MafA is a key regulator of glucose-stimulated insulin secretion

Abstract

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic beta cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.

FIG. 1.

Targeting strategy for mafA mutagenesis and LacZ expression in pancreatic islets in MafA mutant mice. (A) Schematic representation of the wild-type allele, the targeting construct, and the expected product of homologous recombination between them. (B) Southern blot of tail DNA showing NheI-cleaved DNA fragments corresponding to the wild-type (11 kbp) and targeted (16 kbp) mafA alleles. (C) β-Galactosidase expression (blue) in MafA^+/− pancreatic β cells. Immunostaining was performed with antiglucagon (green) and anti-insulin (red) antibodies. Nuclear β-galactosidase staining overlaps that of insulin-expressing cells (merged).

FIG. 2.

Development of diabetes mellitus in MafA^−/− mice. (A) Fasting blood glucose levels of offspring derived from intercrosses of MafA^+/− mice were determined using a semiautomated analyzer. Results represent the mean ± standard error of the mean. (B) Mean body weight ± standard error of the mean of offspring derived from intercrosses of MafA ^+/− mice at the indicated ages (weeks). Both sets of data are from 7 to 22 animals of each genotype. * indicates P < 0.05, while ** represents P < 0.01.

FIG. 3.

Glucose tolerance and arginine tolerance tests and their effects on insulin production. (A) Glucose tolerance tests (ipGTT) after intraperitoneal loading with 2 g d-glucose/kg were performed on 8-week-old male animals of the indicated genotypes following a 12-h fast. Each symbol represents the following: *, P < 0.05, MafA^−/− versus MafA^+/+; **, P < 0.01, MafA^−/− versus MafA^+/+; ***, P < 0.01, MafA^−/− versus MafA^+/−; #, P < 0.05, MafA^+/− versus MafA^+/+; ##, P < 0.01, MafA^+/− versus MafA^+/+. (B) Level of plasma insulin of each MafA genotype during ipGTT. **, P < 0.01, MafA ^−/− versus MafA^+/+; ***, P < 0.05, MafA^−/− versus MafA^+/−; #, P < 0.05, MafA^+/− versus MafA^+/+. (C) Level of plasma insulin after intraperitoneal arginine administration of each MafA genotype. *, P < 0.05, MafA^−/− versus MafA^+/+. (D) Insulin content of wild-type, MafA^+/−, and MafA^−/− mice. All data represent the mean values ± standard error for at least five male mice (8 to 14 weeks of age) of each genotype.

FIG. 4.

Insulin secretion from isolated pancreatic islets in vitro. Insulin secretion in response to the indicated secretagogues. Values are expressed in nanograms of insulin islet⁻¹ h⁻¹, as the mean ± standard error of the mean of at least three male mice (8 to 12 weeks of age) per genotype. * indicates P < 0.05, while ** represents P < 0.01.

FIG. 5.

Histological analysis of pancreatic islets. (A) Insulin (red) and glucagon (green) immunoreactivity in wild-type (+/+) and MafA homozygous mutant (−/−) mice at P1 or 12 weeks of age. Scale bar, 20 μm. (B and C) β-Cell/α-cell ratio of the pancreatic islets from mice of each genotype at P1 (B) and 12 weeks of age (C) (male mice). Pancreatic sections were double stained with anti-insulin and antiglucagon antibodies. Data are the mean β-cell/α-cell ratios ± standard error of the mean for at least three mice of each genotype. * indicates P < 0.05, while ** represents P < 0.01. (D) Morphometric analysis of islet diameter in pancreata from wild-type (+/+), heterozygous (+/−), and MafA homozygous mutant (−/−) 12-week-old male mice. Data represent the mean ratios ± standard error of at least three male mice of each genotype.

FIG. 6.

Comparison of gene expression in pancreatic islets. For quantitative analysis using competitive RT-PCR, pancreatic islets from 8-week-old male mice were used. The amount of each transcript was normalized to the amount of HPRT transcript. Data represent the mean ratios ± standard error of three mice of each genotype.