Molecular interactions between sex hormone-binding globulin and nonsteroidal ligands that enhance androgen activity.

Sex hormone-binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (-)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71-1.80 Šresolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids.

Sex hormone binding globulin during an annual reproductive cycle in the hepatopancreas and ovary of pejerrey (Odontesthes bonariensis).

In the present study, we determined the hepatopancratic shbg transcript abundance and ovarian immunoreactive Shbg (ir-Shbg) localization in pejerrey females at different gonadal stages during an annual ovarian cycle. In the hepatopancreas, shbg expression remains was constant in pre-vitellogenic stages, decreased at final vitellogenesis to increase again in final maturation and atretic stages to previous levels at post-vitellogenic stages. Related to this, also we found a negative significant relation between sex steroid and shbg expression. On the other hand, in the ovary we found ir-Shbg inside of cortical alveoli, from previtellogenic stages to final maturation. This localization of Shbg in a teleost fish ovary suggests a new role for Shbg in oocytes, that may also extend the oocyte fertilization/development process.

Selective serotonin reuptake inhibitor effects on neural biomarkers of perinatal depression.

The effect of perinatal selective serotonin reuptake inhibitors (SSRIs) on brain-derived neurotrophic factor (BDNF) and S100 calcium binding protein B (S100B) has not been investigated. Using a cohort of 86 pregnant women, we found that SSRIs significantly increase BDNF levels in late pregnancy and that S100B, but not BDNF, is associated with maternal depression in SSRI-treated women only. This shows that serum S100B could be a unique biomarker to determine efficacy of SSRIs during gestation.

The human fetal adrenal produces cortisol but no detectable aldosterone throughout the second trimester.

BACKGROUND: Human fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone, estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention through secretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objective of this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzyme expression in normal second trimester human fetuses. METHODS: This was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to 109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study design was balanced to show effects of maternal smoking. RESULTS: Concentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and mass spectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone, dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in all adrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 and CYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-level expression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetal plasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and 16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 and NR5A1 were increased by maternal smoking. CONCLUSIONS: The human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectable aldosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenal regulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia during the second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen in extreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which could have knock-on effects on post-natal health.

Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein.

Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.

Costs and Benefits of Extended Endocrine Strategies for Premenopausal Breast Cancer.

Background: After completing 5 years of adjuvant tamoxifen, women with estrogen receptor (ER)-positive breast cancer benefit from 5 more years of endocrine therapy, either with tamoxifen or an aromatase inhibitor (AI). For premenopausal women, ovarian ablation (OA) would be required before starting an AI (OA/AI). According to the SOFT/TEXT studies, OA/AI improves 5-year disease-free survival compared with tamoxifen alone, suggesting that OA/AI could be superior to tamoxifen as extended endocrine therapy. The long-term costs and consequences of premature menopause from OA are unknown, but could be estimated through a cost-effectiveness analysis. Methods: A Markov chain Monte Carlo simulation model estimated the costs and benefits of 3 extended endocrine strategies in a hypothetical cohort of premenopausal women with ER-positive early breast cancer: (1) no further treatment; (2) tamoxifen for 5 years (extended tamoxifen); or (3) OA/AI for 5 years. Effectiveness was measured in years of life expectancy gain. Sensitivity analyses accounted for uncertainty surrounding various parameters. Monte Carlo simulation estimated the number of adverse events and deaths from each strategy in the US population over a 40-year period. Results: Extended tamoxifen yielded a higher average life expectancy gain than OA/AI (17.31 vs 17.06 years) at lower average cost ($3,550 vs $14,312). For 18,000 premenopausal ER-positive women, the simulation estimated 13,236, 12,557, and 11,338 deaths with no further treatment, extended tamoxifen, and OA/AI, respectively, but an additional 1,897 deaths from OA, for a total of 13,235 deaths associated with OA/AI. After 24.6 years of follow-up, more women are expected to die from OA/AI than extended tamoxifen. Conclusions: For premenopausal women with ER-positive cancer who have completed adjuvant tamoxifen, another 5 years of tamoxifen is the preferable extended endocrine strategy. The potential long-term health consequences of OA could affect overall survival when it precedes the use of an AI.

Sex hormone binding globulin: Expression throughout early development and adult pejerrey fish, Odontesthes bonariensis.

Sex hormone binding globulin (Shbg) is a plasma glycoprotein that binds and transports steroids in the blood of all vertebrate classes apart from birds. In the present study we characterized shbg from pejerrey, a fish species with a well characterized temperature-dependent sex determination. The pejerrey shbg mRNA comprises 1185bp encoding for a 395 amino acid Shbg precursor protein that includes a leader sequence for secretion. Relative quantification of shbg transcript abundance revealed expression early in development coinciding with the sex-determining period and probably in association with temperature leading to male determination. The hepatopancreas was the main site of shbg expression, which varied according to the sex cycle in females. It was also expressed in gills, gonads, gut and taste buds during both larval stages and in adult fish. The presence of Shbg in organs in close contact with the environment such as gills, pseudobranchs, gut and taste buds suggests that these are potential sources of uptake or release of steroids/xenosteroids to and from the aquatic environment.

Genome wide association identifies common variants at the SERPINA6/SERPINA1 locus influencing plasma cortisol and corticosteroid binding globulin.

Variation in plasma levels of cortisol, an essential hormone in the stress response, is associated in population-based studies with cardio-metabolic, inflammatory and neuro-cognitive traits and diseases. Heritability of plasma cortisol is estimated at 30-60% but no common genetic contribution has been identified. The CORtisol NETwork (CORNET) consortium undertook genome wide association meta-analysis for plasma cortisol in 12,597 Caucasian participants, replicated in 2,795 participants. The results indicate that <1% of variance in plasma cortisol is accounted for by genetic variation in a single region of chromosome 14. This locus spans SERPINA6, encoding corticosteroid binding globulin (CBG, the major cortisol-binding protein in plasma), and SERPINA1, encoding α1-antitrypsin (which inhibits cleavage of the reactive centre loop that releases cortisol from CBG). Three partially independent signals were identified within the region, represented by common SNPs; detailed biochemical investigation in a nested sub-cohort showed all these SNPs were associated with variation in total cortisol binding activity in plasma, but some variants influenced total CBG concentrations while the top hit (rs12589136) influenced the immunoreactivity of the reactive centre loop of CBG. Exome chip and 1000 Genomes imputation analysis of this locus in the CROATIA-Korcula cohort identified missense mutations in SERPINA6 and SERPINA1 that did not account for the effects of common variants. These findings reveal a novel common genetic source of variation in binding of cortisol by CBG, and reinforce the key role of CBG in determining plasma cortisol levels. In turn this genetic variation may contribute to cortisol-associated degenerative diseases.

Development of the external genitalia: perspectives from the spotted hyena (Crocuta crocuta).

This review/research paper summarizes data on development of the external genitalia of the spotted hyena, a fascinating mammal noted for extreme masculinization of the female external genitalia. The female spotted hyena is the only extant mammal that mates and gives birth through a pendulous penis-like clitoris. Our studies indicate that early formation of the phallus in both males and females is independent of androgens; indeed the phallus forms before the fetal testes or ovaries are capable of synthesizing androgens. Likewise, pre- and postnatal growth in length of the penis and clitoris is minimally affected by "androgen status". Nonetheless, several internal morphologies, as well as external surface features of the phallus, are androgen-dependent and thus account for dimorphism between the penis and clitoris. Finally, estrogens play a critical role in penile and clitoral development, specifying the position of the urethral orifice, determining elasticity of the urethral meatus, and facilitating epithelial-epithelial fusion events required for proper formation of the distal urethra/urogenital sinus and prepuce. Accordingly, prenatal inhibition of estrogen synthesis via administration of letrozole (an aromatase inhibitor) leads to malformations of the glans as well as the prepuce (hypospadias). The effects of prenatal androgens, anti-androgens and impaired estrogen synthesis correlated with the tissue expression of androgen and estrogen receptors.

Genetic determinants of serum testosterone concentrations in men.

Testosterone concentrations in men are associated with cardiovascular morbidity, osteoporosis, and mortality and are affected by age, smoking, and obesity. Because of serum testosterone's high heritability, we performed a meta-analysis of genome-wide association data in 8,938 men from seven cohorts and followed up the genome-wide significant findings in one in silico (n = 871) and two de novo replication cohorts (n = 4,620) to identify genetic loci significantly associated with serum testosterone concentration in men. All these loci were also associated with low serum testosterone concentration defined as <300 ng/dl. Two single-nucleotide polymorphisms at the sex hormone-binding globulin (SHBG) locus (17p13-p12) were identified as independently associated with serum testosterone concentration (rs12150660, p = 1.2×10(-41) and rs6258, p = 2.3×10(-22)). Subjects with ≥ 3 risk alleles of these variants had 6.5-fold higher risk of having low serum testosterone than subjects with no risk allele. The rs5934505 polymorphism near FAM9B on the X chromosome was also associated with testosterone concentrations (p = 5.6×10(-16)). The rs6258 polymorphism in exon 4 of SHBG affected SHBG's affinity for binding testosterone and the measured free testosterone fraction (p<0.01). Genetic variants in the SHBG locus and on the X chromosome are associated with a substantial variation in testosterone concentrations and increased risk of low testosterone. rs6258 is the first reported SHBG polymorphism, which affects testosterone binding to SHBG and the free testosterone fraction and could therefore influence the calculation of free testosterone using law-of-mass-action equation.

Corticosteroid-Binding Globulin (SERPINA6) Consolidates Sexual Dimorphism of Adult Rat Liver.

Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and 2-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6 gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2710 genes were differentially expressed in wild-type female vs male livers at this age. Importantly, ∼10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.

Characterization and comparison of recombinant full-length ursine and human sex hormone-binding globulin.

Sex hormone-binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell-based expression system for recombinant full-length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10-fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~ 58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in the presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [3 H]DHT were determined to 0.21 ± 0.04 nm for human and 1.32 ± 0.10 nm for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology.

Corticosteroid-binding Globulin (SERPINA6) Establishes Postpubertal Sex Differences in Rat Adrenal Development.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6 gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6 transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.

Differential Changes in Circulating Steroid Hormones in Hibernating Brown Bears: Preliminary Conclusions and Caveats.

Brown bears are obese when they enter the den, and after 6 mo of hibernation and physical inactivity, bears show none of the adverse consequences of a sedentary lifestyle in humans, such as cardiovascular disease, type 2 diabetes, and kidney failure. The metabolic mechanisms that drive hibernation physiology in bears are poorly defined, but systemic endocrine regulators are likely involved. To investigate the potential role of steroid hormones, we quantified the total levels of 12 steroid hormones, the precursor cholesterol, sex hormone-binding globulin (SHBG), and corticosterone-binding globulin (CBG) in paired serum samples from subadult free-ranging Scandinavian brown bears during the active and hibernation states. During hibernation, androstenedione and testosterone were significantly decreased in subadult female bears (n=13), whereas they increased in all males but one (n=6) and therefore did not reach a significant difference. Despite this difference, SHBG increased more than 20-fold during hibernation for all bears. Compared with SHBG concentrations in humans, bear levels were very low in the active state, but during hibernation, levels equaled high levels in humans. The increased SHBG levels likely maintain a state of relative quiescence of the reproductive hormones in hibernating bears. Interestingly, the combination of SHBG and testosterone levels results in similar free bioavailable testosterone levels of 70-80 pM in both subadult and adult sexually active male bears, suggesting a role for SHBG in controlling androgen action during hibernation in males. Dehydroepiandrosterone sulfate, dihydrotestosterone, and estradiol levels were below the detection limit in all but one animal. The metabolically active glucocorticoids were significantly higher in both sexes during hibernation, whereas the inactive metabolite cortisone was reduced and CBG was low approaching the detection limit. A potential caveat is that the glucocorticoid levels might be affected by the ketamine applied in the anesthetic mixture for hibernating bears. However, increased hibernating cortisol levels have consistently been reported in both black bears and brown bears. Thus, we suggest that high glucocorticoid activity may support the hibernation state, likely serving to promote lipolysis and gluconeogenesis while limiting tissue glucose uptake to maintain a continuous glucose supply to the brain.

Diverse roles for sex hormone-binding globulin in reproduction.

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in blood and regulates their access to target tissues. Hepatic production of SHBG fluctuates throughout the life cycle and is influenced primarily by metabolic and hormonal factors. Genetic differences also contribute to interindividual variations in plasma SHBG levels. In addition to controlling the plasma distribution, metabolic clearance, and bioavailability of sex steroids, SHBG accumulates in the extravascular compartments of some tissues and in the cytoplasm of specific epithelial cells, where it exerts novel effects on androgen and estrogen action. In mammals, the gene-encoding SHBG is expressed primarily in the liver but also at low levels in other tissues, including the testis. In subprimate species, Shbg expression in Sertoli cells is under the control of follicle-stimulating hormone and produces the androgen-binding protein that influences androgen actions in the seminiferous tubules and epididymis. In humans, the SHBG gene is not expressed in Sertoli cells, but its expression in germ cells produces an SHBG isoform that accumulates in the acrosome. In fish, Shbg is produced by the liver but has a unique function in the gill as a portal for natural steroids and xenobiotics, including synthetic steroids. However, salmon have retained a second, poorly conserved Shbg gene that is expressed only in ovary, muscle, and gill and that likely exerts specialized functions in these tissues. The present review compares the production and functions of SHBG in different species and its diverse effects on reproduction.

Liver at the nexus of rat postnatal HPA axis maturation and sexual dimorphism.

Normal function of the hypothalamic-pituitary-adrenal (HPA) axis is critical for survival, and its development is choreographed for age-, sex- and context-specific actions. The liver influences HPA ontogeny, integrating diverse endocrine signals that inhibit or activate its development. This review examines how developmental changes in the expression of genes in the liver coordinate postnatal changes in multiple endocrine systems that facilitate the maturation and sexual dimorphism of the rat HPA axis. Specifically, it examines how the ontogeny of testicular androgen production, somatostatin-growth hormone activities, and hypothalamic-pituitary-thyroid axis activity intersect to influence the hepatic gene expression of insulin-like growth factor 1, corticosteroid-binding globulin, thyroxine-binding globulin, 11ß-hydroxysteroid dehydrogenase type 1 and 5α-reductase type 1. The timing of such molecular changes vary between mammalian species, but they are evolutionarily conserved and are poised to control homeostasis broadly, especially during adversity. Importantly, with the liver as their nexus, these diverse endocrine systems establish the fundamental organization of the HPA axis throughout postnatal development, and thereby ultimately determine the actions of glucocorticoids during adulthood.

Homozygous SHBG Variant (rs6258) Linked to Gonadotropin-Independent Precocious Puberty in a Young Girl.

Sex hormone-binding globulin (SHBG) in the blood is a major determinant of bioactivity for key sex steroids such as testosterone and estradiol. Low serum levels of SHBG have been associated with obesity, polycystic ovaries, and metabolic syndrome, and other states associated with hyperandrogenemia. A 9-year, 6-month-old girl presented with a history of peripheral precocious puberty and aggressive behavior. The patient's SHBG level was remarkably low for her age, at less than 5 nmol/L (reference range for a girl with a bone age of 10 years, 73 nmol/L [SEM = 10]) [1]. On genetic and protein analysis, the patient was found to have a homozygous missense potentially pathogenic variant in the SHBG gene (c.554C>T, p.P185L); her parents were asymptomatic heterozygote carriers. Laboratory investigations supported the possible involvement of this genetic alteration in the patient's phenotype. Various analyses of this variant support its pathogenicity, although the exact mechanism remains unclear. In conclusion, we present a genetic SHBG variant in the homozygote state that may have been associated with gonadotropin-independent precocious puberty in a young girl.

Protocol for a cluster randomised trial evaluating a multifaceted intervention starting preconceptionally-Early Interventions to Support Trajectories for Healthy Life in India (EINSTEIN): a Healthy Life Trajectories Initiative (HeLTI) Study.

INTRODUCTION: The Healthy Life Trajectories Initiative is an international consortium comprising four harmonised but independently powered trials to evaluate whether an integrated intervention starting preconceptionally will reduce non-communicable disease risk in their children. This paper describes the protocol of the India study. METHODS AND ANALYSIS: The study set in rural Mysore will recruit ~6000 married women over the age of 18 years. The village-based cluster randomised design has three arms (preconception, pregnancy and control; 35 villages per arm). The longitudinal multifaceted intervention package will be delivered by community health workers and comprise: (1) measures to optimise nutrition; (2) a group parenting programme integrated with cognitive-behavioral therapy; (3) a lifestyle behaviour change intervention to support women to achieve a diverse diet, exclusive breast feeding for the first 6 months, timely introduction of diverse and nutritious infant weaning foods, and adopt appropriate hygiene measures; and (4) the reduction of environmental pollution focusing on indoor air pollution and toxin avoidance.The primary outcome is adiposity in children at age 5 years, measured by fat mass index. We will report on a host of intermediate and process outcomes. We will collect a range of biospecimens including blood, urine, stool and saliva from the mothers, as well as umbilical cord blood, placenta and specimens from the offspring.An intention-to-treat analysis will be adopted to assess the effect of interventions on outcomes. We will also undertake process and economic evaluations to determine scalability and public health translation. ETHICS AND DISSEMINATION: The study has been approved by the institutional ethics committee of the lead institute. Findings will be published in peer-reviewed journals. We will interact with policy makers at local, national and international agencies to enable translation. We will also share the findings with the participants and local community through community meetings, newsletters and local radio. TRIAL REGISTRATION NUMBER: ISRCTN20161479, CTRI/2020/12/030134; Pre-results.

Monosaccharide-induced lipogenesis regulates the human hepatic sex hormone-binding globulin gene.

The liver produces plasma sex hormone-binding globulin (SHBG), which transports sex steroids and regulates their access to tissues. In overweight children and adults, low plasma SHBG levels are a biomarker of the metabolic syndrome and its associated pathologies. Here, we showed in transgenic mice and HepG2 hepatoblastoma cells that monosaccharides (glucose and fructose) reduce human SHBG production by hepatocytes. This occurred via a downregulation of hepatocyte nuclear factor-4alpha (HNF-4alpha) and replacement of HNF-4alpha by the chicken OVA upstream promoter-transcription factor 1 at a cis-element within the human SHBG promoter, coincident with repression of its transcriptional activity. The dose-dependent reduction of HNF-4alpha levels in HepG2 cells after treatment with glucose or fructose occurred in concert with parallel increases in cellular palmitate levels and could be mimicked by treatment with palmitoyl-CoA. Moreover, inhibition of lipogenesis prevented monosaccharide-induced downregulation of HNF-4alpha and reduced SHBG expression in HepG2 cells. Thus, monosaccharide-induced lipogenesis reduced hepatic HNF-4alpha levels, which in turn attenuated SHBG expression. This provides a biological explanation for why SHBG is a sensitive biomarker of the metabolic syndrome and the metabolic disturbances associated with increased fructose consumption.

Characterization and measurement of the plasma alpha- and beta-sex hormone-binding globulin paralogs in salmon.

When the biochemical characteristics of coho salmon SHBG (csSHBG) plasma were examined, two different steroid-binding profiles were obtained corresponding to recombinant csSHBG-alpha and csSHBG-beta. These SHBG paralogs share only 24% sequence identity, and this explains their unique steroid-binding properties. Both proteins bind testosterone, but csSHBG-alpha also binds androstenedione (Kd = 2.8 nm) and ethinylestradiol with high affinity, whereas csSHBG-beta binds estradiol (Kd = 0.8 nm) preferentially. When analyzed by gel filtration, csSHBG-alpha displays the properties of a 153-kDa homodimer, whereas csSHBG-beta elutes as a 68-kDa monomer. The unique steroid-binding properties of csSHBG-alpha and csSHBG-beta allowed us to develop an assay for their measurements in immature (pre-smolt) and mature coho salmon blood. Plasma csSHBG-alpha levels were 3- to 4-fold higher than those of csSHBG-beta irrespective of developmental stage or sex and correlate with each other. The major site of csSHBG-alpha expression in pre-smolts and mature fish is the liver, but low levels of csSHBG-alpha mRNA are present in stomach/intestine of mature fish. In pre-smolts, high levels of csSHBG-beta mRNA are present in gills and ovary, whereas csSHBG-beta mRNA is most abundant in muscle and stomach/intestine of mature fish. Based on the differences in csSHBG-alpha and csSHBG-beta plasma levels and their tissue expression profiles, we conclude that gills and/or muscle contribute mainly to plasma SHBG-beta in coho salmon. The assays we have developed will enable studies of how SHBG-alpha/SHBG-beta biosynthesis is regulated throughout the salmonid life cycle and how they influence steroid hormone action in these fish.