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. 2012 Jun;53(6):1080-92.
doi: 10.1194/jlr.M023382. Epub 2012 Apr 9.

Elevated TCA Cycle Function in the Pathology of Diet-Induced Hepatic Insulin Resistance and Fatty Liver

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Free PMC article

Elevated TCA Cycle Function in the Pathology of Diet-Induced Hepatic Insulin Resistance and Fatty Liver

Santhosh Satapati et al. J Lipid Res. .
Free PMC article

Abstract

The manner in which insulin resistance impinges on hepatic mitochondrial function is complex. Although liver insulin resistance is associated with respiratory dysfunction, the effect on fat oxidation remains controversial, and biosynthetic pathways that traverse mitochondria are actually increased. The tricarboxylic acid (TCA) cycle is the site of terminal fat oxidation, chief source of electrons for respiration, and a metabolic progenitor of gluconeogenesis. Therefore, we tested whether insulin resistance promotes hepatic TCA cycle flux in mice progressing to insulin resistance and fatty liver on a high-fat diet (HFD) for 32 weeks using standard biomolecular and in vivo (2)H/(13)C tracer methods. Relative mitochondrial content increased, but respiratory efficiency declined by 32 weeks of HFD. Fasting ketogenesis became unresponsive to feeding or insulin clamp, indicating blunted but constitutively active mitochondrial β-oxidation. Impaired insulin signaling was marked by elevated in vivo gluconeogenesis and anaplerotic and oxidative TCA cycle flux. The induction of TCA cycle function corresponded to the development of mitochondrial respiratory dysfunction, hepatic oxidative stress, and inflammation. Thus, the hepatic TCA cycle appears to enable mitochondrial dysfunction during insulin resistance by increasing electron deposition into an inefficient respiratory chain prone to reactive oxygen species production and by providing mitochondria-derived substrate for elevated gluconeogenesis.

Figures

Fig. 1.
Fig. 1.
HFD mice have fatty liver with impaired insulin action. (A) Oil red O-stained liver sections (original magnification ×10; n = 2); (B) hepatic insulin sensitivity index; (C) insulin-stimulated phosphorylation of AktT308 and AktS473; (D) insulin-stimulated Foxo1 phosphorylation; (E) endogenous glucose production; (F) total gluconeogenesis; and (G) hepatic anaplerosis or pyruvate carboxylase flux in overnight-fasted mice fed either a control or a high-fat diet for 8, 16, and 32 weeks. Data are presented as the mean ± SE (n = 6–8).
Fig. 2.
Fig. 2.
Hepatic mitochondrial metabolism is elevated in HFD mice. (A) Carbon-13 isotopomer analysis of plasma glucose was used to evaluate hepatic fluxes relative to the TCA cycle after administration of [U-13C]propionate as described by Landau et al. (46) and adapted to NMR analysis (48) for determination of absolute flux (41). See supplementary Table IV for isotopomer data. (B) In vivo hepatic mitochondrial TCA cycle flux in overnight-fasted mice (awake and unrestrained). (C) Oxidation of [1-14C]palmitate in liver homogenates from 16-week-old HFD mice. (D) Determination of in vivo ketogenic flux by apparent turnover of [3,4-13C2]acetoacetate and [U-13C4]β-hydroxybutyrate. (E, F) Suppression of fasting in vivo ketogenesis by (E) euglycemic hyperinsulinemic clamp or (F) feeding. (G) Mitochondrial DNA content determined by real-time quantitative PCR. (H) Isolated mitochondrial respiration measured as O2 consumption with palmitoylcarnitine/malate as substrate in the presence of no ADP (State 2), ADP (State 3), or ADP depletion (State 4) expressed as % difference from their respective age-matched control average (see supplementary Table VII for raw data). Data are presented as the mean ± SE (n = 4–8).
Fig. 3.
Fig. 3.
Markers of oxidative stress and inflammation are elevated with the same timing as increased oxidative metabolism in the liver of HFD mice. (A) Total ceramide levels in the liver measured by LC-MS/MS analysis; (B) superoxide dismutase activity measured in liver homogenates; (C) carbonylation of proteins in lysates from crude liver mitochondria with Coomasie-stained gel as loading control; (D) lipid peroxidation assessed by analysis of TBARS; (E–H) relative gene expression analysis of (E) Il6, (F) Tnfα, and (G) Ptgs2, and (H) Hematoxylin-stained liver sections with arrows indicating macrophage infiltration (original magnification ×20; n = 2) in overnight-fasted mice fed either a control or high-fat diet for 8, 16, and 32 weeks. Data are presented as the mean ± SE (n = 6–8) unless otherwise noted.

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