Effects And Mechanisms Of Metallothionein Induced By Zinc On Hyperlipidemia And Diabetes Mellitus

Objective:This study was to investigate the effects and mechanisms of Zn induced by gavage, metallothionein on the heperlipidemia and diabetes by observing the changes of the major pathophysiological parameters, anti-oxidation indicators, the content of Zn in the main organ and the expression of metallothionein in animal models.Methods:76 Male Wistar rats( 180 220 g),were divided into six groups: normal diet group (NC), high fat diet group (FC), diabetes group (DM), High fat plus Zn group (FZ); Zn diabetes before the intervention group (ZD); Zn after the intervention of diabetes (DZ). NC group was given normal diet, the other five groups were high fat diet. FZ, ZD groups were feed Zn ions (Zn2 +) 15mg·Kg^-1·d^-1 by giving ZnSO4 solution daily gavage, the others is instead of distilled water gavage. Five weeks after the feeding, blood glucose and blood lipid were tested to identify high fat model of animal.Animal fasting 12 h, DM, ZD and DZ animals were injected with STZ 40mg·kg^-1 in the left-sided inferior peritoneal, and others were given citrate buffer. The fasting plasma glucose(FPG) level of experiment group rats was measured on the 3rd and 7th day after given STZ. The rats are determined DM which value of fasting plasma glucose reach 16.7mmol·L^-1.And DZ group at this time given ZnSO4 by gavage. Two weeks after STZ injection, serum from eyeball were collected and main organs (liver, kidney, pancreas, thymus and spleen) were taken for pathological examination.Detection:Take notes about animals'food-intake and volume of drinking water, record animal weigh with electronic scale. Fasting plasma glucose (ARKRAY AY) ,the level of serum total cholesterols (TC),triglyceride (TG),low-density lipoprotein- cholesterols (LDL-C) ,high-density lipoprotein- cholesterol (HDL-C) and liver and Kidney functions (Hitachi Automatic Biochemical Apparatus), serum insulin (radio-immunity),and liver, kidney, heart, pancreas, thymus and spleen of all rats are take out for calculating organ index..The pathological and histological change of liver, pancreas, and kidney are observed in light microscope; the percentage of metallothionein expressing positive cell in organs is investigated by immunohistochemical methods. Antioxygen detect: SOD, GSH-Px in serum and MDA ,T-AOC in liver(Jiancheng , Nanjing); Zn,Cu and Fe content in serum, liver, pancreas, and kidney absorption with atomic absorption after acid digestion.Statistical analysis:Application of SPSS 12.0 statistical software for data analysis. Measurement data to Mean±S.D that the multi-use data to compare variance analysis, some of the information related analysis.Results:1. Hyperlipidemia model was mimicked by feeding rats high fat diet (total cholesterol TC, low LDL-C levels increased, P <0.05), Effect of a one-time model for STZ intraperitoneal injection of 40 mg ? kg-1, caused by high fasting plasma glucose (FPG≥16.7 mmol ? L-1, P <0.01), high blood lipids (TC, TG, LDL-C levels increased , P <0.01), drink more (P <0.01), more food (P <0.01), weight loss (P <0.01) Effect of the symptoms model of DM..2. MDA in liver tissue of hyperlipidemia animal increased (P <0.01); high fat animal model of diabetes in 14 d when there glucose and lipid metabolism disorder: FPG significantly higher (P <0.01) , TC, TG, LDL-C increased. (P <0.01). Liver, kidney indices increased (P <0.01), thymus index decreased (P <0.05), SOD, GSH-Px and liver MDA increased (P <0.01), this display the early damage of liver and kidney function.3. Zn from digestive tract cause SOD increased (P<0.01), GSH-Px lower (P <0.01), liver total antioxidant capability and MDA lower (P <0.01), all of this disclose Zn has that antioxidant-related functions but can't display through the total antioxidant capacity .4. FPG levels of Hyperlipidemia DM show Zn given to the digestive tract after diabetic has few effect to lower FPG (P> 0.05). but through this Zn improve of high blood glucose, high blood lipid levels, under the premise of reducing the amount of insulin secretion (P <0.01), improved insulin sensitivity to insulin receptor; addition to the digestive tract of Zn and high fat Diabetes group model, can improve the high fat diabetic animals "more drink" and the symptoms of weight loss (P <0.01, P <0.05), significantly decreased the level of liver MDA and total antioxidant capacity (P <0.01).5. From the MT mRNA level of detection, in the liver, high fat group MTmRNA levels were significantly more than 5 to 15 times higher than the normal group. High fat diabetic group MTmRNA increased to 10 times the normal group over the Digestive tract to the Zn group compared with the normal group, MTmRNA level three times the increase, but higher than the level of high fat group and high fat diabetic group, even in the high fat in the model after the digestive tract to the establishment of Zn, MT mRNA level but reduced. In the pancreas, high fat group MTmRNA to increase the level of the normal three to five times that of high fat diabetic group MT mRNA levels and close to the normal group, and Zn by the digestive tract to the group about MT mRNA level increased, For the normal group of two to six times. In the kidney, only the high fat in the model after the digestive tract to the Zn group MTmRNA relative increase in the level of three times around the other groups MTmRNA level no significant change.At the same time in different organs found in MT1_mRNA and MT2_mRNA expression of the differences that the two different control mechanisms and Zn nutrition and peroxide-related, but not exactly the same.6. From the comparison of MT protein Immunohistochemical analysis, in the liver, the MT -positive cell rates of pancreas and kidney were significantly different (χ²> 33, P <0.01): in the liver, high fat diabetic group the largest percentage of positive cells, In the pancreas, in the digestive tract to the high fat group Zn positive cells rate relative increase in kidney, the positive rate in the high fat cells to the digestive tract to reduce the Zn group.The results of Pathological image analysis system positive signal (yellow) optical density shows that with normal and high fat group, visible in the liver, the use of blood glucose abnormalities STZ in the three groups MT positive signal increased density in the pancreas, digestive tract to the Zn MT positive signal of the group increased density in the kidney, the groups had no significant difference. These results can Note: in the liver, MT for the induction and expression STZ toxic reaction to stimulate more obvious and in the pancreas, MT induction and practical expression to respond more sensitive to Zn.7. The testing results of the elements in the organs, such as Zn, Cu, Fe shows that among the different organs, there exists difference of Zn and other trace elements, which mainly reflected in the following:(1) ZnComparing with the normal and high fat groups, the Zn level of serum in other groups is lower (P <0.05, or P <0.01); in the liver, Zn in the group of high fat is relatively low, to the digestive tract to the liver Zn group Relatively higher levels (P <0.05); Zn content in the pancreas in each group there are differences, compared with the normal group, the high fat group and high fat for diabetes whether or not the digestive tract and Zn, Zn content increased (P <0.01). Zn content of the kidney and pancreas similar, but in lipid metabolism disorder of blood glucose test animals, and glucose metabolism compared to relatively normal animals, kidney Zn content further increased (P <0.01).(2) Zn/Cu:The ratio between Zn and Cu in the liver showed that there are differences in each group, for the high fat diabetes before and after the digestive tract to the Zn group, the ratio of high fat compared with a higher (P <0.05; P <0.01), other groups In comparison does not indicate a significant difference.(3) Zn/FeThe rules of ratio changes between Serum and Zn /Fe in kidney is almost the same;In the liver, the ratio between Zn and Fe in the tested animals of the blood lipids& blood glucose metabolism disorder is higher than those animals with normal glucose metabolism, which was increased to (P <0.05), and Zn and Fe ratio changes of the content changes are not completely Line: Compared with normal group, high fat content of lower Zn group (P <0.05) while Fe content also decreased significantly (P <0.01), the results of Zn and Fe ratio compared with the normal group did not show a difference (P> 0.05 ), With a similar group and diabetes are high fat and high fat diabetic Zn group, the Zn content of the liver and no difference in the normal group (P> 0.05) and Fe were significantly lower (P <0.01), lead Zn and Fe Compared with the normal ratio increased significantly (P <0.01; P <0.05).In the pancreas, the ration between Zn and Fe in the group of high fat is increased, compared with the normal group (P <0.01), the other groups compared with the normal group there was no significant difference (P> 0.05), and pancreas Zn / Fe ratio of change in the law and Zn content is not entirely consistent: Compared with the high fat, diabetes and high fat content of Zn group relative no difference (P> 0.05), relatively higher Fe content (P <0.01), lead Zn / Fe ratio and high fat Group decreased significantly (P <0.01).8. After making the correlation analysis of elements in the different organs of the tested animals, we can find in serum Zn and liver, kidney, pancreas of Zn was negatively correlated (r <0.000, P <0.05), while kidney and pancreas Zn Zn was positively Related (r = 0.420, P = 0.021); animal liver and kidney Fe Rail was significantly correlated (r = 0.557, P = 0.001); organ of the elements in the ratio of a statistical correlation analysis and found that kidney Zn / Fe ratio And liver Zn / Fe significantly positive correlation (r = 0.800, P = 0.000), and serum Zn / Fe and liver and kidney Zn / Fe significantly negative correlation (r =- 0.519, P = 0.003) (r =- 0.467, P = 0.009).9. The relationship between positive staining MT and levels of trace elements in organs.From the Spearman rank correlation analysis of the MT positive immunohistochemical staining gray value and the organs of the liver, pancreas and kidneys of are a few elements in a, we can see that the liver and liver MT Zn / Fe positive correlation (rs = 1.000, P <0.01) , And Cu contents in the liver, liver Fe content was negatively correlated (rs =- 0.886, P <0.05; rs =- 0.829, P <0.05).Conclusion1. With the usage of feeding Use of Wistar male rats 35 days with high fat, it can cause hyperlipidemia models, after which to give a one-time injection of STZ 40mg·kg^-1 on the abdominal cavity. This can cause high blood lipids, high fasting plasma glucose, and polyphagia, weight losing and other characteristics of the high fat animal model of diabetes.2. Hyperlipidemia model and high fat diabetes animal ones exist to varying degrees of lipid peroxidation injury, which the body can make a response to this injury by adjusting Zn, Fe and other trace elements in the distribution of organs and regulating all kinds of Oxidation-related protein expression to the performance, such as the expression of antioxidant enzyme and MT.3. Feeding with high fat diet, while giving a gavage with ZnSO4 (Zn2 +15 mg·Kg^-1·d^-1), the models of high fat and diabetes glucose and lipid metabolism in disorder can be improved to change the body against the oxidative damage. The anti-oxidation ability of Zn can protect the body through the induction and synthesis of MT.4. In different circumstances of Zn nutrition, the oxidative damage caused by the high fat and high blood glucose can regulate the mobilization of Zn, Fe and other trace elements in the organization and re-distribute the organs. This process relates to not only the sensitivity to the oxidative damage of the physiological functions of the major organs, but the adjustment of oxidation / antioxidant system.5. In different circumstances of Zn nutrition and peroxide, there are differences in the MT1_mRNA and MT2_mRNA expressions of liver, pancreas, kidney, and others, and also different from the protein of synthesized MT. Zn, as the main combination of metals, not only participated in the induced transcription of MTmRNA, but involved in the process of protein synthesis, which is good to the MT synthesis.6. In the liver, the expression of MT is positively correlated with Zn / Fe in the liver, negatively with the contents of Cu and Fe. 7. The level of serum Zn of testing animals was negatively correlated with the content of Zn in the liver, kidney, pancreas, the content of Zn in the kidney is positive with that in pancreas, the content of Fe in the liver is significantly positive with that in kidney.