Free fatty acids (FFA) derived from the breakdown of stored triacylglycerols in adipose tissue are a major energy source for most tissue. The critical enzyme which catalyzes this step is thought to be hormone-sensitive lipase (HSL). HSL activity is tightly regulated by hormones and catecholamines to provide appropriate FFA on demand. Although FFA are essential for tissues as an energy supply, excessive FFA are thought to affect lipid metabolism and insulin resistance. The understanding of HSL activity in adipose tissue has been advanced by examining the mechanisms responsible for its regulation under a variety of physiological conditions, such as fasting, insulin deficiency, etc., and it has been shown that differences in the expression of HSL contribute to regional variations in lipolysis. In addition, direct evidence has been provided that overexpression of HSL in adipose cells in vitro will prevent triglyceride accumulation within fat cells, as well as alter the expression of other adipose cell functions. However, there is no direct evidence that aberrant expression of HSL in adipose tissue directly affects lipid metabolism and insulin resistance in vivo. HSL activity has been shown to be rapidly modulated by hormones via phosphorylation-dephosphorylation reactions. Although this process has recently begun to be explored, the basic mechanisms involved in how phosphorylation-dephosphorylation reactions modulate HSL activity and the structural features essential for HSL actions are not yet fully understood. To address these issues, the overall goals of this project are to establish the structure-function relationships of HSL as a triglyceride lipase in adipose tissue and to understand the mechanisms regulating the expression of HSL in adipose tissue. The specific aims of this project are: to establish the structure-function relationships of HSL in regulating lipolysis in adipose cells by transfecting cells with normal and mutant forms of HSL, to prove that an increased expression of adipose HSL in vivo induces hyperlipidemia and insulin resistance by generating HSL overexpressing transgenic mice, and to explore the mechanisms regulating the rapid modulation of HSL activity in adipose cells using confocal microscopy and the yeast two-hybrid system. The experiments outlined in this proposal will provide a comprehensive examination of the function and mechanisms of action of HSL in adipose tissue.