INTRACELLULAR O GLCNAC AND GLUCOTOXICITY

临床研究和对分离的胰岛或Beta细胞系的研究 表明Beta细胞长期暴露于超生理学 血糖水平会导致β细胞功能受损，这是一种重要的 在2型糖尿病的发病机制中起着重要作用。葡萄糖是如何发挥作用的 这种对Beta细胞的毒性尚不清楚。我们建议， 葡萄糖代谢产物氨基葡萄糖在β细胞功能和 这种葡萄糖毒性。我们得出这个假说是因为我们的 β细胞特异性毒性作用机制的观察 毒素，链脲佐菌素(STZ)。STZ在化学上类似于N- 乙酰氨基葡萄糖(GlcNAc)。此外，我们发现Beta细胞 含有大约100倍的编码这种酶的信使核糖核酸。 谷氨酸氨基转移酶(OGT)。这种酶可以修饰细胞核和细胞骨架。 通过将单糖GlcNAc连接到丝氨酸或苏氨酸来合成蛋白质 蛋白质中的残留物。此时将显示生成的O-GlcNAc修改 修饰转录因子的活性。我们发现STZ 阻止一种从蛋白质中去除o-GlcNAc的酶的活性。 链脲佐菌素对大鼠脑内O-GlcNAc积聚的影响 几个小时前，对胰岛β细胞进行了专门的修饰 β细胞死亡。因为Beta细胞富含OGT， 这些细胞可能最容易受到O-GlcNAc积聚的影响 细胞内的蛋白质。我们还展示了其他类型的细胞， 核内O-GlcNAc对环境葡萄糖浓度敏感。 如果在Beta细胞中也是如此，那么高血糖和STZ可能 两者都增加了Beta细胞O-GlcNAc的含量，从而导致共同的 β细胞毒性的机制。这项研究中提出的实验 GRANT旨在确定高血糖是否真的会导致 β细胞O-GlcNAc增加。我们还将创造转基因小鼠 从葡萄糖合成氨基葡萄糖的模型 或减少。我们将确定这些改变对 氨基葡萄糖代谢对β细胞功能的影响。我们还发现， CAMP依赖的蛋白激酶抑制负责 氨基葡萄糖合成。我们建议研究通过什么机制 氨基葡萄糖的合成受到抑制。总而言之，这些研究将 确定氨基葡萄糖在葡萄糖对Beta细胞毒性中的作用 以及一种控制葡萄糖代谢为氨基葡萄糖的方法。

Clinical studies and studies on isolated islets or Beta cell lines have indicated that chronic exposure of the Beta cell to supraphysiological levels of glucose results in impaired Beta cell function, an important component in the pathogenesis of type 2 diabetes. How glucose exerts this toxicity upon the Beta cell remains unclear. We propose that the glucose metabolite, glucosamine plays a role in Beta cell function and this glucose toxicity. We came to this hypothesis as a result of our observations on the mechanism of toxicity of the Beta cell- specific toxin, streptozotocin (STZ). STZ is chemically analogous to N- acetylglucosamine (GlcNAc). Furthermore, we found that the Beta cell contains approximately 100-fold more of the mRNA encoding the enzyme o- GlcNAc transferase (OGT). This enzyme modifies nuclear and cytoskeletal proteins by linking the monosaccharide GlcNAc to serine or threonine residues in the protein. The resulting O-GlcNAc modification appears to modify the activity of transcription factors. We found that STZ blocks the activity of an enzyme that removes o-GlcNAc from proteins. Treatment of rats with STZ results in the accumulation of the O-GlcNAc modification specifically in the pancreatic Beta cells, hours before Beta cell death. Because the Beta cells are so richly endowed with OGT, these cells may be the most susceptible to an accumulation of O-GlcNAc on intracellular proteins. We have also shown in other cell types, that nuclear O-GlcNAc is sensitive to ambient glucose concentrations. If this is also true in the Beta cell, then hyperglycemia and STZ may both increase Beta cell O-GlcNAc content, thereby leading to a common mechanism of Beta cell toxicity. The experiments proposed in this grant are designed to determine if hyperglycemia indeed result in increased Beta cell O-GlcNAc. We will also create transgenic mouse models in which glucosamine synthesis from glucose is either augmented or decreased. We will determine the effect of these alterations in glucosamine metabolism on Beta cell function. We have also found that cAMP-dependent protein kinase inhibits the enzyme responsible for glucosamine synthesis. We propose to study the mechanism by which glucosamine synthesis is inhibited. Together, these studies will establish the role of glucosamine in glucose toxicity on the Beta cell and a means of controlling glucose metabolism to glucosamine.