- Email: [email protected]
The Trp64Arg polymorphism of the β3-adrenergic receptor gene is associated with hypertension in men with type 2 diabetes mellitus
AJH
2000;13:1027–1031
BRIEF COMMUNICATIONS
The Trp64Arg Polymorphism of the 3Adrenergic Receptor Gene is Associated with Hypertension in Men with Type 2 Diabetes Mellitus Jens Ringel, Reinhold Kreutz, Armin Distler, and Arya M. Sharma
A missense mutation of the 3-adrenergic receptor gene (ADRB3) resulting in a tryptophan/arginine exchange at position 64 (Trp64Arg polymorphism) has recently been associated with greater capacity to gain weight, a low resting metabolic rate, higher blood pressure, and an early onset of type 2 diabetes. These findings prompted us to examine the relationship between this mutation, blood pressure, and vascular complications in German patients with type 2 diabetes. White patients with type 2 diabetes mellitus (n ⴝ 417) were enrolled in the study. The Trp64Arg polymorphism of the ADRB3 gene was detected by polymerase chain amplification and subsequent restriction digest with BstN I. Stepwise logistic regression analysis of the entire study population revealed a significant interaction between gender and genotype (P ⴝ .019). We therefore performed separate analyses for men and women. There was a significant relationship between hypertension and
O
besity and type 2 diabetes mellitus are commonly associated with hypertension and dyslipidemia, and this cluster of abnormalities has also been described as the insulin-resistance or metabolic syn-
Received June 1, 1999. Accepted March 7, 2000. From the Department of Internal Medicine, Division of Endocrinology and Nephrology, and Department of Clinical Pharmacology, Benjamin Franklin Medical Center, Freie Universita¨t Berlin, Germany. Address correspondence and reprint requests to Prof. Dr. Arya M. Sharma, Medizinische Klinik IV, Klinikum Benjamin Franklin, Freie Universita¨t Berlin, Hindenburgdamm 30, D-12200 Berlin, Federal Republic of Germany;e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
the ADRB3 Trp64Arg variant in men (P ⴝ .015), but not in women. Furthermore, blood pressure levels in male patients with the minor allele had higher blood pressure levels (P < .05), despite a significantly greater number of antihypertensive medications (P ⴝ .01). There was no association between ADRB3 genotype and vascular complications in these patients. In conclusion, our data are compatible with a contribution of this genetic variant of ADRB3 to hypertension in male patients with type 2 diabetes. Further studies will be needed to determine the role of this polymorphism as a predictor of hypertension or vascular complications in patients with type 2 diabetes. Am J Hypertens 2000;13:1027–1031 © 2000 American Journal of Hypertension, Ltd.
3-adrenergic receptor, Trp64Arg polymorphism, hypertension, genetic, genes, diabetes, non–insulin-dependent, diabetes, type 2. KEY WORDS:
drome.1,2 Both genetic and environmental factors play important roles in the development of these multifactorial disorders,3– 6 and considerable efforts are currently directed towards identifying genetic risk factors contributing to these disease entities. The 3-adrenergic receptor (ADRB3) is expressed predominatly in adipose tissue and plays an important role in lipid metabolism and metabolic rate.7–11 Therefore, molecular variations of the ADRB3 may lead to insulin resistance, obesity, and type 2 diabetes mellitus. A single-nucleotide missense mutation, resulting in a tryptophan/arginine exchange at position 64 (Trp64Arg polymorphism) of the amino acid chain,12 has been recently associated with abdominal 0895-7061/00/$20.00 PII S0895-7061(00)00290-9
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RINGEL ET AL
obesity and insulin resistance,13 a greater capacity to gain weight,14 and difficulty losing weight.15 A higher prevalence of this variant has also been reported in Pima Indians, in whom it was also associated with early onset of type 2 diabetes mellitus and a lower metabolic rate.12 Recently, Sakane et al16 reported an association between the Trp64Arg variant and diabetic retinopathy in Japanese type 2 diabetes patients. Although some investigators have also reported higher blood pressure levels in individuals carrying the Trp64Arg allele,13,17–19 this has not been confirmed by others.14,20 –22 These findings prompted us to examine the relationship among the ADRB3 Trp64Arg variant, hypertension, vascular, and renal complications in German patients with type 2 diabetes. MATERIALS AND METHODS The protocol of the study was approved by the Ethics Committee of our hospital and informed consent for genetic studies was obtained from all participants. Patient Selection and Clinical Investigation White patients with diabetes mellitus type 2 (n ⫽ 417) were enrolled in the study from one large diabetes clinic and four dialysis centers in Berlin. On selected days, determined by laboratory capacity, all patients with diabetes presenting in the diabetes clinic were approached; of them, more than 70% agreed to participate in the study. Classification of diabetes was based on criteria of the American Diabetes Association (ADA).23 Hypertension was defined as a systolic blood pressure ⬎ 140 mm Hg and a diastolic blood pressure ⬎ 95 mm Hg noted in the medical records on at least two separate occasions, or was based on the prescription of antihypertensive medication excluding diuretics. The diagnosis of nephropathy was based on repeated evidence of albumin excretion of more than 30 mg/24 h or 20 g/min in nonoliguric patients or terminal renal failure necessitating renal replacement therapy (dialysis or transplantation) in patients with end-stage renal failure. Other causes of increased albumin excretion were excluded by appropriate clinical investigation. Past medical history regarding coronary heart disease, stroke, and retinopathy was obtained by review of the medical records of each patient by an investigator unaware of the patient’s genotype. Coronary heart disease was defined as a history of myocardial infarction, coronary angioplasty or bypass surgery, positive coronary angiography, treatment with nitrates, or clinical history of angina pectoris. Stroke was defined as history of stroke in medical records. Diabetic retinopathy was defined as stage III or IV on fundoscopy and history of laser therapy was used as a surrogate marker for the presence of proliferative ret-
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
inopathy. HbA1c was determined by standard laboratory techniques. Genotyping Genomic DNA from each patient was prepared from peripheral white blood cells separated from a 20-mL blood sample using a DNA-selective preparation method (Quiagen, Hilden, Germany). Subsequently, the ADRB3 variant region was amplified with the polymerase chain reaction technique using a flanking primer pair.24 After a restriction digest with BstNI for 2 h at 37°C, the resulting fragments were separated and analyzed on ethidium bromidestained agarose (2%) gels. Statistical Analysis All data are presented as means ⫾ standard deviation or as proportions. Continuous variables were compared by two-sided Student’s t test for independent samples and categorical data were assessed by two-sided 2 statistics. Stepwise logistic regression analysis was used to explore the relationship between clinical parameters and genotype (SPSS 8.0, SPSS Inc., Chicago, IL). A P ⬍ .05 was considered as statistically significant. RESULTS Stepwise logistic regression analysis with hypertension as the dependent variable revealed a significant interaction between the Trp64Arg polymorphism and gender (P ⫽ .019), therefore separate analyses were performed for men and women. Clinical characteristics for each genotype group are presented in Table 1. All subjects with the Trp64Arg polymorphism were heterozygous. Although age, body mass index, duration of diabetes, and HbA1c levels were similar between patients with and without the variant, in men, hypertension was almost twice as common in patients with the variant than in patients homozygous for the wild type (P ⬍ .05). Furthermore, despite a significantly greater proportion of patients treated with multiple antihypertensive medications (P ⫽ .01), both systolic and diastolic blood pressure levels were higher in patients carrying the Trp64Arg allele. In contrast, there was no significant association between genotype and blood pressure in the female patients. When patients were classified with regard to presence or absence of hypertension, hypertensive patients were significantly older, had a longer duration of diabetes, and had a higher prevalence of nephropathy, retinopathy, stroke, and coronary artery disease (Table 2). Again, although the frequency of the Trp64Arg allele was significantly higher in hypertensive than in normotensive men (q Trp64Arg 0.19 v 0.07, P ⬍ .05), the allelic distribution in hypertensive and normotensive women was comparable (q Trp64Arg 0.12 v 0.15). In both groups the genotype distribution of the
POLYMORPHISM OF THE 3-RECEPTOR, DIABETES MELLITUS
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
1029
TABLE 1. PATIENTS’ CHARACTERISTICS Men
Age (years) BMI (kg/m2) Duration of diabetes (years) HbA1c (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Number of antihypetensive drugs (0/1/2/3/⬎ 3 [%]) Nephropathy (%) Retinopathy (%) Hypertension (%) Stroke (%) CAD (%)
Women
Trp64Trp (n ⴝ 183)
Trp64Arg (n ⴝ 25)
Trp64Trp (n ⴝ 181)
Trp64Arg (n ⴝ 28)
59.4 ⫾ 9.5 27.4 ⫾ 4.2 13.8 ⫾ 9.5 8.3 ⫾ 1.6 138 ⫾ 18 80 ⫾ 8
59.3 ⫾ 10.6 26.2 ⫾ 3.6 13.0 ⫾ 7.4 8.7 ⫾ 1.8 148 ⫾ 18* 84 ⫾ 7*
62.0 ⫾ 10.8 28.4 ⫾ 5.8 14.4 ⫾ 11.1 8.8 ⫾ 4.5 141 ⫾ 22 81 ⫾ 11
63.9 ⫾ 9.7 27.3 ⫾ 4.3 13.7 ⫾ 8.0 7.9 ⫾ 1.2 140 ⫾ 20 80 ⫾ 9
64/28/6/1/1 55 26 38 7 23
36/40/16/4/4† 60 24 64* 4 30
50/28/17/4/1 54 21 44 7 23
61/21/18/0/0 50 24 50 8 13
Data are means ⫾ SD. * P ⬍ .05 v patients with Trp64Trp genotype; † P ⫽ .01 v patients with Trp64Trp genotype. BMI ⫽ body mass index; CAD ⫽ coronary artery disease.
Trp64Arg polymorphism of the ADRB3 alleles were in Hardy-Weinberg equilibrium. DISCUSSION The principal finding in this large and ethnically homogeneous group of type 2 diabetes patients was a significant association between hypertension and the ADRB3 Trp64Arg in men. Furthermore, blood pressure levels remained significantly higher in these patients despite a greater number of multiple antihypertensive medications. In contrast, we found no association between ADRB3 genotype and either body mass index or diabetes complications in the whole
group, or between genotype and blood pressure in women. Our findings of a positive association between the Trp64Arg variant of the ADRB3 gene are in agreement with a previous report by Widen et al,13 who observed higher diastolic blood pressure levels in non–insulindependent patients with this variant. Other investigators have also reported a significant relationship between blood pressure and the Trp64Arg allele in white18 and Japanese17,19 patients with and without type 2 diabetes. These findings are also in agreement with our own independent observations in a group of obese subjects, in whom we found significantly higher
TABLE 2. CHARACTERISTICS OF PATIENTS WITH AND WITHOUT HYPERTENSION Men
Age (years) BMI (kg/m2) Duration of diabetes (years) HbA1c (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Number of antihypertensive drugs (0/1/2/3/⬎ 3 [%]) Nephropathy (%) Retinopathy (%) Stroke (%) CAD (%) Frequency of Trp64Arg (%) Abbreviations as in Table 1. Data are means ⫾ SD.
Without Hypertension (n ⴝ 122)
With Hypertension (n ⴝ 86)
57.8 ⫾ 9.9 27.2 ⫾ 4.4 12.4 ⫾ 9.3 8.3 ⫾ 1.7 130 ⫾ 13 78 ⫾ 7
61.6 ⫾ 8.8 27.4 ⫾ 3.9 15.6 ⫾ 9.2 8.4 ⫾ 1.6 151 ⫾ 17 84 ⫾ 2
43 17 3 13 7
8/67/17/5/3 72 37 11 41 19
Women Without Hypertension (n ⴝ 94)
With Hypertension (n ⴝ 115)
.0001 .0001
59.8 ⫾ 11.2 27.3 ⫾ 5.6 12.4 ⫾ 10.0 9.2 ⫾ 6.0 130 ⫾ 14 77 ⫾ 7
64.4 ⫾ 9.6 29.0 ⫾ 5.5 15.8 ⫾ 11.1 8.3 ⫾ 1.5 150 ⫾ 22 84 ⫾ 12
.0001 .001 .001 .0001 .014
38 9 6 7 15
18/45/30/6/1 66 32 8 33 12
P
.005 .015
P
.002 .032 .024 .0001 .0001
.0001 .0001 .0001
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RINGEL ET AL
levels of 24-h ambulatory blood pressure and increased insulin response to an oral glucose load in patients with the Trp64Arg genotype, compared with patients with the Trp64Trp genotype.25 In contrast, several investigators have failed to find a significant association between blood pressure and the Trp64Arg variant of the ADRB3 gene in obese or diabetic patients of white or Japanese ethnicity.14,20,21,26 Furthermore, Fujisawa et al27 found no relationship between this genetic variant and blood pressure in Japanese patients with essential hypertension. The reasons for this discrepancy are not clear. Nevertheless, apart from possible differences in race, environmental background, or other issues pertaining to study design or recruitment of the study populations, it should be appreciated that several negative reports have involved a much smaller number of patients than our present study. In fact, a large study that failed to find a significant relationship between hypertension and the ADRB3 genotype in type 2 diabetes patients also failed to find a relationship between this genotype and obesity, diabetes, and dyslipidemia.21 Why the association between the Trp64Arg allele and hypertension was only apparent in male patients is not clear. However, we may speculate that central obesity is more common in men than in women,28 and is well known to confer a greater risk for the development of hypertension, cardiovascular complications, and type 2 diabetes than the lower-body or gynecoid obesity commonly encountered in women.29 –31 It may also be important to note that expression of the 3adrenergic receptor has been reported to be higher in omental adipose tissue than in lower-body subcutaneous depots.7 Thus, gender-specific differences in adipose-tissue distribution may account for the discrepancy between our findings in men and women. Despite the relationship between the ADRB3 Trp64Arg variant and hypertension in male patients, there was no relationship between this variant and complications of diabetes, including coronary artery disease, stroke, retinopathy, or nephropathy. However, it must be appreciated that the subgroups involved were clearly underpowered to detect such a relationship, and clearly larger studies will be required to test the relationship between this genetic variant and vascular complications in diabetes patients. In conclusion, our findings support the hypothesis that the Trp64Arg polymorphism of the ADRB3 gene is associated with hypertension in white type 2 diabetes patients. The question regarding the utility of this marker for the prediction of hypertension in patients with type 2 diabetes mellitus and whether or not patients with this allele may be at an increased risk for vascular complications remains to be determined.
ACKNOWLEDGMENTS We are grateful to Dr. Elke Austenat of the Diabetes Nachtklinik Tempelhof, Berlin, and to Prof. Wolfgang Pommer of the Humbold Krankenhaus, Berlin, for their support in the recruitment of patients. We also acknowledge Brigitte Egbers, Katrin Kossatz-Eskandani, and Klaus Schlotter for their expert technical help.
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Bjo¨rntorp P: Abdominal obesity and the metabolic syndrome. Ann Med 1992;24:465– 468.
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Mu¨ller-Wieland D, Kotzka J, Knebel B, Krone W: Metabolic syndrome and hypertension: pathophysiology and molecular basis of insulin resistance. Basic Res Cardiol 1998;93(suppl 2):131–134.
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Hsueh WA, Buchanan TA: Obesity and hypertension. Endocrinol Metab Clin North Am 1994;23:405– 427.
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Beck Nielsen H, Groop LC: Metabolic and genetic characterization of prediabetic states. Sequence of events leading to non-insulin-dependent diabetes mellitus. J Clin Invest 1994;94:1714 –1721.
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Lifton RP: Molecular genetics of human blood pressure variation. Science 1996;272:676 – 680.
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Hjermann I: The metabolic cardiovascular syndrome: Syndrome X, Reaven’s syndrome, insulin resistance syndrome, atherothrombogenic syndrome. J Cardiovasc Pharmacol 1992;20(suppl 8):S5–S10.
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Krief S, Lonnqvist F, Raimbault S, Baude B, Van Spronsen A, Arner P, Strosberg AD, Ricquier D, Emorine LJ: Tissue distribution of beta 3-adrenergic receptor mRNA in man. J Clin Invest 1993;91:344 –349.
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Ricquier D, Cassard Doulcier AM: The biochemistry of white and brown adipocytes analysed from a selection of proteins. Eur J Biochem 1993;218:785–796.
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Lonnqvist F, Thome A, Nilsell K, Hoffstedt J, Arner P: A pathogenic role of visceral fat beta 3-adrenoceptors in obesity. J Clin Invest 1995;95:1109 –1116.
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Lowell BB, Flier JS: Brown adipose tissue, 3-adrenergic receptors, and obesity. Annu Rev Med 1997;48:307– 316.
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Strosberg AD: Association of beta 3-adrenoceptor polymorphism with obesity and diabetes: current status. Trends Pharmacol Sci 1997;18:449 – 454.
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Walston J, Silver K, Bogardus C, Knowler WC, Celi FS, Austin S, Manning B, Strosberg AD, Stern MP, Raben N, Sotkin JD, Roth J, Shuldiner AR: Time of onset of non-insulin-dependent diabetes mellitus and genetic variation in the beta 3-adrenergic-receptor gene. N Engl J Med 1995;333:343–347.
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Widen E, Lehto M, Kanninen T, Walston J, Shuldiner AR, Groop LC: Association of a polymorphism in the beta 3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns. N Engl J Med 1995;333:348 –351.
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Clement K, Vaisse C, Manning BS, Basdevant A, Guy Grand B, Ruiz J, Silver KD, Shuldiner AR, Froguel P, Strosberg AD: Genetic variation in the beta 3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity. N Engl J Med 1995;333: 352–354.
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POLYMORPHISM OF THE 3-RECEPTOR, DIABETES MELLITUS
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Yoshida T, Sakane N, Umekawa T, Sakai M, Takahashi T, Kondo M: Mutation of beta 3-adrenergic-receptor gene and response to treatment of obesity. Lancet 1995; 346:1433–1434.
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Sakane N, Yoshida T, Yoshioka K, Nakamura Y, Umekawa T, Kogure A, Takakura Y, Kondo M: Beta 3-adrenoreceptor gene polymorphism: a newly identified risk factor for proliferative retinopathy in NIDDM patients. Diabetes 1997;46:1633–1636.
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Kaburagi Y, Satoh S, Tamemoto H, Yamamoto-Honda R, Tobe K, Veki K, Yamauchi T, Kono-Sugita E, Sekihara H, Aizawa S, Cushman SW, Akanuma Y, Yazaki Y, Kadowaki T: Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes. J Biol Chem 1997;272:25839 –25844. Kurabayashi T, Carey DG, Morrison NA: The beta 3-adrenergic receptor gene Trp64Arg mutation is overrepresented in obese women. Effects on weight, BMI, abdominal fat, blood pressure, and reproductive history in an elderly Australian population. Diabetes 1996; 45:1358 –1363. Sakane N, Yoshida T, Umekawa T, Kondo M, Sakai Y, Takahashi T: 3-adrenergic-receptor polymorphism: a genetic marker for visceral fat obesity and the insulin resistance syndrome. Diabetologia 1997;40:200 –204. Gagnon J, Mauriege P, Roy S, Sjostrom D, Chagnon YC, Dionne FT, Oppert JM, Perusse L, Sjostrom L, Bouchard C: The Trp64Arg mutation of the beta3 adrenergic receptor gene has no effect on obesity phenotypes in the Quebec Family Study and Swedish Obese Subjects cohorts. J Clin Invest 1996;98:2086 –2093. Buettner R, Schaffler A, Arndt H, Rogler G, Nusser J, Zietz B, Enger I, Hugl S, Cuk A, Scholmerich J, Palitzsch KD: The Trp64Arg polymorphism of the beta 3-adrenergic receptor gene is not associated with obesity or type 2 diabetes mellitus in a large populationbased Caucasian cohort. J Clin Endocrinol Metab 1998; 83:2892–2897. Janssen JA, Koper JW, Stolk RP, Englaro P, Uitterlinden AG, Huang Q, van Leeuwen JP, Blum WF, Attanasio AM, Pols HA, Grobbee DE, de Jong FH, Lamberts SW: Lack of associations between serum leptin, a polymor-
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phism in the gene for the beta 3-adrenergic receptor and glucose tolerance in the Dutch population. Clin Endocrinol (Oxf) 1998;49:229 –234. American Diabetes Association: Guide to diagnosis and classification of diabetes mellitus and other categories of glucose intolerance. Diabetes Care 1997;20: S21. Rissanen J, Kuopusjarvi J, Pihlajamaki J, Sipilainen R, Heikkinen S, Vanhala M, Kekalainen P, Kuusisto J, Laakso M: The Trp64Arg polymorphism of the beta 3-adrenergic receptor gene. Lack of association with NIDDM and features of insulin resistance syndrome. Diabetes Care 1997;20:1319 –1323. Kunz I, Schorr U, Barth CA, Distler A, Sharma AM, Klaus S: The beta 3-adrenergic receptor gene and obesity-associated hypertension. Int J Obes Relat Metab Disord 1998;22:S116. Elbein SC, Hoffman M, Barrett K, Wegner K, Miles C, Bachman K, Berkowitz D, Shuldiner AR, Leppert MF, Hasstedt S: Role of the beta 3-adrenergic receptor locus in obesity and noninsulin- dependent diabetes among members of Caucasian families with a diabetic sibling pair. J Clin Endocrinol Metab 1996;81:4422– 4427. Fujisawa T, Ikegami H, Yamato E, Hamada Y, Kamide K, Rakugi H, Higaki J, Murakami H, Shimamoto K, Ogihara T: Trp64Arg mutation of 3-adrenergic receptor in essential hypertension - Insulin resistance and the adrenergic system. Am J Hypertens 1997;10:101–105. Raison JM, Achimastos AM, Safar ME: Sex-dependence of body fat distribution in patients with obesity and hypertension. Clin Exp Hypertens [A] 1992;14:505–525. Williams MJ, Hunter GR, Kekes-Szabo T, Trueth MS, Snyder S, Berland L, Blaudeau T: Intra-abdominal adipose tissue cut-points related to elevated cardiovascular risk in women. Int J Obes 1996;20:613– 617. Scaglione R, Ganguzza A, Corrao S, Parrinello G, Merlino G, Dichiara MA, Arnone S, D’Aubert MD, Licata G: Central obesity and hypertension: Pathophysiologic role of renal haemodynamics and function. Int J Obes 1995;19:403– 409. Selby JV, Friedman GD, Quesenberry CP Jr: Precursors of essential hypertension. The role of body fat distribution pattern. Am J Epidemiol 1989;129:43–53.
2000;13:1027–1031
BRIEF COMMUNICATIONS
The Trp64Arg Polymorphism of the 3Adrenergic Receptor Gene is Associated with Hypertension in Men with Type 2 Diabetes Mellitus Jens Ringel, Reinhold Kreutz, Armin Distler, and Arya M. Sharma
A missense mutation of the 3-adrenergic receptor gene (ADRB3) resulting in a tryptophan/arginine exchange at position 64 (Trp64Arg polymorphism) has recently been associated with greater capacity to gain weight, a low resting metabolic rate, higher blood pressure, and an early onset of type 2 diabetes. These findings prompted us to examine the relationship between this mutation, blood pressure, and vascular complications in German patients with type 2 diabetes. White patients with type 2 diabetes mellitus (n ⴝ 417) were enrolled in the study. The Trp64Arg polymorphism of the ADRB3 gene was detected by polymerase chain amplification and subsequent restriction digest with BstN I. Stepwise logistic regression analysis of the entire study population revealed a significant interaction between gender and genotype (P ⴝ .019). We therefore performed separate analyses for men and women. There was a significant relationship between hypertension and
O
besity and type 2 diabetes mellitus are commonly associated with hypertension and dyslipidemia, and this cluster of abnormalities has also been described as the insulin-resistance or metabolic syn-
Received June 1, 1999. Accepted March 7, 2000. From the Department of Internal Medicine, Division of Endocrinology and Nephrology, and Department of Clinical Pharmacology, Benjamin Franklin Medical Center, Freie Universita¨t Berlin, Germany. Address correspondence and reprint requests to Prof. Dr. Arya M. Sharma, Medizinische Klinik IV, Klinikum Benjamin Franklin, Freie Universita¨t Berlin, Hindenburgdamm 30, D-12200 Berlin, Federal Republic of Germany;e-mail: [email protected]
© 2000 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
the ADRB3 Trp64Arg variant in men (P ⴝ .015), but not in women. Furthermore, blood pressure levels in male patients with the minor allele had higher blood pressure levels (P < .05), despite a significantly greater number of antihypertensive medications (P ⴝ .01). There was no association between ADRB3 genotype and vascular complications in these patients. In conclusion, our data are compatible with a contribution of this genetic variant of ADRB3 to hypertension in male patients with type 2 diabetes. Further studies will be needed to determine the role of this polymorphism as a predictor of hypertension or vascular complications in patients with type 2 diabetes. Am J Hypertens 2000;13:1027–1031 © 2000 American Journal of Hypertension, Ltd.
3-adrenergic receptor, Trp64Arg polymorphism, hypertension, genetic, genes, diabetes, non–insulin-dependent, diabetes, type 2. KEY WORDS:
drome.1,2 Both genetic and environmental factors play important roles in the development of these multifactorial disorders,3– 6 and considerable efforts are currently directed towards identifying genetic risk factors contributing to these disease entities. The 3-adrenergic receptor (ADRB3) is expressed predominatly in adipose tissue and plays an important role in lipid metabolism and metabolic rate.7–11 Therefore, molecular variations of the ADRB3 may lead to insulin resistance, obesity, and type 2 diabetes mellitus. A single-nucleotide missense mutation, resulting in a tryptophan/arginine exchange at position 64 (Trp64Arg polymorphism) of the amino acid chain,12 has been recently associated with abdominal 0895-7061/00/$20.00 PII S0895-7061(00)00290-9
1028
RINGEL ET AL
obesity and insulin resistance,13 a greater capacity to gain weight,14 and difficulty losing weight.15 A higher prevalence of this variant has also been reported in Pima Indians, in whom it was also associated with early onset of type 2 diabetes mellitus and a lower metabolic rate.12 Recently, Sakane et al16 reported an association between the Trp64Arg variant and diabetic retinopathy in Japanese type 2 diabetes patients. Although some investigators have also reported higher blood pressure levels in individuals carrying the Trp64Arg allele,13,17–19 this has not been confirmed by others.14,20 –22 These findings prompted us to examine the relationship among the ADRB3 Trp64Arg variant, hypertension, vascular, and renal complications in German patients with type 2 diabetes. MATERIALS AND METHODS The protocol of the study was approved by the Ethics Committee of our hospital and informed consent for genetic studies was obtained from all participants. Patient Selection and Clinical Investigation White patients with diabetes mellitus type 2 (n ⫽ 417) were enrolled in the study from one large diabetes clinic and four dialysis centers in Berlin. On selected days, determined by laboratory capacity, all patients with diabetes presenting in the diabetes clinic were approached; of them, more than 70% agreed to participate in the study. Classification of diabetes was based on criteria of the American Diabetes Association (ADA).23 Hypertension was defined as a systolic blood pressure ⬎ 140 mm Hg and a diastolic blood pressure ⬎ 95 mm Hg noted in the medical records on at least two separate occasions, or was based on the prescription of antihypertensive medication excluding diuretics. The diagnosis of nephropathy was based on repeated evidence of albumin excretion of more than 30 mg/24 h or 20 g/min in nonoliguric patients or terminal renal failure necessitating renal replacement therapy (dialysis or transplantation) in patients with end-stage renal failure. Other causes of increased albumin excretion were excluded by appropriate clinical investigation. Past medical history regarding coronary heart disease, stroke, and retinopathy was obtained by review of the medical records of each patient by an investigator unaware of the patient’s genotype. Coronary heart disease was defined as a history of myocardial infarction, coronary angioplasty or bypass surgery, positive coronary angiography, treatment with nitrates, or clinical history of angina pectoris. Stroke was defined as history of stroke in medical records. Diabetic retinopathy was defined as stage III or IV on fundoscopy and history of laser therapy was used as a surrogate marker for the presence of proliferative ret-
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
inopathy. HbA1c was determined by standard laboratory techniques. Genotyping Genomic DNA from each patient was prepared from peripheral white blood cells separated from a 20-mL blood sample using a DNA-selective preparation method (Quiagen, Hilden, Germany). Subsequently, the ADRB3 variant region was amplified with the polymerase chain reaction technique using a flanking primer pair.24 After a restriction digest with BstNI for 2 h at 37°C, the resulting fragments were separated and analyzed on ethidium bromidestained agarose (2%) gels. Statistical Analysis All data are presented as means ⫾ standard deviation or as proportions. Continuous variables were compared by two-sided Student’s t test for independent samples and categorical data were assessed by two-sided 2 statistics. Stepwise logistic regression analysis was used to explore the relationship between clinical parameters and genotype (SPSS 8.0, SPSS Inc., Chicago, IL). A P ⬍ .05 was considered as statistically significant. RESULTS Stepwise logistic regression analysis with hypertension as the dependent variable revealed a significant interaction between the Trp64Arg polymorphism and gender (P ⫽ .019), therefore separate analyses were performed for men and women. Clinical characteristics for each genotype group are presented in Table 1. All subjects with the Trp64Arg polymorphism were heterozygous. Although age, body mass index, duration of diabetes, and HbA1c levels were similar between patients with and without the variant, in men, hypertension was almost twice as common in patients with the variant than in patients homozygous for the wild type (P ⬍ .05). Furthermore, despite a significantly greater proportion of patients treated with multiple antihypertensive medications (P ⫽ .01), both systolic and diastolic blood pressure levels were higher in patients carrying the Trp64Arg allele. In contrast, there was no significant association between genotype and blood pressure in the female patients. When patients were classified with regard to presence or absence of hypertension, hypertensive patients were significantly older, had a longer duration of diabetes, and had a higher prevalence of nephropathy, retinopathy, stroke, and coronary artery disease (Table 2). Again, although the frequency of the Trp64Arg allele was significantly higher in hypertensive than in normotensive men (q Trp64Arg 0.19 v 0.07, P ⬍ .05), the allelic distribution in hypertensive and normotensive women was comparable (q Trp64Arg 0.12 v 0.15). In both groups the genotype distribution of the
POLYMORPHISM OF THE 3-RECEPTOR, DIABETES MELLITUS
AJH–SEPTEMBER 2000 –VOL. 13, NO. 9
1029
TABLE 1. PATIENTS’ CHARACTERISTICS Men
Age (years) BMI (kg/m2) Duration of diabetes (years) HbA1c (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Number of antihypetensive drugs (0/1/2/3/⬎ 3 [%]) Nephropathy (%) Retinopathy (%) Hypertension (%) Stroke (%) CAD (%)
Women
Trp64Trp (n ⴝ 183)
Trp64Arg (n ⴝ 25)
Trp64Trp (n ⴝ 181)
Trp64Arg (n ⴝ 28)
59.4 ⫾ 9.5 27.4 ⫾ 4.2 13.8 ⫾ 9.5 8.3 ⫾ 1.6 138 ⫾ 18 80 ⫾ 8
59.3 ⫾ 10.6 26.2 ⫾ 3.6 13.0 ⫾ 7.4 8.7 ⫾ 1.8 148 ⫾ 18* 84 ⫾ 7*
62.0 ⫾ 10.8 28.4 ⫾ 5.8 14.4 ⫾ 11.1 8.8 ⫾ 4.5 141 ⫾ 22 81 ⫾ 11
63.9 ⫾ 9.7 27.3 ⫾ 4.3 13.7 ⫾ 8.0 7.9 ⫾ 1.2 140 ⫾ 20 80 ⫾ 9
64/28/6/1/1 55 26 38 7 23
36/40/16/4/4† 60 24 64* 4 30
50/28/17/4/1 54 21 44 7 23
61/21/18/0/0 50 24 50 8 13
Data are means ⫾ SD. * P ⬍ .05 v patients with Trp64Trp genotype; † P ⫽ .01 v patients with Trp64Trp genotype. BMI ⫽ body mass index; CAD ⫽ coronary artery disease.
Trp64Arg polymorphism of the ADRB3 alleles were in Hardy-Weinberg equilibrium. DISCUSSION The principal finding in this large and ethnically homogeneous group of type 2 diabetes patients was a significant association between hypertension and the ADRB3 Trp64Arg in men. Furthermore, blood pressure levels remained significantly higher in these patients despite a greater number of multiple antihypertensive medications. In contrast, we found no association between ADRB3 genotype and either body mass index or diabetes complications in the whole
group, or between genotype and blood pressure in women. Our findings of a positive association between the Trp64Arg variant of the ADRB3 gene are in agreement with a previous report by Widen et al,13 who observed higher diastolic blood pressure levels in non–insulindependent patients with this variant. Other investigators have also reported a significant relationship between blood pressure and the Trp64Arg allele in white18 and Japanese17,19 patients with and without type 2 diabetes. These findings are also in agreement with our own independent observations in a group of obese subjects, in whom we found significantly higher
TABLE 2. CHARACTERISTICS OF PATIENTS WITH AND WITHOUT HYPERTENSION Men
Age (years) BMI (kg/m2) Duration of diabetes (years) HbA1c (%) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Number of antihypertensive drugs (0/1/2/3/⬎ 3 [%]) Nephropathy (%) Retinopathy (%) Stroke (%) CAD (%) Frequency of Trp64Arg (%) Abbreviations as in Table 1. Data are means ⫾ SD.
Without Hypertension (n ⴝ 122)
With Hypertension (n ⴝ 86)
57.8 ⫾ 9.9 27.2 ⫾ 4.4 12.4 ⫾ 9.3 8.3 ⫾ 1.7 130 ⫾ 13 78 ⫾ 7
61.6 ⫾ 8.8 27.4 ⫾ 3.9 15.6 ⫾ 9.2 8.4 ⫾ 1.6 151 ⫾ 17 84 ⫾ 2
43 17 3 13 7
8/67/17/5/3 72 37 11 41 19
Women Without Hypertension (n ⴝ 94)
With Hypertension (n ⴝ 115)
.0001 .0001
59.8 ⫾ 11.2 27.3 ⫾ 5.6 12.4 ⫾ 10.0 9.2 ⫾ 6.0 130 ⫾ 14 77 ⫾ 7
64.4 ⫾ 9.6 29.0 ⫾ 5.5 15.8 ⫾ 11.1 8.3 ⫾ 1.5 150 ⫾ 22 84 ⫾ 12
.0001 .001 .001 .0001 .014
38 9 6 7 15
18/45/30/6/1 66 32 8 33 12
P
.005 .015
P
.002 .032 .024 .0001 .0001
.0001 .0001 .0001
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RINGEL ET AL
levels of 24-h ambulatory blood pressure and increased insulin response to an oral glucose load in patients with the Trp64Arg genotype, compared with patients with the Trp64Trp genotype.25 In contrast, several investigators have failed to find a significant association between blood pressure and the Trp64Arg variant of the ADRB3 gene in obese or diabetic patients of white or Japanese ethnicity.14,20,21,26 Furthermore, Fujisawa et al27 found no relationship between this genetic variant and blood pressure in Japanese patients with essential hypertension. The reasons for this discrepancy are not clear. Nevertheless, apart from possible differences in race, environmental background, or other issues pertaining to study design or recruitment of the study populations, it should be appreciated that several negative reports have involved a much smaller number of patients than our present study. In fact, a large study that failed to find a significant relationship between hypertension and the ADRB3 genotype in type 2 diabetes patients also failed to find a relationship between this genotype and obesity, diabetes, and dyslipidemia.21 Why the association between the Trp64Arg allele and hypertension was only apparent in male patients is not clear. However, we may speculate that central obesity is more common in men than in women,28 and is well known to confer a greater risk for the development of hypertension, cardiovascular complications, and type 2 diabetes than the lower-body or gynecoid obesity commonly encountered in women.29 –31 It may also be important to note that expression of the 3adrenergic receptor has been reported to be higher in omental adipose tissue than in lower-body subcutaneous depots.7 Thus, gender-specific differences in adipose-tissue distribution may account for the discrepancy between our findings in men and women. Despite the relationship between the ADRB3 Trp64Arg variant and hypertension in male patients, there was no relationship between this variant and complications of diabetes, including coronary artery disease, stroke, retinopathy, or nephropathy. However, it must be appreciated that the subgroups involved were clearly underpowered to detect such a relationship, and clearly larger studies will be required to test the relationship between this genetic variant and vascular complications in diabetes patients. In conclusion, our findings support the hypothesis that the Trp64Arg polymorphism of the ADRB3 gene is associated with hypertension in white type 2 diabetes patients. The question regarding the utility of this marker for the prediction of hypertension in patients with type 2 diabetes mellitus and whether or not patients with this allele may be at an increased risk for vascular complications remains to be determined.
ACKNOWLEDGMENTS We are grateful to Dr. Elke Austenat of the Diabetes Nachtklinik Tempelhof, Berlin, and to Prof. Wolfgang Pommer of the Humbold Krankenhaus, Berlin, for their support in the recruitment of patients. We also acknowledge Brigitte Egbers, Katrin Kossatz-Eskandani, and Klaus Schlotter for their expert technical help.
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