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Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generationin vivoremain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH2/Q ratio, driving excessive mROS production via reverse electron transport (RET) from site IQin complex I. Using multiple complementary genetic and pharmacological modelsin vivowe demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH2/Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Activation of fatty acid oxidation is a promising therapy for NAFLD. Lowering acetyl-CoA carboxylase (ACC) activity depletes malonyl-CoA, activates CPT1, and increases mitochondrial fatty acid delivery. ACC1/2 liver double KO mice (LDKO) are protected from hepatic steatosis and have elevated ketogenesis in the prandial state. However, fasted LDKO mice exhibit an unexpected inhibition of ketogenesis, indicated by lower blood ketones and reduced in vivo ketone turnover of [3,4-13C2]acetoacetate and [U-13C4]β-hydroxybutyrate. Despite impaired fasting ketogenesis in vivo, perfused livers isolated from fasted mice and starved primary hepatocytes had normal or elevated ketone production, suggesting that a substrate-mediated mechanism inhibits ketosis in vivo. There were no differences in plasma NEFAs, but hepatic lactate was elevated during fasting in LDKO mice. The increase in lactate was sufficient to suppress ketone production in isolated perfused liver. An in vivo [U-13C3]lactate/pyruvate challenge was more rapidly diluted in fasted LKDO mice, indicating that alternative endogenous sources of pyruvate were available. Indeed, essential amino acids were elevated in fasted LDKO liver, but not plasma, suggestive of hepatic proteolysis as a source of amino acids. Liver size was 20% larger in LDKO mice, independent of glycogen storage or triglyceride content, but was consistent with increased insulin action and activation of Nrf2 which have been linked to hepatomegaly. Autophagy during fasting was normal or increased, suggesting that hepatomegaly supplies excess amino acids in the fasted state. Injection of exogenous alanine or glutamine in fasted WT mice recapitulated the fasted LDKO phenotype by increasing plasma glucose and lactate and decreasing ketone concentrations. Thus, hepatic growth is increased in the absence of malonyl-CoA synthesis, which provides a surplus of amino acids, anaplerotic maintenance of the TCA cycle and lower ketogenesis during fasting. Disclosure S.Deja: None. S.C.Burgess: n/a. J.A.Fletcher: None. B.Kucejova: None. M.N.Mizerska: None. X.Fu: None. C.Kim: None. J.Browning: None. J.Young: Consultant; Pfizer Inc., Research Support; Merck & Co., Inc., Stock/Shareholder; Metalytics, Inc. J.D.Horton: Board Member; Draupnir Bio, Consultant; Eli Lilly and Company, ESPERION Therapeutics, Inc., Gilead Sciences, Inc., Janssen Pharmaceuticals, Inc., Pfizer Inc., Regeneron Pharmaceuticals Inc. Funding National Institutes of Health (R01DK078184, P41EB015908)

Redox balance is critical for numerous cellular functions, including substrate oxidation, biosynthesis, and antioxidant defenses. Redox homeostasis is dysregulated in many metabolic diseases, including obesity and Type 2 diabetes. In addition to its role in replenishing TCA cycle intermediates, we previously showed that pyruvate carboxylase (PC) -mediated anaplerosis is necessary to regulate redox state, including the NADPH/NADP+ ratio and antioxidant capacity in liver, though the mechanism is unclear. We hypothesized that PC elicits effects on redox by modifying ratios of metabolites involved in redox mediated exchange reactions. Malic enzyme-1 (Me1) catalyzes the reversible, NADPH dependent, decarboxylation of malate to pyruvate. This enzyme plays a vital role in recycling malate and providing NADPH during lipogenesis. We show here that Me1 mediates redox by sensing TCA cycle function through the mass action of malate and pyruvate. We tested the role of Me1, as a sensor of anaplerosis in the TCA cycle and regulator of redox, using acute Me1 knockdown (Me1KD) in liver-specific PC knockout (LPCKO) mice. Stable isotope tracers were used to examine anaplerosis, gluconeogenesis, and pyruvate cycling fluxes, and targeted metabolomics were used to evaluate tissue energy status and redox. Anaplerosis, pyruvate cycling, and gluconeogenesis were lower in the absence of liver PC but were unaffected by Me1KD. Loss of PC decreased the malate/pyruvate ratio, congruent with impaired conversion of pyruvate to TCA cycle intermediates, which resulted in a concomitant decrease in the NADPH/NADP+ ratio. Acute removal of Me1 in LPCKO mice normalized NADPH/NADP+ and restored the malate/pyruvate ratio. Additionally, mitochondrial NAD+/NADH and FAD+/FADH redox states were more oxidized in LPCKO liver and were normalized by loss of Me1. Thus, despite not altering the aforementioned fluxes, Me1 transmits redox state in response to TCA cycle function by sensing alterations in the malate/pyruvate ratio. Disclosure M.R.Inigo: None. J.A.Fletcher: None. B.Kucejova: None. G.Sharma: None. S.Deja: None. X.Fu: None. S.C.Burgess: n/a. Funding Robert A. Welch Foundation Grant (I-1804)

Exercise is an important lifestyle intervention for the prevention and treatment of NAFLD. Importantly, exercise can reduce hepatic steatosis independent of changes in body mass, which may stem from increased oxidative demand in liver during exercise. Increased hepatic gluconeogenesis (GNG) and tricarboxylic acid (TCA) cycle flux have been observed during exercise in rodents, but it is unclear whether this adaptation is chronically maintained outside of the exercise window. Liver metabolism is also altered by nutritional status. Fasting promotes hepatic fat oxidation, ketogenesis and GNG, while feeding promotes glycogen storage and fat synthesis. Hence, fasting and exercise may have a positive interaction, whereas exogenous nutrient intake or glucose infusion is known to reduce the induction of GNG during exercise. Here, we tested whether nutritional status impacts exercise induced hepatic metabolic adaptations. Sprague-Dawley rats were kept sedentary or provided voluntary wheel running (VWR) for 4 weeks in the presence or absence of food restriction during the dark phase when rats spontaneously run. Experiments were conducted one day following the last exercise bout. Hepatic metabolic flux was examined by in vivo stable isotope infusions of [U-13C3]propionate, [3,4-13C2]glucose and 2H2O in rats using mass spectrometry and computational analysis. Chronic VWR in the fed state had no effect on basal hepatic fluxes outside of the exercise window. However, exercise during food restriction chronically induced a 2-fold induction of TCA cycle turnover, increase in GNG from glycerol and lowered ketogenesis outside of the exercise window. Thus, VWR during food restriction triggered an increase in hepatic energy demand that was sustained for at least 24 hours, while the effects of exercise in the fed state dissipated by 24 hours. These data suggest that acute nutritional state may be an important factor in the long-term programming of liver metabolism by exercise. Disclosure S.Deja: None. B.Kucejova: None. A.Maurer: None. M.N.Mizerska: None. X.Fu: None. J.P.Thyfault: None. S.C.Burgess: n/a. Funding National Institutes of Health (R01DK121497, R01DK078184, P41EB015908)

Hepatic gluconeogenesis from amino acids contributes significantly to diabetic hyperglycemia, but the molecular mechanisms involved are incompletely understood. Alanine transaminases (ALT1 and ALT2) catalyze the interconversion of alanine and pyruvate, which is required for gluconeogenesis from alanine. We find that ALT2 is overexpressed in the liver of diet-induced obese and db/db mice and that the expression of the gene encoding ALT2 (GPT2) is downregulated following bariatric surgery in people with obesity. The increased hepatic expression of Gpt2 in db/db liver is mediated by activating transcription factor 4, an endoplasmic reticulum stress-activated transcription factor. Hepatocyte-specific knockout of Gpt2 attenuates incorporation of