Alcohol impairs insulin and IGF-I stimulation of S6K1 but not 4E-BP1 in skeletal muscle

Vinayshree Kumar, Robert A. Frost, and Charles H. Lang

Departments of Cellular and Molecular Physiology and Surgery, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033


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The present study determined whether acute alcohol (ethanol; EtOH) intoxication in rats impaired components of the insulin- and IGF-I-signaling pathway in skeletal muscle. Rats were administered EtOH, and 2.5 h thereafter either insulin, IGF-I, or saline was injected and the gastrocnemius removed. EtOH did not alter the total amount or tyrosine phosphorylation of the insulin receptor, IGF-I receptor, insulin receptor substrate (IRS)-1, or protein kinase B (PKB)/Akt under basal or hormone-stimulated conditions. In contrast, the ability of insulin or IGF-I to phosphorylate T389 and T421/S424 on S6K-1 was markedly diminished by EtOH, and these changes were associated with a reduction in the phosphorylation of the ribosomal protein S6. Under basal conditions, EtOH altered the distribution of eukaryotic initiation factor (eIF)4E, as evidenced by a decreased amount of active eIF4E  · eIF4G complex, an increased amount of inactive eIF4E  · 4E-binding protein (BP)1 complex, and decreased 4E-BP1 phosphorylation. In contrast, EtOH did not impair the ability of either hormone to reverse the changes in eIF4E distribution or 4E-BP1 phosphorylation. Pretreatment with a glucocorticoid receptor antagonist was unable to attenuate either the basal EtOH-induced changes in eIF4E distribution or the impaired ability of IGF-I to stimulate S6K1 and S6 phosphorylation. Hence, acute alcohol intoxication alters selected aspects of translational control under both basal and anabolic hormone-stimulated conditions in skeletal muscle in a glucocorticoid-independent manner.

ethanol; p70 S6; eukaryotic initiation factor 4E; eukaryotic initiation factor 4G; gastrocnemius; rat


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ALCOHOL MUSCLE DISEASE or alcoholic myopathy affects as many as 50-60% of all chronic alcoholics and is one of the most prevalent skeletal muscle disorders (25, 28). The myopathy is characterized by a loss of up to 25% of the skeletal muscle mass. Because skeletal muscle mass comprises ~40% of the whole body mass, this loss in alcoholics is associated with a significant increase in morbidity and mortality. The type II muscle fibers (fast-twitch, anaerobic) are preferentially affected in this disease, whereas the type I fibers (slow-twitch, aerobic) are relatively spared (25, 26). The myopathy is not limited to chronic alcoholics but is also seen after acute intoxication (15, 27). The loss in muscle mass undoubtedly results from disturbances in muscle protein metabolism. The role of protein degradation in the etiology of this myopathy has yet to be completely defined and has yielded conflicting data (16). In contrast, the role of protein synthesis as a mechanism for the development of myopathy has been more clearly defined. Studies from our laboratory (17, 18) as well as others (25-27) have demonstrated a decreased rate of skeletal muscle protein synthesis in response to both chronic alcohol feeding and after acute alcohol intoxication.

Protein synthesis is a multistep, highly regulated process that includes amino acid transport, signal transduction events, transcription, and translation. The actual process of translation involves the association of the 40S and the 60S ribosomal subunits, messenger RNA (mRNA), initiator methionyl-tRNA (met-tRNA<UP><SUB>i</SUB><SUP>met</SUP></UP>), other amino acyl-tRNAs, cofactors (i.e., GTP and ATP), and protein cofactors, collectively known as eukaryotic initiation factors (eIF), eukaryotic 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 highly regulated stages referred to as initiation, elongation, and termination (20). Regulatory control of the process is most often exerted at the level of peptide chain initiation. A critical point of translational control of protein synthesis involves the recruitment of the 43S preinitiation complex to the mRNA, which is mediated by the eIF4F complex (23). The eIF4F complex is a heterotrimeric factor, with each subunit having different functions. One of the subunits, eIF4E, is the least abundant of all initiation factors and in most conditions is considered to be rate limiting in the binding of mRNA to ribosomes (4). eIF4E binds to the mRNA cap structure present at the 5' end of all nuclear-transcribed mRNAs to form an eIF4E  · mRNA complex. During translation initiation, the eIF4E  · mRNA complex binds to eIF4G and eIF4A to form the active eIF4F complex and thereby allows translation to proceed. The binding of eIF4E to eIF4G is controlled in part by the cap-dependent translational repressor protein 4E-binding protein (BP)1. Binding of 4E-BP1 to eIF4E limits the amount of eIF4E available to form the active eIF4F complex (8). This binding of 4E-BP1 to eIF4E in turn is regulated by the phosphorylation status of 4E-BP1 (24). Previous studies have demonstrated that both chronic alcohol consumption and acute intoxication lead to a shift in the equilibrium between eIF4F and its subunits in skeletal muscle, as evidenced by an increased amount of the inactive 4E-BP1  · eIF4E complex and a decreased amount of the active eIF4E  · eIF4G complex under basal conditions (15, 18).

Insulin and insulin-like growth factor (IGF)-I are hormones that are critical for the development of muscle mass and the accretion of lean body mass. Moreover, both hormones appear capable of increasing protein synthesis via changes in the rate of peptide chain initiation (14, 24, 40). Hence, a decrease in the plasma concentration or an impairment in the responsiveness of tissues to these anabolic hormones may mediate, at least in part, the alcohol-induced effects on muscle protein balance. In this regard, previous studies report that the prevailing insulin concentration is normal or even slightly elevated in alcohol-treated rats (15). However, the presence of in vivo insulin resistance, as evidence by the decreased ability of insulin to stimulate muscle glucose uptake, has been observed in response to alcohol administration (38). In contrast, the circulating or tissue concentration of IGF-I appears to be reduced by alcohol (15, 18), but there are no studies in which IGF-I action in skeletal muscle has been assessed in alcohol-treated subjects. Therefore, the purpose of the present study was to determine whether acute alcohol intoxication altered the signal transduction pathways used by insulin and IGF-I to stimulate protein synthesis in skeletal muscle and thereby determine the biochemical loci by which ethanol may produce the observed myopathy. Although insulin and IGF-I have receptors with similar heterotetrameric structure and share many common signaling intermediates (2), the actions of both insulin and IGF-I were assessed separately because these hormones have diverse biological effects, and the responsiveness of animals toward them differs in other catabolic conditions (12, 41). In addition, because acute alcohol intoxication increases the plasma corticosterone concentration in rats (33), and because an elevation in glucocorticoids is a negative regulator of muscle protein synthesis (35-37), the role of endogenous glucocorticoids in mediating the alcohol-induced changes in basal and hormone-stimulated changes in signal transduction was also assessed.


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Experimental protocol. Pathogen-free male Sprague-Dawley rats (200 ± 10 g; Charles River Breeding Laboratories, Cambridge, MA) were used in all studies. The rats were housed in a controlled environment and provided commercial laboratory food and water ad libitum for at least 1 wk before the start of the study. On the day of the study, overnight-fasted rats were divided randomly into two groups (12-16 animals/group); the alcohol-treated group was injected intraperitoneally with ethanol (75 mmol/kg body wt; 20% wt/vol in saline), and the control group was injected intraperitoneally with an equal volume of 0.9% saline. This dose of ethanol was selected because it has been previously reported to significantly decrease muscle protein synthesis (15, 26). Rats were anesthetized with an intraperitoneal injection of pentobarbitol sodium 2.5 h after injection of ethanol or saline, and a midline laparotomy was performed to expose the inferior vena cava. Rats in both groups were injected intravenously with either insulin (10 U/kg body wt), IGF-I (25 mmol/kg body wt), or an equivalent volume of isotonic saline. The insulin dose was selected on the basis of previous studies (5) and on data from a preliminary study in our laboratory that demonstrated a robust stimulation of the insulin receptor and nominal stimulation of the IGF-I receptor with this dose of insulin. The dose of IGF-I was selected after a preliminary dose-response study in our laboratory detected negligible insulin receptor phosphorylation compared with potent IGF-I receptor phosphorylation at this particular dose. After hormone injection, gastrocnemius and blood samples were collected at either 2 or 20 min depending on the particular part of the signaling pathway that was to be analyzed. At the time the rats were killed, the blood alcohol concentration (Analox Instruments, Lunenburg, MA) averaged 322 ± 54 mg/dl in ethanol-treated rats and was undetectable in control rats.

Previous studies have demonstrated that acute alcohol intoxication increases the circulating corticosterone concentration severalfold in rats (33). Therefore, a second study was performed to determine the role of glucocorticoids in mediating the alcohol-induced changes in basal and IGF-I-stimulated signal transduction. Rats were pretreated with the glucocorticoid receptor antagonist RU-486 (Mifepristone; Sigma, St. Louis, MO). RU-486 was dissolved in 95% ethanol to obtain a stock solution of 16 mg/ml. The stock solution was warmed and stirred vigorously while an equal volume of sterile saline was slowly added. Rats were injected intraperitoneally with RU-486 (20 mg/kg) or an equal volume (0.25 ml/100 g) of vehicle 30 min before the injection of ethanol. The dose of ethanol contained in the vehicle did not result in a detectable presence of alcohol in the blood at the time of death nor did it alter various indexes of translation initiation or insulin/IGF-I signaling compared with naive control rats (data not shown). RU-486 is an antiprogestin with antiglucocorticoid properties. RU-486 has a high affinity for cytosolic type II glucocorticoid receptors in various target tissues and exhibits little agonist activity (19). The dose of RU-486 used in the present study attenuates the glucocorticoid-induced increase in muscle catabolism and ameliorates endotoxin- and cytokine-induced changes in the IGF system (17, 44).

All experiments were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine and adhered to the National Institutes of Health (NIH) guidelines for the use of experimental animals.

Reagents. The anti-insulin receptor and anti-insulin receptor substrate (IRS)-1 receptor antibodies were from Upstate Biotechnology (Waltham, MA; nos. 06-492 and 06-526, respectively) or from Santa Cruz Biotechnology (Santa Cruz, CA; nos. 711 and 7200, respectively) and were found to yield comparable results. The anti-IGF-I receptor antibody (no. 713), anti-phosphotyrosine antibody (no. 7202), anti-S6K1 (70-kDa ribosomal protein S6 kinase) antibody (no. 230), anti-phospho-S6K1 (S411; no. 8416) antibody, anti-extracellular signal-regulated kinase (ERK)2 (no. 153), and anti- phosphatidylinositol (PI) 3-kinase antibody (no. 423) were from Santa Cruz. The anti-phospho-S6K1 (T389; no. 9205), anti-phospho-S6K1 (T421/S424; no. 9204), anti-protein kinase B (PKB; no. 9272), anti-phospho-PKB (T308; no. 9275), anti-phospho-PKB (S473; no. 9276), and anti-phospho-ERK1/2 (T202/Tyr204; no. 9106) were from Cell Signaling Technology (Beverly, MA). Anti-phospho-S6 antibody was a generous gift from Dr. Morris Birnbaum (Univ. of Pennsylvania).

Immunoprecipitation and Western blotting. Early components of the signaling pathway were analyzed on muscle obtained 2 min after stimulation with insulin or IGF-I essentially as previously described (5). Briefly, a portion of fresh gastrocnemius was homogenized in a 1:5 ratio of ice-cold homogenization buffer [20 mM HEPES, pH 7.4, 200 mM sodium vanadate, 10 mM sodium pyrophosphate, 160 mM NaF, 8 mM EDTA, 35 mM phenylarsine oxide, 2.5 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and EDTA-free protease inhibitor tablets] by use of a Polytron homogenizer. The remainder of the muscle was freeze-clamped and stored at -70°C. The homogenates were left on ice for 1 h and then centrifuged at 10,000 g for 30 min. The antigens of interest were immunoprecipitated from the homogenates with an excess of precipitating phosphotyrosine (PY) antibody by rotating the sample at 4°C for 12 h. Antibody-antigen complexes were captured with the use of biomagnetic beads (BioMag; Polysciences, Warrington, PA). The samples were placed on a magnetic rack and washed twice with low-salt buffer (20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, and 0.1% beta -mercaptoethanol) and once with high-salt buffer (50 mM Tris · HCl, pH 7.4, 500 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 0.04% beta -mercaptoethanol). After thorough aspiration, the beads were mixed with 1× sample gel buffer (25% of 0.5 M Tris, pH 6.8, 25% glycerol, 25% of a 10% SDS solution, 2.5% beta -mercaptoethanol, 5% of a 4% solution of bromphenol blue, and 17.5% distilled water) and boiled for 5 min. After cooling to room temperature, the tubes were again placed on the magnetic racks, and the samples were removed to a fresh set of microcentrifuge tubes, leaving the beads behind. Immunoprecipitates were electrophoresed on denaturing polyacrylamide gels (6.25-7.5%) and electrophoretically transferred to nitrocellulose. The resulting blots were blocked with 5% nonfat dry milk for 1.5 h and incubated with antibodies against either the insulin receptor, IGF-I receptor, or IRS-1. The blots were washed with Tris-buffered saline (TBS)-T (1× TBS including 0.1% Tween-20) and incubated with secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit) at room temperature for 2 h. The blots were developed with enhanced chemiluminescence (ECL) Western blotting reagents as per the manufacturer's (Amersham) instructions. The blots were exposed to X-ray film in a cassette equipped with a DuPont Lightning Plus intensifying screen. After development, the film was scanned (Microtek ScanMaker IV) and analyzed using NIH Image 1.6 software.

For analyzing changes in PKB phosphorylation, the 10,000-rpm supernatants were mixed with 2× Laemmli sample gel buffer including 0.7% DTT in a 1:1 ratio and boiled for 5 min. After cooling to room temperature, the samples were separated electrophoretically on 10-12.5% polyacrylamide gel, and the proteins were transferred to nitrocellulose membranes. The blots were incubated with either total PKB or phospho-specific PKB (T308 and S473). The blots were developed as described above.

More distal components of the signaling pathway were analyzed on muscle obtained 20 min after injection of insulin or IGF-I. The tissue preparation was essentially the same as described previously (15, 18). Briefly, fresh tissue homogenates were prepared in a 1:7 ratio of ice-cold homogenization buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM beta -glycerophosphate, 1 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, and 0.5 mM sodium vanadate) with a Polytron homogenizer and centrifuged at 10,000 g for 10 min. The supernatant was aliquoted into microcentrifuge tubes, and 2× sample buffer (2 ml of 0.5 M Tris, pH 6.8, 2 ml of glycerol, 2 ml of 10% SDS, 0.2 ml of beta -mercaptoethanol, 0.4 ml of a 4% solution of bromphenol blue, and 1.4 ml of water to a final volume of 8 ml) was added in a 1:1 ratio. The samples were boiled for 5 min and cooled on ice before being used for Western blot analysis. The samples were subjected to electrophoresis on either a 7.5% polyacrylamide gel for S6K1, a 10% polyacrylamide gel for ERK1 and -2, or a 15% polyacrylamide gel for S6. Proteins were electrophoretically transferred to nitrocellulose membranes. The blots were incubated with either a nonselective antibody to total S6K1 or phospho-specific S6K1 (S411, T389, T421/S424), total ERK, phospho-ERK (T202/Tyr204), or phospho-S6 primary antibodies. The blots were developed as described above. The 4E-BP1-eIF4E and eIF4G-eIF4E complexes were quantified as described above. Briefly, eIF4E was immunoprecipitated from aliquots of 10,000-g supernatants with the use of an anti-eIF4E monoclonal antibody (Drs. L. S. Jefferson and S. R. Kimball; Pennsylvania State College of Medicine, Hershey, PA). The antibody-antigen complex was collected using magnetic beads as described above and subjected to electrophoresis using a 7.5% or a 15% polyacrylamide gel. Proteins were then electrophoretically transferred to a nitrocellulose membrane. The blots were incubated with a mouse anti-human eIF4E antibody, a rabbit anti-rat 4E-BP1 antibody, or a rabbit anti-eIF4G antibody for 1 h at room temperature. The phosphorylated forms of 4E-BP1 were measured after immunoprecipitation of 4E-BP1 from the tissue homogenates after centrifugation at 10,000 g. The various phosphorylated forms of 4E-BP1 were separated by SDS-PAGE and analyzed by protein immunoblotting. The blots were then developed with ECL, and the autoradiographs were scanned for analysis as described above.

Statistical analysis. Experimental data for each condition are summarized as means ± SE. Statistical evaluation of the data was performed using ANOVA followed by a Student-Neuman-Keuls test to determine treatment effect. Differences between the groups were considered significant when P < 0.05. The number of animals in each group is indicated in the figure legends.


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Insulin receptor, IGF-I receptor, and IRS-1 tyrosine phosphorylation. In the present study, we examined the effect of acute ethanol on insulin or IGF-I signaling in rat gastrocnemius under in vivo conditions. There were low-to-undetectable basal levels of insulin and IGF-I receptor tyrosine phosphorylation in saline-injected control rats. Acute alcohol intoxication did not produce detectable changes in basal receptor phosphorylation (Fig. 1, A and C). Injection of insulin or IGF-I stimulated receptor tyrosine phosphorylation, and this increase was not altered by alcohol.


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Fig. 1.   A: effect of acute alcohol (ethanol; EtOH) on insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR). SAL, saline. Top: representative immunoblot of tyrosine-phosphorylated (PY) IR and total IR. Bottom: densitometric analysis of PY IR. B: effect of acute EtOH on insulin-stimulated tyrosine phosphorylation of IR substrate (IRS)-1. Top: representative immunoblot of PY IRS-1 and total IRS-1. Bottom: densitometric analysis of PY IRS-1. C: effect of acute EtOH on IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor. Top: representative immunoblot of PY IGF-I receptor. Bottom: densitometric analysis of PY IGF-I receptor. The amount of total IGF-I receptor was not different between groups (data not shown). D: effect of acute EtOH on IGF-I-stimulated tyrosine phosphorylation of IRS-1. Top: representative immunoblot of PY IRS-1. The amount of total IRS-1 was not different between groups (data not shown). Bottom: densitometric analysis of PY IRS-1. Tissue homogenates were prepared and immunoprecipitated with a phosphotyrosine (PY) antibody. The reverse procedure, immunoprecipitation with either an insulin or IGF-I antibody and then Western blot analysis with the PY antibody, yielded comparable results (data not shown). Immunoprecipitates were separated by SDS-PAGE, and IR, IGF-I receptor, or IRS-1 was detected by Western blot analysis. Data are expressed in arbitrary units (AU). Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).

IRS-1 and its isoforms are adaptor proteins that interact with insulin and IGF-I receptors on their activation, thereby permitting the subsequent docking of other signaling molecules and the propagation of the downstream signal. In control rats under basal conditions, there were low levels of tyrosine-phosphorylated IRS-1 (Fig. 1, B and D). An increased tyrosine phosphorylation of IRS-1 was observed in control rats administered either insulin or IGF-I. Acute alcohol intoxication did not alter either basal or hormone-stimulated tyrosine phosphorylation of IRS-1 in muscle. Finally, alcohol also did not alter the total amount of insulin receptor, IGF-I receptor, or IRS-1 protein in skeletal muscle (Fig. 1 and data not shown).

Akt/PKB phosphorylation. PKB, also referred to as Akt, is a downstream substrate of the insulin and IGF-I signal transduction pathway. PKB is a serine/threonine kinase and is activated by phosphorylation of two critical serine/threonine residues. Several studies have established that phosphoinositide-dependent kinase (PDK)1 is the kinase responsible for the phosphorylation of the T308 residue (39). The identity of the kinase that phosphorylates the S473 site (e.g., PDK2) is, as yet, poorly understood. In the basal state, there was essentially no phosphorylation of the T308 site in either the control or ethanol-treated animals (Fig. 2, A and B). When rats were injected with insulin or IGF-I, there was a marked increase in phosphorylation at this site. However, acute ethanol treatment had no effect on the hormone-stimulated phosphorylation of T308 (Fig. 2, A and B). The phosphorylation of S473 appears to be coordinately regulated with the phosphorylation of T308 in skeletal muscle in response to either insulin or IGF-I (data not shown). Furthermore, alcohol also failed to alter hormone-stimulated phosphorylation of S473 (data not shown). There was no difference in the amount of total PKB protein content between the different treatment groups (Fig. 2).


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Fig. 2.   Effect of acute EtOH on insulin- or IGF-I-stimulated phosphorylation of the T308 residue of PKB/Akt. A: insulin-induced changes. B: changes induced by IGF-I. Top: representative immunoblots of phosphorylated (p) T308 and total PKB/Akt. Bottom: densitometric analysis of p-T308 site probed with phospho-specific antibody as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).

S6K1 phosphorylation. S6K1, another serine/threonine kinase downstream in the insulin/IGF-I signaling cascade, is activated by being phosphorylated on at least seven different serine/threonine residues (3). When the kinase is subjected to SDS-PAGE, it resolves into multiple bands with different electrophoretic mobilities dependent on the extent of phosphorylation at these different serine/threonine sites. In this regard, the most slowly migrating forms represent the heavily phosphorylated and thus highly active form of the kinase (34). There was a basal level of S6K1 phosphorylation in control animals treated with saline (Fig. 3A). Acute ethanol alone increased the mobility of the bands, indicating a relative dephosphorylation of S6K1 (Figs. 3A and 4A). Insulin and IGF-I treatment decreased the mobility of the electrophoretic bands, suggesting an increased phosphorylation of the kinase (Figs. 3A and 4A). In contrast, acute ethanol intoxication largely prevented the hormone-stimulated phosphorylation of total S6K1, as evidenced by the increased electrophoretic mobility of the protein (Figs. 3A and 4A).


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Fig. 3.   Effect of acute EtOH on insulin-stimulated phosphorylation of S6K1. A: a representative immunoblot of total S6K1 phosphorylation. Arrow points to higher phosphorylated forms of S6K1, indicative of its activation by insulin. B: a representative immunoblot for the effect of alcohol and insulin on phosphorylation of the S411 site in S6K1. C: a representative immunoblot for the effect of alcohol and insulin on the phosphorylation of the T389 site of S6K1. D: a representative immunoblot for the effect of alcohol and insulin on the phosphorylation of the T421/S424 site in S6K1. E: densitometric analysis of phosphorylated T389 site of S6K1 probed with a phospho-specific antibody as described in METHODS AND MATERIALS. F: densitometric analysis of phosphorylated T421/S424 site of S6K1 probed with a phospho-specific antibody as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).



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Fig. 4.   Effect of acute EtOH on IGF-I-stimulated phosphorylation of S6K1. A: a representative immunoblot of total S6K1 phosphorylation. B: a representative immunoblot for the effect of alcohol and IGF-I on phosphorylation of the S411 site in S6K1. C: a representative immunoblot for the effect of alcohol and IGF-I on the phosphorylation of the T389 site of S6K1. D: a representative immunoblot for the effect of EtOH and IGF-I on the phosphorylation of the T421/S424 site of S6K1. E: densitometric analysis of phosphorylated T389 site of S6K1 probed with a phospho-specific antibody as described in METHODS AND MATERIALS. F: densitometric analysis of phosphorylated T421/S424 site of S6K1 probed with a phospho-specific antibody as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).

We next examined different phosphorylation sites in S6K1 with the use of phospho-specific antibodies. In muscle from fasted control animals, there was essentially no phosphorylation of the T389 site on S6K1 (Figs. 3C and 4C). In response to stimulation by either insulin or IGF-I, there was a dramatic increase in phosphorylation at this site. Acute ethanol decreased insulin-stimulated phosphorylation at this site by ~65% (Fig. 3C). In addition, alcohol completely prevented T389 phosphorylation by IGF-I (Fig. 4C). Comparable results were obtained when the phosphorylation state of T421/S424 was examined in response to either insulin or IGF-I (Figs. 3D and 4D). In contrast, when the phosphorylation state of the S411 site was probed with a phospho-specific antibody, a very high basal level of phosphorylation was observed in the control animals under basal conditions, and there was no detectable effect of insulin, IGF-I, or ethanol on S411 phosphorylation (Figs. 3B and 4B).

Ribosomal S6 phosphorylation. The phosphorylation status of the ribosomal protein (rp)S6, a physiologically relevant S6K1 substrate, was also determined. rpS6 is a component of the 40S ribosome, and previous studies report that phosphorylation of rpS6 is concomitant with an increase in protein synthesis (13). In the present study, rpS6 exhibited a basal level of phosphorylation in muscle from control animals (Fig. 5). Acute alcohol intoxication in the basal state resulted in an ~30% decrease in rpS6 phosphorylation. The administration of insulin or IGF-I significantly increased rpS6 phosphorylation in muscle from control rats (Fig. 5, A and B, respectively). Injection of ethanol attenuated the phosphorylation of rpS6 by IGF-I and, to a lesser extent, by insulin.


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Fig. 5.   Effect of acute EtOH on the stimulation of ribosomal protein (rp)S6 phosphorylation by insulin (A) and IGF-I (B). Top: representative immunoblots of phosphorylated rpS6. Bottom: densitometric analysis of phosphorylated rpS6 probed with a phospho-specific antibody as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).

ERK phosphorylation. Previous studies have shown MAP kinase to be a physiological downstream member of the insulin/IGF-I-signaling pathway, and it is believed to lie on a different arm from the PI 3-kinase/PKB/S6K1 pathway (1). Growth factors can facilitate peptide chain initiation by the phosphorylation of eIF4E, which is believed to be mediated by MAP kinase-integrated kinase (Mnk)-1 (29). There was minimal phosphorylation of ERK1 and -2 in control animals under basal conditions (data not shown). A significant increase in ERK phosphorylation was observed in response to insulin (3.7 ± 0.2-fold) or IGF-I (5.2 ± 2.0-fold) in control animals. Acute ethanol did not significantly alter the insulin-induced increased in phosphorylated ERK (3.1 ± 0.5-fold) but attenuated the increase observed in muscle in response to IGF-I (2.0 ± 0.1-fold). There was no difference in the total MAP kinase protein for any treatment (data not shown).

eIF. The formation of a functional eIF4F complex has been identified to be an important positive regulator of mRNA cap-dependent translation initiation (20). The eIF4F complex is composed of eIF4E, eIF4G, and eIF4A, with the availability of eIF4E in this complex believed to be limiting in skeletal muscle (30). Under basal, nonstimulated conditions, eIF4E is bound to the hypophosphorylated form of 4E-BP1. Mitogen stimulation leads to increased phosphorylation of 4E-BP1 on multiple sites, resulting in the dissociation of 4E-BP1 from eIF4E and allowing binding of eIF4G to free eIF4E. Although in the present study acute alcohol intoxication did not significantly alter the total content of eIF4E in skeletal muscle, alcohol did alter the distribution of eIF4E under basal non-hormone-stimulated conditions. Alcohol decreased the amount of eIF4E bound to eIF4G by ~60% (Figs. 6A and 7A) and increased the amount of eIF4E bound to 4E-BP1 by 75-125% (Figs. 6B and 7B). These changes were associated with an ~40% decrease in the amount of the gamma -, or hyperphosphorylated, isoform of 4E-BP1 (Figs. 6C and 7C). In muscle from control rats, insulin increased the amount of the active eIF4E  · eIF4G complex (2-fold), decreased the amount of the inactive eIF4E  · 4E-BP1 complex (~85%), and increased the gamma -phosphorylated form of 4E-BP1 (2.6-fold) (Fig. 6). Comparable changes in eIF4E distribution and 4E-BP1 phosphorylation were observed in control rats injected with IGF-I (Fig. 7). In general, the ability of either insulin or IGF-I to reverse the alcohol-induced changes in eIF4E distribution and 4E-BP1 phosphorylation was not different from that seen in control animals. The only exception was that IGF-I failed to decrease the amount of the 4E-BP1  · eIF4E complex in alcohol-treated rats to the same extent as that seen in control animals (Fig. 7B).


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Fig. 6.   Effect of acute EtOH on the distribution of eIF4E and phosphorylation status of 4E-BP1 under basal and insulin-stimulated conditions. A: amount of eIF4G bound to eIF4E in the immunoprecipitate. B: amount of 4E-BP1 bound to eIF4E in the immunoprecipitate. C: amount of 4E-BP1 in the hyperphosphorylated gamma -isoform. Left: representative immunoblots. Right: densitometric analysis of immunoblots as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. eIF, eukaryotic initiation factors; BP, binding protein. Means with different letters are statistically different from each other (P < 0.05).



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Fig. 7.   Effect of acute EtOH on the distribution of eIF4E and phosphorylation status of 4E-BP1 under basal and IGF-I-stimulated conditions. A: amount of eIF4G bound to eIF4E in the immunoprecipitate. B: amount of 4E-BP1 bound to eIF4E in the immunoprecipitate. C: amount of 4E-BP1 in the hyperphosphorylated gamma -isoform. Left: representative immunoblots. Right: densitometric analysis of immunoblots as described in METHODS AND MATERIALS. Values are means ± SE; n = 6-8/group. Means with different letters are statistically different from each other (P < 0.05).

Role of endogenous glucocorticoids. Figure 8 illustrates that, under basal conditions, the alcohol-induced decrease in eIF4E  · eIF4G, the increase in eIF4E  · 4E-BP1, and the decrease in the gamma -phosphorylated form of 4E-BP1 were not altered by pretreatment of rats with RU-486. Figure 9 illustrates that the ability of alcohol to prevent or attenuate the IGF-I-induced increase in S6K1 and rpS6 phosphorylation also was not altered by pretreatment with RU-486.


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Fig. 8.   Effect of RU-486 on EtOH-induced changes in basal eIF4E distribution and 4E-BP1 phosphorylation in skeletal muscle. Rats were pretreated with RU-486 30 min before administration of EtOH and killed 2.5 h thereafter, as described in METHODS AND MATERIALS. Values are means ± SE; n = 5-6/group. Means with different letters are statistically different from each other (P < 0.05).



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Fig. 9.   Effect of RU-486 on EtOH-induced changes in the ability of IGF-I to stimulate the phosphorylation of S6K1 and the rpS6 in skeletal muscle. Rats were pretreated with RU-486 30 min before administration of EtOH and killed 2.5 h thereafter, as described in METHODS AND MATERIALS. Values are means ± SE; n = 5-6/group. Means with different letters are statistically different from each other (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Excessive alcohol consumption leads to the development of skeletal muscle myopathy that is due, at least in part, to a reduction in protein synthesis (15, 25, 27). The alcohol-induced impairment in muscle protein synthesis, however, is not restricted to chronic alcohol consumption and also has been observed in rats within hours after acute alcohol intoxication (15, 27). Previous work by our laboratory (15) and others (27) indicates that alcohol alters protein synthesis by impairing mRNA translation at the level of peptide chain initiation, which represents the rate-limiting step in translational control. Growth factor-induced activation of receptors coupled to the PI 3-kinase/PKB/mammalian target of rapamycin (mTOR)-signaling pathway stimulates numerous downstream effector molecules that affect specific components of the translational apparatus. Previous results from in vitro studies (10, 11, 31, 32) indicate that ethanol impairs insulin or IGF-I receptor tyrosine phosphorylation and IRS-1 phosphorylation in cultured myocytes and other nonmuscle cells. However, our data indicate that, under in vivo conditions, acute alcohol intoxication does not impair the proximal portion of the cellular signal transduction pathway in response to either insulin or IGF-I. In this regard, alcohol did not significantly alter the amount of insulin or IGF-I receptor in muscle. Moreover, alcohol did not impair the ability of these hormones to stimulate receptor autophosphorylation, IRS-1 phosphorylation, or PKB phosphorylation. For PKB to be fully activated, two specific sites must be phosphorylated, one in the kinase domain (T308) and the other in the COOH-terminal-regulatory region (S473). The kinase responsible for phosphorylation of T308 has been identified as PDK1 (39). Hence, alcohol does not appear to impair PDK1 activity, at least that activity dependent on phosphoinositol 3,4,5-trisphosphate [PI-(3,4,5)-P3]. Likewise, alcohol did not appear to significantly alter the activity of the enzyme, which at this time has not been definitively characterized, that phosphorylates the hydrophobic COOH-terminal S473 residue of PKB.

Distal to PKB, signals are propagated to the translational apparatus using mTOR as an intermediate (7). Several lines of evidence indicate that mTOR, functioning as a protein kinase, is a common upstream activator of both 4E-BP1 and S6K1. Furthermore, 4E-BP1 and S6K1 appear to lie on parallel pathways (34). The enzymatic activity of S6K1 is regulated by phosphorylation and is pivotal for maintenance of normal rates of protein synthesis. S6K1, in turn, phosphorylates the 40S rpS6, thereby regulating the selective translation of mRNAs containing a tract of oligopyrimidines at their 5'-transcriptional start site (e.g., 5'-TOPs) that encode for components of the translational apparatus. The S6K1 protein has several functional domains with multiple serine/threonine phosphorylation sites (3). There is an NH2-terminal domain rich in acidic residues flanked by a serine/threonine catalytic domain containing the T229 residue. The linker region follows the catalytic domain and contains the critical mitogen-induced phosphorylation residues T389 and S371. Finally, the COOH-terminal domain contains at least four important phosphorylation sites, S411, S418, T421, and S424, and this domain is thought to function as an autoinhibitory domain. The full and complete activation of the S6K1 requires a cascade of ordered phosphorylation of these different serine/threonine sites.

According to the prevailing model of activation of S6K1, the sites in the autoinhibitory domain are phosphorylated by an unknown upstream kinase. This disrupts the interaction between the COOH-terminal and NH2-terminal domains, allowing the molecule to unfurl, thereby exposing the sites in the linker and kinase domain. This is followed by phosphorylation of the T389 residue in the linker domain, and this subsequently promotes phosphorylation of the T229 site. Past studies have demonstrated that the full and functional activation of the kinase requires phosphorylation of the sites in the linker region, especially the T389 site (43). The kinase responsible for phosphorylation of T389 has been tentatively identified as mTOR, but not all data are consistent with this interpretation (7). In the present study, we used several different phospho-specific antibodies directed toward the different sites of S6K1 protein to probe for alcohol-induced defects at specific phosphorylation sites. Our results demonstrate a dramatic drop in the ability of insulin and IGF-I to stimulate phosphorylation of the T421/T424 site (autoinhibitory domain) and the T389 site (linker region) in ethanol-treated rats. In contrast, there was a relatively high constitutive level of basal phosphorylation of S411 that was not increased by insulin or IGF-I and not altered by alcohol. Diminished T389 phosphorylation could potentially be due to a decreased upstream kinase activity, increased upstream phosphatase activity, or decreased accessibility of the site to upstream kinase because of decreased phosphorylation of the sites in the autoinhibitory domain, specifically T421/S424. Additional studies will be required to differentiate between the potential mechanisms.

The physiological regulators of S6K1 phosphorylation are not fully understood, and several lines of evidence indicate that the activity of this central kinase can be modulated by a number of upstream effectors (3). Although in vitro studies have demonstrated that ERK can phosphorylate S6K1 (22), other studies using dominant-negative mutants of the RAS/Raf pathway suggest that ERK is neither necessary nor sufficient for S6K1 activation (21). In this regard, acute alcohol intoxication dramatically decreased the ability of IGF-I, but not insulin, to stimulate phosphorylation of ERK1 and -2 in skeletal muscle. Hence, we cannot exclude the possibility that the alcohol-induced impairment in S6K1 phosphorylation in response to IGF-I is in part due to an inhibition of ERK activity. PDK1, which phosphorylates other substrates including PKB, PKCs, and PKA, appears responsible for phosphorylation of the T229 residue of S6K1 (3). Moreover, the activity of PDK1 toward S6K1 is independent of PI-(3,4,5)-P3. Therefore, potentially, the ability of PDK1 to phosphorylate PKB and S6K1 could be differentially regulated. However, PDK1 has not been shown to be an important regulator of T389 or T421/S424 phosphorylation, and therefore an alteration in the activity of this enzyme is an unlikely mediator of the alcohol-induced decrease in S6K1 phosphorylation at these specific sites.

Receptor tyrosine kinase activation of both S6K1 and 4E-BP1 is impaired by rapamycin (7). Additionally, overexpression of a kinase-dead variant of mTOR also blunts the phosphorylation and activity of S6K1 and 4E-BP1 (9). These data and others suggest that mTOR is an important kinase in the regulation of both S6K1 and 4E-BP1. However, acute alcohol intoxication differentially affected insulin and IGF-I stimulation of S6K1 and 4E-BP1 phosphorylation. That is, alcohol markedly decreased hormone-stimulated S6K1 phosphorylation, but 4E-BP1 phosphorylation appeared unimpaired. These data suggest that alcohol-induced inhibition of mTOR activity is also unlikely to be responsible for the alterations in T389 and T421/S424 phosphorylation of S6K1 in skeletal muscle.

Because the S6K1 branch of the signaling pathway has been shown to be important for protein synthesis, we also examined its downstream substrate, rpS6. This protein is an essential component of the protein synthetic machinery, and its importance is evidenced by the abolition of ribosome biogenesis in livers of mice where the S6 gene has been conditionally deleted (42). Moreover, phosphorylation of rpS6 by S6K1 has been determined to have a positive effect on the synthesis of 5'-TOP proteins (13). Our data clearly demonstrate that acute alcohol intoxication decreases the ability of insulin and IGF-I to phosphorylate rpS6. However, this impairment is not as dramatic as that seen for the inhibitory effect of alcohol on phosphorylation of the T389 and T421/S424 sites of S6K1. These data suggest either that only a minimal amount of phosphorylation of S6K1 is sufficient to elicit a substantial kinase activity or that other kinases (e.g., S6K2) are involved in rpS6 phosphorylation, and the activity of these kinases are less impaired by alcohol.

Considerable evidence indicates that the binding of mRNA to the 43S preinitiation complex mediated by the eIF4F complex can control the overall rate of protein initiation (30). This trimeric complex is composed of the cap-binding protein eIF4E, a large scaffolding protein termed eIF4G that directs the translational machinery to the 5' end of the mRNA, and eIF4A that functions as an RNA helicase. A family of translational repressors, referred to as 4E-BPs, regulates eIF4F formation by competing with eIF4G for an overlapping binding site on eIF4E (8). Moreover, the reversible association between the 4E-BPs and eIF4E can be modulated by phosphorylation. That is, hypophosphorylated 4E-BP1 avidly binds eIF4E, whereas the hyperphosphorylation of 4E-BP1 favors the dissolution of the eIF4E  · 4E-BP1 complex (24). Our results indicate that, in the basal (e.g., no hormone stimulation) state, acute alcohol intoxication decreases the hyperphosphorylated gamma -form of 4E-BP1. This reduced phosphorylation of 4E-BP1 was associated with an increased amount of the inactive 4E-BP1  · eIF4E complex and a decreased amount of the active eIF4E  · eIF4G complex. These data confirm previous observations in rats chronically fed an alcohol-containing diet (18) or after acute alcohol intoxication (15).

In control animals, insulin increased the phosphorylation of 4E-BP1, decreased the amount of the eIF4E  · 4E-BP1 complex, and increased the amount of the eIF4E  · eIF4G complex in skeletal muscle. Similar results have been previously reported in vivo, in the isolated perfused hindlimb, and in cultured myocytes, where these changes are associated with proportional changes in muscle protein synthesis (14, 34, 41). In the present study, IGF-I produced comparable changes in eIF4E availability in muscle from control rats. These data are consistent with results from cultured myocytes (37). However, these data differ from those reported by Vary et al. (40) using the isolated perfused hindlimb. In this earlier study, the IGF-induced increase in muscle protein synthesis and translational efficiency was not associated with an increased phosphorylation of 4E-BP1 or a decreased amount of eIF4E bound to 4E-BP1 but was associated with an increased amount of the eIF4E  · eIF4G complex. The reason for this difference in IGF-I action between the two model systems is not known but may be related to the complete absence of IGF-binding proteins or the lack of basal IGF-I levels in the isolated perfused hindlimb preparation. Regardless of the exact mechanism, our data indicate that acute alcohol intoxication does not impair the ability of either insulin or IGF-I to stimulate the phosphorylation of 4E-BP1, compared with the stimulation observed in control rats. Moreover, alcohol also did not impair the ability of either hormone to increase the amount of the active eIF4E  · eIF4G complex. Collectively, these data suggest that, although alcohol markedly impairs the formation of the eIF4F complex under basal conditions, it does not significantly alter the responsiveness of the system to insulin or IGF-I stimulation.

Acute alcohol intoxication in rodents stimulates the hypothalamic-pituitary-adrenal axis, leading to a rapid elevation in the plasma corticosterone concentration (33). Moreover, glucocorticoid excess has been demonstrated to impair muscle protein synthesis by inhibiting peptide chain initiation (35, 36). In this regard, the synthetic glucocorticoid dexamethasone has been shown to dephosphorylate 4E-BP1, thereby strengthening the interaction of this translational repressor with eIF4E and preventing the formation of the active eIF4E  · eIF4G complex (35, 36). Moreover, elevated glucocorticoids also dephosphorylate S6K1 in muscle, which is evidenced by a selective reduction in phosphorylation of the T389 and T421/S424 sites on S6K1 (37). Therefore, we postulated that the alcohol-induced increase in corticosterone might be an important physiological regulator of alcohol-induced decrease in protein synthesis. However, administration of the glucocorticoid receptor antagonist RU-486 failed to prevent or attenuate the alcohol-induced dephosphorylation of either total S6K1 or the gamma -isoform of 4E-BP1 and did not ameliorate the changes in eIF4E distribution in muscle observed under basal conditions. In addition, RU-486 also did not prevent the ability of ethanol to impair IGF-I-stimulated increases in S6K1 and rpS6 phosphorylation. Finally, the mechanism by which exogenously administered glucocorticoids decrease S6K1 and 4E-BP1 phosphorylation also differs from that observed in response to alcohol, in that glucocorticoids impair the ability of IGF-I to stimulate PKB phosphorylation, whereas alcohol does not (37). Hence, whereas excess exogenous glucocorticoids clearly impair translational efficiency and protein synthesis in skeletal muscle, the alcohol-induced defects in insulin and IGF-I signaling appear to be largely independent of elevations in endogenous glucocorticoids.


    ACKNOWLEDGEMENTS

We thank Xiaoli Liu for technical assistance. We also acknowledge Drs. Leonard S. Jefferson and Scot R. Kimball (Pennsylvania State College of Medicine, Hershey, PA) for generously supplying some of the reagents used in this study. Finally, the antibody to phosphorylated S6 was a generous gift of Dr. M. J. Birnbaum (Univ. of Pennsylvania).


    FOOTNOTES

This work was supported in part by National Institute on Alcohol Abuse and Alcoholism Grant AA-11290.

Address for reprint requests and other correspondence: C. H. Lang, Penn State College of Medicine, Cell. Molec. Physiology (H166), Hershey, PA 17033 (E-mail: clang{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.

July 30, 2002;10.1152/ajpendo.00181.2002

Received 29 April 2002; accepted in final form 11 July 2002.


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