Mitochondrial cholesterol: Metabolism and impact on redox biology and disease.

Cholesterol is a crucial component of membrane bilayers by regulating their structural and functional properties. Cholesterol traffics to different cellular compartments including mitochondria, whose cholesterol content is low compared to other cell membranes. Despite the limited availability of cholesterol in the inner mitochondrial membrane (IMM), the metabolism of cholesterol in the IMM plays important physiological roles, acting as the precursor for the synthesis of steroid hormones and neurosteroids in steroidogenic tissues and specific neurons, respectively, or the synthesis of bile acids through an alternative pathway in the liver. Accumulation of cholesterol in mitochondria above physiological levels has a negative impact on mitochondrial function through several mechanisms, including the limitation of crucial antioxidant defenses, such as the glutathione redox cycle, increased generation of reactive oxygen species and consequent oxidative modification of cardiolipin, and defective assembly of respiratory supercomplexes. These adverse consequences of increased mitochondrial cholesterol trafficking trigger the onset of oxidative stress and cell death, and, ultimately, contribute to the development of diverse diseases, including metabolic liver diseases (i.e. fatty liver disease and liver cancer), as well as lysosomal disorders (i.e. Niemann-Pick type C disease) and neurodegenerative diseases (i.e. Alzheimer's disease). In this review, we summarize the metabolism and regulation of mitochondrial cholesterol and its potential impact on liver and neurodegenerative diseases.

Dietary and genetic disruption of hepatic methionine metabolism induce acid sphingomyelinase to promote steatohepatitis.

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.

STARD1 promotes NASH-driven HCC by sustaining the generation of bile acids through the alternative mitochondrial pathway.

BACKGROUND & AIMS: Besides their physiological role in bile formation and fat digestion, bile acids (BAs) synthesised from cholesterol in hepatocytes act as signalling molecules that modulate hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1) is the rate-limiting step in the alternative pathway of BA generation, the physiological relevance of which is not well understood. Moreover, the specific contribution of the STARD1-dependent BA synthesis pathway to HCC has not been previously explored. METHODS: STARD1 expression was analyzed in a cohort of human non-alcoholic steatohepatitis (NASH)-derived HCC specimens. Experimental NASH-driven HCC models included MUP-uPA mice fed a high-fat high-cholesterol (HFHC) diet and diethylnitrosamine (DEN) treatment in wild-type (WT) mice fed a HFHC diet. Molecular species of BAs and oxysterols were analyzed by mass spectrometry. Effects of NASH-derived BA profiles were investigated in tumour-initiated stem-like cells (TICs) and primary mouse hepatocytes (PMHs). RESULTS: Patients with NASH-associated HCC exhibited increased hepatic expression of STARD1 and an enhanced BA pool. Using NASH-driven HCC models, STARD1 overexpression in WT mice increased liver tumour multiplicity, whereas hepatocyte-specific STARD1 deletion (Stard1ΔHep) in WT or MUP-uPA mice reduced tumour burden. These findings mirrored the levels of unconjugated primary BAs, ß-muricholic acid and cholic acid, and their tauroconjugates in STARD1-overexpressing and Stard1ΔHep mice. Incubation of TICs or PMHs with a mix of BAs mimicking this profile stimulated expression of genes involved in pluripotency, stemness and inflammation. CONCLUSIONS: The study reveals a previously unrecognised role of STARD1 in HCC pathogenesis, wherein it promotes the synthesis of primary BAs through the mitochondrial pathway, the products of which act in TICs to stimulate self-renewal, stemness and inflammation. LAY SUMMARY: Effective therapy for hepatocellular carcinoma (HCC) is limited because of our incomplete understanding of its pathogenesis. The contribution of the alternative pathway of bile acid (BA) synthesis to HCC development is unknown. We uncover a key role for steroidogenic acute regulatory protein 1 (STARD1) in non-alcoholic steatohepatitis-driven HCC, wherein it stimulates the generation of BAs in the mitochondrial acidic pathway, the products of which stimulate hepatocyte pluripotency and self-renewal, as well as inflammation.

MITOCHONDRIAL CHOLESTEROL AND CANCER.

Cholesterol is a crucial component of membrane bilayers that determines their physical and functional properties. Cells largely satisfy their need for cholesterol through the novo synthesis from acetyl-CoA and this demand is particularly critical for cancer cells to sustain dysregulated cell proliferation. However, the association between serum or tissue cholesterol levels and cancer development is not well established as epidemiologic data do not consistently support this link. While most preclinical studies focused on the role of total celular cholesterol, the specific contribution of the mitochondrial cholesterol pool to alterations in cancer cell biology has been less explored. Although low compared to other bilayers, the mitochondrial cholesterol content plays an important physiological function in the synthesis of steroid hormones in steroidogenic tissues or bile acids in the liver and controls mitochondrial function. In addition, mitochondrial cholesterol metabolism generates oxysterols, which in turn, regulate multiple pathways, including cholesterol and lipid metabolism as well as cell proliferation. In the present review, we summarize the regulation of mitochondrial cholesterol, including its role in mitochondrial routine performance, cell death and chemotherapy resistance, highlighting its potential contribution to cancer. Of particular relevance is hepatocellular carcinoma, whose incidence in Western countries had tripled in the past decades due to the obesity and type II diabetes epidemic. A better understanding of the role of mitochondrial cholesterol in cancer development may open up novel opportunities for cancer therapy.

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.

Cartilage oligomeric matrix protein participates in the pathogenesis of liver fibrosis.

BACKGROUND & AIMS: Liver fibrosis is characterized by significant accumulation of extracellular matrix (ECM) proteins, mainly fibrillar collagen-I, as a result of persistent liver injury. Cartilage oligomeric matrix protein (COMP) is largely found in the ECM of skeletal tissue. Increased COMP expression has been associated with fibrogenesis in systemic sclerosis, lung fibrosis, chronic pancreatitis, cirrhosis and hepatocellular carcinoma. We hypothesized that COMP could induce fibrillar collagen-I deposition and participate in matrix remodeling thus contributing to the pathophysiology of liver fibrosis. METHODS: Thioacetamide (TAA) and carbon tetrachloride (CCl4) were used to induce liver fibrosis in wild-type (WT) and Comp-/- mice. In vitro experiments were performed with primary hepatic stellate cells (HSCs). RESULTS: COMP expression was detected in livers from control WT mice and was upregulated in response to either TAA or CCl4-induced liver fibrosis. TAA-treated or CCl4-injected Comp-/- mice showed less liver injury, inflammation and fibrosis compared to their corresponding control WT mice. Challenge of HSCs with recombinant COMP (rCOMP) induced intra- plus extracellular collagen-I deposition and increased matrix metalloproteinases (MMPs) 2, 9 and 13, albeit similar expression of transforming growth factor beta (TGFß) protein, in addition to Tgfß, tumour necrosis factor alpha (Tnfα) and tissue inhibitor of metalloproteinases-1 (Timp1) mRNAs. We demonstrated that COMP binds collagen-I; yet, it does not prevent collagen-I cleavage by MMP1. Last, rCOMP induced collagen-I expression in HSCs via CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway. CONCLUSION: These results suggest that COMP contributes to liver fibrosis by regulating collagen-I deposition. LAY SUMMARY: Cartilage oligomeric matrix protein (COMP) induces fibrillar collagen-I deposition via the CD36 receptor signaling and activation of the MEK1/2-pERK1/2 pathway, and participates in extracellular matrix remodeling contributing to the pathophysiology of liver fibrosis.

Metformin protects rat hepatocytes against bile acid-induced apoptosis.

BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

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.

Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases.

BACKGROUND/AIMS: In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide, primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50 micromol/L) or H2O2 (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining. RESULTS: (1) Menadione (50 micromol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50 micromol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. (2) H2O2 increases necrotic cell death at high concentration or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. (3) PEG-SOD prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. (4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death. CONCLUSIONS: In normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers.

Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.

Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not yet well defined. The aim of this study was to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid [TUDCA]) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA), and TCDCA. The phosphatidylinositol-3 kinase pathway (PI3K) and nuclear factor-kappaB were inhibited using LY 294002 and adenoviral overexpression of dominant-negative IkappaB, respectively. The role of p38 and extracellular signal-regulated protein kinase mitogen-activated protein kinase (MAPK) pathways were investigated using the inhibitors SB 203580 and U0 126 and Western blot analysis. Transcription was blocked by actinomycin-D. Apoptosis was determined by measuring caspase-3, -9, and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry, and nuclear morphological analysis. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to 6 hours of preincubation with TUDCA before addition of GCDCA clearly decreased GCDCA-induced apoptosis. At up to 1.5 hours after exposure with GCDCA, the addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAPK, and PI3K pathways, but independent of competition on the cell membrane, NF-kappaB activation, and transcription. In conclusion, TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways.