Expression of CCAAT/enhancer binding proteins during porcine preadipocyte differentiation

The expression of three CCAAT/enhancer-binding proteins (C/EBPs) was examined with immunocytochemistry and Western blot analysis during preadipocyte differentiation in porcine stromal vascular (S-V) cell cultures. Regardless of treatment and time in culture, immunoreactivity for all three C/EBP isoforms was restricted to cell nuclei. At day 1, 50 +/- 6% of S-V cells were C/EBPdelta positive, whereas 13 +/- 3 and 11.7 +/- 3% of S-V cells were AD-3 and C/EBPalpha positive, respectively. After 3 days of seeding in fetal bovine serum (FBS) and dexamethasone (DEX), C/EBPdelta; AD-3, and C/EBPalpha-positive cells increased to 67 +/- 5, 42 +/- 4, and 32 +/- 3%, respectively. Double staining clearly showed that most of the C/EBPalpha reactive cells had not accumulated appreciable lipid after 3 days of FBS + DEX. Following 3 days of insulin treatment, the percentage of C/EBPdelta cells was 50 +/- 6, whereas the percentage of AD-3- and C/EBPalpha-positive cells was 41 +/- 4 and 31 +/- 3, respectively. After insulin treatment all fat cells were AD-3, C/EBPalpha, and C/EBPdelta positive. Double staining demonstrated that fat cells were C/EBPdelta reactive throughout the culture period. Western blotting showed changes in C/EBP isoform expression that were consistent with the immunocytochemical results. We conclude that C/EBPalpha is a terminal differentiation marker which is expressed later than AD-3 but further studies are needed to determine the relationship between C/EBPdelta and adipogenesis in porcine S-V cultures.