Alcohol and indinavir adversely affect protein synthesis and phosphorylation of MAPK and mTOR signaling pathways in C2C12 myocytes

Ly Q. Hong-Brown, C. Randell Brown, Danuta S. Huber, Charles H. Lang

Research output: Contribution to journalArticle

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Abstract

Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S] methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

Original languageEnglish (US)
Pages (from-to)1297-1307
Number of pages11
JournalAlcoholism: Clinical and Experimental Research
Volume30
Issue number8
DOIs
StatePublished - Aug 1 2006

Fingerprint

Indinavir
Phosphorylation
Muscle Cells
Alcohols
Proteins
Ethanol
Translational Peptide Chain Elongation
Eukaryotic Initiation Factor-4G
Translational Peptide Chain Initiation
Ribosomal Protein S6
Peptide Elongation Factor 2
Eukaryotic Initiation Factors
Peptide Elongation Factors
p38 Mitogen-Activated Protein Kinases
Methionine
Cysteine
Muscle
Elongation
Skeletal Muscle
Western Blotting

All Science Journal Classification (ASJC) codes

  • Medicine (miscellaneous)
  • Toxicology
  • Psychiatry and Mental health

Cite this

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title = "Alcohol and indinavir adversely affect protein synthesis and phosphorylation of MAPK and mTOR signaling pathways in C2C12 myocytes",
abstract = "Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S] methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22{\%}, respectively, while a 35{\%} decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.",
author = "Hong-Brown, {Ly Q.} and Brown, {C. Randell} and Huber, {Danuta S.} and Lang, {Charles H.}",
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Alcohol and indinavir adversely affect protein synthesis and phosphorylation of MAPK and mTOR signaling pathways in C2C12 myocytes. / Hong-Brown, Ly Q.; Brown, C. Randell; Huber, Danuta S.; Lang, Charles H.

In: Alcoholism: Clinical and Experimental Research, Vol. 30, No. 8, 01.08.2006, p. 1297-1307.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Alcohol and indinavir adversely affect protein synthesis and phosphorylation of MAPK and mTOR signaling pathways in C2C12 myocytes

AU - Hong-Brown, Ly Q.

AU - Brown, C. Randell

AU - Huber, Danuta S.

AU - Lang, Charles H.

PY - 2006/8/1

Y1 - 2006/8/1

N2 - Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S] methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

AB - Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S] methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

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