Abnormal glucose tolerance and insulin secretion in pancreas-specific Tcf7l2-null mice

Abstract

Aims/hypothesis: Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis.

Methods: Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols.

Results: From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p < 0.01) or to 17 mmol/l glucose plus 100 nmol/l GLP-1 (44.3 ± 4.9%, p < 0.01) compared with control islets. Glp1r (42 ± 0.08%, p < 0.01) and Ins2 (15.4 ± 4.6%, p < 0.01) expression was significantly lower in pTcf7l2 islets than in controls. Maintained on a high-fat (but not on a normal) diet, pTcf7l2 mice displayed decreased expansion of pancreatic beta cell volume vs control littermates. No differences were observed in plasma insulin, proinsulin, glucagon or GLP-1 concentrations.

Conclusions/interpretation: Selective deletion of Tcf7l2 in the pancreas replicates key aspects of the altered glucose homeostasis in human carriers of TCF7L2 risk alleles, indicating the direct role of this factor in controlling beta cell function.

Figure 1. Generation of pancreas-specific Tcf7l2 null mice.

a) Schematic showing wild-type (top) and conditional inactivation (bottom) of the Tcf7l2 allelle with binding sites for primers used for genotyping (dotted lines). Black boxes represent exons with exon number indicated in white. b) Immunohistochemical analysis of pancreata for Tcf7l2 and insulin. Scale bar = 20 micrometre. c) Real time PCR analysis of gene expression in islets of pTcf7l2 mice (black bar) and wild-type littermate control (dotted/grey bar). Tcf7l2 gene expression was undetected (UD; Ct >40) in pTcf7l2 mice. Ccnd1, cyclin D1 gene.

Figure 2. pTcf7l2 null mice display normal intraperitoneal glucose tolerance.

Male pTcf7l2 mice (grey lines or bars, n=7) and wild-type littermate control (black lines or bars, n=3) were starved for 16 h prior to intraperitoneal glucose tolerance test (1g/kg) at 8 (a, e), 12 (b, f), 16 (c, g), and 20 (d, h) weeks. Time course (a, b, c, d) and area under the curve for the entire test period (e, f, g, h) are shown. *, p<0.05.

Figure 3. pTcf7l2 mice display impaired oral glucose tolerance.

Male pTcf7l2 mice (grey lines or bars, n=11, respectively) and wild-type littermate control (black lines or bars, n= 6 respectively) were subjected to oral glucose tolerance test at 8 (a, e), 12 (b, f), 16 (c, g), and 20 (d, h) weeks. Time course (a, b, c, d) and area under the curve for the entire test period (e, f, g, h) are shown. *, p<0.05.

Figure 4. pTcf7l2 islets display lowered glucose and GLP-1-stimulated insulin secretion.

a) Batches of six size-matched islets, isolated from 20 week old mice, that had been cultured for 10 days in 11 mmol/l glucose and then for 1 h at 3 mmol/l glucose were incubated at the glucose and GLP-1 concentrations indicated for 20 min, after which time the supernatant was collected. Islets were lysed in acid ethanol. Insulin in the islet lysate and supernatant were assayed in duplicate by radioimmunoassay. b) Real-time qPCR results from islets from pTcf7l2 mice (black bars) and wild-type littermate control (grey bars). Data are from 3 separate islet preparations, with measurements in duplicate. c) Plasma GLP-1 concentration was measured from pTcf7l2 mice (grey bar) and wild-type littermate control (black bar). d) Plasma insulin content was measured from 20 week old pTcf7l2 mice (grey line) and control littermates (black line) following glucose administration by intraperitoneal injection. e) Plasma glucagon content was measured from 20 week old pTcf7l2 mice (grey bar) and control littermates (black bar) following 16 h fast. Data are from at least four mice per genotype. *, p<0.05, **, p<0.01.

Figure 5. pTcf7l2 mice on normal diet have normal pancreatic and beta-cell volume.

a) Representative images from optical projection tomography of 16 week old pTcf7l2 mice and wild-type littermate controls. Insulin staining is shown in red. Scale bar = 650 micrmetre. b) Distribution of islet size in pancreata of of pTcf7l2 mice (black bar) and wild-type littermate control (grey bar). c) Relative beta-cell volume and d) total pancreatic volume of pTcf7l2 mice and wild-type littermate control. Cell and tissue volumes were obtained using Volocity™ (Improvision, Coventry) software. Data were obtained from four mice per genotype.

Figure 6. pTcf7l2 mice on high fat diet display decreased insulin sensitivity and impaired glucose tolerance.

a) Intraperitoneal glucose tolerance of pTcf7l2 mice and control littermates following 8 weeks on a high fat diet. Insulin sensitivity in 16 week old pTcf7l2 mice (grey line) and control littermates (black line) following 8 weeks on a high fat diet (b), or maintained on a normal diet (c) following intraperitoneal injection of insulin. Data are from at least six mice per genotype. *, p<0.05.

Figure 7. pTcf7l2 mice on high fat diet display impaired β-cell expansion.

a) Representative images from optical projection tomography of 16 week old pTcf7l2 mice (grey bar, n=3) and wild-type littermate controls (black bar, n=2) following 8 weeks on a high fat diet. Insulin staining is shown in red. Scale bar = 650 micrometre. b) Distribution of islet size in pancreata of of pTcf7l2 mice and wild-type littermate control. c) Relative beta-cell volume and d) total pancreatic volume of pTcf7l2 mice and wild-type littermate control. Cell and tissue volumes were obtained using Volocity™ software (see Fig. 5 legend). *, p<0.05.