Developmental expression and functional activity of β1- and β3-adrenoceptors in murine 3T3-F442A differentiating adipocytes

Abstract

β 1- and β 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 β 3-adrenoceptor transcripts was concomittant with that of the gene encoding adipsin, a very late marker of adipose differentiation. Conversely, the induction of the β 1-adrenoceptor mRNA occurred early after cell commitment towards adipose conversion. Changes in β-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 β 1-adrenoceptor density in day 3 post-confluent cells. The β 3-subtype population became detectable later and represented ∼ 95% of total β-adrenoceptors in day 8 and day 12 post-confluent cells. Adenylyl cyclase activity in response to the β 3-adrenoceptor-selective agonists CGP12177 (4-(3- t-butylamino-2-hydroxypropoxy)-benzimidazol-2-one), ICI201651 ([( R)-4-(2hydroxy-3-phenoxypropylamino-ethoxy)- N-(2-methoxyethyl)phenoxy-acetamidel]) and cyanopindolol was virtually absent in young adipocytes, but dramatically increased in mature cells. The respective contributions of the β 1- and the β 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 β 1- and β 2-adrenoceptor antagonists indicated that the β 1-subtype accounted for the totality of β-adrenoceptor-mediated adenylyl cyclase activation in young adipocytes. In mature adipose cells ∼ 90% of this response was due to an activation of the β 3-adrenoceptor. In addition, ∼ 84% of the maximal norepinephrine-stimulated lipolysis was mediated by the β 3-adrenoceptor in fully differentiated adipocytes. The differentiation-dependent expression of β-subtypes in adipocytes is a biphasic process involving an initial and moderate induction of β 1-adrenoceptors followed by the emergence of a prominent β 3-adrenoceptor population. Compared analysis of both receptor occupancy and cAMP production shows that the β 3-subtype is more efficiently coupled to the adenylyl cyclase system than the β 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.