Alcohol-induced decrease in muscle protein synthesis associated with increased binding of mTOR and raptor: Comparable effects in young and mature rats

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Effect of acute alcohol intoxication on the in vivo rate of protein synthesis in skeletal muscle from young and mature rats. Rats were injected intraperitoneally with either ethanol or saline (control) and protein synthesis in the fast-twitch gastrocnemius muscle was determined 2.5 h thereafter by incorporation of [³H]-phenylalanine (Phe) into protein. Two groups of mature rats were used in this study; rats in group 1 received the same amount of alcohol normalized to body weight as young rats (i.e., 75 mmol/kg), while rats in group 2 were injected with alcohol at a dose of 90 mmol/kg. The approximate blood alcohol concentration achieved in each of the three groups is presented at the bottom of the graph and the absolute values (means ± SEM) are presented in the text. Values for protein synthesis are means ± SEM where the sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 2

Effect of acute alcohol intoxication on the total amount and phosphorylation of ribosomal protein S6 in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Insert at top: representative Western blots of Ser240/Ser244-phosphorylated (P) S6 and total S6 protein in muscle. Top and middle graphs: densitometric analysis of immunoblots of Ser240/244-phosphorylated S6 and total S6, respectively. Bottom graph: ratio of phosphorylated (P) to total S6 protein in muscle. Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 3

Effect of acute alcohol intoxication on 4E-BP1 phosphorylation and the formation of the active eIF4F complex in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Insert above each graph: representative Western blots. Panels A and B: densitometric analysis of immunoblots of γ-phosphorylated 4E-BP1 and total eIF4E in muscle homogenate, respectively, Panels C and D: eIF4E was immunoprecipitated (IP) and the amount of 4EBP1 and eIF4G bound to eIF4E was assessed by immunoblotting (IB). There was no age- or alcohol-induced change in total eIF4E in the immunoprecipitate (data not shown). In panel C, the α- and β-isoforms of 4E-BP1 are indicated. Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 4

Effect of acute alcohol intoxication on the total amount and phosphorylation of eIF4G in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Inset at top: representative Western blots of phosphorylated and total eIF4G. Top and middle graphs: densitometric analysis of immunoblots of Ser1108-phosphorylated eIF4G and total eIF4G, respectively, in muscle. Bottom graph: ratio of phosphorylated (P) to total eIF4G in muscle. Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 5

Effect of acute alcohol intoxication on the binding of mTOR with raptor in skeletal muscle from young and mature rats. Groups are the same as described in Figure 1. Gastrocnemius was collected 2.5 h after administration of alcohol or saline (control). Inset at top: raptor was immunoprecipitated (IP) and the amount of mTOR and raptor determined by immunoblotting (IB). Bottom graph: densitometric analysis of immunoblots of mTOR bound to immunoprecipitated raptor in muscle. There was no difference among the various groups for the amount of immunoprecipitated raptor (mean data not shown). Values (means ± SEM) are expressed relative to the young saline-treated control group. Sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 6

Effect of acute alcohol intoxication on IGF-I concentration and mRNA content in young and mature rats. Inset: representative autoradiographs from a nuclease protection assay (RPA) for IGF-I in liver and gastrocnemius. Graphs A and B: densitometric analysis of RPAs performed on liver and muscle, respectively. Graphs C and D: plasma and muscle IGF-I peptide concentrations were determined by RIA. Groups are the same as described in Figure 1. Values (means ± SEM) where the sample size was 10, 10, 6, 7, 9, and 9 for the six groups, respectively. Values with different letters are significantly different from each other, P < 0.05. Values which share a common letter are not statistically different.

Figure 7

Schematic of possible site for alcohol-induced inhibition of muscle protein synthesis. This paradigm illustrates the central role served by mTOR (mammalian target of rapamycin) at integrating the independent anabolic signals generated by IGF (insulin-like growth factor-I and the amino acid leucine. mTOR is present in a multimeric protein complex termed mTORC1 which consists of itself, raptor, PRAS40 (proline-rich Akt substrate) and GβL (G protein β-subunit-like protein/mLST8). In addition, a second protein complex exists (mTORC2) consisting of mTOR, GβL, rictor, and mSin1 (mammalian stress-activated protein kinase-interacting protein) and this complex is not shown because it does not appear to directly modulate protein synthesis. mTORC1 has kinase activity which phosphorylates both S6K (ribosomal protein S6 kinase)-1 and 4E-BP1 [eukarytoic initiation factor (eIF) 4E-binding protein-1]. The phosphorylation of 4E-BP1 results in the redistribution of eIF4E from an inactive eIF4E-4EBP1 complex to the active eIF4E-eIF4G complex which, along with other proteins, forms the functional eIF4F complex and stimulates mRNA translation. Present data indicates acute alcohol intoxication increases the association of mTOR and raptor suggesting a "closed" inhibitory confirmation which is consistent with the alcohol-induced decrease in 4E-BP1 and S6K1 phosphorylation. Other reports [6] indicate alcohol does not alter phosphorylation of the IGF-I receptor or protein kinase B (PKB; aka Akt), or the formation of the tuberous sclerosis complex (TSC)-1/TSC-2. Although several possible mechanisms have been reported [51,52], the signal transduction pathway used by leucine to stimulate protein synthesis in skeletal muscle is poorly defined but is independent of PKB activation. Solid lines with arrow heads represent stimulation, whereas lines terminating in a perpendicular line represent inhibition. Numerous proteins and cofactors have been omitted from the diagram to simplify and improve clarity.