摘要
目的 探讨己糖胺通路 (HBP)在葡萄糖转运蛋白 1(GLUT1)基因转染系膜细胞功能改变中的作用。方法 利用逆转录病毒载体建立GLUT1基因转染的大鼠系膜细胞 ,以 β 半乳糖苷酶转染细胞 (MCLacZ)为对照。用 2 脱氧 3 H 葡萄糖 (2 DG)测定细胞葡萄糖摄入 ,流式细胞仪分析细胞表型和纤维连接蛋白 (FN)的合成 ,采用比色法测定HBP限速酶———谷氨酰胺 :6 磷酸果糖转氨酶(GFAT)的活性 ,RT PCR检测细胞GFAT基因的表达。结果 MCGT1的 2 DG摄入率明显高于MCLacZ〔(741.0± 6 0 .5 )dpm/ μg蛋白质vs (92 .2± 9.0 )dpm/ μg蛋白质 ,P <0 .0 1〕 ,动力学分析发现MCGT1的Vmax是MCLacZ的 3.7倍 ,而两者Km值无明显差别。MCGT1的GFAT活性明显高于MCLacZ〔(3.2 5± 0 .2 5 )OD3 65·μg蛋白质-1·30min-1·10vs (1.15± 0 .16 )OD3 65·μg蛋白质 -1·30min-1·10〕 ,而两者GFAT的mRNA表达差异无显著性。GFAT的抑制剂———重氮丝氨酸能够改善MCGT1的肥大状态和FN合成。结论 GLUT1的过度表达伴随系膜细胞HBP活性的增加 ,该糖代谢通路的活化与系膜细胞表型及其功能改变有关。
Objective To investigate the role of hexosamine biosynthesis pathway (HBP) in the functionalalterationofmesangialcellstransinfectedwith glucose transporter 1 (GLUT1) gene. Methods Rat mesangial cells were transinfected with the human GLUT1 gene (MCGT1) by retrovirus vector. Mesangial cells transinfected with bacterial β-galactosidase (MCLacZ) were used as control. Glucose uptake was detected with 2-deoxy-〔 3H〕-D-glucose (2-DG). Cell size, RNA/DNA ratio, protein/DNA ratio and the synthesis of fibronectin were evaluated by flow cytometry. The activity of glutamine: fructose-6-P aminotransferase (GFAT), which is the key enzyme of HBP, was assayed by spectrophotometry. The expression of GFAT gene was analyzed by RT-PCR. Results MCGT1 demonstrated higher 2-DG uptake than MCLacZ 〔(741.0±60.5)dpm/μg protein vs (92.2±9.0)dpm/μg protein,P<0.01〕. Kinetic analyses revealed a 3.7-fold higher Vmax in MCGT1 than that in MCLacZ. MCGT1 showed a 2.8-fold greater GFAT activity as compared to MCLacZ 〔(3.25±0.25)OD 365 ·μg protein -1 ·30 min -1 ·10 vs (1.15±0.16)OD 365 ·μg protein -1 ·30 min -1 ·10,P<0.01〕, with no difference in mRNA expression. Inhibition of GFAT by the substrate analogue azaserine ameliorated the cell hypertrophy and the enhanced production of fibronectin in MCGT1. Conclusion GLUT1 overexpression activates HBP in mesangial cells. The excessive flux of glucose metabolism through HBP may contribute to functional alteration of mesangial cells with GLUT1.
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2001年第6期370-373,共4页
Chinese Journal of Endocrinology and Metabolism
基金
国家自然科学基金资助 ( 39870 2 88)
关键词
肾小球
系膜细胞
葡萄糖转运蛋白1
细胞外基质
己糖胺通路
Glomerular mesangium
Mesangial cell
Glucose transporter 1
Extracellular matrix
Hexosamine biosynthesis pathway
