The Role of TCF7L2 in Type 2 Diabetes

The transcription factor 7-like 2 (TCF7L2) (previously TCF4-transcription factor 4) is a member of the T-cell factor/lymphoid enhancer binding factor family (TCF/LEF), a group of transcription factors binding to DNA through a high-mobility group (HMG) domain. The TCF7L2 gene has been the object of much attention due to its strong genetic association with type 2 diabetes (T2D) (1,2). The TCF7L2 gene forms a basic part of the Wnt signaling pathway, which is made up of a complex network of interacting proteins having cellular intercommunications regulated at multiple levels, generating numerous effects, and which was initially related to the biology of development. Proteins involved in the Wnt network, as is the case of TCF7L2, have been related to various and diverse common diseases, as well as to different models of cancer, thus reflecting the importance of this developmental pathway in the pathogenesis of human diseases (3–5).

The human TCF7L2 gene was mapped to chromosome 10q25.3 (6) and initially sequenced in colorectal cancer (CRC) cell lines (7). TCF7L2 has 18 exons, which present a complex splicing pattern in different tissues. This gene has highly conserved sequence regions that correspond to functional domains. The β-catenin binding domain corresponds to exon 1, and the HMG box binding domain corresponds to exons 10 and 11 of human TCF7L2 (hTCF4). Both domains are highly conserved between species. In addition, TCF7L2 possesses highly conserved sequences in the 3′ region of exon 18, with conserved splicing patterns similar to those of the transcription factor T-cell factor 1 (TCF1) gene. These spliced transcripts present two reading frames conserved in Drosophila melanogaster and Caenorhabditis elegans, and two other reading frames are related to the chicken TCF genes. There are two binding motifs in the COOH-terminal region of the TCF7L2 isoforms that are conserved through evolution. The complex TCF7L2 splicing pattern suggests that the alternative use of isoforms in the 3′ region generates distinct protein isoforms with agonistic as well as antagonistic transactivation activities (7) (Fig. 1).

TCF7L2 is the most potent locus for T2D risk and the first locus to have been robustly reported by genomic linkage studies (1) and genome-wide association studies (2). deCODE genetics initially detected linkage to T2D in the Icelandic population on a chromosome 10 locus (8). However, the TCF7L2 at-risk allele present in ∼28% of control subjects and 36% of T2D case subjects did not explain the linkage signal. The exact gene variants contributing to T2D were not determined because some of these variants were in linkage disequilibrium (LD), as well as being located in a noncoding region without evident functional effects. The most probable candidate risk variants with the greatest odds ratio, ∼1.4, were the single nucleotide polymorphisms (SNPs) rs7903146 and rs12255372, in strong LD with the microsatellite marker DG10S478, whereas alleles rs7903146-T and rs12255372-T showed the most statistically significant association with T2D (1). Early in 2007, by analyzing 392,935 SNPs in a French cohort, Froguel and colleagues (2) reported TCF7L2 as a diabetogene. Afterward, in a second cohort they followed up the variants, with the most statistically significant differences between T2D and control subjects. This group confirmed the association of TCF7L2 with T2D risk and found the zinc-transporter gene SLC30A8 and two LD blocks that contain genes insulin degrading enzyme (IDE), kinesin family member 11 (KIF11), and hematopoietically expressed homeobox (HHEX), and exostosin glycosyltransferase 2 (EXT2) and Aristaless-like homeobox 4 (ALX4), associated with T2D. These associations explained a considerable component of disease and established a proof of principle for the genome-wide association study (GWAS) approach to the enlightenment of complex genetic traits (2). Replication of the association in a population of European origin was achieved jointly to the discovery of other diabetogenes (9). With use of genotyping data of 32 European descent GWAS (74,124 T2D case and 824,006 control subjects), >240 loci were reported for T2D, including TCF7L2 (10).

TCF7L2 association with T2D has been consistently replicated in multiple populations with diverse genetic origins, thus representing one of the most powerful genetic associations discovered in studies of complex diseases. A meta-analysis of the genetic associations found in different populations showed that the TCF7L2 variants associated with T2D exert their effects under a multiplicative genetic model. It suggested that TCF7L2 is involved in nearly one-fifth of T2D cases (11).

Soon after the first genetic association of TCF7L2 with T2D risk, other SNPs in the TCF7L2 gene were reported to be associated with this disease by different research groups. Haddad et al. (12) reported in African Americans a new signal in SNP rs114770437, which was independent of the index SNP rs7903146. The variant rs114770437 is monomorphic in the 1000 Genomes Project, which includes European, East Asian, and South Asian populations, thus explaining why it has not been found in previous GWAS of European and Asian ancestry subjects (13). The MEta-analysis of type 2 DIabetes in African Americans (MEDIA) Consortium reported a group of SNPs—SNPs rs7896811, rs11196199, and rs11196203, uncorrelated with the index SNP rs7903146 (r² < 0.05)—that correlate moderately (r² = 0.3) with rs114770437, indicating that they may be tagging the same causal SNP or that they may each one alone or in combination contribute to T2D in the African ancestry population (13).

Chang et al. (14) analyzed TCF7L2 in the Chinese Han population by amplifying 13 SNPs with allelic frequencies >20% and covering >90% of all haplotypes. They found that rs290487-C, located in an LD block close to the 3′ region of the gene, is associated with T2D in the Chinese Han population, in which the allelic frequency is ≤40% and the population-attributable risk fraction (PAR) is 18.7% (14). Another study in the Chinese population replicated the T2D association of SNP rs11196205, which resides within an LD block of 92.1 kb. In the same population, SNP rs11196218, localized downstream of rs11196205, was identified within another LD block, thereby conferring risk in an independent manner (population-attributable risk fraction = 42%) (15). Studies on a Mexican American population also found independent associations with T2D for additional TCF7L2 SNPs in the 5′ and 3′ regions of the same LD block in which the originally identified risk variants are located (16).

Analysis of other ethnic groups has suggested differences among races in terms of the contributions of genetic and environmental factors to T2D (17). In European populations, TCF7L2 SNPs associated with T2D susceptibility vary greatly in terms of allelic frequencies at global levels, with use of the International Haplotype Project (HapMap), Human Genome Diversity Project, Centre d’Etude du Polymorphisme Humain (CEU) databases and The ALlele FREquency Database (ALFRED). In addition, the risk SNPs allelic frequencies vary at regional levels; for example, the frequency of TCF7L2 rs11196218-A is highest in Eurasia, while it is reduced in sub-Saharan African and Native American populations, indicating that these variants may differently contribute to T2D incidence worldwide. Additional studies of gene-gene and gene-environment interactions are needed to elucidate the multifactorial contributions in diverse populations (17).

In a population-based cohort study of U.S. Hispanic/Latino adults (21–76 years old), a protective effect of TCF7L2 rs7930146-T on BMI was detected at ages 21 and 45 years, as well as a significant positive association between rs7903146-T and T2D onset in both middle and late adulthood. The results suggest that a complicated biologic mechanism underlies the functional consequences of TCF7L2 on obesity and T2D across the life course (18).

The mechanisms over which TCF7L2 exerts its effect on T2D are still not well understood. To determine whether the TCF7L2 rs7903146 variant has cis-regulatory properties, a detailed map of cis-regulatory elements within the 92-kb T2D association interval of the GWAS TCF7L2 locus has been made using a systematic cell-based enhancer strategy for cis-regulatory activity in a panel of cell lines. The chromatin state was evaluated in HCT-116 and U2OS cells, and allelic-specific enhancer properties were examined at the associated TCF7L2 rs7903146. A cis-regulatory variation leading to T2D predisposition and SNP rs7903146s allelic-specific effects in multiple cell lines were detected, indicating a peripheral defect in disease etiology (19). The use of the same strategy with additional cell lines will allow a clearer picture of the cis-regulatory elements involved in the disease pathogenesis.

The Wnt signaling pathway consists of 19 secreted glycoproteins containing 22 or 24 highly conserved cysteine residues as well as seven transmembrane domains acting as ligands of receptors mediating human developmental and adult processes (20). The Wnt/β-catenin pathway generates numerous tissue-specific responses, including acting on multiple nuclear-effector proteins, which have different functional properties (e.g., TCF proteins), and regulating gene transcription and transduction in the pathway (Fig. 2). These TCF/LEF transcription factors respond to Wnt signals via a transcriptional interrupter that partially modulates the binding with β-catenin and the recruitment of coactivator complexes; the specificity and fine regulation of this interrupter are achieved by the cellular content–dependent expression of four members of the TCF/LEF family (transcription factor 7 [TCF7], transcription factor 7-like 1 [TCF7L1], TCF7L2, and LEF1) and their numerous isoform variants, exhibiting differences in the affinity and specificity of DNA binding, repression, activation, and regulation (21).

TCF7L2 has fundamental developmental and metabolic roles in adipose tissue. The regulation of adipogenesis by TCF7L2 and Wnt signaling is more complex than previously recognized, and the in vivo inactivation of adipocyte TCF7L2 promotes adipocyte hypertrophy and peripheral and hepatic insulin resistance (22). Qin et al. (23) studied the mechanisms of the Wnt signaling pathway and miRNA-mediated regulation of adipogenesis in the 3T3-L1 cell line. By inhibiting the Wnt signaling with a differentiation cocktail and activating it with the glycogen synthase kinase-3β (GSK-3β) inhibitor, which blocks β-catenin phosphorylation, they identified 18 miRNAs that promote adipogenesis by repressing Wnt signaling and other miRNAs that activate Wnt signaling. In particular, bioinformatics analysis and corroboration experiments predicted that miRNA210 bound to the gene TCF7L2 repressed Wnt signaling, thereby promoting adipogenesis (23) (Fig. 3). Furthermore, miR-181a-5p promotes 3T3-L1 preadipocyte differentiation and adipogenesis by regulating TGFβ/Smad, and it promotes the Wnt signaling pathway by directly targeting Smad7 and Tcf7l2 (24). This information offers a deeper understanding of the mechanisms by which miRNAs regulate adipogenesis.

The expression of miRNAs in adipose tissue can be modulated by inflammation. Proinflammatory factors can impair insulin secretion as well as insulin sensitivity in peripheral organs (25), and these inflammatory processes are associated with the defect of glucose homeostasis that leads to T2D.

TCF7L2 functions as central transcriptional regulator of the adipocyte metabolic program by directly regulating the expression of genes involved in lipid and glucose metabolism (26). TCF7L2 regulates adipocyte size, adipocyte endocrine function, and glucose metabolism. TCF7L2 expression in white adipose tissue is suppressed in genetic and high fat diet–induced models of obesity. Analysis of genome-wide distribution of TCF7L2 binding and gene expression in adipocytes shows that TCF7L2 directly regulates genes implicated in cellular metabolism and cell cycle control. When an animal model of conditional deletion of TCF7L2 in adipocytes is challenged with a high-fat diet, there is progression to impaired glucose intolerance, impaired insulin sensitivity, weight gain, and increased subcutaneous adipose tissue mass. In addition, triglyceride-hydrolase expression is reduced and fasting-induced free fatty acids are released. High-fat feeding results in glucose intolerance after 3 days and reduced insulin sensitivity after 3 months. The findings suggest that the early loss of TCF7L2 predisposes mice to diabetes when challenged with a high-fat diet (26).

While it has been known that Wnt signaling impairs adipogenesis, the TCF7L2 gene was recently shown to fulfill, during adipogenesis, very complex regulatory functions that may depend on several factors, including the balance between TCF7L2 and β-catenin levels, the timing of their interaction, and the interaction between TCF7L2 and other cofactors at specific gene regions (22).

Physiological characterization of TCF7L2 risk variants for T2D will aid understanding of the role of TCF7L2 in the regulation of glycemia and in the secretion of insulin and its action. In addition, the identification of TCF7L2-interacting partners and target genes can lead to the detection of new metabolic pathways potentially involved in pathogenesis of T2D. The majority of the variants known at present to confer risk for T2D and/or continuous glycemic traits primarily impact the function of the pancreatic β-cell rather than sensitivity to insulin (27,28).

When TCF7L2 was discovered, it was known that TCF7L2-knockout mice die 24 h after birth and that they lack the intestinal epithelial stem cell compartment (27). It was also known that TCF7L2 regulates the preproglucagon (GCG) gene that encodes glucagon-like peptide 1 (GLP-1); thus, it was postulated that TCF7L2 variants modifying GLP-1 levels in endocrine cells could influence susceptibility to T2D (29). Successive studies in humans support this hypothesis (30). Silencing TCF7L2 exerts a strong negative effect on glucose-mediated insulin secretion. It has been suggested that the defect may lie in the fusion of insulin-secretory granules. Defective insulin exocytosis may thus underlie increased T2D incidence in carriers of the at-risk TCF7L2 alleles (31). TCF7L2-silencing studies and studies on the in vitro overexpression of gluconeogenic precursors that used chromatin immunoprecipitation combined with massive DNA sequencing (ChIP-Seq) through the genome showed that TCF7L2 is a key regulator of hepatic glucose metabolism; thus, risk variants may affect both fasting and postprandial glycemia in carriers of T2D risk genotypes (32). Most of the TCF7L2 variants are found in noncoding regions, suggesting that they exert their effects by modulating expression. The TCF7L2 molecular mechanism of action was found while exploring the patterns of open chromatin, an evolution-conserved indicator of regulatory activity. The DNA of human islets was analyzed by high-performance sequencing of formaldehyde-assisted isolation of regulatory elements (FAIRE-seq), and the TCF7L2 at-risk alleles were mapped at open chromatin sites, at which rs7903146, one of the TCF7L2 variants with greatest risk for T2D, was selected. The heterozygotes of rs7903146 showed an altered intrinsic enhancer activity, indicating that the variant confers risk through cis regulation and modification of local chromatin structure in the pancreatic islet (33).

Additional studies in different cohorts worldwide have detected the role of the TCF7L2 risk variants in glucose metabolism and T2D. In 2007, the Finnish Diabetes Prevention Study showed that some TCF7L2 variants predict T2D onset and are associated with impaired glucose regulation and insulin secretion (34). In 2011, the French cohort data from the Epidemiological Study on the Insulin Resistance Syndrome showed the impact of TCF7L2 genetic risk on the evolution of T2D: TCF7L2 variants were associated with a more rapid reduction of pancreatic β-cell function, increased glycemia, and decreased insulin secretion. These effects related to familial history of T2D, and TCF7L2 variants indicate the familial genetic predisposition to the development over time of impaired glycemia and T2D (35). Physiological studies found that variant rs290487 contributed to insulin resistance, implicating Wnt signaling in insulin resistance (36).

The GWAS utilizing microarrays used to identify T2D risk variants reported most of the associated variants affecting pancreatic β-cell function by means of molecular mechanisms not yet known in detail. However, we know that one group of these genes affects glucose-stimulated insulin secretion, another group affects incretin-stimulated insulin secretion, and the remaining group has a defect that impairs the conversion of proinsulin into insulin. TCF7L2 risk variants interfere with each of the three above-mentioned mechanisms (37).

Carriers of the TCF7L2 rs7903146-T genotype, in response to an intravenous glucose-tolerance test and a euglycemic-hyperinsulinemic clamp, present phospholipids alterations in plasma metabolic profiles, potentially indicating that phospholipids may mediate metabolic abnormalities prior to the onset of glucose intolerance (38). Of note, phospholipids are the essential components of biological membranes, which provide the cell with a protective barrier that has selective permeability for cellular metabolism. Due to TCF7L2 association with T2D and its key role in fundamental cell functions, several groups have begun to study the role of TCF7L2 variants in biological processes such as natural selection and alternative splicing.

The phylogenetic reconstruction of TCF7L2 intron 4 variants in LD and their related haplotypes, in which T2D risk variants are found, allowed the discovery of two main haplotype lineages: HapA and HapB. Between the two, HapB is the most diverse haplotype, containing all haplotypes carrying the SNP rs7903146 ancestral T allele as a T2D risk variant that is referred to as HapBT2D. The second major lineage, HapA, comprises a cluster of relatively homogeneous haplotypes and is notably divergent from HapB. It exhibits a star-type diversity pattern consistent with a natural selection pattern, and it contains a base haplotype included in 81% of its haploid chromosomes. Among the HapMap populations, the frequency of the HapA lineage presents an unusual degree of divergence that ranges from 10% in the Yoruba population of Nigeria (YRI) to 58% in a population of European ancestry from a Utah pedigree of Centre d’Etude du Polymorphisme Humain (CEU) and 95% in the East Asian group of the Chinese Han of Hong Kong (CHB) and of the Japanese population of Tokyo (JPT). The frequency of the risk variant HapBT2D in the HapMap group of Asians (CHB + JPT) is ∼5% (39).

HapBT2D is the genetic factor conferring the greatest risk for T2D, and it is negatively associated with BMI (39), showing that TCF7L2 may play a role in T2D, which is usually associated with hyperinsulinemia. In in vitro studies, insulin reduced TCF7L2 mRNA expression, and this effect was reduced with free fatty acids. In in vivo studies comparing the subcutaneous adipose tissue (SAT) of insulin-resistant subjects versus insulin-sensitive subjects, TCF7L2 mRNA was significantly more expressed in the SAT of the insulin-resistant individuals, implicating a role for this gene in insulin resistance, although no correlations between TCF7L2 mRNA expression and obesity measures were observed. Also, TCF7L2 expression was higher in visceral adipose tissue than in SAT, and in stratifying for haplotypes, this difference was seen in HapA carriers but not in non–HapA carriers, indicating a possible HapA role in the TCF7L2 expression in visceral adipose (40).

TCF7L2 gene variants may confer a genetic risk for becoming overweight. Men with the HapBT2D variation have a lower BMI, and, in contrast, men with the HapA genetic variation have a higher BMI. Researchers have inferred that individuals with the HapA genetic variation were at an advantage in past centuries because they were able to form fat reserves that allowed them to survive periods of famine (39). To obtain a better picture of the role of natural selection in different ethnic groups, Acosta et al. (41) sequenced the exome of the TCF7L2 gene using the Sanger method. They reported a lack of diversity in intronic region 4–6 in indigenous populations. This characteristic could be an effect of selective sweeps generated by the selection of neighboring rare variants at T2D-associated variations. The abundance of selective peak sweeps in the downstream region of the TCF7L2 gene suggests that the gene is part of a region that is in constant recombination between populations (41).

Alternative splicing is a mechanism by which multiple transcripts are generated from a unique gene, increasing the amount of different functional protein isoforms, some of which are associated with metabolic diseases (42). Prokunina-Olsson et al. (42) described tissue-specific TCF7L2 splicing patterns in various tissues and cells (pancreas, pancreatic islets, skeletal muscle, cutaneous adipose tissue, colon, liver, monocytes, and lymphoblastic cell lines). They identified a unique TCF7L2 “13-13b” splicing form expressed in the pancreas, pancreatic islets, and colon; the “13-13b” splicing form is correlated with the concentration of proinsulin in glucose-stimulated pancreatic islets, suggesting its key role in glucose regulation and thus in the development of T2D. Further, TCF7L2 rs7903146-genotype was associated with alternative splicing patterns in SAT (43). However, when Prokunina-Olsson et al. (44) analyzed TCF7L2 expression in both visceral adipose tissue and SAT in obese individuals, they found that alternative TCF7L2 splicing forms have different functions in each of these tissues. Kaminska et al. (45) showed that weight loss after gastric bypass surgery led to the differential expression of TCF7L2 mRNA isoforms in SAT. Specifically, they found that an adipose tissue–specific variant incorporating exons 12 and 13 was increased after surgery; this splicing form variant is more common in SAT of patients with T2D, and it shows a positive correlation with free fatty acid during insulin clamps. Chen et al. (22) showed that total TCF7L2 expression is reduced ∼20% in adipose tissue of subjects with impaired glucose tolerance and adipose insulin resistance. Nevertheless, they did not find differential expression of transcripts incorporating exon 12 and 13 because this variant also was reduced in the adipose tissue of subjects with impaired glucose tolerance. Another study also showed reduced TCF7L2 mRNA levels in adipose tissue of patients with diabetes. The same pattern is observed in carriers of the rs7903146 T2D risk allele (43). These results support the idea that adipose tissue insulin sensitivity does not regulate TCF7L2 gene splicing (22). More research is needed in this area to define the mechanisms involved in the complicated TCF7L2 splicing pattern that contributes to the pathophysiology of T2D.

TCF7L2 rs12255372-T and rs7903146-T variants modestly influence initial therapeutic success with sulfonylureas, a class of oral hypoglycemic agents stimulating insulin release. In one study, the sulfonylurea therapeutic effect mediated by TCF7L2 rs12255372-T was twofold greater than the treatment failure present in 12% of the population homozygote for the wild-type allele, whereas the association between the TCF7L2 genotype and metformin treatment was very weak (46). The observed differences in treatment response indicate that diabetogenes may be an important contributor to the interindividual treatment response variability (47). In Chinese T2D patients, the intronic TCF7L2 SNP rs290487-CT has been reported to influence the therapeutic efficacy of repaglinide, an oral agent of the meglitinide class of short-acting insulin secretagogues (48). Based on available studies, evaluating the correlation of TCF7L2 gene variants with therapeutic responses is difficult due to differences in study design, definitions of clinical end points, sample sizes, and types and doses of therapies used (47,49).

TCF7L2 is the most potent locus known for T2D risk. The TCF7L2 gene is a downstream effector of the canonical Wnt/β-catenin signaling pathway, which permits TCF7L2 to control a great variety of functions by virtue of the complexity of its expression with numerous mRNA isoforms. This gene has been associated with several fundamental processes, including adipogenesis, natural selection, and alternative splicing, and various and diverse diseases, including different types of cancer. Continued efforts are underway to understand the genomic variation associated with T2D and its functional implications. Future interinstitutional collaborations are needed to guarantee that the clinical translation of this knowledge leads to improved patient care.

Resequencing of the TCF7L2 gene in diverse ethnic groups will allow identification of common and rare genetic variants that contribute to the pathogenesis of T2D. It will be important to follow up on the newly discovered variants with functional and physiological studies to gain insights into diabetes-related molecular pathways. Developing therapies modulating TCF7L2 expression may provide opportunities to treat T2D patients carrying a TCF7L2 risk allele. The role that TCF7L2 variants play in complex regulatory functions has been little explored, and further studies are required. Despite the TCF7L2 genetic role in T2D has been widely confirmed in several populations, the relevance of ethnic differences remains to be determined.

To provide more personalized treatments and improve patients’ quality of life, it is important to explore the role of new common and rare variants at the TCF7L2 locus in diverse ethnic groups in combination with the novel clusters reported.

Funding. E.M.-M. is supported by Consejo Nacional de Ciencia y Tecnología (CONACYT) (258589). C.G. is supported by National Institute of Child Health and Human Development grant 5R01HD086911-02 (Principal Investigator).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.