Lipoprotein lipase (LPL) is a secretory enzyme that has a key role in the metabolism of plasma triglycerides, the distribution of metabolic fuels and perhaps also in the regulation of fat cell size. The enzyme is believed to be synthesized in the parenchymal cells of various tissues and from there transported to its site of action on the capillary endothelium where it is anchored to cell surface heparan sulfate. The regulation of this enzyme is poorly understood and especially so in the human. This proposal will focus on one aspect of regulation, namely, to investigate the mechanism behind the strong inhibitory effect of estradiol on adipose tissue LPL activity that has been well established in experimental animals but less so in the human. One part of the project will be a clinial study to verify whether or not adipose tissue LPL activity in men is inversely correlated to plasma estradiol levels like preliminary studies have shown in women. The main part of the project will be carried out with isolated adipocytes in primary culture. The first hypothesis to be tested is whether various sex steroids affect the biosynthesis of LPL, and in particular, whether estradiol is inhibitory. For these experiments biosynthesis rates for LPL will be calculated from half-maximal labeling times and intracellular LPL mass, determined by double isotope labeling followed by immunoprecipitation, and immunoassay, respectively. The more intriguing hypothesis to be tested is whether sex steroids affect the activity of adipocyte LPL by regulating the biosynthesis of adipocyte surface heparan sulfate anchoring an extracellular pool of the enzyme . For that purpose adipocytes will be incubated with labeled glucosamine and sulfate, followed by isolation and characterization of labeled glycosaminoglycans in order to demonstrate whether or not the cells synthesize heparan sulfate and if so whether synthesis and degree of sulfation are regulated by sex steroids. These experiments will be followed by studies on the binding of exogenous radiolabeled LPL to adipocytes in order to establish whether cells exposed to steroids change their binding capacity for the enzyme.
