Effect And Mechanism Of Rosiglitazone On Prevention Of Diabetes In OLETF Rats

BACKGROUNDThe diabetes incidence rate roars up year by year and diabetes has become the third disease following the cardiovascular diseases and tumor which damage people's health seriously. Nowadays there are more than one hundred and fifty million diabetes patients around the world and the number is supposed to double in 2025. However, although progress has been made in the treatment of diabetes, we can not control the development of diabetes and its complications yet. Prevention of diabetes becomes one of the most important issues in order to solve the problem radically.Among diabetes patients, 90% are type 2 diabetes patients. Almost all the patients suffering type 2 diabetes have got through the phase of impaired glucose tolerance(IGT), which is a phase between NGT and diabetes. It is generally presumed 2hPG of many IGT patients will recover to normal level with properintervention or treatment whereas 10%-15% of those IGT patients develop into diabetes every year without intervention. Intervention in group of IGT is the most effective and economical method in diabetes prevention.Since 1980's, Many researches, such as Daqing Study, Diabetes Prevention Program (DPP), Diabetes Prevention Study (DPS) and STOP-NIDDM, have brought about encouraging answers on prevention of diabetes. Researches mentioned above demonstrated diabetes can be prevented and intervention can result in 31%-58% decrease in incidence rate of type 2 diabetes.Pharmacological intervention is one of the most important methods to prevent diabetes. The medicines used to prevent diabetes include metformin, acarbose and inulin sensitizer. In TRIPOD and DPP, it was found that thiazolidinediones can decrease diabetes incidence rate more effectively compared to other medicines. The research DREAM, in which rosiglitazone is used to prevent diabetes, has already been carried out. That means thiazolidinediones is probably a very promising medicine to prevnt diabetes. Rosiglitazone has less side-effects in thiazolidinediones, may play a more important role in diabetes prevention. So it is very necessary to investigate the mechanism of rosiglitazone on diabetes prevention.The pathogenesis of diabetes has not been understood well yet. B-cell dysfunction and insulin resistance contribute to the development of diabetes mostly. As one of the most important pathogenesis of type 2 diabetes, P-cell dysfunction is coming into spotlight gradually. Some even argued P-cell dysfunction was more important than insulin resistance in the development of diabetes. The renowned research UKPDS showed P-cell dysfunction could lead to the poor control of blood glucose. Many researches has proved that the decreased number of P-cell resulted from P-cell apoptosis could lead to hyperglacemia. Since the 1990's of last century till now, a lot of researches in animal and human beings showed the apoptosis ofP-cell in pancreatic islet happens in the period of pre-diabetes and becomes severer after the onset of diabetes. The apoptosis of P-cell leads to the imbalance of the number of P-cell and the decreased number of P-cell, which result in the deficient insulin secretion and the onset of type 2 diabetes.The present study will observe the effects of rosiglitazone on prevention of diabetes and investigate the the effect and probable mechanism on P-cell apoptosis. OLETF ( Otsuka Long-Evans Tokushima Fatty) rats, an excellent model of spontaneous type 2 diabetes mellitus and their counterparts LETO (Long-Evans Tokushima Otsuka) rats are used in this study.Chapter 1 Effect of Rosiglitazone on prevention of diabetes in OLETF ratsOBJECTIVETo investigate the effect of Rosiglitazone on prevention of diabetes in model of spontaneous type 2 diabetes OLETF rats.METHODS1. OLETF rats, spontaneous type 2 DM model rats, with their lean nondiabetic counterparts, LETO rats, were supplied at 4 weeks of age by the Otsuka Pharmaceutical Company (Tokushima, Japan). All the rats are male and divided into three groups. Forty male OLETF rats were randomly divided into two groups, group administrated with rosiglitazone (Group RSG) and the control group (Group CN). Ten male LETO rats were regarded as normal control group (Group LETO). Rats of Group RSG were given Rosiglitazone(3.0mg-kg"1d"1) intragastrically since age 8 weeks. Except for Group RSG, all rats were given distilled water intragastrically.2. The blood glucose was determined by OGTT (oral glucose tolerance test) todiagnose diabetes. 15 hours before OGTT (5:00 PM, the day before), food was removed from the cage. 2g/kg glucose was administered intragastrically to conscious rats. Blood was obtained from the tail before and 30, 60, 90 and 120 min after the administration of glucose and was analyzed with life scan ultra glucose photometer. Diabetes was diagnosed when both peak blood glucose > 16.7 mmol/L and 120 min blood glucose >ll.lmmol/L. IGT (impaired glucose tolerance) was diagnosed when either was accordant with the standard.3. Record the food intake and body weight every week. Rats were sacrificed at 8, 32 and 40 weeks age. Serum triglyceride(GK — GPO — PAP), serum cholesterol (CHOD-PAP), plasma FFA, plasma insulin(ELIS A) were determined.4. All values are expressed as means ± SD. Statistical analysis was performed using SPSS 10.0 software. The difference between two samples were analyzed by t test and difference amnong three groups by one-way ANOVA. The diabetes incidence rate was analyzed by X2 test. Statistical significance was defined as p<0.05.RESULTS1. The food intake in both OLETF rats groups are significantly higher than group LETO rats (p<0.01). The food intake of rats in Group RSG was higher than that of Group CN since age 10 weeks although it's not significant (p>0.05). Body weight was significantly greater in OLETF rats compared with LETO rats since 6 weeks of age (p<0.01). The body weights of Group RSG were higher than Group CN in most periods and significantly since age 28 weeks (p<0.05).2. The 2hPG of rats in Group CN rised at 23 weeks of age (10.1±1.8mmol/L) significantly than 2hPG at 13 and 18 weeks of age (8.2±0.9,6.7±0.9mmol/L)(p<0.01), rised with weeks age (14.8±3.2mmol/L,40 weeks of age). 2hPG of Group RSG rised at 30 weeks of age and 9.7±2.0mmol/L at 40 weeks. 2hPG of Group LETO atdifferent weeks age didn't change significantly. 2hPG of Group RSG was significantly lower than that of Group CN since 23 weeks of age(p<0.01), whereas no significant difference with Group LETO. The glucose area under curve (AUC) in both Group OLETF rats are higher than that of Group LETO. AUC of Group RSG was lower significatly than that of Group CN in period of the present study (p<0.01).3. OLETF rats in Group CN developed diabetes since 23 weeks of age (4 IGT,1 diabetes) and the incidence rate of diabetes became higher with the time goes by until got to 92.9% at 40 weeks of age. Rats in Group RSG developed diabetes since 30 weeks of age and incidence rate was 28.6% at 40 weeks, significantly lower than Group CN (p<0.01). No rats have developed diabetes or IGT in Group LETO.4. The serum triglyceride, total cholesterol, plasma FFA, plasma insulin was significantly lower in Group RSG than that of Group CN.CONCLUSIONS1. Rosiglitazone can decrease the extent of hyperglacemia and delay the occurrence of hyperglycemia in OLETF rats.2. Rosiglitazone can decrease the incidence rate of type 2 diabetes significantly and prevent diabetes effectively.3. Rosiglitazone can decrease the serum triglyceride, serum cholesterol, plasma FFA and plasma insulin.Chapter 2 Impact of Rosiglitazone on apoptosis of P cells in OLETF ratsOBJECTIVETo observe the apoptosis level of P-cell and the apoptosis related factors in OLETF rats administrated with rosiglitazone, to investigate the effects and probable mechanism of rosiglitazone on P-cell apoptosis.METHODS1. Rats were sacrificed at 8, 32 and 40 weeks of age randomly. Pancreas were removed and weighed with water absorbed away by filter paper after rats were sacrificed. The pancreas were devided and stored in liquid nitrogen, formaldehydum polymerisatum and 3% glutaraldehyde.2. The pancreatic tissues were stained with hematoxylin and eosin to observe the morphlogical change. The paraffin section were stained with the method of immunohistochemistry and TUNEL to examine apoptotic P-cells and examine P-cell mass with anti-insulin antibody and calculate the apoptosis rate of P-cell. At lest 20 islets were counted every rat. The percentage of P-cell apoptosis rate = (number of apoptoticP-cell/number of P-cell) xl00%.3. To count the number of bcl-2 positive cells. The celluar membrane and cytoplasm of positive cells were stained buffy or deeper. At lest 20 islets were counted every rat. The positive rate=(number of positive cells/number of P-cell) xl00%.4. To observe the morphological change of P-cell with electron microscope.5. The expression of Bcl-2 and Bax mRNA in pancreas were determined by semi-quantitative RT-PCR. The levels of Bcl-2 and Bax mRNA expression were presented as IOD values of their electrophoresis band respectively. The IOD values of band were analyzed by UVIBand system.The relative mRNA values were present as ratio of the IOD values of target genes to GAPDH.6. All values are expressed as means ± SD. Statistical analysis was performed using SPSS 10.0 software. The difference between two samples were analyzed by t test and difference amnong three groups by one-way ANOVA. The diabetes incidence rate was analyzed by X2 test. Statistical significance was defined as p<0.05.RESULTS1. Pancreas weight in each group increased with week-age. No significant difference was found among the weight of pancreas in different group as well as the ratio of pancreas/body weight among different groups (p>0.05) before age 32 weeks. The ratio of pancreas/body weight was significantly higher in Group LETO than that of the other two groups in age 40 weeks (p<0.01).2. The structure of islets in age 8 weeks is not different from each other. Hematoxylin and eosin staining disclosed severe fibrosis in pancreatic islet of Group CN rats in 32 weeks of age and it is more severr in 40 weeks of age which featured abnormal round appearance and disorganized islet with atrophy islet observed. There were relatively normal round islet in rats of Group RSG and there were not serious atrophy islets.3. The apoptotic cell was observed with electron microscope, which features nuclear shrek, atrophy organelles and decdreased secretory granules. The p-cell could be observed swelling organelle in Group RSG.4. Apoptosis rates of P-cell in Group LETO in age 8, 32, 40 weeks were 0.08±0.03 0.12±0.04% respectively; 0.22±0.02%, 0.28±0.03%^ 0.26±0.03 %in Group CN; 0.18±0.02%^ 0.17±0.04% in age 32, 40 weeks in Group RSG, significantly different compared to Group CN(p<0.01).5. The positive cell rates of Bcl-2 in Group LETO rats 8, 32, 40 weeks old were 68.45±21.3%* 52.76±12.8%, 53.01+11.3% respectively; 50.92±9.4%^ 36.93±1.4%^33.12±7.1 %in Group CN; 59.60±9.9%, 55.24±9.7% in age 32, 40 weeks in Group RSG, significantly different compared to Group CN(p<0.01).6. The expression level of Bcl-2 mRNA in all groups decreased with age but not significantly. The expression level in age 32 weeks, Group LETO is highest, and Group RSG less, Group CN least, not significantly either(p>0.05). In age 40 weeks, Group CN was lower than Group LETO and Group RSG(p<0.01), but not significant difference between Group LETO and Group RSG(p>0.05).CONCLUSIONS1. The high apoptosis rate of P cells in OLETF rats emergeed before the onset of diabetes and gets higher with the development of diabetes.2.Rosiglitazone can decrease the level of P-cell apoptosis and protect the structure and function of islets as the probable mechanism of diabetes prevention.4 Rosiglitazone could ameliorate the glucotoxity and lipotoxity, relieving the burden of isletp-cells maybe the reasons. Rosiglitazone probably can supresss|3-cell apoptosis directly as well.5 Rosiglitazone can modulate the balance of expression level of apoptosis relatd gene family Bel in protein and mRNA to suppress P-cell apoptosis. Ameliorating glucose and lipid metabolism maybe contribute to this effect.