Foxa2 regulates multiple pathways of insulin secretion (original) (raw)
A prerequisite for understanding the functional role of Foxa2 in the pancreatic β cell is the determination of its transcriptional targets. We have shown previously that Sur1 and Kir6.2 mRNAs were reduced by approximately 75% in islets from _Foxa2_loxP/loxP;Ins.Cre mice (7). In order to interpret the physiological response of _Foxa2_-deficient β cells, it was essential to determine if the remaining Sur1 and Kir6.2 mRNA was present in the _Foxa2_-deficient β cells or if it was due to the contribution of α cells in our islet RNA preparations. To this end, we performed RNA in situ hybridization of pancreas sections from control and mutant P8 mice using digoxigenin-labeled antisense probes (Figure 1). Glucagon mRNA was abundantly expressed in pancreatic α cells, which were localized to the outer edges of control islets (Figure 1, A and E). Although _Foxa2_loxP/loxP;Ins.Cre islets also contained glucagon-secreting α cells, they were not confined to the islet perimeter but were also found in the core, confirming the perturbed islet architecture described previously in these mice (7) (Figure 1, C and G). Sur1 and Kir6.2 mRNAs were expressed in both α and β cells of control islets at approximately equal levels (Figure 1, B and F). The remaining mRNA expression of both KATP channel subunits in the mutant islets is confined to α cells (Figure 1, D and H), which comprise 15–20% of the islet (20). Therefore, β cells lacking Foxa2 are essentially deficient for both subunits of the KATP channel.
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 _Foxa2_loxP/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 _Foxa2_loxP/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.
These findings suggested that Foxa2 acts as a transcriptional activator of Sur1 and Kir6.2 in β cells. To test this possibility, we transfected baby hamster kidney (BHK) cells with Sur1 or Kir6.2 promoter/luciferase reporter constructs along with a Foxa2 expression plasmid. Over 3-fold activation was observed with 1.8 kb of the Sur1 promoter and more than 6-fold activation was measured with the 7.0-kb promoter fragment construct (Figure 2A). Similarly, the addition of Foxa2 resulted in nearly 4-fold activation of Kir6.2 (Figure 2B), confirming that Foxa2 is a potent transactivator of both genes.
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.
KATP channels in the pancreatic β cell couple glucose metabolism to insulin secretion (13). The absence of Sur1 and Kir6.2 mRNA in _Foxa2_-deficient β cells prompted us to investigate further the physiological consequences of this downregulation. Circulating insulin levels in _Foxa2_loxP/loxP;Ins.Cre mice are inappropriately high given their low blood sugar levels, suggesting abnormal regulation of insulin secretion. We had previously investigated the secretory responses of these mice to a variety of stimuli using minced pancreas pieces (7). Newly optimized isolation techniques now allowed us to perform perifusion assays using isolated islets without any potential impairment of stimulus-secretion coupling by surrounding exocrine tissue or the presence of digestive enzymes. After being exposed to a ramp of increasing concentrations of all 20 amino acids, control islets had a negligible insulin secretion response (Figure 3A). Mutant islets, however, displayed a dose-dependent insulin secretion response to amino acids, with peak secretion at 17 mM (Figure 3A). With the addition of 25 mM glucose, control islets responded with robust insulin secretion, while mutant islets showed no additional response (Figure 3A).
_Foxa2_loxP/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, _Foxa2_loxP/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, _Foxa2_loxP/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 _Foxa2_loxP/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.
In a similar islet perifusion, an identical amino acid ramp was applied to control or _Foxa2_loxP/loxP;Ins.Cre islets (Figure 3B). Again, control islets did not respond to amino acid stimulation, but mutant islets showed the same steady increase in insulin secretion, which correlated with amino acid concentration in a dose-dependent manner (Figure 3B). The perifusate was then held at the highest amino acid concentration while the islets were exposed to 300 nM glyburide for 20 minutes. Glyburide is a sulfonylurea that binds to KATP channels in both α and β cells (21, 22) and prevents their opening. This prolonged closure of the KATP channel prevents the outflow of K+ ions from β cells, causing depolarization of their cell membranes and the subsequent opening of voltage-gated calcium channels and insulin secretion. The addition of glyburide to _Foxa2_loxP/loxP;Ins.Cre islets neither blunted nor enhanced the insulin secretion response to amino acids, while control islets exhibited a robust secretory response to glyburide as expected (Figure 3B), confirming that mutant β cells lack functional KATP channels.
We previously reported that glucagon expression is unchanged at the level of both pancreatic content and steady-state mRNA in _Foxa2_loxP/loxP;Ins.Cre mice. The significant decrease in circulating plasma glucagon, therefore, suggests a defect not in the biosynthesis of this hormone but in secretion (7). To investigate the glucagon secretion defect, we exposed islets to an amino acid ramp and determined glucagon release. Even at very low concentrations of the amino acid mixture, control islets were stimulated to secrete glucagon, but _Foxa2_loxP/loxP;Ins.Cre were not. The addition of glucose in conjunction with the maximal concentration of amino acids (17 mM) for 20 minutes did not alter glucagon secretion in control or mutant islets (Figure 3C).
The severely hyperinsulinemic, hypoglycemic phenotype of _Foxa2_loxP/loxP;Ins.Cre mice is usually lethal between P9 and P12. The downregulation of Sur1 and Kir6.2 described above cannot be the sole effect of the β cell–specific deletion of Foxa2, as the phenotype of mice homozygous for a null mutation in either Sur1 or Kir6.2 is mild compared with that of _Foxa2_loxP/loxP;Ins.Cre mice (23–25). Although all three mouse models, _Sur1_–/–, _Kir6.2_–/–, and _Foxa2_loxP/loxP;Ins.Cre, demonstrate KATP channel deficiency in their pancreatic β cells, only mice lacking Foxa2 succumb to severe hyperinsulinemic hypoglycemia by P9–P12. Because these mice do not simply recapitulate the phenotype of the _Sur1_–/– or _Kir6.2_–/– mouse models, we initiated a systematic search for additional pancreas-specific target genes that may contribute to lethality and the abnormal insulin secretion response to amino acids in _Foxa2_loxP/loxP;Ins.Cre mice.
Total RNA was purified from isolated islets pooled from 5–7 P8 mice of like genotype for control and _Foxa2_loxP/loxP;Ins.Cre samples. For quantitative expression studies, it was imperative to use only the highest quality RNA of equal purity. In the representative trace of RNA from isolated islets shown in Figure 4A, the rRNA fluorescence ratio (28S/18S) is about 2.5, which is the theoretical limit, with no evidence of degradation. Although minimal contamination with exocrine cells does not affect assays such as islet perifusions, RNA contributed by non-endocrine cells has the potential to significantly skew microarray or real-time PCR results. We developed a quantitative method for the evaluation of exocrine impurities in islet preparations (for details, see Methods). Amplified islet RNA was quantified, reverse-transcribed, and used in real-time PCR analysis with primers for insulin and amylase (Figure 4C). Using total pancreas RNA for comparison, we were able to calculate the “fold enrichment” of endocrine mRNA for each of our samples and select only the purest for microarray hybridization and real-time PCR. For the representative samples shown, the difference in cycle threshold (Ct) values for insulin and amylase between total pancreas cDNA (Figure 4B) and islet cDNA (Figure 4C) was 12.24 cycles, which “translates” into a 4,837-fold enrichment in endocrine tissue, resulting in about 99% endocrine purity.
Quality and purity assessment of islet RNA for gene expression analysis. (A) RNA Nano 6000 Assay of P8 islet RNA pooled from 5 mutant _Foxa2_loxP/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.
For the microarray analysis, we used the PancChip 4.0, which contains nearly 14,000 elements representing over 10,000 unique genes, most of which are expressed in the endocrine pancreas (26, 27). To analyze gene expression differences between control and _Foxa2_loxP/loxP;Ins.Cre P8 islets, we chose four amplified RNA samples of matched quality and endocrine purity from control and mutant genotypes. Overall, the gene expression profiles were very similar between mutant and control islets, demonstrating that the absence of Foxa2 does not cause a global defect in differentiation of β cells. Only approximately 0.3% of the genes were significantly changed, and for most of them their biochemical function is still unknown. However, three metabolic genes were identified in this screen and confirmed by quantitative real-time PCR analysis as being dependent on Foxa2 (Figure 5 and Table 1): Argininosuccinate synthetase, Fructose-1,6-bisphosphatase, and Hadhsc, the gene that encodes short-chain L-3-hydroxyacyl coenzyme A dehydrogenase (schad). Additional real-time PCR analysis with primers for known regulators of β-cell development and function revealed a significant decrease in Pdx1 mRNA in _Foxa2_loxP/loxP;Ins.Cre islets (data not shown), further confirming Foxa2 as an upstream transcriptional regulator of Pdx1 (28). We also confirmed our earlier experiments that reported unchanged levels of other genes involved in glucose metabolism, namely Glut2 and Glucokinase (ref. 7 and data not shown).
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.
Normalized intensity values for individual control and _Foxa2_loxP/loxP;Ins.Cre RNA samples hybridized to the PancChip 4.0 microarray
Our findings from the microarray experiment prompted us to investigate the mechanism that could explain the abnormal physiology of _Foxa2_loxP/loxP;Ins.Cre islets. We first considered how upregulation of Argininosuccinate synthetase could contribute to the aberrant islet secretion responses seen in our islet perifusions. In pancreatic β cells, this enzyme converts L-citrulline to L-argininosuccinate, which is then metabolized by argininosuccinate lyase to L-arginine. The third member of this biochemical cycle, nitric oxide synthase, metabolizes L-arginine back to L-citrulline, with an additional release of nitric oxide. Overactivation of this citrulline-argininosuccinate-arginine recycling pathway, which produces elevated levels of nitric oxide, has been associated with increases in intracellular calcium concentration in β cells (29). We hypothesized that upregulation of this biochemical pathway may trigger elevated basal intracellular calcium levels and the subsequent high levels of insulin secretion seen in _Foxa2_loxP/loxP;Ins.Cre islets. Real-time PCR analysis (data not shown) revealed modest upregulation of argininosuccinate lyase (P < 0.05) and a trend toward an increase in nitric oxide synthase transcript abundance in mutant islet RNA, suggesting that this pathway is indeed upregulated in _Foxa2_loxP/loxP;Ins.Cre mice.
We further investigated the _Foxa2_-dependent mRNA expression of schad, encoded by Hadhsc, that was identified through our microarray screen (Figure 5 and Table 1). Schad is a soluble mitochondrial matrix protein that plays an essential role in the β-oxidation of short chain fatty acids (30, 31). This enzyme has reportedly high levels of activity in pancreatic islets, suggesting a crucial influence on insulin secretion (32, 33). The strongest evidence for the importance of schad in glucose metabolism came from the discovery that mutations in the corresponding human gene cause PHHI (34–36). Thus, the approximately 3-fold reduction in Hadhsc expression in _Foxa2_loxP/loxP;Ins.Cre mice probably contributes to the severity of the hypoglycemic, hyperinsulinemic phenotype.
Analysis of steady-state mRNA levels does not address the question whether Hadhsc is a direct transcriptional target of Foxa2 in the β cell or whether the reduction in its expression is a secondary consequence of the altered metabolic state of _Foxa2_loxP/loxP;Ins.Cre mice. We analyzed potential _cis_-regulatory elements of the Hadhsc gene for Foxa2-binding sites. Sequence alignments from mouse, rat, and human revealed no areas of conservation within the promoter region of Hadhsc, but detected a span of 28 bases within intron 1 with 93% identity (26 of 28 bases) among all three species (Figure 6A). A transcriptional element search algorithm revealed a putative Foxa2-binding site (TGTTTGTTT) within this region. Although there is no strict consensus Foxa-binding site, this exact sequence had previously demonstrated a high affinity for Foxa2 (hepatocyte nuclear factor 3β [HNF3β]) protein in vitro (37). To investigate whether this site is occupied by Foxa2 in β cells, we performed chromatin immunoprecipitation (ChIP) with a Foxa2-specific antibody on isolated islets. As shown in Figure 6, B and C, the intron of Hadhsc was indeed bound by Foxa2 in vivo. Cotransfection of a luciferase construct containing the Foxa2-binding site within intron 1 of Hadhsc demonstrated that Foxa2 not only bound to Hadhsc but also activated transcription up to 3-fold (Figure 6D). The magnitude of this transactivation is in good agreement with the magnitude of the downregulation of Hadhsc we observed in islets deficient for Foxa2 shown in Figure 5 and Table 1. Thus, we have shown that Hadhsc is a direct target of Foxa2 in pancreatic β cells.
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.