Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor alpha and cAMP

We have used murine 3T3-L1 cells, which differentiate in culture and acquir e morphological and biochemical features of mature adipocytes, as a model f or studying the expression of cyclic-nucleotide phosphodiesterase (PDE) 3B activity, protein and mRNA during differentiation and during long-term trea tment of the cells with tumour necrosis factor alpha (TNF-alpha), a cytokin e associated with insulin resistance, and a cAMP analogue, N-6,2'-O-dibutyr yl cAMP (dbcAMP). PDE3B activity, protein and mRNA could be detected 4 days after the initiation of differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-alpha for 24 h produced a maximal ( 50%) decrease in PDE3B activity, protein and mRNA, which was well correlate d with both activation of protein kinase A (PKA) and stimulation of lipolys is, presumably reflecting an increase in intracellular cAMP concentration. To investigate the effect of cAMP on PDE3B we treated 3T3-L1 adipocytes wit h dbcAMP. After 4 h with 0.5 mM dbcAMP, PDE3B activity was decreased by 80% , which was also correlated with a decrease in PDE3B protein and mRNA. This effect was abolished in the presence of N-[2-(bromocinnamylamino)ethyl]-5- isoquinoline sulphonamide] (H-89), a specific PKA inhibitor. We conclude th at the lipolytic effect of TNF-alpha involves the down-regulation of PDE3B, which is associated with increased activation of PKA, presumably owing to increased levels of cAMP. In addition, the PKA activation induced by dbcAMP resulted in the downregulation of PDE3B. These results, which suggest that PDE3B is a novel target for long-term regulation by TNF-alpha and cAMP, co uld contribute to the understanding of the mechanisms of insulin resistance .