Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats


Journal Title (Medline/Pubmed accepted abbreviation): Am. J. Physiol. Endocrinol. Metab.
Year: 2011
Volume: 300
Page numbers: E986-E992
doi (if applicable):

Summary of Background and Research Design

Background:Muscle protein synthesis is regulated by a protein complex called mTORC1, or simply mTOR. When activated, mTOR initiates a large number of downstream events including phosphorylation of a protein called S6K1. Recently, S6K1 was observed to phosphorylate another protein called programmed cell death 4 (PDCD4). When PDCD4 is not phosphorylated, it binds to an mRNA initiation factor eIF4A, thereby inhibiting mRNA translation (i.e. protein synthesis); thus, phosphorylation of PDCD4 by S6K1 will inactivate it. To date, it was unknown if PDCD4 is expressed in skeletal muscle and, if so, if it is regulated by nutrients. (mTOR has shown to be upregulated by leucine and other amino acids.)

Hypothesis:: Phosphorylation (inactivation) of PDCD4 will lead to increased protein synthesis. This will be observed directly by addressing the collowing objectives: 1) Using rats, nutrient intake will be controlled while monitoring PDCD4 expression in skeletal muscle. 2) In an in vitro muscle cell culture, the rate of protein synthesis in the muscle cell will be observed under a more controlled environment.

Subjects::Young male rats

Experimental design:Randomized

Treatments/Protocol:
Rat study:
The rats were assigned to 1 of the following treatment groups: 1) control group, 2) 48 hrs of food deprivation then immediate analysis, 3) 48 hrs of food deprivation, 2 hrs of refeeding, then analysis, 4) 48 hrs of food deprivation, gavage of amino acids spiked with radioactive phenylalanine, then analysis of protein synthesis 30 min-6 hrs later. All rats had access to water at all times. Rats were euthanized after the procedure. Gastrocnemius (calf) muscles were excised and analyzed for quantity and state of phosphorylation of pertinent enzymes.
Cell culture study:Rat L6 myoblasts were starved for 24 hrs. Some cells were harvested after starvation, others were re-fed. mRNA interference was used to “turn down” the expression of PDC4 mRNA. Cells were then fed for 12 hrs with a radioactive amino acid mixture, then harvested and analyzed for radioactive incorporation of phenylalanine into protein.

Summary of research findings:
  • PDCD4 was detected in rat gastrocnemius muscle.
  • Food deprivation caused the expression of PDCD4 to increase about 60% and the majority was kept its unphosphorylated state, thereby suppressing protein synthesis. Refeeding (and a leucine gavage) activated PDCD4 and decreased the amount of it. Therefore, refeeding with food or leucine activates protein synthesis.
  • There was a direct correlation between activation of mTOR, PDCD4, and other downstream events (S6K1, S6) corroborating the link between these signals.
  • Plasma insulin concentration followed the same trend as the activation of protein synthesis.
  • In the in vitro cell culture, starving the cells had only a small effect on PDCD4 amount and activation state, but refeeding the cells reduced the amount of PDCD4 and induced phosphorylation, thereby activating protein synthesis.
  • When the cell culture was depleted of PDCD4, protein synthesis was higher as indicated by higher incorporation of radioactive phenylalanine into protein.

Interpretation of findings/Key practice applications:

PDCD4 is a regulatory element of protein synthesis. Nutrient availability and type of nutrient affect its expression.

Limitations:

It is unknown whether PDCD4 is directly regulated by nutrient availability or if a signaling molecule upstream is regulated by the nutrients (i.e. mTOR), thereby passing the signal on PDCD4. This is an important distinction in further research targeted at regulating PDCD4.
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