Western Diet Decreases the Liver Mitochondrial Oxidative Flux of Succinate: Insight from a Murine NAFLD Model

Abstract

Mitochondria play an essential role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Previously, we found that succinate-activated respiration was the most affected mitochondrial parameter in mice with mild NAFLD. In this study, we focused on the role of succinate dehydrogenase (SDH) in NAFLD pathogenesis. To induce the progression of NAFLD to nonalcoholic steatohepatitis (NASH), C57BL/6J mice were fed a Western-style diet (WD) or control diet for 30 weeks. NAFLD severity was evaluated histologically and the expression of selected proteins and genes was assessed. Mitochondrial respiration was measured by high-resolution respirometry. Liver redox status was assessed using glutathione, malondialdehyde, and mitochondrial production of reactive oxygen species (ROS). Metabolomic analysis was performed by GC/MS. WD consumption for 30 weeks led to reduced succinate-activated respiration. We also observed decreased SDH activity, decreased expression of the SDH activator sirtuin 3, decreased gene expression of SDH subunits, and increased levels of hepatic succinate, an important signaling molecule. Succinate receptor 1 (SUCNR1) gene and protein expression were reduced in the livers of WD-fed mice. We did not observe signs of oxidative damage compared to the control group. The changes observed in WD-fed mice appear to be adaptive to prevent mitochondrial respiratory chain overload and massive ROS production.

Keywords: mitochondria; nonalcoholic fatty liver disease; oxidative phosphorylation; respirometry; succinate; succinate dehydrogenase.

Figure 1

Representative histopathological samples of epididymal adipose tissue taken from mice fed (a) a control diet (CD30) or (b) a Western-style diet (WD30) for 30 weeks—hematoxylin and eosin staining. Arrows indicate macrophage crown-like structures in the WD30 group.

Figure 2

Representative histopathological samples of livers taken from mice fed a control diet (a,b) or a Western-style diet (c,d) for 30 weeks. Hematoxylin and eosin staining (a,c) and sirius red staining (b,d). Arrows indicate inflammatory infiltrates (c) and fibrosis (d).

Figure 3

Mitochondrial respiration in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. (a) Respiratory protocol 1. (b) Respiratory protocol 2. Liver homogenates were loaded at a protein concentration of 0.15 mg/mL, and substrates, uncoupler, and inhibitors were gradually added according to the protocol: P (pyruvate, 5 mM), M (malate, 2 mM), M.1 (malate, 0.1 mM), G (glutamate, 10 mM), S (succinate, 50 mM), Oct (octanoylcarnitine, 0.5 mM), and Gp (glycerophosphate, 10 mM). The LEAK state was measured in the presence of reducing substrates but in the absence of ADP. The oxidative phosphorylation (OXPHOS) state was measured in the presence of saturating concentrations of ADP (2.5 mM) and defined reduced substrates. The electron transfer system (ETS) state was measured as oxygen consumption in the noncoupled state at the optimum uncoupler concentration (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, 1.5-2 µM), which was obtained by stepwise titration to induce maximum oxygen flux. Capacity indicates respiration in the presence of all substrates. To inhibit NADH-dependent substrate respiration, rotenone (0.5 µM) was added. Data were corrected for residual oxygen consumption (ROX) as the baseline state. ROX is respiration due to oxidative side reactions that continue after inhibition of the ET pathway (antimycin A, 2.5 µM). Outer mitochondrial membrane integrity was assessed by the addition of cytochrome c (10 µM). Results are expressed as the means ± SD; * p < 0.05 (n = 6 each group).

Figure 4

Hepatic succinate dehydrogenase (SDH) in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. (a) Specific activity of SDH. (b) Relative gene expression of SDH subunit A. (c) Relative protein expression of SDH subunit A. (d) Relative gene expression of SDH subunit B. (e) Relative protein expression of SDH subunit B. Results are expressed as the means ± SD; * p < 0.05, ** p < 0.01 (n = 6 each group).

Figure 5

(a) Relative hepatic sirtuin 3 (SIRT3) gene and (b) protein expression. (c) Relative hepatic tumor necrosis factor receptor associated protein 1 (TRAP1) gene and (d) protein expression in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. Results are expressed as the means ± SD; * p < 0.05, ** p < 0.01 (n = 6 each group).

Figure 6

Parameters of liver oxidative stress in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. (a) Liver reduced glutathione (GSH) level. (b) Liver malondialdehyde (MDA) level. (c) Production of reactive oxygen species (ROS) in liver homogenate measured as fluorescence changes in dichlorofluorescein. LEAK, measurements in the absence of ADP; OXPHOS, measurements in the presence of ADP (2.5 mM); NADH-linked substrates (pyruvate, 5 mM; glutamate, 10 mM; malate, 2 mM); S (succinate, 10 mM); capacity (NADH-linked substrates + succinate + octanoylcarnitine, 0.5 mM + glycerophosphate, 10 mM); β-oxidation (octanoylcarnitine + malate). Results are expressed as the means ± SD (n = 6 each group).

Figure 7

(a) Relative hepatic UCP2 gene and (b) protein expression in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. Results are expressed as the means ± SD; ** p < 0.01 (n = 6 each group).

Figure 8

Liver metabolomic analysis in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. (a) Relative level of citrate. (b) Relative level of succinate. (c) Relative level of fumarate. (d) Relative level of malate. (e) Relative level of β-hydroxybutyrate. (f) Relative level of lactate. Results are expressed as the means ± SD; * p < 0.05, ** p < 0.01 (n = 6 each group).

Figure 9

SUCNR1 expression in mice fed a control diet (CD30) or a Western-style diet (WD30) for 30 weeks. (a) Relative gene and (b) protein expression. Results are expressed as the means ± SD; ** p < 0.01 (n = 6 each group).