In response to RFA DK-94-023 entitled "Nutrient Modulation of Cell Integrity and Repair Mechanisms," we propose to study defective insulin regulation of apolipoprotein (apo) B biogenesis in hepatocytes derived from Zucker obese rats in order to elucidate the molecular defect involved in insulin resistance of this pathway. We propose to study the linkage of specific events involved in insulin receptor signal transduction with microsomal events involved in apo B biogenesis with primary focus on the regulatory role of activated phosphatidylinositol 3-kinase (PI 3-K) in the translocation of apo B into the secretory pathway. While the majority of studies involve rat apo B. pilot studies will use the new technology of receptor-mediated gene delivery to study human apo B constructs in primary cultures of rat hepatocytes. Two specific aims are proposed to dissect pathways related to PI 3-K lipid kinase and serine kinase activity both of which are activated by insulin. Aim l is to test the hypothesis that insulin causes localization and activation of lipid kinase of PI 3-K on endoplasmic reticulum (ERA membranes which results in rapid accumulation of highly polar phospholipids which selectively. in a negative fashion. regulate apo B translocation into the secretory pathway. Apo B that fails to translocate and assemble with lipid is trapped within ER membranes and targeted for degradation. This process is responsible for the observed insulin dose-dependent decrease in apo B secretion by hepatocytes. The role of insulin receptor (IR) signaling and PI 3-K activation on apo B secretion will be evaluated in both lean and obese rats, the latter being insulin-resistant in the apo B pathway due to combined effects at the receptor and post-receptor level. - Aim 2 will test the hypothesis that insulin causes activation of the serine kinase of PI 3-K which through localization to the ER or through subsequent activation of S6 kinase. phosphorylates either translocation channel component proteins involved in apo B translocation or newly synthesizing apo B itself hindering apo B translocation. With continued translation, the inability of apo B to translocate modifies normal ribosome-ER interaction and results in the generation of cytoplasmic apo B which is unable to post-translationally translocate and is targeted for intracellular degradation. Studies employ primary and suspended hepatocytes derived from normal rats and from lean and obese Zucker rats and include assays of IR signaling, B-subunit phosphorylation, IRS-I phosphorylation, PI 3-K activation and PI 3-K mass in subcellular fractions. Experiments also employ in vitro apo B translocation assays using streptolysin O permeabilized hepatocytes. Results of proposed studies will allow a more complete understanding of normal and defective apo B biogenesis by liver allowing insights into potential mechanisms involved in the development of hypertriglyceridemia and human obesity and insulin resistance syndromes such as Syndrome X.