Alcohol and Alcoholism Advance Access originally published online on March 29, 2006
Alcohol and Alcoholism 2006 41(3):254-260; doi:10.1093/alcalc/agl017
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CHRONIC ETHANOL CONSUMPTION DECREASES MITOCHONDRIAL AND GLYCOLYTIC PRODUCTION OF ATP IN LIVER
1 Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA and 2 Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA
* Author to whom correspondence should be addressed at: Department ofBiochemistry, Wake Forest University School of Medicine, Winston-Salem, NC 27157-1016, USA. Tel.: +1 336 716-4254; Fax: +1 336 716-7671; E-mail: cunn{at}wfubmc.edu
(Received 18 August 2005; first review notified 19 September 2005; in revised form 26 January 2006; accepted 16 Feburary 2006)
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ABSTRACT |
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Aims: The synthesis of ATP in the liver of the chronic ethanol consumer is suppressed, particularly if the tissue becomes hypoxic. Moreover, the perivenous region of the liver lobule becomes even more oxygen deficient as a result of ethanol consumption. Synthesis of ATP in the perivenous region of the lobule may be depressed in the chronic ethanol consumer due to decreases in both mitochondrial and glycolytic activities. In this study the effects of hypoxia on hepatic ATP levels derived from synthesis by both oxidative phosphorylation and the glycolytic mechanisms were investigated. Methods: Rats were pair-fed liquid diets containing 36% of calories as ethanol or an isocaloric control diet. The contributions of glycolysis and mitochondria to ATP production were assessed employing oligomycin, an inhibitor of oxidative phosphorylation. In order to localize the ethanol-elicited lesion in the glycolytic pathway, the metabolism of [3-3H] D-glucose was followed in hepatocytes from ethanol-fed and control animals. Results: Under both hypoxic and normoxic conditions ATP losses were due to decreases in both glycolytic and mitochondrial ATP production. The rate of production of tritiated water from [3-3H] D-glucose was significantly decreased in hepatocytes from ethanol-fed animals, which indicates there is an ethanol-elicited lesion in glycolysis between glucose and glyceraldehyde-3-phosphate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The development of alcoholic liver disease may progress as a function of the rate of cell damage over the rate at which the damage can be repaired. This would be particularly the case in processes where necrosis is the prominent mode of cell death, as opposed to programmed cell death. It is notable that early in the development of the hepatitis stage of alcoholic liver disease necrotic cell death is more prominent in the perivenous (Zone 3) region of the liver lobule (Ishak et al., 1991
). This may be due partly to a loss in the capacity of the hepatocyte to repair damage elicited by continuous ethanol exposure, which results in the generation of a variety of toxic products such as reactive oxygen species, aldehydic lipid peroxides, acetaldehyde, and the hydroxy ethyl radical. These agents are known to damage cell constituents such as proteins, nucleic acids, and membrane-associated lipids and can be expected to alter cell structure and disrupt cellular homeostasis. The anabolic processes in the hepatocyte have some capacity to repair and/or replace cell constituents that have been damaged as a consequence of ethanol exposure if the energy state of the cell can be maintained. Repair and replacement of cellular macromolecules are energy-requiring processes that can only proceed if there is a sufficiency of ATP. Therefore, conditions that decrease ATP concentrations are likely to exacerbate cell and tissue damage.
It is well established that ethanol consumption damages liver mitochondria such that ATP synthesis is compromised, as has been demonstrated repeatedly with isolated organelles (Cunningham et al., 1990; Hoek, 1994). However, the impact of this damage to mitochondria on the levels of ATP in the hepatocyte has not been evaluated. Furthermore, other studies suggest that chronic ethanol consumption interferes with the glycolytic pathway (Baio et al., 1998; Van Horn and Cunningham, 1999), which might also have an impact on cellular concentrations of ATP, particularly if the tissue becomes hypoxic, which is known to occur in the perivenous region of the liver under conditions of acute ethanol exposure (Sato et al., 1983; Arteel et al., 1996). In this study, the impact of hypoxia on ATP synthesis by both the glycolytic pathway and the mitochondrial oxidative phosphorylation process were investigated in hepatocytes incubated under both hypoxic and normoxic conditions from rats administered an ethanol-containing diet. Metabolism of [3-3H] D-glucose was also compared in hepatocytes from ethanol-fed animals in order to localize the effect of chronic ethanol consumption on glycolysis. This latter study demonstrated an ethanol-elicited lesion in the pathway between glucose and glyceraldehyde-3-phosphate.
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METHODS |
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Feeding regimen, preparation of hepatocytes, and metabolite measurements
Male Sprague–Dawley rats (150–250 g), obtained from Charles River Laboratories, Wilmington, MA, were fed for 31 days a nutritionally adequate diet (Bio-Serv, Frenchtown, NJ) in which ethanol and fat provided 36 and 35% of total calories, respectively. Pair-fed control rats received the same diet, but with maltose–dextrin isocalorically substituted for ethanol. Details of the dietary protocol have been described previously (Bailey and Cunningham, 1998
). Hepatocytes were isolated from these animals as described previously (Spach et al., 1991) and initial viability, as assessed by trypan blue exclusion, was always >90% and was not different in cells from ethanol-fed and control animals. Hepatocyte oxygen utilization and lactate concentrations were determined as described previously (Baio et al., 1998) and ATP concentrations were measured as outlined in Bailey and Cunningham (1999). ATP concentrations were utilized as a measure of liver energy state in this study since it was demonstrated in earlier studies of the effects of chronic ethanol consumption that ATP levels corresponded well to the more rigorous measures of tissue energy state, those being phosphorylation potential and energy charge (Spach et al., 1991).
Incubation conditions for hepatocytes
Estimation of contributions of oxidative phosphorylation and glycolysis to maintenance of ATP concentrations in hepatocytes. In incubations where mitochondrial ATP synthesis was suppressed, oligomycin (10 µg/ml) was added in an incubation mixture which contained 2 x 106 cells/ml. The hepatocytes were then incubated for 60 min while being aerated with either a 5% or <1% O2 atmosphere that contained 5% CO2 to maintain pH, with the balance as nitrogen gas. A 5% O2 atmosphere in the aeration gas mixture establishes an oxygen concentration of 41 µM in the incubation medium, which is very close to the median oxygen concentration of 44 µM estimated for a rat liver in situ (Sato et al., 1983; Fukui et al., 1990). When the medium was saturated with 95% N2:5% CO2, the hepatocyte suspension medium was 
1% O2, which is 8 µM O2 (Bailey and Cunningham, 1999). Further details of incubation conditions are described in Bailey and Cunningham (1999). After incubation, 1 ml aliquots were quick frozen and stored at –80°C until analysed for lactate and ATP.
The values obtained for ATP and lactate in the presence and absence of oligomycin were utilized to estimate the relative contributions of oxidative phosphorylation and glycolysis in maintaining the levels of ATP in hepatocytes. Oligomycin-insensitive ATP concentrations were attributed to glycolytic production of ATP. Oligomycin-sensitive ATP concentrations were attributed to mitochondrial production of ATP and were estimated from the differences between ATP concentrations in the absence of oligomycin and oligomycin-insensitive ATP concentrations. These estimates (oligomycin inhibition, not corrected) were then corrected for oligomycin stimulation of glycolysis utilizing the lactate levels reported in Fig. 1A. In a preliminary experiment it was determined that the rate of lactate production was linear over the entire 60 min incubation period under all the conditions utilized (1% O2, 5% O2, ± oligomycin; data not shown).
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, P Lactate production over the 1 h incubation period in both the presence and absence of oligomycin was determined by subtracting lactate levels in un-incubated cells from concentrations achieved after 1 h. Lactate values obtained for oligomycin incubated cells were divided by the concentrations obtained in the absence of oligomycin to obtain the fold increase in lactate due to the presence of oligomycin. The oligomycin-insensitive ATP concentrations (Fig. 1B) were then divided by the fold increase in lactate to obtain a concentration for ATP that reflects only the basal oligomycin-insensitive (glycolytic) synthesis of ATP. The corrected glycolytic ATP concentrations, thus derived, were subtracted from total ATP measured in the absence of oligomycin to estimate mitochondrial-derived ATP.
In carrying out these calculations it is being assumed that the concentration of ATP is related to the relative rates of synthesis by glycolysis and oxidative phosphorylation. This assumption is valid only if the rate of ATP consumption in the cells does not change with the addition of oligomycin. However, this may not be the case, and, as a result, the values reported have to be considered as estimates.
Incubation in the presence of [3-3H] D-glucose. In experiments where hepatocytes were utilized to assess activities associated with glycolysis (glucose conversion to glyceraldehyde-3-phosphate and lactate production) hepatocytes (5 x 106 cells/ml) were preincubated for 30 min in the presence or absence of glucosamine (40 mM) and in the presence of BSA (25 mg/ml) while being aerated with either a 5% or <1% O2 atmosphere under conditions described above. After the preincubation period the hepatocyte suspensions were brought to 15 mM glucose and 4 µCi of [3-3H] D-glucose (Perkin Elmer, Boston, MA) was added. After an additional 30 min incubation period during which the flasks were capped, 0.5 ml aliquots were mixed with 1.5 ml ice-cold 95% ethanol to terminate glucose metabolism. When [3-3H] D-glucose is metabolized 3H2O is formed due to the exchange of a proton from dihydroxyacetone-P with H2O in the medium, mediated by the active site glutamate in triose phosphate isomerase (Katz et al., 1975; Garrett and Grisham, 1999). Glucose that was not metabolized and radiolabelled products including 3H2O were separated, and product formation was determined as described previously (Katz et al., 1975; Garrett and Grisham, 1999). Because glucose was converted to C3 units during flux through glycolysis, products were expressed as C3 equivalents (1 mol of glucose = 2 mol of C3 equivalents). The details for calculating C3 equivalents are described in Katz et al. (1975).
Statistical analyses
Hepatocytes isolated from rats fed control and ethanol-containing diets will be referred to as control hepatocytes and ethanol hepatocytes, respectively. All values in the bar graphs are reported as the mean ± SEM. Statistically significant differences between treatments were obtained by two-factor ANOVA using the statistical package within the Microsoft Excel program. Diet (control versus ethanol) and inhibitor presence (no inhibitor versus glucosamine or oligomycin) were the two independent variables. The level of statistical significance was set at P < 0.05. When un-incubated hepatocytes were analysed the paired t-test was used to determine statistically significant differences between control and ethanol hepatocytes.
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RESULTS |
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Ethanol-related decreases in mitochondrial and glycolytic production of ATP
It has been demonstrated in several studies that under hypoxic conditions the ATP concentrations in hepatocyte preparations from ethanol-fed rats are significantly lower than that in control cells (Spach et al., 1991
; Ivester et al., 1995; Bailey and Cunningham, 1999). The decreases in cellular ATP concentrations could be due to depressions in both mitochondrial and glycolytic ATP synthesis since there is evidence that both mitochondrial function and glycolytic activity decline in the liver of the alcohol consumer (Van Horn and Cunningham, 1999; Cunningham and Bailey, 2001). In order to evaluate the relative contributions of mitochondrial and glycolytic synthesis of ATP in maintaining the level of ATP in ethanol and control hepatocytes, the oxidative phosphorylation inhibitor, oligomycin was employed. This compound inhibits both ATP synthesis and limits mitochondrial oxygen utilization to the State 4 rate (Devenish et al., 2000). Maximal inhibition of hepatocyte respiration and depression of ATP concentrations was achieved in the presence of oligomycin at 10 µg/ml (data not included).
While oligomycin has no known inhibitory effects on reactions in the glycolytic pathway it could inhibit glycolysis by causing a decrease in ATP concentrations such that flux through the glucokinase catalysed reaction became rate limiting. However, the ATP concentrations in freshly prepared ethanol hepatocytes were several fold above the Km value of glucokinase for ATP (Parry and Walker, 1966). Even after 1 h incubations under hypoxic conditions the ATP remaining in ethanol and control hepatocytes (Fig. 1) would provide for a flux of 
0.4 and 0.7 Vmax for glucokinase, respectively. It was established that the production of lactate is linear over the 60 min incubation period under all the conditions utilized (1% O2, 5% O2, ± oligomycin; data not shown). As demonstrated in Fig. 1A, even higher concentrations of lactate were accumulated in control hepatocytes when oligomycin (10 µg/ml) was included in incubations, demonstrating that lactate production was stimulated, rather than inhibited. While the lactate:pyruvate ratio varies significantly depending on the oxygen environment of both ethanol and control hepatocytes, the concentrations of lactate far exceed those of pyruvate under all conditions utilized in this study (Van Horn and Cunningham, 1999). Therefore, in the present study lactate levels were utilized as one indicator of glycolytic activity.
There was a significant decrease in ATP concentrations in ethanol hepatocytes under both hypoxic and normoxic conditions (Fig. 1B). Moreover, in the presence of oligomycin ATP concentrations were lowered significantly under both hypoxic (P = 0.0001) and normoxic (P = 6 x 10–11) conditions in both control and ethanol hepatocytes (Fig. 1B), illustrating that ATP synthesis via oxidative phosphorylation was being prevented in both control and ethanol hepatocytes. The data in Fig. 1 were analysed as described in the Methods section to correct for oligomycin stimulation of glycolysis. Table 1 contains both uncorrected and corrected estimates for the relative contributions of glycolysis and oxidative phosphorylation in maintaining the steady-state levels of ATP in ethanol and control hepatocytes under both hypoxic and normoxic incubation conditions. Under hypoxic conditions (1% O2) the decreased ATP concentrations in ethanol hepatocytes were due to depressions in both mitochondrial and glycolytic contributions to total ATP. This is illustrated both in the uncorrected and corrected data, but that corrected for oligomycin stimulation of glycolysis suggests a dramatic decrease in glycolytic production of ATP under hypoxic conditions (7.25 versus 1.51 nmol ATP/106 cells in control and ethanol hepatocytes, respectively; Table 1, <1% O2 incubations). Under normoxic conditions (5% O2) the ethanol-elicited decrease in ATP levels was also related to depressions in both mitochondrial and glycolytic production of ATP, as was also illustrated more dramatically when the data were corrected for oligomycin stimulation of glycolysis (oligomycin inhibition, corrected).
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Lesion in glycolytic pathway elicited by chronic ethanol consumption
Previous attempts to localize an ethanol-related lesion in the glycolytic pathway utilizing [U-14C] or [6-3H] D-glucose were unsuccessful (unpublished data). The data in Fig. 2A, generated by following metabolism of [3-3H] D-glucose, demonstrate that in ethanol hepatocytes there is a decrease in flux through the phase 1 portion (Garrett and Grisham, 1999
) of the glycolytic pathway in which glucose is converted to glyceraldehyde-3-phosphate. This portion of the glycolytic pathway can be monitored utilizing [3-3H] D-glucose since tritium is exchanged with H2O at the triose phosphate isomerase step where dihydroxyacetone-P is converted to glyceraldehyde-3-phosphate. This data indicates that there is an ethanol-elicited lesion in the glycolytic pathway between glucose and glyceraldehyde-3-phosphate, which is expressed both when hepatocytes are in a hypoxic or a normoxic environment (Fig. 2A). The tritium-H2O exchange is significantly inhibited by glucosamine, a competitive inhibitor of glucokinase (Van Schaftingen, 1995).
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As was observed in Fig. 1, the data in Fig. 2B demonstrate a dramatic decrease in lactate concentrations in hepatocytes from ethanol consumers under both hypoxic and normoxic conditions. This decrease parallels the observations in Fig. 2A, which indicate there is a lesion in the phase 1 region of the glycolytic pathway. The data in Fig. 2A and B were analysed to determine how much the glucose (15 mM) contributed to the lactate accumulated over the 30 min period after it was added. The C3 units obtained over this 30 min period, representing glucose utilization, were compared with the lactate accumulated over the same period. Under hypoxic conditions the production of lactate compared with the production of C3 metabolites from glucose, as measured from 3H2O levels, indicates that in control and ethanol hepatocytes the added glucose contributed 17 and 63%, respectively, of the lactate, with the remainder from endogenous stores of glucose, e.g. glycogen. When control and ethanol cells were incubated in a 5% O2 atmosphere, added glucose contributed 35 and 100%, respectively, of the lactate. These observations are consistent with a limited source of endogenous glucose in ethanol hepatocytes. Fig. 2C illustrates that ATP concentrations were also depressed in ethanol hepatocytes under conditions where cells were incubated in the presence of added glucose.
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DISCUSSION |
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Chronic consumption of ethanol in high amounts leads to increased production in the liver of reactive compounds, all of which react with cell components to render them non-functional (Rouach et al., 1997
; Cahill et al., 1999; Niemelä, 2001). Under the circumstance where there are increases in intracellular toxins and subsequent damage to macromolecules essential for maintenance of viability, the cell may still be able to repair and/or replace altered components by anabolic mechanisms if it can maintain normal levels of ATP. It has been demonstrated previously that if ATP concentrations in ethanol hepatocytes are maintained at near normal levels the cells will remain viable even while generating high amounts of the lipid peroxide product, malondialdehyde (Cunningham and Ivester, 1999). In contrast, under hypoxic conditions where malondialdehyde levels were much lower in ethanol hepatocytes, viability decreased, which correlated with lowered ATP concentrations (Ivester et al., 1995). These observations inferred that if ATP levels are maintained the viability of hepatocytes, even those from ethanol consumers, could be preserved. They emphasize a role for ATP in the maintenance of hepatocyte viability in an environment where the tissue is being subjected to ethanol-associated toxicity. However, several previous studies have demonstrated that the capacity of hepatocytes to maintain ATP concentrations is depressed as a result of chronic ethanol consumption (Bailey and Cunningham, 1999; Ivester et al., 1995; Spach et al., 1991). The data in the present investigation (Fig 1; Table 1) indicates that chronic ethanol consumption causes a decrease in both mitochondrial and glycolytic production of ATP under both hypoxic and normoxic conditions, which results in significant decreases in total ATP in ethanol hepatocytes.
The observations in this study demonstrate that the ethanol-related lesions in mitochondrial function (Cunningham et al., 1990; Hoek, 1994; Cunningham and Bailey, 2001) translate into depressed ATP production by the oxidative phosphorylation system whether hepatocytes are normoxic or hypoxic. The data also illustrate that there is significant mitochondrial ATP production even under hypoxic conditions. As mentioned earlier, under the low oxygen conditions employed in this study the oxygen content of the incubation medium is in the low micromolar range (Bailey and Cunningham, 1999), which is close to the Km value for O2 for respiration in an intact hepatocyte (Jones and Mason, 1978). Therefore, mitochondrial respiratory activity and ATP synthesis are still possible when O2 is 1% of the gas mixture. This was verified with control hepatocytes where the mitochondrial contribution to hepatocyte ATP concentrations was estimated to be 63% of that observed when hepatocytes were normoxic (Table 1). In contrast, in ethanol hepatocytes maintained under hypoxic conditions the mitochondrial contribution to hepatocyte ATP concentrations was significantly decreased (37% of control) when compared with control hepatocytes kept hypoxic. The more significant decrease in mitochondrial ATP production in ethanol hepatocytes under hypoxic conditions may occur when the combination of lowered oxygen tension and damaged mitochondria (Cunningham et al., 1990; Hoek, 1994) results in a rate of ATP synthesis that is too slow to maintain the steady-state concentrations of mitochondrial-derived ATP observed in cells from normal liver (Table 1). This combination of hypoxia and damaged mitochondria exists in situ in the perivenous region of the liver lobule of the chronic ethanol consumer (Sato et al., 1983; Arteel et al., 1996).
Earlier studies demonstrated clearly that steady-state levels of pyruvate + lactate are significantly lower in ethanol hepatocytes being maintained under either hypoxic or highly oxygenated environments (Baio et al., 1998; Van Horn and Cunningham, 1999). These observations suggested the possibility that either glycolysis is depressed or gluconeogenesis is up-regulated. However, the prevailing evidence indicates that gluconeogenesis is decreased as a result of chronic ethanol exposure (Grimberg et al., 1998; Petersen et al., 1999; Changani et al., 2001). Moreover, the decrease in metabolism of [3-3H] D-glucose in ethanol hepatocytes shown in this study demonstrates directly that there is a depression in flux through the phase 1 portion (Garrett and Grisham, 1999) of the glycolytic pathway in which glucose is converted to glyceraldehyde-3-phosphate. This occurs whether hepatocytes are in a hypoxic or in a normoxic environment.
In addition to the ethanol-induced lesion in the phase 1 portion of the glycolytic pathway, there may be another factor that limits the capacity of ethanol hepatocytes to generate ATP via glycolysis. The levels of glycogen, the endogenous substrate for glycolysis, are dramatically lower in intact liver and hepatocytes in chronic ethanol consumers, whereas they are normal in control animals (Gordon and Lough, 1972; Petersen et al., 1999; Van Horn et al., 2001). Lowered glycogen levels could influence the rate of glycolysis in ethanol consumers by limiting glucose-6-P availability. This observation also predicts that higher levels of glycolytic products would be generated from added glucose in hepatocytes from ethanol consumers since glycogen levels are limiting. Indeed, under normoxic conditions the C3 units generated from added glucose account for 100% of the lactate formed in ethanol hepatocytes, but only 35% in control cells. Moreover, under hypoxic conditions the added glucose was responsible for 63% of the C3 units in cells from ethanol consumers, but only 14% in control hepatocytes. This increase in utilization of exogenous substrate by cells from ethanol consumers suggests that low glycogen levels, in combination with the lesion in phase 1 portion of the pathway, limits glycolytic production of ATP.
ATP concentrations are significantly lower in ethanol hepatocytes maintained under hypoxic conditions, totalling only 8.9 nmol/106 cells as compared with 28 nmol/106 cells in control hepatocytes maintained in a low oxygen environment (Table 1). As mentioned above, this is due to significant reductions in both mitochondrial and glycolytic production of ATP. These combined effects of hypoxia and chronic ethanol consumption might be present in the perivenous region of the liver lobule. Normally there is an oxygen gradient across the liver lobule with the oxygen tension being significantly lower in the perivenous region (Jungermann and Kietzmann, 1997). During ethanol consumption and subsequent metabolism the oxygen tension in the perivenous region is lowered even further, resulting in hypoxia (Sato et al., 1983; Arteel et al., 1996), at least partly due to increased demands for oxygen associated with ethanol oxidation. It is possible that in this region of the lobule ATP synthesis could be limited during ethanol metabolism, particularly in the chronic ethanol consumer since both mitochondrial and glycolytic synthesis appear to be depressed under hypoxic conditions (Table 1, <1% O2). It is interesting to note that the perivenous region is the first to demonstrate hepatocyte necrosis and accompanying inflammation at the alcoholic hepatitis stage of alcoholic liver disease (Ishak et al., 1991). This suggests that an oxygen deficit in perivenous hepatocytes may be a risk factor for the development of alcoholic liver disease due to a resulting decrease in ATP concentration.
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ACKNOWLEDGEMENTS |
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This investigation was supported by a MERIT Award 02887 and a Senior Scientist Award 00279 to C.C.C. and grant 13610 from the National Institute on Alcohol Abuse and Alcoholism. S.M.B. and C.G.V.H. were supported by NIAAA training grant 07565 during their participation in this study.
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