Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, Buenos Aires, 1428, Argentina.
Dev Growth Differ. 2014 Feb;56(2):143-51. doi: 10.1111/dgd.12114. Epub 2014 Jan 20.
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.
脂肪生成是由胰岛素、地塞米松和异丁基甲基黄嘌呤（IBMX）组合刺激 3T3-L1 成纤维细胞的，（I+D+M）。两种转录因子对于获得脂肪细胞表型很重要，即 C/EBPβ（CCAAT 增强子结合蛋白β）和 PPARγ（过氧化物酶体增殖物激活受体γ）。IBMX 增加 cAMP 含量，这可以激活蛋白激酶 A（PKA）和/或 EPAC（cAMP 激活的交换蛋白）。为了研究 IBMX 在分化混合物中的重要性，我们首先评估了添加 IBMX 对 C/EBPβ 和 PPARγ增加的影响，发现这两种蛋白质的含量都增加了。添加 IBMX（I+D+M）或用 cAMP 类似物，二丁酰基-cAMP 或 8-（4-氯苯基硫代）-2-O'- 甲基-cAMP（8CPT-2-Me-cAMP）替代 IBMX，后者激活 EPAC 而不是 PKA，显著增加了 PPARγmRNA。然而，I+D 或任何一种诱导剂单独使用，都不能使 PPARγmRNA 增加到相似的程度，这表明 IBMX 和 I+D 的存在都很重要。还发现添加 IBMX 或 8CPT-2-Me-cAMP 能够增加 C/EBPβ相对于 I+D 的含量。与这些发现一致，微阵列分析表明，分化混合物中存在 8CPT-2-Me-cAMP 或 IBMX 能够上调 PPARγ 和 PPARγ 激活基因以及其他参与脂质代谢的基因。我们的结果证明了 IBMX-cAMP-EPAC 在 3T3-L1 成纤维细胞分化过程中调节脂肪生成基因的参与，因此有助于阐明环 AMP 在这一过程中的作用。
