| Objective: Type 2 diabetes (T2DM) and osteoporosis are(OP) are both systemic metabolic diseases. The insidence of these two diseases increase year after year with the aging of population and changing of life style, but it is not clarified how T2DM influence bone metabolism. What is clear is that T2DM can decrease bone formation and increase fracture risk. Insulin resistance is the pathological basis of T2DM. The suppression of hepatic insulin signaling pathway which means the decreased expression of IGF-1, IRS plays an very important role in the pathogenesis of T2DM and insulin resistance. Besides, the factors involved in insulin signaling pathway also express in bone and participate in bone metabolic regulation. In recent years, the influence of insulin signaling pathway to bone metabolism becomes a hotspot. Researches show that osteoblast can express IGF-1, IRS-1, IRS-2 and osteoclasts only express IRS-2. IGF-1 could promote osteoblast differentiation and the secretion of collagen from mature osteoblasts. IRS-1 plays an important role in maintaining the balance between bone formation and bone resorption while IRS-2 mainly promotes bone formation. The mechanism of how T2DM influence osteoporosis is unclear and there is no report about whether they have common pathogenesis. In this study, we established type 2 diabetes osteoporosis rat model, measured the hepatic gene expression involved in insulin signaling pathway in type 2 diabetic osteoporosis rats and discussed the relationchip of the pathogenesis of these two diseases.Methods: 100 Wistar rats were randomly divided into four groups after 1 week feeding: normal control group (NC,N=24),ovariectomy group(NOVX, N=26) , type 2 diabetes control group(DC,N=24), type 2 diabetic rats under ovariectomy group (DOVX,N=24). NC and NOVX groups were fed with normal feedstuff while DC and DOVX groups were fed with high-sugar-fat feedstuff (mixed with 20% sugar, 2.5% cholesterin, 15% cooked lard) for 8 weeks. T2DM were made by intraperitoneal injection of STZ after 12 hours fasting. At the end of 9th weeks, rats with fasting blood glucose (FBG)≥7.8mmol/L and with insulin resistance were considered as T2DM.Rats chosen for NOVX and DOVX group were ovariectomied under anesthesia 1 week later after T2DM model were done. We randomly selected 5 rats in each group and measured body weight, bone mineral density(BMD) of lumbar 3-6,fasting blood glucose level(FBG) and fasting insulin level(INS) at 0 week,4 weeks,8weeks and 12 weeks after ovariectomy.At 4 weeks,8 weeks,12 weeks after ovariectomy, 5 rats was randomly executed, the liver of which was separated after anesthesia. The expression of IGF-1, IRS-1, IRS-2 at protein level was detected by IHS (immunohistochemistry) while the expression of IGF-1,IRS-1,IRS-2 at RNA level was detected by RT-PCR. The experimental data was presented as mean±standard deviation. Variance Analysis were used to check differences between groups by SPSS13.0, P<0.05 was considered statistically significant.Results:1 Body weight of rats in each group: There were no difference found between the 4 groups right after ovariectomy(0 week). At 4,8,12 weeks after ovariectomy, the body weight of NOVX group is significantly higher than NC group, DC group is significantly lower than NC and NOVX group(P<0.05); and after 8,12 weeks after ovariectomy , the body weight of DOVX group is significantly lower than NOVX group(P<0.05). During the whole course, the body weight of NOVX group increased most obviously and DC group decreased most obviously.2 Bone mineral density(BMD): There is no significant difference between groups at 0 week after ovariectomy, but at 4,,8,12 weeks after ovariectomy, the BMD of OVX groups is significantly lower than control groups(NC and DC groups). With the progression of the experiment, the BMD of OVX groups declines. The BMD of DOVX group declines most obviously, followed by NOVX group. 3 The comparison of serum markers3.1 Fasting plasma glucose(FBG): The FBG and INS level in DC groups and DOVX groups are significantly higher than that in NC and NOVX groups 1 week after STZ were injected, which indicate that the T2DM rat model is successfully established. At 4 weeks after ovariectomy, the FBG in DC and DOVX group is on a downward trend, DOVX group is much higher than NC and NOVX group(P<0.05), but there is no differences between DC group and other groups(P>0.05); At 8 weeks after ovariectomy, the FBG of DC and DOVX group is significantly higher than NC group(P<0.05); At 12 weeks after ovariectomy, the FBG of DOVX group is significantly higher than NC group(P<0.05), but there is no difference between other groups.3.2 Insulin level(INS): The INS of diabetic group (DC group and DOVX group) rats is significantly higher than non-diabetic group (NC group and NOVX group) rats at each period,but there is no statistical differences between DC group and DOVX group, NC group and NOVX group.3.3 Insulin sensitivity index(IAI): At 0,4,8 weeks after ovariectomy, the IAI of DC and DOVX group is significantly lower than NC and NOVX group(P<0.05), At 12 weeks after ovariectomy, the IAI of DC and DOVX group is significantly lower than NC group(P<0.05), but no difference compared with NOVX group(P>0.05).3.4 The estrogen level: at the beginning after ovariectomy, the estrogen level of diabetic groups (DC and DOVX group) is significantly lower than non-diabetic groups(NC and NOVX group),but there is no difference between NC group and NOVX group, DC group and DOVX group(P>0.05); at 4 and 8 weeks after ovariectomy, the estrogen level of NOVX group is lower than NC group, and DOVX group is the lowest(P<0.05); at 12 weeks after ovariectomy, the estrogen in NC group is the highest and DOVX group is the lowest.4 The pathology of the liver: Under microscope, we can see the liver of non-diabetic groups shows clear hepatic lobules, orderly hepatic corDC and clear hepatocyte nucleolis, no steatosis and inflammatory cell infiltration. There is no difference between NC and NOVX group. The liver of diabetic groups shows distortion of hepatic lobules, obvious hepatic steatosis. With the progression of the experiment, steatosis of the hepatocyte becoming more serious, some of which look like signet ring cell, especially in DOVX group.5 The expression level of IGF-15.1 Immunohistochemistry results: IGF-1 expression is located in cytoplasm of the hepatocyte. At 4 weeks after ovariectomy, the expression in DC group and DOVX group is significantly higher than NC and NOVX group (P<0.05), and there is no difference between DC and DOVX group, NC and NOVX group(P>0.05); At 8,12 weeks after ovariectomy, the expression in DC and DOVX groups is much lower than NC and NOVX group, DOVX and NOVX groups is much lower than DC and NC group(P<0.05).5.2 RT-PCR results: At 4 weeks after ovariectomy, the expression in DC and DOVX groups is much higher than NC and NOVX groups, DOVX group is much higher than DC group(P<0.05), there is no statistic differences between NC and NOVX group(P>0.05); At 8,12 weeks after ovariectomy, the expression of IGF-1 in DC and DOVX groups is much lower than NC and NOVX group, DOVX and NOVX groups is much lower than DC and NC group(P<0.05).6 The expression level of IRS-16.1 Immunohistochemistry results: IRS-1 expression is located in cytoplasm of the hepatocyte. At 4 weeks after ovariectomy, the expression of IRS-1 in DOVX group is significantly lower than NC, NOVX, DC groups(P<0.05); at 8 weeks after ovariectomy, the expression in DC and DOVX group is much lower than NC and NOVX group, DC group is much lower than DOVX group, NOVX group is much lower than NC group(P<0.05); at 12 weeks after ovariectomy, the expression in DC group and DOVX group is much lower than NC and NOVX group, NOVX group is much lower than NC group(P<0.05).6.2 RT-PCR results: At 4 weeks after ovariectomy, the expression of IRS-1 in DOVX group is significantly lower than NC, NOVX, DC groups, DC group is significantly higher than NC group(P<0.05), there is no statistic differences between NOVX and DOVX group, NC and NOVX group(P>0.05); at 8 weeks after ovariectomy, the expression in DOVX group is significantly lower than NC, NOVX group, DC group is lower than DOVX group, NOVX group is lower than NC group(P<0.05); at 12 weeks after ovariectomy, the expression in DOVX group is significantly lower than NC and NOVX group, NOVX group is lower than NC group(P<0.05), no statistic differences between DOVX and DC group(P>0.05).7 The expression level of IRS-27.1 Immunohistochemistry results: IRS-2 expression is located in cytoplasm of the hepatocyte. At 4 weeks after ovariectomy, the expression of IRS-2 in DC and DOVX group is significantly lower than NC and NOVX group, DOVX group is significantly higher than DC group(P<0.05),there is no statistic differences between NC group and NOVX group(P>0.05). At 8 weeks after ovariectomy, the expression in DC group is much lower than DOVX, NC, NOVX groups(P<0.05),there is no statistic differences between DOVX, NC and NOVX group(P>0.05). At 12 weeks after ovariectomy, the expression in NC group is much higher than NOVX, DC, DOVX groups(P<0.05),there is no statistic differences between NOVX, DC and DOVX group(P>0.05).7.2 RT-PCR results: At 4,8 weeks after ovariectomy, the expression of IRS-2 in NC and NOVX group is significantly higher than DC and DOVX group, DOVX group is much higher than DC group(P<0.05),there is no statistic differences between NC and NOVX group(P>0.05). At 12 weeks after ovariectomy, the expression in NC group is significantly higher than NOVX, DC and DOVX group(P<0.05),there is no statistic differences between NOVX, DC and DOVX group(P>0.05).Conclusion:1 The expression of IGF-1 and IRS-1 in liver of NOVX group rats is lower than that of NC group rats.2 The expressions of IGF-1, IRS-1 and IRS-2 in liver of DC group rats is lower than that of NC group rats. 3 The expressions of IGF-1 and IRS-1 in liver of DOVX group rats is lower than that of NC,NOVX and DC group rats.4 The inhibition of the expressions of IGF-1 and IRS-1 in liver maybe related to the pathogenesis of Type 2 diabetes with osteroposis.
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