Evidence for neuroendocrine function of a unique splicing form of TCF7L2 in human brain, islets and gut

Abstract

Aims/hypothesis: 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.

Methods: 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.

Results: 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).

Conclusions/interpretation: 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.

Fig. 1

C-terminal alternative exons of TCF7L2 in transcripts expressed in total human brain and pancreatic islets and cellular localisation of TCF7L2 splicing form detected by assay ex13-13b (TCF7L2-NE). a Results of PCR amplification with primers ex10 Forw and ex13b Rev of TCF7L2 (ESM Table 1) in cDNA from total human brain and pancreatic islets. cDNA prepared from 10 ng total RNA was used for all PCR reactions. The PCRs were performed with Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) and the PCR products were resolved on a 2% (wt/vol.) agarose gel. Four types of PCR fragments (forms A–D) were observed based on the combination of alternative exons 12, 13, 13a and 13b. b Schematic representation of C-terminal exons of TCF7L2. Constitutive exons are represented by black rectangles and alternative exons by white rectangles. Black triangles mark alternative translation stops. Arrows indicate positions of PCR primers. Forms A and B use a stop codon within exon 13b and forms C and D use a stop codon within exon 13a. In the forms with alternative exons 13a and 13b (C and D) two in-frame stop codons are separated only by 37 bp. Expression of these forms (assay ex13a-13b) was very low in brain and islets and was not studied further. c Confocal imaging of cellular localisation of the recombinant TCF7L2-NE in human PANC-1 cell line (pancreatic cancer). Upper and lower panels represent images of PANC-1 cells transfected with the TCF7L2-NE Halo-tag expression construct (GenBank FJ010169) [3]. Non-transfected cells serve as controls for specificity of detection. For the expression construct a full-length cDNA for TCF7L2-NE splicing form was cloned into a pFC8A expression vector with a C-terminal Halo-tag (Promega, Madison, WI, USA). The endotoxin-free plasmid was prepared with a EndoFree plasmid Maxi kit (Qiagen, Gaithersburg, MD, USA). For transfection, human PANC-1 (pancreatic cancer) cells were plated on chamber slides (Thermo Fisher Scientific, Rochester, NY, USA) and transfected next day with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, the cells were fixed and incubated with primary rabbit anti-HaloTag antibodies (Promega) and with mouse anti-α-tubulin antibodies ab7291-100 (Abcam, Cambridge, MA, USA). Secondary donkey anti-rabbit and anti-mouse antibodies labelled with Alexa fluor 594 and 488 were used for imaging (Invitrogen). The anti-fade ProLong Gold mounting media with DAPI (Invitrogen) was used to mount the cover slides. The imaging was performed with a confocal microscope LSM 510 Meta (Carl Zeiss Microimaging, Thornwood, NY, USA) with ×63 magnification