placebo, sitagliptin, and glimepiride in patients with type 2 diabetes taking metformin. Diabetes Care http:// Rosenstock, J. et al. Advancing basal insulin replacement in type 2 diabetes inadequately controlled with insulin glargine plus oral agents: a comparison of adding albiglutide, a weekly GLP‑1 receptor agonist, versus thrice-daily prandial insulin lispro. Diabetes Care dc14-0001. Maffioli, P. & Derosa, G. Hypoglycemia, its implications in clinical practice, and possible

ways to prevent it. Curr. Med. Res. Opin. 30, 771–773 (2014). 9. Inzucchi, S. E. et al. Management of hyperglycemia in type 2 diabetes: a patientcentered approach: position statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD). Diabetes Care 35, 1364–1379 (2012). 10. Hauber, A. B. et al. Effect of pill burden on dosing preferences, willingness to pay, and likely adherence among patients with type 2 diabetes. Patient Prefer. Adherence 7, 937–949 (2013).


Functional effects of sex hormone-binding globulin variants Michaël R. Laurent and Dirk Vanderschueren

A new study has found eight single nucleotide polymorphisms in sex hormone-binding globulin that functionally affect its affinity for androgens or estrogens and other biochemical properties. This finding adds to growing concern about the ‘one size fits all’ approach in formulas to calculate free or bioavailable concentrations of estradiol and testosterone. Laurent, M. R. & Vanderschueren, D. Nat. Rev. Endocrinol. 10, 516–517 (2014); published online 22 July 2014; doi:10.1038/nrendo.2014.120

Sex hormone-binding globulin (SHBG) binds androgens and estrogens with nano­ molar affinity: dihydrotestosterone is the preferred ligand, followed by testosterone and estradiol. The sex steroids not bound to SHBG (on average 55% of testosterone and estradiol in healthy adult men and women, respectively) are considered bioavailable because they circulate either freely (~1–3%) or loosely bound to other proteins such as albumin.1,2 In both clinical practice and epidemiology studies, the concentrations of free or bioavailable sex steroids are usually calculated with law of mass action formulas based on the concentrations of testos­terone, estradiol, SHBG and/or albumin, and fixed

affinity constants.3 However, in a recent biochemical investigation, several single nucleo­ tide poly­morphisms (SNPs) in SHBG were identified that decreased affinity for testosterone and dihydro­testosterone, increased binding affinity for estradiol, or both.4 In humans, SHBG is secreted by the liver as a homodimer. Each subunit of SHBG consists of two laminin G‑like domains: the N‑terminal domain contains the st­eroidbinding pocket and binding sites for cal­cium and zinc, whereas the C‑terminal domain contains residues for glycosyla­tion.5 On the basis of the crystal structure of the N‑terminal laminin G‑like domain or evolutionary conservation of certain amino acids, Wu and

Box 1 | Limitations of calculating free or bioavailable sex steroid concentrations ■■ Immunoassayed total concentrations might be inaccurate, especially for low concentrations of testosterone or estradiol, or possibly when single nucleotide polymorphisms affect sex hormone-binding globulin (SHBG) epitopes ■■ SHBG binding sites might be occupied by other ligands, making free testosterone calculation inaccurate in pregnancy, for example ■■ Many different formulas exist, some of which are poorly validated (particularly for estradiol) ■■ Polymorphisms might influence the affinity of SHBG for testosterone, estradiol or both ■■ To what extent the free, bioavailable and SHBG-bound fractions actually contribute to bioactivity is unclear ■■ SHBG might have biological functions beyond restricting the bioactivity of circulating sex steroids ■■ Other metabolites might contribute to, or be better biomarkers of, androgen status ■■ Plasma concentrations of sex steroids do not account for sex steroid metabolism within tissues (‘intracrinology’)

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Hammond prioritized 18 non­synonymous SNPs for an extensive evaluation.4 Using mutated recombinant SHBG proteins, they found three naturally occurring mutants with 2.5–5.0-fold reduced dihydro­testosterone binding affinity. One of these mutants was also secreted at very low levels, probably as a result of altered glyco­sylation and abnor­ mal protein folding. Another mutant had ~60% lower dihydro­testosterone affinity than wild-type SHBG and was also poorly recog­ nized by the monoclon­al antibody used in their immunoassay. Interestingly, three other mutants had normal androgen binding but almost two­ fold increased estradiol binding affinity. Two variants at another position resulted in decreased androgen binding affinity, while estradiol binding was 20–30% better preserved (that is, increased estradiol binding relative to androgen binding). These variants with increased estrogen binding represent a novel class of functional SHBG variants. One particular mutant markedly reduced dimerization and seemed to have defective calcium binding. Apart from providing interesting new insights into the relationship between structure and function in the SHBG protein, this new catalogue of functional SNPs has several implications for clinical chemistry. Measuring low levels of sex steroids with immunoassays might be unreliable,2 but the monoclonal antibodies used in certain immunoassay platforms might also fail to properly recognize particular SHBG variants. Importantly, these results further call into question the blanket use of formulas for calculating free or bioavailable sex st­eroids. Methods of directly measuring the levels of sex steroids that are free (equilibrium dialysis or centrifugal ultrafiltration) or bioavailable (precipitation of SHBG and its bound steroids with ammonium sulphate, for example) correlate quite well with calculated values. However, these techniques are considered too cumbersome and challenging to offer an advantage over the calculations that are normally used in routine clinical chemistry.2,3 Despite widespread use of SHBG in the assessment of the bioactivity of sex steroids, genetic differences that affect its bind­ ing affinity have remained unknown for a long time. This situation changed when a genome-wide meta-analysis identified a nonsynonymous SNP (rs6258, present in 2% of European men) that reduces testos­ terone binding affinity and is associated with lower total testosterone concentrations.6 Hammond originally identified this SNP in

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NEWS & VIEWS a woman with extreme virilization during pregnancy as well as in four other women with hirsutism, polycystic ovarian syndrome or premature ovarian failure, while it was absent in control individuals.7 SHBG levels in this pregnant woman were exceptionally low because rs6258 also impairs SHBG secretion, and her second allele contained a single nucleotide deletion and reading-frame shift. Such cases of SHBG deficiency are extremely rare: only two other examples in women have been reported and, very recently, one in a young man with adult-onset hypogona­ dal symptoms despite normal levels of free testosterone.8 Another SNP (rs6259) has a global minor allele frequency of 8.7% and results in an additional glycosylation site that prolongs the half-life and plasma levels of SHBG, without influencing its steroidbinding properties. This SNP and other variants in the promoter region are not only associated with increased levels of SHBG but also with a reduced risk of type 2 diabetes mellitus, as well as breast and endometrial cancer.5 High levels of SHBG have also been associated with an increased peak bone mass in young men but accelerated bone loss and an increased risk of fracture in ageing men, independent of the concentrations of sex steroids.5,9 However, some of these results might have been confounded by un­recognized function­al SNPs. A more fundamental problem highlighted by this study is the fact that our understanding of sex steroid bioactivity and the role of SHBG seems to be a lot more complex than originally anticipated (Box 1). The free hormone hypothesis postulates that bio­ activity is determined by the free hormone fractions, which are determined by SHBG in the case of sex steroids. However, SHBG has also been reported to have intracellular functions, in the extracellular matrix and at the cell membrane, although the relevance of these mechanisms for human physiology remains controversial.5 SHBG has variably been proposed to reg­ ulate sex steroid bioactivity as an inhibitor, facilitator, or both, as well as decreasing the free androgen to estrogen ratio (because SHBG preferentially binds androgens).9 SHBG might also primarily determine total con­centrations of sex steroids,6 whereas concentrations of free testos­terone might be kept constant in vivo by h­ypothalamic–­pituitary feedback.1 Further­more, testosterone and estradiol are almost exclusively relied on to assess bio­activity, but other metabolites such as dihydro­testos­terone, androstene­dione or urinary glucuronides might be more

important contributors to, or biomarkers of, androgenic status than currently realized. Finally, plasma measure­ments do not account for the increas­ingly recognized autonomous sex steroid metabo­lism that occurs within di­fferent target tissues (‘intracrinology’).10 The prevalence of these functional SNPs identified by Wu and Hammond remains to be determined in different clinical and community-based studies, as do their associations with the concentrations of SHBG and sex steroids and with related clinical outcomes. Still, the study by Wu and Hammond offers a cautionary tale for both clinicians and researchers who use immuno­assayed SHBG values to assess androgenic or estrogenic bioactivity. If we want to achieve more personalized medicine and excellence in clinical chemistry, we will need improved solutions to accommodate interindividual variation in SHBG binding affinities and other components of sex steroid bioactivity. Laboratory of Molecular Endocrinology, Gerontology and Geriatrics (M.R.L.), Clinical and Experimental Endocrinology (D.V.), KU Leuven and University Hospitals Leuven, Herestraat 49, PO Box 902, 3000 Leuven, Belgium. Correspondence to: D.V. [email protected] Acknowledgements The authors would like to thank F. Claessens, B. Decallonne and L. Antonio for helpful discussions related to this work. M.R.L. is a PhD Fellow funded by the Research Foundation Flanders (FWO). D.V. is a Senior Clinical Investigator funded by clinical research funds of the University Hospitals Leuven. The authors would like to acknowledge the support of grant G.0854.13N from the Research Foundation Flanders.

Competing interests The authors declare no competing interests. 1.

de Ronde, W. et al. Serum levels of sex hormone-binding globulin (SHBG) are not associated with lower levels of non‑SHBG‑ bound testosterone in male newborns and healthy adult men. Clin. Endocrinol. (Oxf.) 62, 498–503 (2005). 2. Rosner, W., Auchus, R. J., Azziz, R., Sluss, P. M. & Raff, H. Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J. Clin. Endocrinol. Metab. 92, 405–413 (2007). 3. Vermeulen, A., Verdonck, L. & Kaufman, J. M. A critical evaluation of simple methods for the estimation of free testosterone in serum. J. Clin. Endocrinol. Metab. 84, 3666–3672 (1999). 4. Wu, T. S. & Hammond, G. L. Naturally occurring mutants inform SHBG structure and function. Mol. Endocrinol. 28, 1026–1038 (2014). 5. Hammond, G. L., Wu, T. S. & Simard, M. Evolving utility of sex hormone-binding globulin measurements in clinical medicine. Curr. Opin. Endocrinol. Diabetes Obes. 19, 183–189 (2012). 6. Ohlsson, C. et al. Genetic determinants of serum testosterone concentrations in men. PLoS Genet. 7, e1002313 (2011). 7. Hogeveen, K. N. et al. Human sex hormonebinding globulin variants associated with hyperandrogenism and ovarian dysfunction. J. Clin. Invest. 109, 973–981 (2002). 8. Vos, M. J., Mijnhout, G. S., Rondeel, J. M., Baron, W. & Groeneveld, P. H. Sex hormone binding globulin deficiency due to a homozygous missense mutation. J. Clin. Endocrinol. Metab. jc.2014-2055. 9. Khosla, S. Editorial: Sex hormone binding globulin: inhibitor or facilitator (or both) of sex steroid action? J. Clin. Endocrinol. Metab. 91, 4764–4766 (2006). 10. Labrie, F. All sex steroids are made intracellularly in peripheral tissues by the mechanisms of intracrinology after menopause. J. Steroid Biochem. Mol. Biol. 10.1016/j.jsbmb.2014.06.001.


CPAP effects in sleep apnoea —what should be expected? Patrick Lévy and Jean-Louis Pépin

Patients with obesity and obstructive sleep apnoea have increased morbidity. Now, one study has reported limited beneficial effects of continuous positive airway pressure (CPAP) in patients with both these conditions. However, in another study, the reduction in blood pressure obtained with CPAP was not observed when nocturnal hypoxia was suppressed with supplemental oxygen. Lévy, P. & Pépin, J.‑L. Nat. Rev. Endocrinol. 10, 517–519 (2014); published online 5 August 2014; doi:10.1038/nrendo.2014.131

Obstructive sleep apnoea (OSA) is a wellknown public-health problem owing to its high prevalence and the numerous


consequences of the disorder, including exces­sive daytime somnolence and cognitive impairment, as well as cardiovascular and VOLUME 10  |  SEPTEMBER 2014  |  517

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Reproductive endocrinology: functional effects of sex hormone-binding globulin variants.

A new study has found eight single nucleotide polymorphisms in sex hormone-binding globulin that functionally affect its affinity for androgens or est...
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