To our best knowledge, the current study is the first investigation examining the mediatory role of SHBG, an important biological factor, in explaining sex differences in glucose metabolism and incidence of T2D. Our results indicated a greater level of SHBG in women than in men, which may explain sex differences in glucose levels and incidence of T2D. We observed a high estimated PM by SHBG in the association between sex (women vs. men) and fasting glucose levels (30%) and incidence of T2D (63%).

Our findings on sex differences for SHBG, glucose homeostasis and T2D are in line with previous studies, showing in general higher SHBG levels [22], lower glucose levels [23], lower insulin resistance [23] and lower incidence of T2D [24] in women compared to men.

Our results reinforce previous evidence on the association between SHBG, glucose biomarkers and T2D incidence. The results of a large systematic review and meta-analysis of observational studies on endogenous sex hormones and risk of T2D showed an inverse association of SHBG and risk of T2D in both sexes, although the findings were stronger in women than in men [7]; the same inverse association of SHBG with T2D was found in another systematic review and meta-analysis performed exclusively on women [25]. In support of observational evidence, mendelian randomization studies have also found a causal role of SHBG on T2D [12, 13]. Above all, our study underscores the necessity of developing sex-specific mendelian randomization studies.

A novel finding of our study is the potential mediating role of SHBG on glucose homeostasis. To our knowledge, the current study is the first study that identified SHBG as a potential mediator in the association between sex and glucose hemostasis. We found the mediating effect of SHBG, which was independent of confounding factor and some of potential intermediate factors including age and obesity. Findings of a recent study based on a large population-based cohort on aging, cardiovascular risk and SHBG, found a clear sex-specific pattern of SHBG levels with age. These novel findings highlighted the importance of considering the age-related changes in SHBG levels to avoid controversial results [22]. Thus, we tried to perform the analysis for different subsets of individuals based on median age. Of note, the potentially mediatory role of SHBG on sex differences in glucose levels was observed in participants < 53 years of age and > = 53 years, with a lower PM in those aged 53 and over. The median (IQR) of SHBG levels for individuals aged < 53 were 40.8 (29.6–55.2) and 81.9 (61.4-105.5) and for those > = 53 years were 54.3 (40.2–71.2) and 69.7 (50-97.3) for men and women, respectively. The SHBG levels in men increased with aging, while they decreased in women, resulting in the narrower variation of SHBG levels between men and women in adults aged 53 years and over. This has resulted in lower PM (19–22%) in older individuals compared to higher PM (29–30%) in younger adults.

We also stratified the mediation analysis by BMI categories, given the robust evidence linking overweight and obesity to altered SHBG levels [26]. Interestingly, while the mediation effect of SHBG on sex-differences in glucose levels was strongest in individuals with normal weight, it remained significant across both BMI subsets. The lower PM observed in overweight and obese individuals could be attributed to the well-established reduction in SHBG levels associated with increased adiposity [27, 28].

Various mechanisms have been proposed regarding sex differences in risk of T2D, with steroid hormones being the important ones. Research has indicated that SHBG may interfere with the pathogenesis and development of T2D by regulating the biologic effects of sex hormones (testosterone and estrogen) on peripheral tissues (e.g. muscle, fat and liver).

Increased risk of T2D with low SHBG levels may represent the stronger effects and possible interactions of more bioavailable testosterone and estradiol, and thus, explaining the sex-dependent associations of SHBG. Some studies have proposed a sex-dependent association of SHBG with risk of T2D. For instance, the study done by Haffner et al. indicated that SHBG levels, independent of insulin levels, predict the development of T2D in women but not in men [29]. Another observational study found that SHBG was associated with higher risk of T2D in women rather than men [30]. The findings of a recent mediation analysis on the Masstricht study, investigating the mediatory role of SHBG on the association between intrahepatic lipid content (IHL) and T2D showed greater mediatory role of SHBG in women compared to men, with a PM of 17.2% and 50.9% of SHBG on the association of IHL and T2D for men and women, respectively [31].

Rather than interaction with other sex hormones, there is also a strong evidence supporting independent effect of SHBG on T2D [32]. Studies have found several polymorphisms in the SHBG gene to be associated with insulin resistance and T2D, showing that altered SHBG physiology may trigger the pathogenesis of T2D [11,12,13]. Additionally, it has been shown that SHBG may mediate cell-surface signaling, cellular delivery, and biological action of sex hormones via activation of a specific plasma receptor directly [12, 33]. To test this hypothesis, we repeated the mediation analysis additionally adjusting for testosterone levels in model 1 and 2 and found that the mediatory role of SHBG was still significant and large for sex differences in glucose levels and T2D. In support of some previous literature, our findings suggest that SHBG may play a more significant role in T2D risk rather than previously recognized mechanisms linked to androgens, which warrants further investigations. However, it’s important to exercise caution when interpreting these results, as our mediation analysis model was adjusted for testosterone, a potential mediator, compromising as such the mediation analysis assumptions [17, 18].

Other novel findings of the current study are the inconsistent mediatory role of SHBG on fasting insulin levels, 2 h-glucose levels and HOMA-IR. While we found no significant associations for the DE of sex on these traits, the IE for the above-mentioned outcomes were significant and considerable, which is sometimes called inconsistent mediation [20].

Our findings could provide more insights to implement randomized clinical trials targeting SHBG in women suffering from low levels of SHBG. Although a large number of clinical trials have been investigating the effect of different interventions like dietary interventions [34], medications [35] and hormonal therapy [36] on SHBG levels, to our knowledge, no clinical trial has investigated the effect of SHBG administration on health outcomes.

This current study has several strengths and limitations. This was the first study to report the mediatory role of SHBG on sex differences in glucose metabolism and incidence of T2D. By using data from the KORA study, we were able to investigate the mediatory role of SHBG on the association of sex and glucose homeostasis in a large cohort of individuals both cross-sectionally and longitudinally with a limited proportion of missing values for confounders. OGTT were performed at both baseline and follow-up which helped us to explore the development of clinically diagnosed T2D and also newly OGTT-diagnosed T2D. Having detailed data on menopausal status (e.g. experiencing oophorectomy or hysterectomy) and on hormonal therapy (e.g. anti-estrogens, estrogens, progestin, hormone replacement therapy, androgens, antiandrogens, enzyme inhibitors, gonadotropin releasing hormones, and anabolic steroids) was helpful to account for these important confounding factors which may not be well-collected in other cohort studies. As a limitation, we were not able to repeat the sensitivity analysis stratified by age and BMI categories for T2D due to the limited number of incident cases. Lack of replication and not considering the non-alcoholic fatty liver disease in our statistical models are other limitations of our study. In addition, we could not perform other preferable modeling such as Cox regression analysis, due to the lack of exact time to event data. Finally, this study was conducted among a German population, which limits the generalizability of our findings to other ethnicities.

In conclusion, in a large-scale population-based study we showed that serum SHBG is a potential mediator in the association between sex and glucose levels as well as incident T2D. Whether SHBG could be a target therapy for reducing sex differences in diabetes needs larger and well-designed clinical studies.