Elevation of transcription factor Islet-1 levels in vivo increases β-cell function but not β-cell mass

Abstract

A decrease in the expression of Islet-1 (Isl-1), an islet transcription factor, has been reported in several physiological settings of reduced β-cell function. Here, we investigate whether an increased level of Isl-1 in islet cells can enhance β-cell function and/or mass. We demonstrate that transgenic mice with Isl-1 overexpression display improved glucose tolerance and enhanced insulin secretion without significant changes in β cell mass. From our microarray study, we identify approximately 135 differentially expressed genes in the islets of Isl-1 overexpressing mice that have been implicated to function in numerous biological processes including protein trafficking, metabolism and differentiation. Using real-time PCR we have confirmed upregulation of Caps2, Sec14l4, Slc2a10, P2rx7, Afamin, and Neurogenin 3 that may in part mediate the observed improved insulin secretion in Isl-1 overexpressing mice. These findings show for the first time that Isl-1 is a key factor in regulating adult β cell function in vivo, and suggest that Isl-1 elevation could be beneficial to improve glucose homeostasis.

Keywords: :

• Islet-1
• glucose tolerance
• insulin
• transcription factor
• β-cells

Introduction

Diabetes mellitus is a multi-organ disease that results from the loss of β-cell function in the pancreas and insulin resistance in peripheral tissues. To understand the pathophysiology of diabetes, gene expression analyses of multiple tissues, including pancreatic islets, have been performed in different physiological conditions of altered β-cell function.^1,2 By analyzing changes in gene expression under different conditions such as obesity, strain and age, Keller and colleagues have detected a decrease of Islet-1 (Isl-1) expression in the islets of obese diabetic mice.¹ Similarly, aging can induce a distinct gene expression program in mouse islets, leading to a decrease in the expression of a small number of genes, including Isl-1.^2-5 Taken together, these findings suggest that a decrease in Isl-1 expression might contribute to the pathophysiology of the disease and an increase in Isl-1 expression might be beneficial for a β-cell to maintain its full functionality.

Isl-1, a LIM-homeodomain transcription factor and a known Insulin gene enhancer binding protein, is expressed in the developing pancreas and adult islet cells.^6-9 Using genetic approaches in mice, Isl-1 has been shown to play an important role during early pancreatic development as well as during maturation of the islet cells at the secondary transition.^6,10 Because most of the in vivo analyses for Isl-1 function in the pancreas reported thus far have been focused on studies performed using loss-of function mouse models,^6,10,11 we set out to test whether increasing Isl-1 expression and thus activity in islets would impact adult β-cell function and mass using transgenic overexpression in mice. Our findings suggest that a modest increase of Isl-1 expression in islets is sufficient to enhance β-cell function, but not adequate to increase β-cell mass in vivo.

Results

Increased Isl-1 expression leads to improved β-cell function

To investigate the role of Isl-1 in adult islets and to test the hypothesis that β-cell function can be enhanced upon Isl-1 overexpression, a series of functional tests were performed in control and Isl-1 overexpressing (Pdx1^PB-Isl-1-Myc) mice.¹¹ In this model, a Myc-tagged rat Isl-1 gene was cloned downstream of the Pdx1^PB fragment which has been shown to drive islet-specific expression of Pdx1 and is active as early as E11.5.^12,13 Despite the known early transgenic activity of the Pdx1^PB fragment in the developing islet cells,^12,13 we only detected a few β-cells expressing the transgene at E17.5 (Fig. 1A-B). It was not until adulthood that we observed a significant expression of the transgene in Pdx1^PB-Isl-1-Myc mice demonstrated by Myc immunostaining with substantial islet specific expression of Isl-1-Myc proteins (Fig. 1C-D). We detected approximately one-third of β-cells in 2-mo-old Pdx1^PB-Isl-1-Myc mice with the Isl-1-Myc protein (Fig. 1D).¹¹ We previously reported a 2-fold increase in total Isl-1 protein and mRNA, as detected by western blot and real-time PCR, respectively.¹¹

Figure 1. Physiological increase of Isl-1 expression is sufficient to enhance β-cell function in adult mice. (A-D) Immunohistochemical analysis for insulin and Myc expression in E17.5 and 2-mo-old control and Pdx1^PB-Isl-1-Myc mice. (E) Random Fed blood glucose levels (n > 5). (F) Random Fed blood insulin levels (n > 8). (G) Fasted blood glucagon levels. (H-I) Total pancreatic insulin and glucagon content. (J) Body weight of 2-mo-old control and Pdx1^PB-Isl-1-Myc mice. (K) Glucose tolerance test demonstrates Pdx1^PB-Isl-1-Myc mice exhibit improved glucose clearance (n > 10). (L) Measurements of plasma insulin levels during glucose tolerance test show increased insulin secretion in Pdx1^PB-Isl-1-Myc mice. (M) Static incubation analysis demonstrates enhanced insulin secretion in islets of Pdx1^PB-Isl-1-Myc mice challenged with 16mM glucose. (N) Body weight of 9-mo-old control and Pdx1^PB-Isl-1-Myc mice. (O) Glucose tolerance test demonstrates Pdx1^PB-Isl-1-Myc mice exhibit modest improvement of glucose tolerance (n > 6). (P) Measurements of plasma insulin levels during glucose tolerance test show increased insulin secretion in 9-mo-old Pdx1^PB-Isl-1-Myc mice. (Q) Static incubation analysis demonstrates enhanced insulin secretion in islets of 9-mo-old Pdx1^PB-Isl-1-Myc mice challenged with 16mM glucose and 0.2 μM glyburide (n > 3 animals/group). Data represents the mean ± SEM * P-value < 0.05.

From our examination of the pancreatic sections, we found no obvious changes in the overall appearance of the pancreatic islets in Pdx1^PB-Isl-1-Myc mice when compared with control littermates.¹¹ We also did not detect changes in circulating glucose, insulin or glucagon levels in two-month-old Pdx1^PB-Isl-1-Myc mice (Fig. 1E-G). However, a significant improvement in clearing glucose was observed in Pdx1^PB-Isl-1-Myc mice during glucose tolerance tests (Fig. 1J-K), which was attributed to an enhanced insulin secretory response (Fig. 1L). This enhancement in insulin release was also observed in our in vitro static incubation studies in which isolated islets from control and Pdx1^PB-Isl-1-Myc mice were treated with low and high concentration of glucose (Fig. 1M). As animals aged, 9-mo-old Pdx1^PB-Isl-1-Myc mice continued to show improved insulin secretion; however, despite this increase, we only detected a modest improvement in glucose tolerance tests, suggesting chronic high levels of insulin in Pdx1^PB-Isl-1-Myc mice likely lead to increased body weight and eventual insulin insensitivity (Fig. 1N-P). To further investigate if other insulin secretagogues can also enhance insulin release in Pdx1^PB-Isl-1-Myc mice, isolated islets from both groups were treated for 15 min with 200 μM glyburide, a sulfonylurea that blocks K_ATP channels leading to a fully depolarized plasma membrane, resulting in insulin secretion.^14,15 Compared with the control islets, glyburide-treated islets from Pdx1^PB-Isl-1-Myc mice displayed a robust increase in insulin release similar to what was seen in the 16 mM glucose-treated islets (Fig. 1Q). These data indicate that islets with a higher level of Isl-1 have the ability to release more insulin upon glucose stimulation, which is similar to treatment with high dose of glyburide.

Isl-1 overexpression does not increase β-cell mass

The enhanced response to glyburide observed in the islets of Pdx1^PB-Isl-1-Myc mice pointed to a possibility of increased insulin production. Therefore, we examined total pancreatic insulin content of control and Pdx1^PB-Isl-1-Myc mice, which surprisingly showed no significant differences (Fig. 1H). Total glucagon content was also measured which also showed comparable levels between control and Pdx1^PB-Isl-1-Myc mice (Fig. 1I). Recently, it has been shown that rat islets, when transduced with lentivirus overexpressing Isl-1, increased cell proliferation, which was the result of an upregulation of c-Myc and CyclinD1.¹⁶ Therefore, we tested whether increased Isl-1 expression in vivo affected β- or α-cell mass in the Pdx1^PB-Isl-1-Myc mice. Unlike what was reported, we found no significant differences in β-cell or α-cell mass between control and Pdx1^PB-Isl-1-Myc mice likely due to the lower level of Isl-1 expression (Fig. 2A-F). To investigate whether cell survival and/or proliferation were affected in the Pdx1^PB-Isl-1-Myc mice, we performed immunostaining for Caspase 3 and Ki67, respectively, and found no significant difference (Fig. 2G-H; and data not shown). Real-time PCR analysis of isolated islets for Ki67 and Birc5 expression, markers for proliferating cells,¹⁷ further confirmed that there were no significant changes in proliferation in the islets of Pdx1^PB-Isl-1-Myc mice (Fig. 2I). Taken together, these findings suggest that while a 2-fold increase of Isl-1 is sufficient to enhance β-cell function, this increase is not enough to promote β- or α-cell mass expansion in vivo.

Figure 2. Isl-1 overexpression does not affect β cell mass and cell proliferation in 2-mo-old adult mice. Immunostaining analysis for insulin (A-C) and glucagon (D-F) were performed to measure β- and α-cell mass in control and Pdx1^PB-Isl-1-Myc mice. Immunostaining analysis for Ki67 (G-H) shows no significant changes in β-cell proliferation. (I) Real-time PCR analysis shows comparable mRNA levels of Ki67 and Birc5 in islets between control Pdx1^PB-Isl-1-Myc mice. Bars represent the mean ± SEM.

Altered gene expression in the islets of Pdx1^PB-Isl-1-Myc mice

To evaluate the molecular targets that may mediate improved β-cell function in Pdx1^PB-Isl-1-Myc mice, we performed microarray analysis on purity-matched islets from two-month-old control and Pdx1^PB-Isl-1-Myc mice. We detected 135 differentially expressed genes implicated to function in protein trafficking, metabolism, development, signal transduction, and transcriptional regulation (Fig. 3A and C). To validate results obtained from the microarray study, we chose several candidate genes from each of the six categories and focused specifically on several transcripts that have implicated functions in exocytosis, secretion and transport in other systems (Fig. 3B). From our real-time PCR, we detected an increase in the expression of Afamin,²³ Caps2,^21,22 Slc2a10,^26,27 Sec14l4,^24,25 and P2rx7^18-20 as well as Neurogenin3²⁸ in the Pdx1^PB-Isl-1-Myc islets (Fig. 3B). In the canonical model of insulin secretion, following a rise in blood glucose, ATP/ADP ratio increases which leads to ATP-sensitive K⁺ channels closure. This closure triggers membrane depolarization, which in turn increases intracellular Ca²⁺ concentration leading to insulin granule exocytosis. Interestingly, both Caps2 and P2rx7 have implicated functions in regulating secretion by affecting Ca²⁺ signaling through binding to Ca²⁺ (Caps2) and mediating the effect of ATP (P2rx7), respectively.^18-22 In contrary, while Afamin and Sec14l4 have no known functions in mediating insulin secretion, they have been implicated in transport of α-tocopherol, a form of vitamin E.^23-25 Based on their involvement in transport, these proteins may play crucial roles in regulating exocytosis and vesicular transport in islet β-cells. Additionally, with the implicated functions of Slc2a10 (Glut10), a facilitated glucose transporter and Neurogenin3 in maintaining glucose homeostasis,^26-31 our study suggest that increased levels of these genes potentially lead to the enhanced β-cell function seen in the Pdx1^PB -Isl-1-Myc transgenic mice. Similar to an increase of Arx expression demonstrated previously by real-time PCR,¹¹ an increase in MafA expression was also detected (Fig. 3D), although neither of these genes was detected in the microarray study, likely due to sensitivity issues with gene expression arrays.³² We also attempted to confirm an increase in the expression of several other genes that may be involved in secretion, including glutamate receptor (Grin2c),³³ non voltage-gated sodium channel (Scnn1a),^34,35 and sperm acrosome associated 1 (Spaca1),^36,37 but were unable to validate upregulation of these genes. Taken together, our results suggest that Isl-1 may mediate increased β-cell functionality through a subset of genes involved in protein trafficking, metabolism, development, signal transduction, and transcriptional regulation.

Figure 3. Genome-wide gene expression analysis of 2-mo-old control and Pdx1^PB-Isl-1-Myc islets. (A) Diagram summarizes functions of the 135 differentially expressed genes from the gene expression analysis. A 10% False Discovery Rate was used in this analysis. (B) Real-time PCR analysis confirmation of a subset of upregulated genes chosen from the microarray gene list. Bars represent the mean ± SEM. All changes were significant with p value < 0.05. (C) List of genes that are differentially expressed in the microarray study. (D) Additional genes evaluated by real-time PCR in islets of control and Pdx1^PB-Isl-1-Myc mice.

Discussion

In this study, we investigate whether islet-specific Isl-1 overexpression in vivo can enhance β-cell function and cell proliferation. Although only about one third of β-cells activate the transgene,¹¹ the insulin secretory response in Pdx1^PB-Isl-1-Myc mice was significantly improved. Additionally, an increase of Isl-1 expression in the islet α- or δ-cells might also alter glucose homeostasis in Pdx1^PB-Isl-1-Myc mice. However, based on the small number of these non-β-cells expressing the Isl-1-Myc transgene,¹¹ we conclude that much of the improved glucose tolerance phenotype is due to Isl-1 overexpression in β cells.

A portion of our phenotype observed may be due to an increase in the expression of MafA, which is known to control genes implicated in insulin secretion.^38-40 However, we have also identified ~135 differentially expressed genes from our microarray and were able to confirm an increase in expression in a subset of these genes by real-time PCR in islets of Pdx1^PB-Isl-1-Myc mice. These genes are implicated to function in protein trafficking and metabolism, and may play important roles in β-cell biology. In this analysis, we were able to detect an increase in Slc2a10 (Glut10), a gene that has been implicated in glucose metabolism and type 2 diabetes,^26,27 increased levels of SEC14L4, a factor that has been implicated to be involved in secretion in organs like salivary gland, prostate and pancreas,^24,25 and a 35-fold increase in Afamin levels, a transporter for α-tocopherol in the CNS.²³ Additionally, P2rx7, a purinergic receptor with implicated function for regulating both endocrine and exocrine pancreas function and Caps2, a calcium binding protein were also increased.^18-22 Although the precise function of these genes and the molecular mechanisms by which they impact β-cell physiology remain to be fully characterized, it is tempting to speculate that increased levels of some of these genes may have a positive impact on exocytosis, granule transport and secretion. Future studies manipulating these genes in either animal models or cell culture might provide further insights into the function of these genes in adult β-cells.

Although the Notch/Neurogenin3 pathway has been shown to play critical roles in the maintenance of the pancreatic progenitor pool and in the differentiation into exocrine and endocrine lineages,^29,41,42 it has recently been shown to also play an important role in the regulation of β-cell maturation and function.²⁸ Therefore, a 2-fold increase in Neurogenin3 expression is likely mediating part of the enhanced β-cell function in the Pdx1^PB-Isl-1-Myc mice. In addition, Neurogenin3 has also been implicated in regulating adult β-cell survival as a 4-fold increase in Neurogenin3 expression result in apoptosis.³⁰ Similarly, expression of Neurogenin3 in islets is negatively regulated by Musashi, a RNA binding protein with antiapoptotic roles in adult β-cells.^30,31 Although it is not clear whether a modest increase in Neurogenin3 levels is physiologically relevant for β-cells, our observation fits with the current model that the adult β-cell is likely sensitive to Neurogenin3 levels and that differing levels of Neurogenin3 in β-cells result in activation of different gene programs (e.g., secretion vs. apoptosis).^28,30,31

A large network of islet transcriptional regulators is involved in the regulation of β cell development and function.⁴³ Recently, it has been demonstrated that stress-induced hyperglycemia could be prevented in mice overexpressing Pax4, a β-cell specific gene.⁴⁴ Furthermore, in contrast to what was shown in cell lines, increased expression of Nkx6.1 in mice, another key β-cell transcriptional regulator, had no impact on β-cell mass or glucose clearance.^45,46 Taken together, these findings demonstrate the importance of using gain-of-function studies in vivo to understand the sufficiency of any islet transcription factors.

In summary, microarray analysis provides an ideal approach for comparing changes in gene expression on a large scale. We propose that using an Isl-1 overexpressing transgenic mouse model provides a novel platform for expanding our understanding of how a small increase in transcription factor expression can lead to a significant impact in insulin secretion in adult mice. We have identified a group of genes, many of which have not been fully characterized in islet β-cells, which may contribute significantly to the phenotype we observe. Although we did not find any significant Isl-1 binding within the genomic regions of some of these differentially expressed genes in our Isl-1 ChIP-Seq data set (unpublished data), this observation does not exclude direct binding of Isl-1 to these genes as regulatory domains of many mammalian genes can be far away from the gene itself. Alternatively, these genes might be direct targets of additional transcriptional regulator(s) yet to be identified using PCR-based transcription factor screens or RNA-Seq.³² Furthermore, while Guo and colleagues had reported that Isl-1 can promote cell proliferation and attenuate cell death against oxidative stress in isolated rat islets and HIT-T15 cells,¹⁶ we did not detect significant changes in cell proliferation or cell survival in our transgenic mouse model. The discrepancy between these findings might simply due to the high levels of Isl-1 expression achieved in the lentivirus-based in vitro model, which might lead to activation of different gene programs.¹⁶

In conclusion, these novel findings provide the islet biology community with the gene expression profile of enhanced β-cell function. Future experiments designed to manipulate these genes in mouse models or cell cultures will lead to better understanding of how insulin secretion is regulated and how glucose homeostasis is maintained in adult animals.

Materials and Methods

Animals

Pdx1^PB -Isl-1-Myc male mice used in this study were kept on a C57BL/6J background. Although only one Pdx1^PB -Isl-1-Myc transgenic mouse line was characterized in our study, which we acknowledged it as a caveat, we are confident that the effects we observe in the Pdx1^PB -Isl-1-Myc mice are due to the elevation of Isl-1 expression as Arx and MafA, two genes that are downregulated in Isl-1 deficient pancreata,^10,11 were increased the islets of the Pdx1^PB -Isl-1-Myc transgenic mice. Animal experiments were approved by the Children’s Hospital of Philadelphia’s Institutional Animal Care and Use Committee.

Glucose and insulin measurement

Blood glucose levels were measured from tail vein blood using an automatic glucometer (One Touch Ultra; LifeScan). Plasma was separated from whole blood using heparinized tubes (BD Microtainer, BD, 365958) and total pancreatic proteins were extracted with acid-ethanol. Insulin protein levels were measured using ELISA kit (Mercodia, 10-1113-01).

Immunohistochemistry and immunofluorescence

Pancreata from control and mutant mice (n > 3/group) were removed, weighed, fixed in 4% PFA and embedded in paraffin. For α- and β-cell mass measurements, slides with the maximum footprint (three slides per animal) were subjected to immunohistochemical staining for glucagon or insulin. Hormone positive areas were measured using Aperio software. Cell mass was obtained by hormone-positive area percentage times the weight of the pancreas. The following primary antibodies were used: insulin (1:1000; Linco, 1013), glucagon (1:3000; Linco, Cat. No. 4030-01F) and K_i 67 (1:1000; Leica, NCL-Ki67p). Cy3-conjugated secondary antibody (1:600) was from Jackson ImmunoResearch.

Glucose tolerance test

Following a 16 h fast, animals (n = 10/group) were injected with glucose (2 mg/g body weight) in sterile PBS. Blood glucose levels were monitored at 0, 15, 30, 60, 90 and 120 min after injection.

Islet static incubation

Islets were isolated using the standard collagenase procedure.⁴⁷ About 50 islets from each animal were cultured with islet medium (RPMI 1640, 10%FBS, 1x penicillin/streptomycin, 1 x glutamine, 5 mM Hepes, 5 mM Glucose). After overnight incubation at 5% CO₂, 37°C, the islets were stimulated with low glucose (2.5 mM), then high glucose (16 mM) for 30 min sequentially. Mouse Insulin ELISA was performed to measure insulin levels. Four animals from each group were used for this assay. All values were normalized by total islet insulin content.

Microarray and quantitative real-time PCR

Total RNA from isolated islets was extracted in TRIzol (Invitrogen, 15596-026) and then purified using RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer’s instructions. Four pairs of control and transgenic islets were purity matched,⁴⁸ and hybridized to Agilient Gene expression Array for dual color expression analysis by the DRC Functional Genomics Core of the Penn Institute for Diabetes, Obesity and Metabolism. For real-time PCR, approximately 1 μg RNA was reverse transcribed into cDNA. Real-time PCR reactions were performed using the Brilliant SYBR Green PCR Master Mix (Stratagene, 600828) on Mx3005 Multiplex Quantitative PCR System (Stratagene). All reactions were performed in triplicate with reference dye normalization and medium values were used for analysis. Primer sequences are available upon request.

Acknowledgments

We thank the members of the Molecular Pathology and Imaging Core in the Center for Molecular Studies in Digestive and Liver Diseases (P30-DK050306) for sample processing and Dr Jonathan Schug and the members of the DRC Functional Genomics and Radioimmunoassay/Biomarkers Cores of the Penn Institute for Diabetes, Obesity and Metabolism (DERC: P30-DK19525) for conducting microarray and RIA experiments. We are grateful to Dr Klaus Kaestner and Dr Christine Reid for the critical reading of the manuscript and Dr Changhong Li for the advice on static incubation studies. C.L.M. was supported by NIH-DK078606 and JDRF-2-2007-730.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.