Exchange protein activated by cyclic AMP is involved in the regulation of adipogenic genes during 3T3-L1 fibroblasts differentiation.
Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone and isobutylmethylxanthine, IBMX, (I+D+M). Two transcription factors are important for the acquisition of the adipocyte phenotype, C/EBP beta (CCAT enhancer-binding protein beta) and PPAR gamma (peroxisome proliferator-activated receptor gamma). IBMX increases cAMP content, which can activate protein kinase A (PKA) and/or EPAC (exchange protein activated by cAMP). To investigate the importance of IBMX in the differentiation mixture, we first evaluated the effect of the addition of IBMX on the increase of C/EBP beta and PPAR gamma and found an enhancement of the amount of both proteins. IBMX addition (I+D+M) or its replacement with a cAMP analogue, dibutyryl-cAMP or 8-(4-chlorophenylthio)-2-O'-methyl-cAMP (8CPT-2-Me-cAMP), the latter activates EPAC and not PKA, remarkably increased PPAR gamma mRNA. However, neither I+D nor any of the inducers alone, increased PPAR gamma mRNA to a similar extent, suggesting the importance of the presence of both IBMX and I+D. It was also found that the addition of IBMX or 8CPT-2-Me-cAMP was able to increase the content of C/EBP beta with respect to I+D. In agreement with these findings, a microarray analysis showed that the presence of either 8CPT-2-Me-cAMP or IBMX in the differentiation mixture was able to upregulate PPAR gamma and PPAR gamma-activated genes as well as other genes involved in lipid metabolism. Our results prove the involvement of IBMX-cAMP-EPAC in the regulation of adipogenic genes during differentiation of 3T3-L1 fibroblasts and therfore contributes to elucidate the role of cyclic AMP in this process.
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, Buenos Aires, 1428, Argentina.
Matías
M
Gabrielli
Claudia N
CN
Martini
Javier N
JN
Brandani
Laura J R
LJ
Iustman
Damián G
DG
Romero
María
M
del C Vila
D015056:1-Methyl-3-isobutylxanthine / Q000378:metabolism; D041721:3T3-L1 Cells; D050156:Adipogenesis / Q000235:genetics* / Q000502:physiology; D000704:Analysis of Variance; D000818:Animals; D001391:Azo Compounds; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism; D002452:Cell Count; D002454:Cell Differentiation / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D051379:Mice; D046228:Microarray Analysis; D008856:Microscopy, Fluorescence; D047495:PPAR gamma / Q000378:metabolism
D001391:Azo Compounds; D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D047495:PPAR gamma; D003907:Dexamethasone; D000242:Cyclic AMP; C011049:oil red O; D015056:1-Methyl-3-isobutylxanthine
3T3-L1 cells; CCAT enhancer-binding protein beta; adipogenesis; cyclic AMP; peroxisome proliferator-activated receptor gamma