HIRAI Masashi Tohoku University, Hospital, Research Associate, 医学部附属病院, 助手 (80312578)
YOSHINORI Hinokio Tohoku University, Hospital, Lecturer, 医学部附属病院, 講師 (10282071)
MIYAGI Taeko Miyagi Prefectural Cancer Center, Department of Biochemistry, Director, 生化学部門, 部長(研究職) (50006110)
ISHIHARA Hisamitsu Tohoku University, Hospital, Research Associate, 医学部附属病院, 助手 (60361086)
KATAGIRI Hideki Tohoku University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (00344664)
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¥14,100,000 (Direct Cost : ¥14,100,000)
Fiscal Year 2003 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Fiscal Year 2002 : ¥10,100,000 (Direct Cost : ¥10,100,000)
Gangliosides are a family of sialic acid-containing glycosphingolipids present in the cell surface membranes. Several lines of evidence suggest their important functional roles in regulating a wide range of biological processes including cell growth, cell differentiation, and transmembrane signaling. Plasma membrane-associated sialidase (Neu3) is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions.
We have investigated effects of overexpression in transgenic mice by using the human NEU3 cDNA. We demonstrated that mice overexpressing the human NEU3 develop diabetic phenotype associated with hyperinsulinemia, islet hyperplasia, and increased beta-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation wa
s already attenuated in the younger mice before manifestation of cemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.
We also investigate the possible contribution of single nucleotide polymorphisms (SNPs) in NEU3 to the development of type 2 diabetes. We surveyed SNPs in the NEU3 gene in 298 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations : one consisting of 148 elderly subjects who met stringent criteria for being non-diabetic including age above 60 years and no evidence of diabetes (HbA1c<5.6%), and another 308 subjects with normal glucose tolerance (NGT). We identified eight SNPs with and five SNPs without amino acid substitutions in their coding regions. The allele frequency of one of SNPs was significantly higher in type 2 diabetic patients than in both elderly normal and NGT subjects, whereas the allele frequency of other SNPs was essentially identical in these three groups. Furthermore, in the NGT subjects, the SNP was associated with a significantly lower insulin sensitivity index on oral glucose tolerance test. These results strongly suggest that, in Japanese, the SNP in the NEU32 gene is associated with the development of type 2 diabetes, via reduced insulin signaling. Less