title: Clock-controlled output gene Dbp is a regulator of Arnt/Hif-1β gene expression in pancreatic islet β-cells.
abstract: Aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia inducible factor-1β (HIF-1β) has emerged as a potential determinant of pancreatic β-cell dysfunction and type 2 diabetes in humans. An 82% reduction in Arnt expression was observed in islets from type 2 diabetic donors as compared to non-diabetic donors. However, few regulators of Arnt expression have been identified. Meanwhile, disruption of the clock components CLOCK and BMAL1 is known to result in hypoinsulinemia and diabetes, but the molecular details remain unclear. In this study, we identified a novel molecular connection between Arnt and two clock-controlled output genes, albumin D-element binding protein (Dbp) and E4 binding protein 4 (E4bp4). By conducting gene expression studies using the islets of Wfs1(-/-) A(y)/a mice that develop severe diabetes due to β-cell apoptosis, we demonstrated clock-related gene expressions to be altered in the diabetic mice. Dbp mRNA decreased by 50%, E4bp4 mRNA increased by 50%, and Arnt mRNA decreased by 30% at Zeitgever Time (ZT) 12. Mouse pancreatic islets exhibited oscillations of clock gene expressions. E4BP4, a D-box negative regulator, oscillated anti-phase to DBP, a D-box positive regulator. We also found low-amplitude circadian expression of Arnt mRNA, which peaked at ZT4. Over-expression of DBP raised both mRNA and protein levels of ARNT in HEK293 and MIN6 cell lines. Arnt promoter-driven luciferase reporter assay in MIN6 cells revealed that DBP increased Arnt promoter activity by 2.5-fold and that E4BP4 competitively inhibited its activation. In addition, on ChIP assay, DBP and E4BP4 directly bound to D-box elements within the Arnt promoter in MIN6 cells. These results suggest that in mouse pancreatic islets mRNA expression of Arnt fluctuates significantly in a circadian manner and that the down-regulation of Dbp and up-regulation E4bp4 contribute to direct suppression of Arnt expression in diabetes.
authors.affiliation: Yamaguchi University, Graduate School of Medicine, Division of Endocrinology, Metabolism, Hematological Sciences and Therapeutics, Department of Bio-Signal Analysis, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Nakabayashi
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Ohta
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Susuki
authors.forename: Akihiko
authors.initials: A
authors.lastname: Taguchi
authors.forename: Katsuya
authors.initials: K
authors.lastname: Tanabe
authors.forename: Manabu
authors.initials: M
authors.lastname: Kondo
authors.forename: Masayuki
authors.initials: M
authors.lastname: Hatanaka
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nagao
authors.forename: Yukio
authors.initials: Y
authors.lastname: Tanizawa
mesh_terms: D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000235:genetics / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002940:Circadian Rhythm; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: C497396:ARNT protein, human; C497397:Arnt protein, mouse; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C498260:Dbp protein, mouse; D008565:Membrane Proteins; C497441:Nfil3 protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; C114987:wolframin protein; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator