Anna Fernández

Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH

BACKGROUND/AIMS Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis. METHODS Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls. RESULTS All NASH patients (n=14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p<0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls. CONCLUSIONS FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.

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.

Mitochondrial Cholesterol Loading Exacerbates Amyloid β Peptide-Induced Inflammation and Neurotoxicity

The role of cholesterol in Alzheimers disease (AD) has been linked to the generation of toxic amyloid β peptides (Aβ). Using genetic mouse models of cholesterol loading, we examined whether mitochondrial cholesterol regulates Aβ neurotoxicity and AD pathology. Isolated mitochondria from brain or cortical neurons of transgenic mice overexpressing SREBP-2 (sterol regulatory element binding protein 2) or NPC1 (Niemann-Pick type C1) knock-out mice exhibited mitochondrial cholesterol accumulation, mitochondrial glutathione (mGSH) depletion and increased susceptibility to Aβ1–42-induced oxidative stress and release of apoptogenic proteins. Similar findings were observed in pharmacologically GSH-restricted rat brain mitochondria, while selective mGSH depletion sensitized human neuronal and glial cell lines to Aβ1–42-mediated cell death. Intracerebroventricular human Aβ delivery colocalized with mitochondria resulting in oxidative stress, neuroinflammation and neuronal damage that were enhanced in Tg-SREBP-2 mice and prevented upon mGSH recovery by GSH ethyl ester coinfusion, with a similar protection observed by intraperitoneal administration of GSH ethyl ester. Finally, APP/PS1 (amyloid precursor protein/presenilin 1) mice, a transgenic AD mouse model, exhibited mitochondrial cholesterol loading and mGSH depletion. Thus, mitochondrial cholesterol accumulation emerges as a novel pathogenic factor in AD by modulating Aβ toxicity via mGSH regulation; strategies boosting the particular pool of mGSH may be of relevance to slow down disease progression.

Mechanism of Mitochondrial Glutathione-Dependent Hepatocellular Susceptibility to TNF Despite NF-κB Activation

BACKGROUND & AIMS Nuclear factor kappaB (NF-kappaB) is the master regulator of tumor necrosis factor (TNF) susceptibility. Although mitochondrial glutathione (mGSH) depletion was shown to sensitize hepatocytes to TNF despite NF-kappaB activation, the mechanisms involved, particularly the role of Bax oligomerization and mitochondrial outer membrane (MOM) permeabilization, 2 critical steps in cell death, remained unexplored. METHODS TNF signaling at the premitochondrial and mitochondrial levels was analyzed in primary mouse hepatocytes with or without mGSH depletion. RESULTS Unexpectedly, we observed that TNF activates caspase-8 independently of NF-kappaB inactivation, causing Bid cleavage and mitochondrial Bax oligomerization. However, their predicted consequences on MOM permeabilization, cytochrome c release, caspase-3 activation, and hepatocellular death occurred only on mGSH depletion. These events were preceded by stimulated mitochondrial reactive oxygen species that predominantly oxidized cardiolipin, changes not observed in acidic sphingomyelinase (ASMase)(-/-) hepatocytes. Oxidized cardiolipin potentiated oligomerized Bax-induced MOM-like liposome permeabilization by restructuring the lipid bilayer, without effect on membrane Bax insertion or oligomerization. ASMase(-/-) mice with mGSH depletion by cholesterol loading were resistant to TNF-induced liver injury in vivo. CONCLUSIONS Thus, MOM-localized oligomeric Bax is not sufficient for TNF-induced MOM permeabilization and cell death requiring mGSH-controlled ASMase-mediated mitochondrial membrane remodeling by oxidized cardiolipin generation.

Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis.

Endoplasmic reticulum (ER) is a dynamic organelle that participates in a number of cellular functions by controlling lipid metabolism, calcium stores, and proteostasis. Under stressful situations, the ER environment is compromised, and protein maturation is impaired; this causes misfolded proteins to accumulate and a characteristic stress response named unfolded protein response (UPR). UPR protects cells from stress and contributes to cellular homeostasis re‐establishment; however, during prolonged ER stress, UPR activation promotes cell death. ER stressors can modulate autophagy which in turn, depending of the situation, induces cell survival or death. Interactions of different autophagy‐ and apoptosis‐related proteins and also common signaling pathways have been found, suggesting an interplay between these cellular processes, although their dynamic features are still unknown. A number of pathologies including metabolic, neurodegenerative and cardiovascular diseases, cancer, inflammation, and viral infections are associated with ER stress, leading to a growing interest in targeting components of the UPR as a therapeutic strategy. Melatonin has a variety of antioxidant, anti‐inflammatory, and antitumor effects. As such, it modulates apoptosis and autophagy in cancer cells, neurodegeneration and the development of liver diseases as well as other pathologies. Here, we review the effects of melatonin on the main ER stress mechanisms, focusing on its ability to regulate the autophagic and apoptotic processes. As the number of studies that have analyzed ER stress modulation by this indole remains limited, further research is necessary for a better understanding of the crosstalk between ER stress, autophagy, and apoptosis and to clearly delineate the mechanisms by which melatonin modulates these responses.

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.

Targeting cholesterol at different levels in the mevalonate pathway protects fatty liver against ischemia–reperfusion injury

BACKGROUND & AIMS Liver steatosis enhances ischemia/reperfusion (I/R) injury and is considered a primary factor in graft failure after liver transplantation. Although previous reports have shown a role for qualitative steatosis (macrovesicular vs. microvesicular) in hepatic I/R injury, no studies have compared side by side the specific contribution of individual lipids accumulating in fatty liver to I/R damage. METHODS We used nutritional and genetic models of micro and macrovesicular fatty livers exhibiting specific lipid profiles to assess their susceptibility to normothermic I/R injury. RESULTS Unlike choline-deficient (CD) diet-fed mice, characterized by predominant liver triglycerides/free fatty acids (TG/FFA) accumulation, mice fed a cholesterol-enriched (HC) diet, which exhibited enhanced hepatic cholesterol loading in mitochondria, were highly sensitive to I/R-induced liver injury. In vivo two-photon confocal imaging revealed enhanced mitochondrial depolarization and generation of reactive oxygen species following hepatic I/R in HC-fed but not in CD-fed mice, consistent with decreased mitochondrial GSH (mGSH) observed in HC-fed mice. Moreover, ob/ob mice, characterized by increased hepatic TG, FFA, and cholesterol levels, were as sensitive to I/R-mediated liver injury as mice fed the HC diet. Livers from ob/ob mice displayed increased StAR expression and mitochondrial cholesterol accumulation, resulting in mGSH depletion. Interestingly, atorvastatin therapy or squalene synthase inhibition in vivo attenuated StAR overexpression, mitochondrial cholesterol loading, and mGSH depletion, protecting ob/ob mice from I/R-mediated liver injury. CONCLUSIONS Cholesterol accumulation, particularly in mitochondria, sensitizes to hepatic I/R injury, and thus represents a novel target to prevent the enhanced damage of steatotic livers to I/R-mediated damage.

APP/PS1 mice overexpressing SREBP-2 exhibit combined Aβ accumulation and tau pathology underlying Alzheimer's disease

Current evidence indicates that excess brain cholesterol regulates amyloid-β (Aβ) deposition, which in turn can regulate cholesterol homeostasis. Moreover, Aβ neurotoxicity is potentiated, in part, by mitochondrial glutathione (mGSH) depletion. To better understand the relationship between alterations in cholesterol homeostasis and Alzheimers disease (AD), we generated a triple transgenic mice featuring sterol regulatory element-binding protein-2 (SREBP-2) overexpression in combination with APPswe/PS1ΔE9 mutations (APP/PS1) to examine key biochemical and functional characteristics of AD. Unlike APP/PS1 mice, APP/PS1/SREBP-2 mice exhibited early mitochondrial cholesterol loading and mGSH depletion. Moreover, β-secretase activation and Aβ accumulation, correlating with oxidative damage and neuroinflammation, were accelerated in APP/PS1/SREBP-2 mice compared with APP/PS1 mice. Triple transgenic mice displayed increased synaptotoxicity reflected by loss of synaptophysin and neuronal death, resulting in early object-recognition memory impairment associated with deficits in spatial memory. Interestingly, tau pathology was present in APP/PS1/SREBP-2 mice, manifested by increased tau hyperphosphorylation and cleavage, activation of tau kinases and neurofibrillary tangle (NFT) formation without expression of mutated tau. Importantly, in vivo treatment with the cell permeable GSH ethyl ester, which restored mGSH levels in APP/PS1/SREBP-2 mice, partially prevented the activation of tau kinases, reduced abnormal tau aggregation and Aβ deposition, resulting in attenuated synaptic degeneration. Taken together, these results show that cholesterol-mediated mGSH depletion is a key event in AD progression, accelerating the onset of key neuropathological hallmarks of the disease. Thus, therapeutic approaches to recover mGSH may represent a relevant strategy in the treatment of AD.

Mitochondria, cholesterol and amyloid β peptide: a dangerous trio in Alzheimer disease

The molecular mechanisms of Alzheimer’s disease (AD) are not fully understood. Extensive evidence from experimental models has involved the overgeneration and accumulation of toxic amyloid β peptides (Aβ) in the onset and progression of the disease. The amyloidogenic processing of amyloid precursor protein into pathogenic Aβ fragments is thought to occur in specific domains of the plasma membrane and favored by cholesterol enrichment. Intracellular Aβ accumulation is known to induce oxidative stress, predominantly via mitochondria targeting of toxic Aβ. Recent evidence using mouse models of cholesterol loading has demonstrated that the specific mitochondrial cholesterol pool sensitizes neurons to Aβ-induced oxidant cell death and caspase-independent apoptosis due to selective mitochondrial GSH (mGSH) depletion induced by cholesterol-mediated perturbation of mitochondrial membrane dynamics. mGSH replenishment by permeable precursors such as glutathione ethyl ester protected against Aβ-mediated neurotoxicity and inflammation. Thus, these novel data expand the pathogenic role of cholesterol in AD indicating that in addition to fostering Aβ generation, mitochondrial cholesterol determines Aβ neurotoxicity via mGSH regulation.

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.