Introduction

There is growing awareness that metabolic syndrome (MetS) is one of the most important threats to public health because of its association with type 2 diabetes mellitus, cardiovascular disease and mortality.1, 2, 3 In men, it is well established that endogenous androgens decline with advancing age,4 and low testosterone levels have been associated with insulin resistance,5 type 2 diabetes,6, 7 hypertension8 and increased cardiovascular and all-cause mortality.9, 10 Moreover, men with low testosterone are likely to have more components of MetS in cross-sectional studies,11, 12, 13 and longitudinal studies show that lower total testosterone predicts higher frequency of MetS.14, 15 These data were mostly from studies with Caucasian men in western countries. Regarding Japanese men, one study showed that testosterone was positively correlated with plasma adiponectin.16 However, there are no reports showing a relationship between testosterone and MetS or its components in Japanese men.

Recently, we reported that low testosterone is an independent determinant of endothelial dysfunction in middle-aged men17 and is a predictor of cardiovascular events in men with coronary risk factors,18 suggesting a link between testosterone and cardiovascular pathology. Given these findings, this study investigated the relationship of endogenous testosterone with MetS in middle-aged Japanese men.

Methods

Subjects

Enrollment screening included consecutive, apparently healthy male subjects aged 30–64 years who underwent medical examinations at either our department or at two clinics located in Tokyo. After exclusion of subjects who met the exclusion criteria, 194 subjects (104 from our department and 90 from the clinics) were enrolled. Exclusion criteria included history of cardiovascular disease (stroke, coronary heart disease, congestive heart failure and peripheral arterial disease), malignancy or overt endocrine disease or use of steroid hormones, because these conditions may influence plasma sex hormones and/or the components of MetS. Other exclusion criteria were diabetic subjects on insulin injection or hypoglycemic agent drugs or with hemoglobin A1c >8%, and subjects on β-blockers19 or fibrates. History, physical examination and laboratory tests were performed for all subjects. Of the included subjects, 23% (n=44) were taking anti-hypertensive drugs (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium channel blockers and diuretics), and 22% were taking statins. Each subject gave written, informed consent before study enrollment. The study protocol was approved by the ethics committee of the Graduate School of Medicine at the University of Tokyo.

Assays of metabolic risk factors and plasma hormones

Clinical information was collected at baseline when each patient attended the initial medical examination. Blood sampling and measurement of height, weight, waist circumference and blood pressure were performed in the morning after a 12-h overnight fast. Blood pressure was measured at least twice using an automated, digital electrosphygmomanometer (Omron Healthcare, Kyoto, Japan) on the non-dominant arm in a sitting position, and the average was used for statistical analysis.

Serum total cholesterol and triglyceride were measured enzymatically, and serum high-density lipoprotein (HDL) cholesterol was measured by the heparin-Ca2+Ni2+ precipitation method. Low-density lipoprotein cholesterol was determined using the Friedewald formula or the direct, liquid, selective detergent method when triglycerides were >400 mg per 100 ml. Plasma glucose was assayed by the glucose oxidase method, and hemoglobin A1c was measured by high-performance liquid chromatography. Plasma total testosterone, dehydroepiandrosterone sulfate and estradiol were determined using sensitive radioimmunoassays. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as fasting insulin (μIU ml–1) × fasting plasma glucose (mg per 100 ml)/405. Patients with a fasting plasma glucose >140 mg per 100 ml were excluded from the HOMA-IR calculation because of a lack of data reliability. Serum adiponectin was measured using an enzyme-linked immunosorbent assay (Human Adiponectin ELISA kit, Otsuka Pharmaceutical, Tokyo, Japan). These assays were performed by a commercial laboratory (SRL, Tokyo, Japan). The intra-assay coefficients of variation for the measurements were <5%.

Definition of MetS

We applied both the Japanese criteria20 and the International Diabetes Federation (IDF) criteria for Japanese ethnicity21 for the diagnosis of MetS. In the Japanese criteria, MetS was diagnosed when waist circumference ⩾85 cm and two or more of the following three components were present: (1) HDL cholesterol <40 mg per 100 ml and/or triglyceride ⩾150 mg per 100 ml; (2) systolic blood pressure ⩾130 mm Hg and/or diastolic blood pressure ⩾85 mm Hg and (3) fasting plasma glucose ⩾110 mg per 100 ml. Subjects taking anti-hypertensive medications were considered hypertensive for statistical purposes.

In the IDF criteria for Japanese ethnicity, MetS was diagnosed when waist circumference ⩾85 cm and two or more of the following four components were present: (1) HDL cholesterol <40 mg per 100 ml; (2) triglyceride ⩾150 mg per 100 ml; (3) systolic blood pressure ⩾130 mm Hg and/or diastolic blood pressure ⩾85 mm Hg and (4) fasting plasma glucose ⩾100 mg per 100 ml. Subjects taking anti-hypertensive medications were considered hypertensive for statistical purposes.

Data analysis

Values are expressed as the mean±s.d. in the text unless otherwise stated. Pearson's simple correlation coefficients were calculated between plasma hormones and the number of MetS components. Differences between the quartile groups of sex hormones were analyzed using one-factor ANOVA followed by the Newman–Keuls’ test. Logistic regression analysis was performed to determine the association of sex hormones with the diagnosis of MetS. Furthermore, multiple regression analysis was performed to determine the association between sex hormones and metabolic risk factors for MetS. A value of P<0.05 was considered statistically significant. The data were analyzed using SPSS (Version 17.0, SPSS, Chicago, IL, USA).

Results

Sex hormones and MetS criteria

Characteristics of the study subjects are shown in Table 1. Twenty-three and 32% of the subjects were diagnosed with MetS according to the Japanese criteria and the IDF criteria, respectively. The prevalence is comparable with that reported in middle-aged Japanese men.22, 23

Table 1 Characteristics of study subjects (N=194)
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As plasma total testosterone was negatively correlated with the number of MetS components (Figure 1a), the association of testosterone with MetS was analyzed by quartile of testosterone. As shown in Figure 2a, lower testosterone was associated with a step-wise increase in the number of MetS components. Age-adjusted logistic regression analysis revealed that the hazard ratios for MetS diagnosis by quartile decline of testosterone were 2.02 (95% CI=1.43–2.87) and 1.68 (95% CI=1.25–2.25) according to the Japanese criteria and the IDF criteria, respectively.

Figure 1
figure 1

Scattergrams and regression lines (dotted lines) showing the correlation between testosterone and the number of metabolic syndrome (MetS) components (a) or waist circumference (b).

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Figure 2
figure 2

Number of metabolic syndrome (MetS) components according to quartiles of plasma testosterone (a) and estradiol (b). MetS components were defined according to the Japanese criteria (closed bars) and the IDF criteria for Japanese ethnicity (open bars). Values are expressed as the mean±s.e.m. *P<0.05, **P<0.01 vs. Q1. Cut offs of the quartiles were 14.1, 18.7 and 23.4 nmol l–1 (405, 540 and 674 ng per 100 ml) for testosterone, and 55, 101 and 125 pmol l–1 (15.0, 27.5 and 34.0 pg ml–1) for estradiol.

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Interestingly, plasma estradiol was positively correlated with the number of MetS components (R=0.285, P<0.001); therefore, the association with MetS was also analyzed by quartile of estradiol. As shown in Figure 2b, higher estradiol was associated with a step-wise increase in the number of MetS components. Age-adjusted logistic regression analysis revealed that the hazard ratios for MetS diagnosis by quartile increment of estradiol were 1.48 (95% CI=1.06–2.06) and 1.63 (95% CI=1.20–2.21) according to the Japanese criteria and the IDF criteria, respectively. Dehydroepiandrosterone sulfate was not associated with MetS components or diagnosis (data not shown).

Sex hormones and metabolic risk factors

The associations of plasma sex hormones with each of the metabolic risk factors were analyzed. As shown in Table 2, the unadjusted model shows that testosterone was significantly related to parameters of MetS except for diastolic blood pressure. Testosterone was not related to low-density lipoprotein cholesterol, but this parameter is not included in the definitions of MetS used here. Adjustment for age did not considerably influence the results of the regression analysis, but the association between testosterone and diastolic blood pressure became significant after adjustment for age. In contrast, inclusion of waist circumference into the model weakened the association of testosterone with metabolic risk factors. As a result, systolic blood pressure, triglycerides, fasting plasma glucose and HOMA-IR were significantly related to testosterone. The significant association for diastolic blood pressure, HDL cholesterol, free fatty acids, hemoglobin A1c, insulin and adiponectin were attenuated after adjustment for age and waist circumference. Adjustment for body mass index or waist/hip ratio instead of waist circumference showed similar results (data not shown).

Table 2 Multiple regression analysis determining the impact of plasma testosterone on metabolic risk factors
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As shown in Table 3, estradiol showed weaker association than testosterone with parameters of MetS, but was significantly related to body mass index, waist circumference, systolic blood pressure, HDL cholesterol, insulin, HOMA-IR and adiponectin after adjustment for age. Further adjustment for waist circumference, body mass index or waist/hip ratio (Table 3 and data not shown) eliminated the significant associations between estradiol and these metabolic parameters. Dehydroepiandrosterone sulfate was not significantly related to parameters of MetS in unadjusted or adjusted analyses (data not shown).

Table 3 Multiple regression analysis determining the impact of plasma estradiol on metabolic risk factors
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Discussion

In this study, cross-sectional analysis of 194 middle-aged Japanese men showed that low testosterone is positively related to MetS, MetS components and additional metabolic risk factors. Adjustment for obesity parameters such as waist circumference, body mass index and waist/hip ratio greatly diminished the association, but low testosterone retained weak associations with some metabolic risk factors including systolic blood pressure, triglycerides, fasting plasma glucose and HOMA-IR. Taken together, results in this statistical model suggest that abdominal obesity is an important contributor to the association between low testosterone and MetS, but additional factors may also impact testosterone. To our knowledge, this is the first report showing the significant association between low testosterone and MetS in Japanese men.

Several mechanisms have been suggested for the causal relationship between low testosterone and abdominal obesity. Activation of the lipoprotein lipase and lipolysis24 may explain the effect of testosterone on adipose tissue. Many studies including a medium-sized, randomized controlled trial25 and a meta-analysis26 showed the inverse effect of testosterone on adiposity. Conversely, it has been reported that men with MetS are prone to hypogonadism.27 This finding might be due to elevated leptin levels that interfere with gonadotropin-stimulated androgen production28 and to increased aromatase activity in adipose tissue that leads to higher circulating estradiol and suppression of testosterone production by negative feedback.29 These findings suggest a bi-directional causal relationship between low testosterone and obesity.

After adjustment for waist circumference, testosterone was weakly but significantly related to some metabolic risk factors including systolic blood pressure, triglycerides, fasting plasma glucose and HOMA-IR, which is consistent with earlier reports.5, 6, 8, 12 Testosterone is likely to be involved in the pathogenesis of MetS, irrespective of obesity. For example, testosterone increases the hepatic production of apolipoprotein A-1 and consequently increases HDL cholesterol,30 improves insulin sensitivity and increases muscle strength.31 There was no significant correlation between age and testosterone (R=0.114, P=0.12). This result may be because the cohort was limited to middle-aged men (30–64 years old) . However, age was included in the multivariate analyses in this study, because it is well established that testosterone declines with age.4

The positive association found between testosterone and adiponectin is in agreement with earlier reports.16, 32, 33 However, the direct action of testosterone on adiponectin production/secretion might be different from these findings, because testosterone decreases adiponectin secretion in mice and in adipocytes.34, 35 Accordingly, abdominal obesity may underlie the positive correlation between testosterone and adiponectin in men.

In this study, estradiol was associated positively with MetS and its components, consistent with an earlier report.12 This relationship may be independent of testosterone because estradiol was not correlated with testosterone by simple regression analysis (R=−0.019, P=0.80), and the inclusion of both testosterone and estradiol into the multiple regression model as covariates did not influence the association of each other with MetS parameters (data not shown). The relationship between estradiol and MetS might be attributed to increased aromatase activity and subsequent elevation of circulating estradiol in obese subjects.29 Increased estradiol may subsequently suppress pituitary function,29 and lead to a further decrease in testosterone. Comprehensive assessment of sex hormone, gonadotropin and components of MetS reveal a causal relationship. Unfortunately, we could not measure gonadotropin because of limited plasma. Further investigation is needed to address the mechanistic and pathophysiological interactions between sex hormones and MetS.

There are some limitations to our study. First, the cross-sectional design does not clarify the causal relationship between sex hormones and MetS. As there may be bi-directional causalities as mentioned above, longitudinal follow-up studies and hormone replacement studies should be performed in Japanese populations. Second, active forms of testosterone such as bioavailable and calculated free testosterone were not measured. A direct assay of bioavailable testosterone or of sex hormone-binding globulin (required for free testosterone calculation) was not available for the study. Third, the potential influence of medications on the measured parameters cannot be denied, although the exclusion of subjects on statins (n=40) or anti-hypertensive drugs (n=44) did not seriously affect the association of testosterone with waist circumference (statins, R=−0.304, P<0.01; anti-hypertensives, R=−0.337, P<0.01) and the number of MetS components (statins, R=−0.274, P<0.01; anti-hypertensives, R=−0.278, P<0.01). Fourth, because the sample size (n=194) is relatively small, the finding needs to be confirmed in a larger cohort.

In summary, this study suggests that low testosterone is associated with MetS and its parameters in middle-aged Japanese men. We also found a positive but weaker association between estradiol and MetS. These associations were largely attenuated by adjustment for waist circumference. Our results reinforce the need to address the causal relationship and pathophysiological interactions between sex hormones and MetS.