TCF7L2 in mouse pancreatic beta cells plays a crucial role in glucose homeostasis by regulating beta cell mass
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Common genetic variations of the transcription factor 7-like 2 gene (encoded by TCF7L2), one of the T cell factor/lymphoid enhancer-binding factor transcription factors for the converging wingless-type MMTV integration site family (Wnt)/β-catenin signalling pathway, are known to be associated with type 2 diabetes. Individuals with at-risk alleles of TCF7L2 exhibit impaired insulin secretion. Although previous studies using animal models have revealed the existence of a relationship between the Wnt/β-catenin signalling pathway and glucose homeostasis, it remains unclear whether TCF7L2 in the pancreatic beta cells might be causally involved in insulin secretion in vivo. In this study, we investigated the role of TCF7L2 expressed in the pancreatic beta cells in glucose homeostasis.
Three independent groups of genetically engineered mice (DN mice) were generated, in which expression of the dominant-negative form of Tcf7l2 was driven under a rat insulin promoter. Phenotypes of both adult and newborn mice were evaluated. The levels of genes and proteins expressed in isolated islets were determined by reverse transcription-quantitative PCR and western blot analysis, respectively.
Adult DN mice showed impaired glucose tolerance and decreased insulin secretion in both oral and intraperitoneal glucose tolerance tests. Marked reduction of the beta cell area and whole-pancreas insulin content was observed in both the adult and newborn DN mice. Islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa, consistent with the deleterious effects of the dominant-negative form of Tcf7l2 on beta cell proliferation and insulin production.
TCF7L2 expressed in the pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass.
KeywordsBeta cell proliferation Dominant-negative form Insulin production Insulin secretion TCF7L2 Wnt/β-catenin signalling
- DN mice
ΔN-Tcf7l2 transgenic mice
Glucagon-like peptide 1 receptor
Intraperitoneal glucose tolerance test
v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian)
Quantitative real-time PCR
Rat insulin promoter
Transcription factor 7-like 2
T cell factor/lymphoid enhancer-binding factor
Wingless-type MMTV integration site family
Type 2 diabetes is characterised by peripheral insulin resistance and pancreatic beta cell dysfunction [1, 2]. Since 2006, numerous studies, including genome-wide association studies, have generally confirmed a strong association between single-nucleotide polymorphisms (SNPs) in the human gene encoding transcription factor 7-like 2 (TCF7L2) and the risk of onset of type 2 diabetes [3, 4]. These findings have also been confirmed in Asian populations, including the Japanese population [5, 6, 7]. The TCF7L2 (formerly known as TCF4) at-risk alleles are associated with specific phenotypes characterised by impaired insulin secretion rather than insulin resistance [8, 9]. In addition, it has been shown in human studies that these alleles may modify the insulinotropic actions of the incretin hormones [10, 11].
TCF7L2 is one of the wingless-type MMTV integration site family (Wnt)/β-catenin signalling-associated transcription factors expressed in several tissues, including the pancreas, and vertebrates are known to carry four genes of the TCF/LEF (T cell factor/lymphoid enhancer-binding factor) family (TCF7/TCF-1, LEF1, TCF7L1/TCF3 and TCF7L2/TCF4) [12, 13, 14]. The Wnt/β-catenin signalling pathway has been reported to play a distinct role in the development or function of the pancreas [15, 16]. Deletion of β-catenin from the pancreatic beta cells of embryonic-stage RIP-Ctnnb1-cKO mice generated by crossing floxed Ctnnb1 mice with rat insulin promoter (RIP)-Cre mice resulted in the formation of immature beta cells with reduced pancreatic beta cell area at birth, and glucose intolerance with impaired insulin secretion in the adult stage . Although systemic Tcf7l2 disruption during the embryonic stage by insertion of the hygromycin resistance gene into the exon encoding the DNA-binding high-mobility group (HMG) box caused death in the immediate postnatal period due to the phenotype in the small intestine and liver, the Tcf7l2 −/− mutant mice showed normal development of the endocrine pancreas and normal beta cell proliferation [18, 19]. Furthermore, deletion of the DNA-binding HMG box after weaning in the pancreatic beta cells of RIP-ER T2 -Tcf7l2-cKO mice generated by crossing floxed Tcf7l2 mice with RIP-Cre-ER T2 mice and injection of tamoxifen did not affect the islet architecture or the beta cell function . Considering the different results on the beta cell development in the neonatal state and insulin secretion in the adult stage obtained between the deletion of β-catenin and TCF7L2 in the pancreatic beta cells, it is thought that the Wnt/β-catenin signalling pathway, including TCF7L2, may play an important role in the development or function of the beta cells and be subject to compensatory mechanisms involving other TCF/LEF family proteins.
To elucidate the pathophysiological role of TCF7L2 from the embryonic stage in vivo, we adopted RIP-driven ΔN-TCF7L2 expression and generated genetically engineered mice displaying inhibition of signalling pathways involving other TCF/LEF transcription factors in the pancreatic beta cells. A thorough analysis of these beta cell-specific ΔN-Tcf7l2 transgenic mice (DN mice) revealed that the DN mice showed impaired glucose tolerance associated with decreased insulin secretion. In addition, the beta cell area was markedly decreased and the whole-pancreas insulin content was diminished in both the adult and newborn DN mice. Moreover, the islets from the DN mice showed decreased gene expression of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa. Our results lend support to the hypothesis that the TCF7L2-mediated pathway in pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass during development.
Generation of transgenic mice expressing ΔN-TCF7L2 in the pancreatic beta cells (DN mice)
A murine full-length, short-form Tcf7l2 coding sequence cDNA (NM_009333.2) was prepared by cloning the products of RT-PCR from the islet mRNAs of C57BL/6J mice. To obtain mutant truncated cDNA for the ΔN-TCF7L2 protein, 90 bases were artificially deleted from the 5′ end of the short-form Tcf7l2 by the PCR method [12, 13, 22]. For overexpression in the pancreatic beta cells, the transgene consisted of 757 bases of the rat Ins2 promoter linked to an intron sequence of rabbit β-globin, the 5′ end truncated Tcf7l2 cDNA and a polyadenylation sequence . The construct was inserted into a pBluescript II SK (−) vector (Stratagene, La Jolla, California, USA) and cloned. The purified 3.3 kb fragment digested by NotI was microinjected into the pronuclei of fertilised BDF2 eggs (CLEA Japan, Tokyo, Japan). The recipient eggs were [C57BL/6J × DBA/2] F2 hybrids. Transgenic founder or F2 mice were identified by Southern blot analysis of the tail DNAs using a cDNA probe (from exon1 to exon3 of Tcf7l2) and genotyping PCR (see electronic supplementary material [ESM] Methods for further details). The images of the Southern blot analysis were converted to quantitative data (photo-stimulated luminescence intensity) with an image analyser (BAS 2000, Fuji Film, Tokyo, Japan) . From the six independent lines of transgenic mice obtained, we selected three lines showing obvious expression of ΔN-TCF7L2 and designated them 390, 440 and 469. The founder and transgenic descendants were backcrossed over five generations into a C57BL/6J background. Male transgenic mice and their wild-type littermates were used for the experiments. The transgenic mice served as heterozygotes.
C57BL/6J mice were purchased from CLEA Japan. The mice were housed under a 12 h light–dark cycle and given free access to regular chow (CE-2; CLEA Japan), consisting of 25.6% (wt/wt) protein, 3.8% fibre, 6.9% ash, 50.5% carbohydrates, 4% fat and 9.2% water. The animal care and experimental procedures were approved by the Animal Care Committee of the University of Tokyo.
Western blot analysis
Antibodies to TCF7L2, caspase 3 and cleaved caspase 3 were purchased from Cell Signaling Technology (Tokyo, Japan). Anti-actin and anti-MAFA antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). The islets were sonicated in ice-cold buffer A (25 mmol/l Tris–HCl, pH 7.4, 10 mmol/l Na3VO4, 10 mmol/l NaPPi, 100 mmol/l NaF, 10 mmol/l EDTA, 10 mmol/l EGTA and 1 mmol/l phenylmethylsulfonyl fluoride) with an ultrasonic sonicator. Samples were separated by SDS-PAGE, and immunodetection was performed with an ECL kit (Amersham Biosciences, Piscataway, New Jersey, USA) . Protein was prepared from more than 70 islets from each mouse and 15 μg samples of the proteins were applied to the gel. Liver samples obtained from C57BL/6J mice administered diethylnitrosamine were used as a positive control for detection of the cleaved caspase 3 protein.
TCF/LEF transcriptional activity
The TCF/LEF transcriptional activity in the murine pancreatic beta cell line MIN6 cells  co-transfected with pcDNA3.1(+)-ΔN-Tcf7l2 or a control empty vector pcDNA3.1(+) was measured using the luciferase reporter gene kit (see ESM Methods for further details).
In vivo glucose homeostasis
For the glucose tolerance test, mice were loaded with oral or intraperitoneal glucose (1.5 g/kg body weight) after overnight fasting. Exendin-4 (Sigma, Tokyo, Japan) (24 nmol/kg body weight) was administered 15 min before the intraperitoneal glucose loading. For the insulin tolerance test, the mice were intraperitoneally challenged with 0.75 U/kg body weight of human insulin (Novolin R; Novo Nordisk, Tokyo, Japan) after 1 h of fasting  (see ESM Methods for further details).
Reverse transcription-quantitative real-time PCR
For reverse transcription-quantitative real-time PCR (RT-qPCR), total RNA was prepared from 40 islets of each mouse. Quantitative real-time PCR (qPCR) was performed using the TaqMan Universal PCR Master Mix or SYBR Green PCR Master Mix (Applied Biosystems, Tokyo, Japan)  (See ESM Methods for further details). Commercially available TaqMan Gene Expression Assay IDs and the synthesised primer sequences are listed in ESM Table 1 and ESM Table 2, respectively. The relative expression levels were compared by normalisation to the expression levels of Actb.
Pancreatic islet isolation
Isolation of islets from the pancreas of the mice was carried out as described elsewhere [23, 28]. In brief, after clamping the common bile duct at a point close to the duodenal outlet, 2.5 ml of Hanks’ Balanced Salt Solution (HBSS) (Sigma) containing 0.6 mg Liberase TL (Roche Diagnostics, Tokyo, Japan) and 25 mmol/l HEPES were injected into the duct. The swollen pancreas was removed and incubated at 37°C for 24 min. The pancreatic tissue was then dispersed by pipetting and washed twice with ice-cold HBSS containing 25 mmol/l HEPES and 10% (wt/vol.) FBS. Thereafter, the islets were manually collected through a stereoscopic microscope and used immediately for the experiments.
Insulin assay of the pancreas
For measurement of the insulin content of the whole pancreas, the isolated pancreas was weighed, homogenised in an ethanol/HCl buffer and then incubated overnight at 4°C. At the end of the incubation period, the insulin contained in the buffer was measured with the AlphaLISA insulin kit (PerkinElmer Japan, Yokohama, Japan).
Histological and immunohistochemical analyses of the islets
Isolated pancreases were fixed overnight with 4% paraformaldehyde at 4°C. Tissues were routinely processed for paraffin embedding and 4 μm sections were cut and mounted on silanised slides. The pancreatic sections were stained with anti-insulin antibody (DAKO, Tokyo, Japan) and anti-MAFA antibody . Images of the pancreatic tissues and islet beta cells were viewed on the monitor of a computer through a microscope connected to a camera with a charged-coupled device (Keyence, Osaka, Japan). The areas of the pancreases and the beta cells were traced manually and analysed with WinROOF software ver.5.03 (Mitani, Tokyo, Japan), as previously described . More than 100 islets were analysed per mouse in each group. Bromodeoxyuridine (BrdU) incorporation was analysed with BrdU Labeling and Detection Kit II (Roche Diagnostics)  and apoptotic cells were examined using an in-situ Cell Death Detection kit (Roche Diagnostics) (See ESM Methods for further details).
Results are expressed as means ± SEM. Differences among groups were examined for statistical significance using the Mann–Whitney U test and Kruskal–Wallis test. A p value of less than 0.05 was considered to indicate statistical significance.
Generation of transgenic mice expressing ΔN-TCF7L2 in the pancreatic beta cells
To inhibit the transcriptional activity of Tcf7l2, 90 bases corresponding to the N-terminal β-catenin-binding domain were artificially deleted from the 5′ end of the endogenous full-length, short form of Tcf7l2 cDNA (Fig. 1a). For expression in the pancreatic beta cells, the transgene consisted of the rat Ins2 promoter linked to an intron sequence of rabbit β-globin, ΔN-Tcf7l2 cDNA and a polyadenylation sequence. The purified 3.3 kb fragment digested by NotI was microinjected into the pronuclei of fertilised BDF2 eggs (Fig. 1b). Transgenic founder mice were identified by Southern blot analysis of the tail DNAs using a cDNA probe, and three independent DN mouse lines, 390, 440 and 469, were selected with a clear band of the exogenous transgene (Fig. 1c). The copy number of each line was estimated to be 15, 3 and 3, respectively. To evaluate the expression levels of endogenous Tcf7l2 plus exogenous ΔN-Tcf7l2 mRNA, the primer probe set was designed based on the sequence between exon 5 and exon 6 (ESM Fig. 1a). In the isolated islets of the DN mice, a more than tenfold increase in the exogenous expression level of ΔN-Tcf7l2 was found as compared with the endogenous expression level of Tcf7l2 (Fig. 1d). Similar results were confirmed by western blot analysis and a slight increase in endogenous TCF7L2 was observed in the islets of the DN mice (Fig. 1e–g, ESM Fig. 1b–e). No exogenous expression of ΔN-Tcf7l2 was detected in any other tissues, including the hypothalamus (data not shown). Induction of exogenous ΔN-TCF7L2 suppressed the TCF/LEF transcriptional activity in the murine beta cell line MIN6 cells (Fig. 1h). In addition, the expression levels of Ccnd1, Ccnd2 and Axin2, which are known direct target genes of TCF7L2 [30, 31], were significantly decreased in the islets of the DN mice from line 440 as compared with the corresponding expression levels in the wild-type mice (Fig. 1i).
Adult DN mice developed impaired glucose tolerance associated with decreased insulin secretion as assessed by the OGTT
The incretin effect of exogenous glucagon-like peptide 1 receptor agonist administration observed in DN mice was similar to that in wild-type mice
Beta cell area and whole-pancreas insulin content were diminished in the adult DN mice
Islets from the DN mice showed decreased gene expression of Ins1, Ins2, Irs1, Irs2 and Mafa
DN mice developed impaired glucose tolerance associated with decreased insulin secretion at the time of weaning
The beta cell area and whole-pancreas insulin content were diminished in newborn DN mice
TCF7L2 has been identified as a strong type 2 diabetes susceptibility gene and TCF7L2 at-risk alleles have been shown to be associated with clinical phenotypes of impaired insulin secretion [3, 4, 5, 8, 9]. To elucidate the pathophysiological role of TCF7L2 from the embryonic stage in vivo, DN mice with impaired function of TCF7L2 in the pancreatic beta cells were generated. In a thorough analysis of three independent lines of DN mice, we observed that adult DN mice developed impaired glucose tolerance associated with decreased insulin secretion, as assessed by both OGTT and IPGTT. The beta cell area was markedly decreased and the total insulin content of the whole pancreas was also diminished in the adult DN mice. Moreover, the reduction of the beta cell area and pancreatic insulin content was even observed in the newborn DN mice. Consistent with these findings, islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa, the latter of which is well known to encode for a key transcription factor of beta cell maturation and insulin transcription [32, 33]. These data suggest that the TCF7L2-mediated pathway in the pancreatic beta cells contributes to glucose homeostasis through regulation of the beta cell mass during development.
The Wnt/β-catenin signalling pathway is well known to play a critical role during embryonic development, stem cell differentiation and tumorigenesis [4, 12, 13, 14]. In the pancreatic islets, many splice variants of the TCF7L2 gene are expressed and known to have distinct physiological and pathophysiological effects on the beta cells [34, 35, 36]. In addition, the diversity of vertebrate TCF/LEF isoforms plays an important role in mediating the various functions of Wnt/β-catenin signalling . In fact, expression of Tcf7l1 and Tcf7, as well as of Tcf7l2, has been reported in the pancreas . Genetically engineered animal models of Tcf7l2, generated using different targeting strategies, disrupted exons and Cre mouse strains have shown a variety of phenotypes [18, 19, 31, 39, 40, 41, 42, 43]. In this study, we adopted ΔN-TCF7L2, which theoretically functions as a dominant-negative molecule to inhibit functional TCF7L2 isoforms and other TCF/LEF family proteins at the TCF7L2 binding sites , and confirmed that induction of exogenous ΔN-TCF7L2 suppressed the TCF/LEF transcriptional activity in a pancreatic beta cell line (Fig. 1h). In addition, the expression levels of established target genes of TCF7L2 were significantly decreased in the pancreatic islets of the DN mice (Fig. 1i). Dysregulation of glucose homeostasis and the beta cell area was evident from birth through to the adult stage in the DN mice, since the function of the TCF7L2-mediated pathway may be impaired from the embryonic stage without any compensatory mechanisms. Expression of Tcf7l1 was observed in the pancreatic islets as well as other tissues (ESM Fig. 3). Although the precise role of TCF7L1 in glucose homeostasis and beta cell development is mostly unknown because of the embryonic lethality of systemic Tcf7l1 knockout mice , comparisons of the phenotypes of DN mice and other mice with genetically modified Tcf7l2 suggest that TCF7L1 may compensate for the lack of TCF7L2 actions on the beta cells. Further investigation is warranted to determine whether TCF7L1 might regulate the development of the pancreatic beta cells in coordination with TCF7L2.
Islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1 and Irs2, which may be the cause of the reduction in the beta cell area. Consistent with this notion, previous experiments using the pancreatic beta cell line INS-1 cells demonstrated that TCF7L2 protein bound to the promoter region of Ccnd1 and exogenous ΔN-TCF7L2 indeed inhibited the proliferation of INS-1 cells by 50% as compared with control cells expressing an empty vector . We and others have provided genetic evidence of the role of insulin-signalling pathways in the beta cells for regulating the beta cell mass in vivo [23, 27, 46, 47]. Although the expression level of Insr was unaltered, levels of Irs1 and Irs2 were diminished in the islets of the DN mice. In addition, reduced gene expressions of Mafa and Neurod1, which encode important transcription factors of beta cell maturation and insulin transcription , may contribute to the observed phenotypes of the DN mice. Further study is needed to clarify the possibility of direct regulation of Irs1, Irs2, Mafa and Neurod1 by TCF7L2 in beta cells.
In a study in humans, a positive correlation has been observed between the mRNA levels of TCF7L2 and the insulin gene, although the expression of TCF7L2 mRNA was correlated inversely with the glucose-stimulated insulin secretion rate after normalisation for the total insulin content . Consistent with these data, in the islets of the DN mice, the expression levels of Ins1 and Ins2 were reduced, whereas the expression levels of molecules associated with the secretory machinery were not decreased. The possibility that the TCF7L2 at-risk alleles may modify the insulinotropic actions of incretin hormones in humans is still under debate [10, 11, 48]. In DN mice, a single administration of Ex-4 improved the glucose tolerance, with elevated serum insulin levels. The short-term incretin effect was preserved in the DN mice, probably due to the mild reduction of Glp1r and unaltered secretory machinery in the islets.
It has been reported that rs7903146, a TCF7L2 intronic variant strongly associated with type 2 diabetes, is located in islet-selective open chromatin, consistent with the observation of increased TCF7L2 mRNA expression in the islets isolated from type 2 diabetic patients . However, it has been demonstrated that the protein levels of TCF7L2 are depressed in the islets of patients with type 2 diabetes and animal models of diabetes, while TCF7L2 mRNA levels are increased . In addition to the discrepancy between the increased mRNA and decreased protein expression of TCF7L2, the existence of various TCF7L2 splice variants should be considered to interpret the role of TCF7L2 in the beta cells [34, 36]. Indeed, function-specific transcripts of TCF7L2, which exerted distinct effects on beta cell function and survival, were detected in human beta cells, and increased levels of deleterious TCF7L2 splice variants may explain the impaired insulin secretion in type 2 diabetes . Further analysis of pancreatic beta cells from individuals with at-risk alleles of TCF7L2 or from patients with type 2 diabetes will be required to examine the changes in both the mRNA and protein expression patterns of TCF7L2, including other TCF/LEF family genes and endogenous dominant-negative forms.
In this study, we demonstrated that the expression of the dominant-negative form of TCF7L2 from the embryonic stage in the pancreatic beta cells was associated with a reduction in beta cell area and pancreatic insulin content in the newborn mice, leading to impaired glucose tolerance with decreased insulin secretion in the adult stage. Thus, our findings suggest that TCF7L2 in the pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass during development.
We thank E. Hirata, A. Nagano, Y. Okonogi, M. Henmi and N. Ishikawa of the University of Tokyo for their excellent technical assistance and help with the animal care.
This work was supported by a grant for TSBMI from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-aid for Scientific Research in Priority Areas (A) (16209030), (A) (18209033) and (S) (20229008) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to TKa), and a Grant-in-aid for Young Scientists (B) (23791017) from the Japan Society for the Promotion of Science (to IT).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
IT, NK, KU and TKa were the main contributors in terms of study conception and design, acquisition and interpretation of data and writing the manuscript. KN, KK and SH contributed substantially to conceptual design, acquisition of data and revising the manuscript. TKu, MIn, HK, AO and YS made substantial contributions to analysis and interpretation of data, discussion of the results and drafting the manuscript. EK and MIw contributed substantially to conceptual design, acquisition of data for transcriptional activities and the critical revision of the manuscript for important intellectual content. TKi made substantial contributions to conceptual design, acquisition of data for immunohistochemical analyses and critical revision of the manuscript for important intellectual content. TKa 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 accuracy of the data analysis. All authors gave final approval of the version to be published.
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