Chen, C

Placenta-derived hormones including growth hormone (GH) in humans contribute to maternal adaptation to pregnancy, and intermittent maternal GH administration increases foetal growth in several species. Both patterns and abundance of circulating GH are important for its activity, but their changes during pregnancy have only been reported in humans and rats. The aim of the present study was to characterise circulating profiles and secretory characteristics of GH in non-pregnant female mice and throughout murine pregnancy. Circulating GH concentrations were measured in whole blood (2 μL) collected every 10 min for 6 h in non-pregnant diestrus female C57Bl/6J mice ( n = 9), and pregnant females at day 8.5–9.5 (early pregnancy, n = 8), day 12.5–13.5 (mid-pregnancy, n = 7) and day 17.5 after mating (late pregnancy, n = 7). Kinetics and secretory patterns of GH secretion were determined by deconvolution analysis, while orderliness and regularity of serial GH concentrations were calculated by approximate entropy analysis. Circulating GH was pulsatile in all groups. Mean circulating GH and total and basal GH secretion rates increased markedly from early to mid-pregnancy, and then remained elevated. Pulse frequency and pulsatile GH secretion rate were similar between groups. The irregularity of GH pulses was higher in all pregnant groups than that in non-pregnant mice. Increased circulating GH in murine pregnancy is consistent with an important role for this hormone in maternal adaptation to pregnancy and placental development. The timing of increased basal secretion from mid-pregnancy, concurrent with the formation of the chorioallantoic placenta and initiation of maternal blood flow through it, suggests regulation of pituitary secretion by placenta-derived factors.

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

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

Ghrelin, a gut hormone originating from the post‐translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS‐R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin‐O‐acyltransferase (GOAT), conferring selective binding to the GHS‐R1a receptor via acylated ghrelin. Complete loss of preproghrelin‐derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl‐ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat−/− mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat−/− mice compared to age‐matched wild‐type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH‐dependent liver genes. Of interest, circulating levels of insulin‐like growth factor (IGF)‐1 were elevated in goat−/− mice. This rise in circulating levels of IGF‐1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF‐1 release in goat−/− mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF‐1 release and growth in goat−/− mice.

AbstractGhrelin is a gut hormone processed from the proghrelin peptide acting as the endogenous ligand of the GH secretagogue receptor 1a. The regulatory role of endogenous ghrelin on pulsatile GH secretion and linear growth had to be established. The aim of the present study was to delineate the endogenous actions of preproghrelin on peripheral and central components of the GH axis. Accordingly, the ultradian pattern of GH secretion was measured in young and old preproghrelin-deficient males. Blood samples were collected by tail bleeding every 10 minutes over a period of 6 hours. Analysis of the GH pulsatile pattern by deconvolution showed that GH was secreted in an ultradian manner in all genotypes, with major secretory peaks occurring at about 3-hour intervals. In older mice, the peak number was reduced and secretion was less irregular compared with younger animals. Remarkably, in young Ghrl−/− mice, the amplitude of GH secretory bursts was significantly reduced. In older mice, however, genotype differences were less significant. Changes in GH pulsatility in young Ghrl−/− mice were associated with a tendency for reduced GH pituitary contents and plasma IGF-I concentrations, but with only a minor impact on linear growth. In Ghrl+/− mice, despite reduced Acyl ghrelin to des-acyl ghrelin ratio, GH secretion was not impaired. Ghrelin deficiency was not associated with a reduction in hypothalamic GHRH content or altered response to GHRH stimulation. Therefore, reduction in GHRH production and/or sensitivity do not primarily account for the altered GH pulsatile secretion of young Ghrl−/− mice. Instead, GHRH expression was elevated in young but not old Ghrl−/− mice, suggesting that differential compensatory responses resulting from the absence of endogenous ghrelin is occurring according to age. These results show that endogenous ghrelin is a regulator of GH pulse amplitude in growing mice but does not significantly modulate linear growth.

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1G93A mice throughout the course of ALS disease. Male wild-type and hSOD1G93A mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1G93A mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1G93A mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-μL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

The age‐associated decline in growth hormone (GH) secretion may be a consequence of the reduction in the number of GH‐releasing hormone (GHRH) positive neurones. However, it remains unclear whether an alteration in the number or distribution of somatostatin (SST) neurones contributes to this change. In the present study, we characterised the role of SST in modulating the change in pulsatile GH secretion in male C57Bl/6J mice throughout puberty and into early adulthood. We assessed pulsatile GH secretion in mice at 4, 8 and 16 weeks of age. These ages correspond to early pubertal, early adulthood and adulthood, respectively. We show an elevation in peak, total and pulsatile GH secretion coinciding with periods of rapid linear growth. Using in situ hybridisation and morphometric methods, we mapped the distribution of Sst mRNA expression within the mouse brain relative to this change in pulsatile GH secretion. The results obtained show that altered pulsatile GH secretion in male mice from 4–16 weeks of age does not coincide with a significant change in the number of Sst mRNA expressing neurones or an abundance of Sst mRNA expression throughout the arcuate nucleus (ARC) and periventricular nucleus (PeV). Rather, we observed a progressive decline in Sst mRNA expressing neurones within subnuclei of the paraventricular nucleus at this time. We conclude that structural changes in Sst expression within the PeV and ARC may not reflect the observed decline in pulsatile GH secretion in mice from puberty into early adulthood.

The transition between puberty and adulthood is accompanied by a slowing in linear growth. Although GH is a key factor that drives somatic development into adulthood, early adulthood coincides with a reduction in circulating levels of GH. To this extent, a pathological decline in postpubertal GH secretion is detrimental to attainment of peak lean muscle mass and bone mass and promotes adiposity and increases susceptibility to the development of obesity in adulthood. Here we characterized pulsatile GH secretion in C57BL/6J mice at 12 and 16 wk of age. Deconvolution analysis of these measures reveals a reduction in pulsatile GH secretion between 12 and 16 wk of age. Dietary intervention with high-fat feeding at 8 wk of age results in a significant increase in adiposity, the development of glucose intolerance, and hyperinsulinemia. We show the exacerbation of the age-associated decline in pulsatile GH secretion in high-fat-fed mice after 4 wk of dietary intervention (at 12 wk of age), and a further suppression of pulsatile GH secretion by 8 wk of dietary intervention (at 16 wk of age). Suppressed pulsatile secretion of GH did not coincide with an elevation in circulating free fatty acids. Rather, we observed increased hepatic triglyceride content and an eventual decrease in circulating levels of IGF-I. Given the established role of GH in maintaining healthy aging, we anticipate that an advancing of the age-associated decline in pulsatile GH secretion as a consequence of dietary-induced weight gain may have long-term ramifications on adult health.

GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1G93A transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1G93A transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1G93A mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1G93A mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1G93A mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.