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Endocrinology Vol. 143, No. 8 3152-3161
Copyright © 2002 by The Endocrine Society

ARTICLE

Insulinotropic Hormone Glucagon-Like Peptide-1 Differentiation of Human Pancreatic Islet-Derived Progenitor Cells into Insulin-Producing Cells

Elizabeth J. Abraham, Colin A. Leech, Julia C. Lin, Henryk Zulewski and Joel F. Habener

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114. E-mail: . jhabener{at}partners.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucagon-like peptide-1 (GLP-1) is an intestinal incretin hormone, derived from the processing of proglucagon, that exerts insulinotropic actions on insulin-producing pancreatic islet ß-cells. Recently GLP-1 was shown to stimulate the growth and differentiation (neogenesis) of ß-cells and appears to do so by inducing the expression of the homeodomain protein IDX-1 (islet duodenum homeobox-1; also known as PDX-1, pancreatic and duodenal homeobox gene; and as IPF-1, insulin promoter factor), which is required for pancreas development and the expression of ß-cell-specific genes. Earlier we identified multipotential progenitor cells in the islet and ducts of the pancreas, termed nestin-positive islet-derived progenitor cells (NIPs). Here we report the expression of functional GLP-1 receptors on NIPs and that GLP-1 stimulates the differentiation of NIPs into insulin-producing cells. Furthermore, confluent NIP cultures express the proglucagon gene and secrete GLP-1. These findings suggest a model of islet development in which pancreatic progenitor cells express both GLP-1 receptors and proglucagon with the formation of GLP-1. Locally produced GLP-1 may act as an autocrine/paracrine developmental morphogen on receptors on NIPs, resulting in the activation of IDX-1 and the expression of the proinsulin gene conferring a ß-cell phenotype. GLP-1 may be an important morphogen both for the embryonic development of the pancreas and for the neogenesis of ß-cells in the islets of the adult pancreas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PREVALENCE of diabetes mellitus is increasing throughout the world. Diabetes is caused to a large extent by a reduction in the fully functioning mass of insulin-producing ß-cells that reside within the islets of Langerhans in the pancreas. As a consequent of a reduced mass of pancreatic ß-cells, the amounts of insulin produced are insufficient to meet the body’s needs, and hyperglycemia ensues (1, 2). Although recent studies indicate that islet transplantation may be a cure for diabetes (3), the availability of pancreata as a source for islet transplantation is severely limited. Therefore, it will be necessary to develop alternative sources of islet tissue. One such source may be progenitor cells that can be expanded ex vivo, differentiated into islet tissue, and transplanted.

The glucagon gene encodes a multifunctional proglucagon that is differentially processed by prohormone convertases 1 and 2 in the pancreas and the intestine. In the {alpha}-cells of the pancreas, the major product of proglucagon processing is glucagon, although small amounts of glucagon-like peptide-1 (GLP-1) are produced, whereas in intestinal L cells the major proglucagon-derived products are GLP-1 and GLP-2 (4, 5). However, in streptozotocin-induced diabetic rats there is a robust increase in pancreatic prohormone convertases 1 and 2, resulting in a 2-fold increase in the ratio of amidated GLP-1 to total glucagon immunoreactivity (6), indicating that GLP-1 may play a role in regeneration of ß-cell mass in a diabetic animal model. GLP-1 binds to specific G protein-coupled receptors on pancreatic ß-cells to stimulate insulin secretion via cAMP-dependent pathways (4, 5). When administered to diabetic mice, GLP-1 lowers blood glucose levels and stimulates insulin secretion (7, 8, 9, 10). In addition, GLP-1 increases ß-cell mass by inducing the differentiation and neogenesis of ductal progenitor cells into islet endocrine cells (8, 9, 10, 11, 12). The antidiabetogenic potential of GLP-1 is currently under investigation and shows promise as a therapeutic agent in the treatment of type 2 diabetes (13).

Recently, we identified a distinct population of cells in pancreatic islets and ducts that expresses nestin (14). These nestin-positive islet-derived progenitor cells (NIPs), isolated from adult pancreatic islets, can differentiate in culture into cells with pancreatic exocrine, endocrine, and hepatic phenotypes. We hypothesized that GLP-1 receptors (GLP-1R) must be present on NIPs and that binding of GLP-1 to its receptors on these cells results in activation of the transcription factor IDX-1, a master regulator of endocrine pancreas development (15, 16). IDX-1 then activates the expression of the insulin gene, resulting in a ß-cell phenotype (8, 12).

Here we show the expression of GLP-1 receptors on NIPs and that GLP-1 functionally activates NIPs by virtue of their depolarization and resultant increase in intracellular calcium. Notably, the activation of NIPs by GLP-1 is paradoxically inhibited in conditions of high (20 mM) ambient glucose concentrations, unlike the direct glucose-dependent activation of ß-cells by GLP-1. Further, we show that GLP-1 stimulates the differentiation of NIPs into a pancreatic endocrine phenotype that expresses the homeodomain protein IDX-1 and the hormones insulin, glucagon, and GLP-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
GLP-1-(7–36)amide was obtained from Sigma (St. Louis, MO). Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), leukemia inhibitory factor were obtained from Sigma and Chemicon International (Temecula, CA), respectively. B-27 supplement was obtained from Life Technologies, Inc. (Gaithersburg, MD).

Isolation and culture of NIPs
Human islet tissue was obtained from the Juvenile Diabetes Research Foundation Center for Islet Transplantation, Harvard Medical School (Boston, MA), and the Diabetes Research Institute, University of Miami School of Medicine (Miami, FL). NIPs were isolated as described previously (14). Briefly, islets were washed and cultured in RPMI 1640 medium containing serum, 11.1 mM glucose, antibiotics, sodium pyruvate, ß-mercaptoethanol, and growth factors. Within several days, nestin-positive cells grew out from islets. These cells were cloned and expanded in medium containing 20 ng/ml each of bFGF and EGF. In some instances, long-term passaged cells were maintained in 1000 U recombinant human leukemia inhibitory factor. For differentiation, NIPs were incubated with GLP-1 in the absence of serum, and fresh GLP-1 was added every 48 h without changing the medium. In some experiments differentiation was achieved by culturing NIPs in cell culture medium containing B-27 [DMEM/F-12 (1:1), B-27, bFGF, EGF, and antibiotics] as described by Toma et al. (17) for the culture of skin-derived precursors and for the differentiation of mouse embryonic stem cells (18). Similar to the skin precursors (17), NIPs cultured in B-27 medium generated spherical clusters of cells that were collected, centrifuged, and replated onto laminin-coated 48-well plates [BD Biosciences, Bedford, MA] and cultured in the B-27-supplemented medium now containing 10 nM GLP-1 and no other added growth factors, i.e. bFGF and EGF were absent.

Antibodies
We used rabbit polyclonal antisera to rat IDX-1 (14) and rat GLP-1 receptor (19), which cross-reacts with its human counterpart. The rabbit antihuman nestin was a gift from Dr. C. Messam (NINDS, NIH, Bethesda, MD). Guinea pig antiinsulin and antiglucagon sera were obtained from Linco Research, Inc. (St. Charles, MO). Cy-3- and Cy-2-labeled secondary antisera were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Immunocytochemistry
Cells cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) or gridded coverslips (Bellco Glass, Inc., Vineland, NJ) were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After several rinses in PBS, cells were permeabilized with methanol/Triton-X in some instances, blocked with normal donkey serum for 30 min, and incubated with primary antiserum or preimmune sermum at 4 C. The following day, cells were rinsed with PBS and incubated with secondary antisera (donkey antirabbit and donkey antiguinea pig) labeled with Cy-3 or Cy-2 for 1 h at room temperature. After several washes, coverslips containing cells were mounted onto slides in mounting medium (Vector Laboratories, Inc., Burlingame, CA). Fluorescence images were obtained using a Carl Zeiss (New York, NY) epifluorescence microscope equipped with an Optronics TEC-470 CCD camera (Optronics Engineering, Goleta, CA) interfaced with a Powermac 7100. IP lab Spectrum software (Signal Analytics, Vienna, VA) was used to acquire and analyze images.

RT-PCR
Total cellular RNA prepared from NIP cultures or human islets were reverse transcribed and amplified by PCR for 40–45 cycles as described previously (20). Oligonucleotides for the PCR were as follows: human GLP-1 receptor: forward, 5'-gtgtggcggccaattactac-3'; reverse, 5'-cttggcaagtctgcatttga-3'; and human glucagon: forward, 5'-atctggactccaggcgtgcc-3'; reverse, 5'-agcaatggattccttggcag-3'. An RT-negative control was run for most samples. PCR cycling for glucagon was at 94 C for 1 min, followed by 94 C for 10 sec, 56 C for 10 sec, and 72 C for 1 min (40 cycles), followed by 72 C for 2 min. A hot start PCR was performed for human GLP-1 receptor (GLP-1R) as follows: 94.5 C for 5 min, followed by addition of Taq polymerase and subsequent cycling at 94 C for 10 sec, 54 C for 10 sec, and 72 C for 10 sec (45 cycles). Primer extension at 72 C was performed for an additional 2 min.

Intracellular calcium ([Ca2+]i) measurements
NIPs plated on gridded coverslips were loaded with fura-2 by incubation in standard extracellular saline (138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES) containing 5.6 mM glucose and supplemented with 2% fetal bovine serum, 0.01% pluronic F-127, and 5 µM fura-2/AM. Cells were loaded for 90 min at room temperature, washed with standard extracellular saline, and then transferred to a Peltier temperature-controlled stage at 32 C. Human serum albumin (0.05%) was added as a carrier protein during experiments with GLP-1-(7–36)amide and Exendin-(9–39), a specific antagonist of GLP-1. Calcium measurements were taken at 0.25 Hz using an IonOptix (Milton, MA) imaging system. The grid location was noted, and fluorescence images of the cells were recorded for subsequent identification of the cells for immunohistochemical staining. The solution in the recording chamber was exchanged by a gravity-fed perfusion system.

RIA
Insulin levels in culture media were measured by an ultrasensitive RIA kit purchased from Linco Research, Inc. and Diagnostic Products (Los Angeles, CA). The detection level for the insulin assay was 8 pg/ml. GLP-1 levels in culture media were measured by a GLP-1-specific RIA that uses rabbit antiserum raised against the C terminus of GLP-1-(7–36)amide and does not cross-react with glucagon or proglucagon.

Transfections
A fragment of the rat insulin I gene promoter that spans nucleotides -410 to 49 bp was fused to the coding sequence of luciferase in the pxp2 basic vector to generate the insulin promoter-luciferase construct (INS-LUC) (21). The human insulin promoter factor-1 (IPF-1) cDNA was a gift from Henk-Jan Anstoot (Sophia Children’s Hospital, Rotterdam, The Netherlands). This cDNA was transferred to a cytomegalovirus 5 promoter vector in our laboratory (22). The rat IDX cDNA was cloned previously in our laboratory (23). Adherent NIP cultures plated in 12-well dishes were transfected with 0.6 µg rat INS-LUC and/or 0.125 µg rat IDX-1 cDNA/well for 5 h in serum-free culture medium using Lipofectamine 2000 (2.5 µl/well; Life Technologies, Inc.). A filler plasmid DNA was used to bring the DNA concentration to 1 µg/well. Then, cells were exposed to test substances in medium supplemented with 10% serum. After 20–24 h, cells were lysed, and luciferase activity was measured using a luciferase assay kit (Promega Corp., Madison, WI) in a luminometer (Wallac, Inc., Gaithersburg, MD). These experiments were carried out in duplicate wells and repeated at least three times. In other instances, NIP cultures were plated onto 4-well Lab-Tek chamber slides and transfected with human IPF-1 cDNA (0.2 µg/well) using Geneporter (Life Technologies, Inc.). The following day, transfected cells were incubated with GLP-1 (1–10 nM) in serum-supplemented medium. After 3–4 d, cells were fixed with 4% paraformaldehyde and subjected to immunostaining for IDX-1 and insulin.

Western immunoblot
NIP cultures plated in 10-cm dishes were either transfected with human IPF-1 cDNA using Geneporter or were left untransfected. These cells were subsequently treated with or without 10 nM GLP-1 in serum-supplemented medium for 3–4 d. Then, nuclear extracts were prepared according to the Schreiber method (24), and equal amounts of proteins (20 µg) were loaded and electrophoresed on premade NuPAGE (Invitrogen, Carlsbad, CA) gels according to the manufacturer’s recommendations. The proteins were transferred onto a nitrocellulose membrane and subjected to an IPF-1 immunoblot procedure as described previously (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GLP-1 receptor expression in NIPs
We examined human NIPs for the presence of GLP-1R by immunocytochemistry. Receptor immunoreactivity was detected in the majority of NIPs (>60%; Fig. 1AGo). To further confirm the immunocytochemical identification of GLP-1R in NIPs, we performed an RT-PCR of GLP-1 receptor mRNA prepared from NIP cells and detected the product of the correct size (346 bp) for the GLP-1R (Fig. 1BGo). Clonal variation in the relative amounts of GLP-1R mRNA between lines was seen; receptor expression was lower in some NIP clones than others (see clone 9 vs. 2 in Fig. 1BGo), but was undetectable in only a minority of clones. The expression of GLP-1 receptors in NIPs indicates the potential for GLP-1-mediated regulation of islet progenitor cell differentiation by GLP-1.



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  		Figure 1. Expression of GLP-1R on pancreatic islet-derived stem/progenitor cells. A, NIPs (passages 6–8) plated on gridded coverslips were fixed and subjected to immunocytochemical detection with antiserum to GLP-1R ({alpha}-GLP-1R) (Cy-3, rendered intense white on modifed photomicrograph) or a preimmune serum control. Note the punctate fluorescence on the surface of the cell, typical of receptor aggregation. B, RT-PCR of RNA prepared from different clones of NIPs (passages 4–8) using oligonucleotide amplimers to human GLP-1R give the predicted 346-bp product, which was confirmed by Southern blotting. Human islet tissue was used as the positive control.

 
Functional GLP-1 receptor signaling in NIPs
The application of GLP-1 -(7–36)amide to single isolated NIPs elevates [Ca2+]i. Cells were plated onto gridded coverslips to permit subsequent immunohistochemical staining of the same cells to test for nestin expression. All cells examined that increased [Ca2+]i in response to GLP-1 were nestin positive. In contrast to ß-cells prepared from adult human islets, in human NIPs, GLP-1 stimulated [Ca2+]i at basal (5.6 mM; n = 45 cells) glucose, but had no effect on [Ca2+]i in the presence of high (20 mM) glucose (Fig. 2AGo). These glucose-related effects on [Ca2+]i responses in NIPs were reproduced by forskolin (Fig. 2BGo), suggesting that the effects of GLP-1 on NIPs are mediated via the activation of Gs and cAMP production, the same signaling pathway used by GLP-1 in adult islet-derived ß-cells. However, the glucose dependence of GLP-1 in NIPs differs strikingly from that in adult ß-cells, inasmuch as in adult ß-cells the actions of GLP-1 are markedly directly dependent on glucose concentrations (25, 26). These findings suggest that the coupling of glucose signaling with cAMP signaling (25) in NIP progenitor cells is different from that in adult islet-derived ß-cells. The pretreatment of single isolated NIPs with the peptide exendin-(9–39), a specific antagonist of GLP-1, prevents the increase in [Ca2+]i mediated by GLP-1 (Fig. 2Go, C and D). These inhibitory effects of the GLP-1R antagonist exendin-(9–39) on [Ca2+]i responses suggest that the same isoform of GLP-1R is expressed in NIPs as that expressed in ß-cells. The increase in [Ca2+]i mediated by GLP-1 on NIPs was inhibited by extraellular La3+ (5 µM), indicating that GLP-1 is activating [Ca2+]i influx, consistent with its known role to depolarize ß-cells (Fig. 2EGo). We demonstrate further that tolbutamide (100 µM) stimulates the [Ca2+]i elevation in NIPs, indicating that NIPs must also express ATP-sensitive K+ channels (Fig. 2FGo). These findings suggest that GLP-1 induces membrane depolarization and activation of voltage-dependent Ca2+ channels in NIPs, consistent with its known mechanism of action in ß-cells. However, unlike its known actions in ß-cells, the activation of ion channels in NIPs is inhibited by high (20 mM) glucose.



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  		Figure 2. GLP-1-(7–36)amide and tolbutamide stimulate [Ca2+]i influx in NIPs. A, Fura-2-loaded cells bathed in 5.6 mM glucose show an increase in the [Ca2+]i response to 10 nM GLP-1. Increasing extracellular glucose to 20 mM (20 G) also caused an increase in [Ca2+]i, but application of GLP-1 in 20 mM glucose failed to produce an additional [Ca2+]i response. A third application of GLP-1 on returning to 5.6 mM glucose produced a [Ca2+]i response. These effects of GLP-1 on [Ca2+]i were observed in 45 different cells tested. B, The glucose-dependent effects of GLP-1 were reproduced by 10 mM forskolin, suggesting that the [Ca2+]i elevation is mediated by the cellular cAMP level. C, The GLP-1-mediated increase in [Ca2+]i was reversibly inhibited by 10 nM exendin-(9–39). This inhibitory effect was not due to receptor desensitization (D), because application of GLP-1 in the presence of the GLP-1 receptor antagonist exendin-(9–39) failed to produce a response, whereas subsequent applications of GLP-1 after washout of exendin-(9–39) produced repeated [Ca2+]i elevations. E, The GLP-1-mediated increase in [Ca2+]i is inhibited by 0.5 mM extracellular La3+, an inhibitor of Ca2+ influx, suggesting that GLP-1 stimulates Ca2+ influx. F, NIPs bathed in 5.6 mM glucose were stimulated with 100 mM tolbutamide (Tolbut.) and responded to repeated applications of tolbutamide with increases in [Ca2+]i. Application of 10 nM GLP-1 also stimulated an increase in [Ca2+]i, suggesting that GLP-1 acts by depolarizing the cells. Shown in each panel are representative recordings from single NIPs (clone 006a) that respond to the above-mentioned test substances.

 
GLP-1 induces differentiation of NIPs into insulin-secreting cells
Previous studies demonstrated the insulinotropic actions of GLP-1 as well as its ability to stimulate ß-cell neogenesis in partial pancreatectomized rats (9). Therefore, we determined whether GLP-1 would induce differentiation of human NIPs into insulin-secreting cells. As described previously (14), human islets were cultured in medium containing bFGF and EGF for 14 d. The majority of islets became degranulated, and a monolayer of cells grew out from them. Immunocytochemical analysis of such islet cultures showed that the outgrowing cells were mostly all nestin positive and insulin negative; just a few cells in the monolayer expressed insulin (Fig. 3AGo). NIPs were picked from these cultures and expanded in growth factor-supplemented medium (passage 1) for 3 d as described previously (14). In certain instances NIPs that were expanded for 3–5 d spontaneously expressed insulin. We find that at this stage of passage (30–40 cells/dish) the vast majority of NIPs were nestin positive and insulin negative (Fig. 3BGo). When NIP cultures were expanded for 7–12 d and then treated with GLP-1, a subset of cells became insulin positive (Cy-2; green) and nestin negative (Cy-3; red; Fig. 3Go, C and D). Incubation with exendin-(9–39), a specific antagonist of GLP-1, abolished the appearance of immunostaining for insulin (Fig. 3EGo). The cells treated with GLP-1 also changed their morphology, becoming more rounded and flattened (Fig. 3DGo vs. Fig. 3Go, C and E). The percentage of differentiated cell progeny by virtue of insulin staining varies from 5% or less to 30% depending on the particular cloned NIP culture tested. Treatment with exendin-4, a long-acting analog of GLP-1, induced a 2- to 3-fold increase in insulin secretion, as measured by RIA of the cell culture medium (Fig. 3GGo). The secretory response to exendin-4 was detected in 30% of the NIP clones tested. In some culture wells confluence alone was sufficient to initiate the secretion of small amounts of insulin, indicating that cell confluence can induce differentiation to some extent.



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  		Figure 3. GLP-1 induces differentiation of NIPs into insulin-producing cells. A, Human islets were cultured in growth medium containing bFGF and EGF for 14–18 d. The monolayer of cells that grew out from the islet was fixed with 4% formaldehyde and immunostained with antiserum for insulin (Cy-2, green) and nestin (Cy-3, red). The majority of cells that grew out from islets were nestin positive and insulin negative. B, NIPs that grew out from islets were picked, replated, expanded in the same medium (passage 1) for 3–5 d, fixed, and immunostained for nestin (red) and insulin (green). Most cells at this stage were nestin positive and insulin negative. C, Differentiation of NIP cultures treated with GLP-1. NIP cultures (clone 016f, passage 1) were expanded for 7–12 d. Between d 10 and 12, cultures were replenished with serum-free medium alone (control, C) or containing 10 nM GLP-1-(7–36)amide or GLP-1 plus exendin-(9–39) (100 nM; Ex9–39), a specific antagonist of GLP-1 (D and E, respectively). Seventy-two to 96 h later, cells were fixed and immunostained for nestin (Cy-3, red) and insulin (Cy-2, green). A subset of cells became nestin negative and insulin positive (indicated by white arrows, D). F, To control for background staining, GLP-1-treated cells were incubated with preimmune normal rabbit serum (red) and guinea pig IgG (green). Note the change in cell morphology when cells were treated with GLP-1 (D and F vs. C and E). G, Insulin secretion from NIP cultures treated with exendin-4. NIP cultures (passage 1) were expanded for 7–12 d and treated with 10 nM exendin-4, a GLP-1 agonist, for 48–72 h. Media were collected and assayed for insulin. Values are the mean ± SEM of four wells obtained from two different clones of NIPs.

 
Human NIPs that have been repeatedly passaged lose their ability to secrete insulin in response to GLP-1. However, a modification of the differentiation protocols of these cells can render them again responsive to GLP-1. In these modified experiments, long-term passaged NIPs (<=6 months) were cultured in medium supplemented with B-27, bFGF, and EGF for 4–6 d or more. Similar to skin-derived precursor stem cells (17), NIPs in this medium form floating clusters after 6 d of culture (Fig. 4AGo). For differentiation experiments, these clusters were collected, centrifuged, and plated on laminin-coated dishes in the B-27-supplemented medium now containing 10 nM GLP-1 but no growth factors (bFGF and EGF). A week later, cells grew out from the clusters, which were then fixed and subjected to immunocytochemistry for insulin and IDX-1. A subset of cells became insulin and IDX-1 positive in the wells treated with GLP-1 as opposed to control cells without treatment with GLP-1 (Fig. 4BGo).



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  		Figure 4. Differentiation of long-term passaged (<=6 months) NIPs. A, Confluent NIP cultures (n = 2; clone 009b) were trypsinized and plated in B-27-supplemented medium (see Materials and Methods). The morphology of cells changed as they become more flattened (panel 1), and by 6 d the cells generated clusters (panel 2). B, The clusters were collected, centrifuged, and replated into laminin-coated 48-well plates in B-27 medium alone (control) or supplemented with 10 nM GLP-1. The cells that grew out from the clusters after 1 wk were then rinsed, fixed, and subjected to immunocytochemical detection of IDX-1 (Cy-3, red) and insulin (Cy-2, green).

 
Transfection and expression of IDX-1 in NIPs
The homeodomain protein IDX-1 is critical for pancreas development (27) and plays a major role in transcriptional regulation of the insulin gene (15). It has been shown that GLP-1 agonists induce the expression of IDX-1 (8, 9) and that the expression of IDX-1 is sufficient by itself to induce the expression of insulin in liver cells (28) and in pancreatic ductal cells (12). We previously reported that IDX-1 is expressed in differentiated early passage NIP cell populations (14). We reconfirmed this observation in the current study using DNA binding assays with nuclear extracts prepared from differentiated confluent NIP cultures. A radioactively labeled, synthetic oligonucleotide probe encompassing the cytosine thymidine-II region of the human insulin promoter sequence formed a distinct complex that was eliminated when extracts were incubated with an antiserum for IDX-1, confirming the authenticity of IDX-1 in these cultures (data not shown). However, in long-term NIP cultures there was a loss/diminution of endogenous IDX-1 levels.

Next, we addressed whether GLP-1-induced differentiation of NIPs into insulin-expressing cells might correlate with the expression of IDX-1. Accordingly, we transiently transfected rat IDX-1 cDNA with a fragment of the rat insulin I promoter sequence conjugated to a luciferase construct (INS-LUC) into long-term (>6 months to 1 yr) NIP cultures and treated them with GLP-1 or forskolin. As shown in Fig. 5Go, reexpression of IDX-1 increased basal insulin promoter activity, and this effect was more pronounced when transfected NIPs were treated with GLP-1. In contrast, forskolin enhanced INS-LUC activity regardless of IDX-1 levels, suggesting that the GLP-1 effect on insulin gene expression in NIP cultures may be mediated by increased expression of IDX-1.



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  		Figure 5. GLP-1-induced differentiation of NIPs is mediated through a pancreas-specific transcription factor, IDX-1. A, NIP cultures transfected with -410 INS-LUC (INS-LUC) alone (left) or also with rat IDX-1 cDNA (right) were treated with forskolin (10 µM; Fork), GLP-1 (10 nM), or vehicle (CON) for 20 h. Then cells were lysed and assayed for luciferase activity. Relative light units (RLU) were measured for 10 sec (10s)/sample. Values represent the mean ± SEM of at least four wells from two experiments using clone 06. GLP-1-induced (*, P < 0.05) stimulation of INS-LUC activity is IDX-1 dependent, whereas that produced by forskolin (a*) is IDX-1 independent. Note that transfection with empty vectors (pxp2 and cytomegalovirus 5 promoter) yielded background units that were not altered by either treatment (data not shown).

 
We also hypothesized that a certain concentration of IDX-1 in the cells in conjunction with the presence of GLP-1 is required for NIPs to convert into insulin-producing cells. To test this hypothesis, we treated NIPs transfected with human IDX-1 cDNA or untransfected long-term subconfluent NIP cultures (>3 months) with either 10 nM GLP-1 or vehicle for 3–4 d. Later, cultures were either fixed and immunostained with an antibody against human IDX (red) and insulin (green) or subjected to Western immunoblot analysis for nuclear IDX-1 protein. Immunostaining results show an overall increase in IDX-1 expression levels in transfected cells (four of five times) compared with untransfected cells (Fig. 6Go, A vs. B and C, upper panels). These findings were further confirmed by Western immunoblot analyses, which also suggest that GLP-1 treatment increases IDX-1 levels in NIPs transfected with an IDX-1 expression plasmid (Fig. 6DGo). However, insulin was induced in a subset of transfected and treated NIPs in only two of the above experiments, as shown in a representative experiment in Fig. 6CGo (lower panel) and was not induced in other instances even in the presence of increased IDX-1 expression (Fig. 6BGo, lower panel). Taken together, these experiments suggest that IDX-1 may play a role in GLP-1-induced differentiation of NIPs into insulin-producing cells.



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  		Figure 6. A–C, Immunocytochemical analysis of insulin expression in NIP cultures transfected with human IDX-1. NIP cultures (passages 6–11) plated on Lab-Tek chamber slides were left untransfected (control (CON; A) or were transfected with human IDX-1 expression plasmid (+IDX-1; B and C). Five hours later, cultures were re-fed with medium containing serum. The following day, NIPs were treated with 10 nM GLP-1 (+IDX-1+GLP-1; C) or vehicle control (B). Three days later, cultures were fixed, permeabilized with methanol/Triton-X, and subjected to dual fluorescence immunocytochemical detection of IDX-1 (Cy-3, red) and insulin (Cy-2, green). Shown are representative figures in which NIPS (clone 006a) transfected with IDX-1 remains insulin negative/low (B, lower panel), and a subset of those treated with GLP-1 stains for insulin (C, lower panel). Note that IDX-1 immunostaining is more intense in those cultures that were transfected and treated with GLP-1 than in untransfected, untreated controls (B and C vs. A, upper panels). D, Western immunoblot analysis of IDX-1 expression levels in response to GLP-1. NIP cultures transfected with human IDX-1 cDNA or left untransfected were treated with GLP-1 (10 nM) or vehicle. Then cells were lysed, and nuclear extracts were prepared. Samples were electrophoresed and immunoblotted with an antibody specific for IDX-1. An extract from a rat insulinoma cell line (INS) was used as a positive control. Note the absence of endogenous IDX-1 in this clone of NIPs (>6 months in culture).

 
NIPs express the proglucagon gene and secrete GLP-1
Major regulators of expression of the nestin gene in neural stem cells are the POU homeodomain proteins Brn-2 and Brn-4 (29). Brn-4 is known to be a key activator of the expression of the proglucagon gene by interactions with the G1 element located in the proximal {alpha}-cell-specific expression promoter of the proglucagon gene (30). Therefore, we examined NIPs for expression of the proglucagon gene. Although the proglucagon gene is not expressed in nonconfluent NIPs (Zulewski, H., unpublished observations), when NIPs approach confluence and begin to differentiate, they express the proglucagon gene, as shown by RT-PCR (Fig. 7AGo) and immunocytochemistry (Fig. 7BGo), and secrete GLP-1 into the culture medium (Table 1Go). Because NIPs express functional GLP-1Rs, the later expression of GLP-1 by early differentiating NIPs suggests that GLP-1 may function as an autocrine /paracrine morphogen in the differentiation of multipotential NIPs to pancreatic endocrine cells.



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  		Figure 7. A, Proglucagon is expressed in confluent NIP cultures. RT-PCR was performed of RNA prepared from NIPs (clone 9, passage 4) using oligonucleotide amplimers to human proglucagon giving the predicted 179-bp product. Islets were used as the positive control. B, NIPs were differentiated by culturing them in B-27-supplemented medium and plating them on wells (as described in Materials and Methods). Then cells were rinsed, fixed, and stained with an antiserum to glucagon ({alpha}-glucagon; Cy-2; intense white on modified figure).

 

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  		Table 1. Secretion of GLP-1 by cultured NIPs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings demonstrate the presence of functional GLP-1Rs on pancreatic progenitor cells and suggest a direct role for GLP-1 in the differentiation of NIPs into insulin-producing cells. Differentiation occurs in a subset of NIP cells that is induced to produce insulin. GLP-1 exerts diverse effects on ß-cells, including stimulation of cAMP formation (25, 31) and activation of phosphoinositol 3-kinase (32, 33, 34), which, in turn, activates several downstream signaling targets that stimulate insulin secretion. We characterized the electrophysiological responses of NIPs to GLP-1 and found that NIPs bathed in physiological concentrations of glucose (5.6 mM) show an increase in [Ca2+]i in response to the application of 10 nM GLP-1. Paradoxically, a higher glucose concentration (20 mM) rendered GLP-1 ineffective in evoking a [Ca2+]i response. Like GLP-1, the cAMP agonist forskolin also evoked a similar increase in [Ca2+]i in NIPs at 5.6 mM glucose, and the response to forskolin was abrogated at 20 mM glucose. These results suggest that NIPs contain a glucose-sensing mechanism. In support of these observations, our previous study demonstrates that Glut-2 mRNA is expressed in NIPs (14). Perhaps GLP-1 receptors are more abundant in conditions of physiological glucose concentrations, but are decreased in high glucose concentrations (35). Indeed, a recent study by Hui et al. (12) demonstrates that GLP-1R mRNA in ARIP cells (a pancreatic ductal cell line) is decreased in high glucose concentrations. Similar to islet ß-cells, the NIP pancreatic progenitor cells respond to tolbutamide, a drug that binds sulfonylurea receptors on ß-cells to depolarize them by closing ATP-sensitive K channels and to stimulate insulin secretion (36, 37). These studies demonstrate that functional sulfonylurea receptors are present on NIP pancreatic progenitor cells. Although our studies show the existence of tolbutamide-responsive ATP-sensitive K+ channels on NIPs and a depolarizing response to GLP-1, the apparent loss of augmentation of the response in conditions of high glucose (20 mM) is in contrast to the glucose-responsive sensitivity of ß-cells to GLP-1 (25, 26). These observations suggest that unlike ß-cells that increase their responsiveness to GLP-1 in conditions of acute elevations of glucose concentrations, the response of progenitor cells such as NIPs appears to be impaired in high glucose. It is important to distinguish between long-term glucotoxicity of ß-cells in which cellular function is impaired after several days of exposure to high glucose (21, 38) and the acute impairment of NIP responses observed in just a few minutes. This apparent circumstance of the impairment of NIPs to respond to elevated glucose may be relevant to the deleterious effects of elevated blood glucose levels in individuals with diabetes, such that elevated glucose might impair the neogenesis of new ß-cells from progenitor precursor cells. Such glucotoxicity-mediated impairment of ß-cell neogenesis, is expected to be accompanied by accelerated glucotoxic ß-cell apoptosis (39, 40, 41).

The endocrine cells of the rat pancreas turn over every 40–50 d by processes of apoptosis and neogenesis (42). Neogenesis refers to the differentiation of new islet cells from progenitor cells residing in islets (14) and ducts (43, 44, 45). There have been several reports of the differentiation of pancreatic duct-derived cell lines into insulin-producing cells by growth factors (46), and GLP-1 is also implicated as a differentiation-inducing agent (10, 12). The administration of exendin-4 to rats stimulates ß-cell neogenesis, resulting in increased ß-cell mass (9). GLP-1 is now being considered as a potential new therapeutic agent for type 2 diabetic patients (13).

In their undifferentiated state, NIPs are nestin positive and IDX-1 and insulin negative. When exposed to GLP-1, a subset of cells became nestin negative and IDX-1 and insulin positive. Accordingly, insulin secretion by RIA was also detected in these cells. The differentiation of serially passaged NIP cultures into insulin-producing cells was accelerated by transfecting IDX-1 into NIPs before treating them with GLP-1. These findings are in agreement with those of Hui et al. (12), who also showed that transfection of PANC-1, a ductal cell line with IDX-1, followed by treatment with GLP-1 induced insulin bioynthesis.

The level of IDX-1 in NIPs may be critical; perhaps when NIP cultures are sequentially passaged, the level of endogenous IDX-1 falls, which is then corrected by transfecting in IDX-1. However, transfection with IDX-1 per se did not differentiate NIPs into insulin-producing cells. Treatment of IDX-1-transfected cells with GLP-1 was necessary to induce insulin bioynthesis in a subset of NIPs. Perhaps, transfection of IDX-1 into NIPs up-regulates GLP-1R expression, thus making it more responsive to its ligand. This was demonstrated by Hui et al. (12) using IDX-1 transfected PANC-1 cells. Consistent with our findings Wang et al. (47) demonstrated that the level of IDX-1 expression defines endocrine pancreatic gene expression. Like embryonic stem cells, the clonal variation in NIPs makes these kind of studies challenging in that responses to GLP-1 will depend not only on the presence or absence of its receptor, but also on the level of receptor expression (see Table 2Go for frequencies of events).


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  		Table 2. Events/observations

 
Proglucagon gene expression appears to be restricted to endocrine pancreas, intestine, and brain (4, 5). The posttranslational processing of proglucagon is different in each of these tissues. Although the major proglucagon-derived peptide hormone in the pancreas is glucagon, GLP-1 is also produced. It is surprising that the proglucagon gene appears not to be expressed in nonconfluent passage NIPs, but then becomes expressed when the NIPs become confluent and differentiate. Brn-4 is known to activate nestin gene expression (29) and is a critical {alpha}-cell-specific activator of the proglucagon gene by interactions on the G1 enhancer in the proximal promoter of the gene (30). Of note, we recently reported that the experimental misexpression of Brn-4 in the early developing pancreas (mouse embryonic d 8.5–9.5), directed by the IDX-1 promoter in transgenic mice, results in ectopic expression of the proglucagon gene in the later-developing insulin-producing ß-cells (48). Because Brn-4 appears to play a critical role in the expression of both the nestin gene characteristic of stem cells (29) and the proglucagon gene, and the expression of Brn-4 at the time of activation of the IDX-1 promoter during development activates proglucagon gene expression (48), it is tempting to speculate that NIPs may be precursors of the IDX-1-expressing epithelial cells that appear in the foregut of the early mouse embryo (embryonic d 8.5–9.5) that is destined to give rise to the pancreas. Further, we speculate that the expression of the GLP-1R in NIPs coupled with the expression of proglucagon and resultant GLP-1 may establish an autocrine/paracrine hormonal feedback loop that is important in instructing the differentiation of NIPs into pancreatic endocrine cell lineages, e.g. ß-cells. In addition, our findings are consistent with the reported observations that fetal pancreas-derived progenitor cells give rise to endocrine cells that initially express proglucagon (49) and later coexpress proglucagon and proinsulin (50).


   Acknowledgments
 
We thank Dr. C. A. Messam (NIH) for the gift of the human nestin-specific antiserum, and Hank-Jan Anstoot and Aart Verwest for providing the IPF-1 cDNA. We thank V. Stanojevic for valuable advice on DNA binding assays. We appreciative the Juvenile Diabetes Research Foundation Center for Islet Transplantation at Harvard Medical School and the University of Miami for providing human islets. We thank R. Larraga and D. Hufnagel for expert assistance in the preparation of the manuscript, and K. McManus for performing the RIAs.


   Footnotes
 
This work was supported in part by USPHS (NIH) Grants RO1-DK-30834 and RO1-DK-55365.

J.F.H. is an investigator with the Howard Hughes Medical Institute.

Abbreviations: bFGF, Basic fibroblast growth factor; [Ca2+]i, intracellular calcium; EGF, epidermal growth factor; GLP-1, glucagon-like peptide-1; GLP-1R, GLP-1 receptor; INS-LUC, insulin promoter-luciferase construct; IPF-1, insulin promoter factor-1; NIP, nestin-positive islet-derived progenitor cell.

Received October 22, 2001.

Accepted for publication April 22, 2002.


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