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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.

Atherosclerotic cardiovascular disease (CVD) remains a leading cause of morbidity and mortality in people with type 2 diabetes (T2D) and new therapies are urgently needed. We recently developed an orally available, liver-directed controlled-release mitochondrial protonophore (CRMP) and showed that this agent safely reverses hypertriglyceridemia, fatty liver, and T2D in rodent and nonhuman primate models of obesity by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. To determine the effect of CRMP on atherogenesis, we performed intervention studies in which 12-week Western-diet fed Ldlr -/- mice with established atherosclerosis were treated with CRMP (30 mg/kg-day) or vehicle control for an additional 12 weeks. CRMP-treated mice displayed a significant reduction in fasting plasma and hepatic triglyceride content, independently of changes in body weight, fasting plasma glucose, insulin or cholesterol levels. Morphometric analysis of the aortic root and brachiocephalic artery revealed that CRMP treatment significantly reduced total plaque area and neutral lipid accumulation. CRMP treatment also decreased markers of plaque vulnerability, as evidenced by a reduction in necrotic core area and increase in fibrous cap area ( P Ldlr -/- mice fed a Western diet containing CRMP or vehicle control for 12 weeks. Similarly to the intervention studies, CRMP significantly reduced aortic root plaque area, neutral lipid accumulation and necrotic core area. These changes were associated with a reduction in plasma triglyceride content and improvement in whole-body insulin sensitivity as assessed by hyperinsulinemic-euglycemic clamps (all P Ldlr -/- mice revealed that CRMP reduced the frequency of inflammatory macrophages compared to vehicle (~19% vs ~2%), consistent with an immune-dampening effect of CRMP. Taken together, these data support an anti-atherogenic role for CRMP and suggest the therapeutic potential of liver-directed mitochondrial uncouplers for the treatment of early and late-stage atherosclerosis.

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 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)

Significance Hepatic glycogen synthesis plays a critical role in maintaining normal glucose homeostasis; however, the rate-controlling step regulating this process is unknown. Applying metabolic control analysis in vivo, we show that the regulation of insulin-stimulated hepatic glycogen synthesis under both normal and pathophysiological conditions of fatty liver-associated hepatic insulin resistance is controlled at the glucokinase (GCK) step through GCK translocation., Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Significance Ketogenic diet is an effective treatment for nonalcoholic fatty liver disease (NAFLD). Here, we present evidence that hepatic mitochondrial fluxes and redox state are markedly altered during ketogenic diet-induced reversal of NAFLD in humans. Ketogenic diet for 6 d markedly decreased liver fat content and hepatic insulin resistance. These changes were associated with increased net hydrolysis of liver triglycerides and decreased endogenous glucose production and serum insulin concentrations. Partitioning of fatty acids toward ketogenesis increased, which was associated with increased hepatic mitochondrial redox state and decreased hepatic citrate synthase flux. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by ketogenic diet and highlight hepatic mitochondrial fluxes and redox state as potential treatment targets in NAFLD., Weight loss by ketogenic diet (KD) has gained popularity in management of nonalcoholic fatty liver disease (NAFLD). KD rapidly reverses NAFLD and insulin resistance despite increasing circulating nonesterified fatty acids (NEFA), the main substrate for synthesis of intrahepatic triglycerides (IHTG). To explore the underlying mechanism, we quantified hepatic mitochondrial fluxes and their regulators in humans by using positional isotopomer NMR tracer analysis. Ten overweight/obese subjects received stable isotope infusions of: [D7]glucose, [13C4]β-hydroxybutyrate and [3-13C]lactate before and after a 6-d KD. IHTG was determined by proton magnetic resonance spectroscopy (1H-MRS). The KD diet decreased IHTG by 31% in the face of a 3% decrease in body weight and decreased hepatic insulin resistance (−58%) despite an increase in NEFA concentrations (+35%). These changes were attributed to increased net hydrolysis of IHTG and partitioning of the resulting fatty acids toward ketogenesis (+232%) due to reductions in serum insulin concentrations (−53%) and hepatic citrate synthase flux (−38%), respectively. The former was attributed to decreased hepatic insulin resistance and the latter to increased hepatic mitochondrial redox state (+167%) and decreased plasma leptin (−45%) and triiodothyronine (−21%) concentrations. These data demonstrate heretofore undescribed adaptations underlying the reversal of NAFLD by KD: That is, markedly altered hepatic mitochondrial fluxes and redox state to promote ketogenesis rather than synthesis of IHTG.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1–3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation—all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment—reversing hepatic steatosis and glucose intolerance—were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes. A role and mechanism of action are identified for INSP3R1 in the stimulation of hepatic gluconeogenesis and mitochondrial oxidation by glucagon, suggesting that INSP3R1 may be a target for ameliorating dysregulation of hepatic glucose metabolism.

Significance Metformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin’s glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans.

Fragmentation mechanisms of the singly protonated glutathione ( γ-ECG) and its synthetic analogue peptides (ECG and PPECG) have been investigated by liquid chromatography tandem-mass spectrometry and theoretical calculations. In the mass spectra, similar fragmentation patterns were observed for γ-ECG and ECG, but a completely different one was found in the case of PPECG. The E–C amide bond cleavage is the predominant pathway for the fragmentation of γ-ECG and ECG, whereas the additional N-terminal prolyl residues in PPECG significantly suppress the E–C amide bond cleavage. Theoretical calculations reveal that the fragmentation efficiencies of the E–C bonds in the protonated γ-ECG and ECG are much higher than that in the protonated PPECG, being attributed to their lower barriers of the potential energy; clearly the introduction of two prolyl residues can increase substantially the potential energy barrier. In the proposed mechanism, the protonated E–C amide bonds in the three peptides are first weakened followed by a nucleophilic addition by the glutamyl carboxyl oxygen atom in side chain, leading to the breaking of the E–C amide bonds. However, the processes of E–C bond fragmentation for three protonated analogs were not collaborative. Protonated amide bonds first fragment, then the nucleophilic addition by the side chain of glutamyl carboxyl oxygen atom takes places. On the other hand, the prolyl residues in PPECG can largely diminish the nucleophilic addition, resulting in a much lower efficiency of its E–C amide bond breaking. Distance analysis indicates that breaking the E–C amide bonds in the protonated γ-ECG, ECG, and PPECG ions could not occur without the assistance from the nucleophilic attack, highlighting an asynchronous collaborative process in the bond breakings.

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 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

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 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.

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 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)

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 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.)

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 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.

In the version of this article originally published, the VPC and VCS flux data shown in Fig. 6e,f were inadvertently duplicated from Fig. 5j,k. The correct data are now shown in Fig. 6e,f. In these corrected data, VPC flux in response to chronic oral metformin treatment was still significantly decreased (Fig. 6e), and there was still no impact of metformin on VCS flux (Fig. 6f). Therefore, the text describing these data remains the same and this correction does not change the conclusion of this study.

Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (VCS) and pyruvate carboxylase flux (VPC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of VCS and VPC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-13C]lactate and glucose tracer. PINTA measures VCS and VPC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic VCS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the VPC/VCS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism., Liver mitochondrial metabolism plays an important role for glucose and lipid homeostasis and its alterations contribute to metabolic disorders, including fatty liver and diabetes. Here Perry et al. develop a method for the measurement of hepatic fluxes by using lactate and glucose tracers in combination with NMR spectroscopy.

NAFLD is estimated to affect up to one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance, NASH, and T2D. While weight loss is highly effective at reversing NAFLD, insulin resistance and T2D, it is difficult to sustain and new therapies are required. Our lab has recently developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. While we have previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, and hepatic inflammation/fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess the safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of CRMP treatment on hepatic mitochondrial oxidation and the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in a spontaneous nonhuman primate model of NAFLD and the metabolic syndrome. Using Positional Isotopomer NMR Tracer Analysis (PINTA), we demonstrate that acute CRMP treatment (single dose of 5 mg/kg) significantly increases rates of hepatic mitochondrial fat oxidation by 40% (P Conclusion: These studies provide important proof-of-concept data to support the development of novel liver-targeted mitochondrial uncouplers for the treatment of NAFLD/NASH and T2D in humans. Disclosure L. Goedeke: None. V. Montalvo Romeral: None. G. Butrico: None. M. Kahn: None. S. Dufour: None. X. Zhang: None. G. Cline: None. K. Petersen: Advisory Panel; Spouse/Partner; Aegerion Pharmaceuticals, AstraZeneca, AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk A/S. Consultant; Spouse/Partner; Gilead Sciences, Inc. Consultant; Self; Merck & Co., Inc. Consultant; Spouse/Partner; Nimbus Discoverey. Consultant; Self; Novo Nordisk A/S. K. Chng: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc. Advisory Panel; Spouse/Partner; Merck & Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

NAFLD is estimated to affect up to one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance, NASH, and T2D. While weight loss is highly effective at reversing NAFLD, insulin resistance and T2D, it is difficult to achieve and sustain; therefore, new therapies are urgently required. Our lab has recently developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. While we have previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, and hepatic inflammation/fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess the safety and efficacy in a model highly relevant to humans. Here, we evaluated the safety and impact of chronic CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in two nonhuman primate models of metabolic syndrome. Using Positional Isotopomer NMR Tracer Analysis (PINTA), we demonstrate that acute CRMP treatment (single dose of 5 mg/Kg) increases rates of hepatic mitochondrial fat oxidation by 40%. Importantly, 6-weeks of CRMP treatment significantly reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature or adverse reactions. CRMP treatment was also associated with a significant 20–30% reduction in fasting plasma triglycerides and LDL-C in spontaneously obese dysmetabolic nonhuman primates. Furthermore, oral administration of CRMP significantly reduced endogenous glucose production by 18%, which could be attributed to a ~20% reduction in hepatic acetyl-CoA content (as assessed by whole-body βOHB turnover) and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of novel liver-targeted mitochondrial uncouplers for the treatment of NAFLD/NASH and T2D in humans.

Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association remain under debate. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we applied and validated a stable isotope method to measure the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose) in tumor cells. Using this method, we found that three tumor cell lines associated with obesity (colon cancer [MC38], breast cancer [4T1], and prostate cancer [TRAMP-C3] cells) increase VPDH/VCS in response to physiologic concentrations of insulin. In contrast, three tumor cell lines that are not associated with obesity (melanoma [YUMM1.7], B cell lymphoma [BCL1 clone 5B1b], and small cell lung cancer [NCI-H69] cells) exhibited no oxidative response to insulin. The observed increase in glucose oxidation in response to insulin correlated with a dose-dependent increase in cell division in obesity-associated tumor cell lines when grown in insulin, whereas no alteration in cell division was seen in tumor types not associated with obesity. These data reveal that a shift in substrate preference in the setting of physiologic insulin may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity.

Supplemental material, EMS896708 Supplemental Material for Insight into nucleophilic fragmentation mechanisms by glutamic acid side chain in singly protonated glutathione and related peptidyl ions by Liqun Fan, Jinhu Wang, Chunli Liu, Tiesheng Shi, Xian-Man Zhang, Yanqing Xia, Lina Fan and Yang Liu in European Journal of Mass Spectrometry

In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography/tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously at doses previously used as a tracer, it increased VPyr-Cyc/VMito by 20-30-fold, increased hepatic TCA metabolite concentrations 2-3-fold, and increased endogenous glucose production rates by 20-100%. The physiologic stimuli, glucagon and epinephrine, both increased hepatic glucose production, but only glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable tracer to assess hepatic glycolytic, gluconeogenic, and mitochondrial metabolism in vivo.

Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (V(PDH)) to rates of mitochondrial citrate synthase flux (V(CS)) in muscle. Contrary to this hypothesis we found that high fat diet (HFD)-fed insulin resistant rats did not manifest altered muscle substrate preference (V(PDH)/V(CS)) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased V(PDH)/V(CS) in both muscles in normal and insulin resistant rats. We then examined muscle V(PDH)/V(CS) flux in insulin sensitive and insulin resistant humans and found similar relative rates of V(PDH)/V(CS) following an overnight fast (~20%) and similar increases in V(PDH)/V(CS) fluxes during a HE clamp. Taken together, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.

Summary: Obesity is associated with colon cancer pathogenesis, but the underlying mechanism is actively debated. Here, we confirm that diet-induced obesity promotes tumor growth in two murine colon cancer models and show that this effect is reversed by an orally administered controlled-release mitochondrial protonophore (CRMP) that acts as a liver-specific uncoupler of oxidative phosphorylation. This agent lowered circulating insulin, and the reduction of tumor growth was abrogated by an insulin infusion raising plasma insulin to the level of high-fat-fed mice. We also demonstrate that hyperinsulinemia increases glucose uptake and oxidation in vivo in tumors and that CRMP reverses these effects. This study provides evidence that perturbations of whole-organism energy balance or hepatic energy metabolism can influence neoplastic growth. Furthermore, the data show that glucose uptake and utilization by cancers in vivo are not necessarily constitutively high but rather may vary according to the hormonal milieu. : Wang et al. demonstrate that diet-induced hyperinsulinemia increases colon adenocarcinoma tumor glucose uptake and oxidation in mice. They further demonstrate that reversal of hyperinsulinemia by a liver-specific mitochondrial protonophore is sufficient to reverse the obesity-induced acceleration of tumor growth. Keywords: colon adenocarcinoma, insulin, insulin resistance, glucose metabolism, uncoupling

Caloric restriction without malnutrition (CR) is the most robust and reproducible way to extend lifespan and delay the onset of age-related diseases, such as hepatic steatosis, insulin resistance and type 2 diabetes (T2D). Unfortunately, the strict dietary regime necessary to achieve the beneficial effects of CR limits the use of this treatment to retard aging and disease in humans and new therapies are required. In this regard, our lab has recently developed a new class of therapeutic agents that are designed to safely promote liver-targeted mitochondrial uncoupling with a wide-therapeutic index. Our second-generation compound in this class is controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. While we have previously demonstrated that this agent safely reverses hypertriglyceridemia, fatty liver, and hepatic inflammation/fibrosis in young-adult diet-induced rodent models of obesity, its effects on healthspan and lifespan remain to be determined. Here, we evaluated the impact of CRMP on hepatic steatosis and insulin action in aged 74-week-old male C57BL/6J mice fed a high-fat diet (HFD; 45% fat) for 12 weeks. Specifically, we demonstrate that CRMP treatment significantly reduced liver triglycerides, diacylglycerols, and transaminase levels independently of changes in body weight, whole-body energy expenditure or food intake. Additionally, long-term CRMP treatment reduced fasting plasma glucose concentrations (P Disclosure L. Goedeke: None. J. Camporez: None. A. Nasiri: None. Y. Wang: None. X. Zhang: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Glucagon has been suggested to be both pathogenic and protective against type 2 diabetes (T2D) reflecting our poor understanding of the molecular mechanisms by which glucagon alters hepatic metabolism. Using Positional Isotopomer NMR Tracer Analysis (PINTA) we found that acute glucagon infusion stimulates rates of hepatic gluconeogenesisin glycogen-depleted mice by promoting increases in intrahepatic lipolysis and hepatic acetyl-CoA content (75±6 vs. 101±6 nmol/g, p=0.01), pyruvate carboxylase flux (VPC, 70.9±3.9 vs. 99.9±7.1 µmol/[kg-min], p=0.008) and mitochondrial oxidation (90.0±12.9 vs. 452.1±78.3 µmol/[kg-min], p=0.001) in vivo. Each of these effects was abrogated in liver-specific Inositol Trisphosphate Receptor-I (InsP3R-I) knockout mice demonstrating that intracellular calcium signaling is necessary for these processes. Liver specific knockdown of adipocyte triglyceride lipase abolished acute glucagon stimulation of gluconeogenesis (control 70.5±2.9 vs. 103.3±3.3 µmol/[kg-min], p Disclosure R.J. Perry: None. Y. Wang: None. A.L. Brill: None. L. Peng: None. D. Zhang: None. S. Dufour: None. Y. Zhang: None. X. Zhang: None. Y. Nozaki: None. G. Cline: None. B.E. Ehrlich: None. K. Petersen: Research Support; Spouse/Partner; Gilead Sciences, Inc.. Advisory Panel; Spouse/Partner; AstraZeneca, Merck & Co., Inc., Novo Nordisk A/S. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research & Development, Merck & Co., Inc., Novo Nordisk Inc.. Research Support; Self; Gilead Sciences, Inc..

Exposure to the toxins methylene cyclopropyl acetic acid (MCPA) and methylene cyclopropyl glycine (MCPG) of unripe ackee and litchi fruit can lead to hypoglycemia and death; however, the molecular mechanisms by which MCPA and MCPG cause hypoglycemia have not been established in vivo. To determine the in vivo mechanisms of action of these toxins we infused them into conscious rodents and assessed rates of hepatic gluconeogenesis and ketogenesis, hepatic acyl-CoA and hepatic acetyl-CoA content, and hepatocellular energy charge. MCPG suppressed rates of hepatic β-oxidation as reflected by reductions in hepatic ketogenesis, reducing both short- and medium-chain hepatic acyl-CoA concentrations. Hepatic acetyl-CoA content decreased, and hepatic glucose production was inhibited. MCPA also suppressed β-oxidation of short chain acyl-CoAs, rapidly inhibiting hepatic ketogenesis and hepatic glucose production, depleting hepatic acetyl-CoA content and ATP content, while increasing other short chain acyl CoAs. Utilizing a recently-developed positional isotopomer NMR tracer analysis (PINTA) method we demonstrated that MCPA-induced reductions in hepatic acetyl-CoA content were associated with a marked reduction of hepatic pyruvate carboxylase flux. Taken together, these data reveal the in vivo mechanisms of action of MCPA and MCPG: the hypoglycemia associated with ingestion of these toxins can be ascribed mostly to MCPA- or MCPG- induced reductions in hepatic pyruvate carboxylase flux due to inhibition of β-oxidation of short chain acyl-CoAs by MCPA or inhibition of both short and medium chain acyl-CoAs by MCPG with resultant reductions in hepatic acetyl-CoA content, with an additional contribution to hypoglycemia through reduced hepatic ATP stores by MCPA.

Glucagon promotes hepatic glucose production, lipolysis and amino acid catabolism. Inhibition of glucagon activity, or glucagon resistance, not only lowers blood glucose levels, but also induces non‐alcoholic fatty liver disease and hyperaminoacidemia.

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin’s antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

The failure caused by the corrosion-wear of molten aluminum and its alloys is one of the main problems in aluminum industry. In this work, the resistance behavior of various materials, including Fe-based alloys, ceramics and corresponding high apparatus of corrosion-wear in molten aluminum and its alloys, were reviewed. The synergistic effect of corrosion and wear was discussed based on corrosion and wear mechanics. The effects of dynamic agitation due to rotating of friction pairs, physical property of liquid metal and size of grain etc., on the corrosion-wear resistance performance were investigated. In addition, the characteristics of corrosion-wear resistance performance of materials in molten aluminum and its alloy were summarized. According to our recent progress referred to kinds of materials, especially a TiAl3/Ti3AlC2/Al2O3 composite, the ceramics/metal composites with a co-continuous structure will be of great advantage in the field of corrosion-wear environment of molten aluminum and its alloys.

The transition from the fed to the fasted state necessitates a shift from carbohydrate to fat metabolism that is thought to be mostly orchestrated by reductions in plasma insulin concentrations. Here, we show in awake rats that insulinopenia per se does not cause this transition but that both hypoleptinemia and insulinopenia are necessary. Furthermore, we show that hypoleptinemia mediates a glucose-fatty acid cycle through activation of the hypothalamic-pituitary-adrenal axis, resulting in increased white adipose tissue (WAT) lipolysis rates and increased hepatic acetyl-coenzyme A (CoA) content, which are essential to maintain gluconeogenesis during starvation. We also show that in prolonged starvation, substrate limitation due to reduced rates of glucose-alanine cycling lowers rates of hepatic mitochondrial anaplerosis, oxidation, and gluconeogenesis. Taken together, these data identify a leptin-mediated glucose-fatty acid cycle that integrates responses of the muscle, WAT, and liver to promote a shift from carbohydrate to fat oxidation and maintain glucose homeostasis during starvation.

Palladium (Pd)-catalyzed desulfitative cross-coupling reactions of sodium arylsulfinates with a wide variety of arylboronic acids were achieved in good to excellent yields under simple aerobic conditions. These catalytic reactions could be accelerated by addition of a catalytic amount of Cu(II) salts as co-catalyst. Moreover, these reactions were tolerant to all of the tested functional groups, making them attractive alternatives to the traditional cross-coupling reactions.

Leptin treatment reverses hyperglycemia in animal models of poorly controlled type 1 diabetes (T1D), spurring great interest in the possibility of treating patients with this hormone. The antidiabetic effect of leptin has been postulated to occur through suppression of glucagon production, suppression of glucagon responsiveness or both; however, there does not appear to be a direct effect of leptin on the pancreatic alpha cell. Thus, the mechanisms responsible for the antidiabetic effect of leptin remain poorly understood. We quantified liver-specific rates of hepatic gluconeogenesis and substrate oxidation in conjunction with rates of whole-body acetate, glycerol and fatty acid turnover in three rat models of poorly controlled diabetes, including a model of diabetic ketoacidosis. We show that the higher rates of hepatic gluconeogenesis in all these models could be attributed to hypoleptinemia-induced activity of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in higher rates of adipocyte lipolysis, hepatic conversion of glycerol to glucose through a substrate push mechanism and conversion of pyruvate to glucose through greater hepatic acetyl-CoA allosteric activation of pyruvate carboxylase flux. Notably, these effects could be dissociated from changes in plasma insulin and glucagon concentrations and hepatic gluconeogenic protein expression. All the altered systemic and hepatic metabolic fluxes could be mimicked by infusing rats with Intralipid or corticosterone and were corrected by leptin replacement. These data demonstrate a critical role for lipolysis and substrate delivery to the liver, secondary to hypoleptinemia and HPA axis activity, in promoting higher hepatic gluconeogenesis and hyperglycemia in poorly controlled diabetes.

In this study, chitosan directly cross-linked by PdII cation membranes (Pd-cr-CSM) with good mechanical strength and thermal stabilities have been prepared. Although the prepared Pd-cr-CSM has neither open porous structure nor high specific surface areas, it has similar good catalytic activity and much higher stability as compared with typical prepared chitosan-stabilized palladium heterogeneous catalysts for Heck reactions. It is highly active for the Heck reactions of aryl iodides and bromides with a strong electron-withdrawing group at a palladium catalyst loading of 0.15 mol %. It can be recycled 12 times in dimethyl sulfoxide (DMSO) solution or 7 times in aqueous solution. The high activity and extreme stability of the Pd-cr-CSM catalyst are mainly attributed to the well-entrapped palladium nanoparticles inside the chitosan matrix, which might catalyze the coupling reactions in the free volume holes (open spaces) of the swollen cross-linked chitosan gel networks.

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin's blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

Chlorinated polyvinylchloride (CPVC) nanofiber mats were prepared by an electrospinning technique, and then treated with amines of different chemical structures, followed by immobilization of palladium catalysts (CPVC–NH2–Pd), which have been demonstrated as efficient, stable and easily recyclable heterogeneous catalysts. Their catalytic activities could be correlated with the binding energies of the palladium species with the amine chelating ligands.