Glucose Mediates the Translocation of NeuroD1 by O-Linked Glycosylation

O-Linked GlcNAc modification of nuclear and cytosolic proteins has been shown to regulate the function of many cellular proteins. Increased O-linked glycosylation, observed under chronic hyperglycemia conditions, has been implicated in the pathogenesis of diabetes. However, the exact role of O-GlcNAc modification in regulating glucose homeostasis remains to be established. We report here that the subcellular localization of the pancreatic beta cell-specific transcription factor NeuroD1 is regulated by O-linked glycosylation in the mouse insulinoma cell line MIN6. Under low glucose conditions, NeuroD1 is mainly in the cytosol. However, treatment of MIN6 cells with high glucose results in O-linked GlcNAc modification of NeuroD1 and its subsequent translocation into the nucleus. Consistent with these data, treatment of MIN6 cells with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate, an inhibitor of O-GlcNAcase, causes Neuro-D1 localization to the nucleus and induction of insulin gene expression even on low glucose. Furthermore, we demonstrate that NeuroD1 interacts with the O-GlcNAc transferase, OGT only at high concentrations of glucose and depletion of OGT by using small interfering RNA oligos interferes with the nuclear localization of NeuroD1 on high glucose. On low glucose NeuroD1 interacts with the O-GlcNAcase and becomes deglycosylated, which is likely to be important for export of Neuro-D1 into cytosol in the presence of low glucose. In summary, the presented data suggest that glucose regulates the subcellular localization of NeuroD1 in pancreatic beta cells via O-linked GlcNAc modification of NeuroD1 by OGT. O-Linked GlcNAc modification of nuclear and cytosolic proteins has been shown to regulate the function of many cellular proteins. Increased O-linked glycosylation, observed under chronic hyperglycemia conditions, has been implicated in the pathogenesis of diabetes. However, the exact role of O-GlcNAc modification in regulating glucose homeostasis remains to be established. We report here that the subcellular localization of the pancreatic beta cell-specific transcription factor NeuroD1 is regulated by O-linked glycosylation in the mouse insulinoma cell line MIN6. Under low glucose conditions, NeuroD1 is mainly in the cytosol. However, treatment of MIN6 cells with high glucose results in O-linked GlcNAc modification of NeuroD1 and its subsequent translocation into the nucleus. Consistent with these data, treatment of MIN6 cells with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate, an inhibitor of O-GlcNAcase, causes Neuro-D1 localization to the nucleus and induction of insulin gene expression even on low glucose. Furthermore, we demonstrate that NeuroD1 interacts with the O-GlcNAc transferase, OGT only at high concentrations of glucose and depletion of OGT by using small interfering RNA oligos interferes with the nuclear localization of NeuroD1 on high glucose. On low glucose NeuroD1 interacts with the O-GlcNAcase and becomes deglycosylated, which is likely to be important for export of Neuro-D1 into cytosol in the presence of low glucose. In summary, the presented data suggest that glucose regulates the subcellular localization of NeuroD1 in pancreatic beta cells via O-linked GlcNAc modification of NeuroD1 by OGT. Regulation of insulin production and secretion in accordance with blood glucose levels is critical for maintaining glucose homeostasis (1Permutt M.A. Kipnis D.M. J. Biol. Chem. 1972; 247: 1194-1199Abstract Full Text PDF PubMed Google Scholar, 2Docherty K. Clark A.R. FASEB J. 1994; 8: 20-27Crossref PubMed Scopus (127) Google Scholar). Defects in insulin production are associated with Type 1 as well as Type 2 diabetes. Induction of insulin gene transcription in response to elevated blood glucose levels plays an important role in maintaining normoglycemia. Glucose regulation of insulin gene transcription in the pancreas has been shown to be regulated by three beta cell-specific transcription factors, Pdx-1, MafA, and NeuroD1 (3Melloul D. Marshak S. Cerasi E. Diabetologia. 2002; 45: 309-326Crossref PubMed Scopus (276) Google Scholar, 4Hui H. Perfetti R. Eur. J. Endocrinol. 2002; 146: 129-141Crossref PubMed Scopus (122) Google Scholar, 5Naya F.J. Stellrecht C.M. Tsai M.J. 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O-Linked GlcNAc transferase (OGT) 2The abbreviations used are: OGT, O-linked GlcNAc transferase; MIN6, mouse insulinoma 6; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate; DON, 6-diazo-5-oxonorleucine; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; GFP, green fluorescent protein; HBP, hexosamine biosynthetic pathway; O-GlcNAcase, N-acetyl-β-d-glucosaminidase.2The abbreviations used are: OGT, O-linked GlcNAc transferase; MIN6, mouse insulinoma 6; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino N-phenylcarbamate; DON, 6-diazo-5-oxonorleucine; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; GFP, green fluorescent protein; HBP, hexosamine biosynthetic pathway; O-GlcNAcase, N-acetyl-β-d-glucosaminidase. modifies nuclear and cytoplasmic proteins by attachment of a single N-acetylglucosamine (GlcNAc) sugar to serine and threonine residues of proteins. 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In this report we demonstrate that high concentrations of glucose the O-linked GlcNAc modification of the transcription factor NeuroD1 in pancreatic beta to its translocation from the cytosol into the nucleus and to the of insulin gene to the of O-GlcNAc modification regulates pancreatic beta cell function and insulin gene Cell insulinoma cells of in modified 2 and J. K. E. H. T. Y. Y. K. PubMed Scopus Google Scholar). the glucose regulation MIN6 cells in modified with glucose and with and to low high glucose for the with the O-(2-acetamido-2-deoxy-d-glucopyranosylidene)-amino and 2 from and of of the green the NeuroD1 by using the at with and at with with and the into the of into MIN6 cells by using II with of and of as S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar). NeuroD1 used for in from using the to the using the used and for the mouse insulin promoter to in at for with the DNA to the with the of Cell and from MIN6 cells in 1 and with to 1 of MIN6 cell as S.S. S. Biochem. Biophys. PubMed Scopus Google Scholar). used in this are: NeuroD1 CTD OGT S.S. S. Biochem. Biophys. PubMed Scopus Google Scholar), O-GlcNAcase and by of cells on in and in as K. 2001; PubMed Scopus Google and with the at the cells in to using to the cell and OGT by using a to and in 1 of 1 cells using a and at for at the in 1 of and in of of the for at RNA and RNA and RNA as S.S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). RNA using used as for with to the insulin and used and for the mouse insulin gene H. Biochem. Biophys. 2002; PubMed Scopus Google and and for the gene 2002; PubMed Scopus Google Scholar). are the as S.S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of and oligos from of the oligos into MIN6 cells as S.S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a of 2 of 2 MIN6 cells and of using the and the II to the after cells with glucose and with to OGT levels and localization by using the A are expressed as NeuroD1 to the on NeuroD1 is of the transcription factors important for glucose regulation of insulin gene expression, we changes in glucose levels NeuroD1 binding to the insulin We the binding of NeuroD1 to the insulin promoter by using the with NeuroD1 in MIN6 cells with glucose NeuroD1 associated with the insulin promoter mainly on high glucose in the insulinoma cell line MIN6 NeuroD1 binding to the insulin promoter in low MIN6 of NeuroD1 binding by a in NeuroD1 binding to the insulin promoter in MIN6 cells with glucose Glucose in of we have that NeuroD1 binds to the insulin promoter mainly at high concentrations of glucose. This be due to changes in DNA binding expression subcellular localization of NeuroD1 in response to glucose. this we the levels of NeuroD1 in low and high MIN6 However, we in NeuroD1 levels in MIN6 cells with low high glucose we glucose regulates the subcellular localization of NeuroD1 and its to the insulin this we a in which the to the of of the into the MIN6 cell the cells on low high glucose for and the subcellular localization of NeuroD1 by We that NeuroD1 mainly cytosolic in MIN6 cells with low glucose and nuclear in cells to high glucose and concentrations of NeuroD1 localization that the translocation of NeuroD1 into the nucleus on high glucose due to the of cells with nuclear localization of we that of the high MIN6 cells an nuclear localization of in the of cells NeuroD1 in the cytosol and nucleus In low MIN6 of the cells nuclear and cytosolic localization of NeuroD1 and in of the cells NeuroD1 in the cytosol nuclear localization of NeuroD1 observed in of low localization of NeuroD1 in MIN6 cells at glucose and of cells with glucose a mainly nuclear localization of In MIN6 cells with and 1 insulin in the presence of low NeuroD1 mainly in the cytosol data suggest that to high glucose causes NeuroD1 to from the cytosol into the nucleus and that this translocation of the changes in and insulin secretion of NeuroD1 on Glucose by the hexosamine biosynthetic has been to be a of glucose levels the this important for nuclear localization of we the levels on low glucose by the MIN6 cells with In addition, we the of by the and of this using of MIN6 cells with in translocation of NeuroD1 into the nucleus even on low glucose of the low cells nuclear and cytosolic localization of in of the low glucose and NeuroD1 to the nucleus of with the nuclear translocation of NeuroD1 on high glucose treatment with the of cells with NeuroD1 and in of the cells NeuroD1 in the nucleus and cytosol with high glucose cells of the cells a nuclear localization of NeuroD1 of 2 to MIN6 cells with high glucose in the presence of the inhibitor the nuclear localization of NeuroD1 of the cells nuclear and cytosolic localization of NeuroD1 with high glucose and However, after of to high glucose and NeuroD1 to the nucleus in of the only of the cells nuclear and cytosolic localization data that the the hexosamine biosynthetic the subcellular localization of Increased O-Linked the of NeuroD1 of that the causes the nuclear localization of of the is used as for O-linked which can regulate the subcellular localization of transcription we the role of O-GlcNAc modification in nuclear localization of NeuroD1 in response to high glucose. MIN6 cells with the with PUGNAc, which the of the O-linked and O-linked glycosylation of proteins. shown in NeuroD1 mainly to the nucleus in low MIN6 cells with of the MIN6 cells with low glucose and a nuclear localization of NeuroD1 of the low MIN6 cells a localization of NeuroD1 to the the of cells NeuroD1 only be in the nucleus the we demonstrate that high glucose O-GlcNAc modification glucose with causes NeuroD1 to from the cytosol into the nucleus. the NeuroD1 also its localization in a glucose and O-linked glycosylation MIN6 cells with high low glucose in the presence of these nuclear and with NeuroD1 only a small of NeuroD1 in the nucleus cells with low the of nuclear NeuroD1 after treatment with high glucose low glucose with data demonstrate that the subcellular localization of NeuroD1 changes in response to glucose as observed with the treatment of MIN6 cells with NeuroD1 to to the nucleus even in the presence of low we the binding of NeuroD1 to the insulin promoter after treatment with using the We that NeuroD1 to to the insulin promoter in MIN6 cells with low glucose in the presence of PUGNAc, that nuclear localization of NeuroD1 is for its binding to the insulin NeuroD1 O-GlcNAc in MIN6 with data with MIN6 cells suggest that nuclear localization of NeuroD1 in the presence of high glucose an O-linked glycosylation we NeuroD1 modified by O-linked glycosylation. this MIN6 cells with low high glucose of the the used for with the NeuroD1 proteins with CTD which O-GlcNAc on proteins. these we that NeuroD1 O-linked GlcNAc modified only in high MIN6 Furthermore, treatment of low MIN6 cells with also to the O-GlcNAc modification of NeuroD1 We also the in which we of the proteins using the and for NeuroD1 Consistent with NeuroD1 O-GlcNAc modified mainly on high glucose NeuroD1 with OGT on Glucose and with O-GlcNAcase on data suggest that NeuroD1 becomes O-GlcNAc modified and into the nucleus in response to high on low NeuroD1 is and the nucleus. we the of NeuroD1 with OGT and O-GlcNAcase in the presence of low high glucose. We OGT O-GlcNAcase from and the proteins with NeuroD1 shown in NeuroD1 interacts with OGT mainly on high glucose and with O-GlcNAcase only on low glucose A in which we NeuroD1 with and with OGT O-GlcNAcase results O-GlcNAc of NeuroD1 by OGT for in the of the role of O-GlcNAc modification in nuclear translocation of we oligos to the expression of OGT. in NeuroD1 is to the nucleus in MIN6 cells with a after treatment with high glucose. However, depletion of OGT by treatment the nuclear localization of NeuroD1 by high glucose NeuroD1 to the nucleus in of the MIN6 cells with the and on high glucose. of cells nuclear and cytosolic localization of NeuroD1 of the OGT NeuroD1 in the nucleus and cytosol in of the cells and in the of the cells NeuroD1 mainly in the cells with high glucose with in the of Glucose have that NeuroD1 is to the nucleus also in low MIN6 cells in the presence of PUGNAc, which is an inhibitor of O-GlcNAcase and the of proteins. the nuclear localization of NeuroD1 on low glucose by treatment is to insulin gene expression, we low MIN6 cells with and insulin levels using We observed a in insulin levels in low MIN6 cells after treatment with PUGNAc, which to the insulin levels observed with high MIN6 cells In this report we demonstrate that the insulin gene transcription factor NeuroD1 is O-linked GlcNAc modified in response to high concentrations of which causes its translocation from the cytosol into the nucleus to insulin gene On low NeuroD1 interacts with O-GlcNAcase and becomes deglycosylated, which is likely to be for its nuclear Consistent with these data, treatment of MIN6 cells with inhibitor of to O-GlcNAc modification of NeuroD1 even on low glucose and to its translocation to the nucleus and insulin gene expression on low glucose. depletion of OGT, which is for O-linked GlcNAc modification of proteins by using oligos in MIN6 the nuclear localization of NeuroD1 in response to high glucose. This the of O-GlcNAc modification of NeuroD1 for its translocation into the nucleus. Consistent with this the of high glucose on NeuroD1 localization can be by treatment of low MIN6 cells with This an important role for the hexosamine biosynthetic and O-linked GlcNAc modification in regulation of NeuroD1 in subcellular localization O-GlcNAc modification has been for several transcription factors N.E. Hart G.W. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, T. J. Biochem. Cell Biol. PubMed Scopus Google Scholar). transcription factor which is expressed in cells has been shown to O-GlcNAc modification is in the nucleus J. 2002; PubMed Scopus Google Scholar). transcription factor that the induction of gene expression is also O-GlcNAc modified and only the nuclear of has this modification after Hart N. Cole R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, nuclear localization of transcription factors and has been shown to with O-GlcNAc modification of these proteins K. F.I. Hart G.W. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. L. T. M.J. A. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, T. N. D. M. S. J. Cell. Biochem. 2002; PubMed Scopus Google Scholar, S.M. M. J. Cell. Biochem. PubMed Scopus Google Scholar). of these transcription factors to be mainly in the cytosol. are with data with the as well as with NeuroD1 using nuclear which that NeuroD1 becomes into the nucleus after O-linked GlcNAc modification in response to high glucose. NeuroD1 has been to be as well as by the of NeuroD1 to be important for its binding to the insulin is the of NeuroD1 in the cytosol nucleus Y. M. Huang S. Stein R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). It is likely that NeuroD1 becomes only is in the nucleus. We demonstrate in this report that NeuroD1 into the nucleus and binds to the insulin promoter on low after treatment of the cells with PUGNAc, which NeuroD1 even on low glucose. suggest that NeuroD1 binding to the insulin promoter is regulated by high glucose we binding on low glucose with we that NeuroD1 is in the nucleus becomes by which its binding for the insulin to of insulin gene data that the nuclear localization of NeuroD1 by O-GlcNAc modification in the presence of high glucose the This is the fact that is to which is the used by OGT to proteins by Consistent with these data, depletion of OGT levels by treatment cytosolic localization of NeuroD1 in MIN6 cells with glucose. the has been to as a for of a cell and the has been implicated to regulate many cellular S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. 2000; PubMed Scopus Google Scholar, J. 2002; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). In with the that the is for the nuclear localization of NeuroD1 and for of insulin gene expression, of to MIN6 cells in the presence of low glucose causes the translocation of NeuroD1 into the nucleus. Furthermore, treatment with in the of high glucose insulin gene expression to levels observed under high glucose Although, data are with the that high glucose the O-GlcNAc modification of NeuroD1 and its translocation into the we the that O-linked GlcNAc modification of in glucose nuclear be in nuclear localization of demonstrate the of O-linked GlcNAc modification of NeuroD1 for its nuclear translocation in response to high is to and the modification on translocation of NeuroD1 from the cytosol into the nucleus in response to high glucose has been Stein R. 2002; PubMed Scopus Google Scholar). Furthermore, shown that of the by using the inhibitor the translocation of NeuroD1 into the nucleus. serine in NeuroD1 into in of NeuroD1 in the cytosol Stein R. 2002; PubMed Scopus Google Scholar). these data that phosphorylation of in NeuroD1 is for its translocation into the nucleus. the in NeuroD1 is to be of the high O-GlcNAc modification K. A. R. S. 15: PubMed Scopus Google Scholar). NeuroD1 becomes O-GlcNAc modified by high glucose at this to the translocation of NeuroD1 into the nucleus as Stein R. 2002; PubMed Scopus Google Scholar). is likely that O-GlcNAc modification of is in regulation of NeuroD1 We have shown that the is for glucose induction of expression in the pancreatic beta cell line MIN6 S.S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is also a beta cell-specific transcription factor for insulin gene data that Pdx-1, beta cell-specific insulin gene transcription factor is also modified by O-linked which to regulate the ability of to to the insulin gene promoter Y. J. Hart G.W. Biochem. Biophys. PubMed Scopus Google Scholar). suggest that and O-linked glycosylation an important role in of the function of insulin gene transcription factors by changes in glucose the and O-linked glycosylation to be of glucose in pancreatic beta cells and regulate insulin gene transcription and beta cell function by glucose. In summary, the data presented in this report demonstrate that high concentrations of glucose the O-GlcNAc modification of NeuroD1 via its with OGT in NeuroD1 localization to the nucleus and of insulin gene On low NeuroD1 interacts with O-GlcNAcase and becomes and the nucleus. to of O-linked GlcNAc modification regulates pancreatic beta cell We for the MIN6 cell line and and Stein for the and and for the O-GlcNAcase and We also for with