The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation. To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed; an <i>in vivo </i>study on growing calves and an <i>in vitro</i> C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA). Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (P<0.05). HDG was associated with decreased plasma urea nitrogen and 3-MH concentrations and 3-MH:creatinine output (an index of MF degradation) (P < 0.05). Glucose infusions down regulated the expression of 14-kDa E2 ubiquitin conjugating enzyme and C2 20 S proteasome sub unit, however EAA did not alter the effect of raised plasma insulin on muscle ubiquitin proteasome pathway suggesting that under the conditions employed, EAA do not act synergistically with insulin to decrease myofibrillar protein degradation, <i>in vivo.</i> In the <i>in vitro</i> experiments, amino acid deprivation (0.2 X physiological concentration amino acid; PC AA) of myotubes for 8 h was associated with increased (P < 0.05) proteolysis (measured from TCA soluble <sup>3</sup>H-tyrosine release in the medium), compared to controls (1.0 X PC AA). Addition of insulin inhibited this increase (P < 0.05). Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway. Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05). Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin. In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:288389 |
| Date | January 2003 |
| Creators | Sadiq, Fouzia |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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