Functional characterization of the HNF4α isoform (HNF4α8) expressed in pancreatic β-cells

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Abstract

Mutations in the hepatocyte nuclear factor (HNF) 4α gene cause a form of maturity-onset diabetes of the young (MODY1), which is a monogenic form of type 2 diabetes characterized by impaired insulin secretion by pancreatic β-cells. HNF4α is a transcription factor expressed in the liver, kidney, intestine, and pancreatic islet. Multiple splice variants of the HNF4α gene have been identified and an isoform of HNF4α8, an N-terminal splice variant, is expressed in pancreatic β-cells. However, expression levels of HNF4α protein in pancreatic β-cells and the transcriptional activity of HNF4α8 are not yet understood. In the present study, we investigated the expression of HNF4α in β-cells and examined its functional properties. Western blotting and immunohistochemical analysis revealed that the expression of HNF4α protein in pancreatic islets and INS-1 cells was much lower than in the liver. A reporter gene assay showed that the transactivation potential of HNF4α8 was significantly weaker than that of HNF4α2, which is a major isoform in the liver, suggesting that the total level of HNF4α activity is very weak in pancreatic β-cells. We also showed that the N-terminal A/B region of HNF4α8 possessed no activation function and C-terminal F region negatively regulated the transcriptional activity of HNF4α8. The information presented here would be helpful for the better understanding of MODY1/HNF4α diabetes.

Section snippets

Experimental procedures

Plasmids. The pcDNA3.1-human wild type (WT) and mutant (R127W)-HNF4α2 expression vectors that were used have been described previously [10]. A full-length human cDNA clone encoding HNF4α8 was generated by assembling two separate fragments. The 5′ fragment of HNF4α8 cDNA (228 bp of exon 1D and 134 bp of exon 2) was obtained by PCR with a sense primer (5′-CCTGCTCCTCCATGCCCCCAGCTC-3′) and an anti-sense primer (5′-GAAGAAGCCCTTGCAGCCGTCACA-3′) using a human pancreatic islet cDNA library [11] as the

Expression of HNF4α protein in mouse islets and INS-1 cells

The amino-terminal sequence of HNF4α8, encoded by exon 1D, differed from that of HNF4α2 (Fig. 1A). Miquerol et al. [18] reported that HNF4α transcripts would be more abundant in liver than in islets by RT-PCR, but the actual expression levels of HNF4α protein in pancreatic islets/β-cells are not known. The amount of HNF4α protein expression was investigated by Western blot analysis using αN1.14 and 445 antibodies (Fig. 1B). The antibody for αN1.14 detects the N-terminus of HNF4α1–6, while that

Discussion

In the present study, we demonstrated that the level of HNF4α expression in pancreatic islets was far lower than that in the liver, and that activation by HNF4α8 was much weaker than that by HNF4α2. These results strongly suggest that the total level of HNF4α activity in pancreatic β-cells is very low. MODY1 patients have diabetes associated with impaired insulin secretion by pancreatic β-cells, but do not exhibit severe liver dysfunction [2], [6], [7]. The low level of expression and activity

Acknowledgments

We thank Dr. C.B. Wollheim for providing INS-1E cells. This work was supported by grants (15590938 and 14013038 [Medical Genome Science]) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant from the Ichiro Kanehara Foundation, a grant from Suzuken Memorial foundation, and a grant (1R13 DK067026-01) that is awarded by the Department of Health and Human Services, National Institutes of Health USA, National Institute of Diabetes and Digestive and Kidney

References (26)

  • S. Hata et al.

    Biochim. Biophys. Acta

    (1995)
  • D. Janjic et al.

    Biochem. Pharmacol.

    (1999)
  • B. Wang et al.

    Hepatology

    (2001)
  • L. Miquerol et al.

    J. Biol. Chem.

    (1994)
  • M. Hadzopoulou-Cladaras et al.

    J. Biol. Chem.

    (1997)
  • M.E. Torres-Padilla et al.

    J. Biol. Chem.

    (2002)
  • F.M. Sladek et al.

    Hepatocyte nuclear factor 4α

  • H. Furuta et al.

    Diabetes

    (1997)
  • H. Thomas et al.

    Hum. Mol. Genet.

    (2001)
  • S.F. Boj et al.

    Proc. Natl. Acad. Sci. USA

    (2001)
  • S.K. Hansen et al.

    J. Clin. Invest.

    (2002)
  • M.M. Byrne et al.

    Diabetes

    (1995)
  • K. Yamagata et al.

    Nature

    (1996)
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    Present address: Department of Internal Medicine, Sumitomo Hospital, 5-3-20 Nakanoshima, Kita-ku, Osaka 530-0005, Japan.

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