Cell death and insulin signaling in liver cells: Molecular and systems biology study.

摘要

Liver disorders, such as hepatic insulin resistance, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) are closely related with each other, and their initiation and development are attributed to the dysregulations of the hepatic cellular activities including lipotoxicity and insulin signaling. This thesis focused on the regulatory mechanisms that control these hepatocellular activities.Saturated free fatty acids (FFAs), e.g., palmitate, are known to induce lipotoxicity, which is partially due to apoptosis. Using the molecular and cellular biology techniques, novel mechanisms involved in palmitate-induced apoptosis were identified in this thesis. In brief, double-stranded RNA-dependent protein kinase (PKR) was found, for the first time, to exploit an anti-apoptotic role in human hepatocellular carcinoma cells by regulating the protein level and phosphorylation of Bcl-2. Palmitate suppresses this PKR-mediated anti-apoptotic machinery in HepG2 cells, thereby resulting in apoptosis.It is well recognized that most cellular functions are regulated by networks of genes, proteins, and other small molecules, rather than single, isolated factor(s). Given the complexity of biological systems, a system-level understanding could provide a broader view on the cellular activities and thereby complement the molecular biology studies. In collaboration with statisticians and computational experts, we developed and applied two methodologies for system-level analyses of the palmitate-induced cytotoxicity. These strategies, based on the principles of systems biology, recovered gene networks that are specifically responsible for the phenotype of palmitate-induced cytotoxicity, thereby shedding light into the potential mechanisms of the phenotype.Another part of this thesis focused on the regulation of insulin signaling. This thesis identified a novel player of insulin signaling, PKR, which differentially regulates the two major insulin receptor substrates (IRS1 and IRS2). For IRS1, PKR induces the inhibitory serine phosphorylation at 312, resulting in down-regulation of the tyrosine phosphorylation of IRS1. On the other hand, PKR regulates IRS2 at the transcriptional rather than the post-translational level.In summary, the hepatic insulin signaling and saturated FFA-induced cytotoxicity were investigated in this thesis. The protein PKR was found, for the first time, to be highly involved in regulating these hepatic cellular events, and detailed mechanisms were identified. In addition to the molecular and cellular biology studies, novel systems biology methodologies were developed and implemented to obtain a boarder systems level view of palmitate-induced cytotoxicity. These systems biology approaches suggested potential research targets and novel mechanisms that were supported by the literature as well as our experiments.