Anna Colell

Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species ROLE OF MITOCHONDRIAL GLUTATHIONE

Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C2-ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 μM and maximal at 1-5 μM C2, decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C2, C2-dihydroceramide increased hydrogen peroxide at 1-5 μM. Sphingosine generated hydrogen peroxide at concentrations ≥10 μM, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C2 was not triggered by mitochondrial permeability transition as C2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C2; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C2. Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity.

Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor

BACKGROUND & AIMS Tumor necrosis factor (TNF)-alpha induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-alpha. METHODS Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-alpha, and the extent of oxidative stress, gene expression, and viability were evaluated. RESULTS Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-alpha. The susceptibility to TNF-alpha, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor kappaB activation by TNF-alpha was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-alpha was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH-ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-alpha. CONCLUSIONS These results indicate that mGSH controls the fate of hepatocytes in response to TNF-alpha. Its depletion caused by alcohol consumption amplifies the power of TNF-alpha to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.

Defective TNF-α-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation

Glycosphingolipids, including ganglio‐sides, are emerging as signaling intermediates of extracellular stimuli. Because mitochondria play a key role in the orchestration of death signals, we assessed the interaction of GD3 ganglioside (GD3) with mitochondria and the subsequent cascade of events that culminate in cell death. In vitro studies with isolated mitochondria from rat liver demonstrate that GD3 elicited a burst of peroxide production within 15–30 min, which preceded the opening of the mitochondrial permeability transition, followed by cytochrome c (cyt c) release. These effects were mimicked by lactosylceramide and N‐acetyl‐sphingosine but not by sphinganine or sphingosine and were prevented by cyclosporin A and butylated hydroxytoluene (BHT). Reconstitution of mitochondria pre‐exposed to GD3 with cytosol from rat liver in a cell‐free system resulted in the proteolytic processing of procaspase 3 and subsequent caspase 3 activation. Intact hepatocytes or U937 cells selectively depleted of glutathione in mitochondria by 3‐hydroxyl‐4‐pentenoate (HP) with the sparing of cytosol reduced glutathione (GSH) were sensitized to GD3, manifested as an apoptotic death. Inhibition of caspase 3 prevented the apoptotic phenotype of HP‐treated cells caused by GD3 without affecting cell survival; in contrast, BHT fully protected HP‐treated cells to GD3 treatment. Treatment of cells with tumor necrosis factor increased the level of GD3, whereas blockers of mitochondrial respiration at complex I and II protected sensitized cells to GD3 treatment. Thus, the effect of GD3 as a lipid death effector is determined by its interaction with mitochondria leading to oxidant‐dependent caspase activation. Mitochondrial glutathione plays a key role in controlling cell survival through modulation of the oxidative stress induced by glycosphingolipids.—García‐Ruiz, C., Colell, A., París, R., Fernandez‐Checa, J. C. Direct interaction of GD3 ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability transition, cytochrome c release, and caspase activation. FASEB J. 14, 847–858 (2000)

Tumor Necrosis Factor Increases Hepatocellular Glutathione by Transcriptional Regulation of the Heavy Subunit Chain of γ-Glutamylcysteine Synthetase

Tumor necrosis factor (TNF) is an inflammatory cytokine that causes cell injury by generation of oxidative stress. Since glutathione (GSH) is a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of γ-glutamylcysteine synthetase (γ-GCS-HS), and control of intracellular generation of reactive oxygen species in cultured rat hepatocytes treated with TNF. Exposure of cells to TNF (10,000 units/ml) resulted in depletion of cellular GSH levels (50–70%) and overproduction of hydrogen peroxide (2–3-fold) and lipid peroxidation. However, cells treated with lower doses of TNF (250–500 units/ml) exhibited increased levels of GSH (60–80% over control). TNF treatment increased (70–100%) the levels of γ-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthesis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the γ-GCS-HS gene to a greater extent than control cells, indicating that TNF regulates γ-GCS-HS at the transcriptional level. The capacity to synthesize GSH de novo determined in cell-free extracts incubated with GSH precursors was greater (50–70%) in hepatocytes that were treated with TNF; however, the activity of GSH synthetase remained unaltered by TNF treatment indicating that TNF selectively increased the activity of γ-GCS. Despite activation of nuclear factor-κB (NF-κB) by TNF, this transcription factor was not required for TNF-induced transcription of γ-GCS-HS as revealed by deletion constructs of the γ-GCS-HS promoter subcloned in a chloramphenicol acetyltransferase reporter vector and transfected into HepG2 cells. In contrast, a construct containing AP-1 like/metal response regulatory elements increased chloramphenicol acetyltransferase activity upon exposure to TNF. Thus, TNF increases hepatocellular GSH levels by transcriptional regulation of γ-GCS-HS gene, probably through AP-1/metal response element-like binding site(s) in its promoter, which may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.

Mitochondrial Cholesterol Contributes to Chemotherapy Resistance in Hepatocellular Carcinoma

Cholesterol metabolism is deregulated in carcinogenesis, and cancer cells exhibit enhanced mitochondrial cholesterol content whose role in cell death susceptibility and cancer therapy has not been investigated. Here, we describe that mitochondria from rat or human hepatocellular carcinoma (HC) cells (HCC) or primary tumors from patients with HC exhibit increased mitochondrial cholesterol levels. HCC sensitivity to chemotherapy acting via mitochondria is enhanced upon cholesterol depletion by inhibition of hydroxymethylglutaryl-CoA reductase or squalene synthase (SS), which catalyzes the first committed step in cholesterol biosynthesis. HCC transfection with siRNA targeting the steroidogenic acute regulatory protein StAR, a mitochondrial cholesterol-transporting polypeptide which is overexpressed in HCC compared with rat and human liver, sensitized HCC to chemotherapy. Isolated mitochondria from HCC with increased cholesterol levels were resistant to mitochondrial membrane permeabilization and release of cytochrome c or Smac/DIABLO in response to various stimuli including active Bax. Similar behavior was observed in cholesterol-enriched mitochondria or liposomes and reversed by restoring mitochondrial membrane order or cholesterol extraction. Moreover, atorvastatin or the SS inhibitor YM-53601 potentiated doxorubicin-mediated HCC growth arrest and cell death in vivo. Thus, mitochondrial cholesterol contributes to chemotherapy resistance by increasing membrane order, emerging as a novel therapeutic niche in cancer therapy.

Caveolin-1 deficiency causes cholesterol dependent mitochondrial dysfunction and apoptotic susceptibility

Caveolins (CAVs) are essential components of caveolae, plasma membrane invaginations with reduced fluidity, reflecting cholesterol accumulation. CAV proteins bind cholesterol, and CAVs ability to move between cellular compartments helps control intracellular cholesterol fluxes. In humans, CAV1 mutations result in lipodystrophy, cell transformation, and cancer. CAV1 gene-disrupted mice exhibit cardiovascular diseases, diabetes, cancer, atherosclerosis, and pulmonary fibrosis. The mechanism or mechanisms underlying these disparate effects are unknown, but our past work suggested that CAV1 deficiency might alter metabolism: CAV1(-/-) mice exhibit impaired liver regeneration unless supplemented with glucose, suggesting systemic inefficiencies requiring additional metabolic intermediates. Establishing a functional link between CAV1 and metabolism would provide a unifying theme to explain these myriad pathologies. Here we demonstrate that impaired proliferation and low survival with glucose restriction is a shortcoming of CAV1-deficient cells caused by impaired mitochondrial function. Without CAV1, free cholesterol accumulates in mitochondrial membranes, increasing membrane condensation and reducing efficiency of the respiratory chain and intrinsic antioxidant defense. Upon activation of oxidative phosphorylation, this promotes accumulation of reactive oxygen species, resulting in cell death. We confirm that this mitochondrial dysfunction predisposes CAV1-deficient animals to mitochondrial-related diseases such as steatohepatitis and neurodegeneration.

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.

Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity

Mitochondrial permeability transition (MPT) has been proposed to play a key role in cell death. Downstream MPT events include the release of apoptogenic factors that sets in motion the mitochondrial apoptosome leading to caspase activation. The current work examined the regulation of MPT by membrane fluidity modulated upon cholesterol enrichment. Mitochondria enriched in cholesterol displayed increased microviscosity resulting in impaired MPT induced by atractyloside, a c-conformation stabilizing ligand of the adenine nucleotide translocator (ANT). This effect was dependent on the dose of cholesterol loaded and reversed upon the fluidization of mitochondria by the fatty acid derivative A2C. Mitoplasts derived from cholesterol-enriched mitochondria responded to atractyloside in a similar fashion as intact mitochondria, indicating that a significant amount of cholesterol is still found in the inner membrane. The effects of cholesterol on MPT induced by atractyloside were mirrored by the release of intermembrane proteins, cytochrome c, Smac/Diablo, and apoptosis inducing factor. However, cholesterol loading did not affect the uptake rate of adenine nucleotide hence dissociating the function of ANT as a MPT-mediated protein from its adenine nucleotide exchange function. Thus, these findings indicate that the ability of atractyloside to induce MPT via ANT requires an appropriate membrane fluidity range.

Redox Control of Liver Function in Health and Disease

Reactive oxygen species (ROS), a heterogeneous population of biologically active intermediates, are generated as by-products of the aerobic metabolism and exhibit a dual role in biology. When produced in controlled conditions and in limited quantities, ROS may function as signaling intermediates, contributing to critical cellular functions such as proliferation, differentiation, and cell survival. However, ROS overgeneration and, particularly, the formation of specific reactive species, inflicts cell death and tissue damage by targeting vital cellular components such as DNA, lipids, and proteins, thus arising as key players in disease pathogenesis. Given the predominant role of hepatocytes in biotransformation and metabolism of xenobiotics, ROS production constitutes an important burden in liver physiology and pathophysiology and hence in the progression of liver diseases. Despite the recognized role of ROS in disease pathogenesis, the efficacy of antioxidants as therapeutics has been limited. A better understanding of the mechanisms, nature, and location of ROS generation, as well as the optimization of cellular defense strategies, may pave the way for a brighter future for antioxidants and ROS scavengers in the therapy of liver diseases.