Differentiation of 3T3-L1 fibroblasts to adipo- 3T3-L1 fibroblasts, a subclone of Swiss mouse embryo 3T3 fibroblasts isolated by , are capable of differentiating into adipocyte-like cells engorged with cytoplasmic lipid droplets. The differentiation can be accelerated by treating 3T3-Ll fibroblasts with serum (4), insulin (2), biotin (5), or 1-methyl-3-isobutylxanthine (MIX) (6). Rubin et al. (7) found the combination of MIX and dexamethasone (DEX) to be highly effective. The accumulation oftriglyceride is accompanied by a marked increase in the specific activities of several enzymes involved in fatty acid synthesis and triglyceride synthesis (2, 5, 7-10). There is also an increase in lipoprotein lipase which allows the cells to utilize efficiently the fatty acids from lipoproteins in the medium (11-15). The differentiated 3T3-Ll cells acquire hormone receptors for insulin (7,(16)(17)(18), l3-adrenergic agonists, and adrenocorticotropic hormone (corticotropin) (19). The increase in levels ofthese enzymes and receptors, together with the accumulation ofstored triglycerides, strongly supports the conclusion of Green and coworkers that the 3T3-L1 cell can serve as a model for studies ofadipose tissue metabolism. However, thus far there is no evidence for the expression of the most characteristic and functionally important enzyme of adipose tissue-hormone-sensitive lipase, the neutral lipase activated by cyclic AMP (cAMP)-dependent protein kinase that controls rates of lipid mobilization (20)(21)(22)(23)(24)(25). This enzyme appears to be unique to the adipocyte. The only other tissue in which it has been reported is the adrenal (26, 27). Attempts in this laboratory to demonstrate it in rat heart, skeletal muscle, and liver under various conditions have been negative (unpublished results). Severson (28) reported negative results in rat heart. It has been shown that treatment with dibutyryl cAMP and epinephrine over a period of 2 weeks reduces the accumulation of lipid in 3T3-L1 cells (1), suggesting the presence of a hormone-sensitive lipase. However, the observed reduction in lipid accumulation could be due to inhibition ofthe biosynthesis offatty acid and triglyceride.The studies reported here demonstrate a striking increase (19-fold) in neutral triglyceride lipase during differentiation. Concurrently, basal rate of glycerol release increased by more than 1 order ofmagnitude and glycerol release was further stimulated 3-fold by isoproterenol. Finally, the neutral triglyceride lipase activity in differentiated cells was increased 2-fold by treatment with cAMP-dependent protein kinase.
MATERIALS AND METHODSMaterials. Labeled triolein, diolein, and monoolein (containing [1-'4C]oleic acid distributed randomly among the acylated positions) and cholesteryl [1-'4C]oleate were purchased from Rosechem (Los Angeles, CA). Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, and streptomycin were obtained from Irvine Scientific (Santa Ana, CA). MIX was from Aldrich, isoproterenol hydrochloride was from Br...