Organelle Relationships in Cultured 3 T 3L 1 Preadipocytes 180

In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles . ER cisternae are also situated very close to "rosettes," plasmalemmal specializations found in mature adipocytes in vivo . As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors . The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles ; and (c) a high level of a-naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol. Green and colleagues (20-23) established several cloned lines from disaggregated Swiss mouse embryos (80) that are capable of differentiating into adipocytelike cells (see reference 19 for review). During the course of differentiation, activities of enzymes involved in triacylglycerol biosynthesis increased 50to 100-fold (8, 34, 39), and the cells became responsive to fadrenergic agonists, ACTH (72), and insulin (22, 7l). For these reasons, this cell line is being used as a model for investigations on mammalian adipogenesis and the structure and function of adipocyte organelles . The cytology of the adipocyte has been reviewed often; relatively recent reviews are those of Slavin (77) and of Heindl et al . (27) . A major problem in studying the mature adipocyte has been the difficulty of maintaining the structural integrity of cell organelles during fixation and subsequent processing for microscopy . Cultured 3T3-L l cells possess many of the biochemical characteristics of mature adipocytes and are eminently suited for both light and electron microscope studies because they are easily preserved and because enzyme cytochemistry permits visualization of cell organelles that may be involved in the movement and metabolism of lipid. The observations presented in this communication document the cytological features of cultured 3T3-L1 preadipocytes and provide a basis from which one can begin to study the biochemical functions of adipocyte organelles . MATERIALS AND METHODS Culture conditions for the growth of 3T3-LI cells in our laboratory have been previously described (72) . Cells were originally provided by Dr. Howard Green. Differentiation into adipocytelike cells was stimulated by 48-h treatment with 0.25 pM dexamethasone and 0.5 mM methylisobutylxanthine (71) . As assessed by phase-contrast microscopy, differentiating cells (4-5 d posttreatment) grown on moderately carbon-coated No . 2 coverslips (sterilized by dry heat [200 °C overnight]) were morphologically indistinguishable from cells grown in the usual manner on 100-mm Falcon culture dishes (Falcon Labware, Div. Becton, Dickinson &Co. . Oxnard, Calif.) . All observations were made on cells grown on such coverslips . After culturing, coverslips were transferred with flattened forceps to solutions contained in Columbia staining jars (VWR Scientific Inc., San Francisco, Calif) . They were first rinsed briefly in Earles modified Eagle's Minimal Essential Medium without serum, at room temperature, and then transferred to fixatives