H Y Lin1, Y Yin1, J X Zhang1, H Xuan1, Y Zheng1, S S Zhan1, Y X Zhu1, X Han2. 1. Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China. 2. Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China. hanxiao@njmu.edu.cn.
Abstract
AIMS/HYPOTHESIS: The transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6. METHODS: Chromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP-DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP-DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei. RESULTS: We identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression of A930038C07Rik was significantly decreased and that of Ppa1 was increased in 8-week-old db/db mice. The apoptosis assay showed that overproduction of the protein 'RIKEN cDNA A930038C07' (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis. CONCLUSIONS/ INTERPRETATION: We identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.
AIMS/HYPOTHESIS: The transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6. METHODS: Chromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP-DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP-DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei. RESULTS: We identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression of A930038C07Rik was significantly decreased and that of Ppa1 was increased in 8-week-old db/db mice. The apoptosis assay showed that overproduction of the protein 'RIKEN cDNA A930038C07' (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis. CONCLUSIONS/ INTERPRETATION: We identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.
Authors: T Sivula; A Salminen; A N Parfenyev; P Pohjanjoki; A Goldman; B S Cooperman; A A Baykov; R Lahti Journal: FEBS Lett Date: 1999-07-02 Impact factor: 4.124
Authors: P F Dijkers; R H Medema; C Pals; L Banerji; N S Thomas; E W Lam; B M Burgering; J A Raaijmakers; J W Lammers; L Koenderman; P J Coffer Journal: Mol Cell Biol Date: 2000-12 Impact factor: 4.272
Authors: Kristopher Schwab; Heather A Hartman; Hung-Chi Liang; Bruce J Aronow; Larry T Patterson; S Steven Potter Journal: Dev Biol Date: 2006-04-11 Impact factor: 3.582
Authors: Fang Fang Zhang; Yu Hong Liu; Dan Wei Wang; Ting Sheng Liu; Yue Yang; Jia Min Guo; Yi Pan; Yan Feng Zhang; Hong Du; Ling Li; Liang Jin Journal: Diabetologia Date: 2020-02-01 Impact factor: 10.122
Authors: Yi Dong; Weiwen Chen; Kui Kang; Rui Pang; Yipei Dong; Kai Liu; Wenqing Zhang Journal: Sci China Life Sci Date: 2020-06-18 Impact factor: 6.038