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Title: Investigation of the CAP/e-Cbl Insulin Signalling Pathway in Cultured Human Skeletal Muscle Cells
Author: Xie, Long
ISNI:       0000 0001 3573 7967
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2008
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Insulin stimulates glucose uptake and glycogen synthesis in adipose tissue and muscle. The classical phosphatidylinositol-3-kinase (PI3K) pathway has been suggested to be necessary, but not sufficient, for insulin signal transduction and an alternative CAP (Cbl-associated protein)/c-Cbl (Casitas b-lineage lymphoma) signalling pathway was reported to mediate insulin-stimulated glucose uptake in 3T3-Ll adipocyte-based studies. However, the majority of evidence to date has been collected from adipocytes and currently the importance of the CAP/c-Cbl pathway in multiple insulin-sensitive tissues is being challenged. The aim of the present study was therefore to investigate the expression levels and the roles of CAP and c-Cbl in response to insulin signal transduction and action in cultured human skeletal muscle cells from healthy subjects. Both gene and protein expression levels of CAP and c-Cbl in myoblasts and myotubes were detected using quantitative real-time PCR and Western blotting. c-Cbl expression was found to be significantly decreased in myotubes compared with myoblasts. In addition, 10 min insulin treatment significantly increased tyrosine phosphorylation of c-Cbl at y774 in cultured human muscle cells. CAP gene expression was found to be significantly increased in myotubes. However, it was hard to detect a significant difference in CAP protein expression levels between myoblasts and myotubes due to the non-availability of specific antibodies. Expression of the cCbl gene was then inhibited by siRNA in myoblasts, resulting in a 70% reduction in gene expression level and a 71 % decrease in protein expression level. These cells were subsequently used to study insulin-induced phosphorylation of protein kinase B (PKB) and glycogen synthase kinase-3 (GSK-3), glucose uptake and glycogen synthesis. siRNA-directed c-Cbl silencing significantly inhibited insulin-stimulated phosphorylation of PKB at Ser473 and GSK-3p at Ser9 when compared to the scrambled control. Furthermore, the suppression of c-Cbl expression was found to be associated with a significant decrease in insulin-stimulated glycogen synthesis compared to the scrambled control. However, in contrast, c-Cbl inhibition appeared to have no significant effect on fold increase in glucose uptake in response to insulin. Rosiglitazone is an insulin sensitising agent and has been reported to increase CAP expression in adipocytes. In cultured human muscle cells, Rosiglitazone treatment had no effect on CAP expression. However, Rosiglitazone did increase c-Cbl gene and protein expression in human myoblasts, but not myotubes. Rosiglitazone increased basal and insulin-stimulated glucose uptake at higher doses. As this was observed in both myoblasts and myotubes, a specific role for c-Cbl in this process seems unlikely. In summary, the findings in this thesis demonstrated that c-Cbl gene and protein expression are detectable in cultured human muscle cell and the expression levels of c-Cbl were observed to be increased by Rosiglitazone treatment in human myoblasts. In addition, tyrosine phosphorylation of c-Cbl at Y774 'was enhanced by 10 min insulin stimulation in cultured human muscle cells. Importantly, data from siRNA studies showed a significant effect of c-Cbl gene silencing on insulin-stimulated phosphorylation of PKB and GSK-3p and insulin-induced glycogen synthesis, suggesting a convergent role for c-Cbl on both the PI3K and CAP/c-Cbl pathways in the control of glycogen synthesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available