Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion.
Inhibition of insulin-induced 3T3-L1 preadipocyte differentiation by rapamycin has been attributed to a blockade of the early critical clonal expansion phase of the adipogenic program. Rapamycin binds to, and inhibits, mTOR (mammalian target of rapamycin), leading to diminution of p70 S6 kinase activity and eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) function. Our objective was to determine if rapamycin-sensitive pathways exist subsequent to the clonal expansion phase. We determined that the mitotic clonal 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 potent negative effects on glycerol phosphate dehydrogenase activity, and triacylglycerol accumulation, as well as on the protein expression of adipogenic transcription factors, C/EBPalpha and PPARgamma. Insulin-stimulated p70 S6 kinase activity, and its inhibition by rapamycin, were comparable in preadipocytes at day 0 vs. day 4 post-differentiation. We conclude that a component of the adipogenic program, operating after the completion of clonal expansion, is inhibited by rapamycin, suggesting an ongoing need for mTOR function in this process.
Gagnon
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Department of Medicine and Biochemistry, Ottawa Health Research Institute, Ottawa Hospital, University of Ottawa, Ottawa, Canada.
Lau
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Sorisky
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D016475:3T3 Cells; D017667:Adipocytes / Q000502:physiology*; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D003561:Cytarabine / Q000494:pharmacology; D005993:Glycerolphosphate Dehydrogenase / Q000037:antagonists & inhibitors; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000037:antagonists & inhibitors; D020123:Sirolimus / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000378:metabolism; D014280:Triglycerides / Q000096:biosynthesis
D022763:CCAAT-Enhancer-Binding Protein-alpha; D007328:Insulin; D047428:Protein Kinase Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D014280:Triglycerides; D003561:Cytarabine; D005993:Glycerolphosphate Dehydrogenase; D011494:Protein Kinases; C546843:mTOR protein, mouse; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus