Hepatic Mitochondrial Oxidation Research Articles

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)

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress

BackgroundOxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes. MethodsWe constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo. ResultsHepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress. ConclusionsThis study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed. General significanceRoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

Effects of Acute Hypoxic Stress on Physiological and Hepatic Metabolic Responses of Triploid Rainbow Trout (Oncorhynchus mykiss).

This experiment simulated the hypoxic environment caused by actual production operations in fish farming (i.e., catching, gathering, transferring, and weighting) to study the effects of acute hypoxic conditions on the physiological and metabolic responses of triploid rainbow trout (O. mykiss). Two groups of fish weighting 590 g were sampled in the normoxia group (dissolved oxygen above 7 mg/L) and hypoxia group (dissolved oxygen ranged from 2 to 5 mg/L for 10 min). The results showed that 1) regarding stress response, hypoxia increased plasma levels of cortisol, heat shock protein 70 (HSP-70), lysozyme, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine phosphokinase (CPK); induced the expression of hepatic genes encoding nuclear factor erythroid 2 related factor 2 (Nrf2), interferon γ (IFN-γ) and interleukin-1β (IL-1β). 2) Regarding metabolism response, hypoxia increased plasma levels of globulin (GLOB), glucose (GLU), triglyceride (TG) and lactate dehydrogenase (LDH); upregulated the hepatic gene expression of phosphoenolpyruvate carboxykinase, (PEPCK), pyruvate dehydrogenase kinase (PDK1), acetyl-CoA carboxylase (ACC) and acetyl-CoA oxidase (ACO); downregulated the hepatic gene expression of carnitine palmitoyl transferase 1 (CPT1); and unchanged the expression of hepatic genes in glycolysis and autophagy. 3) In response to hypoxia-inducible factors (HIFs), the hepatic HIF-2α gene was activated in the hypoxia group, but HIF-1α gene expression remained unchanged. Thus, during acute hypoxic stress, triploid rainbow trout were in a defensive state, with an enhanced immune response and altered antioxidant status. Additionally, the hepatic mitochondrial oxidation of glucose- and lipid-derived carbon in trout was suppressed, and hepatic gluconeogenesis and lipid synthesis were activated, which might be regulated by the HIF-2α pathway.

Regulation of Hepatic Lipid and Glucose Metabolism by INSP3R1.

With the rising epidemics of obesity and nonalcoholic fatty liver disease (NAFLD) and its downstream consequences including steatohepatitis, cirrhosis, and type 2 diabetes in the U.S. and worldwide, new therapeutic approaches are urgently needed to treat these devastating conditions. Glucagon, known for a century to be a glucose-raising hormone and clearly demonstrated to contribute to fasting and postprandial hyperglycemia in both type 1 and type 2 diabetes, represents an unlikely target to improve health in those with metabolic syndrome. However, recent work from our group and others' identifies an unexpected role for glucagon as a potential means of treating NAFLD, improving insulin sensitivity, and improving the lipid profile. We propose a unifying, calcium-dependent mechanism for glucagon's effects both to stimulate hepatic gluconeogenesis and to enhance hepatic mitochondrial oxidation: signaling through the inositol 1,4,5-trisphosphate receptor type 1 (INSP3R1), glucagon activates phospholipase C (PKC)/protein kinase A (PKA) signaling to enhance adipose triglyceride lipase (ATGL)-dependent intrahepatic lipolysis and, in turn, increase cytosolic gluconeogenesis by allosteric activation of pyruvate carboxylase. Simultaneously in the mitochondria, calcium transferred through mitochondria-associated membranes activates several dehydrogenases in the tricarboxylic acid cycle, correlated with an increase in mitochondrial energy expenditure and reduction in ectopic lipid. This model suggests that short-term, cyclic treatment with glucagon or other INSP3R1 antagonists could hold promise as a means to reset lipid homeostasis in patients with NAFLD.

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

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)

Chronic Dietary Branched-Chain Amino Acids Induced Lipid Oxidation and Blunted Insulin-Stimulated Glucose Production in Mice With NAFLD

Abstract Objectives Elevated circulating branched-chain amino acids (BCAAs) during insulin resistance are strong predictors of type 2 diabetes mellitus onset. Defects in BCAA degradation are evident in several tissues during insulin resistance and non-alcoholic fatty liver disease (NAFLD). Furthermore, alterations in BCAA metabolism are associated with changes in several aspects lipid metabolism, including lipogenesis, ketogenesis and mitochondrial TCA cycle activity. Considering the crosstalk between BCAAs and lipid metabolism, we hypothesized that chronic supplementation of BCAAs will modulate hepatic insulin resistance and mitochondrial lipid oxidation during NAFLD. Methods Mice (C57BL/6N) were reared on either a low-fat (LF; 10% fat kcal), high-fat (HF; 60% fat kcal or high-fat diet supplemented with BCAA (HFBA; 150% BCAA) for 24 weeks. Metabolic profiling was conducted under fed or overnight fasted (14–16 hrs) conditions. A subset of overnight fasted mice from the HF and HFBA groups were subjected to hyperinsulinemic euglycemic clamps, following implantation of jugular vein catheters. Results Feeding HF and HFBA diets resulted in NAFLD. Circulating BCAAs were higher in ‘fed’ mice consuming HFBA diet (e.g., Valine, µM ± SEM; 311 ± 38 in HF, 432 ± 34 in HFBA, P ≤ 0.05). Overnight fasting significantly reduced BCAA levels in all groups, but the fasting levels of BCAAs remained similar between groups. Fed-to-fasted fold changes in blood glucose, serum insulin and c-peptide were higher in HFBA mice (P ≤ 0.05). Insulin stimulated suppression of glucose production (% ± SEM; HF = 38 ± 11, HFBA = 16 ± 16) was blunted in HFBA mice. Furthermore, fed-to-fasted expression of hepatic genes involved in lipid oxidation, including LCAD, MCAD, PPARa and CPT1a were significantly higher (P ≤ 0.05) in the HFBA mice. Conclusions In summary, chronic BCAA supplementation induced hepatic lipid oxidation gene expression, without any apparent improvements in insulin sensitivity. In conclusion, while the induction of lipid oxidation by BCAAs could explain certain beneficial effects associated with their supplementation, the longer-term impact of the BCAAs on insulin sensitivity need to be further explored. Funding Sources National Institutes of Health (NIH) grant RO1-DK-112865

311-OR: Reduced Hepatic Mitochondrial Metabolism and Markers of Mitochondrial Turnover in Humans Are Linked to NAFLD Progression

Considerable ambiguity exists in the literature regarding the role of hepatic mitochondrial dysfunction in the pathological progression of nonalcoholic fatty liver disease (NAFLD) to steatohepatitis (NASH) and fibrosis. To investigate this relationship, liver biopsies were obtained from patients with morbid obesity undergoing bariatric surgery [n=110, age 44.7±11.7 y, weight 136.3±25.5 kg, BMI 48.7±7.4 kg/m2, AST 35.6±32.1 U/L, ALT 41.0±35.6 U/L, NAFLD activity score 3±2 (NAS; liver injury score: steatosis, ballooning, and inflammation; range: 0-8), fibrosis score 0.5±1.0 (range: 0-4), glucose 104±33 mg/dL] and assessments of mitochondrial function and turnover performed. Data were clustered into four groups based on NAS (No Disease, NAS=0, n=13; Mild NAS=1-2, n=28; Moderate NAS=3-4, n=41; and Severe NAS≥5, n=28). Complete hepatic mitochondrial oxidation of palmitate to CO2 was reduced by ∼50% with Moderate and Severe NAS vs. no disease (p<0.05). This was accompanied by a 50% decrease in hepatic β-HAD activity in all groups with NAFLD (p<0.01), without any differences in citrate synthase activity (p>0.05). In addition to reduced hepatic mitochondrial function, elevated NAS was associated with reduced hepatic mitochondrial biogenesis (PGC1α, SIRT1, PPARα, and AMPKα1 mRNA; p<0.05) and mitochondrial turnover (BNIP3, PARKIN mRNA; p<0.05) markers. Furthermore, hepatic mitochondrial H2O2 emission was elevated in Severe NAS (p<0.01) and positively related to lobular inflammation (r=0.336, p<0.01) and hepatocellular ballooning (r=0.379, p<0.001). Here, we provide novel insight into the relationship between NAFLD severity and hepatic mitochondrial dysfunction. Our findings suggest that compromised hepatic mitochondrial fatty acid metabolism and reduced hepatic mitochondrial biogenesis/turnover may be linked to NAFLD progression in patients with morbid obesity. Disclosure M.P. Moore: None. R. Cunningham: None. G.M. Meers: None. S.A. Johnson: None. A.A. Wheeler: None. R. Ganga: None. J. Pitt: None. N. Spencer: None. A.A. Diaz-Arias: None. A. Swi: None. J.A. Ibdah: None. E.J. Parks: None. R.S. Rector: None. Funding National Institutes of Health (R01DK113701); U.S. Department of Veterans Affairs (I01BX003271)

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)

Glucagon stimulates gluconeogenesis by INSP3R1-mediated hepatic lipolysis.

While it is well-established that alterations in the portal vein insulin/glucagon ratio play a major role in causing dysregulated hepatic glucose metabolism in type 2 diabetes (T2D)1–3, the mechanisms by which glucagon alters hepatic glucose production and mitochondrial oxidation remain poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing hepatic adipose triglyceride lipase activity, intrahepatic lipolysis, hepatic acetyl-CoA content, and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation, mediated by stimulation of the inositol triphosphate receptor-1 (InsP3R-I). Chronic physiological increases in plasma glucagon concentrations increased mitochondrial hepatic fat oxidation and reversed diet-induced hepatic steatosis and insulin resistance in rats and mice; however, the effect of chronic glucagon treatment to reverse hepatic steatosis and glucose intolerance was abrogated in InsP3R-I knockout mice. These results provide new insights into glucagon biology and suggest that InsP3R-I may be a novel therapeutic target to reverse nonalcoholic fatty liver disease and T2D.

Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment 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 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

A mitochondria-targeted fatty acid analogue influences hepatic glucose metabolism and reduces the plasma insulin/glucose ratio in male Wistar rats.

A fatty acid analogue, 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), was previously shown to have hypolipidemic effects in rats by targeting mitochondrial activity predominantly in liver. This study aimed to determine if 1-triple TTA could influence carbohydrate metabolism. Male Wistar rats were treated for three weeks with oral supplementation of 100 mg/kg body weight 1-triple TTA. Blood glucose and insulin levels, and liver carbohydrate metabolism gene expression and enzyme activities were determined. In addition, human myotubes and Huh7 liver cells were treated with 1-triple TTA, and glucose and fatty acid oxidation were determined. The level of plasma insulin was significantly reduced in 1-triple TTA-treated rats, resulting in a 32% reduction in the insulin/glucose ratio. The hepatic glucose and glycogen levels were lowered by 22% and 49%, respectively, compared to control. This was accompanied by lower hepatic gene expression of phosphenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, and Hnf4A, a regulator of gluconeogenesis. Gene expression of pyruvate kinase, catalysing the final step of glycolysis, was also reduced by 1-triple TTA. In addition, pyruvate dehydrogenase activity was reduced, accompanied by 10-15-fold increased gene expression of its regulator pyruvate dehydrogenase kinase 4 compared to control, suggesting reduced entry of pyruvate into the TCA cycle. Indeed, the NADPH-generating enzyme malic enzyme 1 (ME1) catalysing production of pyruvate from malate, was increased 13-fold at the gene expression level. Despite the decreased glycogen level, genes involved in glycogen synthesis were not affected in livers of 1-triple TTA treated rats. In contrast, the pentose phosphate pathway seemed to be increased as the hepatic gene expression of glucose-6-phosphate dehydrogenase (G6PD) was higher in 1-triple TTA treated rats compared to controls. In human Huh7 liver cells, but not in myotubes, 1-triple-TTA reduced glucose oxidation and induced fatty acid oxidation, in line with previous observations of increased hepatic mitochondrial palmitoyl-CoA oxidation in rats. Importantly, this work recognizes the liver as an important organ in glucose homeostasis. The mitochondrially targeted fatty acid analogue 1-triple TTA seemed to lower hepatic glucose and glycogen levels by inhibition of gluconeogenesis. This was also linked to a reduction in glucose oxidation accompanied by reduced PHD activity and stimulation of ME1 and G6PD, favouring a shift from glucose- to fatty acid oxidation. The reduced plasma insulin/glucose ratio indicate that 1-triple TTA may improve glucose tolerance in rats.

Hungry for your alanine: when liver depends on muscle proteolysis.

Fasting requires complex endocrine and metabolic interorgan crosstalk, which involves shifting from glucose to fatty acid oxidation, derived from adipose tissue lipolysis, in order to preserve glucose for the brain. The glucose-alanine (Cahill) cycle is critical for regenerating glucose. In this issue of JCI, Petersen et al. report on their use of an innovative stable isotope tracer method to show that skeletal muscle-derived alanine becomes rate controlling for hepatic mitochondrial oxidation and, in turn, for glucose production during prolonged fasting. These results provide new insight into skeletal muscle-liver metabolic crosstalk during the fed-to-fasting transition in humans.

Regulation of hepatic mitochondrial oxidation by glucose-alanine cycling during starvation in humans.

In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-13C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.

GS-0976 (Firsocostat): an investigational liver-directed acetyl-CoA carboxylase (ACC) inhibitor for the treatment of non-alcoholic steatohepatitis (NASH)

ABSTRACTIntroduction: De novo lipogenesis (DNL) plays a major role in fatty acid metabolism and contributes significantly to triglyceride accumulation within the hepatocytes in patients with nonalcoholic steatohepatitis (NASH). Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl CoA and is a rate-controlling step in DNL. Furthermore, malonyl-CoA is an important regulator of hepatic mitochondrial fat oxidation through its ability to inhibit carnitine palmitoyltransferase I. Therefore, inhibiting ACC pharmacologically represents an attractive approach to treating NASH.Areas covered: This article summarizes preclinical and clinical data on the efficacy and safety of the liver-targeted ACC inhibitor GS-0976 (Firsocostat) for the treatment of NASH. In a phase 2 trial that included 126 patients with NASH and fibrosis, GS-0976 20 mg daily for 12 weeks showed significant relative reduction in liver fat by 29%; however, treatment was associated with an increase in plasma triglycerides with 16 patients having levels >500 mg/dL.Expert opinion: Preclinical and preliminary clinical data support the development of GS-0976 as treatment for NASH. ACC-induced hypertriglyceridemia can be mitigated by fish oil or fibrates, but the long-term cardiovascular effects require further investigations.

Lipid profile variations in non alcoholic fatty liver disease

NAFLD is characterised by fatty infiltration of the liver, mostly in the form of triglycerides, which exceeds 5% of the liver weight. Insulin resistance impairs the suppression of lipolysis, and this leads to an increased release of free fatty acids from adipose tissue so that more are delivered to and taken up by the liver. This excess amount of free fatty acids can overload the hepatic mitochondrial beta oxidation system, the major pathway of fatty acid oxidation in the liver, leading to the accumulation of fatty acids in the liver. The present hospital-based, observational, analytical and comparative study was conducted on 80 subjects including 50 cases of ultrasonographically diagnosed NAFLD and 30 age and gender matched healthy subjects as controls. In this study, lipid profile was assessed in 50 USG diagnosed cases of NAFLD and 30 controls and the results were analysed statistically. The study found a statistically significant change in lipid levels in patients of Non alcoholic fatty liver disease indicating hepatic steatosis. In the present study, increased levels of cholesterol, triacylglycerides, LDL, VLDL and decreased levels of HDL were found. This study concluded that low fat diet, lifestyle modifications, exercises and yoga could be a potential tool to prevent the progression of NAFLD.

Novel Strategies to Treat Hepatic Steatosis and Steatohepatitis.

Concordant with soaring obesity rates, nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. The obesity epidemic demands interventions to reverse obesity-associated hepatic steatosis, NAFLD, and nonalcoholic steatohepatitis, and several new pharmacologic approaches have been developed within the past several years. Steatosis develops when energy delivery to the liver, modulated by rates of hepatic lipogenesis, exceeds the capacity of the liver to utilize or export this energy. Therefore, pharmacologic approaches to reverse hepatic steatosis have focused largely, though not exclusively, on (1) reducing substrate availability to the liver, (2) reducing hepatic lipid synthesis, and (3) increasing hepatic mitochondrial fat oxidation (Figure 1). This Perspective will discuss these three classes of emerging pharmacologic therapies against hepatic steatosis, with the ultimate intent to ameliorate NAFLD and/or nonalcoholic steatohepatitis, and the advantages and pitfalls afforded by each strategy to treat these epidemics of obesity-associated liver disease.

Abstract SY28-02: Hyperinsulinemia mediates the effect of obesity to promote tumor growth in murine breast and colon cancer

Abstract Obesity is associated with an increased incidence of more than one dozen tumor types, and a poorer prognosis of most of these. Intriguingly, interventions which improve the metabolic phenotype of obese individuals – including exercise, weight loss, and treatment with metformin, the most commonly prescribed anti-diabetes drug in the world – slow tumor growth in animals and may have a tumor-slowing effect in humans. The mechanisms for this association have been the subject of numerous studies and are still an area of active debate. Here I will present data arguing that obesity-associated hyperinsulinemia resulting from insulin resistance drives tumor growth in murine models of colon and breast cancer by promoting tumor glucose uptake and oxidation, measured using novel stable isotope methods. In vitro studies reveal that these findings regarding the ability of hyperinsulinemia to drive glucose oxidation are generalizable across multiple human and mouse cell lines from obesity-associated tumors (prostate, breast, ovarian, colon, and pancreatic cancer) but not obesity-independent tumors (small cell lung cancer, melanoma, lymphoma, osteosarcoma). Of particular importance, three insulin-sensitizing and -lowering agents with divergent mechanisms of action slowed breast and colon tumor growth in an insulin-dependent manner: 1) dapagliflozin, a sodium-glucose contransporter-2 (SGLT2) inhibitor, which promotes glycosuria, 2) metformin, which inhibits gluconeogenesis, and 3) a functionally liver-targeted controlled-release mitochondrial protonophore (CRMP), which reverses non-alcoholic fatty liver disease by increasing hepatic mitochondrial oxidation, and thereby improves both hepatic and peripheral insulin sensitivity (the latter due to reduced very low-density lipoprotein export). Restoring hyperinsulinemia via continuous insulin infusion using subcutaneous pellets abrogated the effect of each agent to slow tumor growth. I will further show data indicating that both glucose uptake and oxidation in these obesity-associated tumors is insulin-responsive: diet-induced obesity, causing hyperinsulinemia, increased both glucose uptake and oxidation, whereas insulin-lowering interventions reversed these effects, and restoring hyperinsulinemia by subcutaneous insulin infusion increased tumor glucose uptake and oxidation in mice treated with insulin-lowering agents. These data are contrary to the commonly held view that tumor glucose uptake is constitutively high, and argue that rates of glucose oxidation in tumors may vary dynamically with the hormonal milieu. In summary, I will argue that hyperinsulinemia per semediates the majority of the effect of obesity to drive tumor growth in murine models of colon and breast cancer by increasing tumor glucose uptake and oxidation. These data highlight an important role for insulin-lowering therapies, some of which are already in the clinic (diet and exercise, in particular a ketogenic diet; metformin; SGLT2 inhibitors), and others of which are in preclinical development (liver-targeted mitochondrial uncouplers) to prevent or slow obesity-associated cancer progression. Citation Format: Rachel J. Perry. Hyperinsulinemia mediates the effect of obesity to promote tumor growth in murine breast and colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY28-02.

288-LB: Effects of the Hepatic Mitochondrial Calcium Uniporter on Hepatic Gluconeogenesis and Mitochondrial Metabolism in Awake Mice

Increased hepatic glucose production, due to increased rates of gluconeogenesis, is a contributor to both fasting and postprandial hyperglycemia in patients with T2D. Recent evidence implicates a role for hepatic calcium signaling in mediating glucagon's effect to stimulate glucose production and mitochondrial oxidation; however, the cellular and molecular mechanisms by which glucagon promotes these alterations remain poorly understood. We hypothesize that an acute physiological increase in plasma glucagon concentrations will increase cytosolic and mitochondrial calcium concentrations, promoting increases in rates of hepatic mitochondrial oxidation and gluconeogenesis in a transcription-independent manner. To test this hypothesis, we used mice with a liver-specific loss of the mitochondrial calcium uniporter (MCU), which is the main transporter that allows rapid transport of calcium into the mitochondrial matrix. We found that glucagon increased oxygen consumption rate by 80% (p<0.0001) in WT hepatocytes, while this effect was abrogated in MCU KO hepatocytes. We also measured hepatic mitochondrial oxidation (VCS) in vivo using Positional Isotopomer NMR Tracer Analysis (PINTA) and found a 74% decrease in VCS (p=0.05) in MCU KO mice compared to WT mice. This suggests that tightly regulated mitochondrial calcium signaling is necessary for regulating hepatic mitochondrial oxidation. Next, we measured intrahepatic lipolysis and hepatic gluconeogenesis in vitro and in vivo. We found that glucagon increased lipolysis (p=0.05) and glucose production (p=0.024) by ∼1.5 fold in MCU KO compared to WT hepatocytes. Furthermore, in vivo PINTA results showed that glucagon increased pyruvate carboxylase flux (VPC) by 68% (p=0.08) in MCU KO mice. Taken together, these results suggest that mitochondrial calcium signaling through MCU is necessary to regulate the effect of glucagon to modulate hepatic gluconeogenesis and mitochondrial oxidation. Disclosure J. Lee: None. L. Goedeke: None. Y. Zhang: None. R.J. Perry: Research Support; Self; AstraZeneca. R. Goodman: None. V. Mootha: Advisory Panel; Self; Janssen Pharmaceuticals, Inc. Consultant; Self; 5AM Ventures. Other Relationship; Self; Raze Therapeutics. 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