Expression of the beta 3-adrenoceptor gene polymorphism (Trp64Arg) in obese diabetic and non-diabetic subjects

1. A missense mutation (Trp64Arg) in the beta 3-adrenoceptor (beta 3-AR) gene has been associated with weight gain, insulin resistance and earlier-onset non-insulin-dependent diabetes mellitus (NIDDM), but the strength of these associations varies considerably between populations and the functional significance of Trp64Arg remains unclear. 2. The Trp64Arg mutation was investigated in obese NIDDM (n = 50) and obese non-diabetic (n = 53) subjects by polymerase chain reaction (PCR) amplification of genomic DNA and digestion of the 210 bp product by BstOI. The Arg allele was found in 22.3% of all subjects, but there were no homozygotes for the mutation. Non-diabetic subjects heterozygous for the mutation were more obese and Trp/Arg diabetics had a slightly younger age of onset of NIDDM (47 vs 51 years, respectively), but there were no significant differences in mutation frequency between the two groups. Metabolic parameters (e.g. fasting lipids and glycaemic control) were similar among diabetic subjects with and without the Trp64Arg mutation. 3. In conclusion, the frequency of the Trp64Arg mutation of the beta 3-AR was higher in this obese population compared with some previous studies, but there was no evidence that Trp64Arg confers an increased susceptibility to NIDDM among obese insulin-resistant subjects or that diabetics with the mutation fare worse in terms of lipid or glucose metabolism.     

Trp64Arg variant of the beta3-adrenoceptor and insulin resistance in obese postmenopausal women

There is controversy regarding the role of the Trp64Arg variant of the beta3-adrenergic receptor (beta3AR) gene in the pathogenesis of insulin resistance. The modest effect of the variant as well as differences in study design, gender, age, and genetic background may contribute to divergent results among investigations. Insulin sensitivity (euglycemic clamp and tracers) was measured in 13 obese women (57 +/- 6 yr old) heterozygous for the beta3AR variant and in 14 women (57 +/- 4 yr old) homozygous for the normal gene. Groups were matched for age, body composition, intraabdominal fat, sc abdominal fat, physical activity level, and aerobic capacity. Exogenous glucose infusion during the clamp was significantly lower (P = 0.03) in beta3AR heterozygotes (241 +/- 135 mg/min) vs. normal homozygotes (379 +/- 172 mg/min). Basal endogenous glucose production was not different (P = 0.20) between heterozygotes (175 +/- 27 mg/min) and normal homozygotes (164 +/- 14 mg/min). Endogenous glucose production during hyperinsulinemia was also not different (P = 0.22) between heterozygotes (77 +/- 57 mg/min) and normal homozygotes (56 +/- 16 mg/min). Total glucose disposal adjusted for residual endogenous glucose production was lower (P = 0.049) for heterozygotes (320 +/- 111 mg/min) than for normal homozygotes (441 +/- 183 mg/min). Our results suggest that obese postmenopausal women who are heterozygous for the Trp64Arg variant in the beta3AR gene have greater insulin resistance than age-, body composition-, and physical activity-matched women homozygous for the normal gene.     

Role of the beta 3-adrenergic receptor locus in obesity and noninsulin-dependent diabetes among members of Caucasian families with a diabetic sibling pair

Obesity and insulin resistance are important risk factors for the development of noninsulin-dependent diabetes (NIDDM) and are prevalent among predisposed first degree relatives of diabetic individuals. Recent molecular screening and analysis of a common missense mutation of the beta 3-adrenergic receptor gene suggested this locus as a strong candidate for increased obesity, earlier age of diabetes onset, and insulin resistance. To test the hypothesis that the beta 3-adrenergic receptor locus affects diabetes susceptibility, obesity as measured by body mass index, and components of the insulin resistance syndrome, we examined the role of this region in families ascertained for two or more NIDDM siblings. Linkage analysis was conducted using both parametric and nonparametric analyses, including multipoint sibling pair analysis. We found no evidence for linkage to NIDDM as a dichotomous trait and no evidence for linkage to body mass index, waist/hip ratio, insulin levels, or glucose levels as quantitative traits or to reported age of onset among NIDDM individuals. The Trp64 Arg missense mutation was present in 11% of the population. The mutation was not associated with NIDDM, and Arg64 carriers did not have earlier NIDDM onset, higher body mass index, or higher waist/hip ratio than Trp64 homozygotes. Among relatives, Arg64 carriers had significantly lower fasting glucose levels and lower waist/hip ratios than Trp64 homozygotes, but no characteristics of the insulin resistance syndrome. We conclude that the beta 3-adrenergic receptor locus does not play an important role in NIDDM susceptibility or in the insulin resistance syndrome among members of families with a strong predisposition to NIDDM.     

Meta-analysis of the association of Trp64Arg polymorphism of beta 3-adrenergic receptor gene with body mass index

A possible pathogenic polymorphism in the beta 3-adrenergic receptor gene (Trp64Arg) has been reported to be associated with increased body weight, clinical features of insulin resistance, and early development of type 2 diabetes mellitus in several populations. However, such findings have not been consistent among studies, making the hypothesis that this genetic marker is associated with clinical features controversial. To assess the effect of the genotypes on body mass index (BMI), we performed a meta-analysis of the data from the literature using an extension of ANOVA for continuous measures. In a total of 48 subgroups containing subjects with (n = 2447) and without (n = 6789) the Trp64Arg variant, the summary weighted mean difference in BMI was 0.30 (95% confidence interval, 0.13-0.47) kg/m2, indicating that variant carriers exhibited higher BMI (on the average, 0.30 kg/m2 higher) than normal homozygous subjects. In this case, there was no significant evidence against homogeneity of the effect (P = 0.36). This is the first meta-analysis assessing quantitative phenotypes in relation to a genetic polymorphism, and the results support the hypothesis that the Trp64Arg polymorphism is associated with BMI across diverse population groups, suggesting that the beta 3-adrenergic receptor gene locus plays a role in genetic predisposition to increased body weight in a universal manner.     

Additive effects of the mutations in the beta3-adrenergic receptor and uncoupling protein-1 genes on weight loss and weight maintenance in Finnish women

This study examined whether the Trp64Arg mutation in the beta3-adrenergic receptor (beta3AR) and the A-->G mutation in the uncoupling protein-1 (UCP-1) genes have associations with weight loss and subsequent weight maintenance. Seventy-seven obese (body mass index range, 29-46 kg/m2), clinically healthy, premenopausal women were studied. A 12-wk weight reduction by very low calorie diet (VLCD) was followed by a 40-wk weight maintenance phase. The subjects were divided into four groups according to their beta3AR and UCP-1 genotype: no mutation (control; n=37), only Trp64Arg mutation in the beta3AR gene (n=12), only A-->G mutation in the UCP-1 gene (n=23), and both mutations (n=5). Subjects with both mutations had a lower weight reduction during VLCD than the controls [-10.5+/-0.6 (+/-SEM) vs. -14.0+/-0.5 kg; P=0.051, by ANOVA]. During the maintenance phase, weight in subjects with both mutations increased by 5.8+/-1.5 kg, but remained unchanged in the controls (-0.5+/-0.8 kg; P=0.041). The changes in weight in subjects with only one of the mutation were close to the results in the controls. Resting energy expenditure, adjusted for fat mass, fat-free mass, and maximal aerobic power, did not change differently between the groups throughout the study. The results suggest that a combination of the Trp64Arg mutation in the beta3AR and the A-->G mutation in the UCP-1 genes may be associated with faster weight gain after a VLCD.     

Gender effect of the Trp64Arg mutation in the beta 3 adrenergic receptor gene on weight gain in morbid obesity

Phenotypic expression of the Trp64Arg mutation in the beta 3-adrenoceptor gene (beta 3-AR) has been found to be somewhat variable among different populations, suggesting that it may be influenced by genetic background and environmental factors. As sex may also influence gene allellic expression, we evaluated a potential gender effect of the Trp64Arg mutation in 292 morbidly obese subjects [body mass index (BMI) > or = m/kg2]. Although the 15 mutated obese females were younger than the non-mutated ones, the difference between their current weight and their weight at 20 years was significantly higher (62.4 +/- 20.0 kg versus 47.0 +/- 24.0 kg; p = 0.017). Moreover, in the mutated heterozygous female group, the mean Zscore (individual BMI minus reference French population mean BMI/SD of reference population BMI) was significantly higher (8.0 +/- 2.5 versus 6.0 +/- 2.0 SD of BMI, p = 0.0018), as was the maximal Zscore calculated from the maximal BMI that obese females reached during life (9.0 +/- 3.0 versus 7.0 +/- 2.5, p = 0.005). The regression curves of the Zscore against age showed that the curve of mutated females was shifted to the top, indicating that their BMI was higher regardless of age. These effects were not observed in the male group (the Zscore was 6.7 +/- 3.0 vs. 7.2, p = 0.7 respectively in mutated and non-mutated men). These data reinforce the hypothesis that the expression of the beta 3-AR susceptibility gene depends on additional factors including gender and possibly hormonal status.     

Odontogenic keratocysts: review of the literature and report of a case

The beta 3-adrenergic receptor (beta 3AR) is expressed in visceral fat and is a regulator of resting metabolic rate, thermogenesis, and lipolysis. We genotyped 61 unrelated Mexican Americans for a variant in the beta 3AR gene (codon 64 TGGTrp-->CGGArg; TRP64ARG). The allele frequency was 0.13. The TRP64ARG variant was significantly associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (41.3 +/- 4.6 years vs 55.6 +/- 2.6 years; P < 0.02) and in non-diabetics, with elevated 2-h insulin levels during an oral glucose tolerance test (810 +/- 120 pmol/l vs 384 +/- 6 pmol/l; P < 0.005). Non-diabetic subjects with the variant allele tended to have higher body mass indices (BMI), waist-to-hip ratios, and diastolic blood pressures. The study group was expanded to include 421 related subjects from 31 families in the San Antonio Family Diabetes Study. Using a measured genotype analysis approach to estimate genotype-specific means for each trait, those who were homozygous for the TRP64ARG variant had significantly higher 2-h insulin levels (P = 0.036) and trends towards higher BMI compared to the other two genotypes. We detected no associations of these traits in the TRP64ARG heterozygotes in the larger group. We conclude that the TRP64ARG beta 3AR variant is a susceptibility gene for several features of the insulin resistance syndrome in Mexican Americans. Since its effects are modest, study design (e.g., subject selection, genetic background, and statistical analyses) may influence which traits are associated with this variant and whether or not the effect is detectable in heterozygotes.     

Association of a polymorphism in the beta 3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns

BACKGROUND: Because visceral obesity predicts insulin resistance, we studied whether alterations in the gene encoding for the beta 3-adrenergic receptor in visceral fat are associated with insulin resistance. METHODS: We studied the frequency of a cytosine-to-thymidine mutation that results in the replacement of tryptophan by arginine at position 64 (Trp64Arg) of the beta 3-adrenergic receptor by restriction-enzyme digestion with BstOl in 335 subjects from western Finland, 207 of whom were nondiabetic and 128 of whom had non-insulin-dependent diabetes mellitus (NIDDM). We also determined the frequency of the mutation in 156 subjects from southern Finland. Sensitivity to insulin was measured by the hyperinsulinemic-euglycemic clamp technique in 66 randomly selected nondiabetic subjects. RESULTS: In the subjects from western Finland, the frequency of the mutated allele was similar in the nondiabetic subjects and the subjects with NIDDM (12 vs. 11 percent). The mean age of the subjects at the onset of diabetes was lower among those with the mutation than those without it (56 vs. 61 years, P = 0.04). Among the nondiabetic subjects, those with the mutation had a higher ratio of waist to hip circumference (P = 0.02), a greater increase in the serum insulin response after the oral administration of glucose (P = 0.05), a higher diastolic blood pressure (82 vs. 78 mm Hg, P = 0.01), and a lower rate of glucose disposal during the clamp study (5.3 vs. 6.5 mg [29 vs. 36 mumol] per kilogram of body weight per minute; P = 0.04) than the subjects without the mutated allele. In an analysis of sibling pairs, the siblings with the mutation generally had higher waist:hip ratios (P = 0.05) and higher responses of blood glucose and serum insulin after the oral administration of glucose than their siblings without the mutation (P = 0.02 and P = 0.005, respectively). CONCLUSIONS: The Trp64Arg allele of the beta 3-adrenergic receptor is associated with abdominal obesity and resistance to insulin and may contribute to the early onset of NIDDM:     

Trp64Arg mutation of beta 3-adrenergic receptor and insulin sensitivity in subjects with glucose intolerance

We investigated the relationship between the Trp64Arg mutation in the beta 3-adrenergic receptor gene and insulin sensitivity, which was evaluated by the euglycemic-hyperinsulinemic-clamp technique, in 54 patients with impaired glucose tolerance (IGT) or non-insulin dependent diabetes mellitus (NIDDM) who were not receiving insulin therapy. The frequencies of Trp/Trp, Trp/Arg, and Arg/Arg genotypes in the patients were 63.0, 33.3, and 3.7%, respectively, which did not differ significantly from those of the 227 controls (67.0, 33.3, and 3.7%, respectively, which did not differ significantly from those of the 227 controls (67.0, 31.3, and 1.8%, respectively). The mean glucose infusion rate of the 34 patients with Trp/Trp did not differ from that of the 18 patients with Trp/Arg (4.3 +/- 2.2 and 5.3 +/- 2.4 mg/kg/min, respectively); while that of the 2 patients with Arg/Arg was 11.5 mg/kg/min. There were no differences in the BMI or fat distribution in the abdomen between each genotype of patients, although the frequency of the Arg64 allele tended to increase with body mass index (BMI) in the control subjects under 60 years of age, which suggests that the mutation is involved in weight gain.     

Beta-3 adrenergic receptor agonists cause an increase in gastrointestinal transit time in wild-type mice, but not in mice lacking the beta-3 adrenergic receptor

The effects of beta-3 adrenergic receptor (beta3-AR) agonists on gastrointestinal (GI) motility, as reported by stomach retention and intestinal transit of radiolabelled charcoal, were compared in wild-type (WT) mice and in transgenic mice lacking beta3-AR (beta3-AR[KO]) or having beta3-AR in white and brown adipose tissue only (beta3-AR[WAT+BAT]). After s.c. administration of 3 mg/kg of the selective, rodent specific beta3-AR agonists BRL 35135, CL 316, 243 or ICI 198,157, WT mice exhibited a significant decrease in the extent of movement of radiotracer through the stomach and intestines, indicative of decreased GI motility. These compounds also caused an increase in plasma glycerol levels in the WT mice, suggesting that increased lipolysis in adipose tissue had been evoked. None of these compounds had an effect on GI motility or evoked lipolysis in the beta3-AR[KO] mice. Treatment of WT mice with SR 56811A, a beta3-AR agonist that exhibited a relatively lower affinity for rodent beta3-AR in vitro, did not affect GI motility or plasma glycerol levels in WT or beta3[KO] mice when administered s.c. at 3 mg/kg. Clonidine, an alpha-2 adrenergic receptor agonist, used as a positive control in these GI studies, caused a decrease in GI motility in both WT and beta3-AR[KO] mice. These results are consistent with a postulated role for beta3-AR in regulation of GI motility in the mouse. However, treatment of beta3-AR[WAT+BAT] mice with 3 mg/kg BRL 35135 resulted in elevated plasma glycerol levels, as well as increased stomach retention and decreased intestinal transit of radiotracer. These results suggest that this beta3-AR agonist may exert its effects on the GI tract indirectly, through an unknown signaling mechanism activated by agonism of beta3-AR in adipose tissue.     

UKPDS 19: heterogeneity in NIDDM: separate contributions of IRS-1 and beta 3-adrenergic-receptor mutations to insulin resistance and obesity respectively with no evidence for glycogen synthase gene mutations. UK Prospective Diabetes Study

Insulin receptor substrate-1 (IRS-1), beta 3-adrenergic-receptor (beta 3-AR) and glycogen synthase (GS) genes are candidate genes for non-insulin-dependent diabetes mellitus (NIDDM), insulin resistance, dyslipidaemia and obesity. We studied white Caucasian subjects with NIDDM, 227 being randomly selected, 49 NIDDM within the top two percentiles of insulin resistance; 54 with dyslipidaemia in the top quintile of triglyceride/insulin and the bottom quintile of HDL, and 166 non-diabetic control subjects. We examined the association of the simple tandem repeat DNA polymorphisms (STRPs) near the IRS-1 and GS genes, and the prevalence of mutations at codons of IRS-1 513 and 972, beta 3-AR 64 and GS 464 using restriction fragment length polymorphism (RFLP). The STRP alleles in IRS-1 were significantly different between NIDDM and control subjects (p = 0.015). The IRS-1 972 mutation was significantly different between the four groups with increased prevalence in the insulin resistant and dyslipidaemia subjects (18 and 26% compared with 11% in control subjects; p < 0.0005). Those with or without IRS-1 mutations had similar clinical characteristics and impaired insulin sensitivity. beta 3-AR 64 mutation was not significantly different between the four groups but those with the mutation were more obese, with a test for linear association between number of alleles and degree of obesity in an analysis of variance showing a significant association (p = 0.029). The GS 464 mutation was not detected in any of the diabetic or control subjects and the population association study using GS STRP showed no difference in allelic frequencies between NIDDM patients and control subjects. A mutation in lipoprotein lipase at codon 291, associated in the general population with low HDL cholesterol, was not at increased prevalence in the NIDDM patients with dyslipidaemia. In conclusion, IRS-1 972 had an increased prevalence in subjects with insulin resistance, with or without dyslipidaemia. beta 3-AR 64 was associated with increased obesity but not with insulin resistance or dyslipidaemia. These separate contributions to different features of NIDDM are an example of the polygenic inheritance of this heterogeneous disorder.     

Distribution of myocardial beta-adrenoceptor subtypes and coupling to the adenylate cyclase in children with congenital heart disease and implications for treatment

In congestive heart failure, down-regulation of myocardial beta-adrenoceptors (beta-AR) due to an elevated sympathetic tone is well known. In infancy and childhood, heart failure is usually related to congenital heart disease (CHD). Therefore, 71 samples of right atrial tissue of infants and children with CHD undergoing cardiac surgery were studied for beta-adrenoceptor density and distribution of the beta 1-/beta 2-AR subtypes. In 49 cases, the coupling of the beta-AR to the adenylate cyclase (AC) was examined. In a further study of 19 myocardial samples, AC was selectively stimulated with beta 1- or beta 2-AR whereas the other subtype was blocked by an antagonist. The following results were obtained: (1) Infants and children with severe acyanotic or cyanotic CHD had severely reduced beta-AR densities. (2) In most of the cases, the beta-AR down regulation is beta 1-subtype selective, but in critically ill newborns with congenital aortic valve stenosis or transposition of the great arteries, there is additional significant beta 2-AR down-regulation. In Fallot patients treated with the beta-antagonist propranolol, a significant increased beta-AR number compared with untreated Fallot patients was found. (3) beta-Adrenoceptor reduction in CHD is correlated with elevated noradrenaline plasma levels, thus proving a sympathetic dysregulation. (4) In CHD with moderate hemodynamic load, beta 2-AR coupling to AC was markedly more efficient than beta 1-AR coupling. The small number of myocardial beta 2-AR produced most of the cyclic adenosine monophosphate. (5) In severe acyanotic and cyanotic CHD, a partial decoupling of the beta 2-AR to the AC occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The role of luteinizing hormone-beta gene polymorphism in the onset and progression of puberty in healthy boys

An immunologically anomalous LH with two point mutations in its beta-subunit gene (Trp8Arg and Ile15Thr) has recently been described. This polymorphism is common in Finland; 28% of the population are homo- or heterozygous for the variant allele. To assess the effect of the LH variant on LH action, we correlated its presence in a group of 49 healthy boys with the onset and progression of puberty. This group was followed-up longitudinally from a mean age of 11.7 +/- 0.1 yr for 3 yr at 3-month intervals. In addition, we studied the prevalence of the variant LH in boys with constitutional pubertal delay (testicular volume < or = 4 mL after 13.5 yr of age). The LH beta gene status of each subject in this study was judged from a single venous blood sample using two immunofluorometric LH assays with different combinations of monoclonal antibodies: one detecting both the variant and wild-type LH, and the other detecting only wild-type hormone. Of the boys with pubertal onset at a normal age, 36 (74%) were homozygous for the wild-type LH beta allele, 12 (24%) were heterozygous, and 1 (2%) was homozygous for the variant LH beta allele. Clear differences in pubertal parameters were found between the boys with normal and mutated (homo- or heterozygous) LH genotypes. During the follow-up, the boys with the mutated genotype had smaller testicular volumes (P < 0.03), were shorter (P < 0.02), had slower growth rates (P < 0.04), and had lower serum insulin-like growth factor I-binding protein-3 levels (P < 0.03) than the boys with the normal LH genotype. In the boys with delayed onset of puberty, the frequency of the variant LH beta allele did not differ from that in the reference population, indicating that the variant LH is not associated with conditions due to disturbed control of the reactivation of GnRH secretion. We conclude that during the progression of puberty, the variant LH may be less active in stimulating testicular growth than wild-type LH. Thus, the gene may affect tempo, contributing to the wide normal variation in pubertal progression in healthy boys. Our results also suggest that the variant LH not only affects the course of puberty, but is already involved in the regulation of the GH-insulin-like growth factor I axis during childhood.     

Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway

In both cell culture based model systems and in the failing human heart, beta-adrenergic receptors ( beta-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3'-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by beta-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the beta-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (approximately 50%) in the abundance of beta1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of beta2-AR mRNA was first described, exposure to beta-AR agonist resulted in a significant increase in AUF1 mRNA and protein (approximately 100%). Conversely, agonist exposure significantly decreased (approximately 40%) beta2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3'-untranslated region of the human beta1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to beta1-AR 3'-untranslated region mRNA.     

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.     

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.