title: The influence of dexamethasone and insulin on expression of CCAAT/enhancer binding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: evidence for very early expression of C/EBPalpha.
abstract: Expression of CAAT/enhancer binding protein (C/EBP) isoforms was examined in primary cultures of adipose tissue stromal vascular (S-V) cells before and during preadipocyte differentiation. Immunocytochemistry showed that the proportions and numbers of C/EBPalpha-, C/EBPbeta-, and C/EBPdelta-reactive cells were maximized after seeding and plating from d 0 to 3 in fetal bovine serum (FBS). However, there were few preadipocytes (AD-3+) and fewer cells with lipid and the number of C/EBPalpha-reactive cells clearly exceeded the number of preadipocytes. Seeding and plating in dexamethasone (DEX) + FBS from d 0 to 3 markedly increased the proportions and numbers of preadipocytes and C/EBPalpha-reactive cells compared to seeding and plating in FBS, d 0 to 3. The number of C/EBPalpha- and C/EBPbeta-reactive cells and preadipocyte reactivity for C/EBPbeta decreased with insulin or DEX treatment, d 3 to 6, following FBS, d 0 to 3. However, insulin + DEX treatment, d 3 to 6, maintained the number of C/EBPalpha-reactive cells and either maintained or increased preadipocyte reactivity for C/EBPalpha and C/EBPbeta. DEX and DEX + insulin treatment induced recruitment of a similar number of preadipocytes, but preadipocytes were not reactive for C/EBPalpha and C/EBPbeta in DEX-treated cultures. The number of C/EBPdelta reactive cells did not change from d 3 to 6 and was not influenced by hormone treatment. After DEX + FBS, d 0 to 3, the high numbers of C/EBPalpha-reactive cells and preadipocytes were maintained by insulin treatment alone. Western blot analysis for C/EBPalpha confirmed the immunocytochemical results. Double staining demonstrated that expression of C/EBPalpha protein was maximized before or at the onset of lipid accretion, whereas expression of C/EBPbeta protein was correlated with lipid accretion. These results indicate that coupling or integration of preadipocyte recruitment with C/EBPalpha expression may be a critical step in glucocorticoid-induced adipogenesis.
authors.affiliation: Richard B. Russell Research Center, ARS, USDA, Athens, GA 30604-5677, USA. ghausman@ars.usda.gov
authors.forename: G J
authors.initials: GJ
authors.lastname: Hausman
mesh_terms: D017667:Adipocytes / Q000166:cytology*; D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D022762:CCAAT-Enhancer-Binding Proteins; D002417:Cattle; D002452:Cell Count / Q000662:veterinary; D002454:Cell Differentiation; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D003907:Dexamethasone / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000096:biosynthesis*; D013552:Swine
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007328:Insulin; D009687:Nuclear Proteins; D003907:Dexamethasone