β ‐Adrenoceptor subtype expression and function in rat white adipocytes

The pharmacological features of rat white adipocyte β‐adrenoceptor subtypes were investigated by saturation and β‐agonist competition studies with [ 3 H]‐CGP 12177 and by lipolysis induced by β‐agonists as well as their inhibition by CGP 20712A (selective β 1 ‐antagonist) and ICI 118551 (selective β 2 ‐antagonist) in an attempt to establish a relationship between the functionality and binding capacity of β‐adrenoceptor subtypes. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22±0.07 n m ) and the other with low affinity (23±7 n m ). The low affinity binding sites constituted 90% of the total binding sites. The competition curves, with 15 n m [ 3 H]‐CGP 12177, for the β‐agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective β 3 ‐agonist, BRL 37344 (BRL), were clearly biphasic ( P <0.001). The rank orders of agonist potency (p K i ) in competing for [ 3 H]‐CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28±0.24)>NA (8.90±0.12)>Ad (8.65±0.12)>>BRL (4.53±0.17) and BRL (7.38±0.19)>>Iso (2.96±0.26)NA (2.80±0.17)>Ad (2.10±0.11) indicating the expression of β 1 ‐ and β 3 ‐adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective β 1 ‐agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81±9 fmol mg −1 ) and high p K i value (7.23±0.26) confirming the presence of β 1 ‐adrenoceptors. To assess a possible contribution of the β 2 ‐subtype, procaterol (Proc), a selective β 2 ‐agonist, was used to compete with 2 n m [ 3 H]‐CGP 12177. A single low affinity (4.61±0.07) population of binding sites was identified. The density of these sites (71±12 fmol mg −1 ) was similar to the one obtained with Xam, suggesting that Proc displaced [ 3 H]‐CGP 12177 from the β 1 ‐subtype. The functional potency (pD 2 ) order with BRL (9.07±0.20) and catecholamines (Iso: 7.26±0.06, NA: 6.89±0.02 and Ad: 6.32±0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31±0.06 and 5.66±0.10, respectively. Proc stimulated lipolysis with a low potency (5.59±0.21). The lipolytic response to 0.001 μ m BRL was inhibited by both, selective β 1 ‐ and β 2 ‐antagonist, in a monophasic manner with low potencies (CGP 20712A p K i : <4.5 and ICI 118551 p K i : 5.57±0.13). Similar monophasic profiles were obtained for inhibition of Xam‐ and Dob‐induced lipolysis. In this case, CGP 20712A was more potent (>10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 μ m Iso or 0.13 μ m NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The p K i values for the first sites were 8.41±0.09 and 8.58±0.17, respectively, and for the second population of sites 4.73±0.22 and 4.27±0.27, respectively. The proportion of the first sites was low: 19±4 and 22±5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 μ m Proc (CGP 20712A: p K i 1: 8.17±0.08, site1: 23±6%, p K i 2: 4.77±0.14; ICI 118551: p K i 1: 7.78±0.03, site1: 37±2%, p K i 2: 5.35±0.25). Our results show that the radioligand [ 3 H]‐CGP 12177 allows the characterization of β 1 ‐ and β 3 ‐adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on β 1 ‐ and β 3 ‐adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the β 3 ‐subtype.British Journal of Pharmacology (1997) 120 , 201–210; doi: 10.1038/sj.bjp.0700885