Arx-deficient mice lack glucagon-producing cells. A, C, and E, Immunostaining of glucagon, insulin, and somatostatin in 3- to 6-month-old control and Arx-deficient pancreas. Morphometric analysis indicated 99% reduction in the α-cell mass (B), 1.5-fold increase in the β-cell mass (D), and 2-fold increase in the δ-cell mass (F) in the Arx-deficient animals. Values are mean ± sem from three mice per group. *, P < 0.05 between genotypes.

Arx-deficient mice exhibit increased fat and glycogen in the liver. A, GIR HGP, and Rd were measured during the clamp from 2-month-old male control and Arx-deficient mice. Values are mean ± sem from five mice per group. *, P < 0.05 between genotypes. B, Liver histology of 3- to 6-month-old male control and Arx-deficient paraffin-embedded liver sections stained with H&E or (D) Periodic acid-Schiff (PAS) (magnification, ×40). An enlarged portion of the image is provided in the box. C, Glycogen levels were measured in liver taken from mice at 3–6 months of age after a 16-h fast. Values are mean ± sem from three mice per group. *, P < 0.05 between genotypes. E, Real-time PCR analysis measured relative mRNA levels of genes involved in gluconeogenesis from 3- to 6-month-old male after a 16-h fast. Tat, Tyrosine amino transferase. Values are mean ± sem from four mice per group. *, P < 0.05 between genotypes. F, Western blot analysis using liver samples of 3- to 6-month-old male control and Arx-deficient animals to detect PEPCK (63 kDa), P-CREB (43 kDa), and tubulin (50 kDa). G, Mean intensity of PEPCK and P-CREB normalized to tubulin as quantified from Western blot analysis.

Pdx1-Cre drives pancreas-specific deletion of the Arx gene. A, Representation of wild-type gene sequence (top) and the insertion of the LoxP sites flanking the exon 2 of the Arx gene (middle). Removal of exon 2 after crossing the Arx^fl/fl mice with Pdx1-Cre mice (bottom). B, PCR analysis from genomic DNA obtained from pancreas and liver of P2 control (C), heterozygote (H), and Arx-deficient mutant (M) animals. Presence of the PCR product (402 bp) indicates efficient ablation of the Arx gene in the pancreas but not in the liver. Arrows in A indicate the location of the primers used. C, Real-time PCR analysis of Arx mRNA levels in P7 control and mutant pancreas. Values are mean ± sem from three mice per group. *, P < 0.05.

Arx-deficient mice have no obvious abnormalities and exhibit improved glucose tolerance with age. A, Blood glucose levels of 6-wk-old male mice after 0-, 16-, or 24-h fast. Values are mean ± sem from at least three mice per group. *, P < 0.05 between genotypes. B, Blood glucose levels of 3- to 6-month-old male mice after 0-, 16-, or 24-h fast. Values are mean ± sem from at least three mice per group. *, P < 0.05 between genotypes. C and D, GTT test of 6-wk-old (C) and 3- to -6-month-old (D) male control and Arx-deficient mice. Values are mean ± sem from at least three mice per group. *, P < 0.05 between genotypes. E, Plasma glucagon and insulin levels of 3- to 6-month-old male mice after a 16-h fast. Values are mean ± sem from four mice per group. *, P < 0.05 and between genotypes; **, P < 0.01 between genotypes. Plasma glucagon levels fell below detection threshold (20 pg/ml) for Arx-deficient animals. F, Plasma insulin levels of 3- to 6-month-old male control and Arx-deficient animals was measured during GTT (see inset). Values are mean ± sem from at least three mice per group. *, P < 0.05 between genotypes.

Liver histology from 1-yr-old control and Arx-deficient mice. A, H&E and Oil red O staining of 1-yr-old control and Arx-deficient mice. B, Plasma lipid profile of 4-month-old control and Arx-deficient animals after a 16-h fast. C, Plasma epinephrine and norepinephrine levels of 3- to 6-month-old mice after a 16-h fast. D, Blood glucose levels measured during and after STZ treatment. Day 1–5 was measured after a 4-h fast, d 6 onwards was measured after a 1-h fast. Values are mean ± sem from at least three mice per group. *, P < 0.05 between genotypes.