title: Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes.
abstract: Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Nondifferentiated, transfected cells were screened for HSL overexpression by indirect immunofluorescence microscopy and confirmed by immunoblotting cell extracts with anti-HSL/fusion protein antibodies and by Northern blots for HSL mRNA. Stable transfectants overexpressing HSL were obtained and cloned. Compared with undifferentiated 3T3-F442A cells transfected with pCEP4 not containing the insert (vector alone) where HSL expression was very low, undifferentiated HSL transfectants had up to a 100-fold increase in HSL activity. Likewise, immunoreactive HSL protein and HSL mRNA levels were increased up to 100-fold in HSL transfectants. When confluent cells were allowed to differentiate by exposure to insulin, HSL expression increased in vector alone transfected cells, but remained below that observed in HSL transfectants. A similar degree of differentiation was seen in both vector alone and HSL transfectants when based on the induction of lipoprotein lipase. Cellular triglyceride content increased dramatically in the vector alone transfected cells while triglyceride content was markedly reduced in the HSL transfectants. The expression of late markers of adipocyte differentiation, such as aP2 and GPDH, was diminished and appeared to vary with the degree to which HSL was overexpressed and the cellular triglyceride content was reduced. Thus, the overexpression of HSL in 3T3-F442A cells prevents differentiated adipocytes from taking on the appearance of fat cells, i.e., accumulating triglyceride. Furthermore, the overexpression of HSL directly or indirectly attenuates the expression of several genes that appear during late adipocyte differentiation.
authors.affiliation: Department of Medicine, Stanford University School of Medicine, California 94305, USA.
authors.forename: C
authors.initials: C
authors.lastname: Sztalryd
authors.forename: M C
authors.initials: MC
authors.lastname: Komaromy
authors.forename: F B
authors.initials: FB
authors.lastname: Kraemer
mesh_terms: D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D002454:Cell Differentiation; D002460:Cell Line; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D015870:Gene Expression; D008071:Lipoprotein Lipase / Q000235:genetics; D051379:Mice; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D002787:Sterol Esterase / Q000378:metabolism*; D014162:Transfection; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C082667:Fabp5 protein, mouse; C086936:Fabp7 protein, mouse; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D014280:Triglycerides; D002787:Sterol Esterase; D008071:Lipoprotein Lipase