Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats
© Lang et al; licensee BioMed Central Ltd. 2009
Received: 01 September 2008
Accepted: 20 January 2009
Published: 20 January 2009
Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation. However, these studies have been performed in relatively young rapidly growing rats in which muscle protein accretion is more sensitive to growth factor and nutrient stimulation. Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner. The hypothesis tested in the present study was that young rats will show a more pronounced decrement in muscle protein synthesis than older mature rats in response to acute EtOH intoxication.
Male F344 rats were studied at approximately 3 (young) or 12 (mature) months of age. Young rats were injected intraperitoneally with 75 mmol/kg of EtOH, and mature rats injected with either 75 or 90 mmol/kg EtOH. Time-matched saline-injected control rats were included for both age groups. Gastrocnemius protein synthesis and the activity of the mTOR pathway were assessed 2.5 h after EtOH using [3H]-labeled phenylalanine and the phosphorylation of various protein factors known to regulate peptide-chain initiation.
Blood alcohol levels (BALs) were lower in mature rats compared to young rats after administration of 75 mmol/kg EtOH (154 ± 23 vs 265 ± 24 mg/dL). However, injection of 90 mmol/kg EtOH in mature rats produced BALs comparable to that of young rats (281 ± 33 mg/dL). EtOH decreased muscle protein synthesis similarly in both young and high-dose EtOH-treated mature rats. The EtOH-induced changes in both groups were associated with a concomitant reduction in 4E-BP1 phosphorylation, and redistribution of eIF4E between the active eIF4E·eIF4G and inactive eIF4E·4EBP1 complex. Moreover, EtOH increased the binding of mTOR with raptor in a manner which appeared to be AMPK- and TSC-independent. In contrast, although muscle protein synthesis was unchanged in mature rats given low-dose EtOH, compared to control values, the phosphorylation of rpS6 and eIF4G was decreased.
These data indicate that muscle protein synthesis is equally sensitive to the inhibitory effects of EtOH in young rapidly growing rats and older mature rats which are growing more slowly, but that mature rats must be given a relatively larger dose of EtOH to achieve the same BAL. Based on the differential response in mature rats to low- and high-dose EtOH, the decreased protein synthesis was associated with a reduction in mTOR activity which was selectively mediated via a reduction in 4E-BP1 phosphorylation and an increase in mTOR·raptor formation.
Acute alcohol intoxication decreases muscle protein synthesis in a dose- and time-dependent manner, and this response is largely if not completely abated 24 h after alcohol administration [1, 2]. This alcohol-induced decrease is independent of the oxidative metabolism of ethanol and cannot be explained by the over production of either glucocorticoids or selected proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1 or IL-6 [1, 3, 4]. However, it is clear that alcohol acutely down regulates translational efficiency which is predominantly mediated by a reduction in peptide-chain initiation . Our previous work indicated this change was independent of altered tyrosine phosphorylation of either the insulin or insulin-like growth factor (IGF)-I receptor, or theronine (Thr)-308 phosphorylation of protein kinase B (PKB; aka Akt) in skeletal muscle . In contrast, our data suggested that the mammalian Target Of Rapamycin (mTOR) played a central role in regulating the alcohol-induced decrease in muscle protein synthesis [1, 6, 7]. The activity of this serine (Ser)/Thr kinase is most often assessed by phosphorylation of its immediate down stream substrates namely ribosomal protein S6 kinase (S6K1)-1 and the eukaryotic initiation factor 4E (eIF-4E) binding protein-1 (4E-BP1) . In this regard, acute alcohol intoxication decreases the phosphorylation of both S6K1 and 4E-BP1 as well as the autophosphorylation of mTOR itself [1, 6, 7]. However, the mechanism by which alcohol impairs mTOR activity is poorly defined.
The constitutive or basal rate of muscle protein synthesis is a dynamic process which undergoes marked changes during the life time of the host [9–11]. Muscle protein synthesis rates are elevated in young, rapidly growth animals and then later decrease and reach a relative new steady-state in animals as they mature. However, there remains some controversy regarding whether muscle protein synthesis decreases even further or may actually increase in a compensatory manner in the aged animal or human . Young animals are also especially sensitive to the anabolic actions of growth factors, such as IGF-I and insulin, as well as nutrient signals, such as the branched-chain amino acid leucine [13–15]. Furthermore, in some aspects, young rats appear unusually sensitive to various effects of alcohol [16–18]. Previous investigations of the effect of acute alcohol intoxication on muscle protein synthesis have used relatively young rapidly growing rats and, therefore, it is unknown whether the catabolic effects of alcohol on muscle are also present in older more mature animals.
The present study tests the hypothesis that young rats, which are more sensitive to changes in the prevailing circulating concentrations of growth factors and leucine, will show a more pronounced decrement in muscle protein synthesis in response to acute alcohol intoxication. Moreover, this more precipitous reduction in young rats will be associated with a fall in the content and/or activity (i.e., phosphorylation) of proteins regulating the initiation phase of mRNA translation. As previous studies reported that acute alcohol intoxication does not alter the eIF2/2B system , which controls the binding of met-tRNAi to the 40S ribosomal subunit to form the 43S preinitiation complex, we therefore focused on elucidating the alcohol-induced changes in a second critical locus of translational regulation involving the binding of the 5'-end of cellular mRNA to the 43S preinitiation complex. In general, this reaction is mediated by the cap-binding protein complex eIF4F which is in turn largely mediated by the kinase activity of mTOR.
Acute alcohol intoxication
Fischer 344/NHsd male rats were obtained from the National Institute on Aging at either 2 months or 11 months of age. Rats were then housed for the next 2–3 weeks at a constant temperature, exposed to a 12:12-h light-dark cycle, and maintained on standard rodent chow (Harlan #2018; Madison, WI) and water ad libitum before experiments were performed. The commercial diet consisted of approximately 18% protein, 6% fat and 3.8% fiber (metabolizable energy 3.3 Kcal/g), and the exact macro- and micro-nutrient composition of the diet is available at http://www.teklad.com/globaldiet/g2018.asp. Based on previously described criteria, these groups are considered to represent young adult (3 months) and mature (12 months) for the F344 strain of rat . The current study was not designed to examine the effect of aging per se which would necessitate using rats 24–36 months of age. Analysis of data from rats of this older age can be complicated by secondary changes in food consumption, activity levels, and other pathologies which develop during ageing and thereby indirectly affect muscle protein synthesis . The F344 rat strain was used because it is one of the best characterized models of aging in rats and circumvents the large increase in adiposity which develops in the Sprague-Dawley rat with age .
Young adult rats were injected intraperitoneally with either ethanol (75 mmol/kg body weight) or an equal volume of 0.9% sterile saline. This ethanol dose was selected because it decreases muscle protein synthesis in Sprague-Dawley rats with body weights between approximately 100–300 g [2, 4, 6, 19]. The protein metabolic effect of ethanol injected intraperitoneal is comparable to an equivalent dose administered by oral gavage . The older mature rats were divided into three groups: group 1 – ethanol administered at a dose of 75 mmol/kg; group 2 – ethanol administered at a dose of 90 mmol/kg; and group 3 – time-matched control rats injected with saline. Two different ethanol doses were used in older mature rats because the blood alcohol concentrations were significantly lower in mature vs young rats when injected with a dose of 75 mmol/kg. Therefore, we included a second group of alcohol-treated mature rats which were injected with a higher dose of ethanol (90 mmol/kg) in order to produce equivalent blood alcohol levels between the young and mature rats. Rats in all groups were fasted overnight prior to the injection of alcohol and food was withheld after alcohol administration. Water was available throughout the experimental protocol.
All experimental protocols involving animals 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 guidelines for the use of experimental animals.
Muscle protein synthesis
In vivo protein synthesis in gastrocnemius was determined 2.5 h after injection of either alcohol or saline using the flooding-dose technique . Briefly, overnight fasted rats were anesthetized with intraperitoneal pentobarbitol (100 mg/kg) and a catheter placed in the carotid artery. Arterial blood was removed for measuring the plasma concentration of ethanol and various hormones, and then a bolus injection of L- [2,3,4,5,6-3H]phenylalanine (Phe; 150 mM, 30 μCi/ml; 1 ml/100 g BW) was injected via the jugular vein. Serial blood samples were drawn at 2, 6 and 10 min after Phe injection for measurement of Phe concentration and radioactivity. Immediately after the final blood sample, the gastrocnemius muscle was excised in its entirety and a portion frozen between aluminum blocks pre-cooled to the temperature of liquid nitrogen and the remaining muscle directly homogenized. Blood was centrifuged and plasma was collected. All tissue and plasma samples were stored at -80°C until analyzed. The frozen muscle was powdered under liquid nitrogen and a portion used to estimate the rate of incorporation of [3H]Phe into protein, exactly as described .
Immunoprecipitation and Western blot analysis
The tissue preparation was the same as previously described by our laboratories [1, 6, 7, 19]. Muscle was homogenized in a 1:5 ratio of ice-cold homogenization buffer (pH 7.4) composed of (in mM): 20 mM HEPES, 2 EGTA, 50 NaF, 100 KCl, 0.2 EDTA, 50 β-glycerophosphate, 1 DTT, 0.1 PMSF, 1 benzamidine, 0.5 sodium vanadate, plus one protease inhibitor cocktail tablet from Roche, and clarified by centrifugation. The samples were subjected to SDS-PAGE and the proteins were electrophoretically transferred to PVDF membranes. The blots were incubated with either primary antibodies (unless otherwise noted from Cell Signaling, Beverly, MA) to total (C-20) and Thr1462-phosphorylated TSC2, total 4E-BP1 (Bethyl Laboratories, Montgomery, TX), and total and phosphorylated (Ser1108) eIF4G, total and phosphorylated-S6 (Ser240/Ser244), total and phosphorylated (Thr172) AMP-activated protein kinase (AMPK), as well as total PRAS40 (proline-rich Akt substrate 40; Biosource, Camarillo, CA), GβL (G protein β-subunit-like protein; mLST8) and raptor. In general, blots were washed with TBS-T (1X TBS including 0.1% Tween-20) and incubated with secondary antibody (horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit IgG) at room temperature. The blots were developed with enhanced chemiluminescence (ECL) Western blotting reagents as per the manufacturer's (Amersham, Piscataway, NJ) 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 National Institutes of Health Image 1.6 software.
The eIF4E·4EBP1 and eIF4E·eIF4G complexes were quantified as described [1, 6, 7, 19]. Briefly, eIF4E was immunoprecipitated from aliquots of supernatants using an anti-eIF4E monoclonal antibody (kindly provided by Drs. Jefferson and Kimball; Hershey, PA). Antibody-antigen complexes were collected using magnetic beads, subjected to SDS-PAGE, and proteins transferred to a PVDF membrane. Blots were incubated with a mouse anti-human eIF4E antibody, rabbit anti-rat 4E-BP1 antibody, or rabbit anti-eIF4G antibody.
To maintain potential protein-protein interactions, fresh muscle was also homogenized in CHAPS buffer (pH 7.5) composed of (in mM): 40 HEPES, 120 NaCl, 1 EDTA, 10 pyrophosphate, 10 β-glycerol phosphate, 50 NaF, 1.5 sodium vanadate, 0.3% CHAPS, and 1 protease inhibitor cocktail tablet. The homogenate was mixed on a platform rocker and clarified by centrifugation. An aliquot of the resulting supernatant was combined with either anti-TSC2, anti-mTOR or anti-raptor antibody and immune complexes isolated with a goat anti-rabbit BioMag IgG (PerSeptive Diagnostics, Boston, MA) beads. The beads were collected, washed with CHAPS buffer, precipitated by centrifugation, and subjected to SDS-PAGE as described above. All blots were then developed with ECL and the autoradiographs were scanned for analysis as described above.
Plasma concentrations of alcohol, glucose, amino acids and hormones
The plasma insulin concentration was measured using a commercial radioimmunoassay (RIA) for rat insulin (Linco Research, St. Charles, MO). Additionally, the plasma concentrations of total IGF-I, estradiol, and testosterone were determined using commercial RIA kits (DSLabs, Webster, TX). The plasma glucose and alcohol concentrations were determined by a rapid analyzer (Analox Instruments, Lunenburg, MA). Finally, the branched-chain amino acid concentrations were determined using reverse-phase HPLC after precolumn derivatization of amino acids with phenylisothiocyanate . The plasma concentrations of glucose, insulin, IGF-I, estradiol, testosterone, branched-chain amino acids and alcohol were determined on blood collected immediately prior to injection of radiolabeled phenylalanine (i.e., time 0). Additionally, insulin and glucose were also determined on the blood sample collected 10 min after injection of phenylalanine. The original homeostasis model assessment (HOMA), defined as the [fasting insulin concentration (μU/ml) × glucose concentration (mmol/L)]/22.5, was used as an index of whole-body insulin resistance, as described by Matthews et al . To better assure that a steady-state was achieved, glucose and insulin concentrations were determined at two time points. Because there was no difference in the glucose or insulin concentrations between the 0 time point and the 10-min time point (data not shown), these data were averaged for each rat and HOMA calculated using this average value. The advantages and disadvantages of HOMA for estimating insulin resistance have been reported .
IGF system components
The concentration of free IGF-I was determined by centrifugal ultrafiltration, as originally described . Briefly, samples were diluted 1:5 with Krebs-Ringer bicarbonate buffer (pH 7.4; with 5% BSA) and prefiltered through a 0.22 μm filter (Millex-GV, Millipore, Molsheim, France) to remove debris. The prefiltered samples were then added to Amicon YMT 30 membranes and MPS-1 supporting devices (Amicon Division, W. R. Grace, Co., Beverly MA) and centrifuged at 1500 rpm at 37°C for 100 min. The ultrafiltrate was collected from 40–100 min of centrifugation and used for the IGF-I RIA.
Primer sequences for the determination of IGF binding proteins (IGFBPs)
Forward 5'-GCA GAA TTC GGC TGC AGA AGC TGT ACC TGG A-3'
Reverse 5'-GCA GGT ACC ATT CGA TTG TGG CCC AGC TGC A-3'
Forward 5'-GCA GAA TTC TGA GCT TGC CGA GAG CCC AGA-3'
Reverse 5'-GCA GGT ACC AGA GCC CAG CTT CTC CAT CCA GA-3'
Forward 5'-GCA GAA TTC GGA GAA CCA TGT GGA CGG AAC CA-3'
Reverse 5'-GCA GGT ACC CCC ACG CTG TCC ATT CAG AGA CA-3'
Forward 5'-GCA GAA TTC CTC CTC CGA GTC TAA GCG GGA GA-3'
Reverse 5'-GCA GGT ACC CAG CGG TAT CTA CTG GCT CTG CA-3'
Forward 5'-GCA GAA TTC ATC ACA GGT GCC TGC AGA AGC A-3'
Reverse 5'-GCA GGT ACC TGG AAG TTG CCG TTG CGG TCA-3'
Forward 5'-GCA GAA TTC CGA GTC ATC CCT GCA CCT GAG A-3'
Reverse 5'-GCA GGT ACC CCA CGT TTG CGG CCA CGA GA-3'
Forward 5'-GCA GAA TTC CGG CCT CTG GTG AAG CCC AA-3'
Reverse 5'-GCA GGT ACC CTT CTC CGC ACC CTG TTG TCG-3'
Forward 5'-GCA GAA TTC CTG GCC AAG GTC ATC CAT GAC A-3'
Reverse 5'-GCA GGT ACC GGG GCC ATC CAC AGT CTT CTG-3'
RNA extraction and RPA
Total RNA was extracted from gastrocnemius and liver using TRI Reagent (Molecular Research Center, Cincinnati, OH) and the mRNA content was determined by RPA. An aliquot (2 μl) of template was prepared using T7 Polymerase with buffer (Fermentas, Hanover, MD), NTPs and tRNA (Sigma-Aldrich), RNaisin and DNase (Promega), and 32P-UTP (Amersham Biosciences, Piscatawy, NJ). Unless otherwise noted, the entire RPA procedure including labeling conditions, component concentrations, sample preparation, and gel electrophoresis was as published (BD Pharmingen, San Diego, CA). Hybridization buffer was 80% formamide and 20% stock buffer (200 mM Pipes, pH 6.4, 2 M NaCl, and 5 mM EDTA). Hybridization proceeded overnight at 56°C in a dry bath incubator (Fisher Scientific, Pittsburgh, PA) without the use of mineral oil. Samples were treated with RNAse A+T1 (Sigma) in 1× RNAse buffer (10 mM Tris-HCl pH 7.5, 5 mM EDTA, and 300 mM NaCl) followed by Proteinase K (Fisher Scientific) in 1× Proteinase K buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 1% Tween-20). Following ethanol precipitation, samples were resuspended in 5 ml of loading buffer (98% formamide (v/v), 0.05% xylene cyanol (w/v), 0.05% bromphenol blue (w/v), and 10 mM EDTA). Polyacrylamide gels were run in an S3S Sequencing System (Owl Separation Systems, Portsmouth, NH), transferred to chromatography paper, and dried (FB GD 45 Gel Dryer, Fisher Scientific). Gels were exposed to a PhosphorImager screen (Molecular Dynamics Inc., Sunnyvale, CA). Data were visualized and analyzed using ImageQuant software (Version 5.2, Molecular Dynamics). Signal densities for mRNAs were analyzed in the linear range and normalized to L32 or GAPDH mRNA, which yield comparable results (data not shown).
Experimental data for each condition are summarized as means ± SE where the number of animals in each treatment group is indicated in the legend to the figure or table. Statistical evaluation of the data was performed using ANOVA followed post-hoc by Student-Neuman-Keuls test when the interaction was significant. Differences between the groups were considered significant when P < 0.05.
Plasma alcohol concentration
The concentration of alcohol in the blood 2.5 h after administration of ethanol averaged 265 ± 24 mg/dL in young rats administered 75 mmol/kg. Mature rats given the same amount of alcohol per kg body weight had blood alcohol levels which were 45% lower (P < 0.05) than young animals (154 ± 23 mg/dL; hereafter referred to as the low-dose group). In contrast, mature rats administered 90 mmol/kg alcohol (hereafter referred to as the high-dose group) had a blood alcohol level which was not different from young rats administered 75 mmol/kg of ethanol (281 ± 33 mg/dL; P = NS).
Body and muscle weights
The body weight of the mature rats was 70% greater than animals in the young group (413 ± 13 g vs 239 ± 9 g; P < 0.05). Furthermore, the weight of the gastrocnemius was also significantly increased by approximately 60% in mature animals (428 ± 18 mg wet weight), compared to young rats (266 ± 6 mg). As a result of these changes, the gastrocnemius-to-body weight ratio was not significantly altered between the two age groups (data not shown), suggesting sarcopenia (e.g., age-associated muscle atrophy) had not yet developed in the mature animals. There was also no difference in either the total protein or total RNA content of the gastrocnemius between age groups (data not shown). Finally, because of the short time frame, acute alcohol intoxication did not alter gastrocnemius weight, protein content or RNA content in either young or mature rats (data not shown).
Muscle protein synthesis
Phosphorylation of ribosomal protein (rp) S6
Phosphorylation of 4E-BP1 and formation of the eIF4F complex
Western blot analysis of tissue homogenates showed no age- or alcohol-induced change in the amount of total eIF4E (Figure 3B). However, the function of eIF4E can also be controlled by its binding to a family of cap-dependent translational repressors, of which 4E-BP1 is the most prominent family member in skeletal muscle. Hyperphosphorylation of 4E-BP1 liberates it from eIF4E and allows binding of eIF4E with eIF4G and the stimulation of protein synthesis. There was no difference in the amount of the eIF4E·4EBP1 complex or the eIF4E·eIF4G complex in gastrocnemius between young and mature rats under basal conditions (Figure 3C and 3D). Acute alcohol intoxication resulted in a redistribution of eIF4E from the active to the inactive complex in young rats and mature rats given the high-dose of alcohol. In contrast, no such redistribution of eIF4E was detected in mature rats given the low-dose of alcohol. These alcohol-induced changes in the distribution of eIF4E were independent of a change in the eIF4E content in the immunoprecipitate (see representative blot, Figure 3C and 3D).
mTOR complex 1
TSC and AMPK
mTOR activity is regulated at least in part by the phosphorylation of TSC2 and/or the dimerization of TSC2 with TSC1, which can be modulated independently by insulin and nutrients . However, by Western blot analysis there was no significant age- or alcohol-induced change in either the total amount of TSC1 and TSC2, or the association of TSC1 with TSC2 (data not shown). Although activation of the energy sensor AMPK decreases protein synthesis in muscle via a mTOR-dependent mechanism , we detected no age- or alcohol-induced change in either the total amount or Thr172-phosphorylated AMPK (data not shown).
While there was no age-dependent change in the plasma total IGF-I concentration under basal conditions, acute alcohol intoxication decreased plasma IGF-I to a similar extent (40–45%) in both young and mature rats given high-dose alcohol (Figure 6C). Mature rats given the lower dose of alcohol had a total IGF-I concentration intermediate between two other groups of mature rats. Finally, the concentration of free IGF-I, which is the biologically active form of the hormone, was assessed in skeletal muscle. Although there was no age-dependent change in the basal concentration of free IGF-I in muscle, the concentration of this anabolic hormone was markedly reduced in both young rats administered alcohol and in mature rats given the high-dose of ethanol (Figure 6D).
Effect of acute alcohol intoxication on liver IGFBP mRNA content in young and mature adult male F344 rats
Mature – Low Dose EtOH*
Mature – High Dose EtOH
1.00 ± 0.08a
1.78 ± 0.22b
0.91 ± 0.17a
1.21 ± 0.19a
0.94 ± 0.09a
2.68 ± 0.26c
1.00 ± 0.08a
0.64 ± 0.05b
0.45 ± 0.08b
0.52 ± 0.05b
0.47 ± 0.06b
0.49 ± 0.09b
1.00 ± 0.11
1.05 ± 0.15
0.87 ± 0.11
0.79 ± 0.09
0.84 ± 0.08
0.78 ± 0.14
1.00 ± 0.07
1.18 ± 0.12
0.90 ± 0.08
0.97 ± 0.07
0.89 ± 0.11
0.95 ± 0.09
1.00 ± 0.09a
0.68 ± 0.07b
1.11 ± 0.11a
1.01 ± 0.11a
1.08 ± 0.09a
0.61 ± 0.07b
Effect of acute alcohol intoxication on muscle IGFBP mRNA content in young and mature adult male F344 rats
Mature – Low Dose EtOH*
Mature – High Dose EtOH
1.00 ± 0.08
0.87 ± 0.11
0.81 ± 0.12
0.83 ± 0.11
0.79 ± 0.11
0.77 ± 0.15
1.00 ± 0.12
0.95 ± 0.12
1.24 ± 0.21
1.18 ± 0.19
1.14 ± 0.12
1.13 ± 0.14
1.00 ± 0.06a
0.66 ± 0.06b
0.84 ± 0.07a
0.85 ± 0.06ab
0.89 ± 0.08a
0.49 ± 0.07b
Plasma hormone and substrate concentrations
Effect of acute alcohol intoxication on plasma hormone and metabolic substrate concentrations in young and mature adult male F344 rats
Mature – Low Dose EtOH*
Mature – High Dose EtOH
1.09 ± .09a
0.84 ± .13a
2.13 ± .25b
2.03 ± .21b
2.12 ± .33b
2.09 ± .24b
2.8 ± 0.2a
1.6 ± 0.2b
2.7 ± 0.2a
1.3 ± 0.2b
2.5 ± 0.3a
1.5 ± 0.3b
2.7 ± 0.5
2.1 ± 0.5
3.5 ± 0.4
2.9 ± 0.4
3.6 ± 1.2
2.7 ± 0.4
7.0 ± 0.5a
11.2 ± 1.1b
8.4 ± 0.5a
8.7 ± 0.4a
8.5 ± 0.5a
10.8 ± 0.8ab
8.3 ± 0.8a
10.3 ± 1.3a
19.6 ± 3.3b
21.6 ± 3.1b
19.6 ± 3.4b
23.7 ± 4.3b
95 ± 6a
137 ± 11b
97 ± 4a
107 ± 10a
95 ± 6a
141 ± 12b
81 ± 10
104 ± 11
85 ± 7
91 ± 11
86 ± 8
116 ± 9
138 ± 12
166 ± 21
127 ± 15
134 ± 12
133 ± 14
155 ± 12
Results from the present study indicate muscle protein synthesis in young adult and mature F344 rats is equally sensitive to the suppressive effect of acute alcohol intoxication. However, this effect was only equivalent when the amount of alcohol administered to mature rats was increased in order to produce a blood alcohol level comparable to that seen in young rats. Under conditions where the young and mature rats were administered the same amount of alcohol (75 mmol/kg), the blood alcohol concentration was 40% lower in the mature rats. The lower concentration of alcohol in the older mature rats is consistent with previous in vivo studies in Fischer 344 rats which reported an increased ethanol elimination in animals naive to alcohol [41, 42]. Therefore, the alcohol-induced decrease in muscle protein synthesis produced by acute intoxication is not limited to rapidly growing animals as long as comparisons between different aged rats are matched to the prevailing blood alcohol level and not the dose of alcohol administered.
The mature rats used in the present study should not be categorized as "aged" animals. We purposefully avoided the use of aged rats (e.g., > 18 months of age) because of the potential for sacropenia and increased susceptibility to alcohol toxicity . In this regard, the muscle weight-to-body weight ratios and the rates of muscle protein synthesis were not different between young and mature rats in our study. Hence, these data suggest that overt sacropenia had not yet developed in mature rats and that muscle protein synthesis, at least after an overnight fast, is relatively unchanged. These data are internally consistent with the amount and phosphorylation (i.e., activation) of several protein factors known to regulate mRNA translation. For example, there was no difference in either the total amount or phosphorylation state for 4E-BP1 and the amount of the active eIF4E·eIF4G complex in muscle from young and mature rats under basal fasted conditions. While we did detect a reduction in the total amount of both rpS6 and eIF4G between young and mature rats, the amount of these proteins phosphorylated remained unchanged (e.g. S6) or was even increased (e.g., eIF4G) in young rats compared with mature animals.
Our data clearly demonstrate acute alcohol intoxication increases the association of mTOR bound to raptor. These findings are consistent with data from other in vitro studies using myocytes where a reduction in protein synthesis produced by amino acid or leucine deprivation was associated with an increase in mTOR·raptor formation . Collectively, these data are supportive of alcohol impairing mTOR kinase activity by promoting a "closed conformation" which has been proposed to render it less active . This alcohol-induced change in mTOR·raptor also appears to be mediated by a mechanism which is AMPK- and TSC-independent, although TSC activity per se still needs to be directly assessed in response to alcohol. The mTORC1 complex also binds PRAS40 and GβL via its interaction with raptor [44, 45]; however, there was no detectable change in the total amount or the binding of these two substrates to raptor in response to alcohol. Finally, a decrease in the intracellular leucine concentration might also contribute to the increased mTOR· raptor association, although this possibility seems less likely because the plasma concentration of leucine was elevated in alcohol-treated rats compared to control animals and alcohol does not increase muscle protein degradation .
The prevailing circulating concentration of various hormones or the ability of tissues to respond to these agents can markedly influence muscle protein synthesis. In this regard, our studies provide evidence supporting the importance or lack there of for several hormones. For example, testosterone is a potent anabolic agent capable of increasing protein synthesis and the accretion of lean body mass . However, because alcohol decreased testosterone to the same extent in all three groups, it seems unlikely that a differential regulation of this anabolic hormone is solely responsible for the alcohol-induced decrease in muscle protein synthesis. Similarly, there was no significant alcohol effect on plasma estradiol in any of the three groups, functionally excluding changes in this hormone as a mediator for the decrease in protein synthesis. Changes in the prevailing plasma concentration of insulin can lead to proportional changes in muscle protein synthesis . However, acute alcohol intoxication did not significantly decrease the circulating insulin concentration in any group. Moreover, we calculated an index of insulin resistance (e.g., HOMA) and found that although the mature rats as a group were insulin resistant compare to young animals, there was no difference between mature rats that received either the low- or high-dose of ethanol. Collectively, these data argue against a change in either insulin concentration and/or insulin action as a causative mechanism for the alcohol-induced decrease in muscle protein synthesis. Finally, there was no difference in the plasma concentrations for the three branched-chain amino acids – leucine, isoleucine and valine – between young and mature rats under basal control conditions. Moreover, acute alcohol intoxication produced a comparable increase in the plasma leucine concentration between young rats and mature animals given the high dose of alcohol, and a similar trend was observed for isoleucine and valine. In addition, acute alcohol intoxication does not appear to alter the plasma concentration of total amino acids . Overall, although these data suggest that an acute reduction in the circulating concentration of amino acids in general and branched-chain amino acids in particular is not responsible for the alcohol-induced decrease in muscle protein synthesis, we cannot exclude the possibility that alcohol impairs translation by decreasing the intracellular leucine concentration.
In contrast, although we cannot exclude the possibility that circulating IGF-I mediates the reduction in muscle protein synthesis in response to alcohol, our data demonstrate a close association between the content of IGF-I mRNA and IGF-I protein in muscle and changes in protein synthesis within the same muscle. This is the first report of the concentration of free bioavailable IGF-I in muscle in response to acute alcohol intoxication. The alcohol-induced decrease in free IGF-I within muscle is likely the result of a reduction in the synthesis of IGF-I by muscle as well as the suspected rise in the plasma concentration of IGFBP-1 which is known to sequester free IGF-I. Moreover, elevations in circulating IGFBP-1 have been shown to decrease muscle protein synthesis under both in vivo and in vitro conditions . Although plasma IGFBP-1 concentrations were not directly assessed in the current study, hepatic IGFBP-1 mRNA content is a reliable surrogate marker for this particular binding protein. Finally, alcohol-induced changes in IGFBP-5 mRNA content in skeletal muscle were also directly proportional to changes in IGF-I and protein synthesis. This decrease in muscle IGFBP-5 is consistent with the reduction seen in several other catabolic conditions with accompanying muscle wasting . Because changes in IGF-I produce proportional changes in IGFBP-5 in cultured myocytes , the observed reduction in IGFBP-5 in response to alcohol may occur secondary to the reduction in muscle IGF-I. Hence, the reduction in muscle IGF-I is not caused by the decrease in IGFBP-5 but is instead the mechanism for the reduction in this particular IGFBP. Overall, the mechanism by which the alcohol-induced decrease in autocrine/paracrine produced IGF-I inhibits muscle protein synthesis remains to be determined. Although previous studies have reported acute alcohol does not alter constitutive IGF-I or insulin receptor tyrosine phosphorylation or Thr-308 phosphorylation of Akt , the kinase activity per se of these proteins has not been directly assessed. Hence, it remains possible that alcohol decreases mRNA translation and protein synthesis by impairing IGF-I signal transduction directed via a TSC-independent mechanism. Alternatively, the alcohol-induced decrease in muscle IGF-I may be associated with but not causally related to the reduction in muscle protein synthesis.
Our data indicate that young and mature adult male rats demonstrate the same reduction in muscle protein synthesis when blood alcohol levels are closely matched but, because of the apparently greater rate of ethanol clearance in adult male rats, this requires mature rats be administered a relatively larger dose of alcohol. The differential response observed in the mature rats to the low- and high-dose alcohol suggests that changes in 4E-BP1 phosphorylation, the distribution of eIF4E between active and inactive eIF4F complexes, and the increased association of mTOR and raptor mediates the alcohol-induced decrease in mRNA translation. These changes in translation and protein synthesis in skeletal muscle in response to acute alcohol intoxication were independent of changes in plasma testosterone, estradiol, insulin, and branched-chain amino acids but were associated with the reduction in free muscle IGF-I peptide. Moreover, this potential cellular mechanism by which alcohol inhibits muscle protein synthesis was seen in both young and adult male rats.
This work was supported by grants awarded from the National Institute on Alcohol Abuse and Alcoholism AA-12814 (TCV) and AA-11290 (CHL). We are also grateful to Ms. Gina Deiter and Daunta Huber for their excellent technical assistance. We gratefully acknowledge Dr. Leonard S. Jefferson for kindly providing the eIF4E antibody used in this study.
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