Biochemical and Biophysical Research Communications
Functional characterization of the HNF4α isoform (HNF4α8) expressed in pancreatic β-cells
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
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Present address: Department of Internal Medicine, Sumitomo Hospital, 5-3-20 Nakanoshima, Kita-ku, Osaka 530-0005, Japan.