A representative sample of the civilian noninstitutionalized U.S. population was recruited into NHANES III (1988–1994) with a multistage, stratified sampling design (12). NHANES III oversampled non-Hispanic blacks, Mexican Americans, and adults aged ≥60 years to ensure enough data and reliable estimates in these subpopulations. Response rates were 86% for the household interviews and 78% for the medical examinations. Blood was drawn after an overnight fast for participants in the morning sample. After centrifugation, serum samples were aliquotted and stored at −70°C until they were quantified. The serum samples were shipped on dry ice directly to the assay laboratory. In the present study, NHANES III Survey Phase I (1988–1991) data (n = 1,470 men aged ≥20 years) were analyzed. After exclusion of participants who had fasted <8 h (n = 101) and had missing data on all covariates (n = 143), the analytic sample (n = 1,226, 83.4%) comprises 573 non-Hispanic whites (77.9%), 297 non-Hispanic blacks (9.5%), 307 Mexican Americans (5.1%), and 49 participants with all other race/ethnicity (7.5%).

Fasting insulin (in a natural log scale) was significantly correlated with total testosterone (r = −0.41), SHBG (r = −0.28), CFT (r = −0.22), and CBT (r = −0.24) (all P < 0.0001). Similarly, HOMA-IR (in a natural log scale) was significantly correlated with total testosterone (r = −0.42), SHBG (r = −0.25), CFT (r = −0.26), and CBT (r = −0.28) (all P < 0.0001).

After adjustment for potential confounding effects, the geometric means of fasting serum insulin decreased from the first (lowest) quartile to the fourth (highest) quartile of total testosterone (P < 0.001), SHBG (P < 0.001), CFT (P = 0.02), and CBT (P = 0.02) (Table 1). Similarly, the geometric means of the HOMA-IR decreased from the first quartile to the fourth quartile of total testosterone (P < 0.001), SHBG (P < 0.001), CFT (P = 0.02), and CBT (P = 0.02) (Table 1).

The unadjusted prevalence of metabolic syndrome steadily decreased from the first (lowest) quartile to the fourth (highest) quartile of all four sex hormone measures (all P < 0.001 for linear trend (Fig. 1). However, after adjustment for potential confounding effects, the significant linear trend in the prevalence of metabolic syndrome from lowest quartile to highest quartile remained only for total testosterone (P < 0.001) (Fig. 1A) and SHBG (P = 0.004) (Fig. 1B).

The associations between the continuous sex hormone indexes and metabolic syndrome are shown in Table 2. The results indicated that a 1 SD increase in the logarithmic value of total testosterone, SHBG, CFT, and CBT concentration was associated with ∼17, ∼43, ∼7, and ∼7% decreases in the prevalence of metabolic syndrome after adjustment for age (model 1). With Additional adjustment for race/ethnicity, smoking status, alcohol intake, physical activity level, LDL cholesterol, CRP, and HOMA-IR, the associations remained significant only for total testosterone and SHBG (model 2 and model 3).

The results of Cox regression models with testosterones and SHBG as categorical variables and with age adjustment (Table 2, model 1) showed a significant linear trend for the prevalence ratios of total testosterone (P < 0.001), SHBG (P < 0.001), CFT (P = 0.03), and CBT (P = 0.03) for metabolic syndrome. The age-adjusted prevalence ratios comparing the lowest quartile to the highest quartile (i.e., the referent group) ranged from 1.76 for CFT to 3.77 for total testosterone. After additional adjustment for race/ethnicity, smoking status, alcohol intake, physical activity level, LDL cholesterol, and CRP, the linear trend of the prevalence ratios remained statistically significant for total testosterone (P = 0.01), SHBG (P < 0.001), CFT (P = 0.05), and CBT (P = 0.02) (Table 2, model 2). However, after further adjustment for HOMA-IR (on the basis of model 2), the linear trend of the prevalence ratios was significant for only total testosterone (P < 0.001) and SHBG (P = 0.02) (Table 2, model 3). We replicated the analyses of model 3 in Table 2 by replacing HOMA-IR with fasting serum insulin and obtained similar results (data not shown).

Figure 2 shows the adjusted prevalence ratio and 95% CI of total testosterone, SHBG, CFT, and CBT for the five single metabolic syndrome components. Total testosterone was significantly associated with abdominal obesity (P = 0.01) and high concentrations of triglycerides (P = 0.01) (Fig. 2A). SHBG was also significantly associated with abdominal obesity (P < 0.001 for linear trend) and a high concentration of triglycerides (P < 0.001) (Fig. 2B). CFT was not significantly associated with any metabolic syndrome components (P > 0.10) (Fig. 2C). CBT was significantly associated with abdominal obesity (P = 0.04) and marginally associated with high blood pressure (P = 0.07) (Fig. 2D).

We replicated the analyses after exclusion of men with diagnosed diabetes (n = 60) and those with a fasting glucose concentration ≥126 mg/dl (n = 77). Estimates for the associations between sex hormones and metabolic syndrome were similar to that in the sample including those with diabetes (data not shown).

Our findings from this large representative sample of noninstitutionalized civilian adults in the U.S. indicated that men with lower concentrations of total testosterone and SHBG had a higher likelihood of having metabolic syndrome than those with higher concentrations. This finding was independent of traditional cardiovascular risk factors and surrogate measures of insulin resistance. Further, the effects of total testosterone and SHBG on metabolic syndrome seem to be more significant than those of CBT and CFT.

Because they are not bound to albumin and SHBG, researchers have hypothesized that free testosterone and CBT may be better indexes of biological activity than total testosterone alone (18). Our findings, however, do not support this “free hormone” hypothesis. In our study, total testosterone and SBHG, rather than CFT or CBT, were independently and strongly associated with metabolic syndrome. These findings are consistent with recent reports by others (10,11), in which total testosterone and SHBG were found to be significantly related to metabolic syndrome incidence or prevalence, whereas CFT was not. These population data highlight the independent and important role of SHBG in the pathogenesis of metabolic syndrome.

As with previous studies (5), we found that lower concentrations of total testosterone, SHBG, CFT, and CBT were significantly associated with higher levels of HOMA-IR and fasting serum insulin, two proxy biomarkers of insulin resistance. In particular, the strength of the association between sex hormones and metabolic syndrome decreased appreciably before and after adjustment for HOMA-IR, suggesting that insulin resistance may play a role in the observed association. A previous study has shown that insulin may directly inhibit SHBG secretion from hepatoma cells in vitro (19). In vivo studies show that insulin may stimulate testosterone production and reduce SHBG concentration in both normal-weight and obese men (20). However, polymorphisms in the SHBG gene have recently been shown to affect not only SHBG levels but also type 2 diabetes risks, suggesting a potential causal role in the pathophysiological mechanisms. In particular, carriers of SHBG rs6257 allele (CC or CT) have an increased risk, whereas carriers, both men and women, of an rs6259 variant allele (AA or AG) have a decreased risk of type 2 diabetes (21). Furthermore, the association between low testosterone concentration and insulin resistance may be partially caused by impaired Leydig cell function (22).

Notably, total testosterone and SHBG were significantly associated with abdominal obesity and high triglyceride concentration among the five metabolic syndrome components. Although the exact mechanisms for the association remain unknown, observational studies have suggested that testosterone inhibits lipid uptake, decreases lipoprotein-lipase activity, and reduces visceral adipose tissue accumulation. In a randomized clinical trial, testosterone therapy selectively reduced visceral fat accumulation and increased fat-free mass (23). On the other hand, SHBG has been related to dyslipidemia, possibly by regulating hepatic lipoprotein lipase activity and reducing the release of fatty acids from adipocytes (24). Furthermore, recent clinical trials have shown that testosterone replacement therapy significantly reduces insulin resistance and improves glycemic control and cardiometabolic risk factors in hypogonadal men with type 2 diabetes (25).

Taken together, these findings may have significant implications in clinical practice. Because the total testosterone and SHBG were associated with metabolic syndrome independently of known cardiovascular risk factors, insulin resistance, and/or overall obesity, total testosterone and SHBG may be considered as emerging risk factors for metabolic syndrome in men. Conventionally, SHBG has not been considered as a risk factor in the development of any diseases (i.e., cancer, fracture, heart disease, and type 2 diabetes) because it has been viewed as a hormone sequester to control the bioavailability of steroids. Our results on the independent association of total testosterone and SHBG with metabolic syndrome suggest that SHBG may have important biological effects in a series of adverse metabolic outcomes.

The strengths of our study include the use of a large representative sample of U.S. adult men as well as rigorous and standardized assessment of biochemical analyses and anthropometric indexes in NHANES III. Our results, however, are subject to several limitations. First, using a cross-sectional design, we were unable to establish a temporal sequence in the associations among testosterones, SHBG, and metabolic syndrome. As shown in longitudinal studies, low total testosterone and SHBG concentrations predict the occurrence of metabolic syndrome (10,11). Conversely, individuals with diabetes and/or metabolic syndrome may be more likely to have hypogonadism, testosterone deficiency, or erectile dysfunction than those without. Second, we used calculated rather than directly measured free testosterone and bioavailable testosterone. However, previous studies have demonstrated that calculated free and bioavailable concentrations are valid and reliable (14). Third, surrogate measures of insulin resistance were used in our study. Direct measures of insulin resistance such as steady-state plasma glucose concentration or hyperinsulinemic-euglycemic clamp may be useful to reduce measurement errors.

Our results demonstrate that total testosterone and SHBG concentrations were strongly associated with metabolic syndrome independently of traditional cardiovascular risk factors and surrogate measures of insulin resistance. However, CFT and CBT concentrations were associated with metabolic syndrome but not independently of insulin resistance. Because metabolic syndrome and insulin resistance are risk factors for cardiovascular diseases and type 2 diabetes, early detection of men with decreased total testosterone and SHBG concentrations in clinical settings will be beneficial for preventing future cardiovascular outcomes.