Diabetic complications remain a serious issue in diabetes mellitus. The DCCT and UKPDS studies demonstrate clearly that glycemic control is a key variable in determining the rate of development of complications. However, mechanisms linking hyperglycemia with long-term complications remain poorly understood. One promising hypothesis on the etiology of diabetic complications is the nonenzymatic glycation hypothesis which postulates that diabetes associated pathological changes are a result, at least in part, of the nonenzymatic modification of proteins and some other macromolecules by glucose and its reactive byproducts. The predominant initial intermediates in this process are glucoselysine (GL) and fructoselysine (FL) (a.k.a Early Glycation Products - EGP's) which undergo a series of further rearrangements and reactions to form the irreversible Advanced Glycation Endproducts (AGE's). In our past studies we have obtained evidence that this nonenzymatic glycation process is counteracted in mammalian cells by active deglycation mechanisms. Based on this evidence, we are proposing to modify the nonenzymatic glycation hypothesis of diabetic complications to a Nonenzymatic Glycation/Enzymatic Deglycation hypothesis. Our first postulate is that, in homeothermic organisms nonenzymatic glycation of proteins and some other macromolecules is unavoidable and deleterious. Consequently, our second postulate is that these organisms control the unavodable and damaging effects of non-enzymatic glycation by active deglycation systems. According to our hypothesis, in diabetes, especially during periodic episodes of extreme hyperglycemia, such as observed in the postprandial period, the process of nonenzymatic glycation proceeds unchecked and results, over time, in cumulative and irreversible damage to essential proteins leading ultimately to loss of cell function and viability. Our present research is focused in two areas: 1) Testing the function of FN3K by its overexpression, knockdown of its mRNA and knockout of its gene in appropriate model systems and 2) Study of the FN3K-independent deglycation mechanisms. For the study of FN3K we have drafted a R01 grant proposal to NIDDK (DK62315-01). In order to pursue the higher-risk studies on deglycation we are submitting this R21 grant proposal in which we propose to investigate transglycosylation reactions in which the glucose moiety of glucosamine such as glucoselysine is scavenged away by a more nucleophilic compounds such a reduced glutathione. We already have evidence that such reactions occur readily in vitro and we also have data that the principal stable byproduct of this process, glucosecysteine, is present in urine and is elevated in diabetic individuals. This grant will enable us to further study the transglycation reaction pathway and to determine its importance in normoglycemic and diabetic individuals.