Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity

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.

Keywords: hepatic insulin resistance; mitochondria; nonalcoholic steatohepatitis; steatosis.

Fig. 1.

High-fructose high-trans-fat diet (TFD)-fed mice develop hepatic insulin resistance and simple steatosis by 8 wk that then transitions to nonalcoholic steatohepatitis (NASH) by 24 wk. A: trichrome staining of liver slices reveals normal hepatocyte morphology in mice fed a control diet for 8 or 24 wk. Liver histology of mice fed TFD for 8 wk reveals panacinar steatosis. Liver histology of mice fed TFD for 24 wk reveals microvesicular and macrovesicular steatosis with stage 1b fibrosis. B: expression of genes involved in fibrogenesis was significantly higher in the 24-wk TFD-fed mice compared with their 8-wk counterparts. C: endogenous glucose production fails to get suppressed in 8-wk TFD-fed mice during euglycemic-hyperinsulinemic clamp (insulin stimulation), indicating hepatic insulin resistance. Values are means ± SE (n = 5–7 mice/group). *P ≤ 0.05 between control (C) vs. TFD-fed mice or C vs. C insulin stimulated. ^#P ≤ 0.05 between 8-wk TFD and 24-wk TFD-fed mice.

Fig. 2.

Hepatic mitochondrial tricarboxylic acid (TCA) cycle activity is induced in mice with NASH. Overnight fasting basal levels of endogenous glucose production (A), TCA cycle flux (B), anaplerosis (C), and pyruvate cycling (D), determined using ¹³C-nuclear magnetic resonance (NMR)-based isotopomer analysis, were all elevated in mice fed a TFD for 24 wk compared with their age-matched control counterparts. E: robust relationship between hepatic mitochondrial TCA cycle flux and mitochondrial anaplerosis. Values are means ± SE (n = 6–7/group). *P ≤ 0.05 between C and TFD-fed mice.

Fig. 3.

Principal component analysis provides a snapshot of the overall metabolite profiles during the transition from simple steatosis to NASH. Summary of hepatic profiles of diacylglycerols (A), ceramides (B), acylcarnitines (C), organic acids (D), and amino acids (E) represented as a function of age (8 or 24 wk) and diet (black, TFD; gray, C). Data are means ± SE of metabolite scores from the first principal component (PC1) of variation in each metabolite class.

Fig. 4.

Elevated levels of ceramides and diacylglycerols (DAGs) together with altered acylcarnitine profile in the liver of TFD mice indicate inefficient fat oxidation. Total hepatic DAG content (A), total hepatic ceramide content (B), fold changes in individual hepatic DAGs (C), fold changes in individual hepatic ceramides (D), and fold changes in individual hepatic acylcarnitines (E) relative to their respective age-matched controls. Mice were fed a TFD for either 8 or 24 wk. Values are means ± SE (n = 6–7/group). *P ≤ 0.05 between C vs. TFD-fed mice. ^#P ≤ 0.05 between 8- vs. 24-wk dietary treatments.