Veldhuis, Johannes D.

Abstract Context Neurokinin B (NKB) is obligate for human puberty, but its role in adult female gonadotropin secretion and ovarian follicle growth is unknown. Objective To investigate antagonism of NKB on pulsatile gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion and ovarian follicle development in healthy women. Design Open investigation of the effects of a neurokinin-3 receptor (NK3R) antagonist (NK3Ra) vs a no-treatment control cycle. Setting Clinical research facility. Patients or other participants Healthy women with regular menses (n = 13). Intervention(s) NK3Ra MLE4901 40 mg taken orally twice daily from cycle day 5 to 6 for 7 days. Main outcome measure(s) LH secretion, ovarian follicle growth, and timing of ovulation. Results NK3Ra administration reduced basal LH secretion without a change in pulse frequency and delayed the LH surge by 7 days, the duration of treatment [mean cycle day ± standard error of the mean (SEM), 22 ± 1 days vs 15 ± 1 days in control cycles; P = 0.0006]. Follicle growth (mean diameter at the end of administration of NK3Ra administration ± SEM, 9.3 ± 0.4 mm vs 15.1 ± 0.9 mm in control cycles; P < 0.0001) and rising estradiol concentrations (mean ± SEM, 166 ± 29 pmol/L vs 446 ± 86 pmol/L in control cycles; P < 0.0001) were prevented. After treatment, follicle development resumed and normal preovulatory follicle diameter and estradiol concentrations were demonstrated. Postovulatory progesterone rise was similarly delayed (peak cycle day, 30 ± 2 vs 22 ± 1; P = 0.002) and cycle length was prolonged (35 ± 1 days vs 29 ± 1 days in control cycles; P = 0.0003) but luteal progesterone excretion was unaffected by the NK3Ra (LH surge day +7 mean urinary progesterone levels ± SEM, 58 ± 10 pmol/mol vs 48±7 pmol/mol creatinine in control cycles; nonsignificant). Conclusion These data demonstrate the involvement of NKB-NK3R signaling in the physiological regulation of GnRH/LH secretion, determining normal follicle development in women.

SummaryObjectivePatients with mutations of neurokinin B (NKB) and its receptor show hypogonadotrophic hypogonadism, but there is little evidence for the importance of this pathway in reproductive function in normal men, or its functional hierarchy with kisspeptin.DesignAn open label study wherein men (n = 6) were administered the NK3R antagonist MLE4901 40 mg orally twice a day for 7 days. Kisspeptin‐10 (0.3 μg/kg iv bolus) was given before and on day 7 of NK3R antagonist treatment.PatientsSubjects were healthy men.MeasurementsReproductive hormones were measured before and during the NK3R antagonist administration, including frequent sampling on two occasions for analysis of pulsatile LH secretion.ResultsLH, FSH and testosterone secretion were decreased during NK3R antagonist administration. LH showed a biphasic response, being reduced after 24 hours of treatment (4.5 ± 0.6 IU/L pretreatment to 1.7 ± 0.2 IU/L, P < .05), with partial recovery thereafter, but it was again decreased on day 7 (2.5 ± 0.6 IU/L, P < .05 vs pretreatment). FSH secretion was also suppressed, with a similar temporal pattern to that of LH. Testosterone secretion was decreased from 24 hours (18.4 ± 1.6 pretreatment vs 5.6 ± 1.5 nmol/L, P < .01) and remained suppressed throughout the treatment period. Analysis of LH pulsatility showed that both basal and pulsatile LH secretion were markedly suppressed but there was no detected change in LH pulse frequency. Kisspeptin‐10 stimulated LH secretion, with similar responses before and during NK3R antagonist administration.ConclusionsThese data demonstrate a central role for NKB/NK3R in the physiological regulation of reproductive function in men, and that this is functionally upstream of kisspeptin‐mediated GnRH secretion.

Context Studies on 24-h cortisol secretion are rare. The impact of sex, age and adiposity on cortisol levels, often restricted to one or a few samples, are well recognized, but conflicting. Objective To investigate cortisol dynamics in 143 healthy men and women, spanning 7 decades and with a 2-fold body mass index (BMI) range with different analytic tools. Setting Clinical Research Unit. Design Cortisol concentrations in 10-min samples collected for 24 h. Outcomes were mean levels, deconvolution parameters, approximate entropy (ApEn, regularity statistic) and 24-h rhythms. Results Total 24-h cortisol secretion rates estimated by deconvolution analysis were sex, age and BMI independent. Mean 24-h cortisol concentrations were lower in premenopausal women than those in men of comparable age (176 ± 8.2 vs 217 ± 9.4 nmol/L, P = 0.02), but not in subjects older than 50 years. This was due to lower daytime levels in women, albeit similar in the quiescent overnight period. Aging increased mean cortisol by 10 nmol/L per decade during the quiescent secretory phase and advanced the acrophase of the diurnal rhythm by 24 min/decade. However, total 24-h cortisol secretion rates estimated by deconvolution analysis were sex, age and BMI independent. ApEn of 24-h profiles was higher (more random) in premenopausal women than those in men (1.048 ± 0.025 vs 0.933 ± 0.023, P = 0.001), but not in subjects older than 50 years. ApEn peaked during the daytime. Conclusion Sex and age jointly determine the 24-h cortisol secretory profile. Sex effects are largely restricted to age <50 years, whereas age effects elevate concentrations in the late evening and early night and advance the timing of the peak diurnal rhythm.

Abstract Deletion of the melanocortin-4-receptor (Mc4r) gene in mice causes hyperphagia, followed by hyperinsulinemia, obesity and progressive infertility. Evidence shows that the number of developed corpora lutea is reduced in obese MC4R-knockout (MC4R KO) female mice, but the mechanism is unclear. The effect of hyperphagia and obesity by MC4R KO on pulsatile luteinizing hormone (LH) secretion and ovulation remains unknown. In MC4R KO mice and wild-type littermates (WT LM) during the diestrus period throughout different ages, we examined and monitored their metabolic status, pulsatile LH profiles, follicular morphology and the number of corpora lutea. MC4R KO mice were hyperphagic, obese, hyperglycemic, hyperinsulinemic and demonstrated insulin resistance and hepatic steatosis. Irregular estrous cycles and significant changes in the LH secretion profiles were observed in sexually matured 16- to 28-week MC4R KO mice, without any difference in testosterone levels. In addition, MC4R KO mice at 16 weeks of age had significantly fewer corpora lutea than same age WT LM mice. The ovary examinations of MC4R KO mice at 28 weeks of age showed predominantly antral and preovulatory follicles with no corpora lutea. These findings were consistent with the decrease in total, pulsatile, mass and basal LH releases in MC4R KO mice. The characteristics of hormone profiles in obese MC4R KO mice indicate that MC4R plays an important role in regulating LH release, ovulation and reproductive ability probably via hyperphagia-induced obesity. Further study of correlation between metabolic and reproductive regulatory hormones is warranted to dissect the pathological mechanism underlying obesity-induced infertility. Free Chinese abstract: A Chinese translation of this abstract is freely available at http://www.reproduction-online.org/content/153/3/267/suppl/DC1.

Context: Kisspeptin and neurokinin B (NKB) are obligate for normal gonadotropin secretion, but their hierarchy is unexplored in normal women. Objective: To investigate the interaction between kisspeptin and NKB on estrogen-regulated LH secretion. Design: Women were treated with neurokinin-3 receptor (NK3R) antagonist followed by transdermal estradiol to induce LH secretion 48 hours later, with kisspeptin-10 or vehicle infusion during estrogen administration in a 2-way crossover study. Setting: Clinical research facility. Patients or other participants: Healthy females with regular menses. Intervention(s): NK3R antagonist AZD4901 40 mg twice daily orally was taken from cycle day 4–6 for 6 days (n = 10, with 10 no treatment controls). Transdermal estradiol patches (200 μg/d) were applied after 5 days of NK3R antagonist treatment. At 24-hour estradiol treatment, women were randomized to 7-hour kisspeptin-10 (4 μg/kg/h) or vehicle iv infusion, with the alternate infusion in a subsequent cycle. Main outcome measure(s): Plasma gonadotropin and estradiol secretion. Results: After an initial suppression, LH secretion was increased 48 hours after estradiol treatment. Kisspeptin-10 increased LH secretion during the inhibitory phase, and LH remained elevated beyond the discontinuation of kisspeptin-10 infusion. NK3R antagonist decreased LH pulse frequency (0.5 ± 0.2 vs 0.7 ± 0.2 pulses/h, P &lt; .05) and stimulated FSH response to kisspeptin-10 infusion (10.7 ± 11.0 vs 5.0 ± 3.6 IU/L, P &lt; .05) with a nonsignificant rise in LH. The duration of LH response was blunted, with LH being lower at 48 hours (7.5 ± 4.8 vs 15.0 ± 11.4 IU/L, P &lt; .05). Conclusions: These data demonstrate that NKB signaling regulates GnRH/LH secretion in normal women, and is predominantly proximal to kisspeptin in mediating estrogenic positive and negative feedback on LH secretion.

Germline mutations in the aryl hydrocarbon receptor-interacting protein ( AIP ) gene predispose humans to pituitary adenomas, particularly of the somatotroph lineage. Mice with global heterozygous inactivation of Aip ( Aip +/− ) also develop pituitary adenomas but differ from AIP -mutated patients by the high penetrance of pituitary disease. The endocrine phenotype of these mice is unknown. The aim of this study was to determine the endocrine phenotype of Aip +/− mice by assessing the somatic growth, ultradian pattern of GH secretion and IGF1 concentrations of longitudinally followed male mice at 3 and 12 months of age. As the early stages of pituitary tumorigenesis are controversial, we also studied the pituitary histology and somatotroph cell proliferation in these mice. Aip +/− mice did not develop gigantism but exhibited a leaner phenotype than wild-type mice. Analysis of GH pulsatility by deconvolution in 12-month-old Aip +/− mice showed a mild increase in total GH secretion, a conserved GH pulsatility pattern, but a normal IGF1 concentration. No pituitary adenomas were detected up to 12 months of age. An increased ex vivo response to GHRH of pituitary explants from 3-month-old Aip +/− mice, together with areas of enlarged acini identified on reticulin staining in the pituitary of some Aip +/− mice, was suggestive of somatotroph hyperplasia. Global heterozygous Aip deficiency in mice is accompanied by subtle increase in GH secretion, which does not result in gigantism. The absence of pituitary adenomas in 12-month-old Aip +/− mice in our experimental conditions demonstrates the important phenotypic variability of this congenic mouse model.

Key points Loss of function of the melanocortin 4 receptor (MC4R) results in hyperphagia, obesity and increased growth. Despite knowing that MC4Rs control food intake, we are yet to understand why defects in the function of the MC4R receptor contribute to rapid linear growth. We show that hyperphagia following germline loss of MC4R in male mice promotes growth while suppressing the growth hormone–insulin‐like growth factor‐1 (GH–IGF‐1) axis. We propose that hyperinsulinaemia promotes growth while suppressing the GH–IGF‐1 axis. It is argued that physiological responses essential to maintain energy flux override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. AbstractDefects in melanocortin‐4‐receptor (MC4R) signalling result in hyperphagia, obesity and increased growth. Clinical observations suggest that loss of MC4R function may enhance growth hormone (GH)‐mediated growth, although this remains untested. Using male mice with germline loss of the MC4R, we assessed pulsatile GH release and insulin‐like growth factor‐1 (IGF‐1) production and/or release relative to pubertal growth. We demonstrate early‐onset suppression of GH release in rapidly growing MC4R deficient (MC4RKO) mice, confirming that increased linear growth in MC4RKO mice does not occur in response to enhanced activation of the GH–IGF‐1 axis. The progressive suppression of GH release in MC4RKO mice occurred alongside increased adiposity and the progressive worsening of hyperphagia‐associated hyperinsulinaemia. We next prevented hyperphagia in MC4RKO mice through restricting calorie intake in these mice to match that of wild‐type (WT) littermates. Pair feeding of MC4RKO mice did not prevent increased adiposity, but attenuated hyperinsulinaemia, recovered GH release, and normalized linear growth rate to that seen in pair‐fed WT littermate controls. We conclude that the suppression of GH release in MC4RKO mice occurs independently of increased adipose mass, and is a consequence of hyperphagia‐associated hyperinsulinaemia. It is proposed that physiological responses essential to maintain energy flux (hyperinsulinaemia and the suppression of GH release) override conventional mechanisms of pubertal growth to promote the storage of excess energy while ensuring growth. Implications of these findings are likely to extend beyond individuals with defects in MC4R signalling, encompassing physiological changes central to mechanisms of growth and energy homeostasis universal to hyperphagia‐associated childhood‐onset obesity.

Context: Factors that regulate physiological feedback by pulses of glucocorticoids on the hypothalamic-pituitary unit are sparsely defined in humans in relation to gluco- or mineralocorticoid receptor pathways, gender, age, and the sex steroid milieu. Objective: The objective of the study was to test (the clinical hypothesis) that glucocorticoid (GR) and mineralocorticoid (MR) receptor-selective mechanisms differentially govern pulsatile cortisol-dependent negative feedback on ACTH output (by the hypothalamo-pituitary unit) in men and women studied under experimentally defined T and estradiol depletion and repletion, respectively. Setting: The study was conducted at the Mayo Center for Translational Science Activities. Subjects: Healthy middle-aged men (n = 16) and women (n = 25) participated in the study. Interventions: This was a randomized, prospective, double-blind, placebo- and saline-controlled study of pulsatile cortisol infusions in low cortisol-clamped volunteers with and without eplerenone (MR blocker) and mifepristone (GR blocker) administration under a low and normal T and estradiol clamp. During frequent sampling, a bolus of CRH-arginine vasopressin was infused to assess corticotrope responsiveness. Analytical Methods and Outcomes: Deconvolution and approximate entropy of ACTH profiles were measured. Results: Infusion of cortisol (but not saline) pulses diminished ACTH secretion. The GR antagonist, mifepristone, interfered with negative feedback on both ACTH burst mass and secretion regularity. Eplerenone, an MR antagonist, exerted no detectable effect on the same parameters. Despite feedback imposition, CRH-arginine vasopressin-stimulated ACTH secretion was also increased by mifepristone and not by eplerenone. Withdrawal vs addback of sex steroids had no effect on ACTH secretion parameters. Nonetheless, ACTH secretion was greater (P = .006) and more regular (P = .004) in men than women. Conclusion: Pulsatile cortisol feedback on ACTH secretion in this paradigm is mediated by the glucocorticoid receptor, in part acting at the level of the pituitary, and influenced by sex.

Background: Systemic concentrations of T, estradiol (E2), GH, IGF-1, and IGF binding protein-3 decline in healthy aging individuals. Conversely, T and E2 stimulate GH and IGF-1 production in hypogonadal patients. Hypothesis: Because E2 stimulates GH secretion, putatively via the nuclear estrogen receptor-α and E2 and GH fall with menopause, we postulated that diminished endogenous E2 contributes to low GH output in older women. Location: The study was conducted at the Mayo Center for Clinical and Translational Science. Study Design: This was a randomized, double-blind, controlled study in 60 healthy postmenopausal women treated with the following: 1) double placebo; 2) anastrozole, a potent inhibitor of aromatase-enzyme activity, which mediates E2 synthesis from T; and/or 3) fulvestrant, a selective estrogen receptor-α antagonist. Methods: GH pulse generation was quantified by frequent GH sampling before and after short-term iv somatostatin infusion, thought to induce hypothalamic GHRH-mediated rebound-like GH secretion. Results: On anastrozole, E2 fell from 3.1 ± 0.35 pg/mL to 0.36 ± 0.04 pg/mL, and estrone from 13 ± 1.4 pg/mL to 1.9 ± 0.01 pg/mL (P &lt; .001) by mass spectrometry. Estrogen values were unchanged by fulvestrant. T concentrations did not change. One-hour peak GH rebound after somatostatin infusion declined markedly during both estrogen-deprivation schedules (P &lt; .001). Mean (150 min) maximal GH rebound decreased comparably (P &lt; .001). Measures of GH rebound correlated negatively with computed tomography-estimated abdominal visceral fat (all P &lt; .05). Conclusion: These data suggest a previously unrecognized dependence of hypothalamo-pituitary GH regulation on low levels of endogenous estrogen after menopause.

Context: Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, is characterized by high secretion levels of LH and T. Currently, there is no treatment licensed specifically for PCOS. Objective: The objective of this study was to investigate whether a targeted therapy would decrease LH pulse frequency in women with PCOS, subsequently reducing serum LH and T concentrations and thereby presenting a novel therapeutic approach to the management of PCOS. Design: This study is a double-blind, double-dummy, placebo-controlled, phase 2 trial. Settings: University hospitals and private clinical research centers were included. Participants: Women with PCOS aged 18–45 years participated. Intervention: Intervention included AZD4901 (a specific neurokinin-3 [NK3] receptor antagonist) at a dose of 20, 40, or 80 mg/day or matching placebo for 28 days. Main Outcome Measure: Change from baseline in the area under the LH serum concentration–time curve over 8 hours (area under the curve) on day 7 relative to placebo was measured. Results: Of a total 67 randomized patients, 65 were evaluable. On day 7, the following baseline-adjusted changes relative to placebo were observed in patients receiving AZD4901 80 mg/day: 1) a reduction of 52.0% (95% confidence interval [CI], 29.6–67.3%) in LH area under the curve; 2) a reduction of 28.7% (95% CI, 13.9–40.9%) in total T concentration; and 3) a reduction of 3.55 LH pulses/8 hours (95% CI, 2.0–5.1) (all nominal P &lt; .05). Conclusions: The NK3 receptor antagonist AZD4901 specifically reduced LH pulse frequency and subsequently serum LH and T concentrations, thus presenting NK3 receptor antagonism as a potential approach to treating the central neuroendocrine pathophysiology of PCOS.