Statins Increase Mitochondrial and Peroxisomal Fatty Acid Oxidation in the Liver and Prevent Non-Alcoholic Steatohepatitis in Mice

Abstract

Background: Non-alcoholic fatty liver disease is the most common form of chronic liver disease in industrialized countries. Recent studies have highlighted the association between peroxisomal dysfunction and hepatic steatosis. Peroxisomes are intracellular organelles that contribute to several crucial metabolic processes, such as facilitation of mitochondrial fatty acid oxidation (FAO) and removal of reactive oxygen species through catalase or plasmalogen synthesis. Statins are known to prevent hepatic steatosis and non-alcoholic steatohepatitis (NASH), but underlying mechanisms of this prevention are largely unknown.

Methods: Seven-week-old C57BL/6J mice were given normal chow or a methionine- and choline-deficient diet (MCDD) with or without various statins, fluvastatin, pravastatin, simvastatin, atorvastatin, and rosuvastatin (15 mg/kg/day), for 6 weeks. Histological lesions were analyzed by grading and staging systems of NASH. We also measured mitochondrial and peroxisomal FAO in the liver.

Results: Statin treatment prevented the development of MCDD-induced NASH. Both steatosis and inflammation or fibrosis grades were significantly improved by statins compared with MCDD-fed mice. Gene expression levels of peroxisomal proliferator-activated receptor α (PPARα) were decreased by MCDD and recovered by statin treatment. MCDD-induced suppression of mitochondrial and peroxisomal FAO was restored by statins. Each statin's effect on increasing FAO and improving NASH was independent on its effect of decreasing cholesterol levels.

Conclusion: Statins prevented NASH and increased mitochondrial and peroxisomal FAO via induction of PPARα. The ability to increase hepatic FAO is likely the major determinant of NASH prevention by statins. Improvement of peroxisomal function by statins may contribute to the prevention of NASH.

Keywords: Fatty acid oxidation; Non-alcoholic fatty liver disease; Peroxisomes; Statins.

Fig. 1. Statin treatment attenuates methionine- and choline-deficient diet (MCDD)-induced hepatic steatosis and steatohepatitis. Representative histological images of each experimental group. H&E (×200; scale bar=100 µm), MT (×100; scale bar=50 µm). ND, normal chow diet; MT, Masson's trichrome.

Fig. 2. Histologic scores and hepatic triglyceride (TG) content and plasma alanine aminotransferase (ALT) level. (A) Histologic scores for location and severity of steatosis, inflammation, and fibrosis, according to criteria of Kleiner et al. [18]. (B) Hepatic TG content and plasma ALT level in mice fed normal chow diet (ND) and methionine- and choline-deficient diet (MCDD) with or without various statins for 6 weeks. F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin. ^aP<0.05 compared with ND, ^bP<0.05 compared with MCDD.

Fig. 3. Treatment with statins recovers methionine- and choline-deficient diet (MCDD)-induced suppression of peroxisomal proliferator-activated receptor α (PPARα) and target gene expression levels in the liver. (A) mRNA expression levels and (B) protein expression levels of PPARα. Expression levels of genes involved in mitochondrial and peroxisomal (C) fatty acid oxidation (FAO), and (D) peroxisomal biogenesis factor (Pex) 7. ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin; CPT, carnitine palmitoyltransferase; ACOX, acyl CoA oxidase; DBP, D-bifunctional protein. ^aP<0.05 compared with ND, ^bP<0.05 compared with MCDD.

Fig. 4. Measurement of hepatic fatty acid oxidation (FAO) in mice fed methionine- and choline-deficient diet (MCDD) with or without statins. (A) Mitochondrial FAO. (B) Peroxisomal FAO. DPM, disintegrations per minute; ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin; R, rosuvastatin. ^aP<0.05 compared with ND, ^bP<0.05 compared with MCDD.

Fig. 5. Changes in hepatic lipid peroxidation and gene expression levels of several anti-oxidative enzymes. (A) Hepatic malondialdehyde (MDA) levels in mice fed methionine- and choline-deficient diet (MCDD) and fluvastatin supplementation. (B) mRNA expression levels of catalase, (C) manganese superoxide dismutase (MnSOD), and (D) glutathione peroxidase 1 (GPx-1). ND, normal chow diet; F, fluvastatin; P, pravastatin; S, simvastatin; A, atorvastatin. ^aP<0.05 compared with ND, ^bP<0.05 compared with MCDD.