The impact of cGMP and cAMP dependent pathways on the differentiation of human preadipocytes

Objective: Obesity and the Metabolic Sydrome are correlated with chronic low-grade inflammation characterized by increased plasma levels of proinflammatory mediators. Thus, the induction of inflammation could be used to stimulate adipose tissue formation in tissue engineering approaches. Since nitric oxide (NO) is a key regulator of inflammation and has been demonstrated to have lipogenic properties we investigated the effect of NO and its downstream targets cGMP and cAMP, respectively, on preadipocytes in vitro. Methods: Preadipocytes were isolated from human subcutaneous adipose tissue samples, cultured until confluence, and differentiated by insulin, pioglitazone, dexamethasone, isomethylbutylxanthine, and transferrin. DETA/NO (30–150 μM) was added during proliferation and differentiation. Additionally, the specific guanylyl cyclase inhibitor ODQ, the adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), the cGMP/cAMP-analogues 8-Br-cGMP, 8-pCPT-cGMP, and 8-Br-cAMP, and the adenylyl cyclase activator forskolin were applied. Proliferation was evaluated by microscopy and XTT-test, differentiation was assayed by glycerophosphate dehydrogenase (GDDH) activity. Results: DETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes (p<0.05 compared to control). Proliferation, in contrast, was inhibited in the presence of NO. The application of the downstream targets of NO action, cGMP and cAMP, respectively, increased preadipocyte differentiation even to a higher extend than NO (p<0.01). In contrast, the addition of inhibitors of the underlying pathways resulted in a significant decrease in adipogenic conversion. Conclusion: Our results so far supported the application of NO donors during transplantation of preadipocytes in a three-dimensional setting to accelerate and optimize differentiation of preadipocytes. With the present results, we argue that instead of the rather instable NO, the application of the NO downstream mediators cGMP and cAMP would be even more effective since these substances have a stronger adipogenic effect on preadipocytes and a much higher molecule stability than NO. Also, by applying inhibitors of the underlying pathways the induced inflammatory condition could be modulated to reach the desired level.