help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1142
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yi, F.
Right arrow Articles by Jin, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yi, F.
Right arrow Articles by Jin, T.
Endocrinology Vol. 149, No. 5 2341-2351
Copyright © 2008 by The Endocrine Society

Cross Talk between the Insulin and Wnt Signaling Pathways: Evidence from Intestinal Endocrine L Cells

Fenghua Yi1, Jane Sun1, Gareth E. Lim, I. George Fantus, Patricia L. Brubaker and Tianru Jin

Division of Cell and Molecular Biology (F.Y., I.G.F., T.J.), Toronto General Research Institute, University Health Network; Departments of Medicine (I.G.F., P.L.B., T.J.), Physiology (G.E.L., I.G.F., P.L.B., T.J.), and Laboratory Medicine and Pathobiology (J.S., T.J.); and Banting and Best Diabetes Centre (I.G.F., T.J.), Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1L7

Address all correspondence and requests for reprints to: Tianru Jin, Room 10-354, 10th Floor, Toronto Medical Discovery Tower, MaRS Centre, University Health Network, 101 College Street, Toronto, Ontario, Canada M5G 1L7. E-mail: tianru.jin{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proglucagon gene (glu) encodes the incretin hormone glucagon-like peptide-1 (GLP-1), produced in the intestinal endocrine L cells. We found previously that the bipartite transcription factor β-catenin/T cell factor (cat/TCF), the major effector of the canonical Wnt signaling pathway, activates intestinal glu expression and GLP-1 production. We show here that 100 nM insulin stimulated glu expression and enhanced GLP-1 content in the intestinal GLUTag L cell line as well as in primary fetal rat intestinal cell cultures. Increased intestinal glu mRNA expression and GLP-1 content were also observed in vivo in hyperinsulinemic MKR mice. In the GLUTag cells, insulin-induced activation of glu expression occurred through the same TCF site that mediates cat/TCF activation. Phosphatidylinositol 3-kinase inhibition, but not protein kinase B inhibition, attenuated the stimulation by insulin. Furthermore, nuclear β-catenin content in the intestinal L cells was increased by insulin. Finally, insulin enhanced the binding of TCF-4 and β-catenin to the TCF site in the glu promoter G2 enhancer element, as determined by quantitative chromatin immunoprecipitation assay. Collectively, these findings indicate that enhancement of β-catenin nuclear translocation and cat/TCF binding are among the mechanisms underlying cross talk between the insulin and Wnt signaling pathways in intestinal endocrine L cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CANONICAL WNT signaling pathway was initially identified in colon cancer research and in embryological studies in Drosophila and other species (1, 2, 3, 4, 5, 6). The key effector of the Wnt pathway is the bipartite transcription factor β-catenin/T cell factor (cat/TCF), formed by β-catenin (β-cat) and one of the four TCFs [TCF-1, lymphoid enhancer-binding factor-1 (LEF-1), TCF-3, and TCF-4]. Among them, TCF-4 is the major partner of β-cat in intestinal cells (7, 8). Under nonstimulating conditions, the concentration of free β-cat is tightly controlled by the proteasome-mediated degradation process, with the participation of the tumor suppressor adenomatous polyposis coli (APC), axin, glycogen synthase kinase-3 (GSK-3), and casein kinase-1{alpha} (CK-1{alpha}) (9, 10). In response to Wnt stimulation, free β-cat accumulates, leading to the formation of the cat/TCF complex and activation of cat/TCF downstream target genes. In addition, lithium and other inhibitors of GSK-3 may also stimulate the expression of downstream target genes of the Wnt signaling pathway (11).

The proglucagon gene (glu) is expressed in pancreatic {alpha}-cells, intestinal endocrine L cells, and selected neurons in the brain (12, 13, 14, 15, 16). It encodes the prohormone proglucagon, which can be processed to produce at least three major peptide hormones, glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2. These hormones exert both opposite and overlapping functions. Glucagon, produced in the pancreatic {alpha}-cells, is a major counterregulatory hormone to insulin in the maintenance of glucose homeostasis. In contrast, GLP-1 and GLP-2 are produced by the intestinal endocrine L cells (13). GLP-1 enhances insulin secretion and inhibits glucagon release (12, 13). Other functions of GLP-1 include stimulation of pancreatic β-cell growth and survival and inhibition of gastric emptying (17). In addition, both peripheral and brain administration of GLP-1 inhibits food intake (18, 19, 20). GLP-2 was initially recognized as a growth factor of the small intestinal epithelium (21), although recent studies have suggested that it may also exert overlapping functions with GLP-1 in controlling food intake (22, 23, 24). The opposite biological functions exerted by glucagon compared with the glucagon-like peptides in the regulation of nutrient homeostasis prompted us to propose the existence of cell-type-specific mechanisms underlying intestinal glu expression (25).

We reported recently that glu expression and GLP-1 production are activated by lithium or β-cat overexpression in intestinal endocrine L cells (8, 25). This activation is mediated through a TCF binding site within the glu gene promoter G2 enhancer element, combined with TCF-4 expression in intestinal L cells. In the present study, we demonstrate that insulin also activates glu expression and GLP-1 production in a murine intestinal endocrine L cell line and in primary rat intestinal L cells as well as in the mouse intestine in vivo. These findings stand in marked contrast to the known repressive effect of insulin on expression of the same glu gene in pancreatic {alpha}-cells (26, 27). Our data indicate that activation of glu expression and GLP-1 production by insulin is regulated by the same cis- and trans-elements that mediate activation by the Wnt signaling pathway, supporting the existence of cross talk between the insulin and Wnt signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Insulin was provided by Novo Nordisk (Copenhagen, Demark). Recombinant IGF-I (product number 13769) was purchased from Sigma-Aldrich (St. Louis, MO). Several concentrations of insulin (20, 100, 200, and 800 nM, equivalent to 116 ng, 580 ng, 1.16 µg, and 5 µg per ml, respectively) were used in this study. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and protein kinase B (PKB) inhibitor Akti-1/2 were purchased from Calbiochem (EMD Biosciences, Inc., San Diego, CA).

Plasmids
Construction of the proglucagon-luciferase (GLU-LUC) reporter gene plasmids was reported in previous studies (28, 29, 30). The G2-TK-LUC constructs were generated by inserting one copy of the corresponding element into the parental TK-LUC fusion gene plasmid, as reported previously (8). The S33Y mutant β-cat expression plasmid and TCF-4 dominant-negative retrovirus expression system (pPGS-dnTCF-4 and the empty vector) were gifts from Dr. Eric Fearon (31). The pTOPFLASH reporter plasmid was a gift from Dr. Bert Vogelstein. In this plasmid, expression of a LUC reporter gene is driven by a minimum TK promoter fused with three copies of the TCF binding site (32, 33). The GSK-3β expression plasmid was provided by Dr. James Woodgett (34).

Mice, cell cultures, DNA transfection, and LUC reporter analysis
Hyperinsulinemic MKR mice were kindly provided by Dr. Derek LeRoith (35, 36). All animal experiments were approved by the University of Toronto Animal Care Committee. Age- and sex-matched 10-wk-old MKR and FVB (strain controls; Charles River, St. Constant, Quebec, Canada) mice were used to examine ileal glu mRNA expression and GLP-1 content. Fetal rat intestinal cell (FRIC) L-cell cultures were prepared from 19- to 20-d pregnant Wistar rats (Charles River), as previously described (25, 37). Mouse large intestinal GLUTag (38), small intestinal STC-1 (39), hamster pancreatic InR1-G9 (40), and mouse pancreatic {alpha}-TC-1 cell lines were grown in DMEM (4.5 mg glucose/liter) supplemented with 10% fetal bovine serum. Methods for examining the effect of chemical treatments on GLU-LUC fusion gene expression have been described previously (41).

RNA extraction, Northern blotting, and real-time RT-PCR
RNA was extracted using Trizol reagent (Invitrogen Life Technology, Carlsbad, CA) as per the manufacturer’s instructions. Methods for Northern blotting were described previously (29). cDNAs were generated using the ReverAid kit (Fermentas, Burlington, Onatrio, Canada). Real-time RT-PCR was conducted with Rotor-Gene 3000 (Corbett Research, Dorval, Quebec, Canada), using the QuantiTect SYBR green PCR kit (QIAGEN, Valencia, CA). DNA sequences of the primers used to detect glu mRNA by real-time RT-PCR were forward, 5'-ATGAAGACCATTTACTTTG-3', and reverse, 5'-CGGTTCCTCTTGGTGTTCATCAAC-3', resulting in amplification of a 254-bp glu cDNA fragment in mouse and rat. Expression levels of glu mRNA are presented as relative copy numbers (with the untreated samples or control mice defined as 1), calculated using the mouse glu cDNA inserted into a TA cloning vector as the control and normalized to expression of β-actin mRNA.

Antibodies, Western blotting, and immunohistochemical staining
Rabbit polyclonal anti-Akt, anti-phospho-Akt (Ser473), and anti-phospho-GSK-3{alpha}/β (Ser21/9) antibodies were purchased from Cell Signaling (Cedarlane, Ontario, Canada). The mouse monoclonal anti-GSK-3 (clone 4G-1E), and mouse anti-β-catenin (β-cat, nonphosphorylated, clone 8E4) antibodies were from Upstate Biotechnology (Lake Placid, NY) (42). Another monoclonal anti-β-cat antibody (E-5, sc-7963), and the horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Methods for nuclear protein extraction, whole-cell protein preparation, and Western blotting have been described previously (8). For immunostaining, cells grown on coverslips were fixed with chilled 4% paraformaldehyde in PBS for 15 min and permeabilized with cold methanol for 5 min on ice, followed by incubation with 10% horse serum/0.1% Tween 20 in PBS for 30 min. Slides were then incubated with a rabbit polyclonal anti-TCF-4 (H-125) antibody and a mouse monoclonal anti-β-cat (nonphosphorylated) antibody (clone 8E4) diluted 1:200 in blocking buffer for 3 h at room temperature, followed by Alexa Fluor 546 goat antimouse IgG (H+L) (Invitrogen; 1/200) and antirabbit IgG fluorescein isothiocyanate conjugate (Sigma; 1/100) diluted in blocking buffer for 45 min at room temperature. Nuclei were counterstained with 4',6-diamino-2-phenylindole (DAPI; Sigma), and immunoactivity was viewed using an LSM510 confocal microscope.

Small interfering RNA (siRNA)
siRNA against mouse β-cat and scrambled RNA (as control) were purchased from Dharmacon Research Inc (Lafayette, CO; sequence information included in supplemental Fig. 2Go, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Approximately 1 x 106 cells were seeded in a six-well plate with serum containing medium for 24 h. Cells were then transfected with siRNA (50 nM) using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with different concentrations of insulin for another 8 h before being harvested for Western blotting or real-time RT-PCR.


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 2. Insulin-mediated activation requires the TCF binding site within the G2 element, β-cat, and TCF-4. A and B, Insulin stimulates G2S and G2L expression in the GLUTag cells (A) but not in the InR1-G9 cells (B). Three micrograms of the indicated LUC reporter plasmid were transfected into GLUTag or InR1-G9 cells, and insulin (Ins, 100 nM) was added 12 h before the cells were harvested for LUC reporter analysis. Relative LUC activity was calculated as fold induction with the activity in cells receiving no insulin treatment set to 1-fold (mean ± SD, n = 3). C, Insulin and lithium generated no additive effect on G2S expression. Three micrograms of G2S were transfected into the GLUTag cells, and insulin (Ins, 100 nM) and/or lithium (10 mM) was added 12 h before the cells were harvested for LUC reporter analysis. Relative LUC activity was calculated as fold induction with the activity in cells receiving no treatment set to 1-fold (mean ± SD, n = 3). D, GLUTag cells were transfected with the β-cat siRNA (50 nM). The same amount of scrambled RNA (S-RNA) was used as the negative control. Twenty-four hours after the transfection, cells were harvested for Western blotting. Thirty micrograms of total protein were loaded into each lane, and a mouse monoclonal anti-β-cat antibody (1:1000) was used to determine β-cat expression. The same membrane was stripped, followed by hybridization with a rabbit polyclonal anti-c-myc antibody (1:1000) and a mouse monoclonal anti-β-actin antibody (loading control). E, GLUTag cells were transfected with 50 nM β-cat siRNA, or 50 nM scrambled siRNA (S-RNA) or received no transfection. Twenty-four hours after transfection, cells were treated with the indicated amount of insulin for 8 h, and then harvested. The expression of glu mRNA was quantitatively assessed by real-time RT-PCR. Results are presented as relative glu mRNA expression, with cells receiving no insulin treatment set to 1-fold (mean ± SD, n = 3).

 
RIA
Peptides were extracted from 5 cm of mouse ileum or from cells by extraction with 1 N HCl containing 1% trifluoroacetic acid, 5% HCOOH, and 1%NaCl, followed by passage twice through a cartridge of C18 silica (C18 Sep-Pak; Waters Corp., Milford, MA). RIA for GLP-1 was carried out using an antiserum directed toward the C-terminal end of GLP-1 (Affinity Research Products Ltd., Mamhead, UK) (8, 25, 38). Tissue and cell GLP-1 content was normalized to protein levels, determined by Bradford assay.

Chromatin immunoprecipitation (ChIP)
Approximately 2 x 107 cells were used for each ChIP assay, using the method described previously (8). Briefly, the cells were treated with formaldehyde to cross-link chromatin and nuclear proteins. After sonication, a designated antibody was used (final dilution of 1:500) to precipitate the sheared chromatin. After washing, elution, reverse cross-linking, and purification, approximately 1/20 of the purified DNA (2 µl) was used in each PCR or real-time PCR. A mouse monoclonal anti-TCF-4 antibody (Clone 6H5-3), a mouse monoclonal anti-LEF-1 antibody (C-19), and a rabbit polyclonal anti-β-cat antibody (H-102) were used in the ChIP assay. A mouse monoclonal anti-Flag tag antibody (M5; Sigma) was used as a negative control. The experimental primers (forward, 5'-CAAGGGATAAGACCCTCAAATG-3', and reverse, 5'-GCCTTGCAGATATTACGCTGA-3') amplify a 297-bp DNA fragment that contains the G2 enhancer element of the mouse glu gene promoter, based on the mouse glu gene sequence (GenBank accession no.: NT_039207). The control primers (forward, 5'-TGCTTATAATGCTGGTGCAAG-3, and reverse, 5'-ATTCGTATCCCAGATCAG-3') amplify a 205-bp fragment that is part of exon II of the mouse glu gene. The glu gene G2 element copy number was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) copy number.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin stimulates glu mRNA expression in intestinal glu-expressing endocrine L cells
Insulin has been shown to repress both glu mRNA expression and glucagon production in pancreatic {alpha}-cells (26, 27). Conversely, we have found that insulin stimulates GLP-1 secretion in the GLUTag cell line and a human intestinal GLP-1-producing cell line, NCI-H716 (43). However, the effect of insulin on glu gene expression in intestinal L cells is unknown. Interestingly, we found that insulin at concentrations of 100 nM or higher significantly stimulated glu mRNA expression, in both the intestinal GLUTag L cell line (1.7- to 3.1-fold) and the intestinal STC-1 cell line (1.9- to 2.8-fold, based on results of densitometric analysis). Insulin did not show a significant stimulatory effect at concentrations of 20 nM or lower (data not shown). Representative Northern blotting results are shown in Fig. 1AGo (two left panels). In contrast, insulin exerted no notable effect on glu mRNA expression in the pancreatic {alpha}-cell line InR1-G9 and repressed glu mRNA expression in the {alpha}-TC-1 cell line by 22–52% (Fig. 1AGo, two right panels; results based on densitometric analyses). To quantitatively assess the effect of insulin on glu mRNA expression in the GLUTag cell line, we also conducted real-time RT-PCR analyses. Figure 1BGo shows that 100 nM insulin significantly activated glu mRNA expression by 2.1- to 3.1-fold during the entire 4- to 24-h experimental period. Figure 1CGo shows that treating the GLUTag cell line with 100 nM recombinant IGF-I for 4 h resulted in a similar activation of glu mRNA expression compared with that seen with insulin at the same concentration. At a concentration of 20 nM, both insulin and IGF-I moderately activated glu mRNA expression, but this did not reach statistical significance in either case (data not shown). To further verify whether insulin indeed stimulates glu mRNA expression in intestinal L cells, we also examined the effect of insulin on glu expression in primary FRIC cultures. Figure 1DGo shows that an 8-h treatment with 100 nM insulin generated a 61% increase in glu mRNA expression in FRIC cultures, detected by real-time RT-PCR. Because 100 nM insulin is a supraphysiological concentration, we also determined the potential physiological relevance of these findings by examination of glu mRNA expression in a hyperinsulinemic model, the MKR transgenic mouse (35, 36). MKR mice overexpress a dominant-negative IGF-I receptor construct in muscle cells and therefore develop a compensatory hyperinsulinemia of approximately 1 nM. Compared with FVB control mice, intestinal glu mRNA expression was increased approximately 5-fold in these animals (Fig. 1EGo).


Figure 1
View larger version (45K):
[in this window]
[in a new window]

  		FIG. 1. Insulin stimulates glu mRNA expression in intestinal glu-producing cells. A, GLUTag, STC-1, InR1-G9, and {alpha}-TC-1 cell lines were treated with or without insulin (800 nM) for 4–24 h. The expression of glu mRNA was examined by Northern blotting. Glu, Proglucagon; Tub, tubulin (loading control). B–D, GLUTag cells (B and C) and FRIC cultures (D) were treated with 100 nM insulin (B and D) or 100 nM IGF-I (C) for the indicated time (4 h for C). The expression of glu mRNA was assessed by real-time RT-PCR. Results are presented as relative Glu copy number, with cells that received no treatment as the control (mean ± SD, n = 3). E, glu mRNA expression in ileal sections from hyperinsulinemic MKR and control FVB mice was assessed by real-time RT-PCR (mean ± SE, n = 5).

 
Insulin-mediated activation requires a TCF binding site, β-catenin, and TCF-4
The observations that both the Wnt pathway (8) and insulin activate cell-type-specific glu expression prompted us to examine whether insulin uses the effectors of the Wnt signaling pathway in stimulating glu expression. We first determined the effect of insulin on expression of a LUC reporter driven by a native glu promoter construct containing a TCF binding site as well as on the Wnt-responsive LUC reporter pTOPFLASH. As shown in supplemental Fig. 1Go (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), in the intestinal GLUTag cell line, but not in the pancreatic InR1-G9 cell line, insulin significantly stimulated expression of the TCF binding site-bearing glu promoter and the pTOPFLASH construct by 1.9- and 2.6-fold, respectively, suggesting that insulin may use the effector of the Wnt signaling pathway in stimulating intestinal glu transcription. To further examine whether the TCF site within the G2 enhancer element is necessary for this activation, we examined the effect of insulin on three additional LUC fusion gene constructs, namely G2L-TK-LUC (G2L), G2S-TK-LUC (G2S), and G2M-TK-LUC (G2M), previously described (8). In brief, G2L represents the full-length wild-type G2 enhancer element (–200 to –165 bp), whereas G2S is 8 bp shorter than G2L, although the TCF binding motif remains unchanged. G2M is identical to G2S, except for the presence of a disabling mutation in the TCF motif (8). By using these three LUC reporters, we have demonstrated previously that the TCF binding motif within the G2 element mediates lithium and S33Y (constitutively active)-β-cat-transfection-induced activation of the glu promoter in the intestinal GLUTag cell line (8). Figure 2AGo shows that insulin stimulated expression of both G2L and G2S, but not G2M, when transfected into the GLUTag cell line. In contrast, insulin generated no appreciable effect on expression of either G2S or G2M when transfected into the pancreatic InR1-G9 cell line (Fig. 2BGo). In addition, significant repression by insulin was observed when G2L was examined in the InR1-G9 cells (Fig. 2BGo). This observation suggests that, in addition to the negative cis-elements previously identified within the G1 and G3 regions (27, 44) that mediate the repressive effect of insulin in pancreatic {alpha}-cells, there is an extra negative element within G2 that exerts such a function. Although this clearly merits further investigation, the present study has focused on the mechanisms underlying the cross talk between the insulin and Wnt signaling pathways in intestinal L cells. Figure 2CGo shows that treating the GLUTag cell line with insulin and lithium generated no synergistic or additive effect on G2S expression, further confirming that insulin activates G2S mainly via the TCF binding motif.

The observation that insulin and the Wnt signaling pathway use the same cis-element to stimulate intestinal glu promoter transcription prompted us to examine whether the trans-element, cat/TCF, the major effector of the Wnt signaling pathway, is also involved in insulin-stimulated glu expression in the intestinal L cell line. Supplemental Fig. 2AGo shows the sequence of the siRNA against β-cat used in this study. Transfection of the β-cat siRNA into GLUTag cells significantly attenuated free β-cat expression, accompanied by reduced expression of c-Myc, a known downstream target of the Wnt pathway (Fig. 2DGo). This β-cat siRNA was also shown to knock down β-cat expression in the human HepG2 cell line and to attenuate human β-cat cotransfection-stimulated G2S-TK-LUC expression (supplemental Fig. 2CGo). However, the same amount of scrambled RNA (control) had no effect on the expression of either β-cat or c-Myc in GLUTag cells (Fig. 2DGo). GLUTag cells were therefore transfected with the β-cat siRNA, scrambled RNA, or no RNA (mock transfection) for 24 h, treated with insulin (100, 200, and 800 nM) for an additional 4 h, and examined for glu mRNA expression by real-time RT-PCR. As shown in Fig. 2EGo, all three doses of insulin stimulated glu expression in the mock-transfected cells. In contrast, introduction of β-cat siRNA completely blocked insulin-induced glu activation, whereas the scrambled RNA caused only a small reduction in insulin-activated glu expression.

We have previously demonstrated that expression of dominant-negative TCF-4 (dnTCF-4) in the GLUTag cell line effectively blocked activation of glu promoter expression and GLP-1 production by lithium and S33Y-β-cat transfection (8). To further investigate whether TCF-4 is also involved in insulin-activated glu promoter expression, we assessed the effect of insulin on expression of G2S and pTOPFLASH in dnTCF-4-expressing GLUTag cells. As shown in Fig. 3AGo, expression of both G2S and pTOPFLASH was activated by insulin in the control GLUTag cell line. However, insulin failed to activate expression of these two reporter gene constructs in the dnTCF-4 expressing GLUTag cells. Furthermore, 12 and 24 h insulin treatment also increased the GLP-1 content of the GLUTag cells, by 2.7- and 2.0-fold, respectively, and this was completely abrogated by expression of dnTCF-4 (Fig. 3BGo). Together, these findings implicate both cis-elements (TCF binding motif within G2) and trans-elements (β-cat and TCF-4) in the stimulation of glu mRNA expression and GLP-1 production in response to activation of the Wnt signaling pathway by insulin. Finally, consistent with the finding of insulin-induced enhancement of GLP-1 content in the GLUTag cell line, the intestinal content of GLP-1 in the hyperinsulinemic MKR mice was also increased, by 46.8% (Fig. 3CGo).


Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		FIG. 3. Expression of dnTCF-4 blocks the stimulatory effect of insulin on TCF motif-mediated LUC reporter expression and GLP-1 content. A, GLUTag cells stably transfected with an empty expression vector (V) or dnTCF-4 (dnTCF) were transiently transfected with 3 µg G2S-TK-LUC (G2S) or pTOPFLASH for 12 h. The cells were treated with or without insulin (Ins, 100 nM) for an additional 12 h before harvesting for LUC reporter analysis. Relative LUC activity was calculated as fold induction with the activity in cells receiving no insulin treatment set to 1-fold (mean ± SD, n ≥3). *, P < 0.05; **, P < 0.01. B, The effect of insulin on GLP-1 content in wild-type (WT), empty vector (V)-transfected, and dnTCF-4 (dnTCF)-transfected GLUTag cells was examined by RIA. Relative GLP-1 content was calculated as fold induction compared with cells that received no insulin treatment (mean ± SD, n ≥ 6). *, P < 0.05; **, P < 0.01. C, GLP-1 content of ileal sections from hyperinsulinemic MKR and control FVB mice was determined by RIA and normalized to total tissue protein content [mean ± SD, n = 14 (control) and 11 (MKR)].

 
Inhibition of PI3K, but not of PKB, blocks the stimulatory effect of insulin
To investigate the molecular mechanisms underlying insulin-stimulated glu expression and GLP-1 production in intestinal endocrine L cells, we first examined the involvement of GSK-3, a common negative modulator of both the Wnt and insulin pathways (45, 46, 47). It is well known that insulin activates PKB/Akt via PI3K in many cell lineages, resulting in the phosphorylation of GSK-3. However, no detailed examination has been conducted in glu-expressing endocrine cells. We found that, not only in GLUTag and STC-1 cells, but also in the pancreatic InR1-G9 cells, insulin stimulated GSK-3 phosphorylation (supplemental Fig. 3Go, A–C), suggesting that GSK-3 may not be the key mediator of insulin in the stimulation of glu expression in intestinal endocrine L cells. Supplemental Fig. 3DGo also shows that insulin-stimulated PKB and GSK-3 phosphorylation was inhibited by LY294002, a potent inhibitor of PI3K.

To examine the involvement of PI3K in insulin-stimulated glu expression, GLUTag cells were transfected with G2S or pTOPFLASH for 12 h before the addition of 100 nM insulin, in the presence or absence of LY294002. Cells were harvested after 12 h of insulin treatment, and LUC reporter expression was assessed. As shown in Fig. 4Go, A and B, insulin-activated expression of G2S and pTOPFLASH were completely blocked by PI3K inhibition. We also observed that LY294002 treatment decreased insulin-stimulated expression of G2S and pTOPFLASH below the basal levels (Fig. 4Go, A and B), indicating a possible involvement of PI3K activity in basal expression of Wnt target genes. Figure 4CGo shows that insulin-stimulated G2S expression was also attenuated by the inhibition of MAPK kinase (MEK), a known target of the PI3K signaling pathway. Finally, the involvement of PKB was examined using the chemical inhibitor Akti-1/2 (48). Figure 4DGo shows that 10 µM Akti-1/2 completely blocked the effect of insulin on GSK-3 phosphorylation. However, PKB inhibition did not prevent insulin-induced activation of G2S and pTOPFLASH expression. Indeed, further activation was observed when Akti-1/2 was applied together with insulin (Fig. 4EGo). These observations support the notion that the PKB-GSK-3 signaling cascade is not involved in insulin-induced activation of β-cat in the intestinal L cell.


Figure 4
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 4. PI3K inhibition, but not PKB inhibition, blocks the stimulatory effect of insulin. A–C, GLUTag cells were transiently transfected with 3 µg of the indicated LUC reporter for 12 h. The cells were then treated with or without insulin (Ins, 100 nM) in the presence or absence of LY294002 (LY, 50 µM) or a MEK inhibitor (PD98059, 100 µM, or U1026, 25 µM) for an additional 12 h before harvesting for LUC reporter analysis. Relative LUC activity was calculated as fold change with the activity in cells receiving no treatment set to 1-fold (mean ± SD, n ≥ 3). D, GLUTag cells were pretreated with or without Akti-1/2 (10 µM) for 1 h before the treatment with 100 nM insulin for an additional 2 h. Cells were then harvested for Western blotting against pGSK-3{alpha}/β and GSK-3{alpha}/β. E, GLUTag cells were transiently transfected with 3 µg of the indicated LUC reporter for 12 h. The cells were then treated with or without insulin (100 nM) in the presence or absence of Akti-1/2 (Akti, 10 µM) for an additional 12 h before harvesting for LUC reporter analysis.

 
Insulin stimulates nuclear β-cat content in the GLUTag cell line
It has been reported that in several cancer cell lines and in fibroblasts, insulin does not stimulate free β-cat accumulation in the cytosol, whereas activation of the Wnt pathway does (46). Because nuclear β-cat is the component of the bipartite transcription factor cat/TCF that stimulates gene expression, we tested whether insulin affects nuclear β-cat content by treating both the GLUTag and InR1-G9 cell lines with 20, 100, and 800 nM insulin for 4 h, followed by assessment of nuclear β-cat content by Western blotting. Figure 5AGo shows that insulin markedly enhanced nuclear β-cat content in the GLUTag cell line but reduced nuclear β-cat content in the InR1-G9 cells. Similar effects were also observed in the GLUTag cells when 20 and 100 nM recombinant IGF-I was applied (Fig. 5BGo). We then treated both the GLUTag and InR1-G9 cell lines with 100 nM insulin for 4 h, followed by immunofluorescence staining for TCF-4 and nonphosphorylated β-cat. As shown in Fig. 5CGo, TCF-4 expression was detected in both the nucleus and cytosol of the GLUTag cells, regardless of insulin treatment. In contrast, no β-cat staining was detectable in the nuclei in the absence of insulin, whereas insulin treatment substantially increased nuclear β-cat content. In InR1-G9 cells, TCF-4 immunoactivity was very weak (if not absent). Furthermore, with or without insulin treatment, nuclear β-cat levels were very low in the InR1-G9 cells (Fig. 5DGo). We therefore conclude that in the GLUTag cell line, activation of glu expression by insulin is associated with an increase in nuclear β-cat content.


Figure 5
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 5. Insulin stimulates nuclear β-cat expression. A, GLUTag and InR1-G9 cells were treated with the indicated amount of insulin (Ins) for 4 h before harvesting for nuclear protein extraction. PCNA, Proliferating cell nuclear antigen. Fifteen micrograms of nuclear protein were loaded in each lane, and the expression of nuclear β-cat was detected with a rabbit polyclonal anti-β-cat antibody (1:1000) (n = 3). B, GLUTag cells were treated with the indicated amount of IGF-I for 4 h before harvesting for nuclear protein extraction. Fifteen micrograms of nuclear protein were loaded in each lane, and the expression of nuclear β-cat was detected with a rabbit polyclonal anti-β-cat antibody (n = 3). Histone H3 was used as a loading control. C and D, GLUTag (C) and InR1-G9 (D) cells were treated with insulin for 4 h, followed by detection of TCF-4 and nonphosphorylated β-cat expression by immunofluorescence. Nuclei were localized by 4',6-diamino-2-phenylindole (DAPI) staining.

 
Insulin stimulates binding of β-cat and TCF-4 to the G2 enhancer element
We have previously demonstrated by ChIP assay that antibodies against β-cat, TCF-4, and LEF-1 are able to precipitate the TCF-binding site-bearing G2 enhancer element of the glu gene promoter (8). To quantitatively assess the binding in response to insulin treatment, we therefore conducted quantitative ChIP analyses. In these analyses, the amount of chromatin-associated G2 element detected by real-time PCR is arbitrarily referred to as binding activity. As expected, we found that 4 h of lithium treatment enhanced the binding of TCF-4, LEF-1, and β-cat to the G2 element, by 2.7-, 2.3-, and 5.4-fold, respectively (Fig. 6AGo). We then treated GLUTag cells with either 100 or 800 nM insulin for 4 h and conducted quantitative ChIP with anti-TCF-4 and anti-β-cat antibodies. Figure 6Go, B and C, shows that 100 and 800 nM insulin increased the binding of TCF-4 to the G2 element by 13- and 33-fold, respectively, and increased binding of β-cat to G2 by 6.0- and 5.7-fold, respectively.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		FIG. 6. Lithium and insulin stimulate binding of TCF-4 and β-cat to the glu promoter G2 enhancer element. A, GLUTag cells were treated with 10 mM LiCl for 4 h, followed by ChIP assay with antibodies (Ab) against TCF-4, LEF-1, and β-cat. The amount of G2 element in the chromatin DNA precipitated in each assay was quantitatively assessed by real-time PCR, and the results are presented as fold of relative copy number (with untreated samples defined as 1-fold, mean ± SD). B and C, GLUTag cells were treated with the indicated amount of insulin for 4 h, followed by ChIP assay using antibodies directed against TCF-4 (B) and β-cat (C). The amount of G2 element in the chromatin DNA precipitated in each assay was assessed by real-time PCR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported that lithium treatment or transfection of a constitutively active β-cat mutant stimulates glu expression in intestinal glu-expressing cells and have identified an evolutionarily conserved TCF binding motif within the G2 enhancer element of the glu promoter that mediates the activation by both lithium and constitutively active β-cat (8). Furthermore, we found that expression of dnTCF-4 blocked the stimulatory effect of lithium on glu mRNA expression (8). TCF-4 was also demonstrated to be abundantly expressed in intestinal glu-expressing cell lines but was barely detectable in pancreatic islet {alpha}-cell lines (8), providing at least a partial explanation for the cell-type-specific effect of the Wnt pathway on glu expression. Herein, we further demonstrate that lithium stimulates the interaction of both β-cat and TCF-4 with the glu promoter G2 element (Fig. 6AGo). Our observations collectively suggest that the Wnt signaling pathway stimulates glu mRNA expression in the intestinal L cells. Consistent with our observations, Murtaugh et al. (49) found that loss of β-cat expression during pancreatic development does not significantly perturb islet endocrine cell mass or function, although β-cat expression is essential for pancreatic acinar cell development. In addition, recent genetic linkage analyses have shown an association between several common variants of TCF-4 (TCF7L2) and an increased risk of developing type 2 diabetes mellitus (T2D) (50, 51, 52). Whether intestinal glu expression and GLP-1 production are reduced in individuals carrying the T2D-susceptibility TCF-4 alleles warrants further examination.

Philippe (26, 27) has demonstrated that insulin represses expression of glu mRNA and the glu promoter in pancreatic {alpha}-cells, through a cis-element within the G3 enhancer element. More recently, Schinner et al. (44) reported that PKB/Akt activation is sufficient to mimic the inhibitory effect of insulin on pancreatic glu expression. We show here that in contrast to its repressive effect in pancreatic {alpha}-cell lines, insulin stimulates glu mRNA expression in two intestinal glu-producing cell lines as well as in primary FRIC intestinal L cell cultures. Insulin also enhances GLP-1 content in both the GLUTag and FRIC cultures, and both glu mRNA levels and GLP-1 content are increased in the intestines of hyperinsulinemic MKR mice. Collectively, these observations reveal a potential novel function of insulin as a regulator of the intestinal L cell, at least under conditions of hyperinsulinemia. Additional studies will clearly be required to confirm this effect of insulin on GLP-1 production, particularly during the development and progression of T2D. We have also examined the effect of IGF-I on glu mRNA expression and nuclear β-cat content in the GLUTag cell line. Because the response to IGF-I was similar to that of insulin in these experiments, additional studies will be required to determine whether insulin is acting via its own receptor, via the IGF-I receptor, and/or through a hybrid of the two receptors.

Insulin treatment may lead to enhanced phosphorylation of GSK-3{alpha} and GSK-3β at Ser 21/9 via PKB, thereby attenuating their enzymatic activity. However, Ding et al. (46) suggested that this does not lead to activation of the Wnt pathway because, in their studies, insulin treatment at 800 nM (5 µg/ml) did not result in cytosolic free β-cat accumulation in a battery of cancer cell lines and fibroblasts. In contrast, both Wnt-conditioned medium and lithium induced free β-cat accumulation but did not affect the phosphorylation status of GSK-3 (46). Based on these observations, Ding et al. (46) suggested that insulin and Wnt signals regulate GSK-3 via different mechanisms, leading to distinct downstream events, and that phosphorylation of GSK-3{alpha}/β at Ser 21/9 may not be sufficient to induce free β-cat accumulation. However, studies by other groups have shown that IGF-I or insulin can stimulate β-cat-mediated transactivation (53, 54, 55, 56, 57, 58, 59). The observations made in the present study provide an explanation for the discrepancy between these studies. In brief, we found that insulin treatment increased nuclear β-cat content in the GLUTag cell line, although the source of this β-cat, e.g. cytoplasmic or from the membrane E-cadherin/β-catenin complex, remains to be determined. Furthermore, using quantitative ChIP assays, we found that insulin stimulated binding of β-cat and TCF-4 to the G2 enhancer element of the glu promoter. Enhanced nuclear β-cat translocation and increased binding of cat/TCF-4 to target gene promoters therefore appears to be among the mechanisms underlying the cross talk between the insulin and Wnt signaling pathways.

Interestingly, we found that inhibition of PI3K, but not of PKB, blocked insulin-stimulated glu promoter expression. PI3K-mediated PKB-independent signaling has not been recognized until very recently. Zhang et al. (60) have shown that cigarette smoke-stimulated EGF receptor activation in human bronchial epithelial cells increases expression of the protooncogene FRA-1 through the PI3K-(p21-activated kinase-1, PAK-1)-Raf-MEK-ERK signaling cascade, without the involvement of PKB. Our observation therefore provides another example for the existence of PKB-independent PI3K activity. Whether PAK-1 is involved in stimulating β-cat nuclear translocation and regulating intestinal glu expression deserves examination.

In summary, we have found that insulin stimulates intestinal glu expression and GLP-1 production. This finding reveals a potential novel function of insulin, namely enhancement of the levels of the incretin hormone GLP-1. We also observed that insulin stimulates nuclear β-cat accumulation and binding of β-cat and TCF-4 to the glu gene promoter and suggest that this represents a novel mechanism that underlies cross talk between canonical Wnt signaling and pathways that are triggered by insulin and other growth factors or hormones.


   Acknowledgments
 
We thank Dr. Derek LeRoith for providing the MKR mice, Dr. Eric Fearon for the dominant-negative TCF-4 plasmid, Dr. Bert Vogelstein for the pTOPFLASH reporter plasmid, and Dr. James Woodgett for the GSK-3β expression plasmid. We also thank Ms. Ling Liu for technical assistance.


   Footnotes
 
This work was supported by operating grants from Canadian Institutes for Health Research (CIHR) (68991 to T.J.), Canadian Diabetes Association (CDA1021 to P.L.B.), CIHR (38009 to I.G.F.), a Banting and Best Diabetes Centre (BBDC) Hugh Sellers Postdoctoral Fellowship (to F.Y.), a BBDC Graduate Studentship (to J.S.), and a CIHR Canada Doctoral Award (to G.E.L.). P.L.B. is supported by the Canada Research Chairs Program.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 7, 2008

1 F.Y. and J.S. made an equal contribution in this study. Back

Abbreviations: β-cat, β-Catenin; cat/TCF, β-catenin/T cell factor; ChIP, chromatin immunoprecipitation; dnTCF-4, dominant-negative TCF-4; FRIC, fetal rat intestinal cell; GLP, glucagon-like peptide; GSK-3, glycogen synthase kinase-3; LEF, lymphoid enhancer-binding factor; LUC, luciferase; MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; siRNA, small interfering RNA; TCF, T cell factor; T2D, type 2 diabetes.

Received August 20, 2007.

Accepted for publication January 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kinzler KW, Vogelstein B 1996 Lessons from hereditary colorectal cancer. Cell 87:159–170[CrossRef][Medline]
  2. Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM, Levy DB, Smith KJ, Preisinger AC, Hedge P, McKechnie D, Finniear R, Markhan A. Groffen J, Boguski MS, Altschul SF, Horii A, Ando H, Miyoshi Y, Miki Y, Nishisho I, Nakamura Y 1991 Identification of FAP locus genes from chromosome 5q21. Science 253:661–665[Abstract/Free Full Text]
  3. van Es JH, Barker N, Clevers H 2003 You Wnt some, you lose some: oncogenes in the Wnt signaling pathway. Curr Opin Genet Dev 13:28–33[CrossRef][Medline]
  4. He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW 1998 Identification of c-MYC as a target of the APC pathway. Science 281:1509–1512[Abstract/Free Full Text]
  5. Khor TO, Gul YA, Ithnin H, Seow HF 2006 A comparative study of the expression of Wnt-1, WISP-1, survivin and cyclin-D1 in colorectal carcinoma. Int J Colorectal Dis 21:291–300[CrossRef][Medline]
  6. He X, Saint-Jeannet JP, Woodgett JR, Varmus HE, Dawid IB 1995 Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature 374:617–622[CrossRef][Medline]
  7. Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, Kinzler KW 1997 Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC. Science 275:1787–1790[Abstract/Free Full Text]
  8. Yi F, Brubaker PL, Jin T 2005 TCF-4 mediates cell type-specific regulation of proglucagon gene expression by β-catenin and glycogen synthase kinase-3β. J Biol Chem 280:1457–1464[Abstract/Free Full Text]
  9. Doble BW, Woodgett JR 2003 GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116:1175–1186[Abstract/Free Full Text]
  10. Zeng X, Tamai K, Doble B, Li S, Huang H, Habas R, Okamura H, Woodgett J, He X 2005 A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 438:873–877[CrossRef][Medline]
  11. Stambolic V, Ruel L, Woodgett JR 1996 Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol 6:1664–1668[CrossRef][Medline]
  12. Kieffer TJ 2004 Gastro-intestinal hormones GIP and GLP-1. Ann Endocrinol (Paris) 65:13–21[Medline]
  13. Drucker DJ 2002 Biological actions and therapeutic potential of the glucagon-like peptides. Gastroenterology 122:531–544[CrossRef][Medline]
  14. Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC 1983 Exon duplication and divergence in the human preproglucagon gene. Nature 304:368–371[CrossRef][Medline]
  15. Bell GI, Santerre RF, Mullenbach GT 1983 Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature 302:716–718[CrossRef][Medline]
  16. Heinrich G, Gros P, Lund PK, Bentley RC, Habener JF 1984 Pre-proglucagon messenger ribonucleic acid: nucleotide and encoded amino acid sequences of the rat pancreatic complementary deoxyribonucleic acid. Endocrinology 115:2176–2181[Abstract/Free Full Text]
  17. Brubaker PL, Drucker DJ 2004 Minireview: glucagon-like peptides regulate cell proliferation and apoptosis in the pancreas, gut, and central nervous system. Endocrinology 145:2653–2659[CrossRef][Medline]
  18. Meeran K, O’Shea D, Edwards CM, Turton MD, Heath MM, Gunn I, Abusnana S, Rossi M, Small CJ, Goldstone AP, Taylor GM, Sunter D, Steere J, Choi SJ, Ghatei MA, Bloom SR 1999 Repeated intracerebroventricular administration of glucagon-like peptide-1-(7–36) amide or exendin-(9–39) alters body weight in the rat. Endocrinology 140:244–250[Abstract/Free Full Text]
  19. Turton MD, O’Shea D, Gunn I, Beak SA, Edwards CM, Meeran K, Choi SJ, Taylor GM, Heath MM, Lambert PD, Wilding JP, Smith DM, Ghatei MA, Herbert J, Bloom SR 1996 A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 379:69–72[CrossRef][Medline]
  20. Talsania T, Anini Y, Siu S, Drucker DJ, Brubaker PL 2005 Peripheral exendin-4 and peptide YY(3–36) synergistically reduce food intake through different mechanisms in mice. Endocrinology 146:3748–3756[Abstract/Free Full Text]
  21. Drucker DJ, Erlich P, Asa SL, Brubaker PL 1996 Induction of intestinal epithelial proliferation by glucagon-like peptide 2. Proc Natl Acad Sci USA 93:7911–7916[Abstract/Free Full Text]
  22. Lovshin J, Estall J, Yusta B, Brown TJ, Drucker DJ 2001 Glucagon-like peptide (GLP)-2 action in the murine central nervous system is enhanced by elimination of GLP-1 receptor signaling. J Biol Chem 276:21489–21499[Abstract/Free Full Text]
  23. Wojdemann M, Wettergren A, Hartmann B, Holst JJ 1998 Glucagon-like peptide-2 inhibits centrally induced antral motility in pigs. Scand J Gastroenterol 33:828–832[CrossRef][Medline]
  24. Estall JL, Koehler JA, Yusta B, Drucker DJ 2005 The glucagon-like peptide-2 receptor C terminus modulates β-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation. J Biol Chem 280:22124–22134[Abstract/Free Full Text]
  25. Ni Z, Anini Y, Fang X, Mills G, Brubaker PL, Jin T 2003 Transcriptional activation of the proglucagon gene by lithium and β-catenin in intestinal endocrine L cells. J Biol Chem 278:1380–1387[Abstract/Free Full Text]
  26. Philippe J 1989 Glucagon gene transcription is negatively regulated by insulin in a hamster islet cell line. J Clin Invest 84:672–677[Medline]
  27. Philippe J 1991 Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element. Proc Natl Acad Sci USA 88:7224–7227[Abstract/Free Full Text]
  28. Lu F, Jin T, Drucker DJ 1996 Proglucagon gene expression is induced by gastrin-releasing peptide in a mouse enteroendocrine cell line. Endocrinology 137:3710–3716[Abstract]
  29. Jin T, Drucker DJ 1996 Activation of proglucagon gene transcription through a novel promoter element by the caudal-related homeodomain protein cdx-2/3. Mol Cell Biol 16:19–28[Abstract]
  30. Jin T, Trinh DK, Wang F, Drucker DJ 1997 The caudal homeobox protein cdx-2/3 activates endogenous proglucagon gene expression in InR1–G9 islet cells. Mol Endocrinol 11:203–209[Abstract/Free Full Text]
  31. Kolligs FT, Hu G, Dang CV, Fearon ER 1999 Neoplastic transformation of RK3E by mutant β-catenin requires deregulation of Tcf/Lef transcription but not activation of c-myc expression. Mol Cell Biol 19:5696–706[Abstract/Free Full Text]
  32. Morin PJ, Vogelstein B, Kinzler KW 1996 Apoptosis and APC in colorectal tumorigenesis. Proc Natl Acad Sci USA 93:7950–7954[Abstract/Free Full Text]
  33. Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H 1997 Constitutive transcriptional activation by a β-catenin-Tcf complex in APC–/– colon carcinoma. Science 275:1784–1787[Abstract/Free Full Text]
  34. Liu S, Yu S, Hasegawa Y, Lapushin R, Xu HJ, Woodgett JR, Mills GB, Fang X 2004 Glycogen synthase kinase 3β is a negative regulator of growth factor-induced activation of the c-Jun N-terminal kinase. J Biol Chem 279:51075–51081[Abstract/Free Full Text]
  35. Fernandez AM, Kim JK, Yakar S, Dupont J, Hernandez-Sanchez C, Castle AL, Filmore J, Shulman GI, Le Roith D 2001 Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes Dev 15:1926–1934[Abstract/Free Full Text]
  36. Asghar Z, Yau D, Chan F, Leroith D, Chan CB, Wheeler MB 2006 Insulin resistance causes increased β-cell mass but defective glucose-stimulated insulin secretion in a murine model of type 2 diabetes. Diabetologia 49:90–99[CrossRef][Medline]
  37. Anini Y, Hansotia T, Brubaker PL 2002 Muscarinic receptors control postprandial release of glucagon-like peptide-1: in vivo and in vitro studies in rats. Endocrinology 143:2420–2426[Abstract/Free Full Text]
  38. Drucker DJ, Jin T, Asa SL, Young TA, Brubaker PL 1994 Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line. Mol Endocrinol 8:1646–1655[Abstract/Free Full Text]
  39. Rindi G, Grant SG, Yiangou Y, Ghatei MA, Bloom SR, Bautch VL, Solcia E, Polak JM 1990 Development of neuroendocrine tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of hormone expression. Am J Pathol 136:1349–1363[Abstract]
  40. Takaki R, Ono J, Nakamura M, Yokogawa Y, Kumae S, Hiraoka T, Yamaguchi K, Hamaguchi K, Uchida S 1986 Isolation of glucagon-secreting cell lines by cloning insulinoma cells. In Vitro Cell Dev Biol 22:120–126[Medline]
  41. Chen L, Wang P, Andrade CF, Zhao IY, Dube PE, Brubaker PL, Liu M, Jin T 2005 PKA independent and cell type specific activation of the expression of caudal homeobox gene Cdx-2 by cyclic AMP. FEBS J 272:2746–2759[CrossRef][Medline]
  42. Jamieson CH, Ailles LE, Dylla SJ, Muijtjens M, Jones C, Zehnder JL, Gotlib J, Li K, Manz MG, Keating A, Sawyers CL, Weissman IL 2004 Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML. N Engl J Med 351:657–667[Abstract/Free Full Text]
  43. Lim GE, Brubaker, PL 2006 Glucagon-like peptide 1 secretion by the L-cell. The View from within. Diabetes 55:S70–S77
  44. Schinner S, Barthel A, Dellas C, Grzeskowiak R, Sharma SK, Oetjen E, Blume R, Knepel W 2005 Protein kinase B activity is sufficient to mimic the effect of insulin on glucagon gene transcription. J Biol Chem 280:7369–7376[Abstract/Free Full Text]
  45. Nusse R 1997 A versatile transcriptional effector of Wingless signaling. Cell 89:321–323[CrossRef][Medline]
  46. Ding VW, Chen RH, McCormick F 2000 Differential regulation of glycogen synthase kinase 3β by insulin and Wnt signaling. J Biol Chem 275:32475–32481[Abstract/Free Full Text]
  47. Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR 2000 Requirement for glycogen synthase kinase-3β in cell survival and NF-{kappa}B activation. Nature 406:86–90[CrossRef][Medline]
  48. Fu P, Arcasoy MO 2007 Erythropoietin protects cardiac myocytes against anthracycline-induced apoptosis. Biochem Biophys Res Commun 354:372–378[CrossRef][Medline]
  49. Murtaugh LC, Law AC, Dor Y, Melton DA 2005 β-Catenin is essential for pancreatic acinar but not islet development. Development 132:4663–4674[Abstract/Free Full Text]
  50. Florez JC, Jablonski KA, Bayley N, Pollin TI, de Bakker PI, Shuldiner AR, Knowler WC, Nathan DM, Altshuler D 2006 TCF7L2 polymorphisms and progression to diabetes in the Diabetes Prevention Program. N Engl J Med 355:241–250[Abstract/Free Full Text]
  51. Grant SF, Thorleifsson G, Reynisdottir I, Benediktsson R, Manolescu A, Sainz J, Helgason A, Stefansson H, Emilsson V, Helgadottir A, Styrkarsdottir U, Magnusson KP, Walters GB, Palsdottir E, Jonsdottir T, Gudmundsdottir T, Gylfason A, Saemundsdottir J, Wilensky RL, Reilly MP, Rader DJ, Bagger Y, Christiansen C, Gudnason V, Sigurdsson G, Thorsteinsdottir U, Gulcher JR, Kong A, Stefansson K 2006 Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet 38:320–323[CrossRef][Medline]
  52. Lyssenko V, Lupi R, Marchetti P, Del Guerra S, Orho-Melander M, Almgren P, Sjogren M, Ling C, Eriksson KF, Lethagen AL, Mancarella R, Berglund G, Tuomi T, Nilsson P, Del Prato S, Groop L 2007 Mechanisms by which common variants in the TCF7L2 gene increase risk of type 2 diabetes. J Clin Invest 117:2155–2163[CrossRef][Medline]
  53. Desbois-Mouthon C, Cadoret A, Blivet-Van Eggelpoel MJ, Bertrand F, Cherqui G, Perret C, Capeau J 2001 Insulin and IGF-1 stimulate the β-catenin pathway through two signalling cascades involving GSK-3β inhibition and Ras activation. Oncogene 20:252–259[CrossRef][Medline]
  54. Verras M, Sun Z 2005 β-Catenin is involved in insulin-like growth factor 1-mediated transactivation of the androgen receptor. Mol Endocrinol 19:391–398[Abstract/Free Full Text]
  55. Tian Q, Feetham MC, Tao WA, He XC, Li L, Aebersold R, Hood L 2004 Proteomic analysis identifies that 14-3-3{zeta} interacts with β-catenin and facilitates its activation by Akt. Proc Natl Acad Sci USA 101:15370–15375[Abstract/Free Full Text]
  56. Wilker E, Lu J, Rho O, Carbajal S, Beltran L, DiGiovanni J 2005 Role of PI3K/Akt signaling in insulin-like growth factor-1 (IGF-1) skin tumor promotion. Mol Carcinog 44:137–145[CrossRef][Medline]
  57. Ding Q, Xia W, Liu JC, Yang JY, Lee DF, Xia J, Bartholomeusz G, Li Y, Pan Y, Li Z, Bargou RC, Qin J, Lai CC, Tsai FJ, Tsai CH, Hung MC 2005 Erk associates with and primes GSK-3β for its inactivation resulting in upregulation of β-catenin. Mol Cell 19:159–170[CrossRef][Medline]
  58. Satyamoorthy K, Li G, Vaidya B, Patel D, Herlyn M 2001 Insulin-like growth factor-1 induces survival and growth of biologically early melanoma cells through both the mitogen-activated protein kinase and β-catenin pathways. Cancer Res 61:7318–7324[Abstract/Free Full Text]
  59. Desbois-Mouthon C, Blivet-Van Eggelpoel MJ, Beurel E, Boissan M, Delelo R, Cadoret A, Capeau J 2002 Dysregulation of glycogen synthase kinase-3β signaling in hepatocellular carcinoma cells. Hepatology 36:1528–1536[Medline]
  60. Zhang Q, Adiseshaiah P, Kalvakolanu DV, Reddy SP 2006 A phosphatidylinositol 3-kinase-regulated Akt-independent signaling promotes cigarette smoke-induced FRA-1 expression. J Biol Chem 281:10174–10181[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
P. Wang, Q. Wang, J. Sun, J. Wu, H. Li, N. Zhang, Y. Huang, B. Su, R.-k. Li, L. Liu, et al.
POU Homeodomain Protein Oct-1 Functions as a Sensor for Cyclic AMP
J. Biol. Chem., September 25, 2009; 284(39): 26456 - 26465.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. S. Sirek, L. Liu, M. Naples, K. Adeli, D. S. Ng, and T. Jin
Insulin Stimulates the Expression of Carbohydrate Response Element Binding Protein (ChREBP) by Attenuating the Repressive Effect of Pit-1, Oct-1/Oct-2, and Unc-86 Homeodomain Protein Octamer Transcription Factor-1
Endocrinology, August 1, 2009; 150(8): 3483 - 3492.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
S.-S. Lu, Y.-L. Yu, H.-J. Zhu, X.-D. Liu, L. Liu, Y.-W. Liu, P. Wang, L. Xie, and G.-J. Wang
Berberine promotes glucagon-like peptide-1 (7-36) amide secretion in streptozotocin-induced diabetic rats
J. Endocrinol., February 1, 2009; 200(2): 159 - 165.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
G. E. Lim, G. J. Huang, N. Flora, D. LeRoith, C. J. Rhodes, and P. L. Brubaker
Insulin Regulates Glucagon-Like Peptide-1 Secretion from the Enteroendocrine L Cell
Endocrinology, February 1, 2009; 150(2): 580 - 591.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
W. Kim and J. M. Egan
The Role of Incretins in Glucose Homeostasis and Diabetes Treatment
Pharmacol. Rev., December 1, 2008; 60(4): 470 - 512.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. Jin and L. Liu
Minireview: The Wnt Signaling Pathway Effector TCF7L2 and Type 2 Diabetes Mellitus
Mol. Endocrinol., November 1, 2008; 22(11): 2383 - 2392.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yi, F.
Right arrow Articles by Jin, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yi, F.
Right arrow Articles by Jin, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals