Effects of advanced glycation end-products, diabetes and metformin on the osteoblastic transdifferentiation capacity of vascular smooth muscle cells: In vivo and in vitro studies.

AIMS: Our objective was to study the vascular smooth muscle cells (VSMC) osteoblastic transdifferentiation in AGE exposed cells or those from diabetic animals, and its response to metformin treatment. METHODS: VSMC were obtained from non-diabetic rats, grown with or without AGE; while VSMC of in vivo-ex vivo studies were obtained from non-diabetic control animals (C), diabetic (D), C treated with metformin (M) and D treated with metformin (D-M). We studied the osteoblastic differentiation by evaluating alkaline phosphatase (ALP), type I collagen (Col) and mineral deposit. RESULTS: In vitro, AGE increased proliferation, migration, and osteoblastic differentiation of VSMC. Metformin cotreatment prevented the AGE induced proliferation and migration. Both AGE and metformin stimulated the expression of ALP and Col. AGE induced mineralization was prevented by metformin. VSMC from D expressed a higher production of Col and ALP. Those from D-M showed an ALP increase vs C and M, and a partial decrease vs D. Cultured in osteogenic medium, ALP, Col and mineralization increased in D vs C, remained unchanged in M, and were prevented in D-M animals. CONCLUSION: Both AGE and DM favor VSMC differentiation towards the osteogenic phenotype and this effect can be prevented by metformin.

Incidence and predictive factors of postoperative hypocalcaemia according to type of thyroid surgery in older adults.

PURPOSE: Transient hypocalcaemia after thyroid surgery and its possible predictors have not been extensively described in the elderly. This study aimed to establish the frequency of postsurgical transient hypocalcaemia according to the extent of thyroid surgery in older adults and to assess mineral metabolism biochemical parameters as its predictors. METHODS: All patients ≥60 years undergoing thyroid surgery were prospectively included. Type of surgery (hemithyroidectomy(HT) or total thyroidectomy(TT)); and preoperative 25OH Vitamin D (25OHD) and pre and 6 (only TT), 24 h and 6 months postsurgical serum levels of calcium, magnesium, phosphate and parathormone (PTH) were considered. Postsurgical hypoparathyroidism (hPTpost) was defined at PTH levels ≤11 pg/mL. RESULTS: Out of 46 patients (87% female), age (mean ± SD) 70.1 ± 6.2 years, 24 h postsurgical hypocalcaemia was found in ten patients (22%). In 25 (54%) TT patients, 36% and 16% had postsurgical hypocalcaemia at 6 and 24 h respectively; 28% hPTpost but no definitive hPT was recorded and 44% had 25OHD deficiency. Lower 24 h magnesium levels were found in those TT patients with 24 h hypocalcaemia (1.6 ± 0.1 vs 1.9 ± 0.1 mg/dL (p = 0.005)). Among 21 (46%) HT patients, 28.6% had 24 h postsurgical hypocalcaemia; 9.5% had hPTpost. A positive correlation was observed between preoperative 25OHD and 24 h calcaemia (r:0.51,p = 0.02). 43% of the patients were 25OHD deficient, in whom 55% had 24 h hypocalcaemia vs only 9% in the 25OHD sufficient group (p = 0.049). CONCLUSION: Postsurgical hypocalcaemia was common in elderly thyroidectomized patients. After TT, lower magnesium levels were found in those patients with 24 h hypocalcaemia. In the HT group, preoperative 25OHD deficiency predicted lower postsurgical calcium levels.

Guías Argentinas para el diagnóstico, la prevención y el tratamiento de la osteoporosis 2015 / Argentine guidelines for the diagnosis, prevention and treatment of osteoporosis, 2015

La osteoporosis es una enfermedad en constante crecimiento y que afecta a más de 200 millones de personas a nivel mundial. Nuestras recomendaciones son guías para el diagnóstico, la prevención y tratamiento, pero no normas para las decisiones clínicas en casos individuales. El médico debe adaptarlas a situaciones en la práctica clínica cotidiana, incorporando factores personales que trascienden los límites de estas guías y hacen al saber y al arte de la práctica médica. Como todo conocimiento científico, deben ser actualizadas periódicamente a medida que se adquieran nuevas, mejores y más efectivas herramientas diagnósticas y terapéuticas. (AU)

Osteoporosis is an evolving disease which affects over 200 million people worldwide. Our recommendations are guidelines for its diagnosis, prevention and treatment, but they do not constitute standards for clinical decisions in individual cases. The physician must adapt them to individual special situations, incorporating personal factors that transcend the limits of these guidelines and are dependent on the knowledge and art of the practice of Medicine. These guidelines should be reviewed and updated periodically as new, better and more effective diagnostic and therapeutic tools become available. (AU)

Advanced glycation end products and strontium ranelate promote osteogenic differentiation of vascular smooth muscle cells in vitro: Preventive role of vitamin D.

Advanced glycation end products (AGE) have been demonstrated to induce the osteogenic trans-differentiation of vascular smooth muscle cells (VSMC). Strontium ranelate (SR) is an anti-osteoporotic agent that has both anti-catabolic and anabolic actions on bone tissue. However, in the last years SR has been associated with an increase of cardiovascular risk. We hypothesize that SR can increase the osteoblastic trans-differentiation of VSMC and the induction of extracellular calcifications, an effect that could be potentiated in the presence of AGE and inhibited by simultaneous administration of vitamin D. The present results of our in vitro experiments demonstrate that AGE and SR alone or in combination, stimulate L-type calcium channels, causing an increase in reactive oxygen species and activation of both ERK and NFkB, with the final effect of promoting the osteogenic shift of VSMC. Importantly, these in vitro effects of AGE and/or SR can be prevented by co-incubation with vitamin D.

Effects of fructose-induced metabolic syndrome on rat skeletal cells and tissue, and their responses to metformin treatment.

AIMS: Deleterious effects of metabolic syndrome (MS) on bone are still controversial. In this study we evaluated the effects of a fructose-induced MS, and/or an oral treatment with metformin on the osteogenic potential of bone marrow mesenchymal stromal cells (MSC), as well as on bone formation and architecture. METHODS: 32 male 8week-old Wistar rats were assigned to four groups: control (C), control plus oral metformin (CM), rats receiving 10% fructose in drinking water (FRD), and FRD plus metformin (FRDM). Samples were collected to measure blood parameters, and to perform pQCT analysis and static and dynamic histomorphometry. MSC were isolated to determine their osteogenic potential. RESULTS: Metformin improved blood parameters in FRDM rats. pQCT and static and dynamic histomorphometry showed no significant differences in trabecular and cortical bone parameters among groups. FRD reduced TRAP expression and osteocyte density in trabecular bone and metformin only normalized osteocyte density. FRD decreased the osteogenic potential of MSC and metformin administration could revert some of these parameters. CONCLUSIONS: FRD-induced MS shows reduction in MSC osteogenic potential, in osteocyte density and in TRAP activity. Oral metformin treatment was able to prevent trabecular osteocyte loss and the reduction in extracellular mineralization induced by FRD-induced MS.

[Argentine guidelines for the diagnosis, prevention and treatment of osteoporosis, 2015]. / Guías argentinas para el diagnóstico, la prevención y el tratamiento de la osteoporosis 2015.

Osteoporosis is an evolving disease which affects over 200 million people worldwide. Our recommendations are guidelines for its diagnosis, prevention and treatment, but they do not constitute standards for clinical decisions in individual cases. The physician must adapt them to individual special situations, incorporating personal factors that transcend the limits of these guidelines and are dependent on the knowledge and art of the practice of Medicine. These guidelines should be reviewed and updated periodically as new, better and more effective diagnostic and therapeutic tools become available.

Guías Argentinas para el diagnóstico, la prevención y el tratamiento de la osteoporosis 2015 / Argentine guidelines for the diagnosis, prevention and treatment of osteoporosis, 2015

La osteoporosis es una enfermedad en constante crecimiento y que afecta a más de 200 millones de personas en todo el mundo. Nuestras recomendaciones son guías para el diagnóstico, la prevención y tratamiento, pero no normas para las decisiones clínicas en casos individuales. El médico debe adaptarlas a situaciones en la práctica clínica cotidiana, incorporando factores personales que trascienden los límites de estas guías y hacen al saber y al arte de la práctica médica. Como todo conocimiento científico, deben ser actualizadas periódicamente a medida que se adquieran nuevas, mejores y más efectivas herramientas diagnósticas y terapéuticas.

Osteoporosis is an evolving disease which affects over 200 million people worldwide. Our recommendations are guidelines for its diagnosis, prevention and treatment, but they do not constitute standards for clinical decisions in individual cases. The physician must adapt them to individual special situations, incorporating personal factors that transcend the limits of these guidelines and are dependent on the knowledge and art of the practice of Medicine. These guidelines should be reviewed and updated periodically as new, better and more effective diagnostic and therapeutic tools become available.

Metformin revisited: Does this regulator of AMP-activated protein kinase secondarily affect bone metabolism and prevent diabetic osteopathy.

Patients with long-term type 1 and type 2 diabetes mellitus (DM) can develop skeletal complications or "diabetic osteopathy". These include osteopenia, osteoporosis and an increased incidence of low-stress fractures. In this context, it is important to evaluate whether current anti-diabetic treatments can secondarily affect bone metabolism. Adenosine monophosphate-activated protein kinase (AMPK) modulates multiple metabolic pathways and acts as a sensor of the cellular energy status; recent evidence suggests a critical role for AMPK in bone homeostasis. In addition, AMPK activation is believed to mediate most clinical effects of the insulin-sensitizer metformin. Over the past decade, several research groups have investigated the effects of metformin on bone, providing a considerable body of pre-clinical (in vitro, ex vivo and in vivo) as well as clinical evidence for an anabolic action of metformin on bone. However, two caveats should be kept in mind when considering metformin treatment for a patient with type 2 DM at risk for diabetic osteopathy. In the first place, metformin should probably not be considered an anti-osteoporotic drug; it is an insulin sensitizer with proven macrovascular benefits that can secondarily improve bone metabolism in the context of DM. Secondly, we are still awaiting the results of randomized placebo-controlled studies in humans that evaluate the effects of metformin on bone metabolism as a primary endpoint.

Saxagliptin affects long-bone microarchitecture and decreases the osteogenic potential of bone marrow stromal cells.

Diabetes mellitus is associated with a decrease in bone quality and an increase in fracture incidence. Additionally, treatment with anti-diabetic drugs can either adversely or positively affect bone metabolism. In this study we evaluated: the effect of a 3-week oral treatment with saxagliptin on femoral microarchitecture in young male non-type-2-diabetic Sprague Dawley rats; and the in vitro effect of saxagliptin and/or fetal bovine serum (FBS), insulin or insulin-like growth factor-1 (IGF1), on the proliferation, differentiation (Runx2 and PPAR-gamma expression, type-1 collagen production, osteocalcin expression, mineralization) and extracellular-regulated kinase (ERK) activation, in bone marrow stromal cells (MSC) obtained from control (untreated) rats and in MC3T3E1 osteoblast-like cells. In vivo, oral saxagliptin treatment induced a significant decrease in the femoral osteocytic and osteoblastic density of metaphyseal trabecular bone and in the average height of the proximal cartilage growth plate; and an increase in osteoclastic tartrate-resistant acid phosphatase (TRAP) activity of the primary spongiosa. In vitro, saxagliptin inhibited FBS-, insulin- and IGF1-induced ERK phosphorylation and cell proliferation, in both MSC and MC3T3E1 preosteoblasts. In the absence of growth factors, saxagliptin had no effect on ERK activation or cell proliferation. In both MSC and MC3T3E1 cells, saxagliptin in the presence of FBS inhibited Runx2 and osteocalcin expression, type-1 collagen production and mineralization, while increasing PPAR-gamma expression. In conclusion, orally administered saxagliptin induced alterations in long-bone microarchitecture that could be related to its in vitro down-regulation of the ERK signaling pathway for insulin and IGF1 in MSC, thus decreasing the osteogenic potential of these cells.

Insulin-deficient diabetes-induced bone microarchitecture alterations are associated with a decrease in the osteogenic potential of bone marrow progenitor cells: preventive effects of metformin.

AIMS: Diabetes mellitus is associated with metabolic bone disease and increased low-impact fractures. The insulin-sensitizer metformin possesses in vitro, in vivo and ex vivo osteogenic effects, although this has not been adequately studied in the context of diabetes. We evaluated the effect of insulin-deficient diabetes and/or metformin on bone microarchitecture, on osteogenic potential of bone marrow progenitor cells (BMPC) and possible mechanisms involved. METHODS: Partially insulin-deficient diabetes was induced in rats by nicotinamide/streptozotocin-injection, with or without oral metformin treatment. Femoral metaphysis micro-architecture, ex vivo osteogenic potential of BMPC, and BMPC expression of Runx-2, PPARγ and receptor for advanced glycation endproducts (RAGE) were investigated. RESULTS: Histomorphometric analysis of diabetic femoral metaphysis demonstrated a slight decrease in trabecular area and a significant reduction in osteocyte density, growth plate height and TRAP (tartrate-resistant acid phosphatase) activity in the primary spongiosa. BMPC obtained from diabetic animals showed a reduction in Runx-2/PPARγ ratio and in their osteogenic potential, and an increase in RAGE expression. Metformin treatment prevented the diabetes-induced alterations in bone micro-architecture and BMPC osteogenic potential. CONCLUSION: Partially insulin-deficient diabetes induces deleterious effects on long-bone micro-architecture that are associated with a decrease in BMPC osteogenic potential, which could be mediated by a decrease in their Runx-2/PPARγ ratio and up-regulation of RAGE. These diabetes-induced alterations can be totally or partially prevented by oral administration of metformin.

Strontium ranelate prevents the deleterious action of advanced glycation endproducts on osteoblastic cells via calcium channel activation.

Accumulation of advanced glycation endproducts (AGEs) in bone tissue occurs in ageing and in Diabetes mellitus, and is partly responsible for the increased risk of low-stress bone fractures observed in these conditions. In this study we evaluated whether the anti-osteoporotic agent strontium ranelate can prevent the deleterious effects of AGEs on bone cells, and possible mechanisms of action involved. Using mouse MC3T3E1 osteoblastic cells in culture we evaluated the effects of 0.1mM strontium ranelate and/or 100 µg/ml AGEs-modified bovine serum albumin (AGEs-BSA) on cell proliferation, osteogenic differentiation and pro-inflammatory cytokine production. We found that AGEs-BSA alone decreased osteoblastic proliferation and differentiation (P<0.01) while increasing IL-1ß and TNFα production (P<0.01). On its own, strontium ranelate induced opposite effects: an increase in osteoblast proliferation and differentiation (P<0.01) and a decrease in cytokine secretion (P<0.01). Additionally, strontium ranelate prevented the inhibitory and pro-inflammatory actions of AGEs-BSA on osteoblastic cells (P<0.01). These effects of strontium ranelate were blocked by co-incubation with either the MAPK inhibitor PD98059, or the calcium channel blocker nifedipine. We also evaluated by Western blotting the activation status of ERK (a MAPK) and b-catenin. Activation of both signaling pathways was decreased by AGEs treatment, and this inhibitory effect was prevented if AGEs were co-incubated with strontium ranelate (P<0.01). On its own, strontium ranelate increased both pERK and activated b-catenin levels. In conclusion, this study demonstrates that strontium ranelate can prevent the deleterious in vitro actions of AGEs on osteoblastic cells in culture by mechanisms that involve calcium channel, MAPK and b-catenin activation.

Metformin prevents anti-osteogenic in vivo and ex vivo effects of rosiglitazone in rats.

Long-term treatment with the insulin-sensitizer rosiglitazone reduces bone mass and increases fracture risk. We have recently shown that orally administered metformin stimulates bone reossification and increases the osteogenic potential of bone marrow progenitor cells (BMPC). In the present study we investigated the effect of a 2-week metformin and/or rosiglitazone treatment on bone repair, trabecular bone microarchitecture and BMPC osteogenic potential, in young male Sprague-Dawley rats. Compared to untreated controls, rosiglitazone monotherapy decreased bone regeneration, femoral metaphysis trabecular area, osteoblastic and osteocytic density, and TRAP activity associated with epiphyseal growth plates. It also decreased the ex vivo osteogenic commitment of BMPC, inducing an increase in PPARγ expression, and a decrease in Runx2/Cbfa1 expression, in AMP-kinase phosphorylation, and in osteoblastic differentiation and mineralization. After monotherapy with metformin, with the exception of PPARγ expression which was blunted, all of the above parameters were significantly increased (compared to untreated controls). Metformin/rosiglitazone co-treatment prevented all the in vivo and ex vivo anti-osteogenic effects of rosiglitazone monotherapy, with a reversion back to control levels of PPARγ, Runx2/Cbfa1 and AMP-kinase phosphorylation of BMPC. In vitro co-incubation of BMPC with metformin and compound C-an inhibitor of AMPK phosphorylation-abrogated the metformin-induced increase in type-1 collagen production, a marker of osteoblastic differentiation. In conclusion, in rodent models metformin not only induces direct osteogenic in vivo and ex vivo actions, but when it is administered orally in combination with rosiglitazone it can prevent several of the adverse effects that this thiazolidenedione shows on bone tissue.

Effect of metformin on bone marrow progenitor cell differentiation: in vivo and in vitro studies.

Diabetes mellitus is associated with bone loss. Patients with type 2 diabetes are frequently treated with oral antidiabetic drugs such as sulfonylureas, biguanides, and thiazolidinediones. Rosiglitazone treatment has been shown to increase adipogenesis in bone marrow and to induce bone loss. In this study we evaluated the effect of in vivo and in vitro treatment with metformin on bone marrow progenitor cells (BMPCs), as well as the involvement of AMPK pathway in its effects. The in vitro effect of coincubation with metformin and rosiglitazone on the adipogenic differentiation of BMPCs also was studied. In addition, we evaluated the effect of in vivo metformin treatment on bone regeneration in a model of parietal lesions in nondiabetic and streptozotocin-induced diabetic rats. We found that metformin administration both in vivo and in vitro caused an increase in alkaline phosphatase activity, type I collagen synthesis, osteocalcin expression, and extracellular calcium deposition of BMPCs. Moreover, metformin significantly activated AMPK in undifferentiated BMPCs. In vivo, metformin administration enhanced the expression of osteoblast-specific transcription factor Runx2/Cbfa1 and activation of AMPK in a time-dependent manner. Metformin treatment also stimulated bone lesion regeneration in control and diabetic rats. In vitro, metformin partially inhibited the adipogenic actions of rosiglitazone on BMPCs. In conclusion, our results indicate that metformin causes an osteogenic effect both in vivo and in vitro, possibly mediated by Runx2/Cbfa1 and AMPK activation, suggesting a possible action of metformin in a shift toward the osteoblastic differentiation of BMPCs.

Osteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture.

An association has been previously established between uncompensated diabetes mellitus and the loss of bone mineral density and/or quality. In this study, we evaluated the effects of metformin on the growth and differentiation of osteoblasts in culture. Treatment of two osteoblast-like cells (UMR106 and MC3T3E1) with metformin (25-500 microM) for 24 h led to a dose-dependent increase of cell proliferation. Metformin also promoted osteoblastic differentiation: it increased type-I collagen production in both cell lines and stimulated alkaline phosphatase activity in MC3T3E1 osteoblasts. In addition, metformin markedly increased the formation of nodules of mineralization in 3-week MC3T3E1 cultures. Metformin induced activation and redistribution of phosphorylated extracellular signal-regulated kinase (P-ERK) in a transient manner, and dose-dependently stimulated the expression of endothelial and inducible nitric oxide synthases (e/iNOS). These results show for the first time a direct osteogenic effect of metformin on osteoblasts in culture, which could be mediated by activation/redistribution of ERK-1/2 and induction of e/iNOS.