Effect of bombesin receptor subtype-3 and its synthetic agonist on signaling, glucose transport and metabolism in myocytes from patients with obesity and type 2 diabetes

Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein-coupled receptor (GPCR) member of the bombesin receptor family. Several studies have suggested an association between obesity, alterations in glucose metabolism, diabetes and the BRS-3 receptor. In this study, we focused on patients simultaneously diagnosed with obesity and type 2 diabetes (OB/T2D). The analysis of BRS-3 expression in the skeletal muscle of these patients revealed a marked decrease in the expression of BRS-3 at the mRNA (23.6±1.3-fold downregulation, p<0.0001) and protein level (49±7% decrease, p<0.05) compared to the normal patients (no obesity and diabetes). Moreover, in cultured primary myocytes from patients with OB/T2D, the synthetic BRS-3 agonist, [D-Try6,β‑Ala11,Phe13,Nle14]bombesin6-14, significantly increased the phosphorylation levels of mitogen-activated protein kinase (MAPK), p90RSK1, protein kinase B (PKB) and p70s6K. Specifically, the ligand at 10-11 M induced the maximal phosphorylation of MAPKs (p42, 159±15% of the control; p44, 166±11% of the control; p<0.0001) and p90RSK1 (148±2% of the control, p<0.0001). The basal phosphorylation levels of all kinases were reduced (p<0.05) in the patients with OB/T2D compared to the normal patients. Furthermore, the BRS-3 agonist stimulated glucose transport, which was already detected at 10-12 M (133±9% of the control), reached maximal levels at 10-11 M (160±9%, p<0.0001) and was maintained at up to 10-8 M (overall mean, 153±7%; p<0.007). This effect was less promiment than that attained with 10-8 M insulin (202±9%, p=0.009). The effect of the agonist on glycogen synthase a activity achieved the maximum effect at 10-11 M (165±16% of the control; p<0.0001), which did not differ from that observed with higher concentrations of the agonist. These results suggest that muscle cells isolated from patients with OB/T2D have extremely high sensitivity to the synthetic ligand, and the effects are particularly observed on MAPK and p90RSK1 phosphorylation, as well as glucose uptake. Moreover, our data indicate that BRS-3 may prove to be useful as a potential therapeutic target for the treatment of patients with OB/T2DThis study was supported by a grant from the Instituto de Salud Carlos III (CP08/000158; PIE13/00051), Fundación Eugenio Rodríguez Pascual (12224/001) and Fundación Mutua Madrileña (12224/002), Spain. S.P.-N. is the recipient of a study contract from RETICEF (RD12/0043/008

Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains

Objectives: Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTHrelated protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. Methods: We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells. Results: We showed that both N- and C-terminal PTHrP peptides at 100 nM decreased reactive oxygen species production and forkhead box protein O activation following hydrogen peroxide (H 2 O 2 )-induced oxidation, which was related to decreased lipid oxidative damage and caspase-3 activation in these cells. This was associated with their ability to restore the deleterious effects of H 2 O 2 on cell growth and alkaline phosphatase activity, as well as on the expression of various osteoblast differentiation genes. The addition of Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (a cyclic 3',5'-adenosine monophosphate antagonist) and calphostin C (a protein kinase C inhibitor), or a PTH type 1 receptor antagonist, abrogated the effects of N-terminal PTHrP, whereas protein phosphatase 1 (an Src kinase activity inhibitor), SU1498 (a vascular endothelial growth factor receptor 2 inhibitor), or an anti osteostatin antiserum, inhibited the effects of C-terminal PTHrP. Conclusion: These findings indicate that the antioxidant properties of PTHrP act through its N- and C-terminal domains and provide novel insights into the osteogenic action of PTHrP.This work has been funded by grants from the Fundacion para la Investigacion Osea y Metabolismo Mineral-FEIOMM and the Instituto de Salud Carlos III (PI11/00449, PI15/00340, PI1600065, RD12/0043/0029, RD12/0043/0008 and RD12/0043/0018,). J. A. Ardua, D. Lozano, and S. Portal-Nunez are recipients of postdoctoral contracts from the Ministerio de Economia y Competitividad, Juan de la Cierva program JCI-2011-09548, FPDI-2013-17268, and RETICEF [FEDER “una manera de hacer Europa” (RD12/0043/0008)

Parathyroid hormone-related protein (107-111) improves the bone regeneration potential of gelatin-glutaraldehyde biopolymer-coated hydroxyapatite

Biopolymer-coated nanocrystalline hydroxyapatite (HA) made as macroporous foams which are degradable and flexible are promising candidates as orthopaedic implants. The C-terminal (107-111) epitope of parathyroid hormone-related protein (PTHrP) exhibits osteogenic properties. The main aim of this study was to evaluate whether PTHrP (107-111) loading into gelatin-glutaraldehyde biopolymer-coated HA (HA(GIu)) scaffolds would produce an optimal biomaterial for tissue engineering applications. HA(GIu) scaffolds with and without PTHrP (107-111) were implanted into a cavitary defect performed in both distal tibial metaphysis of adult rats. Animals were sacrificed after 4 weeks for histological, microcomputerized tomography and gene expression analysis of the callus. At this time, bone healing occurred only in the presence of PTHrP (107-111)-containing HA(Giu) implant, related to an increase in bone volume/tissue volume and trabecular thickness, cortical thickness and gene expression of osteocalcin and vascular cell adhesion molecule I, but a decreased gene expression of Wnt inhibitors, SOST and dickkopf homolog 1. The autonomous osteogenic effect of the PTHrP (107-111)-loaded HA(Giu) scaffolds was confirmed in mouse and human osteoblastic cell cultures. Our findings demonstrate the advantage of loading PTHrP (107-111) into degradable HA(GIu) scaffolds for achieving an optimal biomaterial that is promising for low load bearing clinical applications. (C) 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Pleiotrophin-Loaded Mesoporous Silica Nanoparticles as a Possible Treatment for Osteoporosis

Osteoporosis is the most common type of bone disease. Conventional treatments are based on the use of antiresorptive drugs and/or anabolic agents. However, these treatments have certain limitations, such as a lack of bioavailability or toxicity in non-specific tissues. In this regard, pleiotrophin (PTN) is a protein with potent mitogenic, angiogenic, and chemotactic activity, with implications in tissue repair. On the other hand, mesoporous silica nanoparticles (MSNs) have proven to be an effective inorganic drug-delivery system for biomedical applications. In addition, the surface anchoring of cationic polymers, such as polyethylenimine (PEI), allows for greater cell internalization, increasing treatment efficacy. In order to load and release the PTN to improve its effectiveness, MSNs were successfully internalized in MC3T3-E1 mouse pre-osteoblastic cells and human mesenchymal stem cells. PTN-loaded MSNs significantly increased the viability, mineralization, and gene expression of alkaline phosphatase and Runx2 in comparison with the PTN alone in both cell lines, evidencing its positive effect on osteogenesis and osteoblast differentiation. This proof of concept demonstrates that MSN can take up and release PTN, developing a potent osteogenic and differentiating action in vitro in the absence of an osteogenic differentiation-promoting medium, presenting itself as a possible treatment to improve bone-regeneration and osteoporosis scenarios

Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the "race for the surface" theory

Objectives Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the 'race for the surface' theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. Methods We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. Results Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. Conclusions The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence.This work was funded by the following grants from the Spanish MINECO (MAT2013- 48224-C2-2-R and MAT2013-48224-C2-1-R). M. Martínez-Pérez reports funding received from EFORT 2015 congress: travel supported by PFIZER, which is related to this article. J. Esteban and E. Gómez-Barrena report funding received from several companies for travel, expenses and grants, none of which is related to this articl

Local delivery of parathyroid hormone-related protein-derived peptides coated onto a hydroxyapatite-based implant enhances bone regeneration in old and diabetic rats

Diabetes mellitus (DM) and aging are associated with bone fragility and increased fracture risk. Both (1-37) N- and (107-111) C-terminal parathyroid hormone-related protein (PTHrP) exhibit osteogenic properties. We here aimed to evaluate and compare the efficacy of either PTHrP (1-37) or PTHrP (107-111) loaded into gelatin-glutaraldehyde-coated hydroxyapatite (HA-Gel) foams to improve bone repair of a transcortical tibial defect in aging rats with or without DM, induced by streptozotocin injection at birth. Diabetic old rats showed bone structural deterioration compared to their age-matched controls. Histological and -computerized tomography studies showed incomplete bone repair at 4 weeks after implantation of unloaded Ha-Gel foams in the transcortical tibial defects, mainly in old rats with DM. However, enhanced defect healing, as shown by an increase of bone volume/tissue volume and trabecular and cortical thickness and decreased trabecular separation, occurred in the presence of either PTHrP peptide in the implants in old rats with or without DM. This was accompanied by newly formed bone tissue around the osteointegrated HA-Gel implant and increased gene expression of osteocalcin and vascular endothelial growth factor (bone formation and angiogenic markers, respectively), and decreased expression of Sost gene, a negative regulator of bone formation, in the healing bone area. Our findings suggest that local delivery of PTHrP (1-37) or PTHrP (107-111) from a degradable implant is an attractive strategy to improve bone regeneration in aged and diabetic subjects. (c) 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2060-2070, 2016

Role of angiogenesis on bone formation

Angiogenesis and bone formation are coupled during skeletal development and fracture healing. This relationship, although known for some time, has not been properly explored. Advances in the discovery of how angiogenesis is regulated in physiological processes like embryogenesis, endometrial regeneration and wound healing or in pathologies such as cancer have provided a deeper understanding of how angiogenic factors may interact with bone cells to improve bone formation and bone regeneration. The lack of oxygen (hypoxia) and the subsequent generation of angiogenic factors have been shown to be critical in the development of a regular skeleton and achieving successful bone regeneration and fracture healing. Given that vascular status is important for a proper bone homeostasis, defining the roles of osteoblasts, osteoclasts, endothelial cells and angiogenic factors and their interactions in bone is a key issue for the development of new strategies to manage bone pathologies and nonfused fracture

The vertebrae of prematurely aging mice as a skeletal model of involutional osteoporosis

Oxidative stress in bone increases with age, which leads to bone frailty and a high fracture risk. Animal models show that early changes in trabecular structure occur in age-related osteopenia. These models might be valuable to assess the contribution of oxidative stress in age-related bone loss. Premature aging mice (PAM) have previously been characterized as a model of premature immunological and neurological senescence. PAM long bones (mainly consisting of cortical bone) display features of aging bone. Thus, we aimed to evaluate the vertebrae, representing a unique poorly loaded type of trabecular bone in mice, in PAM and no PAM (NPAM) controls. PAM showed an anxious behaviour, based on physical activity evaluation. These mice had decreased bone mineral density (0.078 mg/cm2 in NPAM vs 0.070 g/cm2 in PAM; p<0.05); a decreased number of osteocytes per bone field (404±36 in NPAM vs 320±27 in PAM; p<0.01); and downregulation of various osteoblastic genes and low eroded surface/bone surface, 4.2±0.5 in NPAM vs 1.9±0.2 in PAM; p<0.01). This was associated with increased expression of oxidative stress markers, Foxo1 and GADD45, in PAM vertebrae. Mesenchymal progenitors in the bone marrow of PAM have a poor mineralization capacity (assessed by the number of mineralized nodules and suface), and showed a lower response to an osteogenic input - represented by parathormone-related protein-, compared to NPAM. Collectively, these results indicate that PAM vertebrae show osteopenia related to diminished bone formation and remodeling. Our findings further support the validity of PAM as a suitable model for involutional osteoporosis and its treatment