Tissue-specific deletion of Foxa2 in pancreatic beta cells results in hyperinsulinemic hypoglycemia

Abstract

We have used conditional gene ablation to uncover a dramatic and unpredicted role for the winged-helix transcription factor Foxa2 (formerly HNF-3 beta) in pancreatic beta-cell differentiation and metabolism. Mice that lack Foxa2 specifically in beta cells (Foxa2(loxP/loxP); Ins.Cre mice) are severely hypoglycemic and show dysregulated insulin secretion in response to both glucose and amino acids. This inappropriate hypersecretion of insulin in the face of profound hypoglycemia mimics pathophysiological and molecular aspects of familial hyperinsulinism. We have identified the two subunits of the beta-cell ATP-sensitive K(+) channel (K(ATP)), the most frequently mutated genes linked to familial hyperinsulinism, as novel Foxa2 targets in islets. The Foxa2(loxP/loxP); Ins.Cre mice will serve as a unique model to investigate the regulation of insulin secretion by the beta cell and suggest the human FOXA2 as a candidate gene for familial hyperinsulinism.

Figure 1

The Foxa2^loxP allele is efficiently and specifically deleted in pancreatic β cells by the Ins.Cre transgene. Immunofluourescence analysis of pancreas sections from mice at 16.5 d post coitum (a,b), postnatal day 8 (P8; c,d,g,h), and P22 (e,f), double-labeled for either Foxa2 (red) and insulin (green; a–f) or Foxa2 (red) and glucagon (green; g,h). Foxa2 is normally expressed in all islet cell types, including β cells and α cells, and in some acinar cells (a,c,e,g). (b) Foxa2 is inactivated in β cells as early as embryonic day 16.5 (E16.5) in Foxa2^loxP/loxP; Ins.Cre mice. Cre-mediated deletion of Foxa2 occurs in 85% of β cells in P8 mice (d) and in more than 99% of β cells in P22 mice (f). Arrows (b,d,f) show β cells that have Foxa2 deleted, whereas arrowheads (b,d) show β cells in which Foxa2 protein persists. However, Foxa2 is not deleted in either α cells (h) or acinar cells in Foxa2^loxP/loxP; Ins.Cre mice, indicating the specificity of the Ins.Cre transgene. Images were obtained at 400× magnification using fluorescent confocal microscopy of representative pancreatic sections from wild-type control (Control) mice (a,c,e,g) and mutant Foxa2^loxP/loxP; Ins.Cre mice (b,d,f,h).

Figure 2

Foxa2^loxP/loxP; Ins.Cre mice are growth-retarded and die shortly after birth because of severe hyperinsulinemic hypoglycemia. (a) Eight-day-old (P8) Foxa2^loxP/loxP; Ins.Cre mutant mouse (M; top) and a control littermate (WT; bottom) from a typical litter between Foxa2^loxP/+; Ins.Cre and Foxa2^loxP/loxP mice on a mixed outbred-CD1 background. (b) Growth curve for a litter obtained from mating Foxa2^loxP/+; Ins.Cre and Foxa2^loxP/loxP mice. Foxa2^loxP/loxP; Ins.Cre mutant mice (filled diamond) are growth retarded relative to control littermates (open square). Between P9 and P12, Foxa2^loxP/loxP; Ins.Cre mutant mice lose weight rapidly, and most die with severe hypoglycemia. Values are means ± SEM of n = 5 mice. (c–e,h) Offspring from matings between Foxa2^loxP/+; Ins.Cre and Foxa2^loxP/loxP mice were killed on P8 and blood glucose (c), plasma insulin (d), plasma glucagon (e), and plasma nonesterified (NE) fatty acid (h) concentrations were determined as described in Materials and Methods. Each bar represents the mean ± SEM, with the number indicated in parenthesis. **, P < 0.005; Foxa2^loxP/loxP; Ins.Cre (cross-hatched bars) vs. Foxa2^loxP/+ (stippled bars) or Foxa2^loxP/loxP (horizontal-striped bars) or Foxa2^loxP/+; Ins.Cre (hatched bars). (f) The insulin to glucagon ratio was determined by dividing mean plasma insulin concentrations (d) by mean plasma glucagon concentrations (e). (g) Liver glycogen content was determined in P8 Foxa2^loxP/loxP; Ins.Cre mutant mice (cross-hatched bars) and control littermates (unfilled bars). Each bar represents the mean ± SEM, with the number indicated in parenthesis. **, P < 0.005; Foxa2^loxP/loxP; Ins.Cre vs. all control littermates.

Figure 3

Foxa2^loxP/loxP; Ins.Cre mice show perturbed islet architecture, but islet cell markers are expressed normally. Pancreatic sections from control (a,c) and Foxa2^loxP/loxP; Ins.Cre (b,d) mice were immunostained for two different islet cell lineage markers, and images were captured by confocal microscopy. The markers were insulin for β cells (a–d), glucagon for α cells (a,b), and somatostatin for δ cells (c,d). β Cells normally form the core of the islet with non-β cells positioned on the periphery of the islet, as seen in control mice (a,c). In contrast, islets from Foxa2^loxP/loxP; Ins.Cre mice have non-β cells intermingled in the core of the islet, known as a mixed islet phenotype (b,d). Insulin is not co-expressed with either the α-cell marker (a,b) or the δ-cell marker (c,d) in control or Foxa2^loxP/loxP; Ins.Cre islets. Magnification, 400×.

Figure 4

Quantitative analysis of islet cell area and biosynthesis of insulin and glucagon in Foxa2^loxP/loxP; Ins.Cre mice. (a,b) Pancreatic sections from eight-day-old control (P8; unfilled bars) and Foxa2^loxP/loxP; Ins.Cre (Mutant, cross-hatched bars) mice were immunostained for both insulin and glucagon to determine β-cell area (a) and α-cell area (b), respectively, which were normalized to total pancreatic area (pancreas area). Bars represent means ± SEM, with number indicated in parenthesis. *, P < 0.05; Foxa2^loxP/loxP; Ins.Cre vs. control mice. (c) Reverse-transcription analysis of total RNA isolated from whole pancreata of P8 control and Foxa2^loxP/loxP; Ins.Cre mice. No significant differences between control and mutant mice in insulin 1 (Insulin) or glucagon steady-state mRNA levels were detected when quantified using phosphorImager analysis (data not shown). α-Tubulin served as a loading control. (d,e) Pancreatic insulin (d) and glucagon content (e) were measured in acid-ethanol extracts from P8 control (unfilled bars) and Foxa2^loxP/loxP; Ins.Cre (Mutant, cross-hatched bars) mice by RIA. Bars represent values ±SEM, with number indicated in parenthesis.

Figure 5

Regulation of hormone secretion is defective in Foxa2^loxP/loxP; Ins.Cre mice. All perifusion studies were performed at least three times and representative trace is shown for each experiment. (a) Pancreata from two postnatal day 8 (P8) controls (open squares) and Foxa2^loxP/loxP; Ins.Cre (Mutant, filled diamonds) mice were perifused as described in Materials and Methods with increasing concentrations of glucose from 0 to 15 mM glucose at a rate of 1 mM per minute. Fractions were taken every minute, and the insulin concentration was determined by RIA. (b) Continuation of experiment in a with a glucose ramp decreasing from 26 to 0 mM glucose, changing the glucose concentration at a rate of 1 mM per minute (represented by triangle). At the end of the experiment, 15 mM potassium chloride (KCl) was added to the perifusate, represented by the bar. Fractions were taken every minute, and insulin concentrations were determined by RIA. (c,d) Pancreata from two P8 control (open squares) and Foxa2^loxP/loxP; Ins.Cre (filled diamonds) mice were perifused as described in Experimental Procedures with addition of secretagogue, 15 mM amino acid mixture (AAM) plus 2 mM glutamine or 15 mM KCl (KCl) as indicated. Fractions were taken every minute, and insulin (c) and glucagon (d) concentrations were determined by RIA. c and d represent hormone concentrations of fractions from the same perifusion experiment.

Figure 6

Foxa2 is required in β cells for K_ATP-channel steady-state mRNA expression. Reverse-transcription PCR analysis of Kir6.2 (a) and SUR1 (b) mRNA levels in islet RNA from control (unfilled bars) and Foxa2^loxP/loxP; Ins.Cre (cross-hatched bars) mice, which were normalized to those of HPRT. Radioactive bands were quantified by phosphorimager analysis. Bars represent values ± SEM, with number indicated in parenthesis. *, P < 0.05; Foxa2^loxP/loxP; Ins.Cre vs. control mice.