The Carbonyl stress as an Etiology of Diabetic Microangiopathy. -Gene-targeting of the Enzyme Metabolizing Glucose-derived carbonyl compounds-

2002 Fiscal Year Final Research Report Summary

• Project/Area Number: 13671193
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Metabolomics
• Research Institution: Kobe University
• Principal Investigator: MIYATA Satoshi Kobe University Graduate School of medicine, assistant professor, 大学院・医学系研究科, 助手 (20304090)
• Project Period (FY): 2001 – 2002
• Keywords: diabetes complications / glycation / methylglyoxal / glyoxalase-1 / dicarbonyl compound / gene targeting

Research Abstract

It has been known that the formation of highly reactive dicaibonyl compounds such as metliylglyoxal (MG), an intermediate of the gbycation reaction, is accelerated in diabetic subjects. Several lines of evidence have shown that excess MG influences cell fimctions in vitro, suggesting its involvement in the development of diabetic microangiopatny. On the other hand, it has been proposed that there is a defense system against MG by metabolizing it to an inert agent in vivo. In this regard, previous studies have shown that the glyoxalase system comprising glyoxalase-1 (GLO1) and gtyoxalase-2 plays a major role in it Thus, the present study was designed to clarify the relationship between MG metabolism and alteration in miciovascular tissues by establishing GLO1-knockout mice. In 2001, we first prepared a probe to clone mouse GLO1 cDNAby conducting PCR on mouse liver cDNA library with a set of primer deduced from the known human GLO1 cDNA sequence. Using this probe, we successfully cloned a full-length of mouse GL01 cDNA with an open reading frame of 552 bp whose sequence was 82% identical to that of human. The deduced amino acid sequence exhibits 89% identity with that of human. In 2002, we also found a silent SNP (225T/C) in the cloned GLO1 cDNAs isolated from mouse liver cDNA library. We subsequently screened a genome GLO1 DNA from a mouse genome DNA library with a probe including the start codon. Analysis of its sequence is now in progress. We are going to constmct a targeting vector aiming homologous replacement, followed by establishing GLO1 knockout mice. Finally, the mice will be analyzed in terms of the relationship between MG metabolism and alterations occurring in the imcrovasculatuie tissues.