Pancreatic Glucokinase Is Activated by Insulin-Like Growth Factor-I

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Endocrinology 148(6):2904 –2913 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1149

Pancreatic Glucokinase Is Activated by Insulin-Like Growth Factor-I Kazuya Yoshida, Koji Murao, Hitomi Imachi, Wen M. Cao, Xiao Yu, Junhua Li, Rania A. M. Ahmed, Noriko Kitanaka, Norman C. W. Wong, Terry G. Unterman, Mark A. Magnuson, and Toshihiko Ishida Division of Endocrinology and Metabolism (K.Y., K.M., H.I., W.M.C., X.Y., J.L., R.A.M.A., N.K., T.I.), Department of Internal Medicine, Faculty of Medicine, Kagawa University, 761-0793 Kagawa, Japan; Departments of Medicine and Biochemistry and Molecular Biology (N.C.W.W.), Faculty of Medicine, University of Calgary, Health Sciences Center, Calgary, Alberta, Canada T2N 4N1; Departments of Medicine and Physiology and Biophysics (T.G.U.), University of Illinois at Chicago College of Medicine and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612; and Department of Molecular Physiology and Biophysics (M.A.M.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615 Glucokinase (GK) plays a key role in the regulation of glucose use and glucose-stimulated insulin secretion in pancreatic islet cells. Gene targeting of the IGF-I receptor down-regulated pancreatic islet GK activity. That finding prompted us to examine the potential mechanism that may control GK gene activity using an islet cell line, INS-1, known to express IGF-I receptor. Exposure of these cells to IGF-I induced GK protein expression and activity of the enzyme in a dose-dependent manner. In addition, IGF-I induced activity of a reporter construct containing the GK promoter in parallel with the effect on endogenous GK mRNA levels. The stimulatory effect of IGF-I on GK promoter activity was abrogated by wortmannin and LY294002, specific inhibitors of phosphatidylinositol 3-ki-

nase. Exposure of cells to IGF-I elicited a rapid phosphorylation of Akt and FoxO1, a known target of Akt signaling. Constitutively active Akt stimulates the activity of the GK promoter, and a dominant-negative mutant of Akt or mutagenesis of a FoxO1 response element in the GK promoter abolished the ability of IGF-I to stimulate the promoter activity. Furthermore, cell knockdown of FoxO1 with small interfering RNA disrupted the effect of IGF-I on GK expression. These results demonstrate that the phosphatidylinositol 3-kinase/Akt/FoxO1 pathway contributes to the regulation of GK gene expression in response to IGF-I stimulation. (Endocrinology 148: 2904 –2913, 2007)

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cascade have been identified in pancreatic ␤-cells. Tissuespecific knockout of the insulin or IGF-I receptor (IGF-IR) leads to altered glucose sensing and glucose intolerance in adult mice. The phenotype of knockout mice lacking the insulin receptor showed an age-dependent decrease in pancreatic islet size and ␤-cell mass. These data suggest that both receptors appear to be important for glucose metabolism in tissues but may not play a major role in the early growth and development of the pancreatic islets (8). A recent report showed that selective knockout of the IGF-IR in ␤-cells yielded mice with hyperinsulinemia, glucose intolerance, and reduced expression of pancreatic GK (9). These findings point to the potential importance of IGF-I in regulating GK expression. However, little is known regarding the role of IGF-I in connection with pancreatic GK expression. Our findings show that IGF-I acts through phosphatidylinositol 3-kinase (PI3-K)/Akt and FoxO1 stimulate GK gene expression in INS-1 cells.

N PANCREATIC ␤-CELLS, glucokinase (GK) is a high Michaelis constant (Km) enzyme that phosphorylates glucose. This reaction is the rate-limiting step for glucose metabolism linked to the induction of insulin secretion and hepatic glucose use (1, 2). Small changes in GK activity have large effects on insulin secretion in pancreatic ␤-cells (3). Mutation of GK can lead to maturity-onset diabetes of the young (4) or hyperinsulinemia (5). The central role in regulating glucose homeostasis provides a strong rationale for studying the regulation of this gene. Insulin and IGF-I belong to a family of growth factors that regulate metabolism, growth, and cell differentiation and survival (6). The receptors for these growth factors are present in most mammalian cells. Members of this hormone family bind to distinct transmembrane receptors that activate multiple signaling cascades, which promote protein synthesis and glucose transport and regulate gene expression in many tissues, including skeletal muscle, liver, fat, and pancreas (7). Many components of the insulin/IGF-I signaling

Materials and Methods First Published Online February 22, 2007 Abbreviations: Akt-CA, Constitutively active Akt; Akt-DN, dominant-negative mutant of Akt; DM, diabetes mellitus; FRS, FoxO response sequence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GK, glucokinase; IGF-IR, IGF-I receptor; IR, insulin receptor; Km, Michaelis constant; PBS-T, Tween 20 in PBS; PI3-K, phosphatidylinositol 3-kinase; siRNA, small interfering RNA; Vmax, maximal velocity. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

Materials Wortmannin, LY294002, PD98059, bisindolylmaleimide I, and SB203580 were purchased from Calbiochem (Darmstadt, Germany). Recombinant IGF-I was a gift from Fujisawa Phamaco Co. Ltd. (Osaka, Japan).

Plasmid preparation An expression vector encoding a constitutively active Akt (Akt-CA) and a dominant-negative mutant of Akt (Akt-DN) was described pre-

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Yoshida et al. • Role of IGF-I/Akt/FoxO1 on GK Expression

viously (10). An expression vector encoding a constitutively active p110 subunit was gift from J. Downward (Imperial Cancer Research Foundation, London, UK). The GK reporter gene construct GK (1000/⫹14) was created by cloning the rat GK promoter fragment 1000 to ⫹14 into the promoterless luciferase reporter gene vector pGVB2 basic (Promega, Charbonnier, France) (11, 12). To generate the construct GK [1000/⫹14/ FoxO response sequence (FRS) mut] the FRS within the vector GK (1000/⫹14) was mutated from 582 5⬘-GCCCTCTAGTGTACTCAGATACATAAAATAAGTAAATAAATCTTTAAC-3⬘ 534 to 5⬘-GCCCTCTAGTGTACTCAGATACATAAAATAAGTGCGTAAATCTTTAAC-3⬘ (mutated nucleotides are underlined) by site-directed mutagenesis as previously reported (13). These mutations replace residues that are critical for mediating effects of FoxO1 via related FRS (TTGTTTAC) and include mutations of nucleotides that have been shown to be critical for the binding of recombinant FoxO1 to FRS (14).

Cell culture The INS-1 cells originated from a rat insulinoma cell line developed and propagated at the Division of Biochimie Cliniqe (courtesy of C. B. Wollheim, Geneva, Switzerland). The present experiments were performed using cell passages 61–72, the cells being trypsinized every 10 d. These cells were cultured in RPMI 1640 media (Gibco BRL, Tokyo, Japan) containing 11.2 mm glucose (unless otherwise stated) and supplemented with 10% heat-inactivated fetal bovine serum (Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan), 50 ␮mol/liter 2-mercaptoethanol, 100 U/ml penicillin, and 0.1 mg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37 C. When 80% confluent, the cells were washed twice and incubated with 0.5% fetal bovine serum RPMI 1640 media for 12 h before being stimulated with IGF-I. The cells were treated with varying doses of IGF-I for 24 h before harvesting.

Endocrinology, June 2007, 148(6):2904 –2913

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␮m of each primer. The sequences of the forward and reverse rat GK primers were 5⬘-TATGAAGACCGCCAAGTGA-3⬘ and 5⬘-TTTCCGCCAATGATCTTTTC-3⬘, respectively. The cycling program consisted of initial denaturation for 600 sec at 95 C followed by 55 cycles of 95 C for 5 sec, 62 C for 5 sec, and 72 C for 15 sec, with 20 C/sec slope. Each set of PCRs included water as a negative control and five dilutions of standard. Known amounts of DNA were then diluted to provide standards and a regression curve of crossing points vs. concentration generated with the LightCycler. ␤-Actin was used as housekeeping standard.

Transfection of INS-1 cells and luciferase reporter gene assay To confirm the transcriptional regulation by IGF-I/PI3-K/Akt/ FoxO1 signal transduction pathway of GK expression, we used a construct containing the rat pancreatic GK promoter obtained using PCR and cloned into the luciferase reporter gene as previously described (11). Purified reporter plasmid was transfected into INS-1 (at 80% confluence) using Lipofectamine (Life Technologies, Gaithersburg, MD). Transfected cells were maintained in control media containing 0.5 ␮g/ml (70 nm) IGF-I with or without cotransfection of a vector expressing constitutively active form of Akt (myristylated Akt lacking the pleckstrin homology domain; Akt-CA) or an expression plasmid encoding a dominant-negative mutant of Akt (Akt with a K197M mutation; Akt-DN) for 24 h as previously described (10). Transfected cells were harvested, and ␤-galactosidase activity was measured in an aliquot of the cytoplasmic preparation. Twenty-microliter aliquots were taken for the luciferase assay, which was performed according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).

PCR technique

Immunoblot of Akt and FoxO1

We synthesized cDNA using reverse-transcribed total RNA from INS-1 cells. The expression of IGF-IR was detected by the RT-PCR method as described previously (15). The sense primer for the reaction was 5⬘-CCGGCCTTTCACTCTGTACC-3⬘ and the antisense primer was 5⬘-CCTGGGTTTAGACGGTTAAG-3⬘ corresponding to the published sequences (16). IGF-1R was carried out with a thermal cycler (Sanko Junyaku, Tokyo, Japan) according to a step program of 60 sec at 94 C, 60 sec at 51– 60 C, and 60 sec at 72 C, followed by a 15-min extension at 72 C.

Cells were lysed for 10 min in ice-cold buffer A [50 mmol/liter Tris-HCl (pH 7.5), 1 mmol/liter EDTA, 1 mmol/liter EGTA, 0.5 mmol/ liter Na3VO4, 0.1% of 2-mercaptoethanol, 1% Triton X-100, 50 mmol/ liter NaF, 5 mmol/liter sodium pyrophosphate, 10 mmol/liter sodium glycerophosphate, 0.1 mmol/liter phenylmethylsulfonyl fluoride, 1 ␮mol/liter microcystin, and 1 ␮g/ml each pepstatin, aprotinin, and leupeptin]. The total cell lysates, mostly the cytoplasmic fraction, were centrifuged, and the supernatants were collected. The supernatants containing protein concentration of 20 ␮g/ml were used for immunoblotting according to standard procedures. Akt phosphorylated at Thr308 and FoxO1 phosphorylated at Ser256 was detected by using a phosphospecific Akt polyclonal antibody (diluted 1:500; Upstate Biotechnology, Lake Placid, NY), and a phospho-specific FoxO1 polyclonal antibody (diluted 1:1000; Cell Signaling), and total Akt (diluted 1:500, Upstate Biotechnology, NY), and FoxO1 (diluted 1:1000, Cell Signaling, Beverly, MA) was detected by using phosphorylation-independent antibodies (Upstate Biotechnology). The protein bands were visualized by chemiluminescence.

Western blot analysis INS-1 cells were processed as described previously (17). The proteins were separated using a 10% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane for immunoblotting. The membranes were incubated with 0.1% Tween 20 in PBS (PBS-T) containing anti-IGF-IR antibody (diluted 1:1500; Santa Cruz Biotechnology, Santa Cruz, CA) from whole antiserum as previously described) and anti-GK antibody (diluted 1:1000; Santa Cruz Biotechnology). An antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; diluted 1:1000; Trevigen, Gaithersburg, MD) was used as the internal standard for cytosolic extract. The membranes were then washed with PBS-T and incubated for 1 h at room temperature in PBS-T containing a second antibody linked to horseradish peroxidase. The signal was visualized by using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). For immunoprecipitations, 500 ␮g of protein were incubated with the indicated antibodies and protein A-Sepharose for 16 h at 4 C. Antibodies directed against the subunit of insulin receptor (IR) or IGF-IR were used for their respective immunoprecipitations. Immune complexes were then immunoblotted with an antibody directed against phosphothyrosine.

Real-time RT-PCR PCRs were performed in a final volume of 20 ␮l in LightCycler (Roche, Mannheim, Germany) glass capillaries. The reaction mixture consisted of 2 ␮l LightCycler-FastStart DNA Master SYBR Green I (Roche), 2.4 ␮l 25 mm MgCl2 stock solution, 11.6 ␮l sterile PCR-grade H2O, 2 ␮l of the cDNA template for each gene of interest, and 1 ␮l of 10

GK enzymatic activity INS-1 cells were preincubated for 90 min in 2.8 mm glucose, homogenized, and then centrifuged for 10 min at 12,000 ⫻ g to remove mitochondrial-bound hexokinase (18). DNA content was measured in 10-␮l aliquots ⫻ 3. Glucose phosphorylation was measured by the conversion of NAD⫹ to NADH using exogenous glucose-6-phosphate dehydrogenase. Extract (5 ␮l) was added to 80 ␮l of reaction buffer [50 mm HEPES/ HCl (pH 7.6),100 mm KCl, 7.4 mm MgCl2, 0.05% BSA, 5 mm ATP, 0.5 mm NAD⫹, 15 mm ␤-mercaptoethanol, 0.7 U/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Roche Molecular Biochemicals, Indianapolis, IN; 6 –100 mm glucose] and incubated 90 min at 37 C. The reaction was stopped with 1 ml 500 mm NaHCO3 (pH 9.4). Triplicate samples were measured for glucose concentration (excitation 350 nm/emission 460/nm) and the mean value used as a single observation. The standard curve used glucose-6-phosphate standards (0.3–3.0 nmol) in reaction buffer that contained 100 mm glucose. Glucokinase maximal velocity (Vmax) and Km were calculated by linear regression from an Eadie-Scatchard plot [␯/(s) vs. ␯] after extrapolating the data to 37 C assuming a Q10 of 2 followed by 10 cycles.

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Transfection of small interfering RNA (siRNA) The siRNAs were designed to target the following cDNA sequences: IGF-IR scrambled siRNA, 5⬘-CCAAUGUUAAUACAUCCAACU-3⬘; and IGF-IR siRNA, 5⬘-AAACUCUUCUACAAUUACGCA-3⬘ (19); FoxO1 scrambled siRNA, 5⬘-GGCUUAUUGUUCUUAGUAAGA-3⬘; and FoxO1 siRNA, 5⬘-GGAGAUACCUUGGAUUUUAUU-3⬘ (20). Transfection of the siRNA was performed using siPORT Amine (Ambion, Foster City, CA).

EMSA INS-1 cells were serum starved for 6 h and then incubated with IGF-I (0.5 ␮g/ml) for 24 h, and nuclear extracts were prepared according to a technique described previously (12). Double-stranded oligonucleotides containing the wild-type 5⬘-CATAAAATAAGTAAATAAATCTTTAAC3⬘or mutated sequences 5⬘-CATAAAATAAGTGCGTAAATCTTTAAC-3⬘ (mutated nucleotides are underlined) were end labeled with [␥-32 P]ATP using T4 polynucleotide kinase. Each binding reaction of 20 ␮l contained 25 mm HEPES, 50 mm KCl, 1 mm EDTA, 0.5 mm spermidine, 0.6 mm dithiothreitol, 12% glycerol, 5 ␮g poly (dI 䡠 dC), 1 fmol radiolabeled probe, and 20 ␮g of nuclear extract. In competition analysis, 50- or 200-fold molar excess of unlabeled competitor DNA was added to the reaction before the addition of the nuclear extracts. Bound and free probe were resolved on 6% nondenaturing polyacrylamide gels before autoradiography.

Results IGF-IR expression in pancreatic cells

We used RT-PCR to confirm the expression of IGF-IR in the rat pancreatic islets and the rat insulinoma cell line, INS-1.


Results (Fig. 1A) showed that IGF-IR mRNA was detected in not only rat pancreatic islet but also the INS-1 cells. This observation validates the use of INS-1 cells for the studies below because of its expression of the IGF-I receptor. IGF-I increases abundance and activity of GK and in INS-1 cells

Whether IGF-I induced INS-1 GK expression was tested by exposing the cells to varying concentrations (0.005–5.0 ␮g/ ml) of the growth factor. Western blot analysis (Fig. 1B) showed increased abundance of GK protein in response to rising concentrations of IGF-I with maximum induction observed at 0.5 ␮g/ml of the growth factor. Furthermore, the abundance GK mRNA also increased aftertreatment with IGF-I (Fig. 1C). GK is part of the sensing mechanism within the ␤-cells that monitors ambient glucose for the purpose of regulating the secretion of insulin. GK protein abundance does not tell whether IGF-I affected catalytic activity; therefore, we measured both Vmax and Km of the enzyme (Table 1). GK activity (Fig. 2) extracted from INS-1 cells treated for 24 h with or without IGF-I showed a significant rise in GK Vmax to 135 ⫾ 2% of control (Table 1), but the Km of the enzyme remained unchanged. These results show that IGF-I induces pancreatic GK protein abundance in INS-1 cells.

FIG. 1. IGF-IR expression in pancreatic and INS-1 cells plus effects of IGF-I on GK expression. A, IGF-IR mRNA in rat pancreatic islets and INS-1 cells. Total RNA was isolated from rat pancreas and INS-1 cells and examined using RT-PCR to detect IGF-IR and ␤-actin (internal control) mRNA. Lane 1, Rat pancreas; lane 2, INS-1; lane 3, primer only. B, Effects of IGF-I on GK expression. IGF-I increases GK protein expression. Whole-cell extract from INS-1 cells treated with varying concentrations of IGF-I (shown on top of each of lanes) and probed for GK protein using Western blot analysis. Abundance of GAPDH served as control and is shown on the bottom of each lane, and the ratio of GK to GAPDH is shown as percent of control in the figure. An identical experiment independently performed gave similar results. The asterisk denotes significant difference (P ⬍ 0.001). C, Effect of IGF-I on GK mRNA expression. This graph shows the level of GK mRNA in INS-1 cells treated with 0.5 ␮g/ml IGF-I by the real-time PCR method. Control, No treatment; IGF-I, 0.5 ␮g/ml IGF-I. A graph showing the mean ⫾ SEM of three experiments for each treatment group is shown. The asterisk denotes a significant difference (P ⬍ 0.01).



Effect of IGF-I on GK promoter activity in INS-1 cells

TABLE 1. Kinetic parameters for GK in INS-1 cells

Control IGF-I

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Vmax

Vmax

Km

7.97 ⫾ 0.15 10.8 ⫾ 0.11a

100 ⫾ 2 135 ⫾ 2a

13.2 ⫾ 0.11 14.3 ⫾ 0.40

INS-1 cells were cultured for 24 h (n ⫽ 3) in media with the presence or absence of 0.5 ␮g/ml IGF-I. GK Vmax was measured, and it is expressed as percentage of control INS-1. Statistical significance was determined by comparing the measurements in each group (a P ⬍ 0.01). Vmax and Km values for GK were calculated as described in the text.

Whether changes in the GK promoter activity mediated IGF-1 induction of the gene was tested using transient transfections of INS-1 cells with a reporter that contained the gene segment extending from –1000 to ⫹14 (Fig. 4A). Consistent with the observed changes in the levels of GK protein, IGF-I stimulated GK promoter activity in INS-1 cells. Thus IGF-1 increases GK promoter transcriptional activity. IGF-I induces GK promoter via PI3-K pathway and Akt

Functional role of IGF-IR in GK expression induced by IGF-I

To analyze receptor activation by IGF-I, cells were stimulated with 0.5 ␮g/ml IGF-I for 10 min and 6 h, and protein extracts were immunoprecipitated with antibodies directed against the subunit of IR or IGF-IR and immunoblotted with a phosphotyrosine antibody. IGF-I was able to induce phosphorylation of IGF-IR but did not affect the phosphorylation of IR (Fig. 3A). Although it is likely that IGF-1 acts through its receptor, we sought additional proof by knocking down expression of IGF-1R using siRNA. The siRNA for the receptor or a control was transfected into the cells. Western blot analysis (Fig. 3B) showed that expression of IGF-IR protein was inhibited by specific but not control siRNA (Fig. 3B, lane 3). To further extend this observation, both cells expressing control and specific siRNA were treated for 24 h with IGF-I. Whereas IGF-1 increased GK protein in controls cells (Fig. 3C; compare lanes 1 and 2), abundance of the enzyme did not rise in cells carrying siRNA specific for IGF-IR (Fig. 3C; compare lanes 3 and 4). These findings show that IGF-I induction of GK in INS-1 cells requires IGF-IR. To confirm the above observations, we treated INS-1 cells exposed to IGF-I with a neutralizing antibody against IGF-IR. The treatment with the neutralizing antibody against IGF-IR reduced the effect of IGF-I on GK expression (Fig. 3D).

Interaction of IGF-I with its membrane receptor points to the participation of a signaling pathway. To test this idea, we used known inhibitors of signaling pathways to disrupt the IGF-I induction of GK promoter activity. Therefore, we added compounds known to inhibit the PI3-K (10 ␮m wortmannin, LY294002), a mitogen-activated ERK (10 ␮m PD98059), a protein kinase C (1 ␮m bisindolylmaleimide I), or a p38-MAPK (1 ␮M SB203580) before exposing transfected INS-1 cells to IGF-I. Results (Fig. 4A) showed that inhibitors of either the ERK kinase, protein kinase C, or p38-MAPK pathways had no effect on the action of IGF-I but wortmannin, an inhibitor of PI3-K abrogated the growth factor’s ability to induce GK. These observations suggest that the actions of IGF-I appear to be mediated via the PI3-K cascade. Because Akt is one of the downstream components of the PI3-K cascade, we wondered whether it may participate in the IGF-I induction of GK expression. Therefore, we searched for the presence of phosphorylated Akt after treatment with IGF-I. Because phosphorylation of residues Thr308 and Ser473 in Akt is a prerequisite for the catalytic activity of the protein, we measured the kinetics of this reaction. Results (Fig. 4B) showed that Akt phosphorylation was detected within 5 min after treatment with IGF-I. These findings show that IGF-I induction of GK expression appears to be mediated in part by the PI3-K pathway and activation of this cascade leads to Akt phosphorylation. To further confirm the role of PI3-K and Akt in IGF-I induction of GK expression in the INS-1 cells, we postulated that expressing the p110 catalytic subunit of PI3-K or constitutively active Akt (Akt-CA) should induce GK expression. As in the preceding studies (Fig. 4A), there was a 3-fold rise in luciferase activity after IGF-I stimulation. Consistent with our prediction, both constitutively active PI3-K and Akt increased GK promoter activity (Fig. 4C) without exposure to IGF-I. Furthermore, the effect of IGF-I on GK promoter activity was blocked in cells expressing a dominant-negative form of Akt (Akt-DN) (Fig. 4D). Together, these findings support the idea that PI3-K and Akt are required for IGF-I induction of GK expression in INS-1 cells. IGF-I induces FoxO1 phosphorylation and FoxO1 binds to the GK promoter

FIG. 2. Effect of IGF-I on GK Vmax. The phosphorylation of glucose to glucose-6-phosphate in INS-1 cells was measured during a 90-min incubation at the glucose concentrations shown. IGF-I increased GK catalytic activity. INS-1 cells were cultured 24 h at 11.2 mM glucose in the presence (E-E; n ⫽ 3) or absence (f-f; n ⫽ 3) of 0.5 ␮g/ml IGF-I. Incubations carried out from 6 –100 mM glucose represented GK activity.

To further define the pathway by which IGF-I triggered GK induction, we asked whether the transcription factor, FoxO1, was involved because the GK promoter contains a FoxO1 binding site, FRS. FoxO1 is a member of the FoxO family of transcription factors that contains a conserved forkhead domain and three putative phosphorylation sites for

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FIG. 3. siRNA silencing of IGF-IR. A, Phosphorylation of IR and IGF-IR by IGF-I. Cells were stimulated for 10 min or 6 h with 0.5 ␮g/ml IGF-I. Cell lysates were harvested and subjected to immunoprecipitation with antibodies for the IR or IGF-IR. Immune complexes were then immunobloted with an antibody directed against phosphotyrosine. An identical experiment independently performed gave similar results. B, Immunoblots showing inhibition of IGF-IR expression using siRNA treatment (upper portion). Abundance of GAPDH as a control is shown on the bottom of each lane. Lane 1, No treatment; lane 2, siRNA nonspecific for IGF-IR; lane 3, siRNA specific for IGF-IR. C, Effect of knockdown of IGF-IR on IGF-I-induced GK expression. Western blot analysis was performed to determine the GK expression (upper portion). Abundance of GAPDH as a control is shown on the bottom of each lane. Lanes 1 and 2, siRNA nonspecific for IGF-IR; lanes 3 and 4, siRNA specific for IGF-IR. An identical experiment independently performed gave similar results. D, GK expression after stimulation by IGF-I in the presence and absence of an anti-IGF-IR neutralization antibody. Western blot analysis of total cell protein extracted from INS-1 cells 24 h after treatment with control media containing nonspecific IgG (control IgG) or anti-IGF-IR antibody (anti-IGF-IR; sc461 L; Santa Cruz Biotechnology) with or without 0.5 ␮g/ml IGH-I is shown. Abundance of GAPDH served as a control and is shown on the bottom of each lane. A graph showing the mean ⫾ SEM of three experiments for each treatment group is shown on the right. The asterisk denotes a significant difference (P ⬍ 0.01).

Akt. Perhaps IGF-I activates Akt activity leading to the phosphorylation of FoxO1; as a result, the decreased amount of FoxO1 in the nucleus might release the suppression of GK promoter activity. To test this idea, we measured the kinetics of FoxO1 phosphorylation and showed that it was detected within 5 min after exposure of the cells to IGF-I (Fig. 5A, lane 2). Furthermore, IGF-IR knockdown by siRNA canceled the effect of IGF-I on FoxO1 phosphorylation (Fig. 5B). This finding shows that IGF-I induces a rapid phosphorylation of FoxO1 via IGF-IR. Next we tested whether FoxO1 could bind a putative FRS located (⫺550) upstream from the transcription initiation site within the pancreatic GK promoter. For these studies, we isolated nuclear proteins from INS-1cells and used them in EMSA studies using double-stranded oligonucleotide probes containing the putative FRS and flanking sequences. Results (Fig. 5C) showed that the extract contained FRS binding activity, whereas binding to an oligonucleotide probe containing a mutation within the FRS was greatly reduced (Fig. 5C). The formation of this complex was shifted by preincubation of nuclear extracts with antibody against FoxO1, demonstrating that FoxO1 is present in this complex and neces-

sary for its formation (Fig. 5C), and the treatment of IGF-I decreased the formation of this complex (Fig. 5D). To further show that the role of FoxO1 is important in the activity of the GK promoter, we created a reporter gene construct with/ without a mutation within the FRS motif (1000/⫹14/FRSmut). As shown, the expression of FoxO1 inhibited the transcriptional activity of the GK promoter but not that of the mutant GK promoter (Fig. 5E). The expression of Akt-CA or p110 has showed no effect on the mutant GK promoter activity. IGF-I stimulated the activity of the wild-type promoter activity (Fig. 5F), whereas mutation of the FRS dramatically reduced the ability of IGF-I to stimulate GK promoter function. These results suggest that IGF-I induction of GK promoter activity requires the intact FRS motif. To further characterize the role of FoxO1 in regulating the expression of GK in INS-1 cells, we used siRNA to block FoxO1-expression. Initial studies showed that the expression of FoxO1 was inhibited by FoxO1 siRNA treatment but not by a scrambled siRNA (Fig. 5G). Next, INS-1 cells were exposed to FoxO1 specific or scramble siRNA and then treated with IGF-I. As shown in Fig. 5, G and H, GK promoter activity (Fig. 5G) and protein expression (Fig. 5H) was in-



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FIG. 4. Effect of IGF-I on GK transcriptional activity. A, Effects of a PI3-K inhibitor wortmannin (WM), a MAPK kinase-1 inhibitor PD98059 (PD), a protein kinase C inhibitor bisindolylmaleimide I (BIS), or a p38-MAPK inhibitor SB203580 (SB) on IGF-I-induced GK transcriptional activity in INS-1 cells. Values represent the mean of triplicate determinations. B, IGF-I stimulates the phosphorylation of Akt. Cultured INS-1 cells were exposed to 0.5 ␮g/ml IGF-I for the indicated time, and Akt phosphorylation was detected by Western blot analysis with phospho-specific Akt antibody (P-Akt, upper portion) and total Akt (lower portion). An identical experiment independently performed gave similar results. C, Role of PI3-K/Akt signal transduction pathway on GK promoter activity by IGF-I. INS-1 cells were transfected with pGK-LUC or empty vector, empty vector plus IGF-I-treatment (IGF-I), Akt-CA expression vector (Akt-CA), and P110 expression vector (P110) 24 h before cell harvest. Each data point shows the mean and SEM (n ⫽ 4) of separate transfection. D, Akt-DN blocks IGF-I induction of GK transcription. INS-1 cells were transfected with pGK-LUC and empty vector or Akt-DN and then treated with IGF-I for 24 h. Each data point shows the mean and SEM (n ⫽ 4) of separate transfections performed on separate days. The asterisk denotes a significant difference (P ⬍ 0.01). LY, LY294002; N.S., no significant difference.

creased in cells exposed to scrambled siRNA after stimulation with 0.5 ␮g/ml IGF-I. In contrast, IGF-I induction of GK promoter activity and protein expression was significantly suppressed in cells treated with FoxO1-siRNA. Also, treatment with FoxO1-siRNA (but not scrambled siRNA) increased basal GK promoter activity and protein expression, compared with that of control cells. Additionally, GK activity extracted from FoxO1-knockdown INS-1 cells showed a significant rise in GK Vmax to 126 ⫾ 2.2% of control (Table 2), but the Km of the enzyme remained unchanged. These findings are in keeping with the idea that IGF-I induction of GK expression requires FoxO1. Discussion

In this report, we examined the regulation of GK expression in a rat pancreatic insulinoma cell line, INS-1. GK plays a key role in the pancreatic ␤-cell’s ability to sense ambient

glucose and thus regulate insulin secretion. The participation of GK in these processes makes the enzyme an essential component of glucose homeostasis. The small changes in GK activity have pronounced effects in the glucose regulation of insulin secretion (18, 21–23). Thus a better understanding of how GK expression is regulated may lead to novel targets for augmenting insulin release. This knowledge will be useful in the development of new treatments for diabetes mellitus (DM). Although insulin is an important regulator of GK expression and has overlapping functions and structures with IGF-I, much less is known about whether IGF-I may also affect GK gene activity (19). That IGF-I may have a role in regulating GK comes from recent studies in adults demonstrating that lower baseline levels of the growth factor predicted the subsequent development of impaired glucose tolerance (24). In clinical trials involving a large number of patients with type 2 DM, the

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FIG. 5. Role of FoxO1 on GK promoter activity. A, IGF-I stimulates the phosphorylation of FoxO1. Cultured INS-1 cells were exposed to 0.5 ␮g/ml IGF-I for indicated time, and FoxO1 phosphorylation was detected by Western blot analysis with phospho-specific FoxO1 antibody (p-FoxO1, upper portion) and FoxO1 (lower portion). An identical experiment independently performed gave similar results. B, Effect of knockdown of IGF-IR on IGF-I-induced FoxO1 phosphorylation. INS-1 cells treated with IGF-IR siRNA were exposed to 0.5 ␮g/ml IGF-I for indicated time, and FoxO1 phosphorylation was detected by Western blot analysis with phospho-specific FoxO1 antibody (p-FoxO1, upper portion) and FoxO1 (lower portion). An identical experiment independently performed gave similar results. C, Binding of FoxO1 to GK promoter. Double-stranded-labeled oligonucleotides with the sequence of either the wild-type GK promoter or with mutations within the FoxO1-binding sites (FRSmut) were incubated with nuclear extracts. In competition experiments, the indicated molar excess of the indicated unlabeled double-stranded oligonucleotides were used. The arrowhead (SS) indicates the position of supershifted band. Lane 1, Probe only; lane 2, nuclear extracts of INS-1 cell ⫹ 100-fold molar excess unlabeled probe; lane 3, nuclear extracts of INS-1 cell; lane 4, supershift assay with control IgG; lane 5, supershift assay with anti-FoxO1 antibody; lane 6, probe (FRSmut) only; lane 7, nuclear extracts of INS-1 cell with mutant probe (FRSmut). D, Effect of IGF-I on FoxO1 binding. Lane P, Probe only; lane 1, nuclear extracts of INS-1 cells without treatment; lane 2, nuclear extracts of INS-1 cells with 0.5 ␮g/ml IGF-I. E and F, The site-directed mutagenesis of the Forkhead-binding site abrogates the response to FoxO1 (E) or IGF-I (F). The binding site was disrupted by altering three base pairs (5B⫹c-AAA to 5B⫹c-GCG) in the Forkhead-binding site (GK promoter mutant) derived from the parent construct pGK-LUC (GK promoter) as described in Materials and Methods. Each data point shows the mean and SEM (n ⫽ 3) of separate transfections. The asterisk denotes a significant difference (P ⬍ 0.01). N.S., No significant difference. G, Nuclear extracts of the cells treated with FoxO1 siRNA were subjected to Western blot analysis to examine the FoxO1 expression. Analysis of TFIID as a control is shown on the bottom of each lane. Lane C, Scrambled siRNA; siF, FoxO1 siRNA. An identical experiment independently performed gave similar results. siRNA of FoxO1 (FoxO1 siRNA) or scramble siRNA (control siRNA) and pGK-LUC were transfected into INS-1 cells. These INS-1 cells were treated with 0.5 ␮g/ml IGF-I for 24 h before cell harvest. Each data point shows the mean ⫾ SE (n ⫽ 3) of separate experiments. The asterisk denotes a significant difference (P ⬍ 0.01). N.S., No significant difference. H, Effects of FoxO1-knockdown on GK expression in INS-1 cells. siRNA of FoxO1 or scrambled siRNA (control) was transfected into INS-1 cells and then treated with IGF-I. At 48 h after transfection, the abundance of GK protein level was measured using Western blot analysis (upper panel). The ratio of GK to GAPDH is shown as the percentage of control. Each data point shows the mean ⫾ SE (n ⫽ 3) of separate experiments. The asterisk denotes a significant difference (P ⬍ 0.05). N.S., No significant difference.



TABLE 2. Kinetic parameters for GK in FoxO1 knockdown INS-1 cells

Control siRNA FoxO1 siRNA

Vmax

Vmax

Km

7.14 ⫾ 0.14 9.72 ⫾ 0.16a

100 ⫾ 1.9 126 ⫾ 2.2a

12.8 ⫾ 0.16 137. ⫾ 0.50

INS-1 cells were transfected with control or FoxO1 siRNA (n ⫽ 3). GK Vmax was measured, and it is expressed as percentage of control siRNA transfected INS-1 cells. Statistical significance was determined by comparing the measurements in each group (a P ⬍ 0.01). Vmax and Km values for GK were calculated as described in the text.

infusion of recombinant human IGF-I yielded a great than 3-fold improvement in insulin sensitivity and glucose tolerance when compared with placebo control (25). The preceding information tells us that circulating IGF-I impacts glucose metabolism but does not necessary involve GK. The connection between IGF-I and GK comes from several lines of evidence found in states of DM or glucose intolerance (9, 24). For example, a key role for GK in the pathogenesis of DM that implicates the participation of IGF-I arises from studies of the IGF-IR⫺/⫺ mice. These animals have hyperglycemia, hyperinsulinemia, but not reduced ␤-cell mass of the pancreas. Together these data underscore the importance of both GK and IGF-I in glucose homeostasis. The data also provide strong rationale for undertaking the studies summarized above to define the pathway initiated by the binding of IGF-I to its receptor and regulating GK gene expression. Our first step was simply to show that both the primary cultured rat pancreatic and INS-1 cells contained the IGF-IR (Fig. 1A). The use of RT-PCR with specific primers for rat IGF-IR showed that the mRNA was abundant in both cell types, and this result provided a solid basis for testing the actions of IGF-I using the INS-1 cells. Exposure of these cells to IGF-I induced GK protein abundance in a dose-dependent fashion (Fig. 1B). Both IGF-I and insulin are closely related in terms of structure plus function, and two hormones can act to stimulate glucose uptake in some tissues (26, 27). The lack of functional IGF-IR in knockout mice did not affect ␤-cell mass of the pancreas, but the phenotype had impaired insulin secretion and glucose tolerance (28). In addition, other data have previously shown that IR haploinsufficiency had a strong diabetogenic effect in the setting of a predisposing background (29). Therefore, we devised a series of studies to examine whether an in vitro cell culture model of INS-1 cells mimicked the effect of IGF-IR haploinsufficiency. The studies examined whether IGF-I induction of pancreatic GK expression may be affected by the down-regulation of IGF-IR, thus paralleling a haploinsufficiency state. To create such a model, we analyzed the expression of GK in INS-1 cells after the downregulation of the gene encoding IGF-IR using siRNA. In the presence of specific siRNA against IGF-IR, the ligand had no effect on the expression of GK in INS-1 cells (Fig. 4A). These studies suggest that IGF-IR is needed for IGF-I induction of GK in INS-1 cells. Because IGF-I affected GK gene expression, we wanted to identify the signaling cascade that followed the activation of IGF-IR that mediated this process. To answer this question, we used known signaling pathway inhibitors to block the actions of IGF-I. The results of these studies pointed to the

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PI3-K cascade as one of the avenues that mediated the activities of the hormone (28, 30). Numerous studies have shown that Akt is one of the downstream components of the PI3-K cascade. Therefore, we searched for Akt phosphorylation (Fig. 4B), an indicator of Akt activity, after exposure of INS-1 cells to IGF-I. Additional evidence in support of a role for Akt in mediating the actions of IGF-I comes from the use of an Akt-DN in preventing but constitutively active Akt mimicked IGF-I induction of GK expression (Fig. 4, C and D). These findings are similar to a recent report showing that insulin activates the transcription of the ␤-cell GK gene via the PI3K/Akt pathway (31). The data in this report are the first to show that an analogous role for IGF-I induction of GK gene activity requires the PI3-K/Akt pathway in INS-1 cells. To further define the pathway from Akt to activation of the GK gene, we postulated that the signaling cascade may lead to participation of FoxO1. Two pieces of information prompted us to postulate this possibility: 1) FoxO1 is a known downstream target of Akt phosphorylating activity and 2) there is a potential FoxO1 binding motif within the GK promoter. The FoxO subfamily of forkhead transcription consists of three members, FoxO1 (FKHR), FoxO3a (FKHRL1), and FoxO4 (AFX), which are all inactivated by the actions of Akt (32, 33). FoxO1 phosphorylation by Akt leads to nuclear exclusion and the inhibition of the forkhead proteinmediated transcriptional activation. FoxO1 transcription factors have been implicated in regulating diverse cellular functions including differentiation, metabolism, proliferation, and survival (34 –36). The PI3-K pathway is a partner in this process, when cultured cells are treated with growth factors (i.e. epidermal growth factor, insulin) that are known to activate this cascade, leading to the exclusion of FoxO1 from the nucleus. In contrast, the removal of growth factors from the media used to treat the cells resulted in the accumulation of FoxO1 protein in the nucleus. Furthermore, Akt mediates many of the effect of growth factors downstream of PI3-K (37), and several members of the forkhead family of transcriptional factors are now known substrates of Akt (30, 38 – 40). The GK promoter is comprised of consensus binding motifs for a host of transcription factors (41). Because IGF-I induction of GK expression appears to be mediated by the PI3-K/Akt cascade, we searched and found binding sites for the transcription factor, FoxO1. Whereas a mutation of the FoxO1 binding motif in the GK promoter abrogated the ability of IGF-I to induce activity of the promoter, the presence of the wild-type motif in the promoter was responsive to IGF-I. FoxO1 knockdown increased GK protein expression and canceled the effect of IGF-I on GK expression. Together these findings show that the PI3-K/Akt/ FoxO1 pathway mediate the effect of IGF-I on GK gene expression. Previous reports indicated the similar role of FoxO1 phosphorylation on several gene promoters, i.e. pancreatic duodenal homeobox-1, Foxa2, and peroxisomal proliferator-activated receptor-␥2 (42, 43). Although we reported that the expression of FoxO1 inhibited GK expression, a recent report using FoxO1 transgenic mice or adenoviral FoxO1 expression in isolated hepatocytes confirms that FoxO1 stimulates expression of gluconeogenic genes and suppresses expression of genes involved in glycolysis, the shunt pathway, and lipo-

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genesis, including GK (44). Previous reports also demonstrated that pancreatic GK transcription and activity are regulated by several hormones and vitamins and that this hormonal regulation differs in hepatic and pancreatic genes (45– 47). In this study, it appears that the effect of IGF-I in the GK promoter-luciferase reporter gene is greater than can be accounted for by FoxO1. Mutation of the FoxO1 biding site (Fig. 5, E and F) in the promoter or knockdown of FoxO1 by siRNA (Fig. 5G) both increase expression by approximately 50%, much less than IGF-I, which is about 3-fold. In contrast, GK protein content is increased by IGF-I or FoxO1 knockdown to approximately the same extent (⬃50%, Fig. 5H). This implies some additional mechanisms(s) of IGF-I regulation of the GK promoter or on translation of luciferase. In summary, the results our studies show that IGF-I stimulates the expression of endogenous GK in insulinoma cell line, INS-1. This stimulatory effect of IGF-I on GK promoter is initiated by binding of the growth factor with its receptor and the flow of information from the membrane to the nucleus is mediated by the PI3-K/Akt/FoxO1 cascade. Specific steps in the process include the activation of PI3-K leading to the phosphorylation of Akt. This activated component of the cascade targeted the transcription factor, FoxO1, through phosphorylation. This protein in turn could bind to the FRS motif in the GK promoter, leading to the control of gene activity. These findings raise the possibility that IGF-I may affect the glucose metabolism by controlling GK expression in pancreatic ␤-cells. Acknowledgments Received August 21, 2006. Accepted February 14, 2007. Address all correspondence and requests for reprints to: Koji Murao, M.D., Ph.D., Division of Endocrinology and Metabolism, Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: [email protected]. Disclosure Statement: The authors have nothing to disclose.


11.

12. 13.

14.

15.

16. 17. 18. 19. 20. 21.

22. 23. 24.

25.

References 1. Matschinsky FM 1990 Glucokinase as glucose sensor and metabolic signal generator in pancreatic ␤-cells and hepatocytes. Diabetes 39:647– 652 2. Matschinsky F, Liang Y, Kesavan P, Wang L, Froguel P, Velho G, Cohen D, Permutt MA, Tanizawa Y, Jetton TL 1993 Glucokinase as pancreatic ␤ cell glucose sensor and diabetes gene. J Clin Invest 92:2092–2098 3. Matschinsky FM, Glaser B, Magnuson MA 1998 Pancreatic ␤-cell glucokinase: closing the gap between theoretical concepts and experimental realities. Diabetes 47:307–315 4. Velho G, Froguel P, Clement K, Pueyo ME, Rakotoambinina B, Zouali H, Passa P, Cohen D, Robert JJ 1992 Primary pancreatic ␤-cell secretory defect caused by mutations in glucokinase gene in kindreds of maturity onset diabetes of the young. Lancet 22:444 – 448 5. Glaser B, Kesevan P, Heyman M, Davis E, Cuesta A, Stanley CA, Thornton PS, Permutt MA, Matschinsky FM, Herold KC 1992 Familial hyperglycemia caused by an activating glucokinase mutation. N Engl J Med 338:226 –230 6. Blakesley VA, Butler AA, Koval AP, Okubo Y, LeRoith D 1999 IGF system: biological actions of the IGFs. In: Rosenfeld RG, Roberts CT, eds. The IGF system. Totowa, NJ: Humana Press; 143–164 7. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142 8. Kulkarni RN 2002 Receptors for insulin and insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function. Biochem Soc Trans 30:317–322 9. Kulkarni RN, Holzenberger M, Shih DQ, Ozcan U, Stoffel M, Magnuson MA, Kahn CR 2002 ␤-Cell-specific deletion of the Igf1 receptor leads to hyperinsulinemia and glucose intolerance but does not alter ␤-cell mass. Nat Genet 31:111–115 10. Cao WM, Murao K, Imachi H, Sato M, Nakano T, Kodama T, Sasaguri Y, Wong NC, Takahara J, Ishida T 2001 Phosphatidylinositol 3-OH kinase-Akt/

26. 27. 28.

29. 30.

31.

32. 33. 34. 35.

protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line. Arterioscler Thromb Vasc Biol 21:1592–1597 Moates JM, Shelton KD, Magnuson MA 1996 Characterization of the Pal motifs in the upstream glucokinase promoter: binding of a cell type-specific protein complex correlates with transcriptional activation. Mol Endocrinol 10:723–731 Murao K, Wada Y, Nakamura T, Taylor AH, Mooradian AD, Wong NC 1998 Effects of glucose and insulin on rat apolipoprotein A-I gene expression. J Biol Chem 273:18959 –18965 Ohtsuka S, Murao K, Imachi H, Cao WM, Yu X, Li J, Iwama H, Wong NC, Bancroft C, Ishida T 2006 Prolactin regulatory element binding protein as a potential transcriptional factor for the insulin gene in response to glucose stimulation. Diabetologia 49:1599 –1607 Guo S, Rena G, Cichy S, He X, Cohen P, Unterman T 1999 Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274:17184 –17192 Cao WM, Murao K, Imachi H, Yu X, Dobashi H, Yoshida K, Muraoka T, Kotsuna N, Nagao S, Wong NC, Ishida T 2004 Insulin-like growth factor-I regulation of hepatic scavenger receptor class BI. Endocrinology 145:5540 – 5547 Werner H, Woloschak M, Adamo M, Shen-Orr Z, Roberts Jr CT, LeRoith D 1989 Developmental regulation of the rat insulin-like growth factor I receptor gene. Proc Natl Acad Sci USA 86:7451–7455 Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB 1992 Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130:167–178 Chen C, Hosokawa H, Bumbalo LM, Leahy JL 1994 Regulatory effects of glucose on the catalytic activity and cellular content of glucokinase in the pancreatic ␤ cell. Study using cultured rat islets. J Clin Invest 94:1616 –1620 Da Silva Xavier G, Qian Q 2004 Distinct roles for insulin and insulin-like growth factor-1 receptors in pancreatic ␤-cell glucose sensing revealed by RNA silencing. Biochem J 377:149 –158 Buzzio OL, Lu Z, Miller CD, Unterman TG, Kim JJ 2006 FOXO1A differentially regulates genes of decidualization. Endocrinology 147:3870 –3876 Wang MY, Koyama K, Shimabukuro M, Mangelsdorf D, Newgard CB, Unger RH 1998 Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase, and glucose-stimulated insulin secretion. Proc Natl Acad Sci USA 95:11921–11926 Matschinsky FM 2002 Regulation of pancreatic ␤-cell glucokinase: from basics to therapeutics. Diabetes 51:S394 –S404 Tsakiris D, Ioannou K 2004 An underdiagnosed type of diabetes: the MODY syndromes: pathophysiology, clinical presentation and renal disease progression. J Nephrol 17:637– 641 Xuan S, Kitamura T, Nakae J, Politi K, Kido Y, Fisher PE, Morroni M, Cinti S, White MF, Herrera PL, Accili D, Efstratiadis A 2002 Defective insulin secretion in pancreatic ␤ cells lacking type 1 IGF receptor. J Clin Invest 110: 1011–1019 Moses AC, Young SC, Morrow LA, O’Brien M, Clemmons DR 1996 Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes. Diabetes 45:91–100 Jullien D, Heydrick SJ, Gautier N, Van Obberghen E, Le Marchand-Brustel Y 1996 Effect of IGF-I on phosphatidylinositol 3-kinase in soleus muscle of lean and insulin-resistant obese mice. Diabetes 45:869 – 875 Frick F, Oscarsson J, Vikman-Adolfsson K, Ottosson M, Yoshida N, Eden S 2000 Different effects of IGF-I on insulin-stimulated glucose uptake in adipose tissue and skeletal muscle. Am J Physiol Endocrinol Metab 278:729 –737 Kido Y, Nakae J, Hribal ML, Xuan S, Efstratiadis A, Accili D 2002 Effects of mutations in the insulin-like growth factor signaling system on embryonic pancreas development and ␤-cell compensation to insulin resistance. J Biol Chem 277:36740 –36747 Kitamura T, Kahn CR, Accili D 2003 Insulin receptor knockout mice. Annu Rev Physiol 65:313–332 Dickson LM, Lingohr MK, McCuaig J, Hugl SR, Snow L, Kahn BB, Myers Jr MG, Rhodes CJ 2001 Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic ␤-cells (INS-1). J Biol Chem 276:21110 –21120 Leibiger B, Leibiger IB, Moede T, Kemper S, Kulkarni RN, Kahn CR, de Vargas LM, Berggren PO 2001 Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic ␤ cells. Mol Cell 7:559 –570 Anderson MJ, Viars CS, Czekay S, Cavenee WK, Arden KC 1998 Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 47:187–199 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857– 868 Accili D, Arden KC 2004 FoxOs at the crossroads of cellular metabolism, differentiation, and transformation. Cell 117:421– 426 Skurk C, Maatz H, Kim HS, Yang J, Abid MR, Aird WC, Walsh K 2004 The


36. 37. 38. 39. 40. 41. 42.

Akt-regulated forkhead transcription factor FOXO3a controls endothelial cell viability through modulation of the caspase-8 inhibitor FLIP. J Biol Chem 279:1513–1525 Arden KC, Biggs WH III 2002 Regulation of the FoxO family of transcription factors by phosphatidylinositol-3 kinase-activated signaling. Arch Biochem Biophys 403:292–298 Rena G, Prescott AR, Guo S, Cohen P, Unterman TG Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14 –3-3 binding, transactivation and nuclear targeting. Biochem J 354:605– 612 Medema RH, Kops GJ, Bos JL, Burgering BM 2000 AFX-like forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature 404:782–787 Rena G, Guo S, Cichy S, Unterman T, Cohen P 1999 Phosphorylation of the transcription factor forkhead family member FKHR by protein kinase B. J Biol Chem 274:17179 –17183 Biggs W, Meisenhelder J, Hunter T, Cavenee W, Arden K 1999 Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96:7421–7426 Postic C, Shiota M, Magnuson MA 2001 Cell-specific roles of glucokinase in glucose homeostasis. Recent Prog Horm Res 56:195–217 Armoni M, Harel C, Karni S, Chen H, Bar-Yoseph F, Ver MR, Quon MJ,


43. 44.

45.

46.

47.

2913

Karnieli E 2006 FOXO1 represses peroxisome proliferator-activated receptor-␥1 and -␥2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity. J Biol Chem 281:19881–19891 Buteau J, Spatz ML, Accili D 2006 Transcription factor FoxO1 mediates glucagon-like peptide-1 effects on pancreatic ␤-cell mass. Diabetes 55:1190 –1196 Zhang W, Patil S, Chauhan B, Guo S, Powell DR, Le J, Klotsas A, Matika R, Xiao X, Franks R, Heidenreich KA, Sajan MP, Farese RV, Stolz DB, Tso P, Koo SH, Montminy M, Unterman TG 2006 FoxO1 regulates multiple metabolic pathways in the liver: effects on gluconeogenic, glycolytic, and lipogenic gene expression. J Biol Chem 281:10105–10117 Ferna´ndez-Mejı´a C, Davison MB 1992 Regulation of glucokinase and proinsulin gene expression and insulin secretion in RIN-m5F cells by dexamethasone, retinoic acid and thyroid hormones. Endocrinology 120:1660 –1668 Cabrera-Valladares G, German MS, Matschinsky FM, Wang J, FernandezMejia C 1999 Effect of retinoic acid on glucokinase activity and gene expression in primary cultures of pancreatic islets. Endocrinology 140:3091–3096 Romero-Navarro G, Cabrera-Valladares G, German MS, Matschinsky FM, Wang J, Fernandez-Mejia C 1999 Biotin regulation of pancreatic glucokinase and insulin in primary cultured rat islets and in biotin-deficient rats. Endocrinology 140:4595– 4600

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