RNA-seq analysis of the effects of forskolin treatment on human primary adipocytes

Analysis of forskolin-stimulated cAMP signalling on adipocyte metabolism at gene expression level. Results provide important information of the response of adipocytes to cAMP signaling (a cold-conditioned mimc), such as specific metabolic genes, up- or down-regulated that control expression of putative adipokines. Human white primary preadipocytes (PromoCell, Heidelberg, Germany) were seeded (10,000-15,000 cells/cm2) and grown to confluence (37°C, 5% CO2) in preadipocyte growth medium (0.05ml/ml fetal calf serum, 0.004ml/ml endothelial cell growth supplement, 10ng/ml epidermal growth factor, 1µg/ml hydrocortisone, 90µg/ml heparin). Preadipocytes were differentiated according to supplier’s instructions. Briefly, growth medium was replaced by differentiation media (8µg/ml d-biotin, 0.5µg/ml insulin, 400ng/ml dexamethasone, 44µg/ml isobutylmethylxanthine, 9ng/ml L-thyroxine, 3µg/ml ciglitazone) for 48 hours. Differentiation medium was replaced with adipocyte nutrition media (0.03ml/ml fetal calf serum, 8µg/ml d-biotin, 0.5µg/ml insulin, 400ng/ml dexamethasone) for 12 days until cells were fully differentiated. Media was changed every 48 hours. Human white primary adipocytes were treated with 1 μM forskolin on days 10 - 12 of differentiation or remained untreated (control). Total RNA was then isolated.