The Effect Of Type2Diabetes Mellitus On The Osteoblastogenesis And Osteoclastogenesis In Bone Marrow Micro-environment Of A Mouse Model:an Vivo Study

Objective1. To examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions.2. To investigate whether T2DM disease causes osteoporosis and and the underlying mechanisms.3. To elaborate the mechanisms of thiazolidinedione regulates bone metabolism, we perform this experiment to help us understanding the relationship among Peroxisome Proliferator Activated Receptor gamma (PPARy), its activator rosiglitazone, and the bone metabolism.Methods1. Monocytes were isolated from the bone marrow of the C57BL/6mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6mmol/L), high insulin (1μmol/L), or a combination of high glucose/high insulin (33.6mmol/L glucose and1μmol/L insulin). Cells cultured in a-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including cathepsin K and TRAP were determined by real-time PCR.2. We analyzed the affects of T2DM on bone physiology with a KK/Upj-Ay/J mouse, a mouse model of T2DM. KK/Upj-Ay/J mice develop diabetes in8weeks and exhibit stable diabetes symptoms and signs in10weeks when fed a KK/Upj-Ay/J mice maintenance fodder. And4weeks later, serum levels of glucose, insulin, alkaline phosphatase, osteocalcin, tartrate-resistant acid phosphatase form5b in the two group mice were analyzed. 3. Receptor activator of nuclear factor kB ligand (RANKL) and Osteoprotegrein (OPG) are secreted by osteoblast, and the RANKL/OPG ratio plays an essential role in osteoclastogenesis. In this study, we investigated the effect of RSG on osteoblast and their support for osteoclastogenesis. After treated with pioglitazone, bone marrow stem cell and pre-osteoblastic cell line MC3T3-E1were analyzed; ALP activity, gene expression, osteoclastogenesis by co-culture and protein expression were measured and statistics were performed with SPSS13.0.Results1. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/insulin groups than in normal glucose and high insulin groups. The expression levels of cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment.2. In comparison with the C57BL/6non-diabetes mice, the diabetes mice exhibited hyperglycemia, hyperinsulinemia, and increased body and fat pad weight. Moreover, diabetic mice demonstrated low bone weight and bone mineral density (BMD) in the femur, tibia and fifth lumbar spine. Osteoblastogenesis and osteoclastogenesis in diabetic mice was impaired based on von kossa and TRAP staining, ALP and TRAP activity and gene profiling.3. We found that RSG inhibits differentiation of pre-osteoblast (MC3T3-E1cells) and decreases the OPG/RANKL ratio. Moreover, it attenuates mineralization of extracellular matrix. RSG reduces the osteoclastogenesis in a co-culture system of MC3T3-E1and monocytes.Conclusion1. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.2. These data suggest that bone metabolism is impaired in type2diabetes, resulting in decreased osteoblastogenesis, osteoclastogenesis, and bone mass.3. These results suggest that RSG attenuates the function of osteoblast and reduces the OPG/RANKL ratio in osteoblast, further decreasing the osteoclastogenesis. It could be help for us to explain the diabetic skeletal metabolism or diabetic bone loss.