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Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion
1Department of Medicine and Biochemistry, Ottawa Health Research Institute, Ottawa Hospital, University of Ottawa, Ottawa, Canada.
Abstract:
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.
Insights
Rapamycin inhibits adipogenesis even after cell division, impacting fat cell development. This suggests mammalian target of rapamycin (mTOR) is crucial for later stages of fat cell differentiation.
Area of Science:
- Cell Biology
- Biochemistry
- Metabolism
Background:
- Rapamycin inhibits mammalian target of rapamycin (mTOR), affecting cell growth and proliferation.
- Previous studies linked rapamycin's inhibition of adipogenesis to blocking early clonal expansion in 3T3-L1 cells.
Purpose of the Study:
- To investigate if rapamycin-sensitive pathways are involved in adipogenesis after the clonal expansion phase.
- To determine the role of mTOR in later stages of 3T3-L1 cell differentiation.
Main Methods:
- 3T3-L1 preadipocytes were induced to differentiate.
- Cytosine beta-D-arabinofuranoside (Ara-C) was used to determine the completion of mitotic clonal expansion.
- Rapamycin was administered on day 4 of differentiation to assess its effects on adipogenesis.
- Glycerol phosphate dehydrogenase activity, triacylglycerol accumulation, and adipogenic transcription factor expression were measured.
Main Results:
- Mitotic clonal expansion was completed by day 4 of differentiation.
- Rapamycin treatment initiated on day 4 significantly inhibited adipogenesis markers, including glycerol phosphate dehydrogenase activity and triacylglycerol accumulation.
- Expression of key adipogenic transcription factors (C/EBPalpha and PPARgamma) was reduced by late-stage rapamycin treatment.
- Insulin-stimulated p70 S6 kinase activity and its inhibition by rapamycin were observed at both early (day 0) and late (day 4) stages.
Conclusions:
- A rapamycin-sensitive component of the adipogenic program operates after clonal expansion is complete.
- Mammalian target of rapamycin (mTOR) function is necessary for later stages of adipogenesis in 3T3-L1 cells.
- These findings suggest a more complex role for mTOR in adipogenesis than previously understood.