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. I-125-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 similar to 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-elhoxy)-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 antagonists indicated that the beta(1)-subtype accounted for the totality of beta-adrenoceptor-mediated adenylyl cyclase activation in young adipocytes. In mature adipose cells similar to 90% of this response was due to an activation of the beta(3)-adrenoceptor. In addition, similar to 84% of the maximal norepinephrine-stimulated lipolysis was mediated by the beta(3)-adrenoceptor in fully differentiated adipocytes. The differentiation-dependent expression of P-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.