Osteoporosis With Type 2 Diabetes Mellitus The Basic And Clinical Study

Objective:Osteoporosis(OP) is a bone disease which is threatening to the elder people, especially in postmenopausal women. Therefore, how to prevent and treat OP effectively has became a major concern in the modern society. Postmenopausal osteoporosis(PMOP) is the most common type of OP, which occurs to the postmenopausal women. The epidemiological data about OP is quite limited. Results from the 3rd National Nutrition and Health Survey in USA indicate that, the prevalence of OP is 13%-18%in the women with more than 50years old. A multi-center, randomized study in Canada about OP have shown that, total prevalence of OP in women is about 15.8%, and in lumbar spine and femoral neck were 12.1% and 7.9% respectively. A perspective study performed by 20 European centers in the 90s of 20th century have shown that, the prevalence of OP in European women is 2%-11% after ntertrochanteric femoral neck bone mineral density were measured in women over 50 years old according to WHO criteria. And, 14% to 36% Norwegian women more than 50-year-old have OP. A multi-stage stratified randomized study conducted in China have indicated that, OP prevalence is about 12.4% in five-area measured by DXA, in women is about 15.7%and man is 8.5%respectively .The pathogenesis of OP is complicated. The imbalance of bone remodeling is now considered to be the main mechanism. Bone remodeling is a process in which the old bone is absorbed and replaced with the new bones. Osteoclast (OC) and osteoblast(OB) is the couple both play important role in this process. OC is mainly for bone absorption while OB may promote bone formation. Thus, the bone turn over rate is determined by both bone absorption and formation. The process of bone remodeling can be affected by genotype, endocrinal hormones, age and some cytokines. Menopause is the main cause for OP.In the process of bone remodeling, some cytokines and signal pathways have been considered to be importantly involved. Wnt signaling pathway, which is consisted by Wnt/LRP5/β-catenin, plays importance role in bone formation. Runx2 is the marker for osteoblast differenation and can be modulated by Wnt/LRP5/β-catenin. NF-κB pathways is involved in OC differentiation. And this pathway can be activated after RANKL combing to TRAF6. Actived NF-κB can modulated OC development by its downstream factor NFATc1. Therefore, alterations in gene expression involved in wnt and NF-κB pathways may induce OP occurrence by misbalancing bone formation and absorption.The prevalence of type 2 diabetes mellitus ( T2DM) is rapidly increasing in China, and in elder population, it's get even higher. Data accumulated have indicated that, T2DM may cause increased risk of bone fracture by disturbing bone turnover rate. However, the relationship between T2DM and POMP is still not quite clear. In the present study, we try to characterize the OP with T2DM in rat model by checking BMD and bone turnover markers, the line of osteoblasts and osteoclasts cultured in vitro, the relative gene expression, and also from clinical observations. This study may provide some information about the pathogenesis and possibly the treatment for OP with T2DM.methods:Animal experiment:Part I:After adaptive feeding for 1 week, 100 healthy Wistar female rats were randomly divided into control group (NS, n=24); bilateral ovariectomy group (NOVX, n=26); diabetic group (DS, n=24) and diabetic ovariectomized group (DOVX , n=26). There was no significant differences in body weight of rats in all groups. NS and NOVX rats were fed with normal diet, while type 2 diabetic models were fed with high sucrose-fat diet (normal diet with 20% sucrose, 15% lard, 2.5% cholesterol) for 8 week and then intraperitoneally injected with streptozotocin (STZ, Sigma, USA) 30mg/kg, which was dissolved in sodium citrate solution (pH4.2). NS and NOVX rats only injected the same volume of citric acid buffer. Fasting blood glucose (FBG)≥7.8mmol/L was considered to be T2DM. 1 week after T2DM was set, rats in DOVX and NOVX were underwent bilateral ovariectomy surgery after anesthesia, and sham operation was performed in rats of NS and DS groups. Take the time of ovarian surgery day as 0 week, fasting blood glucose(FBG) and plasma insulin levels (FINS) were measured, and insulin sensitivity index was calculated by formula(IAI)=1/(FBG×FINS). Estrogen (E2) concentration were measured and lumbar spine bone mineral density(BMD) was measured at 0, 4, 8 and 12 weeks.Part II:At 0, 4, 8 and 12 weeks, 5 rats per group were terminated and osteoblasts and osteoclasts were isolated and cultured. Morphological changes were observed under inverted microscopy. The third generation of osteoblasts was identified by alkaline phosphatase staining, and growth activity of osteoblasts was determined by MTT method. Osteoclasts were cultured for 7 days, then cells were fixed, tartrate-resistant acid phosphatase (TRAP) stained. Bone specific alkaline phosphatase(BALP) and tartrate-resistant acid phosphatase 5b (TRACP5b) concentration were measured at the same time point from rats of different groups.Part III:At 4, 8 and 12weeks, rats tibiae were separated and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent according to protocol. Runx2, LRP5, TRAF6,β-catenin, NF-κB, NFATc1 mRNA gene expression were measured by RT-PCR,β-actin was used as house-keeping gene and relative gene expression was calculated.Clinical study:150 postmenopausal women with age 50-70 and 2-14 years menopause duration were selected as controls. While 150 postmenopausal women with type 2 diabetes (age from 48-70 and 1-14 years of menopause) were enrolled. Data of Age, menopause duration (PMD), diabetes duration (DMD), and height, body weight and body mass index (BMI) were collected. Blood glycated hemoglobin (HbA1c), Fasting plasma glucose (FPG), fasting insulin levels (FIns), estrogen (E2), I collagen C telopeptide (CTX-I), tartrate-resistant acid phosphatase 5b(TRACP5b), bone specific alkaline phosphatase(BALP) were also measured. Bone mineral density (BMD) were measured in lumbar spine (L2-4), femoral neck (Neck), greater trochanter (Great T), intertrochanteric (InterTro) by Dual energy X ray absorptiometry instrument (DXA). The subjects have been divided into normal group (NP), reduced bone mass group (DP) and osteoporosis group (OP) according to WHO 1998 criteria for OP.Results:Animal experiments part I:①Fasting blood glucose (FBG): at 0 week, FBG was significantly increased when diabetic group (DS, DOVX) compared with non-diabetic group (NS, NOVX) (p<0.05).②Fasting insulin (INS) and insulin sensitivity index (IAI): at 0 week, FINS concentration was significantly increased while IAI was reduced in both DOVX and DS rats when compared with NOVX and NS animals (p<0.05).③Estrogen (E2): at 0 week, DOVX and DS treatment caused decreased serum E2 levels in rats when compared with NOVX and NS group (p<0.05); E2 levels was found significantly decreased in DOVX group at 4,8 and 12 weeks when compared with NS,NOVX and DS rats at the same time point(p<0.05). and lower E2 levels also found in NOVX when compared with NS rats (p<0.05); significantly decreased E2 levels were only found at 8 and 12 weeks in DS rats when compared with NS rats.④Bone mineral density (BMD): at 4,8 and 12 weeks, compared with DS and NS group, DOVX and NOVX rats had significantly lowed BMD (p<0.05); BMD was significantly decreased at 8 and 12 weeks, when DS rats compared with NS rats (p<0.05).Animal experiments part II:①BALP: at 0 week there was no significant differences in serum BALP levels between groups (p>0.05). At 4 weeks, serum BALP levels in NOVX group was significantly increased when compared with NS and DOVX animals (p<0.05). At 8 weeks, only DS rats have significantly reduced serum BALP levels when compared with DOVX and NS group(p<0.05); no significant differences in serum BALP levels found in all groups at 12 weeks (p>0.05).②TRACP5b: at 0 week, there was no significant differences in serum BALP levels in all groups(p>0.05). At 4 and 8 weeks, TRACP5b was significantly increased in DOVX rats when compared with NS group (p<0.05), there was no significant differences in TRACP5b levels when DOVX compared with DS and NOVX group (p>0.05); NOVX rats had significantly increased TRACP5b compared with NS group (p<0.05); DS rats had significantly increased TRACP5b compared with NS group (p<0.05).At 12 weeks, TRACP5b was significantly increased when DOVX compared with NS, NOVX and DS group (p<0.05); there was no significant differences in TRACP5b levels when NOVX compared with NS group, and DS compared with NS group (p>0.05).③Osteoblast proliferation activity: at 4, 8 and 12 weeks, there was no significantly differences in osteoblasts activity in DOVX when compared with NS group (p>0.05), activity of osteoblasts was significantly decreased in rats of DOVX group when compared with those in NOVX (p<0.05), activity of osteoblasts was significantly increased when DOVX compared with DS rats(p<0.05); compared with NS, the activity of osteoblasts was significantly increased in NOVX rats (p<0.05) while significantly decreased in DS rats (p<0.05).④The number of osteoclast: at 4, 8 and 12weeks, numbers of osteoclast was significantly increased when DOVX compared with NS and DS group (p<0.05); compared with NS, osteoclast numbers was increased in NOVX rats (p<0.05) , but decreased in DS rats (P<0.05).Animal experiments part III:①Gene expression involved in Wnt signaling pathway: at 4, 8 and 12 weeks, DOVX rats had significantly decreased LRP5 andβ-catenin expression when compared with NS group (p<0.05); at 4 weeks, there was no significant difference in Runx2 expression when DOVX compared with NS rats (p>0.05); at 8 and 12 weeks DOVX rats had significantly decreased Runx2 expression when compared with NS group (p<0.05). At 4, 8 and 12 weeks, NOVX rats had significantly decreased LRP5 andβ-catenin expression when compared with NS group (p<0.05); at 4 and 12 weeks, NOVX rats had significantly decreased Runx2 expression when compared with NS group (p<0.05); at 8 weeks, there was no significant difference in Runx2 expression when NOVX compared with NS group (p>0.05). At 4 and 8 weeks, compared with NS group, DS rats had significantly decreased expression of LRP5 andβ-catenin (p<0.05); at 12 weeks, LRP5 expression was significantly decreased when DS compared with NS rats(p<0.05), althoughβ-catenin expression was no significant differences (p>0.05). At 4 and 8 weeks there was no significant differences in Runx2 expression when DS rats compared with NS (p>0.05); at 12 weeks, Runx2 expression was significantly decreased when DS rats compared with NS(p<0.05).②NF-κB signaling pathway factors expression: at 4, 8 and 12 weeks, compared with NS group, DOVX rats had significantly increased TRAF6, NF-κB and NFATc1 expression (p<0.05). At 8 and 12 weeks, compared with NS group, NOVX rats had significantly increased TRAF6, NF-κB and NFATc1 expression (p<0.05); at 4 weeks TRAF6 expression was significantly decreased when NOVX rats compared with NS (p<0.05), while NF-κB expression was significantly increased (p<0.05), there was no significant difference in NFATc1 expression when NOVX rats compared with NS(p>0.05). At 4 weeks, DS rats had significantly decreased TRAF6 expression when compared with NS group (p<0.05), NF-κB and NFATc1 expression was significantly increased when DS rats compared with NS (p<0.05); at 8 weeks there was no significant differences in TRAF6 expression when DS rats compared with NS(p>0.05), although DS rats had significantly decreased NF-κB and NFATc1 expression compared with NS (p<0.05); at 12 weeks DS rats had significantly increased TRAF6 and NF-κB expression compared with NS rats (p<0.05), there was no significant difference in NFATc1 expression when DS rats compared with NS (p>0.05).Clinical study:①The prevalence of osteoporosis was 54% in postmenopausal women with type 2 diabetes mellitus.②There was no significant difference in BMD between postmenopausal women with type 2 diabetes mellitus and without type 2 diabetes.③Compared with NP and DP group, age, menopause duration, duration of diabetes and fasting plasma glucose levels levels were significantly increased, and insulin and estrogen were significantly decreased in postmenopausal osteoporosis women with type 2 diabetes (all p<0.05); BMD was correlated with age, menopause and diabetes duration, fasting plasma glucose negatively but insulin and estrogen levels positively.④CTX-Ⅰ, TRACP5b and BALP concentrations were higher in OP subjects with T2DM when compared with NP and DP separately (OP vs NP, P<0.05; OP vs DP, P<0.05), and CTX-Ⅰconcentration was negatively correlated with the BMD of lumbar spine 2-4 and femur neck (p<0.05), but not greater trochanter and intertrochanteric; TRACP5b, BALP levels were negatively correlated with the BMD of lumbar spine 2-4, neck, greater trochanter and intertrochanteric (p<0.05) respectively, TRACP5b seems more dominant in the correlations to BMD.Conclusions:1 Osteoporosis with type 2 diabetes rat model is successfully set up by ovariectomizing rats with type 2 diabetes;2 Type 2 diabetic rats showed diminished bone mass. There were no significant differences between the BMD of ovariectomized rats with and without T2DM;3 Bone remodeling in diabetic osteoporosis, which is characterized by sTable bone formation and active bone resorption, is affected by both diabetes and ovariectomy;4 Wnt and NF-κB signaling pathway are all involved in the bone remodeling of diabetic osteoporosis. Decreased gene expression ofβ-catenin and increased expression of NF-κB might underlying the pathogenesis of OP;5 The prevalence of osteoporosis was 54% in postmenopausal women with type 2 diabetes mellitus. advanced age, longer duration of menopause and diabetes, higher FPG, poor blood sugar control, lower plasma insulin and estrogen levels are all the risk factors for osteoporosis in postmenopausal women with type 2 diabetes. TRACP5b, a sensitive marker for bone resorption, might be perspective for postmenopausal osteoporosis with type 2 diabetes screening.