The Influence of Adenoviral Infection and the Group VIA Calcium-Independent Phospholipase A2 on Hepatic Lipid Metabolism

THE INFLUENCE OF ADENOVIRAL INFECTION AND THE GROUP VIA CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 ON HEPATIC LIPID METABOLISM By William Palmer Wilkins, III, Bachelor of Science A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2008 Major Director: Suzanne E. Barbour, PhD Professor, Department of Biochemistry and Molecular Biology Sterol regulatory element-binding proteins (SREBP) are transcription factors that regulate genes involved in lipid metabolism especially in the liver. Therefore, hepatic SREBP is significant regulator of systemic lipid metabolism. Evidence demonstrates that insulin and dietary unsaturated fatty acid (UFA) regulate SREBP1 expression and subsequent SREBP1-mediated gene transcription, events that in many instances result in modulation of systemic fatty acid and triglyceride (TG) homeostasis. A series of investigations was designed to uncover novel regulators of SREBP1. Dietary and exogenous addition of UFA has been shown to regulate SREBP function; yet, an xiv endogenous source of UFA capable of modulating SREBP remains elusive. Group VIA calcium-independent phospholipase A2 (iPLA2) releases UFA from the sn-2 position of glycerophospholipids. We hypothesized that iPLA2 provides UFA to suppress SREBP. iPLA2 overexpression and inhibition studies were implemented. iPLA2 inhibition increased SREBP1 expression, SREBP-mediated transcription and the expression of SREBP1 gene targets in vitro. In vivo overexpression of iPLA2 resulted in decreased expression of SREBP1 protein and plasma triglyceride. In contrast, iPLA2 overexpression attenuated SREBP1 expression, SREBP-mediated transcription and expression of SREBP1 targets genes. These data support the hypothesis that iPLA2 generates endogenous UFA that limit SREBP function. Use of a replication-deficient adenovirus 5 (Ad-5) expression vector in the iPLA2 study led to the unexpected observation of hepatic SREBP1 activation following Ad-5 infection. Because of this observation, we tested the hypothesis that replication-deficient Ad-5 might augment lipid synthesis in liver. We demonstrate that first generation Ad-5, a ubiquitous transgene expression vector, induces expression of SREBP1 and its target genes and leads to increases in fatty acid synthesis in vivo and in vitro. The phosphatidylinositol 3-kinase (PI3K) inhibitor, PX-866, suppressed Ad-5-induced SRBEP1 expression and hypertriglyceridemia implicating the PI3K/Akt pathway in Ad-5 activation of SREBP1. Use of PX-866 led to the discovery of a third mechanism of SREBP1 regulation. In vivo studies demonstrate that PX-866 modulates basal lipid metabolism in part through decreasing plasma TG, an increased trend toward decreased SREBP1 expression and a xv significant increase in plasma cholesterol. These studies characterize three distinct novel regulatory mechanisms of SREBP1. CHAPTER 1: General Introduction Cellular lipids serve several roles beyond providing a source of energy and the building blocks of the cell-membrane architecture (1). It is now well appreciated that lipids themselves greatly impact cellular function and homeostasis as studies provide ample evidence that lipids are often bioactive and via intracellular signaling and/or endocrine, paracrine or autocrine action are capable of influencing diverse cellular processes often facilitated by modulation of gene expression (2-9). Consequently, aberrant lipid metabolism itself plays an etiological role in many disease states including diabetes, liver disease, cancer, nervous system dysfunction and atherosclerosis (10-15). The investigation of several distinct elements and their mechanisms that impact lipid metabolism and/or regulate the expression of proteins involved in lipid metabolism is warranted and is the major focus of these studies. The following studies characterize novel regulation of lipid metabolism by first-generation adenovirus-5 (Ad-5), commonly used as an expression vector in research (16-19) and gene therapy (20); the Group VIA calcium-independent phospholipase A2 (iPLA2), an enzyme already appreciated for its roles in lipid metabolism and a novel phosphatidylinositol 3-kinase (PI3K) inhibitor PX-866 (21).