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Leptin Gene Polymorphism Is Associated with Hypertension Independent of Obesity

Department of Geriatric Medicine, Osaka University Graduate School of Medicine (M.S., H.I., Y.K., T.F., M.O., T.K., J.H., T.O.), 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; and Second Department of Internal Medicine, Sapporo Medical University School of Medicine (K.S.), Sapporo 060-8556, Japan

Address all correspondence and requests for reprints to: Dr. Hiroshi Ikegami, Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: . ikegami{at}geriat.med.osaka-u.ac.jp

Abstract

Leptin is an adipocyte-derived hormone that regulates food intake and energy expenditure. Recent functional studies have suggested a direct effect of leptin on blood pressure. In this study we examined the genetic association of the leptin gene polymorphism with obesity, insulin resistance, and hypertension. A highly polymorphic tetranucleotide repeat polymorphism in the 3'-flanking region of the leptin gene was examined. The alleles of the polymorphism consisted of two groups with different size distributions: a shorter one (class I) and a longer one (class II). The frequency of class I/class I genotype was much higher in hypertensive subjects than in control subjects (13.5% vs. 3.4%; P = 0.0027). No significant difference in body mass index was observed with different genotypes in either patients with hypertension or control subjects. Insulin responses to glucose and insulin sensitivity were not different among patients with different genotypes. The leptin gene polymorphism was associated with hypertension independent of obesity. These data together with recent functional data on the direct effect of leptin on blood pressure suggest that the leptin gene and its product, leptin, are an attractive target for studies on the mechanisms of hypertension and for the development of methods for the prediction, prevention, and therapy for hypertension.

OBESITY IS ASSOCIATED with an increased incidence of hypertension and cardiovascular disease (1, 2). These complications have been at least in part attributed to insulin resistance and hyperinsulinemia (3, 4).

Leptin is a hormone mainly produced by adipose tissue. It acts on both energy intake and expenditure to maintain relative stability of body weight and energy storage over long periods of time (5). Mutations in the gene encoding leptin are reported to cause severe obesity in both animal models (6) and humans (7, 8), indicating a direct relationship between the leptin gene and obesity. Although the frequency of such mutations is very low, common polymorphisms of the leptin gene may well contribute to a common form of obesity and, as a consequence, obesity-related hypertension.

In addition to its effects on appetite and energy expenditure, a direct effect of leptin on blood pressure has recently been reported (9, 10, 11). A chronic increase in circulating leptin was reported to cause a sustained increase in arterial pressure in rats. More recently, transgenic skinny mice overexpressing leptin were reported to exhibit elevated blood pressure, suggesting a direct effect of leptin on the pathogenesis of hypertension, independent of obesity. The mechanism by which leptin increases blood pressure is thought to be through sympathetic activation (9, 10, 11, 12).

Because patients with essential hypertension were reported to be hyperleptinemic, and plasma immunoreactive leptin was reported to correlate with blood pressure (13, 14, 15), the leptin gene is a strong candidate gene for hypertension, either through its direct effect on blood pressure or through its effect on obesity.

We previously identified a highly polymorphic tetranucleotide repeat polymorphism in the 3'-flanking region of the leptin gene and found a marginal effect of the polymorphism on obesity (16). In this study we examined the association of the leptin gene polymorphism with hypertension. The results indicated a genetic association of the leptin gene with hypertension independent of obesity, which is consistent with recent functional data on the direct effect of leptin on blood pressure independent of obesity.

Subjects and Methods

Subjects

We investigated 205 Japanese patients with essential hypertension [108 women and 97 men; age, 57.8 ± 12.1 yr (mean ± SD); body mass index (BMI), 24.0 ± 3.3 kg/m²; systolic blood pressure (BP), 169.3 ± 21.6 mm Hg; diastolic BP, 100.1 ± 14.9 mm Hg] with never-treated ambulatory hypertension and 117 normotensive subjects (50 women and 67 men; age, 57.6 ± 12.8 yr; BMI, 22.9 ±2.7 kg/m², systolic BP, 125.4 ± 13.8 mm Hg; diastolic BP, 75.7 ± 9.6 mm Hg). The diagnosis of essential hypertension was established by the presence of elevated BP (>140 mm Hg systolic BP or >90 mm Hg diastolic BP) and the absence of clinical or laboratory evidence suggestive of secondary forms of hypertension. All subjects were free of clinical evidence of coronary artery or cerebrovascular disease. No patients with valvular heart disease, arrhythmias, or renal disease were included. Most of the hypertensive subjects had received antihypertensive medication, which was withdrawn for at least 2 wk before the examination. Informed consent was obtained from all subjects.

Genotyping of leptin gene polymorphism

A tetranucleotide repeat polymorphism in the 3'-flanking region of the human leptin gene was detected by PCR as reported previously (16). Primer sequences were human OB forward (5'-AGT TCA AAT AGA GGT CCA AAT CA; forward primer) and human OB reverse (5'-TTC TGA GGT TGT GTC ACT GGC A; reverse primer). PCR contained 100 ng genomic DNA template, 0.2 µmol/liter of each primer, 2.0 mmol/liter Mg²⁺, 0.8 mmol/liter of each dNTP, 1.5 U Taq polymerase (Perkin-Elmer Corp., Branchburg, NJ), and reaction buffer in a total volume of 10 µl. The PCR were performed for 35 cycles of 30 sec at 94 C, 30 sec at 54 C, and 1 min at 72 C, with initial denaturation of 3 min at 94 C and final extension of 10 min at 72 C. PCR products were run in 9% polyacrylamide gel along with pBR322 DNA-MspI digest (New England Biolabs, Inc., Beverly, MA) as a mol wt marker. Amplified products were visualized by staining with ethidium bromide.

Analysis of insulin secretion and insulin sensitivity

To study the contributions of insulin resistance and hyperinsulinemia as a link between the leptin gene polymorphism and hypertension, insulin secretion and insulin sensitivity were evaluated in a subset of the patients by a 75-g oral glucose tolerance test (OGTT; 93 patients, randomly selected) and the 2-h euglycemic hyperinsulinemic glucose clamp technique (53 patients, randomly selected), respectively. The euglycemic hyperinsulinemic glucose clamp test was performed according to the method of DeFronzo et al. (17) with modification as reported previously (18). Insulin sensitivity was also assessed by homeostasis model assessment (HOMA-R: FBS x fasting IRI/405 mU x g/10 x liter²) (19). Insulin resistance was defined as a HOMA-R score of 1.73 or more (18). The correlation coefficient between the HOMA-R index and the M value in the hypertensive subjects was -0.35 (P = 0.011).

Analysis of serum leptin concentration

To study the relationship between the leptin gene polymorphism and the serum leptin concentration, another group (second group) of patients with essential hypertension (46 women and 61 men; age, 64.2 ± 12.5 yr; BMI, 23.7 ± 3.7 kg/m²) was newly enrolled. Their genotypes were also determined. Serum leptin level was determined with a human leptin immunoassay kit (Biosource Technologies, Inc., Camarillo, CA). Serum was collected in the fasting state in the morning. Frozen samples were kept at -20 C until analysis.

Statistical analysis

Results are given as the mean ± SD. Differences between group means were tested by t test. ² test or Fisher’s exact test was used to compare the frequencies.

Results

The alleles of a microsatellite polymorphism in the 3'-flanking region of the human leptin gene were composed of two groups with different size distributions: a shorter one (termed class I) and a longer one (termed class II), as we reported previously (16). The distribution of genotypes and alleles in patients with hypertension was markedly different from that in the control subjects (Table 1). The frequency of the class I/class I genotype was significantly higher in patients with hypertension (first group) than in normotensive subjects (16.6% vs. 3.4%; P = 0.00078). The frequency of class I allele was also higher in patients with hypertension (first group) than in normotensive subjects (31.0% vs. 23.9%; P = 0.056).

To investigate the roles of insulin resistance and hyperinsulinemia as possible links in the association of the leptin gene polymorphism with hypertension, the insulin response during the 75-g OGTT and insulin sensitivity evaluated by HOMA-R as well as the M value during the euglycemic hyperinsulinemic glucose clamp were compared in hypertensive patients with different genotypes. The plasma insulin level was slightly lower at 30 min and slightly higher at 120 min during the 75-g OGTT in subjects with class I/class I genotype than in those with other genotypes, but the difference was not statistically significant (Table 2). The mean HOMA-R index in patients with the class I/class I genotype (1.8 ± 1.5) was above the cut-off index of 1.73 and was slightly higher than in subjects with other genotypes (1.4 ± 1.5), but the difference between the two groups was not statistically significant (P = 0.30). The M value was slightly lower (3.6 ± 1.5 vs. 4.5 ± 2.3 mg/kg•min) in patients with the class I/class I genotype than in those with other genotypes, but the difference was not statistically significant.

Discussion

The development of hypertension and obesity is suggested to be genetically determined. However, little is known about the genetic factors responsible for hypertension in the general population. Recently, several studies have demonstrated significant correlations between leptin and hypertension (9, 10, 11, 12, 13, 14, 20, 21, 22). In this study we investigated the genetic association of a microsatellite polymorphism in the leptin gene with hypertension and obesity.

The frequency of the class I/class I genotype was markedly higher in patients with hypertension than in normotensive subjects. Despite the strong association of the polymorphism with hypertension, there was no association between the polymorphism and body weight or degree of obesity, as assessed by BMI. These results suggest that the association of the polymorphism with hypertension is independent of obesity. This is consistent with a recent report on transgenic skinny mice that found that chronic hyperleptinemia can lead to a significant elevation of blood pressure without obesity (12).

Several mechanisms other than obesity can be considered links between the leptin gene polymorphism and hypertension. One possibility is insulin resistance and hyperinsulinemia. Hypertension is known to be associated with insulin resistance (23), and overexpression of leptin is reported to increase insulin sensitivity (24). Insulin resistance, however, is unlikely to be the mechanism linking the leptin gene polymorphism and hypertension, because no significant difference was found in insulin sensitivity between the subjects with different genotypes. Moreover, in contrast to the strong association with hypertension, the polymorphism was not associated with type 2 diabetes (16), a well known, insulin-resistant state, indicating that the polymorphism is associated with BP, but not insulin resistance. Considering recent reports on the direct effect of leptin on blood pressure (9, 10, 11, 12), it is reasonable to speculate that the polymorphism contributes to hypertension through its effect on the serum leptin level. The circulating leptin level, however, was not significantly different between hypertensive subjects with the class I/class I genotype and those with other genotypes.

The polymorphism examined in the present study is located in the 3'-untranslated region of the leptin gene. The expression of TNF, another adipocytokine that contributes to insulin resistance in obesity, was reported to be strongly affected by a polymorphism located in the 3'-flanking region of the TNF gene (25). The microsatellite polymorphism in the present study could also affect the expression of the leptin gene. Recent studies have demonstrated leptin expression in nonadipose tissues (26, 27, 28), pointing to a local (noncirculating) action of leptin in autocrine and paracrine manners. The association of the leptin gene polymorphism with hypertension despite the similar level of circulating leptin may therefore be due to the difference in the local expression of the leptin gene. Further studies are necessary to clarify this possibility.

Another possibility is that the polymorphism is in linkage disequilibrium with a mutation or a common polymorphism in the coding or regulatory region of the leptin gene. Previous studies, however, indicated that mutations in the coding region of the leptin gene are very rare (7, 8). Further studies on polymorphisms in the regulatory region of the leptin gene or other nearby genes are necessary to determine whether the association is primary or secondary due to linkage disequilibrium with a mutation in the regulatory region or other nearby genes.

In summary, the leptin gene polymorphism was closely associated with a higher incidence of hypertension independent of obesity. These genetic data together with recent functional data on the direct effect of leptin on blood pressure independent of obesity suggest that the leptin gene and its product leptin are an attractive target for studies on the mechanisms of hypertension and for the development of methods for the prediction, prevention, and treatment of hypertension.

Acknowledgments

Footnotes

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and a Grant for Research on the Human Genome and Gene Therapy from the Ministry of Health and Welfare of Japan.

Abbreviations: BMI, Body mass index; BP, blood pressure; HOMA-R, homeostasis model assessment; OGTT, oral glucose tolerance test.

Received February 13, 2001.

Accepted March 1, 2002.

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