YU, ZK, JT WRIGHT, GJ HAUSMAN. Preadipocyte recruitment in stromal vascular cultures after depletion of committed preadipocytes by immunocytotoxicity. Obes Res. 1997;5:9-15. Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complementmediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 f 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 f 5.1/ unit area) after removal of preadipocytes. Immunocytochemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 f 45/unit area) than FBS alone (10.5 f 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentia-

Growth hormone and thyroid hormones have been implicated as important serum factors for adipocyte development in cell culture. Fetal decapitation removes these factors from serum of the growing fetal pig and results in development of fewer adipocytes than in intact fetuses. These experiments examined the effects of growth hormone or thyroxine supplementation to decapitated fetal pig sera upon pre-adipocyte proliferation and differentiation. Hormones were supplemented to concentrations present in sera from intact pig littermates (reference). Sera +/- hormones were analyzed for their effects upon pre-adipocyte proliferation as determined by [3H]-thymidine incorporation; enzyme expression as determined by sn-glycerol-3-phosphate dehydrogenase activity; and induction of complete differentiation into lipid filled adipocytes as based upon a pre-adipocyte proliferation and enzyme expression than reference sera. Growth hormone had no effect in decap sera upon these parameters. Decap sera permitted detection of 54% more lipid-accumulating, newly formed adipocytes on percol gradients than reference sera, but growth hormone reduced detection to 29% of reference sera. Thyroxine specifically stimulated pre-adipocyte proliferation more than decap sera, but not to the level of reference sera. Complete differentiation, a formation of lipid-accumulating adipocytes was promoted also by thyroxine in comparison to basal decap sera. The results of these experiments indicate thyroid hormones are an important component of fetal sera for regulation of adipocyte development, whereas growth hormone may only affect cellular metabolism and not promote pre-adipocyte growth and development.

The effect of fetal decapitation on adipose tissue development in utero was studied in the pig. Pig fetuses were decapitated at 45 days of gestation, and dorsal subcutaneous adipose tissue was analyzed at 110 days of gestation. The histology, histochemistry, ultrastructure, size and lipoprotein lipase activity of adipocytes from decapitated and sham-decapitated control fetuses were determined. Decapitation resulted in a poorly developed dermis and epidermis, a poorly developed outer layer of adipose tissue and larger fat cells that were metabolically and structurally more mature. Fewer fat cell clusters in decapitated fetuses were associated with fewer blood vessels in the subcutaneous layers. The results of this study demonstrate that fat cell development in subcutaneous tissues may be an integral aspect of the development of skin and associated structures.

The cellular and enzyme-histochemical differentiation of subcutaneous adipose tissue was studied in lean and obese pig fetuses at several ages. Positive reactions for a variety of cytosolic and organellar enzyme markers indicate metabolic competence of fetal adipocytes despite their small size (12 to 15 microns). Reactions for several enzymes decreased with fetal age and may be associated with a qualitative change in activity of adipocyte organelles. Age-associated increases in two lipogenic enzymes were observed in obese adipocytes. Observations on developing cells around hair follicles in the younger fetuses indicated significant temporal lags between the appearance of detectable enzyme activities in adipocytes. Enzyme activities in order of appearance were: dehydrogenases (cytosolic and mitochondrial), lipoprotein lipase and esterase. Esterase activity and several other enzymes were never observed in lipid positive cells that were not spherical. A proportion of hair follicle associated adipocytes in 110-d-old lean fetuses were histochemically and morphologically similar to brown adipocytes in the young rat. There was no evidence for brown adipocyte like cells in obese fetuses. Finally, comparison of the enzyme-histochemical differentiation of lean and obese fetal adipocytes indicates that fetal adipocytes become sensitive to external stimuli between 70 and 90 d of gestation.

Body composition, liver and adipose lipogenesis, and pancreatic insulin release were examined in intact and decapitated fetal pigs on d 110 of gestation. Decapitation was on d 45 of gestation. Decapitated fetuses deposited more body lipid and less body ash compared with intact fetuses. Body weight, water, dry matter and protein remained similar in intact and decapitated fetuses. Hepatic fatty acid esterification and synthesis were two- and threefold greater, respectively, in decapitated than in intact fetuses. Fatty acid synthesis in subcutaneous adipose tissue of decapitated fetuses was three times greater than values obtained in intact fetuses. The data supported the concept that substrate availability from the dam was not the rate-limiting step in fetal pig lipid synthesis and storage. High growth hormone levels in normal fetal pigs may be responsible for inhibiting lipogenesis, while fetal decapitation would remove this inhibition and be associated with greater lipid deposition. However, pancreatic insulin release was greater in decapitated than in intact fetuses; an indication that elevated lipid deposition may also be due to greater fetal insulin secretion.