Pancreatic beta-cells expressing the Arg64 variant of the beta(3)-adrenergic receptor exhibit abnormal insulin secretory activity.
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The Arg64 beta(3)-adrenergic receptor (beta(3)AR) variant is associated with an earlier age of onset of diabetes and lower levels of insulin secretion in humans. The aims of this study were to investigate whether beta(3)AR is expressed by islet cells, if receptor binding affects insulin secretion and, finally, if the beta(3)AR Arg64 variant induces abnormal insulin secretory activity. Human pancreas extracts were subjected to RT-PCR, Western blotting and immunostaining analyses. DNA sequencing and Western blotting demonstrated that the beta(3)AR gene is transcribed and translated in the human pancreas; immunostaining showed that it is expressed by the islets of Langerhans. Cultured rat beta-cells responded to human beta(3)AR agonists in a dose- and time-dependent manner. Transfection of cultured rat beta-cells with the wild-type human beta(3)AR produced an increased baseline and ligand-dependent insulin secretion compared with parental cells. On the other hand, cells transfected with the Arg64 variant of the beta(3)AR secreted less insulin, both spontaneously and after exposure to human beta(3)AR agonists. Furthermore, while transfection with the wild-type beta(3)AR preserved the glucose-dependent secretion of insulin, expression of the variant receptor rendered the host cells significantly less responsive to glucose. In summary, cells express the beta(3)AR, and its activation contributes to the regulation of insulin secretion. These findings may help explain the low levels of insulin secretion in response to an i.v. glucose tolerance test observed in humans carrying the Arg64 polymorphism. (+info)
Mouse beta 3a- and beta 3b-adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways.
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1. This study characterizes the mouse beta(3a)-adrenoceptor (AR) and the splice variant of the beta(3)-AR (beta(3b)-AR) expressed in Chinese hamster ovary cells (CHO-K1). 2. Stable clones with high (approximately 1200), medium (approximately 500) or low receptor expression (approximately 100 fmol mg protein(-1)) were determined by saturation binding with [(125)I]-(-)-cyanopindolol. Competition binding studies showed no significant differences in affinity of beta-AR ligands for either receptor. 3. Several functional responses of each receptor were measured, namely extracellular acidification rate (EAR; cytosensor microphysiometer), cyclic AMP accumulation, and Erk1/2 phosphorylation. The beta(3)-AR agonists BRL37344, CL316243, GR265162X, L755507, SB251023, the non-conventional partial beta-AR agonist CGP12177 and the beta-AR agonist (-)-isoprenaline caused concentration-dependent increases in EAR in cells expressing either splice variant. CL316243 caused concentration-dependent increases in cyclic AMP accumulation and Erk1/2 phosphorylation in cells expressing either receptor. 4. PTX treatment increased maximum EAR and cyclic AMP responses to CL316243 in cells expressing the beta(3b)-AR but not in cells expressing the beta(3a)-AR at all levels of receptor expression. 5. CL316243 increased Erk1/2 phosphorylation with pEC(50) values and maximum responses that were not significantly different in cells expressing either splice variant. Erk1/2 phosphorylation was insensitive to PTX or H89 (PKA inhibitor) but was inhibited by LY294002 (PI3K gamma inhibitor), PP2 (c-Src inhibitor), genistein (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). 6. The adenylate cyclase activators forskolin or cholera toxin failed to increase Erk1/2 levels although both treatments markedly increased cyclic AMP accumulation in both beta(3a)- or beta(3b)-AR transfected cells. 7. These results suggest that in CHO-K1 cells, the beta(3b)-AR, can couple to both G(s) and G(i) to stimulate and inhibit cyclic AMP production respectively, while the beta(3a)-AR, couples solely to G(s) to increase cyclic AMP levels. However, the increase in Erk1/2 phosphorylation following receptor activation is not dependent upon coupling of the receptors to G(i) or the generation of cyclic AMP. (+info)
Physiological antagonism between ventricular beta 1-adrenoceptors and alpha 1-adrenoceptors but no evidence for beta 2- and beta 3-adrenoceptor function in murine heart.
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1. Murine left atrium lacks inotropic beta(2)-adrenoceptor function. We investigated whether beta(2)-adrenoceptors are involved in the cardiostimulant effects of (-)-adrenaline on spontaneously beating right atria and paced right ventricular myocardium of C57BL6 mice. We also studied a negative inotropic effect of (-)-adrenaline. 2. Sinoatrial tachycardia, evoked by (-)-adrenaline was resistant to blockade by beta(2)-selective ICI 118,551 (50 nM) but antagonized by beta(1)-selective CGP 20712A (300 nM). This pattern was unaffected by pretreatment with pertussis toxin (PTX, 600 microg kg(-1) i.p. 24 h) which reversed carbachol-evoked bradycardia to tachycardia. 3. Increases of ventricular force by (-)-adrenaline and (-)-noradrenaline were not blocked by ICI 118,551 but antagonized by CGP 20712A. 4. Under blockade of beta-adrenoceptors, (-)-adrenaline and (-)-noradrenaline depressed ventricular force (-logIC(50)M=7.7 and 6.9). The cardiodepressant effects of (-)-adrenaline were antagonized by phentolamine (1 microM) and prazosin (1 microM) but not by (-)-bupranolol (1 microM). Prazosin potentiated the positive inotropic effects of (-)-adrenaline (in the absence of beta-blockers) from -logEC(50)M=6.2 - 6.8. 5. PTX-treatment reduced carbachol-evoked depression of ventricular force in the presence of high catecholamine concentrations. Inhibition of ventricular function of G(i) protein was verified by 82% reduction of in vitro ADP-ribosylation. PTX-treatment tended to increase the positive inotropic potency of (-)-adrenaline under all conditions investigated, including the presence of ICI 118,551. 6. (-)-Adrenaline causes murine cardiostimulation through beta(1)-adrenoceptors but not through beta(2)-adrenoceptors. The negative inotropic effects of (-)-adrenaline are mediated through ventricular alpha(1)-adrenoceptors but not through beta(3)-adrenoceptors. Both G(i) protein and alpha(1)-adrenoceptors restrain (-)-adrenaline-evoked increases in right ventricular force mediated through beta(1)-adrenoceptors. (+info)
The pharmacokinetics of a thiazole benzenesulfonamide beta 3-adrenergic receptor agonist and its analogs in rats, dogs, and monkeys: improving oral bioavailability.
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The pharmacokinetics and oral bioavailability of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(triflu oromethylphenyl]thiazol-2-yl]benzenesulfonamide (1), a 3-pyridyl thiazole benzenesulfonamide beta3-adrenergic receptor agonist, were investigated in rats, dogs, and monkeys. Systemic clearance was higher in rats (approximately 30 ml/min/kg) than in dogs and monkeys (both approximately 10 ml/min/kg), and oral bioavailability was 17, 27, and 4%, respectively. Since systemic clearance was 25 to 40% of hepatic blood flow in these species, hepatic extraction was expected to be low, and it was likely that oral bioavailability was limited either by absorption or a large first-pass effect in the gut. The absorption and excretion of 3H-labeled 1 were investigated in rats, and only 28% of the administered radioactivity was orally absorbed. Subsequently, the hepatic extraction of 1 was evaluated in rats (30%) and monkeys (47%). The low oral bioavailability in rats could be explained completely by poor oral absorption and hepatic first-pass metabolism; in monkeys, oral absorption was either less than in rats or first-pass extraction in the gut was greater. In an attempt to increase oral exposure, the pharmacokinetics and oral bioavailability of two potential prodrugs of 1, an N-ethyl [(R)-N-[4-[2-[ethyl[2-hydroxy-2-(3-pyridinyl)ethyl]amino]ethyl]phenyl]-4-[4-[4-(t rifluoromethyl)phenyl]thiazol-2-yl]benzenesulfonamide; 2] and a morpholine derivative [(R)-N-[4-[2-[2-(3-pyridinyl)morpholin-4-yl]ethyl]phenyl]-4-[4-[4-(trifluoromethy l)- phenyl]thiazol-2-yl]benzenesulfonamide; 3], were evaluated in monkeys. Conversion to 1 was low (<3%) with both derivatives, and neither entity was an effective prodrug, but the oral bioavailability of 3 (56%) compared with 1 (4%) was significantly improved. The hypothesis that the increased oral bioavailability of 3 was due to a reduction in hydrogen bonding sites in the molecule led to the design of (R)-N-[4-[2-[[2-hydroxy-2-(pyridin-2-yl)ethyl]amino]ethyl]phenyl]-4-[4-(4-trifluo romethylphenyl)thiazol-2-yl]benzenesulfonamide (4), a 2-pyridyl beta3-adrenergic receptor agonist with improved oral bioavailability in rats and monkeys. (+info)
Metabolism of a thiazole benzenesulfonamide derivative, a potent and elective agonist of the human beta3-adrenergic receptor, in rats: identification of a novel isethionic acid conjugate.
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(R)-N-[4-[2-[[2-Hydroxy-2-(pyridin-3-yl)ethyl]amino]ethyl]phenyl]- 4-[4-(4-trifluoro-methylphenyl)thiazol-2-yl]benzenesulfonamide (1) is a potent and selective agonist of the human beta3-adrenergic receptor. We report herein the data from studies of the metabolism and excretion of 1 in rats. Five metabolites were identified in the bile of male Sprague-Dawley rats administered 3H-labeled 1 by either oral gavage (10 mg/kg) or intravenous injection (3 mg/kg). These included a pyridine N-oxide derivative (M2), a primary amine resulting from N-dealkylation and loss of the pyridinyl-2-hydroxyethyl group (M4), a carboxylic acid derived from N-dealkylation and loss of the pyridyl-2-hydroxyethyl amine (M5), and the corresponding taurine and isethionic acid conjugates (M1 and M3). Metabolites M1 and M3 also were identified in rats treated with M5 and were generated in incubations of M5 with rat liver subcellular fractions in the presence of ATP and coenzyme A with supplementary taurine or isethionic acid. These results suggest that M5 is the precursor of M1 and M3 and that the formation of these conjugated metabolites follows similar mechanisms of amino acid conjugation. On the other hand, M2, M4, and M5 were produced from 1 in an NADPH-dependent manner in incubations with liver microsomes from rats, dogs, monkeys, and humans. In human liver preparations, these routes of biotransformation were shown to be catalyzed by cytochrome P450 3A4. In a bidirectional transport assay, transport of 1 across a monolayer of cells expressing P-glycoprotein (Pgp) was observed to be similar to that of vinblastine, which is an established substrate of the transporter protein. This finding, together with the observation that the parent compound was excreted in the feces of bile duct-cannulated animals following intravenous dosing, suggests that 1 is subject to Pgp-mediated excretion from intestine of rats. (+info)
Effect of a 28-d treatment with L-796568, a novel beta(3)-adrenergic receptor agonist, on energy expenditure and body composition in obese men.
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BACKGROUND: Stimulation of energy expenditure (EE) with selective thermogenic beta-adrenergic agonists may be a promising approach for treating obesity. OBJECTIVE: We analyzed the effects of the highly selective human beta(3)-adrenergic agonist L-796568 on 24-h EE, substrate oxidation, and body composition in obese, weight-stable men. DESIGN: In this 2-center, double-blind, randomized, parallel-group study, we measured 24-h EE before and after 28 d of treatment with L-796568 (375 mg/d) or placebo during weight maintenance (ie, without dietary intervention) in nondiabetic, nonsmoking men aged 25-49 y with body mass index (in kg/m(2)) of 28-35 (n = 10 subjects per treatment group). RESULTS: The mean change in 24-h EE from before to after treatment did not differ significantly between groups (92 +/- 586 and 86 +/- 512 kJ/24 h for the L-796568 and placebo groups, respectively). The change in 24-h nonprotein respiratory quotient from before to after treatment did not differ significantly between groups (0.009 +/- 0.021 and 0.009 +/- 0.029, respectively). No changes in glucose tolerance were observed, but triacylglycerol concentrations decreased significantly with L-796568 treatment compared with placebo (-0.76 +/- 0.76 and 0.42 +/- 0.31 mmol/L, respectively; P < 0.002). Overall, treatment-related changes in body composition were not observed, but higher plasma L-796568 concentrations in the L-796568 group were associated with greater decreases in fat mass (r = -0.69, P < 0.03). CONCLUSIONS: Treatment with L-796568 for 28 d had no major lipolytic or thermogenic effect but it lowered triacylglycerol concentrations. This lack of chronic effect on energy balance is likely explained by insufficient recruitment of beta(3)-responsive tissues in humans, down-regulation of the beta(3)-adrenergic receptor-mediated effects with chronic dosing, or both. (+info)
Regulation of adiponectin and leptin gene expression in white and brown adipose tissues: influence of beta3-adrenergic agonists, retinoic acid, leptin and fasting.
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Circulating adiponectin levels fall whereas leptin levels rise with obesity, suggesting that regulation of these two adipocyte-derived hormones may be simultaneously influenced by common obesity-related factors. We examined adiponectin mRNA levels in WAT and in some instances, brown adipose tissue (BAT) following fasting and refeeding, acute and chronic administration of a beta(3)-adrenergic agonist, acute treatment with retinoic acid (RA) and a glucocorticoid, and following chronic infusion of leptin and compared the expression of adiponectin to that of leptin in each circumstance. Serum concentrations of adiponectin were also reported for most of the treatments. Fasting diminished and refeeding reversed both adiponectin and leptin gene expression. Peripheral injection of the beta(3)-adrenergic agonist, CL316,243, suppressed both leptin and adiponectin expression in WAT. A small but significant reduction in adiponectin expression in BAT was also observed following this treatment. Although CL316,23 lowered serum leptin levels markedly, it did not affect serum adiponectin levels. A chronic 7-day infustion of CL316,243 resulted in an elevation of adiponectin expression in WAT and serum concentrations in contrast to suppressions in both mRNA and serum levels of leptin by a similar treatment as previously reported. Chronic administration of leptin did not alter adiponectin synthesis in WAT compared to controls, but prevented the reduction in adiponectin synthesis associated with pair feeding. Food restriction through pair feeding also diminished adiponectin expression in BAT. Collectively, although leptin and adiponectin are inversely correlated with obesity, leptin does not appear to participate directly in adiponectin synthesis. The short-term regulation of the two adipokine expression in WAT is somewhat similar, perhaps subjective to common control of energy balance. The long-term regulation of adiponectin expression in WAT appears to be the opposite of that of leptin and may be more sensitive to changes in adiposity or insulin sensitivity. (+info)
Inhibitory effect of beta3-adrenoceptor agonist in lower esophageal sphincter smooth muscle: in vitro studies.
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We investigated the effects of (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl] - amino]propyl] - 1,3 - benzodioxole - 2, 2 - dicarboxylate (CL 316243) (a typical beta3-agonist) on the spontaneously tonic smooth muscle of the lower esophageal sphincter (LES). Studies were carried out in smooth muscle strips and smooth muscle cells (SMCs) of opossum LES. Isometric tension was recorded in the basal state and after CL 316243, and before and after beta3-antagonist (S)-N-[4-[2-[[3-[-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]be nzenesulfonamide (L 748337) and nonselective antagonist propranolol. In some experiments, the effects of nonadrenergic noncholinergic (NANC) nerve activation by electrical field stimulation (EFS) were also examined. The effects of CL 316243 were compared with those of nonselective beta-agonist isoproterenol. CL 316243 caused a concentration-dependent relaxation of the LES smooth muscle. The relaxant action of CL 316243 was determined to be directly at the smooth muscle because it remained unmodified by the neurotoxin tetrodotoxin and other neurohumoral antagonists, and also was observed in the SMCs. L 748337 selectively antagonized the relaxant effect of CL 316243 and, conversely, had no significant effect on the inhibitory actions of isoproterenol. CL 316243 (1 x 10(-8) M) caused an augmentation of NANC relaxation in the LES. Another beta3-agonist, (S)-4-[hydroxy-3-phenoxy-propylamino-ethoxy]-N-(2-methoxyethyl)-phenoxyacetamide (ZD 7114), also caused concentration-dependent full relaxation of the LES that was selectively antagonized by beta3-anatagonist 3-(2-ethylphenoxy)-1-[(1S)1,2,3,4-tetrahydronaphth-1-ylaminol]-(2S)-2-propanol oxalate (SR 59230A). These studies defined the effects of characteristic inhibitory beta3-adrenoceptors in the spontaneously tonic LES smooth muscle and suggested a potential therapeutic role in the esophageal motility disorders characterized by hypertensive LES. (+info)