Aerobic capacity mediates susceptibility for the transition from steatosis to steatohepatitis

Abstract

Key points: Low intrinsic aerobic capacity is associated with increased all-cause and liver-related mortality in humans. Low intrinsic aerobic capacity in the low capacity runner (LCR) rat increases susceptibility to acute and chronic high-fat/high-sucrose diet-induced steatosis, without observed increases in liver inflammation. Addition of excess cholesterol to a high-fat/high-sucrose diet produced greater steatosis in LCR and high capacity runner (HCR) rats. However, the LCR rat demonstrated greater susceptibility to increased liver inflammatory and apoptotic markers compared to the HCR rat. The progressive non-alcoholic fatty liver disease observed in the LCR rats following western diet feeding was associated with further declines in liver fatty acid oxidation and mitochondrial respiratory capacity compared to HCR rats.

Abstract: Low aerobic capacity increases risk for non-alcoholic fatty liver disease and liver-related disease mortality, but mechanisms mediating these effects remain unknown. We recently reported that rats bred for low aerobic capacity (low capacity runner; LCR) displayed susceptibility to high fat diet-induced steatosis in association with reduced hepatic mitochondrial fatty acid oxidation (FAO) and respiratory capacity compared to high aerobic capacity (high capacity runner; HCR) rats. Here we tested the impact of aerobic capacity on susceptibility for progressive liver disease following a 16-week 'western diet' (WD) high in fat (45% kcal), cholesterol (1% w/w) and sucrose (15% kcal). Unlike previously with a diet high in fat and sucrose alone, the inclusion of cholesterol in the WD induced hepatomegaly and steatosis in both HCR and LCR rats, while producing greater cholesterol ester accumulation in LCR compared to HCR rats. Importantly, WD-fed low-fitness LCR rats displayed greater inflammatory cell infiltration, serum alanine transaminase, expression of hepatic inflammatory markers (F4/80, MCP-1, TLR4, TLR2 and IL-1β) and effector caspase (caspase 3 and 7) activation compared to HCR rats. Further, LCR rats had greater WD-induced decreases in complete FAO and mitochondrial respiratory capacity. Intrinsic aerobic capacity had no impact on WD-induced hepatic steatosis; however, rats bred for low aerobic capacity developed greater hepatic inflammation, which was associated with reduced hepatic mitochondrial FAO and respiratory capacity and increased accumulation of cholesterol esters. These results confirm epidemiological reports that aerobic capacity impacts progression of liver disease and suggest that these effects are mediated through alterations in hepatic mitochondrial function.

Keywords: aerobic capacity; fatty acid oxidation; inflammation; mitochondria; steatohepatitis.

Figure 1. High intrinsic aerobic capacity protects against high‐fat/high‐cholesterol diet‐induced hepatic steatosis and non‐parenchymal cellular infiltration

A, representative H&E images. Scale bars represent 50 μm. B, liver triacylglycerol, total cholesterol, free cholesterol and cholesterol ester determined biochemically. Values are means ± SEM (n = 8). §P < 0.05 interaction; ^* P < 0.05 main effect HCR vs. LCR; †P < 0.05 main effect LFD vs. WD, ^** P < 0.05 HCR vs. LCR within diet.

Figure 2. High intrinsic aerobic capacity attenuates high‐fat/high‐cholesterol diet‐induced expression of markers of liver macrophage infiltration and inflammation

Gene expression in liver was determined by RT‐PCR. Relative mRNA expression of genes for macrophage markers (A), recruitment and activation (B), inflammation initiation and propagation (C), and death receptors (D) were normalized to cyclophilin B (PPIB) and presented as means ± SEM (n = 7–8). §P < 0.05 interaction; ^* P < 0.05 main effect HCR vs. LCR; †P < 0.05 main effect LFD vs. WD; ^** P < 0.05 HCR vs. LCR within diet.

Figure 3. Selection for increased running capacity results in greater hepatic fatty acid oxidation (FAO) and acyl‐carnitine flux

Liver homogenates were incubated with [¹⁴C]palmitate. Complete FAO (A) was determined by trapping ¹⁴CO2, and acid‐soluble metabolite (ASM) production was determined from the reaction buffer and normalized to liver sample wet weight. Parallel incubations of liver homogenate were performed in the presence of the CPT‐1 inhibitor, etomoxir, to determine non‐CPT‐1‐mediated (B) complete FAO and ASM production. Liver acyl‐carnitine concentrations were determined by LC‐MS/MS, and ratios (C) of C2/C4 and C4/C16 were compared to assess complete FAO and β‐oxidation, respectively. (D) Western blot analysis of liver homogenate was performed to assess protein expression of CPT‐1a and HADHA. Values are means ± SEM (n = 8). §P < 0.05 interaction; ^* P < 0.05 main effect HCR vs. LCR; †P < 0.05 main effect LFD vs. WD; ^** P < 0.05 HCR vs. LCR within diet; ††P < 0.05 LFD vs. WD within strain.

Figure 4. Liver mitochondrial content, respiratory capacity and hepatic NAD⁺/NADH ratio is higher in rats with high intrinsic aerobic capacity

A, markers of hepatic mitochondrial content and TCA cycle flux are presented as citrate synthase activity in liver homogenate and 2‐[¹⁴C]pyruvate oxidation to CO₂. B, liver mitochondrial respiration was determined by measurement of O₂ consumption using a Clark electrode system in the presence of glutamate (+malate and succinate) in different respiratory states (basal, state 3 and uncoupled). C, hepatic NAD⁺, NADH, ATP, ADP, and AMP were determined by LC‐MS/MS. The mitochondrial energy state is represented as the ratio of NAD⁺ to NADH, and adenine energy charge. Values are means ± SEM (n = 7–8). §P < 0.05 interaction; ^* P < 0.05 main effect HCR vs. LCR; †P < 0.05 main effect LFD vs. WD; ††P < 0.05 LFD vs. WD within strain.

Figure 5. Protein expression of markers of oxidative stress and anti‐oxidant enzymes in isolated liver mitochondria

Data are presented as means ± SEM (n = 7–8). A, 4‐hydroxy‐2‐nonenal (4‐HNE); B, superoxide dismutase 2 (SOD2); C, uncoupling protein 2 (UCP2); D, glutathione peroxidase 1 (GPx1). §P < 0.05 interaction; ^* P < 0.05 main effect HCR vs. LCR; †P < 0.05 main effect LFD vs. WD; ^** P < 0.05 HCR vs. LCR within diet; ††P < 0.05 LFD vs. WD within strain.

Figure 6. Low intrinsic aerobic capacity increases susceptibility to liver mitochondrial localization of pro‐apoptotic proteins and associated effector caspase activation

A and B, mitochondrial localization of proteins involved in the regulation of mitochondrial outer membrane permeability (MOMP) were determined in isolated liver mitochondria by Western blot analysis. A, anti‐MOMP opening (Bcl‐2 and Bcl‐xL). B, pro‐MOMP opening (Bax, Bak and cleaved BCL‐xL). C, liver expression of active (cleaved) effector caspases, caspase‐3 and caspase‐7, was determined in liver homogenate by Western blot analysis. D, representative blots. All values are presented as means ± SEM (n = 7–8). ^* P < 0.05 main effect HCR vs. LCR, † P < 0.05 main effect LFD vs. WD, ^** P < 0.05 HCR vs. LCR within diet.