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• W1996046875 abstract "It has been shown that IGF-1-induced pancreatic β-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic β-cells, INS-1, suggested that PI3K was not necessary for glucose-induced β-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70S6K), suggested that a major part of glucose-dependent β-cell proliferation requires activation of mammalian target of rapamycin/p70S6K, independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced β-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70S6K signal transduction pathways for a full commitment to glucose-induced pancreatic β-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response. It has been shown that IGF-1-induced pancreatic β-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic β-cells, INS-1, suggested that PI3K was not necessary for glucose-induced β-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70S6K), suggested that a major part of glucose-dependent β-cell proliferation requires activation of mammalian target of rapamycin/p70S6K, independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced β-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70S6K signal transduction pathways for a full commitment to glucose-induced pancreatic β-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response. growth hormone insulin-like growth factor 1 insulin receptor substrate phosphatidylinositol 3-kinase Shc homology 2 extracellular signal-regulated protein kinase phosphatidylinositol 3,4,5-trisphosphate protein kinase B phosphatidylinositol 3,4-diphosphate 3-phosphoinositide-dependent kinase 1 glycogen synthase kinase-3 mammalian target of rapamycin p70-kDa-S6-kinase serum- and glucorticoid-regulated protein kinase inter SH2 of p85α multiplicity of infection adenovirus wild type constitutively active kinase-dead green fluorescent protein luciferase The molecular defects causing obesity-linked type 2 diabetes mellitus are not well defined. However, recently it has become clear that key factors involved in causing type 2 diabetes are both impaired β-cell function (1Swenne I. Borg L.A. Crace C.J. Schnell Landstrom A. Diabetologia. 1992; 35: 939-945Crossref PubMed Scopus (64) Google Scholar) and a failure to increase β-cell mass to compensate for peripheral insulin resistance (2Rhodes C.J. J. Mol. Endocrinol. 2000; 24: 303-311Crossref PubMed Scopus (75) Google Scholar, 3Withers D.J. Gutierrez J.S. Towery H. Burks D.J. Ren J.M. Previs S. Zhang Y. Bernal D. Pons S. Shulman G.I. Bonner-Weir S. White M.F. Nature. 1998; 391: 900-904Crossref PubMed Scopus (1337) Google Scholar). It is therefore critical that strategies be developed to replenish the loss of β-cells and/or to expand existing β-cell mass to compensate for insulin resistance. Such an approach relies on defining the mechanisms involved in regulating β-cell mitogenic signaling pathways in response to growth factors and nutrients. Previous studies have shown that certain growth factors, such as growth hormone (GH)1 and insulin-like growth factor 1 (IGF-1), are important for stimulation of β-cell proliferation (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 5Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In addition, these studies showed that GH and IGF-1 are dependent on glucose at concentrations in the physiologically relevant range of 6–18 mm to increase β-cell mitogenesis. However, the basis of this glucose dependence of β-cell proliferation remains unknown. GH signals via the janus kinase-2/signal transducer and activator of transcription-5 pathway in INS-1 β-cells. Although glucose has no independent effect on the activation of this pathway, it is nonetheless required for GH mitogenic action (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 6Billestrup N. Hansen J.A. Hansen L.H. Moldrup A.H. Galsgaard E.D. Nielsen J.H. Endocr. J. 1998; 45 (suppl.): 41-45Crossref Google Scholar). There appears to be no cross-talk of janus kinase-2 activation to insulin receptor substrate (IRS)-mediated signaling by GH. However, activation of phosphatidylinositol 3-kinase (PI3K) mediated by glucose is at least partially required for full GH-induced β-cell proliferation. IGF-1 has been shown to activate at least two major mitogenic signaling pathways, via PI3K and mitogen-activated protein kinase (7Myers Jr., M.G. Grammer T.C. Wang L.M. Sun X.J. Pierce J.H. Blenis J. White M.F. J. Biol. Chem. 1994; 269: 28783-28789Abstract Full Text PDF PubMed Google Scholar, 8Kadowaki T. Tobe K. Honda-Yamamoto R. Tamemoto H. Kaburagi Y. Momomura K. Ueki K. Takahashi Y. Yamauchi T. Akanuma Y. Yazaki Y. Endocr. J. 1996; 43 (suppl.): 33-41Crossref Google Scholar, 9Benito M. Valverde A.M. Lorenzo M. Int. J. Biochem. Cell Biol. 1996; 28: 499-510Crossref PubMed Scopus (104) Google Scholar, 10Yenush L. White M.F. Bioessays. 1997; 19: 491-500Crossref PubMed Scopus (253) Google Scholar). The mitogenic stimulation of receptors, such as the IGF-1 receptor, results in the phosphorylation and activation of the IRS family of proteins and the SH2-containing protein (Shc). Shc then interacts with Shc homology 2 (SH2) domain-containing proteins, such as the growth factor receptor-bound protein-2, whereas IRS proteins interact with the regulatory subunit of PI3K, p85, as well as growth factor receptor-bound protein-2. The recruitment of growth factor receptor-bound protein-2 and its association to murine sons of sevenless-1 protein (a Ras guanine nucleotide exchange factor) activates Ras, resulting in Raf-1 activation, which phosphorylates the extracellular signal-regulated protein kinase (ERK) kinase, which in turn activates ERK1/ERK2 (8Kadowaki T. Tobe K. Honda-Yamamoto R. Tamemoto H. Kaburagi Y. Momomura K. Ueki K. Takahashi Y. Yamauchi T. Akanuma Y. Yazaki Y. Endocr. J. 1996; 43 (suppl.): 33-41Crossref Google Scholar). Interaction of tyrosine-phosphorylated IRS with the p85 regulatory subunit of PI3K leads to the activation of the catalytic subunit, p110, which in turn phosphorylates phosphoinositides at the 3′ position of the inositol ring, generating PIP3 (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 6Billestrup N. Hansen J.A. Hansen L.H. Moldrup A.H. Galsgaard E.D. Nielsen J.H. Endocr. J. 1998; 45 (suppl.): 41-45Crossref Google Scholar). This increase in phosphorylated phosphoinositides leads to the localization of protein kinase B (PKB) to the membrane through the interaction of PIP3 and PIP2 with the pleckstrin homology domain of PKB (11Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). Full activation of PKB appears to be dependent on the subsequent phosphorylation of two residues, Thr-308 in the activation loop of the kinase domain and Ser-473 in the carboxyl-terminal tail (12Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2517) Google Scholar). The protein kinase shown to phosphorylate Thr-308 is 3-phosphoinositide-dependent kinase-1 (PDK1) (13Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 14Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1048) Google Scholar), whereas the kinase responsible for phosphorylation at the Ser-473 residue has not yet been identified. PKB regulates multiple biological processes, such as cell proliferation and apoptosis, suggesting that it may phosphorylate a number of target proteins (reviewed in Ref 15Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). Glycogen synthase kinase-3 α and β (GSK3α/β) have been shown to be two such targets, and their phosphorylation on Ser-21 and Ser-9, respectively, negatively regulates GSK3α/β activity (16Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4374) Google Scholar). PKB has also been implicated in catalyzing phosphorylation activation of mammalian target of rapamycin (mTOR), which then mediates the phosphorylation of p70-kDa-S6-kinase (p70S6K) (17Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (781) Google Scholar, 18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (412) Google Scholar). In β-cells, glucose and IGF-1 activation of p70S6K via the IRS-mediated signal transduction pathway is dependent on PI3K. However, the important signaling components involved in the regulation of mitogenesis that are downstream of PI3K, such as PDK-1, PKB, mTOR, and GSK3α/β, have not been characterized in β-cells, even though it has been shown that p70S6K is activated by glucose and IGF-1 (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 5Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Therefore, in this study we set out to characterize the signaling elements downstream of PI3K that are regulated by glucose and IGF-1 and found that PKB and p70S6K are differentially activated. These data suggested that a major part of the glucose dependence of β-cell proliferation is via a direct activation of mTOR/p70S6K, independent of PDK-1/PKB activation. The PDK-1 kinase assay kit and PDK-1 antibody were from Upstate Biotechnology Inc. (Lake Placid, NY). The phospho-GSK3α/β (Ser-21/9), total PKB, phosphoThr-308-PKB, phosphoSer-473-PKB antibodies, the GSK3 fusion protein, and the PKB kinase assay kit were from New England Biolabs Inc. (Beverly, MA). Anti-phospho-ERK1/ERK2 was obtained from Promega Corporation (Madison, WI), and the total ERK1/ERK2 antiserum was a gift from Dr. M. Cobb (University of Texas Southwestern Medical Center, Dallas, TX). The p70S6K antisera were generated as described (19Yenush L. Zanella C. Uchida T. Bernal D. White M.F. Mol. Cell. Biol. 1998; 18: 6784-6794Crossref PubMed Scopus (75) Google Scholar). Monoclonal anti-c-Myc clone 9E10 was obtained from Sigma. The total GSK3α/β antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-rabbit and anti-sheep IgG horseradish peroxidase conjugates were from JacksonImmuno Research (West Grove, PA), and the anti-mouse IgG horseradish peroxidase conjugate was from Upstate Biotechnology Inc. Wortmannin and rapamycin were from Calbiochem-Novabiochem. IGF-1 was purchased from Gro Pep Pty Ltd. (Adelaide, Australia). The pUSEamp expression vectors containing Myc-His-tagged mouse PKBα (wild type), myr-PKBα (constitutively active), and PKBα-K179M (kinase-dead) were from Upstate Biotechnology Inc. DNA purification kits and Superfect transfection reagents were purchased from Qiagen (Valencia, CA). Restriction enzymes were from New England Biolabs Inc. The bicinchoninic acid protein assay kit was purchased from Pierce. The [methyl-3H]thymidine and chemiluminescence reagent was from PerkinElmer Life Sciences. All other reagents were of analytical grade from either Sigma or Fisher. The glucose-sensitive pancreatic β-cell line INS-1 (20Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar) was maintained in the complete medium RPMI 1640 (11.2 mm glucose) containing 10‥ (v/v) fetal calf serum, 50 μm β-mercaptoethanol, 10 mm HEPES, 2 mmglutamine, 1 mm sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin and incubated at 37 °C in 5‥ CO2 as described (20Asfari M. Janjic D. Meda P. Li G. Halban P.A. Wollheim C.B. Endocrinology. 1992; 130: 167-178Crossref PubMed Scopus (748) Google Scholar). Thymidine incorporation was measured essentially as described, with minor changes. Briefly, INS-1 cells were cultured on 6-well plates to ∼60‥ confluence and infected with adenovirus as described below. The cells were then counted and ∼1 × 104 cells were added to each well of a 96-well plate. The cells were left to attach overnight and were then made quiescent by incubation in starvation medium for 24 h. The INS-1 cells were then incubated for 24 h in starvation medium with additional glucose ± 10 nm IGF-1 as described. During the final 4 h of this incubation 5 μCi/ml [3H]thymidine was added. The specific incorporation of [3H]thymidine into DNA was then measured by transferring the cell lysates to UniFilter-96 GF/C filter plates using the Packard cell harvester, and the radioactivity on the filters was counted with the Packard cell counter. Cells were subcultured on either 6-well or 10-cm plates to ∼60‥ confluence. The cells were then subjected to 24-h serum and glucose deprivation with starvation medium (RPMI 1640 medium containing 0.1‥ bovine serum albumin, 0.5 mm glucose, 100 units/ml penicillin, and 100 μg/ml streptomycin) or infected with adenovirus (see “Adenovirus Infection”), when appropriate, prior to the 24-h incubation with starvation medium. After the quiescent period the cells were pre-treated for 15 min with or without inhibitors as indicated, followed by incubation with fresh starvation medium with or without inhibitors with 0.5, 3, or 15 mm glucose with or without 10 nm IGF-1 for the times indicated. The medium was removed, the cells were washed once with ice-cold phosphate-buffered saline, and the phosphate-buffered saline was replaced with ice-cold cell lysis buffer consisting of 50 mm HEPES (pH 7.5), 1‥ (v/v) Nonidet P-40, 2 mm activated sodium orthovanadate, 100 mm sodium fluoride, 10 mm sodium pyrophosphate, 4 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μg/ml aprotonin. After sonication insoluble material was removed by centrifugation, and the samples were stored at −80 °C. Cells were stimulated as described (see “Stimulation and Lysis Conditions”). The PKB immunoprecipitation and kinase assay were carried out following the procedures described in the manual, whereas some changes were made to the PDK-1 kinase assay. Active PDK-1 was immunoprecipitated as described in the manual. Briefly, ∼1 mg of cell lysate was pre-cleared with 50 μl of protein G-agarose. The beads were removed, the lysate was then added to the protein G-antibody complex, and the samples were left rotating for 2 h at 4 °C. After two washes with cell lysis buffer, followed by two washes with PDK-1 assay dilution buffer, the immunoprecipitates were resuspended in 30 μl of PDK-1 assay dilution buffer containing 10 μl (1 μg) of GSK3 fusion protein and 10 μl of Mg/ATP mixture. The assay was started by the addition of 10 μl (0.5 μg) of serum- and glucocorticoid-regulated protein kinase (SGK)-inactive recombinant protein. SGK is a member of the AGC subfamily of protein kinases whose kinase domain is most related to that of PKB. However, unlike PKB, SGK does not possess a pleckstrin homology domain enabling the assay to be carried out in the absence of PIP3. SGK is phosphorylated at the residue equivalent to Thr-308 of PKB, resulting in activation of this kinase (21Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (526) Google Scholar). Having established conditions to ensure that the kinase activity was well within the linear portion of the reaction, samples were incubated for 7.5 min at 25 °C with continuous shaking. The reaction was terminated with 30 μl of 3× SDS gel loading buffer, and samples were analyzed by immunoblotting (see “Protein Immunoblot Procedures”) with the phospho-GSK3α/β(Ser-21/9) antibody. The PI3K adenoviruses, inter Src homology region 2 (AdV-iSH2), and the catalytic p110α subunit (AdV-p110) were constructed as described (22Frevert E.U. Kahn B.B. Mol. Cell. Biol. 1997; 17: 190-198Crossref PubMed Scopus (156) Google Scholar). The pUSEamp expression vectors containing Myc-His-tagged mouse PKBα (wild type), myr-PKBα (constitutively active), and PKBα-K179M (kinase-dead) were digested with the restriction enzymesHindIII and Pme1. Each of the cDNAs was inserted between the HindIII and SmaI sites of pBluescript, providing the necessary restriction sites for insertion into pAdTrack-CMV between KpnI and NotI. The PKB adenoviruses were generated and purified as described (23Becker T.C. Noel R.J. Coats W.S. Gomez-Foix A.M. Alam T. Gerard R.D. Newgard C.B. Methods Cell Biol. 1994; 43: 161-189Crossref PubMed Scopus (562) Google Scholar, 24He T.C. Zhou S. da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3250) Google Scholar). The appropriate titer for each adenovirus was determined by the addition of various dilutions of each adenovirus to cells subcultured in 6-well plates (9.5 cm2) to 60‥ confluence (∼2 × 106 cells), giving a multiplicity of infection (m.o.i.) ranging from 50 to 2000 based on 0.5–2.0 × 106 plaque forming units/ml as measured byA 260. The viral stock was replaced with complete medium after 2 h, and the cells were incubated at 37 °C in 5‥ CO2 for ∼16 h. The cells were then treated as described. Cell lysates were normalized for total protein after levels were determined using the bicinchoninic acid protein assay kit. For immunoblot analysis 25–50 μg of protein was separated by SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose, membranes were immersed in blocking buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.1‥ Tween, and 5 or 10‥ (w/v) nonfat dry milk) and incubated with gentle agitation for 1 h. This was followed by incubation overnight at 4 °C with the appropriate primary antibody diluted in primary antibody dilution buffer (10 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.1‥ Tween, and 5‥ bovine serum albumin or 10‥ (w/v) nonfat dry milk). After a series of washes with blocking buffer without milk, the membranes were incubated for 1 h with gentle agitation with horseradish peroxidase-conjugated secondary antibody diluted in blocking buffer. Washes were repeated as before, and the positive signals were visualized with chemiluminescence reagent and x-ray film. It has previously been shown that activation of PI3K is required for glucose and IGF-1-induced β-cell proliferation (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 5Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). As such, it was examined whether bypassing the requirement for PI3K activation by an adenoviral mediated constitutively active form of PI3K would unveil the extent of the requirement for this enzyme activity on glucose and IGF-1-induced β-cell proliferation. Previous studies have shown that adenoviral expression of the iSH2 of the p85α subunit with the catalytic p110α subunit (p110) results in constitutive activation of PI3K (22Frevert E.U. Kahn B.B. Mol. Cell. Biol. 1997; 17: 190-198Crossref PubMed Scopus (156) Google Scholar). Because of the presence of a Myc tag on each of the subunits, INS-1 cells infected with recombinant adenovirus to express iSH2 (AdV-iSH2), p110 (AdV-p110), or iSH2 + p110 (AdV-p110 + AdV-iSH2) could be confirmed by subjecting the lysates to immunoblot analysis with the anti-Myc antibody (Fig. 1 A). Bands at 35 and 115 kDa correspond to the predicted molecular mass of Myc-tagged iSH2 and p110, respectively, whereas the band at 40 kDa is nonspecific (as indicated by its presence in uninfected and AdV-β-Gal-infected control cells). The β-cell mitogenesis was determined in INS-1 cells infected with the control β-Gal (AdV-βGal), AdV-iSH2, AdV-p110, or AdV-p110 + AdV-iSH2. The uninfected and AdV-βGal controls showed similar increases in [3H]thymidine incorporation in response to 3 or 15 mm glucose ± 10 nmIGF-1 (Fig. 1 B), as observed previously (4Cousin S.P. Hugl S.R. Myers Jr., M.G. White M.F. Reifel-Miller A. Rhodes C.J. Biochem. J. 1999; 344: 649-658Crossref PubMed Scopus (81) Google Scholar, 5Hugl S.R. White M.F. Rhodes C.J. J. Biol. Chem. 1998; 273: 17771-17779Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). For uninfected INS-1 cells at 15 mm glucose, β-cell mitogenesis was increased 19-fold (p < 0.01), and in the additional presence of 10 nm IGF-1 was 48-fold (p < 0.005) above the 0.5 mm glucose control (Fig. 1 B). In AdV-p110-infected INS-1 cells β-cell mitogenesis in response to glucose and IGF-1 was not significantly different from the uninfected control. In addition, in AdV-iSH2-infected INS-1 cells glucose-induced β-cell mitogenesis was similar to the uninfected control; however, that at 15 mm glucose + 10 nm IGF-1 was elevated to 68.2 ± 8.4-fold (n = 3) above 0.5 mmglucose (Fig. 1 B; p < 0.02). In AdV-p110/AdV-iSH2-coinfected cells glucose-induced β-cell mitogenesis was not significantly altered compared with that in uninfected or AdV-βGal controls in INS-1 cells (Fig. 1 B). However, IGF-1-induced β-cell mitogenesis in AdV-p110/AdV-iSH2 coinfected INS-1 cells was significantly increased 22.2 ± 0.5-fold (n = 3;p < 0.01) at basal 3 mm glucose and 126.5 ± 12.9-fold (n = 3; p < 0.01) at a stimulatory 15 mm glucose above 0.5 mm glucose control (Fig. 1 B). PDK-1 has been suggested to lie downstream of PI3K in signal transduction pathways (15Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1399) Google Scholar). It was determined whether PDK-1 plays a role in the mitogenic signal transduction in INS-1 cells in response to glucose ± IGF-1. PDK-1 activity was assessed in INS-1 cells incubated with 0.5, 3.0, or 15 mm glucose ± 10 nm IGF-1 for 2, 10, and 40 min. PDK-1 was found to be active at 0.5 mm glucose, but neither an increase in glucose nor the presence of IGF-1 had any significant effect on further increasing PDK-1 activity (Fig. 2 B). Total levels of PDK-1 in each of the samples did not vary, indicating that equivalent amounts of PDK-1 were immunoprecipitated (Fig.2 A). Thus, it would appear that PDK-1 is present in INS-1 cells and is constitutively active. Activation of PI3K leads to downstream activation of PKB (25Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). It has been proposed that PKB translocation to the plasma membrane via its pleckstrin homology domain is increased via interaction with the PIP2 and PIP3 products synthesized by increased PI3K activity at the cell plasma membrane (11Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). PKB membrane localization appears to be sufficient to allow PDK-1-mediated phosphorylation of PKB at Thr-308 and partial activation, although full activation requires additional phosphorylation at Ser-473 by an as yet unidentified kinase (13Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar,14Stokoe D. Stephens L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1048) Google Scholar). PKB Thr-308 and Ser-473 phosphorylation and consequential regulation of PKB activity was examined in INS-1 cells incubated at 0.5, 3.0, or 15 mm glucose ± 10 nm IGF-1 for 2, 5, 10, 20, and 40 min. PKB activity did not change significantly when cells were incubated at basal 3 mm glucose or stimulatory 15 mm glucose (Fig.3 A). In contrast, IGF-1 rapidly increased PKB activity, which reached a maximum after 5 min of IGF-1 treatment and was maintained over the 40-min period at a similar level whether at a basal 3 mm or stimulatory 15 mm glucose level (Fig. 3 A). As such, IGF-1-induced activation of PKB activity did not appear to be glucose-dependent. The PKB phosphorylation state was determined in parallel, and negligible phosphorylation at Thr-308 or Ser-473 was observed in INS-1 cells incubated at 3 or 15 mmglucose, correlating with a lack of glucose-induced PKB activity (Fig.3 B). In contrast, IGF-1 induced a rapid and marked increase in PKB Thr-308 phosphorylation, which was maximal at 2 min and then trailed off but was still significantly above that at time zero by 40 min (Fig. 3 B). IGF-1 also markedly increased phosphorylation of PKB at Ser-473 within 2 min, reaching a maximum between 10 and 20 min, which was maintained at 40 min (Fig. 3 B). In general, the Thr-308/Ser-473 phosphorylation of PKB by IGF-1 correlated with IGF-1-induced PKB activity (Fig. 3 A). Neither 3 or 15 mm glucose affected IGF-1-induced PKB Thr-308 or Ser-473 phosphorylation, also correlating with a lack of influence of glucose on IGF-1-induced PKB activity. The total amount of PKB did not significantly alter with glucose/IGF-1 treatment (Fig.3 B). GSK3α/β has been shown to be a phosphorylation substrate of PKB, where phosphorylation of GSK3α/β by PKB inhibits GSK3α/β activity (26Cohen P. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 485-495Crossref PubMed Scopus (133) Google Scholar). It was determined whether glucose and/or IGF-1 could influence endogenous GSK3α/β phosphorylation in β-cells (Fig.4). INS-1 cells were treated at basal 3 mm glucose or stimulatory 15 mm glucose ± 10 nm IGF-1 for 2, 5, 10, 20, and 40 min and then subjected to immunoblot analysis with the phospho-GSK3α/β antibody or total-GSK3α/β antibody (Fig. 4). Neither 3 or 15 mmglucose had any significant effect on GSK3α/β phosphorylation over the 40-min time course (Fig. 4), correlating with a lack of effect of glucose on PKB activity (Fig. 3 A). In contrast, IGF-1 rapidly and markedly increased GSK3α/β phosphorylation within 2 min that was sustained throughout the 40-min time course and was independent of the glucose activation (Fig. 4). This GSK3α/β phosphorylation pattern correlated with IGF-1-induced PKB activity and phosphorylation (Fig. 3, A and B). The total amount of GSK3α/β did not alter with glucose/IGF-1 treatment (Fig.4). Phosphorylation and activation of p70S6K has also been postulated to be downstream of PKB activation via activation of mTOR by PKB (17Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (781) Google Scholar, 18Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (412) Google Scholar). However, mTOR activation can also be mediated by nutrients such as branch chain amino acids (27Hara K. Yonezawa K. Weng Q.P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1122) Google Scholar, 28Kimball S.R. Shantz L.M. Horetsky R.L. Jefferson L.S. J. Biol. 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