The WNT signalling pathway and diabetes mellitus
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The WNT signalling pathway is involved in many physiological and pathophysiological activities. WNT ligands bind to Frizzled receptors and co-receptors (LDL receptor-related protein 5/6), triggering a cascade of signalling events. The major effector of the canonical WNT signalling pathway is the bipartite transcription factor β-catenin/T cell transcription factor (β-cat/TCF), formed by free β-cat and one of the four TCFs. The WNT pathway is involved in lipid metabolism and glucose homeostasis, and mutations in LRP5 may lead to the development of diabetes and obesity. β-Cat/TCF is also involved in the production of the incretin hormone glucagon-like peptide-1 in the intestinal endocrine L cells. More recently, genome-wide association studies have identified TCF7L2 as a diabetes susceptibility gene, and individuals carrying certain TCF7L2 single nucleotide polymorphisms could be more susceptible to the development of type 2 diabetes. Furthermore, β-cat is able to interact with forkhead box transcription factor subgroup O (FOXO) proteins. Since FOXO and TCF proteins compete for a limited pool of β-cat, enhanced FOXO activity during ageing and oxidative stress may attenuate WNT-mediated activities. These observations shed new light on the pathogenesis of type 2 diabetes as an age-dependent disease.
Keywordsβ-cat/TCF FOXO GLP-1 Stress TCF7L2 WNT
forkhead box transcription factor subgroup O
glycogen synthase kinase-3
lymphoid enhancer-binding factor 1
LDL receptor-related protein
pancreas/duodenum homeobox protein 1
protein kinase B
reactive oxygen species
single nucleotide polymorphism
T cell factor
Introduction to the WNT signalling pathway
In this review, I will first discuss the laboratory experimental studies on the role of the WNT pathway in the development/genesis of mouse pancreatic islets, pancreatic beta cell growth and the production of the incretin hormone glucagon-like peptide-1 (GLP-1). This will be followed by a brief summary of GWA studies of TCF7L2 and the risk of type 2 diabetes. Finally, I will discuss recent findings indicating that forkhead box transcription factor subgroup O (FOXO) and TCF proteins are able to compete for the limited pool of β-cat, and ageing will lead to increased FOXO-mediated gene transcription and reduced TCF-mediated gene transcription. These findings give us new insights into type 2 diabetes as an age-dependent disease.
WNT signalling is involved in the genesis of pancreatic islets and the proliferation of pancreatic beta cells
Rulifson et al. recently examined the effect of WNT signalling in regulating beta cell genesis and proliferation using both in vitro and in vivo approaches . Purified WNT3a (which is known to activate the canonical WNT pathway) stimulated proliferation of both the mouse beta cell line MIN6 and primary mouse pancreatic beta cells, possibly through the cell cycle regulators cyclin D1, cyclin D2 and cyclin-dependent kinase 4, as well as the homeodomain transcription factor Pitx2. Immunohistological examinations of 3-month-old bi-transgenic rat insulin I promoter (RIP)-Cre and β-catactive mice revealed a threefold increase in the production of Ki67 by pancreatic beta cells, which occurred in parallel with a 2.5-fold increase in beta cell mass . Furthermore, axin production led to impaired Pitx2 gene expression, along with impaired beta cell expansion . Taken together, these observations suggest that WNT signalling is necessary and sufficient for pancreatic beta cell proliferation.
The WNT co-receptor LRP5 is essential for normal lipid metabolism and glucose-induced insulin secretion
Besides the effectors of WNT signalling, the co-receptors of the WNT ligands, LRP-5/6 [34, 35], are also important for normal lipid and glucose metabolism. An early study demonstrated that loss of function mutations of LRP5 were associated with the development of the autosomal recessive disorder osteoporosis-pseudoglioma syndrome . The co-receptor was shown to be important in transducing WNT signalling and to play critical roles in modulating bone accrual and eye development . The human LRP5 gene maps within the IDDM4 region on chromosome 11q13, which is linked to type 1 diabetes [21, 22, 23]. A recent GWA study has also shown that polymorphisms in LRP5 are associated with obesity phenotypes . Pancreatic production of LRP5 has been reported [39, 40].
It has been demonstrated that LRP5 can interact with axin, one of the inhibitors of the WNT pathway (Fig. 1). When Lrp5 was expressed in fibroblast cells, the LRP5 protein alone exerted no effect on the WNT pathway, but acted synergistically with the WNT ligands . Furthermore, LRP5 molecules without the extracellular domain were constitutively active. They induced TCF/LEF-mediated transcription and stabilised β-cat . The addition of WNT ligands to the medium triggered the translocation of axin to the cell membrane and enhanced the interaction between axin and LRP5. Finally, the LRP5 domain involved in the interaction with axin is also required for TCF/LEF-mediated transcriptional activation. These observations collectively suggest that binding of axin by LRP5 and its translocation to the cell membrane is an important part of WNT signal activation  (Fig. 1b).
Both WNT and insulin pathways are involved in the production of the incretin hormone GLP-1
GLP-1 is an important incretin hormone that is encoded by the Gcg gene, which is expressed in the intestinal endocrine L cells [42, 43, 44]. In these cells, expression of Gcg mRNA and production of GLP-1 can be activated by lithium, which mimics the function of the WNT ligands , or the overproduction of the constitutively active S33Y β-cat mutant , indicating that Gcg is a downstream target of the WNT signalling pathway. Activation was subsequently attributed to a TCF binding site within the G2 enhancer element of the Gcg promoter and the production of TCF7L2 in the intestinal endocrine L cells . It is well known that Gcg expression and GLP-1 production can be activated by elevations in cAMP levels [46, 47, 48, 49, 50, 51, 52]. Since the G2 enhancer element has been shown to mediate the stimulatory effect of both cAMP and calcium on Gcg promoter activity , it is possible that cAMP pathway cross-talks with the WNT pathway to regulate Gcg expression .
TCF7L2 polymorphisms are associated with the risk of type 2 diabetes
Since TCF7L2 is known as an intestinal cell specific transcription factor  and is an important regulator of intestinal Gcg expression and GLP-1 production , it was suggested that the TCF7L2 SNPs may modify disease susceptibility by affecting intestinal Gcg expression and plasma levels of GLP-1 . More recently, genotyping 1,100 non-diabetic German individuals for the five known TCF7L2 SNPs indicated that TCF7L2 variants are associated with reduced insulin secretion . In contrast, plasma GLP-1 levels during an OGTT were not significantly influenced by the TCF7L2 variants . The CT/TT genotypes of the SNP rs7903146 were shown to strongly predict future type 2 diabetes in two independent Scandinavian cohorts . The risk T allele was associated with impaired insulin secretion, incretin effects, and an enhanced rate of hepatic glucose production . Furthermore, investigators found that islet TCF7L2 expression was increased fivefold in individuals with the TT genotype. Although TCF7L2 expression was positively correlated with the expression of INS, which encodes insulin, it was inversely correlated with glucose-stimulated insulin release . Furthermore, an ex vivo examination demonstrated that TCF7L2 knockdown (with small interfering RNA) increased human pancreatic beta cell apoptosis and reduced beta cell proliferation and glucose-stimulated insulin secretion . Overexpression of TCF7L2, on the other hand, protected islets from glucose- and cytokine-induced apoptosis and from impaired functions . As discussed above, both TCF7L2 and β-cat are required as effectors for GLP-1-stimulated beta cell proliferation .
Although these recent studies indicate that TCF7L2 SNPs may directly affect INS expression and/or insulin secretion, we still do not have a clear picture of how these SNPs affect the function of pancreatic beta cells. As discussed by Schafer and colleagues, the involvement of changes in GLP-1 production and secretion influenced by TCF7L2 variants in the increased risk of type 2 diabetes cannot be eliminated . It should also be pointed out that the participants in their study were non-diabetic individuals. It is possible that a certain compensatory response(s) attenuated the defect in GLP-1 secretion in these TCF7L2 SNP carriers in the pre-diabetic stages.
FOXOs compete with TCFs for the limited pool of β-cat
An evolutionarily conserved interaction between the WNT pathway effector β-cat and FOXOs was discovered in 2005 . In mammalian cells, a yeast two-hybrid screen detected an interaction between β-cat and FOXO1 and FOXO3. The gene bar-1 (also known as C54D1.6) encodes the Caenorhabditis elegans (nematode worm) homologue of β-cat , while the FOXO gene homologue in this organism is daf16 . An interaction between BAR-1 and DAF16 was also detected . In mammalian cells, binding of β-cat to FOXO enhanced the transcriptional activity of FOXO. In C. elegans, the loss of BAR-1 reduced the activity of DAF16 in dauer formation and life span . More importantly, the association between β-cat and FOXO was shown to be enhanced in cells exposed to oxidative stress .
Summary and perspective
Comprehensive in vitro and in vivo studies by multiple laboratories have shown that WNT signalling is important in normal pancreatic islet development, as well as in pancreatic beta cell function and genesis. It appears that the WNT/β-cat pathway plays direct, precise, and even opposing roles during different stages of pancreatic islet development . Obviously, to exert such precise and opposing effects, WNT/β-cat signalling needs to interact with other signalling pathways and regulate the downstream gene expression profiles, both temporally and spatially.
WNT signalling is also important in activating intestinal Gcg transcription and, therefore, the production of the incretin hormone GLP-1 [11, 12, 13], which has been shown to utilise WNT signalling effectors, i.e. β-cat/TCF7L2, to exert its effect on beta cell proliferation . Further examinations are required to verify whether TCF7L2, certain WNT ligands and Frizzled receptors are also involved in the genesis of pancreatic islets and intestinal endocrine L cells.
GWA have revealed relationships between SNPs in LRP5, which encodes a co-receptor of the WNT ligands, and the risk of type 1 diabetes [22, 23], as well as obesity . LRP6 mutations are possibly related to the development of bone loss, coronary disease and the metabolic syndrome [24, 25]. More importantly, extensive recent studies have identified associations between SNPs in TCF7L2 and the risk of type 2 diabetes. These observations suggest that WNT signalling is not only involved in pancreatic islet development during embryogenesis, but also in the function of pancreatic and intestinal endocrine cells during adulthood. Since all the known risk-associated SNPs of TCF7L2 are located within the intronic regions, the effect of these SNPs on TCF7L2 expression should be examined. To ultimately understand why these SNPs affect the risk of type 2 diabetes, we need to explore mechanisms underlying TCF7L2 production in pancreatic and intestinal endocrine cells under both physiological and pathological conditions.
The author thanks Canadian Institutes of Health Research (CIHR grant no. 68991) and Banting and Best Diabetes Centre (BBDC) for supporting his research team in studying the role of WNT signalling in intestinal proglucagon gene expression and GLP-1 production. The author regrets not being able to cite all excellent contributions in the field because of space limitations.
Duality of interest
The author declares that there is no duality of interest associated with this manuscript.
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