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Editorial |
Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Sundeep Khosla, M.D, Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. E-mail: Khosla.Sundeep{at}Mayo.edu.
A long-standing dogma in the field of sex steroid action has been the "free-hormone hypothesis," which holds that it is the fraction of androgens and estrogens not bound to SHBG that is biologically active (1). The underlying premise of this hypothesis is that sex steroids bound to SHBG cannot access target tissues because testosterone and estradiol have relatively high binding affinities for SHBG (2), and the circulating, dimeric form of SHBG has a molecular mass of approximately 90 kDa (thus preventing it from traversing the capillary barrier). This has led to the development of various approaches to estimate non-SHBG-bound ("bioavailable") sex steroids by the ammonium sulfate precipitation method (3) or by using mass action equations (4), or to estimate non-SHBG-, non-albumin-bound ("free") sex steroids by the equilibrium dialysis method or by using mass action equations (4). Although each of these methods has advantages and limitations, the fundamental assumption remains that it is only the non-SHBG-bound steroids that are biologically relevant.
There is considerable indirect and direct evidence in support of the free hormone hypothesis. Thus, in population studies, it is almost always the non-SHBG-bound fraction of testosterone or estradiol that has been most strongly associated with muscle mass, strength, and bone mineral density (BMD) (4). The non-SHBG-bound estradiol concentration has generally been the most robust predictor of BMD in men, with total estradiol and total or non-SHBG-bound testosterone usually showing weaker associations (reviewed in Ref. 5). Because SHBG concentrations more than double over life in men (3), this has led to the plausible hypothesis that the age-related increase in SHBG levels, by limiting the availability of sex steroids, contributes to the observed decline in bone mass in aging men (5).
There is also considerable direct evidence in favor of the free hormone hypothesis (summarized in Ref. 6). Thus, animal as well as in vitro studies have found that SHBG limits access of testosterone to some target tissues such as brain, liver, salivary gland, lymph node, and prostate (6, 7). SHBG also limits the bioavailability of estradiol, particularly in the brain and testis (6, 8, 9), although this effect may be less pronounced in other tissues such as the liver (6).
Despite the evidence in favor of the free hormone hypothesis, scientific dogmas exist, in part, to be challenged, and such is also the case with this hypothesis. Thus, SHBG binding sites with characteristics of receptors have been identified in various cell types (2). However, the most striking, and perhaps controversial, challenge to the free hormone hypothesis came from a paper published in Cell last year by Hammes et al. (10), who found that megalin, an endocytic receptor in reproductive tissues, may mediate the cellular uptake of biologically active androgens and estrogens bound to SHBG. Although the findings and conclusions of this paper have been questioned (11) and await confirmation, these data do challenge the free hormone hypothesis.
It is against this undercurrent of some scientific discomfort with the free hormone hypothesis that the paper by Eriksson et al. (12) in the current issue of JCEM is of particular interest. Thus, in two cohorts of Swedish men consisting of MrOS Sweden (n = 3000; average age, 75.4 yr) and GOOD (n = 1068; average age, 18.9 yr), the authors characterized polymorphisms in the SHBG gene promoter, measured serum levels of SHBG as well as of sex steroids and their metabolites, and related these to BMD at various sites determined by dual-energy x-ray absorptiometry. To complement the human studies, they also assessed BMD in male mice overexpressing human SHBG. The main findings of the study were that SHBG gene promoter polymorphisms were associated with serum testosterone levels and with BMD at all hip bone sites in elderly men. In contrast to previous studies that had found that serum SHBG levels were negatively associated with BMD (13, 14, 15), Eriksson et al. found that serum levels of SHBG were positively associated with BMD. Moreover, the SHBG alleles associated with high serum SHBG levels were also associated with higher BMD. The latter association persisted despite adjustment for serum testosterone levels; although, somewhat disappointingly, the authors did not include serum estradiol (total or non-SHBG bound) in this multivariate analysis. Indeed, because BMD in aging men is generally most strongly associated with estradiol levels (5), lack of this analysis does lead to some concerns about the interpretation of the findings. Nonetheless, consistent with the human association study, the authors also found that the transgenic mice overexpressing human SHBG had increased cortical thickness (but unchanged cortical or trabecular BMD). Because mice do not express the SHBG gene postnatally, these findings need to be interpreted with some caution, and the authors appropriately acknowledge this limitation.
This study poses a significant dilemma—specifically, why were higher levels of SHBG associated with higher BMD in this paper, in contrast to previous papers that reported exactly opposite findings (13, 14, 15)? Although it is always tempting to attribute such conflicting findings to methodological differences, it would be important to formulate a biologically plausible explanation for the conflicting results.
A potential clue to resolution may lie in the hormonal levels in the MrOS Sweden cohort in the paper by Eriksson et al. (12). Specifically, serum total testosterone levels were not significantly different in the elderly and young men, with the elderly men having higher total estradiol levels but relatively similar free estradiol levels as the young men. These findings are strikingly different from virtually all published data on aging men, in which both total testosterone and non-SHBG-bound estradiol levels have been found to decline with age (reviewed in Ref. 16). Indeed, a recent study from the U.S. arm of MrOS also found that, at least within the cohort of men over age 65 yr, testosterone, estradiol, and their free fractions all declined with age (17).
What, then, makes MrOS Sweden different? Because a major regulator of serum LH is estradiol (16), these aging men in MrOS Sweden seem to be responding to rising SHBG levels by appropriately compensating for the rise in SHBG by presumably increasing LH secretion sufficiently so as to maintain non-SHBG-bound estradiol levels in the normal range, with slightly increased total estradiol levels [see Table 1 in Eriksson et al. (12)]. To the extent that the hypothalamic/pituitary axis is responding primarily to free estradiol (6, 8), these findings, in fact, support the concept of the free hormone hypothesis, at least as far as the hypothalamic/pituitary axis and estradiol are concerned. This appropriate compensation for rising SHBG levels may reflect the composition of this cohort, which might have included healthier aging men than other cohorts. Similarly, the SHBG transgenic mice studied by Eriksson et al. (12) have been previously reported (18) to have up to 50-fold higher testosterone levels than their wild-type littermates, resulting almost certainly in normal (or elevated) free sex steroid levels, although the latter have not been measured in either the previous or present study.
With this in mind, one can then propose the following testable hypothesis: SHBG is not simply a passive carrier for sex steroids but serves to amplify effects of sex steroid sufficiency or deficiency. The premise here, based on the work of Hammes et al. (10), is that there are two distinct pathways by which sex steroids can enter the cell: either as free hormones or via the megalin-mediated endocytotic pathway involving SHBG. In the setting of normal free sex steroid levels (for bone, perhaps particularly the free estradiol levels), adequate signaling via the free hormone pathway is augmented by the megalin pathway, as in the case of the aging men in MrOS Sweden or the SHBG transgenic mice, leading to the observed positive associations between SHBG levels and BMD in the men and increased cortical thickness in the bones of the SHBG transgenic mice. By contrast, in the setting of a deficiency in free sex steroids, as in the case of previous studies in aging men with declining free estradiol levels (13, 14, 15), deficient signaling via the free sex steroid pathway cannot be overcome adequately by the endocytotic pathway, and the effects of SHBG in limiting availability of free sex steroids become dominant, leading to the previously observed negative associations between SHBG and BMD (13, 14, 15).
This potential dual role for SHBG—i.e. serving both to augment sex steroid action in the setting of sex steroid sufficiency and to inhibit sex steroid bioavailability in the setting of sex steroid deficiency—might seem paradoxical but may confer some advantages to the organism, particularly during pregnancy and lactation. Thus, SHBG levels are induced by estrogen and rise markedly during pregnancy (6). This is a condition of sex steroid sufficiency (or even excess), and enhancement of the free estrogen pathway via the megalin pathway in various tissues such as bone, the gut, and the kidney may serve, at least in part, to maximize estrogen effects on preserving bone mass and enhancing intestinal and renal calcium conservation for the subsequent period of lactation. By contrast, when estrogen levels fall during lactation (19), SHBG would now serve to further limit sex steroid action on bone, leading to maximal resorption of calcium from bone for entry into breast milk and the calcium needs of the newborn.
Many other issues need further resolution. First, if megalin truly is the SHBG receptor, is megalin expressed and biologically active in bone cells? Second, what is the role of the capillary endothelial barrier? If SHBG and the bound steroids cannot traverse this barrier, how would bone cells ever become exposed to sex steroids that are bound to SHBG? A potential answer comes from recent work by Hauge et al. (20) demonstrating that bone remodeling occurs in highly vascular compartments (bone remodeling compartments) in both cortical and trabecular bone. Bone remodeling compartments are walled-off compartments penetrated by capillaries, and it may well be that the capillary barrier breaks down in these areas of bone remodeling. If so, then both free and SHBG-bound steroids may be able to access and regulate osteoclasts and osteoblasts, along with other cells, such as T cells, that may influence bone turnover.
Unexpected findings, such as those of Hammes et al. (10) and Eriksson et al. (12), should lead us to question traditional dogma. In this case, the dogma will likely survive but may need to be modified. The free hormone hypothesis, which has withstood the test of time, is likely still correct, at least under conditions of declining free hormone levels. On the other hand, the parallel pathway involving SHBG-mediated entry of sex steroids into cells may become more evident in the setting of sex steroid sufficiency. In the end, these apparent contradictory findings reflect the fact that biological processes rarely have absolute truths; it is only our artificial constructs of those truths that tend to be absolute.
Footnotes
Abbreviation: BMD, Bone mineral density.
Received September 11, 2006.
Accepted September 28, 2006.
References
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