Raquel Fucho

Specific contribution of methionine and choline in nutritional nonalcoholic steatohepatitis.Impact on mitochondrial S-adenosyl-L-methionine and glutathione

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-l-methionine (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.

ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading

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(+/+) and ASMase(-/-) mice. Exogenous ASMase, but not neutral SMase, induced ER stress by perturbing ER Ca(2+) 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. CONCLUSIONS Our data indicate that ASMase is critical for alcohol-induced ER stress, and provide a rationale for further clinical investigation in ALD.

ASMase regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage non-alcoholic steatohepatitis

BACKGROUND & AIMS Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. METHODS Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase(-/-) mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. RESULTS ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase(-/-) mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase(-/-) mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase(-/-) mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase(+/+) mice but not in ASMase(-/-) mice. ASMase(-/-) PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. CONCLUSIONS These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.

Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis

BACKGROUND & AIMS Steatohepatitis (SH) is associated with mitochondrial dysfunction and excessive production of superoxide, which can then be converted into H(2)O(2) by SOD2. Since mitochondrial GSH (mGSH) plays a critical role in H(2)O(2) reduction, we explored the interplay between superoxide, H(2)O(2), and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion. METHODS We used isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging. RESULTS In isolated mitochondria and primary hepatocytes, superoxide scavenging by SOD mimetics or purified SOD decreased superoxide and peroxynitrite generation but increased H(2)O(2) following mGSH depletion, despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by SOD mimetics, and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methionine-choline deficient (MCD) diet or MAT1A(-/-) mice exhibited reduced SOD2 activity; in vivo treatment with SOD mimetics increased liver damage, inflammation, and fibrosis, despite a decreased superoxide and 3-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee, but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging when mGSH was depleted transgenic mice overexpressing SOD2 exhibited enhanced susceptibility to MCD-mediated SH. CONCLUSIONS These findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be important in SH.

1265 ACIDIC SPHINGOMYELINASE CONTROLS DIET-INDUCED INSULIN SENSITIVITY, ADIPOSE TISSUE HOMEOSTASIS AND HEPATIC STEATOSIS

Trabajo presentado al 46th Annual Meeting of the European Association for the Study of the Liver (EASL) celebrado en Berlin del 30 de marzo al 3 de abril de 2011.