MOLECULAR BIOLOGY OF SUGAR CATARACT IN LENS CELLS

作为糖尿病的常见并发症，糖性白内障是一种 严重的公共卫生问题，没有已知的有效治疗方法 而不是做手术。高血糖与糖的病因学有关 白内障的形成，但代谢途径及其调控 遗传和生化因素尚未确定。转换 通过醛糖还原酶(AR)将组织葡萄糖转化为山梨醇，已被 可能是导致晶状体糖性白内障的潜在机制。 在过去的六年里，这个实验室的研究已经确定了 高血糖、多元醇途径激活和肌酸代谢之间的联系 肌醇(MI)耗竭。这个实验室最近的研究表明 高渗应激刺激Na/M1基因表达 辅转运蛋白和AR，增加蛋白质合成，提高M1 摄取活性和多元醇的形成。在细胞内的蓄积 这些相容、相互作用的有机渗透分子维持渗透作用 平衡和保护晶状体不受高电压干扰的影响 细胞内电解液的浓度。然而，如果多元醇 积累会持续很长一段时间，它会剥离 通过多个途径限制M1摄取活性的必需M1晶状体 抑制机制：(1)多元醇对M1载体的抑制作用 蛋白质摄取活性，(2)多元醇介导的Na/M1下调 共转运蛋白mRNA和(3)多元醇激活的Mi从细胞到细胞的外流 5~6成熟。在这方面，高张力类似于高血糖，在 前者(通过AR信使核糖核酸的诱导)与后者(通过 提高了底物利用率的优点)引起了 胞内多元醇。对M1运输系统的直接损害 任何一种方法都代表了一种看似合理的共同机制，可以解释 晶状体心肌梗死的耗竭。此竞争对手的续订申请 拟议详细研究以下问题的机制、影响和后果 多元醇和非多元醇诱导晶状体细胞培养和晶状体中M1的耗竭 器官培养。细胞内M1的耗尽而不改变 细胞内多元醇含量将通过采用进料来实现 使用有效的M1摄取竞争性抑制剂L-葡萄糖的方案，a 我们展示的促进细胞内M1耗竭的新技术 晶状体细胞培养不增加多元醇含量。在第一个具体的 目的探讨Na/M1基因表达调控的分子机制 协同转运蛋白在高张和高渗糖尿病条件下将 检查过了。在第二个具体目标中，多元醇和乙醇的相互关系 M1渗透调节机制将被仔细定义。第三个具体问题 AIM将识别和描述调节 M1外排，目前对多元醇诱导的M1机制了解最少 耗尽。第四个具体目标将定义M1摄取的动力学 并将这些结果与已知的动力学相关 晶状体细胞培养的参数。最终的具体目标将利用 免疫电子显微镜定位Na/M1共转运蛋白的研究 完整晶状体中晶状体上皮细胞的基侧或顶端。 在这项竞争性续期拨款申请中提出的研究将 在分子和细胞调控方面产生新的信息 Na/肌醇共转运体和M1摄取活性并提供新的 对我们认为有助于糖形成的机制的洞察 白内障。

As a common complication of diabetes, sugar cataract represents a significant public health problem with no known effective treatment other than surgery. Hyperglycemia has been implicated in the etiology of sugar cataract formation, but the metabolic pathways and their modulation by genetic and biochemical factors have yet to be determined. The conversion of tissue glucose to sorbitol by the enzyme aldose reductase (AR), has been implicated as a potential mechanism leading to sugar cataract in the lens. Over the last six years, studies from this laboratory have defined metabolic links between hyperglycemia, polyol pathway activation and myo- inositol (MI) depletion. Recent studies from this laboratory have shown that hypertonic stress stimulates gene expression of the Na+ /M1 cotransporter and AR, increases protein synthesis and elevates both M1 uptake activity and polyol formation. The intracellular accumulation of these compatible, reciprocal, organic osmolytes operate to maintain osmotic balance and protect the lens against the perturbing effects of high intracellular concentrations of electrolytes. However, if polyol accumulation continues over a protracted period of time, it divests the lens of essential M1 by restricting M1 uptake activity via multiple inhibitory mechanisms; (1) polyol-mediated suppression of m1 carrier protein uptake activity, (2) polyol-mediated downregulation of the Na+ /M1 cotransporter mRNA and (3) polyol-activated efflux of Mi from cell to medium. In this regard hypertonicity is analogous to hyperglycemia, in that the former (by virtue of induction of AR mRNA), like the latter (by virtue of increased substrate availability) elicits an increase in intracellular polyol. The direct impairment of the M1 transport system by either means represents a plausible common mechanism that could account for the depletion of lenticular MI. This competing renewal application proposed detailed study of the mechanisms, implications and consequences of polyol- and non-polyol-induced M1 depletion in lens cell culture and lens organ culture. The depletion of intracellular M1 without alteration of intracellular polyol content will be achieved by employing a feeding regimen with L-glucose, a potent competitive inhibitor of M1 uptake, a novel technique shown by us to promote the depletion of intracellular M1 in lens cell culture without raising polyol content. In the first specific aim the molecular mechanisms which regulate the expression of the Na+/M1 cotransporter in hypertonicity and hyperosmotic diabetic conditions will be examined. In the second specific aim the interrelationship of polyol and M1 osmoregulatory mechanisms will be carefully defined. The third specific aim will identify and characterize the regulatory mechanism which modulates M1 efflux, currently the least understood mechanism of polyol-induced M1 depletion. The fourth specific aim will define the kinetics of M1 uptake in intact bovine lenses and correlate these results with known kinetic parameters from lens cell cultures. The final specific aim will utilize immunoelectron microscopy to localize the Na+/M1 cotransporter tot eh basolateral or apical aspect of lens epithelial cells in the intact lens. The studies proposed in this competitive renewal grant application will yield new information on the molecular and cellular regulation of the Na+/myo-inositol cotransporter and M1 uptake activity and provide new insight into a mechanism we believe contributes to the formation of sugar cataract.