Human Beta-2 Adrenoceptor Gene Polymorphisms Are Highly Frequent in Obesity and Associate with Altered Adipocyte Beta-2 Adrenoceptor Function

Transcription

1 Huma Beta-2 Adreoceptor Gee Polymorphisms Are Highly Frequet i Obesity ad Associate with Altered Adipocyte Beta-2 Adreoceptor Fuctio Valérie Large,* Lea Hellström,* Sigy Reyisdottir,* Fredrik Löqvist,* Per Eriksso, Lars Lafelt, ad Peter Arer* *Departmet of Medicie, Departmet of Geriatric Medicie, ad Research Ceter, Karoliska Istitute, Huddige Uiversity Hospital, Stockholm, Swede; ad Kig Gustav V Research Istitute, Karoliska Hospital, Stockholm, Swede Abstract Catecholamies play a cetral role i the regulatio of eergy expediture, i part by stimulatig lipid mobilizatio through lipolysis i fat cells. The beta-2 adreoceptor (BAR-2) is a major lipolytic receptor i huma fat cells. To determie whether kow polymorphisms i codos 16, 27, ad 164 of this receptor play a role i obesity ad subcutaeous adipocyte BAR-2 lipolytic fuctio, we ivestigated a group of 140 wome with a large variatio i body fat mass. Oly the polymorphisms i codos 16 ad 27 were commo i the study populatio. The Gl27Glu polymorphism was markedly associated with obesity with a relative risk for obesity of 7 ad a odds ratio of 10. Homozygotes for Glu27 had a average fat mass excess of 20 kg ad 50% larger fat cells tha cotrols. However, o sigificat associatio with chages i BAR-2 fuctio was observed. The Arg16Gly polymorphism was associated with altered BAR-2 fuctio with Gly16 carriers showig a fivefold icreased agoist sesitivity ad without ay chage i BAR-2 expressio. However, it was ot sigificatly liked with obesity. These fidigs suggest that geetic variability i the huma BAR-2 gee could be of major importace for obesity, eergy expediture, ad lipolytic BAR-2 fuctio i adipose tissue, at least i wome. (J. Cli. Ivest : ) Key words: fat cell DNA lipolysis restrictio fragmet legth polymorphism polymerase chai reactio Itroductio It is well established that obesity is uder strog geetic ifluece, with up to 40% of the variatio i body fat cotet beig attributed to geetic factors (1). Several of the gees causig obesity i laboratory aimal models have bee characterized recetly (2). Much less is kow about gees ivolved i huma obesity (3), because the obese pheotype i humas is ot oly determied by a umber of differet gees but also by a large umber of evirometal factors. Gees that are ivolved i the regulatio of catecholamie fuctio may be of particular importace for huma obesity Address correspodece to Peter Arer, M.D., Ceter for Metabolism ad Edocriology, M63, Karoliska Istitute at the Departmet of Medicie, S Huddige Uiversity Hospital, Stockholm, Swede. Phoe: ; FAX: Received for publicatio 23 May 1997 ad accepted i revised form 24 September J. Cli. Ivest. The America Society for Cliical Ivestigatio, Ic /97/12/3005/09 $2.00 Volume 100, Number 12, December 1997, because of the cetral role they have i eergy expediture both as hormoes ad eurotrasmitters. A key factor i the eergy balace is the mobilizatio of lipids through lipolysis i fat cells (4). All three kow beta adreoceptor (BAR) 1 subtypes ca promote lipolysis i huma adipose tissue i vivo (5, 6). Recetly, a codig mutatio i the BAR-3 gee was reported which associated with rapid weight gai ad some metabolic complicatios to obesity (7 9). However, this geetic variatio may be of mior importace i obesity sice i most of the populatios studied its frequecy is ot icreased i obesity (10, 11). Furthermore, little or o differece i pheotypic characteristics exists betwee carriers ad ocarriers of this polymorphism (10 12). This mutatio fails to cause a detectable chage i fuctio i both ative huma fat cells (13) ad i recombiat cells (14). I a umber of elegat studies, Liggett ad colleagues have described three polymorphisms i the huma BAR-2 gee that have bee studied i relatio to asthma. These iclude the rare Thr164Ile variat ad two commo Arg16Gly ad Gl27Glu variats (15). Thr164, Arg16, ad Gl27 are cosidered as the wild forms. Whe these are substituted for Ile164, Gly16, or Glu27, the fuctio of the BAR-2 is markedly altered i recombiat cells (16, 17). Eve if i most of the studies oe of these missese mutatios were more prevalet i asthma patiets tha i ormal cotrol subjects, the Gly16 ad Glu27 variats have bee show to be associated with altered pheotypic characteristics i certai forms of asthma (18, 19). It is yet ukow whether these geetic variats are associated with other cliical disorders or if they are liked to chages i ative huma BAR-2 fuctio. I this study, we ivestigated whether the previously described huma BAR-2 gee polymorphisms are associated with obesity ad whether they are of fuctioal importace for the huma fat cell i a study populatio cosistig of 140 wome with a large variatio i body mass idex (BMI). Methods Subjects The study group cosisted of 140 orelated subjects who were ot receivig ay medicatio. Subjects were recruited i two differet ways. Oe group icluded 85 wome who were either healthy, oobese voluteers or otherwise healthy subjects referred to the Departmet of Medicie because of ucomplicated obesity. BMI raged from 17.8 to 52.5 kg/m 2 i this group. The secod group cosisted of 55 wome with a large variatio i body weight (BMI kg/m 2 ), who were referred to the Departmet of Surgery for surgical treatmet of obesity by gastric badig, ucomplicated gallstoes, or abdomial herias. These two groups did ot differ statistically i ay of the followig measures, icludig age, BMI, waist-to-hip ratio (WHR), 1. Abbreviatios used i this paper: BAR, beta adreergic receptor; BMI, body mass idex; WHR, waist-to-hip ratio. Huma Beta-2 Adreoceptor Gee Polymorphisms 3005

2 fat cell volume, ad fastig plasma levels of glucose ad isuli. Therefore, the two cohorts were cosidered as oe uiform group i all subsequet aalyses. Both groups were ivestigated at the hospital after a overight fast. Height ad body weight were first determied. Thereafter, a veous blood sample was obtaied from a atecubital vei for extractio of DNA ad the determiatio of plasma glucose by the hospital s routie chemistry laboratory ad plasma isuli at our ow laboratory by a commercial radioimmuoassay (Pharmacia-Upjoh, Uppsala, Swede). A abdomial subcutaeous fat biopsy was obtaied uder local aesthesia from the medicie subjects ad i coectio with ope surgery from the surgery subjects, exactly as described previously (20). We have demostrated that the adreoceptor fuctio ad lipolysis do ot differ i fat cells obtaied through these two differet procedures (20). Betwee 200 mg ad 1 g of fat was obtaied ad used for immediate aalysis. To determie the frequecy of the ivestigated mutatios, the material was divided ito oobese (58 subjects) ad obese (82 subjects) groups. A BMI of 27 kg/m 2 was used as the cut-off poit. The oobese subjects had a mea BMI of kg/m 2 ad a mea WHR of i compariso with kg/m 2 ad , respectively, for the obese subjects. Both groups were comparable i age; the mea of the ormal weight subjects beig yr ad of the obese group yr. The body fat cotet was determied by a formula derived from age, sex, ad BMI (21). The body fat was also measured o 34 subjects, usig a body fat aalyzer (model TBF-305; Taika, Tokyo, Japa). The BMI was uiformly distributed betwee 25 ad 50 kg/m 2 i these subjects. There was a excellet correlatio betwee both methods of BMI determiatio (r 0.92, slope 1.3). The subjects were iformed i detail about the ivestigatio ad their coset was obtaied. The study was approved by the hospital s Ethical Committee. Aalysis of RFLP Geomic DNA was extracted from leukocytes i samples of whole blood by digestio with proteiase K followed by pheol/chloroform extractio. PCR amplificatio was performed o a automated apparatus (PTC-200; SDS Co., Falkeberg, Swede). PCR amplificatio of the DNA segmet cotaiig codo 27 of the BAR-2 gee. PCR amplificatio of the DNA segmet cotaiig codo 27 of the BAR-2 gee was carried out i a volume of 26 l cotaiig g DNA, 0.38 mm of each deoxyucleoside triphosphate, 10% buffer (100 mm Tris-HCl, 15 mm MgCl 2, 500 mm KCl, ph 8.3), 10% DMSO, 20 pmol of each primer, ad U of Taq DNA polymerase. The forward primer was 5 -GGCCCATGACCA- GATCAGCA-3 ad the reverse primer was 5 -GAATGAGGCT- TCCAGGCGTC-3. PCR was started with deaturatio at 94 C for 4 mi, followed by 30 cycles of deaturatio (94 C, 1 mi), aealig (63 C, 1 mi), ad extesio (72 C, 1 mi), with a fial extesio at 72 C for 10 mi. The PCR product size from these primers is 353 bp. The amplified product was digested at 37 C for 1 h with 0.4 U of Ita I. The fragmets were resolved o a 2% ultra-pure DNA agarose gel with Tris-acetate EDTA (40 mm Tris-acetate, 2 mm EDTA) buffer ad visualized uder ultraviolet illumiatio after staiig with ethidium bromide. This digestio produced fragmets of the followig sizes: 27, 55, 97, ad 174 bp i Gl27 homozygotes; 27, 55, 97, 174, ad 229 bp i Gl27Glu27 heterozygotes; ad 27, 97, ad 229 bp i Glu27 homozygotes. The 27-bp fragmet was too small to be resolved o the gel. PCR amplificatio of the DNA segmet cotaiig codo 16 of the BAR-2 gee. PCR amplificatio of the DNA segmet cotaiig codo 16 of the BAR-2 gee was carried out the same way as for the codo 27 but usig 5 -CTTCTTGCTGGCACGCAAT-3 as the forward primer ad 5 -CCAGTGAAGTGATGAAGTAGTTGG-3 as the reverse primer. The other differeces icluded usig a aealig temperature of 56 C ad excludig DMSO. The PCR product size from these primers is 201 bp. The forward primer is complemetary to the BAR-2 DNA sequece except for oe ucleotide which permits the creatio of a BsrDI restrictio site. The amplified product was digested at 60 C for 1 h with 2 U of BsrDI. The fragmets were resolved o a 3% Meta-Phor agarose gel with Tris-borate EDTA (89 mm Tris, 89 mm boric acid) buffer ad visualized uder ultraviolet illumiatio after staiig with ethidium bromide. This digestio produced fragmets of the followig sizes: 14, 56, ad 131 bp i Arg16 homozygotes; 14, 23, 56, 108, ad 131 bp i Arg16Gly16 heterozygotes; ad 14, 23, 56, ad 108 bp i Gly16 homozygotes. PCR amplificatio of the DNA segmet cotaiig codo 164 of the BAR-2 gee. PCR amplificatio of the DNA segmet cotaiig codo 164 of the BAR-2 gee was carried out the same way as for codo 27 with 5 -GGACTTTTGGCAACTTCTGG-3 as the forward primer, 5 -ACGAAGACCATGATCACCAG-3 as the reverse primer, ad a aealig temperature of 55 C. The PCR product size from these primers is 358 bp. The amplified product was digested at 37 C for 1 h with 2 U of MlI. The fragmets were resolved o a 2% ultra-pure DNA agarose gel with Tris-acetate EDTA (40 mm Tris acetate, 2 mm EDTA) buffer ad visualized uder ultraviolet illumiatio after staiig with ethidium bromide. This digestio produced fragmets of the followig sizes: 38, 114, ad 206 bp i Thr164 homozygotes; 38, 114, 206, ad 320 bp i Thr164Ile164 heterozygotes; ad 38 ad 320 bp i Ile164 homozygotes. Fat cell lipolysis experimets Isolated fat cells were prepared ad icubated as described i detail elsewhere (22). I brief, diluted suspesios (0.2 ml) of isolated fat cells (5,000 10,000 cells/ml) were icubated i duplicate for 2 h i the presece or absece (basal) of icreasig cocetratios of the BAR-1 selective agoist dobutamie or the BAR-2 selective agoist terbutalie. We have demostrated that these two agoists are selective for their desigated receptors i isolated huma abdomial subcutaeous fat cells (5). All icubatios were performed at 37 C i Krebs- Heseleit phosphate buffer (ph 7.4), supplemeted with glucose (1 g/liter) ad BSA (20 g/liter), usig air as the gas phase. The ligads were added simultaeously at the start of the icubatio. The cocetratios used raged from to 10 4 mol/liter. Glycerol release to the icubatio medium after the 2-h icubatio was determied usig a automated biolumiescece assay (23) ad was used as a measuremet of the lipolysis rate, sice this metabolite is ot reused by huma fat cells to ay sigificat extet. Both agoists caused a dose-depedet icrease of glycerol release that always reached a plateau at the highest agoist cocetratios. The sesitivity to agoist actio was defied as the pd 2 value, i.e., the egative logarithm of the EC 50 value. The latter was defied as the cocetratio of each agoist givig a half-maximal effect. These EC 50 values (expressed as log mol/liter) were determied by liear regressio aalysis after log-logit trasformatio of the ascedig part of the idividual cocetratio respose curves. They reflect specific agoist receptor iteractios, sice at this cocetratio the selective agoists have few, if ay, iteractios with other adreoceptor subtypes (24). Lipolysis rates i the presece or absece of maximum effective agoist cocetratios were expressed relative to grams of lipid or icubated cell umber. The methods for lipid extractio ad calculatio of adipocyte cell volume ad umber have bee described i detail previously (22). Fat cell bidig experimets These experimets could oly be performed i a limited umber of subjects because they required very large amouts of fat cells. The BAR-1/BAR-2 bidig experimets were performed as described previously (22). I brief, the oselective BAR atagoist 125 I-cyaopidolol was used i saturatio ad displacemet experimets. Fat cells were icubated for 60 mi, i a cocetratio of 20,000 cells/ml, at 37 C i 0.5 ml of KRP buffer, ph 7.4, with bovie albumi (5 g/liter), glucose (1 g/liter), ad ascorbic acid (0.1 g/liter). I the saturatio experimets, the fat cells were icubated with 10, 50, 100, 250, 500, ad 750 pmol/liter of 125 I-cyaopidolol. Nospecific bidig was determied by additio of propaolol (0.1 mol/ liter). I the displacemet experimets, 125 I-cyaopidolol bidig (100 pmol/liter) was displaced by the highly selective BAR-2 atagoist ICI 118,551 i 12 icreasig cocetratios (0, mol/li Large et al.

3 ter). Nospecific bidig was determied i the presece of 10 4 mol/ liter of ICI 118,551. The saturatio experimets were evaluated by liear regressio aalysis of Scatchard plots givig a estimate of the total umber of bidig sites. The Scatchard plots were always liear. The displacig ligad ICI 118,551 bids to BAR-2 with high affiity ad to BAR-1 with low affiity. Usig a oliear least-squares regressio aalysis of the displacemet curves, it was possible to determie the proportio of bidig sites with high (BAR-2) ad low affiity (BAR-1) for the displacig ligad, respectively. As discussed i detail (22), the cocetratios of 125 I-cyaopidolol used here are ot sufficietly high to idetify BAR-3 bidig sites. Drugs ad chemicals The PCR kit ad the restrictio ezyme ItaI were purchased from Boehriger-Maheim (Maheim, Germay) ad the ultra-pure DNA grade agarose from BIO-RAD (Hercules, CA). The restrictio ezymes BsrDI ad MlI came from New Eglad Biolabs Ic. (Beverly, MA) ad the Meta-Phor agarose from FMC Bioproducts (Rocklad, ME). BSA (fractio V) ad propaolol were obtaied from Sigma Chemical Co. (St. Louis, MO). Terbutalie sulfate came from Draco (Lud, Swede), dobutamie hydrochloride from Eli Lilly & Co. (Idiaapolis, IN), 125 I-cyaopidolol from DuPot/New Eglad Nuclear (Bosto, MA), ad ICI 118,551 from Cambridge Research Biochemicals Ltd. (Cheshire, UK). Figure 1. Detectio of Gl27Glu (A), Arg16Gly (B), Thr164Ile (C) polymorphisms of the BAR-2 by PCR ad aalysis of RFLP. (A) The PCR products (353 bp) were digested with the restrictio ezyme Ita I ad visualized by staiig with ethidium bromide. M, Molecular mass marker; lae 1, Glu27 homozygote; lae 2, Glu27Gl27 heterozygote; lae 3, Gl27 homozygote; laes 4 6, cotrol DNA for laes 1 3, respectively. (B) The PCR products (201 bp) were digested with the restrictio ezyme BsrDI ad visualized by staiig with ethidium bromide. M, Molecular mass marker; lae 1, Gly16 homozygote; lae 2, Gly16Arg16 heterozygote; lae 3, Arg16 homozygote; laes 4 6, cotrol DNA for laes 1 3, respectively. (C) The PCR products (358 bp) were digested with the restrictio ezyme MlI ad visualized by staiig with ethidium bromide. M, Molecular mass marker; lae 1, Thr164 homozygote; lae 2, Thr164Ile164 heterozygote; lae 3, cotrol DNA for lae 1; lae 4, cotrol DNA: Ile164 homozygote. (D) Summary of BAR-2 polymorphism i codos 16, 27, ad 164. Huma Beta-2 Adreoceptor Gee Polymorphisms 3007

4 Statistical aalysis Values are give as the mea SEM. Statistical aalysis was performed usig Studet s upaired t test, the 2 test, ad ANOVA. Relative risk ad odds ratio were calculated as described previously (25). Relative risk (R) is R 1 ( ) = ( ) ad the odds ratio (Ri) is R = ; 2 3 where 1 is the umber of patiets with the characteristic, 2 is the umber of cotrol idividuals with the characteristic, ad 3 ad 4 are the correspodig umbers without the characteristic. Results RFLP. Represetative RFLP aalyses of the geetic variats i codos 16, 27, ad 164 of the huma BAR-2 are show i Fig. 1. A distict patter was foud for each of the polymorphisms. The patters obtaied were idetical to parallel examiatio of cotrol DNA from other subjects carryig the differet polymorphisms (geotyped by Dr. Liggett ad Dr. Hall). Furthermore, idetical results were obtaied whe samples were geotyped by two of the ivestigators idepedetly. For codos 16 ad 27, all kow geetic variats were foud, i.e., homozygous for Arg16 or Gly16, heterozygous Arg16Gly16, homozygous for Gl27 or Glu27, ad heterozygous Gl27Glu27. For codo 164, oly homozygous for Thr164 ad heterozygous Thr164Ile164 were foud. To cofirm the determiatio of the geotype at codos 16 ad 27, the PCR products from radomly chose subjects with the three differet geotypes for both mutatios were submitted to automatic direct DNA sequecig (Cybergee, Stockholm, Swede). The data obtaied by sequecig were i accordace with our results (data ot show). BAR-2 geotypes of obese ad oobese wome. Geotypig of BAR-2 was performed o DNA from all 140 subjects to delieate polymorphisms i amio acids 16, 27, ad 164. The Thr164Ile variat occurred i oly five subjects. Due to the very low polymorphic frequecy at this locus, codo 164 was ot cosidered further. Polymorphisms i codo 27 had a very strog associatio with obesity (Tables I ad II). The clusterig of Glu27 with obesity was highly statistically sigificat by 2 aalysis (P 0.003) with a odds ratio as high as 10.4; 24% of the obese but oly 3% of the oobese subjects carried the homozygous form of Glu27, givig a sevefold differece i frequecy. I additio, the relative risk of beig obese with the homozygous Glu27 geotype was 7. The frequecy of each allele for the two polymorphisms also differed at a highly sigificat level betwee obese ad oobese subjects (P by 2 aalysis). Oly homozygosity for Gl27 was sigificatly associated with obesity. The heterozygotes had a odds ratio of 1.3, ot statistically sigificat differet from 1.0. I the whole material, homozygosity for Gly16 occurred i 49% of the subjects, as compared with 19% for Arg16 homozygosity. Whe the expected umber of subjects i each group was calculated accordig to Hardy Weiberg, there should have bee five homozygotes carryig Glu27Glu27 i the oobese group. However, oly Table I. Distributio of the BAR-2 Gee Polymorphisms i Codos 16 (A) ad 27 (B) ad the Differet Combiatios of Codos 16 ad 27 (C) Geotype Obese subjects Noobese subjects P value Odds ratio (A) Gly16Gly Gly16Arg Arg16Arg (B) Glu27Glu Glu27Gl Gl27Gl (C) Arg16Arg16 ad Gl27Gl Gly16Gly16 ad Glu27Glu Gly16Arg16 ad Glu27Gl Gly16Gly16 ad Glu27Gl Gly16Arg16 ad Gl27Gl Other combiatios 4 2 Values were compared by 2 aalysis., Number of subjects. two carriers of that polymorphism were foud. We have o clear explaatio for this deficit. It might be due to the use of two differet populatios. I the subgroup of 55 wome udergoig elective surgery, the expected umber of oobese subjects who were homozygous for Glu27 was 1 accordig to Hardy Weiberg. We foud oe, which did ot differ from the expected umber i a statistically sigificat way. The combied polymorphisms at codos 16 ad 27 were also examied (Table I). It was evidet that the geetic variability i these two codos was i strog likage disequilibrium, sice 64% of the subjects carried both Gly16 ad Glu27 i homozygous or heterozygous forms. The patter of polymorphisms at both codos 16 ad 27 differed sigificatly betwee the obese ad oobese subjects (P by 2 aalysis). However, the oly major differece i frequecy betwee both groups was a marked overrepresetatio of both Gly16 ad Glu27 homozygosity i the obese subjects, with a odds Table II. Allele Frequecy of BAR-2 Gee Polymorphisms i Codos 16 ad 27 Allele Obese subjects Noobese subjects P value Gly (64%) 76 (66%) Arg16 59 (36%) 40 (34%) Glu27 78 (48%) 35 (30%) Gl27 86 (52%) 81 (70%) Values were compared by 2 aalysis., Number of alleles Large et al.

5 Table III. Stratified Aalysis of the Distributio of the BAR-2 Gee Polymorphisms i Codo 16 (A) i Subjects Carryig (Glu27 ) or Not (Glu27 ) a Glutamic Acid at Codo 27, ad Polymorphisms i Codo 27 (B) i Subjects Carryig (Gly16 ) or Not (Gly16 ) a Glyci at Codo 16 (A) Glu27 Obese subjects Noobese subjects P value Gly16Gly Gly16Arg Arg16Arg Glu27 Gly16Gly Gly16Arg Arg16Arg (B) Gly16 Glu27Glu Glu27Gl Gl27Gl Gly16 Glu27Glu Glu27Gl Gl27Gl Values were compared by 2 aalysis., Number of subjects. ratio as high as % of the obese subjects carried this geetic variat, as compared with oly 3% of the oobese subjects, thus givig a sevefold differece i frequecy. To further evaluate the relative importace of the polymorphism at codo 27 for obesity, we made a stratified aalysis (Table III). First, we divided the samples ito Glu27 (Glu27Glu27 ad Glu27Gl27) ad Glu27 (Gl27Gl27). The we examied the effect of the Arg16 variat o obesity. Neither i Glu27 or i Glu27 subjects did the codo 16 polymorphism associate i a sigificat way with obesity. Secod, we divided the samples ito Gly16 (Gly16Gly16 ad Gly16Arg16) ad Gly16 (Arg16Arg16). The we examied the effect of the codo 27 variatio o obesity. I Gly16 carriers, there was a highly sigificat correlatio betwee the codo 27 polymorphism ad obesity (P 0.009). A tedecy for the same associatio was observed i Gly16 carriers but the P value did ot reach a statistical sigificace. It may be due to the low umber of total subjects sice oly 29 subjects were Gly16. Pheotypic characteristics of differet polymorphisms i codos 16 ad 27. The pheotypic characteristics of the polymorphisms i codo 27 are show i Table IV. As depicted, the Gl27Glu polymorphisms were ot associated with ay alteratio i BAR-1 or BAR-2 fuctio. However, Glu27 homozygotes had, o average, 50% larger fat cells ad 7 kg/m 2 higher BMI tha homozygotes for Gl27. Homozygosity for Glu27 was associated with a excess of 20 kg i body fat, as compared with ocarriers. The Glu27 homozygotes had a higher fastig plasma isuli level tha the Gl27 homozygotes (P 0.03) ad showed sigs of a upper body fat distributio (i.e., icreased WHR). BMI, body fat, fat cell volume, WHR, ad isuli showed slightly higher values for the Glu27 homozygotes tha for the Gl27Glu27 heterozygotes. However the differeces did ot reach a statistically sigificat level. Table V shows the pheotypes of the codo 16 polymorphisms. The geetic variats did ot differ statistically i BMI or i ay other cliical parameters. However, homo- or heterozygosity for Gly i codo 16 was associated with a clear alteratio of adipocyte BAR-2 fuctio. This is illustrated by the mea values for terbutalie actio; the pd 2 value (represetig agoist affiity for the receptor) was log uits higher i Gly16 homozygotes ad i Arg16Gly16 heterozygotes, as compared with Arg16 homozygotes. This implies that mea terbutalie sesitivity was fivefold higher i adipocytes from the two former groups. This is further illustrated i Fig. 2 which shows represetative cocetratio respose curves for the selective BAR-2 agoist terbutalie (three Gly16 homozygotes ad three Arg16 homozygotes). The Gly16 homozygote curves were shifted to the left as compared with the Arg16 homozygote curves, idicatig that the two polymorphisms differed i terbutalie sesitivity. Fig. 2 also shows the cocetratio respose curves obtaied with the BAR-1 selective agoist dobutamie, for the represetative three Arg16 ad three Gly16 homozygotes. Ulike terbutalie, there were o clear differeces betwee the dobutamie curves, or did the mea values for dobutamie pd 2 differ betwee the codo 16 polymorphic groups. There were o statistical differeces i the maximum lipolytic actio of either drug betwee the three geotypes, either whe lipolysis was expressed per gram of lipid or per cell (i.e., mol glycerol/10 6 cells/2 h). The latter values for terbutalie-iduced lipolysis i Arg16 homozygotes, Arg16Gly16 heterozygotes, ad Gly16 homozygotes were , , ad , respectively, ad did ot differ sigificatly. BAR bidig was determied i 48 subjects. Maximum Table IV. Pheotypic Characteristics of BAR-2 Gee Polymorphisms i Codo 27 (A) Gl/Gl (B) Gl/Glu (C) Glu/Glu P value A vs. B A vs. C Age, yr NS NS BMI, kg/m NS Body fat, kg NS Fat cell volume, pl NS WHR NS p-glucose, mmol/liter NS NS p-isuli, mu/liter NS pd 2 Terbutalie NS NS pd 2 Dobutamie NS NS Glycerol release mol/g lipid/2 h: Basal NS NS Terbutalie NS NS Dobutamie NS NS Values are mea SEM. They were compared by Studet s upaired t test., Number of subjects; p, fastig plasma; pd 2, mius log value for half-maximal effective cocetratio. Huma Beta-2 Adreoceptor Gee Polymorphisms 3009

6 Table V. Pheotypic Characteristics of BAR-2 Gee Polymorphisms i Codo 16 (A) Arg/Arg (B) Arg/Gly (C) Gly/Gly P value A vs. B A vs. C Age, yr NS NS BMI, kg/m NS NS Body fat, kg NS NS Fat cell volume, pl NS NS WHR NS NS p-glucose, mmol/liter NS NS p-isuli, mu/liter NS NS pd 2 Terbutalie pd 2 Dobutamie NS NS Glycerol release mol/g lipid/2 h: Basal NS NS Terbutalie NS NS Dobutamie NS NS BAR-1 bidig capacity (amol/10 6 cells) was , , ad for Arg16 homozygotes, Arg16Gly16 heterozygotes, ad Gly16 homozygotes, respectively. These values did ot differ sigificatly (F 0.58 by ANOVA). Correspodig values for BAR-2 maximum bidig were , , ad , respectively. There were o sigificat differeces betwee these values either (F 0.68 by ANOVA). Moreover, there were o sigificat differeces betwee the groups i regard to receptor affiities for the displacig drug ICI 118,551 or for the radioligad 125 I-cyaopidolol (data ot show). The effect o the pheotype of some of the combied polymorphisms i codos 16 ad 27 is demostrated i Table VI. It is apparet that i homozygotes for both Gly16 ad Glu27, most of the pheotypic characteristics were similar to those observed whe Gly16 ad Gl27 homozygosities were ivestigated separately (Tables IV ad V). Thus, they had sigificatly higher BMI, body fat, WHR, fat cell size, ad pd 2 for terbutalie tha the homozygotes for both Arg16 ad Gl27. We also ivestigated the cotributio of codo 16 variats o the pheotype by comparig wome who were homozygous for both Arg16 ad Gl27 with wome who were both heterozygous Gly16Arg16 ad homozygous for Gl27. This latter pheotype was associated with a sigificat icrease i terbutalie pd 2, givig a fivefold icrease i terbutalie sesitivity (P 0.005). However, there were o statistically sigificat chages i ay of the other ivestigated parameters. Ufortuately, it was ot possible to ivestigate separately the pheotypic role of polymorphisms i codo 27 sice oly two subjects who carried the Glu27 variat were homozygous for Arg16. Geotypic ad pheotypic characteristics of a study subgroup. To ivestigate if the use of two populatios had ay importat effect o the distributio of the BAR-2 polymorphism i codos 16 ad 27 or, o the pheotypic characteristics, we did a separate ivestigatio o the 85 wome udergoig biopsy uder local aesthesia, sice they formed the largest subgroup. The fidigs i this cohort were almost idetical with those show for the whole study populatio. The umbers of subjects carryig Glu27Glu27, Glu27Gl27, ad Gl27Gl27 amog the obese wome were 12, 25, ad 10, respectively. The correspodig umbers for the oobese wome were 2, 21, ad 15. The differece betwee obese ad oobese subjects was statistically sigificat (P 0.02). Obese Glu27 homozygotes had a odds ratio of 9.0 ad the obese Glu27Gl27 subjects had a odds ratio of 1.8. O the other had, there was o statistically sigificat associatio betwee the codo 16 polymorphism ad obesity i this subgroup (data ot show). We also examied the pheotypic characteristics of the cohort. Glu27Glu27 was associated with a statistically sigificat icrease i BMI, WHR, body fat, fat cell volume, ad plasma isuli as compared with Gl27Gl27, but there was o differece betwee the two geotypes i regard to BAR agoist sesitivity. The codo 16 polymorphism did ot ifluece the values for BMI, WHR, body fat, fat cell volume, ad plasma Values are mea SEM. They were compared by Studet s upaired t test., Number of subjects; p, fastig plasma; pd 2, mius log value for half-maximal effective cocetratio. Figure 2. Codo 16 BAR-2 polymorphism ad lipolysis. Cocetratio respose curves for terbutalie (top) ad dobutamie (bottom) iduced glycerol release. Fat cells were icubated i the absece ad presece of icreasig cocetratios of the BAR-1 or the BAR-2 selective agoist. Three represetative homozygous Gly16 subjects ad three represetative homozygous Arg16 subjects are depicted Large et al.

7 Table VI. Pheotypic Characteristics of Combied Polymorphisms i Codos 16 ad 27 of the BAR-2 Gee isuli i a statistically sigificat way. Gly16Gly16 was associated with a statistically sigificat icrease i terbutalie sesitivity i compariso with Arg16Arg16. However, o differeces i dobutamie sesitivity betwee the latter two geotypes were foud. Discussio (A) Arg16Arg16 Gl27Gl27 (B) Gly16Gly16 Glu27Glu27 (C) Gly16Arg16 Gl27Gl27 P value A vs. B A vs. C Age, yr NS NS BMI, kg/m NS Body fat, kg NS Fat cell volume, pl NS WHR NS p-glucose, mmol/liter NS NS p-isuli, mu/liter NS NS pd 2 Terbutalie pd 2 Dobutamie NS NS Glycerol release mol/g lipid/2 h: Basal NS NS Terbutalie NS NS Dobutamie NS NS Values are mea SEM. They were compared by Studet s upaired t test., Number of subjects; p, fastig plasma; pd 2, mius log value for half-maximal effective cocetratio. To the best of our kowledge, this study is the first to demostrate a gee polymorphism that is strogly overrepreseted i huma obesity ad that has a marked ifluece o body fat cotet. Aother ovel observatio is that a commo codig mutatio i the huma BAR-2 gee affects receptor fuctio i a ative target cell such as the adipocyte. Substitutio of Gl for Glu i both alleles at codo 27 of the BAR-2 gee had a marked associatio with obesity; the odds ratio was as high as The Glu27 homozygotes had 20 kg of excess body fat ad a 50% icrease i fat cell size, as compared with Gl27 homozygotes. They had also sigs of a upper body type of obesity ad isuli resistace. For all the traits for which Glu27 homozygotes differed from Gl27 homozygotes, however, the Glu27Gl27 heterozygotes had a itermediate value which was ot statistically sigificat. This might suggest that there is also a effect of the heterozygosity o obesity; however, it does ot attai sigificace at the curret sample size. Nocarriers were also slightly obese with a mea BMI of 30 kg/m 2. Therefore, this latter group might have also some obesity-associated abormalities i the cliical measures. Further studies o populatio-based cohorts are eeded to determie whether the Gl27Glu polymorphism i the BAR-2 gee is of importace for obesity-mediated complicatios. No differece i BAR-2 fuctio betwee carriers ad ocarriers of Glu27 was observed. However, it is ot possible to state whether the latter is more apparet tha real, sice geetic variability i codos 16 ad 27 were i likage disequilibrium, as discussed below. Therefore, it is ot kow at preset how Glu27 ca promote obesity. Ayhow, it appears that the Gl27Glu polymorphism is a importat geetic marker for huma obesity, sice it is highly overrepreseted i overweight wome (24% as compared with 3% i cotrols) ad the relative risk of beig obese i subjects homozygous for Glu27 is as high as 7. The codo 16 polymorphism seems to be of marked importace for BAR-2 fuctio, at least i fat cells, but less so for obesity. The polymorphism i codo 16 was ot associated with ay statistically sigificat variatio i obesity variables. However, carriers with oe or two Gly16 alleles had a fivefold icreased agoist BAR-2 affiity. Accordig to classical drug receptor theories (24), a chage i agoist pd 2 but with a preserved maximum effect idicates a alteratio at the receptor level ad ot dowstream i sigal trasductio i a spare receptor system such as the huma adipocyte BAR (26). However, judgig from the results of the radioligad bidig experimets, there was o evidece of a importat effect of the polymorphism o BAR-2 expressio. Therefore, we assume that a substitutio of argiie for glycie at codo 16 i the huma BAR-2 ehaces affiity ad/or couplig of the receptor, at least i fat cells. It was ot possible to explore the molecular mechaism for such Gly16-associated chages i receptor fuctio because of the limited amout of fat tissue that was available. It should be oted that BAR-2 i recombiat cells expressig Gly16 has a icreased sesitivity to log-term agoist-mediated receptor dowregulatio (15). It is possible that the ative huma geotype also carries this feature, which is the characterized by icreased agoist affiity i the restig state presetly ivestigated. Sice heterozygotes ad homozygotes showed the same chage, it appears that Gly16 domiates over Arg16 i BAR-2 fuctio. The polymorphisms i codos 16 ad 27 were i a strog likage disequilibrium, as two-thirds of the wome carried at the same time Gly16 ad Gl27 i a hetero- or homozygous form. Homozygotes for both Gly16 ad Glu27 had a 20 kg body fat excess, 50% elarged fat cells, ad fivefold icreased BAR-2 agoist affiity i compariso with ocarriers. Furthermore, subjects who were homozygotes for Gly16 ad heterozygotes for codo 27 had fivefold higher BAR-2 agoist affiity, but did ot statistically differ i obesity variables whe compared with those who were ocarriers at both loci. Therefore, it is temptig to speculate that the codo 27 variat i some way associates with obesity per se, whereas the codo 16 variat oly modulates BAR-2 fuctio. To further solidify the argumet that codo 27 is primary for the effect o obesity, we also performed a stratified aalysis. I Gly16 carriers, the codo 27 variat associated sigificatly with obesity (P 0.009). O the other had, i Glu27 carriers, the codo 16 variat did ot associate with obesity i a statistically sigificat way. This might further suggest that codo 27 has a primary effect o obesity. To obtai a sufficietly large study group, we pooled data from two differet cohorts, icludig wome who were ivestigated by udergoig either a fat biopsy uder local aesthesia or a fat biopsy durig beig elective geeral surgery. We believe it is relevat to combie these two groups for several reasos. The local aesthesia wome oly varied i their relative body weight. The elective surgery wome were either operated o for obesity or for oedocrie disorders such as Huma Beta-2 Adreoceptor Gee Polymorphisms 3011

8 gallstoes or herias. Thus, the oly major variatio betwee subjects i the two groups was their relative body weight. A direct compariso of cliical variables betwee both groups did ot reveal ay differeces. Furthermore, we have show recetly that whe abdomial subcutaeous adipocytes were obtaied from the same subjects, first uder local aesthesia ad later durig geeral surgery, there were o statistical differeces i adreoceptor fuctio or i lipolysis whe data from the two biopsy procedures were compared (20). Fially, whe oly the local aesthesia wome were ivestigated, the results were essetially the same as for the whole study populatio. Arg16 has bee defied as the wild type, because this variat of the BAR-2 gee was the first to be cloed (27). However, homozygosity for Arg16 has bee show previously to be less commo tha homozygosity for the mutated Gly16 form (28). The same patter was observed here; Arg16 homozygosity occurred i 19% of the subjects as compared with 33% for Gly16 homozygosity. It was demostrated previously that homozygotes for wild-type Gl27 are much more commo tha homozygotes for Glu27 (20). This was also cofirmed i this study; Gl27 homozygosity was about twice as commo as homozygosity for Glu27 i the whole material. Withi the past 3 yr, all of the sigle-gee mutatios resultig i rodet obesity have bee cloed (29). I cotrast, little progress has bee made durig the same period with respect to the geetic basis of huma obesity, as recetly reviewed (3). Most of the earlier associatio studies have bee egative. I the rare positive oes, the sample size was small ad the evidece weak. The earlier likage studies have also bee disappoitig. This egative picture may ow chage. Usig likage aalysis, a major quatitative trait locus determiig fat mass has bee foud which is related to chromosome 2 (30). Our preset study suggests that gees o chromosome 5 (probably the BAR-2 gee) also cotribute to a importat way to variatios i fat mass ad the regulatio of the eergy expediture. However, our results are based o coveiet samples. They eed to be cofirmed o populatio-based samples ad also o other ethic material. Furthermore, we ivestigated wome oly. The importace of geetic variability i BAR-2 for obesity ad receptor fuctio i me remais to be established. I summary, this study shows that commo polymorphisms i the BAR-2 gee are markedly associated with obesity ad BAR-2 fuctio. I the homozygous form, a Glu27 variat is very commo i obesity ad associates with icreased body fat ad elarged fat cells. A Gly16 variat is above all associated with improved adipocyte BAR-2 fuctio. Thus, geetic variability i the huma BAR-2 gee could be of major importace for obesity ad BAR-2 fuctio i adipose tissue, at least i wome. Ackowledgmets The authors thak S. Liggett ad I.P. Hall for sedig DNA from several subjects with sequece prove BAR-2 polymorphism. The valuable advice of Charlotte Forsell regardig geotypig ad the excellet techical assistace of Britt Marie Leijohufvud, Catharia Hertel, Eva Sjöli, ad Kersti Wåhle are greatly appreciated. This study was supported by grats from the Swedish Medical Research Coucil, Swedish Diabetes Associatio, Swedish Medical Society, Swedish Heart ad Lug Foudatio, Commissio of Europea Commuities, INSERM, ALFEDIAM, the Swedish Istitute, Karoliska Istitute ad the foudatios of Novo Nordisk, Bergwall, Söderberg, Old Female Servats, Hedlud, Kig Gustaf V ad Quee Victoria, ad Belvé. Refereces 1. Bouchard, C Log-term programmig of body size. Nutr. Rev. 54: S8 S Spiegelma, B.M., ad J.S. Flier Adipogeesis ad obesity: roudig out the big picture. Cell. 87: Bouchard, C., ad L. Perusse Curret status of the huma obesity gee map. Obesity Res. 4: Lafota, M., ad M. Berla Fat cell adreergic receptor ad the cotrol of white ad brow fat cell fuctio. J. Lipid Res. 34: Eocksso, S., M. Shimizu, F. Löqvist, J. Nordeström, ad P. Arer Demostratio of a i vivo fuctioal 3 -adreoceptor i ma. J. Cli. Ivest. 95: Barbe, P., L. Millet, J. Galitzki, M. Lafota, ad M. Berla I situ assessmet of the role of beta1-, beta2-, ad beta3-adreoceptors i the cotrol of lipolysis ad utritive blood flow i huma subcutaeous adipose tissue. Br. J. Pharmacol. 117: Clemet, K., C. Vaisse, B.J. Maig, A. Basdevat, B. Guy-Grad, J. Ruiz, K. Silver, A.R. Shuldier, P. Froguel, ad A.D. Strosberg Geetic variatio i the 3 -adreergic receptor ad a icreased capacity to gai weight i patiets with morbid obesity. N. Egl. J. Med. 333: Walsto, J., K. Silver, C.J. Bogardus, W. Kowler, F. Celi, S. Austi, B. Maig, D. Strosberg, P. Ster, N. Rabe, J. Sorki, J. Roth, ad A.R. Shuldier Time of oset of o-isuli-depedet diabetes mellitus ad geetic variatio i the 3 -adreergic-receptor gee. N. Egl. J. Med. 333: Widé, E., M. Lehto, T.J. Kaie, J. Walsto, A.R. Shuldier, ad L.C. Groop Associatio of a polymorphism i the 3 -adreergic-receptor gee with features of the isuli resistace sydrome i Fis. N. Egl. J. Med. 333: Sipilaïe, R., M. Uusitupa, S. Heikkie, A. Rissae, ad M. Laakso Polymorphism of the 3 -adreergic receptor gee affects basal metabolic rate i obese Fis. Diabetes. 46: Gago, J., P. Mauriege, S. Roy, D. Sjöström, Y.C. Chago, F.T. Dioe, J.M. Oppert, L. Perusse, L. Sjöström, ad C. Bouchard The Trp64Arg mutatio of the 3 -adreergic receptor gee has o effect o obesity pheotype i the Quebec family study ad Swedish obese subjects cohort. J. Cli. Ivest. 98: Fujisawa, T., H. Ikegami, E. Yamato, K. Takekawa, Y. Nakagawa, Y. Hamada, T. Oga, H. Ueda, M. Shitai, M. Fukuda, ad T. Ogihara Associatio of Trp64Arg mutatio of the 3 -adreergic-receptor with NIDDM ad body weight gai. Diabetologia. 39: Li, L.S., F. Löqvist, H. Luthma, ad P. Arer Pheotypic characterizatio of the Trp64Arg polymorphism i the beta3-adreergic receptor gee i ormal weight ad obese subjects. Diabetologia. 39: Cadelore, M.R., L. Deg, L.M. Tota, L.J. Kelly, M.A. Cascieri, ad C.D. Strader Pharmacological characterizatio of a recetly described huma beta 3-adreergic receptor mutat. Edocriology. 137: Gree, S.A., J. Turki, I.P. Hall, ad S.B. Liggett Implicatios of geetic variability of huma 2 -adreergic receptor structure. Pulm. Pharmacol. 8: Gree, S.A., J.M. Turki, M. Iis, ad S.B. Liggett Amio-termial polymorphisms of the huma 2 -adreergic receptor impart distict agoist-promoted regulatory properties. Biochemistry. 33: Gree, S.A., G. Cole, M. Jacito, M. Iis, ad S.B. Liggett A polymorphism of the huma 2 -adreergic receptor withi the fourth trasmembrae domai alters ligad bidig ad fuctioal properties of the receptor. J. Biol. Chem. 268: Liggett, S.B Geetics of 2 -adreergic receptor variats i asthma. Cli. Exp. Allergy. 25: Turki, J., J. Pak, S.A. Gree, R.J. Marti, ad S.B. Liggett Geetic polymorphisms of the 2 -adreergic receptor i octural ad ooctural asthma. J. Cli. Ivest. 95: Large, V., S. Reyisdottir, L. Eleborg, V. va Harmele, L. Strömmer, ad P. Arer Lipolysis i huma fat cells obtaied uder local ad geeral aesthesia. It. J. Obesity. 21: Gallagher, D., M. Visser, D. Sepulveda, N. Pierso, T. Harris, ad S.B. Heymsfield How useful is body mass idex for compariso of body fatess across age, sex, ad ethic groups. Am. J. Epidemiol. 143: Reyisdottir, S., K. Ellerfeldt, H. Wahreberg, H. Lithell, ad P. Arer Multiple lipolysis defects i the isuli resistace (metabolic) sydrome. J. Cli. Ivest. 93: Hellmer, J., P. Arer, ad A. Ludi Automatic lumiometric kietic assay of glycerol for lipolysis studies. Aal. Biochem. 177: Keaki, T The classificatio of drugs ad drug receptors i isolated tissue. Pharmacol. Rev. 36: Ott, J Aalysis of Huma Geetic Likage. The Johs Hopkis Uiversity Press, Baltimore ad Lodo Arer, P., J. Hellmer, A. Welud, J. Östma, ad P. Egfeldt Large et al.

9 Adreoceptor occupacy i isolated huma fat cells ad its relatioship with lipolysis rate. Eur. J. Pharmacol. 146: Reihaus, E., M. Iis, N. MacItyre, ad S.B. Liggett Mutatios i the gee ecodig for the 2 -adreergic receptor i ormal ad asthmatic subjects. Am. J. Respir. Cell Mol. Biol. 8: Kobilka, B.K., R.A.F. Dixo, T. Frielle, H.G. Dohlma, M.A. Bolaowski, I.S. Sigal, T.L. Yag-Feg, U. Fracke, M.C. Caro, ad F. Lefkowitz cdna for the huma 2 -adreergic receptor: a protei with multiple membrae-spaig domais ad ecoded by a gee whose chromosomal locatio is shared with that of the receptor for platelet-derived growth factor. Proc. Natl. Acad. Sci. USA. 84: Streamso, C., ad R.L. Leibel Obesity gees: molecular ad metabolic mechaisms. Diabetes Rev. 5: Comuzzie, A.G., J.E. Hixso, L. Almasy, B.D. Mitchell, M.C. Mahaey, T.D. Dyer, M.P. Ster, J.W. MacCluer, ad J. Blagero A major quatitative trait locus determiig serum lepti levels ad fat mass is located o chromosome 2. Nat. Geet. 15: Huma Beta-2 Adreoceptor Gee Polymorphisms 3013