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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 E1029 Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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. E1030 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 Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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- Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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) E1031 E1032 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. Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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. Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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. E1033 E1034 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. Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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 Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 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. REFERENCES 1. Anthony JC, Gautsch T, Kimball SR, Vary TC, and Jefferson LS. Orally administered leucine stimulates protein synthesis in skeletal muscle of postabsorptive rats in association with increased eIF4F formation. J Nutr 130: 139–145, 2000. 2. Berrata L, Gingas AC, Svitkin YV, Hall MN, and Sonenberg N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J 15: 658–664, 1996. Downloaded from http://ajpendo.physiology.org/ by 10.220.32.246 on May 13, 2017 p70S6 kinase. Furthermore, the data suggest that the site where alcohol modulates protein synthesis either lies upstream of mTOR or is mediated via a mechanism with dual specificity for both 4E-BP1 and p70S6 kinase. 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