EXPRESSION OF TYPE III PHOSPHODIESTERASE IN NIH 3T3 CELLS

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In isolated rat adipocytes, activation of a CGMP-inhibited cyclic nucleotide phosphodiesterase (CGI PDE) by insulin is an important part of the antilipolytic action of this hormone. This activation is associated with phosphorylation of serine site(s) on the CGI PDE. However, the mechanism and the functional significance of this activation in terms of the cellular action of insulin is not yet well understood. Recently, the CDNA (RcGIP1) for the rat adipocyte CGI PDE has been cloned from rat adipose tissue CDNA libraries. In order to demonstrate that this CDNA encodes an insulin-sensitive CGI PDE we expressed this CDNA in mammalian cells overexpressing the insulin receptor. A recombinant plasmid RcGIP1-pBPV (bovine papilloma virus promoter) was transfected in 3006 cells, a stable cell line of NIH 3T3 fibroblasts overexpressing the insulin receptor. Among fifty clones obtained after screening, twelve exhibited CAMP PDE activity that is inhibited at least 75% by 0.5 microM cilostamide, a selective inhibitor of the CGI PDE family. Specific activities of these positive clones are at least 100-fold higher than the specific activity measured in control cells transfected by pBPV alone. The expressed PDE exhibited the predicted molecular mass (about 135-Kda) and cross-reacted with affinity purified anti-platelet CGI PDE antibody. Treatment of the transfected cells by insulin (10-9M for 15 min) increased the CAMP PDE activity of the crude homogenates by 30 to 100%. Stimulation of 265% by insulin was observed in the microsomal fraction (105,000 g pellet) of one positive clone where the activity of the particulate fraction represents 80% of the total activity of the crude homogenate. These results confirm that the RcGIP1 CDNA encodes for an insulin sensitive membrane-associated CGI PDE. Work is now in progress in order to determine whether the observed insulin stimulation involves phosphorylation of the enzyme.