The pathogenesis and treatment of nonalcoholic steatohepatitis (NASH) are not well established. Feeding a diet deficient in both methionine and choline (MCD) is one of the most common models of NASH, which is characterized by steatosis, mitochondrial dysfunction, hepatocellular injury, oxidative stress, inflammation, and fibrosis. However, the individual contribution of the lack of methionine and choline in liver steatosis, advanced pathology and impact on mitochondrial S-adenosyl-Lmethionine (SAM) and glutathione (GSH), known regulators of disease progression, has not been specifically addressed. Here, we examined the regulation of mitochondrial SAM and GSH and signs of disease in mice fed a MCD, methionine-deficient (MD), or choline-deficient (CD) diet. The MD diet reproduced most of the deleterious effects of MCD feeding, including weight loss, hepatocellular injury, oxidative stress, inflammation, and fibrosis, whereas CD feeding was mainly responsible for steatosis, characterized by triglycerides and free fatty acids accumulation. These findings were preceded by MCD-or MD-mediated SAM and GSH depletion in mitochondria due to decreased mitochondrial membrane fluidity associated with a lower phosphatidylcholine/phosphatidylethanolamine ratio. MCD and MD but not CD feeding resulted in increased ceramide levels by acid sphingomyelinase. Moreover, GSH ethyl ester or SAM therapy restored mitochondrial GSH and ameliorated hepatocellular injury in mice fed a MCD or MD diet. Thus, the depletion of SAM and GSH in mitochondria is an early event in the MCD model of NASH, which is determined by the lack of methionine. Moreover, therapy using permeable GSH prodrugs may be of relevance in NASH.

Caveolins (CAV) are essential components of caveolae; plasma membrane invaginations with reduced fluidity, reflecting cholesterol accumulation [1]. CAV proteins bind cholesterol, and CAV’s ability to move between cellular compartments helps control intracellular cholesterol fluxes [1–3]. In humans, CAV1 mutations result in lipodystrophy, cell transformation, and cancer [4–7]. CAV1 gene-disrupted mice exhibit cardiovascular diseases, diabetes, cancer, atherosclerosis, and pulmonary fibrosis [8, 9]. The mechanism(s) underlying these disparate effects are unknown, but our past work suggested CAV1 deficiency might alter metabolism: CAV1−/− mice exhibit impaired liver regeneration unless supplemented with glucose, suggesting systemic inefficiencies requiring additional metabolic intermediates [10]. Establishing a functional link between CAV1 and metabolism would provide a unifying theme to explain these myriad pathologies [11]. Here, we demonstrate that impaired proliferation and low survival with glucose restriction is a shortcoming of CAV1 deficient cells, caused by impaired mitochondrial function. Without CAV1, free cholesterol accumulates in mitochondrial membranes, increasing membrane condensation and reducing efficiency of the respiratory chain and intrinsic anti-oxidant defence. Upon activation of oxidative phosphorylation, this promotes accumulation of reactive oxygen species resulting in cell death. We confirm that this mitochondrial dysfunction predisposes CAV1 deficient animals to mitochondrial related diseases such as steatohepatitis and neurodegeneration.

Background & aims The pathogenesis of alcohol-induced liver disease (ALD) is poorly understood. Here, we examined the role of acid sphingomyelinase (ASMase) in alcohol induced hepatic endoplasmic reticulum (ER) stress, a key mechanism of ALD Methods We examined ER stress, lipogenesis, hyperhomocysteinemia, mitochondrial cholesterol (mChol) trafficking and susceptibility to LPS and concanavalin-A in ASMase−/− mice fed alcohol. Results Alcohol feeding increased SREBP-1c, DGAT-2 and FAS mRNA in ASMase+/+ but not in ASMase−/− mice. Compared to ASMase+/+ mice, ASMase−/− mice exhibited decreased expression of ER stress markers induced by alcohol, but the level of tunicamycin-mediated upregulation of ER stress markers and steatosis was similar in both types of mice. The increase in homocysteine levels induced by alcohol feeding was comparable in both ASMase+/+ mice and ASMase−/− mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca2+ homeostasis. Moreover, alcohol-induced mChol loading and StARD1 overexpression were blunted in ASMase−/− mice. Tunicamycin upregulated StARD1 expression and this outcome was abrogated by tauroursodeoxycholic acid. Alcohol-induced liver injury and sensitization to LPS and concanavalin-A were prevented in ASMase−/− mice. These effects were reproduced in alcohol-fed TNFR1/R2−/− mice. Moreover, ASMase does not impair hepatic regeneration following partial hepatectomy. Of relevance, liver samples from patients with alcoholic hepatitis exhibited increased expression of ASMase, StARD1 and ER stress markers. Conclusion Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.