The major objective of this project is to extend our knowledge of signaling events that control insulin action. The metabolic and mitogenic actions of insulin are initiated by binding of the hormone to its cell-surface receptor. Insulin stimulation leads to rapid reorganization of the actin cytoskeleton to generate the forces necessary for plasma membrane ruffling and a host of other cellular processes. We have identified phosphoinositide-specific phospholipase C-gamma 1 (PLCg1) as a binding partner to the activated insulin receptor (IR) in various cell lines and in a primary culture of rat hepatocytes. PLCg1 is an enzyme that plays a pivotal role in transmembrane signaling, notably the regulation of extracellular signal-regulated kinases 1/2 (ERK). Our data showed that knockdown of PLCg1 expression by RNA interference significantly reduces ERK activation by insulin, but not that of Akt, in the liver-derived HepG2 cells. Conversely, reconstitution of PLCg1 in PLCg1(-/-) mouse embryonic fibroblasts shows a marked increase in insulin-stimulated ERK activation, suggesting that PLCg1 may be involved in transducing insulin-mediated signals through activation of the Ras/ERK pathway. PLCg1 contains several domains through which it can interact with actin, signaling proteins and lipid products. Through this network of interactions, PLCg1 is activated and redistributed within the cell where it exerts an essential role in mammalian growth and differentiation. Little is known about the signaling cascade involved in the shuttling of PLCg1 between various cellular compartments, and regulation of its activity upon insulin stimulation. Our recent study indicates that the actin-binding protein filamin A binds constitutively to the IR to exert a selective inhibition of signaling cascades leading to the insulin-mediated activation of ERK and its downstream target, the transcription factor Elk-1. Transfection of liver-derived HepG2 cells with a plasmid encoding the C-terminal region of filamin A (FLNa-CT) markedly reduced the association of endogenous filamin A with the IR while causing a selective activation of ERK-mediated transactivation of Elk-1 in response to insulin. Confocal immunofluorescence microscopy showed strong colocalization of filamin A and the IR at the surface of HepG2 cells, whereas ectopically expressed FLNa-CT accumulated at the membrane ruffles, supporting the notion that filamin A has a role in insulin-induced cytoskeletal rearrangement. However, the functions of filamin A with regard to the redistribution and activation of endogenous PLCg1 in response to insulin are not understood. One important function of filamin A is that it tethers plasma membrane receptors to the actin cytoskeleton and, thus, modulates their rate of internalization and directs their intracellular trafficking. It also binds raft-associated caveolin-1, which provides evidence for the organization and clustering of lipid raft microdomains by the actin cytoskeleton. We are currently testing the hypothesis that filamin A alone or together with caveolin-1 modulates insulin-dependent activation of PLCg1 and its intracellular trafficking to promote gene expression and differentiation. The molecular basis for the development of insulin resistance in aging, obesity and in disease states such as type 2 diabetes is complex and remains elusive. Current interventions aimed at reducing hyperglycemia have had disappointing outcomes in clinical trials. Hence, effective new therapeutic interventions to improve insulin responsiveness require a better understanding of protein-protein interactions applied to insulin receptor signaling
| Bernier, Michel; He, Hua-Jun; Kwon, Yong-Kook et al. (2004) The roles of phospholipase C-gamma 1 and actin-binding protein filamin A in signal transduction of the insulin receptor. Vitam Horm 69:221-47 |
| Zhu, Min; Miura, Junnosuke; Lu, Lucy X et al. (2004) Circulating adiponectin levels increase in rats on caloric restriction: the potential for insulin sensitization. Exp Gerontol 39:1049-59 |
| Kwon, Yong-Kook; Jang, Hyeung-Jin; Kole, Sutapa et al. (2003) Role of the pleckstrin homology domain of PLCgamma1 in its interaction with the insulin receptor. J Cell Biol 163:375-84 |
| He, Hua-Jun; Kole, Sutapa; Kwon, Yong-Kook et al. (2003) Interaction of filamin A with the insulin receptor alters insulin-dependent activation of the mitogen-activated protein kinase pathway. J Biol Chem 278:27096-104 |
