Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage sige to the cellsurface. Studies have shown that insulin-activation of PI3K is required for stimulatin of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates anunique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. A proteomic approach was employed to compare the effect of insulin versus PDGF on protein phosphorylation in 3T3-L1 adipocytes, using high-resolution two-dimensional gel electrophoresis (2-DE) in combination with 32P-metabolic labelling and autoradiography. The effect of wortmannin, an inhibitor of PI3K, on insulin-induced phosphorylation was also examined as a further screen for selection of candidate phosphoproteins for insulin-stimulation of GLUT4 translocation. Three subcellular Insulin stimulates glucose uptake in muscle and adipose tissue by translocating glucose transporter 4 (GLUT4) from an intracellular storage site to the cell surface. Studies have shown that insulin-activation of PI3K is required for stimulation of GLUT4 translocation, however, platelet-derived growth factor (PDGF) also activates PI3K in adipocytes without significant effects on GLUT4 localisation. The aim of this study is to examine the hypothesis that insulin stimulates an unique signalling pathway that regulates GLUT4 translocation in adipocytes, and to identify novel signalling candidates in this pathway. fractions were extensively examined, the plasma membrane (PM) fraction, the cytosol fraction and a high speed pellet (HSP) fraction that contains the insulin-responsive pool of GLUT4. These studies showed that phosphorylation of 18 proteins were consistently increased by insulin treatment, with 8 proteins in the cytosol, 10 proteins in the HSP and none in the PM. The phosphorylation of 6 cytosolic and 2 HSP insulin-stimulated phosphoproteins were also significantly increased by PDGF treatment. Wortmannin inhibited the insulin-stimulated phosphorylation of all but 2 proteins. Ten insulin-stimulated phosphoproteins were wortmannin-sensitive and PDGF-insensitive, and were selected as potential candidates for involvement in insulin-stimulated GLUT4 translocation. The molecular identity of insulin-stimulated phosphoproteins were initially examined by immunoblotting. Two cytosolic 2D gel spots (C65 and C79) were identified as mitogen-activated protein kinase (MAPK) isoforms by immunoblotting. Importantly, treatment-induced changes in the phosphorylation of these spots matched those reported for MAPK in adipocytes, confirming that our phosphoprotein mapping accurately reflects cellular phosphorylation. As the observed molecular mass and isoelectric point (pI) of most insulin-regulated phosphoproteins did not match known insulin-signalling proteins, we attempted to identify these by protein sequencing. In agreement with the low abundance of signalling proteins, most of the insulin-regulated phosphoprotein spots were not Coomassie-stained, even on preparative 2D gels. Thus, corresponding spots were pooled from numerous preparative 2D gels, before tryptic digestion and anlaysis by liquid chromatography-coupled tandem mass spectrometry (LC-MS). One of the more abundant candidate phosphoproteins was identified as ATP-citrate lyase (ACL), an enzyme involved in fatty acid synthesis previously shown to be an insulin-regulated phosphoprotein. Insulin treatment also results in the dephosphorylation of several phosphoproteins, one of which was identified as eukaryotic translation elongation factor 2 (eEF2). ACL and eEF2 phosphorylation were found to be regulated in an insulin-specific and wortmannin-sensitive manner in adipocytes. Recent studies suggest that protein kinase B (PKB) is activated downstream of PI3K, and participates in insulin-stimulated glucose uptake. However, the molecular mass and pI of phosphoproteins mapped in this study did not match any of the known PKB isoforms. To investigate the regulation of PKB isoform in adipocytes, studies were performed with PKB antibodies. PKBb expression was found to be induced upon differentiation of 3T3-L1 adipocytes, where insulin, but not PDGF stimulated its phosphorylation. In contrast, PKBa expression was high in fibroblasts, where PDGF was more efficacious than insulin in inducing its phosphorylation. These results suggest that PKBb, rather than PKBa, is the most pertinent isoform for insulin action in adipocytes. PDGF exerted no detectable effect on the phosphorylation of PKBb in 3T3-L1 adipocytes, as determined by phospho-specific antibodies, 32P-labelling and shifts in electrophoretic mobility and pI. Furthermore, insulin stimulated the translocation of PKBb to membrane fractions of adipocytes, whereas PDGF was without effect. Together, these results strongly suggest that PDGF does not activate PKBb in 3T3-L1 adipocytes. Using 2-DE, we have obtained data which support the hypothesis that insulin stimulates an unique signalling pathway in adipocytes. Three potential models are proposed to explain the insulin specificity: targeting of PI3K to pre-assembled signalling complexes in the HSP, activation of specific PI3K isoforms, and activation of two PI3K-dependent signalling pathways. These models remain to be validated in future studies. Candidate phosphoproteins for insulin regulation of glucose metabolism have been mapped using 2-DE. While only abundant metabolic enzymes have been identified so far, methods for phosphoprotein purification and identification have been established. The candidate phosphoproteins mapped in the HSP fraction are likely to be low abundance signalling proteins, and further preparative isolation of these phosphoproteins is required in order to obtain enough protein for identification of these candidates. In conclusion, the present study has demonstrated the successful application of proteomic techniques in cell signalling research.
| Identifer | oai:union.ndltd.org:ADTP/253741 |
| Creators | Hill, Michelle Mei Chih |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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