高血糖动物模型的建立及其影响因素研究
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摘要
研究背景
糖尿病(DM)作为一种以血糖浓度增高、胰岛素缺乏或作用下降为主要特征的慢性疾病已成为严重的社会经济问题,其中2型糖尿病是发病率最高也是危险性最高的一类,通过膳食防治糖尿病已成为社会广泛共识,相应地有关食品调节血糖的功能学研究得以发展。为了探索DM的发病机理、天然食品调节血糖的作用机制及评价等问题,建立能够模拟人类疾病特征的实验动物模型也备受关注。至目前为止用作糖尿病相关研究的动物模型包括手术型、诱导型、自发型、转基因型等。由于2型糖尿病或胰岛素抵抗与糖脂代谢密切相关,肥胖和高能量膳食摄入可增加其发病风险,高脂高糖膳食诱导肥胖型的高血糖动物模型以及在此基础上联合小剂量链脲佐菌素的造模方法因符合人类生存环境及胰岛素发病机理而愈来愈多地被加以应用。但是由于此模型缺少统一的操作手段,对主要影响因素的作用贡献不清,使得不同的造模手法可产生不同的病理生理改变,由此为解释、评价和横向比较食品功能的意义带来困难。为了解不同膳食条件下诱导模型的优势,本研究着重对高脂高糖膳食诱导高血糖动物模型及其影响因素展开了研究。
研究目的
1.分析和探讨正常大鼠血糖相关基础数据及其影响因素
2.确定诱发高血糖动物模型的膳食关键因素及配方
3.确定膳食及化学损伤的作用关系及模型特点
4.探讨动物模型的实用价值和复现性
5.为科学研究和政府需求提供科学依据
材料与方法
1.正常大鼠血糖水平分布及其影响因素
按照严格的入选原则对5年研究中所测定正常Wistar大鼠和SD大鼠空腹血糖和OGTT后2h的血糖测定数据的分布进行统计,分析影响血糖的主要因素；然后采用对比实验设计,对血源(静脉血、动脉血)、血样采集时间(餐后5h、11h)、样品处理时间(立即、30-60min、60-120min、>120mmin)和测试方法(GOD法、血糖仪法)等因素的影响进行估算,并以此对血糖分布进行校核,提出正常大鼠分布上限。
2、高脂高糖饲料诱导胰岛素抵抗及其影响因素
Wistar大鼠适应环境3天后根据体重随机分组：普通饲料组和高脂/高糖饲料组(HFS),HFS饲料依据猪油(F)、蔗糖(S)水平设计为10F10S(含10%猪油10%蔗糖,其余类推)、10F20S、20FOS、20F10S、20F20S,为比较果糖与蔗糖的差异另设10F20Fr组。每周监测体重,于第4、8周测定空腹和OGTT2h后血糖(FBG、PBG)；第8周时同时测定血脂水平。采用重复测量多元分析法探讨脂肪、糖对血糖、血脂的影响。
3高血糖动物模型的建立及影响因素
选择HFS饲料喂养大鼠55只,根据体重、血糖、血脂水平随机分为STZ0组(10只)、STZ20组(15只)、STZ25组(15只)和STZ30组(15只),分别注射0、20、25、30mg/kg,bw STZ,观察血糖与STZ用量的关系,确定合适用量。另选择不同配方饲料喂养大鼠(10F10S、10F20S、20F10S、10F20Fr)按上述确定剂量注射STZ造模,监测5周内的血糖变化,采用多变量方差分析探讨HFS饲料配方与STZ对造模的贡献,并根据大鼠的损伤强度、血糖水平和死亡率评估高血糖和糖尿病的判断标准。
4高血糖动物模型的验证
选择正常对照大鼠(Ctrl.).STZ对照大鼠(STZ-C)、HFS对照大鼠(HFS-C)、模型大鼠(Mod.),各组均随机分为2个亚组,1组每天接受15mg/kg/bw吡咯列酮治疗,另1组给与蒸馏水。14d后根据体重增长和血糖应答反应判断治疗疗效。经过洗脱期后处死大鼠,分析脏器、脂肪垫重量、胰岛素、C肽、糖原等指标。采用因子分析方法对指标进行归类,并评估不同饲料喂养下动物模型的优势。
4动物模型复现性及应用性研究
按照以上建立的造模程序,选择2队试验人员分别在2个动物房复制实验,统计3次重复测量的再现性限。
另取100只大鼠分为3组：第一组进行抗性淀粉(RS)预防胰岛素抵抗实验,即在HFS饲料中加入20%RS,观察10周后血糖、胰岛素、血脂、糖原等指标与HFS对照组的差异；第二组在HFS诱导大鼠胰岛素抵抗成功后,采用20%、30%RS干预6周,观察血糖、胰岛素、血脂等指标的变化；第三组在HFS联合STZ诱导高血糖模型成功后,观察20%、30%RS对血糖、胰岛素、血脂等指标的影响。每组均设正常对照(Ctrl)和模型对照。
研究结果
1.正常大鼠血糖水平分布
正常大鼠475只的FBG和238只的PBG分布范围95%上限分别为6.22mmol/L,10.3mmol/L;对血糖影响因素的分析显示：1)血糖仪法测定血糖的敏感性较低,和葡萄糖氧化酶法仅在血糖水平为7～10mmol/L时结果接近；2)不同来源的血样血糖存在一定差异,腹主动脉血比外周静脉血高1.4倍；3)采样前大鼠禁食时间对血糖的影响为禁食11h血糖低于禁食5h；4)取血后血样在60min内进行分离血糖衰减8%,如超过60min衰减达50%以上。根据以上因素重新整理数据,确定以葡萄糖氧化酶法测定的大鼠正常血糖上限为FBG6.2mmol/L, PBG8.0mmol/L;考虑到血源等不确定因素的影响,统计确定扩大系数1.3倍,由此外推扩大的血糖上限分别为FBG8.0mmol/L, PBG1lmmol/L
2、高脂高糖饲料诱导胰岛素抵抗及其影响因素
经HFS喂养大鼠体重增长明显高于普通饲料组,到4周时出现PBG升高,8周时FBG升高,且PBG超过普通饲料组2个标准差；同时TC、TC升高。当饲料猪油含量为20%时血糖水平最高,多变量分析显示猪油是影响血糖、血脂最大的因素,蔗糖可影响高血糖发生进程,果糖(20%)和蔗糖间没有显著差异。
3高血糖动物模型的建立及影响因素
在HFS饲养基础上,经小剂量STZ造模后HFS大鼠体重迅速下降,3周后血糖趋向稳定。根据血糖的高低、死亡率,确定20～25mg/kg.bwSTZ(?)注射剂量为宜；例料间血糖的差别为20F1OS>10F10S>10F20S=10F20Fr。对STZ与HFS交互作用分析显示HFS对STZ造模效果起到放大作用,其作用时间点仅在STZ注射前,猪油越高血糖越高,蔗糖反之。
4高血糖动物模型的验证
经过吡咯列酮治疗后,HFS大鼠体重回升且餐后血糖升高幅度下降,其中以小剂量STZ(20mg/kg.bw)制造的模型和以20F10S喂养的动物效果最为明显。对生化指标的分析显示模型大鼠出现典型肾肿大、肝糖原升高、胰岛素分泌以及HDL-C下降等生理生化变化。对影响因素的归因分析显示,STZ主要影响心、肾等脏器及糖原；HFS主页影响胰岛素分泌、糖原储存和血脂。
5高血糖动物模型复现性和应用研究
3次重复性检验显示,在规定的实验条件下均可诱导出符合要求的IR模型和T2DM模型,重复性检验相对偏差<20%。在RS预防和干预高血糖的应用研究中,HFS使大鼠PBG升高、空腹胰岛素下降、肝糖原升高、肌糖原下降(与Ctrl相比P<0.05)；给与20%RS的大鼠PBG和肝糖原明显低于HFS组(P<0.05),且空腹胰岛素未与对照组有明显统计学差异。对于餐后血糖和胰岛素升高、且胰岛素敏感指数下降的胰岛素抵抗模型大鼠,给以6周30%RS干预的大鼠虽未见血糖降低,但餐后胰岛素异常分泌下降,胰岛素敏感指数升高(和模型组相比P<0.05)。而对糖脂代谢紊乱的DM模型大鼠,30%RS可使大鼠LDL-C下降、FBG有下降趋势。
结论
1.综合考虑血糖分析方法、血样来源和处理方法、禁食时间等因素的影响,并使用高血糖模型大鼠进行相互验证的结果,确定以葡萄糖氧化酶法测定的大鼠正常血糖上限为FBG6.2mmol/L,PBG8.0mmol/L;扩大上限为FBG8.0mmol/L,PBG11mmol/L,可作为判断高血糖模型成功的依据。
2脂肪、糖、STZ通过不同途径影响血糖,对造模贡献有所不同,其中脂肪是影响血糖、血脂、胰岛素分泌的最重要因素,小剂量STZ可在不影响胰岛素分泌的前提下使血糖迅速上升。
3通过综合对血糖、胰岛素、敏感器官和血脂等相关指标的的归类分析,证实体重增长、脂肪堆积、肝糖原是伴随高血糖最突出的体征和生化改变,以体重和PBG作为分组的指标,以PBG作为高血糖模型判断标准可信度较高。IR模型的判断标准为PBG>8.0mmol/L且超过对照组2个标准差；T2DM的标准为PBG11～30mmol/L。
4本研究证实了所建模型为具有胰岛素抵抗特征的高血糖动物模型,不同的饲料喂养可现示出不同的模型特点,高果糖摄入以增加TG和HDL-C为主,猪油主要影响脂肪堆积和胰岛素分泌；高蔗糖摄入可通过增加肌糖原储存而降低血糖。20mg/kg.bwSTZ因对机体损伤相对较轻、体征变化较轻,对胰岛素增敏剂的敏感性相对较强。
5对模型稳定性和复制性的研究证实所建模型的复现性较强,由于不同阶段的模型具备不同的特点,所以在评价中可能需采用不同的评价指标。由于HFS诱导高血糖是一个缓慢的过程,因此本模型可用于长期的预防性或干预性研究和评价。
本研究的创新之处：
1.首次全面探讨了大鼠正常血糖分布范围和上限,为确定动物高血糖、糖尿病血糖判断标准提供了理论依据。
2.首次探讨了脂肪、糖、STZ对高血糖动物模型的贡献和主要作用点,为解释模型和标化模型提供了依据。
3.首次探讨了模型的稳定性、复现性,对关键技术条件进行了摸索,为修改保健食品辅助降血糖功能评价动物模型的建议提供了参考。
Backgrouds
Diabetes 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.
Objective
1. 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 characteristics
4. To indenty the reproducibility and applicability of the animal models
5. To provide scientific basis for research and meet government's need.
Materials and methods
1. 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.
Results
1. 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.
糖尿病(DM)作为一种以血糖浓度增高、胰岛素缺乏或作用下降为主要特征的慢性疾病已成为严重的社会经济问题,其中2型糖尿病是发病率最高也是危险性最高的一类,通过膳食防治糖尿病已成为社会广泛共识,相应地有关食品调节血糖的功能学研究得以发展。为了探索DM的发病机理、天然食品调节血糖的作用机制及评价等问题,建立能够模拟人类疾病特征的实验动物模型也备受关注。至目前为止用作糖尿病相关研究的动物模型包括手术型、诱导型、自发型、转基因型等。由于2型糖尿病或胰岛素抵抗与糖脂代谢密切相关,肥胖和高能量膳食摄入可增加其发病风险,高脂高糖膳食诱导肥胖型的高血糖动物模型以及在此基础上联合小剂量链脲佐菌素的造模方法因符合人类生存环境及胰岛素发病机理而愈来愈多地被加以应用。但是由于此模型缺少统一的操作手段,对主要影响因素的作用贡献不清,使得不同的造模手法可产生不同的病理生理改变,由此为解释、评价和横向比较食品功能的意义带来困难。为了解不同膳食条件下诱导模型的优势,本研究着重对高脂高糖膳食诱导高血糖动物模型及其影响因素展开了研究。
研究目的
1.分析和探讨正常大鼠血糖相关基础数据及其影响因素
2.确定诱发高血糖动物模型的膳食关键因素及配方
3.确定膳食及化学损伤的作用关系及模型特点
4.探讨动物模型的实用价值和复现性
5.为科学研究和政府需求提供科学依据
材料与方法
1.正常大鼠血糖水平分布及其影响因素
按照严格的入选原则对5年研究中所测定正常Wistar大鼠和SD大鼠空腹血糖和OGTT后2h的血糖测定数据的分布进行统计,分析影响血糖的主要因素；然后采用对比实验设计,对血源(静脉血、动脉血)、血样采集时间(餐后5h、11h)、样品处理时间(立即、30-60min、60-120min、>120mmin)和测试方法(GOD法、血糖仪法)等因素的影响进行估算,并以此对血糖分布进行校核,提出正常大鼠分布上限。
2、高脂高糖饲料诱导胰岛素抵抗及其影响因素
Wistar大鼠适应环境3天后根据体重随机分组：普通饲料组和高脂/高糖饲料组(HFS),HFS饲料依据猪油(F)、蔗糖(S)水平设计为10F10S(含10%猪油10%蔗糖,其余类推)、10F20S、20FOS、20F10S、20F20S,为比较果糖与蔗糖的差异另设10F20Fr组。每周监测体重,于第4、8周测定空腹和OGTT2h后血糖(FBG、PBG)；第8周时同时测定血脂水平。采用重复测量多元分析法探讨脂肪、糖对血糖、血脂的影响。
3高血糖动物模型的建立及影响因素
选择HFS饲料喂养大鼠55只,根据体重、血糖、血脂水平随机分为STZ0组(10只)、STZ20组(15只)、STZ25组(15只)和STZ30组(15只),分别注射0、20、25、30mg/kg,bw STZ,观察血糖与STZ用量的关系,确定合适用量。另选择不同配方饲料喂养大鼠(10F10S、10F20S、20F10S、10F20Fr)按上述确定剂量注射STZ造模,监测5周内的血糖变化,采用多变量方差分析探讨HFS饲料配方与STZ对造模的贡献,并根据大鼠的损伤强度、血糖水平和死亡率评估高血糖和糖尿病的判断标准。
4高血糖动物模型的验证
选择正常对照大鼠(Ctrl.).STZ对照大鼠(STZ-C)、HFS对照大鼠(HFS-C)、模型大鼠(Mod.),各组均随机分为2个亚组,1组每天接受15mg/kg/bw吡咯列酮治疗,另1组给与蒸馏水。14d后根据体重增长和血糖应答反应判断治疗疗效。经过洗脱期后处死大鼠,分析脏器、脂肪垫重量、胰岛素、C肽、糖原等指标。采用因子分析方法对指标进行归类,并评估不同饲料喂养下动物模型的优势。
4动物模型复现性及应用性研究
按照以上建立的造模程序,选择2队试验人员分别在2个动物房复制实验,统计3次重复测量的再现性限。
另取100只大鼠分为3组：第一组进行抗性淀粉(RS)预防胰岛素抵抗实验,即在HFS饲料中加入20%RS,观察10周后血糖、胰岛素、血脂、糖原等指标与HFS对照组的差异；第二组在HFS诱导大鼠胰岛素抵抗成功后,采用20%、30%RS干预6周,观察血糖、胰岛素、血脂等指标的变化；第三组在HFS联合STZ诱导高血糖模型成功后,观察20%、30%RS对血糖、胰岛素、血脂等指标的影响。每组均设正常对照(Ctrl)和模型对照。
研究结果
1.正常大鼠血糖水平分布
正常大鼠475只的FBG和238只的PBG分布范围95%上限分别为6.22mmol/L,10.3mmol/L;对血糖影响因素的分析显示：1)血糖仪法测定血糖的敏感性较低,和葡萄糖氧化酶法仅在血糖水平为7～10mmol/L时结果接近；2)不同来源的血样血糖存在一定差异,腹主动脉血比外周静脉血高1.4倍；3)采样前大鼠禁食时间对血糖的影响为禁食11h血糖低于禁食5h；4)取血后血样在60min内进行分离血糖衰减8%,如超过60min衰减达50%以上。根据以上因素重新整理数据,确定以葡萄糖氧化酶法测定的大鼠正常血糖上限为FBG6.2mmol/L, PBG8.0mmol/L;考虑到血源等不确定因素的影响,统计确定扩大系数1.3倍,由此外推扩大的血糖上限分别为FBG8.0mmol/L, PBG1lmmol/L
2、高脂高糖饲料诱导胰岛素抵抗及其影响因素
经HFS喂养大鼠体重增长明显高于普通饲料组,到4周时出现PBG升高,8周时FBG升高,且PBG超过普通饲料组2个标准差；同时TC、TC升高。当饲料猪油含量为20%时血糖水平最高,多变量分析显示猪油是影响血糖、血脂最大的因素,蔗糖可影响高血糖发生进程,果糖(20%)和蔗糖间没有显著差异。
3高血糖动物模型的建立及影响因素
在HFS饲养基础上,经小剂量STZ造模后HFS大鼠体重迅速下降,3周后血糖趋向稳定。根据血糖的高低、死亡率,确定20～25mg/kg.bwSTZ(?)注射剂量为宜；例料间血糖的差别为20F1OS>10F10S>10F20S=10F20Fr。对STZ与HFS交互作用分析显示HFS对STZ造模效果起到放大作用,其作用时间点仅在STZ注射前,猪油越高血糖越高,蔗糖反之。
4高血糖动物模型的验证
经过吡咯列酮治疗后,HFS大鼠体重回升且餐后血糖升高幅度下降,其中以小剂量STZ(20mg/kg.bw)制造的模型和以20F10S喂养的动物效果最为明显。对生化指标的分析显示模型大鼠出现典型肾肿大、肝糖原升高、胰岛素分泌以及HDL-C下降等生理生化变化。对影响因素的归因分析显示,STZ主要影响心、肾等脏器及糖原；HFS主页影响胰岛素分泌、糖原储存和血脂。
5高血糖动物模型复现性和应用研究
3次重复性检验显示,在规定的实验条件下均可诱导出符合要求的IR模型和T2DM模型,重复性检验相对偏差<20%。在RS预防和干预高血糖的应用研究中,HFS使大鼠PBG升高、空腹胰岛素下降、肝糖原升高、肌糖原下降(与Ctrl相比P<0.05)；给与20%RS的大鼠PBG和肝糖原明显低于HFS组(P<0.05),且空腹胰岛素未与对照组有明显统计学差异。对于餐后血糖和胰岛素升高、且胰岛素敏感指数下降的胰岛素抵抗模型大鼠,给以6周30%RS干预的大鼠虽未见血糖降低,但餐后胰岛素异常分泌下降,胰岛素敏感指数升高(和模型组相比P<0.05)。而对糖脂代谢紊乱的DM模型大鼠,30%RS可使大鼠LDL-C下降、FBG有下降趋势。
结论
1.综合考虑血糖分析方法、血样来源和处理方法、禁食时间等因素的影响,并使用高血糖模型大鼠进行相互验证的结果,确定以葡萄糖氧化酶法测定的大鼠正常血糖上限为FBG6.2mmol/L,PBG8.0mmol/L;扩大上限为FBG8.0mmol/L,PBG11mmol/L,可作为判断高血糖模型成功的依据。
2脂肪、糖、STZ通过不同途径影响血糖,对造模贡献有所不同,其中脂肪是影响血糖、血脂、胰岛素分泌的最重要因素,小剂量STZ可在不影响胰岛素分泌的前提下使血糖迅速上升。
3通过综合对血糖、胰岛素、敏感器官和血脂等相关指标的的归类分析,证实体重增长、脂肪堆积、肝糖原是伴随高血糖最突出的体征和生化改变,以体重和PBG作为分组的指标,以PBG作为高血糖模型判断标准可信度较高。IR模型的判断标准为PBG>8.0mmol/L且超过对照组2个标准差；T2DM的标准为PBG11～30mmol/L。
4本研究证实了所建模型为具有胰岛素抵抗特征的高血糖动物模型,不同的饲料喂养可现示出不同的模型特点,高果糖摄入以增加TG和HDL-C为主,猪油主要影响脂肪堆积和胰岛素分泌；高蔗糖摄入可通过增加肌糖原储存而降低血糖。20mg/kg.bwSTZ因对机体损伤相对较轻、体征变化较轻,对胰岛素增敏剂的敏感性相对较强。
5对模型稳定性和复制性的研究证实所建模型的复现性较强,由于不同阶段的模型具备不同的特点,所以在评价中可能需采用不同的评价指标。由于HFS诱导高血糖是一个缓慢的过程,因此本模型可用于长期的预防性或干预性研究和评价。
本研究的创新之处：
1.首次全面探讨了大鼠正常血糖分布范围和上限,为确定动物高血糖、糖尿病血糖判断标准提供了理论依据。
2.首次探讨了脂肪、糖、STZ对高血糖动物模型的贡献和主要作用点,为解释模型和标化模型提供了依据。
3.首次探讨了模型的稳定性、复现性,对关键技术条件进行了摸索,为修改保健食品辅助降血糖功能评价动物模型的建议提供了参考。
Backgrouds
Diabetes 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.
Objective
1. 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 characteristics
4. To indenty the reproducibility and applicability of the animal models
5. To provide scientific basis for research and meet government's need.
Materials and methods
1. 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.
Results
1. 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.
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24 Niu HS, Liu IM, Cheng JT, et al. Hypoglycemic effect of syringin from Eleutherococcus senticosus in streptozotocin-induced diabetic rats[J].Planta Med.2008 74(2):109-13.
25李聪然,游雪甫,蒋建东.糖尿病动物模型及研究进展[J].中国比较医学杂志,2005,15(1)：59-63
26 WHO. Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia. Gevana, 2002
27 Reaven GM. Role of insulin resistance in human disease. Diabetes,1988;37:1595-1607
28 Knowler WC, Barrett-Connor E, Fowler SE, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin, N. Engl. J. Med.,2002; 346(6):393-403
29 Avramoglu RK, Qiu W, Adeli K:Mechanisms of metabolic dyslipidemia in insulin resistant states:deregulation of hepatic and intestinal lipoprotein secretion. Front Biosci 2003,8:d464-476
30 Astrup A, Finer N:Redifining type 2 diabetes:'diabesity'or'obesity dependent diabetes mellitus'? Obes Rev 2000,1:57-59
31 Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is sufficient to account for the inhibition of the insulin stimulated PKB pathway in C2C12 skeletal muscle cells pretracted with palmitate. J Biol Chem,1999,274(34):24202
32 Savage DB, Peterson KF, Shulman GI. Mechanisms of insulin resistance in humans and possible links with inflammation. Hypertension 2005;45:828-833
33 Lewis GF, Carpentier A, Adeli K, et al. Disordered Fat Storage and Mobilization in the Pathogenesis of Insulin Resistance and Type 2 Diabetes. Endocrine Reviews 2002,23:201-229
34 Bray GA, Lovejoy JC, Smith SR, et al. The Influence of Different Fats and Fatty Acids on Obesity, Insulin Resistance and Inflammation. J. Nutr.2002,132:2488-2491
35 Ebeling P, Koistinen HA, Koivisto VA. Hypothesis:Insulin-independent glucose transport regulates insulin sensitivity. FEBS Letters 1998,436:301-303
36郭啸华、刘志红,李恒等.实验性2型糖尿病大鼠模型的建立[J].肾脏病与透析肾移植杂志2000,9(4)：351-355
37 Basciano H, Federico L, Adeli K. Fructose, insulin resistance and metabolic dyslipidemia. Nutrition & Metabolisn,2005,2:5,1-14
38杨月欣主编.食物血糖生成指数.北京医科大学出版社,2002.
39 http://www.abc.net.au/rn/healthreport/stories/2007/1969924.htm
40王竹,杨月欣,韩军花等.抗性淀粉对饮食诱发葡萄糖耐量异常的预防[J].卫生研究2002
41陈名道.胰岛B细胞的“糖毒性”、“脂毒性”与“糖脂毒性”.中国内分泌代谢杂志,2009,25(2)：5-8
42 WHO. Laboratory Diagnosis and Monitoring of Diabetes Mellitus 2002
43 BelfiorelF,Iannello S. Insulin Resistance in Obesity:Metabolic Mechanisms and Measurement Methods. Molecular Genetics and Metabolism 1998; 65:121-128
44杜瑞琴,李宏亮,杨文英等.吡格列酮对胰岛α细胞胰岛素抵抗的影响[J].中华内分泌代谢杂志,2008,24(2)：29
45 Hallfrisch J. Metabolitic effects of dietary fructose. Faseb J 1990,4:2652-2660
46 Vozzo R, Baker B, Witter GA, et al. Glycemic, hormone, and appetite responses to monsaccharide ingestion in patients with type 2 diabetes. Metaolism 2002,51:949-957
47 Poitout V, Bobertson RP. Minireview:secondary b-cell failure in type 2 diabetes-a convergence of glucotoxicity and lipotoxicity. Endocrinology,2002;143:339-342
48 atsuda M, Defronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing:comparison with the euglycemic insulin clamp. Diabetes Care 1999;22:1462-1470
49苗明三主编.实验动物和动物实验技术.中国中医药出版社1997北京.P241
50 Bray GA, Lovejoy JC, Smith SR, et al. The Influence of Different Fats and Fatty Acids on Obesity, Insulin Resistance and Inflammation. J. Nutr.2002,132:2488-2491
51 Brown IL. Applications and uses of resistant starch. J AOAC Inter,2004; 87(3):727-732
52王竹,杨月欣,周瑞华等.抗性淀粉的代谢及对血糖的调节作用[J].卫生研究2002
53张文青,杨月欣等.抗性淀粉对糖尿病患者的干预研究[J].卫生研究2005
1. INZUCCHI S E. Oral Antihyperglycemic Therapy for Type 2 Diabetes[J]. JAMA,2002, 287:360-372.
2. OLSEN W A, KORSMO H. The intestinal brush border membrane in diabetes studies of sucrase-isomaltase metabolism in rats with streptozotocin diabetes[J]. J Clin Invest,1977, 60:181-188.
3. DYER J, WOOD I S, ALEJWALA P A, et al. Expression of monosaccharide transporters in
4. intestine of diabetic humans[J]. Am J Physiol Gastrointest Liver Physiol,2002, 282:G241-G248.
5. TAKENOSHITA1 M, YAMAJI R, INUI H, et al. Suppressive effect of insulin on the synthesis of sucrase-isomaltase complex in small intestinal epithelial cells, and abnormal increase in the complex under diabetic conditions[J]. Biochem J,1998,329:597-600.
6.王悦欣,张扬,吴疆,等.阿卡波糖对糖耐量受损者糖尿病和心血管疾病病的预防作用[J],中华内分泌代谢杂志,2004,20(4)：323-324.
7. VAN de LAAR FA, LUCASSEN P L B J, RUTTEN GEHM, et al.Alpha-glucosidase inhibitors for type 2 diabetes mellitus:A systematic review[J].中国循证医学杂志 2006,6:335-351.
8. PEREZ C, FERNANDEZ-AGULLO T, DE SOLIS A, et al. Effects of chronic acarbose treatment on adipocyte insulin responsiveness, serum levels of leptin and adiponectin and hypothalamic NPY expression in obese diabetic Wistar rats[J]. J Clin Exp Pharmacol Physiol, 2008,35:256-261.
9. KWON YI, APOSTOLIDIS E, KIM YC, et al. Health benefits of traditional corn, beans, and pumpkin:in vitro studies for hyperglycemia and hypertension management[J]. J Med Food, 2007,10:266-275.
10. McCue P, KWON YI, SHETTY K. Anti-diabetic and anti-hypertensive potential of sprouted and solid-state bioprocessed soybean[J]. Asia Pac J Clin Nutr,2005,14:145-152.
11. FUJITA H, YAMAGAMI T. Fermented soybean-derived Touchi-extract with anti-diabetic effect via alpha-glucosidase inhibitory action in a long-term administration study with KKAy mice[J]. Life Sci,2001,70:219-227.
12. ITOH T, KITA N, KUROKAWA Y, et al. Suppressive effect of a hot water extract of adzuki beans (vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats[J]. Biosci. Biotechnol. Biochem,2004,68:2421-2426.
13.张冉,刘泉,中竹芳,等.应用α-葡萄糖苷酶抑制剂高通量筛选模型筛选降血糖中药[J].中国药学杂志,2007,42：740-743.
14.张哲,王雯,尹鸿萍,等.螺旋藻中α-葡萄糖苷酶抑制剂的提取及动力学[J].生物加工过程,2008,6：39-43.
15.徐林峰,沈忠明,殷建伟.五味子中提取α-葡萄糖苷酶抑制剂的研究[J].中国生化药物杂志,2001,22：127-129.
16.刘志峰,李萍,李慎军,等.5种中药体外α-糖苷酶抑制作用的观察[J].山东中医杂志,2004,23：41-42.
17.罗培,莫止纪.优化山茱萸中α-葡萄糖苷酶抑制剂的提取工艺[J].华西药学杂志,2004,19：273-274.
18.陈海敏,严小军,林伟.α-葡萄糖苷酶抑制剂的构效关系[J].中国生物化学与分子生物学报,2003,19：780-784.
19. ZHANG J, TILLER C, SHEN J, et al. Antidiabetic properties of polysaccharide-and polyphenolic-enriched fractions from the brown seaweed Ascophyllum nodosum[J]. Can J Physiol Pharmacol,2007,85:1116-1123.
20. WIESENFELD P, BALDWIN J 3rd, SZEPESI B, et al. Effect of dietary carbohydrate and phenotype on sucrase, maltase, lactase, and alkaline phosphatase specific activity in SHR/N-cp rat[J]. Proc Soc Exp Biol Med,1993,202:338-344.
21. SHIBANO M, KAKUTANI K, TANIGUCHI M, et al. Antioxidant constituents in the dayflower (Commelina communis L.) and their alpha-glucosidase-inhibitory activity[J]. Nat Med (Tokyo),2008,62:349-353.
22. LI Y, HUANG T H, YAMAHARA J. Salacia root, a unique Ayurvedic medicine, meets multiple targets in diabetes and obesity[J]. Life Sci,2008,82:1045-1049.
23. ELENA LO P, HOLGER S, Nathalie F, Flavonoids for controlling starch digestion:structural requirements for inhibiting human a-Amylase[J]. J Med Chem,2008,51:3555-3561.
24. ISHIKAWA A, YAMASHITA H, HIEMORI M, et al. Characterization of inhibitors of postprandial hyperglycemia from the leaves of Nerium indicum[J]. J Nutr Sci Vitaminol (Tokyo),2007,53:166-173.
25. KONG W H, OH S H, AHN Y R, et al. Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models[J]. J Agric Food Chem,2008, 56:2613-2619.
26.耿鹏,朱元元,杨洋,等.蚕砂总生物碱中a-葡萄糖昔酶抑制剂成分的分析及其生物活性检测[J].中草药,2005,36(s)：159-162.
27. GENG P, YANG Y, GAO Z, et al. Combined effect of total alkaloids from Feculae Bombycis and natural flavonoids on diabetes[J]. J Pharm Pharmacol,2007,59:1145-1150.
28. SHINDE J, TALDONE T, BARLETTA M. et al. Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats[J]. Carbohydr Res,2008,343:1278-1281.
29.卢大胜,吕敬慈,雍克岚,等.用固定化酶筛选模型从天然产物中筛选α-葡萄糖苷酶抑制剂[J].中国新药杂志,2005,14：1411-1414.
30. KIYOSHI M, KAYOKO T, KIYOFUMI T, et al. A novel method for the assay of a-glucosidase inhibitory activity using a multi-channel oxygen sensor[J]. Analytical Sci, 2002,18:1315-1319.
31. CHEN H, YAN X, LIN W, et al. A New Method for Screening a-Glucosidase Inhibitors and Application to Marine Microorganisms[J]. Pharmaceutical Biology,2004,42:416^421.
32. ADACHI T, MORI C, SAKURAI K, et al. Morphological changes and increased sucrase and isomaltase activity in small intestines of insulin-deficient and type 2 diabetic rats[J]. Endocr J, 2003,50:271-279.
2 Armando Arredondo, Alexis ZU'N-IGA. Economic Consequences of Epidemiological Changes in Diabetes in Middle-Income Countries, DIABETES CARE, VOLUME 27, NUMBER 1, JANUARY 2004,104-109
3 Irmgard Schmitt-Koopmann, Matthias Schwenkglenks, Giatgen A. Spinas, Direct medical costs of type 2 diabetes and its complications in Switzerland. E J P H 2004; 14:3-9
4朱熹星主编.现代糖尿病学.上海医科大学山版社.2000,上海.P96
5 Lebovitz HE, Banerji MA. Treatment of insulin resistance in diabetes mellitus. Euro J Pharm, 2004;490:135-146
6 YkiJarvinenH. PathogenesisofnoninsulindependentdiabetesmellitusJ].Lancet.1994,343:9195.
7 Keller KB, Lemberg L. Obesity and the metabolic syndrome. Am J Crit Care,2003,12:167-170.
8 Tuomilehto J, Lindstrom J, Eriksson JG, et al. Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance, N. Engl. J. Med.,2001; 344: 1343-1350
9 American Diabetes Association.Prevention or delay of type 2 diabetes. Diabetes Care,2004;27:S47-S54
10 Clare M. Hasler, The Changing Face of Functional Foods. J Am Coll Nutri,2000, 19(5):499S-506S
11 Alessandra C. Maurizio C, Carlo. Functional foods in European Union:An overview for the sector's main issue.8th Joint Conference on Food, Agriculture and Environment, Wisconsin, 2002.
12 KWON YI, APOSTOLIDIS E, KIM YC, et al. Health benefits of traditional corn, beans, and pumpkin:in vitro studies for hyperglycemia and hypertension management[J]. J Med Food,2007, 10:266-275.
13 McCue P, KWON YI, SHETTY K. Anti-diabetic and anti-hypertensive potential of sprouted and solid-state bioprocessed soybean[J]. Asia Pac J Clin Nutr,2005,14:145-152.
14 FUJITA H, YAMAGAMI T. Fermented soybean-derived Touchi-extract with anti-diabetic effect via alpha-glucosidase inhibitory action in a long-term administration study with KKAy mice[J]. Life Sci,2001,70:219-227.
15 ITOH T, KITA N, KUROKAWA Y, et al. Suppressive effect of a hot water extract of adzuki beans (vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats[J]. Biosci. Biotechnol. Biochem,2004,68:2421-2426.
16张冉,刘泉,中竹芳,等.应用α-葡萄糖苷酶抑制剂高通量筛选模型筛选降血糖中药[J].中国药学杂志,2007,42：740-743.
17张哲,王雯,尹鸿萍,等.螺旋藻中α-葡萄糖甘酶抑制剂的提取及动力学[J.生物加工过程,2008,6：39-43.
18徐林峰,沈忠明,五味子中提取α-葡萄糖甘酶抑制剂的研究[J].中国生化药物杂志,2001,22：127-129.
19刘志峰,李萍,李慎军,等.5种中药体外α-糖苷酶抑制作用的观察[J].山东中医杂志2004,23：41-42.
20罗培,莫正纪.优化山茱萸中α-葡萄糖苷酶抑制剂的提取工艺[J].华西药学杂志,2004, 1 9：273-274.
21王竹,杨月欣.锌和胰岛素、糖尿病关系研究新进展.国外医学卫生学分册2000,
22 Sirintorn Y, Sirichai A, Cheng Yu Y, et al. Slow Acting Protein Extract from Fruit Pulp of Momordica charantia with Insulin Secretagogue and Insulinomimetic activities. Biol. Pharm. Bull. 2006,29(6):1126-1131
23 Patrice F, Paola C, Marie J R, et al. Lipid Peroxidation and Trace Element Status in Diabetic Ketotic Patients:Influence of Insulin Therapy. CLIN CHEM,1993,39(5):789-793.
24 Niu HS, Liu IM, Cheng JT, et al. Hypoglycemic effect of syringin from Eleutherococcus senticosus in streptozotocin-induced diabetic rats[J].Planta Med.2008 74(2):109-13.
25李聪然,游雪甫,蒋建东.糖尿病动物模型及研究进展[J].中国比较医学杂志,2005,15(1)：59-63
26 WHO. Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia. Gevana, 2002
27 Reaven GM. Role of insulin resistance in human disease. Diabetes,1988;37:1595-1607
28 Knowler WC, Barrett-Connor E, Fowler SE, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin, N. Engl. J. Med.,2002; 346(6):393-403
29 Avramoglu RK, Qiu W, Adeli K:Mechanisms of metabolic dyslipidemia in insulin resistant states:deregulation of hepatic and intestinal lipoprotein secretion. Front Biosci 2003,8:d464-476
30 Astrup A, Finer N:Redifining type 2 diabetes:'diabesity'or'obesity dependent diabetes mellitus'? Obes Rev 2000,1:57-59
31 Schmitz-Peiffer C, Craig DL, Biden TJ. Ceramide generation is sufficient to account for the inhibition of the insulin stimulated PKB pathway in C2C12 skeletal muscle cells pretracted with palmitate. J Biol Chem,1999,274(34):24202
32 Savage DB, Peterson KF, Shulman GI. Mechanisms of insulin resistance in humans and possible links with inflammation. Hypertension 2005;45:828-833
33 Lewis GF, Carpentier A, Adeli K, et al. Disordered Fat Storage and Mobilization in the Pathogenesis of Insulin Resistance and Type 2 Diabetes. Endocrine Reviews 2002,23:201-229
34 Bray GA, Lovejoy JC, Smith SR, et al. The Influence of Different Fats and Fatty Acids on Obesity, Insulin Resistance and Inflammation. J. Nutr.2002,132:2488-2491
35 Ebeling P, Koistinen HA, Koivisto VA. Hypothesis:Insulin-independent glucose transport regulates insulin sensitivity. FEBS Letters 1998,436:301-303
36郭啸华、刘志红,李恒等.实验性2型糖尿病大鼠模型的建立[J].肾脏病与透析肾移植杂志2000,9(4)：351-355
37 Basciano H, Federico L, Adeli K. Fructose, insulin resistance and metabolic dyslipidemia. Nutrition & Metabolisn,2005,2:5,1-14
38杨月欣主编.食物血糖生成指数.北京医科大学出版社,2002.
39 http://www.abc.net.au/rn/healthreport/stories/2007/1969924.htm
40王竹,杨月欣,韩军花等.抗性淀粉对饮食诱发葡萄糖耐量异常的预防[J].卫生研究2002
41陈名道.胰岛B细胞的“糖毒性”、“脂毒性”与“糖脂毒性”.中国内分泌代谢杂志,2009,25(2)：5-8
42 WHO. Laboratory Diagnosis and Monitoring of Diabetes Mellitus 2002
43 BelfiorelF,Iannello S. Insulin Resistance in Obesity:Metabolic Mechanisms and Measurement Methods. Molecular Genetics and Metabolism 1998; 65:121-128
44杜瑞琴,李宏亮,杨文英等.吡格列酮对胰岛α细胞胰岛素抵抗的影响[J].中华内分泌代谢杂志,2008,24(2)：29
45 Hallfrisch J. Metabolitic effects of dietary fructose. Faseb J 1990,4:2652-2660
46 Vozzo R, Baker B, Witter GA, et al. Glycemic, hormone, and appetite responses to monsaccharide ingestion in patients with type 2 diabetes. Metaolism 2002,51:949-957
47 Poitout V, Bobertson RP. Minireview:secondary b-cell failure in type 2 diabetes-a convergence of glucotoxicity and lipotoxicity. Endocrinology,2002;143:339-342
48 atsuda M, Defronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing:comparison with the euglycemic insulin clamp. Diabetes Care 1999;22:1462-1470
49苗明三主编.实验动物和动物实验技术.中国中医药出版社1997北京.P241
50 Bray GA, Lovejoy JC, Smith SR, et al. The Influence of Different Fats and Fatty Acids on Obesity, Insulin Resistance and Inflammation. J. Nutr.2002,132:2488-2491
51 Brown IL. Applications and uses of resistant starch. J AOAC Inter,2004; 87(3):727-732
52王竹,杨月欣,周瑞华等.抗性淀粉的代谢及对血糖的调节作用[J].卫生研究2002
53张文青,杨月欣等.抗性淀粉对糖尿病患者的干预研究[J].卫生研究2005
1. INZUCCHI S E. Oral Antihyperglycemic Therapy for Type 2 Diabetes[J]. JAMA,2002, 287:360-372.
2. OLSEN W A, KORSMO H. The intestinal brush border membrane in diabetes studies of sucrase-isomaltase metabolism in rats with streptozotocin diabetes[J]. J Clin Invest,1977, 60:181-188.
3. DYER J, WOOD I S, ALEJWALA P A, et al. Expression of monosaccharide transporters in
4. intestine of diabetic humans[J]. Am J Physiol Gastrointest Liver Physiol,2002, 282:G241-G248.
5. TAKENOSHITA1 M, YAMAJI R, INUI H, et al. Suppressive effect of insulin on the synthesis of sucrase-isomaltase complex in small intestinal epithelial cells, and abnormal increase in the complex under diabetic conditions[J]. Biochem J,1998,329:597-600.
6.王悦欣,张扬,吴疆,等.阿卡波糖对糖耐量受损者糖尿病和心血管疾病病的预防作用[J],中华内分泌代谢杂志,2004,20(4)：323-324.
7. VAN de LAAR FA, LUCASSEN P L B J, RUTTEN GEHM, et al.Alpha-glucosidase inhibitors for type 2 diabetes mellitus:A systematic review[J].中国循证医学杂志 2006,6:335-351.
8. PEREZ C, FERNANDEZ-AGULLO T, DE SOLIS A, et al. Effects of chronic acarbose treatment on adipocyte insulin responsiveness, serum levels of leptin and adiponectin and hypothalamic NPY expression in obese diabetic Wistar rats[J]. J Clin Exp Pharmacol Physiol, 2008,35:256-261.
9. KWON YI, APOSTOLIDIS E, KIM YC, et al. Health benefits of traditional corn, beans, and pumpkin:in vitro studies for hyperglycemia and hypertension management[J]. J Med Food, 2007,10:266-275.
10. McCue P, KWON YI, SHETTY K. Anti-diabetic and anti-hypertensive potential of sprouted and solid-state bioprocessed soybean[J]. Asia Pac J Clin Nutr,2005,14:145-152.
11. FUJITA H, YAMAGAMI T. Fermented soybean-derived Touchi-extract with anti-diabetic effect via alpha-glucosidase inhibitory action in a long-term administration study with KKAy mice[J]. Life Sci,2001,70:219-227.
12. ITOH T, KITA N, KUROKAWA Y, et al. Suppressive effect of a hot water extract of adzuki beans (vigna angularis) on hyperglycemia after sucrose loading in mice and diabetic rats[J]. Biosci. Biotechnol. Biochem,2004,68:2421-2426.
13.张冉,刘泉,中竹芳,等.应用α-葡萄糖苷酶抑制剂高通量筛选模型筛选降血糖中药[J].中国药学杂志,2007,42：740-743.
14.张哲,王雯,尹鸿萍,等.螺旋藻中α-葡萄糖苷酶抑制剂的提取及动力学[J].生物加工过程,2008,6：39-43.
15.徐林峰,沈忠明,殷建伟.五味子中提取α-葡萄糖苷酶抑制剂的研究[J].中国生化药物杂志,2001,22：127-129.
16.刘志峰,李萍,李慎军,等.5种中药体外α-糖苷酶抑制作用的观察[J].山东中医杂志,2004,23：41-42.
17.罗培,莫止纪.优化山茱萸中α-葡萄糖苷酶抑制剂的提取工艺[J].华西药学杂志,2004,19：273-274.
18.陈海敏,严小军,林伟.α-葡萄糖苷酶抑制剂的构效关系[J].中国生物化学与分子生物学报,2003,19：780-784.
19. ZHANG J, TILLER C, SHEN J, et al. Antidiabetic properties of polysaccharide-and polyphenolic-enriched fractions from the brown seaweed Ascophyllum nodosum[J]. Can J Physiol Pharmacol,2007,85:1116-1123.
20. WIESENFELD P, BALDWIN J 3rd, SZEPESI B, et al. Effect of dietary carbohydrate and phenotype on sucrase, maltase, lactase, and alkaline phosphatase specific activity in SHR/N-cp rat[J]. Proc Soc Exp Biol Med,1993,202:338-344.
21. SHIBANO M, KAKUTANI K, TANIGUCHI M, et al. Antioxidant constituents in the dayflower (Commelina communis L.) and their alpha-glucosidase-inhibitory activity[J]. Nat Med (Tokyo),2008,62:349-353.
22. LI Y, HUANG T H, YAMAHARA J. Salacia root, a unique Ayurvedic medicine, meets multiple targets in diabetes and obesity[J]. Life Sci,2008,82:1045-1049.
23. ELENA LO P, HOLGER S, Nathalie F, Flavonoids for controlling starch digestion:structural requirements for inhibiting human a-Amylase[J]. J Med Chem,2008,51:3555-3561.
24. ISHIKAWA A, YAMASHITA H, HIEMORI M, et al. Characterization of inhibitors of postprandial hyperglycemia from the leaves of Nerium indicum[J]. J Nutr Sci Vitaminol (Tokyo),2007,53:166-173.
25. KONG W H, OH S H, AHN Y R, et al. Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models[J]. J Agric Food Chem,2008, 56:2613-2619.
26.耿鹏,朱元元,杨洋,等.蚕砂总生物碱中a-葡萄糖昔酶抑制剂成分的分析及其生物活性检测[J].中草药,2005,36(s)：159-162.
27. GENG P, YANG Y, GAO Z, et al. Combined effect of total alkaloids from Feculae Bombycis and natural flavonoids on diabetes[J]. J Pharm Pharmacol,2007,59:1145-1150.
28. SHINDE J, TALDONE T, BARLETTA M. et al. Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats[J]. Carbohydr Res,2008,343:1278-1281.
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