Selective disruption of Tcf7l2 in the pancreatic β cell impairs secretory function and lowers β cell mass

Abstract

Type 2 diabetes (T2D) is characterized by β cell dysfunction and loss. Single nucleotide polymorphisms in the T-cell factor 7-like 2 (TCF7L2) gene, associated with T2D by genome-wide association studies, lead to impaired β cell function. While deletion of the homologous murine Tcf7l2 gene throughout the developing pancreas leads to impaired glucose tolerance, deletion in the β cell in adult mice reportedly has more modest effects. To inactivate Tcf7l2 highly selectively in β cells from the earliest expression of the Ins1 gene (∼E11.5) we have therefore used a Cre recombinase introduced at the Ins1 locus. Tcfl2(fl/fl)::Ins1Cre mice display impaired oral and intraperitoneal glucose tolerance by 8 and 16 weeks, respectively, and defective responses to the GLP-1 analogue liraglutide at 8 weeks. Tcfl2(fl/fl)::Ins1Cre islets displayed defective glucose- and GLP-1-stimulated insulin secretion and the expression of both the Ins2 (∼20%) and Glp1r (∼40%) genes were significantly reduced. Glucose- and GLP-1-induced intracellular free Ca(2+) increases, and connectivity between individual β cells, were both lowered by Tcf7l2 deletion in islets from mice maintained on a high (60%) fat diet. Finally, analysis by optical projection tomography revealed ∼30% decrease in β cell mass in pancreata from Tcfl2(fl/fl)::Ins1Cre mice. These data demonstrate that Tcf7l2 plays a cell autonomous role in the control of β cell function and mass, serving as an important regulator of gene expression and islet cell coordination. The possible relevance of these findings for the action of TCF7L2 polymorphisms associated with Type 2 diabetes in man is discussed.

Figure 1.

Glucose and insulin tolerance in Tcf7l2^fl/fl::Ins1.Cre⁺mice. Intraperitoneal glucose tolerance was assessed at 8 (A), 12 (B), 16 (C) and 20 (D) weeks of age in Tcf7l2^fl/fl::Ins1.Cre⁺(blue) and Tcf7l2^fl/fl::Ins1.Cre⁻ (black dotted) mice maintained on a normal chow diet (ND), and Tcf7l2^fl/fl::Ins1.Cre⁺(red) and Tcf7l2^fl/fl::Ins1.Cre⁻ (black) maintained on a high fat (HFD; 60%) diet. Glucose tolerance and area under the curve (inset) are shown. Oral glucose tolerance was assessed at 8 weeks in mice maintained on a normal chow diet (E). Intraperitoneal glucose (1 g/kg) tolerance and response to liraglutide (200 µg/kg) treatment (F) and insulin (0.75 units/kg) tolerance (G) was assessed at 20 weeks in mice maintained on a normal chow diet. (H) Fasting plasma glucagon concentration was measured on mice fed on a HFD (see Materials and Methods) n = 7–10 mice; *P ≤ 0.05.

Figure 2.

Tcf7l2 deletion impairs glucose and GLP1-stimulated insulin secretion from isolated islets. (A) Plasma insulin following intraperitoneal injection of glucose from 20-week-old mice that had been maintained on a high fat diet was measured as described in Materials and Methods. (B) Real-time quantitative PCR analysis was performed on islets from 20-weeks-old mice that had been maintained on a normal chow diet. n = 7–10 mice; *P ≤ 0.05. (C and D) Insulin secretion as assessed in isolated islets from 20-week old Tcf7l2^fl/fl::Ins1.Cre⁺(red) and littermate Tcf7l2^fl/fl::Ins1.Cre⁻ (black) mice on normal chow (C) or high fat diet (D). n = 5 mice per genotype; *P ≤ 0.05; **P ≤ 0.01.

Figure 3.

Impact of Tcf7l2 deletion on intra- and inter-cellular Ca²⁺ dynamics. (A and C) Responses to glucose (17 mm; G17) or GLP-1 (100 nm) of β cells within intact islets of Tcf7l2^fl/fl::Ins1.Cre⁺(red) and Tcf7l2^fl/fl::Ins1.Cre⁻ (black) mice maintained on a normal chow diet. (B and D) as for (A and C) but for islets from animals maintained for 12 weeks on a 60% fat diet. Correlation analysis (55,56) for islets from mice maintained on (E) normal diet and (F) high fat diet, and (G) quantitation of changes in typical islets from Tcf7l2^fl/fl::Ins1.Cre⁺ and Tcf7l2^fl/fl::Ins1.Cre⁻ mouse islets as indicated.

Figure 4.

Impact of Tcf7l2 deletion on β and α cell mass. (A) Representative 3D images from OPT (see Material and Methods) of Tcf7l2^fl/fl::Ins1.Cre⁺ and Tcf7l2^fl/fl::Ins1.Cre⁻ pancreata labelled with anti-insulin and anti-glucagon primary antibodies, and revealed using AlexaFluor 568 and 680, respectively. Right hand-most panels are overlaid with the autofluorescence signal revealing the pancreatic ductal system. Quantification of β (B), α (C) and (D) β/α cell mass from Tcf7l2^fl/fl::Ins1.Cre⁺(red) and Tcf7l2^fl/fl::Ins1.Cre⁻ (black) knockout mice pancreata from 20-week-old mice that had been maintained on high fat diet. Quantification was conducted using Volocity software (Invitrogen), N = 4 mice per genotype.