LONG-TERM PHORBOL ESTER TREATMENT DOWN-REGULATES THE BETA(3)-ADRENERGIC RECEPTOR IN 3T3-F442A ADIPOCYTES

The role of protein kinase C (PKC) in the regulation of the beta(3)-adrenergic receptor (beta(3)-AR) gene was examined in murine 3T3-F442A adipocytes, which express this receptor subtype at a high level, We also investigated the involvement of this kinase in the modulation of beta(3)-AR gene expression by insulin, Long term exposure of 3T3-F442A adipocytes to phorbol 12-myristate 13-acetate (PMA) decreased beta(3)-AR mRNA content in a time- and concentration dependent manner, with maximal changes observed at 6 h (6.5-fold decrease) and at 100 nM PMA. This inhibition was selective for beta(3)-AR transcripts, since beta(1)- and beta(2)-AR mRNA content remained unchanged, Also, (-)-[I-125] cyanopindolol saturation and competition binding experiments on adipocyte membranes indicated that PMA induced an similar to 2-fold decrease in beta(3)-AR expression, while that of the two other subtypes was not affected, This correlated with a lower efficacy of beta(3)-AR agonists to stimulate adenylyl cyclase, Conversely, long term exposure to PMA did not alter adenylyl cyclase activity in response to guanosine 5'-O-(3-thiotriphosphate) or forskolin, The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not repress beta(3)-AR mRNA levels. Inhibition of beta(3)-AR mRNA by PMA was suppressed by the PKC-selective inhibitor bisindolylmaleimide, and was not observed in PKC-depleted cells, indicating that PKC was involved in this response, mRNA turnover experiments showed that the half-life of beta(3)-AR transcripts was not affected by long term PMA exposure. When 3T3-F442A adipocytes were pretreated with PMA for 24 h to downregulate PKC, or with bisindolylmaleimide, the insulin-induced inhibition of beta(3)-AR mRNA levels was reduced by 44-67%, These findings demonstrate that sustained PKC activation exerts a specific control of beta(3)-AR gene expression and is involved, at least in part, in the modulation by insulin of this adrenergic receptor subtype.