Identification of a single nucleotide polymorphism showing no insulin-mediated suppression of the promoter activity in the human insulin receptor substrate 2 gene.
OBJECTIVE
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 IRS2transcription was identified by EMSA with (32)P-labelled double-stranded oligonucleotides encompassing regions protected from DNase I digestion by nuclear extract of HepG2 cells. Two SNPs (-765 C/ T and -2062 T/ 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.


CONCLUSIONS
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.
Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University, Japan.
K
K
Iwamoto
H
H
Mori
H
H
Okazawa
M
M
Hashiramoto
M
M
Kasuga
D020121:5' Untranslated Regions / Q000235:genetics; D000368:Aged; D001483:Base Sequence / Q000235:genetics; D002478:Cells, Cultured; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000235:genetics; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics* / Q000378:metabolism*; D004586:Electrophoresis; D005260:Female; D006801:Humans; D007004:Hypoglycemic Agents / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000502:physiology*
D020121:5' Untranslated Regions; D007004:Hypoglycemic Agents; C526297:IRS2 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D003850:Deoxyribonuclease I