Loss of hepatic phosphoenolpyruvate carboxykinase 1 dysregulates metabolic responses to acute exercise but enhances adaptations to exercise training in mice.

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. 2H/13C 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 ß-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.

Acute aerobic exercise reveals that FAHFAs distinguish the metabolomes of overweight and normal-weight runners.

BackgroundResponses of the metabolome to acute aerobic exercise may predict maximum oxygen consumption (VO2max) and longer-term outcomes, including the development of diabetes and its complications.MethodsSerum samples were collected from overweight/obese trained (OWT) and normal-weight trained (NWT) runners prior to and immediately after a supervised 90-minute treadmill run at 60% VO2max (NWT = 14, OWT = 11) in a cross-sectional study. We applied a liquid chromatography high-resolution-mass spectrometry-based untargeted metabolomics platform to evaluate the effect of acute aerobic exercise on the serum metabolome.ResultsNWT and OWT metabolic profiles shared increased circulating acylcarnitines and free fatty acids (FFAs) with exercise, while intermediates of adenine metabolism, inosine, and hypoxanthine were strongly correlated with body fat percentage and VO2max. Untargeted metabolomics-guided follow-up quantitative lipidomic analysis revealed that baseline levels of fatty acid esters of hydroxy fatty acids (FAHFAs) were generally diminished in the OWT group. FAHFAs negatively correlated with visceral fat mass and HOMA-IR. Strikingly, a 4-fold decrease in FAHFAs was provoked by acute aerobic running in NWT participants, an effect that negatively correlated with circulating IL-6; these effects were not observed in the OWT group. Machine learning models based on a preexercise metabolite profile that included FAHFAs, FFAs, and adenine intermediates predicted VO2max.ConclusionThese findings in overweight human participants and healthy controls indicate that exercise-provoked changes in FAHFAs distinguish normal-weight from overweight participants and could predict VO2max. These results support the notion that FAHFAs could modulate the inflammatory response, fuel utilization, and insulin resistance.Trial registrationClinicalTrials.gov, NCT02150889.FundingNIH DK091538, AG069781, DK098203, TR000114, UL1TR002494.

Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-ß-hydroxybutyrate dehydrogenase hepatocyte-deficient mice.

OBJECTIVE: Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-ß-hydroxybutyrate (D-ßOHB). METHODS: Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-ß-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies. RESULTS: Exogenously administered AcAc is reduced to D-ßOHB, which increases hepatic NAD+/NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-ßOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-ßOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia. CONCLUSIONS: Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-ßOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.

Determination of ketone bodies in biological samples via rapid UPLC-MS/MS.

Efforts to enhance wellness and ameliorate disease via nutritional, chronobiological, and pharmacological interventions have markedly intensified interest in ketone body metabolism. The two ketone body redox partners, acetoacetate (AcAc) and D-ß-hydroxybutyrate (D-ßOHB) serve distinct metabolic and signaling roles in biological systems. A highly efficient, specific, and reliable approach to simultaneously quantify AcAc and D-ßOHB in biological specimens is lacking, due to challenges of separating the structural isomers and enantiomers of ßOHB, and to the chemical instability of AcAc. Here we present a single UPLC-MS/MS method that simultaneously quantifies both AcAc and ßOHB using independent stable isotope internal standards for both ketones. This method incorporates one sample preparation step requiring only 7 min of analysis per sample. The output is linear over three orders of magnitude, shows very low limits of detection and quantification, is highly specific, and shows favorable recovery yields from mammalian serum and tissue samples. Tandem MS discriminates D-ßOHB from structural isomers 2- or 4-hydroxybutyrate as well as 3-hydroxyisobutyrate (3-HIB). Finally, a simple derivatization distinguishes D- and L-enantiomers of ßOHB, 3-HIB, and 2-OHB, using the same rapid chromatographic platform. Together, this simple, efficient, reproducible, scalable, and all-encompassing method will support basic and clinical research laboratories interrogating ketone metabolism and redox biochemistry.

Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer.

The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase-selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel's direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor-bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC.

Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.