Establishment Of Hyperglycemia Animal Model And Affected Factors

BackgroudsDiabetes mellitus is a clinical syndrome characterized by elevated plasma glucose levels resulting from absolute or relative insulin deficiency. It is projected that by2010, at least239million people will be affected by this disease globally. The high prevalence of diabetes mellitus as well as its longterm complications has led to an ongoing search for hypoglycemic agents from natural sources. Various types of animal models of type2diabetes derived either spontaneously or induced by treating with chemicals, or dietary or surgical manipulations and combinations. The role of genetic predisposition, aging, obesity and dietetic/sedentary life style are major risk factors involved in the development of type2diabetes. The rats combinated of high-fat diet-fed and low-dose streptozotocin-treated develop obesity, hyperinsulinemia, and insulin resistance and frank hyperglycemia. So we use combination of high-fat diet-fed and low-dose streptozotocin-treated rats as our animal diabetic models. Though chemical-induced diabetic models are the most widely used animal models, however, the mechanism of these models is directly destruct to pancreatic beta cell, so faced to the many and different mechanisms of functional foods/ingredients, these animal models depict symptoms and characteristics typical more of human type1diabetes and are not very suitable for the functional evaluation for all the functional foods/ingredients. Hence, there exists a continued quest for establishing one or some ideal animal models for type2diabetes to complete the functional evaluation procedure.Objective1. To analyze the basic data of normal blood glucose level in rats and related influencing factors.2. To determine the key factors in diet in establishment of insulin resistant animal model and their compatibility. 3. To explore the relationship and cross effects between diet and chemical-injury in hyperglycemia model made in order to obtain reliable model characteristics4. To indenty the reproducibility and applicability of the animal models5. To provide scientific basis for research and meet government’s need.Materials and methods1. Distribution of blood glucose levels in normal rats According to strict principles we analyze the data of fasting blood glucose and2h postload glucose of normal Wistar and Sprague-Dawley(SD) rats in the recent5-year studies of our laboratory, analyze the main factors which affect the blood glucose; then use contrast experimental design to assess the effects of blood sources (venous blood, arterial blood), blood collection time (fasting5h,11h), blood separated time (immediately,30-60min,60-120min,>120min) and testing methods (GOD method, Glucometer method). We also check the distribution of blood glucose, and give the suggestion of the upper limit of blood glucose in normal rats.2Animal models for insulin resistant induced by high-fat-sugar diet After adapt to the environment for3days,130healthy Wistar rats with body wight of290g±20g (50) and180±10g (80) were randomly assigned to normal group and high-fat-sugar feeding group (HFS) based on initial body weight. According to the ratio of lard (F) and sucrose (S) in feeds, HFS diets were repartition to10F10S (representing containing10%lard and10%sucrose in feeds, same below),10F20S,20F0S,20F10S,20F20S. In addition,10F20Fr (frocose)was assigned in order to compare the different effects between fructose and sucrose. Body weight was weekly recorded. At the4th week and8th week, fasting blood glucose and2h postload glucose after oral glucose torlerence test (OGTT) were tested. Simtaniously blood lipid level were detected. The influence of fat and sugar in diets to body weight gain, glucose response and lipid was analyzed by multivariate test with repeated measures.3Hyperglycemia animal models and affected factors Base on body weight, blood glucose, lipid levels,55HFS-fed rats were randomly divided into4groups:STZ0, STZ20, STZ25and STZ30. After fasted for overnitght, each groups were respectively intraperitoneally injected with0,20,25,30mg/kg.bw of streptozotocin (STZ). On the basis of observation on the relationship between dose depended blood glucose response and death, the appropriate dose of STZ used in model made was indentified. Then,5weeks of blood glucose monitor was preceeded in groups of rats with different fomular HFS diet (10F10S,10F20S,20F10S,10F20Fr) after appropriate dose of STZ injection. The influence of dietary regimens and STZ to the animal model was analyzed by univariate analysis. A criterion for judgement of diabetes in rats was envaluated based on integration of injury intensity, blood glucose levels and mortality.4Validation of hyperglycemia animal model Selected normal control rats (Ctrl.), STZ control rats (STZ-C), HFS-fed control rats (HFS-C) and model rats (Mod.) were randomly divided into2sub-groups, each respectively treated per day with pioglitazone (15mg/kg), a kind of insulin sensitizer or distilled water. Forteen days later, curative effect pioglitazone was assessed by of body weight comeback and blood glucose response after OGTT. Once positive effects of pioglitazone were indetified, the rats were exposed to14d washout period, and then killed for collection of aorta abdominalis blood, organs and tissues such as liver, muscle, kidney, heart, fat pad and pancreas.5Reproducibility and application of the animal models Based on the above process, additional2teams of members repeated the model made operation in different anmal laboratories. Reproducibility was statistics based on repeated mean value. In addition, three tests were carried out to observe the effects of resistant starch on prevention or intervention from insulin resistant and diabetes development in rats.Results1. Distribution of blood glucose levels in normal rats The95%upper limit (UL) for FBG distribution in475normal rats (150-600g) and PBG in238normal rats is respective6.2mmol/L and10.3mmol/L. Analysis of blood glucose impact factors showed:1) Glucometer is not a good way to detect blood glucose as compared with typical glucose oxidase method, so that it causes miss evaluation of true value of glycemic results;2) The difference in various blood source is exist, glucose concentrations in abdominalis aorta is1.4times higher than that in peripheral vein;3) Glycemic level after fasting for5h is0.8mmol/L above that after11h;4) Blood glucose will attenuate8%after stored at room temperature in60min and more than50%after stored for6min longer. After re-calibrated the UL of normal blood glucose was corrected to6.2mmol/L for FBG and8.0mmol/L for PBG. And taking into account or uncertain factors like sample source, expansion factor1.3was induced, which extrapolates FBG to8.0mmol/L and PBG to11mmol/L.2Animal models for insulin resistant induced by high-fat-sugar diet After fed with HFS diet, body weight gain fast increased, accompanying with gradually rise of glycemia, which exhibited higher PBG after4weeks, and higher FBG after8weeks with PBG beyond two standard deviation of normal. Similarly, total cholesterol and triglyceride also arose in HFS fed rats. Multivariate analysis showed that lard was the main factor leading to obesity and high blood glucose and lipids level, while sugar only affected the process of hyperglycemia occur.3Hyperglycemia animal models and affected factors On basis of HFS feeding, STZ injection declined rats body weight and run up glycemia rapidly, which trended to stable after3weeks. Accoding to glycemic leval and death rate,20～25mg/kg.bw STZ was thought suitable for animal model made, The difference among various diets in glycemia was20F10S>10F10S>10F20S=10F20Fr. The cross effects of HFS and STZ showed that HFS, which acted before STZ injection, enlarged STZ effects. The more lard in diet, the higher level of glcucose in blood, but sugar contrary.4Validation of hyperglycemia animal model After pioglitazone treatment, rats body weight comeback, and elevated glycemia declined. These changes were most obvious in rats fed20F10S or rats injected lower dose of STZ (20mg/kg.bw). Biochemical analysis showed that the rat model exhibited typical renomegaly, liver glycogen increase, insulin abnormal secretion HDL-C decrease and other physiological and biochemical changes. Univariate analysis variance showed STZ mainly affects organ weight such as heart, kidneys and liver glucose production; lard mainly affects lipid and insulin secretion except for fat accumulation, while high sugar intake can lower blood glucose by increasing muscle glycogen storage.5Reproducibility and application of the animal models3repetitive tests have shown that under the prescript experimental conditions, it could steadily induce the IR model and T2DM model. The relative standard diviation for the reproducibility test is less than20%. In study of preventive and intervention benefits of resistant starch to insulin resistant and diabetes, it was showed that20%RS made HFS fed rats have normal PBG, insulin secreation and glycogen storage,30%RS could normalize postprandial insulin abnormal elevation in IR animals and enhance insulin sensibility. Also,30%RS could decrease LDL-C level in DM model rats, and trend to fall down FBG.Conclusions 1. Distribution of blood glucose levels in normal rats:After fully taking into account the influence of uncertain factors such as analytical method, sample source, handling way, fasting time, etc., we set the normal blood glucose UL to FBG6.2mmol/L and PBG8.0mmol/L, and expansion UL to FBG8.0mmol/L and PBG11.0mmol/L. These values were testified with subsequent hyperglycemia critical.2. The main contribution of fat, sugar, STZ on animal model made were tested through different ways. Fat is the most important factor to disturb glucose and lipid metabolism and insulin secretion. Low-dose of STZ could make rapid increase in blood glucose, without obviouse destroying insulin secretion.3. With consideration of collinearity among biochemical indexes, it was proved by factor analysis that changes in body weight gain, fat accumulation and liver glucogen were prominent signs accompanied with high blood glucose. If using body weight and PBG as grouping indicators, PBG as criteria for judgment of hyperglycemia, the outcome will be more reliable. It suggests that the criteria for IR model should be PBG>8.0mmol/L and higher two standard deviations than control group. As for T2DM animal model, it should PBG range between11mmol/L to30mmol/L.4It has been showed rats will exhibit different characteristics after fed with different HFS fomular, frucost may induce high TG and low HDL-C, lard mainly affectes insulin secretion, and high-sucrose may affect blood glycose rise by accelerating muscle glycogen storage.5The hyperglycemia models have strong consistency in duplication checks, and is suitable for long-term observation in prevention and interervention studies.