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

Inhibition of glucose transporter 1 overexpression in mesangial cells by rhein

目的 通过观察大黄酸对葡萄糖转运蛋白 1(GLUT 1)基因转染系膜细胞 (MCGT 1)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GLUT 1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 ,3 H 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 MCGT 1的 2 DG摄入率明显高于MCLacZ[(741± 6 0 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 ,MCGT 1的3 H 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MCGT 1的糖摄取 [(5 6 0± 6 4)dpm·μgprot 1比 (741± 6 0 5 )dpm·μgprot 1) ,纠正MCGT 1的肥大状态 ,并抑制MCGT 1细胞外基质的合成和表达 ,降低MCGT 1的GFAT活性。结论 GLUT 1的过度表达会明显改变系膜细胞的功能 ,大黄酸能逆转GLUT 1基因转染所致系膜?

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 β 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 spetrophotometry 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 MCLacZ(741±60.5)dpm·μg prot -1 vs (92.2±9)dpm·μg 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·μg prot 1 vs (741±60.5)dpm·μg 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.