Anna Baulies

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.

Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: cause or consequence?

Abstract Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and refers to a spectrum of disorders ranging from steatosis to steatohepatitis, a disease stage characterized by inflammation, fibrosis, cell death and insulin resistance (IR). Due to its association with obesity and IR the impact of NAFLD is growing worldwide. Consistent with the role of mitochondria in fatty acid (FA) metabolism, impaired mitochondrial function is thought to contribute to NAFLD and IR. Indeed, mitochondrial dysfunction and impaired mitochondrial respiratory chain have been described in patients with non-alcoholic steatohepatitis and skeletal muscle of obese patients. However, recent data have provided evidence that pharmacological and genetic models of mitochondrial impairment with reduced electron transport stimulate insulin sensitivity and protect against diet-induced obesity, hepatosteatosis and IR. These beneficial metabolic effects of impaired mitochondrial oxidative phosphorylation may be related not only to the reduction of reactive oxygen species production that regulate insulin signaling but also to decreased mitochondrial FA overload that generate specific metabolites derived from incomplete FA oxidation (FAO) in the TCA cycle. In line with the Randle cycle, reduced mitochondrial FAO rates may alleviate the repression on glucose metabolism in obesity. In addition, the redox paradox in insulin signaling and the delicate mitochondrial antioxidant balance in steatohepatitis add another level of complexity to the role of mitochondria in NAFLD and IR. Thus, better understanding the role of mitochondria in FA metabolism and glucose homeostasis may provide novel strategies for the treatment of NAFLD and IR.

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.

Endoplasmic reticulum stress mediates amyloid β neurotoxicity via mitochondrial cholesterol trafficking

Disrupted cholesterol homeostasis has been reported in Alzheimer disease and is thought to contribute to disease progression by promoting amyloid β (Aβ) accumulation. In particular, mitochondrial cholesterol enrichment has been shown to sensitize to Aβ-induced neurotoxicity. However, the molecular mechanisms responsible for the increased cholesterol levels and its trafficking to mitochondria in Alzheimer disease remain poorly understood. Here, we show that endoplasmic reticulum (ER) stress triggered by Aβ promotes cholesterol synthesis and mitochondrial cholesterol influx, resulting in mitochondrial glutathione (mGSH) depletion in older age amyloid precursor protein/presenilin-1 (APP/PS1) mice. Mitochondrial cholesterol accumulation was associated with increased expression of mitochondrial-associated ER membrane proteins, which favor cholesterol translocation from ER to mitochondria along with specific cholesterol carriers, particularly the steroidogenic acute regulatory protein. In vivo treatment with the ER stress inhibitor 4-phenylbutyric acid prevented mitochondrial cholesterol loading and mGSH depletion, thereby protecting APP/PS1 mice against Aβ-induced neurotoxicity. Similar protection was observed with GSH ethyl ester administration, which replenishes mGSH without affecting the unfolded protein response, thus positioning mGSH depletion downstream of ER stress. Overall, these results indicate that Aβ-mediated ER stress and increased mitochondrial cholesterol trafficking contribute to the pathologic progression observed in old APP/PS1 mice, and that ER stress inhibitors may be explored as therapeutic agents for Alzheimer disease.

Melatonin-induced increase in sensitivity of human hepatocellular carcinoma cells to sorafenib is associated with reactive oxygen species production and mitophagy.

Effects of sorafenib in hepatocellular carcinoma (HCC) are frequently transient due to tumor‐acquired resistance, a phenotype that could be targeted by other molecules to reduce this adaptive response. Because melatonin is known to exert antitumor effects in HCC cells, this study investigated whether and how melatonin reduces resistance to sorafenib. Susceptibility to sorafenib (10 nmol/L to 50 μmol/L) in the presence of melatonin (1 and 2 mmol/L) was assessed in HCC cell lines HepG2, HuH7, and Hep3B. Cell viability was reduced by sorafenib from 1 μmol/L in HepG2 or HuH7 cells, and 2.5 μmol/L in Hep3B cells. Co‐administration of melatonin and sorafenib exhibited a synergistic cytotoxic effect on HepG2 and HuH7 cells, while Hep3B cells displayed susceptibility to doses of sorafenib that had no effect when administrated alone. Co‐administration of 2.5 μmol/L sorafenib and 1 mmol/L melatonin induced apoptosis in Hep3B cells, increasing PARP hydrolysis and BAX expression. We also observed an early colocalization of mitochondria with lysosomes, correlating with the expression of mitophagy markers PINK1 and Parkin and a reduction of mitofusin‐2 and mtDNA compared with sorafenib administration alone. Moreover, increased reactive oxygen species production and mitochondrial membrane depolarization were elicited by drug combination, suggesting their contribution to mitophagy induction. Interestingly, Parkin silencing by siRNA to impair mitophagy significantly reduced cell killing, PARP cleavage, and BAX expression. These results demonstrate that the pro‐oxidant capacity of melatonin and its impact on mitochondria stability and turnover via mitophagy increase sensitivity to the cytotoxic effect of sorafenib.

Myristic acid potentiates palmitic acid-induced lipotoxicity and steatohepatitis associated with lipodystrophy by sustaning de novo ceramide synthesis

Palmitic acid (PA) induces hepatocyte apoptosis and fuels de novo ceramide synthesis in the endoplasmic reticulum (ER). Myristic acid (MA), a free fatty acid highly abundant in copra/palmist oils, is a predictor of nonalcoholic steatohepatitis (NASH) and stimulates ceramide synthesis. Here we investigated the synergism between MA and PA in ceramide synthesis, ER stress, lipotoxicity and NASH. Unlike PA, MA is not lipotoxic but potentiated PA-mediated lipoapoptosis, ER stress, caspase-3 activation and cytochrome c release in primary mouse hepatocytes (PMH). Moreover, MA kinetically sustained PA-induced total ceramide content by stimulating dehydroceramide desaturase and switched the ceramide profile from decreased to increased ceramide 14:0/ceramide16:0, without changing medium and long-chain ceramide species. PMH were more sensitive to equimolar ceramide14:0/ceramide16:0 exposure, which mimics the outcome of PA plus MA treatment on ceramide homeostasis, than to either ceramide alone. Treatment with myriocin to inhibit ceramide synthesis and tauroursodeoxycholic acid to prevent ER stress ameliorated PA plus MA induced apoptosis, similar to the protection afforded by the antioxidant BHA, the pan-caspase inhibitor z-VAD-Fmk and JNK inhibition. Moreover, ruthenium red protected PMH against PA and MA-induced cell death. Recapitulating in vitro findings, mice fed a diet enriched in PA plus MA exhibited lipodystrophy, hepatosplenomegaly, increased liver ceramide content and cholesterol levels, ER stress, liver damage, inflammation and fibrosis compared to mice fed diets enriched in PA or MA alone. The deleterious effects of PA plus MA-enriched diet were largely prevented by in vivo myriocin treatment. These findings indicate a causal link between ceramide synthesis and ER stress in lipotoxicity, and imply that the consumption of diets enriched in MA and PA can cause NASH associated with lipodystrophy.

Lysosomal Cholesterol Accumulation Sensitizes To Acetaminophen Hepatotoxicity by Impairing Mitophagy

The role of lysosomes in acetaminophen (APAP) hepatotoxicity is poorly understood. Here, we investigated the impact of genetic and drug-induced lysosomal cholesterol (LC) accumulation in APAP hepatotoxicity. Acid sphingomyelinase (ASMase)−/− mice exhibit LC accumulation and higher mortality after APAP overdose compared to ASMase+/+ littermates. ASMase−/− hepatocytes display lower threshold for APAP-induced cell death and defective fusion of mitochondria-containing autophagosomes with lysosomes, which decreased mitochondrial quality control. LC accumulation in ASMase+/+ hepatocytes caused by U18666A reproduces the susceptibility of ASMase−/− hepatocytes to APAP and the impairment in the formation of mitochondria-containing autolysosomes. LC extraction by 25-hydroxycholesterol increased APAP-mediated mitophagy and protected ASMase−/− mice and hepatocytes against APAP hepatotoxicity, effects that were reversed by chloroquine to disrupt autophagy. The regulation of LC by U18666A or 25-hydroxycholesterol did not affect total cellular sphingomyelin content or its lysosomal distribution. Of relevance, amitriptyline-induced ASMase inhibition in human hepatocytes caused LC accumulation, impaired mitophagy and increased susceptibility to APAP. Similar results were observed upon glucocerebrosidase inhibition by conduritol β-epoxide, a cellular model of Gaucher disease. These findings indicate that LC accumulation determines susceptibility to APAP hepatotoxicity by modulating mitophagy, and imply that genetic or drug-mediated ASMase disruption sensitizes to APAP-induced liver injury.

Mitochondrial GSH replenishment as a potential therapeutic approach for Niemann Pick type C disease

Niemann Pick type C (NPC) disease is a progressive lysosomal storage disorder caused by mutations in genes encoding NPC1/NPC2 proteins, characterized by neurological defects, hepatosplenomegaly and premature death. While the primary biochemical feature of NPC disease is the intracellular accumulation of cholesterol and gangliosides, predominantly in endolysosomes, mitochondrial cholesterol accumulation has also been reported. As accumulation of cholesterol in mitochondria is known to impair the transport of GSH into mitochondria, resulting in mitochondrial GSH (mGSH) depletion, we investigated the impact of mGSH recovery in NPC disease. We show that GSH ethyl ester (GSH-EE), but not N-acetylcysteine (NAC), restored the mGSH pool in liver and brain of Npc1-/- mice and in fibroblasts from NPC patients, while both GSH-EE and NAC increased total GSH levels. GSH-EE but not NAC increased the median survival and maximal life span of Npc1-/- mice. Moreover, intraperitoneal therapy with GSH-EE protected against oxidative stress and oxidant-induced cell death, restored calbindin levels in cerebellar Purkinje cells and reversed locomotor impairment in Npc1-/- mice. High-resolution respirometry analyses revealed that GSH-EE improved oxidative phosphorylation, coupled respiration and maximal electron transfer in cerebellum of Npc1-/- mice. Lipidomic analyses showed that GSH-EE treatment had not effect in the profile of most sphingolipids in liver and brain, except for some particular species in brain of Npc1-/- mice. These findings indicate that the specific replenishment of mGSH may be a potential promising therapy for NPC disease, worth exploring alone or in combination with other options.

Zinc mitigates renal ischemia-reperfusion injury in rats by modulating oxidative stress, endoplasmic reticulum stress, and autophagy

Oxidative stress is a major factor involved in the pathogenesis of renal ischemia/reperfusion (I/R). Exogenous zinc (Zn) was suggested as a potent antioxidant; however, the mechanism by which it strengthens the organ resistance against the effects of reactive oxygen species (ROS) is not yet investigated. The present study aims to determine whether acute zinc chloride (ZnCl2) administration could attenuate endoplasmic reticulum (ER) stress, autophagy, and inflammation after renal I/R. Rats were subjected to either sham operation (Sham group, n = 6), or 1 hr of bilateral ischemia followed by 2 hr of reperfusion (I/R groups, n = 6), or they received ZnCl2 orally 24 hr and 30 min before ischemia (ZnCl2 group, n = 6). Rats were subjected to 1 hr of bilateral renal ischemia followed by 2 hr of reperfusion (I/R group, n = 6). Our results showed that ZnCl2 enhances renal function and reduces cytolysis (p < 0,05). In addition, it increased significantly the activities of antioxidant enzymes (SOD, CAT, and GPX) and the level of GSH in comparison to I/R (p < 0,05). Interestingly, ZnCl2 treatment resulted in significant decreased ER stress, as reflected by GRP78, ATF‐6,p‐eIF‐2α, XPB‐1, and CHOP downregulaion. Rats undergoing ZnCl2 treatment demonstrated a low expression of autophagy parameters (Beclin‐1 and LAMP‐2), which was correlated with low induction of apoptosis (caspase‐9, caspase‐3, and p‐JNK), and reduction of inflammation (IL‐1ß, IL‐6, and MCP‐1) (p < 0,05). In conclusion, we demonstrated the potential effect of Zn supplementation to modulate ER pathway and autophagic process after I/R.

The 2-oxoglutarate carrier promotes liver cancer by sustaining mitochondrial GSH despite cholesterol loading

Cancer cells exhibit mitochondrial cholesterol (mt-cholesterol) accumulation, which contributes to cell death resistance by antagonizing mitochondrial outer membrane (MOM) permeabilization. Hepatocellular mt-cholesterol loading, however, promotes steatohepatitis, an advanced stage of chronic liver disease that precedes hepatocellular carcinoma (HCC), by depleting mitochondrial GSH (mGSH) due to a cholesterol-mediated impairment in mGSH transport. Whether and how HCC cells overcome the restriction of mGSH transport imposed by mt-cholesterol loading to support mGSH uptake remains unknown. Although the transport of mGSH is not fully understood, SLC25A10 (dicarboxylate carrier, DIC) and SLC25A11 (2-oxoglutarate carrier, OGC) have been involved in mGSH transport, and therefore we examined their expression and role in HCC. Unexpectedly, HCC cells and liver explants from patients with HCC exhibit divergent expression of these mitochondrial carriers, with selective OGC upregulation, which contributes to mGSH maintenance. OGC but not DIC downregulation by siRNA depleted mGSH levels and sensitized HCC cells to hypoxia-induced ROS generation and cell death as well as impaired cell growth in three-dimensional multicellular HCC spheroids, effects that were reversible upon mGSH replenishment by GSH ethyl ester, a membrane permeable GSH precursor. We also show that OGC regulates mitochondrial respiration and glycolysis. Moreover, OGC silencing promoted hypoxia-induced cardiolipin peroxidation, which reversed the inhibition of cholesterol on the permeabilization of MOM-like liposomes induced by Bax or Bak. Genetic OGC knockdown reduced the ability of tumor-initiating stem-like cells to induce liver cancer. These findings underscore the selective overexpression of OGC as an adaptive mechanism of HCC to provide adequate mGSH levels in the face of mt-cholesterol loading and suggest that OGC may be a novel therapeutic target for HCC treatment.