Evidence for neuroendocrine function of a unique splicing form of TCF7L2 in human brain, islets and gut
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Variants in the TCF7L2 gene remain the strongest genetic associations with increased risk of type 2 diabetes. Recently, we identified a unique splicing form of TCF7L2 expressed in pancreatic islets, pancreas and colon and detected by assay ‘ex13-13b’. The expression of ex13-13b strongly correlated with proinsulin in glucose-stimulated pancreatic islets, suggesting a potential role for this form in the development of type 2 diabetes. The goal of this study was to further characterise this unique TCF7L2 splicing form in human tissues.
We used a panel of 34 human tissues and 80 human cell lines to measure the expression of assay ex13-13b with use of quantitative RT-PCR.
The highest expression of assay ex13-13b was detected in several areas of the brain (hypothalamus/thalamus, occipital lobe) and in neuronal cell line SHS5Y5. Low expression was confirmed in pancreatic islets, small intestine, pancreas and colon, while no expression was detected in other human tissues and cell lines. The expression of assay ex13-13b correlated with the gene for cocaine- and amphetamine-regulated transcript (CART, also known as CARTPT) in a panel of human tissues (n = 12, r = 0.85, p = 0.00046), pancreatic islets (n = 23, r = 0.62, p = 0.0016) and colon (n = 98, r = 0.54, p < 0.0001).
The significant correlation between expression of a unique splicing form of TCF7L2, named here TCF7L2-NE, and CART, the gene for an anorexigenic neurohormone expressed in the central and peripheral nervous system, suggests that these transcripts may share neuroendocrine functions important for brain, gut and pancreatic islets.
KeywordsBrain–gut–islets axis CART Gene expression Neuroendocrine regulation TCF7L2 Type 2 diabetes
Transcription factor 7-like 2
Neuroendocrine splicing form of TCF7L2
Transcription factor 7-like 2 (TCF7L2) (also known as TCF-4) plays a central role in the WNT pathway and is involved in a variety of biological processes. Non-coding genetic variants within TCF7L2 gene remain the strongest genetic association with increased risk of type 2 diabetes and dysfunction of pancreatic islets [1, 2]. However, the molecular and cellular mechanisms through which TCF7L2 contributes to these associations are still unclear.
Previously, we described a tissue-specific splicing diversity of the gene resulting from the use of three alternative promoters and six alternative exons . We detected a unique splicing form defined by the presence of an alternative C-terminal exon, 13b, expressed in pancreatic islets, colon and pancreas . Expression of this form strongly correlated with insulin expression in glucose-stimulated pancreatic islets and was decreased in islets of carriers of the type 2 diabetes-associated single nucleotide polymorphisms of TCF7L2, rs7903146 and rs12255372 .
In the current study we aimed to further characterise the TCF7L2 splicing form containing the alternative exon 13b. We provide evidence for a neuroendocrine pattern of expression of this form, named TCF7L2-NE, in human tissues and cell lines. We show that expression of TCF7L2-NE correlated with expression of the gene for cocaine- and amphetamine-regulated transcript (CART, also known as CARTPT), also found in the brain, islets and gut. CART has been shown to play a critical role in glucose sensing, stimulation of hormone secretion, cell survival, thermoregulation and appetite suppression. Based on strong correlation between expression of TCF7L2-NE and CART in a panel of human tissues, islets and colon, these data suggest that TCF7L2-NE may have important neuroendocrine functions.
Samples, cDNA preparation and expression analysis
Samples of total RNA from human tissues were purchased from Clontech (Mountain View, CA, USA) and BioChain Institute (Hayward, CA, USA). Samples of human pancreatic islets, monocytes, colon, pancreas and lymphoblastoid cell lines were previously described . RNA from the NCI-60 cell lines was provided by the Molecular Targets Team, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI/NIH. All other cell lines were purchased from ATCC (Manassas, VA, USA) or Asterand (Detroit, MI, USA). Total RNA was prepared with a MirVana kit (Ambion, Austin, TX, USA) and the quality was evaluated with a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). cDNA from all samples was prepared from 1 μg total RNA, as previously described . All reactions were run in duplicate in 5 μl volumes in 384-well optical plates on an SDS7900 (Applied Biosystems, Foster City, CA, USA). Negative controls and genomic DNA controls were used for all assays. The expression of endogenous control genes, beta-2 microglobulin (B2M) and cyclophilin A (PPIA), measured in separate reactions but in the same cDNA samples was used for normalisation of expression. All assays were tested before analysis and showed comparable amplification efficiencies. The list of expression assays is provided in the Electronic supplementary material (ESM) Table 1.
Average values for technical duplicates were normalised by geometric means of endogenous controls according to the ΔCt method of relative quantification, as previously described . Pearson’s correlation coefficients and two-sided p values were calculated with SPSS 16.0 software (SPSS, Chicago, IL, USA). p values were not adjusted for multiple tests.
Expression of the TCF7L2 splicing form with exon 13b in human tissues and cell lines
Expression of TCF7L2 assay ex13-13b and neurohormone genes
ex13-13b (fold vs pancreas)
SHS5Y5 neuroblastoma cell line
Coexpression of TCF7L2 assay ex13-13b with brain–islet–gut neurohormones
The pattern of expression of TCF7L2 splicing form detected by assay ex13-13b suggested that it might be expressed in neuroendocrine cells of brain, islets and gut. Based on the literature, we selected a number of neurohormones that can be expressed in the brain and enteric nervous system . We tested for correlation in expression of assay ex13-13b with several neurohormone genes, neuropeptide Y (NPY) and agouti-related peptide (AGRP) expressed in glucose-inhibited neurons, pro-opiomelanocortin (POMC) and CART expressed in glucose-activated neurons, a marker of endocrine function chromogranin A (CHRA, also known as CHGA), and several other neuropeptides: ghrelin (GHRE, also known as GHRL), vasoactive intestinal peptide (VIP), gastric inhibitory polypeptide, also known as glucose-dependent insulinotropic peptide (GIP) and somatostatin (SST).
In a panel of 34 human tissues and 80 cell lines (NCI-60 and 20 additional cell lines) the most similar pattern of tissue distribution and level of expression was observed between TCF7L2 assay ex13-13b and CART (r = 0.85, p = 0.00046, Table 1). Both these transcripts were expressed highly in the brain and in neuronal cell line SHS5Y5, low in small intestine, islets, colon and pancreas and not expressed in all other tissues and cell lines tested, even though the expression of endogenous controls B2M and PPIA was equally high in all these samples (data not shown). Based on the observed pattern of expression, we designated the splicing form detected by assay ex13-13b as a neuroendocrine splicing form, TCF7L2-NE. Expression of TCF7L2-NE also showed correlation with CART in human pancreatic islets (n = 23, r = 0.62, p = 0.0016) and colon (n = 98, r = 0.54, p < 0.0001). Expression of both TCF7L2-NE and CART in pancreas (n = 44) was low and not detectable in all samples and was not analysed (data not shown).
The protein encoded by this splicing form is C-terminally truncated and lacks binding sites for the C-terminal binding protein (CtBP) that regulates TCF7L2 post-translationally . Alternative exon 13b contributes only two amino acids before the termination of transcription, making it impossible to distinguish TCF7L2-NE from other TCF7L2 forms at the protein level. We tested cellular localisation of this form by transfecting an expression construct for the full-length TCF7L2-NE splicing form (GenBank FJ010169)  into human PANC-1 cells. The localisation of this form was clearly nuclear (Fig. 1c), as was expected based on the presence of nuclear localisation signals and the role of TCF7L2 as a transcription factor.
Disturbances in brain–gut–islet regulation of satiety and glucose metabolism are associated with development of obesity and type 2 diabetes [6, 7]. In this study we provide evidence that the unique splicing form of TCF7L2 that we named a ‘neuroendocrine form, TCF7L2-NE’, might be a part of this regulation. TCF7L2-NE exhibits a neuronal pattern of expression, as it is highly expressed in the thalamic, hypothalamic and occipital regions of the brain, as well as in a neuronal cell line SHS5Y5. Comparatively lower levels of expression were found in islets and gut (up to 750-fold lower than in brain), while no expression was found in all other human tissues and cell lines tested here. Expression of a similar murine splicing form of TCF7L2 (designated as a form with exons 12-13-14-17-18) was recently located to postmitotic neurons of mouse midbrain .
TCF7L2-NE is highly expressed in the brain areas that contain neurons of the hypothalamic arcuate (ARC) nucleus that regulate energy metabolism and satiety. Glucose-sensing neurons express CART and POMC . In the panel of human tissues used in this study, expression of TCF7L2-NE correlated with CART but not with POMC, possibly marking a subpopulation of glucose-activated neurons activated by blood glucose, insulin and leptin . Expression of TCF7L2-NE did not correlate with NPY and AGRP (markers of glucose-inhibited neurons activated by ghrelin and inhibited by glucose and leptin ). In sum, expression of TCF7L2-NE and CART strongly correlated in a panel of human tissues (n = 12, r = 0.85, p = 0.00046), in islets (n = 23, r = 0.62, p = 0.0016) and colon (n = 98, r = 0.54, p < 0.0001) with p values being significant even after adjustment for multiple tests.
CART has been shown to play a critical role in glucose sensing, stimulation of hormone secretion in pancreatic islets, neuronal survival, thermoregulation and appetite suppression [4, 10, 11]. The functional relationship between TCF7L2-NE and CART is not clear. Based on the strong correlation between expression of TCF7L2-NE and CART, we suggest that these transcripts may have similar functions related to the brain–islet–gut regulation of metabolism. Further studies will be needed to identify the functional role of TCF7L2-NE in different metabolic conditions.
We are grateful to the Molecular Targets Team, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (DCTD), National Cancer Institute (NCI)/National Institutes of Health (NIH) for providing RNA samples from NCI-60 cell lines, to the Islet Cell Resource Centers and the National Disease Research Interchange Network for human islets, to F. S. Collins and M. R. Erdos (National Human Genome Research Institute [NHGRI]/NIH) for sharing samples of pancreatic islets and helpful comments, and to S. Bowell for help with procurement of tissues from the University of Minnesota Tissue Procurement Facility. We would also like to thank M. Carlson and C. Welch for their help in preparing samples for analysis. The study was supported by the intramural research program of NCI/NIH and by the NIH grant 1R21DK078029-01 (J. L. Hall).
Duality of interest
The authors declare that there is no duality of interests associated with this manuscript.
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