Metabolic response to various beta-adrenoceptor agonists in beta3-adrenoceptor knockout mice: evidence for a new beta-adrenergic receptor in brown adipose tissue

The beta3-adrenoceptor plays an important role in the adrenergic response of brown and white adipose tissues (BAT and WAT). In this study, in vitro metabolic responses to beta-adrenoceptor stimulation were compared in adipose tissues of beta3-adrenoceptor knockout and wild type mice. The measured parameters were BAT fragment oxygen uptake (MO2) and isolated white adipocyte lipolysis. In BAT of wild type mice (-)-norepinephrine maximally stimulated MO2 4.1+/-0.8 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (dobutamine 5.1+/-0.3, terbutaline 5.3+/-0.3 and CL 316,243 4.8+/-0.9 fold, respectively); in BAT of beta3-adrenoceptor knockout mice, the beta1- and beta2-responses were fully conserved. In BAT of wild type mice, the beta1/beta2-antagonist and beta3-partial agonist CGP 12177 elicited a maximal MO2 response (4.7+/-0.4 fold). In beta3-adrenoceptor knockout BAT, this response was fully conserved despite an absence of response to CL 316,243. This unexpected result suggests that an atypical beta-adrenoceptor, distinct from the beta1-, beta2- and beta3-subtypes and referred to as a putative beta4-adrenoceptor is present in BAT and that it can mediate in vitro a maximal MO2 stimulation. In isolated white adipocytes of wild type mice, (-)-epinephrine maximally stimulated lipolysis 12.1+/-2.6 fold. Similar maximal stimulations were obtained with beta1-, beta2- or beta3-adrenoceptor selective agonists (TO509 12+/-2, procaterol 11+/-3, CL 316,243 11+/-3 fold, respectively) or with CGP 12177 (7.1+/-1.5 fold). In isolated white adipocytes of beta3-adrenoceptor knockout mice, the lipolytic responses to (-)epinephrine, to the beta1-, beta2-, beta3-adrenoceptor selective agonists and to CGP 12177 were almost or totally depressed, whereas those to ACTH, forskolin and dibutyryl cyclic AMP were conserved.     

Lipolytic effects of beta1, beta2 and beta3-adrenergic agonists in isolated human fat cells from omental and retroperitoneal adipose tissues

The presence of beta1- and beta2-adrenoceptors has been clearly established in human fat cells. There is some controversy about the presence and function of beta3-adrenoceptors. It is well established that there are marked regional variations in catecholamine-induced lipolysis. In this work the possibility that a beta3-adrenoceptor plays a significant role in the control of lipid mobilization is studied and also its importance in comparison to beta1- and beta2-adrenoceptors in isolated human fat cells, is evaluated, by measuring the in vitro lipolysis induced by dobutamine, salbutamol, metaproterenol, BRL 37344 and CGP 12177A. Human adipocytes from omental and retroperitoneal fat deposits exhibited an "atypical" beta-adrenergic response but, given the small lipolytic effect initiated by BRL 37344 and CGP 12177A, they are probably poorly equipped in functional beta3-adrenoceptors.     

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta 4-adrenoceptors in both wild-type and beta 3-adrenoceptor knockout mice

Some blockers of beta1- and beta2-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta4-adrenoceptor) that resembles the beta3-adrenoceptor. It is likely but not proven that the putative beta4-adrenoceptor is genetically distinct from the beta3-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta3-adrenoceptor gene (beta3 KO). (-)-CGP 12177, a beta1- and beta2-adrenoceptor blocker that causes agonist effects through both beta3-adrenoceptors and cardiac putative beta4-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta3 KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 microM) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta3 KO. (-)-[3H]CGP 12177 labeled a similar density of the putative beta4-adrenoceptor in ventricular membranes from the hearts of both WT (Bmax = 52 fmol/mg protein) and beta3 KO (Bmax = 53 fmol/mg protein) mice. The affinity of (-)-[3H]CGP 12177 for the cardiac putative beta4-adrenoceptor was not different between WT (Kd = 46 nM) and beta3 KO (Kd= 40 nM). These results provide definitive evidence that the cardiac putative beta4-adrenoceptor is distinct from the beta3-adrenoceptor.     

Lipolytic action of Buthus occitanus tunetanus venom: involvement of the beta adrenergic pathway

Scorpion venoms contain active neurotoxins known to act selectively at the level of voltage sensitive Na+ and K+ channels on mammal nervous system. In the present report, we show for the first time that the venom of scorpion Buthus occitanus tunetanus (Bot) contains compounds able to activate another cell function in non excitable cells. Addition of this venom to the culture media of 3T3-L1 adipocytes or freshly dissociated rat adipocytes rapidly increases lipolysis as estimated by glycerol release (approximately 3 to 4 fold over basal values) in a dose-dependent manner (EC50 approximately 12 +/- 1.25 micrograms/ml; n = 3). Bot venom effect was lower and not additive to the effect produced by isoproterenol (IPE) (10 microM), a main lipolytic agent, n = 3. In Sephadex G-50 size exclusion chromatography, the lipolytic activity was excluded and not associated to the included neurotoxic fraction. Furthermore, no lipolytic effect could be detected in the Na+ channel specific toxin II purified from Androctonus australis hector (AaHII) or the K+ voltage-dependent channel toxin from Androctonus mauritanicus mauritanicus (KTx). Propranolol (a non selective beta adrenoreceptor (beta AR) antagonist), alprenolol and pindolol (selective beta 1/beta 2 antagonists) totally inhibited in a dose-dependent manner the lipolytic response to Bot venom (IC50 approximately 1 x 10(-7), 7.5 x 10(-8) and 3 x 10(-7)M, respectively), suggesting that venom stimulated lipolysis through the beta AR pathway. The pharmacological profiles of molecules acting more selectively on beta AR subtypes such as CGP 12177 (beta 1/ beta 2 antagonist with beta 3 agonist properties), CGP 20712A (beta 1 antagonist) and ICI 118551 (beta 2 antagonist) strongly suggest that lipolytic action of venom mainly involves the beta 2/beta 1 AR subtypes.     

Desensitization of the beta-adrenergic response in human brown adipocytes

Activation of adenylyl cyclase by beta-adrenergic receptors (betaARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three betaAR subtypes present in adipose tissue, beta3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model of beta-adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions of betaAR subtypes, with beta3AR being the most abundant (approximately 80% of the total). However, beta3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on beta1- and beta2-subtypes, respectively. Upon isoproterenol stimulation, beta1- and beta2AR down-regulated with a half-life of about 3 h and the beta3AR with a half-life of 30-40 h. Long term stimulation with both saturating (micromolar) and nonsaturating (nanomolar) concentrations of beta-adrenergic agonists caused a complete desensitization of the beta-adrenergic response at the adenylyl cyclase level and loss of stimulated protein kinase A activity and CREB phosphorylation. These results suggest that cAMP-dependent processes will be desensitized upon permanent treatment with beta3AR agonists. Further studies should establish whether the beta3AR is coupled to other signaling pathways in human brown adipocytes and whether these may contribute to BAT hypertrophy and/or thermogenesis.     

Hepatitis C viral genome in a subset of primary hepatic lymphomas

Formoterol, a beta2-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of beta3-adrenergic receptors (betaARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that betaAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. beta1AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. beta2ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that beta3AR expression was found in one of three human primary osteoblast cDNAs tested. beta3AR expression was not found in any of the osteoblast-like cell lines. The nonspecific betaAR agonist, isoproterenol, and the beta2AR-specific agonist, formoterol, induced c-fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the beta2AR-specific antagonist, ICI 118551, but not by the beta1AR-specific antagonist, CGP 20712, indicating that induction of c-fos gene expression is specifically mediated by beta2ARs. c-fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c-fos gene expression. Thus, betaARs are expressed in osteoblast-like cells and are coupled to c-fos gene expression via the beta2AR, increases in cAMP levels and activation of a PKA-dependent pathway.     

Control of lipolysis in intra-abdominal fat cells of nonhuman primates: comparison with humans

The mechanisms that control lipolysis in intra-abdominal fat cells from various primate species, the marmoset (Callithrix jacchus), the baboon (Papio papio), and the macaque (Macaca fascicularis), were compared to those of human intraabdominal fat cells. Selective beta 1- or beta 2-adrenoceptor agonists induced lipolysis in all species. Selective beta 3-agonists (BRL 37344, CL 316243, and SR 58611) acted as partial agonists in marmoset but were inefficient in other primates, including humans. alpha 2-Adrenoceptor number ([3H]RX 8210002 binding) equalized (baboon) or exceeded (other primates) beta 1/beta 2-adrenoceptors ([3H]CGP 12177 binding). Baboon fat cell membranes expressed similar amounts of coupled beta- and alpha 2-adrenoceptors. In all species, norepinephrine- or epinephrine-induced lipolysis did not reach the lipolytic effect of isoproterenol but their effects were enhanced after alpha 2-adrenoceptor blockade. N6-phenylisopropyladenosine (PIA) induced a full antilipolytic effect in baboon, macaque, and human adipocytes through adenosine receptors ([3H]DPCPX binding). Peptide YY (PYY) weakly inhibited lipolysis in baboon. Adrenocorticotropic hormone (ACTH) was inactive whereas parathyroid hormone (PTH) partially stimulated lipolysis in primates. Histamine was partially lipolytic in marmoset only. This study emphasizes the similarities of the mechanisms controlling the lipolysis in nonhuman primate and in human adipocytes and suggests that the baboon and the macaque should provide unique models for the study of the regulation of lipolysis.     

Action potential shortening through the putative beta4-adrenoceptor in ferret ventricle: comparison with beta1- and beta2-adrenoceptor-mediated effects

The electrophysiological responses to (-)-CGP 12177 ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative beta4-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min(-1) and compared to those of (-)-noradrenaline and (-)-adrenaline, mediated through beta1- and beta2-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; beta3-adrenoceptor-selective agonists had no effect. (-)-CGP 12177 was the most potent, but (-)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (-)-noradrenaline but not (-)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through beta1- and putative beta4-adrenoceptors may be different.     

3-Pyridylethanolamines: potent and selective human beta 3 adrenergic receptor agonists

The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.     

Evidence for numerous brown adipocytes lacking functional beta 3-adrenoceptors in fat pads from nonhuman primates

Brown adipose tissue (BAT) is involved in the control of energy balance and has been demonstrated to be activated through beta 3-adrenoceptor (beta 3-AR) occupation in rodents. The ability to specifically activate energy expenditure via this receptor is of great interest for the treatment of obesity. Nevertheless, the extent of BAT and the presence of a functional beta 3-AR in humans are now debated, and this situation is difficult to clarify for evident practical and ethical reasons. We investigated the occurrence of brown adipocytes in fat deposits of prepubertal baboons using antibodies raised against uncoupling protein (UCP) in Western blotting and immunocytology experiments. UCP was detected in all types of fat pads studied and was revealed in multilocular cells. Pericardiac and axillary adipose tissues displayed large amounts of UCP and can be assimilated to typical BAT. Most of the other pads looked like white adipose tissue, but exhibited areas with clusters of brown adipocytes and, thus, can be assimilated to the convertible adipose tissue as previously described in rodents. The presence of beta 3-ARs was evaluated by both beta 2-agonist-stimulated lipolysis and messenger ribonucleic acid (mRNA) expression studies. There was no significant lipolytic effect of any of the beta 3-AR agonists tested (SR 58611A, BRL 37344, CGP 12177, or CL 316243) in either white or brown tissues. PCR analysis demonstrated that beta 3-AR mRNA expression is not related to the UCP content of fat pads and that beta 3-AR expression is low. This study demonstrates the presence of great proportions of brown adipocytes in adipose tissue and the heterogeneity of the fat pads in baboons. The lack of a metabolic effect of beta 3-agonists combined with the weak expression of beta 3-AR mRNAs raise the question of the role of beta 3-ARs in adipose tissues of primates.     

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.     

Mice expressing human but not murine beta3-adrenergic receptors under the control of human gene regulatory elements

Beta-adrenergic receptors (ARs) are expressed predominantly in adipose tissue, and beta3-selective agonists are effective anti-obesity drugs in rodents. Rodent and human beta3-ARs differ with respect to expression in white versus brown adipocytes as well as their ability to be stimulated by beta3-AR-selective agonists. Humans express beta3-AR mRNA abundantly in brown but not white adipocytes, while rodents express beta3-AR mRNA abundantly in both sites. To determine the basis for this difference, we have transgenically introduced 74 kilobases (kb) of human beta3-AR genomic sequence into gene knockout mice lacking beta3-ARs. Importantly, human beta3-AR mRNA was expressed only in brown adipose tissue (BAT) of transgenic mice, with little or no expression being detected in white adipose tissue (WAT), liver, stomach, small intestine, skeletal muscle, and heart. This pattern of expression differed from that observed in mice bearing a murine beta3-AR genomic transgene in which beta3-AR mRNA was expressed in both WAT and BAT, but not in other sites. Furthermore, we have transgenically introduced smaller human constructs containing -14.5 and -0.6 kb of upstream sequence into beta3-AR gene knockout mice. Both -14.5 and -0.6 kb constructs were expressed in BAT but not WAT. Thus, human but not murine cis-regulatory elements direct beta3-AR gene expression preferentially to brown adipocytes. Identification of responsible cis-regulatory element(s) and relevant trans-acting factor(s) should provide insight into mechanisms controlling human beta3-AR gene expression. In addition, the beta3-AR agonist, CGP-12177, stimulated oxygen consumption in mice expressing human but not murine beta3-ARs by 91% compared with only 49% in control beta3-AR gene knockout mice, demonstrating that the human beta3-AR can functionally couple with energy expenditure. These "humanized" mice should assist us in the development of drugs that may become effective anti-obesity agents in humans.     

Functional evidence of atypical beta 3-adrenoceptors in the human colon using the beta 3-selective adrenoceptor antagonist, SR 59230A

The role of beta 3-adrenoceptors in human colonic circular smooth muscle was assessed in vitro by use of the beta 3-selective antagonist SR 59230A. Isoprenaline, in the presence of the selective beta-adrenoceptor antagonists CGP 20712A (beta 1) and ICI 118551 (beta 2), both at 0.1 microM, concentration-dependently relaxed the preparation (pEC50 = 5.22). This effect was potently and competitively antagonized by SR 59230A with a pA2 of 8.31, while its R,R enantiomer SR 59483A gave an apparent pKB of 6.21. Relaxation was likewise produced by CGP 12177A (pEC50 = 6.05), but not by BRL 37344. Although only one of these beta 3-selective agonists was effective, the remarkably high potency of SR 59230A as a stereospecific antagonist of non-beta 1 non-beta 2 relaxation of human colonic muscle by isoprenaline provides strong functional evidence of beta 3-adrenoceptors in that tissue.     

Genomic cloning and species-specific properties of the recombinant canine beta3-adrenoceptor

A molecular clone encoding a beta3-adrenoceptor was isolated from a canine genomic library. The cloned receptor exhibited a pharmacological profile similar to that of other species: in particular, high efficiency of the two selective beta3-adrenoceptor agonists, CL 316,243 (disodium(R,R)-5[2[[2-(chlorophenyl)-2hydroxyethyl]-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate) and ICI 201651 ((R)4-(2-hydroxy-3-phenoxypropylaminoethoxy)-N-(2-methoxyethyl)phe noxy acetic acid) and a low affinity for the radioligand (-)-[3-(125)I]-iodocyanopindolol. Interestingly, CGP 12177A ((+/-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one), which is described as a partial agonist for the human receptor, was a full agonist for the canine receptor. After expression and stimulation of the canine beta3-adrenoceptor in stably transfected Chinese hamster ovary cells there was a very low accumulation of cAMP, suggesting weak coupling to Gs-protein and adenylyl cyclase. However, the response was much better in human embryonal kidney cells transfected with the canine beta3-adrenoceptor gene. The cloning of the canine beta3-adrenoceptor and the insights gained from its pharmacological characterization may allow the development of selective compounds for use in the treatment of obese dogs.     

Atypical beta-adrenoceptors in the rat isolated common carotid artery

1. The possible existence of atypical beta-adrenoceptors in vascular smooth muscle of the rat common carotid artery was examined in this study. 2. Isoprenaline produced concentration-dependent relaxation of noradrenaline (10(-7) M) precontracted ring segments of the carotid artery. The relaxation was not affected by endothelial denudation. 3. Propranolol (10(-8) M-3 x 10(-7) M) shifted the isoprenaline curve to the right without suppressing the maximum response. However, the slope (0.74) of the Schild plot was significantly (P < 0.05) less than 1. 4. Salbutamol (beta 2), CGP 12177 and BRL 37344 (beta 3) also concentration-dependently relaxed noradrenaline precontracted artery segments. These relaxations were not affected by propranolol (10(-7) M). Pretreatment of the artery segments with BRL 37344 did not desensitize the tissue to the relaxant effect of isoprenaline, CGP 12177 and salbutamol. 5. It is concluded that atypical beta-adrenoceptors exist in vascular smooth muscle of the common carotid artery.     

Effects of WB4101 and chloroethylclonidine on the positive and negative inotropic actions of phenylephrine in rat cardiac muscle

This study was designed to determine if the positive and negative inotropic actions of alpha-1-adrenergic agonists in rat atrial and ventricular myocardium are mediated via different alpha-1-adrenergic receptor (AR) subtypes. Inotropic effects of phenylephrine were examined in isolated left atrial and papillary muscle before and after treatment with prazosin, WB4101 (N-[2-(2,6-dimethoxyphenoxy)ethyl]-2,3-dihydro-1,4-benzodioxin+ ++-2-methanamine), chloroethylclonidine (CEC) and WB4101 plus CEC. Phenylephrine (10 microM) elicited a monophasic positive inotropic response in left atrial muscle and a triphasic inotropic action in papillary muscle (transient positive, then negative inotropic components preceding a sustained positive inotropic response). CEC, WB4101 and prazosin each antagonized the monophasic response in isolated left atria and the sustained positive inotropic response in papillary muscle. CEC and prazosin each antagonized the transient negative inotropic component in papillary muscle. The transient positive inotropic response was not affected by CEC, WB4101 or CEC plus WB4101, but was antagonized by higher concentrations of prazosin. These data suggest that the sustained positive inotropic effect of alpha-1-adrenergic agonists in rat atrial and ventricular myocardium results from stimulation of alpha-1A and alpha-1B ARs, whereas the transient negative inotropic component of the triphasic response in ventricular preparations is mediated via alpha-1B ARs. However, present data do not exclude the possibility that the CEC-sensitive inotropic responses elicited by phenylephrine may be mediated in part by other recently described alpha-1 subtypes. The receptors involved in the transient positive inotropic action cannot be identified by current results.     

In situ assessment of the role of the beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow in human subcutaneous adipose tissue

1. The involvement of beta 1-, beta 2- and beta 3-adrenoceptors in the control of lipolysis and nutritive blood flow was investigated in abdominal subcutaneous adipose tissue of healthy young adults by use of an in situ microdialysis technique. 2. Dialysis probes were infused either with isoprenaline (non-selective beta-adrenoceptor agonist), CGP 12,177 (selective beta 3-adrenoceptor agonist having beta 1-/beta 2-antagonist properties), dobutamine (selective beta 1-adrenoceptor agonist) or terbutaline (selective beta 2-adrenoceptor agonist). The recovery of each probe used for perfusion was calculated by an in vivo calibration method. The local blood flow was estimated through the measurement of the escape of ethanol infused simultaneously with the drugs included in the probe. 3. Isoprenaline infusion at 0.01 microM had a weak effect while higher concentrations of isoprenaline (0.1 and 1 microM) caused a rapid, sustained and concentration-dependent increase of glycerol outflow; the maximum increase was 306 +/- 34% with 1 microM. Isoprenaline also increased the nutritive blood flow in adipose tissue; a significant effect appeared at 0.1 microM isoprenaline and was greater at 1 microM. 4. CGP 12,177 (10 and 100 microM) increased the glycerol concentration in the dialysate (128 +/- 8 and 149 +/- 12%, respectively) and nutritive blood flow. Terbutaline and dobutamine (100 microM) both provoked rapid and similar increases in glycerol outflow (252 +/- 18 and 249 +/- 18%, respectively). Both, terbutaline and dobutamine increased nutritive blood flow. 5. It is concluded that beta 1- and beta 2-adrenoceptor subtypes are both mainly involved in the mobilization of lipids and in the control of nutritive blood flow. beta 3-Adrenoceptors play a weaker role in the control of lipolysis and nutritive blood flow in human subcutaneous abdominal adipose tissue.     

Nuclear matrix proteins and their potential applications to diagnostic pathology

The sympathetic nervous system controls lipolysis in fat by activation of four adrenergic receptors: beta1, beta2, beta3, and alpha2. During pregnancy, maternal metabolism presents anabolic and catabolic phases, characterized by modifications of fat responsiveness to catecholamines. The contributions of the four adrenergic receptors to adipocyte responsiveness during pregnancy have never been studied. Our aim was to evaluate the influence of pregnancy on adrenergic receptor-mediated lipolysis in rabbit white adipocytes. Functional studies were performed using subtype-selective and non-selective adrenergic receptor agonists. Overall adrenergic responsiveness was measured with the physiological agonist epinephrine. Non-adrenergic agents were used to evaluate different steps of the lipolytic cascade. The alpha2- and beta1/beta2-adrenergic receptor numbers were determined with selective radioligands. Non-adrenergic agents revealed that pregnancy induced an intracytoplasmic modification of the lipolytic cascade in inguinal but not in retroperitoneal adipocytes. Pregnancy induced an increase in beta1- and specially beta3-mediated lipolysis. The amounts of adipocyte beta1/beta2- and alpha2-adrenergic receptors were increased in pregnant rabbits. Epinephrine effects revealed an increased contribution of alpha2-adrenergic receptor-mediated antilipolysis in adipocytes from pregnant rabbits. These results indicate that pregnancy regulates adipocyte responsiveness to catecholamines mainly via the alpha2- and beta3-adrenergic pathways. Pregnancy induces an intracytoplasmic modification of the lipolytic cascade, probably via hormone-sensitive lipase, with differences according to fat location.-Bousquet-Mélou, A., C. Mu?oz, J. Galitzky, M. Berlan, and M. Lafontan. Pregnancy modifies the alpha2-beta-adrenergic receptor functional balance in rabbit fat cells.     

Regulation of endothelial permeability by beta-adrenoceptor agonists: contribution of beta 1- and beta 2-adrenoceptors

The barrier function of cultured, macrovascular endothelial cells derived from bovine aorta was analyzed using confluent monolayers of cells and measuring the exchange of fluorescein dextrans of different molecular masses. The effects of beta-adrenoceptor agonists with different selectivity for beta 1- and beta 2-adrenoceptors (AR) were investigated. Formoterol, a novel high-affinity agonist for beta 2-AR recently introduced in the treatment of bronchial asthma, showed a significant reduction of cell permeability with subnanomolar concentrations, whereas the catecholamines (-)-isoproterenol and (-)-norepinephrine only showed significant effects with micromolar concentrations. In order to elucidate if this difference in potential to regulate cell permeability is related to appropriate changes in the selectivity and affinity of the agonists for beta 2 AR, we investigated the beta AR-coupled adenylate cyclase (AC) in membranes from endothelial cells and compared AC stimulation with the binding of agonists to the receptors using [125I](-)-iodopindolol as radioligand. beta-Adrenoceptors revealed to be closely coupled to AC as assessed by a similar magnitude of effects by receptor agonists in comparison to GTP analogues and direct stimulants of AC activity. AC activity was increased by formoterol in parallel to its receptor occupancy of beta 2AR with nanomolar concentrations which were 50-fold higher than those used for the regulation of cell permeability indicating the existence of spare receptors. In contrast to formoterol, the catecholamines (-)-isoproterenol and (-)-norepinephrine stimulated AC activity through both beta 1AR and beta 2AR. From the overproportional high contribution of beta 1AR to AC stimulation (42%) in comparison to its low fraction (13%) in receptor binding we calculated that beta 1AR is 3-4-fold more effectively coupled to AC than beta 2 AR.     

Agonist interactions with chimeric and mutant beta1- and beta3-adrenergic receptors: involvement of the seventh transmembrane region in conferring subtype specificity

beta1- and beta3-adrenergic receptors (AR) are the predominant beta-AR subtypes in adipocytes, and analysis of native and recombinant beta-AR has revealed several pharmacological and biochemical differences between these subtypes. This study used chimeric and mutated rat beta-AR expressed in Chinese hamster ovary cells to examine the basis of certain characteristic differences in the agonist properties of catecholamines and prototypic beta3-AR agonists. The exchange of sequence beyond transmembrane (TM) region 6 between the beta-AR subtypes had dramatic and reciprocal effects on the affinity and efficacy of the prototypic beta3-AR agonists BRL 37,344 and CL 316,243, without affecting the interactions with catecholamines. Mutation of Phe350 and Phe351 in TM7 of the beta1-AR to Ala and Leu found in the beta3-AR was sufficient to allow activation by prototypic beta3-AR agonists. Interestingly, this mutation did not affect catecholamine action and it did not impair the ability of propranolol to block the actions of isoproterenol or the selective beta3-AR agonists. beta1-AR containing beta3-AR sequence from predicted TM5 through TM6 exhibited reduced affinity for catecholamines without altering agonist potency, suggesting enhanced coupling efficiency. Inclusion of the homologous beta1-AR sequence in the beta3-AR, however, did not produce reciprocal effects. These results are the first to define a major determinant of beta3-AR subtype-selective agonism in TM7 and demonstrate that the determinants of selective phenethanolamines, catecholamines, and propranolol action are distinct.