论文标题:花生四稀酸对胰岛β细胞功能的作用及其机理研究
Effects and Mechanism of Arachidonic Acid on Pancreatic β-cell Functions
论文作者 陆祖谦
论文导师 潘长玉,论文学位 博士后,论文专业 内分泌及代谢疾病研究
论文单位 中国人民解放军军医进修学院,点击次数 464,论文页数 96页File Size5564k
2004-05-01论文网 http://www.lw23.com/lunwen_1159807/ 2型糖尿病,花生四稀酸,胰岛β细胞,软脂酸,硬脂酸,胰岛素,细胞凋亡,Glucokinase,PDX-1
Type 2 Diabetes, arachidonic acid, pancreatic β-cells, palmitic acid, stearic acid, insulin, apoptosis, glucokinase, PDX-1
花生四稀酸对胰岛β细胞功能的作用及其机理研究 糖尿病业已成为全球范围内日益严重的公共健康问题，目前全球糖尿病患者约1.7亿人。我国现有糖尿病患者约4000万人，其中90％以上的患者为2型糖尿病(T2DM)；而且2型糖尿病、肥胖和高脂血症的发生率呈现逐年递增并向年青化发展的趋势。在这些疾病中，胰岛素抵抗是其主要原因，脂代谢异常是它们的共同特点。T2DM中超过80％的人表现为超重或肥胖，而超重肥胖同时又是发生2型糖尿病很强的危险因素。肥胖和T2DM几乎均有不同程度的脂代谢异常，即血浆游离脂肪酸(frec fatty acids, FFA)，胆固醇(Chol)，小而致密的低密度脂蛋白(LDL)和甘油三酯(TG)水平增高，而高密度脂蛋白胆固醇(HDL-C)水平降低。 在典型的2型糖尿病患者中，胰岛β细胞功能的损害不是由高血糖、蛋白的糖化和淀粉样物质的沉积，而是由于脂毒性作用(血清游离脂肪酸浓度增高或／和甘油三酯在胰岛β细胞中的沉积)所致。人体尸检资料显示，胰岛β细胞的脂肪含量出现年龄相关性变化，而胰岛α细胞或导管细胞不出现年龄相关性的改变。令人感兴趣的是，在胰岛素抵抗的患者中，只有三分之一的人发展成为2型糖尿病，而其余的三分之二通过增加胰岛素分泌或释放来代偿胰岛素抵抗。发生这种病理生理的机理是：游离脂肪酸，特别是饱和脂肪酸可减少胰岛β细胞合成和分泌胰岛素；甘油三酯在胰岛β细胞中的沉积增加，导致胰岛β细胞的凋亡增多，引起β细胞分泌胰岛素的功能下降。 T2DM患者常存在肥胖和／或血浆游离脂肪酸增高。体内外实验已经证明，血浆FFA增加，在空腹状态下有刺激胰岛β细胞分泌胰岛素的作用；但长期升高可抑制胰岛素的分泌，造成胰岛素分泌量的减少，并且抑制胰岛素对靶组织，如肝脏和肌肉的生物学效应。例如0.5 mmol／L的FFA有促进胰岛素分泌的作用，而FFA进一步升高却抑制胰岛β细胞分泌胰岛素的功能，称之为β细胞脂毒性作用(Lipotoxicity)。血浆中FFA增高，细胞内长链脂肪酰CoA(LC-CoA)浓度增加。军医进修学院解放军总医院出站报告中文摘要作为一个信号分子，LC一CoA可以调节细胞葡萄糖激酶、葡萄糖一6一磷酸酶，胞吐，高尔基体的融合，内质网钙离子，PKC(蛋白激酶C)，CPT-1(肉碱棕搁酞转移酶一l)、AcC酶(乙酞辅酶A梭化酶)和基因表达等。胰岛p细胞内Lc一CoA浓度增加后可作用于上述胰岛素合成和分泌的各个环节，从而影响胰岛p细胞分泌胰岛素的功能。此外，各种FFA对p细胞分泌胰岛素的功能的作用不完全相同，随碳链的增长和饱和程度增加，它对胰岛p细胞分泌胰岛素的刺激作用增加。 我们既往的研究已经证明，生理浓度1一20倍的AA能剂量依赖性的抑制饱和脂肪酸对颗粒细胞和翠丸Leydig细胞的致凋亡作用，AA浓度高于50倍生理浓度后其本身对细胞也有毒性作用，甚至引起细胞凋亡。这一结果说明血浆中饱和脂肪酸和AA的浓度比例对于细胞的功能非常重要。AA在不同浓度情况下对胰岛日细胞可能有不同的作用。因此，我们推测AA可能具有拮抗饱和脂肪酸引起的胰岛素分泌和合成功能的障碍以及细胞毒性作用。为此，我们选择胰岛p细胞作为研究对象，研究AA对饱和脂肪酸引起的p细胞合成和分泌胰岛素功能的损伤有无保护作用，以及AA是否能拮抗饱和脂肪酸诱导的p细胞凋亡作用。 材料与方法胰岛p细胞株N工T-1在含10%胎牛血清的D入IEM培养基中培养，当NIT-1细胞融合70一80%后，经0.25%胰蛋白酶消化，将细胞接种于24孔培养板内，细胞继续培养24一72h后，分别用饱和脂肪酸，包括软脂酸(PA)和硬脂酸(sA)，单不饱和脂肪酸oA，多不饱和脂肪酸亚麻酸(L nA)和花生四稀酸(AA)以及各种阻断剂，包括环氧合酶、脂氧合酶、蛋白激酶等处理24一%h，然后使用MTT测定活细胞数;或收集细胞培养上清测定胰岛素;或用TRIzol试剂提取细胞RNA检测细胞Glucokinase和PDx-l基因的表达伽orthem blot);或使用流式细胞仪检测细胞凋亡。 结果 1趴、SA、OA和LnA能够时间和剂量依赖性地抑制胰岛仔细胞的存活率，600协mol/L的PA、sA、oA和L叭于培养4天后，其抑制率分别为52%、51%、“%和35%。5拼M的AA即具有刺激细胞生长的作用(外0.05)，而10林M的AA于培养48h后具有明显的刺激细胞生长(P<0 .05);但是，巧协M的AA于培养96h后具有明显的抑制细胞生长的作用，其抑制率为15%，P<0.05;而30协M的人A于培养24h后即具有明显的抑制细胞生长的作用，尸< 0.05。军医进修学院解放军总医院出站报告中文摘要 2.PA与p细胞孵育的同时加入AA(10卿ol/L)，则AA能显著地降低PA对p细胞的抑制作用，尸<0.01;蛋白激酶C阻断剂GFlo92o3X(lo、mol/L)、c扩十阻断剂Nifedipine(10脚ol几)和钙调蛋白阻断剂W一7(20林mol/L)能显著地阻断AA(10林mol/L)的这种保护作用，p<0.01;脂氧合酶阻断剂NDGA(10协mol几)、环氧合酶阻断剂玩domethaein(10仁mol/L)、P45O代谢通路阻断剂Ketoconazole(10林mol几)、p45o代谢产物20一HETE和LB;(80 nmol几)、蛋白激酶A抑制剂H一89(20卿01/L)、酪氨酸蛋白激酶抑制剂Genistein(50娜01/L)、No供体sN好(10协mol/L)以及NF一Kb抑?
Effects and Mechanism of Arachidonic Acid on Pancreatic B-cell FunctionsDiabetes has globally become a serious public health problem, and some 170 million people now have this condition. In China, the people with Diabetes are about 40 million, with more than 90% Diabetes being type 2 Diabetes. The morbidity of type 2 Diabetes, obesity and hyperlipidemia is yearly increasing, and the incidence of Diabetes among the Youngers is increasing alarmingly. Insulin resistance may play a pivotal role in those disorders which are commonly characterized by dyslipidemia. More than 80% of type 2 diabetic individuals are obese or overweight, a strongly risk factor for developing type 2 diabetes. Subjects with obesity and type 2 diabetes exclusively have a disturbance of lipid metabolism, with an increase of plasma FFA, cholesterol, low density lipoprotein, triglyceride, and a decrease of high density lipoprotein.In the typical patient with type 2 diabetes, damage to the P-cells is not caused by hyperglycemia, glycosylation of proteins, or amyloid deposition. Rather, it appears to occur because of lipotoxicity (i.e., elevated serum FFA and depoisition of triglyceride in pancreatic islet), which is also associated with the insulin resistance syndrome. An autopsy study in humans showed that fat content of p-cells, but not a- or duct cells, increased with the age. Interestingly, only one third of insulin-resistant patients developed diabetes; in the other two thirds, insulin production and release increase to overcome the insulin resistance. The pathophysioligical basis for this is increased FFA, especially SFA reducing the biosynthesis and secretion of insulin, and deposition of triglyceride in pancreatic p-cells leads to apoptosis which decreases insulin secretion of islet p-cell.T2DM are characterized of obesity and or elevated plasma FFA level. It is now wellestablished, both in vitro and in vzvo, that short exposure to FFA can stimulate glucose-induced insulin secretion in the fast state, whereas long-term exposure decreases the insulin secretion, and inhibits insulin-mediated glucose uptake in the muscles and liver. FFA concentration of 0.5 mM can stimulate the pancreatic P-cell insulin release, while further elevation of FFA inhibits glucose-induced insulin secretion, referred to as pancreatic p-cell lipotoxicity. A raise of FFA can increase the intracellular content of long-chain acyl-CoA (LC-CoA). LC-CoA, as a signaling molecule, it might modulate the activity of glucokinase or glucose-6-phosphatase, exocytosis, the docking or fusion of the insulin vesicle, endoplasm Ca2+ influx, protein kinase C (PKC), carnitine palmitoyltransfease (CPT-1), acetyl-CoA carboxylase (ACC). An increase in the cytosolic concentration of LC-CoA influences the pancreatic P-cell insulin secretion via the above process. In addition, the effectiveness of individual FFA is different, with increased insulinotropic potency with prolongs of carbon chain and raised degree of saturation.Our previous data indicated that arachidonic acid (AA) more than 1-20 times physiological concentration could inhibit the apoptosis of ovarian granulosa and Leydig cells induced by saturated fatty acids, but AA itself, at the level more than 50 times physiological concentration was cytotoxic, even led to cell apoptosis. It suggested that the ratio of saturated fatty acids and AA might be very important factor for cell function. Different level of AA has different effects on the pancreatic P-cell, Based on our previous data, we hypothesis that AA might antagonize impaired pancreatic P-cell biosynthesis or secretion of insulin and prevent the p-cell cytotoxicity induced by saturated fatty acids. In review of this, we investigate whether AA can relieve the impaired pancreatic p-cell function, decreased insulin biosynthesis and secretion, induced by saturated fatty acids, and whether AA can prevent the pancreatic p-cell apoptosis induced by saturated fatty acids. Material and MethodsPancreatic p-cell line NIT-1 established from a transgenic NOD/Lt mouse were grown in
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