Abstract

Type 2 diabetes manifests when the β-cell fails to secrete sufficient amounts of insulin to maintain normoglycemia and undergoes apoptosis. The disease progression results from an interplay of environmental factors and genetic predisposition. Polymorphisms in T-cell factor 7-like 2 (TCF7L2) strongly correlate with type 2 diabetes mellitus (T2DM). While TCF7L2 mRNA is upregulated in islets in diabetes, protein levels are downregulated. The loss of TCF7L2 induces impaired function and apoptosis. By analyzing human isolated islets, we provide three explanations for this opposite regulation and the mechanisms of TCF7L2 on β-cell function and survival. (i) We found TCF7L2 transcripts in the human β-cell, which had opposite effects on β-cell survival, function and Wnt signaling activation. While TCF7L2 clone B1, which lacks exons 13, 14, 15 and 16 induced β-cell apoptosis, impaired function and inhibited glucagon-like peptide 1 response and downstream targets of Wnt signaling, clones B3 and B7 which both contain exon 13, improved β-cell survival and function and activated Wnt signaling. (ii) TCF7L2 mRNA is extremely unstable and is rapidly degraded under pro-diabetic conditions and (iii) TCF7L2 depletion in islets induced activation of glycogen synthase kinase 3-β, but this was independent of endoplasmic reticulum stress. We demonstrated function-specific transcripts of TCF7L2, which possessed distinct physiological and pathophysiological effects on the β-cell. The presence of deleterious TCF7L2 splice variants may be a mechanism of β-cell failure in T2DM.

INTRODUCTION

TCF7L2 (T-cell factor 7-like 2, formerly known as TCF4) is a Wnt signaling-associated transcription factor expressed in several tissues, including gut and pancreas. Wnt signaling plays an important role in β-cell proliferation and insulin secretion (1,2) and influences synthesis of glucagon-like peptide 1 (GLP-1) in intestinal L-cells (3). Since 2006, the strong association of single nucleotide polymorphisms (SNPs) in the TCF7L2 gene [rs7903146 and rs12255372, located between exons 3 and 4 and exons 4 and 5, respectively (4) with type 2 diabetes mellitus (T2DM)] has been confirmed in numerous studies (5,6). The TCF7L2 at-risk alleles are associated with specific T2DM phenotypes characterized by an early-impaired β-cell function, a reduction in GLP-1 induced potentiation of insulin secretion and fasting and postprandial hyperglycemia in prediabetes (7–9).

Although changes in TCF7L2 expression levels were reported in T2DM, it is not clear whether such changes contribute to the disease progression. In islets, TCF7L2 mRNA is increased in the Zucker diabetic fatty rat (10) and in patients with T2DM or individuals carrying TCF7L2 risk T-alleles (8). This apparent increase in TCF7L2 mRNA in islets in T2DM is in contradiction with the impairment of β-cell function and survival in islets following siRNA-induced loss of TCF7L2 expression (11,12). We addressed this issue in our recent report and demonstrated robust differences in TCF7L2 mRNA and protein expression levels between healthy controls and models of T2DM. While mRNA levels are increased in diabetic islets, TCF7L2 protein levels are decreased. This decrease in the TCF7L2 protein correlates with reduced expression levels of the receptor of GLP-1 and GIP in islets in pancreatic sections from patients with T2DM (13).

The mechanism how such TCF7L2 variations translate into altered biological function as well as the opposite regulation of transcription and translation in the TCF7L2 gene is unknown.

One hypothesis is that the TCF7L2 protein is extremely unstable and rapidly undergoes intracellular protein degradation. There is evidence that the translational control is disturbed in diabetes (14,15) through an inhibition of translation by unfolded protein accumulation. The β-cell is a cell with the capacity to fold and process large amounts of secreted protein (insulin), thereby the β-cell is vulnerable to endoplasmic reticulum (ER) stress activated by the unfolded protein response (16). A possible interaction of WNT signals with the unfolded protein response could activate the ER stress pathway. That way, mRNA would increase as an adaptive compensation for less protein due to translation inhibited by ER stress.

Another possible explanation could be the complex splicing pattern of TCF7L2 and a probable genotype-dependent difference in the resulting transcript profiles, which may impair insulin secretion. The TCF7L2 gene consists of 17 identified exons and is known to display a complex pattern of spliced variants with several alternative exons and splice sites (17), with tissue-specific splicing variants detected in pancreatic islets (18–22). Such tissue-dependent pattern of TCF7L2 splice variants may exert distinct functions, but none of the studies has addressed such hypothesis.

We therefore (i) asked the questions, which TCF7L2 splicing variants are expressed in human β-cells and what is their distinct effect on β-cell survival and function, and explored in this study (ii) the stability of TCF7L2 mRNA and protein under diabetic conditions and (iii) a possible involvement of ER stress.

RESULTS

Identification and cloning of TCF7L2 splice variants in human β-cells

To identify the mechanisms of TCF7L2 regulation, we monitored the documented alternative regions (17,23) in liver, adipose tissue, muscle and pancreas using cDNA from human tissue (MTC panel, BD Biosciences Clontech) by polymerase chain reaction (PCR). We first evaluated the presence or absence of exon 4 in TCF7L2 transcripts and detected PCR products at the expected size for transcripts containing (245 bp) or not containing exon 4 (176 bp) in all analyzed tissues. The relative proportion of transcripts with or without exon 4 was similar in liver, adipose tissue and pancreas, but exon 4 containing transcript expression was lower in muscle (Fig. 1A). Primers located between exons 11 and 17 lead to the amplification of a complex pattern of PCR products demonstrating the high level of TCF7L2 splicing in human tissue. Next, we PCR-amplified and cloned TCF7L2 transcripts using human fluorescence-activated cell sorting (FACS)-sorted β-cells. Rs7903146 genotyping revealed that the two donors used for the analysis were not carrying the at-risk allele of TCF7L2. We screened and sequenced 80 positive clones, which allowed us to identify nine TCF7L2 alternative transcripts, B1–B9 (Fig. 1B). The presence or absence of alternative exon 4 or alternative regions located in exons 7 and 9 did not modify the open reading frame between exons 1 and 12 in all identified alternative transcripts. In contrast, the combination of alternative events in exons 13, 14, 15 and 16 produced different open reading frames (ORFs). Alternative splicing events of exons 14 and 15 modified the reading frame in exon 17, leading to three different C-terminal ends. A short C-terminal end (clones B3 and B5) is created when exons 14 and 15 are both present. Instead, if only one of these two exons is transcribed, then a long C-terminal end is created (TCF4L; B4, B6, B8 and B9). When both exons are lacking, it creates a medium C-terminal end (B1, B2, and B7). With our cloning strategy, we were able to identify isoforms of the TCF4L group (also known as TCF4E; B4, B6, B8 and B9), TCF4M group (also known as TCF4B; B1, B2 and B7) and of the TCF4S group (B3, B5) (21) (Fig. 1C). B7 clone sequencing revealed identical structure to the TCF7L2 clone obtained from the Invitrogen Mammalian Gene Collection previously used (12) and was therefore referred as wt-TCF7L2 in this study.

Figure 1.

Total mRNA from various tissues was reverse transcribed and PCR performed using different combinations of specific primers. (A) Exon 2–5 PCR showing the existence of TCF7L2 with and without exon 4 in human tissues (left panel). Exon-specific PCR combinations showing the existence of multiple spliced hTCF7L2 isoforms (right panel). (1) exons 11–17, (2) exons 12–17, (3) exons 13–17, (4) exons 14–17, (5) exons 15–17. (B) Summary of the gene organization of the TCF7L2 transcripts identified in sorted β-cells according to Duval et al. (17). Alternative exons are represented in red and constitutively used exons in blue. Yellow areas indicate untranslated regions. (C) Diversity in the amino acid sequence at the C-terminal end of TCF7L2 alternative transcripts. Particular exon combination is indicated by numbers in brackets.

Figure 1.

Total mRNA from various tissues was reverse transcribed and PCR performed using different combinations of specific primers. (A) Exon 2–5 PCR showing the existence of TCF7L2 with and without exon 4 in human tissues (left panel). Exon-specific PCR combinations showing the existence of multiple spliced hTCF7L2 isoforms (right panel). (1) exons 11–17, (2) exons 12–17, (3) exons 13–17, (4) exons 14–17, (5) exons 15–17. (B) Summary of the gene organization of the TCF7L2 transcripts identified in sorted β-cells according to Duval et al. (17). Alternative exons are represented in red and constitutively used exons in blue. Yellow areas indicate untranslated regions. (C) Diversity in the amino acid sequence at the C-terminal end of TCF7L2 alternative transcripts. Particular exon combination is indicated by numbers in brackets.

TCF7L2 transcript variants showed distinct effects on β-cell survival

Since wt TCF7L2 had protective effects on β-cell survival and function (12,24), we investigated the effects of the TCF7L2 splice variants on β-cell turnover in human islets. Isolated human islets were transfected with six of the identified TCF7L2 variants of all three groups TCF4S (B3, B5), TCF4M (B1, B2) and TCF4L (B4, B6). Transfected islets with our previously used TCF7L2 full-length clone (Mammalian Gene Collection, Invitrogen, identified in our assay as clone B7) or green fluorescent protein (GFP) were used as controls. Quadruple staining of TUNEL, Ki67, insulin and 4′,6-diamidino-2-phenylindole (DAPI) after 4 days of culture revealed that overexpression of full-length TCF7L2 and B3 had the most protective effects on β-cell proliferation, whereas B1 reduced β-cell survival (Fig. 2A). Wt-TCF7L2 and B3 increased β-cell proliferation 2.4- and 2.3-fold, respectively, compared with the GFP control (Fig. 2B, P < 0.01) and had no effects on β-cell apoptosis (Fig. 2C). In contrast, overexpression of the B1 transcript induced a 2.5-fold increase in β-cell apoptosis (Fig. 2A and C; P < 0.01). All other transcripts did not affect β-cell survival significantly. To further characterize the opposite roles of B1 and B3 transcripts on β-cell survival, we performed western blot analysis of cleaved caspase-3, AKT phosphorylation and eukaryotic initiation factor 2α (eIF2α) phosphorylation. In consistency with results shown in Supplementary Material, Fig. S2A, we did not observe changes in eIF2α phosphorylation by wt-TCF7L2 or any other transcripts (Fig. 2D). Significant increase in AKT phosphorylation was induced by wt-TCF7L2 and B3 transcript overexpression (Fig. 2D; P < 0.01), whereas cleavage of caspase-3 was only induced by B1 transcript overexpression (Fig. 2D; P < 0.005). This was also confirmed in Min6 cells (Fig. 2E).

Figure 2.

Human islets cultured on extracellular matrix-coated dishes were transfected with the following plasmids: GFP (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 and B6. Proliferation measured by the Ki67 antibody stained in red (cy3; A, B) and apoptosis analyzed by the TUNEL assay and stained in black (alkaline phosphate; A, C) were analyzed after the 4-day culture period. Islets were triple-stained for insulin in green (A) and counterstained for DAPI in blue (data not shown). Results are means ± SE of the percentage of Ki67 positive (B) TUNEL-positive (C) β-cells. The average number of β-cells counted were 10 250 for each treatment group in five separate experiments from five different organ donors. (D) Representative western blot from the transfected human islets together with the densitometric analyses of three independent experiments are shown. Membranes were incubated with the different antibodies after stripping. (E) Representative western blot out of three independent experiments from transfected Min6 cells. *P < 0.05 to GFP-transfected control.

Figure 2.

Human islets cultured on extracellular matrix-coated dishes were transfected with the following plasmids: GFP (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 and B6. Proliferation measured by the Ki67 antibody stained in red (cy3; A, B) and apoptosis analyzed by the TUNEL assay and stained in black (alkaline phosphate; A, C) were analyzed after the 4-day culture period. Islets were triple-stained for insulin in green (A) and counterstained for DAPI in blue (data not shown). Results are means ± SE of the percentage of Ki67 positive (B) TUNEL-positive (C) β-cells. The average number of β-cells counted were 10 250 for each treatment group in five separate experiments from five different organ donors. (D) Representative western blot from the transfected human islets together with the densitometric analyses of three independent experiments are shown. Membranes were incubated with the different antibodies after stripping. (E) Representative western blot out of three independent experiments from transfected Min6 cells. *P < 0.05 to GFP-transfected control.

TCF7L2 transcript variants had different effects on β-cell function

To characterize the effects of TCF7L2 splice variants on β-cell function, isolated human islets were transfected with six variants (B1–B6), wt-TCF7L2 and GFP as control for 4 days followed by analysis of glucose-stimulated insulin secretion.

Stimulated insulin secretion induced by 16.7 mm glucose was 5-fold increased in GFP-transfected islets (Fig. 3A). Only the TCF7L2 and B3 transcripts were able to significantly increase insulin secretion when compared with GFP-control conditions (1.6- and 1.7-fold, respectively, P < 0.05). All other isoforms did not change glucose-stimulated insulin secretion (Fig. 3A and B). Recent studies demonstrated that GIP and GLP-1 fail to potentiate glucose-stimulated insulin secretion in patients with TCF7L2 rs7903146 risk alleles as well as in TCF7L2-depleted islets (9,13,25,26). We therefore tested the effect of TCF7L2 splice variants on stimulated insulin secretion. GLP-1 potentiated glucose-stimulated insulin secretion at control GFP-transfected islets (1.3-fold, Fig. 3A, P < 0.05, compared with glucose alone). TCF7L2 and B3 transcript overexpression further increased the effect of GLP-1 (1.6- and 1.9-fold increase, compared with glucose alone, Fig. 3A, P < 0.01). In islets transfected with the other transcripts B1, B2, B4, B5, B6, glucose-stimulated insulin secretion was unaffected, and GLP-1 was unable to further potentiate insulin secretion significantly (Fig. 3A and B). These distinct effects of GLP-1 in the islets overexpressing TCF7L2 transcripts correlated with GLP-1R expression levels. Western blot analysis of islets transfected with the TCF7L2 transcripts showed GLP-1R protein levels significantly upregulated by TCF7L2 and B3 transcripts, whereas GLP-1R expression was unaffected in islets transfected with the other TCF7L2 splice variants (Fig. 3B and C, P < 0.01). In contrast, GIP receptor expression levels were unchanged in islets transfected with different TCF7L2 transcripts. All transcripts showed increased TCF7L2 mRNA (Fig. 3D). GLP-1 mRNA changes were similar to protein levels; while TCF7L2 and B3 significantly increased GLP-1R mRNA, other transcripts had no effects. Pancreatic and duodenal homeobox-1 (PDX-1), a major islet transcription factor, was increased by TCF7L2, B1, B3, B5 and B6, which did not correlate with the survival studies (Fig. 3F). We then investigated the effects of the transcripts on mRNA expression of genes involved in downstream Wnt signaling, including c-myc, menin and survivin (27–29). Overexpression of all TCF7L2 transcripts in human islets resulted in an ∼2-fold induction of c-myc transcription, without any significant differences (Fig. 3G). In contrast, only overexpression of TCF7L2 and B3 transcripts were able to stimulate survivin transcription (Fig. 3H, P < 0.05) and to decrease menin expression significantly. Menin was also decreased by B6 transcript (Fig. 3I, P < 0.05).

Figure 3.

Human islets cultured on extracellular matrix-coated dishes were transfected with the following plasmids: GFP (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 and B6. (A) Basal (2.8 mm), glucose stimulated (16.7 mm) and GLP-1 stimulated (16.7 mm + GLP-1) insulin secretion were normalized to whole islet insulin content, respectively. (B) Stimulatory index denotes the amount of glucose stimulated (16.7 mm glucose) or GLP-1 stimulated (16.7 mm glucose + GLP-1) divided by the amount of basal insulin secretion. Data are shown as mean ± SE from three islet isolations from three different donors. *P < 0.05 to GFP-transfected control at same treatment conditions, +P < 0.05 to glucose stimulated insulin secretion. (C) Representative western blot from the transfected human islets together with the densitometric analyses of three independent experiments are shown. Membranes were incubated with the different antibodies after stripping. (DI) qPCR analysis of TCF7L2 (D), GLP-1R (E), PDX-1 (F), c-myc (G), survivin (H) and menin (I) expression from mRNA isolated from transfected human islets, mRNA levels were normalized to cyclophilin and tubulin with the same result. Data are shown as mean ± SE from four islet isolations from four different donors. *P < 0.05 to GFP-transfected control.

Figure 3.

Human islets cultured on extracellular matrix-coated dishes were transfected with the following plasmids: GFP (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 and B6. (A) Basal (2.8 mm), glucose stimulated (16.7 mm) and GLP-1 stimulated (16.7 mm + GLP-1) insulin secretion were normalized to whole islet insulin content, respectively. (B) Stimulatory index denotes the amount of glucose stimulated (16.7 mm glucose) or GLP-1 stimulated (16.7 mm glucose + GLP-1) divided by the amount of basal insulin secretion. Data are shown as mean ± SE from three islet isolations from three different donors. *P < 0.05 to GFP-transfected control at same treatment conditions, +P < 0.05 to glucose stimulated insulin secretion. (C) Representative western blot from the transfected human islets together with the densitometric analyses of three independent experiments are shown. Membranes were incubated with the different antibodies after stripping. (DI) qPCR analysis of TCF7L2 (D), GLP-1R (E), PDX-1 (F), c-myc (G), survivin (H) and menin (I) expression from mRNA isolated from transfected human islets, mRNA levels were normalized to cyclophilin and tubulin with the same result. Data are shown as mean ± SE from four islet isolations from four different donors. *P < 0.05 to GFP-transfected control.

TCF7L2 transcript variants exerted diverse functions on Wnt/β-catenin signaling

Our findings suggest that TCF7L2 transcripts differentially affect β-cell survival and function. One underlying reason could be differences downstream signaling. Western blot analysis of nuclear and cytosolic extracts from human insulinoma CM cell transfected with the different transcripts showed shifts in the TCF7L2 protein. TCF7L2 is mainly expressed in the nucleus. Besides B6, which resulted in equal TCF7L2 distribution in the nucleus and in the cytosol, all transcripts resulted in mainly nuclear TCF7L2 expression. Transcript B1 only resulted in a very low protein expression. This is in contrast to the high TCF7L2 mRNA expression (2549-fold increase compared with GFP-transfected control). In contrast, B3 is lower expressed at the mRNA level (615-fold increase over GFP), but higher expressed on the protein level than B1. The sizes of the transcripts in the western blot are in accordance with the theoretically predicted sizes (B1: 51 kDa, B2: 53 kDa, B3: 55 kDa, B4: 65 kDa, B5: 52 kDa, B6: 63 kDa, B7: 51 kDa) shown in the nuclear fraction of overexpressed CM cells (Fig. 4A) or in whole cell lysates of Min6 cells (Fig. 4B). An additional band of ∼50 kDa was present in clone B6 in CM and Min6 cells, which could have resulted from post-translational modifications.

Figure 4.

TCF7L2, β-catenin and FOXO1 localization are shown in the β-cell line CM9 which was transfected with either GFP plasmid (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 or B6. After 4 days of culture, nuclear and cytoplasmic fractions were extracted and western blot analysis performed. PARP was used as loading control and purity of fractions for the nuclear extracts and GAPDH for the cytosolic extracts in the same membrane after stripping. No GAPDH was observed in the nuclear and no PARP in the cytosolic extracts. One representative blot out of three experiments is shown. Densitometric analyses of the western blots show nuclear FOXO1 normalized to cytosolic FOXO1 (A, upper panel) and nuclear β-catenin normalized to cytosolic β-catenin (A, lower panel). Western blots are representative of three independent experiments and data are expressed as means ± SE. (B) Min6 cells were transfected with the same transcripts (see above) show similar results. *P < 0.05 to GFP-transfected control.

Figure 4.

TCF7L2, β-catenin and FOXO1 localization are shown in the β-cell line CM9 which was transfected with either GFP plasmid (control), wt-TCF7L2 (clone B7; TCF), B1, B2, B3, B4, B5 or B6. After 4 days of culture, nuclear and cytoplasmic fractions were extracted and western blot analysis performed. PARP was used as loading control and purity of fractions for the nuclear extracts and GAPDH for the cytosolic extracts in the same membrane after stripping. No GAPDH was observed in the nuclear and no PARP in the cytosolic extracts. One representative blot out of three experiments is shown. Densitometric analyses of the western blots show nuclear FOXO1 normalized to cytosolic FOXO1 (A, upper panel) and nuclear β-catenin normalized to cytosolic β-catenin (A, lower panel). Western blots are representative of three independent experiments and data are expressed as means ± SE. (B) Min6 cells were transfected with the same transcripts (see above) show similar results. *P < 0.05 to GFP-transfected control.

β-Catenin, a key molecule in wnt signaling and co-activator for wt-TCF7L2 transcriptional activity, was accumulated in the nucleus in β-cells overexpressing wt-TCF7L2 or B3. In contrast, the pro-apoptotic B1 transcript caused cytoplasmic β-catenin expression (Fig. 4A, P < 0.05). The pro-apoptotic phenotype of the B1 transcript and pro-survival effect of B3 were associated with changes in Foxo1 accumulation. While wt-TCF7L2 and B3 were mainly expressed in the cytosol, B1 caused nuclear Foxo1 accumulation (Fig. 4A, P < 0.005).

TCF7L2 mRNA is unstable in human-isolated islets

To investigate the posttranscriptional regulation of TCF7L2 and its mRNA stability during 96 h of culture, we analyzed the degradation rate of TCF7L2 mRNA. Actinomycin D, which blocks RNA synthesis, reduced TCF7L2 levels already after 8 h. After 96 h, almost no TCF7L2 mRNA was detectable (Supplementary Material, Fig. S1A). Long-term (96 h) glucose and cytokines both induced TCF7L2 mRNA significantly, but protein levels of TCF7L2 were downregulated after 96 h culture. Actinomycin D induced a reduction in the TCF7L2 protein at control and diabetogenic (glucose and cytokine mix) condition. Neither actin nor tubulin levels changed in response to the treatments.

TCF7L2 depletion resulted in GSK-3β activation but not in ER stress signaling

The loss of TCF7L2 could result in impaired translational control through the unfolded protein response, which regulates protein levels by inhibiting protein synthesis or promoting protein degradation. We investigated the effects of TCF7L2 depletion by siRNA in human islets on activation of glycogen synthase kinase 3-β (GSK3-β), eIF2α and TSC2, important regulators of translation and ER stress signaling. Knock-down of TCF7L2 by siRNA reduced p-GSK3β and p-TSC2 but neither affected p-eIF2α (Supplementary Material, Fig. S2A) nor X-box binding protein 1 splicing (Supplementary Material, Fig. S2B). Inhibition of GSK3β phosphorylation leads to GSK3β activation and impaired β-cell proliferation and function (30,31). Therefore, we tested whether GSK3β inhibitors could reverse the deleterious effects of TCF7L2 depletion in isolated human islets. Two specific GSK3β inhibitors 1-AKP (1-Azakenpaullone) (32) and SB216763 (33) effectively restored GSK3β phosphorylation, which was reduced by TCF7L2 depletion (Supplementary Material, Fig. S2C) and significantly improved β-cell function at basal levels and in siTCF7L2 treated islets.

DISCUSSION

The defect in β-cell survival and insulin secretion leading to T2DM results from the interplay between environmental factors and genetic pre-disposition. One such pre-disposition factor is a SNP in the TCF7L2 gene, which has been associated with T2DM (4,34,35). The mechanism how the TCF7L2 genetic variation leads to altered biological function is still largely unknown. Recent studies show that the loss of TCF7L2 correlates with impaired β-cell function and survival (11,12). In T2DM, transcription and translation of the TCF7L2 gene is oppositely regulated (8,13). One possibility for this paradox is that the increase in mRNA levels represents multiple possible transcripts of TCF7L2, which are differently regulated on the post-transcriptional level and could encode less active isoforms with different effects on β-cell function and survival. Indeed, several alternative spliced mRNA transcripts of TCF7L2 exist (17–22) and were identified in human β-cells in our present study.

The combination of alternative events in exons 13, 14, 15 and 16 produced different ORFs, which modified the reading frame in exon 17 and lead to three different C-terminal ends, resulting in three groups of TCF7L2 (TCF4L, TCF4M and TCF4S). For all three groups, we have analyzed β-cell survival and function and demonstrate differential effects of the transcripts on β-cell function and survival. Clone B3, one transcript of the TCF4S group, possessed potent protective activity on β-cell function and survival by strongly promoting glucose and GLP-1-stimulated insulin secretion and by increasing β-cell proliferation. This finding is consistent with a recent publication, which reported that the TCF4S group includes functional isoforms for wnt signaling activation (21). In contrast, B1 transcript overexpression induced β-cell apoptosis and impaired GLP-1-stimulated insulin secretion. In line with our data, Weise et al. identified the same TCF7L2 ‘B1' transcript (called ‘M1'), which had a decreased activation of Wnt/β-catenin target gene promoters. Also the transcript B7, which consists of the same sequence as the previously used TCF7L2 plasmid obtained from the mammalian gene collection (12), has protective effects on β-cell survival. The fact that transcripts B1 (‘anti-survival') and B7 (‘pro-survival') have the same C-terminal end suggests that not the C-terminal end in exon 17 but rather the existence of exon 13 is important for functional regulation. Both ‘pro-survival' transcripts B3 and B7 contain exon 13. In contrast, transcript B1 (‘anti-survival') lacks of exon 13.

Recent reports investigated the potential association of TCF7L2 SNPs and alternative splicing events that may explain the correlation of TCF7L2 modification with the progression of T2DM (18–20,22). The approach used in these previous studies (i.e. quantitative PCR) was not able to distinguish the expression of specific protein isoforms and map the full structure of their respective transcript. The important question remains, whether these studies detected a specific up- or down-regulation of our splice variants B1, B3 and B7, which had distinct effects on β-cell survival?

First, the B3 transcript, which showed proliferative and insulin-secretory phenotypes, is included in the ‘ex13–14', ‘ex11–13', ‘ex12–13' and ‘ex13–13a' expression assays of previous studies (20,22) and positively correlated with insulin mRNA. Secondly, the B1 transcript, which contains exon4 and showed a pro-apoptotic phenotype, is included in the exon4/HbA1c correlation of the Osmark study (18). From these results, one could speculate that the increased TCF7L2 mRNA expression documented in T2DM may reflect an imbalance between the expression of B1 and B3 transcripts, and that increased B1 is present at the expense of B3 and B7. In this case, upregulation of B1 would lead to β-cell apoptosis and downregulation of B3 and B7 transcripts to impaired β-cell proliferation and insulin secretion, ultimately leading to β-cell failure and diabetes. Our results showing B1 highly expressed at the mRNA level, but almost undetectable at the protein level whereas B3 and B7 clones are highly expressed at the protein level with a reduced mRNA level when compared with B1 are in agreement with this hypothesis. An extensive analysis of isolated islets obtained from controls and from patients with diabetes to identify such changes in expression is currently performed in our laboratory.

An imbalance between TCF7L2 gene splicing isoforms with long and short reading frames has already been observed in renal cell carcinoma cells, where it is associated with cancer progression through the inhibition of the apoptotic pathway (23). Also in colorectal cancer cells, a switch in expression levels of specific TCF7L2 isoforms was induced by a frequent TCF7L2 frame-shift mutation (36).

In parallel with the effects on β-cell function and survival, the B3 transcript possessed the ability to promote GLP-1R expression and stimulate Akt activation, β-catenin nuclear translocation and Foxo-1 nuclear exclusion. In contrast, pro-apoptotic B1 increased Foxo-1 nuclear accumulation and did not affect β-catenin nuclear translocation, assuming that the transcripts distinctly influence Wnt signals. This was also confirmed by effects on downstream signals, specifically the decrease in menin and the increase in survivin by B3.

The control of proliferation by menin may occur through suppression of Wnt/β-catenin signaling (37). Conversely, another report showed that menin promotes the wnt/β-catenin signaling pathway in pancreatic endocrine cells (38). In the present study, downregulation of menin was induced by the ‘pro-survival' B3 and B9 transcripts, which also activated Wnt signals. This is in favor of the effect of menin-induced Wnt suppression and supports menin as a TCF7L2 target gene.

Taken together, we demonstrate the existence of function-specific transcripts of TCF7L2, which possessed distinct effects on β-cell function and survival. Our data provide new insights into mechanisms of TCF7L2 genetic variants regulating β-cell function and turnover.

MATERIAL AND METHODS

Islet isolation and cell culture

Human islets were isolated from pancreata of five healthy organ donors at the University of Illinois at Chicago and from INSERM /Université de Lille, Thérapie Cellulaire du Diabète and cultured as described previously (39–41). For long-term in vitro studies, islets were cultured on extracellular matrix-coated plates derived from bovine corneal endothelial cells (Novamed Ltd., Jerusalem, Israel), allowing the cells to attach to the dishes and spread, preserving their functional integrity (42,43). Human insulinoma CM cell line [kindly provided by Dr Paolo Pozzilli, Barts and the London School of Medicine, Queen Mary, University of London, UK (44)] was cultured in RPMI-1640, and the MIN6 mouse insulinoma cell line was grown in DMEM 25 mm glucose medium.

RNA isolation and cloning of TCF7L2 transcript variants, generation of TCF7L2 expression vectors

Human β-cells were sorted by FACS analysis of semi-purified preparations of islet cells using Newport Green, a specific zinc-fluorescent probe (41). Total RNA was extracted using Nucleospin RNA II kit (Macherey Nagel) and treated with DNase 1 (Ambion, Austin, TE, USA) to ensure residual genomic contamination was removed. cDNA from two different subjects was pulled and amplified by standard PCR using the Fast Start Taq (Roche Applied Science). Cloning primers were designed to contain NheI and XhoI restriction sites for subsequent subcloning of amplified TCF7L2 cDNA into pcDNA3 expression vector. We used the following primers located into 3′- and 5′-untranslated region of the human TCF7L2 gene: TCF7L2-NheI forward, 5′-GCCGCTAGCATATCCCCTCCCCTCCTCCCTCT-3′ and TCF7L2-Who1 reverse, 5′-TCACTCGAGGGCACAAAGCAGCGGGGTTCACG-3′. Subsequent cloning of PCR products into the pCR4-TOPO vector was performed using the TOPO TA cloning kit for sequencing (Invitrogen). To generate expression vector of TCF7L2 splice variants, TCF7L2-containing pCR4-TOPO were double digested with NheI and XhoI restriction enzymes and the TCF7L2 corresponding band was subcloned into the pcDNA3 expression vector. Full sequence analysis was then carried out using the BigDye cycle sequencing kit (Applied Biosystems, CA, USA).

RT–PCR analysis of TCF7L2 splicing variants in human tissues

We used commercial cDNAs from the Human MTC panel I for muscle and liver (BD Biosciences Clontech) and RNAs that were reverse transcribed from the pancreas and adipose tissue (Human Adult Normal 5 Donor Pool, BioChain Institute). TCF7L2 exon-specific primers were used in the PCR (see Supplementary Material).

RNA interference and plasmid transfection

Isolated human islets or CM cell lines were exposed to transfection Ca2+-KRH medium (KCl 4.74 mm, KH2PO4 1.19 mm, MgCl26H2O 1.19 mm, NaCl 119 mm, CaCl2 2.54 mm, NaHCO3 25 mm, HEPES 10 mm). After 1 h incubation, lipoplexes [Lipofectamine 2000 (Invitrogen)/DNA ratio 2.5:1, 3 μg DNA of TCF7L2 plasmid (full-length TCF7L2, from the Full-Length Mammalian Gene Collection, Invitrogen), TCF7L2 splicing isoforms plasmids B1, B2, B3, B4, B5 and B6 or a pCMV-GFP control plasmid. or 50 nm siRNA to TCF7L2 (RNAs of 21 nucleotides, designed to target human TCF7L2; Stealth Select™ RNAi, Invitrogen) and scramble siRNA (Ambion)] were added to transfect the islets. Efficient transfection was evaluated based on eGFP-positive cells at control conditions, which resulted in 70% transfection efficiency in β-cells through the whole islets, analyzed by confocal microscopy. TCF7L2 plasmids were not fused to GFP, to avoid a distinct effect of the plasmids due to differences in overexpression, each condition was performed in triplicates and at least three independent experiments were performed, which gave similar results.

Nuclear fractionation

Nuclear and cytoplasmic extractions of CM cell lines were performed according to the instructions of NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA). The purity of fractions was analyzed by incubation of the membranes with anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for cytosolic and anti-PARP for nuclear extracts. For western blot antibodies, see Supplementary Material.

β-cell apoptosis, proliferation and function in human islets

After washing with PBS, 30 islets plated on ECM-coated dishes were fixed with 4% paraformaldehyde followed by permeabilization with 0.5% Triton X-100. Apoptosis was analyzed by the TUNEL technique and β-cell proliferation by an anti-human Ki67 antibody as described before (40). For β-cell function, glucose, GLP-1 and GIP-stimulated insulin secretion were analyzed as described before from 30 islets per dish (13).

RT–PCR

RNA was isolated from 100 islets per dish. For quantitative analysis, we used the Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems) with a commercial kit (Power SYBR Green PCR Master Mix; Applied Biosystems). For primer sequences, please see Supplementary Material.

Statistical analysis

Samples were evaluated in a randomized manner by a single investigator (L.S.) who was blinded to the treatment conditions. Data are presented as means ± SE and were analyzed by paired Student's t-test or by analysis of variance with a Bonferroni correction for multiple group comparisons.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

This work was supported by the European Foundation for the Study of Diabetes (EFSD), the Chinese Diabetes Society (CDS) and Lilly, the University of Bremen Postdoctoral Research Program (to L.S.), the French National Research Agency (ANR-06-BLAN-0236 to P.F.), the German Research Foundation (DFG, Emmy Noether Program MA4172/1-1) and the ERC award #260336. Human islets were provided through the JDRF award 31-2008-413 (ECIT Islet for Basic Research program). Human islets were provided through the JDRF award 31-2008-413 (ECIT Islet for Basic Research program). CM9 cells were kindly provided by Prof. Paolo Pozzilli, Barts and the London School of Medicine, Queen Mary, University of London, UK.

ACKNOWLEDGEMENTS

We thank Jennifer Bergemann and Dr Anke Meier for excellent technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest statement. None declared.

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Author notes

L.S. and O.L.B. contributed equally to the study.