Introduction and Objective: TLC-1180 (1180) is a liver-targeted mitochondrial protonophore in clinical development. Insulin resistance is a major factor in the pathogenesis of T2D and metabolic alterations associated with insulin resistance are linked to heart failure with preserved ejection fraction (HFpEF). This study aims to evaluate the effects of 1180 on tissue specific insulin resistance and exercise capacity. Methods: Male C57Bl6/J DIO mice were randomized to receive either vehicle (VEH) or 1180. Insulin-stimulated tissue specific glucose uptake was assessed using 2-Deoxy-D-Glucose (2-DG) uptake and myocardial pyruvate oxidation through the ratio of pyruvate dehydrogenase flux (VPDH) to citrate synthase flux (VCS) at the end of the hyperinsulinemic-euglycemic clamp (HEC). Cardiac fitness was evaluated using graded exercise testing. Results: Mice treated with 1180 showed lean mass-neutral weight loss and reduced plasma membrane PKCε translocation in liver, skeletal muscle, visceral white adipose tissue (WAT), and myocardium. TLC-1180 improved whole-body insulin sensitivity (+140% increase in glucose infusion rate, p<0.05), predominantly driven by increased insulin-stimulated 2-DG uptake in the gastrocnemius muscle (+43%, p<0.05), heart (+96%, p<0.01) and increased suppression of endogenous glucose production during the HEC. Myocardial pyruvate oxidation improved by +84% (p<0.05) compared to VEH, approaching levels seen in lean controls. These metabolic enhancements were associated with both increased exercise duration (+31%, p<0.05) and maximal running speed (+33%, p<0.05). Conclusion: TLC-1180 reversed DIO-induced defects in liver, muscle, WAT, and myocardium as well as normalized insulin-stimulated myocardial pyruvate oxidation. These improvements were associated with enhanced exercise performance. These preclinical findings suggest that 1180 may have therapeutic potential for improving whole body insulin resistance and cardiac function in obese individuals with HFpEF. Disclosure S. Parikh: None. M. Sharma: None. R. Calais Gaspar: None. X. Huang: None. D. Zhang: None. S. Dufour: None. H.N. Morgan: None. A. Nasiri: None. J. Zheng: None. M. Kahn: None. J. Stack: None. R.P. Myers: Employee; Current; OrsoBio, Inc. G. Subramanian: Employee; Current; OrsoBio. A. Vijayakumar: Employee; Current; OrsoBio, Inc. G. Shulman: Consultant; Current; Novo Nordisk A/S. Research Support; Current; Novo Nordisk A/S. Other - collaboration; Current; Ionis Pharmaceuticals. Advisory Panel; Current; ESPERION Therapeutics, Inc. Research Support; Current; Novo Nordisk Foundation. Advisory Panel; Current; Orsobio. Research Support; Current; Orsobio. Advisory Panel; Current; Village S.S.D. Funding T32DK007356R01DK119968UC2DK134901P30DK045735

Introduction and Objective: The molecular mechanisms by which the noradrenergic system (NES) regulates hepatic mitochondrial metabolism is poorly understood. We examined the hypothesis that the NES regulates hepatic mitochondrial oxidation and gluconeogenesis via increased autophagy mediated by activation of CaMKII during cold exposure (CE) Methods: Male C57BL/6J mice were studied before and during 6h CE (4-6ºC) under 3 conditions: Sympathectomized with 6OHDA, CaMKII antisense oligonucleotide (ASO), ATG7 ASO. Rates of hepatic mitochondrial metabolism were assessed by Q-Flux in combination with stable isotopes to assess glucose, glycerol and βOHB turnover. Results: CE increased hepatic norepinephrine (30%), plasma concentrations of glucose, fatty acids, βOHB, glucagon, corticosterone, and decreased plasma insulin concentrations. These changes were associated with increased rates of glycerol (2-fold) turnover, mitochondrial oxidation (2-fold), autophagy (2-fold) and endogenous glucose production (EGP; 2-fold), which could mostly be attributed to increased glycerol conversion to glucose (60%). 6OHDA treatment decreased hepatic norepinephrine (95%) and caused a 30-60% reduction in all of these parameters independent of changes in plasma insulin, glucagon or corticosterone. Hepatic specific knockdown of CaMKII and ATG7 mimicked all of these effects. Conclusion: These data demonstrate that cold exposure promotes increased EGP and gluconeogenesis mostly through increased glycerol conversion to glucose and supports the hypothesis that the NES regulates hepatic mitochondrial oxidation and gluconeogenesis via increased autophagy mediated by activation of CaMKII. Disclosure H.N. Morgan: None. R. Calais Gaspar: None. A. Schavinski: None. A.P. Assis: None. J. Zheng: None. S. Parikh: None. S. Dufour: None. M. Kahn: None. A. Nasiri: None. M. Perelis: Employee; Current; Ionis Pharmaceuticals. I. Kettelhut: None. L. Navegantes: None. G. Shulman: Consultant; Current; Novo Nordisk A/S. Research Support; Current; Novo Nordisk A/S. Other - collaboration; Current; Ionis Pharmaceuticals. Advisory Panel; Current; ESPERION Therapeutics, Inc. Research Support; Current; Novo Nordisk Foundation. Advisory Panel; Current; Orsobio. Research Support; Current; Orsobio. Advisory Panel; Current; Village S.S.D. Funding Sao Paulo Research Foundation (FAPESP; 2021/05848-4; 2024/03229-3), Novo Nordisk Foundation, and National Institutes of Health (R01DK119968, UC2DK134901 and P30DK045735).

Atherosclerotic cardiovascular disease (ASCVD) remains a leading cause of morbidity and mortality in patients with insulin resistance, and new therapies are urgently needed. We previously developed an orally administered formulation of 2,4-dinitrophenol, here termed controlled-release mitochondrial protonophore (CRMP), and showed that it safely reversed hypertriglyceridemia, hepatic steatosis, and insulin resistance in dysmetabolic rodents and nonhuman primates. Here, we investigated the therapeutic utility of CRMP for treating atherogenesis in a murine model of cardiometabolic syndrome [high-fat cholesterol diet (HFCD)-fed low-density lipoprotein receptor-deficient (Ldlr-/-) mice]. In both early and late disease stages, CRMP treatment diminished total plaque burden and lesion size compared with HFCD. Morphometric analysis of the aortic root revealed that CRMP also decreased neutral lipid and lesional macrophage content and increased plaque stability. Reductions in atheroprogression were associated with lower plasma and hepatic triglyceride levels and improved whole-body insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps. Furthermore, CRMP markedly limited lesional macrophage inflammasome activation and IL-1β release, changes that are consistent with a local immune-dampening effect. Mechanistically, CRMP-mediated reductions in inflammasome activation were driven by mild increases in macrophage mitochondrial inefficiency and lower mitochondrial reactive oxygen species (ROS) production. These effects were context dependent because CRMP failed to curtail lesional IL-1β content and atheroprogression in chow-fed apolipoprotein E-deficient (Apoe-/-) mice. Collectively, these data show that CRMP exerted antiatherogenic effects through uncoupling of oxidative phosphorylation in hepatocytes and macrophages, highlighting the therapeutic potential of mitochondrial uncouplers for treating ASCVD.

Impaired suppression of endogenous glucose production (EGP) drives end-organ damage in insulin resistance and type 2 diabetes. Although the liver is traditionally thought to mediate dysregulated EGP, the role of the renal cortex is less understood. Here, we investigate if high-fat diet (HFD) induces renal cortical insulin resistance while assessing renal glucose production (RGP) and mitochondrial metabolism in male mice. HFD increases plasma membrane sn-1,2-DAGs, PKCε translocation, and Insulin Receptor Kinase (IRK)T1160 phosphorylation while blunting insulin-stimulated pyruvate oxidation and insulin signaling. In HFD mice, RGP is elevated 6.5-fold and accounts for 60% of EGP during hyperinsulinemia. Excess RGP is derived equally from glycerol and mitochondrial sources, chiefly pyruvate. Signaling and flux defects are abrogated in HFD-fed IRKT1150A knockin mice, except for glycerol-derived gluconeogenesis. Our findings implicate the sn-1,2-DAG → PKCε → IRKT1160 axis in renal cortical insulin resistance and highlight renal gluconeogenesis as a driver of dysregulated glucose homeostasis.

Metabolic dysfunction-associated steatohepatitis (MASH) increases liver-related mortality, and new therapies targeting its underlying mechanisms are warranted. We examined whether two lipid-droplet proteins, CIDEB and CGI-58, exert opposing control over MASH by altering cholesterol in liver lipid droplets. Using antisense oligonucleotides, we silenced CIDEB or CGI-58 in the livers of C57BL/6J mice fed a choline-deficient, L-amino acid-defined high-fat diet. CIDEB silencing decreased both triglyceride and cholesterol levels in liver lipid droplets and lowered plasma transaminases and the number of crown-like structures. These protective effects were abrogated by cholesterol supplementation. Conversely, CGI-58 knockdown raised triglyceride and cholesterol levels and exacerbated MASH; bempedoic acid, a cholesterol-synthesis inhibitor, reversed these changes. Dual CIDEB/CGI-58 silencing confirmed that CGI-58 loss abrogated the protective effects of CIDEB knockdown. Our data establish liver lipid-droplet cholesterol as a critical determinant in MASH mediated by CIDEB and CGI-58 and demonstrate that CIDEB knockdown confers protection by enhancing CGI-58-dependent lipolysis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease globally. Disruptions in iron metabolism and mitochondrial oxidative function may cooperatively contribute to its pathogenesis. Ferredoxin reductase (FDXR), a mitochondrial flavoprotein, plays a critical role in mitochondrial respiratory supercomplex formation and iron-sulfur cluster biosynthesis-both essential for efficient oxidative metabolism. However, its role in MASLD remains unclear. Here, we knocked down hepatic Fdxr expression in the liver of C57BL/6 mice using N-acetyl galactosamineconjugated antisense oligonucleotides. [13C5]glutamine tracer infusions revealed that FDXR deficiency disrupted mitochondrial oxidative phosphorylation. In contrast, FDXR deficiency increased hepatic iron accumulation, reactive oxygen species, and lipid peroxidation. Mechanistically, FDXR deficiency disrupted iron-sulfur cluster assembly and reduced mitochondrial proteins such as succinate dehydrogenase complex iron-sulfur subunit B (SDHB), leading to mitochondrial dysfunction and steatosis. FDXR expression was upregulated in both human and murine MASLD livers, suggesting a compensatory protective response. Furthermore, hepatic overexpression of FDXR restored mitochondrial function, enhanced oxidative capacity, and ameliorated steatosis. These findings identify FDXR as a key regulator linking iron metabolism and mitochondrial integrity in MASLD and highlight its potential as a therapeutic target to prevent disease progression.

Introduction and Objective: Insulin resistance plays a pivotal role in the pathogenesis of type 2 diabetes (T2D) and metabolic dysfunction-associated steatotic liver disease (MASLD). Acetyl-CoA is a critical intermediate in metabolic pathways such as the TCA cycle and fatty acid synthesis. We examined the hypothesis that a N-acetylgalactosamine-modified antisense oligonucleotide (ASO) targeting hepatic coenzyme A synthase (Coasy), a key enzyme in CoA biosynthesis, could reduce hepatic acetyl-CoA content, thereby decreasing hepatic steatosis and insulin resistance. Methods: Male C57BL/6J mice were studied under three conditions: regular chow (RC), high-fat diet (HFD) treated with control ASO, or high-fat diet treated with Coasy ASO (HFD-Coasy). Results: HFD-Coasy mice showed significantly reduced hepatic acetyl-CoA and malonyl-CoA levels (p<0.0001 and p<0.05) compared to HFD-fed mice. These changes were accompanied by increased rates of whole-body energy expenditure, reductions in the respiratory quotient, and reduced hepatic triacylglycerol content. The decrease in hepatic fat could be explained by increased hepatic mitochondrial fat oxidation rates, as assessed by a [13C5]glutamine tracer infusion technique (Q-Flux). HFD-fed mice displayed marked whole-body and hepatic insulin resistance compared to RC-fed mice as assessed by a hyperinsulinemic-euglycemic clamp. In contrast, HFD-Coasy mice showed improved whole-body insulin sensitivity, reduced basal and clamp endogenous glucose production rates (EGP; p<0.05 and p<0.001), and enhanced EGP suppression during the clamp compared to HFD-fed mice. These improvements in hepatic insulin sensitivity were associated with increased insulin-stimulated phosphorylation of IRKY1162 and AktS473 in liver. Conclusion: These findings demonstrate that targeting hepatic Coasy may represent a promising therapeutic approach for managing T2D and MASLD. R.C. Gaspar: None. I. Sakuma: None. B.T. Hubbard: None. T.E. LaMoia: None. J. Zheng: None. S. Parikh: None. M. Kahn: None. L. Silveira: None. S. Dufour: None. A. Nasiri: None. M. Perelis: Employee; Ionis Pharmaceuticals. K. Petersen: Consultant; Village S.S.D.r.l. Joy of Movement. V. Samuel: None. G.I. Shulman: Advisory Panel; Novo Nordisk. Consultant; Ionis Pharmaceuticals. Research Support; AstraZeneca, Merck & Co., Inc, ESPERION Therapeutics, Inc., Novo Nordisk. Advisory Panel; OrsoBio, Inc.

Metabolic dysfunction-associated steatohepatitis (MASH) represents a progressive form of steatotic liver disease which increases the risk for fibrosis and advanced liver disease. The accumulation of discrete species of bioactive lipids has been postulated to activate signaling pathways that promote inflammation and fibrosis. However, the key pathogenic lipid species is a matter of debate. We explored candidates using various dietary, molecular, and genetic models. Mice fed a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) developed steatohepatitis and manifested early markers of liver fibrosis associated with increased cholesterol content in liver lipid droplets within 5 d without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides against Coenzyme A synthase (Coasy) or treatment with bempedoic acid or atorvastatin decreased liver lipid droplet cholesterol content and prevented CDAHFD-induced MASH and the fibrotic response. All these salutary effects were abrogated with dietary cholesterol supplementation. Analysis of human liver samples demonstrated that cholesterol in liver lipid droplets was increased in humans with MASH and liver fibrosis and was higher in PNPLA3 I148M (variants rs738409) than in HSD17B13 variants (rs72613567). Together, these data identify cholesterol in liver lipid droplets as a critical mediator of MASH and demonstrate that Coenzyme A synthase knockdown and bempedoic acid are therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis.

CIDEB (cell death-inducing DFF45-like effector B) deficiency is associated with a reduced incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) in humans; however, the underlying mechanism responsible for this protective effect remains unclear. C57BL/6J male mice were fed a high-fat diet (HFD) to recapitulate key aspects of MASLD and hepatic insulin resistance. Cideb knockdown (KD) was achieved using a 2′-O-methoxyethyl (MOE) antisense oligonucleotide (ASO). In vivo rates of hepatic mitochondrial gluconeogenesis and tricarboxylic acid (TCA) cycle flux were assessed by Q-Flux. The Comprehensive Lab Animal Monitoring System (CLAMS) was used to evaluate rates of whole-body energy expenditure. Hepatic and peripheric insulin sensitivity were evaluated using hyperinsulinaemic–euglycaemic clamp studies combined with radio-labelled isotopes. We showed that Cideb ASO treatment increased rates of whole-body energy expenditure by 25

Metabolic dysfunction-associated steatohepatitis (MASH) is a frequent complication of type 2 diabetes. However, the molecular mechanisms responsible for the development of MASH are unknown. Loss of function mutations in the lipid droplet proteins Comparative Gene Identification-58 (CGI-58) and Cell Death-inducing DNA fragmentation factor-like effector B (CIDEB) promote and protect against MASH, respectively. We hypothesized that the discrete impact of these proteins on MASH is due to the alteration of lipid droplet morphology followed by cholesterol content. We tested these hypotheses using antisense oligonucleotides (ASO) to knock down the expression of CGI-58 or CideB in the liver of a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) mouse MASH model. CGI-58 ASO treatment increased plasma ALT, macrophage crown-like structures, and liver inflammation/fibrosis marker expression, which were associated with increased lipid droplet size and liver lipid droplet cholesterol content. Co-treatment with an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (Gpam) decreased liver lipid droplet cholesterol and prevented CGI-58 ASO-induced MASH. In contrast, CideB ASO treatment in CDAHFD mice prevented liver inflammation and fibrosis and reduced liver lipid droplet size and lipid droplet cholesterol content. These protective effects of the CideB ASO were all abrogated with cholesterol supplementation to the diet. Conclusions: Knockdown of CGI-58 promotes liver inflammation and fibrosis by increasing liver lipid droplet size and lipid droplet cholesterol content, whereas knockdown of CideB protects against the development of liver inflammation and fibrosis by reducing liver lipid droplet size and cholesterol content. Furthermore, CIDEB and GPAM are potential therapeutic targets for MASH. Disclosure I. Sakuma: None. R.C. Gaspar: None. A. Nasiri: None. M. Kahn: None. J. Zheng: None. M. Guerra: None. D. Yimlamai: None. S. Murray: Employee; Ionis Pharmaceuticals. M. Perelis: Employee; Ionis Pharmaceuticals. W. Barnes: Employee; Ionis Pharmaceuticals. D.F. Vatner: None. K. Petersen: None. V. Samuel: None. G.I. Shulman: None.

To examine the roles of mitochondrial calcium Ca2+ ([Ca2+]mt) and cytosolic Ca2+ ([Ca2+]cyt) in the regulation of hepatic mitochondrial fat oxidation, we studied a liver-specific mitochondrial calcium uniporter knockout (MCU KO) mouse model with reduced [Ca2+]mt and increased [Ca2+]cyt content. Despite decreased [Ca2+]mt, deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux, α-ketoglutarate dehydrogenase flux, and succinate dehydrogenase flux in vivo. Rates of [14C16]palmitate oxidation and intrahepatic lipolysis were increased in MCU KO liver slices, which led to decreased hepatic triacylglycerol content. These effects were recapitulated with activation of CAMKII and abrogated with CAMKII knockdown, demonstrating that [Ca2+]cyt activation of CAMKII may be the primary mechanism by which MCU deletion promotes increased hepatic mitochondrial oxidation. Together, these data demonstrate that hepatic mitochondrial oxidation can be dissociated from [Ca2+]mt and reveal a key role for [Ca2+]cyt in the regulation of hepatic fat mitochondrial oxidation, intrahepatic lipolysis, gluconeogenesis, and lipid accumulation.

Hepatic insulin resistance (IR) is often said to be "pathway-selective" with preserved insulin stimulation of de novo lipogenesis (DNL) despite attenuated insulin signaling toward glucose metabolism. However, DNL has not been assessed in models of liver-specific IR. We studied mice with differential tissue-specific lipid-induced IR achieved by different durations of high-fat diet (HFD) feeding. Mice with isolated hepatic IR demonstrated markedly reduced DNL, with a rebound seen in mice with whole-body IR. Insr T1150A mice (protected against diacylglycerol-PKCε-induced hepatic IR) maintained normal DNL with HFD feeding. During hyperinsulinemic clamps, hepatic IR reduced DNL, but hyperglycemia augmented DNL in both resistant and sensitive animals. Regulation through SREBP1c did not consistently correlate with changes in DNL. These results demonstrate that hepatic IR is not pathway-selective, highlighting the primacy of lipogenic substrate in stimulation of DNL. Future therapeutics to reduce lipogenesis should target substrate drivers of DNL rather than targeting plasma insulin levels.

Previous studies highlight the potential for sodium-glucose cotransporter type 2 (SGLT2) inhibitors (SGLT2i) to exert cardioprotective effects in heart failure by increasing plasma ketones and shifting myocardial fuel utilization toward ketone oxidation. However, SGLT2i have multiple in vivo effects and the differential impact of SGLT2i treatment and ketone supplementation on cardiac metabolism remains unclear. Here, using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology combined with infusions of [13C6]glucose or [13C4]βOHB, we demonstrate that acute SGLT2 inhibition with dapagliflozin shifts relative rates of myocardial mitochondrial metabolism toward ketone oxidation, decreasing pyruvate oxidation with little effect on fatty acid oxidation in awake rats. Shifts in myocardial ketone oxidation persisted when plasma glucose levels were maintained. In contrast, acute βOHB infusion similarly augmented ketone oxidation, but markedly reduced fatty acid oxidation and did not alter glucose uptake or pyruvate oxidation. After inducing heart failure, dapagliflozin increased relative rates of ketone and fatty acid oxidation, but decreased pyruvate oxidation. Dapagliflozin increased mitochondrial redox and reduced myocardial oxidative stress in heart failure, which was associated with improvements in left ventricular ejection fraction after 3 weeks of treatment. Thus, SGLT2i have pleiotropic effects on systemic and heart metabolism, which are distinct from ketone supplementation and may contribute to the long-term cardioprotective benefits of SGLT2i.

Metabolic dysfunction-associated steatohepatitis (MASH) is highly prevalent among patients with type 2 diabetes. Cholesterol is one of several candidates causing hepatic inflammation and fibrosis. We hypothesized that by interrupting the synthesis of cholesterol, we could prevent a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) C57BL/6J MASH mouse model. We found that cholesterol in liver lipid droplets accumulated on day 1, MASH-associated macrophage markers (Gpnmb and Trem2) mRNA expression and plasma ALT increased on day 2, macrophage crown-like-structures emerged on day 3, and fibrosis markers (Col1a1, αSMA) mRNA expression increased on day 7 in the CDAHFD-treated mice. Filipin staining demonstrated free cholesterol accumulation in liver lipid droplets. We next examined whether a N-acetylgalactosamine-modified antisense oligonucleotide (ASO) against coenzyme A synthase (Coasy), which catalyzes a rate controlling step in hepatic cholesterol synthesis, would reduce cholesterol in liver lipid droplets and inflammation/fibrosis in this model. Coasy ASO treatment prevented accumulation of cholesterol in liver lipid droplets (Control=1.02 ± 0.04 mg/g tissue; Coasy=0.68 ± 0.08, P<0.001) and decreased plasma ALT and liver inflammation/fibrosis markers expression. To examine whether these effects of the Coasy ASO were mediated by reductions in liver lipid droplet cholesterol we examined whether adding 2% cholesterol to the diet would prevent the Coasy ASO protective effect. Cholesterol treatment induced accumulation of cholesterol in liver lipid droplets (Coasy=0.68 ± 0.08 mg/g tissue; Coasy with cholesterol =1.40 ± 0.11, P<0.001) and abrogated the protective effects of the Coasy ASO on liver inflammation and fibrosis. Conclusions: These data demonstrate that cholesterol in liver lipid droplets is a key mediator of MASH and hepatic Coasy knockdown is a potential therapeutic approach. Disclosure I. Sakuma: None. R.C. Gaspar: None. A. Nasiri: None. M. Kahn: None. M. Guerra: None. D. Yimlamai: None. S. Murray: Employee; Ionis Pharmaceuticals. M. Perelis: Employee; Ionis Pharmaceuticals. W. Barnes: Employee; Ionis Pharmaceuticals. D.F. Vatner: None. K. Petersen: None. V. Samuel: None. G.I. Shulman: None.

Inhibition of the ceramide synthetic pathway with myriocin or an antisense oligonucleotide (ASO) targeting dihydroceramide desaturase (DES1) both improved hepatic insulin sensitivity in rats fed either a saturated or unsaturated fat diet and was associated with reductions in both hepatic ceramide and plasma membrane (PM)-sn-1,2-diacylglycerol (DAG) content. The insulin sensitizing effects of myriocin and Des1 ASO were abrogated by acute treatment with an ASO against DGAT2, which increased hepatic PM-sn-1,2-DAG but not hepatic C16 ceramide content. Increased PM-sn-1,2-DAG content was associated with protein kinase C (PKC)ε activation, increased insulin receptor (INSR)T1150 phosphorylation leading to reduced insulin-stimulated INSRY1152/AktS473 phosphorylation, and impaired insulin-mediated suppression of endogenous glucose production. These results demonstrate that inhibition of de novo ceramide synthesis by either myriocin treatment or DES1 knockdown protects against lipid-induced hepatic insulin resistance through a C16 ceramide-independent mechanism and that they mediate their effects to protect from lipid-induced hepatic insulin resistance via the PM-sn-1,2-DAG-PKCε-INSRT1150 phosphorylation pathway.

Muscle sn-1,2-diacylglycerol (DAG) and C18:0 ceramide accumulation in sarcolemmal and mitochondrial compartments have been proposed to regulate muscle insulin sensitivity. Here, we evaluated whether weight loss-induced improvements in insulin sensitivity were associated with changes in muscle sn-1,2-DAG and ceramide content in people with obesity and type 2 diabetes. We measured skeletal muscle insulin sensitivity, assessed by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with stable isotopically labeled glucose tracer infusion, and skeletal muscle sn-1,2-DAG and ceramide contents by using liquid chromatography-tandem mass spectrometry after subcellular fractionation and DAG isomer separation in 14 adults with obesity and type 2 diabetes before and after marked (18.6 ± 2.1%) weight loss. Whole-body insulin sensitivity doubled after weight loss. Sarcolemmal sn-1,2-DAG and C18:0 ceramide contents after weight loss were not different than values before weight loss. In contrast, mitochondrial/ER C18:0 ceramide content decreased by ~20% after weight loss (from 2.16 ± 0.08 to 1.71 ± 0.13 nmol/g, P<0.005). These results suggest a decrease in muscle mitochondrial/ER C18:0 ceramide content could contribute to the beneficial effect of weight loss on skeletal muscle insulin sensitivity.

AGPAT2 (1- acyl-sn- glycerol - 3- phosphate- acyltransferase-2)converts lysophosphatidic acid (LPA) into phosphatidic acid (PA), and mutations of the AGPAT2 gene cause the most common form of congenital generalized lipodystrophy which leads to steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to lipodystrophy and steatohepatitis has not been elucidated. We addressed this question using an antisense oligonucleotide (ASO) to knockdown expression of Agpat2 in the liver and white adipose tissue (WAT) of adult male Sprague- Dawley rats. Agpat2 ASO treatment induced lipodystrophy and inflammation in WAT and the liver, which was associated with increased LPA content in both tissues, whereas PA content was unchanged. We found that a controlled- release mitochondrial protonophore (CRMP) prevented LPA accumulation and inflammation in WAT whereas an ASO against glycerol -3 -phosphate acyltransferase, mitochondrial (Gpam) prevented LPA content and inflammation in the liver in Agpat2 ASO- treated rats. In addition, we show that overnutrition, due to high sucrose feeding, resulted in increased hepatic LPA content and increased activated macrophage content which were both abrogated with Gpam ASO treatment. Taken together, these data identify LPA as a key mediator of liver and WAT inflammation and lipodystrophy due to AGPAT2 deficiency as well as liver inflammation due to overnutrition and identify LPA as a potential therapeutic target to ameliorate these conditions.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, in which prognosis is determined by liver fibrosis. A common variant in hydroxysteroid 17-beta dehydrogenase 13 ( HSD17B13 , rs72613567-A) is associated with a reduced risk of fibrosis in NAFLD, but the underlying mechanism(s) remains unclear. We investigated the effects of this variant in the human liver and in Hsd17b13 knockdown in mice by using a state-of-the-art metabolomics approach. We demonstrate that protection against liver fibrosis conferred by the HSD17B13 rs72613567-A variant in humans and by the Hsd17b13 knockdown in mice is associated with decreased pyrimidine catabolism at the level of dihydropyrimidine dehydrogenase. Furthermore, we show that hepatic pyrimidines are depleted in two distinct mouse models of NAFLD and that inhibition of pyrimidine catabolism by gimeracil phenocopies the HSD17B13 -induced protection against liver fibrosis. Our data suggest pyrimidine catabolism as a therapeutic target against the development of liver fibrosis in NAFLD.