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Diabetologia

, Volume 45, Issue 8, pp 1182–1195 | Cite as

Identification of a single nucleotide polymorphism showing no insulin-mediated suppression of the promoter activity in the human insulin receptor substrate 2 gene

  •  K. Iwamoto
  •  H. Mori
  •  H. Okazawa
  •  M. Hashiramoto
  •  M. Kasuga
Article

Heading

Abstract

Aims/hypothesis. To understand the transcriptional regulation and to investigate the pathological influence upon Type II (non-insulin-dependent) diabetes mellitus of insulin receptor substrate 2 (IRS2), the 5′ flanking region of the human IRS2 gene was cloned and screened in Japanese diabetic patients.

Methods. Luciferase reporter assay and electrophoretic mobility shift assay (EMSA) were combined in HepG2, Fao, RINm5F, and HeLa cells to characterise the human IRS2 promoter region. Single nucleotide polymorphisms (SNPs) were identified in Japanese Type II diabetic patients by sequencing and were genotyped.

Results. The proximal 2399 bp of the 5′ flanking region of the human IRS2 gene was cloned. A core promoter region was extended between nucleotide positions –834 and –557 (relative to the translation initiation site). The region [(–758)AGGGGGAGGG(–749)] that appears important in the positive regulation of IRS2 transcription was identified by EMSA with 32P-labelled double-stranded oligonucleotides encompassing regions protected from DNase I digestion by nuclear extract of HepG2 cells. Two SNPs (–765C/T and –2062T/C), identified by screening Japanese Type II diabetic patients, were not associated with Type II diabetes. IRS2-driven reporter activity in the plasmid containing thymine at –765 was not suppressed by insulin when measured in Fao cells.

Conclusion/interpretation. The 5′ flanking sequence of the human IRS2 was investigated and two SNPs were identified. The SNP at –765 was suggested to be involved in the insulin-mediated regulation of the transcriptional activity of IRS2.

Insulin receptor substrate 2 promoter transcription luciferase assay insulin Fao insulin-responsive element Type II diabetes genetics single nucleotide polymorphism 

Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  •  K. Iwamoto
    • 2
  •  H. Mori
    • 2
  •  H. Okazawa
    • 2
  •  M. Hashiramoto
    • 1
  •  M. Kasuga
    • 2
  1. 1.Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan
  2. 2.Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University, Japan

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