大黄酸对葡萄糖转运蛋白1基因转染系膜细胞功能的影响

目的通过观察大黄酸对葡萄糖转运蛋白1(GLUT1)基因转染系膜细胞(MCGT1)功能的影响,探讨大黄酸治疗糖尿病肾病(DN)的作用机制.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-3H-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,3H-脯氨酸掺入和流式细胞仪分别检测细胞胶原和纤维连接蛋白(FN)的合成,采用比色法测定谷氨酰胺6-磷酸果糖转氨酶(GFAT)的活性,RT-PCR检测细胞胶原Ⅳ、FN和GFAT的表达.结果MCGT1的2-DG摄入率明显高于MCLacZ[(741±60.5)dpm*μgprot-1比(92.2±9)dpm*μgprot-1],同时表现出细胞大小、RNA/DNA和蛋白/DNA明显增加的细胞肥大表型,细胞外基质合成增加,MCGT1的3H-脯氨酸掺入量明显高于MCLacZ[(7.0±0.4)dpm*cell-1比(4.6±0.6)dpm*cell-1],GFAT活性明显增强(约增加1.8倍).大黄酸能够减少MCGT1的糖摄取[(560±64)dpm*μgprot-1比(741±60.5)dpm*μgprot-1),纠正MCGT1的肥大状态,并抑制MCGT1细胞外基质的合成和表达,降低MCGT1的GFAT活性.结论GLUT1的过度表达会明显改变系膜细胞的功能,大黄酸能逆转GLUT1基因转染所致系膜细胞功能的改变.

Inhibition of glucose transporter 1 overexpression in mesangial cells by rhein

OBJECTIVE: To evaluate the effect of rhein on the overexpression of glucose transporter 1(GLUT 1) in mesangial cells transfected with GLUT 1 gene in vitro. METHODS: Rat mesangial cells were transduced with the human GLUT 1 gene(MCGT 1) by retrovirus vector. Mesangial cells transduced with bacterial beta-galactosidase(MCLacZ) were used as control. Glucose uptake was detected by 2-deoxyglucose(2-DG). Cell volume, RNA/DNA ratio and protein/DNA ratio were evaluated by flow cytometry analysis. The synthesis of collagen IV and fibronectin were measured by 3H-proline incorporation and flow cytometry. The activity of glutamine: fructose-6-phosphate aminotransferase (GFAT) was assayed by spectrophotometry method. The expression of collagen IV, fibronectin and GFAT were analyzed by RT-PCR. RESULTS: MCGT 1 exhibited a higher 2-DG uptake with increased Vmax value as compared to MCLaZ(741 +/- 60.5) dpm.microgram prot-1 vs (92.2 +/- 9) dpm.microgram prot-1, (P < 0.01), Even cultured in normal glucose concentration, MCGT1 showed cell hypertrophy, including increased cell volume, RNA/DNA and protein/DNA ratios; increased synthesis of extracellular matrix. 3H-proline incorporation significantly enhanced in MCGT1 compared with that in the control(7.0 +/- 0.4) dpm.cell-1 vs (4.6 +/- 0.6) dpm.cell-1, (P < 0.01); and GFAT activity increased to 1.8 fold. Rhein could inhibit 2-DG uptake of MCGT1(560 +/- 64) dpm.microgram prot-1 vs (741 +/- 60.5) dpm.microgram prot-1, (P < 0.05) in a dose-dependent manner, and reverse the cell hypertrophy of MCGT 1. In addition, rhein could diminish the enhanced GFAT activity of MCGT 1 with no influence in mRNA expression. CONCLUSION: It is suggested that glucose transport activity was an important modulator of cellular glucose metabolism in mesangial cells. Rhein could ameliorate the metabolic derangement of MCGT 1 by decreasing glucose uptake. These findings have led us closer to the identification of therapeutic approaches to abort GLUT 1 overexpression in diabetic nephropathy.