Abstract
Objective. To examine the association of a common ‐2548G/A (rs7799039) promoter variant of the human leptin gene (LEP) with obesity or body mass index (BMI) and its associated phenotypes such as blood pressure variability and the prevalence of hypertension in a sample of the Tunisian population. Design and methods. Two hundred and twenty‐nine obese patients were screened and compared with 251 normal weight subjects. The ‐2548G/A LEP polymorphism was analysed by PCR‐RFLP procedure. Results. No significant association was found between the ‐2548G/A polymorphism and obesity or BMI. However, in obese patients subjects with AA genotype had significantly higher systolic (p = 0.003) and diastolic (p = 0.002) blood pressure compared with those with GA or GG genotypes. Stratified analysis by gender revealed that male patients but not female homozygous for ‐2548A allele exhibited significantly increased systolic (p = 0.01) and diastolic (p<0.001) blood pressure than did carriers of ‐2548G allele. Multiple linear regression analysis revealed that AA genotype significantly affect systolic and diastolic blood pressure in obese men. Additionally, significant association between AA genotype and higher prevalence of hypertension was found in male patients (p = 0.03). Conclusion. The present study showed that the ‐2548G/A LEP polymorphism is associated with blood pressure in obese male patients.
Introduction
Obesity is associated with an increased incidence of hypertension and cardiovascular disease [1], [2]. Although the association of excess body weight with elevated blood pressure has been known for a long time [3], [4], the pathophysiological bases of this association are not completely understood [5]. Leptin, a 16‐kDa polypeptide hormone produced predominantly by white adipose tissue [6], plays an important role in body weight homeostasis through effects on food intake and energy expenditure [7], [8]. In addition to the action of this adipocyte‐derived hormone on satiety through hypothalamic receptors, leptin seems to be implicated in the pathophysiology of hypertension [9]. It is conceivable that leptin‐mediated sympathetic activation in the circulatory system and/or at the renal level may affect blood pressure control and contribute to the occurrence of obesity‐associated hypertension [10], [11]. Rare obesity syndromes are associated with mutations of the human leptin gene (LEP) [12], [13]. Although the frequency of such mutations is very low, common polymorphisms in this gene may contribute to a common form of obesity and, as a consequence, obesity‐related hypertension. One highly polymorphic tetranucleotide repeat polymorphism in the 3′‐flanking region of the leptin gene was reported associated with hypertension, independently of obesity in Japanese patients [14]. Another polymorphism identified in the 5′ untranslated region, a common ‐2548G/A, has been evaluated in different populations, but the association of that variant with obesity have been inconclusive [15–18].
To address the question of the conflicting nature of these studies, and the necessity to conduct additional analyses, we proposed to investigate the possible association of the ‐2548G/A leptin promoter variant with obesity or related phenotypes such as blood pressure variability and the prevalence of hypertension in a sample of the Tunisian population.
Materials and methods
Subjects
Our sample included 251 unrelated normal weight subjects [120 women and 131 men, mean age 49.3±9.3 years, mean body mass index (BMI) 22.5±1.9 kg/m2] living in the community, randomly recruited from the local population register of Tunis city, and 229 unrelated obese patients (131 women and 98 men, mean age 47.6±9.3 years, mean BMI 40±6.7 kg/m2), recruited from the Department of Endocrinology, Rabta University Hospital (Tunis, Tunisia). All participants were homogeneous Tunisian Arab descendants who resided in Tunisia and all were from North Tunisia. The research protocol was approved by the local ethics committee and informed written consent was obtained from each subjects.
Weight and height were measured on the subjects barefooted and lightly clothed. BMI (kg/m2) was calculated and obesity was defined as BMI⩾30 kg/m2[19]. Blood pressure was measured at least at twice with subjects in the sitting position according to the guideline of the American Heart Association [20]. Normal blood pressure was defined as a systolic blood pressure of <140 mmHg and a diastolic blood pressure of <90 mmHg. Hypertension was defined as systolic blood pressure of ⩾140 mmHg and/or diastolic blood pressure of ⩾90 mmHg, or the use of antihypertensive drug treatment or a combination of these. Diabetes mellitus was defined as hyperglycaemia, requiring antidiabetic drugs or testing blood glucose over 7.0 mmol/l. Dyslipidaemia was defined as a total cholesterol level >6.47 mmol/l and/or triglyceride level >2.26 mmol/l. A daily consumption of more than five cigarettes is considered a smoking habit.
DNA analysis
Genomic DNA was obtained from peripheral blood leukocytes by phenol extraction [21]. The genotyping of the ‐2548G/A polymorphism (rs7799039) was carried out using the method of Mammés et al. [16]. The polymorphism was defined by the presence (when G at position ‐2548 from the transcription initiation site of the leptin gene) or the absence (when A) of a Cfo I restriction site [16].
Statistical analysis
Quantitative data were compared among three groups by one‐way analysis of variance, and between two groups by the unpaired Student's t‐test. The chi‐squared tests were used for categorical variables. The potential effect of ‐2548G/A LEP polymorphism on blood pressure was evaluated by multiple linear regression analysis, including age, BMI, gender and ‐2548G/A genotype. We calculated odds ratio (OR) together with their 95% approximate confidence intervals (95% CI) as estimators of the relative risk of hypertension for the ‐2548G/A LEP polymorphism. Univariate regression were used to determine crude OR. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS 11.5 for Windows; SPSS Inc., Chicago, IL, USA). The results were considered statistically significant when the p‐value was ⩽0.05.
Results
Baseline characteristics of the studied groups are shown in Table I. The prevalence of hypertension, diabetes and dyslipidaemia were higher in obese than in normal weight (p<0.001). The means values of systolic and diastolic blood pressure were higher (p<0.001) in obese patients than in control group.
Table I. Anthropometric and clinical characteristics of the study population.
The genotypes frequencies were in agreement with those predicted by the Hardy–Weinberg equilibrium in obese (χ2 = 1.44, p = 0.48) and control groups (χ2 = 1.32, p = 0.51). In obese patients, the genotype frequencies were 42.4% for GG, 44.1% for GA and 13.5% for AA and were 45% for GG, 46.2% for GA and 8.8% for AA in normal weight. No significant differences in genotype and allele frequencies of the ‐2548G/A polymorphism were detected between obese and normal weight subjects.
Anthropometric and clinical characteristics across genotypes of the two groups (obese and controls) were given in Table II. In both groups, age and BMI did not differ among ‐2548G/A genotypes. However, obese patients subjects with AA genotype had significantly higher systolic (p = 0.003) and diastolic (p = 0.002) blood pressure compared with those with GA or GG genotypes. Separate analysis by gender revealed that in obese men, the ‐2548G/A LEP polymorphism was significantly associated with higher systolic (p = 0.01) and diastolic (p<0.001) blood pressure. For obese women, however, systolic and diastolic blood pressure was not related to this LEP variant (Figure 1).
Table II. Anthropometric and clinical characteristics across genotypes of the two groups (obese and control).
Published online:
08 July 2009Figure 1 Variation of systolic and diastolic blood pressure across‐2548G/A genotypes in obese men (a) and women (b).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/iblo20/2008/iblo20.v017.i05-06/08037050802488960/20150517/images/medium/iblo_a_349064_f0001_b.gif"})
Figure 1 Variation of systolic and diastolic blood pressure across‐2548G/A genotypes in obese men (a) and women (b).
The effects of the ‐2548G/A LEP promoter polymorphism on blood pressure was evaluated by multiple linear regression analysis, including age, body mass index and genotype (Table III). The analysis revealed that AA genotype significantly affected systolic and diastolic blood pressure for obese men.
Table III. Effects of ‐2548G/A genotypes on blood pressure in obese patients.
To clarify the effect of the ‐2548G/A polymorphism on blood pressures, we compared the distribution of genotypes between individuals with hypertension and those with normal blood pressure. For obese women, no difference in the distribution of ‐2548G/A genotypes was detected between hypertensive and normotensive groups (data not shown). However, significant association of AA genotype with higher prevalence of hypertension (p = 0.03) was found in obese men. Additionally, significant higher risk of hypertension was observed for carriers of AA genotype (OR = 3.87, 95% CI 1.08–12.8) and carriers of GA genotype (OR = 2.47, 95% CI 1.01–6.03) (Table IV).
Table IV. Genotypes distribution and allele frequencies for the ‐2548G/A LEP polymorphism among obese men according to the presence of hypertension.
Discussion
The regulation of blood pressure involves both the integration of a variety of biological systems that control the structure and tone of the vasculature and the volume and composition of body fluid, as well as the adaptation of these systems to constantly changing physiological needs [22]. Leptin is a 167‐amino acid peptide secreted from the adipocytes into the circulation and communicates the peripheral nutritional status to specific hypothalamic centres [23]. Related to the amount of body fat, leptin is also associated with increased heart rate, blood pressure and sympathetic nervous activity and contributes to platelet aggregation [24]. The common ‐2548G/A LEP promoter variant has been examined for association with obesity or BMI, but with negative results [25], [26]. In contrast to these results, in a Family Heart study, Jiang et al. [27] reported significant association between this LEP polymorphism and obesity. Recently, in Taiwanese population, Wang et al. [17] reported that BMI of the GG genotype was significantly higher than that of GA and AA genotypes in extreme obesity. In this study carried out in Tunisian obese and normal weight subjects, no association was found between the ‐2548G/A LEP polymorphism and degree of obesity as assessed by BMI. However, we showed for the first time a significant association between this common LEP promoter variant and blood pressure in obese men. Particularly, the ‐2548A allele of this polymorphism is associated with higher blood pressure and AA genotype is a predictor of hypertension in males patients. The reason for this gender difference in the relation of ‐2548G/A genotypes to blood pressure remains unclear. It might be attributable, however, at least in part, to the difference in the serum concentration of estrogen between men and women, given that estrogen exerts various favourable effects on vasomotor function, including stimulation of the production of nitric oxide and prostaglandin I2 as well as inhibition of the release of endothelin‐1 by vascular endothelial cells [28].
Several mechanisms other than obesity can be considered as links between the leptin gene polymorphism and blood pressure. One possibility is hyperleptinaemia. Previous studies have shown increased circulating leptin levels in subjects homozygous for ‐2548A allele. The study of Mammés et al. [16] showed that men with the AA genotype exhibited higher serum total leptin levels than did men carriers of the ‐2548G allele. This finding was confirmed by Hoffstedt et al. [29] in 39 non‐obese women. They found that women with the AA genotype had higher serum leptin levels and significantly higher adipose tissue leptin secretion rates than did carriers of the ‐2548G allele. According to Schutte et al. [30], leptin was positively associated with systolic blood pressure in obese but not in lean women. In a rural Chinese population, Hu et al. [31] reported that association between blood pressure and leptin was heavily influenced by body fat mass and distribution. El‐Gharbawy et al. [32] found correlation between leptin and blood pressure in African women with hypertension and obesity, which disappeared after adjustment for other components of the insulin resistance syndrome. Another possibility is insulin resistance and hyperinsulinaemia. Hypertension is known to be associated with insulin resistance [33]. To our knowledge, there are no previous studies that showed an association between the ‐2548G/A LEP variant and insulin variability.
Another explanation is that the polymorphism studied is in linkage disequilibrium with the other polymorphisms of nearby genes that are actually responsible for the development of coronary heart disease and hypertension. Previous studies reported a significant association between a highly polymorphic tetranucleotide repeat polymorphism in the 3′‐flanking region of the leptin gene and hypertension independent of obesity in the Japanese population [14].
One of the weaknesses of this study was the small number of subjects screened. Another was the absence of measurement of leptin levels in subjects, which could potentially affect the relationship between the common leptin variant and blood pressure.
Conclusion
Our present results suggest that ‐2548G/A genotype is a determinant of blood pressure in obese men. Further studies are needed to confirm these data and to precise the underlying mechanisms.
Acknowledgements
This work was supported by a grant from the Ministry of Higher Education, Scientific Research and Technology of Tunisia.
| Variables | Normal weight subjects (n = 251) | Obese patients (n = 229) | p‐value |
|---|---|---|---|
| Age (years) | 49.3±9.3 | 47.9±9.3 | 0.11 |
| BMI (kg/m2) | 22.5±1.9 | 40±6.7 | <0.001 |
| Systolic blood pressure (mmHg) | 118±13 | 139±15 | <0.001 |
| Diastolic blood pressure (mmHg) | 71.5±7.3 | 85.1±10.1 | <0.001 |
| Diabetes (%) | 6.4 | 28.8 | <0.001 |
| Hypertension (%) | 8 | 53.7 | <0.001 |
| Dyslipidaemia (%) | 8 | 27 | <0.001 |
| Smokers (%) | 41.4 | 12.2 | <0.001 |
Data are expressed as means±SD for some and as percent in others variables. BMI, body mass index.
| Variables | Normal weight subjects (n = 251) | Obese patients (n = 229) | ||||||
|---|---|---|---|---|---|---|---|---|
| GG (n = 113) | GA (n = 116) | AA (n = 22) | p‐value | GG (n = 97) | GA (n = 101) | AA (n = 31) | p‐value | |
| Age (years) | 49.1±9.7 | 49.3±9.1 | 49.9±8.6 | 0.93 | 47.6±8.7 | 47.4±10.2 | 48.1±8 | 0.90 |
| BMI (kg/m2) | 22.7±1.9 | 22.4±1.9 | 22.1±1.7 | 0.39 | 40.3±6.8 | 39.8±6.6 | 40.2±6.7 | 0.85 |
| Systolic blood pressure (mmHg) | 120±13 | 116±12 | 123±16 | 0.18 | 136±14 | 139±16 | 149±14 | 0.003 |
| Diastolic blood pressure (mmHg) | 71.8±8.5 | 70.9±6.5 | 73.7±6.2 | 0.52 | 83.5±8.8 | 84.7±9.5 | 92.6±13.3 | 0.002 |
BMI, body mass index.
| Obese men | Obese women | |||
|---|---|---|---|---|
| β‐coefficient | p‐value | β‐coefficient | p‐value | |
| Systolic blood pressure | ||||
| Age (years) | 0.14 | 0.38 | 0.16 | 0.19 |
| Body mass index (kg/m2) | 0.29 | 0.07 | 0.13 | 0.27 |
| ‐2548G/A genotype | ||||
| GG vs AA | 0.65 | 0.001 | 0.29 | 0.01 |
| GA vs AA | 0.45 | 0.034 | 0.19 | 0.12 |
| Diastolic blood pressure | ||||
| Age (years) | 0.10 | 0.54 | 0.09 | 0.44 |
| Body mass index (kg/m2) | 0.26 | 0.11 | 0.28 | 0.02 |
| ‐2548G/A genotype | ||||
| GG vs AA | 0.71 | <0.001 | 0.21 | 0.08 |
| GA vs AA | 0.61 | 0.001 | 0.16 | 0.17 |
β, Standardized regression coefficient.
| Genotypes, n (%) | Normotensive (n = 53) | Hypertensive (n = 45) | OR | 95% CI | p‐value |
|---|---|---|---|---|---|
| GG | 28 (52.8) | 13 (28.9) | 1a | ||
| GA | 20 (37.7) | 23 (51.1) | 2.47 | (1.01–6.03) | 0.04 |
| AA | 5 (9.4) | 9 (20.0) | 3.87 | (1.08–12.8) | 0.03 |
OR, odds ratio; CI, Confidence interval. The χ2 test was used to determine whether significant differences (p‐values) were observed when the hypertensive group was compared with normotensive. 1a, Reference category.
References
- Hsueh W. A., Buchanan T. A. Obesity and hypertension. Endocrinol Metab Clin North Am 1994; 23: 405–427 [Google Scholar]
- Manson J. E., Willett W. C., Stampfer M. J., Colditz G. A., Hunter D. J., Hankinson S. E., et al. Body weight and mortality among women. N Eng J Med 1995; 333: 677–685 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Kannel W. B., Brand N., Skinner J. J. J., Dawber T. R., McNamara P. M. The relation of adiposity to blood pressure and development of hypertension. The Framingham Study. Ann Intern Med 1967; 67: 48–59 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Stamler R., Stamler J., Riedlinger W. F., Algera G., Roberts R. H. Weight and blood pressure. Findings in hypertension screening of 1 million Americans. JAMA 1978; 240: 1607–1610 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Barba G., Ornella R., Siani A., Iacone R., Farinaro E., Gerardi M. C., et al. Plasma leptin and blood pressure in men: Graded association independent of body mass and fat pattern. Obesity Res 2003; 11: 160–166 [Google Scholar]
- Zhang Y., Proenca R., Maffei M., Barone M., Leopold L., Friedman J. M. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425–432 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Campfield L. A., Smith F. J., Guiez Y., Devos R., Burn P. Recombinant mouse OB proteins: Evidence for a peripheral signal linking adiposity and central neural networks. Science 1995; 269: 546–549 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Hallaas J., Gajiwala K. S., Maffei M., Cohen S. L., Chait B. T., Rabinowitz D., et al. Weight‐reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269: 543–546 [Google Scholar]
- Carlyle M., Jones O. B., Kuo J. J., Hall J. E. Chronic cardiovascular and renal actions of leptin: Role of adrenergic activity. Hypertension 2002; 39: 496–501 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Corica F., Corsonello A., Ientile R., Corica F., De Gregorio T., Malara A., et al. Leptin and norepinephrine plasma concentrations during glucose loading in normotensive and hypertensive obese women. Am J Hypertens 2001; 14: 619–626 [Google Scholar]
- Correia M. L., Haynes W. G., Rahmouni K., Morgan D. A., Sivitz W. I., Mark A. L. The concept of selective leptin resistance. Evidence from Agouti yellow obese mice. Diabetes 2002; 51: 439–442 [Google Scholar]
- Montague C. T., Farooqi I. S., Whitehead J. P., Soos M. A., Rau H., Wareham N. J., et al. Congenital leptin deficiency is associated with severe early‐onset obesity in humans. Nature 1997; 387: 903–908 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Strobel A., Issad T., Camoin L., Ozata M., Strosberg A. D. A leptin missense mutation associated with hypogonadism and morbid obesity. Nat Genet 1998; 18: 213–215 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Shintani M., Ikegami H., Fujisawa T., Kawaguchi Y., Ohishi M., Katsuya T., et al. Leptin gene polymorphism is associated with hypertension independent of obesity. J Clin Endocrinol Metab 2002; 87: 2909–2912 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Li W. D., Reed D. R., Lee J. H. Sequence variants in the 5′ flanking region of the leptin gene are associated with obesity in women. Ann Hum Genet 1999; 63: 227–234 [Crossref], [Google Scholar]
- Mammés O., Betoulle D., Aubert R., Herbeth B., Siest G., Fumeron F. Association of the G‐2548A polymorphism in the 5′ region of the LEP gene with overweight. Ann Hum Genet 2000; 64: 391–394 [Crossref], [Google Scholar]
- Wang T. N., Huang M. C., Chang W. T., Ko A. M., Tsai E. M., Liu C. S., et al. G‐2548A polymorphism of the leptin gene is correlated with extreme obesity in Taiwanese aborigines. Obesity 2006; 14: 183–187 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Poitou C., Lacorte J. M., Coupaye M., Bertrais S., Bedel J. F., Lafon N., et al. Relationship between single nucleotide polymorphisms in leptin, IL6 and adiponectin genes and their circulating product in morbidly obese subjects before and after gastric banding surgery. Obes Surg 2005; 15: 11–23 [Google Scholar]
- World Health Organisation. Physical status: The use and interpretation of anthropometry: Report of a WHO expert committee, WHO Technical Report Series, no. 854. WHO, Genova 1995; 321–344 [Google Scholar]
- Perloff D., Grim C., Flack J., Frohlich E. D., Hill M., McDonald M., et al. Human blood pressure determination by sphygmomanometry. Circulation 1993; 88: 2460–70 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Marcadet A., O'Connel P., Cohen D. Standardized Southern blot workshop techniques. Histocompatibility testing, B Dupont. Springer, New York 1987; 587–590 [Google Scholar]
- Lalouel J‐M., Rohrwasser A. Development of genetic hypotheses in essential hypertension. J Hum Genet 2001; 46: 299–306 [Google Scholar]
- Meisel S. R., Ellis M., Pariente C., Pauzner H., Liebowitz M., David D., et al. Serum leptin levels increase following acute myocardial infarction. Cardiology 2001; 95: 206–211 [Google Scholar]
- Konstantinides S., Schäfer K., Koschnick S., Loskutoff D. J. Leptin‐dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity. J Clin Invest 2001; 108: 1533–1540 [Google Scholar]
- Le Stunff C., Le Bihan C., Schork N. J., Bougneres P. A common promoter variant of the leptin gene is associated with changes in the relationship between serum leptin and fat mass in obese girls. Diabetes 2000; 49: 2106–2200 [Google Scholar]
- Portolés O., Sorli J. V., Francés F., Coltell O., Gonzalez J. I., Saiz C., et al. Effect of genetic variation in the leptin gene promotor and the leptin receptor gene on obesity risk in a population‐based case‐control study in Spain. Eur J Epidemiol 2006; 21: 605–612 [Crossref], [Google Scholar]
- Jiang Y., Wilk J. B., Borecki I., Williamson S., DeStefano A. L., Xu G., et al. Common variants in the 5′ region of the leptin gene are associated with body mass index in men from the National Heart, Lung, and Blood Institute Family Heart Study. Am J Hum Genet 2004; 75: 220–230 [Crossref], [Google Scholar]
- Koh K. K. Effects of estrogen on the vascular wall: Vasomotor function and inflammation. Cardiovasc Res 2002; 55: 714–726 [Crossref], [PubMed], [Web of Science ®], [Google Scholar]
- Hoffstedt J., Eriksson P., Mottagui‐Tabar S., Arner P. A polymorphism in the leptin gene promotor region (‐2548g/a) influences gene expression and adipose tissue secretion of leptin. Horm Metab Res 2002; 34: 355–359 [Crossref], [PubMed], [Google Scholar]
- Schutte R., Huisman H. W., Schutte A. E., Malan N. T. Leptin is independently associated with systolic blood pressure, pulse pressure and arterial compliance in hypertensive African women with increased adiposity: The POWIRS study. J Hum Hypertens 2006; 19: 535–541 [Google Scholar]
- Hu F. B., Chen C., Wang B., Stampfer M. J., Xu X. Leptin concentrations in relation to overall adiposity, fat distribution, and blood pressure in rural Chinese population. Int J Obes Relat Metab Disord 2001; 25: 121–125 [Google Scholar]
- El‐Gharbawy A. H., Kotchen J. N., Grim C. E., Kaldunski M., Hoffmann R. G., Pausova Z., et al. Gender specific correlates of leptin with hypertension‐related phentypes in African Americans. Am J Hypertension 2002; 15: 989–993 [Google Scholar]
- Ferranninin E., Buzzigoli G., Bonadonna R., Giorico M. A., Oleggini M., Graziadei L., et al. Insulin resistance in essential hypertension. N Engl J Med 1987; 317: 350–357 [Google Scholar]