OVERPRODUCTION OF VERY LOW-DENSITY LIPOPROTEINS BY LIVERS OF GENETICALLY OBESE RATS
OVERPRODUCTION OF VERY LOW-DENSITY LIPOPROTEINS BY LIVERS OF GENETICALLY OBESE RATS
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DOI：
10.1152/ajplegacy.1971.220.5.1178
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发表时间：
1971-01-01
影响因子：
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通讯作者：
PFLEGER, B
中科院分区：
文献类型：
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作者：
SCHONFELD, G;PFLEGER, B
方法动物。雄性肥胖动物及其正常体重对照（NWC）来自L.扎克。Sprague-Dawley雄性动物（SD）和Dr. Zucker动物自由采食。饲料由Purina大鼠饲料和自来水组成。为了进行比较，还包括了一些饲喂10%果糖代替自来水的SD动物的脂蛋白电泳图（图1）。用戊巴比妥（5 mg/100 g）麻醉动物，从下腔静脉获得用于分析的血液，并取出肝脏用于灌注。灌注。在改良的米勒装置（16）（JR Thebeau，Boston，MA）中进行灌注。在37 ℃，总是在上午10点到11点之间开始。根据以下标准，所有灌注的肝脏在功能上是可行的：大体外观、胆汁流量和灌注液流速。该技术也被认为是足够的外观选定SD肝脏的电子显微镜？灌注液由Krebs-Ringer碳酸氢盐缓冲液pH 7.4（KRB）3 Y ′ o的牛血清白蛋白（BSA，组分V，Sigma批号99 B-0450）、300 mg/dl的葡萄糖和90 mg/dl的氨基酸组成。(The由等重量的21种氨基酸（Sigma Kit LAA-21）制备混合物。加入游离脂肪酸（FFA）作为油酸钠皂（Sigma），使初始灌注液FFA浓度为750 pEq/L（10）。然后在3小时的灌注中再输注496 PEq。(The输注液还含有20 ml KRB和2 g白蛋白）。使用前将所有溶液调至pH 7.4。在5分钟时获得灌注液样品，此后每30-60分钟获得一次。甘油三酯（TG）分泌速率在整个实验期间是稳定的。灌注结束时，将肝脏从装置中取出，立即用冰冷的盐水灌注，并在低温下在Virtis匀浆器中匀浆。然后将等分试样超声处理（Branson Instrument Inc.）用氯仿-甲醇提取，按Carlson（5）法进行分析。肝脏、灌注液、血清和极低密度脂蛋白的甘油三酯采用Carlson方法测定（5），胆固醇采用Leffler方法测定（14），磷脂（PL）测定为脂磷（3）。通过Duncombe（8）的量热法对按照多尔（7）制备的浸提液进行游离脂肪酸定量。采用Narayan的纸片法（17）测定脂蛋白胆固醇（LPE）。通过在生理盐水中，在L型Spinco仪器中，在40.3转筒中，在10 ℃下，将灌注液以其自身密度超离心18小时，获得极低密度脂蛋白。对于气相色谱法，通过薄层色谱法（9）、皂化和甲基化（15）分离肝脏和灌注液提取物的TG。气相色谱法在Beckman GC-5仪器中通过双差示法使用涂有20%l的100-120目Chromosorb W（Beckman）进行。科斯蒂亚诺夫斯基的部门。病理学，华盛顿大学医学院。
METHODSAnimals. Male fatties and their normal weight controls (NWC) were obtained from Dr. L. Zucker. Sprague-Dawley males (SD) and Dr. Zucker’s animals were fed ad libitum. The diet consisted of Purina rat chow and tap water. For purposes of comparison the lipoprotein electropherograms of some SD animals fed 10% fructose in place of tap water are included as well (Fig. 1). The animals were anesthetized with pentobarbital(5 mg/lOO g), blood for analyses was obtained from the inferior vena cava, and livers were removed for perfusion. Perfusion. Perfusions were carried out in a modified Miller apparatus(16)(JR Thebeau, Boston, Mass.) at 37 C and were always started between 10 and 11 AM. All the livers perfused were functionally viable by the following criteria: gross appearance, bile flow, and flow rate of perfusate. The technique was also judged to be adequate by the appearance of selected SD livers by electron microscopy? Perfusates consisted of Krebs-Ringer bicarbonate buffer pH 7.4 (KRB) 3Y’o in bovine serum albumin(BSA, fraction V, Sigma lot no. 99B-0450), 300 mg/dl in glucose and 90 mg/dl in amino acids.(The mixture was prepared from equal weights of 2 1 amino acids, Sigma Kit LAA-2 1.) Free fatty acids (FFA) were added as the sodium soap of oleic acid (Sigma) to make the initial perfusate FFA concentration 750 pEq/liter(10). An additional 496 PEq were then infused over 3 hr of perfusion.(The infusate also contained 20 ml KRB and 2 g albumin.) All solutions were brought to pH 7.4 before use. Samples of perfusate were obtained at 5 min and every 30-60 min thereafter. Triglyceride (TG) secretion rate was steady throughout the experimental period. At the end of perfusion the livers were removed from the apparatus, immediately perfused with ice-cold saline, and homogenized in a Virtis homogenizer in the cold. An aliquot was then sonicated (Branson Instrument Inc.) and extracted with chloroform-methanol by the method of Carlson (5).Analyses. Triglycerides of liver, perfusates, sera, and very low-density lipoproteins were determined by the method of Carlson (5), cholesterol was determined by the method of Leffler (14), and phospholipids(PL) were determined as lipid phosphorus(3). Free fatty acids were quantified by the calorimetric method of Duncombe(8) performed on extracts prepared as per Dole (7). Lipoprotein electrophoreses (LPE) were done by the disc method of Narayan(17). Very low-density lipoproteins were obtained by subjecting the perfusates to ultracentrifugation at their own densities under saline in a model L Spinco instrument in a 40.3 rotor at 10 C for 18 hr. The tubes were sliced and the top layer was extracted as above (22). For gas chromatography the TG of the extracts of livers and perfusates were isolated by thin-layer chromatography(9), saponified, and methylated (15). Gas chromatography was carried out in a Beckman GC-5 instrument by the dual differential method using 100-120 mesh Chromosorb W (Beckman) coated with 20% l Kindly performed by Dr. M. Kostianovsky of the Dept. of Pathology, Washington University School of Medicine.