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The hypothalamus is the central regulator of reproductive hormone secretion. Pulsatile secretion of gonadotropin releasing hormone (GnRH) is fundamental to physiological stimulation of the pituitary gland to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). Furthermore, GnRH pulsatility is altered in common reproductive disorders such as polycystic ovary syndrome (PCOS) and hypothalamic amenorrhea (HA). LH is measured routinely in clinical practice using an automated chemiluminescent immunoassay method and is the gold standard surrogate marker of GnRH. LH can be measured at frequent intervals (e. g., 10 minutely) to assess GnRH/LH pulsatility. However, this is rarely done in clinical practice because it is resource intensive, and there is no user-friendly and accessible method for computational analysis of the LH data available to clinicians. Here we present hormoneBayes, a novel open-access Bayesian framework that can be easily applied to reliably analyze serial LH measurements to assess LH pulsatility. The framework utilizes parsimonious models to simulate hypothalamic signals that drive LH dynamics, together with state-of-the-art (sequential) Monte-Carlo methods to infer key parameters and latent hypothalamic dynamics. We show that this method provides estimates for key pulse parameters including inter-pulse interval, secretion and clearance rates and identifies LH pulses in line with the current gold-standard deconvolution method. We show that these parameters can distinguish LH pulsatility in different clinical contexts including in reproductive health and disease in men and women (e. g., healthy men, healthy women before and after menopause, women with HA or PCOS). A further advantage of hormoneBayes is that our mathematical approach provides a quantified estimation of uncertainty. Our framework will complement methods enabling real-time in-vivo hormone monitoring and therefore has the potential to assist translation of personalized, data-driven, clinical care of patients presenting with conditions of reproductive hormone dysfunction. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The hypothalamus is the central regulator of reproductive hormone secretion. Pulsatile secretion of gonadotropin releasing hormone (GnRH) is fundamental to physiological stimulation of the pituitary gland to release luteinizing hormone (LH) and follicle stimulating hormone (FSH). Furthermore, GnRH pulsatility is altered in common reproductive disorders such as polycystic ovary syndrome (PCOS) and hypothalamic amenorrhea (HA). LH is measured routinely in clinical practice using an automated chemiluminescent immunoassay method and is the gold standard surrogate marker of GnRH. LH can be measured at frequent intervals (e.g., 10 minutely) to assess GnRH/LH pulsatility. However, this is rarely done in clinical practice because it is resource intensive, and there is no user-friendly and accessible method for computational analysis of the LH data available to clinicians. Here we present hormoneBayes, a novel open-access Bayesian framework that can be easily applied to reliably analyze serial LH measurements to assess LH pulsatility. The framework utilizes parsimonious models to simulate hypothalamic signals that drive LH dynamics, together with state-of-the-art (sequential) Monte-Carlo methods to infer key parameters and latent hypothalamic dynamics. We show that this method provides estimates for key pulse parameters including inter-pulse interval, secretion and clearance rates and identifies LH pulses in line with the current gold-standard deconvolution method. We show that these parameters can distinguish LH pulsatility in different clinical contexts including in reproductive health and disease in men and women (e.g., healthy men, healthy women before and after menopause, women with HA or PCOS). A further advantage of hormoneBayes is that our mathematical approach provides a quantified estimation of uncertainty. Our framework will complement methods enabling real-time in-vivo hormone monitoring and therefore has the potential to assist translation of personalized, data-driven, clinical care of patients presenting with conditions of reproductive hormone dysfunction.

Circulating growth hormone (GH) concentrations increase during pregnancy in mice and remain pituitary-derived. Whether abundance or activation of the GH secretagogue ghrelin increase during pregnancy, or in response to dietary octanoic acid supplementation, is unclear. We therefore measured circulating GH profiles in late pregnant C57BL/6J mice and in aged-matched non-pregnant females fed with standard laboratory chow supplemented with 5% octanoic or palmitic (control) acid (n = 4–13/group). Serum total and acyl-ghrelin concentrations, stomach and placenta ghrelin mRNA and protein expression, Pcsk1 (encoding prohormone convertase 1/3) and Mboat4 (membrane bound O-acyl transferase 4) mRNA were determined at zeitgeber (ZT) 13 and ZT23. Total and basal GH secretion were higher in late pregnant than non-pregnant mice (P P = 0.004), but not acyl-ghrelin, and the density of ghrelin-positive cells in the gastric antrum (P = 0.019) were higher, and gastric Mboat4 and Pcsk1 mRNA expression were lower in pregnant than non-pregnant mice at ZT23. In the placenta, ghrelin protein was localised mostly to labyrinthine trophoblast cells. Serum acyl-, but not total, ghrelin was lower at mid-pregnancy than in non-pregnant mice, but not different at early or late pregnancy. In conclusion, dietary supplementation with 5% octanoic acid did not increase activation of ghrelin in female mice. Our results further suggest that increases in maternal GH secretion throughout murine pregnancy are not due to circulating acyl-ghrelin acting at the pituitary. Nevertheless, time-dependent increased circulating total ghrelin could potentially increase ghrelin action in tissues that express the acylating enzyme and receptor.

Context Glucagon increases energy expenditure; consequently, glucagon receptor agonists are in development for the treatment of obesity. Obesity negatively affects the reproductive axis, and hypogonadism itself can exacerbate weight gain. Therefore, knowledge of the effects of glucagon receptor agonism on reproductive hormones is important for developing therapeutics for obesity; but reports in the literature about the effects of glucagon receptor agonism on the reproductive axis are conflicting. Objective The objective of this work is to investigate the effect of glucagon administration on reproductive hormone secretion in healthy young men. Design A single-blinded, randomized, placebo-controlled crossover study was conducted. Setting The setting of this study was the Clinical Research Facility, Imperial College Healthcare NHS Trust. Participants Eighteen healthy eugonadal men (mean ± SEM: age 25.1 ± 1.0 years; body mass index 22.5 ± 0.4 kg/m2; testosterone 21.2 ± 1.2 nmol/L) participated in this study. Intervention An 8-hour intravenous infusion of 2 pmol/kg/min glucagon or rate-matched vehicle infusion was administered. Main Outcome Measures Luteinizing hormone (LH) pulsatility; LH, follicle-stimulating hormone (FSH), and testosterone levels were measured. Results Although glucagon administration induced metabolic effects (insulin area under the curve: vehicle 1065 ± 292 min.µU/mL vs glucagon 2098 ± 358 min.µU/mL, P Conclusions Acute administration of a metabolically active dose of glucagon does not alter reproductive hormone secretion in healthy men. These data are important for the continued development of glucagon-based treatments for obesity.

Context Interleukin-2 (IL-2), a proinflammatory cytokine, has been used to treat malignancies. Increased cortisol and adrenocorticotropin (ACTH) were noted, but growth hormone (GH) secretion was not investigated in detail. Objective We quantified GH secretion after a single subcutaneous injection of IL-2 in 17 young and 18 older healthy men in relation to dose, age, and body composition. Methods This was a placebo-controlled, blinded, prospectively randomized, crossover study. At 20:00 hours IL-2 (3 or 6 million units/m2) or saline was injected subcutaneously. Lights were off between 23:00 and 07:00 hours. Blood was sampled at 10-minute intervals for 24 hours. Outcome measures included convolution analysis of GH secretion. Results GH profiles were pulsatile under both experimental conditions and lower in older than young volunteers. Since the effect of IL-2 might be time limited, GH analyses were performed on the complete 24-hour series and the 6 hours after IL-2 administration. Total and pulsatile 24-hour GH secretion decreased nonsignificantly. Pulsatile secretion fell over the first 6 hours after IL-2 (P = .03), with visceral fat as a covariate (P = .003), but not age (P = .10). Plots of cumulative 2-hour bins of GH pulse mass showed a distinction by treatment and age groups: A temporary GH decrease of 32% and 28% occurred in the first 2-hour bins after midnight (P = .02 and .04) in young participants, whereas in older individuals no differences were present at any time point. Conclusion This study demonstrates that IL-2 temporarily diminishes GH secretion in young, but not older, men.

Background Many hormones display distinct circadian rhythms, driven by central regulators, hormonal bioavailability, and half-life. A set of 11-oxygenated C19 steroids (11-oxyandrogens) and pregnenolone sulfate (PregS) are elevated in congenital adrenal hyperplasia and other disorders, but their circadian patterns have not been characterized. Participants and methods Peripheral blood was collected every 2 h over 24 h from healthy volunteer men (10 young, 18–30 years, and 10 older, 60–80 years). We used mass spectrometry to quantify 15 steroids, including androstenedione (A4), testosterone (T), 11β-hydroxy- and 11-ketotestosterone (11OHT, 11KT),11β-hydroxy- and 11-ketoandrostenedione (11OHA4, 11KA4), and 4 ∆5-steroid sulfates. Diurnal models including mesor (rhythm adjusted median), peak, and nadir concentrations, acrophase, and amplitude were computed. Results 11OHA4 followed a rhythm similar to cortisol: acrophase 8:00 h, nadir 21:00 h and were similar in young and old men. 11KT had similar diurnal patterns, but the peak was lower in older than in young men, as was the case for A4. All four steroid sulfates were higher in young vs older men. PregS and 17-hydroxypregnenolone sulfate (17OHPregS) showed sustained elevations between 8:00 and 18:00 h, and nadirs around midnight, while DHEAS and AdiolS displayed minimal diurnal variations. All 4 11-oxyandrogens correlated tightly with cortisol (r from 0.54 for 11OHT to 0.81 for 11OHA4, P < 0.0001 for all), but very weakly with T, supporting their adrenal origin and ACTH governance. Conclusions 11-Oxyandrogens, PregS, and 17OHPregS display distinct circadian and age variations, which should be accounted for when used as clinical biomarkers.

Quantification of disturbances in glucose-insulin homeostasis has been the cornerstone of appraising insulin resistance and detecting early-stage diabetes. Metabolic homeostasis arises from feedback and feed-forward interactions among (at least) all four of glucose, insulin, glucagon, and cortisol. Quantifying such tetrapartite interactions in the fasting (endogenously regulated) state overnight could elucidate very early regulatory disruption. In the present study, healthy subjects without diabetes (ND; n = 20) and patients with Type 2 diabetes (T2D; n = 21) were investigated by repeated overnight blood sampling of all four of glucose, insulin, glucagon, and cortisol concentrations. To obviate confounding by hormone-specific disappearance rates, analyses were performed at the level of production (glucose) or secretion (insulin, glucagon, and cortisol) rates estimated by regularized deconvolution analysis. Then, a novel method for quantifying the loss of homeostasis among glucose, insulin, and glucagon (and, when available, cortisol) secretion patterns was developed. Potential early stage prediabetic candidates were identified. The new methodology avoids many of the difficulties encountered in the conventional estimation of insulin-glucose sensitivity or resistance, while incorporating the dynamics of the key coregulators under fasting conditions.

Normal reproductive functioning is critically dependent on pulsatile secretion of luteinising hormone (LH). Assessment of LH pulsatility is important for the clinical diagnosis of reproductive disorders, but current methods are hampered by frequent blood sampling coupled to expensive serial immunochemical analysis. Here, we report the development and application of a Robotic APTamer-enabled Electrochemical Reader (RAPTER) electrochemical analysis system to determine LH pulsatility. Through selective evolution of ligands by exponential enrichment (SELEX), we identify DNA aptamers that bind specifically to LH and not to related hormones. The aptamers are integrated into electrochemical aptamer-based (E-AB) sensors on a robotic platform. E-AB enables rapid, sensitive and repeatable determination of LH concentration profiles. Bayesian Spectrum Analysis is applied to determine LH pulsatility in three distinct patient cohorts. This technology has the potential to transform the clinical care of patients with reproductive disorders and could be developed to allow real-time in vivo hormone monitoring., Assessment of luteinising hormone pulsatility is important in the diagnosis of reproductive disorders. Here the authors develop a DNA aptamer-based electrochemical analysis integrated into a robotic platform for high-throughput and sensitive analysis.

Context Sleep loss in men increases cortisol and decreases testosterone, and sleep restriction by 3 to 4 hours/night induces insulin resistance. Objective We clamped cortisol and testosterone and determined the effect on insulin resistance. Methods This was a randomized double-blind, in-laboratory crossover study in which 34 healthy young men underwent 4 nights of sleep restriction of 4 hours/night under 2 treatment conditions in random order: dual hormone clamp (cortisol and testosterone fixed), or matching placebo (cortisol and testosterone not fixed). Fasting blood samples, and an additional 23 samples for a 3-hour oral glucose tolerance test (OGTT), were collected before and after sleep restriction under both treatment conditions. Cytokines and hormones were measured from the fasting samples. Overall insulin sensitivity was determined from the OGTT by combining complementary measures: homeostasis model assessment of insulin resistance of the fasting state; Matsuda index of the absorptive state; and minimal model of both fasting and absorptive states. Results Sleep restriction alone induced hyperinsulinemia, hyperglycemia, and overall insulin resistance (P Conclusion Fixing cortisol-testosterone exposure mitigates the development of insulin resistance and hyperinsulinemia, but not hyperglycemia, from sustained sleep restriction in men. The interplay between cortisol and testosterone may be important as a mechanism by which sleep restriction impairs metabolic health.

Introduction: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. Methods: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5–6 weeks old) or adult (10–19 weeks old) GHS-R KO (Ghsr−/−) and WT (Ghsr+/+) male and female mice. Results: Adult Ghsr−/− male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr−/− mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr−/− females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. Discussion/Conclusion: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.

Obese individuals often show low growth hormone (GH) secretion, which leads to reduced lipid mobilization and further fat accumulation. Pharmacological approaches to increase GH levels in obese individuals by GH injection or GH-releasing hormone receptor agonist showed promising effects on fat reduction. However, side effects on glucose metabolism and the heavy costs on making large peptides hindered their clinical application. Here, we tested whether stimulation of endogenous GH secretion by a synthetic GH secretagogue receptor (GHSR) agonist, hexarelin, improved the metabolism in a hyperphagic obese mouse model. Male melanocortin 4 receptor knockout mice (MC4RKO) were pair-fed and received continuous hexarelin (10.56 μg/day) or vehicle infusion by an osmotic pump for 3-4 weeks. Hexarelin treatment significantly increased the pulsatile GH secretion without detectable alteration on basal GH secretion in MC4RKO mice. The treated mice showed increased lipolysis and lipid oxidation in the adipose tissue, and reduced de novo lipogenesis in the liver, leading to reduced visceral fat mass, reduced triglyceride content in liver, and unchanged circulating free fatty acid levels. Importantly, hexarelin treatment improved the whole-body insulin sensitivity but did not alter glucose tolerance, insulin levels, or insulin-like growth factor 1 (IGF-1) levels. The metabolic effects of hexarelin were likely through the direct action of GH, as indicated by the increased expression level of genes involved in GH signaling pathways in visceral adipose tissues and liver. In conclusion, hexarelin treatment stimulated the pulsatile GH secretion and reduced the fat accumulation in visceral depots and liver in obese MC4RKO mice with improved insulin sensitivity without altered levels of insulin or IGF-1. It provides evidence for managing obesity by enhancing pulsatile GH secretion through activation of GHSR in the pituitary gland.

Context. Interleukin-2 (IL2), a proinflammatory cytokine, is used for treatment of malignancies. Increased cortisol and ACTH were noted, but not investigated in detail. This is the first study in healthy men which uses moderate high IL2 doses as used in cancer treatment. Objective. The goal of this study was to quantify cortisol secretion after a single sc injection of IL2 at 1900 h in young and older healthy men in relation to dose, age and body composition. Design. This was a placebo-controlled, blinded, prospectively randomized cross-over study in 17 young subjects (mean age 24.1 yr, range 19–30 yr) and 18 older subjects (mean age 63.9 yr, range 60–75 yr). The subjects underwent 24 h of blood sampling at 10-min intervals, starting at 1800 h. At 2000 h IL2 (3 or 6 million units per m2 body surface) or saline was injected sc. Lights were off between 2300 and 0700 h. Setting. The study was performed in a Clinical Translational Research Unit. Outcome measures. Mean concentrations of cortisol, deconvolution analysis, and approximate entropy. Abdominal visceral fat (AVF) and total abdominal fat (TAF) were calculated from single slice CT. Results. Cortisol concentrations started to rise at 2300 h. The AUC’s during the lights-on periods were unchanged by IL2. Therefore, most analyses were restricted to the 8 h lights-off period. In young volunteers pulsatile cortisol secretion increased from 52.9±5.8 to 77.0±8.0 µg/L/8h and in older subjects from 60.6±3.8 to 70.6±4.6 µg/L/8h (GLM: treatment P Conclusion. Il2, a paradigm for inflammation, increased pulsatile cortisol secretion, more in young than in older subjects. Higher IL2 doses in young subjects amplified cortisol secretion, but not in older subjects. Cortisol secretion exhibited an advance (earlier) time shift, accompanied by accelerated secretion. Incremental nocturnal cortisol secretion was negatively related to fat mass.

Introduction: In young men, sleep restriction decreases testosterone and increases afternoon cortisol, leading to anabolic-catabolic imbalance, insulin resistance and metabolic, neurocognitive, reproductive, and other adverse effects. Age-related differences in the hypothalamo-pituitary-testicular/adrenal response to sleep restriction could expose older individuals to greater or lesser risk, but this possibility has not been previously studied. Subjects and Methods: Thirty-five healthy young and older men aged 18-30y (n=17) and 60-80y (n=18), underwent blood sampling in the Mayo Clinic Center for Clinical and Translational Science every 10 minutes for 24 hours from 6PM-6PM under two conditions in random order spaced at least 3 weeks apart: awake (no sleep) or sleep (from 10PM to 6AM). Blood was assayed for LH, testosterone (T) and cortisol (F), and then analyzed by automated mathematical deconvolution and with cross approximate entropy statistics to determine hormone secretion and hormone synchrony, respectively. Statistical significance was construed by repeated measures ANOVA using a full factorial model that included age, sleep and the interaction. Results: Sleep deprivation had multiple effects on 24-hour (6PM-6PM) Te secretion with significant reductions in mean concentrations, basal, total and pulsatile secretion, and pulse frequency (each P Conclusion: Sleep restriction decreases morning LH secretion, morning and afternoon Te secretion, and increases afternoon F secretion, especially in older men. This combination of findings could plausibly cause metabolic and reproductive ill-health when accumulated over decades of life, and may explain how chronic sleep loss contributes to metabolic and reproductive diseases that are more prevalent in older men. These preliminary data also suggest a time-of-day dependent uncoupling of the regulatory control of the testicular axis, and of cortisol secretion. Direct verification by interventions that manipulate hormones during the morning and late afternoon in appropriately matched cohorts of young and older men are now required.

While long term fasting is reported to augment the corticotropic function, effect of short term (overnight) fasting on ACTH-cortisol coordinated release pattern and potential effect(s) of nutrient intake is not fully defined. Eleven healthy men (age: 33-70 yrs, BMI 20.4-31.5 kg-m2) were studied after overnight fast on 4 separate days, involving oral ingestion of 300 ml of either water, dextrose, protein, or lipid solutions. Test meals were isocaloric (400 kcal). Sessions were 6.5 h long, starting at 0800-0900 hrs. Blood was collected at 10-min intervals for ACTH (pg per mL), and cortisol (µg per dL) measurements. Linear regression, cross-correlation, deconvolution, and ApEn were used for data analyses. ACTH and cortisol concentration time series during short-term fast (water day) were found not to be chronologically coupled per linear regression (r2= 0.0014, P=0.82), and cross-correlation (r= - 0.156, lag=150 min) statistics. Oral intake of the 3 macronutrients improved the temporal relationship between ACTH and cortisol concentrations, verified by linear regression (r2:P- dextrose 0.54:0.0001, protein 0.65: 0.0001, lipid 0.42:0.0001), and cross-correlation (r:lag in min- dextrose 0.8:10, protein 0.77:10, lipid 0.78:20). Oral ingestion of either macronutrient did not significantly alter mean ACTH and cortisol concentrations and their respective secretion pattern (total, pulsatile, basal) over the period of 6.5 hr. However compared to the control (water) session, dextrose ingestion evoked less frequent and larger ACTH secretory bursts, and more regular ACTH and cortisol secretory patterns. In this study, we have observed lack of concordance between ACTH and cortisol after overnight fasting, which is restored with oral intake of macronutrients. This effect appears to be uniform among the 3 macronutrients, except for less frequent and lager ACTH bursts and more regular ACTH and cortisol release events after dextrose intake. These findings and the specific role of nutrients being direct or via physiologic nutrient-induced hormonal adaptation warrants future investigation.

Insulin and growth hormone (GH) are crucial counter-regulatory hormones in regulating glucose and lipid metabolisms. Insulin promotes fat storage, while GH promotes lipolysis and fat oxidation. In obese individuals, reduced GH secretion (hyposomatotropism) and increased insulin secretion (hyperinsulinemia) co-exist. The imbalance of these two hormones exacerbates fat accumulation. Therapeutic approaches to correct such hormonal imbalance in obesity are limited. The sodium/glucose cotransporter 2 inhibitor (SGLT2i), which promotes urinary glucose excretion, is a novel drug for overt type 2 diabetes (T2D). However, little is known about its efficacy in obese individuals without T2D in the clinic, in particular with hormonal imbalance. By applying SGLT2i (dapagliflozin, 1 mg/kg/d for 10 weeks) to a hyperphagic obese melanocortin 4 receptor knockout (MC4RKO) mouse model, we observed a significant reduction of hyperinsulinemia (fasting: 1.36±0.19 vs. 4.93±1.04 ng/ml, p Acknowledgements: grant (NHMRC, University of Queensland) and scholarship (CSC and UQ International scholarship) Reference: (1) Huang, Zhengxiang, et al. “Dapagliflozin restores insulin and growth hormone secretion in obese mice.” Journal of Endocrinology 245.1 (2020): 1-12. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Context Glucagon-like peptide-1 (GLP-1) potently reduces food intake and augments glucose-stimulated insulin secretion. Recent animal data suggest that GLP-1 may also influence reproduction. As GLP-1 receptor agonists are currently widely used in clinical practice to treat obesity/type 2 diabetes, it is necessary to determine the effects of GLP-1 on the reproductive system in humans. Objective To investigate the effects of GLP-1 administration on the reproductive axis in humans. Design Single-blind, randomized, placebo-controlled crossover study. Setting Clinical Research Facility, Imperial College Healthcare NHS Trust. Participants Eighteen healthy men (mean age 24.7 ± 0.1years, mean BMI 22.1 ± 0.4kg/m2). Intervention Eight-hour intravenous infusion of 0.8 pmol/kg/min GLP-1 or rate-matched vehicle infusion. Main Outcome Measures Number of luteinizing hormone (LH) pulses, LH, follicle-stimulating hormone (FSH), and testosterone levels. Results The number of LH pulses (number of LH pulses/500 min: vehicle 4.2 ± 0.4, GLP-1 4.5 ± 0.3, P = 0.46), LH area under the curve (AUC) (vehicle 1518 ± 88min.IU/L, GLP-1 1524 ± 101min.IU/L, P = 0.95), follicle-stimulating hormone AUC (vehicle 1210 ± 112 min IU/L, GLP-1 1216 ± 112 min IU/L, P = 0.86), and testosterone AUC (vehicle 10893 ± 615 min nmol/L, GLP-1 11088 ± 792 min nmol/L, P = 0.77) did not significantly differ during vehicle and GLP-1 administration. Glucagon-like peptide-1 significantly reduced food intake (vehicle 15.7 ± 1.3 kcal/kg, GLP-1 13.4 ± 1.3 kcal/kg, P = 0.01). Conclusions In contrast to the animal literature, our data demonstrate that acute GLP-1 administration does not affect reproductive hormone secretion in healthy men.

As a model of extreme conditions, eight healthy women, part of a 40-member Nepal mountain-climbing expedition, were monitored for dynamic endocrine adaptations. Endocrine measurements were made at frequent intervals over a 6–10-h period at four altitudes: 450 m, 4,800 m (base camp), 6,050 m, and again at 4,800 m (on descent) after an acclimatization (A) period (4,800 mA). Quantified hormones were growth hormone (GH), prolactin (PROL), cortisol (Cort), thyroid-stimulating hormone (TSH), and free thyroxine. These hormones are important to the anabolic/catabolic balance of the body, and are vital to growth, homeostasis, hypothalamic inhibition, regulation of stress, and metabolism. A key secondary question was the degree to which acclimatization can stabilize hormonal disruption. On the basis of statistical false discovery rates, the present analyses unveil marked adaptive changes in the thyroid axis at the level of pulsatile secretion of the pituitary hormone TSH and its downstream product, free thyroxine; strong effects on the mass of GH, TSH, Cort, and PROL secretion per burst; and prominent pulsatile frequency disruption and recovery for PROL and Cort. Because pulsatility changes reflect de facto perturbations in hypothalamo-pituitary control mechanisms, the present data introduce the concept of both frequency- and amplitude-dependent adaptive control of brain-pituitary neuroendocrine signals under conditions of extreme altitude exertion and exposure.

Study Objectives In young men, sleep restriction decreases testosterone (Te) and increases afternoon cortisol (F), leading to anabolic–catabolic imbalance, insulin resistance, and other andrological health consequences. Age-related differences in the hypothalamo–pituitary–testicular/adrenal response to sleep restriction could expose older individuals to greater or lesser risk. We aimed to evaluate and compare the 24-h and time-of-day effect of sleep restriction on F, luteinizing hormone (LH), and Te in young and older men. Methods Thirty-five healthy men, aged 18–30 (n = 17) and 60–80 (n =18) years, underwent overnight sleep deprivation (complete nighttime wakefulness) or nighttime sleep (10 pm to 6 am) with concurrent 10-min blood sampling in a prospectively randomized crossover study. F, LH, and Te secretion were calculated by deconvolution analysis. Results Sleep deprivation had multiple effects on 24-h Te secretion with significant reductions in mean concentrations, basal, total and pulsatile secretion, and pulse frequency (each p < 0.05), in the absence of detectable changes in LH. These effects were most apparent in older men and differed according to age for some parameters: pulsatile Te secretion (p = 0.03) and Te pulse frequency (p = 0.02). Time-of-day analyses revealed that sleep restriction significantly reduced Te in the morning and afternoon, reduced LH in the morning in both age groups, and increased F in the afternoon in older men. Conclusions These data suggest a time-of-day dependent uncoupling of the regulatory control of the testicular axis and of F secretion. Future studies will need to directly verify these regulatory possibilities specifically and separately in young and older men. Clinical Trial Not applicable.

Context Quantitative studies of the short-term feedback of testosterone (T) on luteinizing hormone (LH) secretion in healthy men are relatively rare. Such studies require the shutting down of endogenous T secretion and the imposition of experimentally controlled IV T addback. Objective To evaluate whether pulsatile and continuous T delivery confers equivalent negative feedback on LH secretion. Design This was a placebo-controlled, blinded, and prospectively randomized crossover study comprising 16 healthy men [age range 23 to 54 years and a body mass index (BMI) between 22.3 and 34.2 kg/m2]. Subjects received ketoconazole to block endogenous T secretion and received continuous or 90-minute pulses of IV T addback. Setting The study was performed in a Clinical Translational Research Unit. Interventions Subjects underwent 14 hours of blood sampling at 10-minute intervals, with a bolus IV injection of 33 ng/kg gonadotropin-releasing hormone (GnRH). Main outcome measures Log-transformed LH and T concentration ratios before and after GnRH administration. Results Despite higher T concentrations during pulsatile T feedback, LH concentrations and secretion rates, whether driven by endogenous or exogenous GnRH, were similar to those during continuous T infusion, indicating diminished pulsatile T feedback. Feedback correlated negatively with BMI. Under controlled T feedback, basal but not pulsatile LH secretion correlated negatively with CT-estimated visceral fat mass. Conclusion Feedback by pulsatile T delivery has diminished inhibitory strength compared with continuous infusion. Feedback is negatively correlated with BMI.

The well-documented hormonal disturbance in a general obese population is characterised by an increase in insulin secretion and a decrease in growth hormone (GH) secretion. Such hormonal disturbance promotes an increase in fat mass, which deteriorates obesity and accelerates the development of insulin resistance and type 2 diabetes. While the pathological consequence is alarming, the pharmaceutical approach attempting to correct such hormonal disturbance remains limited. By applying an emerging anti-diabetic drug, the sodium-glucose cotransporter 2 inhibitor, dapagliflozin (1 mg/kg/day for 10 weeks), to a hyperphagic obese mouse model, we observed a significant improvement in insulin and GH secretion as early as 4 weeks after the initiation of the treatment. Restoration of pathological disturbance of insulin and GH secretion reduced fat accumulation and preserved lean body mass in the obese animal model. Such phenotypic improvement followed with concurrent improvements in glucose and lipid metabolism, insulin sensitivity, as well as the expression of metabolic genes that were regulated by insulin and GH. In conclusion, 10 weeks of treatment with dapagliflozin effectively reduces hyperinsulinemia and restores pulsatile GH secretion in the hyperphagic obese mice with considerable improvement in lipid and glucose metabolism. Promising outcomes from this study may provide insights into drug intervention to correct hormonal disturbance in obesity to delay the diabetes progression.

Context Controlled, blinded studies of sex-hormone replacement in postmenopausal women using natural estradiol (E2) and native progesterone (P) are few. Objective To delineate the effect of E2 alone or with P on lipids and inflammatory markers. Design A placebo-controlled, double-masked, prospectively randomized study of 40 healthy, postmenopausal volunteers assigned to four treatment groups: placebo, intramuscular E2, and/or micronized oral P for 23 (±2) days. Results Treatment with E2 alone compared with placebo lowered total cholesterol (TC; P = 0.006), non–high-density lipoprotein cholesterol (nonHDL-C; P = 0.004), low-density lipoprotein cholesterol (LDL-C; P = 0.012), and apolipoprotein B (Apo B; P = 0.02) levels, and raised HDL-C levels (P = 0.03 vs the 3 other groups). Conversely, addition of P to E2 reduced HDL-C levels (P = 0.015). Triglyceride concentrations manifested no effect on E2 or P. High-sensitivity C-reactive protein (hsCRP) level was highest in women with E2 and P replacement (P = 0.018 vs placebo). Leptin and IL-6 concentrations did not vary. P treatment decreased adiponectin levels (P = 0.019). Serum E2 levels correlated linearly with TC, LDL-C, nonHDL-C, Apo B (all negatively), and SHBG (positively) concentrations. P level correlated negatively with TC (P = 0.029), HDL-C (P = 0.002), and adiponectin (P = 0.002) levels. Conclusion In this study, there were individual and interactive effects of E2 and P on key lipids in postmenopausal individuals., Estradiol given parenterally modifies TC, LDL-C, apo B, and HDL-C, whereas progesterone regulates TC, HDL and adiponectin. Neither hormone alters Lp(a), leptin or IL-6.

Context Exercise elicits incompletely defined adaptations of metabolic and endocrine milieu, including the gonadotropic and corticotropic axes. Objective To quantify the impact of acute exercise on coordinate luteinizing hormone (LH) and testosterone (T) and adrenocorticotropic hormone (ACTH) and cortisol secretion in healthy men in relation to age. Participants and Design Prospectively randomized, within-subject crossover study in 23 men aged 19 to 77 years old. Subjects underwent rest and 30 minutes of mixed exercise at 65% of maximal aerobic capacity with 10-minute blood sampling between 7:00 am and 1:00 pm, 2 weeks apart. Main Outcome Measures Incremental changes in LH, T, ACTH, and cortisol concentrations, the feedforward and feedback strength between exercise and rest, quantified by approximate entropy (ApEn), and bihormonal synchrony, quantitated by cross-ApEn. Results Mean hourly exercise-minus-rest LH and ACTH increments increased from -0.055 ± 0.187 to 0.755 ± 0.245 IU/L (P = 0.003) and from 2.9 ± 2.2 to 71.2 ± 16.1 ng/L (P < 0.0001), respectively, during exercise. T and cortisol increments increased concurrently from -9.6 ± 16.7 to 47.6 ± 17.1 ng/dL (P < 0.0001) and 0.45 ± 0.76 to 7.27 ± 0.64 µg/dL (P < 0.0001), respectively. During exercise, feedforward and feedback LH-T and ACTH-cortisol cross-ApEn decreased markedly quantifying enhanced hormonal coupling. Conclusions Acute moderate mixed exercise in healthy men rapidly enhances feedforward LH-T and ACTH-cortisol coordination and reciprocal feedback within the gonadotropic and corticotropic axes. In principle, enhancement of both LH-T and ACTH-cortisol secretory synchrony by exercise could reflect augmented coupling between brain-testicular and brain-adrenal neural outflow.

Growth hormone is the major regulator of growth and body composition. Pulsatile GH secretion declines exponentially with age. Testosterone replacement is being increasingly offered to older men with age-related low testosterone. Testosterone administration has been shown to stimulate GH secretion. However, little is known about the effect of testosterone aromatization to estradiol on GH pulsatility and its impact on IGF-1 in older men.This randomized controlled proof-of-concept trial investigated the relative effects of testosterone and estradiol on GH pulsatility and IGF-1 in older men with low testosterone.Thirty-seven men, ≥65years with total testosterone350ng/dL were randomized to 5g transdermal testosterone gel (TT), 1mg oral aromatase inhibitor (AI) or placebo daily for 12months. Primary outcome was deconvolution and approximate entropy analyses of pulsatile including basal and entropic modes of secretion performed at baseline and 3months. Secondary outcomes included IGF-1 evaluated at baseline, 3 and 6months.At 3months, mean GH and in IGF-1 were similar between the three groups. At 6months, IGF-1 significantly increased by Δ 15.3±10.3ng/ml in the TT-group compared to placebo (P=0.03). Both intervention groups significantly increased GH pulse frequency (TT-group, P=0.04; AI-group, P=0.05) compared to placebo. The GH secretory-burst mode (duration) significantly decreased in the TT-group (P=0.0018) compared to placebo while it remained unchanged in the AI-group (P=0.059).In older men, testosterone increases GH pulse frequency while the aromatization to estradiol is involved in the rise of IGF-1 levels.

SummaryBackground Gonadotropin-inhibitory hormone (GnIH, human homologue of RFRP-3) suppresses gonadotropin secretion in animal models, but its effects have not been studied in the human. Objective We tested the hypotheses that exogenous GnIH inhibits LH secretion (i) in postmenopausal women and (ii) in men concurrently administered exogenous kisspeptin. Design Following in vitro and in vivo preclinical studies to functionally characterize the GnIH peptide, a dose-finding study (human GnIH: 1·5–150 μg/kg/h, iv for 3 h) was undertaken, and 50 μg/kg/h selected for further evaluation. Five postmenopausal women were administered 50 μg/kg/h iv infusion for 3 h or vehicle on two separate days. Four men were administered kisspeptin-10 (0·3 μg/kg iv bolus) with simultaneous infusion of GnIH (50 μg/kg/h, iv for 3 h) or vehicle. Participants Healthy postmenopausal women (mean age 58 ± 2 years, LH: 30·8 ± 2·9 IU/l, FSH: 78·7 ± 6·4 IU/l, oestradiol

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 athttp://www.reproduction-online.org/content/153/3/267/suppl/DC1.

Background Neurokinin 3 receptor (NK3R) antagonism is a promising novel treatment for menopausal flashes. However, to avoid adverse hormonal effects it is clinically important to first confirm whether gonadotropin and estradiol concentrations change as a result of their administration. Methods Single-center, randomized, double-blind, placebo-controlled, crossover trial of an oral NK3R antagonist (MLE4901) in 28 women aged 40 to 62 years, experiencing >7 hot flashes/24 h; some bothersome or severe (Clinicaltrials.gov, NCT02668185). Weekly serum gonadotropins and estradiol levels were measured using commercially available automated immunoassays a priori. Serum estradiol was also measured post hoc using a highly sensitive direct assay by liquid chromatography tandem mass spectrometry. Hormone levels were compared by the paired sample t tests or by the Wilcoxon matched-pairs signed rank test, as appropriate for the distribution of the data. Results Mean (standard deviation) serum follicle-stimulating hormone (FSH) concentration was not significantly increased when taking MLE4901 (72.07 ± 19.81 IU/L) compared to placebo (70.03 ± 19.56 IU/L), P = .26. Serum estradiol was also not significantly altered, irrespective of which assay method was used (median interquartile range of serum estradiol by immunoassay: placebo 36 ± 3 pmol/L, MLE4901 36 ± 1 pmol/L, P = .21; median serum highly sensitive estradiol: placebo 12 ± 16 pmol/L, MLE4901 13 ± 15 pmol/L, P = .70). However, mean (standard deviation) serum luteinizing hormone concentration significantly decreased with MLE4901 (27.63 ± 9.76 IU/L) compared to placebo (30.26 ± 9.75 IU/L), P = .0024. Implication NK3R antagonists do not increase serum estradiol or FSH despite their reduction in hot flashes. This is clinically significant and highly reassuring for women who have a contraindication to conventional hormone therapy such as prior/existing breast cancer and/or thromboembolism.

Context Central and peripheral administration of peptide YY (PYY) has potent anorectic effects, and PYY analogs are under development as antiobesity treatments. Recent animal data suggest PYY may also influence the reproductive axis; however the effects of PYY on the human reproductive system are unknown. Objective To investigate the effects of PYY administration on the reproductive axis in healthy young men. Design Single-blind, randomized, placebo-controlled crossover study. Setting Clinical Research Facility, Imperial College Healthcare NHS Trust. Participants Eighteen healthy eugonadal men (mean age 24.1 ± 0.9 years, mean body mass index 22.2 ± 0.4 kg/m2). Intervention Eight-hour intravenous infusion of 0.4 pmol/kg/min PYY3-36 or rate-matched vehicle infusion. Main Outcome Measures Number of luteinizing hormone (LH) pulses, LH, follicle stimulating hormone (FSH), and testosterone levels. Results The number of LH pulses (mean number of LH pulses/8 hours: PYY 4.4 ± 0.3 vs vehicle 4.4 ± 0.4, P > .99), LH area under the curve (AUC) (PYY 1503 ± 79 IU.min/L vs vehicle 1574 ± 86 IU.min/L, P = .36), FSH AUC (PYY 1158 ± 513 IU.min/L vs vehicle 1199 ± 476 IU.min/L, P = .49) and testosterone AUC (PYY 10 485 ± 684 IU.min/L vs vehicle 11 133 ± 803 IU.min/L, P = .24) were similar during PYY and vehicle infusions. Conclusions Acute intravenous infusion of 0.4 pmol/kg/min PYY does not affect the reproductive axis in healthy men.

Background Hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurones regulate LH pulsatility. It is widely accepted that the menopausal hot flush (HF) consistently synchronizes with the LH pulse, implicating the hypothalamic KNDy neurones in generating LH pulsatility and HF. Using a modern immunoassay and mathematical modeling, we investigated if the HF and LH pulse were consistently synchronized in menopausal women. Methods Eleven menopausal women (51 to 62 years of age and experiencing ≥7 HF in 24 hours) participated in an 8-hour study. Subjects self-reported HF and underwent peripheral blood sampling every 10 minutes. LH pulsatility was determined using two mathematical models: blinded deconvolution analysis and Bayesian spectrum analysis. The probability that the LH pulse and HF event intervals matched was estimated using the interval distributions observed in our data. Results Ninety-six HFs were self-reported, and 82 LH pulses were identified by blinded deconvolution analysis. Using both models, the probability that the two event intervals matched was low in the majority of participants (mean P = 0.24; P = 1 reflects perfect association). Interpretation Our data challenge the widely accepted dogma that HFs consistently synchronize with an LH pulse and therefore have clinically important therapeutic and mechanistic implications., Using mathematical modeling to determine the relationship between hot flushes and LH pulses in menopausal women challenges the long-held dogma that the menopausal flush synchronizes with the LH pulse.

Background Aging is associated with diminished testosterone (Te) secretion, which may be attributed to Leydig cell dysfunction, decreased pituitary stimulation, and altered Te feedback. Objective To study all regulatory nodes—gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and Leydig cell—in the same cohort of healthy men. Study Design This was a placebo-controlled, blinded, prospectively randomized cross-over study in 40 men, age range 19 to 73 years, and body mass index (BMI) range 20 to 34.3 kg/m2. A submaximal dose of the GnRH antagonist ganirelix was used to assess outflow of GnRH, by calculating the difference between LH output during the control arm and ganirelix arm. Ketoconazole (a steroidogenic inhibitor) was used to estimate feedback, by the difference in LH output during the ketoconazole and control arm. High-dose ganirelix and repeated LH infusions were used to measure testicular responsivity. Blood sampling was performed at 10-minute intervals. Results There were age-related, but not body composition–related decreases in estimated GnRH secretion, the feedback strength of Te on LH, and Leydig cell responsivity to LH, accompanied by changes in approximate entropy. Bioavailable Te levels were negatively related to both age and computed tomography (CT)–estimated abdominal visceral mass (AVF), without interaction between these variables. The LH response to a submaximal dose of GnRH was independent of age and AVF. Conclusion Advancing age is associated with (1) attenuated bioavailable Te secretion caused by diminished GnRH outflow and not by decreased GnRH responsivity of the gonadotrope, (2) diminished testicular responsivity to infused LH pulses, and (3) partial compensation by diminished Te feedback on central gonadotropic regulation.

Background: Oral administration of estradiol (E-2) generally increases GH secretion in postmenopausal women. Oral administration of E-2 is associated with a decrease in IGF-1, whereas parenteral or transdermally administered E-2 may have no effect on GH. The effect of progesterone (P4) on GH secretion has rarely been studied. We hypothesized that moderately increased serum E-2 levels stimulate GH and that P4 modulates E-2-stimulated GH secretion.Study Design: Four parallel groups of randomly assigned postmenopausal women (n = 40). Treatments were saline placebo and oral placebo, saline placebo and oral micronized P4 (3 x 200 mg/d IM), E-2 (5 mg IM) and oral placebo, and E-2 IM and oral micronized P4. Outcome measures were overnight GH secretion (10 hours), stimulated (ghrelin, 0.3 mu g/kg IV bolus) GH secretion, and CT-estimated visceral fat.Results: Intramuscular E-2 administration did not alter nocturnal and ghrelin-stimulated GH secretion. Nocturnal GH secretion was not changed by P4 administration. However, P4 diminished ghrelin-stimulated pulsatile GH release with or without E-2 (average, 7.20 +/- 2.14 and 9.58 +/- 1.97 mu g/L/2 h, respectively; P = 0.045). Respective outcomes for mean GH concentrations and GH peak amplitudes were 0.97 +/- 0.31 and 1.52 mu g/L +/- 0.29 (P = 0.025) and 2.76 +/- 1.04 and 3.95 mu g/L +/- 0.90 (P = 0.031). Ghrelin-stimulated GH secretion correlated negatively with P4 concentration with or without correction for visceral fat area in the regression equation (R = 0.49, P = 0.04, beta = -0.040 +/- 0.016).Conclusions: Low-range physiological E-2 concentrations do not affect spontaneous or ghrelin-stimulated pulsatile GH secretion. Conversely, P4 inhibits ghrelin-stimulated GH secretion in a concentration-dependent fashion. The mechanistic aspects and physiological significance of natural P4's regulation of ghrelin-evoked GH secretion require further study.

Introduction. GH secretion is regulated by hypothalamic neuropeptides, sex steroids, and negative feedback signals across the lifespan. GH secretion in premenopausal women is higher than that in age-matched men, and can be amplified by oral estradiol (E2) in postmenopausal (PM) women. In contrast, the effect of progesterone (P) on GH secretion has rarely been studied in the presence or absence of estrogen repletion. Study design: Four parallel groups of PM women (total 40) received (1) im saline and oral placebo, (2) im saline and oral micronized P (3x200mg/day), (3) E2 im (5 mg) and oral placebo, and (4) E2 im and oral P, respectively. Study parameters were overnight (10 h) and ghrelin-stimulated (0.3 µg/kg) GH secretion. Results: Mean E2 levels increased to 88 and 99 pg/mL and mean P to 10.1 and 20.3 ng/dL. Whereas E2 at this dose did not change GH, P vs placebo reduced pulsatile GH secretion to 7.20±2.14 µg/L/10h vs 9.58±1.97 (P=0.045). Mean GH concentrations and burst amplitudes were respectively 0.97±0.31 and 1.52±0.29 (P=0.025), and 2.76±1.04 and 3.95±0.90 (P=0.031). Visceral fat area, determined by CT, was a strongly negative GH predictor of pulsatile GH secretion, R=-0.53 (P=0.001, β=-0.096±0.026). P levels correlated negatively with GH secretion, with or without AVF or E2 in the regression equation (e.g. pulsatile GH secretion: R=-0.69, P=0.005, β=0.693±0.246). P administration also reduced ghrelin-stimulated GH release (9.6±1.9 vs 7.2±2.1 µg/L, P=0.046). Restricting this analysis to estrogen-treated subjects, P decreased ghrelin-stimulated GH secretion from 10.7±2.7 to 2.6±1.3 µg/L/2 h (P=0.002) in a P concentration-dependent manner, viz., R=-0.49 (P=0.04, β=-0.040±0.018). Conclusions. Early follicular phase E2 levels did not impact spontaneous or ghrelin-stimulated GH concentrations, whereas P inhibited 10-h overnight and 2-h ghrelin-stimulated GH secretion a concentration-dependent fashion. Accordingly, the physiological significance of P on GH regulation requires further studies.

Introduction: Normal fertility requires the presence of adequate nutritional stores. The hormone glucagon-like peptide-1 (GLP-1) is a satiety hormone, which is released by intestinal L-cells during meal ingestion to act as a physiological signal of nutritional intake. GLP-1 reduces appetite and stimulates insulin release. Peripheral GLP-1 crosses the blood brain barrier and GLP-1 receptors (GLP-1R) are present in the arcuate nucleus of the hypothalamus (a neuroendocrine centre that regulates metabolism and reproduction). Both in vitro and in vivo animal studies have demonstrated stimulatory effects of GLP-1 on reproductive hormone release. Thus, GLP-1 acts as a signal of adequate energy intake and may act as a key mediator between metabolic and reproductive systems in animals. As GLP-1R agonists are widely used to treat obesity and diabetes, we sought to determine the effects of GLP-1 on the reproductive axis in humans. Methods: Using a blinded placebo-controlled protocol, 18 healthy men (age 24.7±1yr; mean BMI 22.1±0.4kg/m2) received an 8-hour infusion of 0.8pmol/kg/min of GLP-1 on one study visit and rate-matched vehicle infusion on a separate study visit, in random order. Blood samples were taken every ten minutes during infusions. Visual analogue scales (VAS: 0-10cm) for hunger and nausea were completed by the volunteers pre-, mid- and end-infusion. An ad libitum meal study was performed following the last VAS assessment, after which the infusion was stopped. Luteinizing hormone (LH) pulsatility was determined using blinded deconvolution analysis. Data is presented as mean±SEM. Results: GLP-1 infusion resulted in 14% lower food intake (GLP-1 937±87kcal vs vehicle 1094±87kcal, p=0.03) without increasing nausea (change from baseline VAS score: GLP-1 0.25±0.35cm vs vehicle 0.25±0.22cm, p=0.74). There was no difference in the number of LH pulses over 8hrs (GLP-1 4.7±0.3 vs vehicle 4.4±0.4, p=0.38), LH pulse mass (GLP-1 4.82±0.38IU/L vs vehicle 4.78±0.42IU/L, p=0.94), mean LH (GLP-1 2.86±0.2IU/L vs vehicle 2.75±0.2IU/L, p=0.50), LH AUC (GLP-1 1524±101IU.min/L vs vehicle 1484±88IU.min/L, p=0.70), FSH AUC (GLP-1 1216±112IU.min/L vs vehicle 1210±112IU.min/L, p=0.86) or testosterone AUC (GLP-1 11088±792nmol.min/L vs vehicle 10893±615nmol.min/L, p=0.77). Conclusions: Our results indicate that GLP-1 administration, at a biologically active dose (which reduces food intake), has neither a detrimental nor stimulatory effect on the reproductive axis. This provides important safety data for GLP-1 based therapeutic agents. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Background: Hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurones regulate LH pulsatility. It is widely accepted that the menopausal hot flush (HF) consistently synchronises with the LH pulse. This suggests that the hypothalamic KNDy neurones are implicated in generating LH pulsatility and HF. Using a modern immunoassay and mathematical modelling we investigated if the HF and LH pulse was consistently synchronised in menopausal women. Methods: Eleven menopausal women (51‐62yrs experiencing ≥7 HF/24hrs) attended for an 8hr study where they self‐reported HF and underwent peripheral blood sampling every 10mins. LH pulsatility was determined using two mathematical models. Firstly, blinded deconvolution analysis was used to identify LH peaks and the likelihood that they coincide with self‐reported hot flushes. Secondly Bayesian spectrum analysis was used to determine the probability distribution for LH pulse intervals and the probability of a match between the LH peak and self‐reported hot flush intervals. The probability that the LH pulse and HF event intervals matched was estimated using the interval distributions observed in our data. Results: Ninety‐six HF were self‐reported, and 82 LH pulses were identified by blinded deconvolution analysis. For both methods (BSA and blinded deconvolution analysis) the probability of a match for the intervals between the LH pulses and hot flushes varied greatly between participants (lowest P=0.062 to highest P=0.816, where P=1 reflects a perfect match). For the majority of participants (8/11) the probability of a match was

Normal reproductive functioning is critically dependent on pulsatile secretion of LH. Assessment of LH pulsatility is important for the clinical diagnosis of patients with reproductive disorders, but this is not routinely available in the clinic because of the need for frequent blood sampling coupled with expensive serial immunochemical analysis. The emerging technology of electrochemical aptamer-based (E-AB) sensing could potentially offer a generalizable approach for LH pulsatility measurement in humans. E-AB sensors take advantage of the intrinsic properties of nucleic acid aptamers to specifically bind to a molecular target and undergo a reversible conformational change. The conformational change can be measured through the electrochemical signal response. Aptamers, unlike antibodies, can facilitate low-cost repeat analytical measurements. However, there is currently no aptamer-based LH sensing technology. Here, we report the development and application of a novel antibody-free DNA aptamer-mediated electrochemical analysis to accurately assess LH pulsatility in patients with reproductive disorders. Through selective evolution of ligands by exponential enrichment (SELEX), single-stranded DNA oligonucleotides (aptamers) were identified that bind specifically to LH (and that do not bind to the closely related FSH). The aptamers were integrated into E-AB sensors on a robotic platform (which we term RAPTER) to enable sensitive, rapid and repeatable detection of LH. We next determined if the RAPTER system could measure LH pulsatility in clinical samples from patients with reproductive disorders. We obtained 441 serum samples from 3 patient cohorts - young females with regular menstrual cycles (normal LH pulsatility), menopausal women (disordered LH pulsatility, high LH), and women with hypothalamic amenorrhoea (suppressed/ absent LH pulsatility, low LH). Blood sampling was performed every 10 minutes for 8 hours in these patients and samples were analysed using both the current gold standard LH immunochemical assay and RAPTER. We performed a Bland-Altman analysis and plotted the linear regression to compare the results obtained. The RAPTER assay showed an almost perfect correlation with the clinical immunochemical assay (R2= 0.94). Bayesian Spectrum Analysis was used to determine the effective LH pulse interval from both datasets (clinical assay and RAPTER assay). We are able to distinguish 3 patient cohorts based on the time interval ranges. Taken together, the RAPTER system provides a new approach for LH pulsatility determination by reliably and immediately calculating varying LH concentrations within distinct patient cohorts. This has the potential to transform the clinical care of patients with reproductive disorders as LH pulsatility could be measured easily in clinical practice to aid correct diagnosis. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

The function of the hypothalamo-pituitary-testicular-accessory organ axis is to ensure normal androgenization (including embryonic, infantile, pubertal, and adult sexual maturation), male sexual behavior, and reproductively competent sperm output. Thus the system is important for both the health of individuals and preservation of humankind. All elements in the system acting together as an integrative regulatory network are critical for male reproductive health. Dysfunction may result in ambiguous genitalia, sex reversal, pubertal delay, eunuchism, impaired spermatogenesis, and reduced systemic androgen exposure. Furthermore, partial deficiency of reproductive hormones may contribute to some of the features of male aging, impair recovery from protracted critical illness, and induce visceral adiposity with insulin resistance. This chapter reviews the hypothalamo-pituitary-testicular axis, its components, and their joint regulation. Where practicable, emphasis is on newer discoveries in humans. Hormonal methods to regulate male fertility and to limit age-associated frailty constitute examples of how knowledge of male reproductive physiology is being translated into clinical practice.

CONTEXT: Testosterone (T) increases GH secretion in older men with a relative lack of T, in hypogonadal men of all ages, and in patients undergoing sex reassignment. The role of estradiol (E(2)) in men is less well defined. OBJECTIVE: To assess the contribution of aromatization of T to spontaneous nocturnal and stimulated GH secretion. PARTICIPANTS: Four groups of healthy older men (N = 74, age range 57 to 77 years) were studied. The gonadotropic axis was clamped with the gonadotropin-releasing hormone antagonist degarelix. Three groups received T and one group placebo addback. Two T-replaced groups were treated with anastrozole (an aromatase inhibitor) and either placebo or E(2) addback. MAIN OUTCOME MEASURES: Ten-minute GH concentration profiles were quantified by deconvolution analysis, after overnight (2200 to 0800 hours) sampling, and after combined IV injection of GHRH (0.3 µg/kg) and GHRH-2 (0.3 µg/kg) and withdrawal of a 2-hour somatostatin infusion (1 µg/kg/h). RESULTS: E(2) addback during aromatase inhibition increased basal (P = 0.046), pulsatile (P = 0.020), and total (P = 0.018) GH secretion by 60% to 70%. E(2) did not potentiate GH secretory stimuli. Logarithmically transformed pulsatile GH secretion correlated strongly and positively with concurrent E(2) concentrations overall (P = 0.028) and under anastrozole treatment (P = 0.005). CONCLUSION: E(2) administration in older men transdermally stimulates overnight pulsatile GH secretion. The exact site of E(2) action cannot be ascertained from these experiments but may include hypothalamic loci involved in GH regulation, especially because GH secretagogue effects on somatotrope pituitary cells were not affected.

Context: Interleukin-2 (IL2), a proinflammatory cytokine, is used for treatment of malignancies. Increased cortisol and ACTH were noted, but GH secretion was not investigated in detail. This is the first study in healthy men which uses moderate high IL2 doses as used in cancer treatment. Objective: The goal of this study was to quantify GH secretion after a single sc injection of IL2 in young and older healthy men in relation to dose, age and body composition. Design: This was a placebo-controlled, blinded, prospectively randomized cross-over study in 17 young subjects (mean age 24.1 yr) and 18 older subjects (mean age 63.9 yr). The subjects underwent 24 h of blood sampling at 10-min intervals, starting at 1800 h. At 2000 h IL2 (3 or 6 million units per m2 body surface) or saline was injected sc. Lights were off between 2300 and 0700 h. Outcome Measures: Deconvolution analysis of GH. Abdominal visceral fat (AVF) was calculated from single slice CT. Results: GH secretion (pulsatile and total) was negatively related to AVF (R=0.67, P

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. Abstract: Defects 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.

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.

Optimal blood sampling schemas of GH for the estimation of the 24-hour secretion rate have not been established in acromegalic patients.By censoring available 24-hour GH serum profiles, we investigated the reliability of such simplified schemas. Design, Subjects, and Methods: We used 24-hour serum GH concentration profiles obtained with 10-minute sampling in a large cohort of healthy subjects (n = 130; mean age, 42; range, 18-77 years) and acromegalic patients (n = 87; mean age, 48; range 18-72 years). Patient categories were active disease, surgically cured, and somatostatin analog-treated individuals. The regression coefficients of determination (R(2)) and the linear slopes (β) between 24-hour secretion rates or mean concentrations (144 samples) on the one hand and mean values with less frequent sampling on the other, decreased in controls and in patients a short (1-2 weeks) or long (2-5 years) time after successful surgery. By contrast, the regression parameters remained essentially unchanged in patients with active acromegaly and those under GH suppressive treatment. Excellent correlations (R(2) ≥ 0.90) without GH underestimation existed between mean GH of daytime profiles and mean GH of 24-hour profiles and GH secretion rates estimated by deconvolution in patients with active acromegaly and patients treated with somatostatin analogs.Simplified sampling schemes, particularly a day profile, can be used for the estimation of GH secretion in patients with active acromegaly and under medical treatment. However, in healthy controls and patients after successful surgery, prolonged and frequent sampling schemes, at least at 2-hour intervals, reliably reflect 24-hour secretion.

Neuroendocrine systems control many of the most fundamental physiological processes, e.g., reproduction, growth, adaptations to stress, and metabolism. Each such system involves the hypothalamus, the pituitary, and a specific target gland or organ. In the quantification of the interactions among these components, biostatistical modeling has played an important role. In the present article, five key challenges to an understanding of the interactions of these systems are illustrated and discussed critically.