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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.
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.
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.
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.
Studies have shown that chronic ethanol feeding to young male rats results in a 29% decrease in mitochondrial protein translation (Patel and Cunningham, 2002), a phenomenon that is likely to be responsible for the well documented ethanol-elicited decrease in mitochondrial protein synthesis (Coleman and Cunningham, 1990, 1991). One potential mechanism underlying this lesion may be the impaired assembly of mitochondrial ribosomes (mitoribosomes). Ethanol feeding has been shown to result in the formation of a mitoribosome that possesses a decreased sedimentation coefficient (Patel and Cunningham, 2002), a decreased translational diffusion coefficient (Patel and Cunningham, 2002), an increased hydrodynamic diameter (Patel and Cunningham, 2002) and an altered protein content (Cahill et al., 1996; Cahill and Cunningham, 2000). These ethanol-derived alterations are accompanied by a significant degree of dissociation (8 to 14%) of the intact mitoribosomal monomer into its constituent subunits (Cahill et al., 1996; Patel and Cunningham, 2002).
S-adenosyl-L-methionine (SAM, Adomet) is a ubiquitous methyl donor for a number of methyltransferases involved in methylation of RNA, DNA, proteins, phospholipids, and hormones and, as such, is a pivotal component of the mitoribosomal assembly process. Mitochondrial ribosomes are assembled from mitochondrially encoded rRNA and nuclear-encoded ribosomal proteins. A number of highly conserved regions on both the 12S and 16S rRNA require posttranscriptional methylation by a SAM-dependent rRNA methyltransferase before they can be assembled into competent ribosomal particles (Baer and Dubin, 1981). Other roles for SAM in mitoribosomal assembly include posttranslational methylation of specific mitochondrial ribosomal proteins (MRPs) (Polevoda and Sherman, 2007) and synthesis of ribosome-stabilizing polyamines. SAM is synthesized in the liver from methionine and ATP by methionine adenosyltransferase (MAT) (Kotb et al., 1997). The methionine required for SAM synthesis is obtained from the diet or it can be regenerated from homocysteine by methionine synthase (MS) or, to a lesser extent, by betaine homocysteine methyltransferase (BHMT). Chronic ethanol consumption markedly inhibits MS activity within the first week of feeding, resulting in a 40% depletion in SAM levels (Barak et al., 1996). This is immediately compensated for in the rat by an upregulation in BHMT (Barak et al., 1984) activity leading to restoration of SAM levels after 14 days (Barak et al., 1996). This initial ethanol-elicited decrease in SAM levels may have a deleterious impact on mitoribosomal assembly and may be one of the underlying mechanisms responsible for their impaired structure and increased susceptibility to dissociation.
These studies demonstrate that ethanol-elicited dissociation of mitoribosomes precedes respiratory complex depression and that dietary supplementation of SAM to alcoholic male rats prevents both the ethanol-elicited increases in mitoribosome dissociation and the decreases in mitochondrial respiration.
All reagents were molecular biology grade and obtained from Sigma–Aldrich (St Louis, MO) or Fisher Scientific (Pittsburgh, PA) except where stated.
Male Sprague-Dawley rats (250 g) were maintained on a nutritionally sufficient liquid diet (Lieber and Decarli, 1994) with ethanol constituting 36% of the calories for 4 weeks. Control animals were pair-fed an identical diet but with maltose dextrin isocalorically substituted for the ethanol. S-adenosyl-L-methionine (SAM, toluenesulfonate salt) was administered to ethanol-fed and pairfed animals in the diet at 200 mg/l. SAM was not found to have any effect on the amount of liquid diet (1780 ± 123 ml over the course of the study for all experimental groups) or the amount of ethanol (10.9 ± 0.5 g/kg on day 3, 12.6 ± 0.3 g/kg on day 28) consumed. Animals received humane care according to criteria outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23, Revised 1985).
Hepatic mitochondria were prepared by differential centrifugation (Cohen et al., 1985). Mitoribosomes were isolated in the presence of 20 mM MgCl2 as previously described (Cahill et al., 1995) with the modification that they were not incubated with puromycin prior to sucrose density gradient analyses. In one specific case, i.e., Fig. 2B, gradient-purified mitoribosomes were dissociated into subunits by incubation, in the absence of magnesium, with EDTA (1 mM) for 10 minutes prior to a second sedimentation through a 10 to 30% sucrose density gradient. Sucrosecushion purified mitoribosomes were assayed for activity over a period of 20 minutes using the poly(U)-directed phenylalanine polymerization assay as previously described (Cahill et al., 1995). Previous studies from our group have demonstrated that polymerization proceeds in a linear fashion over this time period (Cahill et al., 1995; Patel and Cunningham, 2002). Soluble mitochondrial translation factors were prepared as previously described (Cahill et al., 1995).
Mitochondria (30 mg) were dissolved in aminocaproic acid (1 M), Tris, pH 7.0 (50 mM) and dodecylmaltoside (3% w/v). Following centrifugation (100,000 × g for 15 minutes), Coomassie Blue (CB) G250 was added to a final concentration of 0.3%. Complexes (300 µg) were loaded onto 5 to 13% polyacrylamide gels (polymerized in 50 mM Bis-Tris, 0.5 mM aminocaproic acid, pH 7.0), run at 30V for the first 30 minutes and then the voltage increased to 80 V. The anode buffer comprised Bis-Tris–HCl, pH 7.0 and the cathode buffer Tricine (50 mM), Bis-Tris–HCl, pH 7.0 containing 0.02% CB. For in-gel assay of complex I, bluenative gels were incubated overnight at room temperature in Tris–HCl (100 mM), NADH (140 µM), and nitro blue tetrazolium (NBT, 1 mg/ml), pH 7.4.
Mitochondria were resuspended (0.5 mg/mL) in respiration buffer (130 mM potassium chloride, 2 mM potassium phosphate, 3 mM HEPES, 2 mM magnesium chloride, 1 mM EGTA, pH 7.2) in a thermally jacketed electrode chamber. Oxygen utilization was measured with a Clarke electrode in the presence of respiratory complex I substrates malate (5 mM) and glutamate (5 mM) or respiratory complex II substrate succinate (10 mM) and amytal (1 mM). Respiration-coupled ATP synthesis was initiated by the addition of ADP (0.6 mM).
Liver tissue (50 to 100 mg) was homogenized in 8 volumes of perchloric acid (0.5N). Following clarification at 12,500 × g, the supernatant was passed through a 0.22 mm filter (Millipore, Bedford, MA) before being injected onto a reverse phase Partisphere 5 C18 column (5 µm particle size, 4.6 mm × 250 mm inner diameter, Whatman Inc., Florham Park, NJ). SAM peaks were detected using a mobile phase consisting of sodium perchlorate (50 mM), sodium acetate pH 3.5 (100 mM), sodium heptylsulfonate (2.4 mM), and acetonitrile (4.2% vol/vol) at a flow rate of 1.3 ml/min with UV detection (254 nm).
Adenine nucleotide levels were measured spectrophotometrically using standard enzymatic analyses as described by Trautschold and colleagues (ATP; Trautschold et al., 1983) and Jaworek and Welsch (ADP and AMP; Jaworek and Welsch, 1983). Total DNA was extracted from liver using the QIAamp DNA mini kit (Qiagen Inc., Valencia, CA) according to manufacturer’s instructions. DNA levels were measured using A260nm UV absorption values and the picogreen quantification assay (Invitrogen Corporation, Carlsbad, CA) according to manufacturer’s instructions. Protein was extracted from liver tissue by homogenizing it in Igepal (1%), deoxycholate (0.5%), sodium dodecyl sulfate (0.1%), complete protease inhibitors (Roche Applied Science, Indianapolis, IN), sodium chloride (150 mM), and Tris–HCl (50 mM), pH 7.4. Protein levels were determined using the Bio-Rad detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as the protein standard. Immunoblotting of prohibitin was performed using a primary mouse anti-Prohibitin polyclonal antibody (1 µg/ml; RDI, Flanders, NJ) and a secondary horseradish peroxidase-conjugated antibody (Pierce, Rockford, IL). Prohibitin bands were detected by chemiluminescence using SuperSignal West Pico (Pierce, Rockford, IL).
Paired t-tests were performed using the Excel software package (Microsoft Corporation, Redmond, WA). Two-way ANOVA analyses were performed using Graphpad Prism version 4.0c for Macintosh (GraphPad Software, San Diego, CA).
Chronic ethanol feeding for 28 days resulted in a 21% decrease in hepatic SAM levels. Co-administration of SAM limited this decrease to 9% (control: 675 ± 69 nmol/liver, ethanol: 533 ± 68*, SAM: 672 ± 49, ethanol plus SAM: 613 ± 17; *p < 0.05, n = 4, significance of difference of ethanol-fed compared with controls using the paired t-test). When these values were normalized for animal body weight it was found that that dietary supplementation of SAM (200 mg/l) did not specifically increase hepatic SAM levels but rather prevented the ethanol-derived decrease in SAM (control 1.84 ± 0.20 nmol/liver/g body weight, ethanol: 1.54 ± 0.19**, SAM; 1.80 ± 0.14, ethanol plus SAM: 1.78 ± 0.04; **p < 0.01, n = 4, significance of difference of ethanol-fed compared with controls using the paired t-test).
Ethanol feeding resulted in a 27% increase in liver/body weight (LBW) ratio in animals that were concomitantly fed SAM in the diet. In the absence of SAM, ethanol feeding resulted in a 20%increase in LBW (Fig. 1C). SAM treatment increased the LBW ratio by 8% in control livers and by 14% in alcoholic livers (Fig. 1C). The larger LBW ratios were primarily caused by increases in liver weight. Ethanol produced similar increases in liver weight when administered alone (14%) or in the presence of SAM (17%). Additionally, SAM was shown to increase control liver weights by 12% (Fig. 1B). In part, this was due to a 6%increase in soluble liver proteins (control; 40.1 ± 0.7 mg/g wet weight liver, SAM; 42.4 ± 0.6**, **p < 0.01, n = 3, significance of difference between controls and SAM-treated assessed using the paired-t-test). No changes in DNA content were detected (data not shown). Two-way ANOVA analyses of these data suggest that both ethanol feeding and SAM administration impact liver size independently of each other (see legend to Fig. 1).
In agreement with our earlier studies (Cahill et al., 2005) ethanol feeding was found to have no effect upon the amount of hepatic mitochondrial protein isolated (454 ± 53 mg/liver, 46 ± 4 mg/g liver, n = 6). Mitochondrial protein levels were not affected by SAM treatment. Levels of the mitochondrial marker prohibitin were also found to be unaffected by ethanol or SAM feeding (data not shown).
Figure 2A shows a sucrose density gradient (10 to 30%) profile of purified mitochondrial ribosomes isolated from a total of 8 rat livers. In a healthy control rat liver, mitochondrial ribosomes exist predominantly as the intact 55S monomer with trace amounts of the unassembled 28S small (SSU) and 39S large (LSU) ribosomal subunits. Confirmation that the 2 peaks appearing above the 55S monomer are indeed 28S and 39S subunits is presented in Fig. 2B. Fractions containing the 55S peak (Fig. 2A) were pooled, sedimented and the purified monomers incubated in a magnesium-free buffer in the presence of EDTA (see Materials and Methods). Magnesium is required for the association of ribosomal subunits and its removal results in dissociation of the monomers. Figure 2B shows a sucrose density gradient of the dissociated 55S monomer. Only 2 peaks arise from dissociation of the intact monomer and these peaks appear in the same fractions as the 2 smaller peaks labeled 28S and 39S in Fig. 2A. Having confirmed the sedimentation patterns of the intact monomers and dissociated subunits, total mitochondrial ribosomal particles were isolated from ethanol-fed animals and their paired controls. Particles were then separated on 10 to 30% sucrose density gradients in the presence of magnesium ions. Figure 2D shows that ethanol-fed animals contain decreased levels of intact 55S monomers and increased levels of 39S subunits when compared with control animals. Further confirmatory evidence as to the identification of specific peaks within the gradients is presented in Fig. 2C where a sedimentation profile of hepatic mitoribosomes isolated from an ethanol-fed animal is superimposed onto a western blot profile of DAP3 (death-associated protein 3). DAP3 (also known as mitochondrial ribosomal protein S29, MRPS29) is a marker protein for the SSU and can be seen to localize specifically to the 28S and 55S peaks (Fig. 2C).
Figures 3A–3D show sucrose density gradients of hepatic mitochondrial ribosomes isolated from a representative paired group of animals fed the following diets; control (Fig. 3A), ethanol (Fig. 3B), SAM (Fig. 3C) and ethanol plus SAM (Fig. 3D). Figure 3B shows a typical sucrose density gradient profile of hepatic mitochondrial ribosomes isolated from ethanol-fed animals (see also Fig. 2C). The ethanol-elicited decrease in the levels of 55S monomers and the increased percentage of ribosomal particles present as 39S subunits is in agreement with Fig. 2C and with previous studies (Cahill et al., 1996; Patel and Cunningham, 2002). This was prevented by dietary supplementation of SAM(Fig. 3D). Statistical analyses of the levels of dissociated 39S LSU, expressed as a percentage of intact 55S monomers and as a percentage of total 39S plus 55S, is represented in Fig. 3F. Ethanol consumption resulted in a significant increase in the levels of dissociated 39S LSUs, an increase that was largely prevented by SAM supplementation. S-adenosyl-L-methionine administration alone resulted in a small, albeit insignificant, decrease in free 39S LSU levels (Fig. 3F). Interestingly, the increase in 39S LSUs was not paralleled by an increase in 28S SSUs. This may be due to differences in the stability of dissociated ribosomal subunits. Large ribosomal subunits are known to be associated with the inner mitochondrial membrane (Szyrach et al., 2003), a phenomenon that may result in a subunit being more resistant to degradation. Ethanol feeding has previously been shown to decrease the sedimentation coefficient of intact 55S monomers (Patel and Cunningham, 2002), a property that can be discerned by superimposing the respective gradients (Cahill et al., 1996; Cahill and Cunningham, 2000). Figure 3E shows that the addition of SAM to an ethanol-containing diet results in mitochondrial ribosomes that sediment further through the gradient than ones isolated from an animal fed solely ethanol. This alteration in mitoribosomal sedimentation properties results in a sedimentation profile that is more consistent with that of a control ribosome (Cahill et al., 1996; Cahill and Cunningham, 2000). In order to ascribe a functional significance to the actions of SAM, mitochondrial ribosomes from control and alcoholic animals were assessed for poly(U)-directed phenylalanine polymerization capability. Figure 4 shows that ethanol feeding resulted in a 30% decrease in mitoribosomal activity, a phenomenon that was prevented by concomitant administration of SAM in the diet.
Figure 5A shows a representative blue-native gel of respiratory complexes I, V, and III isolated from solubilized mitochondria. No significant changes in complex levels were detected in alcoholic animals (Fig. 5A). Similarly, in-gel complex I activities (Fig. 5B) and adenine nucleotide levels (ATP: 2.85 ± 0.17 µmol/g wet weight liver, ADP: 1.10 ± 0.08, AMP: 0.22 ± 0.02, total adenine nucleotides: 4.17 ± 0.23, ATP/ADP: 2.6 ± 0.06, n = 3) were found to be unaltered in animals fed ethanol chronically for 28 days.
In contrast to its effects on respiratory complex levels, adenine nucleotide content and prohibitin levels, 28 days of ethanol feeding was found to have a profound impact on mitochondrial respiration. Ethanol was found to decrease state 3 respiration by 47% and state 4 respiration by 27% (not significant) when complex I substrates were used. This resulted in a decrease in respiratory control ratio (RCR) of 32% (Table 1). Supplementation of the ethanol diet with SAM prevented all of these alterations. When complex II substrates were used, ethanol was found to decrease state 3 respiration by 40% and state 4 respiration by 23%. As with complex I-mediated respiration, supplementation of SAM in the diet significantly prevented the ethanol-elicited depressions in respiration. Two-way ANOVA analysis of the respiratory data for complex I substrates reveals that an interaction exists between ethanol feeding and dietary SAM supplementation (see legend to Table 1).
We have demonstrated that 28 days of chronic ethanol feeding impacts hepatic mitochondria by depressing respiration (Table 1), increasing the dissociation of mitoribosomes (Fig. 2D, Fig. 3A and 3B) and inhibiting mitoribosomal activity (Fig. 4). No changes, however, were detected in the levels of respiratory complexes (specifically I, III, and V, Fig. 5A), the activity of complex I(Fig. 5B), adenine nucleotide levels (see Results text), or mitochondrial protein content (see Results text). These data are the first to delineate ethanol-mediated mitoribosome impairment from respiratory complex depression and lead us to conclude that the damage to the mitoribosome seen in alcoholic animals precedes the well-documented structural impairment of the respiratory chain (Bailey et al., 2006; Coleman and Cunningham, 1990). The shorter ethanol-feeding period has allowed us to determine the sequence of a number of critical alcohol-derived mitochondrial lesions and, in so doing, highlighted an important role for the mitoribosome in ethanol-mediated mitochondrial dysfunction. Interestingly, levels of the mitochondrial chaperone protein prohibitin were also found to be unaffected by ethanol feeding (see Results text). This shows that the elevated levels of prohibitin in the liver only occur when respiratory complex levels are depressed, i.e., during more prolonged periods of ethanol feeding. In cases where ethanol feeding has not impacted respiratory complex levels, such as in this study, prohibitin levels remain unaffected. This suggests either that prohibitin is an important signaling protein for respiratory complex assembly or that it is under the same mechanism(s) of control as other respiratory complex constituents. In addition to mitoribosomal damage, 28 days of ethanol feeding were also found to decrease respiration in the presence of complex I substrates (Table 1). Ethanol feeding has been shown to cause iNOS induction, to increase NO production (Yuan et al., 2006), and to increase the sensitivity of the electron transport chain to inhibition by NO (Venkatraman et al., 2003). Nitric oxide is known to inhibit respiration at cytochrome c oxidase (complex IV) by directly competing with O2 for the reduced binuclear center CuB/a3 (Antunes and Cadenas, 2007).
The fact that chronic ethanol feeding depletes hepatic SAM levels is clear but literature reports differ on the time period over which this lesion manifests itself. Using the Lieber and DeCarli animal model of chronic liver toxicity (Lieber and Decarli, 1994) a study by Bailey et al. (2006) reported significant decreases in hepatic SAM levels in male rats fed ethanol for at least 31 days while an earlier study by Barak et al. found that hepatic SAM levels were unaffected by 4 weeks of ethanol feeding but decreased when the feeding period was extended to 8 weeks (Barak et al., 1987). In our studies, we administered SAM in the liquid diet at a concentration of 200 mg/l, i.e., a 4-fold lower concentration than that utilized in the Bailey study (Bailey et al., 2006) and 2-fold lower than that used in the studies by Fernandez-Checa (Garcia-Ruiz et al., 1995). Interestingly, this concentration did not increase hepatic SAM levels in paired control animals but did prevent the decrease seen in the alcoholic animals (see Results). Reports have indicated that SAM is taken up by the gut in a paracellular fashion without the involvement of a membrane transporter (Mcmillan et al., 2005). This results in limited intestinal absorption and, as a result, a limited availability of SAM for uptake into hepatocytes. Indeed, administering SAM in the diet at a concentration of 800 mg/l (4-fold higher than in our study) to paired control animals only increased hepatic SAM levels by 14% (Bailey et al., 2006). It appears therefore that a range of dietary SAM concentrations (200 to 800 mg/l) have the ability to preserve hepatic SAM levels in alcoholic animals without producing major elevations in paired controls. While it is our belief that dietary SAM supplementation attenuates ethanol-derived mitochondrial damage in the liver by preserving SAM levels, an alternative explanation could be that SAM has a direct impact upon the gut and/or its flora and that this in some way alters ethanol absorption. This latter theory is unlikely, however, as studies have failed to detect any effects of SAM treatment on blood ethanol levels in alcoholic animals (Shelly Lu, USC, personal communication).
Our studies have revealed that the addition of SAM to an ethanol-containing diet prevents not only the dissociation of mitochondrial ribosomes (Figs. 3A–F) but also causes the mitoribosome to sediment at a faster rate through a sucrose density gradient (Fig. 3E) in a manner more consistent with that of a control organelle (Cahill et al., 1996; Patel and Cunningham, 2002). S-adenosyl-L-methionine is synthesized in the cytosol and imported into the mitochondrion via the SAMC transporter (Agrimi et al., 2004). Once inside the mitochondrion, its role as a methyl donor is utilized for posttranscriptional modification of mitochondrial tRNA, rRNA, and possibly mtDNA. In the case of mitochondrial rRNA, methylation of specific residues is necessary for successful binding of MRPs, formation of mitoribosomal subunits, and ultimately, accurate assembly of competent ribosomes. Chronic ethanol feeding results in decreased methionine adenosyltransferase 1A (MAT1A) expression and depletion of hepatic SAMlevels (Lu and Mato, 2005) and this may result in impaired mitochondrial rRNA methylation and formation of improperly assembled mitoribosomes. Supplementation of SAM in the diet prevented the ethanol-derived SAM depletion [see Results text and Bailey et al. (2006), Lieber et al. (1990)] and consequentially may have improved rRNA posttranslational processing and thus promoted accurate mitoribosome assembly. Alternatively, the ethanol-mediated depletion in SAM content may affect the levels of, or posttranslational modification of, specific MRPs that may be required for correct mitoribosome assembly. We have previously shown that chronic ethanol feeding results in the depression of a number of proteins associated with the mitochondrial ribosome (Cahill et al., 1996). Data from the Cunningham laboratory suggests that the ethanol-elicited decrease in mitochondrial ribosome activity is due to lower levels of intact 55S monomers and not due to decreased activity of the monomers (Patel and Cunningham, 2002). This is supported by the data in Fig. 3 and Fig. 4 where it is shown that by preventing ethanol-elicited structural alterations of the mitoribosome their activity can be maintained at control levels.
S-adenosyl-L-methionine administration was also shown to prevent the depression in mitochondrial respiration (Table 1) commonly associated with alcoholic animals (Thayer and Rubin, 1979). This may be due to the modulation effects of SAM on iNOS expression. S-adenosyl-L-methionine has been shown to attenuate the induction of iNOS in the livers of lipopolysaccharide-treated rats (Majano et al., 2001). Ethanol feeding has been shown to induce iNOS expression and to cause increased NO production (Yuan et al., 2006). Attenuation of iNOS expression and NO production by SAM may therefore prevent the inhibition of respiration by NO (Venkatraman et al., 2003).
Although SAM administration was able to prevent a number of ethanol-mediated deleterious mitochondrial lesions from occurring, it had no effect on the increase in liver weight commonly seen in alcoholic animals [Fig. 1B and legend and Britton et al. (1984), Israel and Orrego (1983)]. The hepatomegaly seen in chronic alcoholic animals is caused by an elevation in hepatocyte protein (Baraona et al., 1975; Donohue et al., 1994), water (Israel and Orrego, 1983) and fat content (Navder et al., 1997), phenomena that collectively result in an increase in cell volume. This delineation of hepatomegaly from mitochondrial lesion formation suggests that SAM may have a therapeutic benefit in alcoholic livers that is specific for the mitochondrion. Interestingly, SAM administration alone was found to result in an increase in liver weight (Fig. 1B). This was in part due to an increase in hepatic protein content (see Results text). The precise mechanism responsible for this has yet to be elucidated but one possibility may be that SAM administration causes increased hepatic rRNA methylation. Hepatotoxins such as carbon tetrachloride, ethionine, and D-galactosamine are all known to decrease hepatic protein synthesis via hypomethylation of cytoplasmic rRNA (Clawson et al., 1990). Co-administration of SAM has been found to protect against D-galactosamine-induced hepatocyte damage (Kucera et al., 2006) possible by restoring rRNA methylation.
In conclusion, we have presented novel data showing that ethanol mediated decreases in respiration and impairment of mitoribosome structure and activity precede depressions in respiratory complex levels and ATP depletion in alcoholic animals. Further, we have shown that co-administration of SAM results in prevention of both ethanol-elicited lesions. These data suggest that defects in the mitoribosome may represent a major underlying factor in the mitochondrial dysfunction seen in the early stages of alcohol-induced liver damage.
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