The expression of cyclin D1 during adipogenesis in pig primary stromal-vascular cultures.

Abstract

Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal-vascular (S-V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Western blot analysis was performed on cultured S-V cells using 8% sodium dodecyl sulfate-polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti-mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti-CCAAT/enhancer binding protein (C/EBP alpha) and anti-cyclin D1 together. Cyclin D1 expression was evaluated in 105-day fetal dorsal subcutaneous tissues using paraffin sections. Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S-V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBP alpha beginning at day 3 and remained colocalized with C/EBP alpha through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. The elevated level of cyclin D1 expression colocalized with C/EBP alpha expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid-filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.