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Context FSH may have independent actions on bone remodeling and body fat regulation. Cross-sectionally, we have shown that serum FSH is associated with bone mineral density (BMD) and body fat in older postmenopausal women, but it remains unknown whether FSH predicts bone and fat changes. Objective We examined whether baseline FSH level is associated with subsequent bone loss or body composition changes in older adults. Setting, Design, Participants We studied 162 women and 158 men (mean age 82 ± 4 years) from the Age, Gene/Environment Susceptibility (AGES)-Bone Marrow Adiposity cohort, a substudy of the AGES-Reykjavik Study of community-dwelling older adults. Skeletal health and body composition were characterized at baseline and 3 years later. Main Outcomes Annualized change in BMD and body composition by dual-energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT). Models were adjusted for serum estradiol and testosterone levels. Results There was no evidence for an association between baseline FSH level and change in BMD or body composition by DXA or QCT. For femoral neck areal BMD, adjusted mean difference (95% CI) per SD increase in FSH was 1.3 (-0.7 to 3.3) mg/cm2/y in women, and -0.2 (-2.6 to 2.2) mg/cm2/y in men. For visceral fat, adjusted mean difference (95% CI) per SD increase in FSH was 1.80 (-0.03 to 3.62) cm2/y in women, and -0.33 (-3.73 to 3.06) cm2/y in men. Conclusions Although cross-sectional studies and studies in perimenopausal women have demonstrated associations between FSH and BMD and body composition, in older adults, FSH level is not associated with bone mass or body composition changes.

Objectives: This study aimed to determine the effect of bariatric surgery-induced weight loss on bone marrow adipose tissue (BMAT) and bone mineral density (BMD) in postmenopausal, nondiabetic women. Methods: A total of 14 postmenopausal, nondiabetic women with obesity who were scheduled for laparoscopic Roux-en-Y gastric bypass surgery (RYGB) were included in this study. Vertebral bone marrow fat signal fraction was determined by quantitative chemical shift magnetic resonance imaging, and vertebral volumetric BMD (vBMD) was determined by quantitative computed tomography before surgery and 3 and 12 months after surgery. Data were analyzed by linear mixed model. Results: Body weight [mean (SD)] decreased after surgery from 108 (13) kg at baseline to 89 (12) kg at 3 months and 74 (11) kg at 12 months (P < 0.001). BMAT decreased after surgery from 51% (8%) at baseline to 50% (8%) at 3 months and 46% (7%) at 12 months (P = 0.004). vBMD decreased after surgery from 101 (26) mg/cm 3 at baseline to 94 (28) mg/cm 3 at 3 months (P = 0.003) and 94 (28) mg/cm 3 at 12 months (P = 0.035). Changes in BMAT and vBMD were not correlated (ρ = −0.10 and P = 0.75). Calcium and vitamin D concentrations did not change after surgery. Conclusions: RYGB decreases both BMAT (after 12 months) and vBMD (both after 3 months and 12 months) in postmenopausal, nondiabetic women. Changes in BMAT and vBMD were not correlated. These findings suggest that BMAT does not contribute to bone loss following RYGB.

Background: Interleukin-2 (IL-2), one of the proinflammatory cytokines, is used in the treatment of certain malignancies. In some studies, transient increases in cortisol and ACTH secretion occurred. Thus, this agent may be used as an experimental probe of adrenal cortisol secretion. Objective: This study quantifies the effects of low and moderate doses of IL-2 on cortisol secretion and assesses the modulation by age, dose and body composition. Site: Mayo Clinical Translational Research Unit. Subjects: Study comprised 35 healthy men, 17 young and 18 older. Methods: Randomized prospective double-blind saline-controlled study of IL-2 administration in two doses with concurrent 10-min blood sampling for 24 h. Outcome measures: Deconvolution analysis and approximate entropy of cortisol secretion. Results: Low-dose IL-2 administration increased nocturnal pulsatile cortisol secretion from 1460 ± 160 to 2120 ± 220 nmol/L/8 h in young subjects and from 1680 ± 105 to 1960 ± 125 nmol/L/8 h (treatment P P = 0.02). Comparable results were obtained for total cortisol secretion (P treatment P = 0.005). The higher IL-2 dose caused a large increase in young (P P = 0.90) subjects. This dose also increased approximate entropy from 0.877 ± 0.041 to 1.024 ± 0.049 (P = 0.008), pointing to reduced secretory orderliness. Incremental cortisol (nocturnal) secretion correlated negatively with visceral fat mass (R = −0.41, P = 0.019). Conclusion: In healthy men, IL-2 injection drives pulsatile cortisol secretion in a dose-dependent way in young, but not older, individuals and erodes cortisol secretory orderliness at a higher dose in young subjects. Cortisol responses are diminished with increasing abdominal visceral fat mass.

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.

In this review we discuss skeletal adaptations to the demanding situation of pregnancy and lactation. Calcium demands are increased during pregnancy and lactation, and this is effectuated by a complex series of hormonal changes. The changes in bone structure at the tissue and whole bone level observed during pregnancy and lactation appear to largely recover over time. The magnitude of the changes observed during lactation may relate to the volume and duration of breastfeeding and return to regular menses. Studies examining long-term consequences of pregnancy and lactation suggest that there are small, site-specific benefits to bone density and that bone geometry may also be affected. Pregnancy- and lactation-induced osteoporosis (PLO) is a rare disease for which the pathophysiological mechanism is as yet incompletely known; here, we discuss and speculate on the possible roles of genetics, oxytocin, sympathetic tone and bone marrow fat. Finally, we discuss fracture healing during pregnancy and lactation and the effects of estrogen on this process.

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.

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked migraine pain remain unknown. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. The CGRP-mediated neuronal/Schwann cell pathway is critical to mediate allodynia associated with neurogenic inflammation, thus contributing to the pro-migraine action of CGRP.

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.

Estrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-κB ligand (RANKL). RANKL is secreted by osteoblasts and osteocytes, but more recently bone marrow (pre)adipocytes have also been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT). The aim of this study was to determine the effect of ovariectomy (OVX) on RANKL protein expression by bone marrow adipocytes in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice ( n = 20) were randomized to sham surgery (Sham) or OVX. After 4 wk animals were euthanized. BMAT volume fraction (BMAT volume/marrow volume) was quantified by polyoxometalate-based contrast-enhanced nano-computed tomography. The percentage of RANKL-positive bone marrow adipocytes (RANKL-positive bone marrow adipocytes/total adipocytes) and the percentage of RANKL-positive osteoblasts covering the bone surface (bone surface covered in RANKL-positive osteoblasts/total bone surface) were quantified in the distal metaphysis of immunohistochemically stained sections of the left femur. The effects of OVX were analyzed by Student’s t test or Mann–Whitney U test. RANKL was detected in osteoblasts, osteocytes, and bone marrow adipocytes. OVX significantly increased mean percentage of RANKL-positive bone marrow adipocytes [mean (SD): Sham 42 (18)%; OVX 64 (12)%; P = 0.029] as well as BMAT volume/marrow volume [median (interquartile range): Sham 1.4 (4.9)%; OVX 7.2 (7.3)%; P = 0.008] compared with Sham. We show that OVX increased both the percentage of RANKL-positive bone marrow adipocytes and the total BMAT volume fraction in C3H/HeJ mice. Therefore, RANKL produced by bone marrow adipocytes could be an important contributor to OVX-induced bone loss in C3H/HeJ mice.

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 Follicle-stimulating hormone (FSH) concentrations increase during the perimenopausal transition and remain high after menopause. Loss of bone mineral density (BMD) and gain of bone marrow adiposity (BMA) and body fat mass also occur during this time. In mice, blocking the action of FSH increases bone mass and decreases fat mass. Objective To investigate the associations between endogenous FSH levels and BMD, BMA, and body composition in older adults, independent of estradiol and testosterone levels. Design, Setting, and Participants Older adults from the AGES-Reykjavik Study, an observational cohort study. Main Outcome Measures Areal BMD, total body fat, and lean mass were measured with dual-energy x-ray absorptiometry. Lumbar vertebral BMA was measured by 1H-magnetic resonance spectroscopy. Volumetric BMD and visceral and subcutaneous adipose tissue (VAT, SAT) areas were measured with quantitative computed tomography. The least squares means procedure was used to determine sex hormone–adjusted associations between quartiles of serum FSH and BMD, BMA, and body composition. Results In women (N = 238, mean age 81 years), those in the highest FSH quartile, compared with the lowest quartile, had lower adjusted mean spine integral BMD (−8.6%), lower spine compressive strength index (−34.8%), higher BMA (+8.4%), lower weight (−8.4%), lower VAT (−17.6%), lower lean mass (−6.1%), and lower fat mass (−11.9%) (all P < 0.05). In men, FSH level was not associated with any outcome. Conclusions Older postmenopausal women with higher FSH levels have higher BMA, but lower BMD and lower fat and lean mass, independent of estradiol and testosterone levels. Longitudinal studies are needed to better understand the underlying mechanisms.

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.

Background and Purpose: Muscle weakness and bone fragility are both associated with hip fracture. In general, muscle contractions create forces to the bone, and bone strength adapts to mechanical loading through changes in bone architecture and mass. However, the relationship between impairment of muscle and bone function remain unclear. In particular, the associations of muscle with properties of proximal femur cortical and trabecular bone are still not well understood. The aim of this study was to explore the associations of hip/thigh muscle density (CT attenuation value in Hounsfield units) and size with cortical and trabecular bone mineral density (BMD) of the proximal femur. Materials and Methods: Three-dimensional quantitative computed tomography (QCT) imaging of the lumber, hip and mid-thigh was performed in a total of 301 participants (mean age 68.4 ± 6.1 years, 194 women and 107 men) to derive areal BMD (aBMD) and volumetric BMD (vBMD). Handgrip strength (HGS) and the Timed Up and Go (TUG) test were also performed. From the CT images, cross-sectional area (CSA), and density were determined for the gluteus maximus muscle (G.MaxM), trunk muscle at the vertebrae L2 level, and mid-thigh muscle. Multivariate generalized linear models were applied to assess associations. Results: Total hip (TH) aBMD was associated significantly with G.MaxM CSA (men: P = 0.042; women: P < 0.001) and density (men: P = 0.012; women: P = 0.043). In women, 0.035 cm2 of mid-thigh CSA (95% CI, 0.014–0.057; P = 0.002) increased per SD increase in TH aBMD, but this significance was not observed in men (P = 0.095). Trunk muscle density and CSA were not associated with proximal femur BMD. The associations of hip/thigh muscle parameters with femoral neck BMD were weaker than those with trochanter and intertrochanter BMD. Furthermore, compared to muscle density, muscle CSA showed better associations with vBMD. G.MaxM CSA was associated with trochanter (TR) Cort. vBMD in men (β, 19.898; 95% CI, 0.924–38.871; P = 0.040) and in women (β, 15.426; 95% CI, 0.893–29.958; P = 0.038). Handgrip strength was only associated with TR aBMD (β, 0.038; 95% CI, 0.006–0.070; P = 0.019) and intertrochanter aBMD (β, 0.049; 95% CI, 0.009–0.090; P = 0.016) in men. Conclusions: We observed positive associations of the gluteus and thigh muscle size with proximal femur volumetric BMD. Specifically, the gluteus maximus muscle CSA was associated with trochanter cortical vBMD in both men and women.

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.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose. ispartof: Frontiers In Endocrinology vol:11 ispartof: location:Switzerland status: published

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.

We assessed the associations between changes in total and abdominal fat and changes in biomarkers for breast cancer risk using data of the SHAPE-2 trial. In the SHAPE-2 trial, 243 postmenopausal overweight women were included. The intervention in this trial consisted of 5-6 kg weight loss either by diet only or exercise plus diet. After 16 weeks, we measured serum sex hormones, inflammatory markers, total body fat (measured by DEXA scan) and intra and subcutaneous abdominal fat (measured by MRI). Associations between changes in different body fat depots and biomarkers were analysed by linear regression using the study cohort irrespective of randomisation to make maximal use of the distribution of changes in fat measures. We found that a loss in total body fat was associated with favourable changes in free oestradiol, free testosterone, leptin and sex hormone binding globulin (SHBG). The loss of intra-abdominal fat was associated with a decrease in free testosterone, hsCRP and leptin, and an increase in SHBG. In the multivariable analysis, the best fitted models for the biomarkers free oestradiol, SHBG leptin and adiponectin included only total body fat. For free testosterone, this was subcutaneous abdominal fat, and for hsCRP and IL-6, only intra-abdominal fat change was important. For IL-6 and adiponectin, however, associations were weak and not significant. We conclude that, in our population of healthy overweight postmenopausal women, loss of fat at different body locations was associated with changes in different types of biomarkers, known to be related to risk of breast cancer.

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.

Aggressive spread and liver metastases are predominant features of pancreatic ductal adenocarcinoma (PDAC). This study investigates activation of PDAC circulating tumor cells (CTC) and immunosuppression in the portal venous system.Portal venous and peripheral blood were collected during pancreaticoduodenectomy from patients with PDAC (n = 21) or other non-PDAC pancreatic conditions (n = 20). Circulating tumor cells were isolated by fluorescence-activated cell sorting and characterized for messenger RNA (mRNA) expression and acetylated chromatin encoding K-RAS exon 12 mutation (K-RASmut). Myeloid-derived suppressor cells (MDSC) were identified using flow cytometry.Pancreatic ductal adenocarcinoma K-RASmut mRNA expression in portal venous blood CTC was significantly elevated compared with preoperative and postoperative peripheral blood (P = 0.0123 and P = 0.0246, respectively). There was no significant variation in total CTC numbers between portal and peripheral blood.Portal venous M-MDSC were elevated compared with peripheral blood in PDAC patients (P = 0.0065). M-MDSC increases correlated with K-RASmut mRNA-expressing CTC present in PDAC portal blood (P0.0001).Association of MDSC with active CTC in portal venous blood may support immunosuppression within the portal venous circulation to promote PDAC CTC survival.

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.

Human Prader–Willi syndrome (PWS) is characterised by impairments of multiple systems including the growth hormone (GH) axis and skeletal growth. To address our lack of knowledge of the influence of PWS on skeletal integrity in mice, we have characterised the endocrine and skeletal phenotype of the PWS-IC del mouse model for ‘full’ PWS and determined the impact of thermoneutrality. Tibial length, epiphyseal plate width and marrow adiposity were reduced by 6, 18 and 79% in male PWS-IC del mice, with osteoclast density being unaffected. Similar reductions in femoral length accompanied a 32% reduction in mid-diaphyseal cortical diameter. Distal femoral Tb.N was reduced by 62%, with individual trabeculae being less plate-like and the lattice being more fragmented (Tb.Pf increased by 63%). Cortical strength (ultimate moment) was reduced by 26% as a result of reductions in calcified tissue strength and the geometric contribution. GH and prolactin contents in PWS-IC del pituitaries were reduced in proportion to their smaller pituitary size, with circulating IGF-1 concentration reduced by 37–47%. Conversely, while pituitary luteinising hormone content was halved, circulating gonadotropin concentrations were unaffected. Although longitudinal growth, marrow adiposity and femoral geometry were unaffected by thermoneutrality, strengthened calcified tissue reversed the weakened cortex of PWS-IC del femora. While underactivity of the GH axis may be due to loss of Snord116 expression and impaired limb bone geometry and strength due to loss of Magel2 expression, comprehensive analysis of skeletal integrity in the single gene deletion models is required. Our data imply that thermoneutrality may ameliorate the elevated fracture risk associated with PWS.

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.

Platelets play a pivotal role in stimulating liver regeneration after partial hepatectomy in rodents and humans. Liver regeneration in rodents is delayed when platelets are inhibited. However, the exact mechanisms whereby platelets accumulate and promote liver regeneration remain uncertain. Thrombin-dependent intrahepatic fibrin(ogen) deposition was recently reported after partial hepatectomy (PHx) in mice, but the role of fibrin(ogen) deposits in liver regeneration has not been investigated. We tested the hypothesis that fibrin(ogen) contributes to liver regeneration by promoting intrahepatic platelet accumulation and identified the trigger of rapid intrahepatic coagulation after PHx. PHx in wild-type mice triggered rapid intrahepatic coagulation, evidenced by intrahepatic fibrin(ogen) deposition. Intrahepatic fibrin(ogen) deposition was abolished in mice with liver-specific tissue factor deficiency, pinpointing the trigger of coagulation after PHx. Direct thrombin activation of platelets through protease-activated receptor-4 did not contribute to hepatocyte proliferation after PHx, indicating that thrombin contributes to liver regeneration primarily by driving intrahepatic fibrin(ogen) deposition. Fibrinogen depletion with ancrod reduced both intrahepatic platelet accumulation and hepatocyte proliferation after PHx, indicating that fibrin(ogen) contributes to liver regeneration after PHx by promoting intrahepatic platelet accumulation. Consistent with the protective function of fibrin(ogen) in mice, low postoperative plasma fibrinogen levels were associated with liver dysfunction and mortality in patients undergoing liver resection. Moreover, increased intrahepatic fibrin(ogen) deposition was evident in livers of patients after liver resection but was remarkably absent in patients displaying hepatic dysfunction postresection. The results suggest a novel mechanism whereby coagulation-dependent intrahepatic fibrin(ogen) deposition drives platelet accumulation and liver regeneration after PHx.

In patients with postmenopausal osteoporosis low bone volume is associated with high bone marrow adipose tissue (MAT). Moreover, high MAT is associated with increased fracture risk. This suggests an interaction between MAT and bone turnover, however literature remains equivocal. Estrogen treatment decreases MAT, but the effect of raloxifene, a selective estrogen receptor modulator (SERM) registered for treatment of postmenopausal osteoporosis, on MAT is not known. The aim of this study is 1] to determine the effect of raloxifene on MAT and 2] to determine the relationship between MAT and bone turnover in patients with osteoporosis.Bone biopsies from the MORE trial were analyzed. The MORE trial investigated the effects of raloxifene 60 or 120. mg per day versus placebo on bone metabolism and fracture incidence in patients with postmenopausal osteoporosis. We quantified MAT in iliac crest biopsies obtained at baseline and after 2. years of treatment (n = 53; age 68.2. ±. 6.2. years).Raloxifene did not affect the change in MAT volume after 2years compared to baseline (placebo: 1.89±10.84%, raloxifene 60mg: 6.31±7.22%, raloxifene 120mg: -0.77±10.72%), nor affected change in mean adipocyte size (placebo: 1.45 (4.45) μm, raloxifene 60mg: 1.45 (4.35) μm, raloxifene 120mg: 0.81 (5.21) μm). Adipocyte number tended to decrease after placebo treatment (-9.92 (42.88) cells/mm2) and tended to increase during raloxifene 60mg treatment (13.27 (66.14) cells/mm2) while adipocyte number remained unchanged in the raloxifene 120mg group, compared to placebo (3.06 (39.80) cells/mm2, Kruskal-Wallis p =0.055, post hoc: placebo vs raloxifene 60mg p =0.017). MAT volume and adipocyte size were negatively associated with osteoclast number at baseline (R2 =0.123, p =0.006 and R2 =0.098, p =0.016 respectively). Furthermore adipocyte size was negatively associated with osteoid surface (R2 =0.067, p =0.049). Finally, patients with vertebral fractures had higher MAT volume (50.82 (8.80)%) and larger adipocytes (55.75 (3.14) μm) compared to patients without fractures (45.58 (12.72)% p =0.032, 52.77 (3.73) μm p =0.004 respectively).In conclusion, raloxifene did not affect marrow adipose tissue, but tended to increase adipocyte number compared to placebo. At baseline MAT volume and adipocyte size were associated with bone resorption, and adipocyte size was associated with osteoid surface, suggesting an interaction between bone marrow adipocytes and bone turnover. In addition, we found that high MAT volume and larger adipocyte size are associated with prevalent vertebral fractures in postmenopausal women with osteoporosis, indicating that adipocyte size affects bone quality independent of bone volume.

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.