Download Impaired myocardial protein synthesis induced by acute alcohol

Am J Physiol Endocrinol Metab
279: E1029–E1038, 2000.
Impaired myocardial protein synthesis induced by acute
alcohol intoxication is associated with changes in eIF4F
CHARLES H. LANG, ROBERT A. FROST, VINAYSHREE KUMAR, AND THOMAS C. VARY
Departments of Cellular and Molecular Physiology and Surgery, Pennsylvania State University
College of Medicine, Hershey, Pennsylvania 17033
Received 27 March 2000; accepted in final form 6 July 2000
remains the most common form of drug
abuse in the United States. Between 5 and 10% of the
adult population may be considered heavy alcohol misusers. Alcohol abuse is associated not only with increased morbidity but also with premature mortality.
In addition to its hepatotoxicity, alcohol also causes
myocardial dysfunction. Ingestion of alcohol in exces-
sive quantities induces metabolic and functional abnormalities in the heart (15, 24, 32). Acute alcohol
misuse, binge drinking, leads to a syndrome described
as the “holiday heart” (8, 15), characterized by abnormal cardiac rhythm and changes in other biochemical
and ultrastructural indexes of myocardial function and
metabolism (38). With regard to protein metabolism,
there are conflicting reports describing the effects of
acute ethanol intoxication on cardiac protein synthesis
(44, 59). However, more recent studies have indicated
that acute ethanol exposure depresses protein synthesis in cardiac muscle (35, 39, 45).
The mechanism responsible for the alcohol-induced
inhibition of protein synthesis in cardiac muscle remains unresolved. Synthesis of proteins in the myocardium is achieved through a complex series of enzymatic reactions (for review see Refs. 6, 23, and 50). The
process involves the association of the 40S and 60S
ribosomal subunits, messenger RNA (mRNA), initiator
methionyl-tRNA (met-tRNAimet), other amino acyltRNAs, cofactors (i.e., GTP; ATP), and protein factors,
collectively known as eukaryotic initiation factors
(eIF), elongation factors, and releasing factors, through
a series of discrete reactions that result in the translation of mRNA into proteins. Translation of mRNA on
the ribosome is composed of three phases: 1) initiation,
whereby met-tRNAimet and mRNA bind to 40S ribosomal subunits, and subsequent binding of the 40S
ribosomal subunit to the 60S subunit to form a ribosome complex capable of translation; 2) elongation,
during which tRNA-bound amino acids are incorporated in growing polypeptide chains according to the
mRNA template; and 3) termination, during which the
completed protein is released from the ribosome. However, the biochemical loci where ethanol acts to limit
protein synthesis in cardiac muscle are presently unknown.
The process of peptide-chain initiation involves essentially four major steps (6, 23, 50): 1) dissociation of
the 80S ribosome into 40S and 60S ribosomal subunits,
2) formation of the 43S preinitiation complex with
binding of initiator met-tRNAimet to the 40S subunit, 3)
binding of mRNA to the 43S preinitiation complex, and
Address for reprint requests and other correspondence: C. H.
Lang, Dept. of Cellular & Molecular Physiology (H166), Penn State
College of Medicine, Hershey, PA 17033-0850 (E-mail: clang
@psu.edu).
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. Section 1734
solely to indicate this fact.
cardiomyopathy; peptide-chain initiation; eukaryotic initiation factor 4E; 4E-binding protein 1; eukaryotic initiation
factor 4G
ALCOHOLISM
http://www.ajpendo.org
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society
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Lang, Charles H., Robert A. Frost, Vinayshree Kumar, and Thomas C. Vary. Impaired myocardial protein
synthesis induced by acute alcohol intoxication is associated
with changes in eIF4F. Am J Physiol Endocrinol Metab 279:
E1029–E1038, 2000.—The purpose of the present study was
to examine potential mechanisms for the known inhibitory
effect of acute alcohol exposure on myocardial protein synthesis. Rats were injected intraperitoneally with either ethanol (75 mmol/kg) or saline, and protein synthesis was measured in vivo 2.5 h thereafter by use of the flooding-dose
3
L-[ H]phenylalanine technique. Rates of myocardial protein
synthesis and translational efficiency in alcohol-treated rats
were decreased compared with control values. Free (nonpolysome bound) 40S and 60S ribosomal subunits were increased
50% after alcohol treatment, indicating an impaired peptidechain initiation. To identify mechanisms responsible for this
impairment, several eukaryotic initiation factors (eIF) were
analyzed. Acute alcohol intoxication did not significantly
alter the myocardial content of eIF2␣ or eIF2Bε, the extent of
eIF2␣ phosphorylation, or the activity of eIF2B. Acute alcohol exposure increased the binding of 4E-binding protein 1
(4E-BP1) to eIF4E (55%), diminished the amount of eIF4E
bound to eIF4G (70%), reduced the amount of 4E-BP1 in the
phosphorylated ␥-form (40%), and decreased the phosphorylation of p70S6 kinase and the ribosomal protein S6. There
was no significant difference in either the plasma insulin-like
growth factor (IGF) I concentration (total or free) or expression of IGF-I or IGF-II mRNA in heart between the two
groups. These data suggest that the acute alcohol-induced
impairment in myocardial protein synthesis results, in part,
from an inhibition in peptide-chain initiation, which is associated with marked changes in eIF4E availability and p70S6
kinase phosphorylation but is independent of changes in the
eIF2/2B system and IGFs.
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ALCOHOL AND TRANSLATION INITIATION IN HEART
METHODS AND MATERIALS
Experimental protocol. Specific pathogen-free male
Sprague-Dawley rats (225 ⫾ 8 g; Charles River Breeding
Laboratories, Cambridge, MA) were used in all studies. Rats
were housed in a controlled environment and provided water
and rat chow ad libitum for ⱖ1 wk before the start of the
study. At ⬃0800, one-half of the rats were injected intraperitoneally with ethanol (75 mmol/kg body wt; 20% wt/vol in
saline). Control animals were injected intraperitoneally with
an equal volume of physiological saline. Rats were then
returned to their cages, and food was withheld for the remainder of the study. The in vivo rate of myocardial protein
synthesis was determined 2.5 h after the injection of ethanol
or saline. The ethanol dose, route of administration, and
timing of blood and tissue samples were chosen on the basis
of an extensive number of previous studies demonstrating
that this protocol impairs cardiac and skeletal muscle protein
synthesis (35, 36, 39, 49). Experiments were approved by the
Animal Care and Use Committee of Pennsylvania State
University College of Medicine and adhered to National Institutes of Health guidelines for the use of experimental
animals.
Protein synthesis. Rates of protein synthesis were determined using the flooding-dose technique, as originally described by Garlick et al. (14) and modified in our laboratory
(6, 26, 28, 54, 55). Animals were injected intravenously with
3
L-[2,3,4,5,6- H]phenylalanine (Phe; 150 mM, 30 ␮Ci/ml; 1
ml/100 g body wt). Ten minutes later, rats were decapitated,
and trunk blood was collected in heparinized tubes. The
ventricles were rapidly excised and weighed. A portion of
cardiac muscle was immediately homogenized for measurement of eIF2B activity and analysis of the eIF4E system. The
remaining myocardial tissue was frozen between aluminum
blocks precooled to the temperature of liquid nitrogen and
was subsequently used for analysis of incorporation of radioactivity in cardiac proteins and total RNA content. The frozen tissues were later powdered under liquid nitrogen with a
mortar and pestle and stored at ⫺70°C.
A portion of the powdered myocardium was used to estimate the rate of incorporation of [3H]phenylalanine into total
mixed protein, exactly as described previously (6, 26, 28, 54,
55), and represents the sum of myofibrillar and sarcoplasmic
protein synthesis. Proportional reductions in both myofibrillar and sarcoplasmic protein synthesis have been reported in
other catabolic conditions, which demonstrate a diminished
rate of total protein synthesis (56). The specific radioactivity
of the plasma phenylalanine was measured by HPLC with
supernatant from TCA extracts of plasma. The specific radioactivity was calculated by dividing the amount of radioactivity in the peak corresponding to phenylalanine by the concentration of the amino acid in the same fraction.
Total RNA. Total RNA was measured from homogenates of
tissue samples after alkaline hydrolysis, as previously described (53–55). The concentration of RNA in the alkaline
hydrolysate was determined by measuring the absorbance at
260 nm and correcting for the absorbance at 232 nm. Total
RNA was expressed as micrograms of RNA per gram of
protein.
Isolation of ribosomal subunits. Homogenates from fresh
heart muscle samples were used to isolate 40S and 60S
ribosomal subunits by sucrose density gradient centrifugation, as described previously (53–55). Briefly, supernatants
of a 10,000-g homogenate (0.7 ml) were layered onto 0.44–2.0
M exponential sucrose gradients and subsequently 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.
RNA in fractions corresponding to 40S and 60S ribosomal
subunits was measured as described previously (53–55).
Myocardial eIF2B activity and protein content of eIF2 and
eIF2B. eIF2B activity in cardiac muscle was measured in
postmitochondrial supernatants by the GDP exchange assay
(19, 21). eIF2B activity was measured as the decrease in
eIF2 䡠 [3H]GDP complex bound to nitrocellulose filters. The
rate of exchange of GTP for [3H]GDP in the eIF2 䡠 [3H]GDP
complex 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 䡠 [3H]GDP complex during the assay.
The relative amounts of the ␣-subunit of eIF2 (eIF2␣), the
phosphorylated form of eIF2␣ [eIF2␣(P)], and the ε-subunit
of eIF2B (eIF2Bε) in heart were estimated by protein immunoblot analysis using antibodies specific for either eIF2␣,
ser-51-phosphorylated eIF2␣, or eIF2Bε, as described previously (17, 19, 21). Antibodies were visualized with an enhanced chemiluminescence 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 film was scanned (Microtek ScanMaker IV) and
quantitated using NIH Image 1.6 software. eIF2 and eIF2B
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4) association of the 60S ribosomal subunit to form an
active 80S ribosome. Two of the steps involved in peptide-chain initiation appear important as major regulatory points in the overall control of protein synthesis
in vivo. The first step controlling peptide-chain initiation is the binding of met-tRNAimet to the 40S ribosomal
subunit to form the 43S preinitiation complex. This
reaction is mediated by eukaryotic initiation factor 2
(eIF2) and is regulated by the activity of another initiation factor, eIF2B. The second regulatory step involves the binding of mRNA to the 43S preinitiation
complex, which is mediated by eIF4F, a complex of
several subunits. One of the subunits, eIF4E, binds the
7-methylguanosine 5⬘-triphosphate (m7GTP) cap structure present at the 5⬘-end of many eukaryotic mRNAs
to form an eIF4E 䡠 mRNA complex (41). During translation initiation, the eIF4E 䡠 mRNA complex binds to
eIF4G and eIF4A to form the active eIF4F complex (41,
42, 46). Formation of the active eIF4F complex allows
initiation to proceed. The binding of eIF4E to eIF4G is
controlled in part by the translation repressor protein
4E-binding protein (BP)1. Binding of 4E-BP1 to eIF4E
limits eIF4E availability for formation of the active
eIF4E 䡠 eIF4G complex. The binding of 4E-BP1 to eIF4E
is regulated by phosphorylation of 4E-BP1(as reviewed
in Refs. 10 and 46).
The present investigation was performed to delineate the potential biochemical loci and molecular
mechanisms responsible for the inhibition of myocardial protein synthesis in rats after acute alcohol intoxication. Furthermore, various elements of the insulinlike growth factor (IGF) system were also quantitated
as a possible mechanism for the observed changes in
synthesis and initiation. Our data suggest that alcohol
intoxication impairs translation initiation by modulating the availability of eIF4F without affecting the eIF2
system or IGF-I.
ALCOHOL AND TRANSLATION INITIATION IN HEART
extraction procedure with cryoprecipitation (26, 27). The
plasma concentration of free IGF-I was determined by centrifugal ultrafiltration, as previously described (29). After
both extraction procedures, the IGF-I concentration in the
samples was determined by RIA with recombinant human
[Thr59]IGF-I (Genentech, South San Francisco, CA). The
relative concentrations of IGF binding protein (IGFBP)-1 and
IGFBP-3 were determined by standard Western blot and
ligand blot analysis, respectively, as previously described by
our laboratory (27, 29).
Plasma concentration of glucose, hormones, and alcohol.
The plasma insulin and corticosterone concentrations were
determined by RIA (DPC, Los Angeles, CA). The glucose and
alcohol concentrations in plasma were determined using a
rapid analyzer (model GL5; Analox Instruments, Lunenburg,
MA).
Statistics. Values are presented as means ⫾ SE. The number of rats in each group was eight, unless otherwise indicated. Data were analyzed by Student’s t-test to determine
treatment effect. Statistical significance was set at P ⬍ 0.05.
RESULTS
In vivo protein synthesis. When measured 2.5 h after
the administration of alcohol, the rate of myocardial
protein synthesis was decreased 31%, compared with
time-matched control animals (Fig. 1, top). At this time
point, there were no alcohol-induced changes in either
the wet weight (control ⫽ 659 ⫾ 25 mg vs. alcohol ⫽
670 ⫾ 28 mg) or protein content (control ⫽ 190 ⫾ 22
mg/g vs. alcohol ⫽ 196 ⫾ 5 mg/g wet wt) of heart.
A reduction in tissue protein synthesis may be
caused by a decrease in the number of ribosomes
and/or in the efficiency of mRNA translation. To determine which mechanism was responsible for the alcohol-induced impairment in protein synthesis, the RNA
content and translational efficiency were determined.
Because ⬃85% of the RNA in heart is ribosomal RNA,
changes in total RNA content reflect differing numbers
of ribosomes. As illustrated in Fig. 1 (middle), there
was no significant difference in the RNA content of
hearts from control and alcohol-treated rats. These
data suggest that an alteration in the relative abundance of ribosomes was not primarily responsible for
the alcohol-induced decrease in protein synthesis. The
efficiency of translation, calculated by dividing the
protein synthetic rate by the total RNA content, provides an index of how rapidly the existing ribosomes
are synthesizing protein (23). Our data demonstrate
that there is a 34% decrease in translational efficiency
in hearts from alcohol-treated rats compared with control values (Fig. 1, bottom). Thus acute alcohol intoxication impairs myocardial protein synthesis by limiting translational efficiency.
To determine the nature of the impairment in efficiency of protein synthesis observed in rats treated
with alcohol, the 40S and 60S ribosomal subunits were
isolated using sucrose density gradient centrifugation
(Fig. 2). The number of free 40S and 60S subunits was
increased ⬃50% in hearts from acute alcohol-treated
rats compared with controls. An increase in the content
of free ribosomal subunits in conjunction with decreased rates of protein synthesis indicates an impair-
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were chosen because change in the expression and/or activity
of these eIF correlates with alterations in protein synthesis
(21, 52, 58). eIF2 consists of three subunits, of which the
␣-subunit appears important in regulating protein synthesis.
Likewise, eIF2B is a multimeric protein consisting of five
subunits, with the ε-subunit being representative of the holoenzyme.
Quantification of 4E-BP1 䡠 eIF4E and eIF4G 䡠 eIF4E complexes. The association of eIF4E with either 4E-BP1 or eIF4G
was determined as previously described (18, 22, 26, 28).
Briefly, eIF4E and 4E-BP1 䡠 eIF4E and eIF4G 䡠 eIF4E complexes were immunoprecipitated from aliquots of 10,000-g
supernatants using an anti-eIF4E monoclonal antibody. The
antibody 䡠 antigen complex was collected and 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 nitrocellulose. The membranes
were incubated with a mouse anti-human eIF4E antibody, a
rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-eIF4G antibody. The blots were then developed using ECL, and autoradiographs were scanned and quantitated as described
above.
Phosphorylation of eIF4E, 4E-BP1, p70S6 kinase, and S6.
The phosphorylated and nonphosphorylated forms of eIF4E
in tissue extracts were separated by isoelectric focusing (IEF)
on a slab gel and quantitated by protein immunoblot analysis, as previously described (18, 22, 26, 28). The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from tissue homogenates after centrifugation
at 10,000 g. 4E-BP1 was immunoprecipitated, as described in
the previous section, for immunoprecipitation of eIF4E. The
various phosphorylated forms of 4E-BP1 (designated ␣, ␤,
and ␥) were separated by SDS-PAGE electrophoresis and
quantitated by protein immunoblot analysis as described
above. For determination of the phosphorylation state of
p70S6 kinase, supernatants from a 10,000-g centrifugation
were subjected to SDS-PAGE, transferred to polyvinylidene
difluoride (PVDF) membranes, and immunoblotted with a
polyclonal antibody that recognizes p70S6 kinase (Santa
Cruz Biotechnology, Santa Cruz, CA), as described previously (1, 7). The relative amount of phosphorylated ribosomal
S6 protein was determined by immunoblot analysis using
15% SDS-PAGE and a 1:5,000 dilution of the primary antibody (Dr. M. J. Birnbaum, University of Pennsylvania). The
blots were then developed and quantitated, as described above.
IGF-I and IGF-II mRNA. Total RNA was isolated from
heart using TRI Reagent TR-118 as outlined by the manufacturer (Molecular Research Center, Cincinnati, OH). Total
RNA (20 ␮g) was electrophoresed under denaturing conditions in 1% agarose/6% formaldehyde gels. Northern blotting
occurred via capillary transfer to Zeta-Probe GT blotting
membranes (Bio-Rad Laboratories, Hercules, CA). A 800-bp
probe from rat IGF-I (Dr. P. Rotwein; Portland, OR) and a
551-bp probe from rat IGF-II (Dr. M. Rechler, Bethesda, MD)
were labeled using a Random Primed DNA Labeling kit
(Roche Molecular Biochemicals, Indianapolis, IN). For normalization of RNA loading, a rat 18S oligonucleotide was
end-labeled with 32P-ATP using polynucleotide kinase (Amersham Pharmacia Biotech, Piscataway, NJ). All data were
normalized to ribosomal 18S RNA. Finally, membranes were
exposed to a phosphoimager screen, and the resultant data
were quantitated using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Plasma concentration of IGF-I and IGF-binding proteins.
The concentration of total IGF-I in plasma was determined
using a modified acid-ethanol (0.25 N HCl-87.5% ethanol)
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ALCOHOL AND TRANSLATION INITIATION IN HEART
Fig. 1. Effect of acute alcohol intoxication on in vivo rates of protein
synthesis and translation efficiency in heart. Rats were injected ip
with 75 mmol/kg of ethanol or an equal volume of saline, and tissue
protein synthesis was determined 2.5 h later by the injection of
[3H]phenylalanine (Phe). Total RNA was measured in homogenates
of heart. Translational efficiency was calculated by dividing the rate
of protein synthesis by the RNA content. Values are means ⫾ SE;
n ⫽ 8 for each group. *P ⬍ 0.05 vs. time-matched value from control
animals.
ment of peptide-chain initiation relative to elongation
(23).
eIF. One possible mechanism for the alcohol-induced
inhibition of peptide-chain initiation is by altering the
amount and/or activity of distinct initiation factors.
There were no significant differences in values for 1)
the total amount of eIF2␣ protein, 2) the fraction of
eIF2␣ in the phosphorylated form, 3) the total amount
of eIF2Bε protein, or 4) the functional activity of eIF2B
between control and alcohol-treated rats (Table 1).
Collectively, these data indicate that changes in the
eIF2/2B system are unlikely to explain the alcoholinduced decrease in peptide-chain initiation and protein synthesis in heart.
Another possible mechanism for decreasing translation initiation involves limiting the availability of
eIF4E to bind with eIF4G. Binding of the translational
repressor 4E-BP1 to eIF4E forms an inactive complex.
This is measured on an immunoblot as an increase in
the amount of 4E-BP1 present in the eIF4E immunoprecipitate. Figure 3 illustrates that acute alcohol increased the total amount of 4E-BP1 (sum of ␣- and
␤-forms) associated with eIF4E by 55%. Conversely,
eIF4E immunoprecipitates demonstrated almost a
70% reduction in the amount of eIF4E bound to eIF4G
(Fig. 4). The decreased amount of the eIF4E 䡠 eIF4G
complex did not result from a reduced amount of eIF4E
in the immunoprecipitate between control [4,250 ⫾ 235
arbitrary units (AU)] and alcohol-treated rats (4,350 ⫾
490 AU).
4E-BP1 has at least five potential phosphorylation
states, and the various phosphorylated forms of the
protein are resolved into three bands by SDS-PAGE
(31, 34). These bands have been termed the ␣- (least
phosphorylated and fastest migrating), ␤- (intermediate), and ␥- (most phosphorylated and slowest migrating) forms (Fig. 5, inset). The amount of 4E-BP1 in the
␣- and ␤-forms in heart was not significantly different
between control and alcohol-treated rats (data not
shown). In contrast, the amount of 4E-BP1 in the
␥-form in cardiac muscle was decreased ⬃50% at 2.5 h
after alcohol intoxication (Fig. 5).
To further define potential mechanisms through
which alcohol inhibits translation in heart, the phosphorylation of eIF4E was examined. The percentage of
Table 1. Effect of acute alcohol intoxication on eIF2
and eIF2B in heart
eIF2␣, AU
eIF2␣(P), % of total
eIF2B⑀, AU
eIF2B activity, nmol 䡠 min⫺1 䡠 mg protein⫺1
Control
Alcohol
1,125 ⫾ 58
11 ⫾ 2
666 ⫾ 53
154 ⫾ 22
1,208 ⫾ 152
12 ⫾ 3
606 ⫾ 41
132 ⫾ 15
Values are means ⫾ SE; n ⫽ 8 for both control and alcohol-treated
groups. For both eukaryotic initiation factor (eIF)2␣ and eIF2B⑀,
units are arbitrary densitometric volume units (AU). eIF2␣(P) represents the proportion of eIF2␣ in the phosphorylated state. Values
were quantitated by densitometric analysis, and the percentage of
the phosphorylated form was calculated relative to the total amount
of eIF2 (unphosphorylated ⫹ phosphorylated). For eIF2B activity,
activity was determined by the [3H]GDP exchange assay, and data
are expressed as the nmol of GDP exchanged per min per mg of
tissue protein.
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Fig. 2. Effect of acute alcohol intoxication on levels of free ribosomal
subunits in heart. Ventricular tissue was sampled 2.5 h after ip
injection of ethanol and from time-matched control animals. Free
40S and 60S ribosomal subunits were isolated on sucrose gradients
and analyzed for RNA. Values are means ⫾ SE; n ⫽ 8 per group.
*P ⬍ 0.05 vs. control value.
ALCOHOL AND TRANSLATION INITIATION IN HEART
eIF4E in the phosphorylated state averaged 13 ⫾ 2% in
control hearts and was not significantly altered in rats
administered alcohol (15 ⫾ 3%).
Phosphorylation of p70S6 kinase and ribosomal protein S6. We also examined the extent of phosphorylation of p70S6 kinase in cardiac muscle after acute
alcohol intoxication. p70S6 kinase is activated by multisite phosphorylation that results in phosphorylated
Fig. 4. Effect of acute alcohol intoxication on the amount of eIF4G
associated with eIF4E in heart. Inset: representative immunoblot.
Bar graph: densitometric analysis of total eIF4G bound to eIF4E.
Data are expressed in AU. Values are means ⫾ SE; n ⫽ 8 per group.
*P ⬍ 0.05 vs. control value.
Fig. 5. Effect of acute alcohol intoxication on the phosphorylation
status of 4E-BP1 in heart. Inset: representative immunoblot in which
the ␣-, ␤-, and ␥-forms of 4E-BP1 are identified. Bar graph: densitometric analysis in which data are expressed as the amount of 4E-BP1
in the ␥-form. Data are expressed in AU. Values are means ⫾ SE; n ⫽
8 per group. *P ⬍ 0.05 vs. control value.
forms exhibiting retarded electrophoretic mobility
when subjected to SDS-PAGE (9, 25). We used this
property (assessed by immunoblotting techniques) as
an indicator of the effect of alcohol on the activation of
the kinase. Acute alcohol intoxication reduced the
prominence of the more electrophoretically retarded
bands in the myocardium, indicating a net decrease in
the phosphorylation state of the protein (Fig. 6, top).
The percentage of p70S6 kinase in the least phosphor-
Fig. 6. Effect of acute alcohol intoxication on the phosphorylation of
p70 S6 kinase and the ribosomal protein S6. Homogenates were
obtained from saline (C) and alcohol-treated (A) hearts treated as
described in Fig. 1. All homogenates were subjected to SDS-PAGE,
and the proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes. Top: membranes were exposed
to a polyclonal antibody that recognizes specifically p70 S6 kinase,
and the antibody-antigen complex was visualized using a chemiluminescence detection system. Arrows indicate gel retardation of p70
S6 kinase. Bottom: representative immunoblot for the phosphorylated ribosomal protein S6. All immunoblots shown are representative of hearts from 6 saline-treated and 7 alcohol-treated animals.
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Fig. 3. Effect of acute alcohol (ETOH) intoxication on the amount of
4E-binding protein 1 (4E-BP1) associated with eukaryotic initiation
factor 4E (eIF4E) in heart. Inset: representative autoradiograph of
the ␣- and ␤-forms of 4E-BP1 in the immunoprecipitate as determined by Western blot analysis. Ctl, control. Bar graph: densitometric analysis of total 4E-BP1 bound to eIF4E. Data are expressed in
arbitrary volume units (AU). Values are means ⫾ SE; n ⫽ 8 per
group. *P ⬍ 0.05 vs. control value.
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ALCOHOL AND TRANSLATION INITIATION IN HEART
Table 2. Plasma concentration of hormones, glucose,
and alcohol
Total IGF-I, ng/ml
Free IGF-I, ng/ml
Glucose, mM
Insulin, ␮U/ml
Corticosterone, ng/ml
Alcohol, mg/dl
Control
Alcohol
934 ⫾ 52
91 ⫾ 5
7.8 ⫾ 0.4
22 ⫾ 3
152 ⫾ 11
ND
929 ⫾ 41
104 ⫾ 9
11.9 ⫾ 0.3*
27 ⫾ 5
367 ⫾ 29*
377 ⫾ 56*
Values are means ⫾ SE; n ⫽ 8 for both control and alcohol-treated
groups. IGF-I, insulin-like growth factor I. ND, not determined.
* P ⬍ 0.05 vs. time-matched values from control animals.
Our results provide evidence that acute alcohol intoxication inhibits myocardial protein synthesis
through a reduction in translational efficiency rather
than a change in the abundance of ribosomes. The
diminished translational efficiency after alcohol intoxication may result from an inhibition of peptide-chain
initiation, elongation/termination, or both. Relative
rates of initiation and elongation were assessed by the
measurement of incorporation of phenylalanine and
analysis of the relative abundance of free ribosomal
subunits. The amount of RNA in free ribosomal subunits is reflective of the balance between the rates of
peptide-chain initiation and elongation/termination
(as reviewed in Refs. 6, 23, and 50). Thus a decrease in
the rate of peptide-chain initiation relative to elongation/termination means that the free ribosomal subunits are entering polysomes at a slower rate (initiation), whereas they are moving along mRNA
(elongation) and exiting (termination) at the same rate.
In this scenario, the abundance of 40S and 60S ribosomal subunits in the nonpolysome (i.e., “free”) pool
increases. Analysis of ribosomal subunits revealed an
accumulation of free subunits in conjunction with reduced rates of protein synthesis from hearts of alcoholtreated rats compared with controls. Hence, acute alcohol intoxication limits myocardial protein synthesis
through an inhibition in peptide-chain initiation relative to elongation.
One possible mechanism to account for alcohol-induced inhibition of peptide-chain initiation is via alterations in the amount and/or activity of regulatory eIF
proteins. The cellular content of eIF2 has been correlated with rates of protein synthesis (21, 52). Therefore, we investigated whether the eIF2 content was
decreased in hearts from alcohol-fed rats. There was no
significant difference in the myocardial eIF2 content
between control and alcohol-fed rats. Likewise, the
cellular content of eIF2Bε has been implicated in controlling the overall rate of protein synthesis (57, 58).
Like eIF2, there was no significant difference in the
Fig. 7. Effect of acute alcohol intoxication on the plasma concentration of insulin-like growth factor binding proteins (IGFBP)-1 and -3.
IGFBP-1 (top) was quantitated by Western blot analysis and IGFBP-3 (bottom) by ligand blot analysis. For both blots, samples from
3 representative control (lanes 1–3) and 3 alcohol-treated (lanes 4–6)
rats are shown. All immunoblots shown are representative of plasma
from 6 saline-treated and 7 alcohol-treated animals.
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ylated form increased from 50 ⫾ 3% in hearts from
control animals to 71 ⫾ 3% in hearts from animals
injected with ethanol (P ⬍ 0.05). Conversely, the percentage of p70S6 kinase in the most phosphorylated
form decreased from 13 ⫾ 3% in hearts from control
animals to 3 ⫾ 1% in hearts from animals injected with
ethanol (P ⬍ 0.05). The alcohol-induced change in the
phosphorylation of p70S6 kinase was also reflected by
a significant ⬃30% decrease in the relative amount of
phosphorylated S6 protein [control (c) ⫽ 128 ⫾ 11 AU
vs. alcohol (a) ⫽ 88 ⫾ 13 AU, P ⬍ 0.05; Fig. 6, bottom].
Components of the IGF system. There was no detectable alcohol-induced change in the plasma concentration of total IGF-I (Table 2). Because the bioactivity
and bioavailability of IGF-I can be influenced by
changes in various IGFBPs, plasma levels of two of the
major binding proteins were also determined. As illustrated in Fig. 7 (top), alcohol treatment caused a
greater than eightfold elevation in the circulating concentration of IGFBP-1 compared with control values.
In contrast, the relative concentration of IGFBP-3 was
decreased 35% (c ⫽ 703 ⫾ 85 AU vs. a ⫽ 454 ⫾ 61 AU;
P ⬍ 0.05) in alcohol-treated rats compared with controls (Fig. 7, bottom). However, despite these changes
in circulating IGFBPs, the concentration of free IGF-I
in plasma was unaltered by acute alcohol intoxication
(Table 2).
The relative abundance of both IGF-I and IGF-II
mRNA in hearts was determined by Northern blot
analysis. There was no significant difference in the
relative amount of mRNA for either IGF-I (c ⫽ 1.00 ⫾
0.08 vs. a ⫽ 0.88 ⫾ 0.09 AU: n ⫽ 8) or IGF-II (c ⫽
1.00 ⫾ 0.09 vs. a ⫽ 0.96 ⫾ 0.07 AU: n ⫽ 8) in heart from
alcohol-treated rats compared with control values.
Plasma concentrations of alcohol, glucose, and hormones. The intraperitoneal injection of alcohol raised
blood alcohol levels to ⬃380 mg/dl at the time of tissue
sampling. The blood alcohol content is high relative to
that observed in chronic models of alcohol consumption, but it is comparable to that seen in humans in
response to acute alcohol ingestion (30, 43). Administration of alcohol increased circulating levels of glucose
by 50% but did not produce a concomitant elevation in
plasma insulin levels (Table 2). Finally, acute alcohol
intoxication increased the plasma corticosterone levels
by 140% compared with time-matched control animals.
DISCUSSION
ALCOHOL AND TRANSLATION INITIATION IN HEART
limits cap-dependent mRNA translation by physically
sequestering eIF4E into an inactive complex. In the
present set of experiments, acute alcohol intoxication
caused a ⬃55% increase in the amount of 4E-BP1
associated with eIF4E.
The interaction between 4E-BP1 and eIF4E is regulated by the extent of 4E-BP1 phosphorylation. An
increased amount of the phosphorylated ␥-form of 4EBP1 is associated with the release of eIF4E from the
4E-BP1 䡠 eIF4E complex. In contrast, the hypophosphorylated ␣- and ␤-forms of 4E-BP1 bind to eIF4E
(see Fig. 3 and Ref. 31). Refeeding of starved rats or
insulin treatment of diabetic rats increases 4E-BP1
phosphorylation, causing a dissociation of the 4EBP1 䡠 eIF4E complex, and thereby promotes translation
initiation (1, 18, 20, 22). Presumably, the release of
eIF4E from the 4E-BP1 䡠 eIF4E complex secondary to
increased phosphorylation of 4E-BP1 allows eIF4E to
bind to eIF4G and form the active eIF4E 䡠 eIF4G complex. In perfused skeletal muscle, stimulation of protein synthesis in response to acute insulin administration is associated with a 12-fold increase in the amount
of eIF4G bound to eIF4E (22). In the present set of
experiments, alcohol intoxication decreased the
amount of 4E-BP1 in the highly phosphorylated
␥-form. Thus acute alcohol intoxication may limit myocardial protein synthesis, at least in part, by enhancing
the abundance of 4E-BP1 䡠 eIF4E complex secondary to
decreasing the phosphorylation of 4E-BP1.
Another potential mechanism by which alcohol may
impair protein synthesis is by decreasing translation of
a specific subset of mRNAs containing an oligopyrimidine tract at their 5⬘ terminus. The mRNAs for ribosomal protein S6 and elongation factors eEF1A and
eEF2 are typical examples of such proteins. In this
regard, the ribosomal S6 protein is uniquely positioned
to regulate translation by its location at the tRNA
binding site on the 40S ribosome, and phosphorylation
of this protein enhances translation of mRNA into
protein (9). Ribosomal S6 protein is phosphorylated by
a family of 70-kDa protein kinases referred to as p70S6
kinase (9). The p70S6 kinase, in turn, is activated by
phosphorylation of the protein at multiple serine and
threonine residues catalyzed by FKBP rapamycin-associated protein/mammalian target of rapamycin
(FRAP/mTOR) (4). mTOR appears to represent a functional bifurcation point for signal transduction, because it apparently phosphorylates both 4E-BP1 and
p70S6 kinase (2, 4, 5). Hence, these two modulators
appear to lie on parallel pathways. Alcohol intoxication
resulted in a diminished prominence of the phosphorylated bands of p70S6 kinase. Because activity of p70
S6 kinase is dependent on its phosphorylation, a diminished phosphorylation of p70S6 kinase would be
expected to reduce the phosphorylation of ribosomal
protein S6. In this regard, we observed a decrease in
the amount of phosphorylated S6 protein in response to
acute alcohol intoxication. These results suggest that
the inhibition of cardiac muscle protein synthesis after
acute ethanol intoxication may be mediated, at least in
part, by the dephosphorylation and inactivation of
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myocardial eIF2Bε content between control and alcohol-fed rats. Thus changes in the relative abundance of
eIF2 or eIF2B do not appear responsible for the diminished translation initiation following acute ethanol administration.
We then examined whether acute alcohol intoxication impaired eIF2B activity, which can be regulated
via several mechanisms. eIF2B activity is regulated in
part through phosphorylation of the ␣-subunit of eIF2,
thereby increasing the affinity of eIF2 for eIF2B. The
formation of an eIF2␣(P) 䡠 eF2B complex effectively sequesters available eIF2B and limits its availability.
The extent of phosphorylation of eIF2␣ is inversely
proportional to the rate of protein synthesis under
selective in vivo conditions (23). However, alcohol administration did not increase the extent of eIF2␣ phosphorylation. Likewise, direct measurement of myocardial eIF2B activity revealed no significant difference
between control rats and those injected with alcohol.
Collectively, these data suggest that alterations in the
eIF2/eIF2B system are not responsible for the acute
effects of ethanol on myocardial peptide-chain initiation.
In addition to the eIF2/eIF2B system, considerable
evidence suggests that the binding of mRNA to the 43S
preinitiation complex, which is mediated by eIF4F,
also regulates the overall rate of translation initiation.
One of the subunits of the eIF4F complex, eIF4E, binds
the m7GTP cap structure present at the 5⬘-end of many
eukaryotic mRNAs to form an eIF4E 䡠 mRNA complex
(41, 42). During translation initiation, the eIF4E 䡠
mRNA complex binds to eIF4G and eIF4A to form the
active eIF4F complex (41, 42, 46). The active eIF4E 䡠
eIF4G complex allows binding of mRNA to the 43S
preinitiation complex, and the synthesis of the protein
proceeds. In the present investigation, acute ethanol
administration diminished the abundance of eIF4G
associated with eIF4E by ⬎70%.
During translation initiation, mRNA binds either
directly to eIF4E already associated with 40S ribosomes or to free eIF4E with subsequent binding of the
mRNA 䡠 eIF4E 䡠 eIF4G complex to the ribosome (41, 42,
46). With either scenario, a decreased amount of eIF4E
associated with eIF4G would diminish this association.
Because translation of mRNAs in eukaryotic cells is
heavily dependent upon a cap-dependent process involving eIF4E, it might be expected that modulation of
eIF4E bound to eIF4G would contribute to the inhibition of protein synthesis during acute alcohol intoxication.
The availability of eIF4E is controlled through its
binding to small acid- and heat-stable proteins, termed
4E-BP1, 4E-BP2, and 4E-BP3, forming an inactive
complex (1, 7). In cardiac muscle, the predominant
form of these translation repressor proteins is 4E-BP1.
Hypophosphorylated 4E-BP1 binds to eIF4E to form an
inactive 4E-BP1 䡠 eIF4E complex. When eIF4E is bound
to 4E-BP1, eIF4E binds to mRNA but cannot form an
active eIF4E 䡠 eIF4G complex (16). Thus formation of
the 4E-BP1 䡠 eIF4E complex prevents binding of mRNA
to the ribosome. 4E-BP1 binding to eIF4E essentially
E1035
E1036
ALCOHOL AND TRANSLATION INITIATION IN HEART
known to impair the ability of IGF-I to stimulate protein synthesis in cultured myocytes in a dose-dependent manner (12).
Similarly, a decrease in insulin would also be consistent with essentially all of the alterations in initiation
observed in cardiac muscle of alcohol-treated rats (17,
20). However, at the time tissues were sampled, insulin
concentrations were mildly elevated, albeit not statistically, in alcohol-treated rats. Although only a single
time point determination of insulin was made in the
current study, previous reports indicate that plasma
insulin levels are also not decreased at earlier time
points (3, 33). Therefore, we can exclude an absolute
decrease in plasma insulin as a cause for the observed
changes. However, it is possible that an impairment in
insulin action might be a participating factor. In this
regard, acute alcohol has been reported to produce
insulin resistance in skeletal muscle (47).
Negative nitrogen balance and the erosion of skeletal
muscle mass are also observed in conditions associated
with increased circulating levels of stress hormones,
alone or in combination. Previous work by our laboratory and others suggests that the plasma concentrations of glucagon and catecholamines are probably not
dramatically elevated at this time point in response to
this dose of alcohol (36). Moreover, relatively shortterm increases in either of these hormones do not
appear to impair myocardial protein synthesis (3, 35).
In contrast, acute alcohol intoxication in the rat markedly increases the plasma corticosterone level. However, although excessive glucocorticoids impair protein
synthesis in skeletal muscle, they have not been reported to acutely depress synthesis in the heart (40).
Thus alterations in the classical stress hormones are
unlikely mediators of the decrease in myocardial protein synthesis observed in response to acute alcohol
intoxication. On the basis of studies using the isolated
perfused heart preparation, it seems more probable
that the inhibition of myocardial protein synthesis
results from a direct effect of ethanol on one or more
steps in the translation initiation pathway (44). This
hypothesis is supported by in vivo data indicating that
in rats treated with an inhibitor of alcohol dehydrogenase, 4-methylpyrazole, a depression in cardiac protein
synthesis was still observed (45).
We gratefully acknowledge the generous gifts of the antibody to
phosphorylated S6 (Dr. M. J. Birnbaum, University of Pennsylvania)
and the human recombinant IGF-I (Genentech, South San Francisco,
CA).
This work was supported in part by National Institute on Alcohol
Abuse and Alcoholism Grants AA-11290 and AA-12814, and Grantin-Aid 9950288N awarded by the American Heart Association.
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