Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose
Regulation of intracellular glucose and polyol pathway by thiamine and benfotiamine in vascular cells cultured in high glucose
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DOI：
10.1074/jbc.m600418200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Porta, M
中科院分区：
文献类型：
--
作者：
Berrone, E;Beltramo, E;Porta, M
高血糖是糖尿病血管并发症发生的一个致病因素。过量葡萄糖激活的生化机制之一是多元醇途径，其中的关键酶醛糖还原酶将D-葡萄糖转化为D-山梨醇，导致细胞内稳态失衡。我们的目的是验证硫胺素和苯磷硫胺对高环境葡萄糖下内皮细胞和周细胞的多元醇途径、转酮醇酶活性和细胞内葡萄糖的影响。人脐静脉内皮细胞和牛视网膜周细胞在正常（5.6 mmol/L）或高（28 mmol/L）葡萄糖中培养，添加或不添加硫胺素或苯磷硫胺50或100 μmol/L。通过逆转录PCR测定转酮酶和醛糖还原酶mRNA表达，并通过分光光度法测定其活性；通过气相色谱-质谱法定量山梨醇浓度，通过荧光酶联免疫吸附测定法定量细胞内葡萄糖浓度。在高葡萄糖培养的人内皮细胞和牛视网膜周细胞中，硫胺素和苯磷硫胺可降低醛糖还原酶 mRNA 表达、活性、山梨醇浓度和细胞内葡萄糖，同时增加转酮醇酶（其为辅酶）的表达和活性。硫胺素和苯磷硫胺可纠正血管细胞中高葡萄糖诱导的多元醇途径激活。转酮酶的激活可能会将过量的糖酵解代谢物转移到磷酸戊糖循环中，加速糖酵解通量，并减少细胞内游离葡萄糖，从而防止其转化为山梨糖醇。这种对多元醇途径的影响，连同报道的硫胺素在高葡萄糖条件下的其他有益作用，可以证明测试硫胺素作为预防和/或治疗糖尿病并发症的潜在方法是合理的。
Hyperglycemia is a causal factor in the development of the vascular complications of diabetes. One of the biochemical mechanisms activated by excess glucose is the polyol pathway, the key enzyme of which, aldose reductase, transforms D-glucose into D-sorbitol, leading to imbalances of intracellular homeostasis. We aimed at verifying the effects of thiamine and benfotiamine on the polyol pathway, transketolase activity, and intracellular glucose in endothelial cells and pericytes under high ambient glucose. Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/liter) or high (28 mmol/liter) glucose, with or without thiamine or benfotiamine 50 or 100 mu mol/liter. Transketolase and aldose reductase mRNA expression was determined by reverse transcription-PCR, and their activity was measured spectrophotometrically; sorbitol concentrations were quantified by gas chromatography-mass spectrometry and intracellular glucose concentrations by fluorescent enzyme-linked immunosorbent assay method. Thiamine and benfotiamine reduce aldose reductase mRNA expression, activity, sorbitol concentrations, and intracellular glucose while increasing the expression and activity of transketolase, for which it is a coenzyme, in human endothelial cells and bovine retinal pericytes cultured in high glucose. Thiamine and benfotiamine correct polyol pathway activation induced by high glucose in vascular cells. Activation of transketolase may shift excess glycolytic metabolites into the pentose phosphate cycle, accelerate the glycolytic flux, and reduce intracellular free glucose, thereby preventing its conversion to sorbitol. This effect on the polyol pathway, together with other beneficial effects reported for thiamine in high glucose, could justify testing thiamine as a potential approach to the prevention and/or treatment of diabetic complications.