Uncovering the role of MAFB in glucagon production and secretion in pancreatic α-cells using a new α-cell-specific Mafb conditional knockout mouse model

Abstract

Cre/loxP is a site-specific recombination system extensively used to enable the conditional deletion or activation of target genes in a spatial- and/or temporal-specific manner. A number of pancreatic-specific Cre driver mouse lines have been broadly established for studying the development, function and pathology of pancreatic cells. However, only a few models are currently available for glucagon-producing α-cells. Disagreement exists over the role of the MAFB transcription factor in glucagon expression during postnatal life, which might be due to the lack of α-cell-specific Cre driver mice. In the present study, we established a novel Gcg-Cre knock-in mouse line with the Cre transgene expressed under the control of the preproglucagon (Gcg) promoter without disrupting the endogenous Gcg gene expression. Then, we applied this newly developed Gcg-Cre mouse line to generate a new α-cell-specific Mafb conditional knockout mouse model (Mafb^ΔGcg). Not only α-cell number but also glucagon production were significantly decreased in Mafb^ΔGcg mice compared to control littermates, suggesting an indispensable role of MAFB in both α-cell development and function. Taken together, our newly developed Gcg-Cre mouse line, which was successfully utilized to uncover the role of MAFB in α-cells, is a useful tool for genetic manipulation in pancreatic α-cells, providing a new platform for future studies in this field.

Keywords: Cre/loxP; MAFB; glucagon; pancreatic islet; α-cells.

Fig. 1.

Design of gene targeting for Gcg-Cre knock-in (KI) mice. (A) Schematic illustration of the WT allele, KI vector and KI allele of the Gcg gene. The 2A-NLS-Cre was inserted just before the stop codon of the Gcg locus. (B) The sequence shows the 23-nt CRISPR target sequence (5’-CCTCGTAGGAAATAGGTATTTCA-3’) containing a stop codon and protospacer adjacent motif (PAM). The 5’-homology arm ends at the final coding sequence of the Gcg gene, while the 3’-homology arm starts from the stop codon (TAG) of the Gcg gene.

Fig. 2.

Efficiency and specificity of Gcg-Cre recombination in the pancreatic α-cells of GRR/Gcg-Cre mice. (A) R26GRR mice show ubiquitous EGFP expression and no tdsRed expression without Cre recombination. EGFP is excised, and tdsRed is expressed in the adult pancreas of GRR/Gcg-Cre mice. (B) Colocalization of tdsRed signals with glucagon signals in the pancreatic islets of GRR/Gcg-Cre mice. Nuclei were counterstained with Hoechst 33342. Scale bars, 100 µm. (C) Fraction of glucagon-positive (Glu⁺) and glucagon-negative (Glu⁻) cells among tdsRed-positive (tdsRed⁺) cells in the pancreatic islets GRR/Gcg-Cre mice (n=3).

Fig. 3.

Gcg-Cre recombination in the intestine and brain of GRR/Gcg-Cre mice. Sections from the (A) duodenum, jejunum and ileum of the intestine and (B) the NST of brainstem of GRR/Gcg-Cre mice exhibit tdsRed signal. Nuclei were stained with Hoechst 33342. Scale bars, 100 µm.

Fig. 4.

Deletion of Mafb specifically in α-cells decreases the population of glucagon-positive cells and suppresses α-cell development. (A) Immunostaining of glucagon (green) and insulin (red) in Mafb^ΔGcg (n=3) and control (Mafb^f/f; n=3) pancreatic sections from mice at 6 months of age. Nuclei were counterstained with Hoechst 33342. (B and C) Fraction of glucagon-positive (Glu⁺) (B) and insulin-positive (Ins⁺) (C) cells within islets in Mafb^ΔGcg and control pancreatic sections. (D) Immunostaining of glucagon (green) and ARX (red) in pancreatic sections from Mafb^ΔGcg (n=3) and control (n=3) mice at 6 months of age. Nuclei were counterstained with Hoechst 33342. (E and F) Fraction of ARX-positive (ARX⁺) cells (E) and glucagon-positive α-cells among the total ARX-positive (ARX⁺/Glu⁺) cell population (F) within islets in Mafb^ΔGcg and control mice. Scale bars, 100 µm. ****, P<0.0001.

Fig. 5.

Arginine-induced glucagon stimulation test of Mafb^ΔGcg mice. Plasma glucagon levels were stimulated in overnight-fasted 8-week-old Mafb^ΔGcg (n=3) and control (n=4) mice following an intraperitoneal injection of 1 mg/ml l-arginine. *, P<0.05, ***, P<0.005.