Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion

Inhibition of insulin-induced 3T3-L1 preadipocyte differentiation by rapamy cin has been attributed to a blockade of the early critical clonal expansio n phase of the adipogenic program. Rapamycin binds to, and inhibits, mTOR ( mammalian target of rapamycin), leading to diminution of p70 S6 kinase acti vity and eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) func tion. Our objective was to determine if rapamycin-sensitive pathways exist subsequent to the clonal expansion phase. We determined that the mitotic cl onal expansion was complete by day 4 of the differentiation protocol, based on the response to Ara-C (cytosine beta -D-arabinofuranoside), which only inhibits differentiation when administered during this phase. Treatment of differentiating 3T3-L1 cells with rapamycin, starting on day 4, exerted pot ent negative effects on glycerol phosphate dehydrogenase activity, and tria cylglycerol accumulation, as well as on the protein expression of adipogeni c transcription factors, C/EBP alpha and PPAR gamma. Insulin-stimulated p70 S6 kinase activity, and its inhibition by rapamycin, were comparable in pr eadipocytes at day 0 vs. day 4 post-differentiation. We conclude that a com ponent of the adipogenic program, operating after the completion of clonal expansion, is inhibited by rapamycin, suggesting an ongoing need for mTOR f unction in this process. J. Cell. Physiol. 189: 14-22, 2001. (C) 2001 Wiley -Liss, Inc.