Insulin signaling is critical for maintaining glucose homeostasis, fat synthesis and storage, and for cell growth and differentiation. Perturbations of insulin signaling, e.g., insulin resistance, can have dire consequences resulting in type 2 diabetes mellitus, cardiovascular disease, and cancer. Despite the fact that insulin signaling and diabetes are well studied, limited knowledge is available about the molecular links between insulin resistance and the potential health problems that follow. The purpose of this research is to examine the insulin signaling pathway by studying the consequences of deletion of the Cdc42GAP gene in vivo and in vitro in hepatocytes using a mouse model. Cdc42, a member of the Rho GTPase family, has multiple roles in cell function including actin cytoskeleton reorganization, polarity, intracellular vesicle trafficking, receptor turnover, transcriptional activation, and cell cycle progression. Cdc42GAP is a negative regulator of Cdc42, and Cdc42GAP knockout in mice results in a global gain-of-activity of Cdc42 in various tissues/organs. Cdc42 has been linked, controversially, to the insulin signaling pathway in several capacities, mainly that associated with the 70 kDa ribosomal S6 kinase (S6K1) and phosphatidylinositol 3- kinase (PI3K), both of which act in the pathway of mammalian target of rapamycin (mTOR). In preliminary studies, we found that mice in which Cdc42 is over-active (Cdc42GAP-/-) display a hypersensitivity to insulin challenge. In this study we will examine the hypothesis that Cdc42GAP and Cdc42 play an integral and novel role in insulin/mTOR signaling. In order to achieve these goals we will utilize our gene targeted mouse line (Cdc42GAP-/-) in which loss-of-function of Cdc42GAP results in a constitutuvely active Cdc42.
Aim 1 is to determine the role of Cdc42GAP in insulin/mTOR signaling in vitro by examining the insulin signaling pathway in Cdc42GAP-/- hepatocytes. Effects of Cdc42 activation will be assessed by examining activity changes of components of the insulin signaling pathwayafter insulin stimulation, and molecular connections will be assigned between Cdc42 and S6K1, mTor, and/or JNK.
Aim 2 is to determine the role of Cdc42GAP in insulin-mediated glucose homeostasis in vivo using Cdc42GAP KO mice. Effects of Cdc42GAP knockout on insulin signaling will be assessed by examining glucose homeostasis, e.g. insulin challenge, glucose tolerance test, pyruvate tolerance test. These studies will provide insight into the role of Cdc42 in insulin signaling in liver and hepatocytes, both in vitro and in vivo. Understanding the role of Cdc42GAP and Cdc42 in insulin signaling and glucose homeostasis may allow for modulation of this pathway in the treatment of obesity, diabetes, or cancer.
