Expression of CCAAT enhancer binding proteins during porcine preadipocyte differentiation

The expression of three CCAAT/enhancer-binding proteins (C/EBPs) was examin ed with immunocytochemistry and Western blot analysis during preadipocyte d ifferentiation in porcine stromal vascular (S-V) cell cultures. Regardless of treatment and time in culture, immunoreactivity for all three C/EBP isof orms was restricted to cell nuclei. At day 1, 50 +/- 6% of S-V cells were C /EBP delta positive, whereas 13 +/- 3 and 11.7 +/- 3% of S-V cells were AD- 3 and C/EBP alpha positive, respectively. After 3 days of seeding in fetal bovine serum (FBS) and dexamethasone (DEX), C/EBP delta; AD-3, and C/EBP al pha-positive cells increased to 67 +/- 5, 42 +/- 4, and 32 +/- 3%, respecti vely. Double staining clearly showed that most of the C/EBP alpha reactive cells had not accumulated appreciable lipid after 3 days of FBS + DEX, Foll owing 3 days of insulin treatment, the percentage of C/EBP delta cells was 50 +/- 6, whereas the percentage of AD-3- and C/EBP alpha-positive cells wa s 41 +/- 4 and 31 +/- 3, respectively. After insulin treatment all fat cell s were AD-3, C/EBP alpha, and C/EBP delta positive. Double staining demonst rated that fat cells were C/EBP delta reactive throughout the culture perio d. Western blotting showed changes in C/EBP isoform expression that were co nsistent with the immunocytochemical results. We conclude that C/EBP alpha is a terminal differentiation marker which is expressed later than AD-3 but further studies are needed to determine the relationship between C/EBP del ta and adipogenesis in porcine S-V cultures. (C) 1998 Academic Press.