The influence of dexamethasone and insulin on expression of CCAAT/enhancerbinding protein isoforms during preadipocyte differentiation in porcine stromal-vascular cell cultures: Evidence for very early expression of C/EBP alpha

Expression of CAAT/enhancer binding protein (C/EBP) isoforms was examined i n. primary cultures of adipose tissue stromal vascular (S-V) cells before a nd during preadipocyte differentiation. Immunocytochemistry showed that the proportions and numbers of C/EBP alpha-, C/EBP beta-, and C/EBP delta-reac tive cells were maximized after seeding and plating from d 0 td 3 in fetal bovine serum (FBS). However, there were few preadipocytes (AD-3+) and fewer cells with lipid and the number of C/EBP alpha-reactive cells clearly exce eded the number of preadipocytes. Seeding and plating in dexamethasone (DEX ) + FBS from d 0 to 3 markedly increased the proportions and numbers of pre adipocytes and C/EBP alpha-reactive cells compared to seeding and plating i n FBS, d 0 to 3. The number of C/EBP alpha- and C/EBP beta-reactive cells a nd preadipocyte reactivity for C/EBP beta decreased with insulin or DEX tre atment, d 3 to 6, following FBS, d 0 to 3. However insulin + DEX treatment, d 3 to 6, maintained the number of C/EBP alpha-reactive cells and either m aintained or increased preadipocyte reactivity for C/EBP alpha and C/EBP be ta. DEX and! DEX + insulin treatment induced recruitment of a similar numbe r of preadipocytes, but preadipocytes were not reactive for C/EBP alpha and C/EBP beta in DEX-treated cultures. The number of C/EBP delta reactive cel ls 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/EBP alpha-reactive cells and preadipocytes were maintained by insulin treatment alone. Western blot analysis for C/EBP alpha confirmed the immunocytochemical results. Double staining demonstrated that expression of C/EBP alpha protein was maximized before or at the onset of lipid accretion, whereas expression of C/EBP beta protein was correlated with lipid accretion. These results indicate that c oupling or integration of preadipocyte recruitment with C/EBP alpha express ion may be, a critical step in glucocorticoid-induced adipogenesis.