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Am J Physiol Heart Circ Physiol 288: H121-H128, 2005. First published September 23, 2004; doi:10.1152/ajpheart.00440.2004
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Acute alcohol intoxication enhances myocardial eIF4G phosphorylation despite reducing mTOR signaling

Thomas C. Vary,1 Gina Deiter,1 and Stacy A. Goodman2

1Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey; and 2Department of Biology, Lebanon Valley College, Annville, Pennsylvania

Submitted 11 May 2004 ; accepted in final form 14 September 2004


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acute alcohol intoxication impairs myocardial protein synthesis in rats, secondary to a diminished mRNA translational efficiency. Decreased mRNA translational efficiency occurs through altered regulation of peptide chain initiation. The purpose of the present set of experiments was to determine whether acute alcohol intoxication alters the phosphorylation state of eukaryotic initiation factor (eIF) 4G, eIF4G·eIF4E complex formation, and the mammalian target of rapamycin (mTOR) signaling pathway in the heart. Acute alcohol intoxication was induced by injection of alcohol (75 mmol/kg body wt ip). Control animals received an equal volume of saline. Alcohol administration enhanced phosphorylation of eIF4G (Ser1108) approximately threefold. Alcohol administration lowered formation of the active eIF4G·eIF4E complex by >90%, whereas it increased the abundance of the inactive 4E-binding protein 1 (4E-BP1)·eIF4E complex by ~160%. Phosphorylation of mTOR on Ser2448 and Ser2481 was decreased by 50%. Reduced mTOR phosphorylation did not result from decreased phosphorylation of PKB. Phosphorylation of 4E-BP1 and S6 kinase 1 (Thr389), downstream targets of mTOR, were also reduced after acute alcohol administration. These data suggest that acute alcohol-induced impairments in myocardial mRNA translation initiation result, in part, from marked decreases in eIF4G·eIF4E complex formation, which appear to be independent of changes in phosphorylation of eIF4G but dependent on mTOR.

cardiomyopathy; peptide-chain initiation; eukaryotic initiation factor 4E; 4E-binding protein 1; heart; translational efficiency; p70 S6 kinase; protein kinase B

ALCOHOLISM remains the most common form of drug abuse in the United States. Acute alcohol intoxication, commonly referred to as "binge drinking," causes myocardial dysfunction and produces metabolic and functional abnormalities (11, 19, 29). With regard to protein metabolism, ethanol intoxication depresses protein synthesis in cardiac muscle (23, 24, 36–38).

The mechanisms responsible for alcohol-induced inhibition of protein synthesis in cardiac muscle are beginning to be elucidated. Alcohol consumption does not lower the plasma amino acid levels (25) to rate-limiting concentrations or the energy charge of the myocardium (50), indicating that substrate supply and high-energy phosphates are not limiting protein synthesis. Regulation of protein synthesis can also occur through changes in the abundance of ribosomes and/or translational efficiency. We showed that reductions in the relative abundance of ribosomes are not responsible for inhibition of myocardial protein synthesis in rats treated with ethanol (22–24, 50). Furthermore, the changes in protein synthesis are not the result of diminished mRNA content (50). Instead, the efficiency of translation, calculated by dividing the protein synthesis rates by the total RNA (or mRNA) content, is reduced. Reductions in translational efficiency can occur by inhibition of mRNA translation (peptide chain) initiation or peptide chain elongation (23, 24, 50).

Our studies indicate that alcohol limits mRNA translation initiation through inhibition of binding of mRNA to the 43S preinitiation complex, a process mediated by eukaryotic initiation factor (eIF) 4F (23, 24, 50). eIF4F is a complex of several proteins, including eIF4A (an RNA helicase that functions with eIF4B to unwind secondary structures in the 5'-untranslated region of mRNA), eIF4E (a protein that binds directly to the 7-methyl-GTP cap structure at the 5' end of most eukaryotic mRNAs), and eIF4G (a protein that functions as a scaffold for eIF4E, eIF4A, mRNA, and the ribosome) (43, 44, 47). eIF4G appears to be the nucleus around which the initiation complex forms, because it has binding sites not only for eIF4E but also for eIF4A and eIF3 (21).

Formation of the eIF4E·eIF4G complex may limit mRNA translation initiation. Our laboratory was the first to show that assembly of the eIF4E·eIF4G complex is significantly diminished in hearts from animals treated with alcohol acutely via intraperitoneal injection or through chronic supplementation in the diet (23, 24, 50). Reduced amounts of eIF4E associated with eIF4G after acute or chronic ethanol administration would be expected to diminish the association of mRNA with the ribosome and, hence, limit protein synthesis.

Alcohol appears to alter formation of the active eIF4E·eIF4G complex in the heart through mechanisms other than changes in the myocardial content of eIF4E or eIF4G (23, 24, 50). One potential mechanism for decreased binding of eIF4E to eIF4G involves phosphorylation of eIF4G (31, 41). Increased phosphorylation of eIF4G correlates with conditions known to stimulate protein synthesis (41). Another mechanism involves sequestration of eIF4E by binding with the translation initiation repressor protein 4E-binding protein 1 (4E-BP1) to form an inactive eIF4E·4E-BP1 complex (12). When eIF4E is bound to 4E-BP1, eIF4E cannot bind to eIF4G. Binding of eIF4E to 4E-BP1 is reduced after phosphorylation of 4E-BP1 through a phosphatidylinositol 3-kinase-dependent pathway involving signaling through Akt/PKB and mammalian target of rapamycin (mTOR) (3, 12, 16–18, 27, 28, 34, 48). There is no information available concerning the effect of acute alcohol intoxication on phosphorylation of eIF4G or the mTOR signaling pathway in cardiac muscle. The purpose of the present set of experiments was to determine whether acute alcohol intoxication diminishes the phosphorylation state of eIF4G, eIF4G·eIF4E complex formation, and/or the mTOR signaling pathway in the heart.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
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Animals. Adult male Sprague-Dawley rats weighing 150–225 g were maintained in a controlled environment with a 12:12-h light-dark cycle and provided water and rat chow ad libitum for ≥1 wk before the start of the study. On the night before the experiment, food was withdrawn from the animals. At ~0800, animals were randomly assigned to one of two groups: alcohol or control. The alcohol group was injected with ethanol [75 mmol/kg body wt ip; 20% (wt/vol) in saline]. Animals in the control group 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 ethanol dose, route of administration, and timing of blood and tissue samples were chosen on the basis of previous studies demonstrating that this protocol impairs myocardial protein synthesis (23). 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 is comparable to that seen in humans in response to acute alcohol ingestion (26, 45). 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.

After pentobarbital sodium anesthesia (100 mg/kg body wt), hearts were excised, weighed, and homogenized in 7 vol 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 {beta}-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, and the pellet was discarded. An aliquot of the 10,000-g supernatant was mixed with an equal volume of 2x Laemmli SDS sample buffer and then subjected to protein immunoblot analysis. Another aliquot was used to measure the protein concentration by the Biuret method, with crystalline bovine serum albumin as a standard. A third aliquot was used for determination of the association of eIF4E with eIF4G or 4E-BP1.

Determination of phosphorylation state of eIF4G. To measure the relative extent of phosphorylation of eIF4G, an equal volume of 2x Laemmli SDS buffer (65°C) was added to the homogenate, and proteins were separated by 7.5% SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (PALL Biotech). The membranes were incubated with antibodies specific for eIF4G phosphorylated on Ser1108 (Cell Signaling Technology, Beverly, MA) overnight at 4°C. The blots were then developed using an enhanced chemiluminesence (ECL) Western blotting kit as directed by the manufacturer. Films were scanned using a scanner (ScanMaker III, Microtek) equipped with a transparent media adapter connected to a Macintosh computer. Images were obtained using the ScanWizard Plugin (Microtek) for Adobe Photoshop and quantitated using NIH Image 1.63 software.

After development of the immunoblot, the membranes were treated with a solution containing 62.5 mM Tris·HCl (pH 6.7), 100 mM {beta}-mercaptoethanol, and 2% (wt/vol) SDS to remove antibodies according to the manufacturer’s instructions. This procedure effectively removed all signal resulting from incubation with the phospho-eIF4G antibody. The membranes were blocked with nonfat dry milk and then immunoblotted with the antibody that recognizes eIF4G independently of its phosphorylation state (Bethyl Laboratories, Montgomery, TX). The blots were developed using ECL, and the autoradiographs were scanned and analyzed as described above. The phosphorylated eIF4G signal densities were normalized to the respective total eIF4G signal to reflect the relative ratio of phosphorylated eIF4G to total eIF4G.

Quantification of 4E-BP1·eIF4E and eIF4G·eIF4E complexes. The association of eIF4E with 4E-BP1 or eIF4G was determined in the heart using immunoblot techniques as previously described in our laboratory (48–50). Heart muscle was homogenized in 7 vol of buffer A (see Animals) with a Polytron homogenizer. The homogenate was used directly or centrifuged at 10,000 g for 10 min at 4°C, and the pellet was discarded. The supernatant was used to evaluate regulation of the eIF4E system. An anti-eIF4E monoclonal antibody was used to immunoprecipitate eIF4E, as well as 4E-BP1·eIF4E and eIF4G·eIF4E complexes, from aliquots of 10,000-g supernatants. 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% {beta}-mercaptoethanol, 0.5% Triton X-100, 50 mM NaF, 50 mM {beta}-glycerolphosphate, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium vanadate). The beads were captured using a magnetic sample rack and washed twice with buffer B and once with buffer B containing 500 mM, rather than 150 mM, NaCl. Resuspension in SDS sample buffer and boiling for 5 min eluted protein bound to the beads. The beads were collected by centrifugation, and the supernatants were subjected to electrophoresis on a 7.5% polyacrylamide gel for quantitation of eIF4G or a 15% polyacrylamide gel for quantitation of 4E-BP1 and eIF4E. Proteins were then electrophoretically transferred to a PVDF membrane (PALL, Biotrace). The PVDF membranes were incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-eIF4G antibody overnight at 4°C. The blots were then developed and quantitated as described above for eIF4G.

Determination of the phosphorylation state of 4E-BP1. The phosphorylated forms of 4E-BP1 were measured in heart extracts after an aliquot (200 µl) of the myocardial homogenates was boiled for 5 min. The boiled homogenate was centrifuged in a microcentrifuge at room temperature, and the supernatant was decanted. An equal volume of 2x Laemmli SDS buffer (65°C) was then added to the supernatant. The various phosphorylated forms of 4E-BP1 (designated {alpha}, {beta}, and {gamma}) were separated by SDS-PAGE and quantitated by protein immunoblot analysis as described previously (48–50).

Determination of the phosphorylation of mTOR. Another portion of the homogenate was electrophoresed and transferred as described above for eIF4G. The PVDF membranes were then incubated with an antibody that recognizes mTOR phosphorylated on Ser2448 and on Ser2481 (Cell Signaling Technology). The blots were developed and analyzed as described above for eIF4G. After development of the immunoblot, the PVDF membranes were treated to remove the phosphospecific antibodies as described above for eIF4G. The PVDF membranes were blocked with Tris-buffered saline supplemented with 0.1% Tween containing 5% (wt/vol) nonfat dry milk and then immunoblotted with the antibody that recognizes mTOR independently of its phosphorylation state (Bethyl Laboratories). The blots were developed using ECL, and the autoradiographs were scanned and analyzed as described above. The phosphorylated mTOR signal densities were normalized to the respective total mTOR signal to reflect the relative ratio of phosphorylated mTOR to total mTOR.

Determination of the phosphorylation state of S6 kinase 1. Weng and co-workers (52) provided evidence that, after phosphorylation, protein S6 kinase 1 (S6K1) activity in vivo is most closely related to phosphorylation on Thr389. To examine the phosphorylation of S6K1, homogenates of cardiac muscle were mixed with 2x Laemmli SDS sample buffer and subjected to electrophoresis on 12.5% SDS-polyacrylamide Criterion gels (Bio-Rad, Hercules, CA). Proteins were then electrophoretically transferred to PVDF membranes and blocked with Tris-buffered saline supplemented with 0.1% Tween and containing 5% (wt/vol) nonfat dry milk. Initially, the PVDF membranes were probed with antibody that recognizes only S6K1 phosphorylated on Thr389 (Cell Signaling Technology), the phosphorylation site required for activation of the kinase (52). The blots were developed using an ECL Western blotting kit according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). We used this property as an indicator of the effect of alcohol on the activation of the kinase. After quantification of the relative intensity of the signal for phosphorylation on Thr389, the phosphospecific antibodies were removed from PVDF membranes as described above for eIF4G. The blots were then probed with an antibody (Santa Cruz Biotechnology, Santa Cruz, CA) that recognizes total S6K1 (i.e., phosphorylated and unphosphorylated forms). Results are presented as the ratio of the densitometric analysis of the blot for phosphorylated S6K1 (Thr389) to that for total S6K1 performed on same gel.

Determination of phosphorylation of PKB. Another portion of the homogenate was electrophoresed and transferred as described above for eIF4G. The membranes were then incubated with phosphospecific antibodies that recognize PKB phosphorylated on Thr308 or Ser473 (Cell Signaling Technology). After development of the immunoblot, the PVDF membranes were treated to remove the phosphospecific antibodies as described above for eIF4G. The membranes were blocked with nonfat dry milk and then immunoblotted with the antibody that recognizes PKB independently of its phosphorylation state (Cell Signaling Technology). The blots were developed using ECL, and the autoradiographs were scanned and analyzed as described above. The phosphorylated PKB signal densities were normalized to the respective total PKB signal to reflect the relative ratio of phosphorylated PKB to total PKB.

Statistical analysis. Values are means ± SE. Statistical evaluation of the data was performed using Student’s t-test when a parametric test was indicated or the Mann-Whitney test when a nonparametric test was indicated. Differences among the means were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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The mechanistic changes in myocardial mRNA translation initiation were investigated by analyzing known regulatory steps in the control of translation initiation. The abundance of the eIF4G·eIF4E complex in cardiac muscle was determined by immunoprecipitating eIF4E with a monoclonal antibody followed by immunoblot analysis for eIF4E and eIF4G. Results were normalized to the total eIF4E in the immunoprecipitate. Acute alcohol intoxication decreased the amount of eIF4E associated with eIF4G by >90% (Fig. 1).



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  		Fig. 1. Effect of acute alcohol intoxication changes on abundance of eukaryotic initiation factor (eIF) 4G·eIF4E complex in cardiac muscle. Amount of eIF4G bound to eIF4E was assessed by immunoblotting the immunoprecipitate (IP) for eIF4G and eIF4E. Representative Western immunoblots of eIF4G and eIF4E after immunoprecipitation of eIF4E from cardiac muscle homogenates are shown above bar graphs of means of individual densitometric analysis of immunoblots of eIF4G associated with eIF4E. Arb units, arbitrary units. Values are means ± SE; n = 4–5. *P < 0.05 vs. control.

 
The interaction between eIF4E and eIF4G can be regulated, at least in part, by several mechanisms, including the availability of eIF4E and/or phosphorylation of eIF4G. Increased phosphorylation of eIF4G is associated with enhanced formation of the active eIF4G·eIF4E complex. We examined the phosphorylation state of eIF4G in heart extracts from animals injected with saline or alcohol. Phosphorylation of eIF4G on Ser1108 in heart homogenates was analyzed by Western blot and corrected for the total amount of eIF4G. The extent of phosphorylation of eIF4G was increased >2.5-fold after alcohol injection compared with saline-injected controls (Fig. 2). The increase in phosphorylation of eIF4G during acute alcohol intoxication is not the result of alterations in the myocardial content of eIF4G inasmuch as the total cellular content of eIF4G in animals injected with alcohol is not different from that in controls (data not shown).



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  		Fig. 2. Effect of acute alcohol intoxication on eIF4G phosphorylation in cardiac muscle. Equal amounts of protein in homogenates from hearts described in Fig. 1 were immunoblotted with antibodies specific for the phosphorylated form of eIF4G (Ser1108). Blots were stripped and immunoblotted with an antibody that recognizes phosphorylated and nonphosphorylated forms of eIF4G. Representative Western immunoblots of the phosphorylated form eIF4G from cardiac muscle homogenates are shown above bar graph of means of individual densitometric analysis of immunoblots of eIF4G phosphorylation on Ser1108 in homogenates corrected for total amount of eIF4G in cardiac muscle. Values are means ± SE; n = 5. *P < 0.05 vs. control.

 
Additional mechanisms for decreasing formation of the eIF4G·eIF4E complex involve limiting the availability of eIF4E in the myocardium to bind with eIF4G. Decreasing the availability of eIF4E for formation of the eIF4G·eIF4E complex can occur through binding of the translational repressor 4E-BP1 to eIF4E to form an inactive 4E-BP1·eIF4E complex. Binding of eIF4E to 4E-BP1 prevents formation of an active eIF4E·eIF4G complex, presumably because 4E-BP1 blocks the binding site for eIF4G (39). The eIF4E-to-4E-BP1 association was determined in hearts by immunoprecipitation of eIF4E with a monoclonal antibody followed by immunoblot analysis for eIF4E and 4E-BP1. Results were normalized to total eIF4E in the immunoprecipitate. Acute alcohol intoxication increased formation of an inactive 4E-BP1·eIF4E complex compared with rats receiving an injection of saline (Fig. 3).



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  		Fig. 3. Effect of acute alcohol intoxication on amount of 4E-binding protein 1 (4E-BP1) associated with eIF4E in cardiac muscle. Amount of 4E-BP1 bound to eIF4E was assessed by immunoblotting immunoprecipitate from hearts described in Fig. 1 for 4E-BP1 and eIF4E. Representative Western immunoblots of 4E-BP1 and eIF4E after immunoprecipitation of eIF4E from cardiac muscle homogenates are shown above bar graphs of means of individual densitometric analysis of immunoblots of 4E-BP1 associated with eIF4E. Values are means ± SE; n = 5–10. *P < 0.05 vs. control.

 
The interaction of 4E-BP1 with eIF4E is regulated in part through phosphorylation of 4E-BP1. Formation of the inactive eIF4E·4E-BP1 complex occurs when 4E-BP1 is hypophosphorylated. Therefore, we examined phosphorylation of 4E-BP1 in cardiac muscle after acute alcohol intoxication. 4E-BP1, when phosphorylated, resolves into distinct electrophoretic forms ({alpha}, {beta}, and {gamma}), with the {gamma}-form representing the highest phosphorylated form. The results reveal that ~45% of the total 4E-BP1 was present in the {gamma}-form in hearts from saline-injected animals (Fig. 4). The extent of phosphorylation of 4E-BP1 was significantly reduced after acute injection of alcohol.



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  		Fig. 4. Effect of acute alcohol intoxication on phosphorylation status of 4E-BP1 in cardiac muscle. Equal amounts of protein in homogenates from hearts described in Fig. 1 were immunoblotted with antibodies specific for 4E-BP1. Western immunoblots of 4E-BP1 showing {alpha}-, {beta}-, and {gamma}-forms of 4E-BP1 from cardiac muscle homogenates are shown above a bar graph of means of individual densitometric analysis of immunoblots of proportion of 4E-BP1 in the {gamma}-phosphorylated form. Values are means ± SE; n = 5. *P < 0.05 vs. control.

 
mTOR is a proline-directed serine/threonine protein kinase and is reported to be a common intermediate involved in mRNA translation control produced by growth factors and amino acids (for review see Ref. 14). mTOR is believed to be the upstream kinase responsible for phosphorylating 4E-BP1 (for review see Ref. 24) and S6K1 (6). Phosphorylation of mTOR on Ser2448 and Ser2481 can be used to monitor the activity of mTOR (35). Therefore, we used phosphospecific antibodies as a measure of mTOR autokinase activity after acute alcohol intoxication to examine the phosphorylation of mTOR. Acute alcohol intoxication significantly decreased phosphorylation of mTOR on Ser2448 and Ser2481 by ~50% (Fig. 5). Alcohol did not alter the myocardial content of total mTOR in hearts compared with saline-injected animals (data not shown).



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  		Fig. 5. Effect of acute alcohol intoxication on the extent of mammalian target of rapamycin (mTOR) phosphorylation in cardiac muscle. Equal amounts of protein in homogenates from hearts described in Fig. 1 were immunoblotted with antibodies specific for mTOR phosphorylated on Ser2448 (top) and Ser2481 (bottom). Blots were stripped and immunoblotted with an antibody that recognizes phosphorylated and nonphosphorylated forms of mTOR. Representative Western immunoblots of mTOR phosphorylated on Ser2448 and Ser2481 from cardiac muscle homogenates are shown above bar graphs of means of individual densitometric analysis of immunoblots of mTOR phosphorylation at Ser2448 and Ser2481 in heart homogenates corrected for total amount of mTOR. Values are means ± SE; n = 7–9. *Significantly different from control: P < 0.05 (top) and P < 0.001 (bottom).

 
PKB directly phosphorylates mTOR on Ser2448 (33, 46). Therefore, PKB represents a potential upstream effector in the mTOR-dependent pathway responsible for the alterations in phosphorylation of mTOR after alcohol intoxication in cardiac muscle. Therefore, we examined whether acute alcohol intoxication modulates phosphorylation of PKB, a process associated with activation of PKB (1). The extent of phosphorylation of PKB on Thr308 [ratio of (Thr308) phosphorylated PKB to total PKB = 7.0 ± 1.5 and 9.0 ± 1.1 for control and alcohol, respectively] or Ser473 (9.0 ± 1.8 and 12.0 ± 1.8 for control and alcohol, respectively) was not significantly affected after injection of alcohol. On the basis of these observations, the diminished mTOR phosphorylation would not appear to result from altered PKB activation secondary to changes in PKB phosphorylation.

Because alcohol intoxication diminishes mTOR phosphorylation, we investigated the phosphorylation state of S6K1, another downstream target of mTOR, in hearts after alcohol intoxication. S6K1 is activated by multisite phosphorylation, which results in phosphorylated forms exhibiting retarded electrophoretic mobility when subjected to SDS-PAGE (9, 20). Analysis of multisite phosphorylation of S6K1 indicates that its activity is associated with phosphorylation of Thr389 (51, 52). Acute alcohol intoxication caused a net decrease in S6K1 phosphorylation on Thr389 (Fig. 6). The amount of S6K1 phosphorylated on Thr389 decreased 36% in hearts from animals injected with ethanol compared with controls (P < 0.05).



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  		Fig. 6. Effect of acute alcohol intoxication on phosphorylation status of S6 kinase 1 (S6K1) in cardiac muscle. Equal amounts of protein in homogenates from hearts described in Fig. 1 were immunoblotted with antibodies specific for the phosphorylated form of S6K1 (Thr389). Blots were stripped and immunoblotted with an antibody that recognizes phosphorylated and nonphosphorylated forms of S6K1. Representative Western immunoblots of S6K1 phosphorylated on Thr389 from cardiac muscle homogenates are shown above bar graphs of means of individual densitometric analysis of immunoblots of S6K1 phosphorylation on Thr389 corrected for total amount of S6K1 in homogenates of cardiac muscle. Values are means ± SE; n = 7–9. *P < 0.05 vs. control.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this report, we further examined the possible mechanisms responsible for the alcohol-induced inhibition of peptide chain initiation through alteration of the amount and/or activity of distinct initiation factors. Our previous report indicated that acute alcohol intoxication did not alter 1) the total amount of eIF2-{alpha} protein, 2) the extent of phosphorylation of eIF2-{alpha}, 3) the total amount of eIF2B-{epsilon} protein, or 4) eIF2B activity compared with controls. Collectively, changes in the eIF2/eIF2B system are unlikely to explain the alcohol-induced decrease in peptide chain initiation and protein synthesis in the heart after acute alcohol intoxication (23).

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, can also control the overall rate of translation initiation after alcohol administration (23, 24, 50). One of the subunits, eIF4E, binds the 7-methyl-GTP cap structure at the 5'-end of many eukaryotic mRNAs to form an eIF4E·mRNA complex (40, 43). During translation initiation, the eIF4E·mRNA complex binds to eIF4G and eIF4A to form the active eIF4F complex (40, 43, 47). During translation initiation, mRNA binds directly to eIF4E already associated with 40S ribosomes or to free eIF4E, with subsequent binding of the mRNA·eIF4E·eIF4G complex to the ribosome to allow elongation to proceed (40, 43, 47). With either scenario, a decreased amount of eIF4E associated with eIF4G would diminish the association of mRNA with ribosomes, thereby limiting rates of protein synthesis. Because translation of mRNAs in eukaryotic cells is heavily dependent on 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. Alcohol ingestion reduces the amount of eIF4G bound to eIF4E, and this derangement may influence the extent of mRNA translation initiation. The mechanisms responsible for this observation are beginning to be investigated.

One potential eIF implicated in the regulation of protein synthesis is eIF4G. eIF4G acts as a scaffold protein bringing together mRNA, eIF4E, and eIF4A for binding to the 43S preinitiation complex. Multiple bands observed on immunoblots of eIF4G are consistent with phosphorylated forms of eIF4G. Morley and Traugh (32) reported that increased phosphorylation of eIF4G correlates with accelerated rates of protein synthesis in cell extracts. Furthermore, phosphorylation of eIF4G protein on Ser1108 is associated with enhanced rates of translation (30, 41). Phosphorylation of eIF4G is enhanced by mitogen and serum stimulation and inhibited by rapamycin (30, 41, 42).

In the present investigations, examination of the effects of ethanol on eIF4G phosphorylation serves as a targeted approach for exploring divergent signaling pathways and the subsequent impact on mRNA translation initiation in the heart. If phosphorylation of eIF4G is an important regulator of protein synthesis in cardiac muscle, we should observe a decreased phosphorylation of eIF4G with acute alcohol injection. Instead, the extent of phosphorylation of eIF4G is significantly elevated nearly threefold in hearts from animals injected with alcohol compared with controls in the present set of experiments. The increase in phosphorylation of eIF4G with alcohol is not the result of alterations in the myocardial content of eIF4G inasmuch as the total cellular content of eIF4G in animals injected with alcohol is not different from that in controls (23). Therefore, our data indicate that it is unlikely that changes in phosphorylation of eIF4G are important in causing the reduced formation of the active eIF4E·eIF4G complex after ethanol intoxication. The pathways responsible for increased phosphorylation of eIF4G in hearts from animals injected with alcohol remain unknown.

Alternatively, formation of active eIF4G·eIF4E may be regulated through the availability of eIF4E for binding to eIF4G. The availability of eIF4E to form the active eIF4G·eIF4E complex is controlled through binding to small, acid- and heat-stable proteins, termed 4E-BP1, 4E-BP2, and 4E-BP3, forming an inactive complex. 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·eIF4E complex. When eIF4E is bound to 4E-BP1, eIF4E binds to mRNA but cannot form an active eIF4E·eIF4G complex (12). Consequently, formation of 4E-BP1·eIF4E complexes prevents binding of mRNA to the ribosome. Binding of 4E-BP1 to eIF4E essentially limits cap-dependent mRNA translation by physically sequestering eIF4E into an inactive complex. In the present set of experiments, acute alcohol intoxication caused a ~65% increase in the amount of 4E-BP1 associated with eIF4E in cardiac muscle.

The binding of 4E-BP-1 to eIF4E is regulated through phosphorylation of 4E-BP1 (for review see Refs. 10 and 47). Phosphorylation of 4E-BP1 releases eIF4E from the 4E-BP1·eIF4E complex and allows the eIF4E·mRNA complex to bind to eIF4G and, then, to the 40S ribosome (27, 28). Refeeding of starved rats or insulin treatment in vivo increases phosphorylation of 4E-BP1, causing a dissociation of the 4E-BP1·eIF4E complexes, thereby promoting translation initiation (2, 16–18, 48). Presumably, release of eIF4E from the 4E-BP1·eIF4E complex secondary to increased phosphorylation of 4E-BP1 allows eIF4E to bind to eIF4G and form the active eIF4E·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 (18, 48).

An increased formation of inactive 4E-BP1·eIF4E complexes is measured on an immunoblot as an increase in the amount of 4E-BP1 in the eIF4E immunoprecipitate. 4E-BP1 has at least five potential phosphorylation sites, and the various phosphorylated forms can be resolved into multiple bands by SDS-PAGE, with the most phosphorylated form corresponding to the {gamma}-form. In the present set of experiments, acute alcohol intoxication decreased phosphorylation of 4E-BP1 in the {gamma}-form compared with controls. Thus acute alcohol intoxication appears to limit mRNA translation initiation by enhancing abundance of the 4E-BP1·eIF4E complex secondary to decreasing the proportion of 4E-BP1 in the {gamma}-phosphorylated form.

Phosphorylation of 4E-BP1 is regulated through the mTOR signaling pathway (5, 7, 13). mTOR is a serine/threonine kinase with catalytic domains homologous to those in phosphatidylinostitol 3-hydroxykinase. Direct phosphorylation of 4E-BP1 by mTOR or an association kinase has been demonstrated in vitro (7, 8). Linkage of the FK506 rapamycin-associated protein/mTOR pathway in the heart with changes in phosphorylation of 4E-BP1 would begin to define potential downstream regulators of the responses to alcohol. mTOR undergoes multiple phosphorylations induced not only by itself (autophosphorylation) (35), but also by other cellular kinases, including PKB (33, 35). Phenylephrine-induced stimulation of {alpha}1A-receptor enhances phosphorylation on Ser2481 and mTOR downstream effector molecules, 4E-BP1 and S6K1, in cells in culture (4), indicating a role for Ser2481 phosphorylation in regulation of 4E-BP1 and S6K1 in cells in culture. In the present study, phosphorylation of mTOR on Ser2481 or Ser2448 in cardiac muscle was significantly depressed by ~50% by injection of animals with alcohol compared with controls. Phosphorylation on Ser2481 or Ser2448 is reflective of mTOR kinase activity (33, 35). Decreased phosphorylation on Ser2481 or Ser2448 indicates ethanol limits signaling through mTOR in the heart by decreasing its kinase activity.

FK506 rapamycin-associated protein/mTOR phosphorylates not only 4E-BP1 but also S6K1 (13). Because alcohol intoxication lowered mTOR phosphorylation, we anticipated that acute ethanol intoxication would reduce phosphorylation of S6K1. According to the prevailing model of activation for S6K1, the sites in the autoinhibitory domain (Ser411, Ser418, Thr389, and Ser424) are phosphorylated by an upstream kinase(s), including mTOR (51). These phosphorylation events disrupt the interaction between the COOH- and NH2-terminal domains, thereby permitting S6K1 to unfurl and exposing additional sites in the linker and kinase domains. Subsequently, the Thr389 residue in the linker domain is phosphorylated, and this step has been demonstrated to be necessary for the full and functional activation of S6K1 (52). Acute ethanol administration appreciably decreased phosphorylation of S6K1 (Thr389). The reduced phosphorylation of Thr389 is consistent with a role of diminished mTOR activity in mediating the effects of ethanol.

Taken together, these results suggest that myocardial mTOR kinase activity is directly or indirectly suppressed by acute alcohol intoxication. The site along the mTOR signaling pathway modulated by alcohol most likely lies upstream of 4E-BP1 and S6K1 at the level of mTOR, rather than being a phosphatase with dual specificity for 4E-BP1 and S6K1. However, alcohol does not appear to modulate PKB, an upstream kinase responsible for phosphorylation of mTOR. Therefore, inactivation of mTOR appears to represent an important regulatory nidus in the observed inhibition of myocardial mRNA translation initiation and, hence, protein synthesis after acute ethanol intoxication. The results of the present study not only confirm the above-mentioned findings but also provide novel insights into the possible mechanism for these alterations in the heart. 1) Acute alcohol intoxication decreased mTOR phosphorylation, and this reduction was independent of a change in the total mTOR content in muscle. 2) Acute alcohol administration enhances eIF4G phosphorylation, in contrast to the alcohol-induced decreases in phosphorylation of mTOR, S6K1, and 4E-BP1. 3) Phosphorylation of eIF4G appears independent of mTOR, inasmuch as alcohol intoxication was associated with an enhanced phosphorylation of eIF4G when mTOR phosphorylation was depressed. The modulators involved in regulation of eIF4G phosphorylation in cardiac muscle after alcohol ingestion remain to be resolved.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Institute on Alcohol Abuse and Alcoholism Grant AA-12814 (T. C. Vary).


   ACKNOWLEDGMENTS
 
Dr. Leonard S. Jefferson (Penn State University College of Medicine) kindly provided the antibodies generated to eIF4E used in these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. C. Vary, Dept. of Cellular and Molecular Physiology, Rm. C4710, Penn State Univ. College of Medicine, H166, 500 Univ. Dr., Hershey, PA 17033 (E-mail: tvary{at}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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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