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Long term phorbol ester treatment down-regulates the beta 3-adrenergic receptor in 3T3-F442A adipocytes
B Fève1, F Piétri-Rouxel, K el Hadri
1INSERM Unité82, Hôpital Henri Mondor, Créteil, France.
Abstract:
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, (-)-[125I]cyanopindolol saturation and competition binding experiments on adipocyte membranes indicated that PMA induced an approximately 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 down-regulate 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.
Insights
Protein kinase C (PKC) activation selectively reduces beta 3-adrenergic receptor (beta 3-AR) gene expression in adipocytes. This pathway is also involved in how insulin modulates beta 3-AR levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Protein kinase C (PKC) is a key signaling enzyme involved in various cellular processes.
- The beta 3-adrenergic receptor (beta 3-AR) plays a significant role in adipocyte function and energy metabolism.
- Understanding the regulation of beta 3-AR gene expression is crucial for metabolic research.
Purpose of the Study:
- To investigate the role of PKC in regulating beta 3-AR gene expression in murine 3T3-F442A adipocytes.
- To examine the involvement of PKC in insulin-mediated modulation of beta 3-AR gene expression.
Main Methods:
- Treatment of 3T3-F442A adipocytes with phorbol 12-myristate 13-acetate (PMA) to activate PKC.
- Quantitative analysis of beta 3-AR mRNA levels using RT-PCR.
- Measurement of receptor expression and function via radioligand binding and adenylyl cyclase activity assays.
- Use of PKC inhibitors and PKC-depleted cells to confirm PKC involvement.
Main Results:
- PMA treatment significantly decreased beta 3-AR mRNA and protein expression in a time- and concentration-dependent manner.
- The inhibitory effect of PMA was specific to beta 3-AR, with no significant changes observed for beta 1-AR and beta 2-AR.
- PKC activation reduced the efficacy of beta 3-AR agonists in stimulating adenylyl cyclase.
- PKC depletion or inhibition attenuated the insulin-induced downregulation of beta 3-AR mRNA levels.
Conclusions:
- Sustained activation of PKC specifically controls beta 3-AR gene expression in adipocytes.
- PKC signaling is partially involved in the insulin-mediated regulation of beta 3-AR expression.
- These findings elucidate a novel regulatory mechanism for beta 3-AR in metabolic control.