Cyclic Nucleotide Phosphodiesterase 3B: Regulation in rat adipocytes and 3T3-L1 cells

Abstract: Insulin stimulation of rat adipocytes results in phosphorylation and activation of the cyclic nucleotide phosphodiesterase 3B (PDE3B), a key enzyme in the antilipolytic signalling pathway of this hormone. In this thesis, the site phosphorylated in PDE3B upon stimulation of rat adipocytes with insulin and/or the beta-adrenergic agonist, isoproterenol is identified. In addition, upstream components in the insulin-induced signalling pathway, leading to phosphorylation and activation of PDE3B, are reported. Also, long-term regulation of the expression of PDE3B in response to tumour necrosis factor-alpha (TNF-a) and cyclic adenosine monophosphate (cAMP) were investigated in 3T3-L1 adipocytes. Stimulation of rat adipocytes with insulin and/or isoproterenol results in phosphorylation of PDE3B at a unique site, serine302, which is also associated with activation of the enzyme. One important upstream component in the insulin-mediated signalling pathway, resulting in phosphorylation and activation of PDE3B, include phosphatidylinositol-3 kinase (PI3K), although this kinase is not directly responsible for the insulin-stimulated phosphorylation and activation of PDE3B. Insulin-stimulated PI3K-dependent kinases include the serine/threonine protein kinase B (PKB), mitogen-activated protein kinase (MAPK) and p70 ribosomal S6 kinase (p70S6K). While MAPK and p70S6K are not involved in the insulin-induced antilipolytic signalling pathway, leading to phosphorylation and activation of PDE3B, data are presented in this thesis, suggesting that PKB represents a potential PDE3B kinase. Expression of PDE3B can be detected 4 days after initiation of differentiation of 3T3-L1 cells. Treatment of mature 3T3-L1 adipocytes with TNF-a results in downregulation of the expression of PDE3B , which correlates with both activation of protein kinase A (PKA), presumably reflecting an increase in intracellular cAMP concentrations, and with stimulation of lipolysis. Chronically treatment of 3T3-L1 adipocytes with cAMP also produces a reduction in the expression of PDE3B, an effect mediated via PKA. These results suggest that PDE3B is a novel target for long-term regulation by TNF-a and cAMP, which could contribute to the understanding of the underlying mechanisms for insulin resistance.