Hormone sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides and cholesterol esters. This dual ability of HSL to hydrolyze both triglycerides and cholesterol esters places it in a physiologically important role in the regulation of two independent processes: lipolysis and cholesterol homeostasis. While HSL has been shown to possess intrinsic activity to catalyze both triglyceride and cholesterol ester hydrolysis, its role (function) in these cellular processes has not been definitively established. Nonetheless, based on early physiological studies of lipolysis, it has been well accepted that HSL is the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. HSL activity has been shown to be rapidly modulated by hormones via phosphorylation-dephosphorylation reactions, yet, the basic mechanisms involved in how phosphorylation-dephosphorylation reactions modulate HSL activity are not understood. Moreover, very little information exists on what other mechanisms might be involved in regulating HSL activity other than postranslational modification. To address these issues, the overall goals of this proposal are to understand the mechanisms regulating the expression of HSL in adipose tissue and to establish the structure-function relationships of HSL as a triglyceride lipase in adipose tissue.
The first aim i s to explore the mechanisms regulating the physiological expression of HSL in adipose cells. The mechanisms regulating HSL in vivo in adipose tissue will be examined by following changes in the activity of HSL, the amount of HSL protein, the steady- state mRNA levels, and the rate of transcription of HSL mRNA in fat during metabolic perturbations that include fasting and feeding, and insulin deficient diabetes. In addition, the mechanisms responsible for activation of HSL-mediated lipolysis by lipolytic hormones will be explored in vitro by examining the possible translocation of HSL from an aqueous cytosolic compartment to the lipid droplet.
The second aim i s to establish the structure-function relationships of HSL in regulating lipolysis in adipose cells. Murine adipose cell lines will be transfected to over-express normal HSL and the ability of the adipose cells to accumulate triglycerides and the sensitivity of the adipose cells to lipolytic and anti-lipolytic agents will be examined. These cells will also be used to explore the importance of aberrant HSL expression on the ability of adipose cells to differentiate. A murine adipose cell line will be transfected with an antisense hammerhead ribozyme construct to eliminate HSL expression in order to examine the importance of HSL as the rate limiting enzyme in lipolysis. Finally, CHO cells and adipogenic cell lines will be transfected with mutated forms of rat HSL to examine the function of HSL and the structural relationships of phosphorylation, the region of the putative active site, and the putative lipid binding domain. The results of this proposal will delineate the relationships between structure and function of HSL, the mechanisms controlling its expression, and its contribution to the regulation of lipolysis in adipose cells.
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