2011 ADA Presidents 379-477 - Diabetes€¦ · TOMOAKI INOUE, KUNIHISA KOBAYASHI, MASAKAZU FUJII,...

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ACUTE AND CHRONIC COMPLICATIONS

COMPLICATIONS—HYPOGLYCEMIA

379-PPCorticotrophin Releasing Hormone Receptor Type 2 in Both Ventro-medial Hypothalamus and Medical Amygdalar Nucleus Suppress Counterregulatory Responses to Acute Hypoglycemia in the RatXIAONING FAN, STACEY BROWN, YUYAN DING, ROBERT S. SHERWIN, RORY J. MCCRIMMON, New Haven, CT, Dundee, United Kingdom

Hypoglycemia detection takes place within specialized neurons located within discrete brain regions, e.g. the ventromedial hypothalamus (VMH). We have recently demonstrated the presence of a novel glucose sensing region in the medial amygdalar nucleus (MeA) of the rat. In addition, we also demonstrated that both VMH and MeA glucose-sensing regions were innervated by urocortin 3 (UCN3) containing neurons. UCN3 is an endogenous neuropeptide that acts through binding to the corticotrophin releasing hormone type 2 receptor(CRH-R2), and plays a major role in regulating responses to a variety of physiological and behavioral stresses. To determine whether hypoglycemia-sensing in both VMH and MeA is modulated by CRH-R2-mediated effects we used RNA interference to selectively suppress CRHR2 expression in both brain regions. Ten days prior to study male Sprague-dawley rats were microinjected under general anethesia to either the VMH or MeA with an adeno-associated viral vector (AAV) containing an RNAi targeting CRH-R2 or a control RNAi as well as having chronic vascular catheters inserted. Subsequently, all rats underwent a 90 minute hyperinsulinemic (20 mU/kg/min) hypoglycemic (∼60 mg/dl) clamp study with arterial sampling for counterregulatory hormone responses. During equivalent hypoglycemia, knockdown of CRH-R2 in either the VMH or MeA was found to markedly amplify epinephrine (100% and 88%, respectively) and glucagon (35% and 61%, respectively) responses to hypoglycemia (see Table below; MeA and VMH control studies are combined because CRR responses did not differ between the two; =p<0.05 vs. vehicle).

Control RNAi VMH CRHR2 RNAI MeA CRHR2 RNAiGIR (mg/kg/min) 9.7±0.3 3.7±0.7* 4.5±1.2*Glucagon (ng/l) 164±22 221±29* 264±61Epinephrine (pg/ml) 1990±207 4051±575* 3750±207*

We conclude that acute activation of CRH-R2 neurons in both VMH and MeA diminish counter-regulatory responses to hypoglycemia. We hypothesize that this represents a central feedback mechanism for regulating the magnitude of the physiological response to hypoglycemic stress.

Supported by: JDRF

COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND

HUMAN DIABETES

380-PPAcute Hyperglycemia Markedly Reduces Coronary Flow Reserve: A Study in Nondiabetic Subjects Using Bedside Contrast Echo cardio-graphySAHAR S. ABDELMONEIM, MARY E. HAGEN, EDWARD M. MENDRICK, VISH-WANATH PATTAN, BARBARA J. NORBY, TAMERA M. ROBERSON, TROY K. SZYDEL, RITA BASU, ANANDA BASU, SHARON L. MULVAGH, Rochester, MN

The effect of acute hyperglycemia on coronary perfusion in humans has been poorly studied. To do so, we evaluated the effects of short term hyperglycemia on coronary fl ow reserve (CFR) in healthy volunteers, without diabetes, obesity, smoking, hypertension or hyperlipidemia. We enrolled 15 healthy volunteers [12 female, mean ± SD, age: 46.5 ± 4.6 years, BMI: 25.2 ± 4.4 kg/m2, fasting blood glucose (BG) 5.54± 0.35 mmol/l, HbA1C 5.3 ± 0.3 %] who underwent a two step pancreatic clamp with somatostatin and replacement glucagon and growth hormone infusions. Insulin was infused at 0.75 mU/Kg TBW/min to mimic postprandial plasma insulin concentrations and glucose was infused for 4 hrs to maintain euglycemia (BG 5.19± 0.31 mmol/l for fi rst 2 hrs) followed by hyperglycemia (BG 12.95 ± 1.2 mmol/l for next 2 hrs). Myocardial contrast echocardiography (MCE) was performed at bedside, during the fi nal 30 minutes of each glycemic step using a continuous infusion of Defi nity (1.3 ml diluted in 60 cc 0.9% saline at 200 ml/hr; Lantheus Medical Imaging) at rest and during stress-induced vasodilatation with Regadenoson (Lexiscan 5 ml (400 ug) IV bolus; Astellas Pharma Global Inc.) to quantify myocardial blood fl ow (MBF) and determine CFR [stress MBF/rest MBF] at each glycemic step. Degree of insulin resistance (IR) was assessed from glucose infusion rates (GIR; mg/Kg/min) at euglycemia. CFR during euglycemia vs. hyperglycemia was 3.3±1.5 vs. 2.1 ±1.2 (p=0.003). Mean ±SD of GIR at euglycemia was 5.3 ±2.9 mg/kg/min.

Subjects’ demographics and CFR were also classifi ed by GIR at euglycemia: <5 (n=8) or ≥ 5 mg/kg/min (n=7).

Control RNAi VMH CRHR2 RNAI MeA CRHR2 RNAiGIR (mg/kg/min) 9.7±0.3 3.7±0.7* 4.5±1.2*Glucagon (ng/l) 164±22 221±29* 264±61Epinephrine (pg/ml) 1990±207 4051±575* 3750±207*

Conclusions: CFR, as determined noninvasively by MCE, is signifi cantly decreased during acute hyperglycemia in nondiabetic, healthy volunteers and may be associated with variations in IR.

Supported by: Mayo Foundation

381-PPAtheroprotection Induced by Selective 11β-Hydroxysteroid Dehy-dro genase Type 1 (11βHSD1) Inhibition in Male ApoE-/-MiceTHOMAS HÜBSCHLE, EUGEN FALK, MAIKE GLIEN, THORSTEN SADOWSKI, HARTMUT RUETTEN, Frankfurt, Germany

The concept of a tissue-specifi c, pre-receptor metabolism control of glucocorticoid action regulated via 11βHSD1 seems to open new options in the therapy of diabetic macrovascular complications. Therefore we tested the anti-atherosclerotic effects of a novel, potent (IC50<100nM) and selective 11βHSD1 inhibitor, SAR1, in the ApoE-/- mouse atherosclerosis model.

Male ApoE-/- mice were fed a high fat diet supplemented with or without SAR1 at 30 & 100 mg/kg/d for 16 weeks. Body weight and weekly body weight gain in the SAR1 treated groups did not differ from the high fat control group. Treatment with SAR1 dose-dependently reduced aortic plaque formation by 48% (p=0.0001) and 71% (p<0.0001) (Fig.1A) as analyzed by oil red en face staining. Total serum cholesterol as well as the atherogenic serum lipoproteins were unaltered in the low dose group but signifi cantly diminished by about 35-41% in the high dose group (Fig.1B). Thus, the 11βHSD1 inhibitor SAR1 induces an anti-atherosclerotic effect that goes beyond improvement of systemic dyslipidemia most likely via protective mechanisms that act locally at the vascular wall.

In agreement with the fi ndings above, data from serum ELISA measure-ments of the endothelial infl ammation markers MCP1, VCAM-1 and E-selectin showed reduced levels under SAR1 treatment, again pointing at a local vascular atheroprotection during systemic 11βHSD1 inhibition.

382-PPBreath Biomarkers of Oxidative Stress: Comparison of Exhaled and Plasma Gas ConcentrationsHYUN JI LEE, STACY R. OLIVER, REBECCA L. FLORES, PIETRO R. GALASSETTI, DONALD R. BLAKE, Irvine, CA

While oxidative stress (OS) is a key pathogenetic mechanism for diabetic cardiovascular complications, its determination is laborious, expensive and often inaccurate. Several exhaled gases (isoprene, ethane, n-pentane) were proposed as potential biomarkers of OS, but their clinical application has been very limited. We believe this is due, at least in part, to a fundamental lack of understanding of the relationship between concentrations of these gases in plasma (refl ecting peripheral oxidative metabolism) and breath (where measurements are made). To address this issue, we developed a novel apparatus that can completely extract gases dissolved in plasma with a direct fl ow of helium micro-bubbles, and used it to chemically compare blood and breath gas composition in 7 healthy males (23.7±1.1yrs), all sampled twice. Of the 75 compounds analyzed with gas chromatography, we focused on 6 potential OS breath biomarkers (ratio of breath/degassed plasma is shown in Figure). Our data shows that while concentration patterns (greater concentrations of given gases in breath or plasma) were very consistent across subjects, different compounds displayed very different patterns. In particular, isoprene, previously proposed as a OS marker refl ecting cholesterol synthesis, was surprisingly detected at levels 40 times lower in degassed plasma than in breath, suggesting a complex interaction between peripheral tissue gas production and gas concentrations in exhaled breath. The thorough clarifi cation of all components of this interaction will signifi cantly accelerate development of breath-based, clinically applicable

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testing devices for non-invasive detection/quantifi cation of OS, with considerable relevance for the prevention, monitoring and assessment of treat ment effi cacy of diabetic cardiovascular complications.

Supported by: NIH 1UL1RR031985; K24 DK085223. ADA-Funded Research

383-PPThe Relationship between Coronary Atherosclerotic Burden and Calci -fi ed Coronary Score in Patients with and without Diabetes MellitusHONGXIA ZHANG, JIE YUAN, JIE LIU, XIAOXIAN TANG, WEIPING JIA, Taiyuan, China, Shanghai, China

The purpose of the study is to compare the presence of obstructive and non-obstructive coronary artery disease (CAD) as well as calcifi ed coronary artery plaque burden in patients with and without diabetes mellitus (DM). The study population is consisted of 743 individuals (156 diabetics and 587 non-diabetics) suspected CAD but no history of CAD(prior myocardial infarction or myocardial revascularization procedure). All subjects underwent 64-slice CT coronary angiography (CTCA) as part of a health screening evaluation. The per-patient number of diseased coronary segments were determined and each diseased segment was classifi ed as obstructive lesion (luminal narrowing >50%) or not. Coronary calcium score (CCS) was considered too. Detailed information regarding symptoms, physical examination, medical history and risk factors were collected at the time of presentation. CTCA was performed without complications in all 743 patients: 63% male, 59±17 years. Diabetic patients were older (62±13 years vs. 58±10 years, p=0.002), had a higher BMI (26.7 vs. 25.2, p<0.001), compared to non-diabetics, as well as a larger number of traditional risk factors; 2.6 versus 1.7, including hypertension, hyperlipidemia and obesity, in addition to diabetes mellitus. Diabetics showed a higher number of diseased segments (3.7±2.9 vs. 1.9±2.0; p=0.007); a higher rate of CCS>400(p=0.02), obstructive CAD (26.6% vs. 17.2% of patients; p=0.004), and fewer normal coronary arteries (31.3% vs. 47.6%; p<0.001), as compared to non-diabetics. The percentage of patients with obstructive CAD was increased with CCS in both groups. Diabetes with CCS≤10 had a higher prevalence of coronary plaque (32.7% vs. 20.5%, p=0.01) and obstructive CAD (10.9% vs. 1.6%, p<0.001). CTCA in patients with diabetes shows more plaque and obstructive disease compared with patients without diabetes.Contrary to non-diabetics, a low or negative calcium score does not rule out obstructive coronary artery disease in patients with diabetes mellitus.

COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

384-PPAdipose Triglyceride Lipase Knockdown Enhances Tumor Necrosis Factor α-Induced Intercellular Adhesion Molecule-1 Expression in Human Aortic Endothelial Cells Via Protein Kinase C-Dependent Activation of Nuclear Factor-κBTOMOAKI INOUE, KUNIHISA KOBAYASHI, MASAKAZU FUJII, EIICHI HIRATA, HISASHI YOKOMIZO, YASUTAKA MAEDA, NORIYUKI SONODA, TOYOSHI INO-GUCHI, RYOICHI TAKAYANAGI, Fukuoka, Japan

Heart diseases including ischemic heart disease and heart failure are the major reasons of mortality in diabetics despite the recent development of medical and surgical treatments. It is important to further elucidate the underlying mechanisms and to pick up a new therapeutic target.

Adipose triglyceride lipase (ATGL) was recently identifi ed as a rate-limiting triglyceride (TG) hydrolase. Mutations in the human ATGL gene are associated with triglyceride deposit cardiomyovasculopathy, which shows massive TG accumulation in both coronary atherosclerotic lesions and myocardium. Recent reports show that ATGL expression is decreased in the obese insulin-resistant state and that myocardial TG content is signifi cantly higher in prediabetes or diabetes and related to ventricular diastolic dysfunction. However, it remains to be clear whether TG deposition plays a role in processes of atherosclerosis.

We therefore investigated the effect of decreased ATGL activity on development of atherosclerosis, using human aortic endothelial cells (HAECs). First we found that ATGL knockdown enhanced monocyte adhesion via increasing expression of tumor necrosis factor alpha (TNFα)-induced intercellular adhesion molecule-1 (ICAM-1). Next, we examined which pathways (mitogen-activated protein kinase, protein kinase C (PKC), or nuclear factor-κB (NFκB)) are involved in the ICAM-1 upregulation induced by ATGL knockdown. Both phosphorylation of PKC pathways and degradation of IκBα were increased in ATGL knockdown HAECs. Inhibition of the PKC pathway using the calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. Furthermore, PKC was activated by diacylglycerol synthesis via upregulated CD36 by ATGL knockdown.

In conclusion, ATGL knockdown increased monocyte adhesion to the endothelium through the enhanced TNFα-induced ICAM-1 expression via NFκB and PKC activation. These results suggest that reducing ATGL expression could exert an infl uence on the development of the atherogenic process on neutral lipid storage diseases and obese insulin-resistant state.

COMPLICATIONS—NEPHROPATHY

385-PPAutophagy Defi ciency in Obese and Aging Mice Exacerbates Proteinuria-Related Renal Tubulointerstitial LesionSHINJI KUME, DAISUKE KOYA, SHIN-ICHI ARAKI, KEIJI ISSHIKI, YOSHIHIKO NISHIO, MASAKAZU HANEDA, ATSUNORI KASHIWAGI, TAKASHI UZU, HIROSHI MAEGAWA, Otsu, Japan, Kahoku-Gun, Japan, Asahikawa, Japan

Although obesity and aging are recognized as risk factors for the progression of diabetic nephropathy, the mechanism remains unclear. Auto phagy is an intracellular process through which damaged protein and organelles are degraded via lysosomes and cell homeostasis is controlled. Recent fi ndings have shown that defi cient autophagy is involved in the pathogenesis of diseases associated with obesity and/or aging. We thus examined the role of autophagy on the progression of proteinuria-related tubulointerstitial lesions in the renal proximal tubular cells of obese and aging mice because renal prognosis correlates more closely with tubulointerstitial than glomerular lesions. We evaluated proteinuria-related tubulointerstitial lesions using the protein-overload nephropathy model. Protein-overload exacerbated renal tubular damage, interstitial infl ammation and fi brosis in mice with obesity induced by a high-fat diet (HFD) and in aged mice (24 months old) as compared with control mice. We examined the effects of obesity and aging on autophagy in tissues from GFP-LC3-transgenic mice, in which autophagosomes can be visualized as green dots of LC3. Both HFD-induced obesity and aging suppressed the increase in the numbers of autophagosomal dots in the proximal tubular cells of control mice induced by protein-overload. We investigated whether the defi cient autophagy in obese and in aging mice is involved in the pathogenesis of enhanced tubulointerstitial lesions as follows. We examined tubulointerstitial lesions mediated by protein-overload in mice treated with 3-methyladenine (3-MA), which inactivates autophagy, and in lysosomal-associated membrane

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protein 2 (lamp2)-knockout mice, in which lysosomes do not bind or degrade autophagosomes. Tubulointerstitial lesions induced by protein-overload were exacerbated in both types of mice. These results indicate that obesity and aging suppress proteinuria-induced autophagy in the proximal tubular cells. Decreased autophagy exacerbated tubulointerstitial lesions mediated by proteinuria, suggesting that defi cient autophagy is associated with progressive tubulointerstitial lesions and a poor renal prognosis for diabetic nephropathy.

386-PPBaseline Hyperfi ltration (HF) Is Associated with Accelerated Rate of Decline in Glomerular Filtration Rate (GFR) in Type 1 but Not in Type 2 Diabetes MellitusRICHARD J. MACISAAC, SHILPA VERMA, EROSHA PREMARATNE, CLEMENT LO, GEORGE JERMUS, Heidelberg West, Australia

The aim of this study was to characterise the consequences of HF in type 1 (T1DM) and type 2 diabetes mellitus (T2DM). We examined the relationship between baseline GFR and subsequent decline in GFR over approximately 12.5 years for patients attending a diabetes clinic of a university teaching hospital. Yearly cystatin C measurements were performed and a cystatin C derived GFR (cysGFR) was calculated using the formula: cysGFR = (87/Cystatin C)-4.2. Baseline age unadjusted GFR thresholds (ml/min/1.73m2) were used, i.e., >130 for HF, 90-130 for normofi ltration and <90 for hypofi ltration to initially classify patients. Age adjusted GFR thresholds were then calculated to account for the expected age related decline in GFR of 1 ml/min/1.73m2

/year for patients aged > 40 years. The main outcome was to determine if baseline HF was associated with an accelerated rate of decline of GFR. Mean gradients of decline in GFR were obtained by linear regression. The baseline charateristics of the patients studied are shown in Table 1.

Parameter T1DM (N=98) T2DM (N=143)Age at baseline (years) 37.1 57.7Duration of diabetes (years) 12.2 8.4Duration of diabetes (years) 12.8 12.9Male:Female ratio 58:40 70:73

Baseline HF was seen in 17 % of T1DM and 11 % of T2DM patients.

Age adjusted GFR category

T1 DMbaseline GFR

(ml/min/1.73m2)

Type 1 DMrate of GFR

decline(ml/min/

1.73m2/year)

Type 2 DM basline GFR

(ml/min/1.73m2)

Type 2 DM rate of GFR

decline(ml/min/

1.73m2/year)

Hyperfi ltration 140.9+ 3.5 (n=17) -4.26* 129.5+3.8 (n=16) -2.03Normofi ltration 105.8+1.3 (n=71) -0.95 91.1+1.3 (n=110) -1.63Hypofi ltration 64.1+ 6.2 (n=10) -1.64 62.9+3.0 (n=17) -2.12

* p < 0.001 vs normofi ltration.

Results were similar using age unadjusted threshold for HF. For all HF patients, approximately half showed a true GFR decline with a fi nal GFR <90 ml/min/1.73m2 as opposed to resolution of HF. We conclude that in T1DM but not in T2DM, baseline HF predicts a greater rate of decline in GFR compared to normofi ltration.

387-PPRenal and Retinal Structural Changes in Type 1 Diabetes (T1D)TASMA HARINDHANAVUDHI, MICHAEL MAUER, RONALD KLEIN, LUIZA CARA-MORI, Minneapolis, MN, Madison, WI

Diabetic nephropathy (DN) and retinopathy (DR) are common chronic complications of T1D, often occurring in the same person. While associations between the severity of DR and DN lesions have been described in patients (pts) with T1D, it is not clear whether or not the severity of DR lesions is associated with the incidence of structural changes of DN. Hence, we studied the relationships between DR and DN structural changes over 5 years (yrs). Pts were part of the Renin-Angiotensin System Study (RASS), a 5 yrs randomized, double-blinded trial comparing the effects of enalapril and losartan to placebo on the rates of progression of DN and DR lesions in normotensive, normoalbumiuric T1D pts age ≥ 16 yrs with 2-20 yrs of T1D duration. There were 234 pts who had both baseline and follow-up retinal photographs and kidney biopsies. DN morphometric parameters and fundus photographs (ETDRS protocol) were assessed at baseline and 5 yrs. At baseline, 34% of pts had no DR, 56% had early nonproliferative DR (NPDR), and 10% had moderate to severe NPDR. During the 5 yrs, all DN lesions increased in severity, however this increase varied among pts with differing

severity of baseline DR. Glomerular basement membrane width increased in pts without DR at baseline from those with early NPDR (27.1±70.3 vs. -1.1±64.7 nm; P=0.004), but was not different vs. those with moderate to severe NPDR at baseline (8.3±79.5 nm; P=0.238). On the other hand, changes in cortical interstitial fractional volume were greater in pts with more severe NPDR (0.109±0.084) vs. no DR (0.052±0.055; P=0.001) or early NPDR (0.061±0.052; P=0.002) at baseline. Similarly, changes in the fractional volume of the cortical tubules that were atrophic were also greater in pts with more severe NPDR (0.032±0.109) vs. no DR (0.006±0.017; P=0.021) or early NPDR (0.002±0.027; P=0.006) at baseline. These results did not change when data are adjusted for HbA1C at baseline. This study demonstrates that DR severity is associated with incident structural DN changes. It remains to be seen whether DR will have utility in predicting changes in DN lesions in normoalbuminuric normotensive T1D pts in clinical practice.

COMPLICATIONS—NEUROPATHY

388-PPObstructive Sleep Apnoea Is Independently Associated with Peripheral Neuropathy in Patients with Type 2 DiabetesABD TAHRANI, ASAD ALI, SAFIA BEGUM, KIRAN DUBB, SHANAZ MUGHAL, MILAN PIYA, BIJU JOSE, DEV BANERJEE, ANTHONY H. BARNETT, SHAHRAD TAHERI, MARTIN J. STEVENS, Birmingham, United Kingdom

Diabetic peripheral neuropathy (DPN) causes signifi cant morbidity. Understanding DPN pathogenesis is essential to develop new therapies. Obstructive sleep apnea (OSA) is prevalent in patients with type 2 diabetes (T2D). Since OSA and diabetes complications share common oxidative stress and infl ammatory mechanisms, we hypothesized that OSA is associated with DPN in patients with T2D.

Adults with T2D were recruited randomly from the diabetes clinic of a UK-based hospital. Patients with known respiratory disorders (including OSA) were excluded. DPN was diagnosed using the Michigan Neuropathy Screening Instrument (MNSI). OSA was assessed using home-based portable multi-channel respiratory monitoring (Alice PDX, Philips Respironics, USA). OSA diagnosed when the apnoea-hypopnea index was ≥5 events/hour (OSA+).

Two-hundred and seventy patients recruited to date (204 available for this analysis, age: 59±12 years, diabetes duration: 11(6-17) years, 54.9% had OSA, 42.7% had DPN and 47.6% were Caucasians). OSA+ patients were older and had higher blood pressure (BP) and waist circumference (WC) compared to those without OSA (OSA-).

70% of patients with DPN had OSA. DPN prevalence was higher in OSA+ (vs. OSA-) patients (56.8% vs. 29.3%, p<0.001). OSA+ remained independently associated with DPN (OR: 2.18, 95%CI 1.14-4.17, p=0.018) after adjusting for age, gender, ethnicity, blood pressure, HbA1c, total cholesterol, triglycerides, diabetes duration, smoking, alcohol, renal function, waist circumference, and the use of anti-platelets, anti-diabetes (oral and injectable), lipid-lowering and anti-hypertensive treatments.

In conclusion, we describe a novel association between OSA and DPN in patients with T2D. Ongoing studies are exploring the mechanisms involved. The ability of OSA treatment to impact DPN warrants determination.

Supported by: NIHR, UK

389-PPTransplantation of Mesenchymal Stem Cell-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Improves Diabetic Poly-neuro pathy in MiceTATSUHITO HIMENO, HIDEKI KAMIYA, TETSUJI OKAWA, KEIKO NARUSE, YUSUKE SEINO, MASAKI KONDO, JIRO KATO, ATSUSHI FUJIYA, YOJI HAMADA, ZHAO CHEN, SACHIKO ITO, KENICHI ISOBE, YUTAKA OISO, JIRO NAKAMURA, Nagoya, Japan

Background: Recent studies have shown that mesenchymal stem cells (MSCs) secrete angiogenic factors and ameliorate diabetic polyneuropathy (DPN) in streptozotocin (STZ) induced-diabetic rats. Here we demonstrated differentiation of induced pluripotent stem cells (iPSCs) to MSC like cells (MLCs), and investigated the therapeutic potential of iPS-MLC transplantation on DPN using STZ-diabetic mice.

Research design and methods: C57BL6/J mice were used in this study. Diabetes was induced by intraperitoneal injection of STZ. To differentiate into MLCs, mouse iPSCs that were which induced from bone marrow-derived dendritic cell, were grown on non-adherent plates with retinoic acid for 4 days, following adherent culture for 4 months. MLC characterized by FACS and RT-PCR were transplanted into hindlimb skeletal muscles of 12-week

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diabetic mice by unilateral intramuscular injection (2 × 105 cells/limb). Three weeks after the transplantation, current perception thresholds (CPTs), motor and sensory nerve conduction velocities (MNCV and SNCV, respectively), and plantar skin blood fl ow (PSBF) were evaluated.

Results: FACS analysis showed that MLCs expressed CD44 and Sca-1, but not hematopoietic lineage markers. RT-PCR showed the expression of VEGF, FGF2 and NGF in MLCs. Three weeks after the transplantation, MLCs resided in hindlimb muscles. In 15-week diabetic mice (DM), CPTs in the saline-injected limbs were signifi cantly increased compared with these in normal mice (C), indicating hypoalgesia, and this deterioration was normalized in MLC-transplanted limbs. Decreased MNCV and SNCV in 15-week DM were signifi cantly ameliorated by MLC transplantation (MNCV; C: 44.5 ± 5.2m/s, DM: 34.4 ± 3.2, DM + MLCs: 38.6 ± 2.2, SNCV; C: 38.4 ± 6.8, DM: 26.0 ± 2.7, DM + MLCs: 31.1 ± 6.7). PSBF in 15-week DM was signifi cantly decreased compared with that in normal mice, which was signifi cantly ameliorated by MLC transplantation.

Conclusions: These results suggest that MLC transplantation could have therapeutic effects on DPN through secreting angiogenic and neurotrophic factors.

COMPLICATIONS—OCULAR

390-PPLiver X Receptor Agonist Prevents Endothelial Progenitor Cell (EPC) Dysfunction and Diabetic Retinopathy (DR)MARIA B. GRANT, ASHAY BHATWADEKAR, SUGATA HAZRA, YUANQING YAN, MAKOTO MIYAZAKI, AMRISHA VERMA, XIAOXIN WANG, QIUHONG LI, MOSHE LEVI, Gainesville, FL, Denver, CO

Liver X receptors (LXRs), of the nuclear receptor superfamily of ligand-activated transcription factors, are regulators of cholesterol homeostasis, modulate macrophage function and have potent anti-infl ammatory effects by inhibiting NF-κB, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 and 3 (MCP-1 and MCP-3) and metalloprotease MMP-9. We hypothesized that LXR agonists could correct diabetes induced dysfunction of EPCs to protect against development of the vasodegenerative phase of DR. Diabetic mice, Db/db and STZ treated DBA/2J, or wild type mice were administered 10 mg/kg (orally) of the LXR agonist, GW 3965, daily for 4 months. Mice were then sacrifi ced and retina and bone marrow were isolated. Lin-Sca1+, c-kit+ (LSK) cells from bone marrow were analyzed for migration and for components of renin angiotensin system (RAS), iNOS, and infl ammatory cytokines by qRT-PCR. Retinas were evaluated for number of acellular capillaries and GFAP immunoreactivity. EPCs of diabetic mice treated with GW3965 showed a signifi cant increase (p<0.05) in migration as compared to untreated diabetic mice. EPCs of GW3965-treated mice demonstrated 50-fold (p<0.05) increase in expression of the Mas receptor, the receptor that mediates the benefi cial effects of Ang (1-7), the protective component of RAS. EPCs of GW3965 treated mice showed reduced expression of IL-6,IL-1 β, MCP-1, and NOX2 mRNA while showing increased AT2R expression (by 7-fold) and increased eNOS mRNA (by 50- fold) compared to untreated diabetic mice. A 50% reduction in the number of acellular capillaries (p<0.05) and complete prevention of GFAP expression in Muller glial cells was observed in the retinas of diabetic animals treated with GW 3965 compared to untreated diabetic animals. Therefore, LXR agonists 1) represent promising pharmacological agents for correcting DR and EPC dysfunction and 2) mediate their effect largely by activation of the protective arm of RAS (AT2R and Mas) and by reducing diabetes associated infl ammation.

Supported by: NIH Grants EY007739, U01 HL087366, HL56912, HL102033 ADA-Funded Research

391-PPSight Threatening Retinopathy Is Independently Associated with Obstructive Sleep Apnea in Patients with Type 2 DiabetesABD A. TAHRANI, ASAD ALI, SAFIA BEGUM, PAUL GALSWORTHY, HELEN WHARTON, MILAN K. PIYA, BIJU JOSE, DEV BANERJEE, ANTHONY H. BARNETT, SHAHRAD TAHERI, MARTIN J. STEVENS, PAUL M. DODSON, Birmingham, United Kingdom

Understanding the pathogensis of diabetic retinopathy is essential to develop new therapies. Obstructive sleep apnoea (OSA) is prevalent in type 2 diabetes (T2D). Since OSA and diabetes complications share common oxidative and infl ammatory mechanisms, we hypothesized that OSA is associated with sight threatening retinopathy (STR) in patients with T2D.

Adults with T2D were recruited randomly from a hospital-based diabetes clinic in the UK. Patients with known respiratory disorders (including OSA)

were excluded. STR was assessed utilising 2x45 degrees retinal images as per the English screening programme protocol. STR was defi ned as the presence of preproliferative (R2), proliferative (R3) retinopathy or maculopathy (M1) on the screening images or an ophthalmologist diagnosis. OSA was assessed with home-based multi-channel respiratory monitoring (Alice PDX, Philips Respironics, USA). An apnoea-hypopnea index ≥5 events/hour was considered to be consistent with OSA diagnosis (OSA+).

270 patients recruited to date (198 patients included in this analysis, age: 59(12) years, diabetes duration: 11(6-17) years, 54.9% were OSA+, 32.8% had STR, and 47.6% were Caucasians). OSA+ patients were older, had higher blood pressure (BP) and waist circumference (WC) compared to patients without OSA (OSA-).

73.8% of patients with STR had OSA. STR prevalence was higher in OSA+ (vs. OSA-) patients (44% vs. 19.1%, p<0.001). OSA+ remained independently associated with STR (OR: 3.53, 95%CI 1.68-7.45, p=0.001) after adjusting for age, gender, ethnicity, BP, HbA1c, total cholesterol, triglycerides, diabetes duration, smoking, alcohol, renal function, WC, anti-platelet, anti-diabetes, lipid-lowering and anti-hypertensive treatments.

This data suggest that OSA might play an important role in the development of STR. We described a novel association between STR and OSA in patients with T2D independent of a wide range of possible confounders. Ongoing studies are exploring the mechanisms involved. The ability of OSA treatment to impact STR development/progression warrants determination.

Supported by: NIHR, UK

DIABETIC DYSLIPIDEMIA

392-PPChanges in Total and High Molecular Weight Adiponectin Levels with Intentional Weight Loss Are Associated with Increased HDL-Cholesterol in Persons with Type 2 Diabetes, Independently of Adi-posity, Glucose Control and Fitness Changes: Findings from the Look AHEAD StudyL. MARIA BELALCAZAR, STEVEN M. HAFFNER, WEI LANG, RON C. HOOGEVEEN, KATHERINE M. DONADIO, DAWN C. SCHWENKE, F. XAVIER PI-SUNYER, RUSSELL P. TRACY, ANDREA M. KRISKA, CHRISTIE M. BALLANTYNE, Galveston, TX, Houston, TX, Winston-Salem, NC, Phoenix, AZ, New York, NY, Burlington, VT, Pittsburgh, PA

Persons with type 2 diabetes have low total and high molecular weight adiponectin (HMW-A) levels. Adiponectin and HMW-A are associated with higher HDL cholesterol (HDL-C) levels. The factors that account for this favorable association are not well understood. We evaluated the effects of changes in total and HMW-A and of other metabolic variables in response to a 1-year intensive lifestyle intervention for weight loss (ILI) on HDL-C in a subset of 1,397 Look AHEAD participants. Look AHEAD is a randomized trial evaluating whether ILI will reduce cardiovascular events/mortality in obese diabetic persons, when compared to usual care (Diabetes Support and Education, DSE). Subjects (mean age 57.2 years, BMI 36.3 kg/m2, HbA1c 7.3%, HDL 42.4 mg/dL, submaximal fi tness 5.2 METS) had total and HMW-A levels measured by ELISA (selective protease assay). Baseline total and HMW-A (median [interquartile range]) were 4.6 (3.3, 6.6) and 1.9 (1.1, 3.1) µg/ml. Changes from baseline with ILI at 1 year were: for total adiponectin 12%, HMW-A 21%, HDL-C 10%, fi tness 21%; HbA1c -0.7% and weight -8% (compared to respective changes of 0.2%, 0.9%, 4%, 6%, -0.2% and -0.6% in DSE; p<0.0001 for all, ILI vs. DSE). Separate regression analyses adjusting for demographics, pertinent medical history, medication use, baseline HDL-C level and treatment arm, found that change in weight (kg) (B: -0.13, p<0.0001), fi tness (METS) (B: 0.56, p<0.0001), HbA1c (%) (B: -0.67, p=0.0005) and total and HMW-A (µg/ml; B: 0.52 and 0.76, respectively, p<0.0001 for both) were associated with HDL-C change at 1 year. However, in fully adjusted models, change in total and HMW-A remained highly signifi cant (B:0.46 and 0.66, respectively, each p<0.0001); fi tness and HbA1c changes were no longer associated with HDL-C change (each p ≥0.07) and the contribution of weight change to HDL-C change was considerably attenuated (B: -0.08, p=0.01). Total and HMW-A are independent predictors of HDL-C change in type 2 diabetic persons.

Supported by: HL090514, HL090514S3

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BEHAVIORAL MEDICINE, CLINICAL NUTRITION, EDUCATION, AND EXERCISE

393-PPHigh Glucose Condition Reduced the Expression of Low-Density Lipo protein Receptor-Related Protein 1 (LRP1) in HepG2 Cells, and Rosiglitazone Directly Up-Regulated LRP1 Expression Via Activat-ing the LRP1 PromotorJAE HOON MOON, AE HEE YANG, HYUNG JUN KIM, SAET BYOU KANG, BYUNG WAN LEE, EUN SEOK KANG, HYUN CHUL LEE, BONG SOO CHA, Seoul, Republic of Korea

LRP1 binds to apoE and serves as a receptor for remnant lipoproteins in the liver, thus playing an important role in clearing these atherogenic particles. In this study, we investigated the effect of high glucose condition and rosiglitazone treatment on LRP1 expression in HepG2 cells.

HepG2 cells were incubated in normal or high glucose condition (5.5 vs. 25 mM in culture media) with or without rosiglitazone treatment (0 vs. 0.5 μM in culture media). The LRP1 expression was assessed by immunoblot. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) in the regulation of LRP1.

Rosiglitazone increased LRP1 expression in HepG2 cells in dose dependent manner.

LRP1 expression was reduced in HepG2 cells incubated in high glucose condition, and rosiglitazone treatment recovered this reduced LRP1 expression.

EMSA and Luciferase assay revealed that rosiglitazone treatment induced the binding of PPARγ and PPRE in the promotor region of LRP1 gene, thus resulted in direct activation of the LRP1 promotor.

In conclusion, hyperglycemic condition might reduce the expression of hepatic LRP1 and the clearance of remnant lipoproteins, and PPARγ agonists could recover the reduced hepatic LRP1 expression and the clearance of circulating atherogenic particles.

Supported by: National Research Foundation of Korea Grant (MEST) (NRF-2010-0003277)

FOOT CARE—LOWER EXTREMITIES

394-PPThe Capacity of Type 2 Diabetic Derived Mesenchymal Stem Cells To Promote Impaired Wound HealingLAURA SHIN, DANIEL A. PETERSON, Chicago, IL

Wound healing is the result of a series of well-orchestrated events involving infl ammation, proliferation and remodeling. Type 2 diabetes disrupts the ability of the body to initiate these phases through loss of blood fl ow, and defective signaling of cellular activity and growth factors. Non-healing wounds lead to a higher incidence of infection and ultimately amputations. The type 2 diabetic mouse BKS.Cg-Dock7m +/+ Leprdb/J (db/db) exhibit phenotypes such as obesity, cardiovascular disease, neuropathy and poor wound healing. These animals are used to study the pathophysiology of impaired wound healing and clinical interventions. Bone marrow-derived mesenchymal stem cells (MSCs) offer one therapeutic approach and accelerate closure when engrafted into impaired wounds. Previous studies and our own results demonstrate that grafted exogenous MSCs initiate repair but disappear long before healing is complete, suggesting signifi cant host involvement in the repair process. To utilize the full potential of these cells we must understand the contribution of endogenous cells during healing. MSCs from healthy subjects are well characterized and used clinically, but little is known about the stem cell populations in subjects with type 2 diabetes. This project focuses on the capacity and contribution of diabetic MSCs in wound healing. MSCs derived from db/db mice were used to evaluate proliferation, differentiation, and their therapeutic potential for wound closure. We found that cultured diabetic MSCs underwent a selection process and were capable of self-renewal and induced differentiation similar to healthy MSCs. MSCs from db/db animals were stable in culture for many passages and resistant to freeze/thaw cycles. Topical applications of diabetic MSCs into an excisional splint wound model accelerated wound healing in the diabetic animal indicating the potential for personalized therapies using a patient’s own cells. These results have also shown the benefi ts of expanding and banking a patient’s own cells for future use. A better understanding of the capacity of endogenous stem cells is of clinical relevance and will contribute to the improved management and care of diabetic wounds. ADA-Funded Research

DIABETES EDUCATION

395-PPClinical Outcomes of Pharmacist-Managed Diabetes Clinic in MalaysiaPIK SENG TAN, P.T. THOMAS, SIEW SIANG CHUA, Kuala Lumpur, Malaysia

The prevalence of diabetes mellitus (DM) in Malaysia has almost doubled over the past one decade. Although numerous studies on pharmaceutical care (PC) have been conducted in developed countries, such service is relatively new in Malaysia and hence data on PC is still scarce. Therefore, the aim of this study was to assess the short term clinical outcomes of providing PC to patients with type 2 DM. A total of 222 patients were recruited at a pharmacist-managed diabetes clinic of a government hospital in Malaysia and randomly allocated to intervention and control group. Patients in the intervention group (n = 111) were provided with PC, whereas, patients in the control group (n = 111) received only the usual pharmacy service. Clinical outcomes of patients were evaluated at baseline and after six months. There was no signifi cant difference in the demographic characteristics and clinical outcomes at baseline between the intervention and the control group. Signifi cant reductions (p < 0.05) in clinical outcomes were observed from baseline to 6-month in the intervention group: mean ± standard deviations of HbA1c (9.93 ± 1.76% versus 8.83 ± 1.85%), systolic blood pressure (135 ± 17mmHg versus 127 ± 12mmHg), diastolic blood pressure (81 ± 8mmHg versus 78 ± 8mmHg), total cholesterol (4.87 ± 1.20mmol/l versus 4.51 ± 0.86mmol/l), triglyceride (1.68 ± 0.78mmol/l versus 1.57 ± 0.60mmol/l), low-density lipoprotein (LDL) cholesterol (2.93 ± 0.97mmol/l versus 2.66 ± 0.76mmol/l). Whereas, no signifi cant change was found in the control group. The pharmacist identifi ed 340 pharmaceutical care issues (PCIs) which included inappropriate dose/regimen (83.8%), contraindication (0.6%), side effects/toxicity (9.4%), polypharmacy (0.6%), nonadherence (4.7%) and others (0.9%). In addition, of the 354 PC interventions, 55.1% were as clinically signifi cant while 73.7% were accepted by the physicians. In conclusion, the incorporation of PC into the management of type 2 DM produced positive impact on patients’ clinical outcomes.

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396-PPLong-Term Effects of a Structured, Intensive Diabetes Education Program in Patients with Type 2 Diabetes Based on Diabetes Duration—A 32-Month Follow-Up StudySEUNG HYUN KO, KI HO SONG, JUNG MIN LEE, YONG MOON PARK, HYUK SANG KWON, YU BAE AHN, Seoul, Republic of Korea

Diabetes education is a critical element in diabetes care for type 2 diabetes patients. We investigated whether adherence to lifestyle behaviors prompted by diabetes education would be infl uenced by the duration of diabetes.

Two hundred twenty fi ve people with type 2 diabetes (102 male, 123 female) were recruited and an intensive, collaborative, goal-setting, group-based diabetes education program was delivered. All the participants were followed up every three months thereafter on an outpatient basis with annual reinforcement. We divided the subjects into two groups, according to the duration of their diabetes prior to the education (≤ 1 year vs. ≥3 years). Hemoglobin A1c (A1C) was measured 2–3 times per year. Dietary habits, physical activity, and the frequency of self-monitoring of blood glucose were evaluated by questionnaire before education and at the follow-up end point.

The mean follow-up period was 32.1 months. Mean age and diabetes duration of the subjects were 50.2 ± 10.6 years and 3.4 ± 3.2 months for the ≤1 year group, and 53.3 ± 8.9 years and 8.5 ± 4.9 years for the ≥ 3 years group, respectively. The mean A1C value was signifi cantly lower in the ≤ 1 year group than in the ≥ 3 years group (7.0 ± 1.2 vs. 8.2 ± 1.6%; P < 0.05). Sixty two (45.9%) of the ≤ 1 year group and 15 (16.7%) of the ≥3 years group reached the target A1C level (mean A1C ≤ 7.0%). Self-care behaviors were better adhered to in the recently diagnosed group. Scores for dietary habits (P = 0.004) and physical activity (P < 0.001) were higher at the end point in the ≤1 year group than in the ≥3 years group. Logistic regression analysis revealed that diabetes duration before education was signifi cantly associated with mean A1C level ≥ 7.0% during the follow-up period (≥ 3 years vs. ≤ 1 year, odds ratio 3.758, 95% confi dence interval 1.993–7.087; P < 0.001).

Diabetes duration infl uenced the effect of diabetes education on glycemic control and lifestyle behaviors. More intensifi ed, regular and sustained reinforcement with encouragement may be required for individuals with long-standing type 2 diabetes.

EXERCISE—ANIMAL

397-PPRats Bred for Low Response to Exercise Training Have Insulin Resist -ance and Altered Skeletal Muscle Signal TransductionSARAH J. LESSARD, ANA B. ALVES-WAGNER, MICHAEL F. HIRSHMAN, KRISTIN I. STANFORD, KRISTEN M. HITCHCOX, DONATO A. RIVAS, ROGER A. FIELDING, STEVEN L. BRITTON, LAUREN G. KOCH, LAURIE J. GOODYEAR, Boston, MA, Ann Arbor, MI

Low aerobic exercise capacity is a predictor of mortality and diabetes risk. However, the ability to increase exercise capacity in response to exercise training is a highly variable trait with a strong genetic component. To study the link between the ability to respond to training and disease risk, we developed rat models of high (HRT) and low (LRT) response to exercise training by divergent artifi cial selection. Each generation, rats with the highest (HRT) and lowest (LRT) magnitude of change in maximal running distance as a result of 8 wks of treadmill training were chosen for breeding. Here, we studied female rats from generation 12, where training of HRT rats (n=20) increased running distance by 57±7% and training of LRT rats (n=20) decreased running distance by 29±3%. Rats were then studied after 20 wks of detraining. In this untrained state, LRT had greater body weights (P<0.05), greater gonadal fat pad mass (P<0.05), and impaired insulin sensitivity (HOMA-IR; P<0.05) compared to HRT. Type I fi ber content of plantaris m. was also 40% lower in LRT (P<0.02). We next determined the effects of training response phenotype on exercise-induced gene transcription and signal transduction in skeletal muscle. The rats performed a single bout of exercise (15 m/min, 15% grade, 25 min), which was the same intensity (VO2/VCO2) for LRT and HRT after detraining. Exercise-induced increases in mRNA of the critical metabolic regulators PGC-1α, Nur77 and GLUT4 were similar between LRT and HRT, suggesting that altered transcription of these genes is not responsible for impaired training response in LRT. Exercise also similarly increased AMPK T172, ACC S218 and Akt S473 phosphorylation in skeletal muscle of LRT and HRT. However, exercise-induced activation of JNK, a stress-activated kinase implicated in insulin resistance, was 45% higher in LRT (P=0.007). In conclusion, rats bred for low response to exercise training have elevated risk factors for metabolic disease and altered skeletal

muscle signaling after a bout of exercise at the same intensity. Elevated stress signaling in response to exercise could be a mechanism for the metabolic impairments in LRT.

EXERCISE—HUMAN

398-PPIntramyocellular Lipid Content and Insulin Sensitivity Are Increased Following a Short-Term Low-Glycemic Index Diet and Exercise InterventionJACOB M. HAUS, THOMAS P.J. SOLOMON, LAN LU, JOHN A. JESSBERGER, CHRIS A. FLASK, JOHN P. KIRWAN, Cleveland, OH

The relationship between intramyocellular (IMCL) and extramyocellular lipid (EMCL) accumulation and skeletal muscle insulin resistance is complex and dynamic. We examined the effect of a short-term (7-day) low-glycemic index (LoGI) diet and aerobic exercise training intervention on IMCL and insulin sensitivity in older, insulin resistant humans. Participants (67±1 y; 33.4±0.9 kg/m2) were randomly assigned to a parallel, controlled feeding trial (either a LoGI (LoGI+Ex; N=9) or High-GI (HiGI+Ex; N=9) eucaloric diet), combined with supervised exercise (60 min/d, 85% HRmax). Insulin sensitivity was determined via 40 mU/m2/min hyperinsulinemic euglycemic clamp, and soleus IMCL and EMCL content was assessed by 1H-MR spectroscopy with correction for fi ber orientation. BMI decreased (kg/m2: -0.7±0.2, LoGI+Ex; -0.6±0.1, HiGI+Ex, P<0.0001) after both interventions with no interaction effect for diet composition. Clamp derived insulin sensitivity increased by 0.83±0.21 (LoGI+EX) and 0.35±0.44 mg/kg/min (HiGI+EX); P=0.03. HOMA-IR was reduced by -1.1±0.4 (LoGI+Ex) and -0.2±0.2 (HiGI+Ex); P=0.02. While both interventions increased IMCL content, (Δ: 2.3±1.3, LoGI+Ex; 1.4±0.9, HiGI+Ex, P=0.03), diet composition did not signifi cantly effect the increase. However, the LoGI+Ex group showed a robust increase in the IMCL/EMCL ratio compared to the HiGI+Ex group (Δ: 0.5±0.2 LoGI+Ex vs. 0.07±0.1, P=0.03). The LoGI+Ex group also demonstrated greater reductions in EMCL compared to the HiGI+Ex group (Δ: -5.8±3.4, LoGI+Ex; 2.3±1.1, HiGI+Ex, P=0.03). Changes in muscle lipids and insulin sensitivity were not correlated, however the change in the IMCL/EMCL ratio was negatively associated with the change in FPI (r: -0.78, P=0.002) and HOMA-IR (r: -0.61, P=0.03). These data suggest that increases in the IMCL pool following a low glycemic diet and exercise intervention may represent lipid repartitioning from EMCL. The lower systemic glucose levels that prevail whilst eating a low glycemic diet may promote redistribution of lipid stores in the muscle.

Supported by: NIH RO1 AG12834

NUTRITION—CLINICAL

399-PPA High-Fiber Diet Improves the Vascular Function as Well as Glycemic Control in the Patients with Type 2 Diabetes MellitusYOSHIHIKO NISHIO, KEIKO KONDO, KEIKO NAKAO, TETSUYA HASHIMOTO, ATSUSHI ISHIKADO, SATOSHI UGI, ATSUNORI KASHIWAGI, HIROSHI MAEGAWA, Otsu, Shiga, Japan

Dietary fi ber reduces the glycemic response of carbohydrate resulting in decreasing postprandial glucose level. This effect of dietary fi ber may improve the impairments of vascular function in the patients with type 2 diabetes. To address this possibility, we investigated the vascular function of 8 subjects with normal glucose tolerance (age: 58.0 ± 6.0) and 10 patients with type 2 diabetes (age: 59.2 ± 11.1) with reactive hyperemia using strain-gauge plethysmography. In addition, the diabetic patients were followed for 16 weeks assigned to two periods of 8 weeks of either a high-fi ber diet or control diet in a randomized crossover design. The high-fi ber diet was provided in retort pouch once a day. Before and after each period, vascular function was evaluated and 75g oral glucose tolerance test was performed.

The vascular functions assessed with either peak forearm blood fl ow (416.7 ± 209.3 vs. 800.4 ± 458.4%, p = 0.026) or fl ow debt repayment (FDR) (47.7 ± 18.1 vs. 103.9 ± 68.0%, p = 0.015) was signifi cantly impaired in diabetic patients as compared with control. The intervention of an 8 weeks high-fi ber diet once a day improved 39.2% of peak forearm blood blow (p = 0.075), and 63.5% of FDR (p = 0.006), but control diet did not show any effects. Total energy intake was unchanged through out the study periods. The intake of dietary fi ber in the high-fi ber diet period was higher than that in the baseline or control period (23.9 ± 4.3 vs. 12.8 ± 3.0 vs. 12.4 ± 2.8 g/day, p < 0.0001). Body weight (69.7 ± 12.9 vs. 68.5 ± 13.2 kg, p = 0.007), fasting glucose (135.4 ± 37.7 vs. 124.1 ± 31.1 mg/dL, p < 0.05), glucose AUC for 2 hours (29500 ± 6570

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vs. 27100 ± 7540 mg·min/dL, p < 0.05) and HbA1c (7.0 ± 0.6 vs. 6.8 ± 0.6%, p = 0.036) levels were signifi cantly reduced after the high-fi ber diet period.

Present study demonstrated that taking a high-fi ber diet once a day improved the vascular dysfunction as well as glycemic control in the patients with type 2 diabetes, suggesting the importance of dietary fi ber intake for the prevention of cardiovascular complications of type 2 diabetes.

PSYCHOSOCIAL—BEHAVIORAL MEDICINE

400-PPA Comparative Effectiveness Trial of Two Internet Programs for Youth with Type 1 Diabetes (T1D)ROBIN WHITTEMORE, SARAH JASER, KATHRYN MURPHY, MELISSA FAULKNER, ALAN DELAMATER, MARGARET GREY, New Haven, CT, Philadelphia, PA, Tucson, AZ, Miami, FL

Advances in social media technology and access to the internet have enabled web-based delivery of psychosocial interventions. The purpose of this ongoing multisite clinical trial is to compare the effectiveness of TEENCOPE™ (an internet coping skills training program) with an internet education program (MANAGING DIABETES) for youth with T1D on physiologic (A1C), psychosocial (self-effi cacy, coping, quality of life), and family (confl ict) adaptation. Short-term results (3 month) are reported.

Teens (n=327) with T1D (11-14 yrs) were randomly assigned to the 5-week TEENCOPE™ or MANAGING DIABETES internet programs and completed baseline and 3 month data via the internet on self-report measures. A1C was obtained from clinic records. The mean age of teens was 12.3 +1.1 yrs, mean diabetes duration was 4.5 + 3.5 yrs, mean A1C was 8.29 +1.5%, with 59% on pump therapy. Fifty-fi ve percent were female and 62% were non-Hispanic White (20% Hispanic, 11% Black). Treatment groups were comparable at baseline. There was excellent participation in both programs (83% of youth completed 4 of 5 sessions; 72% completed 5 sessions).

Results of the repeated measures ANOVA indicated that teens in both groups reported better self-effi cacy for diabetes care (p=.001) and less diabetes-related family confl ict (p=.009) over time. For youth with A1C > 8%, there was a signifi cant decrease in A1C in both groups (from 9.33 to 8.99, p=.007). Coping improved in both groups; however this was not statistically signifi cant. There were no differences between groups on any outcomes in this short-term analysis.

Participation in the TEENCOPE™ and MANAGING DIABETES internet programs was excellent, and resulted in improved self-effi cacy and decreased diabetes-related family confl ict after 3 months in ethnically diverse youth with T1D. For youth with poor metabolic control, A1C also improved. These results suggest the use of diabetes-specifi c coping and educational internet programs with young teens improves select outcomes. Longer-term follow-up is indicated.

Supported by: NINR-R01NR04009

401-PPDepression Symptoms, Antidepressant Medicine Use, and Cardio-vascular Disease Risk Factors in the Look AHEAD Clinical Trial of Weight Loss in DiabetesRICHARD R. RUBIN, MARK PEYROT, SARAH A. GAUSSOIN, LOOK AHEAD RESEARCH GROUP, Baltimore, MD, Winston-Salem, NC

We explored associations between depression measures (symptoms, antidepressant medicine [ADM] use) and number of cardiovascular disease (CVD) risk factors in 5,145 overweight adults with type 2 diabetes in the Look AHEAD Trial, a clinical trial comparing an intensive lifestyle intervention (ILI) for weight loss with usual care (diabetes support and education [DSE]). At baseline and annually (study years 1-4), participants completed the Beck Depression Inventory (BDI), reported ADM use, and were assessed for CVD risk factors. Positive CVD risk status for a factor was defi ned as elevated risk factor level, or medication for that risk factor (i.e. elevated HbA1c or insulin use, elevated blood pressure or antihypertensive use, elevated lipid levels or lipid-lowering medication, current smoking, and BMI >30 kg/m2). Overall CVD risk score was the number of positive CVD risk factors.

Linear mixed models assessed the association of current BDI score and ADM use, as time-varying covariates, with overall CVD risk factor score over trial years 1-4, controlling for trial year and baseline characteristics (age, gender, race, education, BDI score, ADM use, and overall CVD risk factor score).

Current ADM use and higher BDI scores (both p<0.0001) were associated with higher overall CVD risk factor scores. Subsequent analysis found a signifi cant interaction between time-varying ADM use and baseline overall

CVD risk factor score (p<0.01): the association was stronger among those with lower baseline overall CVD risk scores. All results were similar for separate analyses of ILI and DSE treatment groups.

In summary, Look AHEAD trial participants who used ADM or had higher depression symptom scores during trial years 1-4 had higher overall CVD risk factor scores; the association with ADM use was stronger in participants with lower baseline overall CVD risk factor scores. The association between depression measures and manifest CVD in this population remains to be determined.

402-PPPredictors of Depression Symptoms and Antidepressant Medicine Use in Diabetic Participants in the Diabetes Prevention Program and the Diabetes Prevention Program Outcomes StudyDAVID G. MARRERO, RICHARD R. RUBIN, YONG MA, WILLIAM KNOWLER, DAVID PRICE, ELIZABETH BARRETT-CONNOR, EDWARD HORTON, MERCEDES CARNETHON, DIABETES PREVENTION PROGRAM RESEARCH GROUP, Indian-apolis, IN, Baltimore, MD, Rockville, MD, Phoenix, AZ, Denver, CO, La Jolla, CA, Boston, MA, Evanston, IL

Diabetes Prevention Program (DPP) and Diabetes Prevention Program Outcomes Study (DPPOS) volunteers in 3 treatment arms [intensive lifestyle (ILS), metformin (MET), placebo (PLB)] were assessed semiannually or annually for diabetes, glucose control, antidepressant use, and depression symptoms based on Beck Depression Inventory (BDI) (score value ranges 0-63). There were 1285 participants who developed diabetes during the DPP/ DPPOS (1996-2008) whose level of depression was measured before and after their diabetes diagnosis (median follow-up 6.0 years

(IQR 3.0-7.9) after diagnosis). We examined whether the diagnosis of diabetes predicted elevated depressive symptoms or antidepressant medicine (ADM) use at the next visit post diagnosis. We also assessed the association between glycemic control (A1C, fasting plasma glucose [FPG], normalization of FPG) and depression symptoms or ADM use after diabetes diagnosis using repeated measures throughout the visits after diagnosis.

Neither depression symptoms nor ADM use increased signifi cantly at the fi rst visit following diabetes diagnosis. However, controlled for potential confounders measured at DPP randomization, during the visits after diabetes diagnosis, higher FPG was associated with greater ADM use in the ILS arm (OR 1.09, 95% CI: 1.04-1.34 per 10 mg/dl higher FPG). In addition, higher FPG, higher A1C (%) and lack of normalization of FPG were associated with higher BDI scores in all three arms. On average, BDI score was 0.073 (95% CI 0.036-0.111, p=0.001) point higher with 10 mg/dl higher FPG, 0.2185 point (95% CI 0.075-0.3620; p=0.0028) higher with 1% higher A1C, 0.3031 point (95% CI 0.0924 to 0.5119; p=0.0045) higher if FPG had not normalized. Assessing BDI as a dichotomous variable (>11 yes or no) did not change these outcomes.

In summary, the diagnosis of diabetes did not affect BDI or ADM use. In contrast, higher glucose levels after diabetes diagnosis were associated with small but signifi cant increases in BDI score and ADM use.

Supported by: NIH

403-PPStructured SMBG Promotes Positive Changes in Self-Management Attitudes in Non-Insulin Treated T2DM: STeP Study ResultsLAWRENCE FISHER, WILLIAM POLONSKY, CHRISTOPHER PARKIN, ZHIHONG JELSOVSKY, ROBIN WAGNER, San Francisco, CA, San Diego, CA, Indianapolis, IN, Tampa, FL

Recent studies suggest that SMBG use in non-insulin treated T2DM is often a discouraging and de-motivating experience, leading to negative attitudes about diabetes self-care. The Structured Testing Protocol (STeP) study investigated the impact of structured SMBG use on changes in HbA1c and diabetes-related attitudes. In this prospective, cluster-randomized, multi-centered clinical trial, 483 poorly-controlled (HbA1c ≥7.5%), insulin-naïve T2DM patients were randomized to structured testing (STG) or active control (ACG). STG subjects used the ACCU-CHEKâ 360° View Blood Glucose Analysis System to collect/interpret 7-point glucose profi les over 3 consecutive days. STG patients completed the tool on a quarterly basis and brought it to medical visits. STG patients and physicians received standardized instruction in SMBG pattern recognition/interpretation. All STG and ACG patients received free blood glucose meters and test strips. At baseline, 1, 3, 6, 9 and 12 months, patients completed self-report measures of diabetes self-effi cacy, diabetes-related autonomous motivation, and SMBG attitudes. Coupled with signifi cantly greater HbA1c improvement in the STG over the 12-month study period, per protocol analyses revealed that STG patients also evidenced greater gains in diabetes self-effi cacy (p<0.05), became more autonomously motivated for diabetes care (p<0.05),

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CLINICAL DIABETES/THERAPEUTICS

and achieved a more positive attitude on the value of SMBG (total SMBG Disinterest Scale [SDS]; p<0.09) than ACG subjects. Of the 3 SDS subscales (SMBG distress, SMBG pointlessness, and SMBG helpfulness), only change in SMBG distress distinguished the two treatment groups at 12 months (p<0.05). HbA1c improvement over the 12 months was linked to gains in diabetes self-effi cacy (p<0.002) and a more positive SMBG attitude (total SDS; p<0.001), with no signifi cant between-group differences. SMBG use is not discouraging or de-motivating when both patients and physicians collaborate to gather, interpret and utilize structured SMBG data; rather, patients feel more confi dent and internally motivated in their diabetes care and are more positive regarding the value of SMBG.

CLINICAL THERAPEUTICS/NEW TECHNOLOGY—GLUCOSE MONITORING AND SENSING

404-PPInitial Experience of Automated Low Glucose Insulin Suspension Using the Medtronic Paradigm Veo SystemTRANG T. LY, JENNIFER A. NICHOLAS, ELIZABETH A. DAVIS, TIMOTHY W. JONES, Subiaco, Australia

Real-time continuous glucose monitoring systems linked to insulin pump therapy with a low glucose suspend (LGS) function cease insulin delivery for 2 hours if the patient does not respond to a low glucose alarm at a preset glucose level. The patient can resume insulin delivery at any stage and if the patient remains unresponsive, insulin delivery will auto-resume after 2 hours. As a part of an intervention trial using the Veo pump in children and adults with type 1 diabetes, we report the initial use of this system.

To date, 13 subjects (mean±SE, age 15.9±1.9y, age range 6.1-30.6y, duration of diabetes 7.9±1.3y, pump duration 4.1±1.0y) have worn the Veo system with LGS set at 60mg/dL for a total of 1021 days. During this time there were 1124 LGS events. Insulin delivery was restarted by patients in the majority of events with 50% of LGS events lasting less than 10 minutes and a further 28% lasting between 10 minutes and 1 hour.

There were 106 full 2 hour LGS events (10% of total) and 85% of these occurred overnight. The suspend patterns differed markedly between patients. Of the nocturnal LGS events, 1 in 5 lasted 2 hours. The mean sensor glucose after the nocturnal 2 hour suspend period was 108.4±0.4mg/dL.

On 6 separate occasions, multiple overnight LGS events led to insulin suspension of 4 hours in one night. These were associated with a fi rst morning fi ngerstick BG of 315±29mg/dL (range 194.4 to 397.8mg/dL). There were no episodes of ketoacidosis requiring hospital admission. There were no episodes of severe hypoglycemia resulting in seizure or coma. Patient satisfaction with the Veo pump was high with 60% of subjects electing to continue using the system after the initial 6 months with the remainder reporting that they would use it if the system was affordable.

The use of the Veo pump with LGS feature appears safe and is well-tolerated. LGS was frequently activated however most events were of short duration. Multiple LGS events can occur overnight leading to extended periods of insulin suspension. The system offers a potentially useful tool in reducing the risk of prolonged severe hypoglycemia, particularly nocturnal hypoglycemia.

405-PPPhysical Activity of Daily Living Predicts CGM Rate of Change in Type 1 DiabetesGIULIA DONA, CHINMAY MANOHAR, CHIARA DALLA MAN, DEBASHIS NANDY, AHMED SAAD, RITA BASU, JAMES A. LEVINE, ANANDA BASU, YOGISH C. KUDVA, CLAUDIO COBELLI, Padova, Italy, Cleveland, OH, Rochester, MN

Although physical activity (PA) has been linked to hypoglycemia in patients with Type 1 Diabetes (T1D), few studies have investigated the effects of activities of daily living on interstitial glucose (IG) dynamics. A better understanding of this interaction would be of great importance to optimize insulin delivery also in an artifi cial pancreas context. Recently, we demonstrated that PA, measured with a Physical Activity Monitoring System (PAMS), is a determinant of IG rate of change in nondiabetic individuals. The aim of this study was thus to test if such a relationship also exists in T1D. Seven T1D patients (3 males; age=43±6 yrs [mean±SE]; BMI= 27±1.6 kg/m2) were studied in the Clinical Research Unit at Mayo Clinic, Rochester MN, for an 88 hour period. Patients were asked to walk on a treadmill at a constant speed for 26 minutes, then sit on a chair for 34 minutes and repeat this exercise several times a day to mimic total daily physical activity performed by people all over the world during normal “free” living. PA was captured with PAMS, a system that has double accelerometers and inclinometers for

highly accurate recording of body postures and movement. IG was measured with CGM Dexcom Seven® Plus. IG derivative was calculated from CGM signal using a deconvolution-based method, in six intervals per subjects (in absence of disturbances, i.e. suffi ciently far from meals and PAMS calibrations). The relationship between the PA and CGM rate of change was assessed using cross-correlation, which takes into account the presence of delay between the signals. Correlation between PA and CGM rate of change was higher than 0.5 (p<0.0001) in 51%, and higher than 0.7 (p<0.0001) in 24% of the analyzed intervals. Five subjects presented at least one interval with correlation higher than 0.7; in the remaining two, the maximum correlation was higher than 0.66. The delay between PA and CGM derivative for intervals with correlation higher than 0.5 was 21±2.6 min. Present results show that PA of daily living captured by PAMS is a determinant of glucose fl uctuations in T1D and this should be considered to optimize open and closed-loop insulin delivery

Supported by: DK85516

CLINICAL THERAPEUTICS/NEW TECHNOLOGY—INSULIN DELIVERY SYSTEMS

406-PPClosed-Loop Control during 6 Overnight Periods in 23 Adult Subjects with Type 1 DiabetesGUIDO FRECKMANN, STEFAN PLEUS, CORNELIA HAUG, STEFAN WEINERT, CAROLINE PATTE, Ulm, Germany, Indianapolis, IN, Burgdorf, Switzerland

Overnight closed-loop control was used in a clinical study to achieve a narrow target range of blood glucose (bG) concentrations before breakfast.

Twenty-three subjects with type 1 diabetes performed 2 overnight experiments on each of 3 visits at the study site. From midnight until get-up (06:00) closed-loop control was based on hourly blood glucose measurements using a proprietary empirical algorithm. Every 10 minutes, the controller made insulin delivery recommendations which had to be approved manually by a healthcare professional. Therapy between dinner and midnight was managed by subjects using their own therapy before the fi rst night and it was managed by the controller based on continuous glucose measurements before the second night. Blood glucose measurements between 01:00 a.m. and 06:00 a.m were evaluated. The controller was set to a point target of 120 mg/dL (6.7 mmol/L) with a target range of 90 – 150 mg/dL (5.0 – 8.3 mmol/L).

Overall, the number of bG measurements within the target range increased from 52.9% (219 out of 414 measurements) during the fi rst night to 72.2% (299 out of 414 measurements) during the second night (p < 0.01, χ²-test). The data further show that the occurrence of interventions related to hypoglycemia was reduced from 14 interventions, the latest occurring at 02:36 a.m., during the fi rst night to 1 intervention occurring at 01:02 a.m. during the second night (p < 0.01, χ²-test). Interventions were performed if bG concentration dropped below 50 mg/dL (2.8 mmol/L) or in case of hypoglycemia symptoms.

During both nights, good glycemic control was established. There was a trend towards better metabolic control during the second nights, suggesting that performance of the controller increased with an adequate run-in time. BG concentrations were shifted towards the target range and hypoglycemia was signifi cantly reduced. Closed-loop control allowed to achieve a narrow range of bG concentrations in the study population.

Supported by: Roche Diabetes Care AG, Switzerland, and Roche Diagnostics, United States

407-PPSensor-Augmented Pump Therapy for A1c Reduction (STAR 3) Study: Results from the 6-Month Continuation PhaseRICHARD M. BERGENSTAL, WILLIAM V. TAMBORLANE, ANDREW AHMANN, JOHN B. BUSE, GEORGE DAILEY, STEPHEN N. DAVIS, CAROL JOYCE, TIM PEOPLES, BRUCE A. PERKINS, JOHN B. WELSH, STEVEN M. WILLI, MICHAEL A. WOOD, Minneapolis, MN, New Haven, CT, Portland, OR, Chapel Hill, NC, La Jolla, CA, Baltimore, MD, St. John’s, Newfoundland and Labrador, Canada, Goleta, CA, Toronto, ON, Canada, Northridge, CA, Philadelphia, PA, Grand Rapids, MI

STAR 3, a 1-yr randomized clinical trial (RCT), compared sensor-augmented insulin pump (SAP) therapy to multiple daily injection (MDI) therapy in 485 subjects with type 1 diabetes. We report here the results of the 6-mo, single crossover (MDI to SAP) continuation phase of STAR 3. Aims were to examine the durability of the effects of SAP for up to 18 mo and to evaluate the effects of switching to SAP in subjects whose metabolic control with MDI had been optimized. In the RCT, baseline A1C levels were 8.3% in both groups but, at study end, A1C values were signifi cantly lower in the SAP group (7.5%) than the MDI group (8.1%) (P<0.001). A total of 443 subjects completed the RCT

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and 420 (95%) enrolled in the continuation phase. As shown in the Figure, A1C remained reduced during the continuation phase in the SAP group. After device training and 3 mo use of the SAP system, A1C decreased to 7.6% in the crossover group (P<0.001). The A1C decrease from 12 mo in the crossover group was signifi cant at both 15 and 18 mo, and among both adult and pediatric subgroups (P<0.05). Rates of severe hypoglycemia were low (19.3 [9 events] and 2.0 [2 events] per 100 patient-yr in prior-SAP and crossover groups, respectively), and not signifi cantly different between groups (P=0.205). Median sensor wear during the continuation phase was higher in the SAP group than in the crossover group (65% vs. 57%, P<0.01). Increased sensor wear was signifi cantly associated with greater A1C reductions in the crossover group (P<0.001). Glycemic benefi ts of SAP therapy persist for at least 18 mo. Both adult and pediatric subjects transitioning from MDI to SAP therapy experienced rapid and safe A1C reductions in the STAR 3 study continuation phase.

Supported by: Medtronic, Inc.

CLINICAL THERAPEUTICS/NEW TECHNOLOGY—PHARMACOLOGIC TREATMENT OF DIABETES

OR ITS COMPLICATIONS

408-PPColesevelam Improves Oral but Not Intravenous Glucose Tolerance by a Mechanism Independent of Insulin Sensitivity and β-Cell FunctionANNA L. MARINA, KRISTINA M. UTZSCHNEIDER, LORENA A. WRIGHT, BRENDA K. MONTGOMERY, SANTICA M. MARCOVINA, STEVEN E. KAHN, Seattle, WA

The bile acid sequestrant colesevelam improves glycemic control in patients with type 2 diabetes, but the mechanism by which it does so is unknown. To determine how colesevelam improves glucose metabolism, we measured insulin sensitivity (SI), β-cell function and glucose tolerance in 20 subjects with impaired fasting glucose (11 men, 9 women; age 60.7±1.9 y (x±sem), BMI 29.4±0.9 kg/m2) at the end of 2-wks of single-blind placebo treatment and again after 8-wks of single-blind treatment with colesevelam 3.75 g once daily with dinner. From a frequently sampled intravenous glucose tolerance test (IVGTT) we quantifi ed SI using the minimal model, the acute insulin response to glucose (AIRg) and intravenous glucose tolerance (Kg), while from a meal tolerance test (MTT) we quantifi ed the incremental area under the curve for glucose (iAUCG) as a measure of oral glucose tolerance and the ratio of iAUC insulin (iAUCI) and glucose (iAUCI/iAUCG) as a measure of insulin release.

Fasting plasma glucose and HbA1c decreased signifi cantly with cole-sevelam treatment (106.4±1.4 to 102.7±1.8 mg/dl, p<0.05 and 5.86±0.06 to 5.76±0.06%, p=0.01, respectively), but fasting insulin did not change (11.0±1.8 to 10.4±1.8 µU/ml). Colesevelam had no effect on IVGTT measures: SI (2.88±0.30 to 2.75±0.35 x10-4 min-1/(µU/ml)), AIRg (361±77 to 394±96 µU/ml.min) and Kg (1.26±0.08 to 1.32±0.10 %.min-1). In contrast, during the MTT both iAUCG (4491±514 to 3581±425 mg/dl.min, p<0.01) and iAUCI (4266±586 to 3316±735 µU/ml.min, p<0.05) decreased signifi cantly with colesevelam. However, β-cell function did not change given that SI remained constant and neither AIRg nor iAUCI/iAUCG (1.16±0.15 to 1.01±0.15 (µU/ml)/(mg/dl)) differed with colesevelam treatment.

In conclusion, colesevelam improves oral but not intravenous glucose tolerance. As insulin sensitivity and β-cell function were not altered, this suggests that either glucose absorption is decreased or a gastrointestinal factor is elaborated that alters glucose metabolism in the liver either directly or by, for example, reducing glucagon release.

Supported by: Daiichi-Sankyo

409-PPComparison of 3 Intensifi ed Insulin Regimens Added to Oral Therapy for Type 2 Diabetes: Twice-Daily Aspart Premixed vs Glargine Plus 1 Prandial Glulisine or Stepwise Addition of Glulisine to GlargineMATTHEW C. RIDDLE, ALEKSANDRA VLAJNIC, BEVERLY A. JONES, JULIO ROSENSTOCK, Portland, OR, Bridgewater, NJ, Lake Mary, FL, Dallas, TX

How to advance from unsuccessful oral to intensifi ed insulin therapy in type 2 diabetes is debated. A 60-wk randomized, open-label study compared effi cacy, hypoglycemia, and body-weight (BW) after adding BID premixed 70/30 protamine-aspart/aspart (PREMIX, n=192), glargine +1 prandial glulisine dose (GLARG+1, n=189), or glargine plus stepwise glulisine (GLARG+0-3, n=191). Mean baseline (BL) A1C was 9.4% after 4-wk run-in on 2-3 oral agents; age 54 y, diabetes duration 9 y, BMI 33.2 kg/m2. Insulin was titrated seeking fasting and preprandial glucose <100 mg/dL with A1C <6.5%. Each regimen reduced A1C (Figure) but more randomized patients had Wk 60 A1C <7% with GLARG+1 (49%, P<0.025) or GLARG+0-3 (45%, P<0.05) than with PREMIX (39%), and more did so without hypoglycemia with GLARG+1 (24%, P<0.05) or GLARG+0-3 (24%, P<0.01) than with PREMIX (14%). Mean ΔA1C at endpoint (last observation carried forward [LOCF]) was -1.8±0.1% with PREMIX vs -2.1±0.1% (P=0.06) and -2.2±0.1% (P<0.01) with GLARG+1 and GLARG+0-3.

Adjusted event-rates per patient-yr (ER) and incidences (%) for categories of hypoglycemia are reported below.

PREMIX GLARG+1 GLARG+0-3 ER % ER % ER %

BG <70mg/dL+ symptoms 12.2±1.7 72±4 7.1±1.0b 63±4a 7.2±1.0b 60±4a

BG <50mg/dL + symptoms 1.9±0.3 46±4 0.8±0.2c 33±4b 0.9±0.2c 32±4b

BG <36mg/dL 0.2±0.1 14±3 0.1±0.0b 9±2 0.2±0.0 11±2Requiring assistance 0.2±0.1 8±2 0.1±0.0 7±2 0.2±0.1 10±2Coma or seizures 2±1 0 0.4±0.4ER±SE, aP<0.05, bP<0.01, cP<0.001 vs PM

ΔBW at Wk 60 was similar among regimens. Conclusion: signifi cantly larger proportions of patients achieved target A1C <7% on both glargine-based regimens with less hypoglycemia than with premixed insulin.

Supported by: sanofi -aventis US

410-PPEffect of Exenatide Therapy on Plasma FGF21 and NAFLD in Type 2 DiabetesPADMA SATHYANARAYANA, RAJA MUTHUPILLAI, MEDHAVI JOGI, SUSAN SAMSON, MANDEEP BAJAJ, Houston, TX

Plasma Fibroblast Growth Factor 21 (FGF21) levels and hepatic FGF21 expression are increased in Non Alcoholic Fatty Liver Disease (NAFLD) and FGF21 has been proposed as a novel biomarker for NAFLD. We examined the effects of exenatide (GLP-1 receptor agonist) therapy on plasma FGF21 levels and hepatic fat content in patients with type 2 diabetes (T2DM). 21 T2DM patients (Age = 52± 3 y, BMI = 32.0 ±1.5, HbA1c = 8.2 ± 0.4%) on diet and/or metformin received additional treatment with either pioglitazone 45 mg/day for 12 months or combined therapy with pioglitazone (45mg/d) and exenatide (10 ug s/c twice daily) for 12 months. Hepatic fat content was determined by MR Spectroscopy. At baseline, hepatic fat content and plasma FGF-21 levels were similar between the two treatment groups. Pioglitazone reduced fasting plasma glucose (P<0.05), fasting FFA (P<0.05), fasting plasma triglyceride (P<0.05) and HbA1c (Δ=1.0%, P<0.01). Hepatic fat content was signifi cantly reduced (Δ=41%, p<0.05) following pioglitazone treatment despite a signifi cant increase in body weight (Δ=3.7 kg). However, fasting plasma FGF21 levels did not change after pioglitazone therapy (1.9 ±

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0.6 ng/mL to 2.2 ± 0.6 ng/mL). Combined pioglitazone and exenatide therapy was associated with a signifi cantly greater decrease in hepatic fat (Δ=61%) versus pioglitazone therapy despite the lack of a signifi cant change in body weight (Δ=0.2 kg). Combined pioglitazone and exenatide therapy was associated with a signifi cant reduction in FGF21 levels (2.3 ± 0.5 ng/mL to 1.1 ± 0.3 ng/mL, p<0.01). Conclusion: In T2DM, pioglitazone therapy does not reduce FGF-21 levels despite a signifi cant reduction in hepatic fat content. However, combined pioglitazone and exenatide therapy is associated with a greater reduction in hepatic fat content and a signifi cant reduction in plasma FGF-21 levels. Further studies are necessary to examine the molecular mechanisms responsible for the effects of exenatide on hepatic fat content and FGF-21 levels in patients with type 2 diabetes and NAFLD.

411-PPLiraglutide as Additional Treatment in Type 1 DiabetesAJAY VARANASI, NATALIE BELLINI, DEEPTI RAWAL, MEHUL VORA, ANTOINE MAKDISSI, SANDEEP DHINDSA, AJAY CHAUDHURI, PARESH DANDONA, Buffalo, NY

In view of the inability of insulin based treatments to control type 1 diabetes adequately in a consistent fashion and to avoid glycemic oscillations, we asked the question whether the addition of liraglutide to insulin treatment would improve the glycemic control since GLP-1 and its agonists are known to suppress post prandial increases in glucagon concentration in type 2 diabetics. Fourteen patients with well controlled type1 diabetes (mean HbA1c: 6.5%) on continuous glucose monitoring and intensive insulin therapy on CSII were included in the study. Their glycemic control was optimized through careful regulation of their carbohydrate intake and insulin regimes for at least two weeks. No signifi cant change in glycemic control or insulin dose was observed. Treatment with liraglutide was then started for either one week (n=14) or 24 weeks (n=14). Mean fasting glucose (130±10 to 110±8 mg/dl; p<0.05) and mean weekly glucose concentrations (138±20 to 115±12 mg/dl; p<0.05) fell signifi cantly while the basal insulin (25±6 to 17±6 U/day; p<0.05) and bolus insulin (23±4 to 16±4 U/day; p<0.05) doses also fell signifi cantly within 1 week. The oscillations in blood glucose concentrations were signifi cantly reduced and the weekly CV for glucose concentrations fell from 39.6±10 to 22.6±7% (p<0.05). The patients who continued on liraglutide for 24 weeks maintained a similar glycemic control but they had further reductions in insulin doses and a signifi cant weight loss (68.0±5 to 63.5±4 Kg; p<0.05). The mean HbA1c was reduced signifi cantly from 6.5±0.5% to 6.1±0.4% (p<0.05). The withdrawal of liraglutide resulted in a rapid reversal of these effects and the restoration of marked glycemic oscillations. The addition of liraglutide to CSII results in a rapid and signifi cant reduction in mean fasting and weekly glucose concentrations, reduction in glycemic oscillations and CV, simultaneously with a reduction in the dose of insulin in type 1 diabetics. In addition, there is a signifi cant weight loss and a reduction in HbA1c.

Supported by: NIH ADA-Funded Research

412-PPMechanisms for the Therapeutic Effect of Fenofi brate on Diabetic Retinopathy in Type 1 Diabetes ModelsYING CHEN, YANG HU, ROBERT MOTT, SCOTT MITCHELL, ALICIA J. JENKINS, ANTHONY C. KEECH, TIMOTHY J. LYONS, JIAN-XING MA, Oklahoma City, OK, Melbourne, Australia, Sydney, Australia

Retinal vascular leakage, infl ammation and neovascularization are features of diabetic retinopathy (DR), a common cause of vision loss. Our previous studies demonstrate that over-activation of the Wnt pathway promotes DR, and that fenofi brate can ameliorate DR in clinical studies of type 2 diabetes patients. As yet there are no studies in type 1 diabetes, nor understanding of the molecular mechanism for the fenofi brate’s retinopathy benefi t, hence we evaluated the effi cacy of fenofi brate on DR in type 1 diabetes models and elucidated the underlying mechanisms. In two Type 1 diabetes models, streptozotocin-induced diabetic rats and Akita mice, oral administration of fenofi brate signifi cantly reduced retinal vascular permeability, retinal vascular leukostasis and retinal infl ammation. Intraocular injection of fenofi brate also robustly reduced retinal vascular leakage and infl ammation in the diabetic models, suggesting that the fenofi brate effects are not dependent on systemic effects. Intravitreal injection of fenofi brate also inhibited ischemia-induced retinal neovascularization in a rat oxygen-induced retinopathy model (OIR). In cultured primary retinal capillary endothelial cells, fenofi brate inhibited tube formation and cell migration. In the retina of diabetic animals and in cultured retinal cells, fenofi brate signifi cantly inhibited phosphorylation of low-density lipoprotein receptor-related protein

6 (LRP6), a co-receptor of the Wnt pathway, and attenuated accumulation, and transcriptional activity of β-catenin, an effector of the canonical Wnt pathway. These results support that fenofi brate can ameliorate DR in type 1 diabetes via blockade of Wnt pathway activation in the diabetic retina.

413-PPSafety and Effi cacy of Linagliptin in Type 2 Diabetes Patients with Severe Renal ImpairmentLANCE SLOAN, JENNIFER NEWMAN, CHRISTOPHE SAUCE, MAXIMILIAN VON EYNATTEN, SANJAY PATEL, HANS J. WOERLE, Lufkin, TX, Ridgefi eld, CT, Reims, France, Ingelheim, Germany, Bracknell, United Kingdom

Renal impairment (RI) is a common complication of type 2 diabetes mellitus (T2DM). However, in patients with RI, disease management is challenging and treatment options are limited. This randomized, double-blind, placebo (PBO)-controlled Phase 3 trial evaluated the effi cacy and safety of the novel dipeptidyl peptidase (DPP)-4 inhibitor linagliptin in patients with T2DM (HbA1c 7.0–10.0%) and severe RI (glomerular fi ltration rate [GFR] <30 mL/min/1.73 m2). Patients were treated with either linagliptin 5 mg qd (n=68) or PBO (n=65); antidiabetic background therapy remained unchanged. The primary endpoint was HbA1c change from baseline at week 12. Mean (±SD) baseline age, HbA1c, and GFR were 64.4±10.3 years, 8.2±1.0%, and 23.5±6.7 mL/min/1.73 m2, respectively. Diabetes duration was >5 years in 96% of patients. The HbA1c change from baseline in the full analysis set and in the subgroup of poorly controlled patients (baseline HbA1c ≥9%) are shown in the fi gure.

Adverse event (AE) and serious AE rates were 86.8% and 23.5% for linagliptin and 81.5% and 26.2% for PBO, respectively. Hypoglycemia occurred in 51.5% of linagliptin and 27.7% of PBO-treated patients. Notably, 61/66 patients receiving linagliptin were on insulin and/or sulfonylurea background; their unchanged doses may account for the hypoglycemic event rate in this arm. Renal function remained stable throughout the study in both treatment arms and cardiovascular deaths in this high-risk population were low: 1 (1.5%) on linagliptin and 3 (4.6%) on PBO. This is the fi rst trial evaluating the safety and effi cacy of a DPP-4 inhibitor exclusively in patients with severe RI. Results confi rm that linagliptin is likely to be a valuable treatment option for T2DM patients with insuffi ciently controlled hyperglycemia in whom renal function is severely impaired. [ClinicalTrials.gov, NCT00800683]

Supported by: Boehringer Ingelheim Pharmaceuticals, Inc.

414-PPSafety, Tolerability, Pharmacokinetics and Pharmacodynamics of TAK-875, a Novel GPR40 Agonist, Following Once-Daily Oral Administration for 2 Weeks in Type 2 Diabetic PatientsECKHARD LEIFKE, JINGTAO WU, PRABHAKAR VISWANATHAN, MARK S. KIPNES, MAJID VAKILYNEJAD, Deerfi eld, IL, San Antonio, TX

GPR40 is highly expressed in pancreatic β cells and mediates free fatty acid-induced insulin secretion. TAK-875 is a novel, highly selective GPR40 agonist that has demonstrated glucose lowering effects in animal models of type 2 diabetes (T2D) and is under development for the treatment of T2D. In this phase 1, double-blind, randomized, placebo-controlled, multiple rising-dose study, safety, tolerability, pharmacokinetics and pharmacodynamics

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of oral TAK-875 taken once daily for 2 wks were examined in 59 patients with T2D, who received either placebo (n=14) or 25, 50, 100, 200 or 400 mg of TAK-875 (n=45). The AUC(0-24) and Cmax of TAK-875 increased approximately dose proportionally. No TAK-875 dose-related adverse events or laboratory abnormalities, including symptomatic hypoglycemia, were observed (1 subject on 400 mg had symptomatic hypoglycemia considered related to prolonged fasting following oral glucose tolerance test [OGTT]). After 2 wks of treatment, TAK-875 showed reductions from baseline in both fasting plasma glucose as well as postprandial glucose following an OGTT. While there were no meaningful changes in fasting insulin or C-peptide levels, TAK-875 showed a dose-dependent increase in C-peptide post-OGTT. TAK-875 rapidly and effectively improves both postprandial and fasting hyperglycemia with low risk of hypoglycemia when given once daily in patients with T2D. Consistent with preclinical data, TAK-875 acts as a glucose-dependent insulinotropic.

415-PPThe DPP-4 Inhibitor Linagliptin Reduces Intra-Myocellular and Hepatic Lipid Accumulation in a Diet-Induced Obesity Rat Model: An MRS-Based Study in Comparison to SibutramineTHOMAS KLEIN, ROLF GREMPLER, ERIC MAYOUX, HEIKO NIESSEN, SHARON CHEETHAM, DETLEF STILLER, MICHAEL MARK, Biberach, Germany, Nottingham, United Kingdom

Linagliptin is a novel dipeptidyl peptidase (DPP)-4 inhibitor, which is currently in late-stage clinical development for the treatment of type 2 diabetes. The effects of 6 weeks’ treatment with linagliptin on body weight, total body fat, intra-myocellular fat, and hepatic fat in a non-diabetic model of diet-induced obesity in comparison to the appetite suppressant sibutramine were investigated. Female Wistar rats were fed a high-fat diet for 3 months and received either vehicle, linagliptin (10 mg/kg), or sibutramine (5 mg/kg) orally, once daily for 6 additional weeks, while continuing the high-fat diet. Magnetic resonance spectroscopy (MRS) analysis of total body fat, intra-myocellular fat, and hepatic fat was performed before treatment and at the end of the study. Sibutramine treatment caused a signifi cant reduction in body weight (–12%) vs control, whereas linagliptin had no signifi cant effect (–3%). Total body fat was also signifi cantly reduced by sibutramine (–12%), but not by linagliptin (–5%). However, both linagliptin and sibutramine caused substantial reductions in intra-myocellular fat (–36% and –55%, respectively). In addition, both linagliptin and sibutramine treatment resulted in substantial decreases in hepatic fat (–39% and –30%, respectively).

Change/Difference

Body weight (%) Total body fat (%)

Liver fat (%) Intra-myocellular

fat (%)Control Control − − − −

Baseline +15 (p=0.016) +11 (p=0.001) +27 (p=0.09) +23 (p=0.49)Linagliptin Control –3 (p=0.56) –5 (p=0.27) –39 (p=0.022) –36 (p=0.14)

Baseline +12 (p=0.001) +5 (p=0.06) –30 (p=0.05) –24 (p=0.039)Sibutramine Control –12 (p=0.018) –12 (p=0.008) –30 (p=0.13) –55 (p=0.037)

Baseline +1 (p=0.64) –0.4 (p=0.86) –29 (p=0.12) –34 (p=0.007)

These results highlight the potential of linagliptin to improve insulin sensitivity by reducing intramyocellular lipids and hepatic fat, independent of body weight reduction, while the effects of sibutramin can be attributed to weight loss.

Supported by: Boehringer Ingelheim Pharmaceuticals, Inc.

416-PPThe Glucagon Receptor Antagonist LY2409021 Attenuates Increases in Hepatic Glucose Output (HGO) and Blood Glucose during Hyper-glucagonemia in Healthy Male SubjectsLAI SAN THAM, EYAS J. ABU-RADDAD, CHAY NGEE LIM, MEI TENG LOH, WEE TECK NG, JANE A. PINAIRE, RONAN P. KELLY, Singapore, Singapore, United States Virgin Islands, Indianapolis, IN

In type 2 diabetes, glucagon levels are inappropriately elevated, promoting HGO and contributing to hyperglycemia. This Phase 1, randomized, subject-blind, placebo (PBO)-controlled, partial crossover study examined the ability of single doses of LY2409021 (LY), a potent, selective glucagon receptor antagonist, to attenuate increases in HGO and blood glucose during hyperglucagonemia in 21 healthy male subjects (HS). Approximately 9 hr after administration of LY or PBO, an infusion of 6,6-[2H2]glucose was started in fasted HS (5 mg/kg for 3 min, then 0.05 mg/kg/min for the next 6 h), followed 3 hr later by a simultaneous “triple infusion” (TI) of 3 h duration of somatostatin (0.1 µg/kg/min), insulin (0.07 mU/kg/min), and glucagon (6 ng/kg/min) to induce exogenous hyperglucagonemia.

There were no clinically signifi cant variations in laboratory tests or vital signs during the study, and the adverse event profi le indicated LY was generally well tolerated.

Before the TI, mean HGO rates and blood glucose concentrations were similar across LY dose levels: 2 to 3 mg/kg/min and 70 to 90 mg/dL, respectively. HGO and blood glucose increased approximately 2- to 3-fold during TI; however, LY attenuated increases in both the mean change from baseline in HGO (0-3 hr; normalized to pre-infusion values as shown) and the mean change from baseline in blood glucose concentration (0-3 hr) in a dose-dependent manner: by 15 to 84% and by 11 to 81%, respectively.

The effects of LY on HGO and blood glucose were highly correlated (r=0.97; p<0.0001), indicating that the majority of the glucose-lowering effect of LY was due to an effect on HGO. These results demonstrate the ability of LY to limit glucagon-induced increases in blood glucose, and support further examination of LY as a potential treatment for T2DM.

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CLINICAL THERAPEUTICS/NEW TECHNOLOGY—TREATMENT OF INSULIN RESISTANCE

417-PPHigh Affi nity Partial Agonist Insulin Receptor Antibodies Stimulate Insulin Activity and Improve Glycemic Control in Murine Models of DiabetesVINAY BHASKAR, JOHN A. CORBIN, IRA D. GOLDFINE, DANIEL H. BEDINGER, HUA F. KUAN, ANGELA LAU, LISA M. GROSS, MASAHISA HANDA, BETTY A. MADDUX, AJAY J. NARASIMHA, SUSAN R. WATSON, RAPHAEL LEVY, NAICHI LIU, XIAORONG WU, DAVID CHEMLA-VOGEL, STEVE R. LEE, CATARINA TRAN, MARK L. WHITE, Berkeley, CA, San Francisco, CA

Targeting the insulin receptor (INSR) with high affi nity, fully human, monoclonal antibodies that act as long-acting insulin analogs is a novel strategy for the treatment of diabetes mellitus. Accordingly, we employed our proprietary phage display technology to produce a panel of fully human monoclonal antibodies to the INSR that had picomolar binding affi nities. In cultured cells, these monoclonal antibodies acted as partial agonists of the INSR. All antibodies elicited robust phosphorylation of Akt, but with an activity that was signifi cantly lower than the maximal activation that was induced by insulin. While the antibodies activated metabolic signaling leading to glucose uptake, they neither induced mitogenesis nor activated the IGF-1R. Biophysical studies in CHO-K1 cells, expressing the human INSR, demonstrated that these activator antibodies did not compete with insulin for binding to INSR. Selected activator antibodies were then evaluated for activity in both hyperinsulinemic and insulinopenic mouse models of type-2 diabetes mellitus (T2DM). In the murine, diet-induced obesity model of insulin resistance, the activator antibodies markedly reduced fasting blood glucose levels and improved glucose tolerance.

Normalization of fasting blood glucose levels and glucose tolerance were also observed in the insulin resistant, insulinopenic multi-low dose streptozotocin, high fat diet T2DM model. After six weeks of treatment there was a signifi cant improvement in HbA1c levels. In these murine models, these partial agonist antibodies improved multiple markers of dyslipidemia. In addition, they stimulated insulin secretion. Hypoglycemia was not observed. This allosteric mechanism of action therefore provides for a unique class of antibody drugs that have the potential to be novel, ultra-long acting insulin analogs that can be used for the treatment of human diabetes mellitus.

HEALTH CARE DELIVERY—ECONOMICS

418-PPEstimating the Long-Term Cost-Effectiveness of Premixed Insulin Analogs Versus Long-Acting Analog Insulin in the USRICHARD POLLOCK, BRADLEY CURTIS, HELEN SMITH, WILLIAM VALENTINE, Basel, Switzerland, Indianapolis, IN, Erl Wood, United Kingdom

A recent AHRQ meta-analysis assessed the relative effectiveness and safety of premixed insulin analogs in type 2 diabetes, concluding that premixed insulin analogs may provide tighter glycemic control than long-acting insulin analogs (LAAIs). In 2007, the American Diabetes Association estimated that US spending on insulin alone had reached USD 3.7 billion per year. Given this substantial expenditure and the ongoing increase in insulin analog prescriptions, we sought to compare the cost-effectiveness of two premixed insulin analogs with LAAI.

A validated model of diabetes was used to evaluate the cost-effectiveness of insulin lispro mix 75/25 (LM75/25) and insulin lispro mix 50/50 (LM50/50) vs a LAAI from a US healthcare payer perspective. Treatment effects were taken directly from the meta-analysis; direct medical costs were obtained from published sources. All costs were expressed in 2010 US dollars and future costs and clinical benefi ts were discounted at 3% per annum. Sensitivity analyses were performed.

LM75/25 and LM50/50 were associated with improvements in life expectancy (+0.07 and +0.09 years, respectively) and quality-adjusted life expectancy (+0.07 quality-adjusted life years [QALYs] and +0.08 QALYs, respectively) when compared with LAAI (Table 1). These clinical benefi ts were accompanied by an increase in cost of USD 1,724 and USD 1,720, yielding incremental cost-effectiveness ratios of USD 28,580 per QALY gained and USD 23,150 per QALY gained, respectively.

Per published evidence, insulin lispro mixtures are likely to be considered cost-effective vs LAAIs in the long-term treatment of type 2 diabetes patients in the US.

Table 1. Long-term cost and clinical outcomes with insulin lispro mix 75/25 and insulin lispro mix 50/50 vs long-acting analog insulin

LAAI LM75/25 Difference LM50/50 Difference Life expectancy (years) 7.82(0.15) 7.89(0.15) +0.07(0.21) 7.91(0.15) +0.09(0.20) Quality-adjusted life expectancy (QALYs) 4.93(0.10) 5.00(0.10) +0.07(0.13) 5.01(0.10) +0.08(0.13) Direct costs (USD) 40,128 41,852 +1,724 41,848 +1,720

USD=2010 US dollars; LAAI=long-acting analog insulin; QALYs=quality-adjusted life years

Supported by: Eli Lilly and Company

419-PPThe Association between Outpatient Visit Frequency and Health Outcomes of Diabetes CareKEIKO ASAO, LAURA N. MCEWEN, JOSEPH V. SELBY, JESSE C. CROSSON, BETH E. WAITZFELDER, WILLIAM H. HERMAN, THE TRANSLATING RESEARCH INTO ACTION FOR DIABETES (TRIAD), Ann Arbor, MI, Oakland, CA, Newark, NJ, Honolulu, HI

Little is known about the optimal frequency of outpatient visits for diabetes care. Frequent visits may unnecessarily increase resource utilization, while infrequent visits may result in missed opportunities for needed care. This study aims 1) to describe the predictors of outpatient visit frequency; and 2) to examine the associations among visit frequency, processes of care, and intermediate health outcomes. We analyzed baseline data from the Michigan site of the TRIAD. The outpatient visit frequency was determined from chart review of participants ≥18 years of age with type 2 diabetes for ≥ 1 year, who had used at least one service during 1999-2000. Only visits to primary care providers (family medicine or general internal medicine) were assessed. Predictors of visit frequency examined were demographic factors (age, sex, race/ethnicity), socioeconomic factors (education, income, smoking, co-pay), diabetes-associated factors (duration, treatment, complications), and co-morbidities. We included 821 adults (417 men, 404 women) with age 63.4 ± 12.1 (mean ± SD) years and duration of diabetes 11.6 ± 9.4 years. The outpatient visit frequency ranged from 1 to 30 per 12 months (geometric mean: 4.7). Being Hispanic, having less education, being a non-smoker, and higher Charlson’s comorbidity index were associated with greater number of visits using multivariate linear regression analysis. Several processes of diabetes care were performed best at the third quartile of visit frequency (4.7 - 7.3 times per year): performance of A1c (80%, 85%, 94%, 90% for the quartile 1, 2, 3, and 4, respectively), LDL cholesterol (65%, 74%, 78%, 75%), and foot examination (62%, 73%, 75%, 72%). The difference remained signifi cant after multiple adjustments. Performance of eye exams and urine protein testing, and the intermediate health outcomes examined (levels of A1c, blood pressure, and LDL cholesterol) were not signifi cantly different by visit frequency. Outpatient visits more frequent than 7 times per year were not associated with the better diabetes care process indices or intermediate outcomes. Other interventions to improve quality of care should be considered.

Supported by: CDC, NIDDK

PEDIATRICS—OBESITY

420-PPExenatide as a Weight-Loss Therapy in Extremely Obese Youth with-out DiabetesAARON S. KELLY, ANDREA M. METZIG, KYLE D. RUDSER, ANGELA K. FITCH, CLAUDIA K. FOX, BRANDON M. NATHAN, JULIA STEINBERGER, MARY DEERING, BETSY L. SCHWARTZ, M. JENNIFER ABUZZAHAB, LAURA M. GAN D RUD, ANTOIN-ETTE MORAN, CHARLES J. BILLINGTON, SARAH JANE SCHWARZENBERG, Minne apolis, MN, St. Louis Park, MN, St. Paul, MN

Extremely obese (BMI ≥99th percentile) youth are at increased risk for T2DM and CVD. Lifestyle modifi cation alone is often inadequate in this population and pharmacologic options are limited. We evaluated the effects of exenatide on BMI and other cardiometabolic risk factors in extremely obese youth without diabetes. Twelve participants (age 12.8 ± 2.0 yrs; 10 girls) were enrolled in a 6-month, randomized, controlled, open-label, crossover, pilot clinical trial consisting of two, 3-month phases: 1) a control phase of lifestyle modifi cation and 2) a drug phase of lifestyle modifi cation plus exenatide (5 mcg BID for 1-month followed by 10 mcg BID for 2-months). BMI, body fat (DXA), blood pressure, lipids, and OGTT were assessed at baseline, 3-, and 6-months. The mean change over each 3-month phase was compared between treatments. Compared to control, exenatide signifi cantly

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reduced BMI, body weight, body fat, and fasting insulin and signifi cantly increased HDL-cholesterol. Compliance with the injection regimen was excellent (≥80% doses administered) and exenatide was well-tolerated (most common adverse event was mild nausea in 33%). These preliminary data suggest that exenatide may be an effective drug therapy for reducing adiposity and improving cardiometabolic risk factors in extremely obese youth.

Covariate Baseline Overall

ΔControl

ΔExenatide

Exenatide vs. Control (95% CI)

P-value

BMI (kg/m2) 37.1 (4.9) 0.9 (1.6) -0.6 (1.2) -1.46 (-2.85, -0.07) 0.040Body Weight (kg) 95.8 (20.5) 2.9 (3.8) -0.8 (3.3) -3.67 (-7.17, -0.16) 0.040Body Fat (%) 48.1 (5.5) 1.6 (2.3) -0.7 (2.4) -2.29 (-4.46, -0.11) 0.040HDL (mg/dL) 37.2 (5.6) -1.1 (5.5) 3.9 (3.4) 4.98 (0.60, 9.35) 0.026Triglycerides (mg/dL) 90.7 (31.7) -2.8 (35.6) 0.1 (30.2) 2.93 (-28.07, 33.92) 0.853Fasting Glucose (mg/dL) 74.5 (3.1) 1.5 (6.5) 3.8 (6.0) 2.25 (-3.58, 8.08) 0.450OGTT Glucose AUC (per 100) 47.5 (22.4) 6.9 (27.8) -16.0 (24.0) -22.98 (-47.35, 1.39) 0.065Fasting Insulin (mU/L) 14.1 (6.3) 6.4 (5.1) -0.6 (8.5) -6.97 (-13.40, -0.54) 0.034OGTT Insulin AUC (per 100) 117 (50.8) 37.6 (86.3) -26.9 (70.7) -64.50 (-142.16, 13.16) 0.104

PEDIATRICS—TYPE 1 DIABETES

421-PPDecline in Residual Insulin Production after Diagnosis of Type 1 Diabetes in Children and AdultsSAMUEL L. ELLIS, VAHRAM GHUSHCHYAN, MICHELLE HAARHUES, ANDREA STECK, PETER A. GOTTLIEB, Aurora, CO

After the diagnosis of type 1 diabetes (T1D), patients enter into a partial remission or “honeymoon” period. Although most patients have relatively short honeymoon phase, some patients remain in partial remission much longer. The purpose of this study is to describe the decline in insulin production and proportion of patients remaining in partial remission during the fi rst 10 years of T1D by using the recently described insulin dose adjusted A1c (IDAA1C) value. The IDAA1C is calculated using both the A1c value and total daily insulin dose as A1c (%) + [4 x insulin dose (units/kg per 24 hours)]. An IDAA1C value ≤ 9 has previously been shown to correlate with a stimulated c-peptide value of >300 pmol/l.

Participants in this study were identifi ed from the BDC database. All patients with T1D who were diagnosed between Jan. 1, 2000 and Nov. 31, 2010 were included. Patients that were pregnant or were >79 years were excluded. We used an average annual A1c and insulin dose to calculate a yearly IDAA1C score.

IDAA1C values were calculated in over 3,000 subjects. The mean IDAA1C value after 1 year was 10.8 ± 2.0 with approximately 20% of patients having a value ≤ 9. The mean IDAA1C remained similar during year 2, but then increased signifi cantly to over 12 by the end of year 4. Figure 1 depicts the proportion of subjects in partial remission by duration of diabetes. The fall in percent of patients with IDAA1c ≤ 9 appears to change after 2 years; although the overall rate of fall appears to be linear.

In this descriptive analysis IDAA1c values suggest a more pronounced decline in residual beta-cell function after 2 years. Future studies need to correlate c-peptide and IDAA1C values in patients with > 1 year duration. Variables associated with prolonged partial remissions and the relationship of IDAA1c to complication rates must also be further assessed.

422-PPIs There any Relationship between Insulin Reserve and Weight Gain in Children with T1aDM?INGRID LIBMAN, MARIBEL CEDILLO, VINCENT ARENA, MASSIMO PIETROPAOLO, DIEGO IZE-LUDLOW, DOROTHY BECKER, Pittsburgh, PA, Ann Arbor, MI, Chicago, IL

We have reported an increase in overweight (BMI%ile ≥85th) after onset of T1DM. It is unknown whether it is associated with changes in insulin reserve. Children <19 years, diagnosed with T1aDM from 2004 to 2006 were included (n=174). Autoantibodies (ICA, insulin, GADA and IA2) were measured. Post-meal c-peptide (CP) levels were assessed at onset and follow-up visits (FUV) 1, 2, 3 and 4 (2.2±0.3, 5.4±0.7, 11.0±0.8 and 13.2±0.9 months after onset resp.). 95% were Caucasian, 43% female, mean age at onset 9.5±3.9 years, BMI%ile (median [interquartile range]) 51.7 [18.5-78.9] increased to 76.6 [56.7-88.9] at FUV1. At onset 20% were overweight, at FUV1 35%. The fi gure shows CP levels at onset (ng/dl) and FUV by BMI status at onset.

43% of the children had CP>0.6 ng/dl. This changed to 79% at FUV1, 68% at FUV2, 52% at FUV3 and 42% at FUV4. In the overweight group, 64% had CP>0.6 ng/dl at onset, 80% at FUV1,82% at FUV2, 60% at FUV3 and 43% at FUV4. Children who were not overweight at onset but became overweight at FUV1 (n=29) did not recover CP any better than those who stayed non-overweight (n=104) (median value at onset 0.69 vs 0.58, FUV1 1.61 vs 1.55, FUV2 1.35 vs 1.35, FUV3 0.88 vs 1.03, FUV4 0.57 vs 0.79 ng/dl resp.; p>0.05. Those that were overweight at onset and remained overweight (n=30) recovered signifi cant amounts of CP at FUV1 and FUV2 (median value at onset 0.94, FUV1 2.31, FUV2 2.05, FUV3 1.11, FUV4 1.05 ng/dl; p< 0.05). These data suggest that CP levels may be quite high especially in overweight children up to 6 months after onset despite the presence of autoimmunity. Children who are overweight at onset have greater insulin reserve initially but end up loosing it at a similar pace after 11 months of diagnosis

Supported by: NHLBI, NIH ADA-Funded Research

PEDIATRICS—TYPE 2 DIABETES

423-PPAbnormal Entero-Insular Axis in Obese and Type 2 Diabetes ChildrenLUISA M. RODRIGUEZ, MOREY W. HAYMOND, AGNETA L. SUNEHAG, JENS J. HOLST, RUBINA A. HEPTULLA, Houston, TX, Copenhagen, Denmark

The incidence of T2DM in the pediatric population is increasing. There is limited information regarding postprandial glucose homeostasis, and the interplay of gut and pancreatic hormones and gastric emptying in children with T2DM. The goal of our study was to examine the entero-insular axis in lean (L), obese (OB) and T2DM children to determine differences in: fasting glucose production rate (GPR), postprandial concentrations of insulin, amylin, glucagon, GLP-1 and gastric emptying. We studied 33 children: 11 L, 11 OB and 11 with T2DM (mean HbA1c 6.5±0.3% and DM duration of 3.3±0.6 yrs). The T2DM group had signifi cant fasting and postprandial hyperglycemia (P<0.002). No signifi cant difference was detected in the overnight GPR among the 3 groups. Fasting insulin was higher in the OB (P=0.003) and T2DM (P<0.0001) vs L. No difference in fasting glucagon, amylin and GLP-1 was found among the 3 groups. After a mixed meal challenge insulin AUC 0-300 min was higher in the T2DM (P=0.003) and OB (P=0.002) vs L controls. Postprandial amylin concentrations did not differ among the 3 groups. In the early postprandial period AUC glucagon 0-120 min was higher in the OB and T2DM vs. L (P<0.05). Glucagon excursions (0-300 min) were highest in the OB compared to L controls (P=0.007), but similar to the T2DM group. Postprandial total GLP-1 excursions were higher in the T2DM subjects vs OB (P=0.005) and L (P=0.024). The acetaminophen absorption test was used to assess gastric emptying. The acetaminophen T max was signifi cantly lower in the T2DM vs L (P=0.05) suggesting accelerated emptying. In conclusion, our data showed

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EPIDEMIOLOGY/GENETICS

signifi cant hyperinsulinemia and hyperglucagonemia in obese euglycemic children. T2DM subjects had signifi cant postprandial hyperglycemia, relative insulin defi ciency, hyperglucagonemia, high total GLP-1 concentrations and accelerated gastric emptying. These are novel fi ndings showing abnormal entero-insular axis of obese and T2DM children.

Supported by: NIDDK K23 DK075931 and Baylor GCRC Grant MO1-RR-00188-34

PREGNANCY

424-PPPre-Pregnancy BMI, but Not Weight Gain during Pregnancy, Is Associated with Maternal Systemic Infl ammation during Pregnancy in Two Independent CohortsSONJA ENTRINGER, JESSICA DE HAENE, CLAUDIA BUSS, LENY MATHEW, NAOMI STOTLAND, PETER HAVEL, PATHIK WADHWA, JENNIFER CULHANE, JANET C. KING, Irvine, CA, Philadelphia, PA, San Francisco, CA, Davis, CA, Oakland, CA

The importance of optimal weight gain during pregnancy for maternal and fetal/newborn/child health outcomes, is emphasized in the new Institute of Medicine Guidelines. Further evaluation regarding the relative importance of gestational weight gain compared to pre-pregnancy body mass index (PPBMI) and key pregnancy and birth outcomes, is warranted.

Systemic infl ammation is increasingly recognized as part of the metabolic pregnancy complications such as preeclampsia and gestational diabetes. The objective of this study was to evaluate the impacts of PPBMI and gestational weight gain (GWG) on systemic infl ammation during pregnancy.

Maternal anthropometry and blood samples were collected at 3 consecu-tive time points over gestation (10,20 and 30wk gestation) in a low-income convenience sample with a normal BMI distribution (mainly African American, n=84). Plasma C-reactive protein (CRP) concentrations were measured as an index of maternal systemic infl ammation. General Linear Model Analyses (GLM), adjusted for race/ethnicity, revealed that PPBMI, but not GWG, was signifi cantly associated with CRP concentrations during pregnancy (F=25.9;p<0.001). Given the increased risk of developing insulin resistance and its consequences in obese women, this association was examined (and replicated) in an independent cohort of overweight/obese pregnant women of mixed race/ethnicity (n=62; PPBMI>27). PPBMI was signifi cantly associated with CRP levels during pregnancy (F=9.7;p<0.01). GWG and fat gain were not associated with CRP. Moreover, IR (estimated by HOMA-IR) during the last half of pregnancy was signifi cantly associated with PPBMI (F=6.9;p<0.01) and mean concentrations of CRP (F=3.0;p<0.05), but not with GWG and fat gain.

These results suggest that maternal body weight before conception may be a stronger determinant of systemic infl ammation and its potential metabolic sequelae (e.g., IR) than GWG or fat gain. PPBMI may be a useful criterion for identifying women who may benefi t from early clinical interventions to reduce metabolic disorders during pregnancy.

Supported by: NIH HD065825 and NIH HD46741

425-PPWomen’s Diabetic and Cardiovascular Risks with GDM during Pregnancy—A Follow Up of HAPO StudyWING-HUNG TAM, RONALD CHING-WAN MA, XILIN YANG, JULIANA CHUNG-NGOR CHAN, TERENCE TZU-HSI LAO, GABRIEL WAI-KWOK YIP, GARY TIN-CHOI KO, MICHAEL HO-MING CHAN, CHI-YIN LI, Hong Kong, China

Chinese women who had participated in the HAPO study between 2001 and 2006 in Hong Kong were followed up at a median of 6 years postpartum. Our objective is to study whether women who have been diagnosed gestational diabetes mellitus (GDM) according to the new HAPO criteria have a higher risk in DM, hypertension, dyslipidemia & arterial stiffness than women with normal glucose tolerance (NGT) during pregnancy. All patient underwent anthropometric & BP measurements and investigated on fasting lipid profi le and 75 gram OGTT. Augmentation index (AI) and pulse wave velocity (PWV) were assessed by using SphygmoCor®PVx. A total of 353 women (286 NGT, 67 GDM) were followed up up till mid-2010 and an interim analysis was performed.

Women with GDM had a higher rate of DM [3%(3/67) vs 0.7% (2/286)] and Impaired glucose regulation (IGR) [31.3%(21/67) vs 11.5%(33/286)] (p<0.001), higher total cholesterol (5.1±0.9 vs 4.8±0.8 mmol/L, p=0.002) & triglyceride levels (3.0±0.8 vs 2.8±0.8 mmol/L, p=0.009). Although there was no signifi cant difference in systolic and diastolic blood pressure, the augmentation index was higher in women with history of GDM (22.6±8.7 vs 18.9±8.5%, p=0.002).

Table 1. Comparison on diabetic and cardiovascular risks between women with NGT and GDM during pregnancy

NGT(n = 286)

GDM(n = 67)

p

Age at follow up 38.0 (4.8) 40.1 (4.5) 0.001BMI (kg/m2) 22.8 (3.2) 23.3 (3.3) 0.2HT 15 (5.2) 1 (1.5) 0.3Glycemic status:IGR (IFG and/or IGT)

DM

33 (11.5)

2 (0.7)

21 (31.3)

2 (3.0)<0.001

Total cholesterol (mmol/L) 4.8 (0.8) 5.1 (0.9) 0.002Triglyceride (mmol/L) 0.9 (0.5) 1.2 (0.8) 0.009LDL-C (mmol/L) 2.8 (0.8) 2.99 (0.8) 0.2Augmentation index % 18.9 (8.5) 22.6 (8.7) 0.002 Pulse wave velocity 6.7 (0.8) 6.9 (1.1) 0.2

HT, hypertension; IGR, impaired glucose regulation, IGT, impaired glucose tolerance; IFG, impaired fasting glycaemia; LDL-C, low density lipoprotein cholesterol

In conclusion, our fi ndings suggest a 4 and 3-fold increase in the risk of DM and IGR in women with GDM. It also suggest that they might have higher risk of subclinical atherosclerosis despite the blood pressure may appear normal at the follow up.

EPIDEMIOLOGY 426-PPDiabetes and Risk of Hearing Impairment: A Meta-AnalysisCHIKA HORIKAWA, SATORU KODAMA, YORIKO HEIANZA, AKI SAITO, REIKO HIRA SAWA, AYUMI SUGAWARA, KUMIKO TOTSUKA, MIHO MAKI, KAZUMI SAITO, HIROHITO SONE, Miya-machi, Mito, Japan

Age-related hearing loss has been reported to be more frequent among persons with than without diabetes. However, the strength of the association between diabetes and risk of hearing impairment (HI) has not been quanti-tatively reviewed. We conducted a meta-analysis of observational studies that investigated the prevalence of HI according to the presence of diabetes. Electronic literature search was conducted using MEDLINE (1966 to 2010 Nov. 23) and EMBASE (1974 to 2010 Nov. 23) with additional manual searches. Studies were included if 1) a cross-sectional design was used; 2) outcome was non-idiopathic progressive HI not due to noise or heredity; 3) HI was defi ned by cut-off values of pure-tone thresholds measured at a frequency range including 2,000 Hz; and 4) data were provided on the number of HI and non-HI cases according to the presence of diabetes. The odds ratio (OR) calculated in each study was pooled with the Mantel-Haenszel method. Data were obtained from 11 eligible studies involving 6,725 HI cases (1,069 cases with diabetes) and 21,734 non-HI cases (2,319 non-cases with diabetes). Three studies used hospital-based and 8 used population-based participants. Five of the 11 included studies were done in the USA. The pooled OR (95% confi dence intervals) of HI for diabetes was 1.99 (1.81-2.20) (P<0.001). A positive association was notable in studies of subjects with mean age <60 years (OR=2.62; P=0.01) or of hospital-based participants (OR=3.27; P=0.04). However, the HI risk associated with diabetes was consistently positive throughout the included studies. Although biological and pathological mechanisms of HI in association with diabetes remain to be investigated, results from this meta-analysis suggest that the diabetic state substantially contributes to progression of HI.

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427-PPGlucose Intolerance Is a Risk Factor for Cancer Death in a General Japanese Population: The Hisayama StudyYOICHIRO HIRAKAWA, YASUFUMI DOI, NAOKO MUKAI, TOSHIHARU NINOMIYA, JUN HATA, MASAYO FUKUHARA, MASANORI IWASE, YUTAKA KIYOHARA, Higashi-ku, Fukuoka, Japan

Purpose: Some epidemiological studies have reported that diabetes is a risk factor for cancer death, but it is uncertain whether prediabetic range, namely impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), increases the risk of fatal cancer.

Methods: A total of 2,438 subjects aged 40-79 years who underwent a 75g oral glucose tolerance test were followed up prospectively for 19 years. At baseline, fasting plasma glucose (FPG) and 2-hour postload glucose (PG) levels were divided into four categories: FPG: <5.6, 5.6-6.0, 6.1-6.9 and ≥7.0 mmol/l; 2-hour PG: <6.7, 6.7-7.7, 7.8-11.0 and ≥11.1 mmol/l. Glucose tolerance status was also defi ned by the 1998 WHO criteria with modifi cation of lower cut-point for IFG from 6.1 to 5.6 mmol/l. During the follow-up, 229 subjects died of cancer.

Results: The age- and sex-adjusted mortality from total cancer signifi cantly increased with elevating FPG and 2-hour PG levels. This association remained robust even after adjustment for age, sex, BMI, total cholesterol, smoking habits, alcohol intake, family history of cancer, physical activity, and dietary factors (P for trend<0.001 for both).

Compared with those with FPG levels of <5.6 mmol/l, subjects with FPG levels of ≥6.1 mmol/l had a signifi cantly higher risk of fatal cancer (6.1-6.9 mmol/l: HR, 1.89, 95%CI, 1.30-2.74, P<0.001; ≥7.0 mmol/l: HR, 2.06, 95%CI, 1.35-3.14, P<0.001). The risk of cancer also signifi cantly increased in subjects with 2-hour PG levels of ≥11.1 mmol/l than in those with 2-hour PG of <6.7 mmol/l (HR, 1.99, 95%CI, 1.34-2.94, P<0.001). In addition, IFG and IGT as well as diabetes were independent risk factors for total cancer death (IFG: HR, 1.49, 95%CI, 1.05-2.11, P=0.02; IGT: HR, 1.52, 95%CI, 1.05-2.22, P=0.03; diabetes: HR, 2.10, 95%CI, 1.41-3.12, P<0.001). In regard to specifi c types of cancer, elevated FPG levels signifi cantly increased the risk of stomach cancer, while elevated 2-hour PG levels were associated with the risks of liver and lung cancer (P for trend<0.05 for all).

Conclusion: Our fi ndings suggest that IFG and IGT as well as diabetes are independent risk factors for cancer death in the general Japanese population.

428-PPLipoprotein-Associated Phospholipase A2 (Lp-PLA2) and Future Risk of Type 2 Diabetes: Results from the Cardiovascular Health StudyTRACY L. NELSON, MARY L. BIGGS, JORGE R. KIZER, MARY CUSHMAN, JOHN E. HOKANSON, CURT D. FURBERG, KENNETH J. MUKAMAL, Fort Collins, CO, Seattle, WA, Ithaca, NY, Burlington, VT, Denver, CO, Winston-Salem, NC, Cambridge, MA

The relationships of circulating lipoprotein-associated phospholipase A2 (Lp-PLA2) mass and activity with incident type 2 diabetes mellitus (T2DM) among older adults have not been examined. We conducted cross-sectional and prospective analyses of 5,489 men and women without previous T2DM from the Cardiovascular Health Study (1989 to 2007). Lp-PLA2 mass and activity were measured in baseline plasma. Diabetes status was ascertained annually with medication inventories and repeated fasting serum glucose measurements. We used general linear models and Cox proportional hazards models with adjustment for multiple confounding factors including BMI and infl ammation. At baseline, the top two quintiles of Lp-PLA2 activity were signifi cantly associated with prevalent T2DM (multivariable RR= 1.37 (95% CI, 1.13-1.67) for quintile 4; RR=1.38 (95% CI, 1.13-1.69) for quintile 5. We also found a signifi cant positive association with both the homeostatic model for insulin resistance (HOMA-IR) and β-cell function (HOMA-β) (β per standard deviation increase in Lp-PLA2 activity = 0.035 (p<0.001) and β = 0.018 (p<0.01)) respectively. In prospective analyses, the risk of T2DM was signifi cantly higher among those in the highest quintile of Lp-PLA2 activity, (multivariable HR = 1.47 (95% CI, 1.01-2.13) compared with the lowest quintile. This association was signifi cantly modifi ed by baseline BMI (p=0.003), with the strongest association among individuals with the highest BMI. Lp-PLA2 mass was not signifi cantly associated with incident diabetes. In conclusion, Lp-PLA2 activity is positively associated with insulin resistance and predicts incident T2DM among older adults independent of multiple factors associated with diabetes pathogenesis.

429-PPMenopause and Risk of Diabetes in the Diabetes Prevention ProgramCATHERINE KIM, SHARON E. EDELSTEIN, JILL P. CRANDALL, DANA DABELEA, ABBAS E. KITABCHI, RICHARD F. HAMMAN, MARIA G. MONTEZ, LEIGH PERRAULT, MARY A. FOULKES, ELIZABETH BARRETT-CONNOR, Ann Arbor, MI, Rockville, MD, Bronx, NY, Aurora, CO, Memphis, TN, San Antonio, TX, La Jolla, CA

Whether menopause per se is associated with greater risk for diabetes among women who already have impaired glucose tolerance (IGT) is unknown. Longitudinal studies have not adjusted for key mediators of IGT, or compared diabetes risk among women with bilateral oophorectomy (BSO), characterized by low testosterone vs. natural menopause. We examined the association between menopause status and diabetes risk among women ages 40-64 years in the Diabetes Prevention Program (DPP), a randomized trial of lifestyle intervention and metformin among adults with IGT. To determine if menopausal status modifi ed response to DPP interventions, menstruating women (n=708) were compared to women in natural postmenopause (n=328) and women with BSO (n=201). Age limits were chosen to exclude premature menopause (<40 years) and vaginal bleeding likely due to a pathologic process (≥65 years). Associations between menopause and diabetes risk were evaluated using Cox proportional hazard models within study arm before and after adjustment for demographic variables (age, race/ethnicity, family history of diabetes, history of gestational diabetes), waist circumference, insulin resistance (1/fasting insulin) and corrected insulin response. Similar models were stratifi ed by type of menopause and hormone therapy use (HT). Before and after adjustment for covariates, natural menopause was not associated with diabetes risk within any intervention arm. In the lifestyle arm, women with BSO had lower diabetes risk compared with menstruating women (adjusted HR 0.19, 95% CI 0.04, 0.94), but observations were too few to determine if this was independent of HT; no signifi cant differences between BSO and diabetes risk were seen in the metformin (adjusted HR 1.29, 95% CI 0.63, 2.64) or placebo arms (adjusted HR 1.37, 95% CI 0.74, 2.55). Natural menopause was not signifi cantly associated with diabetes risk and did not alter response to interventions regardless of HT. BSO was associated with reduced diabetes risk, but only in the lifestyle arm. Study limitations include multiple comparisons. Associations between BSO, HT, and diabetes need confi rmation in other studies.

Supported by: NIH

430-PPSerum 25-Hydroxyvitamin D and Risk of Type 1 Diabetes among US Military PersonnelKASSANDRA L. MUNGER, LYNN I. LEVIN, JENNIFER MASSA, RONALD HORST, TIHAMER ORBAN, ALBERTO ASCHERIO, Boston, MA, Silver Spring, MD, Ames, IA

Vitamin D nutrition may be an important determinant of type 1 diabetes (T1D) in children, but little is known about its possible role in adults. No studies have examined whether serum 25-hydroxyvitamin D (25(OH)D) levels among healthy individuals of any age is associated with risk of developing T1D. We conducted a prospective, nested case-control study among active duty US military personnel who had serum stored in the Department of Defense Serum Repository. We identifi ed 265 individuals who were diagnosed with T1D between 1992 and 2008 and had 3 serum samples collected before the disease onset. Each case was matched to two controls on age, sex, race/ethnicity, branch of military service, and dates of serum sample collection. The samples were collected on average 3.4 years (range <1-16) before clinical onset of T1D. Serum 25(OH)D was measured using a direct, competitive chemiluminescence immunoassay using the DiaSorin LIAISON 25(OH)D vitamin D TOTAL Assay. Analyses were done separately for non-Hispanic whites (160 cases/320 controls), non-Hispanic blacks (57/111), and Hispanics (32/61) because of differences in 25(OH)D levels by race. 25(OH)D levels were adjusted for season of sample collection, age, sex, and laboratory assay batch, and were averaged to estimate long-term vitamin D exposure. We used conditional logistic regression, adjusted for latitude of residence at entry into the military, to estimate the relative risks and 95% confi dence intervals. Among non-Hispanic whites, T1D risk decreased with increasing 25(OH)D levels (RR for a 50 nmol/L increment =0.66, 95% CI: 0.44-0.98; p=0.04). In categorical analyses, compared with individuals with 25(OH)D <75 nmol/L, those with levels >75 nmol/L had a 54% lower risk of T1D (RR=0.46, 95% CI: 0.29-0.74; p=0.001). There was no signifi cant association between pre-onset 25(OH)D levels and risk of T1D among non-Hispanic blacks (p for trend = 0.40) or Hispanics (p for trend = 0.69), possibly due to smaller sample sizes. The results of this prospective study suggest that among non-Hispanic white adults, insuffi cient vitamin D levels may be a potentially modifi able risk factor for T1D.

Supported by: NIH-NINDS (NS042194)

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431-PPSerum Butyrylcholinesterase Is a New Risk Factor of Type 2 DiabetesKYOKO KOGAWA SATO, TOMOSHIGE HAYASHI, NOBUKO HARITA, HIDEO KOH, ISSEKI MAEDA, GINJI ENDO, YOSHIKO NAKAMURA, HIROSHI KAMBE, KANJI FUKUDA, Osaka, Japan

Serum butyrylcholinesterase (BChE) is the enzyme synthesized in liver. A few cross-sectional studies have reported that patients with diabetes or fatty liver had increased BChE. Our purpose is to examine prospectively the relationship bewteen BChE and the risk of type 2 diabetes, independent of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT). Study subjects included 8470 nondiabetic Japanese men aged 40-55 years at entry. Type 2 diabetes was diagnosed if the fasting plasma glucose (FPG) level was ≥126 mg/dL, HbA1c was >6.5%, or if subjects were taking diabetes medications. During the 42227 person-years of follow-up, we confi rmed 868 cases. In Cox proportional hazards models, we fi t a model using BChE, ALT, and GGT categorized by tertiles (Table). After adjusting for age, BMI, the FPG level, alcohol consumption, smoking habit, walk to work, regular leisure-time physical activity, and family history of diabetes, the hazard ratios of type 2 diabetes were 1.00 for BChE of tertile 1, 1.22 (95% CI, 1.02-1.47) for BChE of tertile 2, and 1.33 (1.11-1.60) for BChE of tertile 3. In addition, we examined the model which included BChE, ALT, and GGT simultaneously. Similarly, higher BChE increased the risk of it, independent of ALT and GGT (Table). We concluded that elevated BChE had an increased risk of type 2 diabetes, even after adjustment for both ALT and GGT.

Table Multivariate model of the incidence of type 2 diabetes.

Variables in the model Multiple-adjusted*Hazard Ratio (95% CI)

Butyrylcholinesterase (IU/L) Tertile 1 (56-326) 1.00Tertile 2 (327-381) 1.21 (1.00-1.45)Tertile 3 (382-806) 1.25 (1.04-1.50)

ALT (IU/L) Tertile 1 (3-19) 1.00Tertile 2 (20-30) 1.05 (0.86-1.28)Tertile 3 (31-271) 1.31 (1.06-1.61)

GGT (IU/L) Tertile 1 (7-32) 1.00Tertile 2 (33-64) 1.05 (0.86-1.28)Tertile 3 (65-1530) 1.38 (1.11-1.72)

*Adjusted for age, BMI, the fasting plasma glucose level (<100, 100-109, and 110-125 mg/dL), alcohol consumption (nondrinkers, light drinkers, moderate drinkers, and heavy drinkers), smoking habit (nonsmokers, past smokers, and current smokers), walk to work (0-10, 11-20, and ≥21 minutes), regular leisure-time physical activity, and family history of diabetes.

GENETICS—TYPE 1 DIABETES

432-PPAlterations in Immunoregulation Are Detected by Molecular Signa-ture Analysis Prior to T1D OnsetHIMALA KASHMIRI, MARY KALDUNSKI, SHUANG JIA, SCOTT PAVLETICH, JO-ANNA KRAMER, SANJA GLISIC, XUJING WANG, MARTIN HESSNER, Milwaukee, WI, Birmingham, AL

Type 1 Diabetes (T1D) is an endocrinopathy that results in life-long insulin dependency due to T-cell mediated autoimmunity towards pancreatic beta cells. T1D also has a signifi cant cytokine-based arm. However it is dilute and diffi cult to measure in the periphery, necessitating development of more sensitive and informative biomarkers for studying diabetogenic mechanisms, assessing pre-onset risk, and monitoring therapeutic interventions. Thus, we have refi ned a bioassay whereby infl ammatory factors in serum are used to induce transcription in healthy reporter cells. This, in turn, is measured with a microarray. In cross-sectional studies we fi nd sera of recent onset (RO) T1D patients (n=53) induce a disease-specifi c infl ammatory signature that includes genes regulated by interleukin (IL)-1, relative to longstanding T1D patients (>10 years post onset, n=12), autoantibody negative healthy siblings of probands (n=79) and unrelated healthy controls (n=65). Naturally occurring islet autoantigen-specifi c regulatory T-cells (Tregs) that secrete the immunosuppressive cytokine IL-10 and suppress islet-specifi c T-cell responses have previously been detected in healthy individuals with no family history of T1D. We fi nd that auto-Ab-negative siblings of probands possessing high HLA risk (DR3/DR4 haplotypes, n=32) give rise to an immunoregulatory signature consistent with the presence of IL-10 and/or TGF-B relative to low HLA risk auto-Ab-negative siblings (n=47). This

suggests that immunoregulatory mechanisms are more active in individuals with inherited T1D susceptibility and disease progression may arise through a failure in this mechanism. Further, this elevated activity may result in a higher Treg cell turnover rate, accounting for associations of impaired suppressor function and increased apoptotic rate with RO T1D, as well as increased suppressor function and increased apoptotic rates observed in healthy controls with high-risk HLA haplotypes. In support of this possibility, longitudinal analysis of sera collected from progressors to T1D show induction of infl ammatory transcription and alterations in regulatory transcription prior to autoantibody development and onset.

Supported by: JDRF

433-PPThe Idd9/11 Genetic Locus Regulates the Diabetogenic Activity of CD4 T-Cells in Non-Obese Diabetic (NOD) Mice through an Enhanced Regulatory T-Cell ActivityYI-GUANG CHEN, DAVID V. SERREZE, Milwaukee, WI, Bar Harbor, ME

While the H2g7 major histocompatibility complex (MHC) provides the primary pathogenic component, the development of T-cell-mediated auto-immune type 1 diabetes (T1D) in NOD mice also requires contributions from other susceptibility (Idd) genes. Despite sharing the H2g7 MHC, the closely NOD-related NOR strain remains T1D resistant due to the presence of the protective Idd5.2, Idd9/11, and Idd13 regions. We previously demonstrated that CD4-T cells isolated from the NOR strain were less diabetogenic than those from standard NOD mice, in part due to a T1D resistance gene(s) within the Idd9/11 region. To further determine the underlying mechanism, we asked if CD4-T cells from a NOD stock congenic for the NOR-derived Idd9/11 region (NOD.Chr4NOR ) have enhanced suppressive activity compared to those from standard NOD mice. We fi rst used a co-transfer system to show that CD4-T cells isolated from NOD.Chr4NOR but not standard NOD mice (both expressing the CD45.1 allele) could actively suppress the diabetogenic activity of those from CD45.2 congenic NOD mice. We further demonstrated that the T1D suppressive activity of the NOR-derived Idd9/11 region was completely lost in the CD28-defi cient mice that had greatly reduced numbers of Foxp3+ regulatory T-cells (Tregs). In contrast, genetic ablation of CD1d molecules in NOD.Chr4NOR mice that blocks the development of invariant natural killer T (iNKT)-cells did not abrogate the T1D suppressive activity of the NOR-derived Idd9/11 region. The frequencies of CD4 Treg (Foxp3+CD25+) in spleens were comparable between NOD and NOD.Chr4NOR mice. Therefore, these results indicate that an enhanced T1D suppressive Treg activity contributes signifi cantly to NOR Idd9/11-mediated disease resistance. We have also generated a panel of subcongenic lines carrying distinct intervals of the NOR-derived Idd9/11 region. Diabetes incidence studies indicated that more than one gene within the NOR-derived Idd9/11 region conferred T1D resistance. Future studies will determine the Idd9/11 sub-region that regulates the function of Tregs. ADA-Funded Research

GENETICS—TYPE 2 DIABETES

434-PPCommon Type 2 Diabetes Genetic Variants Have Stronger Pre dis-posing Effects in Lean Compared to Obese PatientsJOHN R.B. PERRY, PHILIPPE FROGUEL, TIMOTHY M. FRAYLING, STEPHANE CAUCHI, DIAGRAM CONSORTIUM, Exeter, United Kingdom, Lille, France

Type 2 diabetes (T2D) is a highly heterogeneous common condition. Patients vary appreciably in the two major risk factors: age and BMI. We hypothesised that type 2 diabetes risk variants, both known and unknown, would have different effects in lean patients (BMI <25Kg/m²) compared to obese patients (BMI >30Kg/m²).

We performed a case-control genome-wide association study (GWAS) using DIAGRAM+ studies, dividing patients by lean and obese BMI status. This included ∼1600 lean cases, ∼4500 obese cases, and ∼ 45,000 healthy controls not matched for BMI. This represents approximately 20% and 40% of the total DIAGRAM+ case sample set respectively. The combined impact of 40 currently known T2D loci was assessed in an independent set of 263 lean cases, 1735 obese cases and 3691 controls.

For each of the known forty T2D loci we compared the effect sizes between the lean and obese strata. Of these 40 loci, 31 had a larger point estimate odds ratio in the lean group compared to obese (P = 0.0007). Six loci had a signifi cant difference (Het P < 0.05) in effect estimates between lean and obese strata, with fi ve stronger in the lean analysis: FTO (lean OR 0.95 vs obese OR 1.25), TCF7L2 (OR 1.58 vs 1.26), ADCY5 (OR 1.25 vs 1.07), NOTCH2 (OR1.25 vs 1.07), VEGFA (OR 1.16 vs 1.07) and SLC30A8 (OR 1.24 vs 1.12).

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IMMUNOLOGY/TRANSPLANTATION

Scoring individuals by number of risk alleles they carried showed signifi cant differences between lean (OR 1.13 (1.09-1.17) per risk-allele) and obese (OR 1.07 (1.05-1.09)) patients.

The highest ranking novel signal in the lean patient GWAS was near the HLA-DQA2 gene locus (P = 1x10-8, OR = 1.3 (95CI 1.19-1.42)). Although there is low correlation between this HLA variant and the Type 1 Diabetes signal, it is unclear whether this association refl ects an auto-immune component to lean type 2 diabetes, or subtle auto-immune case admixture within our cohorts. The highest ranking novel obese GWAS signal was located in HMG20A, P = 8x10-8.

This study provides direct evidence that the known type 2 diabetes variants have stronger effects in lean type 2 diabetic patients compared to obese patients and may refl ect an enrichment of beta cell defects in leaner diabetic patients.

435-PPReplication of Polymorphisms Identifi ed by Type 2 Diabetes Genome-Wide Association Studies in African American and Hispanic Popu-lationsBRIAN H. CHEN, KEI H. CHAN, W. MARK BROWN, CHRISTOPHER S. CARLSON, J. DAVID CURB, CHARLES B. EATON, BARBARA V. HOWARD, REBECCA D. JACKSON, CHARLES L. KOOPERBERG, LEWIS H. KULLER, JOANN E. MANSON, LAWRENCE S. PHILLIPS, BEVERLY M. SNIVELY, ERIC M. SOBEL, HILARY A. TINDLE, LESLEY F. TINKER, NA-CHIEH YOU, ALEX P. REINER, SIMIN LIU, Los Angeles, CA, Winston-Salem, NC, Seattle, WA, Honolulu, HI, Pawtucket, RI, Hyattsville, MD, Columbus, OH, Pittsburgh, PA, Boston, MA, Atlanta, GA

It remains unknown whether genetic variants identifi ed by genome-wide association studies (GWAS) have similar impact on type 2 diabetes (T2D) risk in African American and Hispanic populations. We aimed to identify novel single-nucleotide polymorphisms (SNP) for T2D risk and to replicate previously identifi ed SNPs in a cohort of 8,421 self-identifi ed African American and 3,587 Hispanic women between 50-79 years of age from the Women’s Health Initiative SNP Health Association Resource (WHI SHARe). We identifi ed 1,230 prevalent (1007 AA and 223 HA) and 1,352 incident T2D cases (957 AA and 395 HA) over 5.9 years of follow-up. Genotyping was conducted using the Affymetrix 6.0 platform. Genotype imputation was available only for African Americans using the HapMap CEU and YRI reference panels. We selected 76 SNPs for T2D risk identifi ed by GWAS of European, European American, Japanese, and Chinese populations (p<1x10-5). To account for differences in allele frequencies and linkage disequilibrium (LD) patterns by ancestral groups, we examined regions around each index SNP. Regions were defi ned to be within 500kb and r2>0.8 of the index SNP, which yielded 232 additional SNPs. Logistic regression models were adjusted for age, geographic region, and 3 principal components of global ancestry. The combined 308 index and neighboring SNPs represented 58 independent loci, of which 11 loci in African Americans and 10 loci in Hispanics had at least one SNP with p<0.05. After adjusting for multiple comparisons, only TCF7L2 loci in the African American sample were associated with type 2 diabetes (rs7903146: OR=1.26 95%CI=1.16-1.37, p=3.6x10-8), which confi rmed initial GWAS fi ndings in Europeans. Further, a novel SNP rs11727877 (FLJ46481) was identifi ed for T2D risk among African Americans (OR=1.39, 95%CI=1.24-1.55, p=8.50x10-9). In summary, of the T2D SNPs identifi ed from previous European GWAS, only the TCF7L2 locus was replicated in this cohort of African American women. We also identifi ed a new variant rs11727877 in African Americans that needs to be functionally evaluated in future studies.

436-PPSelection of Extreme Cases and Controls for Whole Genome Se quenc-ing Enriches for Most Known Type 2 Diabetes Susceptibility LociANUBHA MAHAJAN, JOHN PERRY, KYLE J. GAULTON, RICHARD D. PEARSON, NEIL ROBERTSON, NIGEL W. RAYNER, YUHUI CHEN, TIM D. SPECTOR, INGA PROKOPENKO, MARK I. MCCARTHY, ON BEHALF OF THE GOT2D INVESTIGATORS, Oxford, United Kingdom, London, United Kingdom

Sequencing-based studies allow exploration of the role of low frequency (LF) variants in common disease susceptibility.

Given the cost of sequencing, such studies naturally focus on the most informative subjects. Type 2 diabetes (T2D) patients with positive family history and early age of onset are likely to be enriched for LF penetrant loci: selection for relatively lean cases should further enrich for effects on beta-cell function. By analogy, the most informative controls are likely to be those who remain normoglycemic at advanced age, particularly in the face of obesity. The Genetics of T2D (GoT2D) study adopted this sample selection strategy to perform low-pass whole genome, deep exome sequencing and high-density array genotyping of 2650 individuals from the UK, Sweden and Finland.

To establish whether these enrichment effects are visible with known (common) T2D-susceptibility loci, we examined the selected UK GoT2D samples. Cases (n=337) from the Warren 2 collection were selected for fi rst degree family history of T2D, younger age of diagnosis (mean=50.4[SD=8.3] years) and lower BMI (26.5[2.6] kg/m2). Euglycemic controls (n=351) were chosen from the TwinsUK cohort, to be older (61.4[10.0] years) with higher BMI (30.7[5.9] kg/m2).

These samples were genotyped on the 2.5M Illumina OmniChip (1,670,266 polymorphic SNPs passing QC). We compared signal effect size to those previously-reported by the DIAGRAM consortium. TCF7L2 (OR[95%CI], 2.1[1.6-2.7]), DUSP9 (1.7[1.2-2.5]), and ADCY5 (1.5 [1.2-2.0]) showed the largest effect sizes with the TCF7L2 association reaching genome-wide signifi cance (P=1.3x10-9) despite the modest sample size. On average, the point estimate of per-allele risk was ∼7% higher for the selected samples with 8 loci (DUSP9, TCF7L2, ADCY5, CDC123/CAMK1D, RBMS1, CENTD2, HHEX/IDE, GCK) all acting through presumed beta-cell effects showing the most marked increases (20-58%).

Analysis of subjects selected from extremes of the liability distributions achieved the desired effect of enhancing effect size estimates for known common variants. Focusing sequencing efforts in such samples should aid discovery of novel LF associations.

IMMUNOLOGY

437-PPClassifi cation of a Clinical Responder for Evaluation of Treatment Effi cacy in Trials of New-Onset Type 1 DiabetesPAULA L. MCGEE, HEIDI KRAUSE-STEINRAUF, CARLA GREENBAUM, MARK PESCO VITZ, KEVAN HEROLD, LISA SPAIN, JERRY PALMER, JAY SKYLER, JOHN M. LACHIN, THE TYPE 1 DIABETES TRIALNET STUDY GROUP, Rockville, MD, Seattle, WA, Indianapolis, IN, New Haven, CT, Bethesda, MD, Miami, FL

Seven criteria were evaluated to classify clinical responders versus non-responders to a therapy aimed at preservation of beta cell function (C-peptide) in newly diagnosed type 1 diabetes via induction of immunologic tolerance. Two were based on the change in C-peptide from baseline in a study of 126 subjects, two based on within subject variation from a study of 118 subjects with repeat tests, one based on previously reported assay variation, and two based on previously suggested changes in HbA1c and insulin dose (remission and partial-remission). Criteria were compared with respect to the proportion of responders within the Rituximab and placebo groups in the randomized, placebo-controlled TrialNet Rituximab (anti-CD20) trial conducted by TrialNet funded by the National Institute of Diabetes and Digestive and Kidney Diseases (Table 1).

The change and % change in C-peptide from baseline and remission criteria showed low proportions of responders among the Rituximab and control groups. The partial remission criterion showed a high proportion of responders in both groups; whereas the criteria based on variation showed responder proportions closer to 50% with higher fractions in the Rituximab versus placebo groups. The agreement and kappa statistics among the measures based on variation were also higher.

The criterion based on the within-subject coeffi cient of variation appeared to be marginally superior to the others. Subsequent analyses using this criterion in the Rituximab trial show that changes in mechanistic assessments can differentiate responders from non-responders within the Rituximab group.

Since the development of the criteria for response was based on data outside the Rituximab trial, we would expect that these criteria will be generally applicable to other studies in similar populations.

Table 1. Proportion of responders and non-responders by each criterion at 6 months.

Approach Rituximab Control Change 12.6% 10.4%% Change 19.6% 3.5% Within Subject Variation 56.9% 37.9% Within Subject CV 58.8% 37.9% Within Assay CV 54.9% 34.5% Remission 22% 10.3% Partial Remission 84% 44.8%

Supported by: NIDDK

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INSULIN SIGNALING/INSULIN ACTION

438-PPGeneration of Antigen-Specifi c Regulatory T Cells from Human Umbilical Cord BloodZHAO HAN, ROBERT WHITENER, MARK A. ATKINSON, MICHAEL J. HALLER, DESMOND A. SCHATZ, JEFFREY A. BLUESTONE, TODD M. BRUSKO, Gainesville, FL, San Francisco, CA

Regulatory T cells (Tregs) are considered vital to the maintenance of self-tolerance and prevention of autoimmune disease. While proven effective for preventing type 1 diabetes (T1D) in murine models of the disease, analogous methods involving transfer of antigen-specifi c Tregs to prevent and/or reverse the disorder in humans have been hindered by the paucity of naïve Tregs that can be effectively isolated and expanded from peripheral blood (PB). Due to their less mature state and enhanced proliferative capacity, naïve Tregs isolated from umbilical cord blood (UCB) may provide a superior source in adoptive cellular immunotherapy. Using a lentiviral gene delivery system, we expressed an antigen-specifi c T cell receptor (TCR) recognizing glutamic acid decarboxylase (GAD)(555-567) in the context of HLA-DR4 on FACS-isolated UCB naïve Tregs (CD4+CD127-/loCD25+CD45RA+ T cells). Expression of GFP, a downstream reporter, indicated a 55-60% transduction effi ciency using this system. In addition, a 140-fold ex-vivo expansion of transduced Tregs was achieved upon stimulation with αCD3/αCD28-coated microbeads and IL-2 over a 14-day period. Treg phenotype and stable TCR expression were analyzed and confi rmed by fl ow cytometry using FoxP3/Helios and GFP/TCR Vβ5.1 staining, respectively. GFP expression and TCR Vβ5.1 staining of ex-vivo expanded Tregs indicated stable integration and expression of the antigen-specifi c TCR introduced by our lentiviral gene delivery system. Our fi ndings indicate that naïve Tregs isolated from UCB can be engineered to express antigen-specifi c TCRs and carry the capacity for ex-vivo expansion. While further studies to determine the activities and functions of these cells are required, our investigation provides a novel approach for TCR-specifi c gene therapy and carries important implications for the development of therapeutic interventions aimed to restore antigen-specifi c immune tolerance in patients with T1D.

These studies were funded by the JDRF Collaborative Center for Cell Therapy to J.A.B., Cord Blood Center to M.A.A., and Transitional Award to T.M.B.

Supported by: JDRF

439-PPMHC Class I on Pancreatic Islets from Longstanding Diabetes Patients: Persistent Hyperexpression Is Restricted to Type 1 Dia betes and Does Not Correlate with Enteroviral Infection, Infi ltra tion or Insulin DepletionKEN COPPIETERS, ANNA WIBERG, NATALIE AMIRIAN, SALLY KENT, THOMAS W.H. KAY, PETER D. CAMPBELL, MARK A. ATKINSON, GUN FRISK, STEVEN TRACY, MATTHIAS G. VON HERRATH, La Jolla, CA, Worcester, MA, Fitzroy, Australia, Gainesville, FL, Uppsala, Sweden, Omaha, NE

Major Histocompatibility Class (MHC) class I antigens present intracellular peptides to CD8 T cells and render the expressing cell susceptible to killing. Islet cells from recently diagnosed type 1 diabetes patients are known to exhibit upregulated expression of MHC class I for yet unclarifi ed reasons. This study reports on a systematic survey of MHC class I expression patterns and potentially causal pathways in samples obtained via the network for Pancreatic Organ Donors (nPOD).

Freshly frozen pancreas samples were obtained from 39 longstanding type 1 diabetes patients, 14 non-diabetic control individuals, 5 non-diabetic, islet autoantibody positive individuals, 6 type 2 diabetes patients, 1 patient with gestational diabetes and 1 undefi ned case of diabetes. Sections were stained for insulin, MHC class I and CD8 by immunofl uorescence. Consecutive sections from samples with pronounced MHC class I hyperexpression on islets were subjected to PCR analysis and immunofl uorescence for enterovirus species and type I interferon signature genes.

MHC class I hyperexpression on islets was found in four cases and was specifi c to type 1 diabetes. Upregulation may persist for as long as eight years after clinical onset and was observed independent of insulin suffi ciency and CD8+ infi ltration. Despite modulation of type I interferon signature genes, none of the samples showed evidence of chronic enteroviral infection.

This concludes that persistent MHC class I upregulation on pancreatic islets is a type 1 diabetes-specifi c phenomenon that is unlikely to be a consequence of chronic enteroviral infection.

Supported by: JDRF

TRANSPLANTATION

440-PPEffect of Induction on Simultaneous Pancreas-Kidney Re-Trans-planta tion: An Analysis of UNOS DataKAYO WAKI, YASUHIKO SUGAWARA, SUMIHITO TAMURA, NORIYO YAMASHIKI, HIDEO FUJITA, NORIHIRO KOKUDO, TAKASHI KADOWAKI, PAUL I. TERASAKI, Bunkyo-ku, Japan, Los Angeles, CA

The effect of depleting antibody (Ab) induction immunosuppression (IS) in recipients of simultaneous pancreas-kidney (SPK) transplants in which the pancreas was a re-transplant is inconclusive. We analyzed UNOS data for 214 diabetic patients who received such an SPK between 1991 and 2010—using Kaplan-Meier methods to calculate pancreas and kidney graft survival rates, and Cox proportional hazard models to estimate the effect of depleting Ab induction IS adjusted for confounders. We classifi ed the recipients into four groups: those who did not receive induction IS (No-Ind), those who received non-depleting Ab induction IS (Non-Depl), those who received depleting Ab induction IS (Depl), and those who received induction IS other than Ab (Non-Ab). Three-year pancreas graft survival of Depl (72.6%) was similar to that of Non-Depl (72.6%), but signifi cantly higher than that of No-Ind (43.4%) and Non-Ab (60.0%)(p=0.02).

Kidney graft survival rates did not show signifi cant differences between the four groups.

After adjusting for confounders—and using No-Ind as a reference group—three-year pancreas graft survival of Depl (hazard ratio (HR), 0.21; 95% CI, 0.05-0.88) showed lower risk of graft failure. However, the survival of Non-Depl and Non-Ab was similar to the survival of No-Ind. In conclusion, depleting antibody induction IS for SPK recipients with a pancreas re-transplant resulted in improved survival for the re-transplanted pancreas graft after adjustment for confounders.

Supported by: Terasaki Foundation Laboratory

INSULIN ACTION—GLUCOSE TRANSPORT

441-PPRab10 Regulates Glut4 Cycling through Endosomes and Release from Static Retention, but Not Fusion to the Plasma MembraneCYNTHIA CORLEY MASTICK, PAUL D. BREWER, ESTIFANOS N. HABTEMICHAEL, IRINA ROMENSKAIA, Reno, NV

Insulin stimulates the translocation of Glut4 from intracellular compart-ments to the plasma membrane (PM). In basal adipocytes, Glut4 resides in two pools: a non-cycling pool in specialized Glut4 storage vesicles (GSVs) and a cycling pool that overlaps with the general endocytic pathways.

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Insulin has two effects on Glut4 traffi cking: 1) insulin releases Glut4 from the GSVs, increasing the cycling pool 3-10-fold, and 2) insulin increases kex, the rate constant for exocytosis, 3-7-fold. kex is a function of both the rate of traffi cking through the endocytic pathway (ktr), and the rate of fusion to the PM (kfus).

Glut4 traffi cking kinetics in 3T3-L1 adipocytes were measured using quantitative fl ow cytometric assays. These assays allow us to resolve multiple insulin-sensitive steps in the traffi cking itinerary of Glut4, including: 1) release from sequestration, 2) fusion to the PM, 3) endocytosis, and 4) traffi c through the endocytic pathway. We found that AS160 regulates release of Glut4 from sequestration, but not the rate of Glut4 traffi cking (ktr) or fusion (kfus). Rab10 knockdown affects both Glut4 release and ktr, but did not affect kfus. Inhibitor studies show that an additional, unidentifi ed Akt substrate (AS-”X”) regulates kfus.

Our data support the following model: Rab10-GTP is required to make the GSVs competent for fusion. Rab10-GTP binding to either the cycling Glut4 vesicles or the static GSVs converts the vesicles to a prefusion compartment. AS160 inactivates any Rabs that bind to the static GSVs; inactivation/ phosphorylation of AS160 allows association of Rab10-GTP. Once Rab10-GTP binds, effectors are recruited and the fusion step proceeds. The fusion step is regulated through AS-X. Computer simulations show that this model can account for all of the kinetics data observed in AS160 and Rab10 kd cells.

ADA-Funded Research

INSULIN ACTION—INSULIN RESISTANCE IN VITRO

442-PPInsulin-Stimulated Induction of Lipogenic Genes through Atypical PKCs Is Maintained in Insulin-Resistant Primary Rat HepatocytesTHOMAS GALBO, BJØRN QUISTORFF, ERICA NISHIMURA, Måløv, Denmark, Copenhagen, Denmark

Insulin resistance (IR) plays a central role in the pathophysiology of type 2 diabetes (T2D) and is characterized by the classic triad of hyperinsulinemia, hyperglycemia and hypertriglyceridemia with the presence of hyperglycemia in the face of hyperinsulinemia being the defi nition of IR. In liver-insulin receptor knock-out (LIRKO) mice, hyperglycemia and hyperinsulinemia develops, similar to the T2D state. However, plasma triglycerides (TGs) and hepatic TG content are both low, in contrast to what occurs in human T2D. This difference highlights the paradoxical selective hepatic IR that occurs in T2D where the actions of insulin on glucose production are blunted in the liver while insulin’s effects on hepatic lipogenesis are not.

To investigate the molecular mechanisms involved in hepatic IR, primary rat hepatocytes were exposed to 0,5mM palmitate for twenty hours rendering the cells insulin resistant. Insulin-stimulated activation of Akt (Western Blot (WB) p473/total Akt) was inhibited in palmitate-exposed cells while inactivation of Akt downstream targets GSK3α (WB p21/total GSK3α) and FoxO1 (WB p256/total FoxO1) was also impaired. Suppression of the FoxO1-induced gluconeogenic genes (Pepck and G6pc, qPCR) and of de novo glucose production (from 2mM pyruvate) resulted in a right shifted dose-response curve. Insulin-stimulated glucose disposal and glycogen synthesis were also impaired, in line with a relatively enhanced activation of GSK3α leading to reduced GS activity.

Interestingly, similar to what is observed in T2D, the ability of insulin to induce TG accumulation as well as transcription of the enzymes that catalyze de novo lipogenesis and TG assembly was maintained in the presence of

palmitate. Insulin-induction of these genes could, however, be blocked by inhibition of the atypical PKCs, using a myristylated pseudosubstrate, without affecting insulin suppression of G6pc.

Thus, we hypothesize that lipid-induced hepatic IR is essentially a defective insulin-activation of Akt and that elevated compensatory systemic levels of insulin exacerbate the IR by driving lipogenesis and TG assembly via atypical PKC signaling.

INSULIN ACTION—METABOLISM

443-PPInhibition of Hypothalamic Infl ammation Reverts Diet-Induced Insu-lin Resistance in the LiverMARCIANE MILANSKI, PATRICIA O. PRADA, ELIANA P. ARAUJO, MARIO J. SAAD, LICIO A. VELLOSO, Campinas, Brazil

Defective liver gluconeogenesis is the main mechanism leading to fasting hyperglycemia in DM2 and, in concert with steatosis, it is the hallmark of hepatic insulin resistance. Recent studies have shown that experimental obesity results from the installation of an infl ammatory process in the hypothalamus, which leads to resistance to leptin and to the defective regulation of food intake and energy expenditure. Pharmacological or genetic approaches targeting hypothalamic infl ammation restore the sensitivity to leptin and reduce body mass. In the present study, we evaluate the effect of a short-term hypothalamic anti-infl ammatory approach to regulate hepatic responsiveness to insulin. For that, rats fed on a high-fat diet were treated by intracerebroventricular injections, for 7 days, with immunoneutralizing antibodies against TLR4 or TNFα and insulin signal transduction, hepatic steatosis and gluconeogenesis were evaluated. The inhibition of either TLR4 or TNFα reduced hypothalamic infl ammation as determined by the expression of TNFα, IL1β and IL6. This was accompanied by the reduction of hypothalamic resistance to leptin as determined by the leptin-induced reduction of food intake. Upon hypothalamic TLR4 or TNFα inhibition, insulin signal transduction through the insulin receptor, IRS1, Akt and FOXO1 was completely restored in the liver of obese rats. This was accompanied by reduced liver steatosis and reduced hepatic expression of PGC1α, PPARα and fatty acid synthase. In addition, the inhibition of hypothalamic infl ammation restored obesity-associated defective liver glucose production, as determined by a hyperinsulinemic-euglycemic clamp, and led to the reduction of the hepatic expression of PEPCK and G6Pase. All the benefi cial effects of the inhibition of hypothalamic infl ammation on liver responsiveness to insulin were abrogated by vagotomy. Thus, the inhibition of hypothalamic infl ammation in diet-induced obesity results in improved insulin signal transduction in the liver, leading to reduced steatosis and reduced gluconeogenesis. All these effects are mediated by neural signals delivered by the vagus nerve.

Supported by: FAPESP

444-PPRegulation of Hepatic Glucose Metabolism by Brain Insulin ActionCHRISTOPHER J. RAMNANAN, MARTA SMITH, E. PATRICK DONAHUE, BEN FARMER, DOSS NEAL, PHIL WILLIAMS, MARGARET LAUTZ, WANDA SNEAD, DALE S. EDGERTON, ALAN D. CHERRINGTON, Nashville, TN

We previously observed that a 10-fold basal increase in insulin at the brain brought about by head artery insulin infusion, when hepatic insulin and glucagon were clamped at basal levels, caused a modest (∼40%) reduction in net hepatic glucose output (NHGO) and a fall in net hepatic glycogenolytic (NHGLY) fl ux. The aim of the present study was to characterize the effect of a rise in brain insulin when a physiologic rise in insulin also occurs at the liver. Dogs underwent head (carotid and vertebral artery; jugular vein) and liver (femoral artery; portal and hepatic veins) catheterization and a cannula was inserted into the 3rd ventricle (ICV). At -150 min 3H-glucose and somatostatin were infused into a peripheral vein and glucagon and insulin were infused intraportally at basal rates. Following a basal sampling period (-30 to 0 min), artifi cial cerebrospinal fl uid (aCSF; n=6) or the PI3K inhibitor LY294004 (to block brain insulin action; LY; n=5) was infused ICV (0 to 300 min). At 60 min, the portal insulin infusion was increased and carotid and vertebral insulin infusions were initiated so that hyperinsulinemia occurred at the brain (∼10-fold), muscle and fat (∼3-fold), and the liver (∼2-fold). Intralipid and glucose were infused to clamp NEFA and glucose at basal levels. Blockade of brain insulin signaling in the LY group prevented the phosphorylation of both hypothalamic Akt and hepatic STAT3. In addition, insulin-regulated alterations in GK and GSK3β mRNA expression in the aCSF group (7-fold increase and 33% decrease relative to basal control animals, respectively)

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were muted by inhibition of central insulin signaling (4-fold increase and no decrease, respectively, relative to basal controls). Despite these differences, NHGO (Δ1.3±0.2 and 1.2±0.3 mg/kg/min) and EGP (Δ1.1±0.2 and 1.0±0.2 mg/kg/min) decreased similarly in aCSF and LY groups with the decrease being entirely attributable to reduced NHGLY fl ux. These data indicate that under euglycemic conditions, and in the presence of a two-fold rise in insulin at the liver, brain insulin action does not acutely impact hepatic glucose metabolism regardless of its transcriptional effects in the liver. ADA-Funded Research

INSULIN ACTION—SIGNAL TRANSDUCTION

445-PPInsulin Therapy Ameliorates Selective Insulin Resistance in Liver by Inhibiting the FoxO1 and SREBP1c Pathway in Type 2 Diabetic RatsXUAN XIA, JINHUA YAN, YUNFENG SHEN, KUANGXIAO TANG, YANHUA ZHANG, DONGJIE YANG, HUA LIANG, JIANPING WENG, Guangzhou, China

In type 2 diabetes mellitus, insulin failed to decrease hepatic gluco-neogenesis by inhibiting forkhead box protein O1 (FoxO1), while still increased lipid synthesis by activating sterol regulatory element-binding protein 1c (SREBP1c) because of selective hepatic insulin resistance. Whether insulin treatment could ameliorate selective hepatic insulin resistance or not, and the underlying mechanisms need to be clarifi ed. In this study, we investigated the regulation of insulin on molecular expressions involved in hepatic gluconeogenesis and lipogenesis in high-fat-fed and streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) rats. The male SD rats were divided into 3 groups of 6 animals each as follows: NC group (normal control), DM group (untreated diabetic rats), INS group (diabetic rats treated with neutral protamine hagedorn insulin for 5 weeks from third day after STZ injection). Compared with NC group, the fasting glucose levels and plasma triglyceride levels were increased by 142% and 263% in DM group, which were signifi cantly decreased by insulin (P<0.05). Similarly, a decrease of lipid depositions in liver were observed after insulin treatment by using H&E, Sudan III and oil red O staining. The expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) gene were decreased in liver after insulin treatment in mRNA and protein levels. Compared with controls, FoxO1 protein level in DM group was increased by 50%, which was reduced by 65% after insulin therapy (P<0.05), and the inhibition was identical in both fasting and fed conditions. Compared with controls, DM group had a 255% and 85% increase in fatty acid synthesis (FAS) and upstream transcriptional factor SREBP1c protein level in liver, respectively (P<0.05). Levels of FAS and SREBP1c protein were decreased by 72% and 54% respectively in INS group than those in DM group (P<0.05). The data suggested that insulin therapy ameliorate selective hepatic insulin resistance in diabetic rats potentially through inhibition of gluconeogenesis and lipid synthesis in liver.

Supported by: 985 Project PCSIRT 0947

PHYSIOLOGY—ADIPOCYTE BIOLOGY 446-PPCircadian Abnormalities in Obesity Begin in the Adipocyte: Disruption of Rhythmic Transcription of Circadian Clock Genes and AMPK in Metabolic Tissues of Obese MiceMADHU J. PRASAI, PETER J. GRANT, ELEANOR M. SCOTT, Leeds, United Kingdom

Circadian rhythms are prominent in the physiology of metabolic processes. Attenuation of rhythmic transcription of core clock genes is found in obese mice and may be related to the pathogenesis of obesity. The AMP-activated protein kinase (AMPK) both regulates the action of the clock and is itself under transcriptional control of the clock. Its role in metabolism and obesity is well known but no studies have yet explored its metabolic action from a circadian angle.

We postulated differential 24 hour expression profi les between obese and lean mice and across metabolically active tissues (liver, visceral and subcutaneous adipose) of: 1. Core clock genes, 2. Markers of infl ammation, 3. AMPK and associated genes.

Obese male C57BL6J mice fed a high fat diet for 10 weeks and chow fed controls were euthanased every 6 hours and RNA was extracted from liver, visceral and subcutaneous adipose. Diurnal gene transcription was measured by quantitative PCR. Results were analysed by ANOVA with repeated measures and Bonferroni post-hoc adjustment.

We found widespread attenuation of diurnal variation in transcription in obese compared to lean mice. 1. Visceral adipose: Bmal1 (P<0.001) and

Per2 (P<0.001); subcutaneous adipose: Per2 (P<0.05); liver: no disruption. 2. Visceral adipose: increased expression of Complement C3 (P=0.01), and macrophage marker F4-80 (P<0.001) at a single time point; diurnally increased tumour necrosis factor (TNF) (P<0.01). 3. Visceral adipose: AMPK (P<0.01), phosphoenolpyruvate kinase (PEPCK) (P<0.001), hormone sensitive lipase (HSL) (P<0.05); liver: PEPCK (P<0.05) subcutaneous adipose: HSL (P<0.05). GLUT4: reduced expression in visceral adipose at a single time point (P<0.05).

Rhythmic gene transcription was disproportionately attenuated in visceral adipose compared to other metabolically active tissues and correlated with infl ammation. This emphasises the centrality of visceral adipose in obesity and supports observations that clock dysfunction is a pathogenic consequence of obesity. Loss of AMPK transcription rhythm in obesity is interesting and worthy of further study.

447-PPDisruption of Insulin and IGF-1 Signaling in Fat Leads to Impairment in Thermogenesis and Protection Against Obesity and Glucose IntoleranceJEREMIE BOUCHER, MARCELO MORI, KEVIN LEE, CHONG WEE LIEW, YAZMIN MACOTELA, C. RONALD KAHN, Boston, MA

Insulin and IGF-1 signaling play important roles in adipocyte differentiation and overall glucose tolerance and insulin sensitivity. To investigate how insulin/IGF-1 signaling affects adipose tissue development and glucose homeostasis in vivo, we created mice with a tissue specifi c double knockout of both insulin receptor (IR) and IGF-1 receptor (IGF1R) in fat by breeding mice with IR and IGF1R fl oxed alleles with aP2-Cre mice. On normal chow diet, these FIGIRKO mice were signifi cantly leaner than control littermates with a 20% decrease in visceral fat and 50% decrease in subcutaneous fat by 4 months of age. Even more striking was the reduction in brown adipose tissue (BAT), which was decreased in weight by >85% in FIGIRKO mice. Histology revealed no difference in cell size in WAT depots, but markedly reduced lipid content in BAT from FIGIRKO mice. This appears to be due to a failure of brown adipocytes to differentiate, since in vitro, FIGIRKO brown preadipocytes showed a marked impairment in their ability to differentiate into adipocytes, even in the presence of PPARγ agonist rosiglitazone, as assessed by lipid accumulation or expression of adipocyte specifi c markers. In vivo, baseline insulin sensitivity was similar between control and FIGIRKO mice, but FIGIRKO mice were protected against age related glucose intolerance. Energy expenditure as assessed by indirect calorimetry was increased by 16% in FIGIRKO mice compared to control.

Consistent with this increase, FIGIRKO mice were also protected from high fat diet induced obesity and glucose intolerance, despite eating even more food than controls. Basal body temperature was similar at room temperature between control and FIGIRKO mice, but FIGIRKO mice failed to maintain their body temperature when exposed to a 4oC environment with a drop of more than 10oC with a 2 hr cold challenge. Thus, combined absence of insulin and IGF-1 signaling in fat leads to a reduction in WAT and a dramatic decrease in BAT due to defective adipocyte differentiation. This leads to increased energy expenditure and a lean phenotype despite marked impairment of cold-induced thermogenesis.

448-PPGlypican-4 Interacts with and Modulates Insulin/IGF-1 Receptor Signaling and Thereby Adipocyte DifferentiationSIEGFRIED USSAR, C. RONALD KAHN, Boston, MA

Obesity and body fat distribution, especially visceral adiposity, are risk factors for diabetes. We have previously shown that development and patterning genes may play a role in determining fat distribution. In this context, the gene encoding the cell surface proteoglycan Glypican-4 (Gpc4) stands out, since it has marked differential expression between fat depots in both humans and mice, and in humans, Gpc4 expression is highly correlated with body mass index and waist-hip ratio. Furthermore, mutations in the chromosomal region containing glypican-3 and glypican-4 cause the Simpson-Golabi-Behemel syndrome. In this study we show that Gpc4 interacts with and modulates insulin signaling. Thus stable depletion of Gpc4 in preadipocytes by shRNA reduces insulin and IGF-1 receptor activation and signaling. This results in a strong reduction in both the intensity and duration of Akt and Erk activation, specifi c to insulin and IGF-1 stimulation. These defects could not be detected upon activation with FBS or other growth factors, indicating that Gpc4 acts as a specifi c modulator of insulin signaling. The reduced activation of these pathways results in diminished phosphorylation and activation of C/EBPb, causing a failure to induce key adipocyte transcription factors like C/EBPa and PPARg leading to inhibition of adipocyte differentiation in Gpc4

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INTEGRATED PHYSIOLOGY/OBESITY

defi cient cells. Conversely, cells overexpressing non-membrane bound Gpc4 show increased adipogenesis in comparison to control cells. Furthermore, addition of recombinant non-membrane bound Gpc4 rescues the insulin signaling defects in Gpc4 depleted cells and further enhances signaling in control cells. Mechanistically, this appears to be due to an interaction between Gpc4 and both the insulin and IGF-1 receptor, as demonstrated by co-immunoprecipitation of insulin and IGF-1 receptors with Gpc4 in extracts of cultured preadipocytes, as well as freshly isolated murine adipocytes. Thus, Gpc4 acts as a novel regulator of insulin and IGF-1 signaling by interacting with these receptors, modulating their activity and the role of these signaling pathways in adipocyte differentiation.

449-PPThe Ubiquitin E3 Ligase Siah2 Regulates PPARγ Activity in Adipo-cytesGAIL KILROY, RANDALL L. MYNATT, Z. ELIZABETH FLOYD, Baton Rouge, LA

Obesity is an increasingly common disorder that is a major risk factor for developing insulin resistance and type 2 diabetes. The role of obesity in insulin resistance and type 2 diabetes can be linked to factors regulating the expression and activity of the nuclear hormone receptor peroxisome proliferation-activated receptor gamma (PPARγ), a transcription factor that is essential for the formation of adipocytes and the function of mature adipocytes. Treatments for type 2 diabetes include PPARγ activators such as the widely used thiazolidinediones (TZD), which are associated with increased risk of congestive heart failure in men and women and bone fractures in women. Activation of PPARγ by TZDs is linked to modifi cation of PPARγ by the ubiquitin proteasome system, a highly selective signaling pathway that targets proteins to the proteasome by tagging the protein with multiple ubiquitin polypeptides. To understand how activation of PPARγ is linked with destruction of PPARγ proteins in adipocytes, we carried out an RNAi-based screen of ubiquitin E3 ligases, the enzymes of the ubiquitin proteasome pathway that catalyze the transfer of ubiquitin to a substrate. Using this approach, we identifi ed a small set of ubiquitin ligases that regulate PPARγ protein levels in adipocytes, including a mammalian homolog of Drosophilia seven-in absentia, Siah2. Siah2 regulates PPARγ protein levels and PPARγ ubiquitylation in mature adipocytes without altering PPARγ gene expression. In addition, Siah2 is required for TZD-dependent activation of PPARγ in adipocytes. Finally, Siah2 gene expression is upregulated during adipogenesis and Siah2 is required for adipogenesis. The current study identifi es Siah2 as a regulator of PPARγ activity and stability in fully differentiated adipocytes, directly linking post-translational modifi cation of PPARγ by the ubiquitin proteasome system with TZD-dependent activation of PPARγ in adipocytes. The ubiquitin E3 ligase Siah2 may represent a novel target for treatment of insulin resistance via regulation of PPARγ protein degradation in adipocytes.

Supported by: NIH COBRE P20-RR021945 ADA-Funded Research

SIGNAL TRANSDUCTION (NOT INSULIN ACTION)

450-PPEffect of Aging and Resistance Exercise on AMPK Activity and Signaling in Human MuscleMENGYAO LI, LEX B. VERDIJK, KEI SAKAMOTO, LUC J.C. VAN LOON, NICOLAS MUSI, San Antonio, TX, Maastricht, The Netherlands, Dundee, United Kingdom

Aging is associated with an increased incidence of type 2 diabetes (T2D). Alterations in glucose, lipid, and protein metabolism are thought to be responsible for the higher risk of T2D in the elderly. AMP-activated protein kinase (AMPK) is an energy/nutrient-sensitive Ser/Thr kinase whose activation enhances lipid oxidation in muscle by phosphorylating acetyl-CoA carboxylase (ACC) and promotes glucose uptake by increasing GLUT4 translocation through phosphorylation of TBC1D1/4. mTOR is another energy/nutrient-sensitive Ser/Thr kinase that enhances protein synthesis. Animal studies suggest aging leads to altered signaling through AMPK-ACC and mTOR in muscle, which have been implicated in the metabolic abnormalities with aging. Our goals were to determine whether: (1) aging leads to a downregulation of the AMPK-ACC and mTOR pathways in human muscle; and (2) resistance exercise can restore aging-related alterations in AMPK-ACC and mTOR signaling. Measurements were performed in vastus lateralis muscle from 32 healthy, elderly, nondiabetic, community-dwelling subjects [age=72±1 y, fasting plasma glucose (FPG)=104±1 mg/dl, hemoglobin A1c=5.9±0.1%)] and 32 healthy, younger, nondiabetic subjects (age=25±1, FPG=92±1, A1c=5.4±0.1). In older subjects, the muscle samples

were obtained before and after 3 months of resistance training (3 sessions/week). RESULTS: Aging caused signifi cant decreases in AMPKα2 activity (by 25%), and in the phosphorylation of AMPK (by 48%), ACC (by 47%) and mTOR (by 70%). AMPKα1 activity and the protein content of LKB1, an AMPK upstream kinase, were similar between groups. Resistance exercise caused an increase in AMPKα1 activity in older subjects by 1.4-fold (P<0.05). However, AMPKα2 activity, LKB1 protein content, and the phosphorylation of AMPK, ACC, and mTOR were not affected by resistance training. SUMMARY: (i) alterations in the AMPK-ACC and mTOR pathways could explain some changes in glucose, lipid, and protein metabolism that occur with aging; and (ii) resistance exercise causes an isoform-selective (α1) upregulation of AMPK activity, without affecting ACC and mTOR phosphorylation.

Supported by: NH: NIA, NIDDK, American Federation for Aging ADA-Funded Research

INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

451-PPLocal Knockdown of Ventromedial Hypothalamus Insulin Receptors Causes Impaired Insulin Secretory ResponsesSACHIN A. PARANJAPE, OWEN CHAN, WANLING ZHU, ADAM HORBLITT, LAW-RENCE REAGAN, ROBERT SHERWIN, New Haven, CT, Columbia, SC

It is generally believed that insulin regulates glucose homeostasis solely via its effects on liver, muscle and adipose tissues and that circulating metabolic fuels and incretins predominantly regulate insulin secretion. Less attention has been given to the possibility that insulin action and secretion are also mediated at the level of brain, specifi cally the ventromedial hypothalamus (VMH), a key brain glucose-sensing region. We previously reported that knockdown of VMH insulin receptors (InsR) causes glucose intolerance, paradoxical hyperglucagonemia, and hepatic insulin resistance in non-diabetic rats. The current study examines whether these metabolic changes are accompanied by alterations in insulin secretion. For this purpose, we microinjected lentivirus containing an antisense sequence to specifi cally knockdown InsR (or a control lentiviral vector) into the VMH. Insulin secretion was assessed using hyperglycemic clamps (220mg/dL glucose) at 16 wk. Western blots of micro-punched VMH tissue revealed >50% knockdown of VMH InsR. While fasting plasma glucose was unaltered, InsR knockdown rats required 32% less exogenous glucose to maintain identical hyperglycemia (19mg/kg/min VMH InsR knockdown vs 28mg/kg/min controls; p<0.05). This effect on the rate of glucose metabolism in the VMH InsR knockdown rats was mainly attributed to a 40% decrease in insulin release rather than hepatic insulin resistance (215uU/ml in control vs 128uU/ml VMH InsR knockdown; p<0.05). We conclude that suppression of VMH InsR leads to glucose intolerance as a result of impaired insulin secretion and a paradoxical increase in plasma glucagon. Our data suggest that hypothalamic insulin resistance may contribute to the development of type 2 diabetes.

Supported by: JDRF

452-PPPharmacological Activation of GPR40 Reduces Increased Blood Glucose Levels by Modulated Insulin Secretion in a Glucose-Dependent MannerANDREAS W. HERLING, ELISABETH DEFOSSA, GUIDO HASCHKE, VIKTORIA DIETRICH, SIEGFRIED STENGELIN, STEFANIE KEIL, ANGELA DUDDA, MICHAEL WAGNER, PETER RUUS, HARTMUT RUETTEN, Frankfurt, Germany

GPR40 is highly expressed in pancreatic β-cells and to a lesser extent in intestinal L-cells. Agonism of GPR40 mediates FA-induced insulin secretion from β-cells in a glucose-dependent manner, as well as GLP-1 release from L-cells. SAR1 has been identifi ed as a potent and selective GPR40 agonist (EC50<0.1µM).

Specifi city of SAR1 for GPR40 was investigated in wild-type (WT) and GPR40-KO mice. The pharmacological profi le of SAR1 was studied in Wistar (HsdCpb:WU) and female obese (fa/fa) ZDF (ZDF-Leprfa/Crl) rats after oral administration. The improvement of glucose excursions during an oral glucose tolerance test (oGTT) with concomitant measurements of serum insulin levels as well as the effect of fasting glucose profi le was used to characterize SAR1 pharmacologically.

SAR1 improved glucose tolerance in male and female WT mice, but was inactive in GPR40-KO mice, demonstrating the causally link to GPR40. Oral administration of 10mg/kg of SAR1 to Wistar rats improved glucose

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tolerance with a concomitant increase in serum insulin. In a second rat study 30mg/kg of SAR1 did not affect blood glucose (fasting blood glucose profi le) or serum insulin levels, demonstrating that GPR40 agonism did not reduce blood glucose lower than normoglycemia. These results were consistent with the notion that GPR40 mediates insulin secretion in a glucose-dependent manner with no risk of hypoglycemia. As ZDF rats are hyperlipidemic and long-chain FA are the natural ligands of GPR40, we investigated whether SAR1 was still active under these conditions. SAR1 demonstrated a dose-dependent improvement of glucose tolerance combined with a signifi cant dose-dependent increase of serum insulin.

These results confi rm the role of GPR40 in the mediation of glucose-dependent insulin secretion and thereby infl uencing blood glucose and confi rm that SAR1 represents an in vivo active specifi c GPR40 agonist.

INTEGRATED PHYSIOLOGY—LIVER

453-PPBlockade of Mineralocorticoid Receptor Improves Insulin Resist-ance and Steatohepatitis in a Novel Mouse Model of Non-Alcoholic Steatohepatitis (NASH) Utilizing Liver-Specifi c SREBP1c Transgenic MiceTOSHIYASU SASAOKA, YUSUKE MIYASHITA, MOTOHIRO SASAKI, HIROSHI TSUNEKI, TSUTOMU WADA, Toyama, Japan

We previously demonstrated that spironolactone effectively improved insulin resistance, hypertension, dyslipidemia, and fatty liver in C57BL/6 mice fed with 60% fat diet and 30% fructose water (HFFD). The HFFD mice presented simple steatosis without developing into liver fi brosis, possibly by unaltered expression of a potent lipogenic transcriptional factor, SREBP1c. Therefore, we aimed to establish novel mice model of NASH by utilizing liver-specifi c SREBP1c transgenic (Tg) mice fed HFFD. By utilizing the model, we examined the impact of eplerenone (EP), a selective mineralocorticoid receptor (MR) antagonist, administration on the metabolisms and hepatic fi brosis in mice. Mice were divided into four groups; 1) C57Bl/6J fed regular diet (WTRD); 2) SREBP1c Tg mice fed regular diet (TgRD), 3) Tg mice fed HFFD (TgHFFD); and 4) Tg fed HFFD and EP (1.67 g/kg) (TgHFFD+EP). After feeding for 12 weeks, phenotypes of these mice were analyzed. TgHFFD showed increased body weight, epididymal fat weight, elevation of blood pressure, and dyslipidemia without affecting aldosterone level. EP signifi cantly lowered blood pressure and improved dyslipidemia. The elevations of HOMA-R index and blood glucose levels during insulin or pyruvate tolerance tests were effectively ameliorated by EP. Elevated hepatic triglyceride and collagen contents seen in TgHFFD were also reduced. In the histological analysis, TgHFFD showed enlargement of individual adipocyte size with apparent macrophage infi ltration in epididymal fat, and eplerenone markedly ameliorated these changes. Importantly, apparent steatosis with lobular infl ammation and fi brosis in the liver were remarkably improved. Furthermore, mRNA expressions of genes for proinfl ammatory cytokines, lipogenic enzymes, and fi brosis were determined by real-time PCR. Interestingly, elevated hepatic expressions of TNFα, α-Collagen I, and TGFβ in TgHFFD were signifi cantly suppressed by the treatment. These results indicate that blockade of MR appears to be effective as a novel therapeutic approach for diet-induced metabolic syndrome with NASH.

454-PPInsulin-Independent Regulation of Hepatic Lipogenesis by Fatty AcidsSACHIN K. MAJUMDAR, EMIN O. AKGUL, FITSUM GUEBRE-EGZIABHER, RASMUS RABOL, NAOKI KUMASHIRO, DONGYAN ZHANG, GARY CLINE, VARMAN T. SAMUEL, GERALD I. SHULMAN, New Haven, CT

A central paradox of type 2 diabetes (T2D) is the selective hepatic insulin resistance that occurs when insulin fails to suppress hepatic glucose production, yet its effect on hepatic lipogenesis remains unimpaired resulting in dyslipidemia. One explanation for this apparent paradox is that hepatic triglyceride (TG) synthesis is largely driven by substrate delivery, namely esterifi cation of fatty acids into hepatic triglyceride, independent of insulin-stimulation. To test this hypothesis we measured net rates of hepatic triglyceride synthesis and de novo lipogenesis (DNL) in four groups of rats that received either saline or Liposyn/heparin and either basal [0.5 mU/(kg-min)] or high [4 mU/(kg-min)] dose insulin under euglycemic-clamp conditions. Somatostatin was infused in all groups to suppress endogenous insulin secretion. Rates of net hepatic triglyceride synthesis were markedly increased when plasma fatty acid concentrations were raised and were similar independent of changes in plasma insulin concentrations.

Group Plasma Glucose

(mM)

PlasmaInsulin(µU/ml)

Plasma c-Peptide

(pM)

PlasmaFatty Acids

(mM)

Net Rate of Hepatic Triglyceride Synthesis (µg-TG)/(g-liver-min)

Saline + Basal Insulin 6.0 ± 0.2 10 ± 1 46 ± 3 0.8 ± 0.1 0 Liposyn + Basal Insulin 6.1 ± 0.1 10 ± 1 48 ± 4 3.0 ± 0.1 46 ± 2Saline + High Insulin 5.7 ± 0.3 105 ± 3 34 ± 2 0.3 ± 0.1 0Liposyn + High Insulin 6.2 ± 0.2 107 ± 4 41 ± 5 3.2 ± 0.1 53 ± 7

In contrast, rates of DNL were low (<1%) and similar in all groups. Conclusion: These results demonstrate a dominant role for fatty acid delivery in the regulation of hepatic lipogenesis in vivo and suggest that insulin plays a permissive role in this process. Taken together these results demonstrate that hepatic lipogenesis is dependent on fatty acid delivery and may resolve the paradox of selective hepatic insulin resistance that occurs in patients with T2D.

INTEGRATED PHYSIOLOGY—MACRONUTRIENT METABOLISM AND FOOD INTAKE

455-PPMolecular Clock Alterations and Metabolic Abnormalities in Mice Lacking the Secreted Protein Ccdc80FREDERIC TREMBLAY, CHRISTINE HUARD, ANN-MARIE RICHARD, TIFFANY GARESKI, DAVID KUBASIAK, SARAH WILL, JESSIE DOW, MYLENE PERREAULT, QINGCONG LIN, RUTH E. GIMENO, Cambridge, MA

Coiled-coil domain containing 80 (Ccdc80) is a secreted protein highly expressed in white adipose tissue (WAT) whose expression is reduced during fasting and in ob/ob mice, and restored upon treatment of obese mice with a PPARγ agonist. To explore the hypothesis that Ccdc80 modulates glucose and/or energy homeostasis, two separate cohorts of high-fat-fed wild-type (WT) and Ccdc80-/- mice were studied. We initially found that upon high-fat feeding Ccdc80-/- mice developed hyperglycemia and glucose intolerance while displaying reduced glucose-induced insulin secretion when compared to their WT littermates. Interestingly, metabolic abnormalities in Ccdc80-

/- mice were not associated with differences in body weight, food intake or insulin tolerance. In contrast, when a separate cohort of animal was studied with minimal disturbance (i.e. no fasting/refeeding cycles, frequent manipulation or glucose/insulin challenges), Ccdc80-/- mice ate more and became more obese than WT mice. To determine the mechanism by which Ccdc80 regulates metabolism in mice, transcriptional profi ling of WAT, skeletal muscle and pancreas was performed.

Transcripts analysis revealed that components of the molecular clock network were altered in Ccdc80-/- mice. The expression of the core clock member BMAL1/ARNTL but not CLOCK was reduced while that of the oscillating transcription factors TEF and DBP was increased in all tissues examined.

Furthermore, shRNA-mediated knockdown of Ccdc80 in 3T3-L1 cells led to an increase of both TEF and DBP mRNA levels during specifi c phases of adipocyte differentiation suggesting that Ccdc80 regulates these transcription factors in a cell-autonomous manner. In order to correlate these transcriptional alterations with behavioral changes, analysis of feeding patterns was performed. While each group of animals had the same amount of meals each day, Ccdc80-/- mice ate more food in a shorter amount of time during each of these meals resulting in greater daily caloric intake. Taken together, our results suggest that Ccdc80 is a novel modulator of energy homeostasis in part through its ability to regulate molecular clock components in muscle, fat and pancreas.

INTEGRATED PHYSIOLOGY—MUSCLE

456-PPChronic Activation of AMPK Attenuates Contraction-Stimulated GLUT4 Translocation in Mouse Skeletal MuscleHANS PMM LAURIITZEN, MICHAEL F. HIRSHMAN, ALBA GÓMEZ-CABELLO, CLARA PRATS, LEE A. WITTERS, LAURIE J. GOODYEAR, Boston, MA, Copenhagen, Denmark, Dartmouth, NH

Several naturally occurring activating mutations of the g regulatory subunit of AMPK are associated with alterations in glucose uptake and abnormally high glycogen concentrations in skeletal muscle and heart, which can lead to pathological disease. To elucidate mechanisms by which g mutations regulate glucose and glycogen metabolism, we studied transgenic mice expressing a

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mutant g1 subunit of AMPK in skeletal muscle (TG) and their littermate controls (WT). In the basal state, muscle homogenates from TG mice had increased AMPK activity (2-fold; P<0.05) and glycogen concentrations (3-fold; P<0.05) compared to WT. Basal and contraction-stimulated glucose uptake were reduced by 30% in EDL muscles from TG compared to WT mice (P<0.05). To determine if impaired glucose uptake was associated with decreases in GLUT4 traffi cking we used an in vivo imaging method. Superfi cial fi bers of quadriceps muscles were transfected with a GLUT4-EGFP cDNA construct and mice were studied 5 days later under anesthesia. Images were taken before, during, and after contractions (2 Hz, 60 ms,1-3 V), and every 10 min until 90 min post-contractions. Contractions decreased GLUT4-EGFP intracellular depot staining by 70% in WT mice. In contrast, contractions decreased intracellular GLUT4 by only 30% in TG mice (TG vs. WT; P<0.05), and full re-internalization of GLUT4 back to intracellular depots was faster in TG mice (60 vs. 90 min post-contraction; P<0.05). Contractions increased GLUT4-EGFP at the sarcolemma by 3.7-fold in WT, but only by 2.7-fold in TG mice (P<0.05). Contractions in WT mice resulted in a clear striated staining pattern indicating GLUT4 translocation to the t-tubules, whereas this striated pattern did not emerge in TG mice. Interestingly, staining of muscle cross-sections showed increased glycogen particles in TG vs. WT mice, particularly in the t-tubule region. In summary, an activating mutation of AMPK decreases glucose uptake and GLUT4 translocation, and results in elevated glycogen in the t-tubule region of muscle. These data support the hypothesis that increased glycogen can physically impair GLUT4 traffi cking in skeletal muscle, leading to decreased glucose uptake.

457-PPTraining Modifi es Lipid Infusion Effects on Muscle Mitochondrial mRNALISA S. CHOW, K.S. NAIR, ELIZABETH SEAQUIST, Minneapolis, MN, Rochester, MN

Lipid infusion reduces insulin sensitivity. Insulin resistance is associated with mitochondrial dysfunction. Since trained subjects have high muscle mitochondrial function, training may modify lipid infusion effects on insulin sensitivity by reducing muscle mitochondrial mRNA.

Fourteen trained (T) and 14 sedentary (S) subjects were matched for age(22 yrs), sex and BMI(22 kg/m2), with self-report of activity (T:> 45 min running 5d/wk; S:<30 min exercise/wk). Peak VO2, fat free mass (FFM by DEXA) and insulin sensitivity (hyperinsulinemic euglycemic clamp) were measured. Each subject received either a 6 hour lipid (L: 20% Intralipid:90ml/h*6h, no heparin) or glycerol(G: 2.25 g/100 ml :90 ml/hr) infusion with 3 muscle biopsies (Bx1-0, Bx2-2h, Bx3-6h).

Compared with the sedentary group, trained subjects had higher baseline:FFM, peak VO2, insulin sensitivity, mtDNA copy number and Cytochrome c oxidase subunit 3 (Cox3) mRNA levels. Baseline mRNA of NADH dehydrogenase 4 (ND4), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a), and nuclear respiratory factor 1 (NRF-1) were equivalent.

With lipid infusion, both the trained (-35.45% p<0.01) and sedentary (-42.9%:p<0.01) groups had similar eductions in glucose infusion rate compared with respective glycerol controls.

In contrast to the trained glycerol group, the trained lipid group did not increase Cox3 mRNA at the 2nd[-0.07 (L) vs +0.19 (G), p=0.02] or 3rd biopsy [-0.03 (L) vs +0.17 (G), p=0.05] relative to the 1st biopsy. In contrast to the trained glycerol group, the trained lipid group did not increase ND4 mRNA at the 2nd [-0.07 (L) vs +0.18(G)p=0.01] or 3rd biopsy [-0.03(L) vs +0.25 (G), p=0.01] relative to the 1stbiopsy. There was no infusion effect on Cox3/ND4 mRNA in the sedentary group. There was no infusion or training effect on TFAM, PGC-1 or NRF1 mRNA.

Despite similar declines in insulin sensitivity, lipid infusion had a discrepant effect on muscle mitochondrial mRNA between trained and sedentary subjects, suggesting reduction of mitochondrial mRNA plays an important role in lipid induced insulin resistance observed in trained subjects.

Supported by: NIH 5K12RR023247-02

INTEGRATED PHYSIOLOGY—OTHER HORMONES

458-PP11β-Hydroxysteroid Dehydrogenase Type 1 and 2 Enzyme Activity in Subcutaneous Adipose Tissue of Humans; Implications in Obesity and DiabetesBARBARA NORBY, VISHWANATH PATTAN, BETTY DICKE, ANANDA BASU, ROBERT RIZZA, RITA BASU, Rochester, MN

11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD-1) is implicated in obesity and “metabolic syndrome”. We have shown that the liver produces signifi cant amounts of cortisol via 11β-HSD-1 with less contribution from

omental/visceral fat. Direct measurements of cortisol/cortisone production using tracer techniques in adipose tissue have not been done. We determined whether local generation of cortisol and/or cortisone occurs in s.c. fat in people with and without type 2 diabetes.10 nondiabetic (ND) and 11 type 2 diabetic (DM) subjects were studied. Microdialysis catheter was placed in the abdominal s.c. fat following an overnight fast. After 60 minute stabilization period infusions of D7cortisone and D3 cortisol (10mcg/ml/min) were started and continued for four hours. The effl uent was collected before and at the end of the four hours and analyzed via LC-MS/MS for enrichments of various cortisol tracers and metabolites. ND and DM subjects were matched (p=ns) for age (53 ± 4 vs. 60 ± 4 yrs, BMI (28 ± 2 vs. 32 ± 2 kg-m2, and FFM (49 ± 4 vs. 55 ± 3 kg). FPG (88 ± 2 vs. 144 ± 13 mg/dl) and HbA1c (5.4 ± 0.1 vs. 7.1 ± 0.3 %) were signifi cantly higher (p<0.001) in DM as compared to ND subjects. Comparable enrichments of D7 cortisone and D3 cortisol were detected in the infusate and effl uent in both groups of subjects. D3 cortisone was detected and similar in all ND and DM subjects studied (32 ± 5 vs. 35 ± 4 MPE%) indicating that 11β-HSD-2 enzyme (which converts cortisol to cortisone) activity occurs equally in both groups. In contrast D7 cortisol was not detected in any ND and detectable in 6/11 DM subjects (0.0± 0.0 vs. 5 ± 2 MPE% p<0.03) suggesting higher activity of 11β-HSD-1 in DM subjects. These data indicate that in the presence of moderate obesity a) cortisone is produced via 11β-HSD-2 in both ND and DM b) 11β-HSD-1 activity in the s.c. fat is detectable in DM but not in ND c) further studies are required to tease out the effects of leanness vs. obesity on local generation of cortisol in s.c. adipose tissue in humans.

Supported by: NIDDK

459-PPAlbiglutide, a Long Lasting Glucagon-Like Peptide-1 Receptor Agonist, Protects the Rat Heart Against Ischemia/Reperfusion Injury: Evidence for Improving Cardiac Metabolic Effi ciencyWEIKE BAO, KARPAGAM ARAVINDHAN, HASAN ALSAID, THIMMAIAH CHEN-DRI MADA, MARK R. HARPEL, ROBERT N. WILLETTE, JOHN J. LEPORE, BEAT M. JUCKER, King of Prussia, PA

The cardioprotective effects of glucagon-like peptide-1 (GLP-1) have been previously examined. However the effect of albiglutide, a long-acting GLP-1 receptor agonist, on myocardial energetics in the setting of acute myocardial ischemia/reperfusion (I/R) injury is currently unknown. Therefore, we tested the hypothesis that albiglutide may protect the heart against myocardial I/R injury by increasing carbohydrate utilization and improving cardiac energetic effi ciency. Sprague-Dawley rats were treated with albiglutide (1, 3, or 10 mg/kg/day for 3 days, SC) and then subjected to 30 min myocardial ischemia followed by 24 h reperfusion. Albiglutide signifi cantly reduced left ventricle (LV) infarct size in a dose-dependent manner (↓26%, p<0.01) and concomitantly improved post-ischemic LV hemodynamics ( ↑26% of dP/dtmax, p<0.05). Albiglutide markedly increased both in vivo and ex vivo cardiac glucose uptake ( ↑59-67%, p<0.05) while reducing lactate effl ux (↓55%, p<0.05). Analysis of metabolic substrate utilization directly in the heart showed that albiglutide increased the relative carbohydrate versus fat oxidation ( ↑112%, p<0.05) which in part was due to an increase in both glucose and lactate oxidation. Non-invasive cardiac 31P MRS revealed that albiglutide normalized cardiac energetic parameters (PCr, ATP, pH) following I/R injury while preserving cardiac function (Ejection Fraction, EF) (Figure). Metabolic gene expression analysis indicated upregulation of key glucose metabolism genes in the non-ischemic LV by albiglutide.

These fi ndings suggest that albiglutide may have direct therapeutic potential for improving cardiac metabolic effi ciency and energetics resulting in enhanced cardiac function in the setting of myocardial ischemic injury.

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460-PPElevation of Serotonin Activity within the Ventromedial Hypo thala-mus (VMH) Induces the Hypertensive Insulin Resistant State in RatsSHUQIN LUO, MICHAEL EZROKHI, YELENA TRUBITSYNA, ANTHONY H. CIN-COTTA, Tiverton, RI

The infl uence of central nervous system (CNS) serotonin on peripheral metabolism and cardiovascular physiology is complex and poorly understood as is the general CNS pathophysiological interrelation of insulin resistance and hypertension. CNS serotonin activities are site and receptor specifi c and selective serotonin reuptake inhibitor use that produces wide-spread increases in CNS serotonin to treat depression and/or obesity can actually induce weight gain and glucose intolerance. Recently, we have identifi ed increased serotonin release at the VMH, a fuel sensing center that regulates sympathetic function, as a common occurrence among a wide variety of animal models of insulin resistance.

This study therefore investigated the impact of chronic exogenous administration of serotonin site-specifi cally to the VMH on fasting and postprandial glucose and insulin levels as well as blood pressure in young healthy Sprague-Dawley (SD) rats. Cannulas targeting the unilateral VMH were equipped with osmotic pumps to deliver vehicle (control, N=10) or serotonin (N=10) at a rate of 2.5 nmol/hr to increase VMH serotonin levels of healthy (body weight: 249g, age: 10wks) female SD rats to those of insulin resistant rats. Following 2-weeks of such treatment, fasting and post-meal plasma glucose and insulin levels and blood pressure were measured. Relative to controls, VMH serotonin-treated rats exhibited an increase in fasting plasma glucose (from 131±8 mg/dL to 193±25 mg/dL, P=0.026), insulin (from 2.3±0.3 ng/ml to 5.1±1.6 ng/ml, P=0.08) and HOMA-IR (from 18±3 to 59±1/8 µU/mmol*mmol/L, P=0.03) and post meal plasma glucose and insulin levels were increased by 31% and 150%, respectively (P<0.03). VMH serotonin-treated rats also exhibited hypertension relative to controls (systolic blood pressure rise from 138±4 mmHg to 173±6 mmHg, P=0.0003; diastolic blood pressure rise from 100±4 mmHg to 126±5 mmHg, P=0.0009).

These fi ndings are the fi rst to indicate that increased VMH serotonin levels simultaneously induce insulin resistance and hypertension and that such a neurophysiology may be etiological in the coincident hypertensive/insulin resistance state.

461-PPRole of GPR40 in Intestinal Hormone SecretionSEAN ROSS, CHRISTOPHER NYSTROM, JUDI MCNULTY, DONALD ANDERSON, DALLAS CROOM, LIHONG CHEN, YAPING LIU, Research Triangle Park, NC

GPR40 (FFAR1) is a long chain fatty acid receptor that has been implicated in free fatty acid stimulation of insulin secretion. Both, this and another fatty acid receptor (GPR120) have been implicated in regulating secretion of a number of gut peptides. Using a specifi c GRP40 agonist, 313B we examined the regional role of GPR40 in secretion of GLP-1, GIP, PYY and ghrelin in the presence and absence of nutrients. Anesthetized fasted CD rats were surgically cannulated to allow perfusion of approx. 15cm of the duodenum/jejunum region or the colon with 313B. An increase in plasma PYY (peak concentrations 467±109 pg/ml versus 168±72 pg/ml respectively; P<0.05) was observed with colonic infusions of 313B. There was no effect with duodenal infusion. Total GLP-1 (tGLP-1) concentrations tended to increase with duodenal infusion (peak concentrations 180±18pg/ml versus 116±53 pg/ml respectively, NS) and colonic infusion (peak concentrations 280±85pg/ml versus 105±40 pg/ml respectively, NS). Following this discovery, conscious fasted CD rats with either stomach or colon cannulae were infused with 313B for 10mins. There were increases in plasma tGLP-1 from both the stomach and colonic infusions coupled with a rapid decrease in ghrelin. PYY was only increased with colonic infusion. No effect on GIP secretion was observed. Finally, to examine an interaction with nutrient-stimulated secretion, duodenum cannulated rats were infused with glucose in the absence and presence of 313B. On a baseline of glucose infusion, addition of 313B produced a signifi cant increase in active GLP-1 (231±17 pg/ml versus 122±21 pg/ml; P<0.05), PYY (323±27 pg/ml versus 184±29 pg/ml; P<0.05) and GIP (351±61 pg/ml versus 208±45 pg/ml; P<0.05) as well as a signifi cant decrease in ghrelin. These data show that small molecule stimulation of the GPR40 receptor augments secretion of a number of enteroendocrine hormones in both proximal and distal gut, with potential metabolic consequences.

OBESITY—ANIMAL

462-PPPomc-Cre; Sirt1 Conditional Knock-In Mice Present Lean Phenotype Due to Increased Energy ExpenditureTSUTOMU SASAKI, KOSUKE AMANO, TOMOYA KITAZUMI, OSAMU KIKUCHI, MASAKI KOBAYASHI, TADAHIRO KITAMURA, Maebashi, Japan

Sirt1 is a longevity gene that is responsible for improved health and life span mediated by caloric restriction, and encodes NAD+-dependent deacetylase. Sirt1 modulates energy metabolism in various organs, including liver, pancreas, skeletal muscle, adipose tissue, and the brain. Single nucleotide polymorphisms in Sirt1 gene are associated with energy expenditure and obesity. We reported previously that hypothalamic Sirt1 protein level is decreased by fasting via ubiquitination and Sirt1 is stabilized by feeding and that hypothalamic overexpression of Sirt1 by adenoviral microinjection prevents hyperphagia and body weight gain by suppressing orexigenic Agrp expression. However, the contribution of hypothalamic Sirt1 on regulating energy expenditure remained unsolved. In order to address the question, we developed conditional Sirt1 knock-in mice using Rosa26 promoter and Pomc-Cre mice. Male Pomc-Cre; Sirt1 conditional knock-in (KI) mice are signifi cantly leaner than wild-type (WT) littermates on normal chow feeding. KI mice had less perigonadal fat and perigonadal adipocytes were smaller than those of wild-type mice. 24-hour food intake and 2-hour food intake after 24-hour fasting were not different between WT and KI mice. We measured VO2 and heat production of KI mice and WT mice by Oxymax system, and found that KI mice tend to have higher energy expenditure. Meanwhile, there was no increase in locomotor activity, suggesting that the increase in energy expenditure is due the increase in the basic metabolic rate. KI mice had signifi cantly higher plasma T4 level, but no difference in plasma cortisol level was observed. In brown adipose tissue (BAT), the expressions of type 1 beta adrenergic receptor, PGC-1 beta, and UCP2 were increased. Therefore, hypothalamic overexpression of Sirt1 modulates the central melanocortin system and increases energy expenditure in BAT via hypothalamic-pituitary-thyroid axis and sympathetic nervous system.

Supported by: Japan Health Foundation, Kanae Foundation, Banyu Life Science Foundation

463-PPGLP-1 Derived Nonapeptide Attenuates the Development of Insulin Resistance and Metabolic Syndrome in Diet-Induced Obese MiceEVA TOMAS, JENNA A. WOOD, VIOLETA STANOJEVIC, JOEL F. HABENER, Boston, MA

Obesity and insulin resistance are predominant risk factors for the metabolic syndrome. The prevalence of obesity-related diabetes has been increasing world-wide. Pharmacological interventions that exert insulin-like actions in the face of severe insulin resistance are needed. We report that a nonapeptide, FIAWLVKGRamide, GLP-1(28-36)amide, derived from the glucoincretin hormone, glucagon-like peptide-1 (GLP-1) appears to have insulin-like actions. In these initial studies, mice fed a very high fat diet (VHFD) and infused subcutaneously (osmopumps) with GLP-1(28-36)amide for eleven weeks curtailed the rate of weight gain. No discernible effects were seen in mice fed a control low fat diet (LFD) and infused for three weeks. Likewise, in mice fed a VHFD and infused for eleven weeks the fat mass was diminished by 37% when compared to control mice, with no signifi cant difference in lean mass. Surprisingly, VHFD-fed mice receiving GLP-1(28-36)amide infusion consume approximately 25% more calories than those receiving the control vehicle suggesting an increase in energy expenditure. Furthermore, the infusion of GLP-1(28-36)amide for eight weeks in VHFD-fed mice not only attenuated the development of diabetes since both plasma glucose and insulin were decreased close to values obtained in mice fed a LFD but also improved the insulin to glucose ratio by 50%. Livers obtained from peptide-treated mice showed less steatosis that correlated with a 60% decrease in triglyceride accumulation. These fi ndings suggest a potential role for GLP-1(28-36)amide in the improvement of obesity-related insulin resistance and diabetes and may prove to be useful for the treatment of insulin resistance and metabolic syndrome.

464-PPImpact of Mast Cells on Macrophage Phenotype in Adipose TissueJELENA TODORIC, ELISA EINWALLNER, THOMAS STULNIG, HARALD ESTER-BAUER, Vienna, Austria

White adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Adipose tissue macrophages consist of at least two different phenotypes (i.e., classically activated M1 and alternatively activated M2 macrophages). In this study we

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aimed to clarify the potential impact of mast cells on M1/M2 macrophage-polarisation in fat tissue.

Mast cell-defi cient KitW-sh/W-sh and C57BL/6J mice were fed high-fat (HF; 60% calories from fat) and low-fat (LF; 10% calories from fat) diet for 6 months. Mice were monitored by indirect calorimetry over a 72-hour period. Oral glucose tolerance and insulin tolerance tests were performed. Relative mRNA levels of selected genes from epididymal fat tissue were analyzed by real-time PCR.

KitW-sh/W-sh mice gained signifi cantly less weight and showed higher insulin sensitivity than wild-type (WT) controls, both on the HF-diet. Indirect calorimetry analyses revealed that KitW-sh/W-sh mice had signifi cantly greater energy expenditure, O2 consumption and CO2 production than WT mice during the light but not during the dark phase. Gene expression of macrophage marker F4/80 was signifi cantly higher in both KitW-sh/W-sh and C57BL/6J on HF- comparing to the animals on LF-diet. Expression of M1-specifi c genes was signifi cantly elevated in C57BL/6J but not in KitW-sh/W-sh mice on HF- comparing to the littermates on LF-diet. HF-diet resulted in signifi cant increase in expression of M2-specifi c genes in animals of both genotypes.

These fi ndings suggest that mast cells play critical role in the determination of M1 versus M2 polarization of macrophages in WAT.

465-PPLeptin Defi ciency Alters the Distribution of Mast Cells in Abdominal Fat DepotsMEHMET M. ALTINTAS, ALI NAYER, Miami, FL

We have shown that diet-induced obesity is associated with infi ltration of mast cells in adipose tissues. Leptin defi ciency induces obesity and insulin resistance. We explored the effects of leptin defi ciency on the distribution of mast cells in metabolic organs.

Fourteen-week old male ob/ob and control (+/?) mice fed on a standard chow were studied. After overnight fasting, ob/ob mice (n=10) demonstrated signifi cantly higher body weights (60.2±1.2 vs. 33.0±0.7 g), blood glucose (115±8 vs. 64±3 mg/dl), serum insulin (5.8±0.6 vs. 1.1±0.2 ng/ml), and serum cholesterol (228±5 vs. 89±4 mg/dl) concentrations than control mice (n=10). To demonstrate mast cells, tissue sections were stained with toluidine blue.

In lean mice, mast cells were substantially more prevalent in subcutaneous (3.3±0.4 cells/mm2) than in epididymal (0.2±0.04 cells/mm2), mesenteric (0.5±0.1 cells/mm2), and perinephric (0.5±0.1 cells/mm2) adipose tissues. In ob/ob mice, however, the density of mast cells was signifi cantly higher in epididymal (2.1±0.4 cells/mm2) than in subcutaneous (0.3±0.1 cells/mm2),

mesenteric (0.4±0.1 cells/mm2), and perinephric (0.6±0.2 cells/mm2) fat depots. The density of mast cells increased 11-fold in epididymal fat of ob/ob mice, while those of subcutaneous fat declined 13-fold. Mast cells were more prevalent in the gastrocnemius muscle in ob/ob (3.0±0.4 cells/mm2) than lean control mice (2.1±0.2 cells/mm2). Mast cells were exceedingly rare in the liver of both ob/ob and lean mice.

In conclusion, leptin defi ciency was accompanied by a paradoxical change in the density of mast cells in subcutaneous and epididymal fat. A decrease in mast cells in subcutaneous fat may confer this depot protection from obesity-associated adipose tissue infl ammation. Differential distribution of adipose tissue mast cells might partly explain biological diversity of fat in different depots.

OBESITY—HUMAN

466-PPLorcaserin-Induced Weight Loss and Glycemic Control in Patients with Type 2 Diabetes Mellitus: Data from the BLOOM-DM StudyCHRISTEN M. ANDERSON, MATILDE SANCHEZ, JINKUN ZHANG, BRIAN RAETHER, WILLIAM R. SHANAHAN, San Diego, CA

Lorcaserin (Lor) is a selective agonist of the 5HT2C receptor that decreases body weight by regulating hunger and satiety. As part of a drug development program, Lor was evaluated in a randomized, placebo controlled, double blind trial of 604 adults with type 2 diabetes mellitus inadequately controlled with oral agents (“BLOOM-DM”). The primary objective was to evaluate the safety and effi cacy of Lor administered for weight loss for 1 year. Secondarily, changes in glycemic control, safety, and changes in concomitant antidiabetic medications were assessed.

Results for the Lor 10 mg BID (L) group (n=256) and the placebo (P) group (n=252) are presented; enrollment into the Lor QD group (n=95) was halted prematurely to accelerate trial completion. At Baseline, mean HbA1C was 8.1%; 17.6% of patients had HbA1C ≥9%. Mean fasting plasma glucose (FPG) was 162 mg/dL. At baseline 91.7% took metformin, 50.2% took a sulfonylurea (SFU), and 42.0% took both. Using MITT/LOCF analysis, 37.5% of patients on L and 16.1% of patients on P (p<0.001) lost ≥5% of baseline body weight. Changes in glycemic control are presented in the table below. At Baseline, 8% of patients had HbA1C <7%; at Wk 52, 50.4% of patients on L and 26.3% on P had HbA1C <7%; among those with HbA1C<9% at Baseline, 54.9% on L and 28.9% on P were <7% at Wk 52. More patients on metformin than on SFU at Baseline had HbA1C <7% at Wk 52 in both treatment groups. Fewer patients on L vs. P increased (13.5% vs. 22.2%) and more decreased (17.1% vs. 11.7%) use of antidiabetic medications during the study. Symptomatic hypoglycemia was reported by 7.4% of patients on L and 6.3% of patients on P; all events were mild/moderate in severity. Weight loss with L was associated with signifi cant improvement in glycemic control in patients with type 2 diabetes.

Week 24 Week 52L P p-value L P p-value

FPG (mg/dL) -26.9 ±2.7 -16.4 ±2.6 0.0011 -27.4 ±2.5 -11.9 ±2.5 <0.0001 HbA1C (%) -1.02 ±0.06 -0.61±0.06 <0.0001 -0.93 ±0.06 -0.44 ±0.05 <0.0001HOMA-IR -0.5 ±0.1 -0.2 ±0.1 0.01 -0.5 ±0.1 -0.2 ±0.1 0.02Fasting Insulin (µIU/mL) -2.9 ±0.9 -1.1 ±0.9 0.10 -3.0 ±0.7 -1.6 ±0.7 0.12 (LS mean± sem change from baseline)

467-PPReversal of Type 1 Diabetes by Brown Adipose Tissue TransplantDAVID W. PISTON, SUBHADRA C. GUNAWARDANA, Nashville, TN

Current therapies for type 1 diabetes involve insulin replacement or transplantation of insulin-secreting tissue. Here, we show that subcutaneous transplants of embryonic brown adipose tissue (BAT) can correct type 1 diabetes in immune competent mice without insulin treatment. Within two weeks these transplants begin to result in: a) weight gain and reversal of clinical signs of diabetes; b) return to euglycemia (see Fig. 1); c) normalization of glucose tolerance; and d) replenishment of recipients’ subcutaneous white adipose tissue and reduction of infl ammatory cytokines in this tissue.

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ISLET BIOLOGY/INSULIN SECRETION

These results are independent of endogenous insulin, as indicated by persistently low plasma insulin and pancreatic insulin content, and no other exogenous pharmacological treatment is required. Animals with successful BAT transplants maintain were still euglycemic six months post-transplant. At this time, we measure a three-fold increase in plasma adiponectin, a two-fold increase in plasma IGF-1, and a ten-fold increase in plasma leptin leptin, accompanied with a 4-fold reduction of plasma glucagon. These levels are static and not responsive to glucose as is insulin in normal physiology. Further, pharmacological treatment shows that the insulin receptor continues to play a role in these effects. Thus, we propose that the combined action of multiple adipokines establishes a new equilibrium in the animal that allows for chronic glycemic control in the absence of insulin. While the possibility of insulin-independent glucose regulation by adipokines has been established, our data confi rm these effects, and establish the ability of whole adipose tissue to compensate for loss of the insulin secreting beta-cells. Therapies based on this approach could provide a safe and convenient method for type 1 diabetes treatment.

468-PPThe Anorectic Effect of GLP-1 Requires Inhibition of Hypothalamic FoxO1JEAN BUTEAU, MIHAELA BARBUTA-TOBOS, ELENA TIMOFEEVA, DENIS RICHARD, Quebec, QC, Canada

Objective: FoxO1 is an insulin-sensitive transcription factor that regulates critical biological processes. We have previously shown that the gluco-incretin hormone glucagon-like peptide-1 (GLP-1) inhibits FoxO1 via nuclear exclusion to relieve a constraint on beta-cell proliferation. Moreover, FoxO1 mediates the anorectic effect of insulin and leptin, two hormones that play a central role in the maintenance of energy balance. In the present study, we hypothesized that hypothalamic FoxO1 could be responsible for the inhibitory effect of GLP-1 on food intake via the regulation of hypothalamic neuropeptides (AgRP, NPY, POMC).

Reasearch Design And Methods: FoxO1 overexpression in the arcuate nucleus was achieved by stereotaxic injections of AdFoxO1 and AdGFP in adult rats. After an overnight fast, infected rats were injected intraperitoneally with the GLP-1 analog (Exendin4) or saline. We measured food intake, as well as expression of hypothalamic neuropeptides by in situ hybridization. The effect of GLP-1 on AgRP, NPY and POMC expression was confi rmed in embryonic rat hypothalamic cells by qPCR. FoxO1 subcellular localization was studied using a FoxO1-GFP construct and its binding to cognate DNA sequences was investigated by chromatin immunoprecipitation.

Results: Delivery of a constitutively nuclear (CN) mutant FoxO1 in the arcuate nucleus suppressed the action of GLP-1 on food intake. GLP-1 regulated the expression of AgRP, NPY and POMC in hypothalamic cells both in vivo and in vitro, an effect that was inhibited by CN-FoxO1. Finally, GLP-1 provoked FoxO1 nuclear exclusion and reduced its binding to the AgRP promoter.

Conclusion: Our study demonstrates that the anorectic effect of GLP-1 requires inhibition of hypothalamic FoxO1.

Supported by: Natural Sciences and Engineering Research Council of Canada

ISLET BIOLOGY—APOPTOSIS

469-PPC/EBPβ Promotes Pancreatic β-Cell Death during ER-Stress Induced Diabetes by Modulating Protein Synthesis RatesCELVIE L. YUAN, CHARLIE HUANG, COLLEEN M. CRONIGER, MICHELLE A. PUCHO-WICZ, JAN JENSEN, MARIA HATZOGLOU, Cleveland, OH

The pathology of diabetes is associated with the induction of ER stress in pancreatic beta cells. ER stress induces a cellular response program, Unfolded Protein Response (UPR), involving selective mRNA translation and transcription. Depending on the intensity of stress, it can promote survival (short term, low intensity stress) or induce cell death (chronic, high intensity stress). Chronic ER stress of pancreatic beta cells leads to apoptosis and development of diabetes. ER stress causes inhibition of protein synthesis via phosphorylation of the translation initiation factor eIF2α. Translational repression is followed by partial translational recovery, which allows stress-induced mRNAs to be translated into proteins that help cells to adapt and survive the stress conditions. However, we found that in pancreatic beta cells inhibition of protein synthesis is followed by complete translational recovery which increases the burden of the stressed ER, thus accelerating cell death. We hypothesized that the complete translational recovery is associated with the dephosphorylation of eIF2α mediated by the phosphatase GADD34. Transcription of GADD34 is stimulated by the ER stress-induced transcription factors CHOP and ATF4. We have published that a specifi c isoform of the transcription factor C/EBPβ (C/EBPβ-LIP) enhances nuclear localization of CHOP during ER stress. We have used a diabetes mouse model (AKITA) and found that mice heterozygous for AKITA and null for C/EBPβ were protected from the development of diabetes. We are testing the hypothesis that absence of C/EBPβ protects the AKITA mice from diabetes by inhibiting regulation of CHOP-target genes, such as GADD34. This in turns causes decreased protein synthesis in pancreatic islets, despite the continuous presence of the UPR stress response program. Our goal is to identify the potential role of CHOP and C/EBPβ in the translational recovery mechanism and determine their involvement in the loss of pancreatic beta cells during induction of ER stress. We propose that C/EBPβ is essential for the pancreatic beta cell death during ER stress conditions, the in vivo physiological and pathological signifi cance being the induction of diabetes.

470-PPExendin-4 Protects Beta-Cells from Palmitate-Induced Apoptosis by Reducing GPR40 Expression and Interfering with Activation of the MKK4/7-JNK PathwayANNALISA NATALICCHIO, ROSSELLA LABARBUTA, FEDERICA TORTOSA, MAURA ROBERTA ORLANDO, ANNA LEONARDINI, ANGELO CIGNARELLI, MARIANGELA MELCHIORRE, ALESSANDRO PESCHECHERA, PIERO MARCHETTI, SEBASTIO PERRINI, LUIGI LAVIOLA, FRANCESCO GIORGINO, Bari, Italy, Pisa, Italy

Free fatty acids (FFA) induce beta-cell damage when their levels are chronically increased, thus contributing to the pathogenesis of type 2 diabetes. GLP-1 and its receptor agonist exendin-4 (ex-4) increase the survival of beta-cells exposed to various pro-apoptotic stimuli, including FFA. The aim of this study was to investigate the mechanisms of the protective effects of GLP-1 mimetics on FFA-induced beta-cell apoptosis. Exposure of human and rat beta-cells to 0.5 mM palmitate induced a 2.5-fold increase in cell apoptosis measured by evaluation of cytosolic oligosomes and cleaved caspase-3 (p<0.05). Palmitate increased the phosphorylation levels of both JNK and p38 (3.5-fold and 2-fold, respectively; p<0.05) evaluated both by immunoblotting and immunofl uorescence. Inhibition of JNK (using the JNK inhibitors SP600125 and JNKi) or p38 (using the p38 inhibitor SB203580) phosphorylation prevented or reduced palmitate-induced apoptosis, respectively (p<0.05). Treatment with 10 nM ex-4 inhibited JNK and reduced p38 phosphorylation levels, and prevented apoptosis in response to palmitate (p<0.05). Furthermore, ex-4 inhibited palmitate-induced phosphorylation of the upstream kinases MKK4 and MKK7, which are implicated in JNK and p38 activation (p<0.05), and increased the protein content of Islet-Brain 1 (IB1), an endogenous JNK blocker (p<0.05). However, RNAi-mediated suppression of IB1 protein levels did not impair the ability of ex-4 to inhibit JNK and to prevent apoptosis. Finally, ex-4 reduced the mRNA levels of GPR40, a cell-surface FFA transporter (p<0.05). The inhibitory effects of ex-4 on JNK and GPR40 were abrogated in the presence of the PKA inhibitor H89. In conclusion, activation of the stress-kinases JNK and p38 is involved in palmitate-induced apoptosis in rat and human beta-cells. The GLP-1 analog ex-4 counteracts the palmitate effects by reducing GPR40 gene expression and inhibiting MKK7- and MKK4-dependent phosphorylation of JNK and p38 in a PKA-dependent manner.

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ISLET BIOLOGY—BETA CELL—DEVELOPMENT

471-PPDiabetes before HyperglycemiaAFAF GAD EL-SEED ABSOOD, PETER ARVAN, MING LIU, LEANZA LIU, LEENA HAATAJA, DENNIS LARKIN, AHMED B. AL-KHAFAJI, ISRAEL HODISH, Ann Arbor, MI

During early stages of type2 diabetes and certain forms of neonatal diabetes, β-cells are still viable but they exhibit endoplasmic reticulum (ER) crowding and ER-stress. In both diseases, the pathologies are ascribed to defects in proinsulin maturation, such as mutation in the CAPN10 gene leading to type2 diabetes, or the Akita mutation leading to neonatal diabetes. However, these pathologies were documented once hyperglycemia was already present, thus the contribution of glucotoxicity itself could not be excluded as a cause.

Although the mechanism has not been uncovered, growing evidence implies that early insulin therapy can preserve remnant β-cell function during early stages of type2 diabetes. In reality, insulin therapy is introduced only at a later stage when patients have become resistant to oral medications and β-cells are destroyed. This could be of particular importance since preservation of β-cell function is linked to a favorable prognosis.

We hypothesize that ER-crowding and stress preceding onset of hyperglycemia in type2 diabetes can be ameliorated with early insulin therapy.

To test the hypothesis, we have developed transgenic mouse models expressing GFP-labeled proinsulin that allows selective identifi cation of a subset of proinsulin molecules. At a modest expression level, the Akita mutation (disrupting the proinsulin A7-B7 disulfi de bond) does not result in frank diabetes. Rather, mice exhibited impaired glucose tolerance with normal random glucose, more consistent with early type2 diabetes or prediabetes. Nevertheless, the small amount of misfolded proinsulin perturbed maturation of wild-type proinsulin and resulted in ER-stress, ER-crowding and insulin defi ciency. We found that each islet exhibited a heterogeneous population of β-cells with respect to endogenous insulin production versus accumulation of misfolded proinsulin. Early insulin therapy, aiming to limit further proinsulin synthesis, resulted in amelioration of ER-crowding and improved insulin production.

In summary, we have identifi ed β-cell secretory defects in pre-diabetic mice with impaired proinsulin maturation, which is improved by early insulin therapy. These fi ndings should be considered in light of current paradigms for management of prediabetes.

ISLET BIOLOGY—BETA CELL—POSTNATAL GROWTH

472-PPTcf19 Is a Novel Transcription Factor Necessary for β-Cell Prolifera-tionKIMBERLY A. KRAUTKRAMER, JOSHUA I. SUHONEN, LOUISE M. MESKE, GREGORY J. SCHLEIS, TED W. HARRIS, JEREMY A. LAVINE, BRIAN S. YANDELL, ALAN D. ATTIE, DAWN BELT DAVIS, Madison, WI

Tcf19 is a largely uncharacterized transcription factor that is expressed during cell division, beginning at G1/S phase. In a mouse model of obesity-associated diabetes, we identifi ed tcf19 within a group of cell cycle genes whose expression correlates with adaptive islet proliferation. In confi rmation of these microarray data, we observed 3.3-fold upregulation of tcf19 mRNA in islets from obese non-diabetic C57Bl/6J (B6) mice vs. lean (n=5, p=0.01). This obesity-driven upregulation was not observed in islets from the diabetic BTBR strain. We then analyzed islet gene expression in a cohort of 500 obese F2 hybrid (B6 ob/ob x BTBR ob/ob) mice and mapped the expression of multiple cell cycle regulatory genes (expression quantitative trait loci, eQTLs) to mouse chromosome 17. Tcf19 is located directly under the LOD score peak between position 35.649-35.653 Mb, suggesting that a genetic difference within tcf19 may be responsible for controlling differential islet cell cycle gene expression in these different strains. In fact, we have identifi ed a non-coding SNP between the B6 and BTBR strains within exon 1 of tcf19. Together, these data suggest a key regulatory role for tcf19 in obesity-driven islet proliferation. We next examined the direct role of tcf19 in cell cycle regulation, specifi cally within the beta cell. After siRNA-mediated knockdown of tcf19 in INS-1 cells, we identifi ed a signifi cant reduction in the number of viable cells (n=5, p<0.05). More specifi cally, we observed a 40% reduction in proliferation with tcf19 knockdown (n=3, p=0.012) measured by 3H-thymidine incorporation. Tcf19 knockdown caused a decrease in mRNA expression of many cell cycle genes, including A-, B-, and E-type cyclins (17-53%, n=3, p<0.05). In addition, several of the cell cycle genes with eQTLs at the tcf19 locus (mki67, cdca3, bub1, and pbk) were signifi cantly reduced

in response to tcf19 knockdown (33-47%, n=3, p<0.05). In summary, tcf19 is necessary for beta cell proliferation and appears to be a key regulator of obesity-driven proliferation in mouse islets.

ISLET BIOLOGY—BETA CELL—STIMULUS-SECRETION COUPLING AND METABOLISM

473-PPEnergy Production and Insulin Release Are Altered in Pancreatic Islets from Human Type 2 Diabetics but Can Be Normalized by a Gluco kinase ActivatorNICOLAI DOLIBA, WEI QIN, HABIBA NAJAFI, HEATHER COLLINS, DAVID WILSON, JOSEPH GRIMSBY, RAMAKANTH SARABU, CHENGYANG LIU, ALI NAJI, FRANZ M. MATSCHINSKY, Philadelphia, PA, Nutley, NJ

We have reported that isolated human islets from type 2 diabetics show reduced glucose stimulated insulin release. Different factors may contribute to this defect. Among these impaired bioenergetics could play a signifi cant role. To quantitatively assess this possibility, glucose stimulated respiration (VO2), glucose usage and oxidation and insulin release (IR) were measured in pancreatic islets isolated from healthy and type 2 diabetic organ donors. Intracellular Ca2+ measurements were made to supplement this information. Isolated mouse islets were studied for comparison. Human and mouse islets were exposed to a “staircase” glucose stimulus from zero to 3, to 6, to 12 and then to 24 mM while IR and VO2 were measured. Glucose usage and oxidation were determined in batch incubations. VO2 of healthy human islets increased sigmoidally as a function of the stepwise rise in glucose concentrations. Maximal VO2 in normal islets was 0.40 nmol/min/100 islets and the glucose S0.5 was 4.4 mM. In islets from type 2 diabetics, the stimulation of respiration by glucose was reduced to a Vmax of 0.32 nmol/min/100 islets and the S0.5 rose to 5.4 mM. Glucose stimulated IR and the rise of intracellular Ca2+ were also reduced in type 2 islets as compared to controls. The metabolic data allowed us to establish a clear sigmoidal relationship between pancreatic islet energy (ATP) production and IR for human and mouse islets. In the case of normal human islets the IR threshold was at 7.5 pmol/min/islet and the half maximal point was at 13.2 pmol/min/islet of ATP produced. The results of mouse islets were comparable. The glucokinase activator Piragliatin normalized the defective VO2, IR and free calcium response during glucose stimulation in islets from type 2 diabetics. Our data suggest strongly that impaired bioenergetics resulting in reduced ATP production of pancreatic islets are a critical factor in the molecular pathogenesis of type 2 diabetes mellitus.

474-PPFXYD3, a New Regulator of Beta-Cell Glucose Competence That Is Developmentally Regulated by Gluco-Incretin Signalling through DNA MethylationDAVID VALLOIS, SONIA KLINGER, JEAN-YVES CHATTON, ANDREI I. TARASOV, GUY A. RUTTER, BERNARD THORENS, Lausanne, Switzerland, Montreal, QC, Canada, London, United Kingdom

To identify new genes that control beta cell glucose competence, we performed comparative transcriptomic analysis of islets from WT and Gipr-/-;Glp-1r-/- (dKO) mice, which show reduced glucose-stimulated insulin secretion (GSIS) despite normal beta cell mass and insulin content. We found a marked up-regulation of Fxyd3 in dKO as compared to WT islets, which was associated with a fourfold increase in FXYD3 protein expression. FXYD family members are transmembrane proteins that modulate the activity of ions channels such as the Na,K-ATPase, and differentiation or proliferation of certain cell lines. Here, we demonstrate that FXYD3 over-expression in beta-cell lines and primary beta-cells reduced GSIS. This was not due to impaired glucose-induced closure of the KATP channel or activation of Ca++ infl ux, but was associated with a reduced exocytosis of insulin granules as shown by capacitance measurement. When regulated expression of Fxyd3 was evaluated in cell lines or primary islets, no regulation was induced by changes in intracellular cAMP levels following GLP-1 or other pharmacological treatments. Fxyd3 expression in neonatal islets was similar in WT and dKO mice, and this level was the same as found in adult dKO islets. This indicated that Fxyd3 expression is normally down-regulated during postnatal development by gluco-incretin signalling. However, Fxyd3 expression was not regulated in control neonatal islets by short-term GLP-1 treatment, suggesting that gluco-incretin signalling controls postnatal Fxyd3 expression by a non-classical mechanism. We thus investigated whether changes in

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methylation of the Fxyd3 promoter could be involved. Bisulfi te sequencing and targeted pyrosequencing revealed that selected CpG islands present in the proximal promoter region of Fxyd3 were markedly more methylated in WT than in dKO islets. Our data identify Fxyd3 as a novel regulator of glucose competence whose expression is contolled in the postnatal period by gluco-incretin-dependent methylation of its promoter. These data show that GLP-1 can affect long-term function of beta-cells through epigenetic regulation of gene expression.

475-PPKv2.1 Regulates Insulin Secretion from Human β-Cells Independent of Its K+ Channel FunctionXIAO-QING DAI, GREG PLUMMER, MARINA CASIMIR, JOCELYN MANNING FOX, QIAN WANG, CATHERINE HAJMRLE, XING-ZHEN CHEN, PATRICK E. MAC-DONALD, Edmonton, AB, Canada

The voltage-dependent K+ (Kv) channel Kv2.1 mediates a majority of the K+ current thought to contribute to action potential repolarization in β-cells. The role of kv2.1 in human β-cell excitability and secretion has been questioned. We now show that Kv2.1 modulates exocytosis and insulin secretion from human β-cells independent of its role in conducting K+ ions. Expression of either the wild-type, or a non-conducting Kv2.1 pore mutant (W365C/Y380T), directly augmented the exocytotic response of human β-cells to membrane depolarization as assessed by whole-cell capacitance. We fi nd by co-immunoprecipitation that native Kv2.1 channels in human islets interact with the exocytotic SNARE protein syntaxin 1A; furthermore, this endogenous interaction can be disrupted by expression of a C-terminal fragment of the channel (Kv2.1-C1; amino acids 412-633). Disruption of the Kv2.1-syntaxin 1A interaction had no effect on β-cell Kv currents; but acute intracellular dialysis, or transient expression, of Kv2.1-C1 inhibited the exocytotic response to membrane depolarization by 70 and 79% (p<0.001). No effect was seen with either distal C-terminal (C2; aa 634-853) or N-terminal (aa 1-182) fragments of the channel. Disruption of the Kv2.1-syntaxin 1A interaction did not affect secretory granule localization to the plasma membrane or exocytosis in response to direct application of 2 µM Ca2+, suggesting that Ca2+-dependent exocytosis per se remained intact. Rather, the steady-state voltage-dependent Ca2+-channel (VDCC) activity was inhibited. We confi rmed that this effect was specifi c to L-type Ca2+ current. Finally, we fi nd that disruption of the Kv2.1-syntaxin 1A interaction impairs glucose-dependent Ca2+ and insulin secretion responses in human islets.

Conclusion: We demonstrate in human islets a direct interaction between Kv2.1 and the secretory machinery, disruption of which reduces glucose-stimulated insulin release. It results from a role for Kv2.1 in facilitating insulin secretion independent of its K+ conductance. Kv2.1 acts through an interaction with syntaxin 1A to modulate channel activity and exocytosis in human β-cells.

Supported by: National Sciences and Engineering Research Council of Canada

ISLET BIOLOGY—SIGNAL TRANSDUCTION

476-PPActivating Transcription Factor 6 Binds to the A5/Core of the Rat Insulin II Gene PromoterJULIE AMYOT, GHISLAINE FONTÉS, ISMA BENTERKI, DEREK K. HAGMAN, ALLEN VOLCHUK, ERIK JOLY, VINCENT POITOUT, Montreal, QC, Canada, Toronto, ON, Canada

Type 2 diabetes is characterized by impaired insulin secretion from the pancreatic islet β-cells and peripheral insulin resistance. Pancreatic β-cells have a well-developed endoplasmic reticulum (ER) due to their highly specialized secretory function to produce insulin in response to glucose and nutrients. Since it has been previously reported that overexpression of activating transcription factor 6 (ATF6) of the unfolded protein response (UPR) reduces insulin gene expression in part via upregulation of small heterodimer partner, we investigated whether ATF6 directly binds to the insulin gene promoter, and whether its direct binding represses insulin gene promoter activity. A bioinformatics analysis identifi ed a putative ATF6 binding site in the A5/Core region of the rat insulin II gene promoter. Direct binding of ATF6 was confi rmed using several approaches. Electrophoretic mobility shift assays performed in MCF7 cells, isolated rat islets and insulin-secreting HIT-T15 cells showed ATF6 binding to the native A5/Core of the rat insulin II gene promoter. Antibody-mediated supershift analyses revealed the presence of both ATF6 isoforms, ATF6α and ATF6β, in the complex. Chromatin immunoprecipitation assays confi rmed the binding of ATF6α and ATF6β to a region encompassing the A5/Core of the rat insulin II gene

promoter in isolated rat islets. Overexpression of the active p50 fragment of ATF6 inhibited the activity of an insulin promoter-reporter by 50%. However, the inhibitory effect of ATF6 was insensitive to mutational inactivation or deletion of the A5/Core. Therefore, although ATF6 binds directly to the A5/Core of the rat insulin II gene promoter, this direct binding does not appear to contribute to its repressive activity. Inhibition of insulin gene expression by the ATF6 branch of the UPR might represent a protective mechanism to reduce the protein load to the ER under conditions of nutrient excess.

Supported by: NIH, FRSQ

477-PPInhibition of Lysine Deacetylase Activity Protects β-Cells from Infl am ma tory Attack In Vitro and In Vivo by Targeting NF-KB Tran-scriptional ActivityDAN PLOUG CHRISTENSEN, CONNY GYSEMANS, MATTIAS SALLING DAHLLÖF, MORTEN LUNDH, DANIEL NOESGAARD RASMUSSEN, SØREN FISKER SCHMIDT, SUSANNE MANDRUP, NICOLAJ JUUL, CHRISTOPHER WORKMAN, LYKKE BLAABJERG, PAOLO MASCAGNI, CHARLES DINARELLO, NILS BILLESTRUP, CHANTAL MATHIEU, LARS GROTH GRUNNET, THOMAS MANDRUP-POULSEN, Copenhagen, Denmark, Leuven, Belgium, Odense, Denmark, Lyngby, Denmark, Gentofte, Denmark, Milan, Italy, Denver, CO

Pro-infl ammatory cytokines contribute to β-cell demise in diabetes. Lysine deacetylase inhibitors (KDACi) prevent cytokine-induced β-cell damage in vitro, but the mechanisms and the in vivo effi cacy of KDACi in animal models of diabetes are unknown. Here we show that the KDACi suberoylanilide hydroxamic acid and givinostat administered to weaning NOD mice for 120 days reduced diabetes incidence, improved pancreatic insulin contents, suppressed infl ammatory dendritic cell subset levels, and restored regulatory T-cell subset levels. To clarify the mechanisms of protection of KDACi on β-cells, we investigated if KDACi affected infl ammatory β-cell gene expression and signaling.

Time-course microchip array analysis utilizing rat islets revealed i) that KDACi prevented interleukin (IL)-1+ interferon (IFN)γ induction of pro-infl ammatory mediators such as IL-1α and tumor necrosis factor α and ii) central roles of the extracellular regulated kinase (ERK) and nuclear factor - (NF)-κB in top-rated cytokine-induced networks that were affected by KDACi treatment. KDACi potentiated suppressors of cytokine signaling (SOCS) 3 expression in lipopolysaccharide-treated peripheral blood mononuclear cells (PBMC), and SOCS3 siRNA knock-down reduced KDACi-mediated inhibition of PBMC IL-6 secretion. Surprisingly, expression data suggested that the protective mechanism of action of KDACi in β-cells is independent on upregulation of anti-apoptotic proteins such as SOCS3. In insulin producing cells KDACi prevented cytokine-induced phosphorylation of ERK, reduced NF-kB genomic DNA binding (ChIP assay), inhibited NF-κB activity (reporter assay), and blocked NF-kB dependent mRNA expression. We suggest that KDACi counteracts infl ammatory β-cell damage in vivo as in vitro via effects on both immune and β-cells, and that KDACi reduce NF-kB DNA binding and transcriptional activity in β-cells. These data provide a rationale for conducting clinical trials of the effi cacy and safety of KDACi in patients with recent-onset Type 1 diabetes and Type 2 diabetes, and in islet transplantation.

Supported by: JDRF Grant # 26-2008-893 and FWO-KAN 1.5070.09

2011 ADA Presidents 379-477 - Diabetes€¦ · TOMOAKI INOUE, KUNIHISA KOBAYASHI, MASAKAZU FUJII,...

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Transcript of 2011 ADA Presidents 379-477 - Diabetes€¦ · TOMOAKI INOUE, KUNIHISA KOBAYASHI, MASAKAZU FUJII,...