| Insulin resistance (IR) is a state in which a given concentration of insulin produces a less-than-expected biological effect. It is a common risk factor for a variety of metabolic diseases and it often exists in the early stage of these diseases. So the treatment of insulin resistanceit is very important for the prevention and treatment of metabolic diseases, espec-ially diabetes.Obesity (mainly abdominal obesity) was the important risk factor resulting in insulin resistance. Obesity can lead to insulin resistance through endocrine, adipocytokines, infla-ammatory response and intrinsic cell signaling pathways. It is now recognized that free fatty acids (FFA) decomposed by the fat and adipocytokines are often involved in insulin resistance mediated by obesity. These factors can affect the sugar, fat metabolism through different mechanisms, and there are complex linkages among the various factors. Among them, free fatty acid (FFA) and tumor necrosis factor-a (TNF-a) are closely related to insu-lin resistance. TNF-a can promote fat decomposition and FFA concentration. By inhibiting lipoprotein lipase. Increased FFA levels can increase the insulin resistance and can also cause hyperinsulinemia. The liver is the main target organ for insulin. In the metabolic effects of insulin, the main function of liver is in the promotion of liver glycogen synthesis and inhibiting glycogen output. When the liver insulin resistance occurs, the liver glycogen synthesis shoud be reduced and gluconeogenesis shoud be increased, resulting in hypergly-cemia. The insulin receptor substrate-2 (IRS-2) branch signal of Insulin signaling cascade is closely related to insulin sensitivity of liver cells. IRS-2/PI3-K signal pathway is the major signal transduction pathways of insulin playing a physiological effect in the liver. Reduction of insulin receptor substrate-2 (IRS-2) expressions in gene and protein and phosphorylation disorder will affect its the effective signa transmission of its downstream PI3K/Akt pathway, which can cause insulin resistance. At present, a number of factors can be found to lead to liver insulin resistance by affecting the IRS-2 signal transduction. Protein tyrosine phosphatase-1B (PTP-1B) is a major member of the protein tyrosine phosphatase (PTP) family. It could make the tyrosine residues of InsR, IRS and other signaling proteins to be dephosphorylated, and block insulin signaling cascade downstream. PTP-1B is a negative regulator of insulin signaling. So it is supposed that whether we can achieve the purpose of improving insulin resistance by inhibiting PTP-1B expression so as to increase the expression and phosphorylation of IRS-2. This study was carried out under the guidance of Chinese medicine theory of "sour restrains sweet" and "sour enters liver". This study attempts to conduct in-depth study about ursolic acid on the role and mechanism of insulin resistance through animal experiments, and molecular biology tools. The liver will be as a target organ in the study. Hopes to prove the guiding role of traditional Chinese medicine theory of "sour restrains sweet" to the prevention and treatment of insulin resistance and diabetes and provide experimental basis for diabetes prevention and drug discovery.Part I Rats of Insulin Resistance Model Induced by High-Fat-FedObjective:To establish insulin resistance obesity rats model induced by high-fat-fedMethods:60 eight-week-old male Wistar rats, body weight 180-200g, were randomly divided into two groups:normal diet group(n=12) and high-fat diet group(n=48). Normal diet group rats were fed with basic feed(fat10%, carbohydrate67%, protein23%)and high-fat diet group rats were fed with high-fat feed(fat46%, carbohydrate40%, protein14%): basic diet(78.9g) plus lard 10g, cholesterol 1g, egg yolk powder 10g, bile salt 0.1g. Before and after the experiment, the body weight and fasting blood glucose, TC, TG, HDL-C and LDL-C levels were tested; serum insulin levels determined by enzyme-linked immuno-sorbent assay (ELISA); the regulatory function of glucose metabolism determined by the glucose tolerance test and insulin tolerance test; insulin sensitivity evaluated by the insulin sensitivity index (ISI) and insulin resistance index (HOMA-IR).Results:Compared with the normal diet group rats, the fasting blood glucose of high-fat diet group rats is not obvious or mildly elevated, while the body weight, blood lipids (TC, TG, LDL-C), fasting plasma insulin levels were significantly higher. The high-fat diet group rats appear impaired glucose tolerance and insulin tolerance and decline in insulin sensitivity.Conclusion:Insulin resistance obesity rats model can be successfully established by 9-week high-fat diet feeding. The model's characteristics is similar to human insulin resistance caused by obesity and we think it is a economic and practical animal model for the study of obesity and insulin resistance.PartⅡEffects of ursolic acid on glucose, lipid metabolism and FFA, TNF-a levels in insulin resistance ratsObjective:To observe the effects of ursolic acid on glucose, lipid metabolism and free fatty acid (FFA), tumor necrosis factor-a (TNF-a) levels in insulin resistance rats.Methods:60 rats were randomly divided into five groups:normal group (normal diet+ intragastric administration with PBS solution), model group (high-fat diet+intragastric administration with PBS solution), metformin group (high-fat diet+intragastric administration with metformin,200mg/kg·d), ursolic acid high-dose group(high-fat diet+ ursolic intragastric administration with ursolic acid,300mg/kg·d), and ursolic acid low-dose group (high-fat diet+ursolic intragastric administration with ursolic acid, 150mg/kg-d). Each group is 12. At point 4 weeks and point 8 weeks after administration of each group, to observe the changes of fasting blood glucose (FBG), fasting serum insulin (FINS), blood lipids, body weight, liver weight, abdominal adipose weight, glucose tolerance, insulin tolerance, liver glycogen content and serum free fatty acids (FFA), tumor necrosis factor-a (TNF-a); to calculate the liver index(LI), abdominal adipose index(AI), insulin sensitivity index (ISI) and insulin resistance index (HOMA-IR); to observe the liver tissue pathological changes by light microscopy (HE staining).Results:Ursolic acid and metformin treatment can reduce serum FINS, TC, TG, LDL-C and FFA, TNF-a levels in insulin resistance rats, also they can lower body weight, liver index and abdominal adipose index; reduce intra-abdominal fat accumulation; improve glucose tolerance, insulin tolerance; add liver glycogen content; increase insulin sensitivity index (ISI) and lower insulin resistance index (HOMA-IR). Compared with the model control group, There were significant difference between treatment group and model control group (P<0.05 or P<0.01). There were no significant difference between ursolic acid high-dose group and low-dose group.Conclusion:Ursolic acid can reduce body weight, regulate glucose and lipid metabolism, reduce the levels of FFA, TNF-a in insulin resistance rats and improve insulin resistance.PartⅢEffects of ursolic acid on expressions of PTP-1B and IRS-2 in the liver of insulin resistance rats.Objective:To observe the effects of ursolic acid on expressions of PTP-1B and IRS-2 in the liver of insulin resistance rats and explore the role of PTP-1B and IRS-2 in the process of insulin resistance.Methods:60 rats were randomly divided into five groups:normal group, model group, metformin group, ursolic acid high-dose group, and ursolic acid low-dose group. Each group is 12(administration same to PartⅡ). The mRNA expressions of PTP-1B and IRS-2 in liver were detected by real-time fluorescent quantitative RT-PCR. Relative mRNA quantification methods was used for real-time PCR data analysis. The protein expressions of PTP-1B, IRS-2 and IRS-2 tyrosine phosphorylation in liver were detected by Western blot.Results:The content of liver PTP-1BmRNA expression of model control group detected by real-time fluorescent quantitative RT-PCR is higher than that of the normal control group, while IRS-2mRNA is lower than that of the normal control group. After the therapy of ursolic acid (high-dose and low-dose) and metformin, PTP-1BmRNA levels were decreased and IRS-2mRNA levels were increased. There were significant difference between treatment group and model control group (P<0.05 or P<0.01). Similar protein results were detected in Western blot assay. Western blot results showed that PTP-1B protein expression was decreased; IRS-2 protein expression and tyrosine phosphorylation was increased.Conclusion:PTP-1B expression was increased and IRS-2 expression was decreased in liver of insulin resistance rats. Ursolic acid and metformin can inhibit PTP-1B expression in the liver, thereby contributing to increased IRS-2 expression and IRS-2 tyrosine phosphorylation. That may be one of the molecular mechanisms of ursolic acid improving insulin resistance. |
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