IMetabolic BioPharma Corporation Research Articles

849-P: Antidiabetic Effects of TLC-3595, a Selective ACC2 Inhibitor, in ZDF Rats

Background: TLC-3595 is a potent (IC50=14nM), selective, small molecule, allosteric inhibitor of acetyl-CoA carboxylase 2 (ACC2). TLC-3595 provides a novel therapeutic approach for type 2 diabetes (T2D), via reduction in intramyocellular lipid (IMCL) content, that is complementary to approved therapies. Reduced IMCL strongly correlates with insulin sensitivity (IS). Here, we demonstrate antidiabetic effects of TLC-3595 in Zucker Diabetic Fatty (ZDF) rats. Methods: IMCL was measured by 1H-MRS. T2D progression and IS by hyperinsulinemic-euglycemic clamp were assessed in 30% diet-restricted ZDF rats treated with TLC-3595 (1.67, 5, or 15mg/kg) or pioglitazone (Pio, 0.75mg/kg) daily for 5-12 weeks. Results: TLC-3595 or an analog caused rapid and sustained IMCL reduction (~50%) in lean and dysmetabolic rodents. TLC-3595 enhanced systemic fatty acid oxidation (increased plasma beta-hydroxybutyrate) and reduced cardiac lipid in ZDF rats. TLC-3595 dose-dependently delayed β-cell failure in ZDF rats and was non-inferior to Pio for reductions in hyperglycemia (15-25% lower glucose AUC) and HbA1c (0.6-1.6% vs. vehicle) (Fig A, B). TLC-3595 improved IS and increased clamp glucose infusion rate by 29%, (Fig C). Conclusion: TLC-3595 reduced IMCL, improved IS, and delayed T2D progression comparable to Pio in ZDF rats. These data provide strong rationale for evaluation of TLC-3595 to treat insulin resistance and T2D. Disclosure A. Vijayakumar: Employee; OrsoBio Inc, Gilead Sciences, Inc. E. Murakami: Employee; OrsoBio. R.S. Huss: Employee; OrsoBio, Inc. Stock/Shareholder; OrsoBio, Inc. N. Sroda: None. A. Shimazaki: Employee; Shionogi & Co. Ltd. Y. Kashiwagi: Employee; Shionogi & Co. Ltd. R.P. Myers: Employee; OrsoBio, Inc. Stock/Shareholder; OrsoBio, Inc. M. Subramanian: Other Relationship; OrsoBio Inc. G.I. Shulman: Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC. Consultant; Novo Nordisk. Other Relationship; iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc. Consultant; DiCerna Pharmaceuticals, Inc. Other Relationship; Equator Therapeutics, Inc., Generian Pharmaceuticals. Stock/Shareholder; Levels Health, Inc. Other Relationship; Fortress Biotech, Inc. Consultant; Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc. Other Relationship; OrsoBio, Aro Biotherapeutics Company.

1558-P: The Mitochondrial Calcium Uniporter Regulates Hepatic Mitochondrial Oxidation and Intracellular Redox In Vivo

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the TCA cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed reduced mitochondrial calcium ([Ca2+]mt; p<0.05) and increased cytosolic calcium ([Ca2+]cyt; p<0.01). Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux (VIDH; p<0.01), α-ketoglutarate dehydrogenase flux (VOGDH; p<0.01), and succinate dehydrogenase flux (VSDH; p<0.05) in vivo, using a novel [13C5]glutamine tracer infusion technique (Q-Flux). Further, we show that rates of [14C16]palmitate oxidation (p<0.01) and intrahepatic lipolysis (p<0.05) were significantly increased in liver slices collected from MCU KO mice compared to WT mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p<0.01). Hepatocellular redox state was also altered with MCU KO, as indicated by a decreased cytosolic redox ratio ([lactate]:[pyruvate]; p<0.05) and increased mitochondrial redox ratio ([β-hydroxybutyrate]:[acetoacetate]; p<0.01). Conclusion: Here we show that increases in [Ca2+]cyt potently increase hepatic mitochondrial fat oxidation and alters hepatic redox balance to reduce hepatic lipid content independently of [Ca2+]mt in vivo and targeting [Ca2+]cyt may represent a novel therapeutic target to treat NAFLD. Disclosure T.E.Lamoia: None. B.T.Hubbard: None. M.Guerra: None. R.Goodman: None. M.H.Nathanson: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. Funding National Institutes of Health (F31DK126362, T32GM007324)

1510-P: Lipid-Induced Renal Cortical Insulin Resistance Perturbs Gluconeogenic and Oxidative Metabolism via an sn-1,2-diacylglycerol-PKCe-Insulin Receptor Kinase Axis In Vivo

Recently, our group demonstrated that 3-week high fat diet feeding (HFD) impairs insulin signaling in the renal cortex at the level of the insulin receptor kinase (IRK). Specifically, IRKY1162 and AKTS473 phosphorylation were reduced by ~50% during hyperinsulinemic-euglycemic clamp (HEC); however, it is unknown how renal cortical insulin resistance (IR) may affect gluconeogenic (GNG) and oxidative metabolism. To address this, here we measure renal cortical GNG fluxes in vivo using Q-Flux. In regular chow (RC) fed mice, hyperinsulinemia significantly suppressed mitochondrial pyruvate carboxylase (PC), as PC flux relative to α-ketoglutarate dehydrogenase flux (VPC/VOGDH) was 1.25 under basal conditions, but decreased by ~50%, to 0.57 during HEC. Similarly, PEPCK flux relative OGDH flux (VPEPCK/VOGDH) was reduced by ~60% during HEC. In contrast, insulin failed to suppress both PC and PEPCK fluxes with HFD. Using a [13C6]glucose tracer, we also find insulin-stimulated mitochondrial pyruvate oxidation to be impaired by ~30% with HFD. Mechanistically, we previously found HFD led to a 2-fold increase in plasma membrane (PM) sn-1,2-diacylglycerol (DAG) and a 4-fold increase in Protein Kinase Cε (PKCε) translocation, suggesting renal cortical IR may be mediated by an sn-1,2-DAG-PKCε-IRK axis. We further interrogated this possibility here by assessing inhibitory phosphorylation at IRKT1150, the key target of PKCε, finding a 42% increase with HFD. In addition, IRKT1150A knockin mice, whose IRK cannot be phosphorylated by PKCε, were protected from HFD-induced signaling and pyruvate oxidation defects. Conclusion: HFD inhibits renal cortical insulin signaling at the level of the IRK, which is associated with perturbed mitochondrial GNG and oxidative metabolism. IRKT1150A mice are protected from HFD-induced defects, indicating a PM sn-1,2-DAG-PKCε-pIRKT1150 mechanism for lipid-induced renal cortical IR. Disclosure B.T.Hubbard: None. R.C.Gaspar: None. D.Zhang: None. M.Kahn: None. A.Nasiri: Other Relationship; Medtronic. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.

192-OR: Lipid-Induced Insulin Resistance in Brown Adipose Tissue Is Mediated by the sn-1,2 DAG-PKCe-IRKT1150 Phosphorylation Pathway

While there is substantial evidence that brown adipose tissue (BAT) is insulin-responsive, it remains unclear whether BAT becomes insulin resistant with high fat diet (HFD) feeding. Here, we examined the hypothesis that the sn-1,2-diacylglycerol (DAG)/Protein Kinase C epsilon (PKCε)/insulin receptor kinase (IRK)T1150 phosphorylation pathway may trigger HFD-induced BAT insulin resistance. To address this hypothesis we first studied two groups of male Sprague Dawley rats fed either a regular chow diet (RC) or a HFD. HFD-fed rats manifested BAT insulin resistance as reflected by reduced insulin-stimulated phosphorylation of IRKT1158 (p=0.03), AKTT308 (p=0.003), PDHS232 (p=0.0002), and PDHS300 (p=0.0004) compared to RC rats during a hyperinsulinemic-euglycemic clamp (HEC). These reductions in insulin signaling occured at the level of the IRK and were associated with increased sn-1,2 diacylglycerols (p=0.03) and increased PKCε translocation (p<0.004). These defects in insulin signaling were also associated with a 35% (p<0.009) reduction in insulin-stimulated BAT mitochondrial pyruvate dehydrogenase/citrate synthase flux (VPDH/VCS) as assessed by LC-MS/MS and GC-MS measurements of C4C5 glutamate (m+2)/pyruvate (m+3) during a HEC where [13C6]glucose was the infusate. In order to assess the role of PKCε-induced IRKT1150 phosphorylation in this process we next studied the effects of HFD feeding in: 1) wild type mice fed a regular chow diet (WT), 2) wild type mice fed a HFD (WT-HFD) and 3) IRKT1150A knockin mice fed a HFD (IRKT1150A-HFD) and found that IRKT1150A-HFD mice have preserved levels of insulin-stimulated AKTS473 phosphorylation compared to WT-HFD mice (p=0.04). Conclusion: These results demonstrate a critical role of the sn-1,2-DAG-PKCε-IRKT1150 phosphorylation pathway in mediating HFD-induced insulin resistance and mitochondrial dysfunction in BAT. Disclosure R.C.Gaspar: None. B.T.Hubbard: None. I.Sakuma: None. T.E.Lamoia: None. D.Zhang: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.

1569-P: Lysophosphatidic Acid Mediates Inflammation in Liver and White Adipose Tissue in a Rat Model of 1-acyl-sn-glycerol-3-phosphate Acyltransferase 2 Deficiency

AGPAT2 (1-acyl-sn-glycerol-3-phosphate acyltransferase 2) converts lysophosphatidic acid (LPA) into phosphatidic acid (PA) and mutations of the AGPAT2 gene are responsible for the most common form of congenital generalized lipodystrophy (CGL) which leads to diabetes and steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to these abnormalities has not been elucidated. We explored this question using an antisense oligonucleotide (ASO) to knock down expression of AGPAT2 in liver and white adipose tissue (WAT) of adult male SD rats. AGPAT2 ASO treatment induced lipodystrophic features in which adipose and hepatic inflammation were associated with increased LPA content in WAT (Control ASO=0.4 ± 0.1 nmol/g tissue; AGPAT2 ASO=0.8 ± 0.4, P<0.05) and liver (Control ASO=2.7 ± 0.4 nmol/g tissue; AGPAT2 ASO=4.2 ± 0.9, P<0.01) whereas PA content was unchanged. We hypothesized that increased LPA content may lead to inflammation in these tissues. We tested if either a controlled-release mitochondrial protonophore (CRMP) or an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) would reduce LPA content and inflammation in these tissues in AGPAT2 ASO treated rats. CRMP treatment (20 mg/[kg-day]) primarily prevented LPA accumulation in WAT (Control ASO=0.4 ± 0.1 nmol/g tissue; AGPAT2 ASO=0.9 ± 0.1; AGPAT2 ASO+CRMP=0.6 ± 0.1) (AGPAT2 ASO vs AGPAT2+CRMP P<0.05) and prevented WAT inflammation. In contrast GPAM ASO treatment primarily prevented hepatic LPA accumulation (Control ASO=3.6 ± 0.4 nmol/g tissue; AGPAT2 ASO=4.8 ± 0.2; AGPAT2+GPAM ASO=2.7 ± 0.4) (AGPAT2 ASO vs AGPAT2+GPAM ASO P<0.01) and prevented hepatic inflammation. Conclusion: These results demonstrate a key role of LPA acid in mediating inflammation in liver and WAT in AGPAT2 deficient animals and identify novel therapeutic targets to treat CGL in patients with AGPAT2 deficiency. Disclosure I.Sakuma: None. R.C.Gaspar: None. P.Luukkonen: None. M.Kahn: None. S.Murray: Employee; Ionis Pharmaceuticals. V.Samuel: None. K.Petersen: Advisory Panel; Novo Nordisk, Research Support; Merck Sharp & Dohme Corp., Gilead Sciences, Inc. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc.

191-OR: Deletion of the Type 2 Diabetes Candidate Gene SLC16A11 Reduces Peripheral Insulin Sensitivity in Mice

Background and Aims: Recent GWAS in large human populations identified the monocarboxylate transporter SLC16A11 as a candidate gene for T2D. While previous studies postulated that SLC16A11 plays a role in hepatic lipid and glucose metabolism, the existing data are controversial and exact mechanisms of how SLC16A11 affects T2D are missing. Here, we aimed at elucidating the role of SLC16A11 in the development of metabolic disease. Methods: HEK293 cell lines expressing murine and human SLC16A11 were established. Moreover, using CRISPR/Cas9, a SLC16A11 knockout mouse model was generated. Finally, SLC16A11 mRNA expression was measured in various insulin responsive tissues of ob/ob and db/db mice. Results: In HEK293 cells, substrates of the transporter were identified. mRNA expression of SLC16A11 was reduced in skeletal muscle of ob/ob and db/db mice (-69.8% and -22.8%; p<0.05) compared to littermate controls. SLC16A11 KO and WT littermate mice fed NCD or HFD did not differ in body weight and composition, energy expenditure or food intake. While basal glucose and insulin in SLC16A11 KO and WT littermates on a HFD were not different, glucose infusion rate during hyperinsulinemic euglycemic clamp was lower in SLC16A11 KO mice (SLC16A11 WT= 36.4±3 mg/kg/min; SLC16A11 KO= 24.7±2 mg/kg/min; P<0.01), indicating reduced whole-body insulin sensitivity. No changes were observed in hepatic glucose production, liver fat content or hepatic mitochondrial function using PINTA method. Instead, clamped 2-deoxy-glucose uptake was significantly reduced in skeletal muscle (-41%; p<0.05) of SLC16A11 KO, together with increased DAGs levels and PKCθ activation, indicating skeletal muscle insulin resistance. Conclusions: Our data show for the first time that in contrast to previous assumptions, SLC16A11 impairs skeletal muscle insulin sensitivity without affecting hepatic metabolism. Taking also human GWAS into account, we propose that SLC16A11 may be an interesting target for the treatment of T2D. Disclosure N.N.El-agroudy: None. T.Schumann: None. A.Kurzbach: None. G.Sancar: None. L.Sandforth: None. C.Herrmann: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. A.L.Birkenfeld: None.

222-OR: Metformin Reduces Fasting Glycemia in Well-Controlled Type 2 Diabetes by Promoting Aerobic Glycolysis Independent of Decreasing Endogenous Glucose Production

It is well established that metformin improves glycemic control in individuals with poorly-controlled type 2 diabetes (T2D) by reducing rates of endogenous glucose production (EGP) mediated by inhibition of hepatic gluconeogenesis. However, the mechanism by which metformin reduces blood glucose levels in well-controlled individuals with T2D is still unknown. To address this question, we examined rates of EGP, lactate turnover and hepatic triglyceride (TAG), ATP and glycogen content in near-normoglycemic individuals with T2D (n=10, HbA1c 7±1%) with (M+; 1000 mg bid x 2 wks) and without (M-; 2 wks) metformin treatment using the PINTA method with [2H7]glucose and [3-13C]lactate tracer infusions in combination with 1H- /31P- /13C- magnetic resonance spectroscopy. Metformin treatment did not affect rates of EGP, but reduced fasting plasma glucose concentrations by 30% (p<0.01 vs. M-), which could be attributed to increased rates of glucose clearance (+18%, p<0.005 vs. M-) resulting in increased lactate production (+14%, p<0.01 vs. M-). These changes were associated with decreased hepatic ATP content (-20%, p<0.05 vs. M-), increased hepatic glycogen content (+40%, p<0.05 vs. M-) and no changes in hepatic TAG content. Conclusion: In contrast to the glucose-lowering effect of metformin in poorly-controlled T2D, which is due to reductions in gluconeogenesis, this study demonstrates that in well-controlled T2D metformin reduces fasting glycemia - independent of reductions in EGP - primarily by increasing rates of aerobic glycolysis as reflected by increased peripheral glucose clearance and lactate production as well as reduced hepatic ATP content, which is consistent with metformin-induced inhibition of mitochondrial electron transport chain activity. Disclosure T.Sarabhai: None. T.E.Lamoia: None. S.Friesl: None. M.Jonuscheit: None. K.Petersen: Advisory Panel; Novo Nordisk, Research Support; Merck Sharp & Dohme Corp., Gilead Sciences, Inc. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. M.Roden: Advisory Panel; Eli Lilly and Company, Consultant; TARGET PharmaSolutions, Inc., Research Support; Boehringer-Ingelheim, Novo Nordisk, Novartis, Sanofi.

122-LB: Effect of Dapagliflozin on Mitochondrial Metabolism and Cardiac Function in the Failing Heart

Several studies highlight the potential for SGLT2 inhibitors (i) to exert their cardioprotective effects by shifting myocardial fuel utilization towards ketone oxidation. However, direct in vivo measurements of cardiac mitochondrial substrate oxidation in awake rodents during heart failure are lacking, and whether SGLT2i-mediated increases in ketone availability and oxidation are able to improve cardiac efficiency remains unclear. Here, we examined the effect of acute SGLT2 inhibition with dapagliflozin (dapa) on cardiac function and mitochondrial metabolism in awake male Sprague Dawley rats 2 weeks after induction of heart failure by permanent ligation of the left anterior descending coronary artery. Echocardiography and in vivo metabolic flux studies were performed 4-6 h after oral dapa (1.5 mg/kg) or vehicle treatment in sham-surgerized rats and rats with myocardial infarction (MI) . Acute dapa treatment caused a 15% decrease in fasting plasma glucose concentrations and 50% increase in fasting plasma NEFAs, whole-body βOHB turnover and plasma βOHB levels in sham and MI rats (all P<0.05 vs. VEH) . Dapa treatment decreased left ventricular (LV) [14C]deoxyglucose uptake and relative rates of pyruvate oxidation to total mitochondrial oxidation (VPDH/VCS) by 40-60%. Dapa treatment also caused a 60% increase in relative rates of LV ketone oxidation to total mitochondrial oxidation (VβOHB/VCS) in sham and MI rats (all P<0.05 vs. VEH) , independently of changes in cardiac metabolic gene expression. Interestingly, the dramatic shift in substrate utilization caused by acute dapa treatment did not significantly alter cardiac output or LV ejection fraction. Acute dapa treatment also did not significantly alter myocardial acetyl-CoA content, malonyl-CoA content, ATP/ADP ratios or ATP/AMP ratios in the LV myocardium remote from the infarct. Conclusion: Taken together, these results demonstrate that single-dose dapa treatment, which acutely decreased LV myocardial pyruvate oxidation and increased LV ketone oxidation, was not sufficient to alter cardiac output or LV ejection fraction. Disclosure L. Goedeke: Other Relationship; The Liver Company, Inc. . G. I. Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc., Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . Y. Ma: None. J. Zhang: n/a. N. Guerrera: None. X. Wu: None. D. Zhang: None. M. Kahn: None. X. Zhang: None. L. H. Young: None. Funding NIH (P30 DK045735 and K99 HL150234) , AstraZeneca (NCR-19-20028)

219-LB: The Mitochondrial Calcium Uniporter Regulates Hepatic Lipid Accumulation and Mitochondrial Oxidation

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the tricarboxylic acid (TCA) cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed significantly reduced mitochondrial calcium ([Ca2+]mt, p<0.05) and increased cytosolic calcium ([Ca2+]cyt, p<0.01) in isolated hepatocytes. Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU resulted in increased rates of isocitrate dehydrogenase flux (VIDH, p<0.01) , α-ketoglutarate dehydrogenase flux (VOGDH, p<0.01) , and succinate dehydrogenase flux (VSDH, p<0.05) , using a novel in vivo [13C5]glutamine tracer technique. Further, we report that rates of [14C16]palmitate oxidation (p<0.01) and intrahepatic lipolysis (p<0.05) were significantly increased in precision-cut liver slices collected from MCU KO mice compared to WT, consistent with [Ca2+]cyt activation of CAMKII (p<0.01) and ATGL (p<0.001) in MCU KO mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p<0.01) . Taken together, these data demonstrate that hepatic mitochondrial activity can be dissociated from mitochondrial calcium concentrations and reveal a key role for cytosolic calcium and mitochondrial anaplerosis in the regulation of hepatic mitochondrial fat oxidation. Disclosure T. E. Lamoia: n/a. B. T. Hubbard: None. M. Guerra: None. R. Goodman: None. M. H. Nathanson: None. V. Mootha: Advisory Panel; 5AM Ventures, LLC, Janssen Research & Development, LLC. G. I. Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc. , Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . Funding F31 DK126362/DK/NIDDK NIH HHST32 GM007324/GM/NIGMS NIH HHS

195-OR: A Novel 13C5 Glutamine Tracer Method (Q Flux) Reveals a Key Role of Succinyl CoA Anaplerosis in Promoting Increased Rates of Hepatic Gluconeogenesis during Hyperglucagonemia

Hyperglucagonemia is a hallmark of type 2 diabetes mellitus (T2DM) and contributes to increased rates of endogenous glucose production (EGP) , mostly through increased rates of hepatic gluconeogenesis (GNG) , but the anaplerotic pathways and respective substrates that support this process are unclear. To address this question, we developed a novel and extensively validated 13C5 glutamine-based in vivo metabolic flux analysis method (Q Flux) , which allows for quantitation of both the relative and absolute rates of hepatic gluconeogenesis from pyruvate (via pyruvate carboxylase [PC]) , succinyl CoA (via methylmalonyl CoA mutase [MUT]) , glutamine (via glutaminase [GLS2]) , and glycerol. Male Sprague-Dawley rats (∼300 g) were infused for 90 minutes with either low-dose (LD) glucagon (1.25 ng/[kg-min]) or high-dose (HD) glucagon (ng/[kg-min]) , plus insulin (0.5 mU/[kg-min]) , somatostatin (4 μg/[kg-min]) , and 13C5 glutamine (3 μmol/[kg-min]) after a 16-hour fast to deplete hepatic glycogen. HD animals displayed significantly increased plasma glucose concentrations and EGP relative to LD controls. Analyses of liver tissues collected at sacrifice revealed that in LD animals, 70% of mitochondrial GNG was from pyruvate, 15% from succinyl CoA, and 15% from glutamine. In HD animals, 59% of mitochondrial GNG was derived from pyruvate (p=0.vs. LD) , 26% from succinyl CoA (p=0.0vs. LD) , and 15% from glutamine (p=0.99 vs. LD) . Conclusion: We find an unexpected role for succinyl CoA anaplerosis and methylmalonyl CoA mutase flux in supporting increased rates of mitochondrial GNG during hyperglucagonemia. Furthermore, relative contributions of glutamine to hepatic gluconeogenesis were unchanged, contrary to previous reports. Taken together, these results identify novel targets to reduce hyperglucagonemia-induced increases in rates of gluconeogenesis and hyperglycemia in T2DM. Disclosure B.T.Hubbard: None. G.I.Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc. , Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. .

1336-P: FoxO1-ATGL-Sirt1-FoxO1 Feed Forward Signaling Mediates Effects on Hepatic Gene Expression and Glucose Homeostasis in LIRKO Mice

FoxO1 is a major target of insulin action and regulates expression of ATGL and its inhibitor, G0S2, in the liver (Cell Rep 15:349, 2016) . Here, we asked whether ATGL mediates effects of FoxO1 in liver-specific insulin receptor knockout (LIRKO) mice. Studies in LIRKO and IR/FoxO1 double knockout (LIRFKO) mice show ATGL expression is increased and G0S2 is suppressed in a FoxO1-dependent fashion when insulin signaling is disrupted in the liver. ATGL knockdown (KD) reduces high plasma glucose and insulin concentrations, HOMA-IR and fatty acid oxidation, and improves glucose tolerance in LIRKO mice. Hyperinsulinemic-euglycemic clamp studies show ATGL KD improves insulin sensitivity due to effects on both HGP and glucose utilization, reflecting changes in WAT lipolysis, BAT metabolism and hepatic gene expression. The expression of FoxO1-regulated gluconeogenic genes (PEPCK, G6Pase, PCO, PGC-1α) , hepatokines (e.g., follistatin) and IGFBP-1 is increased in LIRKO mice and reduced by ATGL KD, reflecting changes in acetylation of FoxO1 and its co-activator, PGC-1α (decreased in LIRKO, increased by ATGL KD) . Rictor acetylation, which promotes activation of Akt and phosphorylation of FoxO1, also is increased by ATGL KD in LIRKO mice and isolated hepatocytes. Since ATGL promotes activation of Sirt1 (Mol Cell 77:810, 2020) and Sirt1 deacetylates FoxO1, PGC-1α and rictor, these studies suggest FoxO1 promotes increased ATGL and Sirt1 activity, and that Sirt1 activation enhances the effects of FoxO1 on gene expression and glucose homeostasis in a feed forward fashion (FoxO1 -> ATGL -> Sirt1 -> FoxO1) . Targeting the FoxO1/ATGL/Sirt1 pathway may provide a useful strategy for improving glucose homeostasis when insulin signaling is impaired in the liver. Disclosure I.O-sullivan: None. S.Bhattacharyya: None. C.Liew: None. S.M.Lee: None. J.Cordoba-chacon: None. R.J.Perry: None. G.I.Shulman: Advisory Panel; 89bio, Inc., AstraZeneca, Equator Therapeutics, Inc., Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; DiCerna Pharmaceuticals, Inc., Novo Nordisk, Other Relationship; Generian Pharmaceuticals, iMetabolic Biopharma Corporation, Maze Therapeutics, The Liver Company, Stock/Shareholder; Levels Health, Inc. . T.Unterman: None. Funding Department of Veterans Affairs (5I01BX001968)

282-OR: The Effect of Glucagon on Rates of Hepatic Mitochondrial Oxidation and Pyruvate Carboxylase Flux in Man Assessed by Positional Isotopomer NMR Tracer Analysis (PINTA)

Dual GLP-1/glucagon agonists are now in clinical trials for the treatment of T2D and NASH. In addition to promoting further reduction in food intake on top of GLP-1, the glucagon component of these dual agonists may also promote increased rates of hepatic mitochondrial fat oxidation. In order to investigate this possibility we assessed the glucagon-specific effects of a dual agonist on rates of hepatic mitochondrial oxidation (VCS) and pyruvate carboxylase flux (VPC) in humans by infusing glucagon to achieve a physiological increase in plasma glucagon concentrations in combination with a novel 13C PINTA method to assess rates of hepatic VCS and VPC in 15 healthy volunteers using a paired study design. Following an overnight fast a 3-h IV infusion of [3-13C]lactate (4.3 μmol/Kg-min) and [1,2,3,4,5,6,6-2H7]glucose (0.22 μmol/Kg-min) was begun and during the last 2 hours glucagon (GLG) was co-infused [6 ng/(Kg-min)] or no GLG (CON). The GLG infusion resulted in a ~2.4 fold increase in plasma GLG conc. (from 75±11 to 183±20 pg/mL; P<0.0001), which was associated with an ~20% increase in both plasma C-peptide (from 1.89±0.17 to 2.30±0.26 ng/mL; P=0.02) and plasma insulin conc. (from 9.9±2.0 to 12.0±2.4 μU/mL; P=0.06). Plasma glucose conc. increased from 4.90±0.16 to 5.64±0.19 mmol/L (P=0.0001) which was associated with a tendency for increased rates of glucose production (CON:1042±123 vs. GLG:1148±122 μmol/min; p=0.086) despite no change in rates of VPC flux (CON: 403±69 vs. GLG: 373±55 μmol/min; p=NS). In contrast, this physiological increase in plasma GLG concentrations caused an 85% increase in rates of hepatic VCS (P<0.05). Conclusion: Taken together these studies demonstrate that a physiological increase in plasma glucagon promotes increased rates of hepatic mitochondrial oxidation in humans, which may provide additional benefits to the anorexic effects of dual GLP-1/glucagon agonists to reduce hepatic steatosis. Disclosure K. Petersen: Advisory Panel; Spouse/Partner; AstraZeneca, iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; Spouse/Partner; Aegerion Pharmaceuticals Inc., Novo Nordisk, Research Support; Self; Gilead Sciences, Inc., Merck & Co., Inc. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company. Funding Merck & Co., Inc. (MISP583402); National Institutes of Health (R01DK113984)

501-P: Lower Plasma Membrane Sn-1,2-Diacylglycerol Content and PKCepsilon/theta Activity Explain the Athlete’s Paradox

Increases in intramyocellular lipids (IMCL) are strongly associated with muscle insulin resistance. An important exception to this observation occurs in athletes, who manifest increased IMCL content despite increased muscle insulin sensitivity. This phenomenon has been coined the “Athlete’s Paradox” and is not well understood. Here, we examined the hypothesis that the Athlete’s Paradox can be explained by lower plasma membrane (PM) associated sn-1,2-diacylglycerol (DAG) content leading to lower PKCε and PKCθ translocation in skeletal muscle despite increased muscle triglyceride (TAG) content. To address this hypothesis we studied 3 groups of male C57BL/6J mice following 6 weeks of: 1) Regular chow feeding (RC), 2) High-fat diet feeding (HFD); 3) RC-feeding and running wheel exercise (EX). DAG stereoisomers were assessed in five subcellular compartments [PM, endoplasmic reticulum (ER), mitochondria (Mito), and lipid droplet (LD)] using a novel liquid chromatography-tandem mass spectrometry/cellular fractionation method. Consistent with the Athlete’s Paradox we found that EX mice manifested improved glucose tolerance and muscle insulin sensitivity compared to HFD mice, as assessed by glucose tolerance and hyperinsulinemic-euglycemic clamp studies, despite similar increases in muscle TAG content (RC=5.7 ± 3.2 µg/mg; HFD=17.8 ± 6.0 µg/mg; EX=13.7 ± 5.2 µg/mg) compared to RC mice (RC vs. HFD P<0.01, RC vs. EX P<0.01 and HFD vs. EX P=NS). However, in contrast to the EX and RC mice the HFD group had a 2-fold increase in PM sn-1,2-DAG content compared to the RC and EX mice (both P<0.01), which was associated with a 2-3 fold increase in PKCε and PKCθ translocation (both P<0.05 compared to HFD) and reduced insulin-stimulated InsrY1162/AKT473 phosphorylation and glucose transport (all P<0.01 vs. HFD). Conclusion: Lower PM sn-1,2-DAG content and PKCε/PKCθ activity provides an explanation for the preserved insulin sensitivity in EX mice and thus the Athlete’s Paradox. Disclosure R. C. Gaspar: None. M. Kahn: None. G. Cline: None. J. R. Pauli: None. R. J. Perry: None. K. Petersen: Advisory Panel; Spouse/Partner; AstraZeneca, iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., Consultant; Spouse/Partner; Aegerion Pharmaceuticals Inc., Novo Nordisk, Research Support; Self; Gilead Sciences, Inc., Merck & Co., Inc. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company. K. Lyu: None. B. T. Hubbard: None. B. Leitner: None. P. Luukkonen: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Research Support; Self; Novo Nordisk Foundation. S. Hirabara: None. I. Sakuma: None. A. Nasiri: Employee; Spouse/Partner; Medtronic. D. Zhang: None. Funding São Paulo Research Foundation (2019/11338-9, 2017/20542-3); National Institutes of Health (R01DK116774, P30DK045735)

323-OR: SGLT2 Inhibition Promotes Myocardial Ketone Utilization in the Normal and Failing Heart

Recent clinical outcome studies demonstrate that SGLT2 inhibitors (i) significantly reduce major adverse cardiovascular events and heart failure in patients with and without type 2 diabetes; however, the mechanisms by which SGLT2i exert their cardiovascular benefits remain unclear. Here, we aimed to elucidate the acute effects of SGLT2i (dapagliflozin, dapa) on cardiac mitochondrial substrate oxidation. Using LC-MS/MS methodology combined with an infusion of [13C6]glucose and [13C4]βOHB, we assessed the ratio of cardiac mitochondrial pyruvate (VPDH) and βOHB (VβOHB) oxidation rates to rates of mitochondrial citrate synthase flux (VCS) in awake male Sprague Dawley (SD) rats. Metabolic studies were performed 4-6 h after oral dapa (1.5 mg/kg) or vehicle treatment in control rats and after induction of heart failure induced by permanent ligation of the left anterior descending coronary artery. Acute dapa treatment led to marked glycosuria with a 15% reduction in fasting plasma glucose concentrations and 50% increase in whole-body βOHB turnover and plasma βOHB concentrations (all P<0.05 vs. VEH). Dapa caused a 40-60% decrease in myocardial [14C]deoxyglucose uptake and pyruvate oxidation (VPDH/VCS) and a 60% increase in myocardial ketone oxidation (VβOHB/VCS) (all P<0.05 vs. VEH). Similar effects were observed in heart failure rats. Dapa treatment caused a 15% decrease in fasting plasma glucose concentrations, 50% increase in plasma βOHB levels and 60% increase in left ventricular VβOHB/VCS (all P<0.05 vs. VEH). Conclusion: Collectively, these studies demonstrate that dapa shifts both the normal and failing heart to increased mitochondrial ketone oxidation, which may be mediated by increased hepatic ketogenesis and βOHB availability. Disclosure L. Goedeke: None. X. Zhang: None. R. J. Perry: None. G. Cline: None. L. H. Young: None. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company. J. Lee: None. Y. Ma: None. X. Hu: None. J. Zhang: n/a. J. Dong: None. K. D. Galsgaard: None. N. Guerrera: None. S. Haedersdal: None. Funding National Institutes of Health (R01DK113984, R01DK045735, K99HL150234); AstraZeneca

335-OR: Lipid-Induced Insulin Resistance in the Renal Cortex Is Associated with Plasma Membrane Sn-1,2-diacylglycerol Accumulation and PKCe Translocation

While there is substantial evidence that the kidney is insulin responsive, it is unclear whether the organ becomes insulin resistant. Here, we investigate whether the kidney is susceptible to lipid-induced insulin resistance and, if so, whether insulin resistance may be mediated by increases in plasma membrane sn-1,2-diacylglycerol (DAG) content and activation of Protein Kinase Cε (PKCε). We studied 14 week old C57BL/6J mice fed either regular chow (RC) or a high fat diet (HFD) for three weeks. Infusion of [13C6]glucose under hyperinsulinemic-euglycemic clamp (HEC) conditions revealed HFD fed mice to have an ~40% reduction (P<0.01) in renal cortical mitochondrial pyruvate dehydrogenase flux relative to citrate synthase flux (VPDH/VCS) compared to RC mice. Next, we infused RC mice with a lipid emulsion (Intralipid® 20%) (RC+IL) during HEC. Despite matched plasma NEFAs at the clamp conclusion, renal cortical VPDH/VCS was significantly higher in RC+IL mice than in HFD mice (P<0.02). These findings suggest that reductions in insulin-stimulated mitochondrial VPDH/VCS observed in HFD mice are intrinsic to the kidney and cannot be attributed to alterations in fatty acid flux to the kidney. Consistent with our mitochondrial flux data, we found that feeding a HFD induced a defect in insulin signaling at the level of the insulin receptor (INSR) as evidenced by ~50% reductions in both insulin-stimulated INSRY1162 (P<0.05) and AKTS473 phosphorylation (P<0.001). HFD-induced reductions in insulin signaling were associated with an ~2-fold increase (P<0.01) in plasma membrane sn-1,2-DAG content, which was associated with an ~4-fold increase (P<0.05) in PKCε translocation. Conclusion: HFD feeding induces insulin resistance in the renal cortex at the level of INSRY1162 phosphorylation. This defect in INSR signaling is associated with reduced insulin-stimulated pyruvate oxidation and may be mediated by a sn-1,2-DAG-PKCε-INSR mechanism. Disclosure B. T. Hubbard: None. R. C. Gaspar: None. D. Zhang: None. M. Kahn: None. A. Nasiri: Employee; Spouse/Partner; Medtronic. X. Zhang: None. G. Cline: None. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company.

281-OR: Endothelial Cell Cd36 Regulates Systemic Glucose and Lipid Metabolism

The transmembrane protein, cluster of differentiation 36 (CD36), facilitates tissue fatty acid uptake and is highly expressed in a variety of different cell types, including endothelial cells (ECs). Loss of EC Cd36 reduces parenchymal long-chain fatty acid uptake and improves whole-body glucose tolerance and insulin sensitivity; however, the mechanisms by which this occurs remain unclear. Here, we report that EC-specific deletion of Cd36 in chow-fed male mice (EC-Cd36-/-) leads to significant reductions in whole-body fat utilization during fasting, subsequently increasing plasma non-esterified fatty acid levels by 30% (P<0.05 vs. Cd36fl/fl). We performed hyperinsulinemic-euglycemic clamp studies, using [13C6]glucose as the infusate, and found that loss of EC Cd36 improved whole-body insulin sensitivity by 15% (P<0.05 vs. Cd36fl/fl) due to a 40-50% increase in insulin-stimulated glucose uptake in heart and brown adipose tissue (both P<0.05 vs. Cd36fl/fl). Consistent with enhanced rates of glucose transport, EC-Cd36-/- mice displayed a significant 20% increase in relative rates of myocardial pyruvate dehydrogenase oxidation to citrate synthase flux (VPDH/VCS) compared to Cd36fl/fl controls; in contrast, mitochondrial VPDH/VCS was unaffected in liver and skeletal muscle. Interestingly, improvements in whole-body insulin sensitivity due to loss of EC Cd36 was also associated with 20% reductions in fasting plasma glucose concentrations and hepatic glucose production (both P<0.05 vs. Cd36fl/fl), which could be attributed to a significant 20% decrease in hepatic acetyl-CoA content. Conclusion: Taken together, these results demonstrate a key role for EC Cd36 in regulating parenchymal fuel selection and hepatic glucose production and provide insight into the mechanisms by which EC Cd36 controls systemic glucose and lipid metabolism. Disclosure L. Goedeke: None. N. Son: None. T. E. Lamoia: None. A. Nasiri: Employee; Spouse/Partner; Medtronic. M. Kahn: None. X. Zhang: None. G. Cline: None. I. J. Goldberg: Advisory Panel; Self; Akcea Therapeutics, Arrowhead Pharmaceuticals, Inc., Consultant; Self; Esperion. G. I. Shulman: Consultant; Self; 89bio, Inc., BridgeBio, Ionis Pharmaceuticals, Maze Therapeutics, Novo Nordisk, Other Relationship; Self; AstraZeneca, Esperion Therapeutics, Inc, Generian Pharmaceuticals, Inc., Gilead Sciences, Inc., iMetabolic Biopharma Corporation, Janssen Research & Development, LLC, Merck & Co., Inc., The Liver Company. Funding National Institutes of Health (HL150234, R01DK113984, R01DK045735)

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<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<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 & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & 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.)

220-LB: Glucagon Promotes Hepatic Autophagy by AMPK-Mediated mTORC1 Inhibition

Glucagon plays an important role in hepatic glucose metabolism as well as protein/amino acid metabolism but detailed knowledge about glucagon’s role in hepatic autophagy is limited. We hypothesized that glucagon regulate hepatic protein metabolism partly through activation of AMP-kinase (AMPK) that in turn lead to inhibition of mammalian target of rapamycin complex1 (mTORC1) and activation of Uncoordinated-51 like autophagy activating kinase-1 (ULK1) resulting in increased autophagy. In order to test this hypothesis, we infused chronically catheterized awake mice with [13C5]-glutamine [0.225 mg/(kg-min)] for 120 min after which a second line was connected infusing somatostatin [4 µg/(kg-min)] and insulin [0.1 mU/(kg-min)], with (n=7) or without glucagon [10 ng/(kg-min)] (n=6) for 90 min. This raised plasma glucagon concentration ~18-fold compared to control. Glucagon infusion increased phosphorylation of hepatic: AMPKThr172, RaptorSer792, ULK1Ser555 (P=0.03, P=0.03, and P=0.06, respectively, compared to control), and resulted in hepatic Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II accumulation (P=0.02), a marker of autophagy, assessed by immunoblotting. Glucagon treatment also increased the 13C enrichment (m+3) in liver glucose (P<0.0001), indicating increased conversion of [13C5]-glutamine to glucose as well as increased plasma urea concentrations (P=0.04). Consistent with these in vivo results, parallel studies performed in liver slices obtained from overnight fasted male rats (n=6) incubated in Krebs-Henseleit bicarbonate buffer supplemented with [3-13C]-alanine (1 mM), glucagon (250 µM) treatment increased: glucose production (P=0.01), urea production (P=0.0005), and intracellular levels of leucine (P=0.045). In conclusion, our data suggest that glucagon promotes hepatic proteolysis and ureagenesis by AMPK-mediated suppression of mTOC1 activity, leading to increased hepatic autophagy. Disclosure K.D. Galsgaard: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & 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. K. Petersen: Advisory Panel; Spouse/Partner; Merck Sharp & Dohme Corp. Research Support; Self; Merck Sharp & Dohme Corp., National Institute of Diabetes and Digestive and Kidney Diseases. Research Support; Spouse/Partner; National Institute of Diabetes and Digestive and Kidney Diseases. A. Nasiri: Employee; Spouse/Partner; Medtronic. G. Cline: None. X. Zhang: None. J. Lee: None. B.T. Hubbard: None.

224-LB: Role of the Mitochondrial Calcium Uniporter in the Regulation of Hepatic Mitochondrial Metabolism and Gluconeogenesis

The mitochondrial calcium uniporter (MCU) is a key regulator of mitochondrial calcium, however, the role of MCU in mediating hepatic mitochondrial metabolism and gluconeogenesis has received limited attention. In order to address these questions, we examined rates of mitochondrial oxidation in both liver slices and in awake liver-specific MCU knockout (MCU KO) mice. We found that deletion of MCU significantly increases glucagon-induced intrahepatic lipolysis (p<0.04) and glucose production (p<0.04) in ex vivo liver slices. We also observed significantly reduced liver triglyceride accumulation (p<0.05) and increased hepatic long chain acyl-CoA (p<0.02) and acetyl-CoA (p=0.05) content in MCU KO mice, consistent with increased intrahepatic lipolysis and fat oxidation. This effect was not due to changes in white adipose tissue lipolysis. Deletion of MCU also impaired glucose tolerance in chow-fed mice, indicating dysregulated hepatic glucose production in these mice. These metabolic alterations could be attributed to reduced mitochondrial buffering of cytoplasmic calcium, leading to a 77% increase in cytoplasmic calcium (p<0.0001). This led to activation of CAMKII (p<0.04) and subsequent increase in ATGL phosphorylation (p<0.04) in hepatocytes lacking MCU. In conclusion, these results demonstrate a critical role for MCU in mediating hepatic mitochondrial oxidation and hepatic gluconeogenesis. Disclosure T.E. LaMoia: None. J. Lee: None. M. Guerra: None. R. Goodman: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & 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 (T32GM0007324)

205-OR: Hepatic Protein Kinase C-e Is Necessary and Sufficient in Mediating Lipid-Induced Hepatic Insulin Resistance

Nonalcoholic fatty liver disease (NAFLD) is strongly associated with hepatic insulin resistance (HIR), however the key lipid species and molecular mechanisms linking these conditions are widely debated. In order to address these questions, we examined the metabolic impact of a liver-specific antisense oligonucleotide against PKCε and found that liver-specific PKCε knockdown ameliorated high-fat diet-induced HIR in rats, reflected by ∼2x increase in insulin-stimulated hepatic glycogen synthesis rate and improved insulin-mediated suppression of endogenous glucose production during a hyperinsulinemic-hyperglycemic (HH) clamp. This was associated with increased IRK Y1162 and Akt S473 phosphorylation and could be attributed to decreased PKCε-mediated IRK T1160 phosphorylation. Overexpressing a constitutively active PKCε (A159E) in the liver induced HIR in regular chow-fed rats, reflected by reduced insulin-stimulated glycogen synthesis rate during an HH clamp. This was associated with impaired IRK Y1162 and Akt S473 phosphorylation and could be attributed to increased PKCε-mediated IRK T1160 phosphorylation. We also developed a subcellular fractionation method to quantify diacylglycerol (DAG) stereoisomers and ceramides in the endoplasmic reticulum, mitochondria, plasma membrane (PM), lipid droplet and cytosol and applied this method to a cohort of obese human subjects with and without NAFLD and HIR. Liver PM sn-1,2 DAG content was ∼5x higher in HIR individuals with NAFLD compared to insulin-sensitive individuals without NAFLD, associated with ∼3x higher IRK T1160 phosphorylation, supporting the importance of PM sn-1,2 DAG-PKCε-IRK pT1160 pathway to mediate HIR in humans with NAFLD. In contrast there were no significant differences in ceramide content in any of the subcellular compartments. Conclusion: These data identify PM sn-1,2 DAGs as the key lipids that activate PKCε, and that hepatic PKCε is both necessary and sufficient in mediating HIR. Disclosure K. Lyu: None. D. Zhang: None. M. Kahn: None. M.R.S. Rodrigues: None. S. Hirabara: None. P. Luukkonen: None. S. Lee: None. S. Bhanot: None. J. Rinehart: None. N. Blume: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M. Rasch: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. M.J. Serlie: None. J. Bogan: None. G. Cline: None. V. Samuel: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, LLC, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & 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 U.S. Public Health Service (R01DK116774, R01DK119968, R01DK113984, P30DK045735, R01 DK092661); U.S. Department of Veternas Affairs (I01BX000901)