The anabolic effect of insulin on muscle protein synthesis is acutely sensitive to amino acids . Studies on muscle protein degradation have suggested that amino acids also influence insulin action but few studies have investigated the specific cellular pathway involved . The ATP-dependent ubiquitin-proteasome pathway is the most likely target for control by insulin and amino acids since proteasome-dependent-proteolytic activity and expression of components of this pathway are stimulated by starvation and inhibited by refeeding and insulin . However, a previous study of protein degradation in C2C12 myotubes could only find evidence of alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media . We therefore carried out a study to examine the effect of more physiological alterations in amino acid concentration on the effect of insulin on the ubiquitin-proteasome pathway in C2C12 myotubes.
This study is the first to report that the amino acids alone, or additively with insulin, decrease muscle protein degradation by acting on the ubiquitin dependent proteasome pathway. We also report that varying the concentration of leucine whilst maintaining all other amino acids at a low level, does not mimic the effect of varying all amino acid concentrations suggesting that the response to amino acids is more than a response to alterations in leucine as has been previously suggested .
Increasing the media amino acid concentration from 0.2 × physiological levels to 1.0 × PC AA decreased myotube proteolysis as assayed by the release of protein-bound labelled tyrosine into the medium, but these effects were abolished by addition of the proteasome inhibitor MG132. Furthermore, the expression of both the 14-kDa E2 and C2 sub unit of 20 S proteasome was significantly increased during incubations with the sub-physiological amino acid incubation medium. The effect of amino acids was additive with insulin: maximal stimulation of protein degradation and expression of 14-kDa E2 and C2 were observed in the absence of insulin and at 0.2 × amino acids. Addition of insulin in the presence of amino acids strongly inhibited proteolysis and expression of 14-kDa E2 and C2. Depletion or supplementation of media leucine concentration influenced proteolysis but only in the presence of insulin. These results therefore strongly argue that insulin and amino acids interact to suppress myotube proteolysis by acting on the ubiquitin-proteasome pathway. The tyrosine release assay measures total myotube protein degradation and is therefore more representative of muscle protein degradation and is also able to determine small differences in turnover that are not possible with the measurement of specific proteins or their rate of ubiquitlylation.
A previous study in C2C12 cells  was unable to find any effects of amino acid or leucine starvation in the media on proteasome-dependent proteolysis or expression of components of this pathway. Our current study differed from the previous study in two ways: firstly, we examined the effect of physiological changes in amino acid concentrations by decreasing to 0.2 × physiological levels or in the case of leucine, altering the concentrations whilst maintaining the others at 0.2 × physiological levels. Secondly, the effects of amino acids were examined alone or in combination with insulin. Others have reported the involvement of both lysosomal and ubiquitin-proteasome pathways during incubation of rat skeletal muscle in vitro in the presence of 10 mM leucine . This suggests the possibility that the immediate cellular response towards amino acid withdrawal is autophagy either through sequestration of protein substrates to lysosomes or through modification of mRNA levels for lysosomal proteases, but is followed by longer term activation of ubiquitin-proteasome pathway. The activation of lysosomal pathway in response to total amino acid deprivation as reported by the previous study is more likely due to cell stress .
In vitro studies using L6 or human muscle cells have confirmed the inhibitory effects of insulin on muscle protein degradation [25, 26]. Furthermore the mechanism of insulin action appears to be mediated by a decrease in the activity of the ubiquitin-proteasome pathway . Both in vitro  and in vivo  studies have demonstrated that insulin down regulates the expression of 14-kDa E2 ubiquitin conjugating enzyme and ubiquitin, respectively. The present study demonstrates that the inhibitory action of insulin on expression of components of the proteasome pathway is decreased when either amino acids or leucine concentrations in the media are decreased to 0.2 × physiological concentrations. In vivo, the starvation-induced increase in muscle proteasome-dependent proteolysis is only slowly reversed over 12 h of refeeding rats and acute infusion of amino acids over 3 h fail to alter the expression of components of the proteasome pathway . A 6-hour hyperinsulinaemic, hyper-aminoacidaemic infusion study significantly reduced the mRNA expression for ubiquitin in fast twitch and mixed skeletal muscle . These data combined with our own suggest that under normal fed conditions insulin and amino acids act together to suppress the ubiquitin-proteasome pathway and that the fasting-induced increase in muscle protein degradation is due to lowered insulin and amino acid circulating concentrations resulting in enhanced expression of members of the proteasome pathway.
Numerous studies have demonstrated that insulin and amino acids regulate muscle protein synthesis and hepatic autophagy through the mTOR [18–29]. Inhibition of the mTOR pathway by rapamycin abolishes the effect of leucine media starvation on autophagic proteolysis in C2C12 myotubes . Here, we demonstrate that rapamycin blocked the inhibitory effects of increasing amino acid concentration from 0.2 to 1.0 × PC AA, on protein degradation, but did not block the inhibitory effect of either leucine or insulin. In agreement with the present results, previous studies have demonstrated that leucine suppresses proteolysis in skeletal muscle and hepatocytes by an mTOR independent mechanism[15, 30, 31]. Since our observations demonstrated that amino acid mixtures suppressed proteolysis in an mTOR dependent manner, this suggests that the mTOR pathway is influenced by amino acids other than the branched chain amino acid group. It has been proposed that glutamate and glutamine have suppressive effects on proteolysis in muscle  and studies in other cell types have demonstrated that these amino acids influence the mTOR signalling pathway [33, 34]. Phoshorylation targets for mTOR have been proposed to be S6 kinase and 4E-BP1 as well as the phosphorylation dependent control of the degradation of many F-box proteins. Phosphorylation of S6 kinase by insulin greatly depends on amino acid availability  and leucine alone has been reported to induce S6 activation [36, 37]. We were also able to demonstrate that rapamycin inhibited amino acid or insulin-dependent p70 S6 kinase phosphorylation. These changes were paralleled by a consistent trend for an increase in mRNA for 14-kDa E2 enzyme. The expression of several muscle-specific E3 ubiquitin ligases is consistently increased in conditions causing muscle wasting and mouse knock out studies have demonstrated that these are required for muscle atrophy . Insulin and insulin-like growth factor-1 acting through the PI3-k/AKT pathway, suppress the expression of E3 ligases, MuRF1 and MAFbx/atrogin-1 . Taken together, the present study argues that amino acids act through mTOR to regulate myofibrillar proteasome-dependent proteolysis but further studies are necessary to establish the down stream effector pathways responsible for changes in the expression of components of the proteasome complex.