Bone Metabolic Characters In Diabetic Rats

Objective:To characterize the bone metabolism in type 2 and type 1 diabetic rats induced by high-fat feed combined with low dose of streptozotocin and high dose of streptozotocin.Non-diabetic Wistar rats and Goto-Kakizaki(GK) rats,a spontaneous type 2 diabetic model,were enrolled as negative and positive controls respectively. Bone mineral density(BMD),bone histomorphometric and biomechanical properties, as well as the bone biochemical indices and cytokines were observed,and correlations between bone metabolism and insulin level,metabolic control were analyzed to seek the possible mechanism for bony metabolic disturbance.Methods:Male Wistar rats were randomly served as non-diabetic controls(NC), inducible type 2 diabetic group(T2D) and type 1 diabetic group(T1D),with sex-and course of disease- matched GK rats served as positive controls.Type 2 diabetes was established by high fat and high-sugar feeding for eight weeks combined with one injection of small-dose(30 mg/kg body weight) of streptozotocin(STZ).The diabetes was confirmed by an intraperitoneal glucose tolerance test(IPGTT) one week after the injection of STZ.Type 1 diabetes was established by one injection of large-dose (60 mg/kg body weight) of STZ.The diabetes was confirmed with random blood glucose levels≥16.7 mmol/L detected 72 hours later.Double labeling for bone histomorphometry was conducted by an intraperitoneal injection of tetracycline 14 days before the sacrifice.The rats were killed 20 weeks after the onset of diabetes. Urine was collected from the rats for calcium,creatinine and hydroxyproline analyses. Blood specimens were collected from carotid arteries for fasting blood glucose, glycosylated hemoglobin,calcium,phosphate,osteocalcin and tartrate-resistant acid phosphatase activity analyses.The femur,tibia and the fifth lumbar vertebra were harvested and freed of soft tissue attachments carefully.Analyses for bone mineral density was performed by dual energy X-ray absorptiometry on femur and the fifth vertebral body respectively followed by the biomechanical studies of three-point bending test and compressive test.The metaphyseal tibiae were embedded in methylmethacrylate to obtain the undecalcified sections.Histomorphometry analysis was performed on 5μm(stained with Von Kossa) and 7μm bone sections with an analyze software for multimedia pathological image.Results:①Fasting plasma glucose,glycosylated hemoglobin,insulin concentrations, and HOMA-IR of the T2D rats were higher than the NC group significantly(P＜0.01), companied by significantly increased serum levels of TG,TC and LDL.②All the diabetic rats showed significantly deceased ratio of bone ash and dry weight,as well as the bone density determined on femur and lumber vertebra(P＜0.05).There was no significant difference among the diabetic groups(P＞0.05).③The maximal load, maximal energy,maximal strain,cross-sectional moment of inertia and coefficient of stiffness in the three-point bending test of the femora decreased significantly(P＜0.05或P＜0.01) in the three groups of diabetic rats,among which the T1D group had the most evident changes(P＜0.05).The maximal load and elastic modulus of vertebral bodies significantly decreased in the diabetic rats(P＜0.01),but there was no significant difference in the elastic modulus of femur among the groups.④Histomorphometrical study showed decreased trabecular bone volume,trabecular thickness,osteoid surface,osteoid thickness and osteoid volume in all the diabetic rats (P＜0.05或P＜0.01),as well as erosion surface in the T1D group and GK rats(P＜0.05).Mineralizing surface,mineral apposition rate and bone formation rate also decreased in all the diabetic rat(P＜0.01)s,along with an increase in osteoid maturation time in the T1D group and GK rats(P＜0.05).There was no significant difference among the diabetic groups(P＞0.05).⑤There was no significant difference in the serum calcium and phosphate levels among all the groups(P＞0.05). Compared with the healthy controls,the T2D rats had significantly lower serum osteocalcin and insulin-like growth factor-1 concentrations,and higher 24h urinary calcium,hydroxyproline exclusion and serum tartrate-resistant acid phosphatase activity(P＜0.01).The serum levels of IGF-1 were significantly lower in the T1D group than in the T2D groups(P＜0.01).⑥Compared to healthy controls,diabetic rats had significantly increased interleukin 1βand 6,and tumor necrosis factorαconcentrations in the serum(P＜0.05 or 0.01).There was no significant difference among the diabetic groups(P＞0.05).⑦Partial correlation analysis showed that the levels of GHb were significantly correlated with the BMD,biomechanical properties and BV/TV,and the serum levels of IL-1β,IL-6 and TNF-αwere negatively correlated with the BMD in the control and diabetic rats.Conclusions:1.Wistar rats induced by high fat and high-sugar feeding combined with one injection of small-dose streptozotocin can be the type 2 diabetes model successfully,which were charactered by high blood glucose,hypefinsulinemia, insulin resistant and metabolic disturbance of blood fat.2.This rat model without obesity excludes the interference of obesity on bone and can be the ideal animal model for study on abnormality of bone metabolism under type 2 diabetes.3.The diabetic rats had decreased bone density,bone mass and biomechanical forces.4.the diabetic rats had disturbance of bone metabolic markers in the blood and urine and abnormal bone histomorphometry.Dysfunction of osteoblasts might be the major mechanism for bone metabolic disturbance under diabetic status,in which osteoclasts may involve.5.The bone mineral density,bone biomechanical and histomorphometric indices were inversely correlated with the glycosylated hemoglobin significantly in the diabetic rats,indicating that hyperglycemia and advanced glycation endproducts might involve in the bone metabolic disturbance in diabetic rats. Objective:To observe the expression of RAGE,OPG,RANKL and IGF-1 in bone from the STZ-treated diabetic rats in order to study the possible mechanisms of hyperglycemia and advanced glycation endproducts on bone metabolism.Methods:Male Wistar rats were randomly divided into control group(NC),untreated diabetic group(T1D) and insulin-treated diabetic group(DI).Diabetes was established as previously detailed in part one.Rats in the DI group were treated with insulin to control the blood glucose level under 10 mmol/L.All the rats were killed 20 weeks after the onset of diabetes.Urine was collected from the rats for creatinine and hydroxyproline analyses.Blood specimens were collected for fasting blood glucose, glycosylated hemoglobin,osteocalcin and tartrate-resistant acid phosphatase activity analyses.Analyses for bone mineral density was performed by dual energy X-ray absorptiometry on the left femora.RNA samples were extracted directly from the right femora.The expression of RAGE,OPG,RANKL and IGF-1 mRNA were detected by real-time reverse transcription polymerase chain reaction(RT-QPCR). The expression of RAGE protein was detected by immunohistochemieal analysis on the tibiae sections embedded in methylmethacrylate with an analyze software for multimedia pathological image.Results:1.Compared with the healthy controls,the untreated T1D diabetic rats had significantly higher optical density value of RAGE protein in the tibiae(P＜0.01).The expression of IGF-1 mRNA significantly decreased(P＜0.01),and both RAGE and RANKL mRNA significantly increased(P＜0.05 or 0.01) in the untreated T1D diabetic rats.The change in the expression of OPG mRNA was undetectable.Then the ratio of OPG/RANKL decreased significantly in the untreated diabetic rats(P＜0.01).Insulin improved all the above changes in the diabetic rats(P＜0.01).2.The optical density value of RAGE protein and expression of RAGE mRNA were positively correlated with the glycosylated hemoglobin in the diabetic rats(r=0.656, P＜0.01;r=0.535,P＜0.05).The expression of RAGE mRNA was negatively correlated with the femoral BMD,serum OCN and BV/TV(r=-0.583,P＜0.01;r= -0.519,P＜0.05;r=-0.564,P＜0.05),while positively correlated with ES/BS(r =0.535,P＜0.05).There was no correlation between the expression of RAGE and hydroxyproline exclusion and serum tartrate-resistant acid phosphatase activity.The expression of RAGE mRNA was positively correlated with the expression of RANKL mRNA and RANKL/OPG(r=0.555,P＜0.05;r=0.651,P＜0.01),and negatively correlated with the expression of IGF-1 mRNA(r=-0.459,P＜0.05).There were negative correlations between the expression of IGF-1 mRNA and RANKL mRNA and RANKL/OPG(r=-0.672,P＜0.01;r=-0.567,P＜0.05).Conclusions:1.AGEs accumulated in the diabetes induce both the protein and mRNA expression of its receptor,RAGE.2.The increased RAGE in bones might play a role in the bone loss of the diabetic rats through stimulating the osteoclasts activities and inhibit the osteoblasts by increasing the expression of RANKL.3.The increased expression of RAGE may be associated with the decreased expression of IGF-1.The latter impacts the bone remodeling directly or indirectly through the OPG/RANKL/RANK axis in diabetic rats.