No abstract

Mitochondria generate reactive oxygen species (ROS) as byproducts of molecular oxygen consumption in the electron transport chain. Most cellular oxygen is consumed in the cytochrome-c oxidase complex of the respiratory chain, which does not generate reactive species. The ubiquinone pool of complex III of respiration is the major site within the respiratory chain that generates superoxide anion as a result of a single electron transfer to molecular oxygen. Superoxide anion and hydrogen peroxide, derived from the former by superoxide dismutase, are precursor of hydroxyl radical through the participation of transition metals. Glutathione (GSH) in mitochondria is the only defense available to metabolize hydrogen peroxide. A small fraction of the total cellular GSH pool is sequestered in mitochondria by the action of a carrier that transports GSH from the cytosol to the mitochondrial matrix. Mitochondria are not only one of the main cellular sources of ROS, they also are a key target of ROS. Mitochondria are subcellular targets of cytokines, especially tumor necrosis factor (TNF); depletion of GSH in this organelle renders the cell more susceptible to oxidative stress originating in mitochondria. Ceramide generated during TNF signaling leads to increased production of ROS in mitochondria. Chronic ethanol-fed hepatocytes are selectively depleted of GSH in mitochondria due to a defective operation of the carrier responsible for transport of GSH from the cytosol into the mitochondrial matrix. Under these conditions, limitation of the mitochondrial GSH pool represents a critical contributory factor that sensitizes alcoholic hepatocytes to the prooxidant effects of cytokines and prooxidants generated by oxidative metabolism of ethanol. S-adenosyl-L-methionine prevents development of the ethanol-induced defect. The mitochondrial GSH carrier has been functionally expressed in Xenopus laevis oocytes microinjected with mRNA from rat liver. This critical carrier displays functional characteristics distinct from other plasma membrane GSH carriers, such as its ATP dependency, inhibitor specificity, and the size class of mRNA that encode the corresponding carrier, suggesting that the mitochondrial carrier of GSH is a gene product distinct from the plasma membrane transporters.

Glycosphingolipids, including gangliosides, 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.

Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.

Cell survival reflects a balance between death and protective pathways. Because sphingolipids have emerged as putative death signals and because transcription factor-kappa B (NF-κB) activates a survival pathway, we analyzed the role of C2-ceramide (C2) and GD3 ganglioside (GD3) on the generation of reactive oxygen species (ROS), NF-κB activation, and survival of rat hepatocytes or HepG2 cells. Although both C2 and GD3 generated similar dose-dependent levels of ROS derived from mitochondria, hepatocytes displayed a selective sensitivity to GD3 treatment. Consistent with this finding, C2 and GD3 differed in the activation of NF-κB as C2, unlike GD3, and enhanced the DNA binding of NF-κB despite the fact that both sphingolipids signaled the degradation of IκB-α. Glucosylceramide (GluCer), lactosylceramide (LactCer), and ganglioside GM1 (GM1) mimicked the repressing effect of GD3 on NF-κB activation, which suggests the presence of common structural features among these glycosphingolipids. Competent DNA binding NF-κB complexes were observed predominantly in the cytosol of GD3-stimulated cells, which paralleled the absence of NF-κB p65 in the nuclei and indicated that GD3 blocks the nuclear translocation of NF-κB complexes. Pretreatment of hepatocytes with a sublethal dose of GD3 blocked the activation of NF-κB and subsequent κB-dependent gene expression induced by WXPRU QHFURVLV IDFWRU . 71)-α), which sensitized hepatocytes to TNF-α-induced apoptosis. Thus, gangliosides are efficient death effectors by a dual mechanism that involves mitochondrial recruitment and suppression of the NF-κB-dependent survival pathway, which may be of potential therapeutic use in conditions aimed to control apoptosis resistance.Key words: mitochondria • necrosis • oxidative stress • sphingolipids poptosis plays an essential role in development and tissue homeostasis, although its dysregulation is increasingly recognized as a key event in the pathogenesis of many diseases. Cell fate in response to many apoptosis stimuli, such as tumor necrosis factor . (TNF-α) and members of the TNF-.family, including CD95 (Fas), is determined by a balance A between death-and survival-signaling pathways. These are triggered by the inducing stimuli (1-3). Recent results have shown that transcription factor-kappa B (NF-κB), which is activated by a wide range of signals, including TNF-α, is at least partly responsible for the resistance to cell killing induced by a diverse array of apoptosis-inducing triggers (1,2,4,5). Indeed, TNF-α simultaneously activates both apoptosis and anti-death pathways, and the TNF-stimulated antideath activity, unlike TNF-induced cell death, depends on novo protein synthesis, which indicates that a gene or set of genes, is induced on TNF-α receptor activation that downregulates apoptosis.Sphingolipids have emerged as putative signaling lipid intermediates that play a role in the stress response and cell death (6, 7). Ceramides or their permeable analogs (e.g., C2 ceramide) have been shown to target mitochondria and induce a burst o...

Inflammation is an adaptive response in pursuit of homeostasis reestablishment triggered by harmful conditions or stimuli, such as an infection or tissue damage. Liver diseases cause approximately 2 million deaths per year worldwide and hepatic inflammation is a common factor to all of them, being the main driver of hepatic tissue damage and causing progression from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), cirrhosis and, ultimately, hepatocellular carcinoma (HCC). The metabolic sensor SIRT1, a class III histone deacetylase with strong expression in metabolic tissues such as the liver, and transcription factor NF-κB, a master regulator of inflammatory response, show an antagonistic relationship in controlling inflammation. For this reason, SIRT1 targeting is emerging as a potential strategy to improve different metabolic and/or inflammatory pathologies. In this review, we explore diverse upstream regulators and some natural/synthetic activators of SIRT1 as possible therapeutic treatment for liver diseases.

Mitochondrial glutathione plays an important role in maintaining a functionally competent organelle. Previous studies have shown that ethanol feeding selectively depletes the mitochondrial glutathione pool, more predominantly in mitochondria from perivenous hepatocytes. Because S-adenosyl-L-methionine (SAM) is a glutathione precursor and maintains the structure and function of biological membranes, the purpose of the present study was to determine the effects of SAM on glutathione and function of perivenous (PV) and periportal (PP) mitochondria from chronic ethanol-fed rats. SAM administration resulted in a significant increase in the basal cytosol and mitochondrial glutathione in both PP and PV cells from both pair-fed or ethanol-fed groups. When hepatocytes from ethanol-fed rats supplemented with SAM were incubated with methionine plus serine or N-acetylcysteine, mitochondrial glutathione increased in parallel with cytosol, an effect not observed in cells from ethanol-fed rats without SAM. Feeding equimolar N-acetylcysteine raised cytosol glutathione but did not prevent the mitochondrial glutathione defect. In addition, SAM feeding resulted in significant preservation of cellular adenosine triphosphate (ATP) levels (23% to 43%), mitochondrial membrane potential (17% to 25%), and the uncoupler control ratio (UCR) of respiration (from 5.1 +/- 0.7 to 7.3 +/- 0.6 and 2.1 +/- 0.3 to 6.1 +/- 0.7) for PP and PV mitochondria, respectively. Thus, these effects of SAM suggest that it may be a useful agent to preserve the disturbed mitochondrial integrity in liver disease caused by alcoholism through maintenance of mitochondrial glutathione transport.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi‐Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol‐mediated sensitization of the liver, by Anna Colell, Carmen Garcia‐Ruiz, Neil Kaplowitz, and Jose C. Fernandez‐Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S‐adenosylmethionine affects tumor necrosis factor‐α gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi‐Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFα gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut‐derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.