The expression of dominant negative TCF7L2 in pancreatic beta cells during the embryonic stage causes impaired glucose homeostasis

Abstract

Objective: Disruption of TCF7L2 in mouse pancreatic β-cells has generated different outcomes in several investigations. Here we aim to clarify role of β-cell TCF7L2 and Wnt signaling using a functional-knockdown approach.

Methods: Adenovirus-mediated dominant negative TCF7L2 (TCF7L2DN) expression was conducted in Ins-1 cells. The fusion gene in which TCF7L2DN expression is driven by P TRE3G was utilized to generate the transgenic mouse line TCF7L2DN Tet . The double transgenic line was created by mating TCF7L2DN Tet with Ins2-rtTA, designated as βTCFDN. β-cell specific TCF7L2DN expression was induced in βTCFDN by doxycycline feeding.

Results: TCF7L2DN expression in Ins-1 cells reduced GSIS, cell proliferation and expression of a battery of genes including incretin receptors and β-cell transcription factors. Inducing TCF7L2DN expression in βTCFDN during adulthood or immediately after weaning generated no or very modest metabolic defect, while its expression during embryonic development by doxycycline feeding in pregnant mothers resulted in significant glucose intolerance associated with altered β-cell gene expression and reduced β-cell mass.

Conclusions: Our observations support a cell autonomous role for TCF7L2 in pancreatic β-cells suggested by most, though not all, investigations. βTCFDN is a novel model for further exploring the role of TCF7L2 in β-cell genesis and metabolic homeostasis.

Keywords: GLP-1; GSIS; Incretin receptors; Pancreatic β-cells; TCF7L2; TCF7L2DN; Wnt.

Figure 1

TCF7L2DN expression in the Ins-1 cell line attenuates GSIS, inhibits cell growth and represses the expression of a battery of β-cell specific genes. A) A schematic of TCF7L2DN (75 kDa) and wild type TCF7L2 (78 kDa). β-cat, the β-cat interaction domain; HMG, the HMG-box DNA binding domain. B) Detection of TCF7L2 and TCF7L2DN in virus infected Ins-1 cells by Western blotting. GFP, the control virus. C) The expression of TCF7L2DN but not wild type TCF7L2 inhibited GSIS. LG, low glucose (2.8 mM), HG, high glucose (16.7 mM). Ad-GFP, the control virus. n = 4 for each virus infection. D) TCF7L2DN expression inhibited Ins-1 cell growth, assessed by MTT. n = 8. E) qPCR assessment shows that TCF7L2DN suppressed the expression of a panel of β-cell specific genes.

Figure 2

Impaired glucose homeostasis in βTCFDN offspring when the pregnant mothers were fed with doxycycline to induce TCF7L2DN expression. A) Body weight comparison. B–C) Impaired response to OGTT (B) but normal response to IPITT (C). D–E) Increased blood glucose levels after oral glucose gavage D) and attenuated plasma insulin levels after glucose gavage in βTCFDN mice. n = 4 to 5 for panels A–E.

Figure 3

Reduced β-cell mass, Pdx-1 and Nkx6.1 positive islet cells in βTCFDN offspring when pregnant mothers were fed with doxycycline to induce TCF7L2DN expression. A–B) Assessment of β-cell mass (A) and α-cell (B) mass. n = 3. C–D) Immunostaining shows reduced percentage of Pdx-1 positive islet cells in βTCFDN mice. Immunostaining shows reduced percentage of Nkx6.1 positive islet cells in βTCFDN mice. The percentages were determined by counting 2,200–3,000 islet cells from βTCFDN mice and the Ins2-rtTA control mice (n = 3 for each group).

Figure 4

βTCFDN mouse islets show reduced β-cell specific gene expression and attenuated response to GLP-1 treatment. A) qPCR assessment for β-cell specific gene expression. n = 3 for both βTCFDN and control ins2-rtTA mice. B) Assessment of insulin secretion by GLP-1 treatment in isolated βTCFDN mouse islets. n = 3 for both βTCFDN and control ins2-rtTA mice.