Role Of CAMP Signaling In Adipocyte Differentiation Of Human Mesenchymal Stem Cells Derived From Adipose Tissue

Adpocytes have a crucial role in regulating the energy balance, glucose homostasis and imflammation not only because they are the main cellular component of energy storage, but also because of their function as endocrine cells. Adipocytes are derived from Mesenchymal stem cells. Adipogenesis is a highly controlled process which is modulated by various hormons and nutritional signals. Dysregulation of this process will result in dysfunction of adipose tissue, which may lead to metabolic disease such as insulin resistance, obesity, type Ⅱ diabete, cardiovascular disease and hypertension. Therefore understanding the cellular and molecular basis of adipogenesis is an important area of research that may lead to the development of therapies to treat or prevent obesity.In this study, we investigated the effects of cAMP signalings PPAR ligands and various kinds of fatty acids on the adipogenic differentiation of human mesenchymal stem cells derived from adipose tissue (hMADS). Firstly, we characterized the cellular and molecular events that take place during adipogenic differentiation of hMADS cells in serum free condition. By using real-time RT-PCR, We detected the expression change of adipogenic genes including PPARγ2s C/EBPα、FABP4and LPL during the differentiation. The results indicated adipogenic differentiation of hMADS cells can be characterized into three phases, while Dex、IBMX and insulin are required for the early differentiation which is from dayO to day3.Secondly, we investigated the role of cAMP signaling in adipocyte conversion of hMADS cells. We show that elevated levels of introcellular cAMP is required to promote adipocyte differentiation of hMADS cells. In the absence of elevated levels of cAMP, only very few cells underwent differentiation in the presence of of Dex and Insulin. Most intracelluar effects of cAMP are mediated by Protein kinase A (PKA) and Exchange protein directly activated by cAMP (Epac). Although the critical role of PKA in adipogenesis has been well accepted, the role of Epac in adipogenesis of human mesenchymal stem cells is not understood yet. Thus we tested wether the selective Epac-activating analog007, alone or in combination with the selective PKA-activating analog MB, could mimic the effects of IBMX in stimulating adipogenesis. Our results show that the adipogenic effect of cAMP requires activation of both Epac and PKA in hMADS cells. Furthermore, we inhibitd the endogenous Epac/Rapl signaling by transducing hMADS cells with a retroviral vector expressing a dominant negative Epac1. hMADS cells expressing dnEpac1can not be differentiated into adipocytes, which indicated that Epacl signaling is required for adipogenesis. All together these results proved that cAMP signaling involving the simultaneous activation of both PKA-and Epac-dependent pathway is critical for adipogenic differentiation of hMADS cells.Thirdly, we characterized the temporal contribution of the different PPAR isoforms in adipogenesis of hMADS cells. Our results show that PPARy is the most critical isoform for adipogenesis, while PPARσ can promote adipogenic differentiation only by prolonged treatment. Only weak differentiation was observed in cells treated with the PPARa agonist. Finally, we tested the ability of a series of fatty acids to promote adipogenesis of hMADS cells. In murine preadipocytes, fatty acids has been shown to promote adipogenesis by binding and activating PPARs. However, in our results, none of the tested fatty acids could promot adipocyte differentiation of hMADS cells by replacing the PPAR ligand of induction media. Except working as PPAR ligands, n-6PUFA can be metablized into prostaglandins including PGI2, while PGI2could promte differentiation of the murine preadipocytes by stimulating intracellular cAMP production. Our results shown that in hMADS cells, fatty acids could not promote adipogenesis by replacing the cAMP elevating reagent in the induction media. However, carbaprostacyclin, a stable analog of PGI2, can sufficiently rescue adipocyte differentiation of hMADS cells in the absence of IBMX, suggesting that PGI2synthesis or release may be impaired in hMADS cells. In summary, our study emphasize the need for cAMP signaling in concert with treatment with a PPARy ligand to secure efficient adipocyte differentiation of human mesenchymal stem cells. More than that, we proved that activation of both PKA-and Epac-dependent pathways are necessary to promote adipocyte differentiation of hMADS cells. Unlike the case for murine preadipocytes cell lines, fatty acids including arachidonic acid did not promote adipocyte differentiation of hMADS cells, thereby clearly distinguishing the human mesenchymal stem cells from murine preadipocytes cell lines. Our findings have provide further insights to understand the role of cAMP siganaling in controling the adipogenesis of hMADS cells. These information will be useful to develop novel therapies to treat or prevent obesity.