| Low physical activity and hyperalimentation are lifestyle factors associated with an increased risk of type 2 diabetes mellitus (T2DM). The incidence rate of diabetes melhtus roars up year by year, diabetes has become the third disease following the cardiovascular diseases and tumor which damage people's health seriously. With the knowledge of insulin, the development and application of oral hypoglycemic agents, includine insulin secretagogues, biguanides, insulin sensitizing agents, etc., are available in the therapy of diabetes. However, we still can not cure it. Whether diabetes could be prevented and what is the most effective way becomes a big problem which must to be tackled. It is indicated that T2DM which accounts for over 90% of all diabetic patients could be prevented in the data of Evidence-based medicine (EBM) and the progression of diabetes risk would be decreased by 31%-58%. So it is necessary and significant to conduct prevention in the high risk population of diabetes mellitus.The mechanism of T2DM has not been acknowleged yet. β -cell dysfunction and insulin resistance are the most two important reasons. In recent years, cross-sectional and prospective studies demonstrated increased concentration of inflammatory cytokines in patients with T2DM. Inflammatory reaction has been shown to precede the onset of T2DM. A large number of studies have been revealed inflammation was closely linked with diabetes. The role of chronic inflammation in T2DM has been recognized in these few years. Salicylates, including sodium salicylate and aspirin, are used to treat inflammatory condition. Historically, it has been known that high doses of salicylates are able to lower blood glucose concentrations since 1950. It is reported that some proinflammatory cytokines included tumor necrosis fact, interleukin 1, interleukin 6, interleukin 8, interferon ,et al.,effected the glucose homeostasis. In the past decade, clinical, laboratory, and epidemiological research have coalesced to give rise to a new paradigm for understanding T2DM. It showed that the mechanism of T2DM was linked with inflammation, and T2DM was conducted by inflammatory meditors, that is to say, T2DM may be one of the innate immunue systemic disease, inflammation is crucially important for the oneset of diabetes.Rosiglitazone is one of the strongest thiazolidinediones. The mechanism of its action involves binding to the peroxisome proliferators-activated receptor y, a member of nuclear receptor family of transcription factors, regulates the expression of specific genes especially in fat cells but also in other tissues. Thiazolidinediones have been shown to interfere with expression and release of mediators of insulin resistance originating in tissue. It is reported that PPARγ could inhibit the expression of inflammatory meditors indepented with reducing glucose. Thus, inflammatory reaction may have important roles in the development of insulin resistance and T2DM. We therefore designed a study within the preventive intervention of rosiglitazone onOLETF (Otsuka Long- Evans Tokushima Fatty) rat, a spontaneously diabetic rat with polyuria, polydipsia, and mild obesity, to observe the effect of intervention rosiglitazone on diabetes including inflammation. See the following three parts.Part 1 The Effects of Rosiglitazone Preventive Intervention onDiabetes in OLETF RatsOBJECTIVETo investigate the effects of pharmacologic intervention with rosiglitazone on metabolic changes and prevention diabetes in OLETF rats.METHODS1. OLETF rats and their control counterpart Long-Evans Tokushima Otsuka(LETO) male rats at 4 weeks of age were kindly supplied by the Tokushima Research Institute, Otsuka Pharmaceutical(Tokushima Japan). Forty male OLETF rats were randomly divided into two groups at 5 weeks of age: diabetic control group (control group) and rosiglitazone treated group (intervention group). Ten male LETO rats were in diabetic negative control group (LETO group). Intervention group was maintained on the rosiglitazone (3mg ? kg'd1) by intragastric administration from 8 weeks of age before the onset of diabetes, until the end of the study at 40 weeks of age.2. The blood glucose was determined by OGTT (oral glucose tolerance test) to diagnose diabetes. An OGTT was performed for 16 hours before the test. Glucose(2g/kg) solution was given intragastricly to conscious rats, and glucose levels were measured before, 30, 60, 90 and 120 min after the administration by tailed. Diabetes was diagnosed when both peak blood glucose > 16.7 mmol/L and 120 min blood glucose > 11.1 mmol/L. IGT (impaired glucose tolerance) wasdiagnosed when either was determined.3. Body weight and food intake were monitored every week. Rats were sacrificed at 8, 32 and 40 weeks. The rats were treated with sodium pentobarbital and the abdomen was quickly opened to remove abdominal aorta, pancreas, hear, liver, spleen, lung, kidney, adipose tissue, skeletal muscle. All tissues were collected and frozen immediately in liquid nitrogen. The fasting serum glucose, serum triglyceride, serum cholesterol, plasma FFA (free fatty acid), plasma insulin were determined.RESULTS1. Control group and intervention group consumed more food than LETO group (p<0.05), and there was no different between control group and intervention group in food intaking. And thereafter remained and nearly the same level until the end of the study. Body weight of control and intervention group were significantly heavier than LETO group at 6 weeks old (161. 46 + 6. 52g vs 185. 21 ± 12. 75g, p<0. 001) .The body weight of both control and intervention group increased progressively with age, reaching a plateau at 37 to 40 weeks of age. Compared with the control group, the body weight of intervention group was significantly greater from 30 to 36 weeks old(p>0.05).2. At week 40, the cumulative incidences of control group with DM or IGT were 92.5% (13/14) or 7.5% (1/14), respectively. No rats have developed DM or IGT in LETO group.3. At the age of 8 weeks, there were no difference of fasting serum triglyceride(TC), serum cholesterol(CHOL), serum free fatty acid(FAA) between three groups(p>0.05), and except of fasting serum glucose other biochemical target increased progressively with age. Until 40 weeks age, all biochemical taget levels were higher in control group than in LETO group. Intervention group dignificantly decreased serum TC, serum CHOL, serum FAA at 32 weeks of age(p<0.05).Part 2 The Effects of Rosiglitazone Intervention on InflammatoryMeditors in OLETF RatsOBJECTIVETo observe the relationship between inflammatory meditors and T2DM, and investigate the effect of rosiglitazone intervention on serum inflammatory meditors.METHODS1. Animal grouping see part 1.2. Serum hsCRP was determined by the immunonephelometry assay. Serum TNF-a, EL-6, MCP-1 were measured by Enzyme-linked immunoadsordent assay (ELISA). Insulin sensitivity index (ISI) was calculated according to the fasting blood glucose and fasting plasma insulin concentration.3. To observe the changes of serum inflammatory meditors from NGT to IGT andT2DM.4. To observe the effect of rosiglitazone intervention on serum inflammatory meditors.5. To analyze the relationship between ISI and inflammatory meditors.RESULTS1. The serum inflammatory meditors of control group increased progressively with age, at 40 weeks age, were significantly higher than those of LETO group (p<0.01, p<0.001). At 32 weeks age,the level of ISI of control group was lower than that of LETO group (p<0.05), and the level of intervention group of ISI was significantly higher than control goup (p<0.05).2. At 40 weeks age, the serum inflammatory meditors of intervention group were all significantly lower than those of control group (p<0.01, p<0.001).3. At 32 and 40 weeks age, ISI was negtively related with serum hsCRP and IL-6Part 3 The Effects of Rosiglitazone Intervention on the Expression of PPARy mRNA in OLETF RatsOBJECTIVETo observe the effect of rosiglitazone intervention in spontaneous diabetic OLETF rats and develope a method for absolute quantification of peroxisome proliferator-activated receptor gamma (PPARy) mRNA by using of TaqMan real-time polymerase chain reaction (PCR) in tissues.METHODS1. Animal grouping see part 1.2. PPARy mRNA was detected with TaqMan absolute quantification RT-PCR iin various tissue of three groups.3. To investigate the effect of rosiglitazone intervention on the expression of PPARy mRNA.RESULTS1. PPARy mRNA was present in all the tissues, with the greatest expression in adipose tissue, with values 10-100 times more than that in other tissues. High level of PPARy were detected in abdominal aorta, pancreas, kidney, lung, while, on the other hand, only very low levels were ditected in skeletea muscle, heart, liver and spleen.2. Statistical analysis indicated that the expression of PPARy mRNA of control group were significantly lower than those of LETO group, especially in abdomial aorta and adipose tissues (p<0.001).3. Statistical analysis indicated that the PPARy mRNA expression was significantly increased in intervention group, in abdomial aorta, pancreas, skeletal muscle and kidney tissues, respectively (P < 0. 05).CONCLUSIONS1. This study successfully established spontaneous type 2 diabetic model, and observed the whole pathophysiological changes of OLETF rats from NGT, IGT to T2DM.2. Rosiglitazone can decrease the extent of and delay the occurrence of hyperglycemia in OLETF rats. Rosiglitazone can decrease the incidences of DM and IGT significantly which doesn't depend on reducing food intake or losing weight. Rosiglitazone can decrease the serum triglyceride, serum cholesterol, plasma FFA and plasma insulin.3. The serum of hsCRP, TNF- a ,IL-6, MCP-1 of control group were significantly increased (p<0.01, p<0.001), it is suggested inflammatory meditors participated in the occurrence and progress of T2DM and the levels of those may be a further hemostatic markers of underlying insulin resistant and T2DM, and possible account for the predictive power of inflammatory meditors with respect to T2DM. The serum hsCRP and IL-6 level were negatively correlated with ISI, it revealed insensitivity resistant may be associated with inflammation.4. Rosiglitazone can decrease the serum of inflammatory meditors, it suggested that rosiglitazone had the effect of anti-inflammation and immune regulation.5. The technology of TaqMan real-time quantitative RT-PCR can be used to detect the PPAR Y mRNA of various tissues of rats. PPARy is lower expression in tissues of OLETF rat than that of LETO. It suggested that the decrease of PPARymRNA may be associated with the pathogeny of DM of OLETF rat.6. Rosiglitazone can increase the expression of PPAR Y mRNA, It is postulated that the effect of prevention of T2DM and inhibition inflammation of rosiglitazone may be associated with the increased expression of PPAR Y .7. Rosiglitazone could increase the expression of PPARymRNA in abdomial aorta. It is supposed that the function of PPARy may be linked with diabetic cardiovascular complication.
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