ENZYMATIC CONTROLS OF NONENZYMATIC GLYCATION

描述(由申请人提供)： 除了高血糖的作用外，很明显还有遗传和代谢因素使个体易患糖尿病并发症。甲基乙二醛(MG)是一种强有力的糖基化试剂，是糖基化终末产物(AGEs)的前体，在糖尿病并发症的发生发展中起重要作用。此外，细胞内环境中高浓度的葡萄糖和赖氨酸必然导致葡萄糖介导的美拉德反应，其早期和晚期产物也对细胞功能有害。我们已经证明，在加速性糖尿病肾病中，MG的产生和氧化应激增加。这种MG产量的增加可能是由于下游甘油醛磷酸脱氢酶(GAPDH)的遗传或获得性缺陷造成的。果糖胺-3-激酶(FN3K)的纯化和鉴定是控制高等生物中美拉德反应的潜在系统的第一个迹象，也可能在肾病的易感性中发挥作用。这项工作的广泛目标是检查影响AGE前体产生和解毒的因素，并探索酶机制在非酶化学反应中所起的作用。第一个目标是阐明肾病易感个体MG产生增加的机制。我们将对活检证实为糖尿病肾病进展或不进展的两大组的GAPDH活性、基因表达和突变进行检测。为了检查可能的遗传因素，我们将在40对表现出相似程度的肾病的兄弟姐妹中进行类似的实验。氧化应激、糖基化和NAD+水平对GAPDH活性的作用也将被评估。第二个目标是在蛋白质、mRNA和基因组水平上鉴定人类FN3K亚型，并开发体内酶活性的特异性标记物，用于易发生并发症和耐药的糖尿病患者。这项建议利用独特的研究人群来提供对糖化机制的严格测试，这些机制决定了对糖尿病肾病发展的抵抗或易感性。如果我们要找到有效的策略来预防糖尿病肾病，增加对决定二酮和Amadori产物水平及其导致的AGEs的酶机制所起作用的了解是至关重要的。

DESCRIPTION (provided by applicant): In addition to the role of hyperglycemia, it is clear that there are genetic and metabolic factors which predispose an individual to develop diabetic complications. Methylglyoxal (MG) is a powerful glycating agent and precursor of advanced glycation end products (AGEs) which appears to play an important role in the development of diabetic complications. In addition, high concentrations of glucose and lysine in the intracellular milieu leads inevitably to glucose-mediated Maillard reactions, the early and advanced products of which are also harmful to cell function. We have shown that MG production and oxidative stress are increased in accelerated diabetic nephropathy. This increased MG production could be secondary to genetic or acquired defects in downstream glyceraldehyde phosphate dehydrogenase (GAPDH). The purification and characterization of fructosamine-3-kinase (FN3K), which is capable of deglycating fructoslysine on glycated proteins, is the first indication of a potential system for controlling the Maillard reaction in higher organisms and may also play a role in susceptibility to nephropathy. The broad goal of this work is to examine factors that effect the production and detoxification of AGE precursors and to explore the role played by enzymatic mechanisms in chemical reactions that are non-enzymatic. The first objective is to elucidate the mechanisms responsible for increased MG production in nephropathy prone individuals. We will measure GAPDH activity, gene expression and mutations from two large groups who show biopsy proven progression or non-progression of diabetic nephropathy. To examine possible genetic factors we will do similar experiments in 40 sibling pairs who show similar degrees of nephropathy. The role of oxidative stress, glycation, and NAD+ levels, on GAPDH activity will also be evaluated. The second objective is to identify human FN3K isoforms at the protein, mRNA and genomic levels and to develop specific markers of in vivo enzyme activity, in complication-prone and resistant diabetic patients. This proposal utilizes unique study populations to provide a rigorous test of glycation mechanisms that determine resistance or susceptibility to the development of diabetic nephropathy. Increased understanding of the role played by enzymatic mechanisms that determine levels of dicarbonyls and Amadori products and their resulting AGEs is essential if we are to find effective strategies to prevent diabetic nephropathy.