The goal is to identify site of regulation of hepatic production of apo B-lipoproteins. Emphasis is placed on the role of insulin in models of altered insulin states including hyperinsulinemia produced by insulin pumps, streptozotocin-induced hypoinsulinemic diabetes, and fasting- hypoinsulinemia in rats. Insulin and the opposing effects of counterregulatory hormones will be examined in vitro in primary hepatocyte cultures in established animal models of altered insulin action. Levels of hepatic regulation of apo B production will be identified by measurement of apo B MRNA (edited and unedited), apo B synthesis including apo Bh (B100) and apo BL (B48), apo B degradation, apo B pool size, apo B secretion and apolipoprotein and lipid composition of secretory lipoproteins. Studies will identify transcriptional, translational, and pre-secretory events. Evaluation of apo E will be compared with apo B.
Specific Aim 1 : Studies will investigate transcriptional regulation of apo B and apo E in altered insulin states. Studies include measurement of hepatic apo B MRNA (total, GLN, UAA) with comparison to MRNA of other proteins including apo E. Preliminary evidence indicates that increased apo B MRNA are present with insulin treatment of hypoinsulinemic diabetes with preferential expression of apo B MRNA UAA (apo BL). Studies will explore whether insulin in other states induces apo B MRNA expression. Whole liver analysis will be compared to hepatocytes and in vitro effects of insulin and counterreulatory hormones will be assessed in cultures extending to 72h. Apo BH, apo BL and apo E synthesis will be studied in hepatocytes using steady-state labeling and pulse-chase labeling experiments to evaluate secretory pathways. Translation rates for proteins will be studied using dual label techniques combined with immunoprecipitation of subcellular fractions.
Specific Aim 2 : Studies will investigate intracellular degradation of apo B in altered insulin states in vitro. Intracellular degradation is evaluated using pulse-chase protocols with amino acid label. Studies will include use of protease inhibitors to distinguish lysosomal and non-lysosomal pathways. Effects of perturbations will be evaluated with respect to the apo B intracellular pool. Subcellular fractionation studies will determine the kinetics of intracellular apo B movement.
Showing the most recent 10 out of 35 publications
