ABSTRACT
En este trabajo, estudiamos el efecto de una Diabetes inducida por destrucción parcial de la masa de células beta pancreáticas, sobre el compromiso osteogénico de células progenitoras de médula ósea (CPMO), y su modulación por el tratamiento oral con Metformina. Para ello utilizamos ratas Sprague Dawley, divididas en cuatro grupos: controles [C], controles tratadas con Metformina [M], diabéticas [D], y diabéticas tratadas con Metformina [DM]. La inducción de Diabetes se realizó, por inyección intraperitoneal sucesiva de ácido nico-tínico y estreptozotocina. Sobre los cultivos de CPMO se evaluó la actividad específica de Fosfatasa Alcalina (FAL) y la producción de Colágeno tipo 1 (Col-1) en estado basal y en medio de diferenciación osteogénico luego de 15 días. A los 21 días, se evaluaron los depósitos de mineral extracelular. La FAL y el Col-1 de CPMO basales, no mostraron diferencias significativas entre los cuatro grupos experimentales. Al cabo de 15 días, las CPMO de ratas M mostraron un incremento en el Col-1 de 122 % respecto de C; D 30 % respecto de C y DM 68 % respecto de C. La FAL expresó un 171 % para M, 34 % para D; y 125 % para DM todos respecto de C. Luego de 21 días, se observó una disminución en la mineralización de las CPMO de D (65 % respecto del grupo C). El tratamiento con metformina incrementó la mineralización de las CPMO en todos los casos. En conclusión, en nuestro modelo experimental de Diabetes, ésta disminuye el potencial osteogénico de las CPMO, un efecto que es parcialmente revertido por el tratamiento oral con Metformina. Estos hallazgos podrían explicar, al menos en parte, las alteraciones óseas descriptas en el hueso asociadas con la Diabetes. Los autores declaran no poseer conflictos de interés.
Diabetes mellitus is associated with an increased incidence of skeletal abnormalities, resulting in lower bone formation and/or remodeling. Osteopenia, osteoporosis and an increased incidence of non-traumatic fractures has been particularly observed in patients with type 2 diabetes. Recently, we have demonstrated that metformin has in vitro and in vivo osteogenic effects: (a) it stimulates the proliferation, differentiation and mineralization of osteoblasts in culture, and (b) in non-diabetic rats, it increases the repair of minimal bone lesions and improves femoral trabecular bone microarchitecture. In this study, we evaluated in rats the effect of diabetes induction by a partial destruction of pancreatic beta cells, on the osteogenic commitment of bone marrow progenitor cells (BMPC), and the modulation of this effect by orally administered metformin. We used young male Sprague Dawley rats (200 g), divided into four groups: untreated non-diabetic controls [C], non-diabetic rats treated for 2 weeks with metformin administered in drinking water (100 mg/kg/day) [M], untreated diabetic rats [D], and diabetic rats treated for 2 weeks with metformin (100 mg/kg/day) [DM]. Induction of Diabetes was performed one week prior to treatment with metformin, by successive intraperitoneal injections with 75 mg/kg body weight of nicotinic acid and 60 mg/kg body weight of streptozotocin. At the end of all treatments, blood samples were obtained to confirm the development of Diabetes, after which the animals were sacrificed by cervical dislocation under anesthesia. Femora and/or tibiae were dissected, and bone marrow cells were collected by flushing the bone diaphysal canal with Dulbecco's modified essential medium (DMEM) under sterile conditions. Adherent cells were grown to confluence in DMEM-10 % fetal bovine serum (FBS), after which we assessed alkaline phosphatase specific activity (ALP) by an enzymatic kinetic method, and type 1 collagen production (Col-1) by a Sirius Red colorimetric method (basal osteoblastic differentiation of BMPC). Subsequently, BMPC were submitted to an osteogenic induction for 15 days with an osteogenic medium (DMEM-10 % FBS containing ascorbic acid and sodium beta-glycerophosphate), after which ALP and Col-1 were evaluated. Basal ALP activity and type 1 collagen production (BMPC without osteogenic differentiation) showed no significant differences between the four experimental groups. After 15 days of culture in osteogenic medium, BMPC from control rats increased their expression of ALP (5 times compared to baseline) and collagen production (11 times compared to baseline). BMPC from diabetic rats after 15 days culture in osteogenic medium, also showed a significant (although smaller) increase in ALP (2-3 fold over basal activity) and collagen production (4-fold compared to baseline). BMPC obtained from rats treated with metformin (groups M and DM) and submitted to osteogenic induction for 15 days, showed an approximately 2-4-fold increase in both ALP and Col-1 (when compared with groups C and D, respectively). After 21 days of osteogenic induction, a decrease was observed in the mineralization of BMPC obtained from group D (65 % of that for group C). Treatment with metformin increased the mineralizing capacity of BMPC in all cases, including a reversal of the inhibitory effect of Diabetes on this parameter. In conclusion, we have found that our model of Diabetes reduces the osteogenic potential of bone marrow progenitor cells, and that this effect is partially reverted by orally administered metformin. These findings could explain, at least in part, the bone alterations that have been associated with Diabetes mellitus. No financial conflicts of interest exist.
ABSTRACT
El Sndrome Metablico (SM) se ha asociado recientemente con una disminucin en la densidad mineral sea, y con un aumento en la incidencia de fracturas osteoporticas. Recientemente encontramos que la Metformina por va oral en ratas, promueve la diferenciacin osteognica de clulas progenitoras de mdula sea e incrementa la reparacin de lesiones seas. En este trabajo evaluamos los efectos del SM inducido por Fructosa sobre la microarquitectura sea en ratas, y la modulacin de estos efectos por Metformina administrada en forma oral. Utilizamos ratas Sprague Dawley macho jvenes: C (control sin tratamiento), C+M (100mg/kg/da Metformina en el agua de bebida), F (10 % Fructosa en el agua de bebida) y F+M (Fructosa+Metformina en el agua de bebida). Los tratamientos se continuaron por 3 semanas luego de lo cual se tomaron muestras de sangre, previas al sacrificio de los animales. Se disecaron los fmures para evaluacin histomorfomtrica de la microarquitectura metafisaria por tincin con Hematoxilina-Eosina (H-E). Se observ un incremento en la glucemia y trigliceridemia en el grupo F versus el C, compatible con el desarrollo de SM. El anlisis de las metfisis femorales mostr un aumento en la densidad osteoctica trabecular para el grupo C+M (118 % del control, p<0,05). El tratamiento con Fructosa sola disminuy la densidad osteoctica (79 % del control, p<0,05), mientras que el co-tratamiento Fructosa+Metformina (grupo F+M) revirti parcialmente este descenso (88 % del control). Similarmente, el porcentaje de hueso trabecular en la metfisis femoral aument luego del tratamiento slo con Metformina (129 % respecto del control), se redujo en las ratas tratadas con Fructosa (89 % respecto del control), y fue intermedia en el grupo F+M (94 % respecto del control). Estos resultados muestran que el SM inducido por Fructosa en ratas altera la microarquitectura metafisaria femoral; y que estos efectos deletreos pueden ser parcialmente prevenidos por un tratamiento oral con Metformina.
Several clinical studies have demonstrated that the Metabolic Syndrome (MS) is associated with a decrease in bone mineral density, and with an increased risk for non-vertebral osteoporotic fractures. We have recently found that orally administered Metformin induces osteogenic effects in rats, promoting osteoblastic differentiation of bone marrow progenitor cells and increasing the repair of bone lesions. In the present work we have evaluated the effects of Fructose-induced MS on bone micro-architecture in rats, and the possible modulation of these effects by orally administered Metformin. We utilized young male Sprague-Dawley rats, divided into four groups: C (non-treated controls); C+M (100 mg/kg/day of Metformin in drinking water); F (10 % of Fructose in drinking water); and F+M (Fructose+Metformin in drinking water). After three weeks of all treatments blood samples were taken, after which animals were sacrificed by cervical dislocation under anaesthesia. Femurs were then dissected for evaluation of metaphyseal micro-architecture after Haematoxilin-Eosin staining of 5 μm histological slices of decalcified bone. In particular, osteocytic density and relative trabecular volume were determined. An increase in serum glucose and triglycerides was observed in Fructose-treated rats, in accordance with the development of MS. In rats treated with Metformin alone (group C+M), the analysis of femoral metaphyses showed an increase in trabecular osteocytic density (118 % of control [group C], p<0.05). Treatment with Fructose alone (group F) significantly decreased ostecytic density (79 % of control, p<0.05), while co-treatment with Fructose and Metformin partially reverted this decrease (group F+M, 88 % of control). Similarly, the relative trabecular volume of femoral metaphysic was increased by treatment with Metformin alone (129% of control), was reduced in Fructose-treated rats (89 % of control), and tended to revert back to control values after Fructose-Metformin co-treatment (94 % of control). These results show for the first time that (a) Fructose-induced MS in rats alters their femoral metaphysis micro-architecture; and that (b) these deleterious effects can be partially prevented by orally administered Metformin.