Role of Wnt signaling pathway in type 2 diabetes mellitus: An overview

Introduction

Transcription factor 7 like 2 gene (TCF7L2, previously known as T-cell factor 4, or TCF-4) is considered as one of the most important candidate genes which plays a major role in blood-glucose homeostasis and beta-cell function.¹ TCF7L2 gene which spans about 215.9 Kb with 17 exons is a high mobility group box containing transcription factors involved in the Wnt signaling pathway, playing a key role in cell development and regulatory mechanisms.^1,2  TCF7L2 is a ubiquitous protein that belongs to a family of TCF/ lymphoid enhancer factor (LEF) transcription factors. TCF7L2 was known as an important component of Wnt signaling pathway and as a regulator of the proliferative cellular compartment in the intestine.¹

TCF7L2 gene encodes for a transcription factor involved in the canonical β-catenin-dependent Wnt signaling pathway,Wnt signaling pathway regulates cell survival, migration, proliferation, differentiation and in organ development. Heterodimerization of TCF7L2 with β-catenin in the nucleus forms a transcriptionaly productive complex to activate the expression of Wnt-signaling target genes, including intestinal proglucagon. The proglucagon is the precursor of gut-derived insulinotropic hormone, glucagon-like peptide-1 (GLP-1). The active Wnt signaling stimulates the multiplication of beta cells.³

Introduction of Wnt signaling pathway:

In 1982, Nusse and Varmus discovered the first Wnt ligand-encoding gene, Int-1, in their breast cancer research as well as in embryonic developmental studies of Drosophila xenopus and other organisms.⁴ This pathway involves not only a large battery of Wnt ligands, receptors and coreceptors, but also a number of proteins that can regulate the production of the Wnt ligands. This proto-oncogene was later renamed Wnt-1, since it shares strong amino acid sequence homology with the drosophila Wingless (wg), which is important for segment polarity of the insect. There are 19 Wnt genes identified in rodents and humans. Wnt ligands are secreted glycoproteins which mainly exert their functions via the selective interaction with more than a dozen seven-transmembrane domain Frizzled receptor as well as the co-receptor known as low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6).⁵

The key effector of the Wnt signaling pathway is the bipartite transcription factor β-cat/TCF, formed by free β-cat and a member of the TCF family (TCF-1/TCF7, LEF-1, TCF-3/TCF7L1 and TCF-4/TCF7L2). Free β-cat concentration in the cytosol of resting cells is tightly controlled by the proteasome-mediated degradation process through the actions of adenomatous polyposis coli (APC), axin/conductin, and the serine/threonine kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1a (CK1a).⁶ APC and axin serve as the scaffold, while GSK-3 and CK1a phosphorylate certain serine (Ser) residues at the N-terminus of β-cat, including the Ser33position. Once β-cat is phosphorylated at the N-terminal positions, it is ubiquitinated and degraded by the proteasome. Following binding of a Wnt ligand to the frizzled receptor and LRP5/6 co-receptor, an association is made between the Wnt receptors and Dishevelled (Dvl), an event that triggers the disruption of the complex that contains APC, axin, GSK-3, and β-cat, thus preventing the phosphorylation-dependent degradation of β-cat. This leads to the translocation of β-cat into the nucleus, the formation of the β-cat/ TCF complex, and the activation of β –cat/TCF (or Wnt) downstream target genes.⁵

GSK-3 has been recognized as an important negative modulator of the Wnt signaling pathway. Lithium and other inhibitors of GSK-3 can mimic the function of Wnt ligands in stimulating the expression of Wnt downstream target genes. Furthermore, the Wnt effector β-cat/TCF may also function as an effector for other signaling cascades, including insulin, insulin-like growth factor-1 (IGF-1), glucagon-like peptide-1 (GLP1) and a number of other peptide hormones and neurotransmitters that use cAMP as a second messenger. In a number of cell lineages, activation of protein kinase A (PKA) was shown to stimulate β-cat phosphorylation at Ser 675, an event that is positively associated with the activation of β-cat/ TCF-mediated Wnt target gene expression.⁷

Although the prototype of the TCF family, TCF7 (TCF-1), was originally isolated as a lymphoid transcription factor, members of this family are now well recognized to be transcriptional regulators of many developmental processes, and in the adult organism to be functional in many other cell lineages and organs. Because the highmobility group (HMG) boxes of the TCF7L1, TCF7L2, TCF7 sequences show striking similarity.⁸ In 2000, Duval and colleagues presented the genomic structure of the human TCF7L2 gene and mapped it to chromosome 10q 25.3 by fluorescence in situ hybridization.⁹

Biochemistry of TCF7L2

Transcription factor 7 like 2 also known as TCF-4, is a nuclear factor for CTNNBI (β-catenin), which in turn mediates the canonical Wnt signaling pathway. Wnts are ligands secreted by different cells; about 19 Wnts have been identified illustrating the complexity of this signaling pathway,¹⁰ which is one of the key developmental and growth regulatory mechanisms of the cell.¹¹  Wnt signaling is critical for glucagon-like peptide-1 (GLP-1) secretion by the intestinal endocrine L-cells. Thus, alteration in this pathway could lead to reduced secretion of GLP-1 which in turn, could have effect on both insulin secretion after a meal and the generation of new β cells from the ductal precursor cells. Wnt signaling pathway is important for:

•  Normal embryogenesis and cell proliferation

•  Causation of several cancers

• Normal self renewal of stem cells

•  Regulating myogenesis, adipogenesis and adipose cell differentiation

•  Normal development of pancreas

Unless Wnt signaling is inhibited, committed pre-adipocytes will not differentiate into mature adipose cells. The potential increase in Wnt signaling in carriers of TCF7L2 risk variants could be expected to influence adipose tissue growth and development and, thus BMI.¹¹ TCF7L2 might play some role in regulation of adipogenesis by altering transcriptional regulation of the genes encoding CCAAT/enhancer-binding protein-a (CEBPA) and peroxisome-proloferator activator receptor – gamma (PPARG).¹² TCF7L2 is involved in the activation of mRNA expression of the proglucagon and the glucagon-like peptide-1 (GLP-1) genes in the gut endocrine cells. Glucagon-like peptide-1, produced in the gut and brain, lowers blood glucose levels through.¹³  

•  Stimulation of insulin secretion and biosynthesis

•  Inhibition of glucagon release and gastric emptying

• Enhancement of peripheral insulin sensitivity

• Induction of satiety

Predisposition of type 2 diabetes mellitus (T2DM) is the result of reduced insulin secretion rather than reduced insulin action, making the pancreatic β cell the most likely primary cell target of the altered TCF7L2 activity.¹⁴

The role of the Wnt signaling pathway in adipogenesis and the function of adipocytes:

The Wnt signaling pathway positively regulates bone formation and negatively regulates adipogenesis. In addition to the involvement of the Wnt signaling pathway in adipogenesis, Wnt ligands produced by adipocytes may function as paracrine or endocrine factors. Adult adipocytes express different kinds of Wnt ligands5. The carriers of TCF7L2 risk alleles, the Wnt signaling is potentially increased and thereby the preadipocytes are not differentiated into the mature adipocytes and probably influence the growth and development of adipose tissue and alter the BMI. Thus, TCF7L2 might play some role in the adipogenesis resulting in the deposition of triglycerides in peripheral tissues leading to insulin resistance.²

Expression and function of TCF7L2 in and intestinal endocrine cells and pancreatic cells

TCF7L2 in Intestinal Endocrine l cells

In the intestinal endocrine L cells, expression of the proglucagon gene leads to the production of the incretin hormone GLP-1. In 2003, Ni et al. examined whether proglucagon is a downstream target of the Wnt signaling pathway. They found that both lithium (which mimics the function of the Wnt ligands) and constitutively active β-cat (the S33Y mutant) stimulated the activity of the proglucagon promoter. Lithium was also shown to stimulate endogenous proglucagon mRNA expression and GLP-1 production in the mouse intestinal GLUTag and STC-1 cell lines, as well as in the fetal rat intestinal cell cultures.¹⁵

Yi et al. found that the stimulatory effect of lithium on proglucagon expression occurred in intestinal endocrine L cells, but not in pancreatic α-cells. Activation of proglucagon promoter activity is dependent upon a TCF binding site within the G2 enhancer element of the proglucagon gene promoter. It is well known that insulin inhibits proglucagon expression in pancreaticαcells, and this inhibition is physiologically important because proglucagon expression in the pancreas leads to the production of glucagon, the primary counter-regulatory hormone of insulin.¹³

TCF7L2 in pancreatic β-cells

Rulifson et al. examined the effect of Wnt signaling in regulating pancreatic β-cell genesis and proliferation in both in vitro and in vivo approaches. They found that purified Wnt-3a stimulated β-cell proliferation in both the mouse Min-6 cell line and primary mouse pancreatic islets, possibly through the cell cycle regulators cyclin D1, cyclin D2, and CDK-4, as well as the homeobox gene Pitx2. In the three month-old bi-transgenic rat insulin promoter (RIP)-cre and β-cat active mice, immunehistological examinations revealed increased levels of β-cat in both the cytoplasm and nuclei of pancreatic β-cells, along with a significant increase in β-cell mass. Their observations collectively suggest that Wnt signaling is necessary and sufficient for islet cell proliferation. However, it is still not clear how Wnt is mechanistically involved in the embryonic genesis of pancreatic β-cells.¹⁶

Liu and Habener examined the role of Wnt signaling in pancreatic β-cells from different angle. They demonstrated that both TCF7L2 and β-cat function as effectors of the incretin hormone GLP-1 in stimulating β-cell proliferation.¹⁷

The Wnt signaling pathway and tcF7L2 regulate the expression and function of the incretin hormones:

The expression of the proglucagon gene (gcg) and production of GLP-1 were stimulated in intestinal endocrine L cells by lithium, which mimics the activation of Wnt signaling by Wnt ligands. The positive regulation of gut gcg expression by Wnt signaling was then confirmed using a constitutively active β-cat mutant and dominant negative TCF7L2, as well as the use of the Wnt ligand Wnt -3a. Mechanistically, the activation is due to the binding of β-cat/TCF7L2 to the G2 enhancer element of gcg promoter, demonstrated by quantitative chromatin immunoprecipitation.¹³

Another incretin hormone, GIP, is produced by intestinal endocrine K cells. Wnt signaling and its effectors β-cat and TCF proteins are involved in the production of both GLP-1 and GIP, two known incretin hormones.

Pancreatic β-cells are the most important targets of GLP-1 and GIP. GLP-1 and its longacting analogue exendin-4 (Ex-4) not only stimulate insulin secretion by β-cells, but also up-regulate pro-insulin gene expression, active β-cell proliferation, and protect β-cell from stress induced apoptosis [4]. Thus, Wnt signaling and its effectors β-cat and TCF proteins are important for both the production and function of the incretin hormone GLP-1.^18,19

TCF7L2 and the Wnt signaling pathway regulate hepatic gluconeogenesis:

TCF7L2 is expressed in organs other than the gut and pancreatic islets, including liver, brain, muscle and fat tissues. As these organs are involved in mediating metabolic homeostasis as well, it is necessary and interesting to examine the metabolic function of TCF7L2 and Wnt signaling in those organs.⁵

Lyssenko et al. found that the CT/TT genotypes of SNP rs7903146 were associated with enhanced rates of hepatic glucose production. The Wnt signaling pathway is known to be important in the development and zonation of the embryonic liver.

Liu et al. found that starvation induced the expression of mRNAs that encode different Wnt isoforms in hepatocytes. They also demonstrated using loss and gain of function models that β-cat is a positive regulator of hepatic glucose production.²⁰ Briefly, β-cat ablation improved glucose disposal and reduced the expression of the rate-limiting gluconeogenic genes, including phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase. Over expression of β-cat, however, produced reciprocal effects on hepatic gluconeogenesis.²⁰ TCF7L2 silencing led to increased basal levels of hepatic glucose production in a rat hepatic cell line, associated with the over-expression of gluconeogenic genes.²¹ Thus, TCF7L2 is a potential negative regulator gluconeogenesis.

Potential metabolic effect of TCF7L2 in other organs:

Wnt signaling negatively regulates adipogenesis and positively regulates bone formation. Wnt ligands released by adipocytes stimulate insulin secretion. TCF7L2 is expressed in adipocytes and its expression can be down-regulated by insulin. Insulin resistant human subjects express higher levels of TCF7L2 in subcutaneous adipose tissue. TCF7L2 is also expressed in skeletal muscle, although its role in glucose uptake or insulin signaling is currently unknown.

TCF7L2 is expressed in the brainstem, hypothalamus and other areas of brain. TCF7L2 knockout mice show abnormalities in their pituitary gland. Since both incretin hormones and their receptors are expressed in brain neurons and brain GLP-1signaling controls satiety and peripheral insulin signaling, it is important to examine whether brain TCF7L2 play a role in energy and glucose homeostasis.⁵

TCF7L2 polymorphism and type 2 diabetes mellitus:

As early as 1999, Duggirala et al. reported that a region on chromosome 10q was linked to type 2 diabetes mellitus in Mexican Americans.²² Reynisdottir et al. also found evedence for a suggestive linkage of type 2 diabetes to 10q in an Icelandic population. In 2006, Grant et al, reported their discovery of the potential linkage between the polymorphisms in TCF7L2 and the risk of type 2 diabetes. Microsatellite, DG 10S478 gene, located within intron 3 of the TCF7L2 gene, was found to be associated with type 2 diabetes.^23,24

Molecular mechanism of effect:

TCF7L2 is one of family of HMG box transcription factors that have been identified as down-stream effectors of the Wnt signaling pathway. Aberrant Wnt signaling has been linked to familial and sporadic colon cancers, but it is a novel candidate pathway for the development of type 2 diabetes mellitus. There are number of ways by which changes to Wnt signaling could increase the risk of disease. The TCF7L2 knockout mouse dies within 24 h due to inadequate intestinal epithelium, but with no other reported organ abnormalities, whilst mis-expression of Wnts leads to significant abnormal development of foregut structures, including the pancreas.²⁵ Thus, disruption in the pathway could reduce both pancreatic size and number of insulin secreting cells. Low levels of TCF7L2 expression could cause a failure to develop adequate gut endocrine tissue, which would lead to disturbance of enteroendocrine function, which in turn could lead to type 2 diabetes mellitus.²⁵

Conclusion:

Wnt signaling pathway controls hormone gene expression and mediates the function of certain hormones including GLP-1, GIP and insulin, which are critically important in glucose and energy homeostasis. Polymorphisms in certain Wnt ligands as well as the Wnt effector TCF7L2, are strongly linked with type 2 diabetes mellitus risk.

Although the TCF7L2 polymorphisms are associated with the risk of type 2 diabetes mellitus in different ethnic groups, molecular mechanisms underlying this association are far from understood at this time. Since no risk SNP of type 2 diabetes mellitus has been identified within the coding region of TCF7L2. In the future, more efforts should be made to assess whether polymorphisms within the coding region and the promoter region of TCF7L2 are associated with the risk of type 2 diabetes, other metabolic diseases, and colorectal tumors.