Decreased TCF7L2 protein levels in type 2 diabetes mellitus correlate with downregulation of GIP- and GLP-1 receptors and impaired beta-cell function

Abstract

Recent human genetics studies have revealed that common variants of the TCF7L2 (T-cell factor 7-like 2, formerly known as TCF4) gene are strongly associated with type 2 diabetes mellitus (T2DM). We have shown that TCF7L2 expression in the beta-cells is correlated with function and survival of the insulin-producing pancreatic beta-cell. In order to understand how variations in TCF7L2 influence diabetes progression, we investigated its mechanism of action in the beta-cell. We show robust differences in TCF7L2 expression between healthy controls and models of T2DM. While mRNA levels were approximately 2-fold increased in isolated islets from the diabetic db/db mouse, the Vancouver Diabetic Fatty (VDF) Zucker rat and the high fat/high sucrose diet-treated mouse compared with the non-diabetic controls, protein levels were decreased. A similar decrease was observed in pancreatic sections from patients with T2DM. In parallel, expression of the receptors for glucagon-like peptide 1 (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP-R) was decreased in islets from humans with T2DM as well as in isolated human islets treated with siRNA to TCF7L2 (siTCF7L2). Also, insulin secretion stimulated by glucose, GLP-1 and GIP, but not KCl or cyclic adenosine monophosphate (cAMP) was impaired in siTCF7L2-treated isolated human islets. Loss of TCF7L2 resulted in decreased GLP-1 and GIP-stimulated AKT phosphorylation, and AKT-mediated Foxo-1 phosphorylation and nuclear exclusion. Our findings suggest that beta-cell function and survival are regulated through an interplay between TCF7L2 and GLP-1R/GIP-R expression and signaling in T2DM.

Figure 1.

Reciprocal changes in TCF7L2 mRNA and protein in human and rodent type 2 diabetes mellitus. (A) TCF7L2 protein expression assay in human pancreatic sections. Triple staining for TCF7L2 in red, insulin in green and DAPI in blue in human pancreatic sections from seven poorly controlled T2DM patients and seven healthy controls. (B–E) C57Bl/6J mice were fed a ND (normal diet) or HFD (high-fat/high sucrose diet) and intraperitoneal glucose tolerance tests (IPGTTs) were performed after 12 weeks of diet, their islets isolated and protein and mRNA extracted. (F–I) Eight-week-old db/db and their heterozygous db/+ littermates and (J–M) 8–10-week-old Vancouver diabetic fatty (VDF) Zucker rats (fa/fa) and their lean littermates (Fa/?) were used for the experiments. (E, I) Intraperitoneal or oral (M) glucose tolerance tests (IPGTT/OGTT) were performed after 12 h fast with 1 g/kg body weight glucose. Tests were performed in four independent experiments with a total of 16 mice in each group. *P < 0.01 to control animals at all time points during the IPGTT/OGTT. (B–D,F–H,J–L) Islets were isolated from 16 mice each group and western blot (B,C,F,G,J,K) and quantitative RT-PCR (D,H,L) analyses of TCF7L2 protein and mRNA were performed in four independent experiments. (C,G,K) show densitometric analyses of western blot results, normalized to GAPDH and expressed as change of control. The levels of TCF7L2 mRNA expression were normalized against β-actin and tubulin with similar results and shown as change from control. Results are means ± SE. *P < 0.05 to non-diabetic control animals.

Figure 2.

Depletion of TCF7L2 results in decreased GLP-1R and GIP-R expression in human islets. (A) Triple staining for GLP-1R (left)/GIPR (right; red), insulin in blue and glucagon in green in human pancreatic sections from seven poorly controlled type 2 diabetic patients (T2DM; lower panel) and seven healthy controls (upper panel). (B–H) Human islets transfected with scrambled control (siScr) or TCF7L2 siRNA (siTCF7L2) or with pCMV-TCF7L2 or an empty control plasmid under the same promoter, were cultured on extracellular matrix-coated dishes for 4 days and exposed to increasing glucose concentrations (5.5, 33.3 mm) or the mixture of 2 ng/ml IL-1β+1000 U/ml IFN-γ (ILIF). Islet lysates were collected for western blot analysis of GLP-1R (B,C) and GIPR (D,E) and TCF7L2 (F,G). (C,E,G) show densitometric analyses of western blot results, normalized to actin and expressed as change of control ± SE. Western blots are representative of three (B–E) or seven (F–H) independent experiments from three/seven different organ donors. (H) Quantitative mRNA analysis of the downregulation of TCF7L2 achieved by the siRNA (left panel) and upregulation achieved by pCMV-TCF7L2 (right panel). *P < 0.05 to siScr-treated islets, +P < 0.05 to islets transfected with empty control vector at the same treatment conditions.

Figure 3.

TCF7L2 is important for regulation of AKT activation and FOXO1 trafficking. Isolated human pancreatic islets were cultured on extracellular matrix-coated dishes and exposed to siTCF7L2 or scrambled control siRNA (siScr) for 4 days with or without exposure to 100 nm GLP-1 or 10 nm GIP for the whole 4-day culture period (B–F) or for the last 4 h of prior to addition of 2 ng/ml IL-1β+1000 U/ml IFN-γ (ILIF) for the last 2 h of the experiment (G,H). Western blot analysis for p-AKT, Ser473 (A–H) or p-FOXO1 (I,J) was performed, actin was used as loading control on the same membrane after stripping. (B,D,F,H,J) show densitometric analyses of western blot results, normalized to actin and expressed as change of control ± SE. (K) Fixed islets were triple-stained for Foxo1 in red, DAPI in blue and insulin in green. (L,M) FOXO1 localization is shown in treated islets by western blot analysis after extraction of nuclei and cytoplasm. (M) shows densitometric analyses of the western blots of cytosolic FOXO1 normalized to nuclear FOXO1 and expressed as change of control ± SE. Western blots are representative of three independent experiments from three different organ donors. *P < 0.05 to siScr-transfected islets at the same treatment; **P < 0.05 to siScr-transfected control islets.

Figure 4.

Depletion of TCF7L2 results in impaired β-cell function through suppression of GLP-1 and GIP-stimulated insulin secretion. Human pancreatic islets were cultured on extracellular matrix-coated dishes and exposed to siRNA sequences to TCF7L2 (siTCF7L2) or scrambled control siRNA (siScr) for 4 days. Basal and stimulated insulin secretion indicate the amount secreted during 1-h incubations at 2.8, 16.7 mm glucose, 16.7 mm plus 100 nm GLP-1 (A) or 16.7 mm plus 10 nm GIP (C), following the 4-day culture period, normalized to whole islet insulin content. (B,D) Stimulatory index which denotes the amount stimulated divided by the amount of basal insulin secretion. (E) Human islets transfected with scrambled control (siScr) or TCF7L2 siRNA (siTCF7L2) were placed in perifusion chambers in aliquots of 20 islets of equal size, respectively. The perifusate contained 2.8 mm glucose for 1 h (measured for the last 30 min), 16.7 mm glucose for 30 min, 16.7 mm glucose plus 10 nm GIP for 30 min, 2.8 mm glucose for 30 min (the effluent was collected in 5-min intervals) and 10 min 20 mm KCl (the effluent was collected in 2-min intervals). Insulin secretion is expressed as percentage of content. Experiments were performed in triplicate. (F,G) For KCl and IBMX/Forskolin-stimulated insulin secretion analysis, basal and stimulated insulin secretion indicate the amount of secreted insulin during 1-h incubations at 2.8 mm glucose (basal), and 20 mm KCl or IBMX (100 µm)/Forskolin (10 µm) following the 4-day culture period, normalized to whole islet insulin content. (G) Stimulatory index which denotes the amount of KCl or IBMX/Forskolin-induced insulin secretion divided by the amount of basal insulin secretion. Data represent results of three different experiments from three different organ donors in triplicate. Results are means ± SE. *P < 0.05 siTCF7L2 versus siScr; +P < 0.05 16.7 versus 2.8 mm glucose; **P < 0.05 GLP-1/GIP+16.7 versus 16.7 mm glucose; #P < 0.05 KCl or IBMX/Forskolin versus 2.8 mm glucose alone. (H) Transfected human islets were placed in perifusion chambers in aliquots of 20 islets of equal size. The perifusate contained 2.8 mm glucose for 1 h, 16.7 mm glucose for 30 min (not shown) and 16.7 mm glucose plus 100 nm GLP for 30 min (shown here). The effluent was collected in 1-min intervals. Data represent results from one representative experiment in six different perifusion chambers out of three independent experiments from three organ donors and show that TCF7L2 depletion leads to a decreased mass, but not frequency of insulin secretory bursts.