Negative cooperativity across 1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 1-adrenoceptor binding conformation

At the β1-adrenoceptor, CGP 12177 potently antagonizes agonist responses at the primary high-affinity catecholamine conformation while also exerting agonist effects of its own through a secondary low-affinity conformation. A recent mutagenesis study identified transmembrane region (TM)4 of the β1-adrenoceptor as key for this low-affinity conformation. Others suggested that TM4 has a role in β1-adrenoceptor oligomerization. Here, assessment of the dissociation rate of a fluorescent analog of CGP 12177 [bordifluoropyrromethane-tetramethylrhodamine-(±)CGP 12177 (BODIPY-TMR-CGP)] at the human β1-adrenoceptor expressed in Chinese hamster ovary cells revealed negative cooperative interactions between 2 distinct β1-adrenoceptor conformations. The dissociation rate of 3 nM BODIPY-TMR-CGP was 0.09 ± 0.01 min−1 in the absence of competitor ligands, and this was enhanced 2.2- and 2.1-fold in the presence of 1 µM CGP 12177 and 1 µM propranolol, respectively. These effects on the BODIPY-TMR-CGP dissociation rate were markedly enhanced in β1-adrenoceptor homodimers constrained by bimolecular fluorescence complementation (9.8- and 9.9-fold for 1 µM CGP 12177 and 1 µM propranolol, respectively) and abolished in β1-adrenoceptors containing TM4 mutations vital for the second conformation pharmacology. This study suggests that negative cooperativity across a β1-adrenoceptor homodimer may be responsible for generating the low-affinity pharmacology of the secondary β1-adrenoceptor conformatio

Fibroblast growth factor 21 mediates specific glucagon actions

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways

Markedly reduced effects of (−)-isoprenaline but not of (−)-CGP12177 and unchanged affinity of β-blockers at Gly389-β(1)-adrenoceptors compared to Arg389-β(1)-adrenoceptors

1. Substitution of arginine by glycine at position 389, a frequent β(1)-adrenoceptor polymorphism, reduces adenylyl cyclase stimulation by (−)-isoprenaline. β(1)-Adrenoceptors mediate the effects of catecholamines and nonconventional partial agonists ((−)-CGP12177) through different sites. We investigated the influence of the 389 polymorphism on β blocker affinity, as well as on the responses to (−)-isoprenaline and the nonconventional partial agonist (−)-CGP12177 on cyclic AMP levels in CHO cells expressing recombinant Arg389-β(1)-adrenoceptors (101 fmol mg(−1) protein) or Gly389-β(1)-adrenoceptors (94 fmol mg(−1)). 2. The affinity of β-blockers and partial agonists, estimated from competition binding with (−)-[(125)I]-cyanopindolol, was not different for Arg389-β(1)-adrenoceptors and Gly389-β(1)-adrenoceptors. 3. The maximum cAMP increases by (−)-isoprenaline and (−)-CGP12177 at Gly389-β(1)-adrenoceptors were reduced by 97 and 46%, but the potencies enhanced 2 and 0.5 log units, respectively, compared to Arg389-β(1)-adrenoceptors. The intrinsic activity of (−)-CGP12177 with respect to the (−)-isoprenaline was 0.057 at Arg389-β(1)-adrenoceptors and 1.05 at Gly389-β(1)-adrenoceptors. 4. We confirm in intact CHO cells that responses to (−)-isoprenaline are markedly reduced at Gly389-β(1)-adrenoceptors compared to Arg389-β(1)-adrenoceptors. However, the 389 polymorphism reduces considerably less the agonist responses to (−)-CGP12177, indicating that coupling to G(s) protein is different for β(1)-adrenoceptors activated by catecholamines than for receptors activated by nonconventional partial agonists. The affinity of β-blockers is conserved across the Arg389Gly polymorphism