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

Lang CH, Pruznak AM, Nystrom GJ, Vary TC - Nutr Metab (Lond) (2009)

Bottom Line: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation.Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. clang@psu.edu.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: The phosphorylation of S6K1 and 4E-BP1 is routinely use as surrogate markers of mTOR kinase activity [8]. However, under basal conditions in vivo, there is little constitutive Thr389-phosphorylation of S6K1 which is necessary for full activation of the enzyme. Therefore, we assessed the phosphorylation of rpS6 at Ser240/Ser244, a site specifically phosphorylated by S6K1 and S6K2 [29]. As illustrated in Figure 2 (middle graph), we detected a 30% decrease in the total amount of rpS6 in skeletal muscle of mature compared with young rats. Despite the reduction in total rpS6 in muscle from mature rats, the extent of S6 phosphorylation was not different between young and mature rats under basal conditions (Figure 2, top graph). Moreover, the alcohol-induced decrease in S6 phosphorylation was comparable between mature rats receiving either the low- or high-dose of ethanol. The ratio of the phosphorylated S6 to total S6 protein in muscle showed an alcohol-induced change comparable to that reported for S6 phosphorylation alone (Figure 2, bottom graph). Similar changes in Ser235/Ser236 phosphorylation, which represents an initiating phosphorylation event, were also observed in muscle from young and mature rats (data not shown).


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

Lang CH, Pruznak AM, Nystrom GJ, Vary TC - Nutr Metab (Lond) (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651172&req=5

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.
Mentions: The phosphorylation of S6K1 and 4E-BP1 is routinely use as surrogate markers of mTOR kinase activity [8]. However, under basal conditions in vivo, there is little constitutive Thr389-phosphorylation of S6K1 which is necessary for full activation of the enzyme. Therefore, we assessed the phosphorylation of rpS6 at Ser240/Ser244, a site specifically phosphorylated by S6K1 and S6K2 [29]. As illustrated in Figure 2 (middle graph), we detected a 30% decrease in the total amount of rpS6 in skeletal muscle of mature compared with young rats. Despite the reduction in total rpS6 in muscle from mature rats, the extent of S6 phosphorylation was not different between young and mature rats under basal conditions (Figure 2, top graph). Moreover, the alcohol-induced decrease in S6 phosphorylation was comparable between mature rats receiving either the low- or high-dose of ethanol. The ratio of the phosphorylated S6 to total S6 protein in muscle showed an alcohol-induced change comparable to that reported for S6 phosphorylation alone (Figure 2, bottom graph). Similar changes in Ser235/Ser236 phosphorylation, which represents an initiating phosphorylation event, were also observed in muscle from young and mature rats (data not shown).

Bottom Line: Acute alcohol (EtOH) intoxication decreases muscle protein synthesis via inhibition of mTOR-dependent translation initiation.Furthermore, some in vivo-produced effects of EtOH vary in an age-dependent manner.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. clang@psu.edu.

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.

No MeSH data available.


Related in: MedlinePlus