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Purpose: Undercarboxylated-Osteocalcin (ucOCN), acting on its putative receptor GPRC6A, was shown to stimulate testosterone (T) production by Leydig cells in rodents, in parallel with the hypothalamus-pituitary-gonadal axis (HPG) mediated by luteinizing hormone (LH). The aim of this cross-sectional study was to evaluate the association among serum ucOCN, rs2247911 polymorphism of GPRC6A gene and the endocrine/semen pattern in a cohort of infertile males, possibly identifying an involvement of the ucOCN-GPRC6A axis on testis function., Methods: 190 males, including 74 oligozoospermic subjects, 58 azoosperminc patients and 58 normozoospermic controls, were prospectively recruited at the Orient Hospital for Infertility, Assisted Reproduction and Genetics in Syria (Study N. 18FP), from July 2018 to June 2020. Outpatient evaluation included the clinical history, anthropometrics and a fasting blood sampling for hormonals, serum OCN (both carboxylated and undercarboxylated), glycemic and lipid profile and screening for rs2247911 GPRC6A gene polymorphism., Results: Higher serum ucOCN associated with higher T and HDL-cholesterol (respectively: r = 0.309, P < 0.001 and r = 0.248, P = 0.001), and with lower FSH (r =  - 0.327, P < 0.001) and LDL-cholesterol (r =  - 0.171; P = 0.018). Patients bearing the GG genotype of rs2247911 had higher sperm count compared to GA genotype (P = 0.043) and, compared to both AG and AA genotypes, had higher serum T (P = 0.004, P = 0.001) and lower triglycerides levels (P = 0.002, P < 0.001). Upon normalization for LH levels and body mass index, rs2274911 and ucOCN were significantly associated with higher serum T at linear stepwise regression analysis (P = 0.013, P = 0.007)., Conclusions: Our data suggest the involvement of ucOCN-GPRC6A axis in the regulation of T production by the testis, subsidiary to HPG., (© 2022. The Author(s), under exclusive licence to Italian Society of Endocrinology (SIE).)

During the last decade, the disclosure of systemic effects of osteocalcin (OCN) in its undercarboxylated form contributed to switch the concept of bone from a merely structural apparatus to a fully endocrine organ involved in the regulation of systemic functions. Since that time, the role of OCN as osteokine has been more and more widened appreciated and detailed by the major use of animal models, starting from the original function in the bone extracellular matrix as Gla-protein and spanning from the protective effects towards weight gain, insulin sensitivity and glucose homeostasis, to the anabolic and metabolic roles in skeletal muscle, to the stimulating effects on the testis endocrine function and male fertility, to the most recent preservation from anxious and depressive states through a direct activity on the central nervous system. In this review, experimental data supporting the inter-organ communication roles of this protein are discussed, together with the available data supporting the consistency between experimental data obtained in animals and those reported in humans. In addition, a specific session has been devoted to the possible significance the OCN as a template agonist on its receptor GPRC6A, for the development of novel therapeutic and pharmacological approaches for the treatment of dismetabolic states and male infertility., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Increasing evidence disclosed the existence of a novel multi-organ endocrine pathway, involving bone, pancreas and testis, of high penetrance in energy metabolism and male fertility. The main mediator of this axis is undercarboxylated osteocalcin (ucOC), a bone-derived protein-exerting systemic effects on tissues expressing the metabotropic receptor GPRC6A. The recognized effects of ucOC are the improvement of insulin secretion from the pancreas, the amelioration of systemic insulin sensitivity, in particular in skeletal muscle, and the stimulation of the global endocrine activity of the Leydig cell, including vitamin D 25-hydroxylation and testosterone production. The supporting evidence of this circuit in both animal and human models is here reviewed, with particular emphasis on the role of ucOC on testis function. The possible pharmacological modulation of this hormonal circuit for therapeutic aims is also discussed., (© 2017 American Society of Andrology and European Academy of Andrology.)

The undercarboxylated form of osteocalcin (ucOC) regulates male fertility and energy metabolism, acting through the G protein-coupled receptor (GPRC)6A, thus forming a new pancreas-bone-testis axis. Recently, GPRC6A has also been suggested to mediate the nongenomic responses of free testosterone (T). However, these data did not consider the physiological scenario, where circulating T is mainly bound to sex hormone-binding globulin (SHBG) and only a small percentage circulates freely in the blood. Here, by the use of computational modelling, we document the existence of similar structural moieties between ucOC and SHBG that are predicted to bind to GPRC6A at docking analysis. This hypothesis of competition was assessed by binding experiments on human embryonic kidney-293 cells transfected with human GPRC6A gene. Unliganded SHBG specifically bound the membrane of human embryonic kidney-293 cells transfected with GPRC6A and was displaced by ucOC when coincubated at 100-fold molar excess. Furthermore, specific downstream Erk1/2 phosphorylation after stimulation of GPRC6A with ucOC was significantly blunted by 100-fold molar excess of unliganded SHBG. Intriguingly previous incubation with unliganded SHBG, followed by incubation with T, induced Erk1/2 phosphorylation in a dose-dependent manner. Neither binding nor stimulating activities were shown for SHBG saturated with T. Experiments on mutation constructs of GPRC6A strengthened the hypothesis of a common binding site of ucOC and SHBG. Given the role of GPRC6A on energy metabolism, these data agree with epidemiological association between SHBG levels and insulin sensitivity, suggest GPRC6A as a likely SHBG receptor, and add bases for the possible regulation of androgen activity in a nonsteroidal manner.

Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells.

Osteocalcin (OCN) is a small noncollagenous protein mainly produced by osteoblasts and is highly represented in bones of most vertebrates. Human OCN contains up to three gamma-carboxyglutamic acid (Gla-OCN) residues at positions 17, 21 and 24 which are thought to increase calcium binding strength, improving mechanical properties of the bone matrix. Recent studies revealed that OCN exerts also important endocrine functions, affecting energy metabolism and male fertility. The latter effect seems to be mediated by the uncarboxylated form of OCN (Glu-OCN). We employed human and mouse OCN as models of fully carboxylated and uncarboxylated OCN forms to investigate, by the use of circular dichroism and molecular dynamics simulations, the respective conformational properties and Ca2+ affinity. Ca2+ binding was found to trigger a similar conformational transition in both Glu-OCN and Gla-OCN, from a disordered structure to a more compact/stable form. Notably, gamma-carboxylation increases the affinity of OCN for Ca2+ by > 30 fold suggesting that, in physiological conditions, Gla-OCN is essentially Ca2+-bound, whereas Glu-OCN circulates mainly in the Ca2+-free form.

Background: Although vascular-calcification mechanisms are only partially understood, the role of circulating calcifying cells and non-collagenous bone matrix proteins in the bone-vascular axis is emerging. In spite of the fact that platelets represent a cellular interface between hemostasis, inflammation and atherosclerosis, and have a myeloid precursor, a possible involvement in the modulation of vascular calcification has rarely been investigated. We investigated if osteocalcin (OC) is released by platelets and described OC expression in patients with carotid artery occlusive disease., Methods: Expression and release of OC were determined by Western blot, immunofluorescence, fluorescence-activated cell sorting (FACS) and ELISA in human resting and activated platelets and megakaryocytes. Co-localization of platelet aggregates, macrophages, OC and calcifications was studied in carotid endarterectomy specimens and normal tissues., Results: Human platelets expressed OC and co-localized with CD63 in δ-granules. Upon activation with an endogenous mechanism, platelets released OC in the extracellular medium. Expression of OC in megakaryocytes suggested lineage specificity. The OC count in circulating platelets and the released amount were significantly higher in patients with carotid artery occlusive disease than in healthy controls (P < 0.0001) in spite of similar serum levels. In atherosclerotic plaques, OC strongly overlapped with CD41+ platelets in the early stage of calcification, but this was not seen in normal tissues. CD68+OC+ cells were present at the periphery of the calcified zone., Conclusions: Given the active role played by platelets in the atherosclerotic process, the involvement of OC release from platelets in atherosclerotic lesions and the impact of genetic and cardiovascular risk factors in mediating bone-marrow preconditioning should be investigated further., (© 2012 International Society on Thrombosis and Haemostasis.)

Objective: Androgens inhibit adipogenic differentiation through an androgen receptor (AR)-mediated pathway, increase lipolysis and reduce lipid accumulation in adipocytes. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion and is released by adipose tissue (AT). Our objective was to investigate, ex vivo and in vivo, the role of androgens on osteocalcin (OCN) modulation in human AT. DesiGN, PATIENTS, SETTING: Omental AT (OAT) for in vitro study and blood samples from 91 male patients of Padova University Hospital were used., Measurements: Omental AT was treated with dihydrotestosterone (DHT) in presence and in absence of flutamide. cOCN and ucOCN release by AT in a simple growth medium was evaluated by ELISA. OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA., Results: After 24-h DHT stimulation, the release of both cOCN and ucOCN by OAT was statistically increased (P < 0·05). Co-incubation with flutamide blunted OCN production. Overweight and obese patients had lower total and free testosterone (T), associated with lower ucOCN and ucOCN/OCN ratio. Free T was negatively correlated to BMI (ρ = -0·706, P < 0·05) and positively correlated to ucOCN/OCN ratio (ρ = 0·223, P < 0·05)., Conclusions: Our data suggest that androgens modulate OCN release by OAT in vitro. In addition to the anti-adipogenic role of androgens, they support a novel mechanism by which androgens could exert a protective effect in energy metabolism. This hypothesis appears even more significant considering that sexual hormones' levels are greatly altered in obesity and that AT is both highly involved in their clearance and able to produce OCN., (© 2011 Blackwell Publishing Ltd.)

Objective: Endothelial dysfunction is considered a key factor in the development of cardiovascular diseases. Endothelial regeneration is necessary for the maintenance of endothelial function and circulating endothelial progenitor cells (EPC) participate of it in both direct and indirect manner. The molecular phenotype of EPC is not univocally defined and recent studies identified an osteocalcin (OCN)-positive (EPC-OCN+) subpopulation of EPC highly correlated with atherosclerosis progression., Aim: Considering that hypogonadism is a risk factor for cardiovascular diseases and atherosclerosis, we investigated the circulating levels of EPC-OCN+ in hypogonadal patients., Subjects and Methods: Ten hypogonadotropic hypogonadal (HH) male patients and 30 healthy eugonadal men were evaluated for clinical status and hormonal levels. Circulating levels of CD34+/CD133+/kinase insert domain-receptor+ EPC and EPC-OCN+ were also determined by flow cytometry., Results: Compared to controls, HH patients displayed lower FSH, LH, estradiol, testosterone, and EPC levels. On the contrary, EPC-OCN+ were significantly increased., Conclusions: The observed association of low levels of circulating EPC and increased values of EPC-OCN+ sub-population in hypogonadal men strengthens the significance of hypogonadism as cardiovascular risk factor.

Context: The adipose tissue (AT), which is an endocrine organ, is linked to several metabolic abnormalities. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion., Objective: Our objective was to investigate the involvement of OCN in obesity and to evaluate, in vitro and ex vivo, the role of AT in the modulation of this endocrine circuit., Design, Patients, and Setting: This transversal study involved 83 male subjects, divided according to the World Health Organization body mass index classification, evaluated at Padova's Obesity Outpatient Clinic., Methods: OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA. OCN mRNA expression and protein production were measured by quantitative RT-PCR and immunohistochemistry during in vitro adipogenesis and in sc AT (SAT) and omental AT (OAT) from normal adult men. cOCN and ucOCN release by AT in a simple growth medium was verified by ELISA., Results: Overweight and obese patients had a lower ucOCN and ucOC/OCN ratio. In the whole cohort, ucOCN/OCN ratio was negatively correlated to body mass index (rho = -0.233; P < 0.05). OCN mRNA was present in SAT and OAT and during all stages of adipogenesis, with higher expression in the first steps. Immunohistochemistry confirmed the expression of OCN protein. Both SAT and OAT were able to release cOCN and ucOCN., Conclusions: Our data support a pathophysiological link between ucOCN and cOCN balance and obesity. OCN is present in the first phases of adipogenesis but also in human AT ex vivo. AT releases, in vitro, both ucOCN and cOCN, suggesting a possible link between AT and OCN in the regulation of metabolism.

Introduction: Erectile dysfunction (ED) was shown to be the expression of a systemic vascular disease that can precede coronary artery disease of some years. Endothelial progenitor cells (EPCs) are a population of circulating cells with endothelial-regenerative potential that may be reduced in ED and coronary patients. Recently, increased levels of osteocalcin (OCN)-positive EPC have been reported in coronary patients., Aim: Investigate the correlation between OCN-positive EPC and cavernous atherosclerotic lesion in ED patients., Methods: A total of 35 subjects (20 ED patients and 15 controls) were evaluated in our andrological center and enrolled in the study., Main Outcome Measure: All subjects underwent routine clinical examination. Patients were also evaluated with high resolution echo color doppler of penile districts (intima media thickness [IMT] before and after intracavernous alprostadil injection) and circulating levels of progenitor cells (PC), EPC, and OCN-positive fraction of EPC., Results: A progressive reduction of circulating EPC with the severity of cavernous artery atherosclerosis was found. Conversely circulating OCN-positive EPC levels undergo to a significant increase with cavernous atherogenesis progression., Conclusions: OCN-positive EPC levels in association with penile-color Doppler ultrasound evaluation of cavernous IMT could be predictive markers of subsequent coronary artery disease in ED patients.