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Glucose-stimulated Preproinsulin Gene Expression and Nucleartrans-Location of Pancreatic Duodenum Homeobox-1 Require Activation of Phosphatidylinositol 3-Kinase but Not p38 MAPK/SAPK2*

Open AccessPublished:May 26, 2000DOI:https://doi.org/10.1074/jbc.275.21.15977
      Exposure of islet β-cells to elevated glucose concentrations (30 versus 3 mm) prompts enhanced preproinsulin (PPI) gene transcription and thetrans-location to the nucleoplasm of pancreaticduodenum homeobox-1 (PDX-1; Rafiq, I., Kennedy, H., and Rutter, G. A. (1998) J. Biol. Chem. 273, 23241–23247). Here, we show that in MIN6 β-cells, over-expression of p110.CAAX, a constitutively active form of phosphatidylinositol 3-kinase (PI3K) mimicked the activatory effects of glucose on PPI promoter activity, whereas Δp85, a dominant negative form of the p85 subunit lacking the p110-binding domain, and the PI3K inhibitor LY 294002, blocked these effects. Similarly, glucose-stimulated nucleartrans-location of endogenous PDX-1 was blocked by Δp85 expression, and wortmannin or LY 294002 blocked thetrans-location from the nuclear membrane to the nucleoplasm of epitope-tagged PDX-1.c-myc. By contrast, SB 203580, an inhibitor of stress-activatedprotein kinase-2 (SAPK2)/p38 MAP kinase, had no effect on any of the above parameters, and PPI promoter activity and PDX-1.c-myc localization were unaffected by over-expression of the upstream kinase MKK6 (MAP kinase kinase-6) or wild-type p38/SAPK2, respectively. Furthermore, no change in the activity of extracted p38/SAPK2 could be detected after incubation of cells at either 3 or 30 mm glucose. These data suggest that stimulation of PI3K is necessary and sufficient for the effects of glucose on PPI gene transcription, acting via a downstream signaling pathway that does not involve p38/SAPK2.
      PPI
      preproinsulin
      PDX-1
      pancreatic duodenum homeobox-1
      PI3K
      phosphatidylinositol 3-kinase
      SAPK2
      stress-activated protein kinase-2
      MAP kinase
      mitogen-activated protein kinase
      Erk
      extracellular-regulated protein kinase
      N:C
      nucleus:cytosol ratio
      ATF-2
      activating transcription factor-2
      CMV
      cytomegalovirus
      SUMO-1
      smallubiquitin-related modifier-1
      Glucose homeostasis in mammals requires the proper regulation of insulin secretion from pancreatic islet β-cells. The primary signal for this activated secretion is an elevation in blood glucose concentrations, which enhances the release of stored insulin (
      • Ashcroft F.M.
      • Rorsman P.
      ), the transcription (
      • Nielsen D.A.
      • Welsh M.
      • Casadaban M.J.
      • Steiner D.F.
      ,
      • Permutt M.A.
      • Kipnis D.M.
      ,
      • Efrat S.
      • Surana M.
      • Fleischer N.
      ,
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.O.
      ) and translation (
      • Ashcroft S.J.
      • Bunce J.
      • Lowry M.
      • Hansen S.E.
      • Hedeskov C.J.
      ,
      • Itoh N.
      • Okamoto H.
      ,
      • Welsh M.
      • Nielsen D.A.
      • MacKrell A.J.
      • Steiner D.F.
      ,
      • Welsh M.
      • Brunstedt J.
      • Hellerstrom C.
      ) of the preproinsulin (PPI)1 gene, and the stability of PPI mRNA (
      • Welsh M.
      • Nielsen D.A.
      • MacKrell A.J.
      • Steiner D.F.
      ). However, the mechanisms by which glucose influences the expression of the preproinsulin gene are less well understood than those that control insulin secretion acutely.
      The 5′ flanking region of the PPI gene has been extensively studied and a number of important regulatory elements and trans-acting factors identified (
      • German M.
      • Ashcroft S.
      • Docherty K.
      • Edlund H.
      • Edlund T.
      • Goodison S.
      • Imura H.
      • Kennedy G.
      • Madsen O.
      • Melloul D.
      • Moss L.
      • Olson K.
      • Permutt M.A.
      • Philippe J.
      • Robertson R.P.
      • Rutter W.J.
      • Serup P.
      • Stein R.
      • Steiner D.
      • et al.
      ). Particular interest has centered around thehomeodomain transcription factor, PDX-1 (pancreatic duodenal homeobox-1), previously referred to as IPF-1 (
      • Ohlsson H.
      • Karlsson K.
      • Edlund T.
      ), STF-1 (
      • Leonard J.
      • Peers B.
      • Johnson T.
      • Ferreri K.
      • Lee S.
      • Montminy M.
      ), IDX-1 (
      • Miller C.P.
      • McGehee R.E.J.
      • Habener J.F.
      ), and IUF-1 (
      • Boam D.S.
      • Clark A.R.
      • Docherty K.
      ). PDX-1 binds to a region upstream of the insulin gene, termed the A3 (−216CTAATG) box (
      • Petersen H.V.
      • Serup P.
      • Leonard J.
      • Michelsen B.K.
      • Madsen O.D.
      ), and this binding is increased as glucose concentrations are increased in the near physiological range (0.5–30 mm) (
      • MacFarlane W.M.
      • Read M.L.
      • Gilligan M.
      • Bujalska I.
      • Docherty K.
      ,
      • Marshak S.
      • Totary H.
      • Cerasi E.
      • Melloul D.
      ). Although the signaling mechanisms involved are largely unclear, recent data (
      • MacFarlane W.M.
      • Smith S.B.
      • James R.F.L.
      • Clifton A.D.
      • Doza Y.N.
      • Cohen P.
      • Docherty K.
      ) have implicated the MAP kinase family member p38 MAP kinase (the mammalian homologue of the yeast HOG1 gene, also called reactivating kinase, stress-activated protein kinase-2, SAPK2, CSBP, and Mxi2) (
      • Han J.
      • Lee J.D.
      • Bibbs L.
      • Ulevitch R.J.
      ) in the response of the PPI gene to glucose. These authors reported a profound inhibition of glucose-activated PPI gene expression with the pyridinyl imidazole inhibitor of p38/SAPK2, SB 203580 (
      • Cuenda A.
      • Rouse J.
      • Doza Y.N.
      • Meier R.
      • Cohen P.
      • Gallagher T.F.
      • Young P.R.
      • Lee J.C.
      ).
      We have previously shown that high glucose concentrations cause thetrans-location of epitope-tagged PDX-1 from the nuclear periphery to the nucleoplasm with a concomitant increase in insulin gene transcription (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ). Subsequent subcellular fractionation studies have also shown nuclear trans-location of endogenous PDX-1, although in this case from the cytoplasm to the nucleus (
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ). Arsenite, a metabolic stress that activates p38/SAPK2 (
      • Ludwig S.
      • Hoffmeyer A.
      • Goebeler M.
      • Kilian K.
      • Hafner H.
      • Neufeld B.
      • Han J.
      • Rapp U.R.
      ), was able to mimic the effect of glucose, suggesting a role for this protein kinase in the nuclear trans-location of PDX-1 (
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ).
      In the present study, we demonstrate, by expression of dominant negative and dominant positive forms of the enzyme, that phosphatidylinositol 3-kinase (PI3K) activity is important in glucose regulation of the PPI gene but that p38/SAPK2 is unlikely to be involved. We also provide evidence that elevated glucose concentrations may activate a related, SB 203580-inhibitable stress-activated protein kinase but that this activity is unlikely to be involved in the regulation of the preproinsulin gene.

      EXPERIMENTAL PROCEDURES

      Materials

      Monoclonal anti-c-myc antibody 9E:10 was the kind gift of Dr. Gerard Evan (Imperial Cancer Research Fund, UK). Inhibitors PD 98059, Ro-31–8220, wortmannin, LY 294002, and SB 203580 were all purchased from Calbiochem. Rabbit polyclonal anti-firefly luciferase antiserum and plasmid pRL.CMV, encoding Renilla reniformisluciferase under cytomegalovirus immediate early gene promoter control, were from Promega (Madison, WI). All other reagents were obtained from Promega, Molecular Probes (Eugene, OR), Sigma, or Life Technologies, Inc.

      Methods

      Construction of Plasmids

      PDX-1.c-myc and pINS (260–60).Luc were prepared as described previously (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ). Plasmid pcDNA3.MKK6* contained cDNA expressing full-length MKK6, in which Ser151 and Thr155 were mutated to Glu (
      • Han J.
      • Lee J.D.
      • Jiang Y.
      • Li Z.
      • Feng L.
      • Ulevitch R.J.
      ) under the control of the cytomegalovirus (CMV) immediate-early gene promoter. cDNAs encoding intact full-length p38/SAPK2 and a nonphosphorylatable form of the enzyme in which Thr180 and Tyr182 in the regulatory TGY motif were mutated to Ala and Phe, respectively, were cloned into pEXV3 (
      • Miller J.
      • Germain R.N.
      ) to generate plasmids pEXV3.CSBP2.WT and pEXV3.CSBP2.TAYF, respectively. A chimera between the trans-activation domain of PDX-1 (residues 2–144) (
      • Petersen H.V.
      • Peshavaria M.
      • Pedersen A.A.
      • Philippe J.
      • Stein R.
      • Madsen O.D.
      • Serup P.
      ) and the yeast Gal4 DNA binding domain was generated by polymerase chain reaction amplification (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ) using primers 5′-T.TTT.GGA.TCC.GTA.ACA.GTG.AGG.AGC.AGT.AC (BamHI site underlined) and 5′-T.TTT.GAG.CTC.GGG.TTC.CGC.TGT.GTA.AGC (SacI site underlined), and the product was subcloned into the vector pSG424 (
      • Sadowski I.
      • Ptashne M.
      ). All plasmids were purified on a CsCl gradient (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ).

      Cell Culture and Transfection

      MIN6 cells (
      • Miyazaki J.
      • Araki K.
      • Yamato E.
      • Ikegami H.
      • Asano T.
      • Shibasaki Y.
      • Oka Y.
      • Yamamura K.
      ) (passages 15–25) were continuously cultured in Dulbecco's modified Eagle's medium containing 25 mm glucose, as described previously (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ), and transferred into medium containing 3 mm glucose 24 h prior to the experiments. Jurkat cells were cultured in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum, 100 units/ml−1 penicillin and 100 μg/ml−1streptomycin. Pressure microinjection was performed as described previously (
      • Rutter G.A.
      • Burnett P.
      • Rizzuto R.
      • Brini M.
      • Murgia M.
      • Pozzan T.
      • Tavaré J.M.
      • Denton R.M.
      ,
      • Kennedy H.J.
      • Viollet B.
      • Rafiq I.
      • Kahn A.
      • Rutter G.A.
      ) using glass borosilicate capillaries and a total plasmid concentration of 0.3–0.5 mg/ml−1 in 1 mm Tris-HCl, 0.1 mm EDTA. Lipoamine-mediated transfection was performed using 2.5 μg of DNA/35-mm dish and a 3:1 charge ratio of TFx-50 (Promega):DNA in serum-free medium. Cells were transfected by incubation for 2 h at 37 °C with the DNA mixture before replacing with complete medium. Transfection efficiency was typically 1–5%.

      Photon-counting Digital Imaging

      Photon-counting digital imaging was performed using a cooled intensified photon camera (Photek, Lewes, UK) and an Olympus IX-70 microscope (×10, 0.4 numerical aperture objective) as described previously (
      • Kennedy H.J.
      • Viollet B.
      • Rafiq I.
      • Kahn A.
      • Rutter G.A.
      ,
      • Rutter G.A.
      • Kennedy H.J.
      • Wood C.D.
      • White M.R.H.
      • Tavaré J.M.
      ). Briefly, firefly luciferase activity was measured by imaging for 5 min in the presence of 1 mm luciferin, and the sum of firefly and R. reniformis luciferases were assessed by a further 5-min imaging period in the presence of 5 μm coelenterazine. Individual firefly and Renilla activities for each cell were subsequently obtained by off-line image analysis.

      Immunocytochemistry and Confocal Imaging

      In all cases, MIN6 cells were maintained in 0.5 mm glucose for 24 h prior to transfection with PDX-1 or PDX-1.c-myc. After stimulation, cells were fixed and permeabilized with 4% (v/v) paraformaldehyde and 0.2% Triton X-100. A monoclonal antibody to c-myc (9E:10, 1:200) and rabbit polyclonal antibodies to PDX-1 (1:50) (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ) and firefly luciferase (1:200; Promega) were revealed with fluorescein isothiocyanate-conjugated anti-mouse antibody (1:500) in 0.2% (v/v) bovine serum albumin. Cells were washed with phosphate-buffered saline between incubations and mounted on coverslips with Mowiol before analysis. Confocal images were captured using an upright Leica TCS 4D/DM IRBE™ laser scanning confocal microscope as described previously (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ).

      Incubation and Assay of p38/SAPK2 in Cell Extracts by Phosphorylation of ATF2

      MIN6 cells were grown to ∼80% confluency on 100-mm diameter Petri dishes before incubation with stimuli, as indicated in the legend to Fig. 4. Lysates were prepared by washing with ice-cold phosphate-buffered saline and adding 1 ml of 50 mm Tris-HCl, pH 7.4, containing 1% (v/v) Nonidet P-40, 0.25% Na+ deoxycholate, 150 mm NaCl, 1 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml−1 each aprotinin, leupeptin, and pepstatin, 1 mm Na3VO4, 1 mm NaF, and 1 μm microcystin. Cell debris was removed by centrifugation at 14,000 × g for 5 min and the supernatant taken for enzyme assay as follows. p38/SAPK2 was precipitated by adding 3 μl of rabbit polyclonal anti-p38 antibody and 20 μl of protein A (100 mg/ml−1) and tumbling for 2 h at 4 °C. The protein A pellet was subsequently washed twice in assay buffer comprising 20 mm Hepes, pH 7.5, 20 mm β-glycerophosphate, and 1 mm EDTA and resuspended in 10 μl of complete assay buffer (the above was supplemented with 12 mm mercaptoethanol and 2 mm protein kinase A inhibitor20; Sigma). 10 μl of ATF2 (1.25 mg/ml−1) was added, and the phosphorylation reaction was started by the addition of 5 μl of 0.5 mm [32P]AT (65 MBq/μmol−1) in 50 mm MgCl2. Incubation was continued for 15 min at 30 °C before the addition of 10 μl of SDS-based loading buffer, and electrophoresis by SDS-polyacrylamide gel (10% polyacrylamide). The gel was dried, and autoradiography (Hyperfilm™, Amersham Pharmacia Biotech) was performed as described (
      • Moule S.K.
      • Denton R.M.
      ). Quantification was achieved using ImageQuant™ software.
      Figure thumbnail gr4
      Figure 4Effect of changes in p38/SAPK2 activity on PDX-1 .c-myc localization (A andB) and effect of glucose on p38/SAPK2 activity (C). A, after transfection with PDX-1.c-myc, cells were cultured for 24 h in 0.5 mm glucose. Culture was then continued for 1 h at 3 mm glucose in the additional presence of 10 μm SB 203580 and then at 3 or 30 mm in the continued presence of the inhibitor as shown. Immunocytochemistry was performed and quantified as described under “Experimental Procedures” and the legend to Fig. . Values are given as percent of (%) nucleoplasmic localization ± S.E. for three separate experiments. Statistical significance was calculated by two-tailedt test: ***, p < 0.001, and **,p < 0.01 when compared with 3.0 mmglucose, respectively. B, cells were co-transfected with either empty pcDNA3 (Con) or with plasmid pEXV3.CSBP2.WT (p38), as indicated. Cells were then cultured for a further 24 h in 0.5 mm glucose before stimulation at the indicated glucose concentrations for 2 h. C,lanes 1–4: activity of p38/SAPK2 in extracts of MIN6 cells incubated for 2 h in the presence of 3 (
      • Permutt M.A.
      • Kipnis D.M.
      ) or 30 mmglucose (
      • Rutter G.A.
      • Burnett P.
      • Rizzuto R.
      • Brini M.
      • Murgia M.
      • Pozzan T.
      • Tavaré J.M.
      • Denton R.M.
      ), 3 mm glucose plus 0.1 m sorbitol (S), or 0.1 mm sodium arsenite (Ar);lanes 5 and 6 report p38 activity in extracts of Jurkat cells exposed either to 0.5 m sorbitol (JS) or 100 nm anisomycin (Jan) for 15 min. Phosphorylation of ATF2 and SDS-polyacrylamide gel electrophoresis were performed as described under “Experimental Procedures.”

      Statistical Analysis

      All single-cell data were obtained from three independent experiments, with the number of single cells indicated. Unless indicated otherwise, pooled data are presented as the means (± S.E., given in error bars) for the total number of cells examined in these experiments.

      DISCUSSION

      In this study, we have examined signaling pathways involved in changes in preproinsulin gene transcription and the subcellular location of endogenous and over-expressed PDX-1 in response to changes in glucose concentration.

      Regulation of PPI Gene Expression by PI3K

      Over-expression of PI3K led to a doubling of the insulin promoter activity at low glucose concentrations (3 mm) and a 1.6-fold increase at high glucose concentrations. A truncated form of PI3K (Δp85) lacking the inter-SH2 domain needed for interaction with the catalytic subunit of PI3K, and thus acting as a dominant negative, completely abolished this increase in insulin promoter activity. Moreover, Δp85 expression actually led to a reversal of the glucose response of the insulin promoter, that is, inhibition of promoter activity at high glucose concentrations (Fig. 2). This ability of high glucose to act in the presence of PI3K blockade as an inhibitor of the PPI promoter suggests that the sugar may normally activate both activatory and inhibitory signaling pathways. Only the former pathway, which normally predominates, would appear to require PI3K activation.

      Regulation of Trans-location of Over-expressed and Endogenous PDX-1 by Glucose Role for PI3K

      We have previously shown that an elevation in the concentration of glucose prompts thetrans-location of PDX-1 tagged with a c-mycepitope, from the nuclear periphery to the nucleoplasmic region (
      • Rafiq I.
      • Kennedy H.
      • Rutter G.A.
      ). In the present studies we demonstrate that, like PDX-1.c-myc, endogenous PDX-1 resides largely within the nucleoplasm in cells maintained at either 3 or 30 mmglucose. Unlike PDX-1.c-myc, a small but detectable proportion of endogenous PDX-1 resides in the cytosol at 3 mm glucose. At elevated glucose concentrations, a further recruitment to the nucleoplasm of endogenous PDX-1 is apparent. However, endogenous PDX-1 does not show any evident association with the nuclear periphery in cells incubated at either high or low glucose levels (Fig. 2). Thus, a physiological importance for masstrans-location of PDX-1 between the nuclear periphery and the nucleoplasm seems unlikely at normal levels of PDX-1 expression. We suggest instead that the glucose-sensitive association with the nuclear membrane displayed by PDX-1.c-myc (and by over-expressed, untagged PDX-1) may reflect an interaction with nuclear import/export machinery normally involved in trafficking PDX-1 across the nuclear membrane. In nontransfected cells, this interaction presumably leads to the expulsion from the nucleus of the low levels of endogenous PDX-1 in 3 mm glucose but may become saturated when PDX-1 is over-expressed. Thus, the imaging of the subcellular localization of over-expressed PDX-1.c-myc may provide a convenient assay of the interaction between the endogenous transcription factor and the nuclear membrane, which is not normally evident. Although the composition and identity of the putative nuclear membrane binding protein(s)/complex is unknown, the recent finding by 2-hybrid analysis that the glucose-sensitive Saccharomyces cerevisiaerepressor Mig1 is associated with the nuclear exportin Msn5 raises the intriguing possibility that PDX-1 may be associated with a mammalian Msn5 homologue in β-cells.
      Consistent with the view that the interaction of PDX-1.c-mycwith the nuclear periphery may be important in controlling the nuclear:cytosolic partitioning of endogenous PDX-1, both parameters were regulated similarly by changes in PI3K activity (Fig. 2). In particular, the cytosol → nucleus trans-location of endogenous PDX-1 and trans-location from the nuclear periphery to the nucleoplasm of over-expressed PDX-1.c-mycboth occurred via a wortmannin/LY 294002-sensitive pathway. Further, the present studies show that in the presence of either inhibitor, elevated glucose concentrations provoke a decrease in the proportion of PDX-1.c-myc located in the nucleus. This behavior may therefore provide an explanation of the inhibitory effect of high glucose on the PPI promoter during PI3K blockade (see Fig. 1 and text above).

      Regulation of PPI Promoter Activity and PDX-1 Trans-location by Glucose Involvement of p38/SAPK2

      Recent studies of both PPI promoter regulation (
      • MacFarlane W.M.
      • Smith S.B.
      • James R.F.L.
      • Clifton A.D.
      • Doza Y.N.
      • Cohen P.
      • Docherty K.
      ) and PDX-1 localization (
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ) have described an inhibition with the p38/SAPK2 inhibitor SB 203580 of the effects of glucose. In the present studies, neither incubation of cells with SB 203580 (Fig. 3 A) nor with arsenite at 3 mmglucose (results not shown) had any effect on PPI promoter activity. Furthermore, over-expression of a constitutively active form of MKK6, a kinase immediately upstream of p38/SAPK2 (
      • Han J.
      • Lee J.D.
      • Jiang Y.
      • Li Z.
      • Feng L.
      • Ulevitch R.J.
      ), exerted no effect on PPI promoter activity (Fig. 3 B), whereas expression of this construct strongly trans-activated Gal4 promoter activity in a SB 203580-dependent manner (Fig. 3, C andD). These observations concur with the results of other recent studies (
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.O.
      ) that reported an absence of any effect of SB 203580 on the stimulation of PPI gene expression at 16 mm glucose. Similarly, we observed no effect of manipulating p38/SAPK2 activity on the subcellular localization of endogenous PDX-1 or PDX-1.c-myc (see above). Although we have no clear explanation for the discrepancy between these and the previously published results of MacFarlane and colleagues (
      • MacFarlane W.M.
      • Read M.L.
      • Gilligan M.
      • Bujalska I.
      • Docherty K.
      ,
      • MacFarlane W.M.
      • Smith S.B.
      • James R.F.L.
      • Clifton A.D.
      • Doza Y.N.
      • Cohen P.
      • Docherty K.
      ,
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ), it is possible that differences in experimental protocols (e.g. in studies of PPI promoter activity:microinjection versustransfection; pre-culture in 3 mm rather than 0.5 mm glucose; normalization to the activity of a nonregulated promoter) may be involved. A further possibility, which may explain the effects of SB 203580 observed in earlier studies, may be a nonspecific inhibitory effect on protein kinase B, acting downstream of PI3K.
      C. Marshall, personal communication.
      Nevertheless, the present study clearly indicates that elevated glucose concentrations can robustly stimulate the PPI promoter in the absence of significant activation of p38/SAPK2.
      We also observed in these studies that elevated glucose concentrations stimulate the activity of an SB 203580-sensitive pathway, which leads to the trans-activation of the yeast Gal4.ATF2 chimera (Fig.3 C). Because elevated glucose was found to have no detectable effect on p38/SAPK2 activity measured in MIN6 cell extracts (Fig. 4 C), as also observed for the glucose-responsive INS-1 β-cell line (
      • Khoo S.
      • Cobb M.H.
      ) and human islets,
      G. A. Rutter and M. Dickens, unpublished observation.
      these data suggest that a distinct member of the SAPK family may be activated by glucose in β-cells. This activity may be more sensitive to the mild osmotic stress imposed by glucose (because of the intracellular generation of glycolytic intermediates and other metabolites) (
      • Miley H.E.
      • Sheader E.A.
      • Brown P.D.
      • Best L.
      ) than p38/SAPK2. This alternative SAPK2 activity may be related to a protein kinase of molecular mass 63 kDa recently identified through the use of an in-gel kinase assay (
      • da Silva Xavier G.
      • Dickens M.
      • Rutter G.A.
      ). Whether this activity represents SAPK3/Erk6 (
      • Cuenda A.
      • Goedert M.
      • Craxton M.
      • Jakes R.
      • Cohen P.
      ) is a possibility that remains to be resolved. It now seems feasible that the activation by glucose of MAP kinase activating protein kinase-2, an immediate downstream target of p38/SAPK2 (
      • Rouse J.
      • Cohen P.
      • Trigon S.
      • Morange M.
      • Alonso-Llamazares A.
      • Zamanillo D.
      • Hunt T.
      • Nebreda A.R.
      ), observed by MacFarlaneet al. (
      • MacFarlane W.M.
      • Smith S.B.
      • James R.F.L.
      • Clifton A.D.
      • Doza Y.N.
      • Cohen P.
      • Docherty K.
      ) may have involved this or another glucose/stress-activated protein kinase.

      Regulation of PI3K by Glucose

      The present work indicates that the activation of PI3K is important for the regulation of PDX-1 and for stimulated preproinsulin gene transcription. Indeed, our results suggest that activation of this enzyme is both necessary and sufficient for PDX-1 trans-location and necessary for insulin promoter activation. Supporting this view, GLP-1 (glucagon-like peptide-1) was shown to increase PI3K activity in INS-1 cells and to increase insulin mRNA and the DNA binding activity of PDX-1, an effect inhibited by LY 294002 (
      • Buteau J.
      • Roduit R.
      • Susini S.
      • Prentki M.
      ).
      Because PI3K is a downstream target of insulin receptor substrate-1 and -2, and insulin exerts its effects via PI3K in “classical” insulin-responsive cells (
      • Alessi D.R.
      • Downes C.P.
      ), a role for insulin has been suggested in the activation of the preproinsulin gene (
      • Xu G.G.
      • Rothenberg P.L.
      ,
      • Rutter G.A.
      ). Direct evidence for this role of insulin came from a study in which insulin secreted from the β-cell (both rat islets and HIT-T15 cells) was shown to have a feed forward effect on its biosynthesis by enhancing preproinsulin gene transcription (
      • Leibiger I.B.
      • Leibiger B.
      • Moede T.
      • Berggren P.O.
      ). This autocrine response was further shown to involve the insulin receptor PI3K, p70S6 kinase, and calmodulin kinase pathways. The involvement of PDX-1 in this novel pathway of PPI gene activation is uncertain, although unpublished observations by MacFarlane et al. (
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ) have supported a role for insulin in activation of PDX-1 and the PPI gene via a pathway involving PI3K and p38/SAPK2 but not a rapamycin-sensitive pathway. We have also examined the effect of insulin on PPI promoter activity in MIN6 cells but found no discernible effect at concentrations of 100 nm (
      • Kennedy H.J.
      • Rafiq I.
      • Pouli A.E.
      • Rutter G.A.
      ). Similarly, no change in the transcriptional response to glucose was observed after the near complete blockade of insulin secretion (
      • Kennedy H.J.
      • Rafiq I.
      • Pouli A.E.
      • Rutter G.A.
      ). Thus, the role of insulin secretion in the regulation of insulin gene transcription remains controversial (
      • Rutter G.A.
      ). Furthermore, we have recently noted that the trans-location to the plasma membrane of the exchange factor ARNO (ADP-ribosolyation factornucleotide opening site) (
      • Venkateswarlu K.
      • Oatey P.B.
      • Tavare J.M.
      • Cullen P.J.
      ), which is mediated by binding to a pleckstrin homology domain of phosphatidylinositol 3,4,5-trisphosphate generated by PI3K, is weakly stimulated by glucose but unaffected by insulin concentrations below 1 μm.
      I. Rafiq, K. Venkateswarlu, G. A. Rutter, unpublished results.
      These observations support the view that the activation of PI3K by glucose is likely to involve a direct intracellular signaling mechanism that is independent of insulin secretion.

      Conclusion

      Glucose stimulates PPI promoter activity via an intracellular signaling pathway involving PI3K and protein kinase C but not p38/SAPK2 or Erk-1/2. Part of this response involvestrans-location to the nucleoplasm of PDX-1. The molecular mechanisms involved in triggering the shift of PDX-1 remain to be elucidated. Although it has been proposed (
      • MacFarlane W.M.
      • McKinnon C.M.
      • Felton Edkins Z.A.
      • Cragg H.
      • James R.L.
      • Docherty K.
      ) that a shift in the apparent molecular mass of PDX-1 (from ∼31 → 46 kDa) may also be involved in PDX-1 nuclear trans-location, we have observed no such change in the apparent molecular mass of either endogenous or over-expressed PDX-1.c-myc by Western analysis of MIN6 cell extracts, with PDX-1 migrating at a molecular mass close to 46 kDa in extracts of cells incubated at either 3 or 30 mm glucose (results not shown). Further, although these data suggest that PDX-1 (calculated apparent molecular mass close to 31 kDa) exists as a covalently modified species in MIN6 cells, we have recently found that this is unlikely to be the result of attachment of the candidate molecule SUMO-1 (small ubiquitin-relatedmodifier-1; apparent molecular mass ∼12 kDa) (
      • Mahajan R.
      • Delphin C.
      • Guan T.
      • Gerace L.
      • Melchior F.
      ) involved in targeting RanGAP to the nuclear pore complex protein, RanBP2. Thus, antibodies to SUMO-1 fail to immunoprecipitate PDX-1, and conversely, anti-PDX-1 polyclonal antibody does not immunoprecipitate SUMO-1.
      I. Rafiq and G. A. Rutter, unpublished observation.
      The current studies demonstrate that PI3K activity has a direct effect on the control of insulin gene transcription and the regulation of PDX-1 localization. Whether PI3K is activated directly by glucose, or indirectly by secreted insulin (
      • Rutter G.A.
      ), remains to be determined.

      Acknowledgments

      We thank Drs. Peter Cullen and Jeremy Tavaré (University of Bristol, UK) for plasmids Δp85 and p110.CAAX, respectively, and Dr. Martin Dickens (University of Leicester, UK) for plasmids pCMV.MKK6*, Gal4.luciferase, and Gal4.ATF2. We thank Dr. Chris Marshall (Chester Beatty Institute, UK) for useful discussion. We are grateful to Dr. Mark Jepson and Alan Leard of the Bristol Medical Research Council Cell Imaging Facility for technical support.

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      1. da Silva Xavier, G., Leclers I., Salt, I., Doiron, B., Hardie, D. G., Kahn, A., and Rutter, G. A. Proc. Natl. Acad. Sci. U. S. A., in press