β₃ Receptors: Role in Cardiometabolic Disorders

Abstract

Pharmacological and molecular approaches have shown that an atypical β-adrenoceptor (AR), called β₃-AR, that is distinct from β₁-ARs and β₂-ARs, exists in some tissues in heterogeneous populations such as β_3a-ARs and β_3b-ARs. β₃-ARs belong to a superfamily of receptors linked to guanine nucleotide binding proteins (G proteins). The β₃-AR gene contains two introns whereas the β₁-AR and β₂-AR genes are intronless, leading to splice variants. β₃-ARs can couple to G_i and G_s and they are reported to be present in brown adipose tissue, vasculature, the heart, among other tissues. β₃-ARs cause vasodilation of microvessels in the islets of Langerhans and may participate in the pathogenesis of cardiac failure, during which modification of β₁-AR and β₂-AR expression occurs. The development of β₃-AR agonists has led to the elaboration of promising new drugs, including antiobesity and antidiabetic drugs. This article reviews the various pharmacological actions of β₃-ARs and their clinical implications for diabetes and cardiovascular diseases.

Introduction

The pressor effect of adrenal extracts was first shown by Oliver and Schafer in 1895. The active principle was named epinephrine by Abel in 1899. The existence of more than one adrenergic receptor was first proposed by Ahlquist in 1948. He proposed the terms α and β for receptors on smooth muscle where catecholamines produce excitatory and inhibitory responses respectively. Almost 50 years after Ahlquist first discovered evidence of the heterogeneity of adrenergic receptors [Ahlquist, 1948], the number of receptor subtypes is still unclear. β-Adrenoceptors (β-ARs) were later subdivided into β₁ and β₂, which are present in the myocardium and smooth muscle respectively. Pindolol, a nonselective β-AR antagonist with significant agonist activity, was found to cause relaxation of canine-isolated perfused mesenteric vessels [Clark and Bertholet, 1983] and rat aorta precontracted with potassium chloride [Doggrell, 1990]. In both instances, the vasorelaxant effect of pindolol was not significantly antagonized by propranolol, suggesting the presence of a β-AR subtype different from the conventional β₁-ARs and β₂-ARs. The effect of isoprenaline was ascribed not only to activation of β₁-ARs and β₂-ARs, but also to that of an additional adrenoceptor [Doggrell, 1990; Clark and Bertholet, 1983]. Later on, the existence of a third β-AR came into light and Gauthier et al. [1996] found that stimulation of β₃-AR in human cardiac muscle, in contrast with β₁- and β₂-AR stimulation, resulted in a profound dose-dependent negative inotropic effect and hence suggested the participation of β₃-AR in the pathogenesis of cardiac failure. Moreover, various in vivo studies have also demonstrated that positive β₃-AR-related chronotropic effects were prevented by β₁- or β₂-AR antagonists and are likely due to baroreflex activation in response to β₃-adrenoceptor agonist- induced vasodilation [Wheeldon et al. 1994; Takayama et al. 1993; Wheeldon et al. 1993; Tavernier et al 1992].

Studies using molecular and biochemical techniques are likely to provide additional new and unexpected insights into the role of AR subtypes in both normal physiologic functions and diseases. Initially the presence of β₃-ARs was demonstrated in vasculature and heart, but later they were shown in adipocytes. β₃-ARs mediate lipolysis in white adipose tissues and thermogenesis in brown adipose tissues [Lönnqvist et al. 1993; Langin et al. 1991; Zaagsma and Nahorski, 1990]. β₃-ARs represent a heterogeneous population, such as β_3a-ARs and β_3b-ARs, as suggested by the studies in Chinese hamster ovary cells. Furthermore, the activation and signal transduction of such a β_3a-AR and β_3b-AR complex may prevent the full potency of the β₃-AR agonist [Hutchinson et al. 2002]. The regulation of adrenergic receptors by receptor-specific agonists and antagonists has been actively studied for many years and is important clinically because alterations in these receptors have been suspected in many pathological states. The development of β₃-AR agonists has led to the elaboration of promising new drugs and they are a target for antiobesity and antidiabetic drugs [Lowell and Flier, 1995; Pietri-Rouxel and Strosberg, 1995]. Although considerable information is available on β₃-AR physiology in fat, there are many other areas in which β₃-ARs are involved. The existence of an atypical β-AR, called β₃-AR, distinct from β₁-ARs and β₂-ARs, has been demonstrated in various tissues by pharmacological [Berlan et al. 1993; Holloway et al. 1992; Tavernier et al. 1992; Langin et al. 1991; Mc Laughlin and MacDonald, 1990; Hollenga and Zaagsma, 1989; Bojanic et al. 1985] and molecular approaches [Granneman et al. 1991; Muzzin et al. 1991; Tate et al. 1991; Emorine et al. 1989]. The overview of location of β₃ receptors is given in Table 1. This review aims to provide an overview of the presence of β₃-ARs in various tissues, with special emphasis on the clinical implications for diabetes and cardiovascular diseases.

Table 1.

Location of β₃-adrenoceptors.

Organ	Species	Reference
Heart
Atria	Human	Krief et al. (1993); Berkowitz et al. (1995)
Ventricle	Human	Gauthier et al. (1999)
Vascular smooth muscles
Veins		Viard et al. (2000)
Cutaneous vascular smooth muscles	Canine	Berlan et al. (1994)
Vasculature		Tavernier et al. (1992); Shen et al. (1994);
		Rohrer et al. (1999)
Thoracic aorta	Rat	Trochu et al. (1999)
Internal mammary artery	Human	Rozec et al. (2005)
Non-vascular smooth muscles
Gastrointestinal tract, brain, and prostate		Granneman et al. (1991); Emorine et al. (1989);
		Bensaid et al. (1993); Rodriguez et al. (1995)
Ileum	Rat	Roberts et al. (1995); Roberts et al. (1999)
Rectum and IAS membranes	Western blot studies	Rathi et al. (2003)
Urinary tract		Tomiyama et al. (1998)
Near-term myometrium	Human	Bardou et al. (2000)
Brown adipose tissues		Emorine et al. (1994)

Molecular structure and signal transduction mechanism of β receptors

ARs are members of a large superfamily of receptors linked to guanine nucleotide binding proteins (G proteins). All G-protein coupled receptors share structural features, such as extracellular amino terminals with sites for N-linked glycosylation, seven α-helical domains that span the plasma membrane, and intracellular carboxy terminals containing amino acid sequences that indicate probable sites of phosphorylation by one or more protein kinases. The G proteins, linked to adrenergic receptors, are heterotrimeric proteins with α, β, and γ subunits. Each subunit is part of a family consisting of multiple members [Simon et al. 1991]: approximately 20 α subunits that have been divided into four subfamilies — α_s, α_i, α_q and α₁₂; at least five β subunits (β_1–5); and at least six γ subunits (γ_1–6). Although several hundred different subunit combinations (heterotrimers) are theoretically possible, the repertoire of G proteins used by a particular receptor system is limited [Hescheler and Schultz, 1994]. Each type of G protein can be used for signaling by more than one type of receptor. β₁, β₂ and β₃ share approximately 60% amino acid sequence identity within the presumed membrane spanning domains. The β₃-AR gene contains two introns [Lelias et al. 1993; Granneman et al. 1992] in contrast to β₁-AR and β₂-AR genes, which are intronless. This structure leads to splice variants. The B and C isoforms contain 12 and six additional amino acids, respectively, at their C terminus in comparison with the A isoform [Lelias et al. 1993; Granneman et al. 1992]. In rat adipocytes, a unique isoform is expressed that is close to the B isoform, whereas in human brown adipocytes, the C isoform is predominant [Lelias et al. 1993; Van Spronsen et al. 1993]. It was hypothesized that the physiological response to β₃-AR stimulation differs depending on the isoform expressed in a given species [Levasseur et al. 1995]. The human β₃-AR exists in at least two different agonist conformations with a similar high-affinity and low-affinity pharmacology analogous to β₁-AR. Both conformations are present in living cells and can be distinguished by their pharmacological characteristics [Baker, 2005].

All β-AR subtypes signal by coupling to the stimulatory G protein G_αs, leading to activation of adenyl cyclase and accumulation of the second messenger cAMP [Emorine et al. 1989; Frielle et al. 1987; Dixon et al. 1986]. G_s can directly enhance the activation of voltage-sensitive Ca²⁺ channels in the plasma membrane of skeletal and cardiac muscle. However, some recent studies indicate that, under certain circumstances, β 3-AR can couple to G_i as well as to G_s [Gauthier et al. 1996; Xiao et al. 1995; Chaudry et al. 1994]. Multiple mechanisms control the signaling and density of G-protein-coupled receptors. Catecholamines, which are hydrophilic, do not bind to the highly charged extracellular domains of the receptors, as might be expected, but bind in the more hydrophobic membrane-spanning domains [Caron and Lefkowitz, 1993; Jasper and Insel, 1992].

On the basis of many pharmacological and molecular studies, the existence of a fourth β-AR subtype was postulated [Brodde and Michel, 1999; Galitzky et al. 1998; Strosberg et al. 1998; Kaumann, 1997; Strosberg, 1997; Summers et al. 1997; Strosberg and Pietri-Rouxel, 1996; Barnes, 1995; Arch and Kaumann, 1993]. To date, at least nine subtypes of adrenergic receptors (three subtypes each of α₁-ARs, α₂-ARs, and β-ARs) have been identified. The precise function of all these receptors has not yet been defined, in part because of a dearth of highly specific agonists and antagonists. An alternative way to examine receptor function is to use molecular genetic techniques to overexpress or to knock out the expression of particular subtypes in laboratory animals [Milano et al. 1994a, 1994b; Bertin et al. 1993].

Pharmacological actions

Adipose tissue and diabetes

β₃-ARs mediate lipolysis in white adipose tissues and thermogenesis in brown adipose tissues [Lönnqvist et al. 1993; Langin et al. 1991; Zaagsma and Nahorski, 1990]. The presence of the Arg64 allele in the first intracellular loop of the β₃-AR gene may predispose patients to abdominal obesity, which may in turn predispose them to insulin resistance and the earlier onset of type 2 diabetes mellitus (T2DM) [Widén et al. 1995]. A naturally occurring variation, Trp⁶⁴Arg β₃-AR mutation, found in about 8% of Europeans and North Americans, actually restores the arginine residue in humans, which is found present in animals [Strosberg, 1997]. This variation was found to be associated with the following:

1. An increased capacity of obese French patients to gain weight [Clément et al. 1995].

2. An early onset of T2DM in obese Pima Indians by altering the balance of energy metabolism in visceral adipose tissue and tend to have a lower resting metabolic rate [Walston et al. 1995]. Similar observations were also reported in Japanese participants [Fujisawa et al. 1996].

3. An early onset of T2DM and clinical features of the insulin resistance syndrome in Finns [Widén et al. 1995].

The Trp⁶⁴Arg β₃-AR genotype is associated with mild gestational diabetes and this polymorphism is associated with increased weight gain during pregnancy [Festa et al. 1999]. The increased amount of adipose tissue after menopause is considered to elevate estradiol production, which in turn increases the risk for breast cancer. Thus, genetic traits that are related to obesity may influence the risk of postmenopausal breast cancer in an indirect manner [Huang et al. 2001]. A missense mutation in codon 64 of the β₃-AR gene that results in substitution of tryptophan by arginine [Trp⁶⁴ → Arg] in the first intracellular loop of the receptor protein has been reported in various ethnic groups, including the Japanese [Kadowaki et al. 1995]. A review [Arner and Hoffstedt, 1999] identified a link between obesity and the Trp64 → Arg polymorphism in 13 studies. Also a polymorphism in codon 27 of the ADRβ₂ gene that features a replacement of glutamine by glutamic acid [Gln27 → Glu] is linked with obesity [Large et al. 1997]. Thus, β₃-ARs may constitute a target for antiobesity and antidiabetic drugs [Lowell and Flier, 1995; Pietri-Rouxel and Strosberg, 1995].

BRL 26830 (see Table 2), a selective β₃-AR agonist, caused a marked increase in blood flow to brown adipose tissue in the anesthetized rat [Takahashi et al. 1992]. The increase in blood flow may well be secondary to an augmented metabolic process [Shen and Claus, 1993], since BRL 37344 (see Table 2) causes marked increases in the plasma levels of free fatty acids and insulin. In vitro studies have demonstrated that in rat [Granneman, 1992] and dog [Galitzky et al. 1993] fat cells, catecholamines stimulate β₃-ARs at higher concentrations than those required to activate β₁-ARs or β₂-ARs. Similar results were demonstrated in dog in vivo studies [Pelat et al. 2003]. Also, BRL 26830A causes stimulation of insulin secretion in pancreas β cells [Yoshida et al. 1991].

Table 2.

Agonists and antagonists of β₃-adrenoceptors (ARs).

β3-Agonist	Chemical name
BRL 26830A	methyl 4-[2-[[2-hydroxy-2-phenethyl]amino]propyl]benzoate-2-butanedioate
BRL 35135	methyl 4-[2-[2-hydroxy-2-[3-chlorophenyl]ethylamino] propyl] phenoxyacetate
BRL 37344	4-[-[[2-hydroxy-[3-chlorophenyl] ethyl]- amino] propyl] phenoxyacetate
CGP 12177	4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2-one
CGP 20712A	2-hydroxy-5-[2-[{2-hydroxy-3-[4-[1-methyl-4-trifluoromethyl-2-imidazolyl]phenoxy]propyl}amino]ethoxy] benzamide
CL 316243	disodium[R,R]-5-[2[[2-[3-chlorophenyl]-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate
FR-149175	ethyl-[[S]-8-[[R]-2-[3-chlorophenyl]-2-hydroxyethylamino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yloxy]acetate monohydrochloride monohydrate
ICI 198157	methyl [4-[2-[[2-hydroxy-3-phenoxypropyl]amino]ethoxy]phenoxy]acetate
ICI D7114	[S]-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-[2-methoxyethyl]phenoxyacetamide
L 742,791	[S]-N-[4-[2-{[3-[4-hydroxyphenoxy]-2-hydroxypropyl]amino}ethyl]phenyl]-4-iodobenzenesulfonamide
L 750355	3-pyridyloxypropanoloamine derivative
L755,507	4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4-hydroxyphenoxy) propyl] amino]ethyl]phenyl]-benzenesulfonamide
L 770,644	[R]-4-[4-[3-cyclopentylpropyl]-4,5-dihydro-5-oxo-1 H-tetrazol-1-yl]-N-[4-[2-[[2-hydroxy-2-[3-pyridinyl]ethyl]amino] ethyl]phenyl]- benzenesulfonamide
L 796568	[R]-N -[4-[2-[[2-hydroxy-2-[3-pyridinyl]ethyl]amino]ethyl]-phenyl]-4-[4-[4-[trifluoromethyl]phenyl]thiazol-2-yl]-benzenesulfonamide, dihydrochloride
LY 79771	[R-[R*, S*]] alpha-[[[3-[4-hydroxyphenyl]-1-methylpropyl]amino]methyl]benzenemethanol
RO363	[-]-1-[3,4-dimethoxyphenethylamino]-3-[3,4-dihdroxyphenoxy]-2-propanol] oxalate
SB 251023	[4-[1-{2-[S]-hydroxy-3-[4-hydroxyphenoxy]-propylamino} cyclopentyl methyl]phenoxymethyl]phenylphosphonic acid lithium salt
SR 58611	[RS]-N-[[25]-7-ethoxycarbonylmethoxy-1,2,3,4-tetrahydronapth-2-yl]-[2R]-2-[3-chlorophenyl]-2-hydroxyethanamine hydrochloride
SR 58611A	[N2S]-7-carbethoxymethoxy-1,2,3,4-tetrahydronaphth-2-yl-[2R]-2-hydroxy-2-chlorophenyl ethanamine hydrochloride
SR 59104A	N-[[6-hydroxy-1,2,3,4-tetrahydronaphthalen-[2R]-2-yl]methyl]-[2R]-2-hydroxy-2-[3-chlorophenyl]ethanamine hydrochloride
SR 59119A	N-[[7-methoxy-1,2,3,4-tetrahydronaphthalen-[2R]-2-yl]methyl]-[2R]-2-hydroxy-2-[3-chlorophenyl]ethanamine hydrochloride
ZD2079	4-[2-[[(2R)-2-Hydroxy-2-phenylethyl] amino]ethoxy]-benzeneacetic acid hydrochloride
ZD 7114	[S]-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-[2-methoxyethyl]phenoxyacetamide
ZM 215001	[S]-4-[2-hydroxy-3-phenoxy-propylamino-ethoxy] phenoxyacetic acid [R]-N-[4-[2-[[2-hydroxy-2-[3-pyridinyl]ethyl]amino]ethyl]phenyl]-1-[4-octylthiazol-2-yl]-5-indolinesulfonamide
β3-Antagonist	Chemical name
L 748328	[S]-N-[4-[2-{[3-[3-{aminosulfonyl}phenoxy]-2-hydroxypropyl]-amino}ethyl] phenyl]benzenesulfonamide
SR 59230A	3-[2-ethylphenoxy]-1[1 S]-1,2,3,4-tetrahydronaphth-1-ylaminol-[2S]2-propanol oxalate

In diabetic ZDF rats, CL 316243, a β₃ selective agonist (see Table 2) did not reduce hyperglycemia when given under acute conditions (single intravenous injections or subcutaneous infusions for a few days). However, long-term treatment of CL 316243 progressively normalized glycemia, reduced insulinemia and decreased the levels of circulating free fatty acids in obese diabetic ZDF rats. This treatment also markedly improved their glucose and insulin responses during an intravenous glucose tolerance test [Liu et al. 1998]. Hyperinsulinemic-euglycemic clamps combined with the [2-³H]deoxyglucose method revealed that chronic CL 316243 treatment markedly increased insulin responsiveness in obese rats and that it increases glucose uptake in brown adipose tissue, white adipose tissue, the diaphragm, and skeletal muscles, but not in the heart. The maximal capacity of various tissues for glucose uptake in CL 316243-treated animals varied in the following order: brown adipose tissue > heart > diaphragm > skeletal muscles > white adipose tissue [Liu et al. 1998]. This sequence of potencies agrees with previous observations for normal rats treated with insulin [Vallerand et al. 1987] or norepinephrine [Liu et al. 1994] as well as with cold-exposed animals [Vallerand et al. 1990]. Acute treatment of obese rodents with CL 316243 causes a number of diverse metabolic effects, including an increase in oxygen consumption and insulin levels, and a decrease in food intake. The mechanism by which CL 316243 increases insulin secretion in rodents is not known. Nevertheless, the stimulus for insulin secretion is extremely potent as it causes a 50 to 100-fold increase in insulin levels, which remain elevated (24-fold increase) despite the presence of hypoglycemia. This effect cannot be mediated by the direct effects of CL 316243 on pancreatic β cells because pancreatic islets appear not to express detectable levels of β₃-AR mRNA and because insulin secretion is not stimulated following addition of CL 316243 to cultured pancreatic islets [Grujic et al. 1997].

In another study by Fu and colleagues [Fu et al. 2007], CL 316243 and BRL 37344 downregulated adiponectin, but upregulated adiponectin receptor 2 (not receptor 1) in epididymal and/or subcutaneous white adipose tissue and in brown adipose tissue. Tumor necrosis factor-α (TNF-α) expression was upregulated only in epididymal adipose tissue, which suggests that upregulation of TNF-α and downregulation of adiponectin by β -AR activation may contribute to the pathogenesis of catecholamine-induced insulin resistance, and that upregulation of adiponectin receptor 2 may be a feedback result of reduced adiponectin [Fu et al. 2007]. In addition, the effects of CL 316243 were investigated in obese diabetic KKAy mice by Fu and colleagues [Fu et al. 2008]. Two weeks’ subcutaneous administration of CL 316243 reduced serum levels of glucose, insulin, triglyceride, free fatty acid and TNF-α, and increased adiponectin. CL 316243 recovered the mRNA expressions of adiponectin, adiponectin receptors and β₃-ARs, which were reduced in epididymal white adipose tissue in KKAy mice. Meanwhile, CL 316243 suppressed the overexpressed mRNA level of TNF-a in both epididymal white adipose tissue and brown adipose tissue. These data suggest that the normalization of adiponectin, adiponectin receptors and TNF-α may result in the amelioration of obesity-induced insulin resistance [Fu et al. 2008].

β₃-Agonists appear to be of significance not only in obesity but also in terms of the risks of cardiovascular disorders because visceral obesity is the most dangerous form of regional fat accumulation, the form of obesity that is more directly linked to β₃-AR activity [Arner, 1995].

Vascular smooth muscles

β₃-ARs produce sustained peripheral vasodilation that is predominant in skin and fat [Shen et al. 1994; Berlan et al. 1993]. A study showed that the relaxation of rat thoracic aorta was caused by selective β₃-AR agonists like CGP 12177 [Mohell and Dicker, 1989], cyanopindolol [Engel et al. 1981], ZD 2079 [Grant et al. 1994], ZM 215001 [Tesfamariam and Allen, 1994], and SR 58611 [Trochu et al. 1999] (see Table 2), further supporting the presence of β₃-ARs [Brawley et al. 2000a, 2000b]. A β₃-AR-mediated vasorelaxation was also observed in the canine pulmonary artery, an effect that was exerted through a cAMP-dependent pathway [Tagaya et al. 1999]. In the rat carotid artery, the selective β₃-AR agonist BRL 37344 and the selective β₂-AR agonist, salbutamol, were not antagonized by propranolol (100 nM), and pretreatment of the artery segments with BRL 37344 did not desensitize the tissue to the relaxant effect of isoprenaline and salbutamol [Oriowo, 1994]. In the same tissue, MacDonald and colleagues [MacDonald et al. 1999] confirmed the presence of β₃-AR by the relaxant effects of two selective β₃-AR agonists, BRL 37344 and ZD 2079. In normal dogs, the infusion of BRL 37344 or CL 316243 or CGP 12177 induced an increase in heart rate and cutaneous blood flow (evaluated in the internal part of the ear) [Berlan et al. 1994].

The Trp⁶⁴Arg mutation of β₃-AR has been suggested to confer susceptibility to essential hypertension [Morris et al. 1994] and this was confirmed by Tonolo and colleagues [Tonolo et al. 1999]. These authors concluded that the Trp⁶⁴Arg polymorphism of the β₃-AR gene is associated more often with high blood pressure than with normal blood pressure. Isoproterenol, BRL 37344 and CGP 12177 are reported to produce a reduction in arterial blood pressure. In sinoaortic denervated animals, isoproterenol infusion provoked tachycardia and hypotension [Tavernier et al. 1992]. It was demonstrated that a higher dose of isoproterenol is required to obtain in vivo β₃-mediated vasodilation than that necessary for β₁-or β₂-mediated vasodilation. The reason for this may be that β₃-AR is a ‘back-up’ receptor activated during extreme or stressful conditions [Pelat et al. 2003].

Significant increases in systolic blood pressure and Doppler stroke distance occurred with BRL 37344 and salbutamol, which were unaffected by pretreatment with bisoprolol and completely blocked by nadolol, in keeping with the β ₂-mediated effects. BRL 37344 and salbutamol produced significant chronotropic effects, which were unaffected by β₁-AR blockade [Wheeldon et al. 1994]. In a clinical study, isoprenaline produced an increase in systolic blood pressure and left ventricular stroke distance that was not attenuated by a dose of nadolol, which produced complete blunting of β₁- and β₂-mediated responses but not of β₃-mediated effects [Wheeldon et al. 1993].

Experimental in vivo studies have demonstrated that positive β₃-AR-related chronotropic effects were prevented by β₁-AR or β₂-AR antagonists and are likely due to baroreflex activation in response to β₃-AR agonist-induced vasodilation [Tavernier et al. 1992; Takayama et al. 1993]. Apart from this, positive chronotropic effects were not observed in denervated animals and thus it was concluded that tachycardia resulted from a baroreceptor-mediated reflex in response to a drop in blood pressure caused by the vasodilating action of β₃-AR agonists [Berlan et al. 1994; Shen et al. 1994]. In normal dogs, infusion of isoproterenol, BRL 37344 or CGP 12177 increased heart rate with the following order of potency: BRL 37344 > isoproterenol >> CGP 12177. Isoproterenol stimulated adenylate cyclase activity in heart membranes from normal dogs, whereas CGP 12177 and BRL 37344 were without any stimulating action [Tavernier et al. 1992]. In vitro studies are more suitable in analyzing the cardiac effects of β₃-ARs. A typical example of the masking effects of baroreflex activation lies with 1,4-dihydropyridines, which induce a negative inotropic effect in vitro, but a positive chronotropic and inotropic effect in vivo as a consequence of vasodilation [Piepho, 1991].

The presence of β₃-ARs has also been reported in veins. In the rat portal vein, activation of β₃-ARs stimulates L-type Ca²⁺ channels through a Gα_s-induced stimulation of the cyclic AMP/ protein kinase, a pathway and the subsequent phosphorylation of the channels [Viard et al. 2000]. In rats, the selective β-AR agonist CL 316243 induced marked increases in islet blood flow and plasma insulin level, and these increases were stopped by bupranolol, a β₁-AR, β₂-AR and β₃-AR antagonist, but not by nadolol, a β₁-AR and β₂-AR antagonist, indicating that β₃-ARs caused a vasodilation of microvessels in the islets of Langerhans [Atef et al. 1996].

Cardiac effects

β₃-AR stimulation of the human cardiac muscle, in contrast with β₁-AR and β₂-AR stimulation, resulted in a profound dose-dependent negative inotropic effect. This unexpected finding suggests that β₃-ARs may participate in the pathogenesis of cardiac failure, during which modification of β₁-AR and β₂-AR expression occurs [Brodde, 1993]. Functional β₃-ARs stimulation, which occurs in the normal left ventricle, causes direct inhibition on (Ca²⁺)_iT and I_Ca,L(L-type Ca channels) and produces a negative inotropic action [Cheng et al. 2001]. In another study, it was found that β₃-AR activation inhibits the L-type Ca²⁺ channel in both normal and heart failure rat myocytes. In heart failure, β₃-AR stimulation-induced inhibition of Ca²⁺ channels is enhanced, which is responsible for reduced inotropic response [Zhang et al. 2005]. β₃-AR agonists induce negative inotropic effects with the following order of potency: BRL 37344 > SR 58611 = CL 316243 > CGP 12177, similar to that observed in Chinese hamster ovary cells transfected with human β₃-ARs [Pietri-Rouxel and Strosberg, 1995; Dolan et al. 1994]. In another study, the mechanical effects of BRL 37344 were not modified by pretreatment with metoprolol (β₁-AR antagonist) or nadolol, indicating that this effect was not mediated by β₁-ARs or β₂-ARs. By contrast, bupranolol, which possesses β₃-AR antagonist properties [Pietri-Rouxel and Strosberg, 1995; Galitzky et al. 1993; Sugasawa et al. 1992], antagonized the negative inotropic effects of BRL 37344 with a pA₂ value similar to that determined in adipocytes [Galitzky et al. 1998; Pietri-Rouxel and Strosberg, 1995].

In heart failure, increased activity of the sympathetic nervous system leads to downregulation of cardiac β₁-ARs and β₂-ARs [Brodde, 1993] resulting from their phosphorylation by cAMP-dependent protein kinase or β-AR kinase. Reduced β₁-ARs and β₂-ARs lead to a decrease in the contractile response to β-AR agonists [Strosberg, 1993]. Contrary to β₁-ARs and β ₂-ARs, the abundance of the negatively inotropic β₃-ARs increases in the failing myocardium [Moniotte et al. 2001]. β₃-ARs lack phosphorylation sites for cAMP-dependent protein kinase or β-AR kinase [Strosberg, 1993], and thus may not be downregulated in heart failure. According to this hypothesis, the high adrenoceptor tone during heart failure may alter the cardiac contractile activity as a result of unmasked β₃-AR stimulation in the presence of reduced β₁-ARs and β₂-ARs [Gauthier et al. 1996]. Overstimulation of the relatively desensitization-resistant β₃-AR [Liggett et al. 1993] after increased sympathetic tone and norepinephrine release in the setting of heart failure in humans may further decrease cardiac inotropy [Moniotte et al. 2001]. The levels of β₃-AR mRNA and proteins show an increase in the failing heart compared with the nonfailing heart. The β₃-AR agonist BRL 37344 was found to markedly aggravate the cardiac function and stimulate cardiac myocytes apoptosis in the failing heart. If the levels of β₃-AR are too high, they might contribute to the loss of cardiac function and be the foundation of the functional degradation of heart failure [Kong et al. 2004]. Moreover, another study in isoproterenol-induced chronic heart failure rats suggests that the myocardial upregulation of β₃-AR in heart failure is associated with increased oxidative stress [Kong et al. 2010]. These studies open the perspective for correcting the disordered adrenergic regulation of the failing heart with specific antagonists of the human cardiac β 3-AR. By contrast, Rasmussen et al. (2009) reported that as increased intracellular myocyte Na+ levels represent a key adverse pathophysiological feature of heart failure, and the β₃-AR mediates the stimulation of the only export route for Na+- the Na+-K+ pump — the upregulation of this receptor may also represent a useful compensatory mechanism. Data from animal studies and circumstantial observations from clinical trials suggest that β₃-AR activation is beneficial in severe heart failure, and that β₃-AR agonists are a promising therapeutic option for the treatment of this disease (Rasmussen et al. 2009). In transgenic mice with cardiac-specific overexpression of protein of the human β₃-AR (TGβ₃ mice), the human β₃-AR is quiescent until stimulated with a selective agonist L 755,507, at which point there is a marked augmentation in left ventricular contractility. In addition, because β3-AR is relatively insensitive to catecholamines, it would be minimally activated by endogenous catecholamines. This approach could have important therapeutic potential in patients with heart failure, in which delivery of the human β₃-AR by gene therapy could provide a functionally inactive signaling protein that becomes activated only when a highly selective agonist is exogenously administered [Kohout et al. 2001].

In congestive heart failure (CHF), β₃-AR expression is increased. This augmentation is proposed to exacerbate the dysfunctional [Ca²⁺]_i regulation, enhance inhibition of cardiac contraction and relaxation, and lead to worsening of cardiac failure [Cheng et al. 2001]. In CHF, when marked increases in sympathetic tone and cardiac norepinephrine release have rendered the positive inotropic β₁-AR system relatively unresponsive, the upregulated β₃-AR pathways would continue to exhibit a negative inotropic effect. This altered balance between opposing inotropic influences of β₁-ARs and β₃-ARs in CHF may contribute to progressive cardiac dysfunction in CHF. The enhanced response to β₃-AR stimulation in CHF may also be related to increased numbers of β₃-ARs or an altered signal transduction [Cheng et al. 2001]. As shown in Figure 1, in CHF, NO-cGMP signaling may be altered [Mohan et al. 1996], thereby altering CHF myocyte response to β₃-AR stimulation [Cheng et al. 2001]. The enhanced contractile response to β₃-AR stimulation in CHF myocytes of dogs may be coupled to G_i through both NO-dependent and NO-independent mechanisms [Cheng et al. 2001]. The activation of G_i also has the potential to couple β₃-ARs to other important signaling pathways such as mitogen-activated protein kinase [Soeder et al. 1999]. The increase in other neurohormonal activation, such as TNF-α, endothelin 1, and angiotensin II, may also differentially modulate β₃-AR expression and function. These studies indicate that using β₃-AR agonists for the treatment of obesity and diabetes [Arch et al. 1984] may have cardiac side effects, especially in patients with CHF [Cheng et al. 2001]. Also, these studies suggest several novel therapeutic strategies for the treatment of CHF, such as the use of β₃-AR antagonists or G_i inhibitors. Gan and colleagues [Gan et al. 2007a] reported that a β₃-AR antagonist SR 59230A can block the β₃-AR—nitric oxide synthase (NOS)—cyclic GMP pathway and improve cardiac function in heart failure in rats if administered long term. SR 59230A can also attenuate cardiac remodeling by inhibition of interstitial fibrosis to a certain degree, which may help to improve cardiac function in heart failure [Gan et al. 2007b]

Figure 1.

Postulated changes in β-adrenoceptor signaling in cardiomyocytes from nonfailing to failing myocardium. eNOS, endothelial nitric oxide synthase.

β₃-ARs are involved in the vasomotor control of the internal mammary artery and thus open new fields of investigation in coronary bypass graft management, heart failure, and hypertension [Rozec et al. 2005]. In the hearts of long-term diabetic rats, the expression of β₁-ARs decreases, whereas that of β₃-ARs increases. This may suggest that a decrease in β₁-AR together with an increase in β₃-AR expression might be involved in the development of diabetes-induced cardiac dysfunction [Dinçer et al. 2001].

In cardiac myocytes, repolarization of the action potential is produced by several potassium currents [Barry and Nerbonne, 1996] like very slow activating and deactivating delayed rectifier potassium current (IKs). This current represents the predominant repolarizing current during increased heart rate [Zeng et al. 1995; Jurkiewicz and Sanguinetti, 1993; Varnum et al. 1993]. The channel underlying IKs is formed by the assembly of two transmembrane proteins, the KvLQT1 and MinK protein [Barhanin et al. 1996; Sanguinetti et al. 1996]. The IKs current amplitude in the heart is increased by catecholamines, which are mediated by β-ARs [Lo and Numann, 1998; Sanguinetti et al. 1991; Duchatelle-Gourdon et al. 1989]. Catecholamines develop negative inotropic effects and shorten the human cardiac action potential through β₃-ARs [Gauthier et al. 1996]. The shortening of the human cardiac action potential under β₃-AR stimulation may be because they can couple functionally to the KvLQT1/MinK potassium channel in the Xenopws oocyte expression system, which involves G proteins [Kathofer et al. 2000]. The shortening of cardiac action potentials is expected to affect the repolarization process, thereby potentially triggering arrhythmias. The coupling of the KvLQT1/MinK channel to the β₃-AR may have important implications for arrhythmogenesis in the heart and thus may open new perspectives for the prevention and treatment of cardiac arrhythmias [Kathofer et al. 2000]. The findings of Zhou and colleagues [Zhou et al. 2008] suggested that β-AR blocking agents with β₃-AR agonist properties might be useful for cardiac arrhythmia control after myocardial infarction, especially in treating ventricular tachycardia storms.

Endothelium

After L-NAME treatment or removal of endothelium, relaxant responses to isoprenaline were found to be unaffected by propranolol, suggesting that they were mediated by β₃-ARs and/or the low-affinity state of β₁-ARs, formerly proposed as putative β₄-ARs [Brawley et al. 1998].

In the rat thoracic aorta, β₃-ARs act in conjunction with β₁-ARs and β₂-ARs to mediate relaxation through activation of an NOS pathway and subsequent increase in cyclic GMP levels [Trochu et al. 1999]. In human vessels, β₃-AR relaxation was also found to be mediated partly through NO production. This was evidenced by its complete abrogation by NOS inhibition under circumstances when both prostanoids and endothelium-derived hyperpolarizing factors (EDHFs) are inoperative (that is, after cyclooxygenase inhibition and preconstriction with high potassium chloride respectively) [Dessy et al. 2004]. This may be caused by functional coupling of β₃-AR agonists to NO production in whole human ventricular muscle through G_ai proteins [Moniotte et al. 2001; Gauthier et al. 1998]. Endothelial cells produce a hyperpolarization leading to vascular muscle relaxation through activation of calcium-dependent K⁺ channels [Busse et al. 2002]. Dessy and colleagues [Dessy et al. 2004] demonstrated vessel hyperpolarization in response to β₃-AR agonists and the abrogation of β₃-AR-mediated relaxation after vessel pretreatment with the K⁺ channel inhibitors charybdotoxin and apamin, two signatures of an EDHF response. These results are also in agreement with the recent proposition of β₃-AR-mediated relaxation through K⁺ channel activation in rat aorta [Rautureau et al. 2002]. Functional β₃-AR vasorelaxation mediated in part by EDHFs in human coronary resistance arteries may have a major bearing on our understanding of regulating coronary perfusion in circumstances such as dyslipidemia, diabetes and atherosclerosis, all associated with decreased NO production and/or bioavailability.

Conclusions

Almost 50 years after Ahlquist first uncovered evidence of the heterogeneity of adrenergic receptors, the number of receptor subtypes is still unclear, although nine subtypes are well documented (three subtypes each of α₁-ARs, α₂-ARs and β-ARs). Adrenergic receptors are members of a large superfamily of receptors linked to G proteins. The identification of new subtypes of receptors offers the promise of new therapeutic agents. β₃-ARs, which are found at unique sites such as in brown adipose tissue and the gallbladder, are potential targets for antiobesity drugs. Although considerable information is available on β₃-AR physiology in fat, there are many other areas in which β₃-ARs are involved. The presence of β₃-ARs in vasculature and heart provides new avenues for the development of innovative type-specific drugs. Since alterations in adrenergic receptors have a role in many clinical settings, the development of such agonists and antagonists may give therapeutic potential for the treatment of various disorders, including diabetes mellitus, hypertension, dyslipidemia, cardiac arrythmias, heart failure and diabetes-induced cardiac dysfunction. It is known that using β₃-AR agonists to treat obesity and diabetes may have cardiac side effects, especially in patients with CHF. However, with the knowledge that there are two types of β₃-ARs (β_3a and β_3b), it may be possible to develop subtype-specific drugs that are more effective and have fewer side effects than those currently available.