ASMase regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage non-alcoholic steatohepatitis.
ABSTRACT: 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.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.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.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.
Project description: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.
Project description:Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% energy from lipids) for 16 weeks, drinking melatonin (10 mg/kg) or not. Indirect calorimetry, glucose tolerance, steatosis, inflammation, ER stress, mitochondrial changes, autophagy and microRNA-34a-5p expression were estimated. Melatonin improved hepatic metabolism and steatosis, influenced ER stress and mitochondrial shape, and promoted autophagy in WT HFD mice. Conversely, melatonin was ineffective in HET HFD mice, maintaining NASH changes. Indeed, autophagy was inconsistent in HET HFD or starved mice, as indicated by LC3II/LC3I ratio, p62/SQSTM1 and autophagosomes estimation. The beneficial role of melatonin in dietary induced NAFLD/NASH in mice was related to reduced expression of microRNA-34a-5p and sterol regulatory element-binding protein (SREBP1) but only in the presence of full SIRT1 availability.
Project description:Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries. NAFLD describes a wide range of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is distinguished from simple steatosis by inflammation, cell death and fibrosis. In this study we found that mice lacking triacylglycerol hydrolase (TGH, also known as carboxylesterase 3 or carboxylesterase 1d) are protected from high-fat diet (HFD) - induced hepatic steatosis via decreased lipogenesis, increased fatty acid oxidation and improved hepatic insulin sensitivity. To examine the effect of the loss of TGH function on the more severe NAFLD form NASH, we ablated Tgh expression in two independent NASH mouse models, Pemt(-/-) mice fed HFD and Ldlr(-/-) mice fed high-fat, high-cholesterol Western-type diet (WTD). TGH deficiency reduced liver inflammation, oxidative stress and fibrosis in Pemt(-/-) mice. TGH deficiency also decreased NASH in Ldlr(-/-) mice. Collectively, these findings indicate that TGH deficiency attenuated both simple hepatic steatosis and irreversible NASH.
Project description:The present study tested a hypothesis that excess accumulation of sphingolipid, ceramide, its metabolites, or a combination contributes to the development of obesity and associated kidney damage. Liquid chromatography/mass spectrometry analysis demonstrated that C57BL/6J mice on the high-fat diet (HFD) had significantly increased plasma total ceramide levels compared with animals fed a low-fat diet (LFD). Treatment of mice with the acid sphingomyelinase (ASMase) inhibitor amitriptyline significantly attenuated the HFD-induced plasma ceramide levels. Corresponding to increase in plasma ceramide, the HFD significantly increased the body weight gain, plasma leptin concentration, urinary total protein and albumin excretion, glomerular damage index, and adipose tissue ASMase activity compared with the LFD-fed mice. These HFD-induced changes were also significantly attenuated by treatment of mice with amitriptyline. In addition, the decline of plasma glucose concentration after an intraperitoneal injection of insulin (0.15 U/kg b.wt.) was more sustained in mice on the HFD with amitriptyline than on the HFD alone. Intraperitoneal injection of glucose (3 g/kg b.wt.) resulted in a slow increase followed by a rapid decrease in the plasma glucose concentration in LFD and HFD plus amitriptyline-treated mice, but such blood glucose response was not observed in HFD-fed mice. Immunofluorescence analysis demonstrated a decrease in the podocin and an increase in the desmin in the glomeruli of HFD-fed mice compared with the LFD and HFD plus amitriptyline-treated mice. In conclusion, our results reveal a pivotal role for ceramide biosynthesis in obesity, metabolic syndrome, and associated kidney damage.
Project description:Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). In humans, only 20%-40% of patients with fatty liver progress to NASH, and mice fed a high-fat diet (HFD) develop fatty liver but are resistant to NASH development. To understand how simple steatosis progresses to NASH, we examined hepatic expression of anti-inflammatory microRNA-223 (miR-223) and found that this miRNA was highly elevated in hepatocytes in HFD-fed mice and in human NASH samples. Genetic deletion of miR-223 induced a full spectrum of NAFLD in long-term HFD-fed mice including steatosis, inflammation, fibrosis, and HCC. Furthermore, microarray analyses revealed that, compared to wild-type mice, HFD-fed miR-223 knockout (miR-223KO) mice had greater hepatic expression of many inflammatory genes and cancer-related genes, including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ-binding motif (Taz), two well-known factors that promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR-223 and that overexpression of miR-223 reduced their expression in cultured hepatocytes. Hepatic levels of miR-223, CXCL10, and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR-223 targeted genes as well as several proinflammatory, cancer-related, and fibrogenic genes. Conclusion: HFD-fed miR-223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model; miR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression and may be a therapeutic target for the treatment of NASH.
Project description:In order to investigate the possible involvement of endoplasmic reticulum (ER) stress in the developmental origins of hepatic steatosis associated with undernourishment in utero, we herein employed a fetal undernourishment mouse model by maternal caloric restriction in three cohorts; cohort 1) assessment of hepatic steatosis and the ER stress response at 9 weeks of age (wks) before a high fat diet (HFD), cohort 2) assessment of hepatic steatosis and the ER stress response on a HFD at 17 wks, cohort 3) assessment of hepatic steatosis and the ER stress response at 22 wks on a HFD after the alleviation of ER stress with a chemical chaperone, tauroursodeoxycholic acid (TUDCA), from 17 wks to 22 wks. Undernourishment in utero significantly deteriorated hepatic steatosis and led to the significant integration of the ER stress response on a HFD at 17 wks. The alleviation of ER stress by the TUDCA treatment significantly improved the parameters of hepatic steatosis in pups with undernourishment in utero, but not in those with normal nourishment in utero at 22 wks. These results suggest the pivotal involvement of the integration of ER stress in the developmental origins of hepatic steatosis in association with undernourishment in utero.
Project description:UNLABELLED:Nonalcoholic fatty liver disease (NAFLD) is a common liver disease that ranges from simple steatosis to nonalcoholic steatohepatitis (NASH). So far, the underlying mechanism remains poorly understood. Here, we show that hepatic carboxylesterase 2 (CES2) is markedly reduced in NASH patients, diabetic db/db mice, and high-fat diet (HFD)-fed mice. Restoration of hepatic CES2 expression in db/db or HFD-fed mice markedly ameliorates liver steatosis and insulin resistance. In contrast, knockdown of hepatic CES2 causes liver steatosis and damage in chow- or Western diet-fed C57BL/6 mice. Mechanistically, we demonstrate that CES2 has triglyceride hydrolase activity. As a result, gain of hepatic CES2 function increases fatty acid oxidation and inhibits lipogenesis, whereas loss of hepatic CES2 stimulates lipogenesis by inducing endoplasmic reticulum stress. We further show that loss of hepatic CES2 stimulates lipogenesis in a sterol regulatory element-binding protein 1 (SREBP-1)-dependent manner. Finally, we show that hepatocyte nuclear factor 4 alpha (HNF-4?) plays a key role in controlling hepatic CES2 expression in diabetes, obesity, or NASH. CONCLUSION:CES2 plays a protective role in development of NAFLD. Targeting the HNF-4?/CES2 pathway may be useful for treatment of NAFLD. (Hepatology 2016;63:1860-1874).
Project description:Endoplasmic reticulum (ER) stress is associated with the pathogenesis of hepatic steatosis. Alisma orientale Juzepzuk is a traditional medicinal herb for diuretics, diabetes, hepatitis, and inflammation. In this study, we investigated the protective effects of methanol extract of the tuber of Alisma orientale (MEAO) against ER stress-induced hepatic steatosis in vitro and in vivo. MEAO inhibited the tunicamycin-induced increase in luciferase activity of ER stress-reporter constructs containing ER stress response element and ATF6 response element. MEAO significantly inhibited tunicamycin-induced ER stress marker expression including GRP78, CHOP, and XBP-1 in tunicamycin-treated Human hepatocellular carcinoma (HepG2) cells and the livers of tunicamycin-injected mice. It also inhibited tunicamycin-induced accumulation of cellular triglyceride. Similar observations were made under physiological ER stress conditions such as in palmitate (PA)-treated HepG2 cells and the livers of high-fat diet (HFD)-induced obese mice. MEAO repressed hepatic lipogenic gene expression in PA-treated HepG2 cells and the livers of HFD obese mice. Furthermore, MEAO repressed very low-density lipoprotein receptor (VLDLR) expression and improved ApoB secretion in the livers of tunicamycin-injected mice or HFD obese mice as well as in tunicamycin or PA-treated HepG2 cells. Alismol, a guaiane-type sesquiterpenes in Alisma orientale, inhibited GRP78 expression in tunicamycin-treated HepG2 cells. In conclusion, MEAO attenuates ER stress and prevents hepatic steatosis pathogenesis via inhibition of expression of the hepatic lipogenic genes and VLDLR, and enhancement of ApoB secretion.
Project description:Mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) is an integral component of the respiratory chain, and recent studies have suggested that it plays an important role in hepatic glucose homeostasis. However, its function in hepatic lipid metabolism is unclear. Here, we identified a role for mGPDH in nonalcoholic fatty liver disease (NAFLD). Specifically, mGPDH expression and activity were lower in fatty livers from patients and mice with NAFLD (ob/ob, high-fat diet [HFD] and db/db). Liver-specific depletion of mGPDH in mice or mGPDH knockdown in cultured hepatocytes exacerbated diet-induced triglyceride accumulation and steatosis through enhanced lipogenesis. RNA-sequencing revealed that mGPDH regulated endoplasmic reticulum (ER)-related proteins and processes. mGPDH deletion exacerbated tunicamycin (ER stress inducer)-induced hepatic steatosis, whereas tauroursodeoxycholic acid (ER stress inhibitor) rescued mGPDH depletion-induced steatosis on an HFD. Moreover, ER stress induced by mGPDH depletion could be abrogated by the intracellular Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane N,N,N´,N´-tetraacetic acid acetoxymethyl ester, mitochondrial permeability transition pore (mPTP) inhibitor cyclosporine A, or cyclophilin-D (Cyp-D) knockdown. mGPDH promoting Cyp-D ubiquitination was also observed. Finally, liver-specific mGPDH overexpression attenuated hepatic steatosis in ob/ob and HFD mice. Conclusion: mGPDH is a pivotal regulator of hepatic lipid metabolism. Its deficiency induces ER stress by suppressing Cyp-D ubiquitination, a key regulator of the mitochondrial Ca2+ conductance channel mPTP, and results in hepatic steatosis. mGPDH may be a potential therapeutic target for the treatment of NAFLD.
Project description:The aim of our work was to investigate the role of interleukin-33 (IL-33) and its receptor ST2 in the progression of diet-induced nonalcoholic steatohepatitis (NASH) in mice, and the characteristic expression in livers of patients with NASH. Mice were fed with high-fat diet (HFD) or methionine-choline 4-deficient diet (MCD) and injected intraperitoneally with IL-33. Both mRNA and protein expression levels of IL-33 and ST2 were up-regulated in the livers of mice fed with HFD or MCD. Treatment with IL-33 attenuated diet-induced hepatic steatosis and reduced activities of ALT in serum, as well as ameliorated HFD-induced systemic insulin resistance and glucose intolerance, while aggravated hepatic fibrosis in diet-induced NASH. Furthermore, treatment with IL-33 can also promote Th2 response and M2 macrophage activation and beneficial modulation on expression profiles of fatty acid metabolism genes in livers. ST2 deficiency did not affect hepatic steatosis and fibrosis when fed with controlling diet. IL-33 did not affect diet-induced hepatic steatosis and fibrosis in ST2 knockout mice. Meanwhile, in the livers of patients with NASH, IL-33 was mainly located in hepatic sinusoid, endothelial cells, and hepatic stellate cells. The mRNA expression level of IL-33 and ST2 was elevated with the progression of NASH. In conclusion, treatment with IL-33 attenuated diet-induced hepatic steatosis, but aggravated hepatic fibrosis, in a ST2-dependent manner.