect on glycemic control, insulin reduces the risk of diabetic A low dose of insulin helps to improve peripheral nerve dysfunction in autonomic neuropathy by increasing the levels of Insulin-Like Growth Factor

animals with streptozotocin (STZ)-induced diabetes without affecting I (IGF-I), a neurotrophic factor that stimulates nerve regeneration in animal POSTERS glycemia. The precise mechanisms of the neuroprotective effects of insulin models of diabetes. Complications in this model remain unknown, but altered peripheral nerve insulin signaling We evaluated 129 patients with type 1 diabetes (T1D) from the Wisconsin Acute and Chronic might be one cause. We caused diabetes in 10-week-old male Wistar rats by Diabetes Registry study (mean age at diagnosis=10.0 years, 51% male), who injecting them with streptozotocin (45 mg/kg) and then assigned them to one were followed from diagnosis to up to 20 years, had baseline measurements group that received half of an insulin implant (∼1 U/day; I-group; n = 11) and of IGF-I in plasma, and had ≥1valid deep breathing test (expiration-inspiration another that remained untreated (U-group; n = 10) for 6 weeks. The controls difference [EID] in beats per minute [bpm]) conducted at ≥10 years of age. were age- and sex-matched non-diabetic Wistar rats (C-group; n =12). A low Cross-sectional associations (at baseline) were evaluated using the Pearson’s dose of insulin did not alter hemoglobin A1c and serum insulin levels, which correlation coeffi cient (r). Longitudinal measurements of EID were regressed were increased and decreased by 132% and by 92%, respectively, in the on baseline IGF-I, adjusting for age at diagnosis, diabetes duration, body U-group compared with the C-group. Serum free fatty acid levels increased weight at exam, and glycated hemoglobin A1c (HbA1c averaged over one by 39% in the U-group relative to the C-group and were decreased by 18% year before each exam) using random effects models. We tested the IGF-I- (p = 0.0424) in the I-group compared with the U-group. Thermal hypoalgesia by-duration interaction to determine whether the growth factor modifi ed the and mechanical hyperalgesia developed in the U-group, but not in the rate of change of EID. I-group. Sensory and motor nerve conduction velocities were depleted in the Mean IGF-I level was 144.4 ng/ml (standard deviation [SD] = 95.3 ng/ U-group, whereas sensory nerve conduction velocity increased by 7% (p = mL). At baseline, IGF-I was higher with greater age and body weight (r=0.67 0.0351) in the I-group compared with the U-group. Western blots revealed and r=0.68, p<0.001), was higher in women than in men (164.9 vs. 124.9 ng/ unaltered total insulin receptor (IR) and p44/42 MAPK protein levels, but mL, p<0.05), and was inversely correlated with HbA1c (r=-0.38, p<0.001). a 29% decrease and 3.7- and 4.6-fold increases in phosphorylated IR, In the longitudinal analysis (mean duration over follow-up=8.4 years), EID p44 and p42 MAPK protein levels, respectively, in sciatic nerves from the decreased 0.3 bpm per year of disease duration and a higher baseline IGF-I U-group compared with the C-group. Levels of phosphorylated IR protein independently predicted a higher mean EID (1.6 bpm per 1 SD of IGF-I, were further decreased by 40% (p = 0.009) in the I-group compared with p=0.029); however, IGF-I did not modify the rate of cardiovagal function the U-group, and phosphorylated p44/42 MAPK protein also decreased (p decline (p-value for interaction=0.997). < 0.0001) to control levels. Thus, a low dose of insulin deactivated p44/42 Our results support a protective role of circulating IGF-I levels on MAPK and ameliorated peripheral sensory nerve dysfunction in rats with cardiovagal function among young patients with T1D, independent of STZ-induced diabetes. These fi ndings support the notion that an insulin glycemic control. This fi nding suggests the existence of alternative defi ciency rather than hyperglycemia functions in the pathogenesis of type neuroprotective mechanisms of insulin in human diabetes. 1 diabetic neuropathy. Supported by: NIH Grants R01 DK36904, R01 HL62897 and R29 DK47270 Supported by: Japan Society for the Promotion of Science (18590520) 654-P 652-P Oxidative Stress and Chronic Infl ammation in Brain Is Associated Metanx Alleviates Diabetes-Induced Large and Small Fiber Neuro- with Cognitive Dysfunction in Diabetic db/db Mice pathies and Promotes Small Sensory Nerve Fiber Regeneration EIICHI HIRATA, NORIYUKI SONODA, YOHEI MINAMI, YASUTAKA MAEDA, TAKA- IRINA G. OBROSOVA, PIERRE WATCHO, HANNA SHEVALYE, Baton Rouge, LA HIRO KATO, YOSHIHIRO SEKI, AKIRA MONJI, RYOICHI TAKAYANAGI, TOYOSHI Metanx (PamLab L.L.C.) is a prescription combination medicine containing INOGUCHI, Fukuoka, Japan L-methylfolate, pyridoxal 5’-phosphate, and methylcobalamin. Metanx Oxidative stress contributes to the development of diabetic complications. increases production of tetrahydrobiopterin, a cofactor of endothelial Increasing epidemiologic evidence suggested that diabetes mellitus is nitric oxide synthase (eNOS), which, theoretically, should counteract eNOS associated with dementia and cognitive decline. However, underlying uncoupling, generation of superoxide, superoxide-induced upregulation of mechanism is not fully understood. We have reported the superoxide inducible nitric oxide synthase (iNOS), and peroxynitrite formation. We, overproduction through protein kinase C (PKC)-dependent activation of therefore, evaluated Metanx on diabetic peripheral neuropathy (DPN) using NAD(P)H oxidase in several tissues of diabetic models. Therefore, in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes. Metanx was present study, we evaluated cognitive function and its association with administered to 15 week old ZDF and ZDF lean rats at either 4.87 mgkg-1d-1 oxidative stress using diabetic db/db mouse model. (the dose used in clinical practice) or 24.35 mgkg-1d-1, 2 times a day, for 4 Reactive oxygen species (ROS) production of 20-week aged db/db weeks. Neuropathy was evaluated by sciatic motor (MNCV) and hind-limb mice brain was markedly increased compared to control db/+ mice by digital sensory (SNCV) nerve conduction velocities, thermal response dihydroethidium (DHE) staining that is oxidized to the fl uorescent dye latency (Hargreaves method), mechanical withdrawal threshold (Randall- ethidium by superoxide. Furthermore, db/db mice exhibited a prominent Selitto test), tactile response threshold (fl exible von Frey fi lament test), accumulation of lipid peroxidation products in brain. To further characterize and intraepidermal nerve fi ber density (fl uorescent immunohistochemistry the changes that occur in brain with overproduction of ROS, we profi led with confocal microscopy). Sciatic nerves were used for nitrite/nitrate and pro-infl ammatory and ROS production related transcript expression in db/ nitrotyrosine assays (ELISA). 15 week old ZDF rats displayed hyperglycemia, db and db/+ mice brain at this age. Real-time PCR results revealed that MNCV and SNCV defi cits, thermal and mechanical hypoalgesia, tactile expression of NAD(P)H oxidase components including Nox 2 and Nox 4 and allodynia, and ∼26% loss of intraepidermal nerve fi bers. Both doses of pro-infl ammatory cytokines were upregulated in db/db mice compared to Metanx alleviated SNCV defi cit and thermal and mechanical hypoalgesia, in control mice, whereas anti-infl ammatory cytokines were downregulated the absence of any reduction of hyperglycemia. Low dose Metanx treatment in diabetic mice. However, both db/db and db/+ mice at this age did not induced small sensory nerve fi ber regeneration and increased intraepidermal show amyloid beta accumulation in brain. In correlation with these fi ndings, nerve fi ber density in ZDF rats. At the end of the study, sciatic nerve Morris water maze test results showed that working memory of db/db mice nitrotyrosine and nitrite/nitrate concentrations were lower in the Metanx- was signifi cantly impaired compared to the controls at 20-week age. treated ZDF rats compared with the untreated group. In conclusion, Metanx, Taken together, our results suggested that NAD(P)H oxidase activation and at the dose currently employed in clinical practice, alleviates sensory large consequently elevation of oxidative stress and infl ammatory cascade might and small fi ber DPN and induces small sensory nerve fi ber regeneration, be a causative pathogenic mechanism in diabetes-associated cognitive by mechanisms that are likely to involve inhibition of oxidative-nitrosative impairment in diabetic rodent model and that regulation of redox state may stress. Evaluation of Metanx on eNOS uncoupling and iNOS expression are open up new approaches to augmenting diabetes-associated dementia. in progress. Supported by: Research Grant from PamLabs L.L.C

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A179 COMPLICATIONS—NEUROPATHY

655-P 657-P Protection from Neuropathy: Lessons from Patients with Long The Combination Effect of Pioglitazone and alpha Lipoic Acid on the Duration Type 1 Diabetes Peripheral Nerves in Experimental Diabetic Rats UAZMAN ALAM, HASSAN FADAVI, IOANNIS PETROPOULOS, GEORGIOS TAE SUN PARK, HEUNG YONG JIN, KYUNG AE LEE, KYUNG TAEK PARK, HEA MIN PONIRAKIS, OMAR ASGHAR, ANDREW MARSHALL, MITRA TAVAKOLI, ANDREW YU, HONG SUN BAEK, Jeon Ju, Republic of Korea JM BOULTON, RAYAZ A. MALIK, Manchester, United Kingdom There are few data about combination therapeutic approach using Subjects with more than 50 yrs of Type 1 DM are a special group known as pathogenically benefi cial drugs in the diabetic peripheral neuropathy (DPN).

POSTERS the ‘Gold Medallists’ who may have minimal/delayed microvascular compli- Therefore, in present study, we have investigated the combined effect of

Complications ca tions. alpha lipoic acid (ALA) and pioglitazone (PIO) on the peripheral nerves in the Acute and Chronic We have assessed neuropathy (Neuropathy symptom profi le (NSP), diabetic rats. Animals were divided into 8 groups (N=6-8) and designated neuropathy disability score (NDS), vibration perception threshold (VPT), as follows: Normal, Normal+ALA(100mg/kg/day), Normal+PIO(10mg/kg/ peroneal/sural nerve electrophysiology, warm and cold thresholds (WT/CT) day), Normal+ALA(100mg/kg/day)+PIO(10mg/kg/day), DM,DM+ALA(100mg/ (°C), sudomotor function (Neuropad), Corneal confocal microscopy (CCM) kg/day), DM+PIO(10mg/kg/day), DM+ALA(100mg/kg/day)+PIO(10mg/kg/day). and corneal sensitivity) in 12 gold medallists, 11 patients with <30 years of Statistical analyses were performed about current perception threshold(CPT), Type 1 DM and 9 controls (C). oxidative stresses, intraepidermal nerve fi ber density(IENFD), gastric Gold medallists v shorter duration Type 1 patients v C: age (66.5±8.3 v mucosal nerve fi ber quantity, and axonal morphology in sciatic nerve. In 55.5±10.8 v 53.3±3.1yrs), duration of diabetes (54.7±3.1 v 28.8±8.3 v 0yrs), results, blood glucose levels were not affected, however CPT was reduced HbA1c(%) (8.1±1.2 v 9.5±1.7 v 5.8±0.2, P<0.0001), total cholesterol (4.6±0.9 at 2000Hz in the DM+ALA+PIO group compared with other DM groups (DM, v 4.1±1.1 v 5.2±0.6, P=0.03), HDL (1.7±0.6 v 1.7±0.6 v 1.5±0.2), LDL (2.3±0.9 DM+ALA, DM+PIO,respectively) (70.56±12.6 vs 75.4±9.8 vs 73.3±12.3 vs v 1.8±0.5 v 3.1±0.5, P=0.002) and triglycerides (1.2±0.6 v 1.3±0.8 v 1.3±0.8). 73.1±11.5, P<0.05). IENFD was less reduced signifi cantly in the DM+ALA+PIO Diabetic patients were abnormal compared to controls, but there were no group than other DM groups (7.63±0.97 vs 5.21±0.87 vs 6.78±1.67 vs difference comparing gold medallists with shorter duration diabetes: NSP 6.51±0.97, P<0.05)(Figure 1). Besides, the number of gastric small nerve fi bers (9.6±7.6 v 3.9±3.8 v 0, P=0.0004), NDS (4.5±2.7 v 5.1±3.7 v 0, P=0.001); VPT penetrating into the mucosa at 100um horizontal line from the luminal side (21.9±9.7 v 24.1±15.1 v 7.6±6.1, P=0.001), Sural amplitude(μV) (5.0±3.3 v (4.6±1.1 vs 1.9±0.7 vs 2.9±1.5 vs 3.1±1.6, P<0.05) and mean myelinated axonal 4.9±2.4 v 16.4±9.1, P=0.002), Sural NCV(m/s) (39.8±2.6 v 40.4±8.2 v 48.2±1.8, area in the sciatic nerves (62.5±2.34 and 51.1±2.12 vs 56.5±3.12 vs 57.5±2.54, P=0.001), peroneal NCV (39.6± 5.4 v 36.7±3.5 v 47.2±3.9, P=0.002); Cold P<0.05) were higher signifi cantly in the DM+ALA+PIO group than other DM threshold (22.5±6.7 v 16.9±11.6 v 29.4±1.4, P=0.001) and % Neuropad response groups. Our results demonstrated that combination using pioglitazone and (55.5±41.4 v 58.0±41.6 v 87.5±21.9). Both WT (41.2±3.6 v 46.1±3.7 (P=0.04) v ALA is more benefi cial than ALA or pioglitazone monotherapy in the respect 38.7±1.8, P=0.0006) and corneal sensitivity (mBar) (0.7±0.5 v 3.2±4.6 (p=0.03) of peripheral nerve preservation in diabetes. Moreover, pioglitazone is v 0.6±0.4, P=0.04) were signifi cantly better in the gold medallists. Corneal preferentially considered in DPN patients taking ALA and who need more nerve fi bre density(no.mm2) (16.5±11.4 v 16.9±7.1 v 37.2±4.4, P=0.001), intensive glucose control at the same time. branch density(no.mm2) (31.5±26.8 v 33.2±13.4 v 108.3±41.3, P=0.001) and length(mm/mm2) (13.1±8.1 v 13.8±5.8 v 28.6±3.0, P=0.001) were signifi cantly lower than controls with no differences between diabetic groups. The ‘Gold Medallist’ have preserved measures of neuropathy comparable to diabetic patients with ∼25 yrs lesser duration of diabetes, despite comparable glycaemia and lipid profi les. Alternative risk factors merit exploration to explain this fi nding. Supported by: JDRF

656-P Role of Cardiac Autonomic Dysfunction in Hypertension and in In creased Coronary Risk in Nondiabetic Obese Patients ISABELA BANU, SABRINA CHIHEB, MINH TUAN NGUYEN, EMMANUEL COSSON, PAUL VALENSI, Bondy, France Cardiac autonomic dysfunction (CAD) was shown to contribute to elevate blood pressure and cardiovascular risk in diabetic patients. Obesity is often associated with CAD. The aim of the present study was to evaluate the role of CAD in hypertension and the respective role of CAD and hypertension in the elevated coronary risk in obese or overweight patients without known diabetes. We included 377 patients (BMI 39±7 kg/m²), aged 38.3±14.6 years. An oral glucose tolerance test was performed. CAD was defi ned by at least one 658-P abnormal out of 3 standard tests of heart rate variability (deep-breathing, The Relation of Heart Rate Variability with Glycemic Variability in lying-to-standing, Valsalva) that depend mostly on vagal control. CAD was Type 1 Diabetic Patients considered as severe when 2 or 3 tests were abnormal. The 10-y UKPDS risk SOLANGE HOUSSAY, JORGE HORACIO ALVARIÑAS, GUILLERMO BURLANDO, score for coronary events was calculated. CLAUDIO GONZALEZ, ALICIA GARCIA, SILVIA VASTA, Caba, Argentina CAD was present in 158 patients (44%) and hypertension in 131 patients Objective: To study Heart Rate Variability(HRV) as a marker of Diabetic (35%). The prevalence of hypertension was signifi cantly higher in the patients Autonomic Neuropathy(DAN) and it´s relation with Glycemic Variability(GV) with severe CAD (50%) than in those without (30%) or with only one (38%) in Type 1 Diabetes. abnormal CAD test (p=0.01). The UKPDS risk score differed signifi cantly in the Patients and method: A total of 54 T1DM patients(pts) with a median age 4 following groups (p<0.001): normotensive patients without CAD (2.3±2.1%), of 37,35 ± 10,8 years without chronic complications and no signs of DAN 2 normotensive patients with CAD (3.2±4.5%), hypertensive patients without were included. The mean Body Mass Index(BMI) = 24± 2,6 kg/m ; waist CAD (6.0±6.4%), hypertensive patients with CAD (9.1±5.5%). circumference = 80 ± 9,3 cm; normal levels of Total Colesterol, HDL, LDL In a multivariate analysis taking CAD, dysglycemia (diabetes or prediabetes and Triglycerols; all normotensive. All pts were receiving NPH Insulin or a defi ned by impaired fasting glycemia and/or impaired glucose tolerance) prolonged action analog and aspartic insulin boluses premeals according to and pulse pressure (an index of artery stiffness) as independent variables, premeal glycemias and carbohydrate counting. The presence of Subclinic these three factors were associated independently and signifi cantly with Autonomic dysfunction was analyzed with Power Spectral Analisys of the UKPDS score. HRV in a DI, EKG trace, processing the RR consecutive intervals using Fast These data suggest that in obese or overweight patients free of known Fourier Rapid Transformation. Ninety blood capillary glucose tests per pt diabetes, CAD might contribute to increase blood pressure due to sympathetic were performed within a month, pre and 2 hs post prandially. Mean blood predominance and to aggravate coronary risk in those with hypertension. glucose(MBG), Standard Deviation of blood glucose(SD), Hyperglycemic risk(HBGI: elevated blood glucose index) and hypoglycemic risk(LBGI: low blood glucose index) were calculated.

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A180 COMPLICATIONS—OCULAR

Results: 92,2% of T1DM pts, non symptomatic for DAN with good (p=0.007) and DME (p<0.0001). Of patients with DR, 52%, 17% & 14% metabolic control and without chronic complications had reduced HRV. reported being aware (p<0.0001) and 66%, 60% & 46% reported appropriate HbA1c was associated with elevated resting heart rates (r= 0,44 p= 0,001). follow-up (p<0.0001) based on DR severity when seen by a retina specialist, HBGI was associated with elevated resting heart rates (r= 0,38 p= 0,04). SD comprehensive ophthalmologist or optometrist, respectively. These data of glycemias correlated positively with faster resting heart rates (r= 0,28 p= suggest that selection of eye care provider is important and that efforts 0,03) and greater amplitude of Low Frequency Component(LF) in Frequency to enhance successful transfer of knowledge from eye providers to their Domain Power Spectral Analysis of HRV (r = 0,30 p= 0,02) patients as well as to the patient’s medical doctors are urgently needed. Conclusions: Subclinic DAN is a frequent chronic complication in T1DM. POSTERS The possitive association of LF component of HRV with Glycemic Variability & 661-P Complications suggests an early vagal dysfunction and sympathetic predominance in pts Role of Extravasated Lipoproteins in Muller Cell Death in Diabetic Acute and Chronic with greater Glycemic Variability. These pts have an enhanced sympathetic Retinopathy response in relation to glycemic drops. MINGYUAN WU, SHIHE YANG, MICHAEL ELLIOT, DONGXU FU, KENNETH WILSON, JING ZHANG, MEI DU, JUNPING CHEN, TIMOTHY J. LYONS, Oklahoma City, OK COMPLICATIONS—OCULAR Modifi ed low density lipoprotein has been associated with pathogenesis of diabetic retinopathy (DR) including apoptotic pericyte loss. Here our [See also: Presidents Posters 390-PP– to 391-PP, page A108.] goal is to investigate whether modifi ed low density lipoprotein induced apoptosis in human retinal Muller cells and whether oxidative stress and endoplasmic reticulum (ER) stress are implicated. Human Muller cell line Guided Audio Tour: Ocular Complications (Posters 659-P to 666-P), MIO-M1 were treated with highly oxidized glycated low density lipoprotein see page 13. (HOG-LDL) or native LDL (N-LDL) at 200 mg/L for 1,6,12 and 24h. In further & 659-P experiments, Muller cells were pretreated for 1 hr with NAC (a blocker of Protective Effect of HDL on Modifi ed LDL-Induced Endoplamic oxidative stress) and 4-PBA (a blocker of ER stress) before spiking HOG-LDL Reticulum Stress and Apoptosis in Retinal Pigment Epithelial Cells or N-LDL. Cell counting kit-8, western blot for caspase related proteins, MEI DU, MINGYUAN WU, JING ZHANG, KENNETH WILSON, DONGXU FU, SHIHE and TUNEL immunostaining were performed to detect apoptosis. Western YANG, TIMOTHY J. LYONS, Oklahoma City, OK blots were conducted to detect oxidative stress and ER Stress. HOG- LDL leakage and modifi cation in the retina are implicated in the pathogenesis LDL induced apoptosis as shown by deceased cell viability (n=3, p<0.01), of diabetic retinopathy (DR). In atherosclerosis, HDL is protective (reverse increased TUNEL positive cells, increased protein levels of Caspase 3 and cholesterol transport, anti-oxidant, anti-infl ammatory functions), but its role 12, and increased Bax/Bcl-2 ratio. HOG-LDL also increased oxidative stress in DR is unknown. In this study, possible protective effects of HDL in human as shown by increased levels of 3-nitrotyrosine, Nox-4 and SOD2, and (hTERT) Retinal Pigment Epithelial (RPE) cells were investigated. Cells were decreased levels of GP-1. In parallel, HOG-LDL induced ER stress as shown by pretreated with native HDL (N-HDL, 200, 500, 1000 µg/ml) or ‘Highly Oxidized increased levels of ER stress markers eIF2-a, KDEL, ATF6, and Chop. Further, Glycated’ HDL (HOG-HDL, 200, 500, 1000 µg/ml) for 1h, and then exposed pretreatment with NAC or 4-PBA partially attenuated apoptosis, oxidative (1-24h) to N-LDL or HOG-LDL (200 µg/ml). Morphological changes (optical stress, and ER stress. In comparison, N-LDL did not induce any changes. We microscopy), cell viability changes (CCK-8), endoplasmic reticulum (ER) stress concluded that modifi ed LDL induced Muller cell death, suggesting that, in markers (KDEL, ATF6, eIF2α, CHOP), autophagy markers (LC-3II, Atg12, Beclin-1) addition to hyperglycemia, exposure of Muller cells to oxidized and glycated (western blot and immunohistochemistry), and apoptosis (TUNEL assay) were extravasated lipoprotein is implicated in DR. measured. HOG-LDL (200 µg/ml, 24hr) caused a 74% reduction (vs SFM) in cell viability, but cell viability increased to 150% by pre-treatment with 500 µg/ml & 662-P N-HDL for 1h (n=3, P<0.05). Pre-treatment with N-HDL increased the level of Intensive Glycemic Control (INT) and Ocular Outcomes in the antioxidant enzyme GPX-1(p<0.05) and suppressed HOG-LDL-induced ER stress Veterans Affairs Diabetes Trial (VADT) as evidenced by decreased levels of KDEL and P-eIF2α (P<0.05). Pre-treatment NASRIN AZAD, LILY AGRAWAL, NICHOLAS V. EMANUELE, RONALD KLEIN, with N-HDL also suppressed HOG-LDL-induced autophagy and apoptosis by GIDEON BAHN, DERRICK KAUFMAN, THOMAS MORITZ, DOMENIC REDA, WILLIAM decreased levels of LC-3II, Atg12, and cleaved-PARP, but pre-treatment with DUCKWORTH, CARLOS ABRAIRA, Hines, IL, Milwaukee, WI, Phoenix, AZ, Miami, FL HOG-HDL showed no such benefi cial effects. In conclusion, in RPE cells, HOG- VADT was a prospective, randomized, controlled trial of 1,791 subjects LDL induced cytoxicty and apoptosis, mediated by ER stress and autophagy. with DM2 to determine if INT would reduce CV events. Microvascular N-HDL mitigated these effects, but HOG-HDL failed to do so. The data suggest data were also collected. The effects of INT and baseline variables on eye that extravasated HDL may be protective in the retina, but protective effects complications are reported here. The baseline variables were diabetes are lost if HDL is modifi ed. duration, age, gender, smoking status, HbA1c, basic lipid parameters, & 660-P fi brinogen, PAI-1, albuminuria, eGFR, and history of retinopathy or macular Retinopathy Awareness and Appropriateness of Eye Care Follow- edema (ME). The analysis was performed based on a model utilizing a Up among Patients with Diabetes composite outcome (overall incidence, 36.7%) which included incidence of PAOLO S. SILVA, JERRY D. CAVALLERANO, JENNIFER K. SUN, ANN M. TOLSON, retinopathy (18.5%), or progression of retinopathy (13.9%, progression of 2 LLOYD M. AIELLO, LLOYD PAUL AIELLO, Boston, MA or more steps on the ETDRS scale), or incidence of ME (5.4%), or panretinal Prevention of visual loss from diabetes is critically dependent upon patient or focal/grid photocoagulation (8.3%), or vitrectomy (3.7%). Though INT self-understanding of eye disease and appropriately scheduled ophthalmic itself was not related to the development of the composite ocular outcome follow-up. We assessed awareness of diabetic retinopathy (DR) and timing (OR INT vs. STD 0.97; CI= 0.85, 1.30; p = 0.25), 2 factors were, baseline HbA1c of next eye care follow-up upon fi rst presentation to a teleophthalmology (OR per 1%=1.17; CI= 1.04, 1.31; p < 0.01) and smoking status (OR=0.57; CI= program (Joslin Vision Network [JVN]) in a tertiary academic diabetes 0.36, 0.91; p < 0.02). In addition, there were two signifi cant interactions and center. From 12/15/2009-12/31/2010, all JVN patients with diabetes (DM) one nearly signifi cant interaction between treatment and certain baseline without previous eye care at Joslin Diabetes Center were evaluated. Self- variables. INT was associated with less worsening of ocular outcome reported DR status, time since last eye exam, and time to next scheduled in those with serum total cholesterol above ∼185 mg/dl (p < 0.005), urine eye exam were recorded and retinal images were graded for DR severity. albumin/creatinine below ∼7 mg/g (p < 0.03), and PAI-1 below ∼ 17 ng/dl Of 3,006 patients, median age was 56yrs (Q1,Q3:44,66), DM duration 9yrs (p = 0.0506). (3,16), HbA1c 7.8% (7,9), 54% were male, 67% white, and 66% type 2 DM. In conclusion, poorer glycemic control prior to entry into VADT was No DR was present in 59% (1,763), any DR present in 40% (1,201) and 1% (42) associated with a 17% higher incidence of a composite ocular outcome. were ungradable. Vision threatening DR (vtDR: moderate nonproliferative However, in the main trial, INT was not statistically different than standard DR or worse, or diabetic macular edema [DME]) was present in 25% (764). control on individual ocular variables. Paradoxically, current smokers were Only 18% (209) of patients with any DR and only 23% (176) with vtDR 43% less likely to develop the composite eye outcome, possibly due to were aware they had any DR despite 71% (848) and 73% (569), respectively, selective survival. Certain baseline characteristics did modify the ocular reporting an eye exam within the past year. Nearly 48% (369) with vtDR had response to treatment generating the hypothesis that INT may have follow-up interval longer than recommended for their DR severity. Patients benefi cial effects on eye disease in some patients with long standing DM2. with vtDR who were aware of their DR were younger (p=0.01), had longer Supported by: VA Cooperative Studies Program, NIH, and Pharma DM duration (p<0.0001), worse vision (p=0.005), more systemic disease ADA-Funded Research

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A181 COMPLICATIONS—OCULAR

& 663-P & 665-P Signifi cance of Diabetes-Induced Outer Blood-Retina Breakdown The Association between Neuropathy, Corneal Hypoesthesia and and Its Relevance to Diabetic Macular Edema Dry Eye Syndrome in Patients with Type 2 Diabetes YUN-ZHENG LE, YAN LIU, MEILI ZHU, HUIZHUO XU, Oklahoma City, OK NIKOLAOS TENTOLOURIS, VASILIOS ACHTSIDIS, IOANNA ELEFTHERIADOU, Outer blood-retina barrier (BRB) separates the neural retina from choroidal EMMANOUIL VAIKOUSIS, PANAYIOTIS G. THEODOSSIADIS, Athens, Greece circulation, which is responsible for approximately 80% of blood circulation Subjects with diabetes are at increased risk to develop dry eye syndrome. in the eye. To investigate the relevance of outer BRB breakdown to diabetic In addition, reduced corneal sensation due to neuropathy may predispose POSTERS

Complications macular edema, a major cause of vision loss in diabetic patients, we developed to corneal irritation, erosions and ulcer development. In this study we

Acute and Chronic an imaging assay and explored the therapeutic potential of treating outer BRB examined the association between diabetic peripheral neuropathy with leakage in various rodent models. Fluorescein isothiocyanate (FITC)-dextran corneal sensitivity and dry eye in subjects with type 2 diabetes. leaked through outer BRB was visualized and quantifi ed by fl uorescent A total of 61 patients with type 2 diabetes (mean age 63.72±9.51 years; microscopy. Inhibiting outer BRB breakdown biochemically and genetically 27 without and 34 with peripheral neuropathy) and in 38 age- and gender- was investigated in mice using this imaging assay. Substantial leakages matched controls we examined. Peripheral neuropathy was diagnosed of macromolecules through the outer BRB in diabetic and ischemic rodents using the neuropathy disability score (NDS), neuropathy symptoms score were detected. The number of severe breaking points in the outer BRB is (NSS) and vibration perception threshold (VPT). Corneal sensitivity was inversely proportional to the size of macromolecules. Outer BRB-specifi c measured using the Chochet-Bonnet esthesiometer. Symptoms of dry eye leakage was signifi cantly reduced in mice with tissue-specifi c knockout of were assessed according to the International Dry Eye Workshop (2007) vascular endothelial growth factor (VEGF) or its receptor (VEGF-R2) and in recommendations. Eye dryness was determined using the Schrimer test and mouse model with biochemical inhibition of extracellular-signal-regulated the tear fi lm break up time (BUT). kinase (ERK) pathway. For the fi rst time, a microscopic imaging assay was Controls and diabetic patients without neuropathy tended to have less developed to visualize and quantify macromolecules leaked through the often symptoms of dry eye in comparison with diabetic patients with outer BRB in diabetic and ischemic rodents. Our results suggest that outer neuropathy (31.6%, 29.6% and 52.9%, respectively, P=0.09). The mean BRB breakdown contributes signifi cantly to overall diabetes- and ischemia- corneal sensitivity was lower in diabetic patients with neuropathy in induced vascular leakage, which is regulated through an autocrine VEGF comparison with patients without neuropathy and controls (3.50±0.81 vs. signaling mechanism. Pre-clinical treatments of outer BRB breakdown with 4.46±0.42 and 4.71±0.34, P<0.001). The mean values of Schirmer test were biochemical and genetic approaches were effective. Therefore, our study also lower in patients with neuropathy than in those without neuropathy may have signifi cant implications to the mechanism and diagnosis of diabetic and controls (10.05±6.75mm vs. 13.61±6.31mm and 10.05±6.75mm, P=0.01); retinopathy. In addition, our imaging assay will be very useful to screening the same was valid for the mean BUT values (7.08±2.56sec vs. 9.69±3.68sec drugs for diabetic macular edema. and 11.36±2.92sec, respectively, P<0.001). All indices of neuropathy were Supported by: NIH Grant R01EY20900, Grants from BIMR, FFB, OCAST correlated signifi cantly with measures of corneal sensitivity and eye dryness ADA-Funded Research (P<0.05). Dry eye syndrome and corneal hypoesthesia are associated with peripheral & 664-P neuropathy in subjects with diabetes. Our data suggest that subjects with Differential Role and Regulation of AlphaA- and AlphaB-Crystallins diabetic peripheral neuropathy may be screened for eye dryness to prevent in the Retina during Diabetes corneal irritation and erosions. PATRICE E. FORT, Ann Arbor, MI The goal of this study was to characterize and compare the role and & 666-P regulation of alphaA- and alphaB-crystallin proteins in the retinal cells during Diabetic Retinopathy in the SEARCH for Diabetes in Youth Cohort: diabetes. We previously showed that alpha-crystallins are expressed in the A Pilot Study retina and over-expressed during diabetes. We also demonstrated that they ELIZABETH J. MAYER-DAVIS, RONALD KLEIN, BARBARA E. KLEIN, CRALEN DAVIS, have neuroprotective functions in retinal neurons but that these functions JINAN SAADDINE, RALPH B. D’AGOSTINO, JR., RONNY A. BELL, DANA DABELEA, could be altered by diabetes by a mechanism that remained unclear. LAWRENCE DOLAN, SEEMA GARG, JEAN M. LAWRENCE, BARBARA LINDER, Mice lacking either one, or both, of the alpha-crystallins were used to SANTICA M. MARCOVINA, CATHERINE PIHOKER, BEATRIZ L. RODRIGUEZ, Chapel analyze the regulation in the retina of the remaining crystallins as well as Hill, NC, Madison, WI, Winston-Salem, NC, Atlanta, GA, Aurora, CO, Cincinnati, OH, the impact on retinal cell survival in response to diabetes. Protein changes Pasadena, CA, Bethesda, MD, Seattle, WA, Honolulu, HI were analyzed using western-blot and immunohistochemistry analysis Current data are scant on the prevalence and correlates of diabetic while cell death was assessed using a DNA fragmentation Elisa. Alpha- retinopathy (DR) in youth. The SEARCH for Diabetes in Youth study comprises crystallins were isolated from retina of control and diabetic animal models the largest contemporary cohort of youth with diabetes in the US inclusive by immunoprecipitation methods and post-translational modifi cations were of youth diagnosed with DM younger than age 20 yrs whose diabetes was then characterized using mass spectrometry analysis. prevalent in 2001 or incident in the years thereafter. A pilot study to assess We demonstrated that the diabetes-induced crystallin upregulation is not DR was conducted from Dec 2009-Sept 2010 among 226 T1D (180 NHW, 46 a general stress response leading to the induction of all heat-shock proteins. other race/ethnicity) and 69 T2D (19 NHW, 50 other) with > 5 yrs DM duration It is rather an adaptive mechanism that becomes negated through different who participated in SEARCH. DR status was determined by grading of digital post-translational modifi cations. Using alpha-crystallin knock-out mice we fundus retinal photographs taken of both eyes using a non-mydriatic camera demonstrated that the absence of alphaA-crystallin led to a larger increase under a standardized protocol. DR severity status was based on the worse in retinal cell death during diabetes. We also showed that both alphaA- and eye and categorized as none, minimal non-proliferative DR (NPDR), mild- alphaB-crystallins are targeted for post-translational modifi cations in the moderate NPDR, or proliferative (PDR). The prevalence of any DR was 17% retina during diabetes. for T1D and 49% for T2D (OR=2.16, 95% CI 1.05, 4.47; p=0.04 adjusted for Using biochemical and proteomic method, we identifi ed and studied race/ethnicity and DM duration; see Figure). Among T1D and T2D, DR was specifi c alphaA- and alphaB-crystallin phosphorylation sites. less frequent in NHW than other race/ethnicities but was only statistically This study demonstrates the crucial but differential neuroprotective signifi cant in T1D (p=0.004). Concurrent data on A1C and SBP were available function that both alphaA- and alphaB-crystallin play in retinal degenerative for 225 T1D and 43 T2D (mean age, 16.8 yr (SD 4.6); duration 6.8 yrs (SD 1.0)). diseases such as diabetic retinop athy. This stud y also point s out the specifi cit y A1C was signifi cantly higher among those with DR (adjusted mean, 9.1) vs no of the response in regard to heat shock proteins as well as how this function DR (8.3) (p=0.01). SBP and duration of diabetes did not differ by DR status. is altered in these diseases. Understanding alpha-crystallins function and In conclusion, the overall prevalence of DR in this sample of youth was their regulation in these conditions could lead to the development of new substantial, particularly among youth with T2D and among minority youth. therapies. Further detailed study of DR in this contemporary cohort of individuals with Supported by: JDRF and Pennsylvania Lions Sight Conservation and Eye childhood onset T1 and T2D is required to advance our understanding of the Research frequency and correlates of this important microvascular complication.

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A182 COMPLICATIONS—OCULAR

of 3-month experiments. Retinas were analyzed for apoptosis by TUNEL staining and Roche Cell Death ELISA. Signaling pathways related to cell death and cell survival were assessed by kinase assays, microarray, qRT-PCR and immunohistochemistry. Phloridzin reduced systemic and ocular blood glucose levels but periocular insulin did not. Both treatments reduced cell death rates and restored insulin receptor and Akt1 kinase activity to control values. Local insulin partially reduced glial cell activation whereas phloridzin fully reversed this POSTERS

stress response. Local insulin normalized 10 of the 15 mRNAs of a diabetic Complications

retinopathy biomarker set while phloridzin reversed 8 mRNAs, only fi ve of Acute and Chronic which being common. These data indicate that increased ocular insulin signaling and reduced systemic glucose have direct and distinct effects on the development of neuroretinal defects in early diabetes, and suggest that both hormone and nutrient levels may infl uence the development of early diabetic retinopathy. Supported by: R01 EY020582 ADA-Funded Research

669-P Diabetic Retinopathy at Diagnosis of Type 2 Diabetes in Scotland Supported by: CDC/DDT and NIH/NIDDK HELEN C. LOOKER, DAVID CROMIE, ON BEHALF OF THE SCOTTISH DIABETIC RETINOPATHY SCREENING COLLABORATIVE, Dundee, United Kingdom, Hamilton, United Kingdom 667-P A nationwide Diabetic Retinopathy Screening (DRS) programme was A Pilot Study of Dissemination of Teleretinal Imaging To Limit launched in Scotland in 2006. The aim of this study was to assess the Disparities in Diabetic Retinopathy in Rural North Carolina success of this programme in delivering prompt screening to people with KATRINA E. DONAHUE, PAUL BRAY, DOYLE M. CUMMINGS, CHRISTINA OLLIS, newly diagnosed type 2 diabetes (T2D) and to document their prevalence MADELINE MITCHELL, RICHARD DAVIS, Chapel Hill, NC, Greenville, NC, Windsor, NC of retinopathy. In this process, we also studied a number of issues which Early detection and treatment of Diabetic Retinopathy (DR) can signifi cantly may have direct health policy implications, including the effect of delayed reduce severe vision loss. However DR screening rates remain low, especially screening, the effect of sex in the screening process, and the risk factors in areas where access to an ophthalmology specialist is diffi cult. Here we for early eye disease. examine the feasibility of teleretinal imaging via point of care screening for DR Data were extracted from the Scottish Care Information-Diabetes amongst population groups with type 2 diabetes, and the difference between Collaboration (SCI-DC), a clinical diabetes database which hosts information the primary care based evaluation of digital retinal photos compared to that on >99% of patients with diabetes in Scotland. It captures diabetes-related of an ophthalmologist. data, including body mass index (BMI), HbA1c, and blood pressure, from both Teleretinal imaging was placed in a rural primary care practice. Patients primary care and hospital clinics. with diabetes were offered a point of care digital imaging exam at their 46,739 people were diagnosed with T2D between January 1st 2005 and offi ce visit. A licensed practical nurse (LPN) trained by an ophthalmologist May 31st 2008. As of December 2009, the DRS has successfully screened screened the retinal images for DR based on the International Classifi cation 85.3% of the cohort. The median time from diagnosis to fi rst DRS screening of Diabetic Retinopathy and this reading was in parallel to a remote fell from 688 days for those diagnosed in 2005 (prior to the launch of the ophthalmologist. DRS) to 83 days for those diagnosed in 2008. Overall, 17.8% had evidence Of 87 patients offered screening, 82 consented (94.2%) and seventy-seven of diabetes-related eye disease at fi rst screening while the prevalence of (88.5%) underwent imaging. The eyes of 5 patients could not be dilated. The referable diabetic eye disease (retinopathy or maculopathy) was 1.9%. average age was 57.2 years (range: 26-86); 53.2% were female; 71.4% were Retinopathy at fi rst screening was associated with male sex, lower BMI, African American. Of the patients evaluated, 23.4% had abnormal retinal higher HbA1c, and higher systolic blood pressure at diagnosis. Longer time images requiring ophthalmologic referral, and over one-third of abnormal to screening was associated with a higher prevalence of diabetes related images showed moderate to severe retinopathy. Agreement in readings eye disease. People screened within a year of diagnosis had prevalences of between the LPN and ophthalmologist reader was assessed using weighted 17.1% for any retinopathy and 1.5% for referable diabetic eye disease versus kappa. The weighted kappa for the left eye ratings was .90 and .93 for the 18.7% and 2.2% respectively for people who were screened more than a right eye. Two patients (2.4%) rated as having mild NPDR by the LPN were year after diagnosis (p<0.0001 for both). assessed by the ophthalmologist as having no retinopathy. No patient having The DRS has improved time from diagnosis to screening for people with some degree of retinopathy was missed by the LPN. newly-diagnosed T2D. The overall prevalence of referable retinopathy at Retinal assessments within the primary care setting appear acceptable fi rst screening is lower than seen in past studies suggesting that diabetes is to patients and successful with the majority of those consented. A large now being screened for and diagnosed at an earlier stage than previously. number (23.4%) of patients evaluated had retinal disease. There was Supported by: Scottish Government, Wellcome Trust (Scottish Health Informatics excellent agreement in readings between the trained LPN and those of the Programme) ophthalmologist. This study shows the feasibility of point of care screening in identifi cation of patients from rural populations at signifi cant risk for DR. Supported by: National Center for Research Resources, #UL1RR025747 670-P Endothelium-Derived Reactive Oxygen and Nitrogen Species Impair Myogenic Tone in Type-2 Diabetic Rat Ophthalmic Artery 668-P YAGNA P. JARAJAPU, ISAMU ITO, DENNIS L. GUBERSKI, MARIA B. GRANT, Both Hyperglycemia and Insulin Defi ciency Contribute to Early Gainesville, FL, Tokyo, Japan, Worcester, MA Neural Defects in Diabetic Retinopathy Pressure-induced constriction or myogenic tone (MT) in arterioles and THOMAS W. GARDNER, PATRICE E. FORT, WILLARD M. FREEMAN, STEVEN F. small arteries, originates from vascular smooth muscle and is modulated ABCOUWER, Ann Arbor, MI, Hershey, PA by endothelium. MT determines the autoregulation of blood fl ow in The aim of this study was to investigate the separate contributions different vascular beds and has been shown to be altered in pathological of hyperglycemia and insulin defi ciency in the development of diabetic conditions such as diabetes. We have previously reported that myogenic retinopathy, by comparing the effects of blood glucose reduction following tone in ophthalmic artery is impaired in obese Type-2 diabetic BBZDR/Wor phloridzin administration to reduce hyperglycemia and subconjunctival rat (T2D). In this study, we have delineated the role of endothelium-derived insulin injections to increase local insulin signaling. reactive oxygen and nitrogen species in the attenuated myogenic response. Diabetic and non-diabetic control male Sprague-Dawley rats received Ophthalmic arteries were isolated from BBZDR/Wor rats with 4–5 months systemic insulin by subcutaneous implants, subconjunctival injections of duration of diabetes and the age-matched lean non-diabetic rats (control). insulin (0.03 U/100 g body weight) once daily, or intraperitoneal injections Arterial segments were cannulated in an arteriograph and inner diameter of phloridzin (0.2 mg/100 g body weight) twice daily for the last 4 days was continuously recorded over a range of intraluminal pressures. Arterial

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A183 COMPLICATIONS—OCULAR

segments were treated with pharmacological agents either intraluminally Fenofi bric acid (25 mmol/l and 100 mmol/l) was added (1 application/day) the (N-acetyl cysteine, diphenyliodonium, apocynin and ebselen) or abluminally last 3 days of the experiment. RPE permeability was evaluated by measuring (1400W, carbachol, bradykinin and L-NAME). Pressure-induced constriction apical-basolateral movements of FICT-dextran (40 kDa). The expression of in T2D arteries was signifi cantly lower compared to that in control-arteries tight junction proteins and AMPK phosphorylation was assessed by Western in the range of 50–150 mmHg. Dilations to carbachol or bradykinin were blot. Immunohistochemical studies of tight junction proteins and siRNA signifi cantly lower and constriction to L-NAME (non-specifi c nitric oxide transfection to AMPK were also performed in ARPE-19 monolayers. synthase inhibitor) was higher in T2D artery compared to the control at the Results: Treatment of ARPE-19 cells with fenofi bric acid signifi cantly intraluminal pressure of 70 mmHg. MT in T2D artery was restored to normal reduced the increment of permeability and the breakdown of the ARPE cell POSTERS

Complications over the pressure range of 50–150 mmHg with the intraluminal application monolayer induced by 25 mmol/l D-glucose + IL-1β (10 ng/ml) in a dose- Acute and Chronic of 1400W (10 µM). Similar effects were observed when T2D arteries were dependent manner. This effect was unrelated to changes in the content of treated intraluminally with a scavenger of reactive oxygen species, N-acetyl tight junction proteins. Fenofi bric acid prevented the activation of AMPK cysteine (10 µM), NADPH oxidase inhibitors diphenyliodonium or apocynin induced by IL-1β and the hyperpermeability induced by IL-1β was blocked (100 µM) or peroxynitrite scavenger ebselen (100 µM). These results support by silencing AMPK. that endothelial generation of superoxide by NADPH oxidase and NO by Conclusions: RPE disruption induced by IL-1β is prevented by fenofi bric iNOS was higher in T2D arteries. Peroxynitrite that was overproduced from acid due to its ability to suppress AMPK activation. This mechanism could be superoxide and NO, attenuates MT in smooth muscle and impairs pressure- involved in the benefi cial effects of fenofi brate on DME development. dependent autoregulation of blood fl ow in the ocular circulation of T2D. Supported by: ISCIII (CIBERDEM) and MCIN (SAF2009-07408) Supported by: EY 007739; EY012601 673-P 671-P Infl ammatory Cytokines and Diabetic Vascular Complication in Exendin-4 and GLP-1 Decreases TNF-α and Glycated Albumin In- Obese Korean Type 2 Diabetes duced Expression of ICAM-1, VCAM-1 and RAGE in Human Retinal JUNGJIN LEE, MOONSUK NAM, SO HUN KIM, SEONGBIN HONG, YONGSUNG Pigment Epithelial Cells (HRPE) KIM, Incheon, Republic of Korea MARIOLA DORECKA, TOMASZ M. FRANCUZ, WOJCIECH GARCZORZ, WANDA Diabetes and obesity are the major culprit of various vascular complica- ROMANIUK, JAN GMINSKI, Katowice, Poland tions. Chronic low grade infl ammation may be a pivotal link between There is a growing body of evidence that advanced glycation end products obesity and diabetes. Most of the studies of development and pro gression (AGE) through their receptor (RAGE) plays an important role in developing of diabetic vascular complications were done in obese type 2 diabetic of ocular complications in diabetes. HRPE plays an important role in retinal patients, which makes it diffi cult to differentiate the effect of obesity homeostasis. The aim of presented study was to investigate whether GLP- and type 2 diabetes on vascular complication. We compared the effects 1 and Exendin-4 can prevent the deleterious effects of AGE on HRPE. In of adipokines on vascular complications in type 2 diabetic patients with our study we measured RAGE, ICAM-1 and VCAM-1 expression in HRPE and without obesity. 761 subjects with type 2 diabetes recruited from the stimulated with TNF-α (as an potent infl ammatory inducer) and glycated outpatient clinic at Inha university hospital diabetes center, were divided albumin (GlyAlb). into two groups – non-obese group (body mass index (BMI)<25 kg/m2 ) Cells were stimulated for 24h with TNF-α (2,5pg/L) or GlyAlb (500mg/L) and obese group (BMI≥25 kg/m2). Circulating infl ammatory cytokines-total and co-treated with GLP-1 (10 and 100nM) or Exendin-4 (1 or 10nM). Next, adiponectin, HMW adiponectin, tumor necrosis factor-α (TNF-α) and IL-6 cells were harvested, and cell lysates were used for determining RAGE were measured. Severity of retinopathy and nephropathy and carotid intima protein expression; ICAM-1 and VCAM-1 were determined in cell culture media thickness and presence of carotid plaque(s) were investigated. Obese medium by ELISA. type 2 diabetes had signifi cantly and lower total, HMW adiponectin and As expected, we have found that TNF-α slightly increased (14%, p<0.05), higher hs-CRP compared with non-obese diabetic patients. In the obese whereas GlyAlb decreased (25%, p<0.01) RAGE expression in compare to group, levels of total and HMW adiponectin were signifi cantly higher in control group. In cells co-treated with GLP-1 or Exendin-4 the results were as patients with macroalbuminuria compared with normoalbuminuria and follows: GLP-1 decreased RAGE expression by 40% (p<0.01) in TNF-α treated levels of total adiponectin, HMW adiponectin and TNF-α were signifi cantly cells, and by 10% (p<0.05) in GlyAlb group; Exendin-4 decreased RAGE by higher in patients with proliferative retinopathy compared with no apparent 44% (p<0.01) and 33% respectively (p<0.01). ICAM-1 was decreased by retinopathy after adjustment of covariates. Only IL-6 was signifi cantly higher 21% in GLP-1 group, and by 32% in Exendin-4 group (both results were in patients with proliferative retinopathy in non-obese group. There were statistically signifi cant). VCAM-1 expression was lowered by 15% (p<0.05) no signifi cant difference of carotid IMT and presence of plaque between and 27% (p<0.01) in GLP-1 and Exendin-4 treated groups respectively. In all two groups. In obese group, logistic regression analysis revealed that among tested groups Exendin-4 has exerted much more potent effect than GLP-1. infl ammatory adipokines, only total adiponectin signifi cantly infl uenced on Observed effects of GLP-1 and Exendin-4 can play an important role proliferative retinopathy (OR= 1.209, p= 0.038). In obese type 2 diabetic in prevention of diabetes induced cytotoxity on HRPE cells. Decreased patients, total and HMW adiponectin and TNF-α were associated with expression of RAGE can make cells more resistant to circulating AGEs, and proliferative retinopathy. Among them, total adiponectin had infl uence on decreased expression of adhesion molecules (ICAM-1, VCAM-1) can be the proliferative retinopathy. Further studies are needed to test the signifi cance result of anti-infl ammatory properties of incretins and decreased expression of these fi ndings. of RAGE. Both effects suggest that tested incretins may improve the HRPE cell dysfunction associated with development of microvascular diabetic 674-P complications. RAGE/ERK Mediates AdvancedWITHDRAWN Glycation End Products-BSA- Induced Bovine Retinal Pericytes Migration 672-P KI MO KIM, JUNGHYUN KIM, NAN HEE KIM, CHAN-SIK KIM, JIN SOOK KIM, Fenofi bric Acid Prevents Retinal Pigment Epithelium Disruption Daejeon, Republic of Korea Induced by Interleukin-1β by Supressing AMPK Activation Although the migration of retinal pericytes contributes to the development RAFAEL SIMO, MARTA VILLARROEL, MARTA GARCIA-RAMÍREZ, LIDIA CORRALIZA, of diabetic retinal pericytes loss, the signal regulating migration of these CRISTINA HERNÁNDEZ, Barcelona, Spain cells is poorly understood. In this study, we investigated that bovine Aims: Diabetic macular edema (DME) is the main cause of visual impairment retinal pericytes (BRP) migration is stimulated by advanced glycation end for diabetic patients. Vascular leakage caused by the breakdown of the products-modifi ed bovine serum albumin (AGE-BSA) through receptor for blood retinal barrier (BRB) is the main event involved in the pathogenesis of advanced-glycation end products (RAGE) and extracellular signal-regulated DME. In the FIELD study on diabetic retinopathy, treatment with fenofi brate kinase (ERK) pathways. The impact of AGE-BSA performed by intravitreal (a PPAR-a agonist) reduced the need for laser treatment for DME by 30%. injection to mice eye showed that pericytes migration and levels of focal However, the mechanisms involved in the benefi cial effects of fenofi brate adhesion kinase (FAK), and paxillin were increased. Bovine retinal pericytes on the development and progression of DME remain to be elucidated. To treated with AGE-BSA also increased the levels of receptor for advanced- shed light on this issue we have explored the effect of fenofi bric acid on the glycation end product (RAGE) mRNA and protein, and vascular endothelial barrier function of human retinal pigment epithelial (RPE) cells. growth factor (VEGF) mRNA. These increases of AGE-BSA-induced pericytes Methods: ARPE-19 cells (a human RPE line) were cultured for 18 days under migration were inhibited by the PD98058 (ERK1/2 specifi c inhibitor) excluding standard conditions and under conditions leading to the disruption of the p38 inhibitor. Furthermore, AGE-BSA increased focal adhesion kinase (FAK) monolayer [25 mmol/l D-glucose + IL-1β (10 ng/ml) added at days 16 and 17]. and paxillin phosphorylations in a dose and time dependent manners which

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A184 DIABETIC DYSLIPIDEMIA were attenuated by PD98059 and ERK1/2 small interfering RNA (siRNA). Our time dependent change in A1c, MBG, or A1c statistically controlled for MBG data suggest that AGE-BSA may induce the migration of pericytes by RAGE- over the course of the DCCT. Development or progression of retinopathy was a ERK-FAK-paxillin signaling pathways. sustained change from baseline of three steps in the Early Diabetic Retinopathy Treatment Study score at any retinal exam during the DCCT. Multiple paired A1c and MBGs were obtained at study visits from each participant. In a model unadjusted for MBG, a 1 unit (%) difference in A1c was associated with an increase of 51% in the cumulative risk of retinopathy (p=0.0004). In a model containing A1c and MBG simultaneously, thus POSTERS

statistically removing A1c from its dependence on MBG, a 1 unit (%) increase Complications

in A1c was associated with a 42% increase in retinopathy risk (p=0.0004). Acute and Chronic Thus indicating that A1c retained most of its association with retinopathy when adjusted for MBG. MBG was also an independent predictor, a 50 mg/dL increase being associated with 10% increase in retinopathy risk (p=0.004). HGI had an intra-class correlation of 90% with A1c adjusted for MBG. Thus A: Pretreatment of PD98058 (ERK1/2 specifi c inhibitor) inhibits AGE- HGI is virtually synonymous with MBG-independent variation in A1c. BSA-induced FAK and paxillin phosphorylation, but not p38 inhibitor. B: We conclude 1) Variation in A1c includes both MBG-dependent and ERK1/2 Knock down cells inhibited AGE-BSA-stimulated FAK and paxillin MBG-independent components; 2) Risk of retinopathy is predicted by both phosphorylation. MBG-independent and MBG-dependent components; 3) HGI is a practical way to facilitate study of the clinical implications of the MBG-independent Supported by: Grants [L09030, K10040] from the Korea Institute of Oriental component of A1c. Medicine 677-P 675-P Tyrosine Nitration of Prostacyclin Synthase Is Associated with En- Serum Angiogenin Infl uences Progression of Diabetic Retinopathy hanced Retinal Cell Apoptosis in Diabetes MARTIN URSLI, CLEMENS HOEBAUS, SIMON BRUNNER, RENATE KOPPEN- HONGLIANG LI, MING-HUI ZOU, CHAOYONG HE, MINGKAI LIN, TIMOTHY J. STEINER, JOHANNA-MARIA BRIX, GUNTRAM SCHERNTHANER, GERIT-HOLGER LYONS, ZHONGLIN XIE, Oklahoma City, OK SCHERNTHANER, Vienna, Austria Loss of retinal pericytes is central to the development of diabetic Diabetic Retinopathy (DR) is the leading cause of blindness in working- retinopathy. The underlying mechanism, however, is not fully understood. age individuals in the developed world. Since the detailed mechanisms There is evidence that the risk of diabetic retinopathy is associated with conducting the aberrant retinal neovascularization are still unknown, we the presence of both oxidative stress and toxic eicosanoids. Whether investigated infl ammatory and angiogenic proteins under suspicion to play oxidative stress actually causes diabetic retinopathy via the generation of a decisive role in DR. toxic eicosanoids, however, remains unknown. The aim of the present study Serum levels of Vascular Endothelial Growth Factor (VEGF), Angiogenin was to determine whether tyrosine nitration of prostacyclin synthase (PGIS) (ANG), Brain Derived Neurotrophic Factor (BDNF) and Interleukin-18 (IL-18) contributes to retinal cell death in cultured human retinal pericytes and the were measured in 193 patients with Diabetes mellitus (DM, HbA1c 8.0±1.7 retinas of diabetic Akita mice. We observed oxidative stress, PGIS nitration, rel.%, BMI 27.1±4.9 kg/m2) and 66 age-and sex matched healthy controls (CO, and retinal cell apoptosis in human retinal pericytes treated with heavily BMI 26.2±5.2 kg/m2) by ELISA. All DM patients underwent eye-examination oxidized and glycated low-density lipoprotein (HOG-LDL) and in Akita mice and staging for retinopathy: 66 non-proliferative DR (NPDR), 37 proliferative treated with tempol. Exposure of human retinal pericytes to HOG-LDL, but DR (PDR), 90 without DR (WDR). not native forms of low-density lipoprotein (N-LDL), for 24 h signifi cantly Patients with DM had lower serum ANG levels compared to CO (343 increased pericyte apoptosis, accompanied by increased tyrosine nitration [215-400] vs 410 [237-489] ng/ml). Highest levels of ANG were measured of PGIS and decreased PGIS activity. Inhibition of the thromboxane receptor in patients with PDR, and were signifi cant higher compared to WDR (438.0 or cyclooxygenase-2 dramatically attenuated HOG-LDL-induced apoptosis [262-472] vs 323.9 [192-398] ng/ml, p=0.009), as well as NPDR (438.0 [262- without restoring PGIS activity. Administration of superoxide dismutase (to 472] vs 316.9 [215-356] ng/ml, p=0.011). VEGF levels were signifi cant higher scavenge superoxide anions) or L-NG-Nitroarginine methyl ester (L-NAME, in patients with PDR compared to WDR (490.5 [319-578] vs 402.3 [205-542] a non-selective nitric oxide synthase inhibitor) restored PGIS activity and ng/ml, p=0.045). Correlation analysis of retinopathy staging and all available attenuated HOG-LDL-enhanced pericyte apoptosis. In Akita mouse retinas, quantitative variables revealed VEGF (R=0.146, p=0.043), ANG (R=0.169, diabetes induced oxidative stress, as evidenced by increased intraretinal p=0.019) and DM Duration (R=0.271, p<0.001) as signifi cant associates. A oxidized LDL content, 3-nitrotyrosine formation, and PGIS nitration. logistic regression for the difference between WDR and PDR, demonstrated Diabetes also enhanced apoptotic cell death and impaired blood–retinal DM Duration (odds: 1.057, p=0.008) and ANG (odds: 9.383, p=0.022) as only barrier function in Akita mouse retinas. Chronic administration of tempol, signifi cant predictors. VEGF, BDNF and IL-18 did not infl uence relations. a superoxide scavenger, reduced intraretinal oxidized LDL staining, iNOS Subsequent calculations resulted in an 88% increase of PDR per 100ng/ml induction, PGIS nitration, and retina cell apoptosis, and increased the serum ANG increase. integrity of blood-retinal barriers in Akita mice. These results suggest that Anti-VEGF therapy proves to be effective in patients with DR. However, oxidized LDL-mediated PGIS nitration and associated thromboxane receptor by far not all patients are thereby saved from this threatening diabetes stimulation might be important in the initiation and progression of diabetic complication. We are the fi rst to demonstrate that Angiogenin seems to be retinopathy. involved as well. In our patient cohort Angiogenin, but not VEGF withstood Supported by: NIH, OCAST ADA-Funded Research multivariate modeling.

676-P DIABETIC DYSLIPIDEMIA The Importance of the Mean Blood Glucose (MBG)-Independent Component of Hemoglobin A1c (A1c) for Developement of Retinopathy [See also: Presidents Posters 392-PP to 393-PP, page A108.] in the Diabetes Control and Complications Trial (DCCT) ROBERT J. MCCARTER, SHAUNA BUTLER, JAMES M. HEMPE, STUART A. CHALEW, Washington, DC, New Orleans, LA Guided Audio Tour: Lipid Metabolism (Posters 678-P to 685-P), see A1c and MBG are highly correlated and frequently used interchangeably page 13. as assessments of glycemia. However, we found that in addition to the & 678-P infl uence of MBG, variation in the level of A1c also has an MBG-independent Mammalian Cyclin-Dependent Kinase-8 Regulates Lipid Metabo- component which can be quantifi ed using a Hemoglobin Glycation Index lism by Suppressing Lipogenic Gene Expression (HGI). HGI controlled for MBG was predictive for the development of XIAOPING ZHAO, DAORONG FENG, ELLEN S. YANG, JEFFREY E. PESSIN, FAJUN retinopathy and nephropathy in the DCCT(Diabetes Care 27: 1259, 2004). The YANG, Bronx, NY HGI concept has been challenged (Diabetes 56: 1913, 2007) which prompted Transcriptional cofactors are critically involved in the precise control of us to reexamine the hypothesis with this new analysis. gene expression, and thus play important roles in human diseases, including We used Cox Proportional Hazards Regression to examine the relationship diabetes. One such cofactor is the multi-subunit protein complex named between development or worsening of diabetic retinopathy as a function of Mediator, which bridges gene activator signaling to the transcription

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A185 DIABETIC DYSLIPIDEMIA

apparatus. Cyclin-dependent kinase-8 (CDK8) is a subunit of the Mediator was kept comparatively. Compared to non DM model, WT mice increased complex. However, the biological functions of CDK8 remain poorly under- plasma TG (5.7 fold), FFA (2.6 fold), beta-hydroxybutyric acid (11.2 fold) stood. Here, we identify CDK8 as a negative regulator of lipogenic gene remarkably, however HSL KO mice increased these moderately (1.6-2.9 fold). expression by suppressing the SREBP-1c transcription factor, which is a key This difference was observed clearly under ad libitum feeding. The analysis activator of rate-limiting enzymes in the de novo lipogenesis (DNL) pathway. of northern blotting or quantitative PCR showed that in diabetic WT mice, CDK8 knockdown in hepatocytes results in SREBP-dependent up-regulation the expression of adipose triglyceride lipase (ATGL) which was a potent TG of lipogenic genes and an increase of lipid accumulation. Interestingly, CDK8 lipase in WAT increased to about 2 fold, and the expression of lipoprotein can phosphorylate SREBP-1c, regulating its stability. Knocking down CDK8 lipase (LPL) which contributed to lipid uptake to adipocyte decreased about POSTERS

Complications in mouse livers in vivo also leads to increased expression of SREBP-target 60%. However both those were hardly changed in diabetic HSL KO mice. The

Acute and Chronic genes. As a result, hepatic CDK8 knockdown causes acceleration of DNL and comparison between MF diet (5.3% fat content) and fat free diet revealed accumulation of lipids in mouse livers. Strikingly, hepatic CDK8 knockdown in that only diabetic WT mice fed MF diet increased plasma TG remarkably. mice also results in a dramatic increase of plasma triglycerides. Consistent It was suspected that diabetic dyslipidemia depended on a meal greatly, with the repressive role of CDK8 in DNL, hepatic CDK8 proteins in mouse and HSL contributed to intestinal lipid absorption. These results conclude, models of obesity and insulin resistance are signifi cantly lower than normal. in the condition with impaired insulin action, HSL reduces WAT weight via Dysregulation of lipid metabolism causes fatty liver and dyslipidemia, not only one’s own lipolytic ability, but also cooperating with ATGL and which are prevalent health problems in developed countries and are closely LPL. In addition, HSL causes dyslipidemia via accelerating intestinal lipid associated with insulin resistance and type 2 diabetes. Thus, CDK8 may play absorption. a role in the development of fatty liver, dyslipidemia and diabetes. & 681-P & 679-P Evaluation of 4-Hydroxynonenal Modifi ed LDL as a Marker of Plaque Inhibition of Ceramide Synthesis Reduces Plasma Lipids, Adiposity Progression in Type 2 Diabetes Mellitus and Hepatic Lipogenesis in the Insulin Resistant/Hyperlipidemic RYO SAIKI, RIE TADOKORO, TORU IIZAKA, KENJI IWAKU, SHOTARO SATO, Hamster Model FUMIKO OOTSUKA, YASUYOSHI TAKAHASHI, YOSHIHIKO SUZUKI, TSUTOMU MARK J. DEKKER, CHRIS L. BAKER, MARK NAPLES, KHOSROW ADELI, Toronto, HIRANO, MATSUO TANIYAMA, Yokohama, Japan, Tokyo, Japan ON, Canada 4-hydroxynonenal (4HNE) is a lipidperoxidation product and known as Recent evidence indicates that ceramides play an important role in a specifi c marker of oxidative stress. Its cytotoxicity has been considered regulating the insulin resistant phenotype observed following high fat diet to be a great risk of atherosclerosis. 4HNE modifi ed LDL (4HNE-LDL) is one feeding. However, less is known about the regulatory role of ceramides on of oxidized LDLs but its clinical application has not been matured. This in vivo lipid metabolism following a lipogenic diet. In this study, we have study was conducted to assess the utility of 4HNE-LDL for the marker of employed our well characterized high fat, high fructose, high cholesterol atherosclerosis and to compare with the other atherosclerotic markers in (FFC) hamster model of dyslipidemia and insulin resistance. To study the patients with type 2 diabetes mellitus (T2DM). role of ceramides, we treated chow and FFC fed hamsters vehicle or 0.3 Ninety-fi ve middle aged patients with T2DM with a mean age of mg/kg myriocin, a potent inhibitor of ceramide synthesis, by intraperitoneal 54.7±0.99 years were enrolled. Elderly subjects aged over 65 were excluded. injection every other day for 2 weeks after which plasma and tissue samples Ultrasonography (US) of the carotid arteries was performed to examine the were collected. Although body mass was not different between groups, progression of atherosclerosis; mean carotid intima media thickness (CIMT), FFC diet increased (p<0.050) epididymal fat pad mass while treatment with max CIMT were measured, and stenosis was assessed by ECST method. myriocin decreased (p<0.05) fat pad mass in both dietary groups. Myriocin Furthermore, as a major marker of atherosclerosis, LDL, malondialdehyde signifi cantly lowered hepatic ceramide concentrations (p<0.05). Myriocin modifi ed LDL, small dense LDL and 4HNE-LDL were determined after signifi cantly lowered plasma TG and cholesterol (p<0.05) in both chow overnight fasting. 4HNE-LDL was measured by sandwich ELISA assay using and FFC hamsters. Although FFC signifi cantly increased hepatic TG and monoclonal antibody to 4HNE and polyclonal antibodies to apoB. Spearman cholesterol (p<0.05), these lipids were not lowered by myriocin treatment. correlation coeffi cient (r) was used to evaluate the correlation between Real-time PCR evaluation of the de novo lipogenic transcription factor SREBP- the serum markers and the results of US. Multiple liner regression analysis 1c showed an increase with FFC feeding and a normalization to near chow (MRA) was performed with stenosis. levels with myriocin treatment (p<0.05). Hepatic expression of SCD-1 was No signifi cant relations were found between mean CIMT and atherosclerotic signifi cantly increased with FFC treatment and in chow + myriocin hamsters markers. In max CIMT and stenosis, there was signifi cant association only (p<0.05). Taken together, these preliminary data suggest that 1) inhibition with 4HNE-LDL (r=0.23, p=0.03; r=0.24, p=0.03 respectively). Moreover of ceramide synthesis can reduce circulating lipids in hamsters following in subjects without administration of antioxidative agent pioglitazone chow and FFC which may suggest that lipid secretion is reduced following (n=71), the association was more apparent (r=0.27, p=0.02; r=0.30, p=0.01 myriocin treatment 2) myriocin can reduce adiposity without altering body respectively). In MRA, 4HNE-LDL had a highest standard regression mass following chow and FFC 3) Hepatic lipids are not reduced with myriocin coeffi cient (0.29) with stenosis. treatment, but SREBP-1c mRNA is normalized with myriocin, suggesting that Mean CIMT is a major indicator of the progression of atherosclerosis de novo lipogenesis is decreased. which is progressed with aging, but it showed no signifi cant relation to atherosclerotic markers in this study. On the other hand max CIMT and & 680-P stenosis represents plaque size and contributes to the early progression of Hormone-Sensitive Lipase Contributes to Diabetic Weight Loss atherosclerosis. Our fi ndings suggests that these markers, especially 4HNE- Via Cooperating with ATGLWITHDRAWN and LPL, and Causes Dyslipidemia Via LDL, are available marker for the early progression of atherosclerosis. Intestinal Lipid Absorption MIKIO TAKANASHI, MOTOHIRO SEKIYA, JUN-ICHI OSUGA, KEISUKE OHTA, & 682-P YOKO IIZUKA, KEN OHASHI, HIROAKI OKAZAKI, NOBUHIRO YAMADA, HITOSHI Effects of GLP-1 and GLP-2 Co-Administration on Regulation of SHIMANO, SHUN ISHIBASHI, NAOYA YAHAGI, TAKASHI KADOWAKI, Tokyo, Intestinal Lipid Metabolism and Chylomicron Production in Syrian Japan, Tochigi, Japan, Tsukuba, Japan Golden Hamsters Hormone-sensitivity lipase (HSL) catalyzes the hydrolysis of triacylglycerol GUSTAVO J. HEIN, CHRISTOPHER L. BAKER, JOANNE HSIEH, KHOSROW ADELI, (TG), and is regulated by insulin. It is reported that plasma TG or free fatty Toronto, ON, Canada acid (FFA) concentration of HSL defi ciency (HSL KO) mice is low compared to Postprandial hypertriglyceridemia and hyper-chylomicronemia are mayor wild type (WT) mice. In the condition with impaired insulin action such as complications of insulin resistant states. Glucagons-like peptides (GLP-1) type 1 diabetes mellitus (DM), body weight loss, dyslipidemia, and ketosis and GLP-2 are derived from post-translational processing of the proglucagon are observed. However, why such symptoms occur is still not elucidated polypeptide and are co-secreted in equimolar amounts. There is evidence suffi ciently. We expect that HSL is involved in these symptoms because indicating that while GLP-1 exerts an acute suppressive effect, GLP-2 that works for lipolysis and FFA supply. We examined to seek novel roles triggers marked stimulation of fatty acid absorption and chylomicron (CM) or involvement level of HSL by using streptozotocin (STZ) induced DM production. The interplay between these two peptides in the regulation of model. WT mice lost 90% of white adipose tissue (WAT) weight within 7 intestinal CM is intriguing, but their relative contribution to the CM pathway days after STZ injection. Interestingly, WAT weight of HSL KO mice was is currently unknown. To determine the contribution of GLP-1 and GLP-2 to 50% remained. Histological examination showed adipocyte of diabetic regulation of intestinal lipid metabolism and CM production, we analyzed WT mice miniaturized remarkably, but cell size of diabetic HSL KO mice the effects on the in vivo production of ApoB48, triglyceride-rich lipoprotein

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A186 DIABETIC DYSLIPIDEMIA

(TRL)-triglycerides and TRL-cholesterol levels and fatty acid absorption after sitagliptin signifi cantly increased macrophage-derived 3H-cholesterol fecal an intravenous (IV) infusion of physiological levels of GLP-1, GLP-2 or both excretion by 39%. To investigate whether sitagliptin promotes RCT through in Syrian golden hamsters. GLP-1 decreased intestinal ApoB48 production, a reduction of intestinal cholesterol absorption, 14C-cholesterol labelled TRL-triglycerides and TRL-cholesterol (p<0.05) and decreased fatty acid olive oil was then administered orally and 14C-tracer plasma appearance absorption (p<0.05), while GLP-2 increased these parameters (p<0.05). On was measured over 6 hours. A signifi cant reduction of 14C-tracer plasma the other hand, these parameters were signifi cant increased (p<0.05) with appearance over time was observed with sitagliptin, indicating that this simultaneous infusion of GLP-1 and GLP-2. Peripheral co-administration of drug may promote RCT through lower intestinal cholesterol absorption. both peptides reveals a predominant role for GLP-2 promote CM production In conclusion, sitagliptin stimulates RCT in CETP-apoB100 transgenic POSTERS

(late phase: 60-120 minutes) when co-infused with GLP-1 at equimolar mice. This suggests that DPP-4 inhibition might be benefi cial in preventing Complications concentrations. cardiovascular risk in type 2 diabetes. Acute and Chronic This would suggest that physiologically, GLP-1 effect seems to be respon- sible for inhibiting CM production in early stages while GLP-2 increases CM & 685-P production over a prolonged period. Small Molecule Dual FABP4/5 Inhibitors Ameliorate Dyslipidemia in Mice with Diet-Induced Obesity & 683-P HONG LAN, CLIFF C. CHENG, TIMOTHY J. KOWALSKI, LING PANG, LIXIN SHAN, A1 Adenosine Receptors Do Not Play a Signifi cant Role in the CHENG-CHI CHUANG, JAMES JACKSON, ALBERTO ROJAS-TRIANA, LORETTA Regulation of Lipogenic Gene Expression in Hepatocytes BOBER, LI LIU, JOHANNES VOIGT, PETER ORTH, XIANSHU YANG, GERALD W. MING YANG, RUTH CHU, JEFFREY W. CHISHOLM, HOLGER DOEGE, LUIZ BELAR- SHIPPS, JOSEPH A. HEDRICK, Kenilworth, NJ, Cambridge, MA DI NELLI, ARVINDER K. DHALLA, Palo Alto, CA FABP4 and FABP5 are two closely related fatty acid binding proteins Activation of A1 adenosine receptors (A1AdoRs) in adipose tissue inhibits expressed primarily in adipose tissue and macrophage. Mice with double lipolysis and lowers circulating free fatty acid (FFA) levels. Given the role of FFA deletion of Fabp4 and Fabp5 genes are resistant to diet-induced obesity (DIO), in the pathophysiology of insulin resistance, it has been suggested that FFA- dyslipidemia, insulin resistance and type 2 diabetes. A small molecule FABP4 lowering by A1 agonists would have considerable clinical benefi ts. However, inhibitor BMS309403 was previously reported to improve insulin sensitivity recently it was reported that activation of A1AdoRs leads to increase in fatty in leptin-defi cient Lepob/Lepob (ob/ob) mice. However, the compound was acid synthesis in AML-12 cells, a mouse hepatic cell line, by increasing the not extensively characterized in the more physiologically relevant DIO mice. gene expression of sterol regulatory binding protein 1c (SREBP-1c) and fatty Here, we report the discovery of a novel series of FABP4/5 dual inhibitors, acid synthase (FAS). Because these fi ndings have important implications for represented by Compounds 1-3. These compounds are triazolopyrimidine drug discovery, we sought to examine the role of A1AdoRs in the regulation derivatives without a carboxylic acid moiety. Compared with BMS309403, of lipogenic gene expression and to characterize the expression of A1AdoRs the compounds had signifi cant in vitro potency towards both FABP4 and in human and mouse livers and multiple hepatic cell lines, including AML-12, FABP5. In cell-based assays, BMS309403 and Compounds 2, 3 inhibited HepG2, Huh7 and Hep3B. Relative gene expression levels were measured basal and isoproterenol-stimulated lipolysis in 3T3 L1 adipocytes and in by quantitative RT-PCR. cAMP production was measured by a DiscoveRx primary human adipocytes, with the dual inhibitors being slightly more assay. Radioligand binding assays with membranes from cells or livers potent. They also inhibited basal and lipopolysaccharide (LPS)-stimulated were performed using [3H]-DPCPX. Treatment of both AML-12 and HepG2 MCP-1 release from THP-1 macrophage and primary human macrophage. cells with CPA (a full A1 agonist) or GS-9667 (a partial A1 agonist) for 24h When chronically administered to DIO mice, BMS309403 or Compound 3 caused no increase in either SREBP-1c or FAS gene expression, although reduced plasma triglyceride and free fatty acid levels, but no signifi cant treatment with TO901317 (LRX agonist) and 9-cis-retinoic acid (RXR agonist) change was observed on insulin, glucose or glucose tolerance. Compound 3 strongly induced the expression of both genes. In AML-12 and HepG2 cells, reduced plasma free fatty acids at all three dose levels (3 mg/kg, 10 mg/kg CPA was unable to antagonize the forskolin-stimulated cAMP production, and 30 mg/kg), while the effect of BMS309403 was only observed at 30 mg/ a characteristic of A1AdoR activation. Consistent with this fi nding, A1AdoR kg. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia mRNA and protein levels were absent or very low in the hepatic cell lines in DIO mice, and that dual FABP4/5 inhibition appears to be more effective used in this study. Radioligand binding studies also showed very low A1AdoR than selective FABP4 inhibition. expression in mouse and human livers. Finally, in in vivo studies, treatment Supported by: Merck with GS-9667 for 6h did not increase SREBP-1c or FAS gene expression in livers of Sprague Dawley rats. Taken together, we conclude that the 686-P expression levels of A1AdoRs in hepatic cells are insuffi cient to function as a “Intra-Individual Variability” of Fasting Lipids in Primary Hyper- major regulator of hepatic lipogenesis. lipidemia Is Associated with Fatty Liver, Hyperapobetalipo protein- emia and Advanced Coronary Artery Disease, but Not with Insulin & 684-P Resistance DPP-4 Inhibitor Sitagliptin Improves Reverse Cholesterol Transport ATSUSHI NOHARA, KUNIMASA YAGI, TOHRU NOGUCHI, HAYATO TADA, MIKA through Reduced Intestinal Cholesterol Absorption in Obese Insulin MORI, CHIAKI NAKANISHI, MASA-AKI KAWASHIRI, AKIHIRO INAZU, JUNJI Resistant CETP-apoB100 Transgenic Mice KOBAYASHI, MASAKAZU YAMAGISHI, HIROSHI MABUCHI, Kanazawa, Japan FRANCOIS BRIAND, RÉMY BURCELIN, THIERRY SULPICE, Labege, France, Toulouse, Intra-individual variability (IIV) of lipid-phenotype is most common criterion France for diagnosis of familial combined hyperlipidemia (FCHL) in many countries, Type 2 diabetes is characterized by lower HDL-cholesterol (HDL-c) levels, but backgrounds of IIV are not well described. It is well known that subjects which would impair macrophage-to-feces reverse cholesterol transport with FCHL are frequently accompanied by insulin resistance. (RCT) and thus increase cardiovascular risk. Dipeptidyl peptidase-4 (DPP- Methods: Two hundreds and fi fty fi ve patients (M/F=127/128, mean age 4) inhibitors improve glycemic control in type 2 diabetes, but their benefi ts 58.3 ± 14 ys) with primary hyperlipidemia were investigated. Lipid profi les on RCT remain to be determined. We therefore evaluated the effects of with ultracentrifugation, HOMA-R, HOMA-beta, oral glucose insulin DPP-4 inhibitor sitagliptin on RCT in obese insulin resistant CETP-apoB100 sensitivity index (OGIS) with 75gOGTT, post-heparin lipoprotein lipase transgenic mice, which exhibit a human-like lipoprotein profi le. activities (PHLA), and FCHL associated SNP in USF1 gene (rs3737787) were CETP-apoB100 mice were made obese, insulin resistant and dyslipidemic analyzed. Coronary angiography was performed in 121 patients, and 2 to with a 60% high fat diet over 3 months. Mice were then treated over 4 weeks 3vessels with >75% stenosis was defi ned as advanced coronary artery with sitagliptin 500mg/kg/day in drinking water or vehicle (n=7/group). Two disease (CAD). IIV positive (+) was defi ned as change among type IIA, IIB, other groups of mice were also treated with vehicle or metformin 300mg/kg/ and IV lipoprotein phenotype in fasting without lipid lowering drugs. day orally as a reference compound. Results: IIV(+) was 87 patients (34%), and showed no gender difference. Both metformin and sitagliptin improved oral glucose tolerance and Compared with IIV(-) primary hyperlipidemia, plasma apoB (144 ± 25 vs. 127 signifi cantly reduced hyperglycemia, hyperinsulinemia and HbA1c levels. ± 36mg/dL, p<0.0001) and frequencies of fatty liver with ultrasonography Both drugs had no effect on total cholesterol and HDL-c levels, but sitagliptin (59 vs 38%, p<0.05) were signifi cantly higher in IIV(+). BMI, HbA1c, HOMA- reduced triglycerides levels by 25% (p<0.01 vs. vehicle). Sitagliptin, but not indexes, OGIS, and PHLA were not signifi cant. Risk allele of USF1 SNP was metformin, increased fecal cholesterol mass excretion by 132% (p<0.001 vs. associated with signifi cant increase of HbA1c and HOMA-R, and decrease vehicle), suggesting a potent effect on cholesterol metabolism. of OGIS, but not with IIV. Familial history of hyperlipidemia was strongly To c o n fi r m t his e f f e c t in v i v o, mic e w e r e t h e n inje c t e d i.p. w i t h 3H-cholesterol associated with IIV(+), and presence of type IIB hyperlipidemia in the family labelled macrophages to measure RCT over 48 hours. Compared to vehicle, was higher in patients with IIV(+) (40% vs. 10%, p<0.0001). Mean HbA1c was

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A187 DIABETIC DYSLIPIDEMIA

6.1 ± 1.2%, and 21% was diagnosed as diabetes mellitus with 75gOGTT, but history of hypertension (69.4% vs. 47.4%, p=0.001). Total cholesterol (TC) exclusion of these subjects did not signifi cantly change results. Advanced (4.76±0.91mmol/L vs. 4.52±0.83mmol/L, p=0.04), triglycerides(TG) [1.51 CAD was more frequent in IIV(+) compared with IIV(-) (49 vs. 26%, p<0.05). (1.08-2.35)mmol/L vs 1.27 (0.93-2.06)mmol/L, p=0.028], HbA1c [6.8 (6.1- Conclusion: Presence of IIV was strongly associated with hyperapobeta- 7.68)% vs 6.4 (5.55-7.45)%, p=0.024] and urinary albumin creatinine ratio lipoproteinemia, presumably based on hepatic apoB overproduction, and can (UAC) [17.52 (7.38- 61.01)mg/mmol vs 9.07 (3.84-55.69)mg/mmol, p=0.045] be a noteworthy sign of susceptibility to CAD. Insulin resistance seems not were higher in PSQ patients. Patients with pain-disturbed sleep (n=32) had a to be the fundamental background of this characteristic phenotype of FCHL. higher PSQI score (8.7±4.7 vs. 5.6±4.2, p=0.001). When these were excluded from the analysis, TC (4.81± 0.94mmol/L vs 4.51±0.83mmol/L, p=0.018), TG POSTERS Complications 687-P [1.49 (1.11-2.28)mmol/L vs 1.25 (0.92–2.07)mmol/L, p=0.026] and HbA1c [6.8 Acute and Chronic Age, Abdominal Obesity, and Baseline hsCRP Are Associated with (6.05–7.7)% vs 6.4 (5.6–7.3)%, p=0.029) remained signifi cantly higher in PSQ LDL-C, Non-HDL-C and ApoB Responses to Lipid-Lowering Therapy patients. Patients were divided into short-sleep (3 to 5 hours, n=39), mid- in Patients with Metabolic Syndrome and Moderately High/High range sleep (6 to 8 hours, n=149) and long-sleep duration (> 8 hours, n=21). CHD Risk TC was highest in long sleep (5.02±0.96mmol/L) followed by short-sleep JENNIFER G. ROBINSON, CHRISTIE M. BALLANTYNE, WILLA A. HSUEH, JEFFREY (4.94±1.07mmol/L) and mid-range sleep duration (4.5±0.8mmol/L), p=0.002. B. ROSEN, JIANXIN LIN, ARVIND K. SHAH, JOANNE E. TOMASSINI, ROBERT S. TG were highest in short-sleep [1.87 (1.26-2.56)mmol/L] followed by long- LOWE, ANDREW M. TERSHAKOVEC, Iowa City, IA, Houston, TX, Coral Gables, FL, sleep [1.45 (0.99–2.24)mmol/L] and mid-range sleep duration [1.27 (0.94– Whitehouse Station, NJ 2.05)mmol/L], p=0.023. In summary, sleep duration and/or quality may have Metabolic syndrome (MetS) is associated with increased cardiometabolic an impact on diabetes control and lipid profi le in Caucasians with diabetes. risk (CMR). LDL-C, non-HDL-C and ApoB treatment targets have been There appears to be a U-shaped relationship between sleep duration and TC recommended for management of dyslipidemia in MetS patients with CMR, & TG. We conclude that consideration of sleep quality and duration may be however response to therapy may be infl uenced by patient demographic and useful as part of the overall diabetes management programme. baseline (BL) characteristics. This post hoc analysis of a multicenter, double- blind, randomized, 6-wk parallel group study in >1000 hypercholesterolemic 689-P MetS patients with moderately high/high risk; designed to identify factors Comparison of High-Density Lipoprotein Subspecies between Type 1 affecting treatment effi cacy of ezetimibe/simvastatin (E/S) or atorvastatin Diabetes and Type 2 Diabetes without/with Intensive Insulin Therapy (A). Treatment was a signifi cant predictor for change in all variables, with TOMOYASU FUKUI, TSUTOMU HIRANO, Tokyo, Japan E/S >A for many. Age ≥65, abdominal obesity, and lower BL hsCRP were The reduced levels of high-density lipoprotein (HDL)2-cholesterol in signifi cant predictors for reductions in LDL-C, non-HDL-C, ApoB, total C, diabetes and other metabolic disorders associated with a high risk of triglycerides (TG) and VLDL, but not for changes in HDL-C or Apo AI. Age cardiovascular disease are well established. Few studies, however, have ≥65 was associated with signifi cantly greater attainment of LDL-C and non- compared the HDL subspecies in type 1 diabetes (T1D) with those in type HDL-C targets; abdominal obesity and lower BL hs-CRP also had an effect. 2 diabetes (T2D) treated with insulin or oral anti-diabetic agents (OAD). We MetS, blood pressure, fasting glucose, HOMA IR tertiles, race and diabetes examined the concentrations of HDL2 and HDL3 in T1D patients treated with were not predictive of any treatment response, and effects of gender, BL TG insulin (M/F:17/10), 33 T2D patients treated with insulin alone or insulin plus or HDL-C were limited. In conclusion, this post hoc study of at risk patients OAD (M/F:19/14), 36 T2D patients treated with OAD alone (M/F:21/15), and with MetS identifi ed age ≥65, abdominal obesity, and lower BL hsCRP as key 25 non-diabetic subjects (M/F:11/14). Insulin was injected four times daily in factors which signifi cantly infl uence changes in LDL-C, non-HDL-C and ApoB, a basal-bolus manner for both T1D and T2D. Subjects treated with fi brates or the current treatment targets for reduction of CMR. The clinical benefi t of niacin were excluded. Plasma levels of cholesterol (C), apolipoprotein (apo) ezetimibe has not been proven. AI, and AII were determined in HDL2 and HDL3 by the single precipitation method, and small dense LDL-C was measured by a homogenous method both established by our group. HDL-C levels were signifi cantly higher in T1D (77±21mg/dl) and lower in T2D (50±16), compared with the controls (63±15). Insulin-treated T2D had higher HDL-C than T2D treated with OAD alone (59±16 vs.43±10). T1D had higher HDL2-C and HDL2-apo AI levels (51±16 and 105±16) than the controls (38±15 and 88±28) or T2D (25±13 and 69±28). Insulin-treated T2D had higher HDL2-C and -AI levels than T2D without insulin therapy (33±13 vs.18±8 and 85±25 vs.54±23). HDL3 levels were comparable among controls, T1D, and T2D with or without insulin therapy. Thus, the HDL2/HDL3 ratio was the highest in T1D and the lowest in T2D treated with OAD. HDL2-C and -AI levels were both inversely associated with triglyceride, small dense LDL-C, LDL, HbA1c, and BMI, and positively associated with insulin therapy. Multiple regression analysis revealed that insulin therapy is independently associated with HDL2. In conclusion, T1D had remarkably higher HDL2 levels, and insulin-treated T2D had higher HDL2 levels than T2D without insulin therapy. These results suggest that intensive insulin therapy has favorable effects on HDL metabolism, irrespective of the presence or absence of insulin resistance.

690-P Double-Blind Randomized Clinical Trial of the Effects of Ezetimibe 688-P on Postprandial Hyperlipidaemia and Hyperglycaemia Association between Poor Sleep Quality and Cardiovascular Risk KAORI KIKUCHI, YASUO TERAUCHI, Yokohama, Japan Factors in Diabetes Ezetimibe inhibits cholesterol absorption, thereby lowering serum WAN AIZAD WAN MAHMOOD, MOHD SHAZLI DRAMAN, LUCY ANN BEHAN, cholesterol, however its effect on glucose tolerance, insulin sensitivity and TOMMY KYAW TUN, JOHN MCDERMOTT, SEAMUS SREENAN, Blanchardstown, postprandial hypertriglyceridaemia in human is not understood. Ireland We investigated the effects of ezetimibe on postprandial hyperlipidaemia Sleep restriction has been shown to contribute to reduced glucose and hyperglycaemia in obese subjects with dyslipidaemia in a double-blind tolerance and insulin sensitivity in non-diabetic subjects and to impact on randomized crossover trial. 20 obese men (BMI ≥ 25 or waist circumference HbA1c in African Americans with type 2 diabetes (T2DM). We used the ≥ 85cm) whose triglyceride(TG) level was 150-400 mg/dl were assigned Pittsburgh Sleep Quality Index questionnaire (PSQI score) to assess sleep randomly to ezetimibe- or placebo-precedence treated group. Subjects in quality in 241 Caucasian patients with diabetes. Blood pressure, fasting the ezetimibe group took ezetimibe 10mg/day for the fi rst 4 weeks, followed lipids and HbA1c were also measured. Looking at the whole group, 133 by 4 week interval and placebo for another 4 week. The placebo group took (55%) had good sleep quality (GSQ, score ≤5) and 108 (45%) had poor sleep in reverse order. For the study, the subjects were requested to fast for at quality (PSQ, score >5). There were higher proportion of female in the PSQ least 12 hours and received a standard meal (1300kcal; protein:48g; fat:50g; group, 57.4% vs 38.3%, p=0.003. PSQ patients were more likely to have a carbohydrate:170g). Blood samples were collected at 0, 0.5, 1, 2, 4, 6 and

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A188 DIABETIC DYSLIPIDEMIA

8 h after the start of the meal at Day 0, 28, 56, 84 for measuring lipid and between cholesterol absorption markers and LDL-C levels. After 12 weeks glucose metabolism markers. All data were analyzed by determining paired treatment in Ez monotherapy, signifi cant reduction of LDL-C (159→130 t test with the SPSS software. mg/dl), RLP-C (6.1→5.1 mg/dl), hs-CRP (592→488 ng/ml), and cholesterol Ezetimibe signifi cantly decreased postprandial serum TG excursion (p=0.01) absorption markers, and signifi cant increase of HDL-C (58→61 mg/dl) and and fasting serum LDL-cholesterol, RLP and ApoB48 (p<0.05). Postprandial rathosterol (2.3→3.5 mg/dl) were observed. Since FPG and A1C did not glucose excursion and serum GIP was not affected by ezetimibe signifi cantly, change, IRI (7.8→6.6 µU/ml) and HOMA-R (1.9→1.6) decreased signifi cantly but postprandial serum insulin levels tended to be reduced (p=0.08). These after Ez treatment. In patients with high basal ALT (≥32 IU/L) levels, ALT and results were consistent with the animal results that ezetimibe improved AST/ALT ratio were signifi cantly decreased and increased, respectively after POSTERS insulin resistance. Reduction of LDL-cholesterol and RLP were regarded as Ez treatment, while those changed oppositely in patients with normal basal Complications the results of the inhibited cholesterol absorption. Reduction of ApoB48 ALT levels. IRI and HOMA-R tended to decrease in patients with high ALT Acute and Chronic may be associated with a decrease in chylomicron generation, however the levels. A signifi cant negative correlation between change of AST/ALT ratio reduction of postprandial serum TG excursion by ezetimibe was not fully and HOMA-R was observed. Insulin sensitivity was improved especially in explained by the minimally decreased chylomicron level, because the TG the patients with liver dysfunction and low AST/ALT ratio, suggesting that Ez peak was 4 h after the start of the meal. Release of VLDL from liver may also improves insulin sensitivity through the improvement of fatty liver. be involved in this alteration. In conclusion, ezetimibe restored postprandial dysregulation of the lipid and glucose metabolism in RCT. We will also report 693-P the impact of ezetimibe on the oxidized cholesterol profi le during the meal Fenugreek with Reduced Bitterness Prevents Diet-Induced Metabolic tolerance test. Disorders in Rats ETSUKO MURAKI, HIROSHIGE CHIBA, SHOHEI HOSHINO, KEIKO TAKETANI, 691-P NOBUAKI TSUGE, YASUHIRO TAKENOUCHI, NOBUYO TSUNODA, KEIZO KASONO, Evaluation of Abnormal HDL Function in Patients with and without Keyakidai, Sakado, Japan, Takanodai, Yotsukaido, Japan Type I Diabetes Mellitus Several therapeutic effects of Fenugreek (Trigonella foenum-graecum; FS) STEPHANIE SMOOKE PRAW, CECILIA MORGANTINI, ANDREW J. DREXLER, on metabolic disorders have been reported. However, the bitterness of FS ALAN M. FOGELMAN, SRINIVASA T. REDDY, Los Angeles, CA, Pisa, Italy prevents human to eat suffi cient dose for achieving therapeutic effects. This Type I Diabetes Mellitus (TIDM) has long been associated with an increased study was undertaken to evaluate the effects of FS with reduced bitterness risk of coronary artery disease (CAD) despite the presence of higher than (FRB) on metabolic disorders in rats. expected levels of high-density lipoproteins (HDL), thought to be protective FS contains bitter saponins such as protodioscin. FRB was prepared by under normal circumstances. The anti-infl ammatory characteristics of HDL treating FS with beta-glucosidase. Forty SD rats were fed with high-fat high- can become dysfunctional in states of chronic disease and lead to alterations sucrose (HFS) diet for 12 wk to induce mild glucose and lipid disorders. Then, in three risk factors of CAD - reverse cholesterol transport, oxidation of low rats were divided into 5 groups. In the experiment 1, each group (n = 8) was density lipoproteins (LDL), and vascular infl ammation. The present study fed with the HFS, HFS containing 2.4% FS, 1.2, 2.4 and 4.8% FRB for 12 wk. In was designed to determine the status of HDL function in patients with TIDM the experiment 2, we examined the effects of lower dose of FRB (0.12, 0.24, when compared to healthy controls. Forty patients with Type I Diabetes 1.2%) using the same protocol (n = 7 in each groups). Mellitus and 16 age and sex-matched controls were studied. The ability of In the experiment 1, FRB decreased food intake, body weight gain, the patient’s HDL to prevent oxidation of normal LDL (cLDL) was measured epididymal white adipose tissue and soleus muscle weight in a dose- by a cell free assay in which the change in fl uorescence intensity resulting dependent manner. FRB also decreased plasma and hepatic lipid levels from oxidation of DCF-DA by LDL in the presence or absence of test HDL is and increased fecal lipid levels in a dose-dependent manner. Plasma total quantifi ed. Values for the fl uorescence intensity of DCFH activated by LDL cholesterol levels (mmol/L) in three FRB and HFS groups were 1.58 ± 0.09, cholesterol alone were normalized to 1.0 to create the HDL Infl ammatory 1.45 ± 0.05*, 1.29 ± 0.07*, 2.00 ±0.18, respectively (*; p<0.05 vs. HFS). Index (HII). Patients with and without TIDM were well-matched in terms of Hepatic total cholesterol levels (mmol/g liver weight) were 0.116 ± 0.011, age (37 vs. 33 years), BMI (25.4 vs. 23 kg/m2), gender and cholesterol profi les. 0.112 ± 0.006, 0.099 ± 0.007*, 0.144 ± 0.012, respectively (*; p<0.05 vs. HFS). Of the patients with TIDM, 16% had retinopathy, 5% neuropathy and 12% The calculated insulin resistance indices, plasma insulin level x fasting blood nephropathy with an average HbA1c 7.7%. HDL infl ammatory index was not glucose level (the relative value to normal rats) were 0.81 ± 0.07*, 0.74 ± signifi cantly different between diabetic patients and healthy controls, (0.25 0.10*, 0.69 ± 0.08*, 1.71 ± 0.24, respectively (*; p<0.05 vs. HFS). Any FRB ±0.18 vs. 0.20±0.12 HDL/c LDL, p=n.s). There was no signifi cant difference groups did not show the adverse effect on the liver, kidney and hematological in HDL function between patients and healthy controls. The protective functions. In the experiment 2, no signifi cant difference of food intake was function of HDL remained intact. The absence of pro-infl ammatory HDL in observed, whereas only 1.2% FRB group showed almost same effects on our patients may be a refl ection of patient’s age distribution (relatively young glucose and lipid metabolism as in the experiment 1. <40), duration of TIDM (17 ± 11 years), well-controlled TIDM, and absence We demonstrated that FRB (1.2∼4.8%) prevented diet-induced metabolic of cardiovascular complications. In the future, we will study TIDM patients disorders including insulin resistance, dyslipidemia and fatty liver. >40 years of age to determine whether an alteration of HDL function exists in TIDM. 694-P Impaired Generation of 12-Hydroxylated Bile Acids Links Hepatic 692-P Insulin Signaling with Dyslipidemia Ezetimibe Improves Serum Lipid Profi les and Insulin Sensitivity in REBECCA A. HAEUSLER, DOMENICO ACCILI, New York, NY Hypercholesterolemia Patients The association of insulin-resistant (type 2) diabetes with elevated YUTA MORINAGA, HIROAKI UENO, KAZUHIRO NAGAMINE, WAKABA TSUCHI- plasma triglyceride and very low-density lipoproteins, and intrahepatic MOCHI, NOBORU TAMAKI, TOMOMI SHIIYA, MASAMITSU NAKAZATO, Kiyotake, lipid accumulation represents a pathophysiological enigma and an unmet Miyazaki, Japan therapeutic challenge. Recently it has come to light that defective lipid Since Ezetimibe (Ez) has began to use for hypercholesterolemia, its metabolism in type 2 diabetes may be due to a detrimental admixture of treatment effects including pleiotropic effect have not been fully elucidated. insulin resistance and insulin sensitivity in selective hepatic signaling To evaluate the effects of Ez on lipid and glucose metabolism in Japanese pathways. FoxO1 is a transcription factor that is tightly regulated by insulin, patients with high serum low-density lipoprotein-cholesterol (LDL-C) level, and it has an established role in regulating the glucose production branch of we investigated physical and blood examinations before and 12 weeks hepatic insulin signaling. Mice lacking FoxO1 in liver (L-FoxO1) are insulin- after Ez treatment. The including criteria of patients were over 20 years-old sensitive and display low glucose production. outpatients and hyper LDL-cholesterolemia. Before and after Ez treatment, Here we show that, surprisingly, when challenged with a western diet, fasting plasma glucose (FPG), insulin (IRI), A1C, HOMA-R, serum lipid profi les L-FoxO1 mice have considerable defects of lipid metabolism including [LDL-C, triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), steatosis and, when crossed with Ldlr-/- mice, high serum lipids. Using remnant-like particle cholesterol (RLP-C)], high sensitivity C-reactive protein gene expression and metabolomic analyses, we identifi ed alterations in (hs-CRP), liver function (AST, ALT, γ-GTP), cholesterol absorption markers bile acid composition in L-FoxO1 mice, with a defi ciency in production of (sitosterol, campesterol, cholestanol), and cholesterol synthesis marker 12-hydroxylated bile acids. As bile acids are known to affect triglyceride (rathosterol) were measured. We studied 95 patients of Ez monotherapy and and cholesterol homeostasis, we hypothesized that the lipid metabolic 16 patients of Ez plus statin. Plasma rathosterol level was signifi cantly lower abnormalities were due to impaired feedback inhibition of lipid metabolism in Ez plus statin than Ez monotherapy. There were positive correlations by bile acids. Supplementing L-FoxO1 mice with the major product of

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A189 DIABETIC DYSLIPIDEMIA

12-hydroxylation, cholic acid, rescued their hyperlipidemia and gene were randomized 1:1 to twice-daily (BID) SITA/MET or MET for 18 wks. SITA/ expression defects. This work reveals a novel mechanism by which insulin MET and MET were up-titrated over 4 wks to 50/1000 mg BID and 1000 mg regulates triglyceride and cholesterol metabolism through its ability to BID, respectively. At Wk 18, a higher percentage of pts in the SITA/MET modulate bile acid composition. group had A1C levels of ≤6.5% and <7% than those in the MET group within each baseline A1C subgroup (table). Of pts who initiated SITA/MET with 695-P a baseline A1C of >7.5-9.0%, 48.6% achieved an A1C of ≤6.5% at Wk 18 Impaired Postprandial Suppression of Endogenous VLDL-TG Secretion compared with 23.1% of pts who initiated MET monotherapy (p<0.001). In pts with a baseline A1C of >9.0%, 24.0% on SITA/MET achieved an A1C of POSTERS in Type 2 Diabetic Men Complications ESBEN SØNDERGAARD, LARS P. SØRENSEN, IBEN RAHBEK, LARS C. GORMSEN, ≤6.5% compared with 12.8% on MET (p<0.001). In summary, in pts with a Acute and Chronic JENS S. CHRISTIANSEN, SØREN NIELSEN, Aarhus, Denmark baseline A1C of >7.5-9.0%, substantially more pts achieved the AACE/ACE Hypertriglyceridemia is a prominent feature of diabetic dyslipidemia A1C goal of ≤6.5% with initial treatment with dual therapy (SITA/MET) than and strongly related to diabetes comorbidity. Chronic hyperinsulinemia in with initial monotherapy (MET), in agreement with the AACE/ACE treatment type 2 diabetic patients is associated with increased VLDL-TG secretion. algorithm. In contrast, acute hyperinsulinemia leads to a rapid inhibition of hepatic BBaselineaseline AA1C1C ≤77.5%.5% BBaselineaseline AA1C>7.5-9.0%1C>7.5-9.0% BBaselineaseline >>9.0%9.0% VLDL-TG secretion. This has been demonstrated in hyperinsulinemic clamp studies, but the effect of physiological hyperinsulinemia after a meal remains WWkk 1818 WWkk 1818 WWkk 1818 WWkk 1188 WWkk 1818 WWkk 1188 unknown. We studied 8 type 2 diabetic men and 8 healthy men were studied [[N]N] A1CA1C≤66.5%.5% AA1C<7%1C<7% [[N]N] A1CA1C≤66.5%.5% AA1C<7%1C<7% [[N]N] A1CA1C≤66.5%.5% AA1C<7%1C<7% after ingestion of a fat-free test meal (40% of REE). VLDL-TG kinetics was TTreatmentreatment n ((%)%) n ((%)%) n ((%)%) n (%)(%) n ((%)%) n (%)(%) assessed using a primed-constant infusion of ex-vivo labeled [1-14C]triolein VLDL-TG using non-steady-state calculations. As expected, type 2 diabetic MMETET [[37]37] 1155 ((40.5)40.5) 2299 ((78.4)78.4) [[182]182] 4422 ((23.1)23.1) 8855 ((46.7)46.7) [[345]345] 4444 ((12.8)12.8) 9 ((22.9)22.9) men had higher basal VLDL-TG concentration [Diabetic men: 1.1±0.4 mmol/L; SSITA/METITA/MET [[35]35] 2244 ((68.6)*68.6)* 3300 ((85.7)85.7) [[183]183] 8899 ((48.6)**48.6)** 112727 ((69.4)**69.4)** [[341]341] 8822 ((24.0)**24.0)** 111818 ((34.6)**34.6)** Healthy men: 0.4±0.3 mmol/L (p<0.01)] and secretion rate [Diabetic men: **p<0.01p<0.01 vvss MMET;ET; ***p<0.001*p<0.001 vvss MMETET 137±61 μmol/min; Healthy men: 78±30 μmol/min (p=0.03)]. Postprandially, VLDL-TG concentration differed with an increase in diabetic men and a decrease in healthy men (p=0.02). VLDL-TG secretion rate decreased 697-P postprandially in healthy men (p<0.01), but remained unchanged in diabetic Insulin Sensitivity Modifi es the Relationship between Thyroid men (p=0.47). The difference in VLDL-TG secretion over time was barely Function and Lipid Profi le in Euthyroid Type 1 Diabetic Patients signifi cant between the two groups (p=0.06) (Figure). The relative change in TOMISLAV BULUM, LEA DUVNJAK, NIKICA CAR, Zagreb, Croatia VLDL-TG secretion rate between the two groups was signifi cantly different Overt hypothyroidism, as well as subclinical hypothyroidism, is associated (p=0.01). Basal VLDL-TG clearance was signifi cantly lower in diabetic men with dyslipidemia and increased atherosclerotic vascular disease. It has [Diabetic men: 133(49-390) ml/min; Healthy controls: 215(137-933) ml/ been suggested that this association might be present already at the stage min (p<0.05)]. After meal ingestion clearance decreased in healthy men of normal thyroid function through altered insulin sensitivity. To investigate (p<0.01), but was unchanged in diabetic men (p=1.00). We conclude, that the complex interplay between insulin resistance and lipid profi le in type 1 type 2 diabetic men have a reduced postprandial suppression of VLDL-TG diabetes we divided 304 patients with normal thyroid function according to secretion and a lower VLDL-TG clearance compared to healthy men, which median estimated glucose disposal rate (eGDR=9.72 mgkg-1min-1) into lower both contribute to the increased postprandial lipidemia. (n=153) and higher-insulin sensitivity (n=151) group. None showed signs of adrenal, thyroid, renal or cardiovascular disease and received drugs, apart from insulin, that could attenuate glucose metabolism, insulin resistance or thyroid function. Estimated glucose disposal rate was calculated using the equation: eGDR=24.3-(12.22xWHR)-(3.29xHT)-(0.57xHbA1c). The units were mgkg-1min-1, WHR=waist to hip ratio, HT=hypertension. TSH, fT3 and fT4 were determined by fl uoroimmunoassay. Cholesterol and triglycerides in serum were measured by an enzymatic colorimetric method. Patients with lower insulin sensitivity (eGDR=7.67±1.39 mgkg-1min-1) in comparison to patients with higher insulin sensitivity (eGDR=11.01±0.80 mgkg-1min-1) showed signifi cantly higher total, LDL, VLDL cholesterol (5.23 vs 4.83; 2.98 vs 2.64; 0.5 vs 0.35 mmol/L, p< 0,001), and triglycerides level (1.1 vs 0.76 mmol/L, p<0.001) and lower HDL cholesterol level (1.58 vs 1.76 mmol/L, p< 0.001). In a multiple regression analysis in subjects with lower insulin sensitivity TSH was independently associated with total (β=0.221, p=0.008), and LDL-cholesterol (β=0.160, p=0.023). The independent relation of eGDR with TSH and total and LDL cholesterol suggests that insulin sensitivity might infl uence thyroid function already at the normal stage. Whether the detection of increased TSH level within the normal range in insulin resistant type 1 diabetic patients has predictive value for development of dyslipidemia and increased cardiovascular risk needs to be assessed in further follow-up studies. 696-P Initial Therapy with the Fixed-Dose Combination (FDC) of Sitagliptin ™ 698-P and Metformin (JANUMETWITHDRAWN) in Patients with Type 2 Diabetes Lipoproteins Measured by Conventional Methods vs. Nuclear Mellitus (T2DM) Provided Superior Glycemic Control vs. Metformin Magnetic Resonance (NMR) Spectroscopy and Association with Alone, Based on the AACE/ACE Diabetes Algorithm Glucose Tolerance Status: The Insulin Resistance Atherosclerosis THOMAS L. SECK, SAMUEL S. ENGEL, GREGORY T. GOLM, ALAN G. MEEHAN, Study (IRAS) KEITH D. KAUFMAN, BARRY J. GOLDSTEIN, Rahway, NJ ANDREAS FESTA, CARLOS LORENZO, LYNNE E. WAGENKNECHT, ANTHONY J.G. The AACE/ACE diabetes treatment algorithm recommends a stratifi ed HANLEY, STEVEN M. HAFFNER, Vienna, Austria, San Antonio, TX, Winston-Salem, approach to initial therapy to achieve an A1C goal of ≤6.5% in pts with NC, Toronto, ON, Canada T2DM who have inadequate glycemic control on diet and exercise alone: Lipoprotein abnormalities increase cardiovascular disease (CVD) risk. monotherapy for A1C ≤7.5%; dual therapy for A1C >7.5-9.0%; and dual or The extent of lipoprotein abnormalities may be better assessed by NMR triple therapy (if asymptomatic) or insulin (if symptomatic) for A1C >9.0%. spectroscopy than conventional methods. To determine CVD risk conferred We conducted a post hoc analysis of a large, randomized, double-blind study by lipoprotein abnormalities in relation to glucose tolerance (GT) status, we comparing initial dual therapy with sitagliptin and metformin (SITA/MET; measured lipoproteins and apolipoproteins by conventional methods and administered as an FDC tablet) to initial monotherapy with metformin (MET) by NMR spectroscopy in 1,386 patients in the IRAS who were not taking in drug-naïve pts with T2DM (A1C ≥7.5%) to examine A1C goal attainment lipid-lowering medications. GT status was defi ned by the 2003 ADA criteria based on these stratifi ed A1C categories. 1250 pts (mean baseline A1C=9.9%) and the WHO criteria. One-way ANCOVA was used to determine statistical

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A190 DIABETIC DYSLIPIDEMIA differences after adjusting for age, sex, ethnicity and center. LDL cholesterol did not differ by GT status (Table). Increased triglyceride concentration was associated with IGT, but a wider range of alterations was detected by NMR spectroscopy such as higher small LDL and large VLDL particles and increased VLDL size. Apolipoprotein B and total LDL particles were increased in individuals with isolated IFG but not in those with isolated IGT. HDL cholesterol and large HDL particles were signifi cantly decreased in the “IFG+IGT” and diabetes categories (DM). Further adjustment for BMI POSTERS and insulin resistance as measured by FSIGT explained little variance in Complications the lipoproteins associated with GT status, except for HDL cholesterol. In Acute and Chronic summary, NMR spectroscopy detects atherogenic lipoprotein abnormalities that are not apparent with a standard lipid panel. The type of lipoprotein abnormalities in IGT as compared with IFG may partially explain the greater CVD risk associated with IGT. The “IFG+IGT” and “DM” have, to a large extent, a similar lipoprotein profi le.

Supported by: Abbott

700-P Low-Density Lipoprotein Particle Number (LDL-P) Distribution in Type 2 Diabetes at LDL Cholesterol (LDL-C) < 50 mg/dL RAY POURFARZIB, DAVID G. ROBERTSON, Raleigh, NC, Atlanta, GA Background: Many patients with diabetes have relatively normal levels of LDL cholesterol (LDL-C) yet have increased numbers of atherogenic lipoproteins (LDL-P) leading to residual risk. Differences in lipoprotein subclasses with type 2 diabetes (T2DM) who have achieved very low levels Data are means. of LDL-C (Less than 50 mg/dL) have not been extensively examined. Normal glucose tolerance as reference for comparison: * p <0.05; † p <0.01; Objective: The aim of this study was to assess variations in lipids, ‡ p <0.001. lipoprotein particle concentration in diabetic patients. Supported by: NHLBI and GCRC Grants Methods: Data were selected from a large single laboratory database. Cases were patients with a T2DM diagnosis code. Lipoprotein particle 699-P concentrations were analyzed by nuclear magnetic resonance (NMR) Long-Term Effi cacy and Safety of Fenofi bric Acid in Combination with spectroscopy. Lipids (calculated LDL-C) were measured at a central Statins in Patients with Mixed Dyslipidemia and Type 2 Diabetes laboratory certifi ed for lipid analysis by the Standardization Program of the Mellitus Centers for Disease Control. MAUREEN T. KELLY, KAMLESH THAKKER, CAROLYN M. SETZE, INDERJIT MANDAIR, Results: Among the 1,970 patients with T2DM, the mean age was 60.62 PATRICK AUBONNET, DARRYL J. SLEEP, Abbott Park, IL, Allschwil, Switzerland for males and 63.22 for females (51.2% male). At LDL-C concentrations of less Mixed dyslipidemia (MD) characterized by elevated LDL-C and triglycerides than 50 mg/dL (triglyceride less than 150 mg/dL and high-density lipoprotein (TG) and reduced HDL-C is common in patients with type 2 diabetes cholesterol (HDL-C) greater than 40 mg/dL), the LDL-P concentration mellitus (T2DM). Short-term treatment with fenofi bric acid (FA) + statin distributions were 41% less than 700 nmol/L, 45% between 700 to 1000 nmol/L, comprehensively improves multiple lipid parameters more effectively than 12% between 1001 to 1300 nmol/L, and 2% greater than 1301 nmol/L. either monotherapy in patients with MD and T2DM. The long-term effi cacy Conclusion: Despite attainment of LDL cholesterol goals less than 50 mg/ and safety of FA + statin in this population was assessed. dL, these patients retained considerable residual coronary heart disease risk, Patients included had MD and T2DM, completed 12 weeks treatment with 59% of them having LDL-P concentration greater than 700 nmol/L. in 1 of 3 controlled studies (evaluating FA + statin vs corresponding-dose monotherapies), and subsequently were treated with open-label FA 135mg + 701-P moderate-dose statin (simvastatin 40mg, atorvastatin 40mg, or rosuvastatin Niacin Extended-Release/Simvastatin (2000/40 mg) Combination 20mg) for 52 weeks. Effi cacy measurements were calculated from baseline Therapy Produces Larger Favorable Changes in Lipoprotein Particles (start of the controlled studies) to specifi c time points across the controlled Compared to Atorvastatin Monotherapy (40 mg) and open-label studies. Adverse events occurring during either the controlled PETER TOTH, THAO T. DOAN, PING JIANG, Sterling, IL, Abbott Park, IL studies or the extension study were included in the safety analysis. Atherogenic dyslipidemia, in patients with insulin resistance and A total of 413 patients with MD and T2DM received combination therapy diabetes and characterized by multiple abnormalities in lipoprotein particle and were included in this analysis. Treatment with FA + statin led to sustained concentration and size, is associated with increased risk for coronary heart improvements in all effi cacy variables (Figure 1). Mean % changes from disease (CHD). Studies have shown a strong negative correlation between baseline to week 64 were: LDL-C (-43.0), HDL-C (+17.9), non-HDL-C (-47.5), high-density lipoprotein particle (HDL-P) concentration/large particle size Total-C (-37.7), VLDL-C (-50.0), and ApoB (-43.8). Median % changes from and risk for CHD. A strong association has also been shown between high baseline to week 64 in TG and hsCRP were -50.3 and -32.6, respectively. levels of small LDL-P and CHD risk. Overall, 117 (28.3%) patients experienced treatment-related adverse events This is a post-hoc analysis of 137 patients with dyslipidemia from a 12wk, (AEs), and 49 (11.9%) patients discontinued therapy due to an AE. No cases randomized, study of patients treated with daily NER/simvastatin (NER/S, of rhabdomyolysis or treatment-related deaths were reported. Simcor) (N=74, 1000/40 mg x 4 wks; 2000/40 mg x 8 wks) or atorvastatin (Atv, Long-term therapy with FA + statin resulted in sustained improvements Lipitor) (N=63, 40 mg x 12 wks). 137 patients had particle concentration and in multiple lipid values and was generally well tolerated in patients with size measurements at baseline (BL) and 12wk by NMR spectroscopy. MD and T2DM. Patients in both treatment groups had comparable particle values at BL. Treatment with NER/S resulted in a greater increase in large HDL-P concentration (P=0.078), signifi cantly greater decrease in small HDL-P concentration (P<0.05), and signifi cantly greater increase in HDL-P size (P<0.001), HDL-C and apoA-I vs Atv. Treatment with NER/S also signifi cantly decreased the concentration of total LDL-P (P<0.05) and small LDL-P (P<0.05) and signifi cantly increased LDL-P size (P<0.01) vs Atv (Table). Compared with Atv 40 mg monotherapy, combination therapy NER/S at 2000/40 mg results in multiple favorable changes in both HDL and LDL metrics. These data show that NER/S treatment promotes a greater shift toward less atherogenic lipoprotein subclasses than Atv.

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A191 DIABETIC DYSLIPIDEMIA

Table. Median % Change from BL at Wk 12 µM) for 1-12hs, cell viability assay (CCK-8) showed 4-HNE induced a time NER/S Atv and dose-dependent cytotoxic effect, 4-HNE at 40 µM caused 31.5±3.5%, N=74 N=63 69.6±4.6% reduction in cell viability at 1, 6hr compared with SFM-treated % % cells (assigned as 100%, n=4, p<0.05). Pretreated with berberine (0.5, 1, 5 HDL-P µM, 1h) then exposed to 4-HNE (40 µM for 6h) improved cell viability by 13.9- 21.8% vs. 4-HNE treatment alone (n=3, p<0.05). In addition, pretreatment of Total 10.4 6.4 AMPK inhibitor-Compound C (10 µM, 1h), then berberine (5 µM, 1h) and 4-HNE Small -1.8* 4.2 (40 µM, 6h) attenuated the benefi cial effects of berberine on cell viability in POSTERS

Complications Medium -26.9 -32.8 4-HNE-treated cells. In whole cell extracts, and in nuclear and cytoplasmic Acute and Chronic Large 102.4 39.2 preparations, analyses of AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor α (PPARα), ERK1/2 and NFκB were performed HDL-P size 6.0*** 1.3 by western blot in berberine and 4-HNE treated cells with or without HDL-C totala 33.6*** 11.4 Compound C pretreatment, NFκB was detected by immunofl uorescence. Apo-AIa 10.1*** 1.4 Berberine promoted AMPK and ERK1/2 phosphorylation, down-regulated nuclear NF- B expression, and up-regulated PPAR translocation in a dose- Total LDL-Pa -51.6* -42.7 κ α dependent manner, and these effects were partially blocked by Compound a Small LDL-P -55.0* -44.7 C. The fi ndings suggest that berberine has protective effects on 4-HNE- LDL-P sizea 2.7** 1.0 induced cytotoxicity in Müller cells at least in part via activation of AMPK- *P<.05, **P<.01, ***P<.001; NER/S vs Atv using Wilcoxon Rank-Sum test. PPARα-NFκB phosphorylation, stimulating ERK1/2 signaling pathway. aData presented in Vasc Health and Risk Manag 2010;6:1065-1075. 704-P Supported by: Abbott Relationship between Glucose Metabolism and Chylomicronemia Syndrome 702-P SABINE FISCHER, ALBRECHT LIEBAU, ULRICH JULIUS, ULRIKE SCHATZ, VIRGINIA Postchallenge Insulin and Proinsulin Levels in Normoglycemic KAMVISSI, STEFAN R. BORNSTEIN, Dresden, Germany Men and Women Are Mainly Associated with Saturated or Mono- In this investigation we examined the relationship between the unsaturated Fatty Acid Containing Di- and Triacylglycerides chylomicronemia syndrome as a specifi c type of hyperlipoproteinemia, and JUERGEN GRAESSLER, DOMINIK SCHWUDKE, PETER E.H. SCHWARZ, RONNY the disorders of the glucose metabolism. HERZOG, KAI SCHUHMANN, ANDREJ SHEVCHENKO, STEFAN R. BORNSTEIN, There were 117 patients with chylomicronemia syndrome (defi ned as Dresden, Germany triglycerides > 10 mmol/l in the medical history) included in the study. In 46 The objective of this study was to evaluate the effect of postchallenge patients (39.3%) there was an established diabetes mellitus type 2 (DM2). In levels of insulin and proinsulin on lipidomic profi le in men and women with 71 patients (60.7%) there was no diabetes mellitus known; in those patients normal glucose tolerance. we performed a standardized 75g glucose tolerance test after an 8h fasting Routine lipid parameters and lipidomic screens by mass spectrometry, period. According to the result of the glucose tolerance test the patients covering 95 lipid species of 9 major lipid classes, were analyzed in 70 men were divided in the following groups (ADA/WHO criteria): patients with and 56 women, which underwent a standardized oral glucose tolerance test normal glucose tolerance (NGT, fasting plasma glucose ≤ 6.0 mmol/l and (OGTT). plasma glucose 120 min after glucose ingestion ≤ 7.8 mmol/l) and patients In men, insulin was correlated directly with routine triglycerides (TG) and with impaired glucose tolerance (IGT, fasting plasma glucose ≤ 6.0 mmol/l inversely with HDL-cholesterol (HDL-C) at baseline and 2h OGTT. Lipidomic and plasma glucose 120 min after glucose ingestion 7.8 - 11.0 mmol/l). Three analysis revealed signifi cant correlation of insulin level with tri- (TAG: [50:3], patients with impaired fasting glucose, were excluded from the study. The [52:2-4], [54:3-6]) and diacylglycerol (DAG: [36:2&3]) metabolites. Insulin insulin secretion was calculated with the form: (30 min insulin – fasting at 2h was directly correlated with some phosphatidylcholine (PC) and insulin)/ (30 min plasma glucose – fasting plasma glucose). The insulin phosphatidylethanolamine (PE) metabolites. Proinsulin during OGTT was resistance (HOMA) was calculated with the form: (fasting plasma glucose correlated with several TAG metabolites (e.g. [46:1&2], [48:2&3]; [50:3&4]), x fasting insulin) / 22.5. but not with routine lipids. The patients with chylomicronemia syndrome and DM2 had a mean BMI of In women, triglycerides, but not HDL-C or LDL-C, measured by routine 29.6 kg/m2 and a mean age of 56.9 years. The patients with chylomicronemia clinical chemistry, were directly correlated with insulin levels at all OGTT syndrome and no known DM2 had a mean BMI of 28.6 kg/m2 (n.s. vs DM2) time points. Appropriate lipidomic screens revealed a direct correlation with and a mean age of 49.9 years (p<0.05 vs DM2). In the group of patients TAG´s preferentially containing saturated or monounsaturated fatty acids with chylomicronemia syndrome and no DM2, 32 patients had a NGT and 21 (e.g.: [46:0-2], [48:0-3], [49:1&2]). Strong direct correlations were detected for patients had an IGT. There were signifi cant differences in insulin secretion insulin at 90min OGTT with DAG´s [34:1], [36:2&3], [38:2], PC´s [34:3], [36:3], (NGT: 130.3, IGT 80.7 DIns/DPG), but we found no differences in insulin [38:3], PE´s [36:2], [38:5], and cholesterol ester [18:3]. As insulin, proinsulin resistance between the two groups. was correlated with the same TAG and DAG metabolites. In addition, a Our investigations show, that a high percentage of patients with a signifi cant inverse correlation was found for proinsulin at 2h OGTT with chylomicronemia syndrome have a DM2 or IGT. An important reason for this several PC-O and PE-O species. seems to be the disturbance of insulin secretion. In contrast, the two groups These data demonstrate that already in individuals with an apparent with NGT and IGT showed no signifi cant differences in insulin resistance. normal glucose tolerance individual glucose-induced response patterns of insulin and proinsulin were closely associated with derangements of the 705-P lipidomic profi le indicating beginning insulin resistance which is mainly Relationships between Composition of Major Free Fatty Acids (FFA) refl ected by the increase of saturated and monounsaturated fatty acid and Fat Distribution, and Insulin Resistance in Japanese containing plasma TAG´s and DAG´s. TOSHIHIDE KAWAI, YUKO OGUMA, FUMINORI KATSUKAWA, HIROSHI HIROSE, KUMIKO TANAKA, KOICHIRO AZUMA, SHU MEGURO, HIDEO MATSUMOTO, 703-P HIROSHI ITOH, Tokyo, Japan Protective Effects of Berberine on 4-HNE-Induced Cytotoxicity in The aim of this study was to evaluate the relationships between the Human Müller Cells through AMPK-PPARα-NFκB Activation composition of FFA and metabolic parameters, including body fat distribution. JING ZHANG, MINGYUAN WU, SHIHE YANG, DONGXU FU, MEI DU, KENNETH The study subjects were 83 Japanese who underwent 75g-OGTT. BMI (kg/ WILSON, TIMOTHY J. LYONS, Oklahoma City, OK m2), blood pressure, 24 fractions of fasting serum FFA, lipids, and visceral Berberine, an isoquinolone alkaloid of the genera coptis and berberis, has and subcutaneous fat areas as determined by CT scanning at the umbilical been shown to modulate glucose and lipid metabolism in animal and human level were measured. Based on the fi ndings of 75g-OGTT, the subjects were clinical studies. Its effects on retinal cells exposed to modifi ed lipoproteins, divided into NGT, IGT and DM groups. Data including major fraction of FFA as occurs in diabetic retinopathy (DR) are unknown. Modeling exposure of (%) in each group are shown in the table. In the subjects with diabetes, retinal cells to oxidized lipids in DR, we investigated effects of berberine on percentages of linoleic acid in serum were signifi cantly lower than the NGT human retinal Müller cell responses to 4-hydroxynonenal (4-HNE). Confl uent and IGT. cells were starved overnight, then treated with 4-HNE (0, 5, 10, 20 and 40

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A192 FOOT CARE—LOWER EXTREMITIES

NGT IGT DM p FOOT CARE—LOWER EXTREMITIES n (male/female) 27 (17/10) 37 (15/22) 19 (14/5) [See also: Presidents Poster 394-PP, page A109.] Age (yr) 48.0±11.9 53.2±12.6 57.4±9.7 0.024 BMI (kg/m2) 26.0±5.9 25.5±4.8 26.0±4.1 0.724 707-P total FFA (µg/ml) 2111.6±573.1 2250.1±721.1 2545.3±648.6 0.064 Study of the Risk of Developing Diabetic Foot and Glycemic Control Linoleic acid (%) 31.5±4.9 29.9±4.5 28.4±3.1 0.009 in Patients with Diabetes mellitus

ANA CARLA POZZI OLIVEIRA, CAIO JORDÃO TEIXEIRA, TALITHA FERNANDES POSTERS

Palmitic acid (%) 22.6±2.3 22.4±2.6 22.1±2.3 0.775 STEFANELLO, MÁRCIA APARECIDA CARRARA, IONE TAKAKI, ROBERTO BARBOSA Complications Oleic acid (%) 20.4±4.0 20.2±3.0 19.2±3.8 0.543 BAZOTTE, MÁRCIA REGINA BATISTA, Maringá, Brazil Acute and Chronic Docosahexaenoic acid (%) 4.0±1.7 4.7±1.8 5.8±1.9 0.002 Persistently high blood glucose levels are harmful and results in Visceral fat area (cm2) 110.8±58.2 112.2±54.4 136.9±45.2 0.248 uncontrolled prolonged chronic complications, including damage to various tissues, loss of normal function and failure of various organs. The diabetic Concerning relationships between major FFA and other metabolic foot is one of the most disabling chronic complications stemming from poor parameters, visceral fat area (VFA) were signifi cantly negatively correlated control of Diabetes mellitus. The aim of this study was to compare the risk with linoleic acid (r= -0.455, p<0.0001), positively correlated with palmitic of developing diabetic foot and glycemic control (fasting plasma glucose acid (r=0.355, p<0.01) and oleic acid (r=0.346, p<0.01). HOMA-IR were also (FPG) and glycated hemoglobin (HbA1C)) in patients with type 2 diabetes. signifi cantly negatively correlated with linoleic acid (r= -0.519, p<0.0001) To evaluate the risk, we performed a sensitivity test on the feet, based on and positively correlated with palmitic acid (r=0.376, p<0.01), but not with the National Hansen’s Disease Program (HRSA - U.S.), and patients were oleic acid (r=0.186, p=0.093). ISI (comp) were positively correlated with divided into four categories, according to the loss of sensitivity observed. linoleic acid (r=0.427, p<0.0001), negatively correlated with palmitic acid (r= This program establishes that the higher the risk the greater the chance of -0.329, p<0.01) and oleic acid (r= -0.251, p<0.05). This might be associated developing diabetic foot. The study was conducted at the Clinical Analysis with negative correlation between percentages of linoleic acid and VFA or Teaching and Research Laboratory of the State University of Maringá, from HOMA-IR, and positive correlation between linoleic acid and ISI (comp). August to December 2010. We evaluated 29 patients, 79.3% women, mean In conclusion, these data suggest that linoleic acid level was inversely age was 59.4 ± 8.8 years. The table below shows the results. correlated with accumulation of visceral fat in relation to a reduction of insulin resistance in Japanese. Table 1. Categories of risk of developing diabetic foot and glycemic control in patients with Diabetes mellitus type 2, represented by mean and standard 706-P deviation. The Effects of Ezetimibe on Cholesterol Absorption and Synthesis Risk Categories % patients FGP (mg/dL) HbA1C (%) Markers in Hyperlipidemic Patients with or without Type 2 Diabetes 0 38.0 136.8 ± 26.3 7.1 ± 1.2 Mellitus (Result from E-CAP Study) 1 24.1 119.3 ± 32.9 7.1 ± 1.1 MASUMI AI, MASAYUKI YOSHIDA, OSAMU TOMONAGA, ECAP STUDY GROUP, 2 27.5 153.7 ± 97.2 8.3 ± 2.2 Tokyo, Japan Background: Hypercholesterolemia (HC) is an additional risk for coronary 3 10.4 344.7 ± 79.1 9.0 ± 0.8 heart disease in patients with type 2 diabetes (DM). The increase of The results showed that the category of highest risk was more lack of cholesterol absorption in intestine leads to HC and ezetimibe (EZ) inhibits glycemic control, refl ected by the FPG and HbA1C levels. But the second this pathway and reduces serum cholesterol level. We aimed to examine category requires attention, the fact that 27.5% of patients with plasma whether there is a distinct effect of EZ on serum levels of cholesterol and glucose levels were signifi cantly changed. At this stage these patients cholesterol absorption and synthesis markers (CASM) in those with DM and should be advised and informed about the risk of progressing to the third obesity. category and develop the diabetic foot. This study expected that patients Methods: Subjects were consisted with 100 patients with HC. Half of who were in the third category would present a more adequate glycemic them also had DM and the other half were normal glucose tolerant (NGT). control, because they have had injuries and amputations on both legs. Thus, In all subjects, 46 were obese (OB) and 54 were non-obese (NOB). All we conclude it is necessary to guide and monitor the patient’s risk in the participants took EZ 10 mg/day for 6 weeks. Serum samples were obtained second category to prevent progress to the third category. before and after the medication to measure serum levels of lipids including total cholesterol (TC) and CASM such as campesterol (Cam), sitosterol (Sit), and lathosterol (Lat). 708-P Impaired Foot Ulcer Healing in Diabetic Rats Results: At baseline, mean levels of Cam/TC, Sit/TC, and Lat/TC in JIRO KATO, HIDEKI KAMIYA, EITARO NAKASHIMA, TATSUHITO HIMENO, patients with DM were similar to those in NGT participants. EZ reduced YUSUKE SEINO, TETSUJI OKAWA, ATSUSHI FUJIYA, MASAKI KONDO, KEIKO TC, Cam/TC and Sit/TC levels while it raised Lat/TC level both in DM and NARUSE, SHIN TSUNEKAWA, NOBUAKI OZAKI, YOJI HAMADA, YUTAKA OISO, NGT subjects (TC; -14% vs -14%, Cam:-35% vs -39%, Sit: -28% vs -32%, Lat: JIRO NAKAMURA, Nagoya, Japan +55% vs +51%, respectively) and no signifi cant difference was observed Background: Diabetes mellitus is one of the main causes of non-traumatic between them. Regarding obesity, mean levels of Cam/TC and Sit/TC were amputation of lower extremities, which substantially impair quality of life. signifi cantly lower in OB than NOB, while mean Lat/TC level in OB was Therefore, the elucidation of the mechanisms of diabetic foot ulcer is required. similar to that in NOB at baseline. However the effects of EZ on TC, Cam/ Abnormalities of macro- and micro-vasculature, and peripheral nerves and TC, Sit/TC, and Lat/TC in OB were similar to those in NOB (TC;-13% vs -14%, infection have been proposed to delay wound healing in diabetic patients. Cam:-35% vs -38%, Sit: -28% vs -31%, Lat: +52% vs +55%, respectively). However the mechanisms of impaired wound healing in diabetes have not Conclusion; Serum levels of CASM in patients with DM were similar to been clarifi ed in details yet. This study was conducted to establish a foot ulcer those in NGT individuals. Serum cholesterol absorption markers were lower model using diabetic rats and to elucidate the mechanisms of delayed wound in OB than NOB population. No difference was found in the effects of EZ healing using keratinocytes cultured under the high glucose condition. on serum TC levels and CASM 1) between in DM and NGT individuals, or 2) Methods: 1) Diabetes was induced by intraperitoneal injection of Streptzotocin between in OB and NOB individuals. Our data point a previously unrecognized to 6-week-old male SD rats. 2) After 8 weeks, a punch hole 4mm was created unique cholesterol metabolism in obese condition that may play a role in Φ by BIOPSY PUNCH in the hind paw of normal (N) and diabetic rats (D). 3) Wounds dyslipidemia in obesity. were photographed and the size of the remaining wound were mesured every Supported by: Grant from Tokyo Medical and Dental University other day. 4) Blood fl ow on the wound beds (BF) was measured by laser blood fl ow meter. 5) Primary human keratinocytes (HKC) were cultured under the normal (6mM glucose :NG) or high glucose (20mM glucose :HG) condition and the proliferation activities were evaluated by MTS and BrdU assays. Results: 1) D showed hyperglycemia (N: 6.8±0.2 mM, DM: 24.1±2.2) and reduced body gain (N: 471±17g, DM: 242±29). 2) Delayed wound healing was observed in D. Complete wond healing was observed at day 11.3±0.5 in N and day 17.0±1.9 in D. 3) BF was signifi cantly decreased in D at day 0 (N: 18.0±0.8 ml/min/100g, DM: 12.3±0.9) and day 3 (N: 18.1±0.9, DM:13.3±0.9).

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A193 FOOT CARE—LOWER EXTREMITIES

4) Proliferation activities of HKC were signifi cantly impaired in D (MTS NG: 711-P 0.045±0.002, HG: 0.019±0.002) (BrdU NG: 0.144±0.008, HG: 0.113±0.015). New Arterial Stiffness Measurement in Type 2 Diabetic Patients Conclusions: These data indicate that delayed wound healing observed with and without Foot Lesions in diabetic rats would be induced by not only decreased blood fl ow but also MARIA SAMBATARO, GIANNI MORANA, FABRIZIO FARNETI, PIERGIORGIO impaired re-epithelialization under the diabetic condition. SCOTTON, DANIELLA PIZZOLATO, AGOSTINO PACCAGNELLA, Treviso, Italy In diabetes arterial hypertension, but also β stiffness impairment (reduced 709-P arterial diameter modifi cation with sistolic wave passage) are frequent. A

POSTERS Role of Lower Limb Angiography in Patients with Charcot Neuro- derived index CAVI (cardio-ankle vascular index) from VASERA 1000 FUKUDA Complications osteoarthropathy and Non-Healing Foot Ulcers instrument describes systolic pulse wave velocity in arterial peripheral tree. Acute and Chronic ROBERT BEM, ALEXANDRA JIRKOVSKA, MICHAL DUBSKY, VERONIKA In diabetic foot, transcutaneous oximetry (TCPO2) is a valid prognostic WOSKOVA, VLADIMIRA FEJFAROVA, JELENA SKIBOVA, Prague, Czech Republic amputation parameter, but its relationship with arterial indirect blood fl ow Association between Charcot neuroosteoarthropathy (CNO) and measurements as ankle/radial ratio systolic pressure (Ankle Brachial Index peripheral arterial disease (PAD) is not fully understood. The hypothesis of ABI) is still controversial. In type 2 diabetic patients with (T2R) and without the pathogenesis of CNO is mainly based on diabetic neuropathy. In clinical (T2) distal cronical limb ischemia (CLI), CAVI index and two ABI indexes were practice, it is frequently observed that CNO patients treated for non-healing derived: oscillometric ABIO obtained during CAVI measurement and sonografi c diabetic foot ulcers (DFU) are under clinical suspicion on PAD as well. ABID from doppler CW or vascular ecodoppler examination (ratio of posterior The aim of our study was to assess the role of lower limb angiography in and anterior tibial artery mean sistolic pressure/ omolateral radial artery patients with CNO and DFU with the respect to pathological fi ndings and systolic pressure). TCPO2 was measured basal and four week later in T2R possibility of vascular intervention. Patients treated for non-healing DFU which underwent baloon distal revascularization and this parameter was with CNO (n=33; CNO group) or without CNO (n=54; controls) who underwent compared with CAVI, ABIO e ABID. 25 T2 and 14 T2R with good prognosis angiography indicated for suspicion on PAD (clinical signs or transcutaneous for limb salvage were similar for age (67±10 e 70±9 (years±DS)), peripheral oxygen tension) were enrolled into the study in our foot clinic during 18 neuropathy, lipid and glucose control. 9 healthy controls (C). were different months period. The diagnosis of CNO was based on clinical and radiological for age (34±6 anni). In T2, T2R e C, CAVI was respectively : dx 9.4±1.5, 9.9±1.5, examination; before or up to 12 months after angiography. There were no 6.5±1, sx 9.5±.1.5, 10.0±1.5, 6.5±1 (p<0,001 T2 e T2R vs C); ABIOdx :1.05±0.11, signifi cant differences in abnormal angiographic fi ndings on lower limb 0.87±0.15, 1.06±0.07, ABIOsx: 1.04±0.1, 0.85±013, 1.05±0.043 (p<0,002 T2R arteries (stenosis >70% or oclusion) between the CNO group (29/33 - 87.9%) vs T2 and C), ABIDmdx: 1.16±0.14, 0.94±0.26, 1.05±0.06 ABIDmsx 1.17±0.14, and controls (48/54 - 88.9%). Of these PAD patients, a signifi cant difference 1.01±0.19, 1.05±0.052 (p<0,03 T2R vs T2). In T2R ΔTCPO2 of revascularized between groups was only in the presence of most severe fi ndings: 1 or none leg (basal 24.8±6 mm Hg) (mean±SE)) was signifi cant increased (+18 mmHg); patent crural artery had 20/29 (69%) of patients in CNO group vs. 43/48 and correlated with CAVI (R2 0,6 p<0,01) and ABIO (R2 0,48 p<0,03) but not (89.6%) of controls; p<0.05. Revascularization rate based on angiographic with ABID. CAVI index identifi es a possible variable for limb salvage and fi ndings was comparable between groups – 26 (21 percutaneous transluminal describes a role for distal reperfusion. In diabetes, l’ oscillometric ABI is not angioplasty - PTA; 5 bypasses)/29 (89.7%) of patients in CNO group vs. 42 affected by tonaca media calcifi cations and better descibes CLI for values (33 PTA, 9 bypasses)/48 (87.5%) in controls, NS. Our results indicate that <0.9 if oximetry is <30-50 mm Hg. patients with CNO and non-healing DFU may have benefi t from lower limb Supported by: Italian Society of Diabetologia-Regional Section angiography, because it leads to equaly frequent revascularization as for non-CNO patients. Nevertheless, the differences in angiographic fi ndings 712-P are plausible. Control of Lower Extremity Edema in Persons with Diabetes with Supported by: MZO 00023001 Mild Compression Diabetic Socks: A Pilot Study STEPHANIE C. WU, JESSICA MINDER, RYAN T. CREWS, Chicago, IL 710-P It is common for persons with diabetes to present with coexisting lower Relationship between Lipid Parameters in Diabetic Foot Gangrene extremity edema that may lead to ulcerations and subsequent infections. EIKO KITAMOTO, KUNIMASA YAGI, NAOKO ITO, AZUSA OOBATAKE, AYA Compression therapy is considered the standard treatment for edema. FUJIMOTO, MIYUKI KUBOTA, KAORU NAKANO, YOSHIYUU TAKEDA, JYUNJI However, since over 50% of persons with diabetes have concomitant KOBAYASHI, MASAKAZU YAMAGISHI, Kanazawa, Japan peripheral arterial disease, compression therapy is often contraindicated. Both atherosclerosis and peripheral neuropathy contribute to the This pilot study sought to assess whether diabetic socks with mild occurrence and progression of diabetic foot gangrene (DFG). However, compression (18-25mmHg) can reduce lower extremity edema in persons few studies have examined the relationship between atherosclerotic with diabetes without negatively impacting vascularity. risk factors and clinical features of DFG. Therefore, we examined the 14 subjects (7 males, 7 females) aged 62 ± 10 years with diabetes, lower relationship between the severity of DFG and the laboratory data on the extremity edema, and ankle-brachial index (ABI) ≥ 0.6 were enrolled. lipoprotein metabolism and nutrition status. We examined 11 patients with Subjects were sized per manufacturer’s directions and instructed to wear DFG. Severity of DFG was evaluated following the Wagner classifi cation; the socks during all waking hours. Follow-up visits occurred weekly for 4 grade 0 to 5. Whole subjects were separated into 2 groups; one group (G1-3) consecutive weeks. Edema was quantifi ed through midfoot, ankle, and calf of 6 subjects with Wagner grade from G1 to G3 and another group (G4-5) circumferences and via interstitial fl uid measurements. Vascular status was of 5 subjects with Wagner grade severer than G4. We compared these 2 tracked via ABI. groups with various clinical parameters including Cre, blood urea nitrogen, Repeated measures ANOVA and LSD post hoc analyses were used for data total cholesterol (TC), triglyceride (TG), HDL-C, leukocytes counts, neutrophil analyses. Calf circumferences showed a statistically signifi cant decrease of counts, serum C-reactive protein (CRP), total protein (TP), albumin (Alb) and 1.4 ± 0.4cm after just one week and the measurements remained signifi cantly HbA1c. These parameters were examined at 3 time points; before the onset (p < 0.05) smaller than baseline throughout the study. Foot circumferences of DFG, after the onset of DFG, and after curing from DFG with medications decreased gradually, with weeks 3 and 4 demonstrating signifi cantly smaller and/or operation. Clinical profi les of the subjects were shown as below; 6 values than baseline (reductions of 1.3 ± 0.4cm and 1.2 ± 0.4cm respectively). males and 5 females, age 62.5±17.5 yrs, one type 1 and ten type 2 diabetics The mean ankle circumference and mean interstitial fl uid measurements according to the diagnosis guideline. Clinical parameters in G1-3 vs. G4-5 demonstrated consistent trends of reduction (by week 4: 1.9 ± 0.4cm and 1.9 after the onset of DFG were respectively shown as follows; TC 200±25mg/ ± 1.0 respectively) but were not statistically signifi cant. The ABI signifi cantly dL vs. 116±23mg/dL (p=0.0139), TG 116±19mg/dL vs. 94±11mg/dL (ns), differed (p=.01) from baseline only at week 3 (increased 0.16 ± .05). HDL-C 37±2mg/dL vs. 32±12mg/dL (ns), TP 7.3±0.13g/dL vs. 6.2±0.31g/dL This initial study indicates that mild compression diabetic socks may help (p=0.0142), Alb 4.2±0.12g/dL vs. 3.1±0.45g/dL (p=0.0369), HbA1c 7.6±0.62% diabetic persons with lower extremity edema. Additionally, the socks did vs. 7.5±1.7% (ns). Moreover, the difference in TG between before and after not appear to compromise lower extremity vascularity. ABI differed from the onset of DFG negatively correlated with the difference in CRP (γ=-0.995, baseline only at week 3 and at that point the ABI was actually elevated p=0.0049). Severity of the DFG didn’t show the signifi cant correlation with indicating increased blood fl ow to the lower extremity relative to the upper other parameters including renal function and infl ammation. Additionally, no extremity. signifi cant differences were observed in various parameters including TC, Supported by: Partially by T35DK074390 Grant from NIDDK, and by Sigvaris Inc. TP and Alb between G1-3 and G4-5 after curing from DFG. Severity of DFG positively correlated with the reduced level of TC, TP and Alb. These results strongly suggested the existence of hyponutritional state in DFG.

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713-P 715-P Geographic Distribution of Bioburden in Chronic Non-Infected Limb Salvage with Syme Amputation: Experience in 54 Cases Diabetic Ulcers over Time ROBERT G. FRYKBERG, CHRISTOPHER WERNER, EDWARD TIERNEY, ARTHUR STEPHANIE C. WU, DAVID ARENS, KRISTY L. SHANAHAN, Chicago, IL TALLIS, Phoenix, AZ Chronic diabetic ulcerations are often contaminated with bacterial Syme’s ankle disarticulation amputation is often overlooked as an pathogens and can develop into bacterial infections. The purpose of this alternative to below-knee amputation in cases of life and limb threatening study was to assess variations in geographic distribution of bacterial foot infections and gangrene. This study examines pre-operative criteria contaminants over time both in vitro and in vivo. and outcomes of the Syme amputation to help ascertain predictors that may Five aerobic microorganisms commonly found in diabetic ulcers were determine greater success of the operation. diluted to the ratio 1:1,000,000 and inoculated to a blood agar plate either as In this retrospective study, pre-operative data and post-operative results a 100uL aliquot of a mixture of the fi ve bacteria or as 20uL of each bacteria were assessed for 54 consecutive patients (mean age 64) who underwent a after dilution. The plates were divided into 46 1cm2 sections to identify Syme amputation for non-healing ulceration, osteomyelitis, and/or gangrene. and defi ne the location of the given microbe and observed over fi ve days. Fifty (93%) patients had diabetes mellitus for an average of 17 years. HbA1c For the inoculation of 20uL of each bacteria, after 24 hours P. Aeruginosa ranged from 5.6 to 13.4 with a mean of 8.0%. Thirty-fi ve (65%) patients had grew to encompass 15 of the 21 sections identifi ed to have growth, primarily peripheral vascular disease (PVD) of which 15 (53%) of these patients underwent growing from the center of the inoculation site. One colony of S. Aureus, preliminary lower extremity bypass grafting. Seventeen (41%) patients had an

three of MRSA, and two of E-coli were located at the periphery of the plate. ankle-brachial index (ABI) of less than 0.9 and 24 (49%) were greater than 1.2. POSTERS Plates inoculated with 100uL of the mixture of bacteria had similar results Of those with skin perfusion pressures (SPP) at the ankle, 38% of patients had with minimal variation over time. values < 30 mmHg. Forty-three (80%) patients either were current smokers or 11 consecutive patients with 12 chronic, UTSA grade 1A wounds were smoked in the past, and 24 (44%) patients had a positive methicillin-resistant included. Wounds were debrided and irrigated to help remove superfi cial Staphylococcus aureus (MRSA) culture originating from their affected foot. Behavioral Medicine, Clinical contaminants and geographically divided into 1cm2 sections. Daily tissue All Patients were followed for an average of 130 weeks until healing, major Nutrition, Education, and Exercise cultures were obtained from each individual area for 5 consecutive days, amputation, or death. Thirty- seven (69%) healed and were ulcer free for >30 grown on separate blood, chocolate, Columbia CAN, and MacConkey agar days, 31 (57%) are currently ambulating with a Syme prosthesis, 16 (30%) plates then isolated for identifi cation. Quantitative growth was determined patients required more proximal amputation (12 BKA, 4 AKA), and 23 (43%) using semi-quantitative analysis. patients died at an average of 150 weeks after Syme amputation. Of those that Wounds contaminated with P. Aeruginosa behaved differently over died, 5 (22%) patients had a more proximal amputation. Of the patients with SPP time than wounds without. Despite minor daily fl uctuations, P. Aeruginosa < 30mmHg, 43% needed a proximal amputation compared with 45% of those showed the strongest growth and dominated the central aspect of the with values ≥30mmHg (p=0.173). Nonetheless, SPP≥30mmHg predicted a nearly wounds while gram positive bacteria remained in the periphery. In wounds 5-fold increased probability for healing (OR 4.8, 95% CI 1.13-20.46, P=0.034) not contaminated with P. Aeruginosa, gram positive bacteria expanded from Syme amputation can be a viable alternative to major amputation when the the periphery overpowering gram negative bacteria to dominate the entire leg is at risk and SPP values are at least 30mmHg at the ankle. Nonetheless, wound. Of the gram positive bacteria, MRSA showed aggressiveness in 3 year mortality (43%) is high in these complicated patients. growth. The results of this initial study may provide some insight into bacteria behavior over time in clinically non-infected wounds, this may prove different DIABETES EDUCATION in infected wounds where the microbiota may change. Supported by: NIDDK [See also: Presidents Posters 395-PP to 396-PP, page A109.]

714-P The Impact of Footwear and Walking Distance on Gait and Dynamic Guided Audio Tour: Educating Providers to Improve Diabetes Management Stability in Patients with Diabetes and Peripheral Neuropathy in Primary Care Settings (Posters 716-P to 722-P), see page 11. BIJAN NAJAFI, TAHIR KHAN, ADAM FLEISCHER, JAMES S. WROBEL, Chicago, IL Normal walking requires sensory input to adapt and modify motor patterns & 716-P and muscle output to carry out the desired task. Several studies have Treating Diabetes, Missed Opportunities addressed gait alterations among diabetes. However most of these studies SYEDA SADIA ZAIDI, RUSSELL L. ROTHMAN, DAVID ESKIND, MICHAEL E. explored gait in a laboratory condition with space restriction, which may not BOWEN, TOM A. ELASY, KERRI L. CAVANAUGH, Nashville, TN replicate true conditions that the subjects are active in. This study explored Primary care providers provide the majority of diabetes care for patients gait differences among patients with diabetes and peripheral neuropathy with diabetes. However, studies detailing the discussion of diabetes (DPN) and aged matched control subjects over short and long walking between primary care providers and patients are limited. distances as well as during barefoot and shod conditions. Twelve DPN and In a cross-sectional study, we audio-recorded 99 adult primary care eight healthy control subjects were asked to walk at habitual speed in two physician-patient encounters in an academic internal medicine clinic. conditions: 1) barefoot, 2) regular shoes over a short (7m, SWD) and long (20m Encounters were transcribed and coded using 60 mutually exclusive LWD) walking distance to replicate indoor and outdoor walking. A validated categories. Demographic and clinical variables were collected from patient body worn sensor was used for extracting spatio-temporal parameters of self-report and medical record abstraction. gait. Based on statistical inter-cycle fl uctuation of gait velocity, gait steady Total conversation time averaged (SD) 20.3 (8.7) minutes per encounter. state and gait initiation phases were analyzed separately. Both groups On average patients were 56.1 (10.1) years old, 51% female, 49% African walked slower during barefoot and SWD respectively compared to shod American, BMI was 34 (9), average A1C was 7.8% (2) and 79% were cared for and LWD (p<0.05). During the shod condition, gait initiation was 21% longer by medical residents. Glycemic issues, including glucose control, monitoring in DPN and 3% shorter in control subjects compared to barefoot condition. and diet, were discussed on average 16% (13) of total conversation time, In DPN, gait velocity was slower than control subjects during both gait averaging 3.4 (3.4) minutes of discussion per visit. Eight-percent of time initiation and steady state especially during barefoot and LWD conditions. was allotted to discussing the prevention and treatment of diabetes-related Interestingly, inter-cycle gait variability was signifi cantly higher in DPN co-morbid conditions including hypertension, hyperlipidemia, and obesity. subjects only during barefoot and LWD, while no signifi cant difference was However, hypertension was not discussed in 40% of encounters, lipids observed during SWD and shod condition. Additionally results revealed a in 47% and obesity in 65%. Other specifi c topics with limited discussion signifi cant improvement in gait inter-cycle variability by wearing shoes included hypoglycemia (discussed in 27% of total visits), vaccinations in DPN group (31%, p<0.05), while gait steadiness was not infl uenced by (35%), physical activity (39%), and carbohydrate monitoring (8%). Overall, footwear condition in healthy control group. This study suggests that gait time spent discussing non-diabetes related topics accounted for 62% (19) alterations due to DPN should be carefully assessed over ample walking of the visit, and less diabetes-related talk was associated with older age distances and during both shod and barefoot conditions. The results also (p=0.043), white race (p=0.058), and having participated in a diabetes revealed the benefi t of wearing footwear for improving gait steadiness in education class (p=0.003). Patients with higher A1C had a greater portion of DPN subjects. conversation time spent discussing glycemic issues (r=0.25; p=0.013), even Supported by: American Podiatric Medical Students’ Association when adjusting for age, sex, race, provider level of training and concurrent endocrinology care. But actual time spent with patients with worse glycemic control (A1C>7%) was only 75 seconds more.

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