Diabetic Bone Research Articles

CGRP regulates the dysfunction of peri-implant angiogenesis and osseointegration in streptozotocin-induced diabetic rats.

Diabetes is a chronic systematic disease which results in neuropathy and dysfunctional bone metabolism and microcirculation. Calcitonin gene related peptide (CGRP) is an important neuropeptide that is involved in bone formation and vascular response. This study aimed to elucidate the role of CGRP in diabetic peri-implant angiogenesis and osteogenesis, which is yet to be reported. In vivo, we injected streptozotocin into SD rats to establish an experimental diabetes model. We then implanted 1mm×5mm Ti implants into rat tibiae and injected lentivirus into the bone marrow cavity to overexpress or silence the peri-implant CGRP expression. We also applied overexpression lentivirus and silencing short hair RNA (shRNA) in rat bone marrow mesenchymal stem cells (BMSCs) to investigate the biological effects of CGRP in vitro. Through the investigation of diabetic neurons, blood, and peri-implant bone, we could observe that diabetes led to decreased synthesis and expression of CGRP, and high CGRP expression were only seen in peri-implant tissues in the early-to-middle phase of diabetic bone integration. Microfil perfusion followed by micro-CT analysis showed that the overexpression of CGRP enhanced peri-implant angiogenesis via increased vessel volume and thickness. Regarding osteogenesis, CGRP was found to improve the impaired osseointegration, as observed through micro-CT reconstruction and H&E staining. Similarly, overCGRP alleviated the hyperglycemia-triggered decrease in mineralization, and rescued ALP activity and the mRNA and protein expression of VEGF-A, ALP, and OPN. CGRP also attenuated the high glucose-induced production of reactive oxygen species (ROS). Our results demonstrate the potential promotive role of CGRP in early-to-middle phase of osseointegration, as CGRP could regulate the diabetes-induced dysfunctions in peri-implant angiogenesis and osseointegration. Our study provides a new insight into the diabetic peri-implant vasculature and the potential positive effect of CGRP on diabetic peri-implant vessels and bone.

535-P: Neurotensin Improves Endothelial Progenitor Cell Function in Diabetic Wound Healing

Diabetic neuropathy plays an important role in the development of chronic wounds. Endothelial progenitor cells (EPCs) are known important regulators of wound angiogenesis. However, the role of neuropeptides in EPC function, and particularly Neurotensin (NT), is unknown in diabetic wound healing. EPCs isolated from normal mice were cultured in high glucose (25 mM) or normoglicemia (5.5 mM) and treated with NT (25-400 nM) for 3 days. Moreover, EPCs isolated from streptozotocin (STZ)-induced diabetic mice were treated with NT (100 nM) for 3 days. Subsequently, EPC viability, cell adhesion and reactive oxygen species (ROS) levels were evaluated. To the in vivo wound healing model, 6 mm wounds were created in the dorsum of diabetic mice and wound progression was followed up to day 10. The wound edges were injected with ex-vivo diabetic bone marrow derived EPCs treated in culture with NT (100 nM) for 3 days, or EPCs without NT treatment, or vehicle. Angiogenesis was evaluated by immunohistochemistry. The viability of EPCs cultured under high glucose or EPCs from diabetic mice was improved by NT treatment. Also, EPCs from diabetic mice showed decreased adhesion (58% of control, p<0.05) and increased ROS levels (148% of control, p<0.05) when compared to control EPCs, and these effects were reversed by NT treatment. In addition, NT-treated EPCs, from diabetic mice, improved wound closure (day 10, 4% of original wound size) when compared with non-treated EPCs or vehicle (day 10, 32% and 35% of original wound size, respectively, p<0.05). Furthermore, NT-treated EPCs, from diabetic mice, increased neovascularization at the wound site (153% of vehicle, p<0.05) when compared with non-treated EPCs (115% of vehicle) or vehicle. These results show that neurotensin is able to improve the function of EPCs from diabetic animals, which are able to improve wound healing by promoting angiogenesis. The restoration of EPC function by neurotensin could improve their therapeutic effect for the treatment of chronic diabetic wounds. Disclosure E.C. Leal: None. A. Figueiredo: None. E. Carvalho: None. Funding Portuguese Foundation for Science and Technology; European Foundation for the Study of Diabetes; Portuguese Society of Diabetology

MiR-1908/EXO1 and MiR-203a/FOS, regulated by scd1, are associated with fracture risk and bone health in postmenopausal diabetic women.

Background: Stearoyl–coenzyme A desaturase-1 (SCD1) can inhibit the development of diabetic bone disease by promoting osteogenesis. In this study, we examined whether this regulation by SCD1 is achieved by regulating the expression of related miRNAs.Methods: SCD1 expression levels were observed in human bone-marrow mesenchymal stem cells (BM-MSCs) of patients with type 2 diabetes mellitus (T2DM), and the effect of SCD1 on osteogenesis was observed in human adipose-derived MSCs transfected with the SCD1 lentiviral system. We designed a bioinformatics prediction model to select important differentially expressed miRNAs, and established protein–protein interaction and miRNA–mRNA networks. miRNAs and mRNAs were extracted and their differential expression was detected. The SCD1–miRNA–mRNA network was validated.Findings: SCD1 expression in bone marrow was downregulated in patients with T2DM and low-energy fracture, and SCD1 expression promotes BM-MSC osteogenic differentiation. The predictors in the nomogram were seven microRNAs, including hsa-miR-1908 and hsa-miR-203a. SCD1 inhibited the expression of CDKN1A and FOS, but promoted the expression of EXO1 and PLS1. miR-1908 was a regulator of EXO1 expression, and miR-203a was a regulator of FOS expression.Interpretation: The regulation of BM-MSCs by SCD1 is a necessary condition for osteogenesis through the miR-203a/FOS and miR-1908/EXO1 regulatory pathways.

Thrombomodulin Functional Domains Support Osteoblast Differentiation and Bone Healing in Diabetes in Mice.

Thrombomodulin (TM) is a transmembrane glycoprotein that contains five functional domains. Soluble TM (sTM), comprising extracellular domains TMD1 (lectin-like), TMD2 (epidermal growth factor [EGF]-like repeat containing), and TMD3 (serine-threonine rich), can be shed from cells by the intramembrane protease rhomboid-like-2 (RHBDL2). TM is expressed by osteoblasts, yet its role there has not been determined. Herein we aimed to investigate the properties of TM and its domains in osteoblast function and bone repair following injury in diabetes. In response to a scratch injury of cultured osteoblast-like MG63 cells, expression of TM and RHBDL2 was enhanced, with increased release of sTM. Conditioned media from the injured cells promoted osteoblast migration, an effect that was lacking with conditioned media from MG63 cells in which TM was silenced by shRNA. Exogenous recombinant TMD1 had no effect on osteoblast activities or on bone repair in vivo. However, TM domains 2 and 3 (TMD2/3), induced MG63 cell migration, proliferation and mineralization in vitro, and when locally administered in mice, improved in vivo healing of injured calvarium. This beneficial effect of TMD2/3, mediated via fibroblast growth factor receptor (FGFR)/ERK signaling pathways, was also observed in vitro under high glucose conditions where endogenous TM expression was reduced, and in vivo in diabetic mice following tibia fracture or calvarium injury, where the osteoblastic response and healing were otherwise dampened. Taken together, osteoblast TM participates in bone healing, and recombinant TMD2/3 holds promise as a novel therapy for diabetic bone defect healing. © 2020 American Society for Bone and Mineral Research.

Effects of Targeted Delivery of Metformin and Dental Pulp Stem Cells on Osteogenesis via Demineralized Dentin Matrix under High Glucose Conditions.

High glucose condition inhibited osteoblast differentiation could be a main mechanism contributing to the decreased bone repair associated with diabetes. Metformin, a widely prescribed antidiabetic drug, was shown to have osteogenic properties in our previous study. Transplanted mesenchymal stromal cells (MSCs) may differentiate into osteoblasts and promote bone regeneration. Our study aimed to combine the benefits of metformin and MSCs transplantation on osteogenesis in high glucose conditions. We developed demineralized dentin matrix (DDM) as a carrier to target deliver metformin and dental pulp-derived MSCs (DPSCs). We collected clinically discarded teeth, isolated DPSCs from the dental pulp, and prepared the DDM from the dentin. The DDM was observed by scanning electron microscopy and was found to have well-distributed tubes. Then, metformin was loaded into the DDM to form the DDM-Met complex (DDM-Met); DDM-Met released metformin at a favorable concentration. The DPSCs seeded with the DDM-Met in a high glucose medium showed satisfactory attachment and viability together with increased mineralization and upregulated osteogenesis-related genes, including alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). A possible mechanism of the enhanced osteogenic differentiation of DPSCs was explored, and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway was found to play a role in the enhancement of osteogenesis. DDM-Met appeared to be a successful metformin and DPSC carrier that allowed for the local delivery of metformin and DPSCs in high glucose conditions. DDM-Met-DPSC construct has promising prospects to promote osteogenesis and enhance the much-needed diabetic bone regeneration.

A Meta-analysis of immune signaling pathways between human type 2 diabetic tissue and mouse bone repair

A Meta-analysis of immune signaling pathways between human type 2 diabetic tissue and mouse bone repair

Progression and Etiology of Cortical Porosity in Diabetic Bone Disease

Progression and Etiology of Cortical Porosity in Diabetic Bone Disease

Spatiotemporal characterization of microdamage accumulation and its targeted remodeling mechanisms in diabetic fatigued bone

The skeleton of type 1 diabetes mellitus (T1DM) has deteriorated mechanical integrity and increased fragility, whereas the mechanisms are not fully understood. Load-induced microdamage naturally occurs in bone matrix and can be removed by initiating endogenous targeted bone remodeling. However, the microdamage accumulation in diabetic skeleton and the corresponding bone remodeling mechanisms remain poorly understood. Herein, streptozotocin-induced T1DM rats and age-matched non-diabetic rats were subjected to daily uniaxial ulnar loading for 1, 4, 7, and 10 days, respectively. The SPECT/CT and basic fuchsin staining revealed significant higher-density spatial accumulation of linear and diffuse microdamage in diabetic ulnae than non-diabetic ulnae. Linear microcracks increased within 10-day loading in diabetic bone, whereas peaked at Day 7 in non-diabetic bone. Moreover, diabetic fatigued ulnae had more severe disruptions of osteocyte canaliculi around linear microcracks. Immunostaining results revealed that diabetes impaired targeted remodeling in fatigued bone at every key stage, including increased apoptosis of bystander osteocytes, decreased RANKL secretion, reduced osteoclast recruitment and bone resorption, and impaired osteoblast-mediated bone formation. This study characterizes microdamage accumulation and abnormal remodeling mechanisms in the diabetic skeleton, which advances our etiologic understanding of diabetic bone deterioration and increased fragility from the aspect of microdamage accumulation and bone remodeling.

AdipoRon promotes diabetic fracture repair through endochondral ossification-based bone repair by enhancing survival and differentiation of chondrocytes

Diabetic bone defects may exhibit impaired endochondral ossification (ECO) leading to delayed bone repair. AdipoRon, a receptor agonist of adiponectin polymers, can ameliorate diabetes and related complications, as well as overcome the disadvantages of the unstable structure of artificial adiponectin polymers. Here, the effects of AdipoRon on the survival and differentiation of chondrocytes in a diabetic environment were explored focusing on related mechanisms in gene and protein levels. In vivo, AdipoRon was applied to diet-induced-obesity (DIO) mice, a model of obesity and type 2 diabetes, with femoral fracture. Sequential histological evaluations and micro-CT were examined for further verification. We found that AdipoRon could ameliorate cell viability, apoptosis, and reactive oxygen species (ROS) production and promote mRNA expression of chondrogenic markers and cartilaginous matrix production of ATDC5 cells in high glucose medium via activating ERK1/2 pathway. Additionally, DIO mice with intragastric AdipoRon administration had more neocartilage and accelerated new bone formation. These data suggest that AdipoRon could stimulate bone regeneration via ECO in diabetes.

Bone Mineral Is More Heterogeneously Distributed in the Femoral Heads of Osteoporotic and Diabetic Patients: A Pilot Study.

ABSTRACTOsteoporosis is associated with systemic bone loss, leading to a significant deterioration of bone microarchitecture and an increased fracture risk. Although recent studies have shown that the distribution of bone mineral becomes more heterogeneous because of estrogen deficiency in animal models of osteoporosis, it is not known whether osteoporosis alters mineral distribution in human bone. Type 2 diabetes mellitus (T2DM) can also increase bone fracture risk and is associated with impaired bone cell function, compromised collagen structure, and reduced mechanical properties. However, it is not known whether alterations in mineral distribution arise in diabetic (DB) patients’ bone. In this study, we quantify mineral content distribution and tissue microarchitecture (by μCT) and mechanical properties (by compression testing) of cancellous bone from femoral heads of osteoporotic (OP; n = 10), DB (n = 7), and osteoarthritic (OA; n = 7) patients. We report that though OP cancellous bone has significantly deteriorated compressive mechanical properties and significantly compromised microarchitecture compared with OA controls, there is also a significant increase in the mean mineral content. Moreover, the heterogeneity of the mineral content in OP bone is significantly higher than controls (+25%) and is explained by a significant increase in bone volume at high mineral levels. We propose that these mineral alterations act to exacerbate the already reduced bone quality caused by reduced cancellous bone volume during osteoporosis. We show for the first time that cancellous bone mineralization is significantly more heterogeneous (+26%) in patients presenting with T2DM compared with OA (non‐DB) controls, and that this heterogeneity is characterized by a significant increase in bone volume at low mineral levels. Despite these mineralization changes, bone microarchitecture and mechanical properties are not significantly different between OA groups with and without T2DM. Nonetheless, the observed alterations in mineral heterogeneity may play an important tissue‐level role in bone fragility associated with OP and DB bone. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Elevated glucose acts directly on osteocytes to increase sclerostin expression in diabetes

Bone quality in diabetic patients is compromised, leading to weaker bones and increased fracture risk. However, the mechanism by which this occurs in diabetic bone remains to be fully elucidated. We hypothesized that elevated glucose and glucose variation would affect the function of osteocytes, essential regulators of bone homeostasis and quality. To first test this hypothesis, we used the IDG-SW3 osteocyte-like cell line to examine the effects of glucose levels on osteocyte function and viability in vitro. We confirmed our in vitro findings using the in vivo streptozotocin-induced (STZ) diabetic rat model and ex-vivo cultured osteocytes from these rats. IDG-SW3 cells cultured under high glucose conditions displayed significantly increased Sost mRNA(100-fold) and sclerostin protein, a negative regulator of bone formation(5000-fold), compared to cells in control media. mRNA expression of osteoblast markers such as Osx, Ocn and Col1a1 was unaffected by glucose. Factors associated with osteoclast activation were affected by glucose, with Rankl being upregulated by low glucose. Opg was also transiently upregulated by high glucose in mature IDG-SW3 cells. Induction of diabetes in Sprague-Dawley rats via a single dose of STZ (70 mg/kg) resulted in elevated maximum glucose and increased variability compared to control animals (670/796 vs. 102/142 mg/dL). This was accompanied by increased Sost/sclerostin expression in the osteocytes of these animals. These results show that glucose levels directly regulate osteocyte function through sclerostin expression and suggest a potential mechanism for the negative impact of diabetes on bone quality.

Possible osteoprotective effects of myricetin in STZ induced diabetic osteoporosis in rats

Myricetin is a flavonoid which has many pharmacological effects. However, to date there is no evidence study on the effect of myricetin in diabetic condition. This study was aimed to investigate whether myricetin could protect against diabetic osteoporosis in streptozotocin induced rats.Female Wistar rats were randomly allocated to four equal groups: diabetic group (DG), diabetic group with myricetin (50 mg per kilogram per day), (D) diabetic group with myricetin (100 mg/kg/day) and normal control group (CG). Body weight was recorded once a week. After treatment with myricetin for 12 weeks, serum biochemical analyses, the microarchitecture of femora, and histological changes were evaluated.We found that the bone mineral density (BMD) of myricetin (100 mg per kilogram per day)treatment group significantly increased than in the diabetic group (P < 0.05). The alkaline phosphatase and osteocalcin were markedly blocked in diabetic rats relative to normal control group (P < 0.05); however, the inhibition was prevented by the myricetin treatment group. Results also showed that myricetin treatment could dramatically improve trabecular bone microarchitecture through increasing bone mass such as trabecular number (Tb.N), bone volume per tissue volume (BV/TV), and decreasing that of structure model index (SMI) and trabecular separation (Tb.Sp), comparing with the control group. We also found that myricetin could significantly lower the oxidative damage and up-regulate the activity of superoxide dismutase (SOD) and catalase activity.In summary, we showed that myricetin can effectively improve abnormal bone metabolism in streptozotocin induced rats, which may provide a beneficial medicine on diabetic bone disease.

Evaluation of the Effects of Low-Level Laser Therapy on Diabetic Bone Healing.

The aim of the present study was to evaluate the effects of low-level laser therapy (LLLT) and biphasic alloplastic bone graft material on diabetic bone healing. Induction of diabetes was performed in 14 male Sprague-Dawley rats by intraperitoneal injection of a 50 mg/kg dose of streptozotocin. Two bilaterally symmetrical non-critical-sized bone defects were created in the parietal bones in each rat. Right defects were filled with biphasic alloplastic bone graft. Rats were randomly divided into 2 groups, with 1 group receiving 10 sessions of LLLT (GaAlAs, 78.5 J/cm, 100mW, 0.028 cm beam). The LLLT was started immediately after surgery and once every 3 days during postoperative period. At the end of treatment period, new bone formation and osteoblast density were determined using histomorphometry. Empty (control), graft-filled, LLLT-treated and both graft-filled and LLLT-treated bone defects were compared. New bone formation was higher in the graft treatment samples compared with the control (P = 0.009) and laser samples (P = 0.029). In addition, graft-laser combination treatment samples revealed higher bone formation than control (P = 0.008) and laser (P = 0.026) samples. Osteoblast density was significantly higher in the laser treatment (P <0.001), graft treatment (P = 0.001) and graft-laser combination treatment (P <0.001) samples than control samples. In addition, significantly higher osteoblast density was observed in the graft-laser combination treatment samples compared to the graft treatment samples (P = 0.005). The LLLT was effective to stimulate osteoblastogenesis but failed to increase bone formation. Graft augmentation for treatment of bone defects seems essential for proper bone healing in diabetes, regeneration may be supported by the LLLT to enhance osteoblastogenesis.

Effects of mechanical vibration on cell morphology, proliferation, apoptosis, and cytokine expression/secretion in osteocyte-like MLO-Y4 cells exposed to high glucose.

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well-recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO-Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO-Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG-induced apoptosis in MLO-Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG-incubated MLO-Y4 cells were observed after mechanical vibration via the F-actin fluorescence staining. The real-time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β-catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor-κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme-linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E2 and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL in the supernatant of HG-treated MLO-Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.

Impaired Gastric Hormone Regulation of Osteoblasts and Lysyl Oxidase Drives Bone Disease in Diabetes Mellitus.

ABSTRACTDiabetic bone disease is a complication of type I and type II diabetes, both of which are increasing in the United States and elsewhere. Increased hip and foot fracture rates do not correlate well with changes in bone mineral density (BMD), whereas studies support the importance of collagen structure to bone strength. Extracellular lysyl oxidase (LOX) catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagens resulting in aldehydes that subsequently form critically important biosynthetic crosslinks that stabilize functional collagens. Although LOX‐dependent biosynthetic crosslinks in bone collagen are deficient in diabetic bone, the expression and regulation of bone LOXs in diabetes have not been comprehensively studied. Here, we found that LOX is profoundly downregulated in bone in diabetes. Moreover, we have identified a novel metabolic regulatory relationship that is dysregulated in diabetes using mouse models. Data indicate that the incretin (gastric hormone) known as glucose‐dependent insulinotropic polypeptide (GIP) that is anabolic to osteoblasts strongly upregulates LOX, and that this regulation is disrupted in the streptozotocin‐induced model of diabetes in mice. In vivo and in vitro studies support that diabetes results in elevated circulating peripheral dopamine, likely also derived from the gut, and is responsible for blocking GIP signaling and LOX levels in osteoblasts. Moreover, peripheral administration of the dopamine D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of LOX. Taken together our data identifies a novel metabolic relationship between the gut‐derived hormone GIP and bone‐derived LOX, and points to the importance of LOX dysregulation in the pathology of diabetic bone disease. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Correlation between diabetic retinopathy and bone mineral density in type 2 diabetic patients

Correlation between diabetic retinopathy and bone mineral density in type 2 diabetic patients

Photobiomodulation therapy compensate the impairments of diabetic bone marrow mesenchymal stem cells.

Pathophysiologic conditions associated with diabetes mellitus affect mesenchymal stem cells (MSCs), and this phenomenon may lead to some diabetic secondary complications. The present study was conducted to evaluate the impact of photobiomodulation (PBM) on rat diabetic MSC (DMSC) behavior in vitro. For the purpose of PBM, we used helium-neon laser with a wavelength of 632.8nm at three different energy densities (0.5, 1, 2J/cm2) and radiation periodicity of once, twice, and thrice. The survival, proliferation, and apoptosis in the normal MSCs (NMSCs), DMSCs, and diabetic MSCs, which were laser irradiated (DMSCs+L), were assessed using MTT assay, Ki67 immunofluorescence staining, and TUNEL assay, respectively. Our results demonstrated that DMSCs have significantly lower survival (P < 0.05) and proliferation rates (P < 0.001), and dramatically higher population doubling time (PDT, P < 0.001) and apoptosis rates (P < 0.001) as compared to NMSCs. Moreover, PBM with energy density of 1J/cm2 and the periodicity of 1 or 2 times could improve diabetic MSC capabilities in the term of survival, proliferation, and apoptosis. Considering these findings, it is suggested that PBM could improve the ability of diabetic MSCs in vitro prior to transplantation or may rise their capabilities in their native niche in vivo.

The effect of metformin and P38 MAPK inhibitor on diabetic bone porosity in non-obese type 2 diabetic rats

Hyperglycaemia enhance bone resorption and impairment. Controlling blood glucose via metformin benefits bone cells. Hyperglycaemia enhances basal phosphorylation of p38 MAPK, which aggravates bone resorption. Therefore, the aim of this study was to assess the osteoprotective effects of metformin and p38 MAPK inhibitor in non-obese T2DM rats. In this study, non-obese T2DM (Goto-kakizaki, GK) rats were divided into four groups including DM group, metformin treatment, SB203580 treatment, and metformin combined with SB203580. Wistar rats were used as control group. Femur, tibia and iliac rat bones were collected to determine bone porosity via synchrotron radiation microtomography. Primary osteoblasts were isolated from calvarias to investigate cell proliferation and osteoblast function including alkaline phosphatase (ALP) expression and calcium deposition. The results showed that diabetes increase bone porosity. Treatment with metformin significantly reduced porosity in trabecular and cortical bone of the femur, tibia and iliac while SB203580 significantly reduced porosity in cortical bone. A combination group showed significantly reduced bone porosity only in trabecular bone of the femur. Isolated osteoblasts showed lower growth rates. Treatment with metformin significantly increased cell proliferation, ALP expression and calcium deposition. In summary, metformin treatment improved bone quality by reducing bone porosity, increasing cell proliferation and improving osteoblast characteristics.

1202-P: Glucose Lowering with an SGLT2I Improves Trabecular Bone but Does Not Correct Bone Brittleness in TallyHo Model of T2D

Higher fracture risk in T2D is attributed to disease specific deficits in micro-structural and material properties of bone. The TallyHO (TH) mouse is a polygenic model of early-onset T2D/obesity analogous to adolescent-onset T2D in humans. Male TH mice commence rapid weight gain at 4 weeks of age and by ∼10-14 weeks insulin resistance progressing to frank hyperglycemia is apparent in ∼75% of mice. Compared to SWR/J control strain, TH mice exhibit lower: 1) bone formation markers; 2) trabecular bone volume; and 3) fracture resistance. Utilizing this model, we examined the impact of glucose-lowering therapy by SGLT2I on diabetic bone. TH male mice (8-10 weeks) with confirmed hyperglycemia and age-matched, nondiabetic, male SWR/J mice were monitored ± canagliflozin (CANA: 125 ppm in chow) for 10 weeks of treatment. At study end, measurements of body weight, BG, HbA1c, serum insulin, and urine calcium/CR were obtained; femurs were harvested for analysis by µCT and three-point bending. At 20 weeks, these severely hyperglycemic TH mice (BG: 687±106 vs. 151±26 mg/dL; p&amp;lt;0.0001) were obese and had longer femurs with increased cortical bone (↑Ct.Ar, Ct.Th; p&amp;lt;0.001 for all) compared to SWR/J. However, trabecular bone was reduced (↓Tb.BV/TV, Conn.D, Tb.N, Tb.TMD; p&amp;lt;0.001 for all) and TH bones were more brittle (↓toughness; p&amp;lt;0.0001). CANA treatment of TH mice significantly lowered BG and HbA1c while increasing weight gain and insulin secretion (p&amp;lt;0.0001 for all). With glycemic improvement, trabecular bone improved (↑Tb.BV/TV, p=0.0002; Tb.Th, p&amp;lt;0.0001; Tb.TMD; p=0.002) compared to untreated TH mice, but toughness was not altered. Unexpectedly, CANA treatment of SWR/J mice lowered toughness of the femur diaphysis. Hypercalciuria was a consistent feature of both groups of TH mice; CANA also increased Ca excretion in SWR/J mice. CANA therapy may protect against elevated fracture risk in T2D with respect to trabecular rich sites but may not be beneficial if bone becomes brittle with duration. Disclosure K.M. Thrailkill: None. R. Bunn: None. S. Uppuganti: None. P.D. Ray: None. I. Popescu: None. E. Kalaitzoglou: None. J. Fowlkes: None. J.S. Nyman: Advisory Panel; Self; ActiveLife Scientific, Inc. Consultant; Self; Sanofi Genzyme. Research Support; Self; ActiveLife Scientific, Inc., Sanofi Genzyme. Funding National Institutes of Health (R21AR070620, R01DK084045); U.S. Department of Veterans Affairs (1l01BX001018)

Scopoletin and Coumarin Attenuate Aberrant Bone Remodeling in Glucose-exposed Osteoblasts and Osteoclasts (P06-062-19)

ObjectivesDiabetes mellitus is a complex disease with harmful effects on the osteoblast and osteoclast activity. Type 2 diabetic patients have normal or high bone mineral density but associated with an increased risk of fractures. Coumarins (1-benzopyran-2-one) are chemical compounds in the benzopyrone class of organic compounds found in many plants. The purpose of this study was to identify that hyperglycemia resulted in impaired bone remodeling and to examine whether coumarins were capable of preventing diabetic osteoporosis. MethodsThe in vitro study employed osteoblastic MC3T3-E1 cells that were exposed to 33 mM glucose for 6 days in the presence of 20 μM coumarins of scopoletin, aesculetin and coumarin. Alkaline phosphatase (ALP) activity was quantitatively determined in osteoblastic MC3T3-E1 cells by using stable p-nitrophenyl phosphate. In addition, murine macrophage Raw 264.7 cells were differentiated with receptor activator of nuclear factor-κΒ ligand (RANKL) in 33 mM glucose and 20 μM coumarins for 5 days. Tartrate-resistant acid phosphatase (TRAP) activity was measured using an assay kit. ResultsHigh glucose attenuated the ALP activity of osteoblastic MC3T3-E1 cells, which was enhanced by treating scopoletin, aesculetin and coumarin to cells. In addition, 33 mM glucose diminished TRAP activity in RANKL-differentiated Raw 264.7 macrophages, indicating that bone remodeling was impaired in diabetic osteoclasts. In contrast, scopoletin and coumarin elevated osteoclastic differentiation and activation. Interestingly, coumarin was highly effective in balancing bone turnover of osteoblasts and osteoclasts. ConclusionsThese results demonstrate that glucose evoked abnormal bone remodeling leading to diabetic bone disorder. In addition, coumarins such as scopoletin and coumarin ameliorated bone remodeling impaired in diabetic osteoblasts and osteoclasts. These findings suggest the possibility that coumarins might be a potential agent for the treatment of diabetic osteoporosis. Funding SourcesThis work (Grants No. C0501612) was supported by project for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Ministry of SMEs and Startups in 20.