GATA4 and GATA6 play essential, but redundant, roles in pancreas formation in mice, and GATA6 mutations cause pancreatic agenesis in humans. GATA6 mutations have also recently been linked to adult-onset diabetes, with subclinical or no exocrine insufficiency, suggesting an important role for GATA6 in human β-cell physiology. To investigate the role of GATA6 in the adult endocrine pancreas, we generated mice in which Gata6 is specifically inactivated in the pancreas. These mice develop glucose intolerance. Islets deficient in GATA6 activity display decreased insulin content and impaired insulin secretion. Gata6-deficient β-cells exhibit ultrastructural abnormalities, including increased immature insulin granules, swollen mitochondria, and disorganized endoplasmic reticulum. We also demonstrate that Pdx1 expression in adult β-cells depends on GATA sites in transgenic reporter mice and that loss of GATA6 greatly affects β-cell–specific gene expression. These findings demonstrate the essential role of GATA6 in β-cell function.

The formation of β-cells during embryonic development is regulated by a notable number of transcription factors that activate lineage-specific genes. Interestingly, many of these transcription factors are also important in pancreas function during adult life. Members of the GATA zinc finger transcription family are crucial for pancreas organogenesis in both human and mouse (13). Exome sequencing studies have revealed that heterozygous GATA6 mutations are the most common cause of pancreatic agenesis (3,4). Mutations in GATA4 have also been linked to neonatal diabetes and pancreatic agenesis in humans (5,6). In the mouse, GATA4 and GATA6 play redundant roles in embryonic pancreas formation. Single inactivation of Gata4 or Gata6 in pancreatic progenitor cells does not have a major effect in pancreas organogenesis. However, the simultaneous inactivation of both Gata4 and Gata6 results in pancreatic agenesis due to defects in pancreatic progenitor cell proliferation and differentiation (1,2).

The expression of these two GATA transcription factors is not restricted to the embryonic stages of pancreas formation. Thus, in the adult pancreas, Gata4 is expressed in the acinar compartment, whereas Gata6 is expressed in the endocrine and exocrine compartments. Interestingly, recent genetic studies have revealed that GATA6 mutations are also linked to adult-onset diabetes with subclinical or no exocrine insufficiency. Moreover, GATA6 deficiency in human pluripotent stem cells altered in vitro directed differentiation toward β-like cells as well as their subsequent functionality (79). Taken together, these studies indicate that in addition to pancreas development, GATA6 plays a critical role in β-cell function that remains uncharted. Here, we thoroughly analyze mice with single inactivation of Gata6 in the pancreas. We show that although Gata6-deficient mice are apparently normal at birth, as they age they develop glucose intolerance. Furthermore, loss of Gata6 causes impairment of β-cell insulin biosynthesis and secretion and markedly affects the islet transcriptome.

Mice

Gata6flox/flox, Pdx1-Cre, Pdx1-wt-lacZ, and Pdx1-mut-lacZ mice have been previously described (1,10,11). All experiments using animals were approved by the University of Seville Institutional Animal Care and Use Committee, Seville, Spain.

Histology, Immunohistochemistry, and Immunofluorescence

Histological and immunohistochemical analyses were performed, as previously described (12), on at least four different sections from five different mice. Primary antibodies were used at the indicated dilution: anti-GATA6 (1:1,000, AF 1700; R&D Systems), anti-Pdx1 (1:200, ab47308; Abcam), anti-Nkx6.1 (1:50, F55A10; DSHB), anti-Nkx2.2 (1:25, 74.5A5; DSHB), anti-glucagon (1:100, G2654; Sigma-Aldrich), anti-insulin (1:500, I2018; Sigma-Aldrich), anti-GLUT2 (1:300, 07-1402; Millipore), anti–β-galactosidase (1:500, 559762; MP Biochemicals), anti–C-peptide (1:100, 05-1109; Millipore), anti-MafA (1:50; Bethyl Laboratories), anti-somatostatin (1:1,000; Santa Cruz Biotechnology), anti-pancreatic polypeptide (1:100; Millipore), and anti-inducible nitric oxide synthase (iNOS; 1:100; Thermo Fisher Scientific).

Transmission Electron Microscopy and Insulin Granules Quantification

For transmission electronic microscopy (TEM) analysis, isolated pancreatic islets were processed using a standard Spurr protocol (13). TEM images were taken with an EMCCD camera (TRS 2k × 2k). Quantifications of insulin granules were performed on 20 TEM images obtained from isolated islets of control and Gata6 knockout (KO) mice (n = 6), as previously described (14,15), using ImageJ software.

Islets Isolation

Islets were isolated from pancreata of male Gata6 KO and control mice by the collagenase digestion method (16). Islets were handpicked after several purification steps and used for RNA isolation or kept in culture overnight for insulin secretion tests.

Glucose and Insulin Tolerance Test

After a 16-h fast, mice were weighed, and an Accu-Chek Aviva glucometer was used to measure glucose levels. Mice were injected intraperitoneally with glucose (2 g/kg of body weight). Blood glucose levels were measured every 30 min. Blood for in vivo insulin measurements was collected from the tail vein before and at 5 and 30 min after the glucose injection. Insulin concentration was measured using the Insulin ELISA kit (Mercodia) following the manufacturer’s instructions. The insulin tolerance test was performed as previously described (17).

Insulin Secretory Response

For in vitro insulin secretion, isolated islets were incubated in 2.8 mmol/L glucose or 16.7 mmol/L glucose for 1 h at 37°C. Ten matched-size islets were used per triplicate per condition. After incubation, media was collected to measure insulin using the Insulin ELISA kit (Mercodia). Islets were collected, spun down, and lysed using a 30-gauge syringe with Tris-EDTA buffer for measuring total insulin content.

C-Peptide and Proinsulin Content

Blood was collected from the tail vein of mice after a 16–h fast, and C-peptide and proinsulin levels were measured using the C-peptide ELISA kit (Crystal Chem) and Proinsulin ELISA kit (Mercodia), respectively, following the manufacturers’ recommendations.

β-Cell Area Quantification

β-Cell area quantification was performed as described (18). Immunohistochemistry images were taken with a Nikon microscope and processed using ImageJ software. β-Cell area was calculated as the percentage of insulin area–to–total pancreatic area (marked by DAPI staining).

Pancreas and Islets Insulin Content

Dissected pancreata were minced using a Polytron homogenizer in Tris-EDTA buffer, and isolated islets were lysed in Tris-EDTA buffer using a 30-gauge syringe. Debris was spun down, and the supernatant was collected to measure insulin, as described above. Total pancreatic protein content and islets DNA was measured using Bradford reagent (Sigma-Aldrich) and the Quant-iT PicoGreen dsDNA Assay kit (Invitrogen), respectively.

Microarray Analyses and Quantitative PCR

Isolated islets from three control or three Gata6 KO mice were pooled to obtain RNA. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). Gene profile was performed in three independent pools of control and four pools of Gata6 KO islets using the Affymetrix GeneChip Mouse Gene 2.0 ST Array. Differentially expressed genes were defined as those for which the nominal P was 0.05. Gene expression data are available through the Gene Expression Omnibus database (accession number GSE106316) (19). Quantitative (q)PCR was performed from at least seven independent pools of control and Gata6 KO islets. qPCR was performed using the following TaqMan probes (Applied Biosystems): Ins1: Mm01950294_s1; Ins2: Mm00731595_gH; Slc2a2: Mm00446224_m1; Pdx1: Mm00435565_m1; Abcc8: Mm00803450_m1; Nkx6.1: Mm00454961_m1; Nkx2.2: Mm03053916_s1; Pcsk1: Mm00479023_m1; Gcg: Mm00801714_m1; Sst: Mm00436671_m1; Gata6: Mm00802632_m1; Gata4: Mm00484689_m1; Irs2: Mm03038438_m1; Cacna1c: Mm01188822_m1; Nos2: Mm0040502_m1; and β-actin: Mm02619580_g1. The following primers were used to quantify gene expression by SYBR Green: MafA: forward 5′-GAGGAGGTCATCCGACTGAAA-3′, reverse 5′-GCACTTCTCGCTCTCCAGAAT-3′; Snap25: forward 5′-GAGAACCTGGAGCAGGTGAG-3′, reverse 5′-AGCATCTTTGTTGCACGTTG-3′.

Statistical Analysis

Data in scatter plots and graph bars are presented as mean ± SD and mean ± SEM, respectively. The Student t test was performed with a level of significance of P < 0.05. Two-way ANOVA with repeated measures with Bonferroni correction were used to analyze intraperitoneal glucose tolerance test data.

Pancreas-Specific Inactivation of Gata6 Leads to Glucose Intolerance With Aging

To elucidate the role of GATA6 in β-cell function, we generated pancreas-specific Gata6 KO mice by crossing mice with a conditional (flox) allele of Gata6 (Gata6flox/flox) with mice that express the Cre recombinase under the control of the Pdx1 promoter (Pdx1-Cre mice) (1). Efficient Gata6 inactivation in the pancreas was demonstrated by immunohistochemistry and qPCR (Fig. 1A–C). Clear elimination of GATA6 was observed in exocrine and islet cells of Gata6flox/flox; Pdx1-Cre mice (Gata6 KO mice, hereafter) (Fig. 1B). Gross morphology and histological analysis did not reveal any apparent pancreatic abnormalities in newborn (Fig. 1D and E) or 2-month-old (Fig. 1F and G) Gata6 KO mice. As mice aged, we observed mild loss of acinar cells, acinar- to ductal-metaplasia lesions, and pancreatic lipomatosis in Gata6 KO mice (Fig. 2H–O), in agreement with previous studies describing GATA6 as an essential factor for the maintenance of acinar identity (20). However, acinar cell morphology was not largely disrupted, as assessed by cell polarity markers (Supplementary Fig. 1). Similarly, overall exocrine pancreatic function was not compromised, as determined by plasma amylase activity (Supplementary Fig. 1). Histological (Fig. 2A and H) and immunohistochemical analysis for pancreatic hormones revealed that although the overall architecture of Gata6 KO islets was not markedly affected, insulin levels appeared lower in Gata6-deficient β-cells (Fig. 2C–H). To determine the functional requirement for GATA6 in the adult endocrine pancreas, we examined glucose homeostasis in Gata6 KO mice. Male and female Gata6 KO mice at 2 and 4 months old displayed normal glucose tolerance (Fig. 3A–D and Supplementary Fig. 2). However, by age 6 months, male and female Gata6 KO mice displayed a significant impairment in glucose tolerance compared with control mice (Fig. 3E and F and Supplementary Fig. 2), a phenotype that was maintained at 12 months of age (Fig. 3G and H). Despite the abnormal glucose tolerance, no differences in basal glycemia under fasting conditions were observed between Gata6 KO and control mice (Fig. 3I). Because Gata6flox/flox, Gata6flox/+; Pdx1-Cre, Gata6flox/+, and Pdx1-Cre mice exhibited similar phenotypes in glucose tolerance (Supplementary Fig. 3), we only included Gata6flox/flox littermates as controls in all subsequent experiments. The glucose intolerance phenotype of Gata6 KO mice was not associated to insulin resistance, as shown by insulin tolerance tests (Fig. 3J). Thus, our data indicate that Gata6 inactivation in the pancreas compromises glucose homeostasis.

Gata6 KO Mice Display Reduced Pancreatic Insulin Content and Defects in Insulin Secretion

The glucose intolerance phenotype observed in 6-month-old Gata6 KO mice could be due to decreased β-cell numbers. Nonetheless, quantification of β-cell area did not reveal significant differences between 6-month-old Gata6 KO and control littermates (Fig. 4A). Similarly, no differences in β-cell apoptosis and proliferation were observed between Gata6 KO and control islets (Supplementary Fig. 4). Because immunofluorescence analysis revealed a potential reduction in insulin accumulation in β-cells (Fig. 2C–H), we measured total pancreatic insulin content. A significant decrease in insulin levels was observed in the Gata6 KO pancreas compared with the control pancreas (Fig. 4B). Moreover, the insulin content of isolated Gata6 KO islets was reduced by 2.5-fold compared with isolated control islets (Fig. 4C).

We next investigated whether Gata6 inactivation affected insulin secretion. To this aim, we measured circulating insulin levels in 6-month-old mice subsequent to an intraperitoneal glucose tolerance test. Gata6 KO mice exhibited impaired insulin secretion under basal conditions and upon glucose challenge (Fig. 4D). To directly analyze β-cell function, insulin secretion assays were performed on islets isolated from 6-month-old control and Gata6 KO mice. Similar to what we observed in vivo, insulin secretion from Gata6 KO islets was reduced compared with control islets under basal and high glucose concentrations (Fig. 4E). Nonetheless, the difference between basal and high glucose was maintained in Gata6-deficient compared with control islets, suggesting a dampened yet preserved glucose-induced insulin response (Fig. 4E). To confirm these findings, we measured intracellular calcium flux in isolated islets at different glucose concentrations using fluorescent calcium fluorescence sensor Fura-2. No marked differences between Gata6 KO and control islets were observed (Supplementary Fig. 5). Altogether, our results indicate that the glucose intolerance phenotype of Gata6 KO mice might be due to defects in both insulin production and secretion.

Key Genes in Insulin Biosynthesis and Secretion Are Downregulated in Gata6-Deficient Islets

To gain insight into the role of GATA6 in islet function, we performed microarray analysis comparing Gata6 KO and control islets. To avoid any potential interference of glucose intolerance in islet expression, gene expression was analyzed in islets collected from 2-month-old mice when they still display normal glucose tolerance. The most downregulated genes were genes involved in different aspects of β-cell function, such as glucose sensing (Slc2a2, encoding for GLUT2 transporter), insulin biosynthesis (Pcsk1), insulin secretion (Abcc8; Cacna1c; SNAP-25), and major transcriptional regulators of adult β-cell function such as Pdx1, Nkx2.2, Nkx6.1, and MafA (2124). Insulin transcripts (both Ins1 and Ins2) of Gata6 KO islets also displayed a notable decrease compared with control islets, in agreement with the observed reduction in pancreatic and islet insulin content observed in 6-month-old mice (Fig. 2B and C). Gene expression analysis by qPCR confirmed the marked downregulation of these key mature β-cell markers in Gata6-deficient islets (Fig. 5A). However, the expression of other islet hormone genes, such as glucagon and somatostatin, was not decreased in Gata6 KO islets (Fig. 5A). Of note, levels of Gata4 transcripts were similar between Gata6-deficient and control islets (Fig. 5A).

To determine whether these changes in gene expression in Gata6 KO islets correlated with changes in protein accumulation, we performed immunohistochemical and immunofluorescence analyses in 2-, 4-, and 6-month-old mice. At both 2 and 4 months of age, a mild reduction in insulin and C-peptide was observed in Gata6-deficient islets compared with control islets. However, plasma insulin and C-peptide levels were normal in fasted 2-month-old Gata6 KO mice (Supplementary Figs. 6 and 7). Moreover, no apparent decrease in the accumulation of other β-cell markers, such as Pdx1, Glut2, Nkx2.2, and Nkx6.1, was observed in Gata6 KO islets compared with control islets (Supplementary Fig. 6). At 6 months of age, Pdx1, Glut2, Nkx2.2, Nkx6.1, and MafA levels were markedly diminished in Gata6 KO islets compared with control islets (Fig. 5B–I). In addition, islets lacking GATA6 displayed a notable reduction in C-peptide (Fig. 5J and K). Circulating levels of insulin and C-peptide were also decreased in Gata6 KO mice (Supplementary Fig. 7). Thus, Gata6 inactivation significantly affects the expression of critical β-cell–specific genes.

We next determined whether Gata6 depletion in adult β-cells affected β-cell gene expression. To this end, we inactivated Gata6 in isolated islets from 2-month-old Gata6flox/flox mice by infection with adenovirus expressing Cre. Although an efficient Gata6 inactivation was achieved, the expression of key β-cell genes was unaffected in Gata6-depleted islets, in contrast to what it was observed in GATA6 KO mice of the same age (Fig. 2 and Supplementary Fig. 6).

Gata6-Deficient β-Cells Exhibit Increased Immature Insulin Granules, Swollen Mitochondria, and Disorganized Endoplasmic Reticulum

To further explore the role of GATA6 in β-cell function we used the Ingenuity Pathway Analysis software to identify pathways affected by Gata6 inactivation. Consistent with our previous results, pathways related to insulin and β-cell function, such as “maturity-onset diabetes of the young signaling” and “insulin receptor signaling,” were found among the top canonical pathways (Fig. 6A). Other pathways identified were related to translational control and, interestingly, mitochondrial dysfunction (Fig. 6A). Indeed, several genes involved in mitochondrial function, including respiratory chain components (i.e., Cox6a2, Cyp4f39) and mitochondrial transporters (i.e., Slc25a12, Slc25a22), were downregulated in Gata6 KO islets (Supplementary Table 1). Because a link between mitochondrial dysfunction and defective β-cell function has been proposed (25,26), we used TEM to perform ultrastructural analysis of 6-month-old Gata6 KO and control β-cells. Mitochondria of Gata6 KO β-cells appeared swollen, with disorganized inner-membrane cristae (Fig. 6C and E), in contrast to the dense and organized cristae of mitochondria in control β-cells (Fig. 6B and D). TEM analysis revealed additional ultrastructural abnormalities in Gata6-deficient β-cells compared with control mice (Fig. 6F and G). Thus, extension of the endoplasmic reticulum (ER) was observed in Gata6 KO β-cells, indicative of ER stress (Fig. 6G). An association among mitochondrial dysfunction, ER stress, and induction of iNOS has been reported (27,28). Consistent with this notion, we observed a marked increase in iNOS expression in Gata6 KO islets as assessed by immunofluorescence and qPCR analysis (Fig. 7). TEM analysis also showed a marked reduction in the total number of insulin granules in Gata6-deficient β-cells compared with control cells (Fig. 6B–K). The secretory insulin granules from control β-cells featured an electron-dense core and a translucent halo appearance, indicative of mature secretory granules (Fig. 6B, D, F, and H). However, Gata6 KO β-cells displayed a decreased number of dense granules and a prominent number of gray low-dense granules (Fig. 6C, E, G, I, and J), indicating an increase in immature insulin granules. Despite the increase of immature secretory granules, no differences in the ratio of proinsulin to insulin circulating levels were found between Gata6 KO and control mice (Supplementary Fig. 7). Altogether, our results indicate that GATA6 is necessary to preserve the integrity of the ER and mitochondria and the formation of insulin secretory vesicles in β-cells.

GATA6 Directly Regulates Pdx1 Expression in Adult Islets

We previously showed that GATA factors directly regulate Pdx1 expression in the pancreas during embryonic stages by binding to two deeply conserved GATA sites in area III of Pdx1 regulatory sequences (1). We decided to determine whether the GATA sites in area III also contribute to Pdx1 expression in adult islets by analyzing Pdx1-wt-lacZ mice, transgenic mice that harbor the cis-regulatory region of Pdx1-containing areas I, II, and III fused to the lacZ reporter gene. This transgenic construct faithfully recapitulated the endogenous expression of Pdx1 in adult islets in three independent Pdx1-wt-lacZ transgenic lines. Thus, β-galactosidase localization directed by Pdx1-wt-lacZ completely overlapped with PDX1 accumulation in pancreatic islets (Fig. 8A–D and I–L). A similar pattern of β-galactosidase expression was observed in islets of transgenic mice containing mutations in Pdx1-area III GATA sites at 11 weeks of age in two independent transgenic lines analyzed (Fig. 8E–H). However, mutations in Pdx1-area III GATA sites resulted in a dramatic decrease of β-galactosidase expression in 6-month-old mice, with only a few β-galactosidase–positive cells found in the Pdx1 expression domain (Fig. 8M–P). These results indicate that GATA factors directly regulate Pdx1 expression in adult islets through GATA sites present in area III of the Pdx1-cis–regulatory region.

Previous studies have shown that GATA4 and GATA6 play essential, albeit redundant, roles during embryonic pancreas development (1,2). Here we show that conditional inactivation of Gata6 in the pancreas results in compromised glucose tolerance, mainly due to defects in insulin production and secretion.

Gata6-deficient mice showed a significant reduction in β-cell insulin content, including decreased insulin transcription. Impaired formation of insulin secretory vesicles could also contribute to the reduced insulin secretion of Gata6-deficient islets. TEM analysis showed that β-cells lacking GATA6 displayed decreased insulin granules but, more interestingly, increased immature insulin secretory vesicles. Increased immature insulin secretory vesicles can be associated to defective insulin processing. However, we failed to observe significant changes in the ratio of proinsulin to insulin blood levels in Gata6 KO mice. Consistent with the defective insulin secretion phenotype, a decrease in the expression of SNAP-25, a t-SNARE protein that participates in the exocytosis of secretory granules (29), was observed in Gata6-deficient islets. Gata6-deficient islets also displayed reduced expression levels of Slc2a2 and Abcc8 that encode GLUT2 and SUR1, respectively, two key proteins critical for glucose-stimulated insulin secretion (30,31). However, although basal insulin secretion was diminished, stimulation of secretion by glucose appeared to be unaffected by Gata6 inactivation. In agreement with this, the intracellular calcium response to glucose was not affected in Gata6-deficient β-cells, indicating that GLUT2 and SUR1 expression was not reduced to levels low enough to affect glucose-induced insulin secretion.

Gata6 inactivation results in decreased expression of β-cell–enriched transcription factors, including Pdx1, Nkx6.1, and MafA, which are essential for insulin transcription and secretion (23,32,33). Thus, we cannot rule out that the effects of GATA6 activity on β-cell function could be indirect. We previously demonstrated that GATA6 directly regulates Pdx1 expression during early pancreatic development through GATA sites present in area III of the Pdx1 regulatory sequence (1). We now show that, interestingly, these GATA sites are also required for Pdx1 expression in adult β-cells. Altogether, our data reveal GATA6 as a key component of the transcriptional network regulating β-cell function.

Despite multiple defects in insulin transcription, biosynthesis, and secretion, Gata6 KO mice do not become overtly hyperglycemic. We did not observe any compensatory increase in β-cell area or insulin sensitivity that could explain the lack of hyperglycemia, but we cannot rule out other compensatory mechanisms such as neural regulation or incretin hormone secretion. Interestingly, inactivation of other β-cell transcription factors, such as Rfx6 and NeuroD, also results in β-cell dysfunction and glucose intolerance without developing overt hyperglycemia (34,35).

Our microarray analysis revealed a marked aspect of the gene expression profile of Gata6 KO islets, the impairment of mitochondrial gene expression. In agreement with these results, TEM analysis revealed swelling of mitochondria, exhibiting disrupted cristae in Gata6-deficient β-cells. Similar mitochondrial alterations have been associated to β-cell failure, as reviewed by Supale et al. (25). Thus, mitochondrial dysfunction might also underlie the impairment of β-cell function in Gata6 KO mice. ER abnormalities were also observed in Gata6-deficient β-cells in concordance with the increasing body of evidence suggesting that mitochondrial and ER function are interconnected in β-cells (36). Interestingly, a marked increase in iNOS expression was found in Gata6 KO islets, a protein associated with mitochondrial dysfunction and ER stress (iNOS) has been reported (27,28). The mTOR signaling may also play a role in the interplay between mitochondrial and ER function according to recent reports (37,38) Interestingly, Ingenuity Pathway analysis predicted activation of the mTOR signaling pathway in Gata6 KO islets. Further studies will be required to clarify the direct role of GATA6 in the regulation of these pathways in β-cells.

Our results are in apparent contradiction with two reports describing inactivation of Gata6 in mouse β-cells. The inactivation of Gata6 using a Ptf1a-Cre line did not have any effect in glucose homeostasis in 30-week-old mice (20). Similarly to the Gata6flox/flox;Pdx1-Cre mice used in this study, the inactivation of Gata6 in Gata6flox/flox;Ptf1a-Cre mice also occurred early in pancreatic progenitors but at slightly different times (39). A possible explanation to the discrepancy in the glucose tolerance phenotype between these mouse models is that GATA6 might be required for the proper maturation and function of adult β-cells during a specific developmental time window in which Pdx1, but not Ptf1a, is expressed. Differences in Cre recombinase efficiency in endocrine cells of the two mouse models might also contribute to the observed discrepancies (40).

In other study, the inactivation of Gata6 specifically in adult β-cells (using the Ins2-CreERT mouse strain) did not affect glucose homeostasis (41). However, the authors report inefficient Gata6 deletion that could explain the lack of metabolic phenotypes on their mice. In contrast, we observed substantial Gata6 inactivation using the Pdx1-Cre line. An alternative possibility is that Pdx1-Cre–mediated Gata6 inactivation during embryonic stages causes the observed defects later in adult β-cells. In this regard, we performed in vitro Gata6 depletion in adult mouse islets without any apparent effect on β-cell gene expression. Although these experiments are far from conclusive regarding a possible developmental origin of Gata6 KO defects, they suggest the Gata6 inactivation does not have an immediate effect on β-cell function. Interestingly, despite the early inactivation of Gata6 by using the Pdx1-Cre line, Gata6 KO mice do not become clearly glucose intolerant until 6 months of age. The progressive defect in glucose homeostasis is not uncommon in mouse models of β-cell dysfunction and might be due to increased metabolic stress associated to age and concomitant increase in body weight. However, 6-month-old Gata6 KO mice develop exocrine defects that could potentially affect islet function. Although we cannot formally rule out this effect, we consider this an unlikely possibility based on the following observations. First, compromised expression of β-cell genes is found in 2-month-old mice, before overt exocrine defects are observed. Second, plasma amylase levels in 2- and 6-month-old Gata6 KO mice are not increased, indicating the functional effect of pancreatic injury was minor. Finally, 30-week-old Gata6flox/flox;Ptf1a-Cre mice display extensive pancreatic exocrine abnormalities but no defects in glucose homeostasis.

Recent genetic studies have revealed that GATA6 mutations can cause adolescent/adult-onset diabetes with subclinical or no exocrine insufficiency. Unfortunately, little is known about deficits in β-cell function in this specific group of patients. However, two recent studies using in vitro–directed differentiation of β-like cells from human pluripotent stem cells indicate a critical role for GATA6 in β-cell function (7,8). GATA6 mutant β-like cells display impaired insulin secretion and downregulation of key genes involved in β-cell function. Our results are in agreement with these reports demonstrating that GATA6 activity is necessary for β-cell function in mice. The elucidation of the mechanisms underlying diabetes onset on patients with GATA6 mutations will shed light on the differential requirements of GATA6 for pancreas development versus adult β-cell function.

In summary, our results show that loss of Gata6 markedly decreases the expression of insulin, key components of insulin synthesis and secretion machinery, and β-cell–enriched transcription factors, demonstrating an essential role for GATA6 in β-cell function.

Acknowledgments. The authors thank Antonio Cárdenas and Alberto Morante, from Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), for their technical assistance. The authors also thank Juan Luis Ribas, from the University of Seville, for the electronic microscopy technical services, and Eloisa Andújar and Mónica Pérez, from the Genomic Unit of CABIMER. The authors thank the Andalusian Bioinformatics Platform at the University of Malaga (www.scbi.uma.es) for providing access to Ingenuity Pathway Analysis software.

Funding. L.V. was supported by a contract from Spanish Ministry of Economy and Competitiveness (RYC-2013-14533). E.R.-S. was supported by a “Juan de la Cierva” postdoctoral fellowship from Spanish Ministry of Economy and Competitiveness (IJCI-2014-19251). This work was supported by grants from ISCIII cofunded by Fondos FEDER (PI14/01015, RD/0019/0028, and RD16/0011/0034 to B.S. and PI14/0804 to A.R.) and “Ramón y Cajal” program from the Spanish Ministry of Economy and Competitiveness (PI14/0804, RYC-2013-14533 to A.R.). P.M. was supported by a Juan de la Cierva fellowship from Spanish Ministry of Science and Innovation and by a Stand-Alone Grant from the Austrian Science Fund (FWF P27361-B23). Work by I.Q. was supported by the Spanish Ministry of Economy and Competitiveness (BFU2016-77125-R). Work by D.A.C. was supported by the Nicolás Monardes program of Andalusian Ministry of Health (C-0015-2014) and the Andalusian Ministry of Science and Innovation (CTS-7478).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. L.V., E.R.-S., R.A., M.C., E.B.-T., and J.M.M.-G. performed the experiments. B.R.G., P.M., I.Q., B.S., F.M., D.A.C., and A.R. conceived the data. I.Q., D.A.C., and A.R. analyzed the data. D.A.C. and A.R. wrote the manuscript. All authors edited the manuscript. A.R. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Carrasco
M
,
Delgado
I
,
Soria
B
,
Martín
F
,
Rojas
A
.
GATA4 and GATA6 control mouse pancreas organogenesis
.
J Clin Invest
2012
;
122
:
3504
3515
[PubMed]
2.
Xuan
S
,
Borok
MJ
,
Decker
KJ
, et al
.
Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis
.
J Clin Invest
2012
;
122
:
3516
3528
[PubMed]
3.
Allen
HL
,
Flanagan
SE
,
Shaw-Smith
C
, et al
.
GATA6 haploinsufficiency causes pancreatic agenesis in humans
.
Nat Genet
2011
;
44
:
20
22
[PubMed]
4.
De Franco
E
,
Shaw-Smith
C
,
Flanagan
SE
, et al
.
GATA6 mutations cause a broad phenotypic spectrum of diabetes from pancreatic agenesis to adult-onset diabetes without exocrine insufficiency
.
Diabetes
2013
;
62
:
993
997
[PubMed]
5.
D’Amato
E
,
Giacopelli
F
,
Giannattasio
A
, et al
.
Genetic investigation in an Italian child with an unusual association of atrial septal defect, attributable to a new familial GATA4 gene mutation, and neonatal diabetes due to pancreatic agenesis
.
Diabet Med
2010
;
27
:
1195
1200
[PubMed]
6.
Shaw-Smith
C
,
De Franco
E
,
Lango Allen
H
, et al
.
GATA4 mutations are a cause of neonatal and childhood-onset diabetes
.
Diabetes
2014
;
63
:
2888
2894
[PubMed]
7.
Tiyaboonchai
A
,
Cardenas-Diaz
FL
,
Ying
L
, et al
.
GATA6 plays an important role in the induction of human definitive endoderm, development of the pancreas, and functionality of pancreatic β cells
.
Stem Cell Reports
2017
;
8
:
589
604
[PubMed]
8.
Shi
ZD
,
Lee
K
,
Yang
D
, et al
.
Genome editing in hPSCs reveals GATA6 haploinsufficiency and a genetic interaction with GATA4 in human pancreatic development
.
Cell Stem Cell
2017
;
20
:
675
688.e6
[PubMed]
9.
Lorberbaum
DS
,
Sussel
L
.
Gotta have GATA for human pancreas development
.
Cell Stem Cell
2017
;
20
:
577
579
[PubMed]
10.
Sodhi
CP
,
Li
J
,
Duncan
SA
.
Generation of mice harbouring a conditional loss-of-function allele of Gata6
.
BMC Dev Biol
2006
;
6
:
19
[PubMed]
11.
Hingorani
SR
,
Petricoin
EF
,
Maitra
A
, et al
.
Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse
.
Cancer Cell
2003
;
4
:
437
450
[PubMed]
12.
Rojas
A
,
Kong
SW
,
Agarwal
P
,
Gilliss
B
,
Pu
WT
,
Black
BL
.
GATA4 is a direct transcriptional activator of cyclin D2 and Cdk4 and is required for cardiomyocyte proliferation in anterior heart field-derived myocardium
.
Mol Cell Biol
2008
;
28
:
5420
5431
[PubMed]
13.
Spurr
AR
.
A low-viscosity epoxy resin embedding medium for electron microscopy
.
J Ultrastruct Res
1969
;
26
:
31
43
[PubMed]
14.
Wijesekara
N
,
Dai
FF
,
Hardy
AB
, et al
.
Beta cell-specific Znt8 deletion in mice causes marked defects in insulin processing, crystallisation and secretion
.
Diabetologia
2010
;
53
:
1656
1668
[PubMed]
15.
Tong
X
,
Kono
T
,
Anderson-Baucum
EK
, et al
.
SERCA2 deficiency impairs pancreatic β-cell function in response to diet-induced obesity
.
Diabetes
2016
;
65
:
3039
3052
[PubMed]
16.
Gonzalez
A
,
Merino
B
,
Marroquí
L
, et al
.
Insulin hypersecretion in islets from diet-induced hyperinsulinemic obese female mice is associated with several functional adaptations in individual β-cells
.
Endocrinology
2013
;
154
:
3515
3524
[PubMed]
17.
Puri
S
,
Cano
DA
,
Hebrok
M
.
A role for von Hippel-Lindau protein in pancreatic beta-cell function
.
Diabetes
2009
;
58
:
433
441
[PubMed]
18.
Muñoz-Bravo
JL
,
Hidalgo-Figueroa
M
,
Pascual
A
,
López-Barneo
J
,
Leal-Cerro
A
,
Cano
DA
.
GDNF is required for neural colonization of the pancreas
.
Development
2013
;
140
:
3669
3679
[PubMed]
19.
Barrett
T
,
Wilhite
SE
,
Ledoux
P
, et al
.
NCBI GEO: archive for functional genomics data sets--update
.
Nucleic Acids Res
2013
;
41
:
D991
D995
[PubMed]
20.
Martinelli
P
,
Cañamero
M
,
del Pozo
N
,
Madriles
F
,
Zapata
A
,
Real
FX
.
Gata6 is required for complete acinar differentiation and maintenance of the exocrine pancreas in adult mice
.
Gut
2013
;
62
:
1481
1488
[PubMed]
21.
Cissell
MA
,
Zhao
L
,
Sussel
L
,
Henderson
E
,
Stein
R
.
Transcription factor occupancy of the insulin gene in vivo. Evidence for direct regulation by Nkx2.2
.
J Biol Chem
2003
;
278
:
751
756
[PubMed]
22.
Taylor
BL
,
Liu
FF
,
Sander
M
.
Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells
.
Cell Rep
2013
;
4
:
1262
1275
[PubMed]
23.
Iype
T
,
Francis
J
,
Garmey
JC
, et al
.
Mechanism of insulin gene regulation by the pancreatic transcription factor Pdx-1: application of pre-mRNA analysis and chromatin immunoprecipitation to assess formation of functional transcriptional complexes
.
J Biol Chem
2005
;
280
:
16798
16807
[PubMed]
24.
Zhang
C
,
Moriguchi
T
,
Kajihara
M
, et al
.
MafA is a key regulator of glucose-stimulated insulin secretion
.
Mol Cell Biol
2005
;
25
:
4969
4976
[PubMed]
25.
Supale
S
,
Li
N
,
Brun
T
,
Maechler
P
.
Mitochondrial dysfunction in pancreatic β cells
.
Trends Endocrinol Metab
2012
;
23
:
477
487
[PubMed]
26.
Lu
H
,
Koshkin
V
,
Allister
EM
,
Gyulkhandanyan
AV
,
Wheeler
MB
.
Molecular and metabolic evidence for mitochondrial defects associated with beta-cell dysfunction in a mouse model of type 2 diabetes
.
Diabetes
2010
;
59
:
448
459
[PubMed]
27.
Jeon
MJ
,
Leem
J
,
Ko
MS
, et al
.
Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes
.
Exp Mol Med
2012
;
44
:
562
570
[PubMed]
28.
Lee
JW
,
Kim
WH
,
Yeo
J
,
Jung
MH
.
ER stress is implicated in mitochondrial dysfunction-induced apoptosis of pancreatic beta cells
.
Mol Cells
2010
;
30
:
545
549
[PubMed]
29.
Jeans
AF
,
Oliver
PL
,
Johnson
R
, et al
.
A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse
.
Proc Natl Acad Sci U S A
2007
;
104
:
2431
2436
[PubMed]
30.
Guillam
MT
,
Dupraz
P
,
Thorens
B
.
Glucose uptake, utilization, and signaling in GLUT2-null islets
.
Diabetes
2000
;
49
:
1485
1491
[PubMed]
31.
Seghers
V
,
Nakazaki
M
,
DeMayo
F
,
Aguilar-Bryan
L
,
Bryan
J
.
Sur1 knockout mice. A model for K(ATP) channel-independent regulation of insulin secretion
.
J Biol Chem
2000
;
275
:
9270
9277
[PubMed]
32.
Kataoka
K
,
Han
SI
,
Shioda
S
,
Hirai
M
,
Nishizawa
M
,
Handa
H
.
MafA is a glucose-regulated and pancreatic beta-cell-specific transcriptional activator for the insulin gene
.
J Biol Chem
2002
;
277
:
49903
49910
[PubMed]
33.
Matsuoka
TA
,
Artner
I
,
Henderson
E
,
Means
A
,
Sander
M
,
Stein
R
.
The MafA transcription factor appears to be responsible for tissue-specific expression of insulin
.
Proc Natl Acad Sci U S A
2004
;
101
:
2930
2933
[PubMed]
34.
Piccand
J
,
Strasser
P
,
Hodson
DJ
, et al
.
Rfx6 maintains the functional identity of adult pancreatic β cells
.
Cell Rep
2014
;
9
:
2219
2232
[PubMed]
35.
Gu
C
,
Stein
GH
,
Pan
N
, et al
.
Pancreatic beta cells require NeuroD to achieve and maintain functional maturity
.
Cell Metab
2010
;
11
:
298
310
[PubMed]
36.
Malhotra
JD
,
Kaufman
RJ
.
ER stress and its functional link to mitochondria: role in cell survival and death
.
Cold Spring Harb Perspect Biol
2011
;
3
:
a004424
[PubMed]
37.
Kim-Muller
JY
,
Fan
J
,
Kim
YJ
, et al
.
Aldehyde dehydrogenase 1a3 defines a subset of failing pancreatic β cells in diabetic mice
.
Nat Commun
2016
;
7
:
12631
[PubMed]
38.
Chau
GC
,
Im
DU
,
Kang
TM
, et al
.
mTOR controls ChREBP transcriptional activity and pancreatic β cell survival under diabetic stress
.
J Cell Biol
2017
;
216
:
2091
2105
[PubMed]
39.
Burlison
JS
,
Long
Q
,
Fujitani
Y
,
Wright
CV
,
Magnuson
MA
.
Pdx-1 and Ptf1a concurrently determine fate specification of pancreatic multipotent progenitor cells
.
Dev Biol
2008
;
316
:
74
86
[PubMed]
40.
Puri
S
,
García-Núñez
A
,
Hebrok
M
,
Cano
DA
.
Elimination of von Hippel-Lindau function perturbs pancreas endocrine homeostasis in mice
.
PLoS One
2013
;
8
:
e72213
[PubMed]
41.
Sartori
DJ
,
Wilbur
CJ
,
Long
SY
, et al
.
GATA factors promote ER integrity and β-cell survival and contribute to type 1 diabetes risk
.
Mol Endocrinol
2014
;
28
:
28
39
[PubMed]
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Supplementary data