Insulin/IGF-1 is required for differentiation of 3T3-L1 adipose cells. Downstream targets of insulin/IGF-1 that lead to adipocyte differentiation appear to include Ras, phosphatidylinositol (PI) 3-kinase, Raf, and mitogen-activated protein kinase. We have tested whether protein kinase B (PKB), a serine/threonine kinase activated by PI 3-kinase, is sufficient for 3T3-L1 preadipose cell differentiation. A plasmid vector encoding a version of PKB that is constitutively activated (Gag-PKB) was expressed in 3T3-L1 preadipose cells (Gag-PKB cells). Spontaneous morphological changes indicative of adipocyte differentiation were observed in Gag-PKB cells. The cells assumed a spherical shape and they acquired characteristic lipid droplets that stained positively for Oil Red O. Northern blot analysis detected upregulation of LPL and aP2 mRNA, specific indicators of adipocyte differentiation. Our data demonstrate that constitutive activation of PKB is sufficient to trigger adipocyte differentiation.
Apoptosis is critical for mammalian tissue homeostasis, and its disruption has been linked to a wide variety of disorders, including cancer, neurodegenerative disease, autoimmune disease and diabetes. This review will focus on recent investigations that have begun to address the potential role of apoptosis in adipose tissue growth. Evidence for apoptosis occurring in mature adipocytes has been obtained through the use of in vitro cell culture models as well as in vivo studies in rodents and humans. Preadipocytes, ®broblast-like adipocyte precursor cells, can also undergo apoptotic cell death. As they differentiate, preadipocytes acquire a relative resistance to apoptosis. The levels of the cell survival proteins Bcl-2 and neuronal apoptosis inhibitory protein (NAIP) have been observed to increase during adipogenesis. Further research on the effect of apoptosis on adipose tissue cellularity should clarify its in¯uence on adipose tissue mass and distribution.
We have used the 3T3-L1 and 3T3-F442A preadipocyte cell lines to examine the expression and regulation of neuronal apoptosis inhibitory protein (NAIP) during adipocyte differentiation. When 3T3-L1 preadipocytes differentiated into adipocytes, they developed resistance to apoptosis induced by growth factor deprivation, as assessed by terminal deoxynucleotide transferase (TdT)-mediated dUTP nick end labeling. Protein expression of NAIP was markedly elevated in 3T3-L1 and 3T3-F442A adipocytes compared with that in their fibroblast-like precursors. NAIP was also present in rat white adipocytes. In 3T3-L1 cells, the increase in NAIP occurred by day 4 of the 8-day differentiation protocol, which includes exposure of confluent preadipocytes to insulin, dexamethasone, and isobutylmethylxanthine. Incubation of confluent 3T3-L1 preadipocytes with any of these components alone had no effect on NAIP expression. When 3T3-C2 cells, a control cell line that does not differentiate, were subjected to the differentiation protocol, the low NAIP levels remained unaltered. Addition of rapamycin, a p70 S6 kinase inhibitor that blocks adipocyte differentiation, to the 3T3-L1 differentiation medium prevented the rise in NAIP expression. These data demonstrate for the first time that NAIP is expressed in adipocyte cell lines and primary adipocytes. The differentiation-dependent augmentation of NAIP protein levels in 3T3-L1 adipocytes is closely correlated with the development of resistance to apoptosis induced by growth factor deprivation, suggesting a potential role for NAIP in these cells.
OBJECTIVE: Little is known about the regulation of apoptosis in the adipocyte. Using the 3T3-L1 cell model of adipogenesis, we investigated whether induction of apoptosis by serum-starvation is differentiation-speci®c. METHODS: Apoptosis was assessed by phase-contrast microscopy, Hoechst staining and DNA fragmentation. Protein expression levels were evaluated by immunoblot analysis. RESULTS: Following serum deprivation, Hoechst staining revealed chromatin condensation and formation of apoptotic bodies in 3T3-L1 preadipocytes, but not in differentiated adipocytes. Similarly, although cell cultures of serum-starved 3T3-L1 preadipocytes displayed extensive apoptotic DNA fragmentation, this was barely detectable for cell cultures subjected to differentiation medium, in which 70 ± 90% of the cells had assumed the mature phenotype. Consistent with the ability of the adipocytes to resist apoptosis, immunoblot analysis revealed that differentiated cell cultures expressed Bcl-2 and the endonuclease DNase I at higher and lower levels, respectively. CONCLUSION: Expression of cell survival genes is modulated during 3T3-L1 adipocyte differentiation, potentially contributing to the state of resistance to apoptosis observed for mature 3T3-L1 adipocytes upon growth factor deprivation.
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