Role Of FOXO1 In Pancreatic Beta Cell And Its Molecular Mechanism

Type 2 diabetes is characterized by a progressive decline inβ-cell function and chronic insulin resistance. Pancreaticβ-cells produce insulin and secrete it in response to elevations in circulating blood glucose and to other signaling molecules, providing a key contribution to glucose homeostasis and to the coordination of metabolism within the body. Defects ofβ-cell function in diabetes are complex and include reduced insulin secretion and alterations ofβ-cell mass. Regulation of theβ-cell mass involves a balance ofβ-cell replication and apoptosis, as well as development of new islets from exocrine pancreatic ducts. Disruption of any of these pathways ofβ-cell formation or increased rates ofβ-cell death could cause a decrease inβ-cell mass. Previous studies showed a reduction of islet and/or insulin-containing cell mass or volume in type 2 diabetes, indicating the importance of apoptosis as the cause of pancreaticβcell loss in the development of insulin deficiency and the onset and/or progression of the disease. However, the mechanisms regulatingβcell mass and their adaptations to inRsulin resistance are not complelely defined.FOXO transcription factors control several fundamental cellular processes, including metabolism, cell differentiation, cell cycle arrest, DNA repair, and cellular stress. FOXO1, one of the O subfamily members of FOXO factors, was identified in liver, adipose tissue andβ-cells. Quantitative PCR experiments of FOXO1 mRNA level were significantly higher in islets isolated from type 2 diabetic compared to the matched nondiabetic cadaveric organ donors, indicating that increased FOXO1 was associated withβcell dysfunction. However, the exact funtion of FOXO1 inβcells remains to be further characterized. In this study, the expression and cellular localization of FOXO1 were detected by immunohistochemistry and laser confocal microscopy. Adenoviral vectors expressing a constitutively active form of FOXO1 (FOXO1-AAA) and its small interfering RNA (siRNA) were prepared. The effects of FOXO1 onβcell proliferation and apoptosis were investigated with rat insulinoma cell line (INS-1E) by introducing either active FOXO1 or its siRNA. Furthermore, DNA arrays were carried to reveal the possible mechanisms mediated by the downstream target genes under the control of FOXO1. The major results are summarized below:1. To detect the distribution and subcellular localization of FOXO1 protein in adult rat pancreatic islets, SD rats aged 4-6 weeks were fed for one mouth, then sacrificed and their pancreatic islets were subjected for cryostat section. Distributions of FOXO1 protein in rat pancreatic islets were observed by immunohistochemical staining using anti-FOXO1 antibody. Subcellular localization of FOXO1 was further analyzed by double immunofluorescent staining with anti-FOXO1 and anti-insulin antibody under a laser confocal microscope. Results showed that FOXO1 proteins expressed in pancreatic islet and co-localized with insulin inβcells.2. To evalue overexpression of FOXO1 onβcell function, a constitutively active mutant FOXO1 (FOXO1-AAA) with single-amino-acid-substitution on the three main phosphorylation sites, Thr24/Ala, Ser256/Ala, and Ser319/Ala corresponding to human FOXO1 was used. FOXO1-AAA was subcloned into the pAd-Track vector coexpressing GFP. Adenoviruses carrying the FOXO1-AAA constructs were produced using the pAdeasy adenovirus-packaging system. Expression of FOXO1 on the recombinatant adenovirus can thus be easily detected by fluorescent microscope. The titer of recombinant adenovirus was assayed (about 5×1012U/ml). To test the expression capabilities of recombinant adenoviruses, HepG2 cells were infected with adenovirus carring FOXO1 and the cell lysates were assayed by Western blot, results demonstrated that the FOXO1 remarkablely overexpressed to 9.8 times.3. To investigate down regulation of FOXO1 onβcell function, four siRNA sites specifically targeting FOXO1, named si-FOXO1-1, si-FOXO1-2, si-FOXO1-3 and si-FOXO-pan, were designed using an online software (www.ambion.com). The corresponding double strands of oligodeoxynucleotides sequences for si-FOXO1s and a negative control, a stretch of scrambled sequence not targeting any gene, were synthesized by automatic DNA synthesis machine. After annealing and disgesting with restriction enzymes, the four si-FOXO1s DNA sequences were inserted into the shuttle vector pAd-Track-CMV carrying green fluorescent protein (GFP). Recombinant adenovirus encoding si-FOXO1s were generated after linearisation of pAd-Track-CMV-si-FOXO1s and cotransformed with the adenoviral plasmid pAd-easy-1 in E. coli BJ5183. Kanamycin was used for selection. After linearisation by restriction enzyme PacI, the recombinant plasmids were transfected into HEK 293-cells and amplified to give large quantities of infectious recombinant adenovirus carrying the sequences of si-FOXO1s, designated Ad-si-FOXO1-1, Ad-si-FOXO1-2, Ad-si-FOXO1-3, Ad-si-FOXO-pan and Ad-NC, respectively. The titers as potency of infection were determined in HEK 293 cells (about 5×1012pfu/ml). The efficiency of si-FOXO1s to downregulate the endogenous FOXO1 gene expression were detected by Western blotting in HepG2 cells. Results demonstrated that endogenouse FOXO1 was efficiently suppressed by 45-65% with Ad-si-FOXO1s and 65% with Ad-si-FOXO1-3.4. To test downregulation of FOXO1 on its transcriptional activity, an established FOXO1 target gene, phosphoenolpyruvate carboxykinase (PEPCK), was tested. The 592bp 5′flanking region of PEPCK promoter was amplified with genome DNA of HepG2 by PCR and the segment was cloned into the eukaryotic expression vector pGL3-Basic. HepG2 cells were transfected with plasmid PGL3-PEPCK, followed by infection with Ad-si-NC and Ad-si-FOXO1s recombinant adenoviruses. Effects of si-FOXO1s on the transcriptional activity of PEPCK promoter were analyzed by measuring luciferase activity. The expression level of PEPCK was decreased to 40% by Ad-si-FOXO1-1, 55% by Ad-si-FOXO1-2, 67% by Ad-si-FOXO1-3 and 68% by Ad-si-FOXO-pan.5. To test the expression activities of Ad-si-FOXO1 and Ad-FOXO1-AAA inβcells function, INS-1E cells, a rat insulinoma cell line, were infected with Ad-si-NC, Ad-si-FOXO1-3 or Ad-FOXO1-AAA recombinant adenoviruses for 24h. Endogenous FOXO1 expression levels were determined with Western blotting. Results demonstrated that FOXO1 decreased by 74% with si-FOXO1-3, no statistical change with Ad-si-NC and increased about 8.7 times with Ad- FOXO1-AAA.6. To test effects of up- or down-regulation of FOXO1 onβcell proliferation or its apoptosis, INS-1E was assessed. By infection of the cells with Ad-FOXO1-AAA or Ad-si-FOXO1, 3H-TdR incorporation experiment was performed as a test for cell growth. Results showed that decreased expression of FOXO1 significantly promotes INS-1E growth, whereas increased expression of FOXO1 significantly inhibits cell growth. MTT experiment showed the same effects of FOXO1 expression levels on the survival of INS-1E cells. Furthermore, the effect of FOXO1 on INS-1E apoptosis was assessed by fluorescence activated cell sorter (FACS) and results showed an increased apoptosis with higher levels of FOXO1 expression and decreased apoptosis with lower levels of FOXO1. Insulin secretion of INS-1E cells by radioimmunoassay did not signaficantly change with infection of Ad-si-NC, Ad-si-FOXO1-3 or Ad-FOXO1-AAA.7. To further understand the molecular mechanisms mediating FOXO1 effects onβcell functions, gene array studies were carried out. INS-1E cells were infected with Ad-si-NC or Ad-FOXO1-AAA, and total RNAs were isolated. The cRNA probes prepared from total RNA were hybridized with Agilent rat gene array. After signal detection, image comparison and data analysis, the differentially expressed genes were screened. 254 significantly differentially expressed (with the fold differences of >2, or <0.5) genes were detected by genechips, 107 of them were ESTs (Expressed Sequenee Tag) and the others were genes with complete sequences. 135 showed higher expressions and 33 showed lower expressions. Of them, 16 target genes with a potential role in cell proliferation or apoptosis were discussed.These data demonstrates that FOXO1 proteins expressed in pancreatic islet and co-localized with insulin inβcells. Increased expression of FOXO1 significantly inhibits INS-1E cell growth and survival, but increases cell apoptosis. Sixteen target genes with a potential role in cell proliferation or apoptosis were detected by genechips. Taken together, these data demonstrates that FOXO1 involves in the regulation of survival, proliferation and apoptosis of pancreaticβcells. Dysregulation of FOXO1 expression may thus conttributes to pathogenesis of type 2 diabetes which is closely linked to decreasedβcell mass.