Departments of Cellular and Molecular Physiology, and Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
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ABSTRACT |
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The present study examined potential cellular
mechanisms responsible for the inhibition of protein synthesis in liver
after chronic alcohol consumption. Rats were maintained on an
alcohol-containing diet for 14 wk; control animals were fed
isocalorically. Hepatic ATP content was not different in alcohol-fed
and control animals. No alcohol-induced reduction in total hepatic RNA
content (an estimate of ribosomal RNA) was detected, suggesting that
alcohol decreased translational efficiency. Alcohol feeding increased the proportion of 40S and 60S ribosomal subunits in the
nonpolysome-associated fraction by 30%. To identify mechanisms
responsible for the impairment in initiation, several eukaryotic
initiation factors (eIF) were analyzed. Alcohol feeding decreased
hepatic eIF2B activity by 36%. This reduction was associated with a
20% decrease in eIF2B content and a 90% increase in eIF2
phosphorylation. Alcohol also dramatically influenced the distribution
of eIF4E. Compared with pair-fed control values, alcohol feeding
increased the amount of eIF4E present in the inactive 4E-binding
protein 1 (4E-BP1) · eIF4E complex by 80% and
decreased binding of eIF4G to eIF4E by 70%. However, the
phosphorylation status of 4E-BP1 and eIF4E was not altered by alcohol.
Although the plasma concentrations of threonine, proline, and
citrulline were mildly decreased, the circulating amount of total amino
acids was not altered by alcohol feeding. In summary, these data
suggest that chronic alcohol consumption impairs translation initiation
in liver by altering eIF2B activity as well as eIF4F function via
changes in eIF4E availability.
eukaryotic initiation factor 2; eukaryotic initiation factor 4E; ribosomal subunits; amino acids; liver; rats
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INTRODUCTION |
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THE LIVER PLAYS A CENTRAL ROLE in regulating
circulating amino acid and protein concentrations, which are necessary
for maintenance of whole body metabolic homeostasis and host defense.
Alcohol is known to have diverse effects on hepatic protein turnover
(35). The large majority of in vitro studies indicate that addition of
ethanol to isolated hepatocytes inhibits protein synthesis (6, 23, 46).
It also appears that, for at least physiologically relevant
concentrations, the ability of ethanol to inhibit protein synthesis is
attributable to its metabolism (23). Furthermore, ethanol also
decreases protein (i.e., albumin) synthesis in isolated perfused rabbit
liver (27). Recent studies indicate that both acute alcohol
intoxication (31, 42) and chronic (6 wk) feeding of an
alcohol-containing diet (32, 41) significantly decrease hepatic protein
synthesis in vivo.
The mechanism by which alcohol reduces the synthesis of hepatic protein remains largely unexamined. Early studies indicated that chronic alcohol consumption in rats decreases both protein synthesis and total RNA in liver (32). Because the large majority (>80%) of total liver RNA is ribosomal, these data were interpreted to mean that the alcohol-induced impairment in protein synthesis occurred secondary to a reduced number of ribosomes. However, the decrease in protein synthesis in this early study was proportionately greater than the decrease in RNA. Hence, an impairment in translational efficiency most likely exists as well. Translational efficiency reflects how well the existing protein synthetic machinery is functioning. The translational phase of protein synthesis can be categorized into three phases: peptide-chain initiation, elongation, and termination (2). Indirect evidence based on the abundance of polysomes suggests that alcohol impairs translational efficiency by decreasing peptide-chain initiation (24). Translation initiation is regulated by a large number of protein factors, termed eukaryotic initiation factors (eIFs). One of these initiation factors, eIF2, mediates the first step in initiation, which involves the attachment of the initiator methionyl-tRNA (met-tRNAmeti) to the 40S ribosomal subunit to form the 43S preinitiation complex (2). A second regulatory step in initiation involves the binding of mRNA to the 43S preinitiation complex, which is mediated by eIF4F (34). Both of these regulatory steps are altered in other conditions that are associated with decreases in protein synthesis (2, 34, 48). However, there is no information on the effects of chronic alcohol on either of these two steps involved in peptide-chain initiation. Therefore, the purpose of the present study was to determine whether chronic alcohol consumption in rats impairs regulation of the initiation process by modulating eIF2 and/or eIF4F in liver.
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METHODS AND MATERIALS |
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Experimental protocol.
Specific pathogen-free, male Sprague-Dawley rats (Charles
River Breeding Laboratories, Cambridge, MA) were maintained on an ethanol-containing agar block diet for 14 wk (1, 20). Initially, all
rats were provided agar without alcohol for 2 days. Thereafter, one-half of the animals were fed agar containing 10% alcohol, and the
others were provided agar containing an equal caloric amount of
dextrin-maltose. The ethanol content of the agar was increased to 20%
and then finally to 30% at 1-wk intervals. Rats were maintained on the
30% ethanol-agar block for the remainder of the experimental protocol.
Calculated ethanol intake during the final 10 wk of the experimental
protocol averaged 16 ± 3 g · kg1 · day
1.
Control animals were provided the same amount of agar, but isocaloric dextrin-maltose was substituted for the ethanol.
Determination of total RNA. Total RNA was measured from homogenates of liver samples (44). Briefly, 0.3 g of fresh liver was homogenized in five volumes of ice-cold 10% trichloroacetic acid. The homogenate was centrifuged at 9,000 g for 11 min at 4°C. The supernatant was discarded, and 2.5 ml of 6% perchloric acid (PCA) were added to the remaining pellet. The sample was centrifuged at 9,000 g for 6 min, the supernatant was discarded, and the procedure was repeated. Next, 1.5 ml of 0.3 N KOH was added to the pellet, and the samples were heated for 1 h at 50°C. Samples were then mixed with 5 ml of 4 N PCA and centrifuged at 9,000 g for 11 min. The concentration of RNA in the supernatant was determined by absorbance at 260 nm corrected by the absorbance at 232 nm. Total RNA was expressed as milligram RNA per gram tissue.
Isolation of ribosomal subunits. Fresh liver was used to isolate 40S and 60S ribosomal subunits by sucrose density gradient centrifugation, as described previously (9). Briefly, liver was homogenized in a Dounce homogenizer in seven volumes of homogenization buffer [in mM: 25 HEPES (pH 7.5), 2 magnesium acetate, 250 KCl, 0.5 dithiothreitol (DTT), 0.08 EDTA, and 250 sucrose]. The homogenate was centrifuged at 10,000 g for 15 min, and the supernatant was recovered. Aliquots of the samples (0.5 ml), to which 0.1 vol of 10% (wt/vol) Triton X-100 and deoxycholate solution had been added, were then layered onto 0.44-2.0 M exponential sucrose gradients. The samples were centrifuged at 167,000 g in a SW41 rotor (Beckman Instruments) for 20 h to resolve the 40S and 60S ribosomal subunits. The absorbance of the gradients was monitored at 254 nm, and fractions were collected using a density gradient fractionator (Instrumentation Specialties, Lincoln, NE).
Relative amounts of eIF2 and eIF2B.
The relative amounts of the -subunit of eIF2 (eIF2
) and the
-subunit of eIF2B (eIF2B
) in liver were estimated by protein immunoblot analysis, as described previously (10, 14, 45). eIF2 and
eIF2B were chosen because changes in these initiation factors correlate
with alterations in protein synthesis (2). eIF2 consists of three
subunits, of which the
-subunit appears important in regulating
protein synthesis (48). Previous studies have established that the
expression of the
-subunit is representative of the other subunits
of eIF2 (47). Likewise, eIF2B is a multimeric protein consisting of
five subunits, with the
-subunit being the catalytic subunit (47).
Therefore, the relative abundance of eIF2B
was taken as
representative of the eIF2B holoenzyme. Briefly, liver was homogenized
in 7 vol of buffer composed of (in mM) 20 Tris (pH 7.4), 250 sucrose,
100 KCl, 0.2 EDTA, 1 DTT, 50 NaF, 50
-glycerolphosphate, 1 phenylmethylsulfonyl fluoride (PMSF), 1 benzamidine, and 0.5 sodium
vanadate. The samples were mixed with 2× Laemmli sodium dodecyl
sulfate (SDS) buffer, boiled for 3 min, and centrifuged. Equal amounts
of protein (~160 µg) from liver homogenates were electrophoresed at
60 mA in a 12.5% polyacrylamide gel. After electrophoresis, proteins
in the gel were transferred to nitrocellulose. After blocking for 30 min with nonfat milk (5% wt/vol) in 25 mM Tris (pH 7.6)-0.9% saline containing 0.01% Tween 20 (Tris-NaCl-Tween), the membranes were washed
extensively in Tris-NaCl-Tween. The nitrocellulose was incubated for 1 h at room temperature with an antibody specific for either eIF2
or
eIF2B
(47). Antibodies were visualized using an enhanced
chemiluminescence (ECL) procedure, with the secondary antibody linked
to horseradish peroxidase (Amersham). The blots were exposed to X-ray
film in a cassette equipped with a Du Pont Lightning Plus intensifying
screen. After development, the X-ray film was scanned (Microtek
ScanMaker IV) and quantitated using NIH Image 1.6 software.
Measurement of phosphorylation state of eIF2.
The relative amount of eIF2
present in the phosphorylated form,
eIF2
(P), was estimated by immunologic visualization of proteins after separation by use of slab-gel isoelectric focusing (IEF) (43).
Livers were homogenized in the same buffer as described above for eIF2.
A 75-µl aliquot of the homogenate was mixed with 42.9 mg of urea and
300 µl of IEF sample buffer [9.5 M urea, 2% Nonidet P-40,
ampholytes (BHD Resolyte 4-8), and 0.7 M
-mercaptoethanol]. The samples were electrofocused and then
transferred electrophoretically onto polyvinylidene fluoride membranes.
The membranes were subsequently incubated with an eIF2
monoclonal
antibody, and eIF2
was visualized as described above. The proportion
of eIF2
present in the phosphorylated state was measured by
densitometric scanning of the membranes and is expressed as a
percentage of the total eIF2
content (i.e., phosphorylated + unphosphorylated). Previous studies have demonstrated that eIF2
(P)
can be determined on frozen tissue samples (43).
Determination of eIF2B activity. Hepatic eIF2B activity was measured in postmitochondrial supernatants by use of a [3H]GDP-GDP exchange assay, as previously described (10, 14). Fresh tissue was homogenized in 1 vol of buffer consisting of (in mM) 45 HEPES (pH 7.4), 0.375 magnesium acetate, 95 KOAc, 0.075 EDTA, and 10% glycerol. The homogenate was then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was assayed immediately for eIF2B activity, as described previously (10, 14). Briefly, aliquots of the reaction mixture were analyzed for eIF2B activity by measuring the decrease in [3H]GDP bound to eIF2. The rate of exchange was linear over the time points measured (data not shown). Under these conditions, ~50% (0.3 pmol) of the [3H]GDP was released from the eIF2 · [3H]GDP complex during the 1.5-min assay. eIF2B activity was expressed as picomoles of GDP exchanged per minute per milligram protein.
Quantification of 4E-BP1 · eIF4E and
eIF4G · eIF4E complexes.
The association of eIF4E with 4E-binding protein 1 (4E-BP1) and eIF4G
was determined as previously described (5, 15). Briefly, liver was
homogenized in seven volumes of buffer
A (20 mM HEPES, pH 7.4, 100 mM KCl, 0.2 mM EDTA, 2 mM
EGTA, 1 mM DTT, 50 mM NaF, 50 mM -glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, 0.5 mM sodium vanadate, and 1 µM microcystin LR) with
a Polytron homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. eIF4E and
4E-BP1 · eIF4E and eIF4G · eIF4E
complexes were immunoprecipitated from aliquots of 10,000 g supernatants by use of an anti-eIF4E
monoclonal antibody. The antibody-antigen complex was collected by
incubation for 1 h with BioMag goat anti-mouse IgG beads (Perseptive
Biosystems, Framingham, MA). Before use, the beads were washed in 1%
nonfat dry milk in buffer B (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1%
-mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM
-glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium
vanadate). The beads were captured using a magnetic sample rack and
were washed twice with buffer B and
once with buffer B containing 500 mM,
rather than 150 mM, NaCl. Proteins remaining bound were eluted by
resuspending the beads in SDS-sample buffer, followed by boiling for 5 min. The beads were collected by centrifugation, and the supernatants were subjected to electrophoresis either on a 7.5% polyacrylamide gel
for quantitation of eIF4G or on a 15% polyacrylamide gel for quantitation of 4E-BP1 and eIF4E. Proteins were then
electrophoretically transferred to a nitrocellulose membrane as
previously described (15). The membranes were incubated with a mouse
anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a
rabbit anti-eIF4G antibody for 1 h at room temperature. The blots were
then developed using an ECL Western blotting kit according to the
manufacturer's instructions. Films were scanned and quantitated as
described in Relative amounts of eIF2 and
eIF2B.
eIF4E, 4E-BP1, and p70 S6 kinase phosphorylation. The phosphorylated and nonphosphorylated forms of eIF4E in liver extracts were separated by IEF on a slab gel and quantitated by protein immunoblot analysis, as previously described (5, 13). The various phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from liver homogenates after centrifugation at 10,000 g (5, 13). 4E-BP1 was immunoprecipitated as described in the previous section for immunoprecipitation of eIF4E. The immunoprecipitates were solubilized with SDS sample buffer. The various phosphorylated forms of 4E-BP1 were separated by electrophoresis and quantitated by protein immunoblot analysis, as described previously (5, 13). For detection of p70 S6 kinase, an aliquot of cell homogenate was combined with an equal volume of SDS sample buffer, and the diluted samples were subjected to electrophoresis on a 7.5% polyacrylamide gel, as previously described (13). The samples were then analyzed by protein immunoblot analysis by use of a rabbit anti-rat p70 S6 kinase antibody.
Tissue ATP content. An aliquot of powdered liver was extracted in cold PCA, neutralized, and used for the determination of adenosine triphosphate (ATP) by standard fluorometric methods.
Liver enzyme and plasma alcohol and amino acid levels. Aspartate aminotransferase (AST; EN 2.6.1.1) activity in plasma was determined using a standard enzymatic assay (Sigma). The alcohol concentration in plasma samples was determined using a rapid alcohol analyzer (model GL5; Analox Instruments, Lunenburg, MA). Plasma was deproteinized with sulphosalicylic acid and the supernatant used for amino acid analysis by ion-exchange HPLC (model 6300, Beckman Instruments, Fullerton, CA). Absorbance was measured at 440 and 570 nm after postcolumn Ninhydrin treatment. (S)-2-aminoethyl-L-cysteine was used as an internal standard, and data acquisition and management were performed by Beckman System Gold 8.10.
Statistics. Values are presented as means ± SE. The number of rats per group is indicated in the legends to the figures and tables. Data were analyzed by Student's t-test to determine treatment effect. Statistical significance was set at P < 0.05.
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RESULTS |
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Whole body composition and liver weight.
The body weight was 16% lower in animals fed the alcohol-containing
diet for 14 wk, compared with pair-fed control animals (427 ± 8 vs.
517 ± 12 g, respectively; P < 0.05). Despite the same caloric intake, the average weight gain
in control animals was 4.4 g/day, compared with 3.5 g/day for the
alcohol-fed group (P < 0.05).
Estimates of body composition indicated that alcohol feeding decreased
the amount of lean body mass (312 ± 12 vs. 399 ± 9 g;
P < 0.05) without significantly
altering the amount of body fat (115 ± 6 vs. 118 ± 5 g;
P < 0.05). However, estimates of
lean body mass based on electrical conductance and related methodologies may be influenced by perturbations in the hydration state
of the subject (3, 8). The effect of chronic alcohol consumption on the
volume and ionic composition of various body fluid compartments was not
directly measured in the current study. However, there are several
lines of evidence that suggest our alcohol-fed rats do not have major
changes in fluid compartments. First, tissue water content was
essentially identical between control and alcohol-fed rats for both
skeletal muscle (control, 0.747 ± 0.004 ml/g; alcohol, 0.745 ± 0.006 ml/g) and liver (see Table 1).
Second, there was no significant difference in hematocrit between the
two groups (control, 43 ± 2%; alcohol, 42 ± 2%), indicating that
non-red blood cell volume was most likely unaffected by alcohol feeding.
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Total RNA content and ribosomal subunits.
A decreased rate of protein synthesis may result from alterations in
either the number of ribosomes or the efficiency of translation. Ribosomal RNA accounts for as much as 80% of the total RNA in liver.
Hence, changes in total tissue RNA content presumably reflect changes
in ribosomal RNA. There was no detectable difference in the total RNA
content of liver between control and alcohol-fed rats (Fig.
1A).
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Relative amounts of eIF2 and eIF2B, and eIF2B activity.
One possible mechanism for the alcohol-induced decrease in translation
is via alterations in the amount and/or activity of specific eIF
proteins. There was no significant difference in the amount of total
eIF2 in liver from control and alcohol-fed rats (data not shown).
However, the percentage of eIF2
in the phosphorylated form was
increased almost twofold in alcohol-consuming rats (Fig.
2).
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Regulation of eIF4E.
Another potential mechanism for decreasing hepatic protein synthesis
involves altered regulation of eIF4E. Binding of the translational
repressor 4E-BP1 to eIF4E forms an inactive complex. This is visualized
on an immunoblot as an increase in the amount of 4E-BP1 present in an
eIF4E immunoprecipitate. Figure
5A
illustrates that alcohol feeding increased the amount of both the -
and
-forms of 4E-BP1 in the immunoprecipitate. Densitometric
analysis of both bands indicates an 80% increase in the total amount
of 4E-BP1 associated with eIF4E in liver from alcohol-fed rats (Fig.
5B).
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Plasma amino acid concentrations.
Table 2 presents the plasma amino acid
concentrations in control and alcohol-fed rats. There was no
significant difference between the two groups for the various
gluconeogenic, branched-chain, or aromatic amino acids. Alcohol feeding
decreased the concentration of threonine (26%), proline (18%), and
citrulline (39%), as well as increasing the concentration of taurine
(114%), 3-methylhistidine (45%), and
-amino-n-butyric acid (5-fold).
However, the plasma concentration of total amino acids was essentially
identical between control and alcohol-fed rats.
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ATP content. There was no difference in the hepatic ATP concentration between control and alcohol-fed rats (3.17 ± 0.12 vs. 3.21 ± 0.09 µmol/g wet weight, respectively).
AST and blood alcohol. Alcohol-induced hepatic injury was assessed by measuring plasma AST activity. AST levels were elevated 68% in alcohol-fed rats compared with control values (227 ± 32 vs. 135 ± 21 U/ml; P < 0.05). Plasma alcohol levels were 54 ± 20 mg/dl in alcohol-fed rats and not detectable in control animals at the time they were killed.
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DISCUSSION |
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Recent work has demonstrated that chronic alcohol consumption decreases the in vivo rate of hepatic protein synthesis (32, 40, 42). However, there is a paucity of data related to the cellular mechanism responsible for this impairment. Inhibition of protein synthesis can result from a decreased number of ribosomes and/or a reduction in translational efficiency. Because 80-90% of the cellular RNA is ribosomal, changes in liver RNA content primarily reflect changes in ribosomal content. In the present study, we were unable to confirm an alcohol-induced decrease in the total hepatic RNA content described previously by Preedy and Peters (32). The reason for this difference is unclear. However, an impairment in translational efficiency is a commonality of both studies.
The impaired translational efficiency may result from a reduction in either peptide-chain initiation or elongation/termination (2). Analysis of the distribution of ribosomal subunits between free subunits (i.e., nonpolysome associated) and polysomes was used to estimate the rate of initiation relative to elongation. In general, the amount of RNA in free 40S and 60S ribosomal subunits is indicative of a balance between the two processes. That is, when the rate of initiation is decreased relative to elongation/termination, free ribosomal subunits are binding to mRNA at a slower rate (initiation) than they are moving along (elongation) and exiting (termination) mRNA. The net result of this defect is an increase in the abundance of free ribosomal subunits. Our analysis of subunits indicated a moderate increase in the accumulation of free 40S and 60S ribosomal subunits in liver from alcohol-fed rats. This finding is consistent with the previous demonstration that alcohol decreases hepatic polysome aggregation (27). Collectively, this ribosomal redistribution is characteristic of a decrease in the rate of initiation of peptide chains relative to the rate of elongation/termination (4).
eIF2 (a heterotrimer consisting of -,
-, and
-subunits)
represents a major regulatory control point for initiation (34). The
first step in initiation is the formation of a ternary complex consisting of eIF2, GTP, and
met-tRNAmeti. eIF2 mediates the binding
of met-tRNAmeti to the 40S ribosomal
subunit to form the 43S preinitiation complex. A reduction in the
tissue content of eIF2 protein, therefore, could lead to a decrease in initiation. In several nonmuscle tissues (including liver), the cellular eIF2 content is linearly related to rates of protein synthesis
(17, 43). However, in the present study, there was no detectable
difference in eIF2 content (as assessed by Western blot analysis of
eIF2
) of liver between control and alcohol-fed rats. Thus the defect
in translation initiation is not mediated by reduced hepatic eIF2 content.
Alternatively, a decrease in the activity of another eIF, eIF2B, can
also decrease the ability of eIF2 to form the ternary complex. eIF2 is
bound to GDP as an inactive complex when it is released from the
ribosome, and this GDP must be exchanged for GTP before binding another
molecule of met-tRNAmeti. The replacement
of GDP for GTP on eIF2 is catalyzed by eIF2B, a guanine nucleotide
exchange factor, under physiological conditions. This initiation factor
is important in the recycling and activation of eIF2. Hence, a decrease
in eIF2B activity would ultimately limit translation initiation by
reducing the amount of eIF2 · GTP that is available
to bind to tRNAmeti. Livers from alcohol-fed rats clearly demonstrated a decrease in eIF2B activity (Fig. 9). These data are consistent with
previous studies in other conditions that have reported a proportional
decrease in both eIF2B activity and protein synthesis in liver (17,
43). Therefore, a decrease in eIF2B-mediated guanine nucleotide
exchange appears to be at least partially responsible for the
alcohol-induced decrease in initiation.
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Several mechanisms are known to regulate eIF2B activity (48). A major
mechanism involves phosphorylation of the -subunit of eIF2, which
increases the affinity of eIF2 for eIF2B (18). Phosphorylation of
eIF2
effectively converts eIF2 from a substrate into a competitive
inhibitor, thereby limiting the recycling of the
eIF2 · GDP complex. Previous work by Kimball et al.
(16) demonstrated that the eIF2B-to-eIF2 ratio in liver is ~0.6. Thus the formation of the highly stable eIF2
(P) · eIF2B
complex would be expected to sequester available eIF2B. The net result
of this sequestration is a reduction in guanine nucleotide exchange
activity and impairment of initiation. In selected in vitro systems,
the extent of eIF2
phosphorylation has been demonstrated to be
inversely proportional to the rate of protein synthesis (21, 36). In the present study, chronic alcohol consumption almost doubled the
amount of phosphorylated eIF2
.
Theoretically the alcohol-induced increase in eIF2(P) could result
from an increase in protein kinase or a decrease in phosphatase activity. In this regard, ethanol has been shown to decrease ternary complex formation in rabbit reticulocyte lysates by the increased activation of a heme-controlled repressor (49), which has eIF2 kinase
activity (28). The second mechanism by which eIF2B activity can be
regulated is by decreasing the amount of hepatic eIF2B. Our data
demonstrate a significant, albeit small, 20% decrease in the relative
content of eIF2B in liver from alcohol-fed rats (Fig. 9). eIF2B content
was assessed by Western blot analysis of the
-subunit, which is
critical for binding to eIF2 (12). Finally, changes in the cellular
redox potential can also potentially regulate eIF2B activity
allosterically. Increases in the
NAD+/NADH ratio would be expected
to inhibit eIF2B activity (10). However, alcohol metabolism results in
a well-characterized decrease in the hepatic
NAD+/NADH ratio (19). Hence, an
alcohol-induced alteration in the redox potential is unlikely to be an
important regulator of hepatic eIF2B activity in our experimental
model. Therefore, our data indicate that the alcohol-induced decrease
in hepatic eIF2B activity results from both a decrease in eIF2B content
and an increase in the extent of eIF2
phosphorylation.
A second regulatory step controlling peptide-chain initiation involves the binding of mRNA to the 43S preinitiation complex, which is mediated by eIF4F (34). One of the components of the eIF4F complex, eIF4E, binds directly to the m7GTP cap structure present at the 5'-end of all eukaryotic mRNA and stimulates mRNA binding to the small ribosomal subunit. During translation initiation, the eIF4E · mRNA complex binds with eIF4G and eIF4A to form the functional eIF4F cap-binding complex. One mechanism for modulating the formation of the eIF4F complex is by regulating the relative distribution of eIF4E between inactive and active protein complexes with other proteins. 4E-BP1 (also called PHAS-I) binds to amino acid residues in eIF4E that also bind eIF4G to form the active eIF4F complex. Thus 4E-BP1 functions as a repressor of translation initiation. In the current study, the amount of 4E-BP1 bound to eIF4E was markedly increased in liver from alcohol-fed rats (Fig. 9). We also observed a concomitant decrease in the amount of eIF4E bound to eIF4G in these animals. These data strongly suggest that alcohol feeding decreases initiation, at least in part, by an impairment in eIF4F function secondary to a decrease in eIF4E binding to eIF4G.
eIF4E activity can be regulated by phosphorylation of either 4E-BP1 or
eIF4E (18). Phosphorylation of 4E-BP1 releases eIF4E from the
4E-BP1 · eIF4E complex, thereby permitting the
eIF4E · mRNA complex to bind first to eIF4G and then
ultimately to the small ribosomal subunit. Hence, stimuli that limit
translation initiation and protein synthesis are associated with a
decreased percentage of 4E-BP1 in the phosphorylated -form (15).
However, there was no detectable alteration in the phosphorylation
state of 4E-BP1 in liver from alcohol-fed rats. We cannot exclude the possibility that the analytical methods used lacked sufficient sensitivity to detect a change in phosphorylation of 4E-BP1. However, we also failed to detect a shift in the electrophoric mobility of p70
S6 kinase in alcohol-fed rats. Because both 4E-BP1 and p70 S6 kinase
are phosphorylated and activated by a rapamyocin-sensitive pathway
involving the upstream regulator mTOR (13, 29), these data suggest that
alcohol-induced changes in eIF4E are not mediated by a change in the
phosphorylation state of 4E-BP1.
Changes in the phosphorylation state of eIF4E can also influence eIF4E binding to mRNA. Although both phosphorylated and nonphosphorylated eIF4E bind to the mRNA cap structure, phosphorylation of eIF4E enhances the affinity of the factor for the m7GTP cap severalfold (22). Moreover, in vitro studies have demonstrated that increases in phosphorylation are proportional to increases in translation (40). However, again, chronic alcohol consumption failed to produce a detectable alteration in the phosphorylation state of eIF4E.
Alterations in hepatic protein balance can be induced by changes in either the rate of protein synthesis or the rate of proteolysis. Although our results indicate that alcohol impairs peptide-chain initiation (and likely protein synthesis), we cannot exclude the possibility that a portion of the alcohol-induced decrease in liver weight results from an increase in hepatic protein degradation. However, this scenario appears unlikely. Ethanol has previously been shown to have either no effect on protein degradation in isolated hepatocytes (46) or to actually inhibit proteolysis when determined in the isolated perfused rat liver or under in vivo conditions (30).
Amino acids can regulate translation initiation and protein synthesis
by modulating various signaling pathways involving eIF2 and eIF4E (13,
29). However, chronic alcohol ingestion produced only mild changes in a
small number of individual amino acids and did not change the total
concentration of amino acids in the blood. Therefore, it appears
unlikely that an alcohol-induced decrease in plasma amino acids was
responsible for any of the observed changes in initiation. Although
acute administration of ethanol can dramatically decrease a large
number of amino acids (7), our data are consistent with those of a
previous study that failed to detect major changes in plasma amino
acids in rats fed a nutritionally adequate liquid diet containing
alcohol for 8-10 wk (39). There were changes in two amino acids,
however, that are noteworthy. First, alcohol-fed rats demonstrated a
fivefold elevation in
-amino-n-butyrate compared with
control animals. A similar finding has been previously reported (37)
and appears to result from an alcohol-induced stimulation of hepatic
production of this amino acid (38). In contrast, the plasma
concentration of
-amino-n-butyrate
(and that of the branched-chain amino acids) is decreased in animals
and humans by dietary protein deficiency (37). These data further
support the conclusion that dietary protein intake was adequate in
alcohol-fed rats and that a difference in nutrient consumption was not
a primary mediator of the observed changes in hepatic translation
initiation. Second, alcohol-fed rats also demonstrated a 45% increase
in the plasma concentration of 3-methylhistidine. Concentrations of
this amino acid in blood or urine have been used as an indirect
estimate of myofibrillar protein degradation. In rodents, the primary
sites of production for this amino acid appear to be the intestine and
skeletal muscle (26). Hence, elevated circulating levels of
3-methylhistidine suggest an enhanced rate of proteolysis in these
tissues and are consistent with the decreased lean body mass in
alcohol-fed rats.
In summary, our data suggest that chronic alcohol consumption in rats
results in an imbalance in hepatic protein metabolism mediated, at
least in part, by a reduction in peptide-chain initiation. The
alcohol-induced inhibition of translation initiation appears to result
from a decrease in eIF2B activity. This diminished activity is
associated with a moderate reduction in eIF2B and a larger increase
in eIF2
phosphorylation. In addition, alcohol-fed rats also had an
impairment in eIF4F function as evidenced by the increase in 4E-BP1
bound to eIF4E and the corresponding decrease in eIF4E bound to eIF4G.
This redistribution of eIF4E could not be explained by changes in the
phosphorylation status of either 4E-BP1 or eIF4E. As illustrated in
Fig. 9, these data indicate that alcohol alters a variety of key
regulatory steps in translation initiation that would be expected to
impair hepatic protein synthesis.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the National Institute on Alcohol Abuse and Alcoholism (AA-1290, to C. H. Lang), the National Institute of Diabetes and Digestive and Kidney Diseases (DK-13499, to L. S. Jefferson), and the American Heart Association (to T. C. Vary).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: C. H. Lang, Dept. of Cell. Molec. Physiology (H166), Penn State College of Medicine, Hershey, PA 17033-0850 (E-mail: clang{at}psu.edu).
Received 11 January 1999; accepted in final form 9 June 1999.
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