ABSTRACT
The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by(13)C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction (P< 0.05) of mitochondrial fluxes (µmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of "lipotoxic" metabolites in the liver and could hasten inflammation and the metabolic transition to NASH.
Subject(s)
Citric Acid Cycle/physiology , Fatty Acids, Nonesterified/metabolism , Insulin Resistance , Liver/metabolism , Mitochondria, Liver/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , RNA, Messenger/metabolism , Animals , Carbon Isotopes , Carnitine/analogs & derivatives , Carnitine/metabolism , Ceramides/metabolism , Chromatography, Liquid , Dietary Fats , Dietary Sucrose , Diglycerides/metabolism , Disease Models, Animal , Fructose , Glucose Clamp Technique , Inflammation , Liver/pathology , Magnetic Resonance Spectroscopy , Metabolome , Mice , Non-alcoholic Fatty Liver Disease/pathology , Oxidation-Reduction , Real-Time Polymerase Chain Reaction , Severity of Illness Index , Tandem Mass Spectrometry , Trans Fatty Acids , TranscriptomeABSTRACT
The goal of this research was to find the most comprehensive lipid extraction of blood plasma, while also providing adequate aqueous preparation for metabolite analysis. Comparisons have been made previously of the Folch, Bligh-Dyer, and Matyash lipid extractions; furthermore, this paper provides an additional comparison of a phospholipid removal plate for analysis. This plate was used for lipid extraction rather than its intended use in lipid removal for polar analysis, and it proves to be robust for targeted lipid analysis. Folch and Matyash provided reproducible recovery over a range of lipid classes, however the Matyash aqueous layer compared well to a typical methanol preparation for polar metabolite analysis. Thus, the Matyash method is the best choice for an untargeted biphasic extraction for metabolomics and lipidomics in blood plasma.
