Abstract
BackgroundThe ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysisResultsIncubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA), relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p < 0.05). The proteasome inhibitor MG132 blocked the rise in proteolysis observed in the 0.2 × PC AA media. Addition of insulin to the medium inhibited proteolysis at both 0.2 and 1.0× PC AA and the expression of 14-kDa E2 proteins and C2 sub unit of 20 S proteasome (p < 0.05). Incubation of myotubes with increasing concentrations of leucine in the 0.2 × PC AA media inhibited proteolysis but only in the presence of insulin. Incubation of rapamycin (inhibitor of mTOR) inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation, blocked (P < 0.05) the inhibitory effects of 1.0 × PC AA on protein degradation, but did not alter the inhibitory effects of insulin or leucineConclusionIn a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.
Highlights
The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosomedependent proteolysis in response to complete starvation of amino acids or leucine from the media
Insulin inhibited the release of TCA soluble radioactivity from C2C12 myotubes pre-labelled with L[ring -3, 5-3H]-tyrosine at all of the concentrations employed with the half-maximal effect on proteolysis of approximately 10 -8 M (P < 0.01) (Figure 2)
In conclusion we provide evidence that depletion of a mixture of amino acid in the media of C2C12 myotubes increases proteasome-dependent-proteolysis and the expression of the components of the ubiquitin-proteasome pathway
Summary
The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosomedependent proteolysis in response to complete starvation of amino acids or leucine from the media. BMC Molecular Biology 2007, 8:23 http://www.biomedcentral.com/1471-2199/8/23 the latter is considered the predominant biological mechanism controlling protein degradation in myofibrillar proteins in skeletal muscle [3]. This proteolytic pathway comprises of a cascade of ATP dependent enzymatic reactions in which ubiquitin protein is activated by ubiquitin activating enzyme, E1, which transfers ubiquitin to ubiquitin conjugating enzyme, E2. Increased expression of 14-kDa E2 and E3 ubiquitin ligases, atrogin-1/MAFbx and MuRF1 has been observed in atrophying muscle [5,6]
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