OVEREXPRESSION OF HORMONE-SENSITIVE LIPASE PREVENTS TRIGLYCERIDE ACCUMULATION IN ADIPOCYTES

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydr olyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct e vidence to prove this concept has been lacking, The present study was designed to establish the function of HSL in adipocytes, A 2360-bp fra gment containing the entire HSL coding region was cloned into the vect or pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line, Nondifferentiated, transfected cells were screened for HSL overexpres sion by indirect immunofluorescence microscopy and confirmed by immuno blotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA, Stable transfectants overexpressing HSL w ere obtained and cloned, Compared with undifferentiated 3T3-F442A cell s transfected with pCEP4 not containing the insert (vector alone) wher e HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity, Likewise, immunoreactive HS L protein and HSL mRNA levels were increased up to 100-fold in HSL tra nsfectants, When confluent cells were allowed to differentiate by expo sure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants, A simila r degree of differentiation was seen in both vector alone and HSL tran sfectants when based on the induction of lipoprotein lipase, Cellular triglyceride content increased dramatically in the vector alone transf ected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differenti ation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglycerid e content was reduced, Thus, the overexpression of HSL in 3T3-F442A ce lls prevents differentiated adipocytes from taking on the appearance o f fat cells, i.e., accumulating triglyceride, Furthermore, the overexp ression of HSL directly or indirectly attenuates the expression of sev eral genes that appear during late adipocyte differentiation.