CONVERGENCE AND DIVERGENCE OF THE SIGNALING PATHWAYS FOR INSULIN AND PHOSPHOINOSITOLGLYCANS

Phosphoinositolglycan molecules isolated from insulin sensitive mammal ian tissues have been demonstrated in numerous in vitro studies to exe rt partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heter ogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale f rom the core glycan of a glycosyl-phosphatidylinositol-anchored membra ne protein from Saccharomyces cerevisiae has recently been shown to st imulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 mu m) of the maximal ins ulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Muller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intr acellular signaling with that of insulin. The activation of glucose tr ansport by both PIG-P and insulin in isolated rat adipocytes and diaph ragms was found to require stimulation of phosphatidylinositol (PI) 3- kinase but to be independent of functional p70S6kinase and mitogen-act ivated protein kinase. The increase in glycerol-3-phosphate acyltransf erase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggests that the signaling pathways fur PIG-P and insulin to glucose transport and meta bolism converge at the level of PI 3-kinase. A component of the PIG-P signaling pathway located upstream of PI 3-kinase was identified by de sensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell v iability and the insulin-mimetic activity of sodium vanadate but compl etely blunted the insulin response. Incubation of the cells with eithe r trypsin or NaCl alone was ineffective. The desensitized adipocytes w ere reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 value s for PIG-P (2 to 5 mu m) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/s alt extract was demonstrated to bind in a functional manner to the adi pocyte plasma membrane of desensitized adipocytes via bipolar interact ions. An excess of trypsin/salt extract inhibited PIG-P action in untr eated adipocytes in a competitive fashion compatible with a receptor f unction for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolat ed rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling b y PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocy tes with PIG-P as well as with other agents exerting partially insulin -mimetic activity, such as PI-specific phospholipase C (PLC) and the s ulfonylurea glimepiride, triggered tyrosine phosphorylation of the cav eolar marker protein caveolin, which was apparently correlated with st imulation of lipogenesis. Strikingly, in adipocytes subjected to combi ned trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride fa iled completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins loc ated in caveolae/detergent-insoluble complexes.