Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development.

Triiodothyronine regulates beta 3-adrenoceptor expression in 3T3-F442A differentiating adipocytes.

The effect of thyroid hormones on the beta 3-adrenoceptor expression was studied in the preadipose 3T3-F442A cell line. As assessed by molecular and pharmacological analyses, triiodothyronine addition to differentiating 3T3-F442A cells caused a 2.3-fold increase in beta 3-adrenoceptor mRNA levels, which was correlated with a parallel induction of beta 3-adrenoceptor number and of beta 3-adrenoceptor coupling to the adenylate cyclase system. Nuclear transcription experiments showed that triiodothyronine did not significantly alter the transcription rate of the beta 3-adrenoceptor gene. By contrast, the hormone increased by 36% the half-life of beta 3-adrenoceptor mRNA. Triiodothyronine exhibited a discrete effect on beta 3-adrenoceptor expression when added to mature 3T3-F442A adipocytes. This study indicates that thyroid hormones exert a differentiation-dependent and post-transcriptional regulation of beta 3-adrenoceptor expression in adipocytes.

Developmental expression and functional activity of beta 1- and beta 3-adrenoceptors in murine 3T3-F442A differentiating adipocytes.

Beta 1- and beta 3-adrenoceptor mRNA and protein expression, and contribution of each subtype to the catecholamine-sensitive adenylyl cyclase system were studied during the adipose conversion of the murine 3T3-F442A cell line. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that emergence of beta 3-adrenoceptor transcripts was concomittant with that of the gene encoding adipsin, a very late marker of adipose differentiation. Conversely, the induction of the beta 1-adrenoceptor mRNA occurred early after cell commitment towards adipose conversion. Changes in beta-subtype gene expression were accompanied by parallel modifications in receptor expression and function. 125I-cyanopindolol saturation and competition binding experiments showed a 3-fold increase in beta 1-adrenoceptor density in day 3 post-confluent cells. The beta 3-subtype population became detectable later and represented approximately 95% of total beta-adrenoceptors in day 8 and day 12 post-confluent cells. Adenylyl cyclase activity in response to the beta 3-adrenoceptor-selective agonists CGP12177 (4-(3-t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one), ICI201651 ([(R)-4-(2 hydroxy-3-phenoxypropylamino-ethoxy)-N-(2- methoxyethyl)phenoxy-acetamide]) and cyanopindolol was virtually absent in young adipocytes, but dramatically increased in mature cells. The respective contributions of the beta 1- and the beta 3-subtypes to the production of cAMP were resolved by an Eadie-Hofstee computer analysis of isoproterenol and norepinephrine concentration-response curve of adenylyl cyclase activity. Agonist response curves in the presence of beta 1- and beta 2-adrenoceptor antagonist indicated that the beta 1-subtype accounted for the totality of beta-adrenoceptor-mediated adenylyl cyclase activation in young adipocytes. In mature adipose cells approximately 90% of this response was due to an activation of the beta 3-adrenoceptor. In addition, approximately 84% of the maximal norepinephrine-stimulated lipolysis was mediated by the beta 3-adrenoceptor in fully differentiated adipocytes. The differentiation-dependent expression of beta-subtypes in adipocytes is a biphasic process involving an initial and moderate induction of beta 1-adrenoceptors followed by the emergence of a prominent beta 3-adrenoceptor population. Compared analysis of both receptor occupancy and cAMP production shows that the beta 3-subtype is more efficiently coupled to the adenylyl cyclase system than the beta 1-adrenoceptor. Thus in mature adipose cells this receptor subtype represents the core of cAMP-dependent regulation of the lipolytic, antilipogenic and thermogenic effects of catecholamines.

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, (-)-[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.