Acute alcohol intoxication increases REDD1 in skeletal muscle.
BACKGROUND
The mechanism by which acute alcohol (EtOH) intoxication decreases basal muscle protein synthesis via inhibition of the Ser/Thr kinase mammalian target of rapamycin (mTOR) is poorly defined. In this regard, mTOR activity is impaired after over expression of the regulatory protein REDD1. Hence, the present study assessed the ability of REDD1 as a potential mediator of the EtOH-induced decrease in muscle protein synthesis.


METHODS
The effect of acute EtOH intoxication on REDD1 mRNA and protein was determined in striated muscle of rats and mouse myocytes using an RNase protection assay and Western blotting, respectively. Other components of the mTOR signaling pathway were also assessed by immunoblotting. For comparison, REDD1 mRNA/protein was also determined in the muscle of rats chronically fed an alcohol-containing diet for 14 weeks.


RESULTS
Intraperitoneal (IP) injection of EtOH increased gastrocnemius REDD1 mRNA in a dose- and time-dependent manner, and these changes were associated with reciprocal decreases in the phosphorylation of 4E-BP1, which is a surrogate marker for mTOR activity and protein synthesis. No change in REDD1 mRNA was detected in the slow-twitch soleus muscle or heart. Acute EtOH produced comparable increases in muscle REDD1 protein. The EtOH-induced increase in gastrocnemius REDD1 was independent of the route of EtOH administration (oral vs. IP), the nutritional state (fed vs. fasted), gender, and age of the rat. The nonmetabolizable alcohol tert-butanol increased REDD1 and the EtOH-induced increase in REDD1 was not prevented by pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole. In contrast, REDD1 mRNA and protein were not increased in the isolated hindlimb perfused with EtOH or in C2C12 myocytes incubated with EtOH, under conditions previously reported to decrease protein synthesis. Pretreatment with the glucocorticoid receptor antagonist RU486 failed to prevent the EtOH-induced increase in REDD1. Finally, the EtOH-induced increase in REDD1 was not associated with altered formation of the TSC1*TSC2 complex or the phosphorylation of TSC2 which is down stream in the REDD1 stress response pathway. In contradistinction to the changes observed with acute EtOH intoxication, REDD1 mRNA/protein was not changed in gastrocnemius from chronic alcohol-fed rats despite the reduction in 4E-BP1 phosphorylation.


CONCLUSIONS
These data indicate that in fast-twitch skeletal muscle (i) REDD1 mRNA/protein is increased in vivo by acute EtOH intoxication but not in response to chronic alcohol feeding, (ii) elevated REDD1 in response to acute EtOH appears due to the production of an unknown secondary mediator which is not corticosterone, and (iii) the EtOH-induced decrease in protein synthesis can be dissociated from a change in REDD1 suggesting that the induction of this protein is not responsible for the rapid decrease in protein synthesis after acute EtOH administration or for the development of alcoholic myopathy in rats fed an alcohol-containing diet.
Lang
Charles H
CH
Department of Cellular & Molecular Physiology, Penn State University College of Medicine, Hershey, Pennsylvania 17033, USA. clang@psu.edu
Frost
Robert A
RA
Vary
Thomas C
TC
D000428:Alcohol Drinking / Q000378:metabolism; D000435:Alcoholic Intoxication / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D032448:Myoblasts, Skeletal; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C469992:Ddit4 protein, mouse; C000632146:Ddit4 protein, rat; C499526:Eif4ebp1 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C546843:mTOR protein, mouse; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases