Investigations on the metabolic role of the Wnt signaling pathway and hepatic transcription factor 7-like 2 (TCF7L2) have generated opposing views. While some studies demonstrated a repressive effect of TCF7L2 on hepatic gluconeogenesis, a recent study using liver-specific Tcf7l2−/− mice suggested the opposite. As a consequence of redundant and bidirectional actions of transcription factor (TCF) molecules and other complexities of the Wnt pathway, knockout of a single Wnt pathway component may not effectively reveal a complete metabolic picture of this pathway. To address this, we generated the liver-specific dominant-negative (DN) TCF7L2 (TCF7L2DN) transgenic mouse model LTCFDN. These mice exhibited progressive impairment in response to pyruvate challenge. Importantly, LTCFDN hepatocytes displayed elevated gluconeogenic gene expression, gluconeogenesis, and loss of Wnt-3a–mediated repression of gluconeogenesis. In C57BL/6 hepatocytes, adenovirus-mediated expression of TCF7L2DN, but not wild-type TCF7L2, increased gluconeogenesis and gluconeogenic gene expression. Our further mechanistic exploration suggests that TCF7L2DN-mediated inhibition of Wnt signaling causes preferential interaction of β-catenin (β-cat) with FoxO1 and increased binding of β-cat/FoxO1 to the Pck1 FoxO binding site, resulting in the stimulation of Pck1 expression and increased gluconeogenesis. Together, our results using TCF7L2DN as a unique tool revealed that the Wnt signaling pathway and its effector β-cat/TCF serve a beneficial role in suppressing hepatic gluconeogenesis.
Following pivotal studies indicating that the transcription factor 7-like 2 (TCF7L2) is an important risk gene for the development of type 2 diabetes (T2D) (1), great efforts have been made to explore its role as a Wnt signaling molecule in pancreatic β-cells and other tissues including liver (2–16). Although several investigations suggested that TCF7L2 negatively regulates hepatic gluconeogenesis (8,10,11,17), one recent study (7) reported that liver-specific knockout of TCF7L2 reduced hepatic glucose production (HGP), while hepatic overexpression of TCF7L2 increased HGP. As a result, the controversial issues surrounding the metabolic function of TCF7L2 have been extended from pancreatic β-cells to liver and hepatocytes specifically (4–6,9,13,18–20).
TCF7L2 or other transcription factor (TCF) members can interact with β-catenin (β-cat), forming the bipartite transcription factor β-cat/TCF, which serves as the important effector of the Wnt signaling pathway. The role of Wnt signaling has been intensively studied in many tissues, including the liver, using various transgenic animal models. Although overexpression of a given Wnt ligand or the expression of constitutively active S33Y mutant β-cat has provided solid evidence for the involvement of this signaling cascade in liver development, zonation, cell proliferation, tumorigenesis, and the susceptibility to oxidative stress (21–26), the hepatic role of Wnt signaling in metabolic homeostasis still remains unclear. The complexity of the Wnt signaling pathway is reflected by the existence of multiple Wnt ligands, receptors, and coreceptors, as well as various intracellular modulating elements (27,28). Furthermore, the function of TCFs is bidirectional, depending on their interaction partners as well as the availability and phosphorylation status of their cofactor β-cat. Finally, β-cat also interacts with FOXOs, key effectors of the stress and aging signaling pathway (29). The pathophysiological contribution of the interaction between FOXOs and β-cat was demonstrated previously in bone diseases (30,31) and very recently in hepatic gluconeogenesis (32). Thus, the function of TCF members is not only directly controlled by its cofactor β-cat, nuclear coactivators, and corepressors, but also indirectly regulated by the stress signaling pathway effectors FOXOs.
Here, we generated a transgenic mouse model, LTCFDN, in which dominant-negative (DN) TCF7L2 (TCF7L2DN) was expressed specifically in the liver (33). TCF7L2DN has been shown to repress Wnt target gene expression in vitro and in vivo by our team and other investigators (12,34–36). The observations that LTCFDN mice exhibit progressive impairment of pyruvate and glucose tolerance along with the upregulation of the gluconeogenic gene program and glucose output in primary hepatocytes indicate that the Wnt signaling cascade negatively regulates hepatic gluconeogenesis.
Research Design and Methods
The LTCFDN mouse model was generated by cloning the mouse serum albumin 2.4-kb promoter/enhancer construct (provided by Dr. Richard Palmiter, University of Pennsylvania) upstream of the 1.6-kb human TCF7L2DN long-isoform cDNA sequence (12,33–35). FVB mouse zygote pronuclear microinjection of the linearized DNA construct and implantation into pseudopregnant recipients were then performed by the Toronto Centre for Phenogenomics Transgenic Core (12). Male heterozygous LTCFDN mice were consistently bred with female wild-type (WT) FVB mice to produce heterozygous mice (labeled as LTCFDN) and control WT littermates. Male C57BL/6 mice (at 7 weeks of age) were purchased from Charles River Laboratories (St. Laurent, Quebec, Canada) as previously described (37). The mice were housed on a 12-h light-dark cycle at ambient room temperature with free access to normal chow diet and water. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University Health Network.
In Vivo Tolerance Tests and Glucose Production Assay
For glucose and pyruvate tolerance tests, mice were fasted for 16 h prior to intraperitoneal injection of either glucose (2 g/kg body wt) or pyruvate (2 g/kg body wt) (38). For the insulin tolerance test, mice were starved for 6 h prior to intraperitoneal insulin (0.75–1.0 units/kg body wt) injection (38). The method for the glucose production assay has been previously described and is detailed in the Supplementary Experimental Procedures (38).
Isolation of Mouse Primary Hepatocytes and Cell Culture
The WT TCF7L2 and TCF7L2DN cDNA were originally provided by Dr. Eric Fearon (University of Michigan Medical School) (35). The adenoviruses (Ads) Ad-green fluorescent protein, Ad-TCF7L2WT, and Ad-TCF7L2DN were generated using the AdEasy XL Adenoviral Vector System (Agilent Technologies), as detailed in the Supplementary Experimental Procedures.
RNA Isolation, Reverse Transcription, and Quantitative PCR
Isolation of RNA, cDNA synthesis by reverse transcription, and real-time PCR were performed as previously described (38). Nonquantitative PCR was performed using the Fast DNA Polymerase PCR Mix (Kapa Biosystems) followed by agarose gel electrophoresis. Specific genes were quantified by amplification using specific primers listed in Supplementary Table 1. All instructions of the manufacturers were followed.
Whole-cell lysates were prepared from Hepa1-6 cells in radioimmunoprecipitation assay buffer. Two micrograms of β-cat, FoxO1, or control IgG antibody (Santa Cruz Biotechnology) were mixed with 500 μg lysates overnight at 4°C with agitation followed by incubation with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) for 2 h at 4°C with agitation. Beads were washed four times in radioimmunoprecipitation assay buffer by centrifugation and removal of supernatant, then were suspended in 50 μL 1× sample buffer and boiled for 5 min. Samples were then subjected to SDS-PAGE for Western blotting. Lysates not subjected to the immunoprecipitation procedure were used as the input controls. The HEK293 cell line was provided by the American Type Culture Collection. Cells were infected by Ad-TCF7L2WT or Ad-TCF7L2DN for 36 h. Two micrograms of hemagglutinin (HA)-tagged antibody were used for immunoprecipitation, followed by Western blotting with the indicated antibody.
Plasmid DNA Transfection, Luciferase Reporter Analysis, and Electrophoretic Mobility Shift Assay
The methods for plasmid DNA transfection and luciferase (LUC) reporter analyses of TOPflash and Pck1(−595/+67)-LUC were described previously, with Hepa1-6 cells cultured in six-well plates (10,34). Cells were harvested 18 h after plasmid transfection (1 µg reporter plasmid or 1 µg reporter plasmid plus 0.5 µg indicated cDNA or control plasmid) for LUC analysis (10). The procedure for electrophoretic mobility shift assay is detailed in the Supplementary Experimental Procedures.
Serum insulin levels were measured using the Rat Insulin Radioimmunoassay Kit (Millipore) according to the manufacturer’s instructions.
Data are presented as the mean ± SEM. Significance was determined using the Student t test or one-way ANOVA followed by Bonferroni post hoc test as appropriate for single or multiple comparisons, respectively. Differences were considered statistically significant when P < 0.05.
Verification of the DN Action of TCF7L2DN and the Generation of the LTCFDN Mouse Model
The albumin-TCF7L2DN fusion gene (Fig. 1A) was constructed in which the expression of human TCF7L2DN (12) is driven by a mouse albumin promoter/enhancer construct, which was provided by Dr. Richard Palmiter (University of Pennsylvania) (33). Lacking the β-cat interaction domain (Fig. 1A), TCF7L2DN acts as a DN molecule to block, in theory, the function of any TCF7L2 isoforms and other TCFs such as TCF7 and TCF7L1, which are known to be expressed in hepatocytes (10). In our previous in vitro and in vivo investigations (12,34), TCF7L2DN attenuated basal as well as Wnt-stimulated gut proglucagon gene expression. To verify the DN action of TCF7L2DN at the molecular level, we infected HEK293 cells with either Ad-TCF7L2WT or Ad-TCF7L2DN. The expression of each exogenous TCF7L2 protein was verified by detection of the HA tag (Supplementary Fig. 1A). Both WT TCF7L2 and TCF7L2DN were bound to the consensus TCF binding site (Supplementary Fig. 1B), and the binding in both cases was attenuated by the addition of an unlabeled “cold” probe (Supplementary Fig. 1C). Precipitation of the HA tag also pulled down β-cat in HEK293 cells infected with Ad-TCF7L2WT but not in HEK293 cells infected with Ad-TCF7L2DN (Supplementary Fig. 1D and E). Thus, TCF7L2DN possessed the ability to bind to the TCF binding site on target promoters but failed to recruit the coactivator β-cat, as intended.
We generated the LTCFDN mouse model via insertion of the albumin-TCF7L2DN fusion gene. Among the five transgene-positive founders (Supplementary Fig. 2A), the offspring of founder C showed germ-line transmission and substantial adult hepatic TCF7L2DN expression (Supplementary Fig. 2B and Fig. 1B), and were thus used for all further studies. Liver-specific expression of TCF7L2DN was confirmed as this exogenous protein, which is 3 kDa smaller than the 78-kDa endogenous isoform, was not detected in other adult organs (Fig. 1C). While TCF7L2DN was also expressed in the liver of 2-week-old LTCFDN mice, it was undetectable in the liver of newborn mice (Fig. 1D). Interestingly, both WT and LTCFDN newborn mice lacked endogenous hepatic TCF7L2 protein expression (Fig. 1D), although three TCF members were detectable at the mRNA level in the newborn mouse liver (Supplementary Fig. 2C). In LTCFDN hepatocytes, the expression of Axin2, a known Wnt pathway downstream target gene in hepatocytes, was significantly reduced (Fig. 1E), indicating that TCF7L2DN expression in hepatocytes indeed attenuated the Wnt signaling pathway. The expression levels of the gluconeogenesis genes, including Pck1, G6pc, Fbp1, and Ppargc1a, were increased in the liver tissue of LTCFDN mice in comparison with WT mice (Fig. 1F). The majority of our study was performed in adult male mice. When analyzing newborn and 2-week-old mice, sex was ignored.
LTCFDN Mice Exhibit Progressive Defects in Pyruvate Tolerance
On chow diet for up to 42 weeks, LTCFDN mice showed no significant alterations in body weight (Supplementary Fig. 3A), or in fasted or fed glucose and insulin levels (Supplementary Fig. 3B and C), while 12-week-old LTCFDN mice showed a modest but significant increase in liver weight (Supplementary Fig. 3D). At the age of 9 weeks, LTCFDN mice displayed significant impairment of tolerance to pyruvate, a major gluconeogenic precursor (Fig. 2A and Supplementary Fig. 3E). This defect was exacerbated at 23 and 40 weeks of age (Fig. 2B and C and Supplementary Fig. 3F and G). We did not observe an impaired tolerance to glucose challenge in 10-week-old LTCFDN mice (Fig. 2D and Supplementary Fig. 3H). However, 24-week-old LTCFDN mice were glucose intolerant (Fig. 2E and Supplementary Fig. 3I). Importantly, at 11 or 41 weeks of age, we did not detect any abnormal responses to insulin challenge (Fig. 2F and G and Supplementary Fig. 3J and K). Consistently, hepatocytes isolated from 12-week-old LTCFDN mice produced higher levels of glucose from gluconeogenic precursors (Fig. 2H). While the canonic Wnt ligand Wnt-3a repressed glucose production in WT hepatocytes, this effect was absent in hepatocytes isolated from LTCFDN mice (Fig. 2H and Supplementary Fig. 3L). Furthermore, there were no defects in Akt S473 phosphorylation in response to intraperitoneal insulin injection in the livers of LTCFDN mice (Supplementary Fig. 3M and N). Finally, we examined the hepatic expression levels of FoxO1, S256 FoxO1, β-cat, as well as S675 β-cat. No appreciable differences were observed between LTCFDN and WT littermates, suggesting that hepatic expression of TCF7L2DN did not directly affect the expression or phosphorylation levels of these two components, at least in the absence of a challenge (Supplementary Fig. 4).
Ad-Mediated Expression of TCF7L2DN but Not WT TCF7L2 Increases Gluconeogenesis In Vitro
We generated Ads to elicit the expression of WT TCF7L2 or TCF7L2DN in vitro (Fig. 3A). Treatment of adult C57BL/6 hepatocytes with these viruses caused >95% cell infection (Supplementary Fig. 5A). Primary hepatocytes infected by Ad-TCF7L2DN, but not Ad-TCF7L2WT, showed significantly increased glucose production (Fig. 3B) and the expression of a panel of gluconeogenic genes, including Pck1, G6pc, Fbp1, and Ppargc1a (Fig. 3C). In addition, hepatocytes infected with either Ad-TCF7L2WT or Ad-TCF7L2DN showed significantly reduced expression of Axin2, a known Wnt pathway downstream target (Fig. 3D). The repressive effect of WT TCF7L2 on Axin2 expression has further indicated the bidirectional nature of TCF members.
β-Cat and FoxO1 Exert Opposite Effects on TOPflash Expression
The function of TCF is largely determined by its interaction partners, including β-cat. The TOPflash reporter system is a useful tool in assessing Wnt pathway activation or β-cat/TCF activity (40). Treatment of Hepa1-6 cells with Wnt-3a, which increases nuclear β-cat content, stimulated the expression of TOPflash (Fig. 4A). Although S33Y β-cat cotransfection also increased TOPflash activity, FoxO1 cotransfection repressed the expression of TOPflash (Fig. 4B). Interestingly, FoxO1-mediated repression of TOPflash was not attenuated by S33Y β-cat cotransfection (Fig. 4B).
Peptide hormones such as GLP-1 and insulin stimulate β-cat S675 or S552 phosphorylation and hence increase β-cat/TCF activity (10,41). We observed previously in mice that feeding increased hepatic TCF7L2 expression while in vitro insulin treatment increased β-cat S675 phosphorylation in hepatocytes (10). We show here that feeding also increased hepatic β-cat S675 and S552 phosphorylation in C57BL/6 mice (Fig. 4C and D), further supporting the notion that β-cat/TCF mediates the hepatic function of peptide hormones in response to food consumption (10).
As the function of TCFs can be controlled by the competition between TCF members and FoxO proteins for a limited reservoir of β-cat, we verified the physical interaction between β-cat and FoxO1 in hepatocytes by coimmunoprecipitation. FoxO1 interacted with β-cat in mouse hepatocytes as both β-cat and FoxO1 could be detected after precipitation of either β-cat or FoxO1 in the Hepa1-6 cell line (Fig. 4E). The above findings collectively indicate the role of FoxO1 in interfering with β-cat/TCF activity in hepatocytes.
Increased Binding of FoxO1 and β-Cat to the Pck1 Gene Promoter in LTCFDN Hepatocytes
There is no functional TCF binding motif that has been identified within the 5′ flanking region of the Pck1 gene, although a previous study (8) identified a TCF motif within the 3′ region of the gene. A functional FoxO binding site, however, is located within the proximal promoter regions of human and rodent Pck1 genes (Fig. 5A) (42,43). To further elucidate the mechanism underlying the increased levels of hepatic gluconeogenesis observed in the LTCFDN mouse model, we assessed the binding of FoxO1 and β-cat to the Pck1 gene promoter by ChIP. We detected binding of β-cat and FoxO1 to the Pck1 promoter in both WT and LTCFDN hepatocytes (Fig. 5B). Importantly, quantitative ChIP revealed that the interactions of FoxO1 and β-cat with the Pck1 promoter were increased in LTCFDN hepatocytes (Fig. 5C). These observations collectively suggest that TCF7L2DN-mediated inhibition of Wnt signaling causes preferential interaction of β-cat with FoxO1 and increased binding of β-cat/FoxO1 to the Pck1 FoxO binding site, resulting in the stimulation of Pck1 expression.
To further verify the dual function of the β-cat molecule, we tested the effect of S33Y β-cat transfection and Wnt-3a treatment directly on Pck1 promoter activity in Hepa1-6 cells. The profound stimulatory effect generated by β-cat cotransfection (Fig. 5D) indicates the importance of the FoxO binding motif within the Pck1-LUC reporter construct. Wnt-3a treatment significantly repressed Pck1-LUC expression (Fig. 5E), which is consistent with our observations that Wnt-3a represses gluconeogenesis (Fig. 2H) and Pck1 mRNA levels in mouse hepatocytes (10). Together, these observations illustrate the repressive effect of Wnt signaling and β-cat/TCF on gluconeogenesis, involving the competition between TCF and FoxO for their common cofactor β-cat as well as an undetermined intrinsic repressive property of β-cat/TCF.
Although a few investigations (8,10,11,17) have suggested a repressive effect of Wnt signaling activation or TCF7L2 itself on hepatic gluconeogenesis, a recent study (7) using liver-specific TCF7L2 knockout mice and a TCF7L2 overexpression model presented an opposite view. Our studies here were designed to address discrepancies regarding the metabolic function of the Wnt signaling pathway as well as the mechanistic role of TCF7L2 as a T2D risk gene. We propose that, because of the bidirectional function of the TCF molecules as well as the cross talk between Wnt and the stress FoxO signaling pathway, knockout of a single TCF member is an inadequate strategy to reveal the complete role of the Wnt signaling pathway in metabolic homeostasis. We thus conducted our in vitro and in vivo investigations primarily using a DN blocker, TCF7L2DN, and explored two other components in this pathway, β-cat and FoxO1. There are several advantages of our LTCFDN mouse model. First, the albumin promoter is not expressed in the fetus (Fig. 1D), and thus the potential effect of TCF7L2DN on embryonic liver development is avoided. Second, the functional knockdown approach circumvents the potential problems caused by the bidirectional and redundant actions of TCF7L2 and other TCF members, which would be experienced using regular overexpression or knockout approaches. Finally, this approach allowed us to dissect the contribution of β-cat and FoxO1, as TCF7L2DN lacks the interaction motif for β-cat. Nevertheless, a relatively lower level of transgene expression in our system is a potential drawback.
The LTCFDN transgenic mice generated for this study developed a progressive impairment of pyruvate tolerance in the absence of appreciable whole-body or hepatic insulin resistance (Fig. 2F and G and Supplementary Fig. 3M and N), suggesting that liver-specific TCF7L2DN expression may directly cause the elevation of hepatic gluconeogenesis. Based on these results and those obtained from the use of Ad-TCF7L2WT and Ad-TCF7L2DN in mouse primary hepatocytes, we conclude that the Wnt signaling pathway effector β-cat/TCF negatively regulates hepatic gluconeogenesis. It is necessary to note that, in the absence of pyruvate injection, basal plasma glucose levels were similar in LTCFDN and WT littermates. Furthermore, LTCFDN mice remained glucose tolerant at 10 weeks old (Fig. 2D). This is likely because of the existence of compensatory mechanisms in controlling glucose homeostasis in the absence of challenge. Nevertheless, the negative regulation of gluconeogenesis represents a novel mechanism by which metabolic hormones control hepatic glucose metabolism in response to physiological or nutritional changes. During fasting, glucagon, which was previously shown to increase hepatic β-cat S675 phosphorylation (10), stimulates the stress signaling pathway effector FoxO1 and gluconeogenic gene expression while hepatic TCF7L2 levels are relatively low. In response to food intake, the level of insulin elevates. Insulin not only inactivates FoxO1 via the PI3K/Akt signaling cascade, but also increases the expression of TCF7L2 and β-cat S675 and S552 phosphorylation, leading to the repression of hepatic gluconeogenic gene expression and gluconeogenesis (Fig. 5F).
Genome-wide association studies revealed that certain TCF7L2 single nucleotide polymorphisms are strongly associated with susceptibility to T2D (1,44–46). Following this milestone discovery in diabetes research, the metabolic function of TCF7L2 has been intensively investigated. Initial studies were primarily conducted in pancreas or pancreatic β-cells as impaired GLP-1–induced insulin secretion was observed in TCF7L2 T2D risk single nucleotide polymorphism carriers (3). Many studies (3–5,13,36,47) suggested that TCF7L2 exerts beneficial effects on β-cell proliferation, viability, and insulin secretion. Boj et al. (7), however, demonstrated very recently that β-cell–specific deletion of TCF7L2 in mice generated no deleterious effects on these parameters, which is in contrast to the findings of a study presented by da Silva Xavier et al. (9), showing an impairment of glucose homeostasis and β-cell function in mice with β-cell–specific Tcf7l2 deletion. This discrepancy can be attributed to subtle experimental details, such as the usage of different β-cell–specific promoters (Pdx1 vs. Ins2) to drive Cre recombinase–mediated excision of LoxP-flanked Tcf7l2 sequences in the β-cell lineage. However, we cannot exclude the possibility that, because of the extreme complexity of the Wnt signaling pathway, knockout or overexpression of a given member may not always be sufficient to reveal the true function of this signaling cascade. TCF7L2 and other TCF members may possess certain compensatory functions as they carry very similar DNA binding and β-cat interaction domains. In addition, all TCF members can recruit either nuclear coactivators, such as CBP, or nuclear corepressors, such as Groucho and CtBP (48–50).
We have previously expressed TCF7L2DN in vitro as well as in vivo to assess the role of Wnt signaling in gut and brain proglucagon gene expression, GLP-1 production, and function (12,34). Here, we further explored the characteristics of the TCF7L2DN protein in binding to the consensus TCF binding motif by electrophoretic mobility shift assay and the inability to recruit β-cat by coimmunoprecipitation. In mouse pancreatic islets, liver, and adipocytes, there are two major isoforms of TCF7L2 of size 78 and 58 kDa, both of which possess common DNA and β-cat interaction domains. Further functional exploration of these two major isoforms in hepatic metabolic homeostasis is necessary. Very recently, Takamoto et al. (36) generated a transgenic mouse model in which the short isoform of TCF7L2DN (58 kDa) was driven by the Ins2 gene promoter. These transgenic mice showed impaired glucose homeostasis and reduced β-cell mass and insulin secretion. Thus, TCF7L2DN indeed represents a powerful alternative approach to explore the metabolic functions of β-cat/TCF and the Wnt signaling pathway.
We and others have investigated the metabolic function of hepatic TCF7L2 during the past few years. Norton et al. (8) found that the silencing of TCF7L2 in hepatocytes induced a marked increase in basal glucose production and gluconeogenic gene expression, while TCF7L2 overexpression reversed this phenotype and reduced glucose production. An in vivo study presented by Oh et al. (11) also suggested a crucial role of TCF7L2 in reducing HGP. We demonstrated the expression of three TCF members in mouse hepatocytes and revealed that hepatic TCF7L2 expression was stimulated by feeding in C57BL/6 mice or by insulin treatment in hepatocytes in vitro, while Wnt-3a treatment repressed gluconeogenesis in mouse primary hepatocytes (10). Most recently, Neve et al. (17) replicated our findings on the repressive effect of TCF7L2 on gluconeogenic gene expression. Evidence from the current study using the unique LTCFDN mouse model and Ad-TCF7L2DN expression in vitro further prompts us to conclude that Wnt and β-cat/TCF repress hepatic gluconeogenesis in response to nutrient intake.
It is worth highlighting that our observations also suggest that the cofactor β-cat, but not TCFs, serves as a limiting factor in regulating hepatic Wnt activity, at least in certain settings. Overexpression of WT TCF7L2 produced no appreciable effect, while TCF7L2DN generated a stimulatory effect on gluconeogenic gene expression in primary hepatocytes. Furthermore, although Ad-TCF7L2DN repressed Axin2 expression, Ad-TCF7L2WT also repressed Axin2 expression, supporting the notion of bidirectional function of TCF members. Indeed, in the absence of β-cat, TCFs are known to recruit nuclear corepressors (48–50).
Although Norton et al. (8) located a TCF binding motif within the Pck1 gene, this potential cis element is positioned in the 3′ region of the gene, and its contribution to Pck1 gene expression remains unknown. Hence, we decided to conduct our investigation on the role of Wnt signaling and β-cat/TCF on Pck1 expression by assessing the contribution of the known FoxO binding site within the proximal promoter region. The stimulatory effect of S33Y β-cat and the repressive effect of Wnt-3a on Pck1 promoter activity further indicate that β-cat cooperates with FoxO1 in stimulating hepatic gluconeogenesis (32). In addition, we speculate that Wnt activation by Wnt-3a exerts an intrinsic repressive effect on gluconeogenesis via an undetermined atypical TCF binding site within the proximal promoter region, a hypothesis that requires further exploration. Indeed, starvation reduced the interaction between TCF7L2 and β-cat, and blunted the expression of a panel of Wnt targets (32). In further support of this notion, we demonstrated that β-cat accumulation was increased on the Pck1 promoter in LTCFDN hepatocytes. Interestingly, FoxO-mediated repression of TOPflash reporter activity was not attenuated by S33Y β-cat cotransfection (Fig. 4B), suggesting that other mechanisms exist besides the competition between TCF and FoxO1 for the common cofactor β-cat (30,31). One may speculate that β-cat could recruit FoxO to the TCF binding site, followed by nuclear corepressor recruitment. Alternatively, the repressive effect of FoxO may simply be overwhelming in this particular in vivo reporter gene transfection system.
In summary, we applied a novel concept and a unique animal approach to address an important yet controversial issue. Using TCF7L2DN as a tool for both in vivo and in vitro investigations, we demonstrated that the blockage of Wnt signaling led to increased gluconeogenesis and gluconeogenic gene expression. This observation was at least partially attributed to the increased occupancy of the gluconeogenic gene promoter Pck1 by β-cat/FoxO1.
Z.C. is currently affiliated with the Department of Endocrinology and Metabolism, The Third Affiliated Hospital of Sun Yat-sen University and Guangdong Provincial Key Laboratory of Diabetology, Guangzhou, People’s Republic of China.
Funding. W.I. is a recipient of the Canadian Institutes of Health Research Doctoral Canada Graduate Scholarship, Ontario Graduate Scholarship, and the Banting & Best Diabetes Centre-University Health Network Graduate Award. This work was supported by operating grants from the Canadian Institutes of Health Research to M.B.W. (MOP-102588) and T.J. (MOP-89987 and MOP-97790).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. W.I. designed and conducted the experiments, analyzed the data, and wrote the manuscript. W.S., Z.S., and Z.C. conducted the experiments. M.B.W. provided research material, assisted with the experimental design, and edited the manuscript. T.J. designed and conducted the experiments and wrote the manuscript. T.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.