Hepatic Insulin Sensitivity Research Articles

Role of Patatin-Like Phospholipase Domain-Containing 3 Gene for Hepatic Lipid Content and Insulin Resistance in Diabetes.

The rs738409(G) single nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 (PNPLA3) gene associates with increased risk and progression of nonalcoholic fatty liver disease (NAFLD). As the recently described severe insulin-resistant diabetes (SIRD) cluster specifically relates to NAFLD, this study examined whether this SNP differently associates with hepatic lipid content (hepatocellular lipids [HCL]) and insulin sensitivity in recent-onset diabetes. A total of 917 participants in the German Diabetes Study (GDS) underwent genotyping, hyperinsulinemic-euglycemic clamps with stable isotopic tracer dilution, and MRS. The G allele associated positively with HCL (β = 0.36, P < 0.01), independent of age, sex, and BMI across the whole cohort, but not in the individual clusters. Those with SIRD exhibited lowest whole-body insulin sensitivity compared with those with severe insulin-deficient (SIDD), moderate obesity-related (MOD), moderate age-related (MARD), and severe autoimmune diabetes (SAID) clusters (all P < 0.001). Interestingly, the SIRD group presented with higher prevalence of the rs738409(G) SNP compared with other clusters and the glucose-tolerant control group (P < 0.05). HCL was higher in the SIRD group (median 13.6% [1st quartile 5.8; 3rd quartile 19.1] compared with the MOD (6.4 % [2.1; 12.4], P < 0.05), MARD (3.0% [1.0; 7.9], P < 0.001), SAID (0.4% [0.0; 1.5], P < 0.001), and glucose-tolerant (0.9% [0.4; 4.9), P < 0.001) group. Although the PNPLA3 polymorphism did not directly associate with whole-body insulin sensitivity in SIRD, the G-allele carriers had higher circulating free fatty acid concentrations and greater adipose tissue insulin resistance compared with noncarriers (both P < 0.001). Members of the SIRD cluster are more frequently carriers of the rs738409(G) variant. The SNP-associated adipose tissue insulin resistance and excessive lipolysis may contribute to their NAFLD.

High Glucose-Aggravated Hepatic Insulin Resistance: Role of the NLRP3 Inflammasome in Kupffer Cells.

This study aimed to investigate whether the NLRP3 inflammasome in Kupffer cells (KCs) can be activated in response to high glucose (HG) and toevaluate its influence on hepatic insulin sensitivity. Primary KCs and hepatocytes were isolated from mice, and lipid accumulation, glucose output, and insulin sensitivity of hepatocytes were investigated after culturing either alone or with KCs exposed to HG. The influence of HG-induced NLRP3 inflammasome activation in KCs on insulin sensitivity of hepatocytes was examined. Treatment with gadolinium trichloride caused KC depletion, and, subsequently, a streptozotocin-induced hyperglycemic mouse model was used to confirm the influence of KCs on hepatic insulin sensitivity. Hepatocytes cocultured with KCs showed enhanced lipid accumulation, glucose output, and impaired insulin sensitivity when exposed to HG. Enhanced NLRP3 inflammasome activation was also evident in both hepatocytes and KCs. Moreover, KCs that were pretreated with caspase-1 inhibitor, NLRP3 inhibitor, and NLRP3 smallinterfering RNA corrected coculture-induced aberrances in insulin action and NLRP3 inflammasome activation in hepatocytes. KC coculture also increased interleukin-1β (IL-1β)-mediated nuclear factor-κB (NF-κB) activation in hepatocytes. In hyperglycemic mice, KC depletion inhibited NLRP3 inflammasome activation and improved hepatic insulin sensitivity. NLRP3 inflammasome activation impaired insulin sensitivity through KC-derived IL-1β-mediated NF-κB activation in hepatocytes exposed to HG.

Inhibition of prolyl hydroxylases increases hepatic insulin and decreases glucagon sensitivity by an HIF-2α-dependent mechanism

ObjectiveRecent evidence indicates that inhibition of prolyl hydroxylase domain (PHD) proteins can exert beneficial effects to improve metabolic abnormalities in mice and humans. However, the underlying mechanisms are not clearly understood. This study was designed to address this question. MethodsA pan-PHD inhibitor compound was injected into WT and liver-specific hypoxia-inducible factor (HIF)-2α KO mice, after onset of obesity and glucose intolerance, and changes in glucose and glucagon tolerance were measured. Tissue-specific changes in basal glucose flux and insulin sensitivity were also measured by hyperinsulinemic euglycemic clamp studies. Molecular and cellular mechanisms were assessed in normal and type 2 diabetic human hepatocytes, as well as in mouse hepatocytes. ResultsAdministration of a PHD inhibitor compound (PHDi) after the onset of obesity and insulin resistance improved glycemic control by increasing insulin and decreasing glucagon sensitivity in mice, independent of body weight change. Hyperinsulinemic euglycemic clamp studies revealed that these effects of PHDi treatment were mainly due to decreased basal hepatic glucose output and increased liver insulin sensitivity. Hepatocyte-specific deletion of HIF-2α markedly attenuated these effects of PHDi treatment, showing PHDi effects are HIF-2α dependent. At the molecular level, HIF-2α induced increased Irs2 and cyclic AMP-specific phosphodiesterase gene expression, leading to increased and decreased insulin and glucagon signaling, respectively. These effects of PHDi treatment were conserved in human and mouse hepatocytes. ConclusionsOur results elucidate unknown mechanisms for how PHD inhibition improves glycemic control through HIF-2α-dependent regulation of hepatic insulin and glucagon sensitivity.

1934-P: Branched-Chain Amino Acids Are Poor Markers of Insulin Resistance in Women: The Federal Women’s Study

The data relating branched chain amino acids (BCAAs) with obesity and type 2 diabetes is conflicting but may be further clarified by examining its relationship to race/ethnicity and menopause. Therefore, we determined the relationship of BCAA with markers of insulin sensitivity (SI) in 122 federally employed women (49 White, 73 Black: 58 African American and 15 African Immigrant, age 44±10 (mean±SD) (range 24-62y); BMI 30.2±5.7 (range 20.3-45.2 kg/m2); 33% menopausal) using linear regression models adjusted for race and menopause. Fasting BCAAs were measured by nuclear magnetic resonance (NMR) spectroscopy, whole-body SI derived from the minimal model, basal hepatic insulin sensitivity (HISI) using [6,62H2]glucose, and hepatic and visceral adipose tissue (VAT) by proton NMR. BCAAs were similar in black and white women (leucine:104±30 vs. 114±37, valine:196±40 vs. 204±34, isoleucine:41±12 vs. 38±12 μmol/L, all P≥0.1) and did not differ by menopause (P&amp;gt;0.5). Total and individual BCAAs were associated with lower whole-body SI (Figure), weakly associated with VAT (AdjR2=0.1, P=0.01), liver fat (AdjR2=0.01, P=0.01), and there was no association with HISI (P&amp;gt;0.2). Overall, plasma BCAAs were not robust markers of insulin resistance in women of white or African ancestry and higher levels in women with insulin resistance may reflect a composite effect of dietary and metabolic processes. Disclosure S. Matta: None. C.K. Cravalho: None. A. Villalobos-Perez: None. A. Meyers: None. L. Mabundo: None. A.B. Courville: None. M.L. Sampson: None. A.E. Sumner: None. S.T. Chung: None. Funding National Institutes of Health (to S.T.C., L.M., A.B.C.)

1832-P: Palmitic Acid Esters of Hydroxy Stearic Acids Reduce Endogenous Glucose Production through GPR43 Activation

Palmitic Acid esters of Hydroxy Stearic Acids (PAHSAs), bioactive lipids with anti-inflammatory and antidiabetic effects, improve systemic and hepatic insulin sensitivity in insulin-resistant mice. PAHSAs augment insulin action on endogenous glucose production (EGP) through direct and indirect actions involving inter-tissue communication between liver and adipose tissue. The direct effect of PAHSAs to inhibit EGP is mediated through a cAMP dependent pathway involving Gα/i protein-coupled receptors. Here, we investigated which receptor mediates PAHSA effects on EGP. We found that 9-PAHSA activates GPR43 in a Ca+2 flux assay. 4-CMTB (0.4μM), a selective agonist-allosteric modulator of GPR43, reduced basal EGP by 23% and glucagon-stimulated EGP by 20% in primary hepatocytes ex vivo. Acute 9-PAHSA treatment (40μM) inhibited basal EGP to the same extent as 4-CMTB. 9-PAHSA suppression of basal EGP was abolished in primary hepatocytes from whole body GPR43 KO mice. Intravenous infusion of 9-PAHSA (9μg/hr) for 3 hrs decreased ambient EGP in chow-fed mice studied 5 hours after food removal (WT: vehicle= 24.2±1.9 vs. PAHSA= 17.2±0.7 mg/kg/min p&amp;lt;0.05). This effect was absent in GPR43 KO mice (KO: vehicle= 25.3±4.2 vs. PAHSA= 23.4±2.7 mg/kg/min). We also examined whether GPR43 activation is required for the direct effect of 9-PAHSA on WAT lipolysis. Treatment with the β-adrenergic agonist isoproterenol increased FFA release from WAT explants by 3.8-fold, and 9-PAHSA inhibited isoproterenol-induced lipolysis by 21% which was similar to the effects of insulin and 4-CMTB. In WAT explants from GPR43 KO mice, 9-PAHSA suppression of isoproterenol-induced FFA release was abolished while insulin still suppressed lipolysis. Sum: 9-PAHSA activates GPR43, which is involved in its direct effects to reduce hepatic glucose production and WAT lipolysis. Conclusion: We have identified a new receptor that mediates some of the beneficial metabolic effects of PAHSAs. Disclosure A. Santoro: None. P. Zhou: None. Y. Zhu: None. O.D. Peroni: None. A.T. Nelson: None. D. Siegel: None. B. Kahn: Advisory Panel; Self; Harrington Discovery Institute, Janssen Pharmaceuticals, Inc., National Institute of Diabetes and Digestive and Kidney Diseases. Funding American Diabetes Association (1-18-PDF-134 to P.Z.); National Institutes of Health (DK43051, DK57521, DK106210, DK112622); JPB Foundation; American Heart Association

1698-P: Obesity-Induced Increase in Cystatin C Alleviates Tissue Inflammation and Hepatic Insulin Resistance

Removal of one kidney (uninephrectomy; UniNx) in mice reduced high fat-diet (HFD)-induced adipose tissue inflammation thereby improving adipose tissue and hepatic insulin sensitivity. Such finding was accompanied by increased circulating cystatin C levels in UniNx compared to sham-operated mice. Importantly, cystatin C may have anti-inflammatory properties, and circulating cystatin C levels were reported to positively correlate with obesity as well as type 2 diabetes. However, the causal relationship of such correlation remains unclear. Herein, HFD feeding of cystatin C-deficient (CysC KO) mice aggravated obesity-associated adipose tissue dysfunction and inflammation, as reflected by an accumulation of pro-inflammatory monocytes and elevated expression of pro-inflammatory mediators. Moreover, markers of adipocyte differentiation were decreased. Similarly to findings in adipose tissue, expression of pro-inflammatory cytokines was increased in liver and skeletal muscle of HFD-fed CysC KO mice. In line, HFD-induced hepatic insulin resistance and impairment of glucose tolerance were further aggravated in knockout mice. Consistently, chow-fed CysC KO mice were more susceptible to lipopolysaccharide (LPS)-induced adipose tissue inflammation. In people with obesity, circulating cystatin C levels correlated negatively with mRNA expression of pro-inflammatory cytokines such as Hif1α as well as IL-6. Moreover, healthy (i.e., insulin-sensitive) subjects with obesity depicted significantly higher mRNA expression of cystatin C in subcutaneous white adipose tissue. In conclusion, cystatin C is upregulated under obesity conditions and thereby counteracts inflammation of peripheral insulin-sensitive tissues and, thus, obesity-associated deterioration of glucose metabolism. Disclosure M.A. Dedual: None. S. Wueest: None. T. Challa: None. F. Lucchini: None. T. Aeppli: None. M. Borsigova: None. A.A. Mauracher: None. S. Vavassori: None. J. Pachlopnik Schmid: Advisory Panel; Self; Novartis AG, Swedish Orphan Biovitrum. M. Blüher: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk A/S, Sanofi. Speaker’s Bureau; Self; Amgen, Novartis AG. D. Konrad: Research Support; Self; Novo Nordisk A/S. Funding Children’s Research Centre; University Children’s Hospital Zurich; University of Zurich (FK-18-027); Swiss National Science Foundation (310030-160129, 10030-179344); Wolfermann-Nägeli Foundation

1743-P: Metabolic Characteristics of Diabetes among Low-BMI Individuals in India

Diabetes among low BMI individuals has been recognized globally for over 60 years, and classified by WHO as malnutrition-related diabetes mellitus in 1985. Despite its prevalence in low and middle income countries, this condition has never been comprehensively studied. State of the art metabolic studies were performed in 20 South Indian men with Low BMI Diabetes (LD; BMI 18.3 ± 0.9 kg/m2), after excluding monogenic and immune diabetes, and visible pancreatic pathology. Other groups were studied: T1D, T2D, and nondiabetic subjects (BMI-matched to LD and T2D). Insulin secretion was assessed by C-peptide deconvolution following a mixed-meal tolerance test. Peripheral and hepatic insulin sensitivity were analyzed using tracer-based (6,6-deuterated glucose), stepped hyperinsulinemic-euglycemic pancreatic clamps following correction of glucose toxicity. LD subjects were distinct from both T1D and T2D. Insulin secretion was significantly lower in LD than in T2D subjects, yet higher than in T1D. LD subjects were more insulin sensitive than T2D; glucose uptake and endogenous glucose production did not differ from lean nondiabetic or T1D subjects. These comprehensive studies are the first to demonstrate that LD individuals are characterized by markedly impaired insulin secretion yet preserved insulin sensitivity. Thus, diabetes in low BMI individuals is a distinct entity that warrants further investigation. Disclosure E. Lontchi Yimagou: None. R. Das Gupta: None. S. Anoop: None. S. Kehlenbrink: None. J. Moy: None. A. Manavalan: None. P.M. Mathias: None. A. Goyal: None. M. Carey: None. D.T. Stein: None. N. Thomas: None. M. Hawkins: None. Funding National Institutes of Health (R01DK069861)

2010-P: Adipocyte Clusterin Is a Link between Cardiometabolism and Alzheimer’s Disease

Background: Obesity heightens the risk for both cardiometabolic complications and Alzheimer’s disease (AD). Although the reasons have yet to be fully elucidated, weight loss improves cognition. Given this link, factors overproduced by obese adipose tissue are ideal candidates for novel investigation into AD pathogenesis. We have recently shown that clusterin from the adipocyte is related to multiple factors of the metabolic syndrome (insulin resistance, dyslipidemia, NAFLD, and atherosclerosis). Clusterin is also involved in CNS lipid trafficking and has been proposed as an AD biomarker. Rationale: Our goals were to characterize the effect of high-fat diet (HFD) feeding on CNS clusterin and neuronal insulin signaling in an AD mouse model (5XFAD); and furthermore, determine the relationship of adipocyte clusterin with neurocognition and insulin sensitivity in obese humans at baseline and following bariatric surgery. Methods: We treated 5XFAD and WT mice with normal chow and HFD for 4 mo., measured CNS clusterin, and determined its effect on neuronal insulin signaling. We next measured subcutaneous adipocyte (SAd) clusterin expression, and performed neuropsychological evaluation and a hyperinsulinemic-euglycemic clamp procedure in obese human subjects (n=11) before and after sleeve gastrectomy. Results: In the 5XFAD mice, HFD increases brain clusterin levels compared to WT, and in primary cortical neurons clusterin attenuates insulin signaling in a dose-dependent manner. In humans, SAd clusterin is related to hepatic insulin resistance, higher fasting glucose, and reduced working memory/attention. After sleeve gastrectomy SAd clusterin expression is reduced, with a significant inverse association between clusterin and working memory/attention and hepatic insulin sensitivity. Conclusions: Our data suggests that SAd clusterin is a potential player in AD pathology, providing a critical link between excess adiposity, T2D, insulin resistance, and heightened AD risk. Disclosure D. Bradley: None. J.Z. Liu: None. A.M. Blaszczak: None. V.P. Wright: None. R. Tarawneh: None. Y. Liu: None. J. Ryu: None. S. Yoon: None. W. Hsueh: None. Funding American Diabetes Association (1-16-ICTS-049 to W.H.); National Institutes of Health (KL2TR001068, R01HL135622)

459-P: Liver-Targeted Mitochondrial Uncoupling by CRMP Improves Whole-Body Insulin Sensitivity and Attenuates Atherosclerosis in A LDLR-/- Mouse Model of Metabolic Syndrome

Progress in preventing cardiovascular disease (CVD) has been stalled by the growing epidemic of type 2 diabetes, which increases the relative risk of developing atherosclerosis four-fold compared to nondiabetic individuals. We recently developed an orally available, liver-targeted controlled release mitochondrial protonophore (CRMP) that promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency and shown that this agent safely reverses dyslipidemia, fatty liver, and insulin resistance in rodents and nonhuman primates. While these studies suggest that CRMP may be a novel therapy for the major components of metabolic syndrome, its therapeutic utility for the treatment of CVD remains unknown. To assess the effect of CRMP on atherogenesis, we performed progression studies in Ldlr-/- mice fed a high-fat cholesterol diet and treated with CRMP (30 mg/kg-day) or vehicle control for 12 weeks. Here, we show that CRMP-treated mice display a 30% reduction in fasting plasma triglycerides (which could be attributed to a 20% decrease in VLDL-production) and a significant improvement in both hepatic and peripheral insulin sensitivity, due to reductions in hepatic and muscle ectopic lipid content. Importantly, CRMP-mediated decreases in hypertriglyceridemia and improvements in whole-body insulin sensitivity were associated with a 30% reduction in aortic root plaque area and neutral lipid accumulation (P&amp;lt;0.05). More detailed analysis of these plaques revealed that CRMP treatment also significantly lowered markers of plaque vulnerability, as evidenced by a 30% reduction in necrotic core area and increase in fibrous cap area (P&amp;lt;0.05). Conclusion: Taken together, these results support an anti-atherogenic role for CRMP and suggest the therapeutic potential of liver-targeted mitochondrial uncouplers for the treatment of cardiometabolic disorders. Disclosure L. Goedeke: None. N. Rotllan: None. K. Toussaint: None. A. Nasiri: Employee; Spouse/Partner; Medtronic. X. Zhang: None. J. Lee: None. X. Zhang: None. C. Fernández-Hernando: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. Funding National Institutes of Health (R01DK119968, R01DK116774, P30DK045735 to G.I.S.), (K99 HL150234 to L.G.)

1861-P: Short-Term Overfeeding with Dairy Fat Impairs Insulin Sensitivity in Muscle and Liver but Increases Adipose Tissue Insulin Sensitivity and Suppresses Postprandial Glucose Rise in Healthy Nonobese Men

Previous studies have demonstrated that saturated fat intake increases the risk of type 2 diabetes (T2DM) due to reduction of insulin sensitivity. On the other hand, the consumption of dairy fat that predominantly includes saturated fat is reported to be protective against T2DM. However, its underlying mechanism has not been fully understood. To address this, we recruited 21 healthy non-obese men and evaluated metabolic changes before and after 6-day high-calorie, high-fat diet (HCHFD) consisting of a regular diet plus 45% energy excess by supplementation with dairy fat. We measured intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) in tibialis anterior muscle by 1H-MRS and metabolic rate during sleeping by metabolic chamber. One day after the measurement, we performed meal test, and the next day, we measured tissue specific insulin sensitivity by 2-step hyperinsulinemic euglycemic clamp. After the HCHFD, body weight was increased by 0.4 kg, and IMCL and IHL were also increased by 47% and 200%, respectively. On the other hand, hepatic and muscle insulin sensitivity showed only a modest decrease by 8% and 4%, respectively. Consistently, fasting endogenous glucose production elevated by only 3% without altering insulin level; however, unexpectedly, fasting free fatty acid (FFA) greatly decreased by 43%, suggesting increased adipose tissue insulin sensitivity (ATIS), and total ketone body levels was decreased by 53% and glucose oxidation during sleeping was consistently increased. Further, post-prandial glucose rise during the meal test was suppressed after the HCHFD, although insulin secretion was lower during the test. In conclusion, short-term HCHFD by dairy fat supplementation increased ATIS and glucose oxidation, and suppressed post-prandial glucose rise with lowered insulin secretion. These metabolic changes by dairy fat may be beneficial to prevent T2DM. Disclosure D. Sugimoto: None. Y. Tamura: Advisory Panel; Self; Astellas Pharma Inc. Research Support; Self; Kowa Company, Ltd. Speaker’s Bureau; Self; Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd. S. Kadowaki: None. Y. Someya: None. H. Kaga: None. R. Suzuki: None. S. Kakehi: None. N. Yamasaki: None. M. Sato: None. A. Kanazawa: Speaker’s Bureau; Self; Novartis Pharma K.K., Sanofi, Takeda Pharmaceutical Company Limited. R. Kawamori: None. H. Watada: Advisory Panel; Self; Abbott, Ajinomoto, Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Fuji Film, Janssen Pharmaceuticals, Inc., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. Research Support; Self; Astellas Pharma Inc., Bayer Yakuhin, Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi-Aventis, Sanwa Kagaku Kenkyusho, Shionogi &amp; Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Yakult. Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Bayer Yakuhin, Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly Japan K.K., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi-Aventis, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. Funding Ministry of Education, Culture, Sports, Science and Technology of Japan (18H03196)

5-LB: Consuming High-Fructose Corn Syrup or Sucrose-Sweetened Beverages Increases Hepatic Lipid Content and Decreases Insulin Sensitivity in Young Adults

Rodent studies demonstrate that compared with diets sweetened with sucrose, high fructose corn syrup (HFCS) produces greater metabolic dysfunction. However, the effects of sustained consumption of HFCS and sucrose have not been adequately compared in humans. The objective of this study was to compare HFCS-, sucrose-, or aspartame- sweetened beverage (-SB) consumption on hepatic lipid content and insulin sensitivity in young adults. In a parallel, double-blinded study, adult men and women (18-40 yrs) were assigned to beverage groups matched for sex, BMI and fasting triglycerides. Inpatient magnetic resonance imaging for hepatic lipid content and oral glucose tolerance tests were performed at baseline and intervention 15 days later. For 12 outpatient days subjects consumed their usual diets with 3 servings/day of HFCS- (n=28) or sucrose-SB (n=24) providing 25% energy requirement (Ereq), or aspartame-SB (n=23). Matched standardized diets were provided during inpatient testing with the sugar in beverages replacing complex carbohydrate during intervention. Hepatic lipid content was increased by consumption of HFCS-SB (LS means of absolute Δ±SEM: 0.5%±0.2, P&amp;lt;0.05) and sucrose-SB (Δ: 0.5%±0.2, P&amp;lt;0.01) compared with baseline levels and compared with aspartame-SB (Δ: -0.2%±0.2, P&amp;lt;0.05 vs. HFCS- or sucrose-SB). The 3-hr Matsuda insulin sensitivity index was decreased by consumption of HFCS-SB (Δ: -0.3±0.2) and sucrose-SB (Δ: -0.5±0.2, P&amp;lt;0.01 vs. baseline) compared with aspartame-SB (Δ: 0.3±0.2, P&amp;lt;0.05 vs. HFCS- or sucrose-SB). The 2-hr Oral Glucose Insulin Sensitivity index was also decreased by consumption of HFCS-SB (Δ: -13.6±7.5) and sucrose-SB (Δ:-11.5±7.9) compared with aspartame-SB (Δ: +17.9±8.3, P&amp;lt;0.05 vs. HFCS- or sucrose-SB). Consumption of SB providing 25% of Ereq as HFCS or sucrose for 2 weeks increased hepatic lipid content and reduced insulin sensitivity in young adults compared with consumption of aspartame-SB. Disclosure D. Sigala: None. B. Hieronimus: None. C. Price: None. V. Lee: None. M. Nunez: None. V. Medici: None. A.J. Chaudhari: None. J.P. McGahan: None. Y. Abdelhafez: None. N.L. Keim: None. M.I. Goran: Advisory Panel; Self; Yumi. Other Relationship; Self; Penguin Random House. G. Pacini: None. A. Tura: None. P.J. Havel: Research Support; Self; Arrowhead Pharmaceuticals, Inc., Magnamosis, Inc. Other Relationship; Self; None. K.L. Stanhope: None. Funding National Institutes of Health (R01HL091333, R01HL107256)

204-OR: Inhibition of Hepatic ACC on a High-Fat Diet Results in Hyperglycemia and Hepatomegaly Due to Excess Energy Generation

Liver triglyceride accumulation associated with insulin resistance is partially mediated by elevated de novo lipogenesis (DNL); thus, limiting this pathway is a promising therapeutic strategy for NAFLD. Targeted suppression of acetyl-CoA carboxylase (ACC) activity depletes malonyl-CoA resulting in lower DNL and a simultaneous increase in fat oxidation due to activation of CPT-1. Recently developed ACC1/2 liver specific double KO mice (LDKO) are protected from hepatic steatosis and closely recapitulate pharmacological inhibition of ACC1/2 in humans with NAFLD. However, hepatic ACC inhibition causes surprising elevations in circulating triglyceride and glucose concentrations. LDKO mice had worsened glucose tolerance, which was most consequential during a HFD and especially prominent during fasting. Glucose labeling from 2H2O and [U-13C3] lactate was higher in LDKO hepatocytes, an effect that was dependent on the availability of long chain fatty acids, suggesting that loss of malonyl-CoA and activation of CPT-1 promote gluconeogenesis (GNG). Ketogenesis was increased in LDKO hepatocytes and showed limited response to insulin. Acetyl-CoA was elevated in the fed state, as previously reported, but was normal during fasting when hyperglycemia was most prominent. LDKO mice exhibited massive accumulation of liver citrate and a high fasted hepatic energy charge, which are both known to activate GNG. Hence, despite improved hepatic lipid profile and insulin sensitivity, LDKO liver activates GNG. The resulting hyperinsulinemia partially ameliorates the glycemia phenotype, but may also induce hepatomegaly through canonical signaling pathways and effects on energy status. Thus, activation of fat oxidation in liver lowers lipid accumulation, but has downstream metabolic effects on energetically dependent pathways, such as GNG, that may promote elevated glucose levels in contexts of reduced insulin action, such as fasting and diet induced obesity. Disclosure S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. C. Kim: None. J. Browning: None. J.D. Horton: Board Member; Self; Pfizer Inc. Consultant; Self; Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Merck &amp; Co., Inc. Stock/Shareholder; Self; Catabasis Pharmaceuticals. Other Relationship; Self; Regeneron Pharmaceuticals, Sanofi. S.C. Burgess: None. Funding National Institutes of Health (R01DK078184, P41EB015908); Robert A. Welch Foundation (I-1804)

1834-P: Neuronal Nitric Oxide Synthase Activity and Hepatic Insulin Resistance

Objective: Neuronal nitric oxide synthase (nNOS) contains PZD domain through which NOS1AP was connected. We reported previously that NOS1AP regulated hepatic insulin sensitivity depending on its PZD binding domain suggesting an involvement of nNOS in NOS1AP effect. Thus, in the present study, we aimed to explore the role of nNOS on hepatic glucose and lipid metabolism, and the mechanism underlined. Methods: Liver specific nNOS overexpression was achieved by tail-vein-injection of adenovirus-nNOS in C57BL/6 mice fed with a high-fat diet (HFD). nNOS specific inhibitor (L-NPA) was used to change the activity of nNOS. Western blot was used to evaluate the protein expression. Real-time PCR was used to detect the gene expression. Results: nNOS expressed in livers from humans and mice, and its phosphorylation in Ser1417 increased in the livers of ob/ob and HFD-induced obese mice. Overexpression of nNOS in the livers of HFD-induced obese mice further impaired the glucose tolerance, increased the intrahepatic lipid accumulation, decreased the glycogen storage, and blunted insulin-induced phosphorylation of IRβ and AKT in livers (all P &amp;lt;0.05). Similarly, overexpression of nNOS increased the triglyceride production, decreased the glucose utilization, and insulin-induced phosphorylation of IRβ, AKT and GSK3β in HepG2 cells (all P &amp;lt;0.05). In contrast, inhibition of nNOS with L-NPA in ob/ob mice improved the glucose tolerance and increased insulin-induced phosphorylation of IRβ and AKT in livers (all P &amp;lt;0.05). Moreover, overexpression of nNOS in HepG2 cells increased phosphorylation of P38 MAPK, whereas, inhibition of P38 MAPK with SB203580 significantly reversed the defect in insulin signaling activation -induced by nNOS overexpression. Conclusions: Hepatic nNOS inhibited insulin signaling pathway through the activation of P38 MAPK, suggesting a role of nNOS as a potential therapeutic target for the prevention and treatment of type 2 diabetes. Disclosure C. Wang: None. Q. Li: None. Y. Zhu: None. Funding National Natural Science Foundation of China (81670707)

580-P: Role of Bile Acids in Hepatic Lipid Metabolism: Characterization of the Fasting Lipid and Lipoprotein Profile of Tgr5-/- Hamsters

Introduction: A major complication of insulin resistant states is fasting dyslipidemia, a lipid profile characterized by elevated triglycerides, increased small dense LDL and low HDL-cholesterol. Recently, bile acid signaling has been suggested to improve hepatic insulin sensitivity and lipid accumulation through activation of the Takeda G protein-coupled receptor 5 (TGR5). Using CRISPR/CAS9 gene editing, and in collaboration with the Wang laboratory at Utah State University, we have developed a novel Tgr5-/- hamster model. As the role of TGR5 in regulating hepatic lipid and lipoprotein metabolism has not been explored, the present study was performed to characterize the fasting lipid profile of this model in normal and mild insulin resistant states and further examine bile acid signaling within these hamsters. Methods/Results: Eight-week-old Tgr5+/+ and Tgr5-/- hamsters were fed a high fat, high fructose and low cholesterol (FFLC) diet for 6 weeks. Glucose tolerance and fasting plasma lipids were examined at baseline, and following 6-weeks of FFLC consumption. Unlike the Tgr5+/+ hamsters, who exhibited impaired glucose tolerance following 6-weeks of FFLC-feeding, Tgr5-/- hamsters did not. This alteration in glucose tolerance was not associated with any significant differences in body weight or food consumption, or hepatic triglyceride or cholesterol between genotypes. Lipoprotein profiling was performed via FPLC analysis. Preliminary results suggest that Tgr5-/- hamsters exhibit reduced VLDL-triglyceride and -cholesterol, and HDL-cholesterol. Finally, chow fed hamsters received intraduodenal injections of deoxycholic acid (DCA), a bile acid with a strong affinity for TGR5. DCA administration was associated with reduced VLDL-triglyceride secretion in Tgr5+/+ hamsters, an effect that was blunted in Tgr5-/- hamsters. The present research will set a framework to expand our knowledge of the role of TGR5 signaling in hepatic lipogenesis. Disclosure D.C.L. Alvares: None. S.S. Hoffman: None. K. Adeli: None. Funding Heart and Stroke Foundation of Canada

1744-P: Hepatic and Disposal Insulin Sensitivity with Single Tracer Method: Use of Naturally Abundant 13C-Polysaccharide

We have recently demonstrated successful estimation of postprandial glucose fluxes using a novel triple tracer technique after ingestion of a mixed meal containing complex carbohydrates (CC) that is naturally enriched with 13C polysaccharide ([13C]-PS). We also reported higher insulin sensitivity (SI) with CC than simple carbohydrate (SC) containing mixed meals. The current analysis was designed to determine if [13C]-PS per se could directly measure hepatic (SIH) and disposal SI (SID) during the CC containing mixed meal. Sixteen healthy subjects (9M, age=33±2 y, BMI=23±1 kg/m2) received, in random order, either SC (labeled with [13C]-glucose) or CC (naturally containing [13C]-PS) mixed meals. All meals were isocaloric and contained 50g carbohydrates with similar macronutrient composition. Plasma glucose, [13C]-glucose and insulin were measured and the Single Tracer Oral Minimal Model applied to estimate SIH and SID. The model well predicted the data. SIH was significantly higher, while SID was numerically but not statistically higher in CC than SC meals (Figure 1). This implies that the higher SI with CC meals is likely due to higher hepatic insulin sensitivity. Taken together, the data suggest that a single, naturally occurring [13C]-PS tracer can measure postprandial SI and its components without the need for the triple tracer method. Disclosure C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. R. Visentin: None. M. Schiavon: None. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. C. Cobelli: None. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. Funding National Institutes of Health (DK085516, DK94331, DK29953); Italian Minister for Education

1752-P: Discovery of Targets for Insulin Resistance by Microfluidic Organ-on-Chip Models

Metabolism of glucose and lipids is essential for nutrient balance and metabolic homeostasis, and is dynamically regulated by adipose tissue and liver. Dysfunction in regulation of these molecules as well as the secreted factors results in metabolic syndrome and insulin resistance (IR), which is a precursor to type 2 diabetes. Studies have shown that as the main site of adaptive thermogenesis, brown adipose tissue (BAT) improves hepatic insulin sensitivity, whereas white adipose tissue (WAT) can induce hepatic IR. However, how these organs communicate to regulate insulin sensitivity is not completely understood due to the lack of representative experimental models. Animals models are useful but suffer from confounding effects from other organs; traditional in vitro models have limited physiological representation. We have developed an organ-on-chip platform to study the interactions between BAT/WAT and liver under different metabolic states. The adipose-liver organ-chip consists of two interconnected microfluidic devices - brown/white adipocytes were cultured in one device and hepatocytes (insulin sensitive and IR) in the other, both under perfusion. We show that (a) adipocytes exhibited higher intracellular lipid and fatty acid secretion and (b) the hepatocytes secreted higher albumin compared to traditional co-cultures. We found that brown adipocytes secretions restored insulin sensitivity, and improved glucose/lipid metabolism in IR hepatocytes. In contrast, white adipocytes worsened insulin sensitivity and glucose/lipid metabolism. IR hepatocytes lowered Akt-phosphorylation and glucose uptake while upregulated adipocyte fatty acids secretion. Through RNA-seq, we have identified adipose genes responsible for improving hepatic IR. We have discovered potential new targets for improving hepatic insulin sensitivity. The novel organ-chip platform represented the cells physiologically and will be useful in the discovery and validation of new therapeutic targets for T2D. Disclosure N. Tanataweethum: None. A. Trang: None. L. Annepureddy: None. J. Mehta: None. R.N. Cohen: None. A. Bhushan: None. Funding DRTC (P30DK020595)

1654-P: Deletion of the Mammalian Indy Homolog (Slc13a5) Improves Hepatic Insulin Sensitivity through Vagal Nerve Signaling

Background: INDY (I’m Not Dead Yet) is a plasma membrane citrate transporter and is highly expressed in liver and brain. In mammals, whole body deletion of the coding gene (mIndy, Slc13a5) increased energy expenditure and protected mice from diet-induced obesity and insulin resistance. Generation of neuronal mIndy-KO (NINKO) mice revealed improved insulin sensitivity (IS) in these mice, mediated through better hepatic IS. Gene expression studies in wildtype C57Bl/6 mice revealed high expression of mIndy in the hypothalamus (HTM), which is the main brain area regulating hepatic glucose metabolism. One mechanisms is the regulation through AMPK. Reduced hypothalamic AMPK phosphorylation is known to reduce hepatic glucose output, probably mediated via vagal nerve signaling. Hypothesis: Hepatic glucose production is regulated via mIndy expression in the HTM. Methods: Hyperinsulinemic-euglycemic clamp studies were performed to assess IS after hepatic vagotomy of NINKO mice. NestinCre+ controls were sham-denervated. Ex vivo brain slices of C57Bl/6 mice and the neuronal hypothalamic cell line CLU183 were acutely stimulated with 1 mM sodium citrate to investigate the effect of citrate on the brain and neurons. Results: Acute stimulation of brain slices and CLU183 cells with citrate decreased cellular respiration (-19.7%, p≤0.01), ATP production (-37.7%, p≤0.01) and increased AMPK phosphorylation (+24.7%, p≤0.001). In line with that, AMPK phosphorylation in the HTM of NINKO mice was significantly reduced (-68.1%, p≤0.05). With hepatic branch vagotomy, no differences in hepatic IS could be observed anymore (suppression of basal hepatic glucose output (%); NestinCre+: 100.0±27.2, NINKO: 99.4±8.8). Conclusion: These data suggest that neuronal mIndy is a critical regulator of glucose homeostasis in mammals, probably regulated via AMPK phosphorylation in the HTM and vagal nerve signaling. Further studies will address the exact mechanisms involved in the effect. Disclosure A. Kurzbach: None. M. Schell: None. D.M. Willmes: None. N. El-Agroudy: None. A. Kleinridders: None. A.L. Birkenfeld: None.

4261 Insulin Sensitizing Effects of Vitamin D Mediated through Reduced Adipose Tissue Inflammation and Fibrosis: Evidence from a Human Randomized Trial and Mice Studies

OBJECTIVES/GOALS: Vitamin D [25(OH)D], known to have anti-inflammatory and anti-fibrotic effects in other tissues, may also impact adipose tissue. We designed parallel studies in humans and rodents to define the effects of vitamin D on adipose tissue inflammation and fibrosis, and on systemic insulin resistance. METHODS/STUDY POPULATION: We performed a randomized, double-blinded placebo-controlled trial to examine the effects of repleting vitamin D at to two levels (to &gt;30 ng/ml and to &gt; 50 ng/ml) in 25(OH)D-deficient (&lt;20 ng/ml), insulin resistant, overweight-to-obese humans (n = 19). A comprehensive study of whole-body insulin action was undertaken with euglycemic stepped hyperinsulinemic clamp studies, both before (1st visit) and after administration of vitamin D or placebo (2nd visit and 3rd visit). Adipose tissue fibrosis and inflammation were quantified by ‘real-time’ rt-PCR and immunofluorescence. To determine whether vitamin D’s effects are mediated through adipocytes, we performed hyperinsulinemic clamp studies and adipose tissue analysis in an adipocyte-specific vitamin D receptor knockout (VDR KO) mouse model. RESULTS/ANTICIPATED RESULTS: 25(OH)D repletion (to &gt;30 ng/ml) was associated with reductions in adipose tissue expression of inflammatory (0.6-0.7-fold decreased expression of TNF-α, IL-6, iNOS and PAI-1) and pro-fibrotic (0.4-0.8-fold decreased expression of TGF-β1, HiF1α, Collagen I, V, VI and MMP7) factors, decreased collagen VI immunofluorescence (p = 0.02) and improved hepatic insulin sensitivity in humans, with suppression of endogenous glucose production (EGP) (1.28 ± 0.20 vs 0.88 ± 0.18 mg/kg/min, p = 0.03). Compared to wild type (WT), VDR KO mice exhibited increased adipose tissue expression of several pro-inflammatory (Tnf-α, iNos, Pai-1, Mcp-1 and F4/80; 4-10 fold) and pro-fibrotic genes (Tgf-β1, Collagen VI, and Tsp1; 2-4 fold), in concert with hepatic insulin resistance (EGP 10 ± 3 vs 3 ± 2 mg/kg/min in WT, p = 0.021). DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these complementary human and rodent studies establish a beneficial role of vitamin D to improve hepatic insulin resistance, likely by restraining adipose tissue inflammation and fibrosis. Thus, normalizing 25(OH)D levels could have metabolic benefits in targeted individuals. CONFLICT OF INTEREST DESCRIPTION: N/A

Intrahepatic fat, irrespective of ethnicity, is associated with reduced endogenous insulin clearance and hepatic insulin resistance in obese youths: A cross-sectional and longitudinal study from the Yale Pediatric NAFLD cohort.

To evaluate whether intrahepatic fat accumulation contributes to impaired insulin clearance and hepatic insulin resistance across different ethnic groups. The intrahepatic fat content (HFF%) was quantified by magnetic resonance imaging in a multi-ethnic cohort of 632 obese youths aged 7-18 years at baseline and after a 2-year follow-up. Insulin secretion rate (ISR), endogenous insulin clearance (EIC) and hepatic insulin resistance index (HIRI) were estimated by modelling glucose, insulin and C-peptide data during 3-hour, 9-point oral glucose tolerance tests. African American youths exhibited the lowest HFF% and a prevalence of non-alcoholic fatty liver disease (NAFLD) less than half of that shown by Caucasians and Hispanics. Furthermore, African Americans had lower EIC and glucose-stimulated ISR, despite similar HIRI and plasma insulin levels, compared with Caucasians and Hispanics. EIC and HIRI were markedly reduced in individuals with NAFLD and declined across group-specific HFF% tertiles in all ethnic groups. Consistently, the HFF% correlated with EIC and HIRI, irrespective of the ethnic background, after adjustment for age, sex, ethnicity, adiposity, waist-hip ratio, pubertal status and plasma glucose levels. An increased HFF% at follow-up was associated with decreased EIC and increased HIRI across all groups. Intrahepatic lipid accumulation is associated with reduced insulin clearance and hepatic insulin sensitivity in obese youths, irrespective of their ethnic background.

Monounsaturated fat rapidly induces hepatic gluconeogenesis and whole-body insulin resistance.

BACKGROUNDWhile saturated fat intake leads to insulin resistance and nonalcoholic fatty liver, Mediterranean-like diets enriched in monounsaturated fatty acids (MUFA) may have beneficial effects. This study examined effects of MUFA on tissue-specific insulin sensitivity and energy metabolism.METHODSA randomized placebo-controlled cross-over study enrolled 16 glucose-tolerant volunteers to receive either oil (OIL, ~1.18 g/kg), rich in MUFA, or vehicle (VCL, water) on 2 occasions. Insulin sensitivity was assessed during preclamp and hyperinsulinemic-euglycemic clamp conditions. Ingestion of 2H2O/acetaminophen was combined with [6,6-2H2]glucose infusion and in vivo 13C/31P/1H/ex vivo 2H-magnet resonance spectroscopy to quantify hepatic glucose and energy fluxes.RESULTSOIL increased plasma triglycerides and oleic acid concentrations by 44% and 66% compared with VCL. Upon OIL intervention, preclamp hepatic and whole-body insulin sensitivity markedly decreased by 28% and 27%, respectively, along with 61% higher rates of hepatic gluconeogenesis and 32% lower rates of net glycogenolysis, while hepatic triglyceride and ATP concentrations did not differ from VCL. During insulin stimulation hepatic and whole-body insulin sensitivity were reduced by 21% and 25%, respectively, after OIL ingestion compared with that in controls.CONCLUSIONA single MUFA-load suffices to induce insulin resistance but affects neither hepatic triglycerides nor energy-rich phosphates. These data indicate that amount of ingested fat, rather than its composition, primarily determines the development of acute insulin resistance.TRIAL REGISTRATIONClinicalTrials.gov NCT01736202.FUNDINGGerman Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, Portugal Foundation for Science and Technology, European Regional Development Fund, and Rede Nacional de Ressonancia Magnética Nuclear.