Multiligand specificity and wide tissue expression of GPRC6A reveals new endocrine networks

Emerging evidence supports the hypothesis that the skeleton is an endocrine organ that regulates energy metabolism through the release of the osteoblast-derived hormone, osteocalcin (Ocn). This bone-pancreas endocrine network is controversial because important gaps remain to be filled in our knowledge of the physiological effects of Ocn in multiple organs and the complex alterations in other hormonal networks induced by Ocn administration. A key step toward understanding the integrative regulation of energy metabolism by bone is the identification of GPCR family C group 6 member A (GPRC6A) as the Ocn receptor. GPRC6A is an amino acid-sensing G protein-coupled receptor highly expressed in β-cells and is activated by recombinant Ocn in vitro and in vivo but that is widely expressed in tissues other than the pancreas and is capable of sensing multiple structurally unrelated ligands, including l-amino acids, cations, and anabolic steroids in addition to Ocn. The broad expression and multiligand specificity of GPRC6A is identifying both systemic and paracrine regulation of seemingly disparate biological processes, ranging from energy metabolism, sexual reproduction, hypothalamic-pituitary function, bone formation, and prostate cancer. Consistent with the existence of more complex endocrine networks, ablation of GPRC6A in Gprc6a(-/-) mice results in complex metabolic abnormalities, including obesity, glucose intolerance, hepatic steatosis, insulin resistance, hyperphosphatemia, osteopenia, plus several hormonal abnormalities, including decreased circulating testosterone, IGF-I, and insulin and increased estradiol, LH, GH, and leptin. Recombinant Ocn also regulates testosterone production by the testes and male fertility through a GPRC6A-dependent mechanism, and testosterone regulation of LH secretion is abnormal in Gprc6a(-/-) mice. Thus, GPRC6A, as the biologically relevant receptor for Ocn, defines not only a molecular mechanism for linking bone metabolism with metabolic regulation of β-cells and sexual reproduction but also as a receptor shared by testosterone and dietary factors, and it is also involved in multiple endocrine networks integrating the functions of pancreas, muscle, liver, fat, testes, bone, and the hypothalamic-pituitary axis with alterations in both environmental and endogenous ligands.     

A paradigm of integrative physiology, the crosstalk between bone and energy metabolisms

Thanks to integrative physiology, new relationships between organs and homeostatic functions have emerged. This approach to physiology based on a whole organism approach has allowed the bone field to make fundamental progress.In the last decade, clinical observations and scientific evidences in vivo have uncovered that fat with leptin controls bone mass through brain including a hypothalamic relay and sympathetic nervous system.The finding that energy metabolism affects bone remodelling suggested that in an endocrine perspective, a feedback loop should exist. Beside its classical functions, bone can now be considered as a true endocrine organ secreting osteocalcin, a hormone pharmacologically active on glucose and fat metabolism. Indeed osteocalcin stimulates insulin secretion and β-cell proliferation. Simultaneously, osteocalcin acts on adipocytes to induce Adiponectin which secondarily reduce insulin resistance. This cross regulation between bone and energy metabolism offers novel therapeutic targets in type 2 diabetes and osteoporosis.     

Exploring the role of ghrelin as novel regulator of gonadal function.

In mammals, proper gonadal function critically relies on a complex regulatory network of systemic (endocrine) and locally-produced (paracrine and autocrine) signals. Among others, a number of factors primarily involved in the control of energy balance and metabolism have been proven as putative modulators of the gonadal axis, thus providing the basis for the well-known link between energy homeostasis and fertility. Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R), has been recently demonstrated as a pleiotropic regulator involved in a large array of endocrine and non-endocrine functions, including food intake and energy balance. However, the potential reproductive role of this newly discovered molecule has remained largely neglected. Yet, we review herein several lines of evidence which strongly suggest the involvement of ghrelin in the control of some aspects of gonadal function. Thus, expression of ghrelin has been demonstrated in mature Leydig cells of rat and human testis, as well as in steroidogenically active luteal and interstitial hilus cells of the ovary. In addition, expression of the functional ghrelin receptor, the GHS-R type 1a, has been shown in Sertoli and Leydig cells of the testis, and in follicular, luteal, surface epithelial and interstitial hilus cells of the ovary. In terms of function, ghrelin has been proven to dose-dependently inhibit testicular testosterone secretion in vitro, and to modulate Leydig cell proliferation in vivo, as well as the expression of relevant testicular genes, such as that encoding stem cell factor. Moreover, extragonadal actions of ghrelin upon the reproductive axis have been also reported, as ghrelin was able to suppress LH secretion in vivo and to decrease LH responsiveness to GnRH in vitro. In summary, the data so far available strongly suggest that, through local and/or systemic actions, ghrelin operates as a novel regulator of gonadal function that may contribute to the integrated control of energy balance and reproduction.     

Spatiotemporal regulation of the two atrial natriuretic peptide receptors in testis

By interacting with a guanylyl cyclase (GC) activity-containing receptor, termed GC-A, atrial natriuretic peptide (ANP) acts as a regulator of blood pressure and fluid volume homeostasis. High expression levels of GC-A in the testis and reported effects of ANP on testosterone secretion by Leydig cells are indicative of important local functions in this organ. Here we show, based on radioligand receptor labeling and immunological approaches, that seminiferous tubules rather than Leydig cells are the predominant GC-A expression sites in the rat testis. Functional activity was proved by ANP- induced cGMP accumulation in isolated seminiferous tubules. Although ontogenetic studies revealed a massive increase in GC-A levels during sexual maturation, the so-called natriuretic peptide clearance receptor, another type of ANP receptor proposed to locally control the availability of natriuretic peptides, was found to be expressed predominantly before puberty, exceeding the level of GC-A expression at this time. Natriuretic peptide clearance receptor also shows a distinct distribution pattern surrounding the seminiferous tubules. These findings raise the possibility of novel physiological roles for ANP and cGMP in the testis related to germ cell maturation and/or the regulation of the onset of puberty and suggest that the two ANP receptors function in a coordinated manner at this target organ.     

Endocrine regulation of testicular function in men: implications for contraceptive development

The testicle has two important and complimentary functions in the adult male: the production of sperm and the synthesis of testosterone. Testicular function is directed by the central nervous system through its effects on the pituitary gland. Precise regulation of testicular function is accomplished by an elegant endocrine feedback loop in which the secretion of pituitary gonadotropins is stimulated by the pulsatile release of GnRH from the hypothalamus and modulated by testicular hormones. Inhibin, testosterone and its metabolites inhibit the secretion of the gonadotropins both directly at the pituitary and centrally at the level of the hypothalamus. In the testis, LH mainly stimulates testosterone production and FSH promotes spermatogenesis, but the exact details of these actions are only now being elucidated. The discovery of novel mutations in gonadotropin structure and signaling provide unique insight into the roles of these crucial hormones in the development and function of the male reproductive axis. A key goal of research into hormonal regulation of testicular function is the development of reversible, safe and effective male hormonal contraceptives. Recent promising trials of hormonal contraceptive combinations may soon bring the promise of male contraception to fruition.     

Reproductive hormone secretion and spermatogenic function in thyroidectomized rams receiving graded doses of exogenous thyroxine

This study aimed to obtain a better understanding of the relationship between circulating thyroxine (T4) concentrations and reproductive endocrine function in the ram. Mature Merino rams were thyroidectomized and supplemented with 0, 30, 100 and 300% of normal T4 for 10 weeks. Thyroidectomy had no apparent effect on spermatogenic function but interfered with sperm maturation, the latter being returned to normal by 30% T4 replacement. Circulating testosterone levels were reduced by thyroidectomy and restored to control levels by 30% T4; when T4 levels were supranormal (300%), circulating testosterone levels were again reduced. The lowered circulating testosterone levels in thyroidectomized rams occurred as a result of suppressed testosterone secretion from the testis, observed under basal conditions and also following LH-releasing hormone (LHRH) and human chorionic gonadotrophin injection. In thyroidectomized rams, sex hormone binding globulin (SHBG) levels were depressed without changes in testosterone clearance rate (TCR), while in rams with supranormal T4 levels, TCR was increased without changes in SHBG levels. Subnormal levels of T4 also restored to normal the reduced LH pulse frequency in thyroidectomized rams. Reduced LH pulse frequency, together with diminished LH release following LHRH injection in thyroidectomized rams, suggested effects of T4 at the hypothalamo-pituitary axis. The present study demonstrates that complete lack of thyroid hormones suppresses normal reproductive endocrine function in the ram, but that this can be restored to normal by 30% T4 replacement. The results support the theory that T4 plays a permissive rather than a regulatory role in reproductive function in males.     

Leptin in male reproduction: the testis paradigm

Leptin, the adipocyte-derived hormone that plays a key role in body weight homeostasis, has recently emerged as a relevant neuroendocrine mediator in different systems, including the reproductive axis. Thus, compelling evidence points out a major role of leptin in the regulation of female pubertal development and fertility, both in humans and experimental animals. The contribution of leptin to the proper functioning of the male reproductive system has been less clear. However, data gathered in recent years, from independent groups and through a variety of experimental approaches, strongly suggest that leptin is able to act at different levels of the hypothalamic-pituitary-testicular axis. Herein, we review the biological effects and potential mechanisms of action of leptin upon rodent testis. Leptin appears to act as a direct inhibitory signal for testicular steroidogenesis, which may be relevant to explain the link between decreased testosterone secretion and hyperleptinaemia in obese men. Analysis of the molecular basis for leptin-induced inhibition of testosterone secretion revealed the potential involvement of decreased gene expression of several up-stream factors (e.g. SF-1, StAR and P450scc) in the steroidogenic pathway. In this context, testicular expression of leptin receptor (Ob-R) gene shows a complex pattern of alternative splicing with generation of multiple variants, including the functional leptin receptor type-b (Ob-Rb) and several short isoforms. Moreover, Ob-R mRNA expression in rat testis was regulated by homologous (leptin) as well as heterologous (gonadotropins) signals. Overall, the current data indicate that the testis is a direct target for leptin actions. Furthermore, the available evidence is suggestive of a tightly regulated, complex mode of action of leptin at different levels of the male gonadal axis that involves not only stimulatory but also inhibitory effects.     

Newly discovered endocrine functions of the liver

The liver, the largest solid visceral organ of the body, has numerous endocrine functions, such as direct hormone and hepatokine production, hormone metabolism, synthesis of binding proteins, and processing and redistribution of metabolic fuels. In the last 10 years, many new endocrine functions of the liver have been discovered. Advances in the classical endocrine functions include delineation of mechanisms of liver production of endocrine hormones [including 25-hydroxyvitamin D, insulin-like growth factor 1 (IGF-1), and angiotensinogen], hepatic metabolism of hormones (including thyroid hormones, glucagon-like peptide-1, and steroid hormones), and actions of specific binding proteins to glucocorticoids, sex steroids, and thyroid hormones. These studies have furthered insight into cirrhosis-associated endocrinopathies, such as hypogonadism, osteoporosis, IGF-1 deficiency, vitamin D deficiency, alterations in glucose and lipid homeostasis, and controversially relative adrenal insufficiency. Several novel endocrine functions of the liver have also been unraveled, elucidating the liver’s key negative feedback regulatory role in the pancreatic α cell-liver axis, which regulates pancreatic α cell mass, glucagon secretion, and circulating amino acid levels. Betatrophin and other hepatokines, such as fetuin-A and fibroblast growth factor 21, have also been discovered to play important endocrine roles in modulating insulin sensitivity, lipid metabolism, and body weight. It is expected that more endocrine functions of the liver will be revealed in the near future.     

Hypophyseal-Leydig function in the diabetic

Experimental diabetes in animals induces marked alterations of gonadal androgenic functions: reduction in testis weight, morphological alterations of Leydig cells, decrease of plasma testosterone levels and reduction of the ability of the Leydig cells to secrete testosterone in vitro. hCG treatment restores the testicular endocrine function; in addition several morphological and functional changes are observed in the hypothalamic-hypophyseal-gonadal axis which are probably responsible for the testicular lesions found in untreated experimental diabetes mellitus. In diabetic men, the hypothalamic-hypophyseal-gonadal axis seems to be normal (with exception of individual cases); Mean plasma levels of testosterone, LH, FSH and the responses of the gonadal axis to hCG and LHRH are normal.     

Acute suppression of circulating testosterone levels by cortisol in men

The effect of acute activation of the ACTH-adrenal axis on circulating testosterone (T) levels was investigated. Elevation of circulating cortisol resulting from insulin-induced hypoglycemia or the administration of hydrocortisone was followed by a rapid decrease in serum T levels, without accompanying changes in LH or PRL. These findings suggest that hypercortisolism of endogenous or exogenous sources suppresses T secretion by a direct action on the testis. This adrenal-testicular axis may have biological implications on the reproductive adaptation to stress.     

Bone and the regulation of global energy balance

The skeleton, populated by large numbers of osteoblasts and long‐lived osteocytes, requires a constant supply of energy‐rich molecules to fuel the synthesis, deposition and mineralization of bone matrix during bone modelling and remodelling. When these energetic demands are not met, bone acquisition is suppressed. Recent findings suggest that key developmental signals emanating from Wnt low‐density lipoprotein‐related receptor 5 and hypoxia‐inducible factor pathways impact osteoblast bioenergetics to accommodate the energy requirements for bone cells to fulfil their function. In vivo studies in several mutant mouse strains have confirmed a link between bone cells and global metabolism, ultimately leading to the identification of hormonal interactions between the skeleton and other tissues. The hormones insulin and leptin affect postnatal bone acquisition, whilst osteocalcin produced by the osteoblast in turn stimulates insulin secretion by the pancreas. These observations have prompted additional questions regarding the nature of the mechanisms of fuel sensing and processing in the osteoblast and their contribution to overall energy utilization and homeostasis. Answers to such questions should advance our understanding of metabolic diseases and may ultimately improve management of affected patients. In this review, we highlight recent studies in this field and offer a perspective on the evolutionary implications of bone as a metabolic endocrine organ.     

TSH stimulates leptin secretion by a direct effect on adipocytes

Leptin is a circulating hormone secreted by adipose tIssue which acts as a signal to the central nervous system where it regulates energy homeostasis and neuroendocrine processes. Although leptin modulates the secretion of several pituitary hormones, no information is available regarding a direct action of pituitary products on leptin release. However, it has been pointed out that leptin and TSH have a coordinated pulsatility in plasma. In order to test a direct action of TSH on in vitro leptin secretion, a systematic study of organ cultures of human omental adipose tIssue was performed in samples obtained at surgery from 34 patients of both sexes during elective abdominal surgery. TSH powerfully stimulated leptin secretion by human adipose tIssue in vitro. In contrast, prolactin, ACTH, FSH and LH were devoid of action. These results suggest that leptin and the thyroid axis maintain a complex and dual relationship and open the possibility that plasmatic changes in TSH may contribute to the regulation of leptin pulses.     

Stimulatory action of follice-stimulating hormone on estradiol-17 beta secretion by hypophysectomized rat ovaries in organ culture.

Ovaries of rats explanted in organ culture 2-3 days after hypophysectomy at 26 days of age synthesize ans secrete estradiol-17beta into the culture medium at approximately linear rates for 48 h. Addition of testosterone (5 times 10- minus 7M) to the culture medium occasionally caused small increases of up to 50 percent in rate of estradiol production. Significant increases (60-200 percent) in estradiol secretion resulted from addition of two different FSH preparations, in concentrations as low as 0.25 mug/ml in the absence of testosterone. In the presence of testosterone, the same FSH preparation caused much more marked increases in estradiol secretion of up to 900 percent. In the absence of testosterone, two different luteinizing hormone preparations failed to increase estradiol secretion significantly, although in the presence of testosterone, small increases were sometimes observed. It is concluded that FSH regulates estradiol biosynthesis in the hypophysectomized rat ovary by a specific stimulatory action on the aromatizing enzyme system.     

The relationship of serum osteocalcin concentration to insulin secretion, sensitivity, and disposal with hypocaloric diet and resistance training

CONTEXT: Bone has recently been described as exhibiting properties of an endocrine organ by producing osteocalcin that increases insulin sensitivity and secretion in animal models. OBJECTIVE AND DESIGN: We aimed to evaluate circulating osteocalcin in association with insulin sensitivity and insulin secretion in three different studies in nondiabetic subjects: one cross-sectional study in 149 men (using minimal model), and two longitudinal studies in two independent groups (one formed by 26 women, and the other by 9 men and 11 women), after a mean of 7.3 and 16.8% weight loss, and after a mean of 8.7% weight loss plus regular exercise. RESULTS: In the cross-sectional study, circulating osteocalcin was associated with insulin sensitivity, mainly in lean subjects, and with insulin secretion (only in lean subjects). A mean of 16.8%, but not 7.3% weight loss, led to significant increases in circulating osteocalcin. However, a mean of 8.7% weight loss plus regular exercise led to the more pronounced effects on the serum osteocalcin concentration, which increased in parallel to reduced visceral fat mass, unchanged thigh muscle mass, and increased leg strength and force. The postintervention serum levels of osteocalcin were associated with both insulin sensitivity (r = 0.49; P = 0.03) and fasting triglycerides (r = -0.54; P = 0.01). The change in visceral fat was the parameter that best predicted the change in serum osteocalcin, once age, body mass index, and insulin sensitivity changes were controlled for (P = 0.002). CONCLUSION: Circulating osteocalcin could mediate the role of bone as an endocrine organ in humans.     

Effects of pinealectomy on the secretion of luteinizing hormone, testosterone and prolactin in rams exposed to various lighting régimes.

Two experiments were carried out to study the effects of controlled lighting régimes on plasma levels of LH were low in all groups of rams, which made the detection of significant effects of any treatment very unlikely. Pinealectomy reduced the effects of changes in the daily photoperiod on the patterns of secretion of testosterone and prolactin. These findings establish the pineal gland as an organ which influences the endocrine responses of rams to photoperiodic stimuli and it is concluded that the pineal gland is probably important as a mediator of seasonal reproductive changes in these animals.     

Impairment of testicular function in adult male Japanese quail (Coturnix japonica) after a single administration of 3-methyl-4-nitrophenol in diesel exhaust particles

The effects of 3-methyl-4-nitrophenol (PNMC), a component of diesel exhaust, on reproductive function were investigated in adult male Japanese quail. The quail were treated with a single i.m. dose of PNMC (78, 103 or 135 mg/kg body weight), and trunk blood and testes were collected 1, 2 or 4 weeks later. Various levels of testicular atrophy were observed in all groups treated with PNMC. Sperm formation, cloacal gland area, and plasma LH and testosterone concentrations were also reduced in birds with testicular atrophy. To determine the acute effect of PNMC on gonadotrophin from the pituitary, adult male quail were administrated a single i.m. injection of PNMC (25 mg/kg), and plasma concentrations of LH were measured at 1, 3 and 6 h. This dose significantly lowered plasma levels of LH at all three time points. These results suggest that PNMC acts on the hypothalamus-pituitary axis, by reducing circulating LH within a few hours of administration and subsequently reducing testosterone secretion. In addition, in order to investigate the direct effects of PNMC on the secretion of testosterone from testicular cells in quail testes, cultured interstitial cells containing Leydig cells were exposed to PNMC (10(-6), 10(-5) or 10(-4) M) for 4, 8 or 24 h. These quantities of PNMC significantly reduced the secretion of testosterone in a time- and dose-dependent manner. The present findings also suggest a direct effect of PNMC on the testis to reduce testosterone secretion. This study clearly indicates that PNMC induces reproductive toxicity at both the central and testicular levels, and disrupts testicular function in adult male quail.     

Onset of the response to chorionic gonadotropin in the chick embryo testis

The stage of development of the chick embryo testis when it begins to respond to gonadotropin stimulation was investigated. The testosterone secretion in vitro, measured by radioimmunoassay, was employed to evaluate the response to hCG in testis from 8 to 16 days of incubation. At 8 to 10 days of the chick embryo development, the testis secreted testosterone, but no increment in the steroid production has been observed after hCG treatment. On the contrary, at 12, 14, and 16 days a clear increase in testosterone secretion has been demonstrated when hCG was added to the culture medium. The absence of hCG response before 12 days of incubation agrees with the hypothesis of an early independence period between testis and adenohypophysis during embryonic development.     

Thyroid gonadal interactions in the male domestic duck in relationship with the sexual cycle

The annual variations of the plasma testosterone concentrations (PT), and the metabolic clearance rate (MCR) of ³H-testosterone, that permits evaluation of the secretion rate (SR = PT × MCR) of the hormone, have been measured in Pekin ducks reared outdoors. Three salient features were observed: (1) during the progressive phase of the reproductive cycle, there is no close parallelism between the testis weight (spermatogenetic activity) and the SR of testosterone; in 1971–1972 both rhythms followed a particularly asynchronous course; (2) in late May the PT falls down to almost autumnal values when the testis weight is still high; (3) this breakdown of the PT is concomitant with a 80% rise in the MCR of testosterone, which contributes, together with a depressed SR to lower the PT.The two latter events (lowered PT in association with an enhanced MCR) can be reproduced by thyroxine administration (0.1 mg or 1 mg/day over 9 days) either during the vernal progressive phase, or during autumn in ducks photo-stimulated by “long days.”The recently evidenced increase in thyroid function in May–June in ducks reared under similar conditions led to the assumption that a seasonal hyperthyroidism may, at least partially, account for the characteristic pattern of testosterone secretion and metabolism at the onset of the regressive phase of the sexual cycle.The postnuptial molt occurs once this peculiar endocrine balance (hyperthyroidism associated to hypoandrogenism) is fulfilled.