This proposal focuses on signal transduction events that mediate adipocyte development. Adipose tissue is subject to complex modulation by growth factors at the level of adipocyte precursor cell proliferation and differentiation. Insulin-like growth factor-I (IGF-I) stimulates growth of proliferating preadipocytes, but upon growth arrest, IGF-I stimulates differentiation into adipocytes. Defining the signaling pathways mediating this dual role of IGF-I is the subject of our proposal. In our preliminary studies, we have made novel observations associating the change in function with a change in IGF-I signaling. IGF-I's mitogenic effect is dependent on activation of the mitogen-activated protein kinases (MAPKs), ERK1 and ERK2. ERK activation, in turn, occurs via IGF-I receptor (IGFR)mediated phosphorylation of the adaptor protein Shc, and not IRS-1. We tested the hypothesis that Src family kinases (SFKs) mediate IGF-I mitogenic signaling. The Src family kinase inhibitor, PP 1, blocks IGF-I-mediated Shc and MAPK activation in proliferating 3T3-L 1 cells. Transient transfection of a dominant-negative, kinase-dead Src mutant inhibits IGF-I activation of MAPKs. IGF-I stimulates the activities of SFK members c-Src and Fyn in proliferating 3T3-Ll cells. Although SFKs are known to modulate IGFR-mediated cell transformation and mitogenic signaling by other growth factors, our studies are novel in identifying a role for SFKs in IGF-I-mediated preadipocyte growth. The present proposal is aimed at defining the role of SFKs in IGF-I-mediated preadipocyte proliferation. We hypothesize that IGF-I signaling through Src is uncoupled when preadipocytes undergo growth arrest, thus allowing IGF-I to act as a differentiation-inducing factor. We will use two established models for adipogenesis, the 3T3-L1 cell line and primary cultures of mouse preadipocytes, to achieve the following specific aims: 1. Characterize Src-mediated mitogenic pathway downstream from IGF-I, and determine if the role of Src is specific to IGF-I. 2. Elucidate the mechanism associated with growth arrest that uncouples Src kinases from IGF-I mitogenic signaling. 3. Determine if loss of Src activation is required for IGF-I-mediated 313-Li differentiation, and what role SFKs play in pathways that mediate the differentiation effects of IGF-I. These studies will better define IGF-I signaling pathways that mediate adipogenesis, and may identify targets for the modulation of obesity.
