α-硫辛酸对胰岛素抵抗HepG2细胞糖代谢紊乱的改善作用
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篇名: α-硫辛酸对胰岛素抵抗HepG2细胞糖代谢紊乱的改善作用
TITLE: Improvement Effects of α-lipoic Acid on Glucose Metabolism Disorder of Insulin Resistant HepG 2 Cells
摘要: 目的:研究α-硫辛酸对胰岛素抵抗HepG2细胞糖代谢紊乱的改善作用。方法:采用MTT法测定25~1000µmol/Lα-硫辛酸对人肝癌细胞HepG2存活率的影响,以确定α-硫辛酸给药浓度。实验设阴性对照组、胰岛素抵抗组(1×10-7mol/L胰岛素)、联合抵抗组(30µmol/L亚砷酸钠+1×10-8mol/L胰岛素)和α-硫辛酸低、中、高浓度组。按分组加入α-硫辛酸作用HepG2细胞12h后,再分别给予相应浓度的亚砷酸钠或/和胰岛素继续培养24h。采用葡萄糖氧化酶法检测细胞葡萄消耗量,比色法检测细胞己糖激酶、丙酮酸激酶活性,蒽酮法检测细胞糖原含量,Westernblot法检测细胞中葡萄糖转运体4(GLUT4)、磷酸化糖原合成激酶3β(p-GSK3β)、GSK3β蛋白表达水平和磷酸化蛋白激酶B(p-Akt)/Akt、p-GSK3β/GSK3β比值。结果:25、50、100µmol/L的α-硫辛酸对细胞存活率无明显影响(P>0.05),且细胞存活率均大于96%,故将其作为后续研究的低、中、高浓度。与阴性对照组比较,胰岛素抵抗组、联合抵抗组细胞葡萄糖消耗量、己糖激酶和丙酮酸激酶活性、糖原含量、GLUT4和p-GSK3β蛋白表达水平、p-Akt/Akt和p-GSK3β/GSK3β比值均显著降低,GSK3β蛋白表达水平均显著升高(P<0.05)。与联合抵抗组比较,α-硫辛酸各浓度组细胞葡萄糖消耗量(α-硫辛酸低浓度组除外)、己糖激酶(α-硫辛酸低、中浓度组除外)和丙酮酸激酶(α-硫辛酸低、中浓度组除外)活性、糖原含量、GLUT4(α-硫辛酸低浓度组除外)、p-GSK3β蛋白表达水平和p-Akt/Akt(α-硫辛酸低、中浓度组除外)、p-GSK3β/GSK3β(α-硫辛酸低浓度组除外)比值均显著升高,GSK3β(α-硫辛酸低、中浓度组除外)蛋白表达水平均显著降低(P<0.05),且α-硫辛酸高浓度组细胞糖原含量、GLUT4蛋白表达水平、p-GSK3β/GSK3β比值和α-硫辛酸中、高浓度组细胞p-GSK3β蛋白表达水平的改善效果更明显(P<0.05)。结论:α-硫辛酸对胰岛素抵抗HepG2细胞的糖代谢紊乱具有一定的改善作用,其机制可能与增加葡萄糖消耗,增强糖代谢相关酶活性,提高糖原含量,上调Akt、GSK3β蛋白的磷酸化水平和GLUT4、p-GSK3β蛋白的表达水平,下调GSK3β蛋白的表达水平有关。
ABSTRACT: OBJECTIVE:To study the i mprovement effects of α-lipoic acid on glucose metabolism disorder of insulin resistant HepG2 cells. METHODS :The effects of 25-1 000 µmol/L α-lipoic acid on survival rate of human hepatoma cell HepG2 were determined by MTT assay so as to determine the concentration of α-lipoic acid. Negative control group ,insulin resistance group (1× 10-7 mol/L insulin ),combination resistance group (30 µmol/L sodium arsenite+ 1×10-8 mol/L insulin ),α-lipoic acid low- concentration,medium-concentration and high-concentration groups were set up. HepG 2 cells were treated with α-lipoic acid for 12 h and then cultured with corresponding concentration of sodium arsenite or/and insulin for 24 h. The glucose oxidase method was used to detect the glucose consumption ,colorimetric method was used to detect hexokinase activity and pyruvate kinase activity , and anthrone method was used to detect glycogen content. Western blot assay was used to detect the protein expression of GLUT 4, p-GSK3β and GSK3β as well as the ratio of p-Akt/Akt and p-GSK3β/GSK3β. RESULTS:25,50,100 µmol/L α-lipoic acid had no significant effect on the survival rates of HepG 2 cells(P>0.05),and survival rates of H epG2 cells were higher than 96%,so they were used as the low ,medium and high concentration for follow-up study. Compared with negative control group ,glucose consumption,the activities of hexokinase and pyruvate kinase ,glycogen content ,protein expression of GLUT 4 and p-GSK 3β,the ratio of p-Akt/Akt and p-GSK 3β/GSK3β were decreased significantly in insulin resistance group and combined resistance group, while the protein expression of GSK 3β was increased significantly(P<0.05). Compared with combination resistance group ,the glucose consumption (except for α-lipoic acid low- concentration group ),the activities of h exokinase(except for α-lipoic acid low-concentration and medium-concentration groups ) andpyruvate kinase (except for α-lipoic acid low-concentration com and medium-concentration groups ), glycogen contents , protein expression of GLUT 4 (except for α-lipoic acid mail:bliang163@163.com low-concentration group )and p-GSK3β,the ratio of p-Akt/ Akt(except for α-lipoic acid low-concentration and medium-concentration groups )and p-GSK 3β/GSK3β(except for α-lipoic acid low-concentration groups )were increased significantly in α-lipoic acid groups ,while protein expression of GSK 3β(except for α-lipoic acid low-concentration and medium-concentration groups ) was decreased significantly (P<0.05);glycogen content , protein expression of GLUT 4 and the ratio of p-GSK 3β/GSK3β in α-lipoic acid high-concentration group as well as the protein expression of p-GSK 3β in α-lipoic acid medium-concentration and high-concentration groups were improved significantly (P<0.05). CONCLUSIONS:α-lipoic acid can improve the disorder of glucose metabolism in insulin resistant HepG 2 cells,the mechanism of which may be associated with the increase of glucose consumption ,the activities of glucose metabolism related enzymes and glycogen content ,and expression up-regulation of the phosphorylation levels of Akt and GSK 3β protein,the expression of GLUT 4 and p-GSK 3β proteins,down-regulation of the expression of GSK 3β protein.
期刊: 2021年第32卷第14期
作者: 安仙蓉,管俊欢,杨秀丽,吴先进,梁冰
AUTHORS: AN Xianrong ,GUAN Junhuan ,YANG Xiuli,WU Xianjin ,LIANG Bing
关键字: α-硫辛酸;人肝癌细胞HepG2;亚砷酸钠;胰岛素抵抗;糖代谢紊乱;磷脂酰肌醇3-激酶/蛋白激酶B
KEYWORDS: α-lipoic acid ;Human hepatoma cell HepG 2;Sodium arsenite ;Insulin resistance ;Glucose metabolism disorder ;
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