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The progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) to metabolic-dysfunction-associated steatohepatitis (MASH) involves complex alterations in both liver-autonomous and systemic metabolism that influence the liver's balance of fat accretion and disposal. Here, we quantify the relative contribution of hepatic oxidative pathways to liver injury in MASLD-MASH. Using NMR spectroscopy, UHPLC-MS, and GC-MS, we performed stable-isotope tracing and formal flux modeling to quantify hepatic oxidative fluxes in humans across the spectrum of MASLD-MASH, and in mouse models of impaired ketogenesis. We found in humans with MASH, that liver injury correlated positively with ketogenesis and total fat oxidation, but not with turnover of the tricarboxylic acid cycle. The use of loss-of-function mouse models demonstrated that disruption of mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting step of ketogenesis, impairs overall hepatic fat oxidation and induces a MASLD-MASH-like phenotype. Disruption of mitochondrial β-hydroxybutyrate dehydrogenase (BDH1), the terminal step of ketogenesis, also impaired fat oxidation, but surprisingly did not exacerbate steatotic liver injury. Taken together, these findings suggest that quantifiable variations in overall hepatic fat oxidation may not be a primary determinant of MASLD-to-MASH progression, but rather, that maintenance of hepatic ketogenesis could serve a protective role through additional mechanisms that extend beyond quantified overall rates of fat oxidation.

Regular exercise elicits adaptations in glucose and lipid metabolism that allow the body to meet energy demands of subsequent exercise bouts more effectively and mitigate metabolic diseases including fatty liver. Energy discharged during the acute exercise bouts that comprise exercise training may be a catalyst for liver adaptations. During acute exercise, liver glycogenolysis and gluconeogenesis are accelerated to supply glucose to working muscle. Lower liver energy state imposed by gluconeogenesis and related pathways activates AMP-activated protein kinase (AMPK), which conserves ATP partly by promoting lipid oxidation. This study tested the hypothesis that AMPK is necessary for liver glucose and lipid adaptations to training. Liver-specific AMPKα1α2 knockout ( AMPKα1α2 fl/fl +AlbCre ) mice and littermate controls ( AMPKα1α2 fl/fl ) completed sedentary and exercise training protocols. Liver nutrient fluxes were quantified at rest or during acute exercise following training. Liver metabolites and molecular regulators of metabolism were assessed. Training increased liver glycogen in AMPKα1α2 fl/fl mice, but not in AMPKα1α2 fl/fl +AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2 fl/fl +AlbCre mice. Deletion of AMPKα1α2 attenuated training-induced declines in liver diacylglycerides. In particular, training lowered the concentration of unsaturated and elongated fatty acids comprising diacylglycerides in AMPKα1α2 fl/fl mice, but not in AMPKα1α2 fl/fl +AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2 fl/fl +AlbCre mice. These lipid responses were independent of differences in tricarboxylic acid cycle fluxes. In conclusion, AMPK is required for liver training adaptations that are critical to glucose and lipid metabolism. NEW & NOTEWORTHY This study shows that the energy sensor and transducer, AMP-activated protein kinase (AMPK), is necessary for an exercise training-induced: 1 ) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, 2 ) decrease in liver glycerolipids independent of tricarboxylic acid (TCA) cycle flux, and 3 ) decline in the desaturation and elongation of fatty acids comprising liver diacylglycerides. The mechanisms defined in these studies have implications for use of regular exercise or AMPK-activators in patients with fatty liver.

Acute exercise increases liver gluconeogenesis to supply glucose to working muscles. Concurrently, elevated liver lipid breakdown fuels the high energetic cost of gluconeogenesis. This functional coupling between liver gluconeogenesis and lipid oxidation has been proposed to underlie the ability of regular exercise to enhance liver mitochondrial oxidative metabolism and decrease liver steatosis in individuals with nonalcoholic fatty liver disease. Herein we tested whether repeated bouts of increased hepatic gluconeogenesis are necessary for exercise training to lower liver lipids. Experiments used diet-induced obese mice lacking hepatic phosphoenolpyruvate carboxykinase 1 (KO) to inhibit gluconeogenesis and wild-type (WT) littermates. 2 H/ 13 C metabolic flux analysis quantified glucose and mitochondrial oxidative fluxes in untrained mice at rest and during acute exercise. Circulating and tissue metabolite levels were determined during sedentary conditions, acute exercise, and refeeding postexercise. Mice also underwent 6 wk of treadmill running protocols to define hepatic and extrahepatic adaptations to exercise training. Untrained KO mice were unable to maintain euglycemia during acute exercise resulting from an inability to increase gluconeogenesis. Liver triacylglycerides were elevated after acute exercise and circulating β-hydroxybutyrate was higher during postexercise refeeding in untrained KO mice. In contrast, exercise training prevented liver triacylglyceride accumulation in KO mice. This was accompanied by pronounced increases in indices of skeletal muscle mitochondrial oxidative metabolism in KO mice. Together, these results show that hepatic gluconeogenesis is dispensable for exercise training to reduce liver lipids. This may be due to responses in ketone body metabolism and/or metabolic adaptations in skeletal muscle to exercise. NEW & NOTEWORTHY Exercise training reduces hepatic steatosis partly through enhanced hepatic terminal oxidation. During acute exercise, hepatic gluconeogenesis is elevated to match the heightened rate of muscle glucose uptake and maintain glucose homeostasis. It has been postulated that the hepatic energetic stress induced by elevating gluconeogenesis during acute exercise is a key stimulus underlying the beneficial metabolic responses to exercise training. This study shows that hepatic gluconeogenesis is not necessary for exercise training to lower liver lipids.