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Global 13C tracing and metabolic flux analysis of intact human liver tissue ex vivo

ABSTRACT: Liver metabolism is central to human physiology and influences the pathogenesis of common metabolic diseases. Yet, our understanding of human liver metabolism remains incomplete, with much of current knowledge based on animal or cell culture models that do not fully recapitulate human physiology. Here, we performed in-depth measurement of metabolism in intact human liver tissue ex vivo using global 13C tracing, non-targeted mass spectrometry and model-based metabolic flux analysis. Cultured liver tissue exhibited normal anatomical structure and retained canonical liver functions such as glucose production, albumin and VLDL synthesis at near-physiological rates. Isotope tracing with a highly 13C-labeled medium generated 13C enrichment in hundreds of compounds, allowing qualitative assessment of a wide range of metabolic pathways within a single experiment. This confirmed well-known features of liver metabolism, but also revealed unexpected metabolic activities such as de novo creatine synthesis and branched-chain amino acid transamination, where human liver appears to differ from rodent models. Metabolic flux analysis identified glycogenolysis as the main source of glucose production, which could be suppressed by pharmacological inhibition of glycogen phosphorylase. Glucose production ex vivo also correlated with donor plasma glucose, suggesting that cultured liver tissue retains individual metabolic phenotypes. Moreover, liver tissue responded to postprandial levels of nutrients and insulin by suppressing glucose production and increasing nutrient uptake. Isotope tracing ex vivo allows measuring human liver metabolism with great depth and resolution in an experimentally tractable system.

ORGANISM(S): Homo sapiens

PROVIDER: GSE271041 | GEO | 2024/07/08

REPOSITORIES: GEO

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Project description:Background: Heart failure involves metabolic alterations including increased glycolysis despite unchanged or decreased glucose oxidation. The mitochondrial pyruvate carrier (MPC) regulates pyruvate entry into the mitochondrial matrix, and cardiac deletion of the MPC in mice causes heart failure. How MPC deletion results in heart failure is unknown. Methods: We performed targeted metabolomics and isotope tracing in wildtype (fl/fl) and cardiac-specific Mpc2-/- (CS-Mpc2-/-) hearts after in vivo injection of 13C-glucose. Cardiac glycogen was measured biochemically and by transmission electron microscopy. Cardiac glucose uptake of 2-deoxyglucose was measured and western blotting performed to analyze insulin signaling and enzymatic regulators of glycogen synthesis and degradation. Isotope tracing and glycogen analysis was also performed in hearts from mice fed either low-fat diet or a ketogenic diet previously shown to reverse the heart failure in CS-Mpc2-/- mice. Cardiac glycogen was also assessed in mice infused with angiotensin-II that were fed either low-fat or ketogenic diet. Results: Failing CS-Mpc2-/- hearts contained normal levels of ATP and phosphocreatine, suggesting their heart failure is not caused by energetic stress. These hearts displayed increased enrichment from 13C-glucose and increased glycolytic metabolite pool sizes. 13C enrichment and pool size was also increased for the glycogen intermediate UDP-glucose, as well as increased enrichment of the glycogen pool. Glycogen levels were increased ~6-fold in the failing CS-Mpc2-/- hearts, and glycogen granules were easily detected by electron microscopy. This increased glycogen synthesis occurred despite enhanced inhibitory phosphorylation of glycogen synthase and reduced expression of the priming enzyme glycogenin-1. In young, non-failing CS-Mpc2-/- hearts, increased glycolytic 13C enrichment occurred, but glycogen levels remained low and unchanged compared to fl/fl hearts. Feeding a ketogenic diet to CS-Mpc2-/- mice reversed the heart failure and normalized the cardiac glycogen and glycolytic metabolite accumulation. Cardiac glycogen levels were also elevated in mice infused with angiotensin II, and both the cardiac hypertrophy and glycogen levels were improved by ketogenic diet. Conclusions: Our results indicate that loss of MPC in the heart increases glycolytic metabolism and ultimately glycogen accumulation and heart failure, while a ketogenic diet can reverse both the glycogen accumulation and heart failure. We conclude that maintaining mitochondrial pyruvate import and metabolism is critical for the heart, unless cardiac pyruvate metabolism is dramatically reduced by consumption of a ketogenic diet.

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Global 13C tracing and metabolic flux analysis of intact human liver tissue ex vivo

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