Lisa Gianesello

Evidence for osteocalcin production by adipose tissue and its role in human metabolism.

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 Padovas 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.

Mutations in the Insulin-Like Factor 3 Receptor Are Associated With Osteoporosis

Introduction: Insulin‐like factor 3 (INSL3) is produced primarily by testicular Leydig cells. It acts by binding to its specific G protein–coupled receptor RXFP2 (relaxin family peptide 2) and is involved in testicular descent during fetal development. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. The aim of this study was to verify whether reduced INSL3 activity could cause or contribute to some signs of hypogonadism, such as reduced BMD, currently attributed to testosterone deficiency.

Relaxin stimulates osteoclast differentiation and activation

Relaxin is a pleiotropic hormone with actions in reproductive and non-reproductive tissues, and has a role in tumor biology. It can promote growth, differentiation and invasiveness of different tumors, especially those that give bone metastases, and relaxin serum concentrations are increased in patients with bone metastasis. In osteolytic metastasis the destruction of bone is mediated by osteoclasts that are multinucleated cells derived from hematopoietic progenitors. We found that human hematopoietic precursors and mature osteoclasts express the relaxin receptor RXFP1. Then, we investigated the effects of relaxin on the differentiation, activation and gene expression of osteoclasts during in vitro osteoclastogenesis from human hematopoietic progenitor cells. Relaxin alone was able to induce the multistep differentiation process of human osteoclastogenesis with timing similar to that obtained with the classical stimulators of osteoclastogenesis RANKL, M-CSF and PTH. The expression profile of several osteoclast genes was studied with quantitative RT-PCR during the entire process of osteoclastogenesis. This analysis showed that relaxin induced genes that are implicated in the differentiation, survival and activation of osteoclasts. Relaxin-induced osteoclasts were fully differentiated, positive for tartrate resistant acid phosphatase and vitronectin receptor, expressing a typical F-actin ring and able to resorb the bone. Furthermore, relaxin induced the expression of its specific receptor RXFP1 in osteoclasts. This study demonstrates for the first time that relaxin is a potent stimulator of osteoclastogenesis from hematopoietic precursors and regulates the activity of mature osteoclasts, opening new perspectives on the role of this hormone in bone physiology, diseases and metastasis.

Profiling Insulin Like Factor 3 (INSL3) Signaling in Human Osteoblasts

Background Young men with mutations in the gene for the INSL3 receptor (Relaxin family peptide 2, RXFP2) are at risk of reduced bone mass and osteoporosis. Consistent with the human phenotype, bone analyses of Rxfp2 −/− mice showed decreased bone volume, alterations of the trabecular bone, reduced mineralizing surface, bone formation, and osteoclast surface. The aim of this study was to elucidate the INSL3/RXFP2 signaling pathways and targets in human osteoblasts. Methodology/Principal Findings Alkaline phosphatase (ALP) production, protein phosphorylation, intracellular calcium, gene expression, and mineralization studies have been performed. INSL3 induced a significant increase in ALP production, and Western blot and ELISA analyses of multiple intracellular signaling pathway molecules and their phosphorylation status revealed that the MAPK was the major pathway influenced by INSL3, whereas it does not modify intracellular calcium concentration. Quantitative Real Time PCR and Western blotting showed that INSL3 regulates the expression of different osteoblast markers. Alizarin red-S staining confirmed that INSL3-stimulated osteoblasts are fully differentiated and able to mineralize the extracellular matrix. Conclusions/Significance Together with previous findings, this study demonstrates that the INSL3/RXFP2 system is involved in bone metabolism by acting on the MAPK cascade and stimulating transcription of important genes of osteoblast maturation/differentiation and osteoclastogenesis.

Effect of Relaxin on Human Sperm Functions

Relaxin is a circulating hormone with functions in pregnancy, parturition, and other aspects of female reproduction. It is also secreted from the prostate gland into the seminal fluid; however, the role of relaxin in male reproduction is debated. Studies conducted in the past have suggested possible actions on human spermatozoa, but the data were contrasting. Here, we show that the relaxin receptor RXFP1 (Relaxin Family Peptide Receptor 1) is expressed in human spermatozoa, and it mainly localizes in the astrodome. In vitro studies on human sperm demonstrated that this hormone attenuates the natural decline in sperm motility and maintains higher mitochondrial activity and lower apoptosis level. Furthermore, relaxin induced an increase in sperm hyperactivation, intracellular calcium and cAMP, and acrosome reaction. These effects were abolished by the use of the specific anti-RXFP1 antibody. Relaxin concentrations were low in the blood (x ± SD, 0.16 ± 0.03 nM) and very high in the seminal plasma (x ± SD, 10.3 ± 4.0 nM), confirming its secretion mainly by the prostate. Taken together, these data demonstrate that relaxin influences positively many sperm functions linked to fertilizing ability, and it preserves sperm functionality, with possible practical value in assisted reproduction techniques.

New roles for INSL3 in adults.

Insulin‐like factor 3 (INSL3) is produced primarily by testicular Leydig cells and acts by binding to its specific G‐protein‐coupled receptor, RXFP2 (relaxin family peptide 2). INSL3 is involved in testicular descent during fetal development, and mutations in the INSL3 and RXFP2 genes cause cryptorchidism. The physiological role of INSL3 in adults is not known, although substantial INSL3 circulating levels are present. After extensive clinical, biochemical, and hormonal investigations, including bone densitometry with dual energy X‐ray absorptiometry, on 25 young men (age, 27–41 years) who have the well‐characterized T222P mutation in the RXFP2 gene, we found that 16 of them (64%) had significantly reduced bone density. No other cause of osteoporosis was evident in these subjects, whose testosterone and gonadal function were normal. Expression analysis of INSL3 and RXFP2 on human bone biopsy and human and mouse osteoblast cell cultures performed by reverse transcription‐PCR and immunohistochemistry showed the presence of RXFP2 in these cells. Real‐time cAMP imaging analysis and proliferation assays under the stimulus of INSL3 showed a dose‐ and time‐dependent increase in cAMP and cell proliferation, and specific osteoblast gene activation was observed by real‐time PCR after INSL3 stimulation. Lumbar spine and femoral bone of Rxfp2‐deficient mice were studied by static and dynamic histomorphometry and micro‐computed tomography, respectively, and showed decreased bone mass, mineralizing surface, bone formation, and osteoclast surface compared to wild‐type littermates, compatible with a functional osteoblast impairment. This study identified for the first time a role for INSL3 in adults, demonstrating a modulating effect on bone metabolism and linking RXFP2 gene mutations with human osteoporosis.

Effects of type 5-phosphodiesterase inhibition on energy metabolism and mitochondrial biogenesis in human adipose tissue ex vivo

Objective: An excess of adipose tissue (AT) in obese individuals is linked to increased cardiovascular risk and mitochondria have been shown to be defective in the muscle and AT of patients with metabolic disorders such as obesity and Type 2 diabetes. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays a role in mitochondrial biogenesis through cyclic-GMP (cGMP). AT harbors the whole molecular signaling pathway of NO, together with type 5-phosphodiesterase (PDE-5), the main cGMP catabolising enzyme. Aim: Our aim was to evaluate the effect of the modulation of NO pathway, through PDE-5 inhibition, on energy metabolism and mitochondria biogenesis in human omental AT. Methods and measurements: Cultured human omental AT was stimulated with PDE-5 inhibitor, vardenafil, at different concentration for 24 and 72 h. Analysis of the expression of both key-regulator genes of adipocyte metabolism and mitochondria- biogenesis markers was performed. Results: We found an increased gene expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), adiponectin, and proliferator- activated receptor gamma coactivator- 1 α (PGC- 1α) after a 24- h stimulation with vardenafil at the lowest concentration employed compared to controls (p<0.05). After 72 h of stimulation, a significant increase of mitochondrial DNA was found compared to control samples (p<0.05). Conclusion: Our data suggest that PDE-5 inhibition could have an impact on mitochondrial content of human AT suggesting a positive effect on energy metabolism and adding new elements in the comprehension of AT pathophysiology.

Androgens modulate osteocalcin release by human visceral adipose tissue.

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.

INSL3 Plays a Role in the Balance between Bone Formation and Resorption

We have recently demonstrated that the disruption of the INSL3 hormonal system, as observed in humans with mutations in the RXFP2 gene and in Rxfp2‐deficient mice, might affect the equilibrium of the osteoblast–osteoclast system, resulting in an imbalance between bone formation and bone resorption that may result in reduced bone mass. In the present study we have better characterized the in vitro effects of INSL3 on human osteoblasts. Stimulation of human primary osteoblasts with INSL3 at serial concentrations (1 pM, 1 nM, and 1 μM) induced a dose‐dependent increase in osteoblast proliferation and expression of specific osteoblast genes.

Role of Relaxin in Human Osteoclastogenesis

Recently, we have demonstrated that INSL3 is important for bone metabolism, and in this study we analyzed the possible role of the cognate hormone relaxin on human osteoclasts. In fact, previous studies showed an effect of relaxin on peripheral blood mononuclear cells (PBMCs), the precursors of osteoclasts. Analysis of the expression of the relaxin receptor RXFP1 and RLN‐2 mRNA in primary cell cultures of human osteoclasts obtained from PBMCs showed by reverse transcriptase PCR and immunofluorescence only the presence of RXFP1 transcripts. Analysis of the in vitro effects of relaxin on osteoclastogenesis showed that relaxin is able alone to induce the differentiation of PBMCs into mature osteoclasts. This study shows, for the first time, that relaxin has an effect on bone metabolism, facilitating the differentiation of osteoclasts. A possible role for relaxin in osteolytic bone metastasis is also proposed.