Overexpression of dominant negative mutant hepatocyte nuclear factor (HNF)-1alpha inhibits arginine-induced insulin secretion in MIN6 cells.
OBJECTIVE
To explain the mechanisms whereby mutations in the HNF-1alpha gene cause insulin secretory defects.


METHODS
A truncated mutant HNF-1alpha (HNF-1alpha288t) was overexpressed in hepatoma cells (HepG2) and murine insulinoma cells (MIN6) using a recombinant adenovirus system and expression of the HNF-1alpha target genes and insulin secretion were examined.


RESULTS
Expression of phenylalanine hydroxylase and alpha1-antitrypsin genes, the target genes of HNF-1alpha, was suppressed in HepG2 cells by overexpression of HNF-1alpha288t. In MIN6 cells, overexpression of HNF-1alpha288t did not change insulin secretion stimulated by glucose (5 mmol/l and 25 mmol/l) or leucine (20 mmol/l). Potentiation of insulin secretion by arginine (20 mmol/l, in the presence of 5 mmol/l or 25mmol/l glucose) was, however, reduced (p < 0.0001 and p = 0.027, respectively). Similarly reduced responses were observed when stimulated with homoarginine. Expression of the cationic amino acid transporter-2 was not reduced and insulin secretory response to membrane depolarization by 50 mmol/l KCl was intact.


CONCLUSIONS
The HNF-1alpha288 t, which is structurally similar to the mutant HNF-1alpha expressed from the common MODY3 allele, P291fsinsC, exerts a dominant negative effect. Suppression of HNF-1alpha in MIN6 cells severely impaired potentiation of insulin secretion by arginine, whereas glucose-stimulated and leucine-stimulated insulin secretion was intact. Our findings delineate the complex nature of beta-cell failure in patients with MODY3. This cell model will be useful for further investigation of the mechanism of insulin secretory defects in these patients.
Tanizawa
Y
Y
Third Department of Internal Medicine, Yamaguchi University, School of Medicine, Japan.
Ohta
Y
Y
Nomiyama
J
J
Matsuda
K
K
Tanabe
K
K
Inoue
H
H
Matsutani
A
A
Okuya
S
S
Oka
Y
Y
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology; D015870:Gene Expression; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D007328:Insulin; D009687:Nuclear Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D001120:Arginine