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. 2004 Aug;114(4):512-20.
doi: 10.1172/JCI21149.

Foxa2 regulates multiple pathways of insulin secretion

Kristen A Lantz  1 , Marko Z Vatamaniuk, John E Brestelli, Joshua R Friedman, Franz M Matschinsky, Klaus H Kaestner
Affiliations

Foxa2 regulates multiple pathways of insulin secretion

Kristen A Lantz et al. J Clin Invest. 2004 Aug.

Abstract

The regulation of insulin secretion by pancreatic beta cells is perturbed in several diseases, including adult-onset (type 2) diabetes and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). The first mouse model for PHHI has a conditional deletion of the gene encoding the winged-helix transcription factor Foxa2 (Forkhead box a2, formerly Hepatocyte nuclear factor 3beta) in pancreatic beta cells. Using isolated islets, we found that Foxa2 deficiency resulted in excessive insulin release in response to amino acids and complete loss of glucose-stimulated insulin secretion. Most PHHI cases are associated with mutations in SUR1 (Sulfonylurea receptor 1) or KIR6.2 (Inward rectifier K(+) channel member 6.2), which encode the subunits of the ATP-sensitive K(+) channel, and RNA in situ hybridization of mutant mouse islets revealed that expression of both genes is Foxa2 dependent. We utilized expression profiling to identify additional targets of Foxa2. Strikingly, one of these genes, Hadhsc, encodes short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase, deficiency of which has been shown to cause PHHI in humans. Hadhsc is a direct target of Foxa2, as demonstrated by cotransfection as well as in vivo chromatin immunoprecipitation experiments using isolated islets. Thus, we have established Foxa2 as an essential activator of genes that function in multiple pathways governing insulin secretion.

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Figures

Figure 1
Figure 1
Sur1 and Kir6.2 mRNAs are undetected in Foxa2-deficient β cells. (A–H) RNA in situ hybridization using paraffin-embedded pancreatic sections and digoxigenin probes for Glucagon, Sur1, and Kir6.2. Glucagon is expressed in the α cells on the perimeter of both control islets (A and E) and mutant Foxa2loxP/loxP; Ins.Cre islets (C and G). Sur1 (B) and Kir6.2 (F) are expressed throughout control islets, but are confined to the α cells of Foxa2loxP/loxP;Ins.Cre islets (D and H). Note the similar staining pattern between Glucagon and both KATP subunits in mutant islets (C versus D and G versus H). Magnification, ×40 for all images.
Figure 2
Figure 2
Foxa2 activates the promoters of Sur1 and Kir6.2. (A) Cotransfection of a Foxa2 expression plasmid (pHD-Foxa2) results in stimulation of luciferase activity from Sur1 promoter constructs containing 1.8 kb [pGL3-Sur1(1.8)] and 7.0 kb [pGL3-Sur1(7.0)] of promoter sequence in BHK cells. (B) Cotransfection with pGL3-Kir6.2, containing the entire Kir6.2 promoter region, reveals Foxa2-dependent activation. For each condition, n = 3. *P ≤ 0.05 and ***P ≤ 0.0001 by ANOVA.
Figure 3
Figure 3
Foxa2loxP/loxP;Ins.Cre islets exhibit misregulated hormone secretion in response to glucose and amino acids. (A) Control islets (filled symbols) immediately secrete insulin in response to 25 mM glucose but do not respond to amino acids alone. In contrast, Foxa2loxP/loxP;Ins.Cre islets (open symbols) secrete insulin in a dose-dependent manner upon exposure to increasing concentrations of a mixture of all 20 amino acids (0.55 mM/min for 30 minutes up to 17 mM) followed by 20 minutes at 17 mM, with no additional response to 25 mM glucose. Data shown are mean ± SEM of 2 identical experiments. Similar responses were seen in 8 additional trials (data not shown). (B) Control islets do not respond to amino acid stimulation, but 300 nM glyburide closes KATP channels and leads to insulin secretion. As in A, Foxa2loxP/loxP;Ins.Cre islets respond to the identical amino acid ramp, but glyburide has no additional effect. Trace shown is representative of 2 similar experiments. (C) Control islets secrete glucagon in response to low concentrations of an amino acid ramp (0.55 mM/min for 30 minutes up to 17 mM), but Foxa2loxP/loxP;Ins.Cre islets do not. While the amino acid concentration is held constant at 17 mM, additional exposure to 25 mM glucose for 20 minutes has no effect on secretion from either control or mutant islets. Mean ± SEM of two identical experiments is shown. Downward arrows indicate times of reagent administration.
Figure 4
Figure 4
Quality and purity assessment of islet RNA for gene expression analysis. (A) RNA Nano 6000 Assay of P8 islet RNA pooled from 5 mutant Foxa2loxP/loxP;Ins.Cre mice using the Agilent 2100 Bioanalyzer. Islet RNA was quantified and evaluated for evidence of degradation using the ratio of 28S rRNA peaks to 18S rRNA peaks. In the representative sample shown, the 28S/18S ratio is approximately 2.5 with a concentration of 79 ng/μl and no evidence of degradation. (B) Real-time PCR analysis of total pancreas cDNA for endocrine and exocrine content using insulin (open circles) and amylase (filled squares) as markers and TBP, TATA-box binding protein (filled triangles), a “housekeeping” gene, as an internal control. (C) Representative real-time PCR analysis of cDNA from isolated islets. In relation to the total pancreas cDNA shown in B, the insulin/amylase ratio has been enriched for 12.24 cycles to achieve a purity of about 99%. dRn, raw fluorescence normalized for baseline and reference dye intensities by MX4000 software.
Figure 5
Figure 5
Gene expression changes discovered by microarray analysis are confirmed by real-time quantitative PCR. Fold changes in gene expression calculated from microarray data (black bars) are similar in value to those calculated from quantitative real-time PCR results (gray bars). All fold changes refer to deviations from gene expression in control islets. *P ≤ 0.05 and **P ≤ 0.01 by Student’s t test.
Figure 6
Figure 6
Identification of a conserved Foxa2-binding site in intron 1 of the Hadhsc gene, encoding schad. (A) Mouse Hadhsc is located on chromosome 3 and contains 8 exons. Exons are represented as vertical black bars, with bar width indicative of exon size. The 28-base region of the 1st Hadhsc intron shown is 93% conserved (26 of 28 bases) among mouse, rat, and human and includes the identical Foxa2-binding site shown in bold. (B) ChIP using mouse islets and a Foxa2 antibody followed by PCR confirmed the binding site on Hadhsc shown in A. Glut2 (a known Foxa2 target) served as a positive PCR control and MyoD is the negative PCR control. (C) Real-time PCR of purified DNA from ChIP eluates using primers for Hadhsc confirms the occupancy of this intron enhancer by Foxa2. Input (filled squares) and Foxa2 (open circles) Ct values were approximately 25 and 30, respectively. IgG (filled triangles) served as the ChIP control. (D) Cotransfection experiments with a luciferase construct containing 100 bp of Hadhsc intron 1 (pGL3-Hadhsc) and a Foxa2 expression plasmid (pHD-Foxa2) result in 3-fold activation compared with transfections with antisense Foxa2 (pHD-Foxa2 AS). n = 3 and ***P ≤ 0.0001 by Student’s t test.
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