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this laboratory to demonstrate it in rat heart, skeletal muscle, and liver under various conditions have been negative (unpublished results).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...

Because many or most lipid-laden foam cells in atheromas and in xanthomas derive from macrophages, it is important to understand how they accumulate lipids and how they can divest themselves of lipids. The mobilization of stored triglycerides from macrophages was studied in cell cultures. Mouse resident peritoneal macrophages and J774 macrophages increased their triglyceride content six-to tenfold during a 24-hour incubation with free fatty acids complexed to albumin. Subsequent incubation in fresh medium containing free fatty acid-poor albumin was accompanied by a fall in cell triglyceride content (50% in 20 hours) and a corresponding increase in mediumfree fatty acid. Release of free fatty acid was linear as a function of time, provided fresh medium was added hourly. When medium was not changed, release rates fell off rapidly, probably due to re-uptake of released free fatty acid. 1 -4 In the case of eruptive xanthomas associated with hyperchylomicronemia, the lesions can appear over a relatively short time and disappear almost as rapidly, 5 presumably because these cells have the capacity to metabolize or mobilize the stored lipids rapidly. The possibility that the cells move out of the lesions back into the circulation should also be considered, but it seems unlikely that this can oc-

High levels of neutral triglyceride lipase activity have been demonstrated In several types of macrophages (J774 cells, human monocyte/macrophages, rabbit alveolar macrophages, and resident mouse peritoneal macrophages). The pH optima ranged from 6.5 to 7.4 depending upon the buffer and the conditions of incubation. The addition of bovine serum albumin stimulated activity at low concentrations, as expected for a fatty acid-releasing reaction, but strongly inhibited at higher concentrations; maximal activity was observed In the presence of 0.625 mg/ml of bovine serum albumin. The enzyme was remarkably thermostable, showing no apparent loss of activity at 50°C for as long as 6 hours. The lipase was Inhibited 80% by 0.1 M NaCI. Assayed under optimal conditions, the specific activity of the neutral triglyceride lipase from J774 cells was more than 100-fold greater than the activity of llpoproteln lipase or neutral cholesterol esterase from those cells; this activity was 10-fold greater than the levels of hormone- T he lipid that accumulates in eruptive xanthomatosis, mostly triglycerides, is found in histiocytes or foam cells, cells that are probably derived from circulating monocytes.1 In patients with hyperchylomicronemia, these triglyceride-rich cells are found in the spleen and liver as well as in the skin; similar triglyceride-rich cells are seen in severe forms of diabetic-hyperlipemia.2 When the hyperlipemia is brought under control, the eruptive xanthomata may resolve very rapidly, sometimes within a day or two. Serial bfopsy studies by Parker et al. 2 showed that the triglyceride content of the lesions fell much more rapidly than the cholesterol ester content. Electron microscopic studies show that most of the stored lipid in foam cells is contained in cytoplasmic vesicles lacking a limiting membrane.3 A number of lines Received May 12, 1983; revision accepted August 17, 1983. of evidence suggest that these cytoplasmic lipid droplets result from lysosomal degradation of lipids taken up from chylomicrons (and/or very low density lipoproteins) and then are resynthesized in the cytoplasmic compartment from products escaping the lysosome. 4' 5 Mobilization of these lipid stores presumably involves either movement back into the lysosomal compartment for hydrolysis or the action of cytoplasmic hydrolases.Previous studies in this laboratory have demonstrated the presence of a neutral cytoplasmic cholesterol esterase in homogenates of several types of macrophages.6 These cells were also shown to contain and to secrete a lipoprotein lipase similar in most respects to postheparin plasma lipoprotein lipase. 78Being a triglyceride lipase, the latter enzyme might theoretically play a role in the mobilization of stored triglycerides, but lipoprotein lipase in adipose tissue and other tissues functions exclusively in the uptake of plasma lipoprotein triglycerides, not in the mobilization of stored triglycerides. Our initial attempts to demonstrate additional neutral triglyceride lipase activities in macrophages were un...

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