糖尿病小型猪三磷酸腺苷结合盒转运体A1表达的变化

用贵州小香猪建立2型糖尿病动物模型,探讨糖尿病小型猪三磷酸腺苷结合盒转运体A1(ABCA1)表达的变化.采用高脂高蔗糖饲料喂养贵州小香猪,建立2型糖尿病动物模型.血浆总胆固醇、甘油三酯、高密度脂蛋白胆固醇和葡萄糖的浓度均用氧化酶法测定,血浆游离脂肪酸(FFA)用比色法测定, 采用逆转录-聚合酶链反应、蛋白质印迹和免疫组织化学分别检测ABCA1mRNA和蛋白质的表达.喂养6个月后,实验组与正常对照组比较,空腹血糖值明显升高;空腹胰岛素水平在头3个月轻度升高,在第6个月末其水平降低; 血清总胆固醇、甘油三酯和游离脂肪酸水平升高;肝组织、冠状动脉、肾组织ABCA1表达上调,同时观测到糖尿病小型猪肝组织LXRα表达上调.结果提示高脂高蔗糖饲料可引起小型猪脂质和糖代谢紊乱, 并导致肝组织、冠状动脉和肾组织ABCA1表达上调以及肝组织LXRα表达上调.     

高脂高糖饲料联合低剂量链脲佐菌素(STZ)诱导广西巴马小型猪2型糖尿病动物模型的建立

本研究的目的是应用高脂高糖饲料联合低剂量STZ建立理想的广西巴马小型猪2型糖尿病模型。挑取8头雌性巴马小型猪体重为(21.64±0.62)kg分成对照组和实验组2组,每组4头。对照组标准饲喂9个月(control,CTR);实验组高脂高糖饲料饲喂6个月后按照60 mg/kg给予注射链脲佐菌素(STZ),接着高脂高糖饲喂至9个月(HFHC+STZ,HS);每个月检测空腹血糖、空腹胰岛素、甘油三酯和胆固醇的变化。静脉注射葡萄糖耐实验检测葡萄糖利用率。高胰岛素血症与葡萄糖耐受损判定为胰岛素抵抗。结果显示:与对照组相比,实验组猪均发生二型糖尿病症状,包括胰岛素抵抗和葡萄糖耐受损。实验结束时,HS组与CTR组空腹血糖分别为(13.83±0.74 vs 4.28±0.09)mmol/L;空腹胰岛素分别为(40.67±0.80 vs 38.94±1.75)m U/L。本研究通过高脂高糖饲料联合低剂量STZ成功建立广西巴马小型猪2型糖尿病模型,为后续糖尿病的临床研究和药物治疗奠定了基础。     

高脂饮食对西藏小型猪胰岛素抵抗及肝胰岛素受体底物1、2表达的影响

目的探讨高脂饮食对西藏小型猪胰岛素抵抗(IR)及肝胰岛素受体底物(IRS)1、2表达的影响。方法将10只西藏小型猪随机分为正常对照组(Ctr)5只饲喂普通饲料、IR模型(IR model)组5只饲喂高脂饲料,连续造模12周。造模12周后,称重并测量体长,计算体质量指数(BMI),空腹取前腔静脉血测定总胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)、甘油三酯(TG)、游离脂肪酸(FFA)、空腹血糖(FBG)和胰岛素(insulin),计算胰岛素抵抗指数(homeostasis model assessment-estimated insulin resistance,HOMA-IR);同时进行糖耐量试验,并计算糖耐量曲线下面积(AUC);取肝组织检测IRS-1和IRS-2基因和蛋白表达,并行油红O、PAS和HE染色,分别观察肝脂质沉积、糖原及组织病理变化。结果与正常对照组比,IR模型组体重、BMI指数、TC、LDL-C、HDL-C、FFA、FBG、insulin和HOMA-IR指数均显著升高(P0.05,P0.01);糖耐量试验显示血糖和胰岛素水平曲线下降延缓,而AUC_(血糖)和AUC_(胰岛素)均明显升高(P0.05,P0.01);肝组织中出现脂质沉积、糖原增加和局部肝细胞浊肿、部分胞核消失或被挤向一端,偶见淋巴细胞浸润;同时肝组织中IRS-1和IRS-2 mRNA和蛋白表达均显著降低(P0.05,P0.01)。结论高脂饮食可引起西藏小型猪胰岛素抵抗,肝组织IRS-1和IRS-2表达降低是高脂饮食影响西藏小型猪胰岛素敏感性的分子机制之一。     

不同时间脂肪乳输注对大鼠葡萄糖输注率的影响

目的观察不同时间脂肪乳输注使血浆游离脂肪酸（FFA）升高对大鼠葡萄糖输注率（GIR）的影响。方法分别给大鼠输注脂肪乳2、5、7和48 h,行高胰岛素-正血糖钳夹试验评价葡萄糖输注率,测定血浆葡萄糖、FFA。结果与生理盐水输注组比较,2 h脂肪乳输注使血浆FFA升高了2倍（P〈0.01）,GIR下降27%（P〈0.05）,脂肪乳输注5 h GIR降低了52%（P〈0.01）,7 h GIR降低56%（P〈0.01）,输注48 h脂肪乳GIR降低58%（P〈0.01）,5 h组7、h组及48 h脂肪乳输注组间GIR差异没有统计学意义。结论大鼠输注脂肪乳使FFA浓度达到基础值的3倍左右,可复制出脂毒性胰岛素抵抗的模型,而且胰岛素抵抗达到一定程度后保持恒定。     

动脉粥样硬化小型猪三磷酸腺苷结合盒转运体Al表达的变化

用贵州小香猪建立动脉粥样硬化动物模型,探讨动脉粥样硬化小型猪三磷酸腺苷结合盒转运体Al(ABCAl)表达的变化．采用血管内膜损伤法加高脂高胆固醇饲料喂养贵州小香猪,建立动脉粥样硬化动物模型．血浆总胆固醇、甘油三酯和高密度脂蛋白胆固醇的浓度均用氧化酶法测定,采用逆转录聚合酶链反应检测ABCAlmRNA水平,蛋白质印迹和免疫组织化学检测ABCAl蛋白质的表达．喂养12个月后,实验组与正常对照组比较,空腹血浆总胆固醇、甘油三酯和高密度脂蛋白胆固醇水平升高;实验组小型猪主动脉、髂动脉、颈总动脉和冠状动脉可见动脉粥样硬化斑块和脂质条纹;实验组小型猪肝组织、主动脉、小肠组织ABCAl表达上调．结果提示,采用血管内膜损伤法加高脂高胆固醇饲料喂养小型猪可建立动脉粥样硬化动物模型．动脉粥样硬化小型猪肝组织、主动脉和小肠组织ABCAl表达上调．     

燕麦纤维改善高脂饮食诱导的肥胖小鼠胰岛素抵抗的机制研究

目的：研究燕麦纤维对高脂饮食诱导小鼠胰岛素抵抗的影响。方法：采用8w龄C57BL/6J雄性小鼠高脂喂养16w,同时预防性给药,监测血液生化指标,进行糖耐量实验,ELISA法测胰岛素并计算HOMR-IR指数,解剖分离小鼠的内脏脂肪组织并称重,以及取部分组织做HE染色进行形态学观察,研究燕麦纤维对高脂诱导小鼠肥胖和胰岛素抵抗的影响。结果：与模型组比较,阳性药小檗碱组与燕麦纤维组的血糖（GLU）、甘油三酯（TG）、游离脂肪酸（FFA）、总胆固醇（TC）均能显著降低;燕麦纤维组低密度脂蛋白胆固醇（LDL-C）也显著降低,血浆胰岛素水平极显著降低,能增强胰岛素敏感性,改善胰岛素抵抗;血浆中炎症因子TNF-α、IL-6、L-1β及MCP-1显著降低,分别降低了27%、81%、31%、50%。小鼠体质量和内脏脂肪显著减少。脂肪细胞面积减小。结论：燕麦纤维通过减少小鼠内脏脂肪,减少FFA和炎症因子的分泌,改善由高脂饮食诱导的胰岛素抵抗,增加胰岛素敏感性。     

慢性高剂量胰岛素刺激猪脂肪细胞脂肪分解

为研究慢性高剂量胰岛素对猪脂肪细胞脂肪分解的影响及其分子机制, 分化的猪脂肪细胞在PKA(Protein kinase A)或ERK(Extracellular signal-related kinase)抑制剂预处理或不处理的情况下, 再用不同浓度的胰岛素(0、200、400、800、1600 nmol/L)处理不同时间(24、48、72、96 h), 通过测定甘油释放量检测脂肪细胞的脂解率; 采用RT-PCR和Western blotting检测perilipin A和PPARg2的mRNA和蛋白表达。结果显示, 慢性高剂量胰岛素以剂量和时间依赖性的方式刺激猪脂肪细胞的脂肪分解, 并削弱脂肪细胞对异丙肾上腺素刺激的脂解应答; 同时显著下调perilipin A和PPARg2的mRNA及蛋白表达; 另外, PKA和ERK抑制剂均显著抑制胰岛素刺激的脂肪分解, 但仅ERK抑制剂显著逆转perilipin A基因表达的下调。由此推测, 慢性高剂量胰岛素通过ERK通路抑制perilipin A的表达, 进而刺激猪脂肪细胞的脂肪分解。     

肥胖患者载脂蛋白M水平及其与炎症因子的关系研究

目的：观察肥胖患者载脂蛋白M水平并探讨其与炎症因子的关系。方法：58例体重正常者和36例肥胖患者常规测量体重、身高,计算体重指数,抽取空腹静脉血检测血脂、血浆载脂蛋白M（apoM）、白细胞介素-6（IL-6）、C反应蛋白（CRP）、肿瘤坏死因子α（TNF—α）。结果：肥胖患者血浆apoM、高密度脂蛋白胆固醇（HDL-C）降低（P〈0．05）,IL-6、TNF—α、CRP水平升高（P〈0．05）,肥胖患者血浆αpoM与HDL-C正相关,血浆αpoM与IL-6、TNF—α、CRP水平负相关。结论：肥胖患者血浆apoM显著降低,αpoM水平与CRP、TNF-α、IL-6水平密切相关,apoM可能受到这些炎症因子的调控。     

不同剂量链尿佐菌素诱导2型糖尿病大鼠模型的研究

目的:探讨不同剂量链脲佐菌素(Streptozotocin,STZ)联合高糖高脂饮食对2型糖尿病大鼠模型建立的影响。方法:90只8周龄SD雄性大鼠随机平均分为六组:普通饲料喂养+缓冲液组、高糖高脂饲料喂养+缓冲液(H.E组)、高糖高脂饲料喂养+35mg/kg链尿佐菌素组(H.E+35 mg/kg STZ组)、高糖高脂饲料喂养+45 mg/kg链尿佐菌素组(H.E+45 mg/kg STZ组)、高糖高脂饲料喂养+55 mg/kg链尿佐菌素组(H.E+55 mg/kg STZ组)及高糖高脂饲料喂养+65 mg/kg链尿佐菌素组(H.E+65 mg/kg STZ组),高糖高脂饲料喂养4周后诱导胰岛素抵抗,继之腹腔注射STZ,建立2型糖尿病大鼠模型。检测体重、胰岛素、空腹血糖、血脂、胰岛素敏感指数(ISI)。结果:与常规饮食组相比,高糖高脂饮食各组大鼠出现空腹血浆胰岛素(FINS)、空腹血糖(FBG)、血清甘油三脂(TG)、总胆固醇(TC)、游离脂肪酸(FFA)显著升高(P0.01),ISI显著下降(P0.01)。不同剂量STZ注射,H.E+45 mg/kg STZ组成模率最高且无自愈现象。结论:通过STZ腹腔注射联合高糖高脂饮食可成功复制出实验性2型糖尿病动物模型,45 mg/kg为STZ理想注射剂量。     

槟榔碱对2型糖尿病大鼠肝脏胰岛素抵抗的作用

目的：探讨槟榔碱对2型糖尿病大鼠肝脏胰岛素抵抗的影响及其机制。方法：采用高果糖饲料饲养Wistar大鼠12周制备2型糖尿病大鼠模型,实验动物随机分为5组（n=8）：对照组、模型组、模型＋不同浓度的槟榔碱（0,0．5,1,5mg／kg）组。4周后通过检测血糖、血脂、胰岛素、RT-PCR检测肝脏组成型雄甾烷受体（CAR）、孕甾烷x受体（PXR）、糖代谢相关基因：葡萄糖-6-磷酸酶（G6Pase）、磷酸烯醇式丙酮酸羧激酶（PEPCK）和炎症相关因子：白细胞介素-6（IL-6）、肿瘤坏死因子α（TNF-α）mRNA表达,Western blot检测大鼠肝内p-AKT和葡萄糖转运体4（GLUT4）蛋白表达。结果：1,5mg／kg槟榔碱显著降低糖尿病大鼠体重、空腹血糖、空腹胰岛素、血脂和糖代谢相关基因及炎症相关因子mRNA水平,提高CAR、PXR mRNA水平及p-AKT、GLUT4蛋白水平。结论：槟榔碱可能通过提高CAR和PXR的表达,导致肝脏糖代谢关键酶PEPCK、G6Pase基因表达或者炎性因子肿瘤坏死因子-α（TNF-α）、白介素-6（n-6）表达降低,改善2型糖尿病大鼠肝脏胰岛素抵抗。     

A Lipoprotein Lipase–Promoting Agent,NO-1886, Improves Glucose and Lipid Metabolism in High Fat,High Sucrose–Fed New Zealand White Rabbits

The synthetic compound NO-1886 is a lipoprotein lipase activator that lowers plasma triglycerides and elevates high-density lipoprotein cholesterol (HDL-C). Recently, the authors found that NO-1886 also had an action of reducing plasma glucose in high-fat/high-sucrose diet–induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on insulin resistance and β-cell function in rabbits. Our results showed that high-fat/high-sucrose feeding increased plasma triglyceride, free fatty acid (FFA), and glucose levels and decreased HDL-C level. This diet also induced insulin resistance and impairment of acute insulin response to glucose loading. Supplementing 1% NO-1886 into the high-fat/high-sucrose diet resulted in decreased plasma triglyceride, FFA, and glucose levels and increased HDL-C level. The authors also found a clear increased glucose clearance and a protected acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 suppresses the elevation of blood glucose in rabbits induced by feeding a high-fat/high-sucrose diet, probably through controlling lipid metabolism and improving insulin resistance.     

NO-1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs

It is widely believed that high density lipoprotein-cholesterol (HDL-C) functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport (RCT), a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP binding cassette transporter called ABCA1 mediates the first step of RCT. NO-1886 has been proven to be highly effective at increasing HDL-C and reducing atherosclerosis. However, the mechanism of atherosclerosis inhibition for NO-1886 is not fully understood. In this study, the effects of NO-1886 on ABCA1 were investigated in high-fat/high-sucrose/high-cholesterol-fed Chinese Bama minipigs. Administration of NO-1886 (0.1 g/kg body weight/day) in the diet for 5 months significantly reduced atherosclerosis lesions and significantly increased plasma HDL-C and apolipoprotein A-I levels. The mRNA and protein levels of ABCA1 in the liver, retroperitoneal adipose tissue, and aorta were increased by NO-1886 as well. Multivariate linear regression analysis showed that the levels of LPL in plasma and the levels of ABCA1 in aorta were independently associated with the atherosclerotic lesion area. In addition, NO-1886 upregulated liver X receptor alpha and affected the expression of scavenger receptor class B type I in the liver. These results demonstrate that the mechanism of atherosclerosis inhibition for NO-1886 is associated with its effect on ABCA1.     

Lipoprotein lipase activator ameliorates the severity of dietary steatohepatitis

Dietary model of steatohepatitis was established by feeding mice a methionine choline deficient (MCD) diet. Mice on MCD or control diet for 3 weeks were treated with or without NO-1886, a newly synthetic lipoprotein lipase (LPL) activator. In a separate experiment, NO-1886 was given after pre-treatment with 3 weeks of MCD diet. NO-1886 significantly reduced MCD-induced inflammation by repressing levels of hepatic lipid peroxides and pro-inflammatory tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2). In addition, NO-1886 dampened hepatic steatosis via accelerating fatty acid oxidation caused by enhanced expression of PPARalpha, cytochrome P450-10 (Cyp4a10), and Acyl-CoA oxidase (ACO). It failed to regulate genes of fatty acid uptake and synthesis pathways. In conclusion, NO-1886 ameliorated and induced regression of experimental steatohepatitis via increasing endogenous LPL activation resulting in suppression on pro-inflammatory factors and reduction of hepatic fatty acids. These findings indicate that NO-1886 is a potential therapeutic agent for steatohepatitis.     

Low-dose GH supplementation reduces the TLR2 and TNF-alpha expressions in visceral fat

The increased population of TLR2/TNF-α co-expressing adipocytes is associated with the development of insulin resistance. We have herein shown the significance of low-dose growth hormone (GH) supplementation for the regulation of TLR2 and TNF-α expressions in visceral fat using different kinds of mouse models fed with a high-fat diet. Low-dose GH supplementation reduced the increased population of TLR2/TNF-α co-expressing adipocytes in high-fat fed mice. The neutralization of IGF-1 abolished the effect of GH supplementation on the TLR2 expression using GH-overexpressing mice. IGF-1, but not GH, inhibited the FFA-induced TLR2 and TNF-α expression in 3T3-L1 cells. Finally, low-dose GH supplementation reduced the TLR2 expression without an obvious change in the visceral fat volume in ob/ob mice. These results indicate that low-dose GH supplementation possibly inhibits the high-fat induced change of the adipocytes to TLR2/TNF-α co-expressing cells through the action of IGF-1.     

Initiation of hyperinsulinemia and hyperleptinemia is diet dependent in C57BL/6 mice.

C57BL/6 female mice were fed high fat diets containing different types of carbohydrate (sucrose or corn starch) and contents of cholesterol (0.03 % or 1 %) to identify early metabolic changes leading to increases in leptin levels and eventual insulin resistance. Under identical dietary fat conditions, type of carbohydrate and cholesterol content contributed to the timing of leptin increases. Mice fed a high-fat, high-sucrose diet showed early (4 weeks) and robust increases in circulating insulin and leptin levels (2-fold and 5-fold, respectively). In contrast, mice fed this diet with added cholesterol or with sucrose substituted by corn starch led to marked delays (8-10 weeks) in the elevations of insulin and leptin, although body weight gains were nearly identical among test diet groups. Thus, sucrose in combination with saturated fat played a specific role in initiating early metabolic changes associated with elevated leptin and insulin levels. Because leptin levels were most reflective of changes in insulin, our data support a role for insulin in determining plasma leptin levels in mice.     

Running Exercise Improves Metabolic Abnormalities and Fat Accumulation in Sucrose-Induced Insulin-Resistant Rats

NARA, MAKOTO, MASAKI TAKAHASHI, TSUGIYASU KANDA, YOUNOSUKE SHIMOMURA, ISAO KOBAYASHI. Running exercise improves metabolic abnormalities and fat accumulation in sucrose-induced insulin-resistant rats. Insulin resistance and hyperinsulinemia are observed in rats fed a high sucrose diet. Insulin resistance is thought to be related to abnormal fat distribution. We previously reported the metabolic characteristics and the fat distribution in rats with sucrose-induced insulin resistance. This study was designed to examine the effects of exercise in these rats. The rats were divided into three groups: those receiving a starch-based diet (control), those receiving a high-sucrose diet (sucrose fed), and those receiving a high-sucrose diet and wheel-running exercise (exercised). Animals were killed after 4 weeks or 12 weeks. After 4 weeks, the three groups did not differ with respect to gain in adipose tissues. The portal vein (PV) insulin concentration was significantly increased in the sucrose-fed and the exercised rats compared with the control rats. The inferior vena cava (IVC) glucose concentration and the PV free fatty acid (FFA) were significantly lower in the exercised rats than in the sucrose-fed rats. After 12 weeks, the exercised rats had significantly lower mesenteric fat (MS) and subcutaneous fat (SC) and a lower MS:SC ratio than the sucrose-fed rats. The glucose levels in IVC, PV, and FFA in PV were significantly reduced in the exercised rats as compared with the sucrose-fed rats. These findings suggest that long-term exercise improves insulin resistance by reducing the accumulation of MS as well as SC. It is also suggested that short-term exercise improves glucose metabolism without change of fat accumulation.     

The adipose triglyceride lipase,adiponectin and visfatin are downregulated by tumor necrosis factor-α (TNF-α) in vivo

Inflammatory cytokines have been linked to obesity-related insulin resistance. To investigate the effect of TNF-α, an inflammatory cytokine, on insulin action, C57BL/6J mice were treated with TNF-α for 7 days after which we examined the in vivo effects of TNF-α on glucose tolerance and insulin sensitivity with IV glucose tolerance tests and hyperinsulinemic-euglycemic clamps. In addition, we analyzed the in vivo effect of TNF-α on several metabolism-related genes and adipocytokines implicated in the development of insulin resistance. TNF-α treatment resulted in markedly increased fasting blood glucose, insulin and free fatty acids (FFA) levels and reduced glucose tolerance. During the clamps, the rates insulin-stimulated whole body (G_Rd) and skeletal muscle glucose uptake (MGU) and insulin’s ability to suppress hepatic glucose production (HGP) were decreased in TNF-α treated animals, indicating insulin resistance. In addition, both PPARγ and ATGL mRNA expression in adipose tissues as well as ATGL protein levels in plasma were downregulated. Moreover, adipose mRNA expression and plasma protein levels of adiponectin and visfatin were significantly down-regulated. We conclude that the alterations of PPARγ, ATGL, adiponectin and visfatin may contribute to the development of insulin resistance mediated by TNF-α.     

Variation in Gene Expression of Inflammatory Cytokines in Leukocyte-Derived Cells of High-Fat-Diet-Induced Insulin-Resistant Rats

A high-fat diet is thought to enhance inflammation in various tissues by increasing insulin resistance. In this study, we determined the mRNA levels of inflammatory cytokines in leukocyte-derived cells in the blood of rats with high-fat-diet-induced insulin resistance. Feeding rats a high-fat diet for 77 d induced moderate insulin resistance, which was determined by increased plasma glucose and insulin concentrations, following an oral glucose tolerance test. The interleukin (IL)-1β mRNA level was higher in the insulin-resistant rats than in control rats at the fasting stage, whereas the tumor necrosis factor (TNF)-α mRNA level was greatly elevated at 180 min after glucose administration in the insulin-resistant rats. The results suggest that feeding rats a high-fat diet enhances the expression of fasting IL-1β and postprandial TNF-α genes in leukocyte-derived cells.     

Effects of Dietary Korean Proso-Millet Protein on Plasma Adiponectin,HDL Cholesterol,Insulin Levels,and Gene Expression in Obese Type 2 Diabetic Mice

We investigated the effect of dietary Korean proso-millet protein concentrate (PMP) on glycemic responses, plasma lipid levels, and the plasma level and gene expression of adiponectin in obese type 2 diabetic mice under normal and high-fat feeding conditions. The findings were that the feeding of PMP clearly elevated plasma high-density lipoprotein cholesterol (HDL cholesterol) and adiponectin levels and brought about effective reduction in the levels of glucose and insulin in mice under high-fat diet conditions as compared with a control diet. Gene expression study revealed that the diet up-regulated expression of adiponectin and down-regulated tumor necrosis factor-α (TNF-α). Considering the central role of adiponectin and HDL cholesterol in improving and ameliorating type 2 diabetes, obesity, and cardiovascular disease, our findings imply that PMP may have potential for therapeutic intervention in type 2 diabetes.