RESUMO
OBJECTIVE: The aims of this study were: first, to evaluate the possible effects of a fructose rich diet (FRD)-induced metabolic syndrome (MS) on different aspects of long bone histomorphometry in young male rats; second, to investigate the effects of this diet on bone tissue regeneration; and third, to correlate these morphometric alterations with changes in the osteogenic/adipogenic potential and expression of specific transcription factors, of marrow stromal cells (MSC) isolated from rats with fructose-induced MS. MATERIALS/METHODS: MS was induced in rats by treatment with a FRD for 28 days. Halfway through treatment, a parietal wound was made and bone healing was evaluated 14 days later. After treatments, histomorphometric analysis was performed in dissected femoral and parietal bones. MSC were isolated from the femora of control or fructose-treated rats and differentiated either to osteoblasts (evaluated by type 1 collagen, Alkaline phosphatase and extracellular nodule mineralization) or to adipocytes (evaluated by intracellular triglyceride accumulation). Expression of Runx2 and PPARγ was assessed by Western blot. RESULTS: Fructose-induced MS induced deleterious effects on femoral metaphysis microarchitecture and impaired bone regeneration. Fructose treatment decreased the osteogenic potential of MSC and Runx2 expression. In addition, it increased the adipogenic commitment of MSC and PPARγ expression. CONCLUSIONS: Fructose-induced MS is associated with deleterious effects on bone microarchitecture and with a decrease in bone repair. These alterations could be due to a deviation in the adipogenic/osteogenic commitment of MSC, probably by modulation of the Runx2/PPARγ ratio.
Assuntos
Adipócitos/metabolismo , Células da Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Frutose/administração & dosagem , Frutose/farmacologia , Síndrome Metabólica/metabolismo , Osteoblastos/metabolismo , Edulcorantes/administração & dosagem , Edulcorantes/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Regeneração Óssea , Osso e Ossos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Dieta , Fêmur/metabolismo , Frutose/metabolismo , Masculino , Síndrome Metabólica/patologia , Osteogênese/efeitos dos fármacos , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Edulcorantes/metabolismo , Fatores de Tempo , Triglicerídeos/metabolismoRESUMO
Introducción: En la Diabetes Mellitus se ha descripto un incremento en el riesgo de fracturas, las cuales podrían asociarse a la acumulación de productos de glicación avanzada (AGEs) que alteran la función de los osteoclastos (Oc), células gigantes multinucleadas encargadas de resorber el hueso. Los bifosfonatos (BP), drogas ampliamente usadas en enfermedades áseas, inhiben la actividad resortiva de los Oc, aunque su uso en pacientes diabéticos es controversial. Objetivo: Estudiar el efecto de AGEs y alendronato sobre el desarrollo de Oc en cultivo, así como los posibles mecanismos involucrados en la acción de estos agentes. Materiales y Métodos: Se cocultivaron macráfagos Raw264.7 y osteoblastos UMR-106 durante 8 días, con BSA o AGE (50-200 µg/ml), con o sin alendronato (10-8-10-4M). Se evaluá el efecto de estas condiciones de cultivo sobre la formación de Oc (número de los mismos, y actividad de fosfatasa ácida tartrato-resistente [TRAP] ), la expresión de RAGE (Receptor de AGEs) en los Oc, y la expresión del ligando de RANK (RANKL) en los osteoblastos por inmunofluorescencia indirecta. Resultados: Los AGEs (50-200 µg/ml) inhibieron en forma dosis-dependiente la TRAP (10-30 %) y el número de osteoclastos generados (55 %), similarmente a lo inducido por bajas dosis de alendronato (10-8M-10-6M). La coincubación de bajas dosis de alendronato con 100 µg/ml de AGEs no indujo una inhibición adicional a la de los AGEs sobre la actividad de TRAP o el número de Oc. Altos niveles de alendronato (10-5M-10-4M) inhibieron la actividad TRAP (20-25 % respecto a BSA y 17 % respecto de AGEs), así como el número de Oc desarrollados en presencia de AGEs (16 % con respecto a AGEs). Los Oc incubados en presencia de 100 µg/ml AGEs mostraron un incremento significativo en la expresión de RAGE (152 % respecto de BSA), situación similar a la observada postincubación con alendronato 10-8M (130 % respecto de BSA). Por el contrario, altas dosis de alendronato (10-5M) no modificaron la expresión del RAGE en los cocultivos incubados con BSA (95 % respecto de BSA). Por otro lado, bajas dosis de alendronato en presencia de AGEs no alteraron la "up-regulation" del RAGE inducida por los AGEs (145 % respecto de BSA). Sin embargo, cuando los Oc se incubaron con AGEs y Ale 10-5M, esta dosis del bifosfonato bloqueá el efecto estimulante de los AGEs sobre la expresión de RAGE (105 % respecto de BSA). La incubación con 100 µg/ml AGE produjo una inhibición (50 % respecto de BSA), en la expresión del RANKL en los osteoblastos. El alendronato (10-8M-10-5M) indujo también una inhibición del RANKL en forma dosis dependiente (65-47 % respecto de BSA). Por otro lado en presencia de AGEs, el alendronato (10-8M-10-5M) no modificá la inhibición de la expresión del RANKL inducida por los AGEs (59-45 % del BSA). Conclusiones: Los AGEs y el alendronato inhiben el número y diferenciación de Oc en cultivo, con un efecto aditivo entre ambos a altas concentraciones de alendronato. También reducen la expresión de RANKL en osteoblastos, lo cual podría explicar parcialmente sus efectos sobre el reclutamiento y la maduración de Oc. Los AGEs y bajas dosis de alendronato aumentan la expresión de RAGE en Oc.
Introduction: Patients with Diabetes mellitus frequently show osteopenia and/or osteoporosis, as well as an increase in low-trauma fracture risk. This has been postulated to be caused partially by the accumulation of advanced glycation endproducts (AGEs) in bone extracellular matrix. AGEs could affect the homeostasis of bone cells, such as osteoblasts, osteocytes and osteoclasts. Osteoclasts (Oc) are multi-nucleated cells specialized in resorbing bone. Bisphosphonates (BP) are drugs widely used for treatment of bone diseases, and their principal mechanism of action is to inhibit the resorptive action of Oc. However, the use of BP for the treatment of patients with Diabetes-related bone disease is still controversial. Objective: To study the effect of AGEs and Alendronate (an N-containing BP) on the development of Oc in culture, as well as possible mechanisms of action involved in these effects. Materials and Methods: RAW264.7 macrophages and UMR106 osteoblasts were co-cultured for 8 days, with BSA or AGEs (50-200 µg/ml), with or without Alendronate (10-8-10-4M). The effect of these culture conditions on Oc formation was evaluated (number of Oc, cell-associated tartrate-resistant acid phosphatase [TRAP] activity), as well as Oc expression of RAGE (receptor for AGEs), and osteoblastic expression of RANK ligand (RANKL). These last two parameters were evaluated by indirect immunofluorescence, in order to estimate the expression and sub-cellular distribution of both membrane-associated proteins. Results: AGEs (50-200 µg/ml) dose-dependently inhibited TRAP activity (10-30 %) and the number of multinucleated Oc (55 %). Similar results were observed for low doses of Alendronate (10-8M-10-6M). The co-incubation of low doses of Alendronate with 100 mg/ml of AGEs, did not induce an additional inhibition of TRAP activity or Oc number, to that observed for AGEs alone. High levels of Alendronate (10-5M-10-4M) inhibited Oc TRAP activity (20-25 % inhibition versus BSA, and 17 % versus AGEs), and also decreased the number of Oc formed in the presence of AGEs (16 % inhibition versus AGEs). Oc incubated in the presence of 100 µg/ml AGEs, showed a significant increase in the expression of RAGE (152 % versus BSA). Similar results were found after incubating with 10-8M Alendronate (130 % versus BSA). On the contrary, high doses of Alendronate (10-5M) did not affect the expression of RAGE in co-cultures incubated with BSA (95 % versus BSA). On the other hand, low doses of Alendronate in the presence of AGEs, did not affect the up-regulation of RAGE induced by AGEs (145 % versus BSA). However, when the Oc were co-incubated with AGEs and 10-5M Alendronate, this dose of BP was able to block the stimulation of RAGE expression induced by AGEs (105 % versus BSA). In osteoblasts, incubation with 100 µg/ml AGEs induced an inhibition in the expression of RANKL (50 % versus BSA). Alendronate (10-8M-10-5M) also inhibited RANKL expression in a dose-dependent manner (65-47 % versus BSA). However, Alendronate (10-8M-10-5M) did not modify the AGEs-induced inhibition in osteoblastic RANKL expression (59-45 % versus BSA). Conclusions: AGEs and Alendronate inhibit the number and differentiation of Oc in culture, with an additive effect for both agents at high Alendronate concentrations. AGEs and Alendronate also reduce the osteoblastic expression of RANKL, which could partially explain their effects on the recruitment and maturation of Oc. In addition, AGEs and low doses of Alendronate increase the expression of RAGE in cultured Oc, and this effect correlates with their inhibition of Oc development.
RESUMO
Introducción: En la Diabetes Mellitus se ha descripto un incremento en el riesgo de fracturas, las cuales podrían asociarse a la acumulación de productos de glicación avanzada (AGEs) que alteran la función de los osteoclastos (Oc), células gigantes multinucleadas encargadas de resorber el hueso. Los bifosfonatos (BP), drogas ampliamente usadas en enfermedades áseas, inhiben la actividad resortiva de los Oc, aunque su uso en pacientes diabéticos es controversial. Objetivo: Estudiar el efecto de AGEs y alendronato sobre el desarrollo de Oc en cultivo, así como los posibles mecanismos involucrados en la acción de estos agentes. Materiales y Métodos: Se cocultivaron macráfagos Raw264.7 y osteoblastos UMR-106 durante 8 días, con BSA o AGE (50-200 Ag/ml), con o sin alendronato (10-8-10-4M). Se evaluá el efecto de estas condiciones de cultivo sobre la formación de Oc (número de los mismos, y actividad de fosfatasa ácida tartrato-resistente [TRAP] ), la expresión de RAGE (Receptor de AGEs) en los Oc, y la expresión del ligando de RANK (RANKL) en los osteoblastos por inmunofluorescencia indirecta. Resultados: Los AGEs (50-200 Ag/ml) inhibieron en forma dosis-dependiente la TRAP (10-30 %) y el número de osteoclastos generados (55 %), similarmente a lo inducido por bajas dosis de alendronato (10-8M-10-6M). La coincubación de bajas dosis de alendronato con 100 Ag/ml de AGEs no indujo una inhibición adicional a la de los AGEs sobre la actividad de TRAP o el número de Oc. Altos niveles de alendronato (10-5M-10-4M) inhibieron la actividad TRAP (20-25 % respecto a BSA y 17 % respecto de AGEs), así como el número de Oc desarrollados en presencia de AGEs (16 % con respecto a AGEs). Los Oc incubados en presencia de 100 Ag/ml AGEs mostraron un incremento significativo en la expresión de RAGE (152 % respecto de BSA), situación similar a la observada postincubación con alendronato 10-8M (130 % respecto de BSA). Por el contrario, altas dosis de alendronato (10-5M) no modificaron la expresión del RAGE en los cocultivos incubados con BSA (95 % respecto de BSA). Por otro lado, bajas dosis de alendronato en presencia de AGEs no alteraron la "up-regulation" del RAGE inducida por los AGEs (145 % respecto de BSA). Sin embargo, cuando los Oc se incubaron con AGEs y Ale 10-5M, esta dosis del bifosfonato bloqueá el efecto estimulante de los AGEs sobre la expresión de RAGE (105 % respecto de BSA). La incubación con 100 Ag/ml AGE produjo una inhibición (50 % respecto de BSA), en la expresión del RANKL en los osteoblastos. El alendronato (10-8M-10-5M) indujo también una inhibición del RANKL en forma dosis dependiente (65-47 % respecto de BSA). Por otro lado en presencia de AGEs, el alendronato (10-8M-10-5M) no modificá la inhibición de la expresión del RANKL inducida por los AGEs (59-45 % del BSA). Conclusiones: Los AGEs y el alendronato inhiben el número y diferenciación de Oc en cultivo, con un efecto aditivo entre ambos a altas concentraciones de alendronato. También reducen la expresión de RANKL en osteoblastos, lo cual podría explicar parcialmente sus efectos sobre el reclutamiento y la maduración de Oc. Los AGEs y bajas dosis de alendronato aumentan la expresión de RAGE en Oc.(AU)
Introduction: Patients with Diabetes mellitus frequently show osteopenia and/or osteoporosis, as well as an increase in low-trauma fracture risk. This has been postulated to be caused partially by the accumulation of advanced glycation endproducts (AGEs) in bone extracellular matrix. AGEs could affect the homeostasis of bone cells, such as osteoblasts, osteocytes and osteoclasts. Osteoclasts (Oc) are multi-nucleated cells specialized in resorbing bone. Bisphosphonates (BP) are drugs widely used for treatment of bone diseases, and their principal mechanism of action is to inhibit the resorptive action of Oc. However, the use of BP for the treatment of patients with Diabetes-related bone disease is still controversial. Objective: To study the effect of AGEs and Alendronate (an N-containing BP) on the development of Oc in culture, as well as possible mechanisms of action involved in these effects. Materials and Methods: RAW264.7 macrophages and UMR106 osteoblasts were co-cultured for 8 days, with BSA or AGEs (50-200 Ag/ml), with or without Alendronate (10-8-10-4M). The effect of these culture conditions on Oc formation was evaluated (number of Oc, cell-associated tartrate-resistant acid phosphatase [TRAP] activity), as well as Oc expression of RAGE (receptor for AGEs), and osteoblastic expression of RANK ligand (RANKL). These last two parameters were evaluated by indirect immunofluorescence, in order to estimate the expression and sub-cellular distribution of both membrane-associated proteins. Results: AGEs (50-200 Ag/ml) dose-dependently inhibited TRAP activity (10-30 %) and the number of multinucleated Oc (55 %). Similar results were observed for low doses of Alendronate (10-8M-10-6M). The co-incubation of low doses of Alendronate with 100 mg/ml of AGEs, did not induce an additional inhibition of TRAP activity or Oc number, to that observed for AGEs alone. High levels of Alendronate (10-5M-10-4M) inhibited Oc TRAP activity (20-25 % inhibition versus BSA, and 17 % versus AGEs), and also decreased the number of Oc formed in the presence of AGEs (16 % inhibition versus AGEs). Oc incubated in the presence of 100 Ag/ml AGEs, showed a significant increase in the expression of RAGE (152 % versus BSA). Similar results were found after incubating with 10-8M Alendronate (130 % versus BSA). On the contrary, high doses of Alendronate (10-5M) did not affect the expression of RAGE in co-cultures incubated with BSA (95 % versus BSA). On the other hand, low doses of Alendronate in the presence of AGEs, did not affect the up-regulation of RAGE induced by AGEs (145 % versus BSA). However, when the Oc were co-incubated with AGEs and 10-5M Alendronate, this dose of BP was able to block the stimulation of RAGE expression induced by AGEs (105 % versus BSA). In osteoblasts, incubation with 100 Ag/ml AGEs induced an inhibition in the expression of RANKL (50 % versus BSA). Alendronate (10-8M-10-5M) also inhibited RANKL expression in a dose-dependent manner (65-47 % versus BSA). However, Alendronate (10-8M-10-5M) did not modify the AGEs-induced inhibition in osteoblastic RANKL expression (59-45 % versus BSA). Conclusions: AGEs and Alendronate inhibit the number and differentiation of Oc in culture, with an additive effect for both agents at high Alendronate concentrations. AGEs and Alendronate also reduce the osteoblastic expression of RANKL, which could partially explain their effects on the recruitment and maturation of Oc. In addition, AGEs and low doses of Alendronate increase the expression of RAGE in cultured Oc, and this
RESUMO
Patients with long-standing Diabetes mellitus can develop osteopenia and osteoporosis. We have previously shown that advanced glycation endproducts reduce the bone-forming activity of osteoblasts. Bisphosphonates are used for the treatment of various bone disorders, since they reduce osteoclastic function and survival, and stimulate osteoblastic bone-forming capacity. In this work we have investigated whether bisphosphonates are able to revert advanced glycation endproducts-induced deleterious effects in osteoblasts. MC3T3E1 and UMR106 osteoblastic cells were incubated with control or advanced glycation endproducts-modified bovine serum albumin, in the presence or absence of different doses of the bisphosphonates Alendronate, Pamidronate or Zoledronate. After 24-72 h of culture, we evaluated their effects on cell proliferation and apoptosis, type-1 collagen production, alkaline and neutral phosphatase activity, and intracellular reactive oxygen species production. Advanced glycation endproducts significantly decreased osteoblast proliferation, alkaline phosphatase activity and type 1 collagen production, while increasing osteoblastic apoptosis and reactive oxygen species production. These effects were completely reverted by low doses (10(-8) M) of bisphosphonates. High doses of bisphosphonates (10(-4)-10(-5) M) were toxic for osteoblasts. Nifedipine (L-type calcium channel blocker) did not affect the advanced glycation endproducts-induced decrease in osteoblastic proliferation, although it blocked the reversion of this effect by 10(-8) M Alendronate. Both advanced glycation endproducts and Alendronate inhibited the activity of intracellular neutral phosphatases. In conclusion, we show that bisphosphonates revert the deleterious actions of advanced glycation endproducts on osteoblastic cells, and that these effects of bisphosphonates depend on: (a) Ca(2+) influx through L-type voltage-sensitive channels, and (b) blockage of advanced glycation endproducts-induced reactive oxygen species generation.