The dissection of insulin signaling isoforms in vivo: Molecular mechanisms of hepatic insulin action

Type 2 diabetes affects more than 18 million Americans; it costs the United States more than {dollar}132 billion a year. The work compiled in this dissertation seeks to understand the physiologic and molecular mechanisms of insulin action in the liver, an organ vital to glucose homeostasis. Toward this end, we dissected the functions of the molecular isoforms of the proximal insulin signaling pathway with a variety of techniques and demonstrated that the isoforms of the IRS proteins and PI3K regulatory subunits have specific and unique functions that are critical to hepatic insulin action in vivo. In Chapter 2, we analyze the functional differences between the two major IRS isoforms, IRS-1 and IRS-2, with a novel adenovirus-mediated RNA interference (RNAi) technique. We show that while IRS-1 and IRS-2 have complementary roles in the maintenance of hepatic PI3K and Akt activity in vivo, they have unique roles in insulin-regulated gene expression, where IRS-1 is more associated with gluconeogenic gene expression and IRS-2 is more closely linked to lipogenic gene expression. We extend these findings in chapter 3 to show that the hepatic expression of IRS-1 and IRS-2 may represent an important checkpoint in the preventing the onset of diabetes in mice with a genetic predisposition to diabetes. In chapter 4, we analyze the isoforms of the regulatory subunit of phosphomositide 3-kinase (PI3K) via a liver-specific knockout of Pik3r1. Here we show that a liver-specific deletion of p85alpha recapitulates the enhanced insulin sensitivity previously observed in whole body p85alpha deletions. L-Pik3r1KO mice exhibited increased insulin-dependent Akt activation in the liver of KO mice, despite substantial reductions in total PI3K activity and p85 binding to IRS-1 and IRS-2. The dramatic discrepancy between hepatic PI3K activity and Akt activation was resolved by analyzing levels of the P13K lipid product, phosphatidylinositol(3,4,5)-trisphosphate (PIP3), as KO animals displayer increased accumulation of PIP3 in response to insulin. The regulation of PIP3 levels by p85alpha may occur via a reduction in insulin-dependent activation of the cdc42/c-Jun N-terminal kinase (JNK) pathway.; We then crossed L-Pik3r1KO mice with Pik3r2 null mice to create animals with no functional regulatory subunits in liver (L-p85DKO), which effectively ablated all PI3K function in liver. In chapter 5, we show that the functions of PI3K recapitulate nearly all of insulin's known regulatory functions in the liver. These data demonstrate the pivotal role of the IRS-PI3K pathway in the mediating hepatic insulin action in vivo, and the mechanisms by which specific isoforms of this pathway mediate specific biological functions.