Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver

BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.     

Renal mitochondrial damage and protein modification in type-2 diabetes

Although mitochondrial reduction-oxidation (redox) stress and increase in membrane permeability play an important role in diabetic-associated renal microvasculopathies, it is unclear whether the intra-renal mitochondrial oxidative stress induces mitochondrial protein modifications, leading to increase mitochondrial membrane permeability. The hypothesis is that mitochondrial oxidative stress induces mitochondrial protein modification and leakage in the mitochondrial membrane in type-2 diabetes. The present study was conducted to determine the involvement of intra-renal mitochondrial oxidative stress in mitochondrial protein modifications and modulation of membrane permeability in the setting of type-2 diabetes. Diabetes was induced by 6-week regimen of a high calorie and fat diet in C57BL/6J mice (Am J Physiol 291:F694–F701, 2006). Subcellular fractionation was carried out in kidney tissue from wild type and diabetic mice. All fractions were highly enriched in their corresponding marker enzyme. Subcellular protein modifications were determined by Western blot and 2-D proteomics. The results suggest that diabetes-induced oxidative stress parallels an increase in NADPH oxidase-4 (NOX-4) and decrease in superoxide dismutase-1, 2 (SOD-1, 2) expression, in mitochondrial compartment. We observed loss of mitochondrial membrane permeability as evidenced by leakage of mitochondrial cytochrome c and prohibitin to the cytosol. However, there was no loss in control tissue. The 2-D Western blots for mitochondrial post-translational modification showed an increase in nitrotyrosine generation in diabetes. We conclude that diabetes-induced intra-renal mitochondrial oxidative stress is reflected by an increase in mitochondrial membrane permeability and protein modifications by nitrotyrosine generation. G. K. Kartha and K. S. Moshal have contributed equally to this article.     

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

Background & Aims: Tumor necrosis factor (TNF)-α 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-α. Methods: Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-α, 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-α. The susceptibility to TNF-α, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor κB activation by TNF-α 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-α 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-α. Conclusions: These results indicate that mGSH controls the fate of hepatocytes in response to TNF-α. Its depletion caused by alcohol consumption amplifies the power of TNF-α to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.GASTROENTEROLOGY 1998;115:1541-1551     

Effect of Acute Ethanol Exposure on Cultured Fetal Rat Hepatocytes: Relation to Mitochondrial Function

Studies from our laboratory have shown that short-term ethanol exposure inhibits epidermal growth factor-dependent replication of cultured fetal rat hepatocytes, along with a drop in ATP level, and that these effects could be caused, at least in part, by ethanol-induced oxidative stress. In these prior studies, mitochondrial morphology was abnormal and membrane lipid peroxidation products were increased, along with reduced transmembrane potential and enhanced permeability to sucrose. To define the effects of ethanol on mitochondrial function further, the present study examines the impact of ethanol exposure on mitochondrial electron transport chain components. A 24-hr exposure of cultured fetal rat hepatocytes to ethanol (2.5 mg/ml) reduced mitochondrial complex I activity by 16% ( p < 0.05), complex IV by 28% ( p < 0.05), and succinate dehydrogenase by 23% ( p < 0.05). This reduction was paralleled by lower ADP translocase activity (24%, p < 0.05) and diminished mitochondrial glutathione (GSH) (20%, p < 0.05). Pretreatment with 0.1m m S-adenosyl methionine, before ethanol exposure, normalized mitochondrial GSH along with activities of complex I, complex IV, and succinate dehydrogenase. A 3-hr exposure of isolated mitochondria (which do not metabolize ethanol) to ethanol (2.5 mg/ml), inhibited the activities of complex I (19%, p < 0.05), complex IV (24%, p < 0.05), and of ATP synthesis (20%, p < 0.05). It is concluded that: (1) short-term ethanol exposure modestly decreases ATP synthesis by inhibiting activities of fetal hepatic mitochondrial electron transport chain components; (2) ethanol, in the absence of acetaldehyde (as in the isolated mitochondria), can directly impair mitochondrial respiratory chain components; and (3) protection from some of the adverse effects of ethanol by pretreatment with S -adenosyl methionine suggests that these effects are secondary to depletion of mitochondrial GSH.     

Difluoromethylornithine decreases long-lasting protein oxidation induced by neonatal ethanol exposure in the hippocampus of adolescent rats

BACKGROUND: Ethanol exposure and withdrawal during central nervous system development can cause oxidative stress and produce severe and long-lasting behavioral and morphological alterations in which polyamines seem to play an important role. However, it is not known if early ethanol exposure causes long-lasting protein oxidative damage and if polyamines play a role in such a deleterious effect of ethanol. METHODS: In this study we investigated the effects of early ethanol exposure (6 g/kg/d, by gavage), from postnatal day (PND) 1 to 8, and of the administration of difluoromethylornithine (DFMO, 500 mg/kg, i.p., on PND 8), a polyamine biosynthesis inhibitor, on the extent of oxidative modification of proteins. Indices of oxidative modification of proteins included protein carbonyls, 3-nitrotyrosine (3-NT), and protein bound 4-hydroxynonenal (HNE) in the hippocampus, cerebellum, hypothalamus, striatum, and cerebral cortex of Sprague-Dawley rats at PND 40. RESULTS: Both ethanol and DFMO administration alone increased protein carbonyl immunoreactivity in the hippocampus at PND 40, but the combination of DFMO and ethanol resulted in no effect on protein carbonyl levels. No alterations in the content of protein-bound HNE, 3-NT, or carbonyl were found in any other cerebral structure. CONCLUSIONS: These results suggest that the hippocampus is selectively affected by early ethanol exposure and by polyamine synthesis inhibition. In addition, the results suggest a role for polyamines in the long-lasting increase of protein carbonyls induced by ethanol exposure and withdrawal.     

Ethanol‐Induced Oxidative Stress and Mitochondrial Dysfunction in Rat Placenta: Relevance to Pregnancy Loss

Background: Ethanol consumption during pregnancy increases the risk of early pregnancy loss and causes intrauterine growth restriction. We previously showed that chronic gestational exposure to ethanol impairs placentation, and that this effect is associated with inhibition of insulin and insulin growth factor signaling. Since ethanol also causes oxidative stress and DNA damage, we extended our investigations to assess the role of these pathological processes on placentation and placental gene expression. Methods: Pregnant Long Evans rats were pair‐fed liquid diets containing 0% or 24% ethanol by caloric content. Placentas harvested on gestation day 16 were used to examine DNA damage, lipid peroxidation, apoptosis, mitochondrial gene/protein and hormonal gene expression in relation to ethanol exposure. Results: Gestational exposure to ethanol increased fetal resorption, and trophoblast apoptosis/necrosis, oxidative stress, DNA damage, and lipid peroxidation. These adverse effects of ethanol were associated with increased expression of pro‐apoptotic (Bax and Bak) and reduced levels of the anti‐apoptotic Bcl‐2 protein. In addition, increased trophoblast apoptosis proneness was associated with p53‐independent activation of p21, reduced mitochondrial gene and protein expression, and dysregulated expression of prolactin (PRL) family hormones that are required for implantation and pregnancy‐related adaptations. Conclusions: Chronic gestational exposure to ethanol increases fetal demise due to impaired survival and mitochondrial function, increased oxidative stress, DNA damage and lipid peroxidation, and dysregulated expression of prolactin family hormones in placental trophoblasts.     

S-adenosyl-L-methionine co-administration prevents the ethanol-elicited dissociation of hepatic mitochondrial ribosomes in male rats

Background:  Chronic ethanol feeding to male rats has been shown to result in decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates. In addition, ethanol consumption has been shown to result in an increased dissociation of mitoribosomes. S-adenosyl-L-methionine (SAM) is required for the assembly and subsequent stability of mitoribosomes and is depleted during chronic ethanol feeding. The ability of dietary SAM co-administration to prevent these ethanol-elicited lesions was investigated.
Methods:  Male Sprague-Dawley rats were fed a nutritionally adequate liquid diet with ethanol comprising 36% of the calories according to a pair-fed design for 28 days. For some animals, SAM was supplemented in the diet at 200 mg/l. Liver mitochondria were prepared and mitoribosomes isolated. Respiration rates, ATP levels, respiratory complex levels, and the extent of mitoribosome dissociation were determined.
Results:  Twenty-eight days of ethanol feeding were found to result in decreased SAM content, depressed respiration, and increased mitoribosome dissociation. No changes in mitochondrial protein content; levels of respiratory complexes I, III, and V; complex I activities; and ATP levels were detected. Co-administration of SAM in the diet was found to prevent ethanol-induced SAM depletion, respiration decreases and mitoribosome dissociation.
Conclusions:  Taken together, these findings suggest (1) that mitoribosome dissociation precedes respiratory complex depressions in alcoholic animals and (2) that dietary supplementation of SAM prevents some of the early mitochondrial lesions associated with chronic ethanol consumption.     

INTERMITTENT HYPOXIA CONDITIONING PREVENTS OXIDATIVE BRAIN INJURY IN ETHANOL WITHDRAWN RATS

Cyclic bouts of intermittent, normobaric hypoxia (IH) have been found to protect the central nervous system from ischemic and excitotoxic injury. We investigated whether IH mitigates oxidative brain injury in male rats subjected to ethanol intoxication and abrupt ethanol withdrawal (EW). We assessed the effects of IH on superoxide generation, protein oxidation, and mitochondrial membrane swelling and rupture as a marker of mitochondrial permeability transition pore (PTP) opening. Male rats consumed dextrin control diet or 6.5% ethanol diet for 5 weeks. During the last 20 days of the diet, rats were treated with repetitive (5-8/day), brief (5-10 min) periods of hypoxia (9.5-10% inspired O₂) separated by 4 min exposures to room air. Cerebellum, cortex and hippocampus were biopsied at the end of the ethanol diet or at 24 hours of EW. Superoxide and protein carbonyl contents in tissue homogenates and rate of absorbance decline at 540 nm in mitochondrial suspensions were measured as indicators of oxidative stress, protein oxidation and PTP opening, respectively. EW increased superoxide and protein carbonyl contents and accelerated PTP opening in all three brain areas in a manner that was ameliorated by IH. These results suggest that antecedent IH conditioning during chronic ethanol consumption attenuates subsequent oxidative damage to the brain in ethanol withdrawn rats. (supported by NIAAA/AA013864, NIAAA/AA015982, NCCAM/AT003598).     

Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells

BACKGROUND & AIMS: Alcohol consumption exacerbates liver injury in chronic hepatitis C, and enhanced mitochondrial oxidative stress is one possible mechanism. The aim of this study was to determine whether hepatitis C virus core protein and alcohol-inducible cytochrome P450 2E1 contribute to reactive oxygen species production and cytotoxicity in human hepatoma cells. METHODS: Huh-7 cells expressing core protein, cytochrome P450 2E1, or both were exposed to 0.1 mmol/L tertiary butyl hydroperoxide, tumor necrosis factor alpha, and/or 25 mmol/L ethanol. Cytotoxicity, reactive oxygen species production, glutathione content, and mitochondrial membrane potential were measured. RESULTS: Expression of core/cytochrome P450 2E1 synergistically enhanced cell death induced by either tertiary butyl hydroperoxide or tumor necrosis factor alpha. After tertiary butyl hydroperoxide treatment, total reactive oxygen species production was increased more than 3-fold compared with cells that did not express core and cytochrome P450 2E1. Mitochondrial depolarization and reduced glutathione depletion occurred as well, and cell death was prevented by inhibition of mitochondrial permeability transition or caspase activity. Confocal microscopy showed that the mitochondria themselves were the origin of the reactive oxygen species. In the absence of core/cytochrome P450 2E1 expression, mitochondrial changes and cell death did not occur. Ethanol treatment further decreased mitochondrial reduced glutathione content and exacerbated mitochondrial reactive oxygen species production, depolarization, and cell death. All these effects were prevented by the antioxidant N -acetylcysteine. CONCLUSIONS: Mitochondrial reactive oxygen species production is induced by hepatitis C virus core and cytochrome P450 2E1, resulting in a reduction of mitochondrial antioxidant capacity and sensitivity to oxidants and tumor necrosis factor alpha. Alcohol further depletes mitochondrial reduced glutathione, which exacerbates depolarization and cell death. Sensitization of mitochondria to oxidative insults is thus a potential mechanism for alcohol-related exacerbation of liver injury in chronic hepatitis C.     

Ethanol and Protein Metabolism

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Carol C. Cunningham and Victor R. Preedy. The presentations were (1) Ribosomal content, ribosomal localization and the levels of ribosomal protein mRNA and rRNA in rat skeletal muscle exposed to ethanol, by Alistair G. Paice, John E. Hesketh, Timothy J. Peters, and Victor R. Preedy; (2) Altered hepatic mitochondrial ribosome structure after chronic ethanol administration, by Vinood B. Patel and Carol C. Cunningham; (3) Clinical aspects of hepatic protein metabolism and alcohol, by Elena Volpi; and (4) Effects of oral intake of alanine plus glutamine on ethanol metabolism and ethanol-related depression in motor activity, by Kazunori Mawatari, H. Masaki, M. Mori, and Kunio Torii.     

Lysocardiolipin acyltransferase 1 (ALCAT1) controls mitochondrial DNA fidelity and biogenesis through modulation of MFN2 expression

Oxidative stress causes mitochondrial fragmentation and dysfunction in age-related diseases through unknown mechanisms. Cardiolipin (CL) is a phospholipid required for mitochondrial oxidative phosphorylation. The function of CL is determined by its acyl composition, which is significantly altered by the onset of age-related diseases. Here, we examine a role of acyl-CoA:lysocardiolipin acyltransferase lysocardiolipin acyltransferase 1 (ALCAT1), a lysocardiolipin acyltransferase that catalyzes pathological CL remodeling, in mitochondrial biogenesis. We show that overexpression of ALCAT1 causes mitochondrial fragmentation through oxidative stress and depletion of mitofusin mitofusin 2 (MFN2) expression. Strikingly, ALCAT1 overexpression also leads to mtDNA instability and depletion that are reminiscent of MFN2 deficiency. Accordingly, expression of MFN2 completely rescues mitochondrial fusion defect and respiratory dysfunction. Furthermore, ablation of ALCAT1 prevents mitochondrial fragmentation from oxidative stress by up-regulating MFN2 expression, mtDNA copy number, and mtDNA fidelity. Together, these findings reveal an unexpected role of CL remodeling in mitochondrial biogenesis, linking oxidative stress by ALCAT1 to mitochondrial fusion defect.     

Effect of 40% restriction of dietary amino acids (except methionine) on mitochondrial oxidative stress and biogenesis, AIF and SIRT1 in rat liver

Previous studies have shown that the decrease in mitochondrial reactive oxygen species (mitROS) generation and oxidative damage to mitochondrial DNA (mtDNA) that occurs during life extending dietary restriction also occurs during protein or methionine restriction, whereas it does not take place during carbohydrate or lipid restriction. In order to study the possible effects of other amino acids, in this investigation all the dietary amino acids, except methionine, were restricted by 40% in male Wistar rats (RESTAAS group). After 6–7 weeks, experimental parameters were measured in the liver. Amino acid restriction did not change the levels of the methionine metabolites S-adenosylmethionine and S-adenosylhomocysteine, mitochondrial oxygen consumption and ROS generation, oxidative damage to mtDNA, amounts of the respiratory complexes I–IV, and the mitochondrial biogenesis factors PGC-1α and NRF-2. On the other hand, adenylate energy charge, mitochondrial protein oxidation, lipooxidation and glycooxidation, the degree of mitochondrial fatty acid unsaturation, and the amount of the apoptosis inducing factor (AIF) were decreased in the RESTAAS group. Amino acid restriction also increased SIRT1 protein. These results, together with previous ones, strongly suggest that the decrease in mitROS generation and oxidative damage to mtDNA that occurs during dietary restriction is due to restriction of a single aminoacid: methionine. They also show for the first time that restriction of dietary amino acids different from methionine decreases mitochondrial protein oxidative modification and AIF, and increases SIRT1, in rat liver.     

Effect of chronic ethanol ingestion on alveolar type II cell: glutathione and inflammatory mediator-induced apoptosis

BACKGROUND: In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome, a syndrome that requires alveolar type II cell proliferation and differentiation for repair of the damaged alveolar epithelium. We previously showed in a rat model that chronic ethanol ingestion decreased the antioxidant glutathione (GSH) in type II cells and exacerbated endotoxin-mediated acute lung injury. We hypothesized that this GSH depletion by ethanol, particularly mitochondrial GSH, predisposed type II cells to inflammatory mediator-induced apoptosis. METHODS: Adult male rats were fed the Lieber-DeCarli diet for 2, 6, or 16 weeks. Alveolar type II cells were then isolated and treated with hydrogen peroxide or TNF-alpha. The effect on glutathione (cytosolic and mitochondrial), apoptotic events, and necrosis were determined. In other studies, rats were fed ethanol for 6 weeks and were treated with endotoxin and apoptosis of type II cells determined by the TUNEL method. RESULTS: Chronic ethanol ingestion alone resulted in a progressive decrease in mitochondrial GSH and a progressive increase in the basal apoptosis and necrosis rate (p < or = 0.05). Furthermore, there was a progressive increase in the sensitivity of the cells to H2O2 or TNF-alpha induced cytochrome c release, caspase 3 activation, apoptosis, and necrosis (p < or = 0.05). Finally, there was a 2-fold increase in apoptotic type II cells in vivo when chronic ethanol ingestion was superimposed on endotoxemia. CONCLUSIONS: These results suggested that chronic ethanol ingestion resulted in a progressive depletion of mitochondrial GSH and sensitization of type II cells to inflammatory mediator-induced apoptosis and necrosis. These effects may be particularly relevant during acute stress when proliferation and differentiation of these cells are critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of acute respiratory distress syndrome in patients with a history of alcohol abuse.     

Acute ethanol administration induces oxidative changes in rat pancreatic tissue.

BACKGROUND--There is mounting clinical evidence that ethanol toxicity to the pancreas is linked with glutathione depletion from oxidative stress but there is not experimental proof that this occurs. AIMS AND METHODS--The effect of acute ethanol ingestion (4 g/kg) on the pancreatic content of reduced (GSH) and oxidised (GSSG) glutathione, malondialdehyde (MDA), and carbonyl proteins were therefore studied in the rat. RESULTS--Ethanol caused a significant reduction in GSH (p < 0.02) and an increase in GSSG (p < 0.005), MDA (p < 0.05), and carbonyl proteins (p < 0.05) in the rat pancreas. The GSH/GSSG ratios were significantly decreased after ethanol, especially in rats pretreated with diethylmaleate (DEM), a GSH blocker. Administration of ethanol after DEM further increased the rate of lipid and protein oxidation. Pretreatment with cyanamide (an inhibitor of aldehyde dehydrogenase) but not with 4-methylpyrazole (an alcohol dehydrogenase inhibitor) caused higher production of GSSG and MDA. CONCLUSIONS--These findings indicate that acute ethanol reduces the pancreatic content of GSH, which seems to be protective against ethanol toxicity, since its depletion is accompanied by increased oxidative damage to cell structures. The further increase of lipid peroxidation and GSSG production in the presence of cyanamide suggests that acetaldehyde might be responsible for the oxidative changes that occur in pancreatic cells after ethanol administration.     

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency, on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1(+/+)) and Sod1 knockout (Sod1(-/-)) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1(-/-) mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1(+/+) mice fed ethanol showed mild steatosis; both Sod1(+/+) and Sod1(-/-) mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1(-/-) mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in Sod1(+/+) and Sod1(-/-) mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1(-/-) mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1(-/-) mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1(-/-) mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol-induced liver injury.     

Alcohol increases tumor necrosis factor alpha and decreases nuclear factor-kappab to activate hepatic apoptosis in genetically obese mice

Both obesity and alcohol can cause oxidative stress, cytokine induction, and steatohepatitis. To determine the consequences of their combination, we compared the hepatic effects of moderate ethanol binges in lean and obese ob/ob mice. Mice received water or ethanol (2.5 g/kg) by gastric intubation daily for 4 days, and were killed 2 hours after the last administration. Some obese mice also received pentoxifylline, an inhibitor of tumor necrosis factor-alpha (TNF-alpha) production, before each ethanol administration. In lean mice, these moderate ethanol doses did not increase plasma TNF-alpha and hepatic caspase-3 activity, but triggered some apoptotic hepatocytes. Naive ob/ob mice had a few necrotic and apoptotic hepatocytes, but exhibited little oxidative stress, possibly because of adaptive increases in manganese superoxide dismutase, heat shock protein 70 (Hsp70), mitochondrial cytochrome c, and mitochondrial DNA. Alcohol administration to ob/ob mice did not increase oxidative stress despite increased CYP2E1, but increased plasma TNF-alpha, further increased Hsp70, and profoundly decreased p65 nuclear factor kappaB (NF-kappaB) protein and DNA-binding activity in nuclear extracts. Caspase-3 was activated, and more apoptotic hepatocytes were found in intoxicated obese mice than naive obese mice. In intoxicated obese mice, pentoxifylline fully prevented the increase in plasma TNF-alpha the decrease in nuclear NF-kappaB activity, and the increase in hepatic caspase-3, and it also decreased hepatic triglycerides. In conclusion, obese mice develop adaptations that may limit oxidative stress. Moderate ethanol intoxication does not increase oxidative stress in obese mice, but increases TNF-alpha and also decreases nuclear NF-kappaB activity, thus unleashing the apoptotic effects of TNF-alpha.     

Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons

The protein kinase A (PKA) and the cAMP response element (CRE) binding protein (CREB) signaling pathways mediate plasticity and prosurvival responses in neurons through their ability to regulate gene expression. The PKA-CREB signaling mechanism has been well characterized in terms of nuclear gene expression. We show that the PKA catalytic and regulatory subunits and CREB are localized to the mitochondrial matrix of neurons. Mitochondrial CRE sites were identified by using both serial analyses of chromatin occupancy and chromatin immunoprecipitation. Deferoxamine (DFO), an antioxidant and iron chelator known to inhibit oxidative stress-induced death, activated mitochondrial PKA and increased mitochondrial CREB phosphorylation (Ser-133). DFO increased CREB binding to CRE in the mitochondrial D-loop DNA and D-loop CRE-driven luciferase activity. In contrast, KT5720, a specific inhibitor of PKA, reduced DFO-mediated neuronal survival against oxidative stress induced by glutathione depletion. Neuronal survival by DFO may be, in part, mediated by the mitochondrial PKA-dependent pathway. These results suggest that the regulation of mitochondrial function via the mitochondrial PKA and CREB pathways may underlie some of the salutary effects of DFO in neurons.     

S-adenosyl-L-methionine attenuates alcohol-induced liver injury in the baboon

Chronic ethanol consumption by baboons (50% of energy from a liquid diet) for 18 to 36 mo resulted in significant depletion of hepatic S-adenosyl-L-methionine concentration: 74.6 +/- 2.4 nmol/gm vs. 108.9 +/- 8.2 nmol/gm liver in controls (p less than 0.005). The depletion was corrected with S-adenosyl-L-methionine (0.4 mg/kcal) administration (102.1 +/- 15.4 nmol/gm after S-adenosyl-L-methionine-ethanol, with 121.4 +/- 11.9 nmol/gm in controls). Ethanol also induced a depletion of glutathione (2.63 +/- 0.13 mumol/gm after ethanol vs. 4.87 +/- 0.36 mumol/gm in controls) that was attenuated by S-adenosyl-L-methionine (3.89 +/- 0.51 mumol/gm in S-adenosyl-L-methionine-methanol vs. 5.22 +/- 0.53 mumol/gm in S-adenosyl-L-methionine controls). There was a significant correlation between hepatic S-adenosyl-L-methionine and glutathione level (r = 0.497; p less than 0.01). After the baboons received ethanol, we observed the expected increase in circulating levels of the mitochondrial enzyme glutamic dehydrogenase: 95.1 +/- 21.4 IU/L vs. 13.4 +/- 1.8 IU/L; p less than 0.001, whereas in a corresponding group of animals given S-adenosyl-L-methionine with ethanol, the values were only 30.3 +/- 7.1 IU/L (vs. 9.6 +/- 0.7 IU/L in the S-adenosyl-L-methionine controls). This attenuation by S-adenosyl-L-methionine of the ethanol-induced increase in plasma glutamic dehydrogenase (p less than 0.005) was associated with a decrease in the number of giant mitochondria (assessed in percutaneous liver biopsy specimens), with a corresponding change in the activity of succinate dehydrogenase, a mitochondrial marker enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)