Abstract 320 Poster Session 1, Saturday, 5/1 (poster 158)

A complex interplay of molecular mechanisms has been identified in regulating the differentiation of adipocytes. Limited information exists however, concerning apoptosis of adipocytes. We have recently reported that inhibition of adipocyte differentiation by treatment with TNF-alpha is associated with not only alterations in the expression of the retinoblastoma proteins, p130 and p107, but also results in adipocyte apoptosis. Given that apoptotic signalling pathways might represent novel targets for therapeutic interventions to reduce hyperplastic adipose tissue stores, we undertook this study to examine the expression of three other proteins known to be involved in apoptosis in other cell lines, the cyclin kinase inhibitors p21 and p27 and the tumor suppressor p53. We first examined the expression of each protein via Western analysis during typical 3T3-L1 differentiation in which post-confluent cells are treated with insulin, dexamethasone and isobutylmethyxanthine. Significant amounts of p21 were identified in both quiescent 3T3-L1 preadipocytes prior to treatment and following hormonal induction. p21 remained elevated to Day 3 when most cells have differentiated into mature adipocytes. p27 was not detected in cells prior to treatment but was first identified at Day 1, a timepoint when cells have re-entered the cell cycle prior to terminal differentiation. p27 remained elevated out to Day 3. The tumor suppressor protein p53 was not detected in quiescent cells but was detected at Day 1 and was abundant by Days 2 and 3. Following hormonal treatment of 3T3-L1 cells in the additional presence of 10 nM TNF-alpha, a dramatic reduction in p21 expression was identified on Day 1, a critical time during which these cells re-enter the cell cycle and undergo clonal expansion prior to terminal differentiation. Levels of p21 were also significantly reduced on Day 2 but minimally on Day 3. Less dramatic reductions were identified in p27 expression following TNF-alpha treatment. Interestingly, no p53 was detected on Day 1 following TNF-alpha treatment and levels were reduced on Day 2. However p53 levels on Day 3 were little changes from that observed during differentiation. Given the dramatic reduction in p21 expression on Day 1 after TNF-alpha treatment, these results suggest early alterations in p21 expression are important in diverting preadipocytes to undergo apoptosis. Similarly, given that p21 transcriptional activity is p53-dependent, early alterations in p53 following TNF-alpha treatment may also be involved in regulation of p21.