Induction of insulin resistance in normal adipose tissue by uremic human serum.
Induction of insulin resistance in normal adipose tissue by uremic human serum.
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尿毒症人血清诱导正常脂肪组织中的胰岛素抵抗。
DOI：
10.1038/ki.1984.33
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发表时间：
1984
影响因子：
19.6
通讯作者：
Lockwood,DH
中科院分区：
文献类型：
--
作者：
McCaleb,ML;Mevorach,R;Freeman,RB;Izzo,MS;Lockwood,DH
方法材料。雄性Sprague-Dawley大鼠获自Charles River Laboratories（威尔明顿，马萨诸塞州）。Crys-talline猪胰岛素由Eli Lilly Company（Indianapolis，印第安纳州）赠送。沃辛顿生物化学公司，Free-hold，新泽西提供粗胶原酶（1型），牛白蛋白组分V（BSA）从Reheis，坎卡基，伊利诺伊州获得。所有放射性同位素均获自Amer-sham Corp.，伊利诺伊州，阿灵顿高地。SpectraiPor 6，截留分子量为3，500的透析管，由亚瑟托马斯公司提供，宾夕法尼亚州，费城。牛PTH（6800 IU/mg）的合成N-末端片段[1-34]获自Beckman，Palo Alto，加州。胰高血糖素由Sigma Chemical Co.，圣路易斯，密苏里州。患者特征。血清样本来自17名男性和12名女性患者，年龄范围为21至88岁。然而，在一天的不同时间抽血，通常是在吸收后状态。根据既定标准，这些患者被诊断为尿毒症[9]。他们的尿素氮和肌酸酐水平范围分别为45至200（Xsd，109 42）和3.6至15（7.3 3.2）mg/dl。患者的更多特征见结果。这些患者没有接受糖皮质激素治疗或任何其他已知改变葡萄糖耐量的药物。根据既定标准[10]，他们均未接受透析治疗或有任何糖尿病证据（例如，隔夜空腹血糖水平低于140 mg/dl）。所有患者均被告知本研究并获得知情同意。从20例年龄范围为20 - 70岁的受试者中获得合并对照血清，这些受试者已知无肾脏疾病和其他胰岛素抵抗状态，如肥胖或糖尿病。脂肪组织培养和细胞分离。使用纤维素透析管将血清在帕克氏培养基199（100倍过量体积）中透析18小时（4 ℃）.雄性大鼠附睾脂肪组织（180至230 g）合并，切碎，并与人血清预孵育3 h（50%vol/vol），以及培养基199，其含有1g/dl BSA和0.3mg Hepes/ml，pH 7.4，在37 ℃下使用95%O2/5%CO2的潮湿气氛[4]。然后在含有3 g/dl BSA的Krebs-Ringer磷酸盐缓冲液（pH 7.4）中通过胶原酶消化[11]分离脂肪细胞。在通过三次洗涤去除胶原酶后，将细胞悬浮液保持在该缓冲液中。如前所述[4]，用对照受试者的透析血清替代含有BSA的培养基199不会影响胰岛素对脂肪细胞的后续作用。对照和尿毒症血清预处理细胞的胞质空间[12]（分别为0.08 ± 0.02和0.08 ± 0.02 td/105细胞，X SEM）与用确定的培养基预孵育的细胞（0.07 ± 0.01，td/105细胞）无显著差异。葡萄糖代谢。用对照或尿毒症血清预处理的脂肪细胞在37 ℃下孵育，
Methods Materials. Male Sprague-Dawley rats were obtained from Charles River Laboratories, Wilmington, Massachusetts. Crys-talline porcine insulin was a gift of the Eli Lilly Company, Indianapolis, Indiana. Worthington Biochemical Corp., Free-hold, New Jersey, supplied crude collagenase (type 1), and bovine albumin fraction V (BSA) was obtained from Reheis, Kankakee, Illinois. All radioisotopes were obtained from Amer-sham Corp., Arlington Heights, Illinois. SpectraiPor 6, dialysis tubing with a molecular weight cutoff of 3,500, was supplied by Arthur Thomas Co., Philadelphia, Pennsylvania. The synthetic N-terminal fragment [1—34] of bovine PTH (6800 lU/mg) was obtained from Beckman, Palo Alto, California. Glucagon was supplied by Sigma Chemical Co., St. Louis, Missouri. Patient characteristics. Samples of serum were obtained from 17 male and 12 female patients, who ranged in age from 21 to 88 years. Blood was drawn at various times of the day, however, usually in the postabsorptive state. These patients were diagnosed as having uremia according to established criteria [9]. Their ranges of urea nitrogen and creatinine levels were 45 to 200 (Xsd, 109 42) and 3.6 to 15 (7.3 3.2) mg/dl, respectively. Further characteristics of the patients are presented in Results. These patients were not receiving glucocorticoid treatment or any other drugs known to alter glucose tolerance. None of them had received either dialysis therapy or had any evidence of diabetes mellitus according to established criteria [10](for example, overnight fasting blood glucose levels were less than 140 mg/dl). All patients were informed of the study and consent was obtained. The pooled control serum was obtained from 20 subjects, ranging in age from 20 to 70 years, who were known to be free of renal disease and other insulinresistant states, such as obesity or diabetes mellitus. Adipose tissue culture and cell isolation. Sera were dialyzed 18 hr (4 C) against Parker's Medium 199 (100-fold excess volume) using cellulose dialysis tubing. Epididymal adipose tissue derived from male rats (180 to 230 g) was pooled, minced, and preincubated for 3 hr with human serum (50% vol/vol), from either normal subjects (control) or uremic patients, and Medium 199, which contained I g/dl BSA and 0.3 mg Hepes/ml, at pH 7.4, using a humidified atmosphere of 95% 02/5% CO2 at 37 C [4]. The adipocytes were then isolated by collagenase digestion [11] in Krebs-Ringer phosphate buffer, pH 7.4, that contained 3 g/dl BSA. The cell suspensions were maintained in this buffer after the removal of collagenase by three washings. As noted previously [4], substitution of the Medium 199 containing BSA with dialyzed serum from control subjects did not influence the subsequent action of insulin on adipocytes. The cytosolic space [12] of control and uremic-sera pretreated cells (0.08 0.02 and 0.08 0.02 td/l05 cells, X SEM, respectively) were not significantly different than that of cells preincubated with the defined culture medium (0.07 0.01, tdIlO5 cells). Glucose metabolism. Adipocytes, which had been pretreated with either control or uremic sera, were incubated at 37 C with