| Class IA PI3Ks are signaling molecules that control cell survival, growth, proliferation and metabolism. Dysregulated PI3K signaling is found in patients with diseases such as diabetes and cancer. The two aims of my thesis research were (1) to determine the distinct roles of the PI3K isoforms p110alpha and p110beta in regulating hepatic lipid and glucose metabolism and (2) to investigate the regulation of p110alpha and p110beta by the heterotrimeric G protein Galphaq. For aim 1, mice with liver-specific gene deletion of p110alpha or p110beta were generated. My studies found that mice lacking hepatic p110alpha were largely protected from high-fat diet-induced liver steatosis, whereas p110beta ablation did not attenuate triglyceride accumulation in the liver. The protective effect of p110alpha ablation is probably due to decreased liver uptake of long chain fatty acids. High-fat diet-induced increases in mRNA and protein levels of liver fatty acid binding protein were blunted in the p110alpha-null liver. On the other hand, mice lacking hepatic p110beta developed glucose intolerance and hyperinsulinemia. Higher cAMP levels and increased expression of adenylyl cyclase 5 correlated with increased gluconeogenesis and glycogenolysis in p110beta-/- hepatocytes. Mice with p110alpha-null liver did not exhibit glucose intolerance or hyperinsulinemia. Furthermore, ablation of p110alpha decreased insulin signaling in the liver, whereas deletion of p110a had relatively minor effects on this signaling pathway.;Aim 2 of my thesis research investigated the mechanism by which Galpha q inhibits PI3Ks. My studies used purified recombinant proteins and fluorescence spectroscopy to demonstrate that Galphaq directly binds to p110alpha and blocks Ras binding to p110alpha. In addition, in vitro PI3K activity assays revealed that Galphaq inhibits the four PI3K enzyme complexes p110alpha/p85alpha, p110alpha/p85beta, p110beta/p85alpha and p110beta/p85beta. It was determined that Galpha q binds to the p85-binding domain of p110alpha and does not appear to directly interact with the catalytic domain. Further, I found that Galpha q can bind to free p85 in the iSH2 region independently of p110 binding.;In summary, findings from my thesis research indicate that p110a and p110a have differential effects on hepatic lipid and glucose metabolism and that activated Galphaq can directly bind and inhibit PI3K complexes. |
