This study examines the effects of insulin in adipose tissue on: a) the availability of lipoprotein lipase (LPL) specific mRNA at the polysomal sites and, b) the membrane mediated modifications of the newly formed LPL nascent chain(s). To achieve this goal, we determine the effects of feeding and fasting and of injecting insulin to overnight fasted rats. Studies are also performed in adipose tissue incubated with physiological concentrations of insulin in vitro. Total RNA and poly A-mRNA are translated in a cell-free rabbit reticulocyte lysate system containing L-[35S]-methionine. The incorporation of radioactivity into nascent LPL molecules is identified and quantified by immunoprecipitation using a monospecific antibody directed to the rat adipose tissue enzyme and 1D slab polyacrylamide gel electrophoresis coupled with fluorography. To examine the relative contribution of the nucleus and cytosol to the availability of LPL specific mRNA during insulin induction, fat tissue pieces are incubated in vitro with insulin (0.3 mU/m1) with or without cordycepin (0.1 mg/ml) or Alpha-amanitin (10 Mug/ml). We also establish an in vitro translation system to study the processing and modification of nascent LPL using microsomal membranes from dog pancreas. The extent to which insulin influences the membrane processing system is tested in adipose tissue membranes pre-incubated with insulin. Prolactin RNA is translated simultaneously as a control to assess the activity of the microsomal membrane preparation. Glycosylation of LPL is examined by pretreating the microsomal membrane with tunicamycin or by the addition of Nikkol to the assay during translation. The data obtained from this project will allow the development of a model system to explain a mode of insulin action distinct from the hormonal effects on transmembrane transport and substrate oxidation.
