Phosphorylation Marks IPF1/PDX1 Protein for Degradation by Glycogen Synthase Kinase 3-dependent Mechanisms

The transcription factor IPF1/PDX1 plays a crucial role in both pancreas development and maintenance of β-cell function. Targeted disruption of this transcription factor in β-cells leads to diabetes, whereas reduced expression levels affect insulin expression and secretion. Therefore, it is essential to determine molecular mechanisms underlying the regulation of this key transcription factor on mRNA levels and, most importantly, on protein levels. Here we show that a minor portion of IPF1/PDX1 is phosphorylated on serine 61 and/or serine 66 in pancreatic β-cells. This phosphorylated form of IPF1/PDX1 preferentially accumulates following proteasome inhibition, an effect that is prevented by inhibition of glycogen synthase kinase 3 (GSK3) activity. Oxidative stress, which is associated with the diabetic state, (i) increases IPF1/PDX1 Ser61 and/or Ser66 phosphorylation and (ii) increases the degradation rate and decreases the half-life of IPF-1/PDX-1 protein. In addition, we provide evidence that GSK3 activity participates in oxidative stress-induced effects on β-cells. Thus, this current study uncovers a new mechanism that might contribute to diminished levels of IPF1/PDX1 protein and β-cell dysfunction during the progression of diabetes. The transcription factor IPF1/PDX1 plays a crucial role in both pancreas development and maintenance of β-cell function. Targeted disruption of this transcription factor in β-cells leads to diabetes, whereas reduced expression levels affect insulin expression and secretion. Therefore, it is essential to determine molecular mechanisms underlying the regulation of this key transcription factor on mRNA levels and, most importantly, on protein levels. Here we show that a minor portion of IPF1/PDX1 is phosphorylated on serine 61 and/or serine 66 in pancreatic β-cells. This phosphorylated form of IPF1/PDX1 preferentially accumulates following proteasome inhibition, an effect that is prevented by inhibition of glycogen synthase kinase 3 (GSK3) activity. Oxidative stress, which is associated with the diabetic state, (i) increases IPF1/PDX1 Ser61 and/or Ser66 phosphorylation and (ii) increases the degradation rate and decreases the half-life of IPF-1/PDX-1 protein. In addition, we provide evidence that GSK3 activity participates in oxidative stress-induced effects on β-cells. Thus, this current study uncovers a new mechanism that might contribute to diminished levels of IPF1/PDX1 protein and β-cell dysfunction during the progression of diabetes. The homeodomain transcription factor IPF1/PDX1 (insulin promoter factor 1/pancreas duodenum homeobox 1) operates as a master of pancreas development and also ensures differentiated β-cell function. Homozygous disruption of the Ipf1/Pdx1 gene results in pancreatic agenesis in both mice (1Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1570) Google Scholar, 2Offield M.F. Jetton T.L. Labosky P.A. Ray M. Stein R.W. Magnusson M.A. Hogan B.L.M. Wright C.V.E. Development. 1996; 122: 983-995Crossref PubMed Google Scholar) and humans (3Stoffers D.A. Zinkin N.T. Stanojevic V. Clarke W.L. Habener J.F. Nat. Genet. 1997; 15: 106-110Crossref PubMed Scopus (931) Google Scholar). Moreover, targeted disruption of the Ipf1/Pdx1 gene selectively in β-cells leads to diabetes in mice (4Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (784) Google Scholar), whereas partial reductions of IPF1/PDX1 expression levels in genetically modified mouse models affect insulin expression (4Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (784) Google Scholar) and insulin secretion (5Brissova M. Shiota M. Nicholson W.E. Gannon M. Knobel S.M. Piston D.W. Wright C.V.E. Powers A.C. J. Biol. Chem. 2002; 277: 11225-11232Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and predispose islets to apoptosis (6Johnson J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Investig. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar). In humans, heterozygosity for an inactivating mutation in the Ipf1/Pdx1 gene is associated with development of (MODY 4) maturity onset diabetes of the young (7Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Nat. Genet. 1997; 17: 138-139Crossref PubMed Scopus (8) Google Scholar), a monogenetic form of type 2 diabetes that results from impaired β-cell function characterized by perturbed insulin secretion. Moreover, missense mutations in the Ipf1/Pdx1 gene, in conjunction with other mutations, appear to predispose to the adult onset form of type 2 diabetes (8Cockburn B.N. Bermano G. Boodram L.L. Teelucksingh S. Tsuchiya T. Mahabir D. Allan A.B. Stein R. Docherty K. Bell G.I. J. Clin. Endocrinol. Metab. 2004; 89: 971-978Crossref PubMed Scopus (41) Google Scholar, 9Elbein S.C. Karim M.A. Diabetes Care. 2004; 27: 1968-1973Crossref PubMed Scopus (15) Google Scholar, 10Hani E.H. Stoffers D.A. Chevre J.C. Durand E. Stanojevic V. Dina C. Habener J.F. Froguel P. J. Clin. Investig. 1999; 104: R41-R48Crossref PubMed Scopus (268) Google Scholar, 11Hansen L. Urioste S. Petersen H.V. Jensen J.N. Eiberg H. Barbetti F. Serup P. Hansen T. Pederson O. J. Clin. Endocrinol. Metab. 2000; 85: 1323-1326PubMed Google Scholar). These results strongly support an essential role for IPF1/PDX1 in pancreas development and β-cell function that is conserved from mice to humans. The participation of IPF1/PDX1 in the transcription of several genes essential for glucose sensing and insulin synthesis underlines its key role in differentiated β-cells. IPF1/PDX1 has been shown to bind and regulate the promoter activity of specific β-cell genes such as insulin (12Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (774) Google Scholar), Glut2 (13Waeber G. Thompson N. Nicod P. Bonny C. Mol. Endocrinol. 1996; 10: 1327-1334Crossref PubMed Scopus (325) Google Scholar), glucokinase (14Watada H. Kajimoto Y. Miyagawa J. Hanafusa T. Hamaguchi K. Matsuoka T. Yamamoto K. Matsuzawa Y. Kawamori R. Yamasaki Y. Diabetes. 1996; 45: 1826-1831Crossref PubMed Google Scholar), and islet amyloid polypeptide (15Bretherton-Watt D. Gore N. Boam D.S. Biochem. J. 1996; 313: 495-502Crossref PubMed Scopus (47) Google Scholar, 16Carty M.D. Lillquist J.S. Peshavaria M. Stein R. Soeller W.C. J. Biol. Chem. 1997; 272: 11986-11993Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). At the level of the insulin gene promoter, IPF1/PDX1 acts in concert with other transcription factors, such as Beta2/Neuro D1 and the E2A family proteins, and coactivators including p300 and Bridge-1 (17Ohneda K. Mirmira R.G. Wang J. Johnson J.D. German M.S. Mol. Cell Biol. 2000; 20: 900-911Crossref PubMed Scopus (168) Google Scholar, 18Qiu Y. Guo M. Huang S. Stein R. Mol. Cell Biol. 2002; 22: 412-420Crossref PubMed Scopus (155) Google Scholar, 19Thomas M.K. Yao K.-M. Tenser M.S. Wong G.G. Habener J.F. Mol. Cell Biol. 1999; 19: 8492-8504Crossref PubMed Scopus (64) Google Scholar, 20Stanojevic V. Habener J.F. Thomas M.K. Endocrinology. 2004; 145: 2918-2928Crossref PubMed Scopus (55) Google Scholar, 21Stanojevic V. Yao K.-M. Thomas M.K. Mol. Cell Endocrinol. 2005; 237: 67-74Crossref PubMed Scopus (25) Google Scholar). Thus, it is the accurate protein-protein interactions between transcription factors and coactivators that define the level of expression of the insulin gene. Despite a clearly established role for IPF1/PDX1 in pancreas development and β-cell function, less is known about its regulation, particularly at the post-translational level. Previous reports indicated nuclear translocation of IPF1/PDX1 upon glucose stimulation that correlate with an increase in insulin-promoter activity (22Rafiq I. Kennedy H.J. Rutter G.A. J. Biol. Chem. 1998; 273: 23241-23247Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 23Macfarlane W.M. McKinnon C.M. Felton-Edkins Z.A. Cragg H. James R.F.L. Docherty K. J. Biol. Chem. 1999; 274: 1011-1016Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Others have reported a nutrient-dependent regulation of insulin promoter activity through increased DNA binding capacity of IPF1/PDX1 (24Petersen H.V. Peshavaria M. Pederson A.A. Philippe J. Stein R. Madsen O.D. Serup P. FEBS Lett. 1998; 431: 362-366Crossref PubMed Scopus (73) Google Scholar, 25Macfarlane W.M. Smith S.B. James R.F.L. Clifton A.D. Doza Y.N. Cohen P. Docherty K. J. Biol. Chem. 1997; 272: 20936-20944Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 26Wu H. Marfarlane W.M. Tadayyon M. Arch J.R.S. James R.F.L. Docherty K. Biochem. J. 1999; 344: 813-818Crossref PubMed Scopus (81) Google Scholar). IPF1/PDX1 has also been shown to be potentially modified by post-translational mechanisms such as phosphorylation (24Petersen H.V. Peshavaria M. Pederson A.A. Philippe J. Stein R. Madsen O.D. Serup P. FEBS Lett. 1998; 431: 362-366Crossref PubMed Scopus (73) Google Scholar, 27Fernandez-Alvarez J. Barbera A. Nadal B. Barcelo-Batllori S. Piquer S. Claret M. Guinovart R. 2004; PubMed Scopus Google Scholar, S. S.C. Y. German M.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), Y. Biochem. 2003; PubMed Scopus Google Scholar), and A. T. Y. H. A. J. 2003; PubMed Scopus (81) Google Scholar). the post-translational the this the underlying and the effect on IPF1/PDX1 function synthase kinase 3 (GSK3) glycogen synthase kinase insulin is an kinase that has been in including and and J. Cell 2003; PubMed Scopus Google Scholar). of GSK3 by and that and in most protein GSK3 is in and is stimulation by factors such as and GSK3 a of including such as and as as transcription factors including and J. Cell 2003; PubMed Scopus Google Scholar). The current study to post-translational in the of the pancreatic transcription factor IPF1/PDX1 function. we that IPF1/PDX1 be phosphorylated in on serine 61 and/or serine 66 in pancreatic β-cells. also show that this phosphorylation the protein for degradation by the proteasome and that it is the that oxidative stress, which in the diabetic state, increases serine 61 and/or serine 66 which with an increased degradation rate and a half-life for the IPF1/PDX1 protein. provide evidence of a new mechanism that leads to reduced levels of IPF1/PDX1 protein and β-cell dysfunction during the development of diabetes. and from other from the of the a the of to protein a to the to of has been (12Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (774) Google Scholar). from from phosphorylation on serine of and serine of and from Cell and IPF1/PDX1 expression and a by an the the and mutations the a to the The insulin for the insulin promoter from to of the gene. of the by and by DNA The mouse for from an and Cell by of pancreas B. E. H. U. F. 1997; Full Text PDF PubMed Scopus Google Scholar). the islets for at in modified 3 the islets at in modified with 3 The islets as with and in The mouse and in modified and 2 with in at The in modified 2 and as (4Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (784) Google Scholar). At islets for glucose (12Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (774) Google Scholar) and and with in and of and of by The protein with as of by and the The in and for at The with in by with in with the with and of IPF1/PDX1 by in and of with for 2 at for at with and of the in to of and both for at The by and in protein expression and with to the the indicated of expression to a of DNA protein expression the and protein as the with and the to the The as activity by activity to for the of the at in of the of IPF1/PDX1 in the pancreatic β-cell by an a at and a minor of molecular phosphorylation has been as a post-translational for IPF1/PDX1 (24Petersen H.V. Peshavaria M. Pederson A.A. Philippe J. Stein R. Madsen O.D. Serup P. FEBS Lett. 1998; 431: 362-366Crossref PubMed Scopus (73) Google Scholar, 27Fernandez-Alvarez J. Barbera A. Nadal B. Barcelo-Batllori S. Piquer S. Claret M. Guinovart R. 2004; PubMed Scopus Google Scholar, S. S.C. Y. German M.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we that the might to a phosphorylated form of this IPF1/PDX1 and to the and the be the the that the protein is a phosphorylated form of IPF1/PDX1 protein expression in mouse islets and in pancreatic The phosphorylated and of IPF1/PDX1 protein in islets and pancreatic β-cell in the pancreatic that a minor portion of IPF1/PDX1 is phosphorylated in pancreatic β-cells. of the we conserved phosphorylation Ser61 and that also have been by as phosphorylation for IPF1/PDX1 V. Habener J.F. Thomas M.K. Endocrinology. 2004; 145: 2918-2928Crossref PubMed Scopus (55) Google Scholar, S. S.C. Y. German M.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, we a specific that phosphorylation of IPF1/PDX1 on Ser61 and/or Ser66 the that to the molecular by the IPF1/PDX1 (12Ohlsson H. Karlsson K. Edlund T. EMBO J. 1993; 12: 4251-4259Crossref PubMed Scopus (774) Google Scholar), by the which phosphorylated serine by and to study an of IPF1/PDX1 Ser61 and/or Ser66 we to and mutations in the Ipf1/Pdx1 in and the expression of the proteins, with phosphorylation The type a protein of with a minor of molecular the by the the and to the phosphorylated IPF1/PDX1 whereas the that also be by the The of and the as a protein In both the by the results that Ser61 Ser66 phosphorylation on IPF1/PDX1 that phosphorylation on Ser61 is to of and that Ser61 phosphorylation is to Ser66 Moreover, results support that IPF1/PDX1 is phosphorylated in on Ser61 and/or Ser66 in pancreatic β-cells and that Ser61 and/or Ser66 to be for The of IPF1/PDX1 has been to be by glucose in a At IPF1/PDX1 to be from the and in the whereas to glucose of and in a translocation of IPF1/PDX1 to the W.M. McKinnon C.M. Felton-Edkins Z.A. Cragg H. James R.F.L. Docherty K. J. Biol. 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D. 1994; Full Text PDF PubMed Scopus Google Scholar) on IPF1/PDX1 phosphorylation and protein levels. shown in of with to a of phosphorylated IPF1/PDX1 protein increase and increase whereas in the IPF1/PDX1 protein level. with the protein synthesis the of phosphorylated that this is post-translational This that the phosphorylated form of IPF1/PDX1 is targeted for degradation by the proteasome IPF1/PDX1 an to in IPF1/PDX1 Ser61 and/or Ser66 we with kinase and IPF1/PDX1 phosphorylation of IPF1/PDX1 phosphorylation following with the the kinase the shown to mRNA expression of the IPF1/PDX1 gene insulin S. S.C. Y. German M.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. T. Mol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Clin. Investig. 1996; PubMed Scopus Google Scholar, Y. H. H. K. M. Wong T. J. J. Biochem. 2000; 971-978Crossref PubMed Scopus Google Scholar). of with the GSK3 V. L. Biol. 1996; Full Text Full Text PDF PubMed Google Scholar), in a in IPF1/PDX1 phosphorylation by IPF1/PDX1 protein expression in IPF1/PDX1 also reduced IPF1/PDX1 phosphorylation by with IPF1/PDX1 and an increase in IPF1/PDX1 phosphorylation with with IPF1/PDX1 This level with the increase in protein Moreover, the of reduced the IPF1/PDX1 phosphorylation results that GSK3 activity is to IPF1/PDX1 phosphorylation on Ser61 and/or results that phosphorylation of IPF1/PDX1 might be a for degradation by the proteasome we inhibition of GSK3 activity of the phosphorylated IPF1/PDX1 protein following proteasome shown in prevented the of phosphorylated the that phosphorylation of Ser61 and/or Ser66 of IPF1/PDX1 the phosphorylated protein for degradation by Oxidative IPF1/PDX1 is evidence that oxidative stress, which is in the diabetic state, is in the progression of β-cell J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). IPF1/PDX1 phosphorylation is by the oxidative state, we to oxidative by with The of to a of IPF1/PDX1 phosphorylation results in mouse islets as shown in of IPF1/PDX1 phosphorylation an level of phosphorylated IPF1/PDX1 increase with the increase by following 2 of with which with a in the IPF1/PDX1 protein that phosphorylation IPF1/PDX1 protein for we that a increase in IPF1/PDX1 phosphorylation affect the protein we IPF1/PDX1 protein in with protein synthesis has been by in expression of IPF1/PDX1 in with with with of the that IPF1/PDX1 protein half-life in in an increase in the degradation rate of IPF1/PDX1 following shown in a of phosphorylated IPF1/PDX1 in in in increased the of phosphorylated IPF1/PDX1 with and evidence that phosphorylation of which is by the results that oxidative stress, which leads to and phosphorylation of results in IPF1/PDX1 protein IPF1/PDX1 protein in 3 The following the with for 2 with for the indicated and of by and to the indicated of the shown in B. expression levels of IPF1/PDX1 in at with for for 2 IPF1/PDX1 phosphorylation with IPF1/PDX1 expression is The results the of with for with for with for 2 reports have shown an of GSK3 in to of such as DNA and stress, that GSK3 might a role in effects of P. L. A. Johnson U. S. A. 2002; PubMed Scopus Google Scholar, L. Huang S. D. O. M. C. Y. A. Genes Dev. 2004; PubMed Scopus Google Scholar, Y. J. Cell 2005; PubMed Scopus Google Scholar). provide evidence for a role for GSK3 in oxidative effects in β-cells. In we to a in and an increase in GSK3 in in mouse islets and in with prevented the IPF1/PDX1 the of prevented the of phosphorylated IPF1/PDX1 in following proteasome inhibition to GSK3 in oxidative IPF1/PDX1 protein we the effect of on IPF1/PDX1 protein as shown in the of to prevented the of IPF1/PDX1 protein strongly an role for GSK3 in IPF1/PDX1 Ser61 and Ser66 IPF1/PDX1 IPF1/PDX1 Ser61 and/or Ser66 phosphorylation its activity. we the effect of on the insulin gene promoter activity. to IPF1/PDX1 phosphorylation IPF1/PDX1 protein and effect with to insulin promoter activity At IPF1/PDX1 phosphorylation increased and by reduced levels of IPF1/PDX1 whereas effect with to activity of with in reduced insulin promoter as a of reduced levels of IPF1/PDX1 protein. of insulin promoter activity following IPF1/PDX1 which levels of IPF1/PDX1 Ser61 and Ser66 phosphorylation we also to in insulin promoter activity the effect reported for IPF1/PDX1 and its coactivators on insulin promoter activity be by a effect E2A family on insulin promoter this for the IPF1/PDX1 and the results that Ser61 and/or Ser66 phosphorylation IPF1/PDX1 activity. The IPF1/PDX1 transcription factor plays a crucial role in pancreas development and β-cell function. Despite a of the molecular mechanisms IPF1/PDX1 mRNA has been to define molecular mechanisms IPF1/PDX1 expression at the protein level. of protein expression post-translational In the we that IPF1/PDX1 protein be phosphorylated in in pancreatic β-cells and that this phosphorylation of IPF1/PDX1 the protein for degradation by the proteasome have serine and in the of IPF1/PDX1 and conserved that be phosphorylated in in pancreatic β-cells. results that phosphorylation on Ser61 is an Ser66 phosphorylation of of Ser61 phosphorylation prevented the phosphorylation of the and Ser66 whereas Ser61 phosphorylation the to and Ser66 that Ser61 and, Ser66 phosphorylation might also phosphorylation of the protein on other In this of the we to the at IPF1/PDX1 phosphorylation on several levels of phosphorylation on IPF1/PDX1 protein. Moreover, it to be the molecular phosphorylated IPF1/PDX1 that accumulates in the of this study reported that phosphorylation of IPF1/PDX1 on with degradation of the phosphorylated protein by the proteasome P. E. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, phosphorylation might be to IPF1/PDX1 for The that phosphorylation of Ser61 and/or Ser66 decreases IPF1/PDX1 protein that (i) of phosphorylated IPF1/PDX1 Ser61 and/or following proteasome inhibition, (ii) of IPF1/PDX1 phosphorylation by prevented both the of the phosphorylated protein following proteasome inhibition and the degradation of and protein level increased degradation and half-life of IPF1/PDX1 protein and IPF1/PDX1 phosphorylation on Ser61 and/or Ser66 and from is the that mechanisms Ser61 and/or Ser66 phosphorylation of phosphorylation of serine to affect IPF1/PDX1 protein is that GSK3 has been in the protein of other in the regulation of including for C. M. Huang E. D. 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Previous have shown a of oxidative on insulin gene expression and insulin secretion. translocation of IPF1/PDX1 has been D. Kajimoto Y. H. Y. Y. T. H. Yamasaki Y. M. Diabetes. 2003; PubMed Scopus Google Scholar). the of with we in IPF1/PDX1 of with and we at with to affect which the of the Others have shown a in of IPF1/PDX1 to the insulin promoter of with H. G. N. S. S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) the of for Kajimoto Y. H. H. M. Y. Y. T. Kawamori R. Yamasaki Y. J. Clin. Investig. 1997; PubMed Scopus Google Scholar). IPF1/PDX1 mRNA and protein and DNA binding activity might have been the of IPF1/PDX1 protein levels. In the in insulin promoter activity of reduced IPF1/PDX1 protein expression Thus, it that oxidative might to reduced IPF1/PDX1 protein transcription of IPF1/PDX1 diabetic mice a a IPF1/PDX1 β-cell with that in diabetic mice on a H. Kajimoto Y. Miyagawa Matsuoka Y. Y. Hanafusa T. Matsuzawa Y. Yamasaki Y. M. 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In we provide evidence that a minor portion of IPF1/PDX1 is phosphorylated in β-cells that the of the protein. increased and phosphorylation of IPF1/PDX1 as for during oxidative stress, protein synthesis might be to the of IPF1/PDX1 protein of and a in IPF1/PDX1 protein levels is reduced levels of expression of IPF1/PDX1 have been shown to insulin expression and secretion as as β-cell (4Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (784) Google Scholar, M. Shiota M. Nicholson W.E. Gannon M. Knobel S.M. Piston D.W. Wright C.V.E. Powers A.C. J. Biol. Chem. 2002; 277: 11225-11232Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Investig. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar), it that oxidative stress, by IPF1/PDX1 protein to β-cell GSK3 as for diabetes S. D. K. C. A. Y. J. B. J. J.C. J. E. 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