León Schurman

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.

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.

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.

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.

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-1β 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.

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.