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Original Article |
Whittier Institute and Department of Pediatrics (J.M., G.M.B., A.D.L., A.H.), University of California, San Diego, La Jolla, California 92037; and Laboratoire de Physiopathologie de la Nutrition (J.M.), Centre National de la Recherche Scientifique UMR 7059, 75251 Paris, France
Address all correspondence and requests for reprints to: Alberto Hayek, M.D., Islet Research Laboratory, Department of Pediatrics, University of California, San Diego Medical School, 9894 Genesee Avenue, La Jolla, California 92037.
Abstract
In addition to stimulating insulin secretion, glucagon-like peptide and its long-acting analog exendin 4 have been reported to increase ß-cell mass by both differentiation/neogenesis of precursor cells and enhanced replication of existing ß-cells. Here, we investigated the effect of exendin 4 in the growth and differentiation of ß-cells from undifferentiated precursors in islet-like cell clusters (ICCs) derived from human fetal pancreases. Our results show that the addition of exendin 4 to the culture media stimulates PDX 1 expression in ICCs as shown by immunofluorescence staining. The up-regulation of PDX 1 was not accompanied by changes in insulin expression because we did not find a significant difference in the number of insulin-positive cells in the exendin 4-treated ICCs, compared with controls. We also tested the effects of exendin 4 in the glucose-induced insulin secretion of human ICCs transplanted under the kidney capsule of athymic rats. In the exendin 4-treated rats (given ip during 10 d) 8 wk after the beginning of the treatment, insulin was released in response to glucose as detected by the measurement of circulating human C-peptide. In control (saline-treated) rats, the basal levels of human C-peptide did not change significantly after glucose stimulation. Thus, exendin 4 induces functional maturation of fetal ß-cells in response to glucose. In these rats, serial sections of the kidney-bearing grafts were examined histologically for insulin containing cells. We found a significant increase in ß-cell number, compared with the control rats. Overall, these results show that in vivo exendin 4 causes growth and differentiation of human fetal ß-cells from undifferentiated precursor cells. It also accelerates the functional maturation of fetal ß-cells as evidenced by their glucose-stimulated insulin secretion.
DEVELOPMENT OF THE human endocrine pancreas is regulated at several levels by cell/cell and cell/matrix interactions and locally produced and circulating peptides that generate diverse intracellular signals leading, in the case of the ß-cell, to a cell able to synthesize and release insulin in response to appropriate secretagogues. We previously reported that growth and differentiation of endocrine precursor cells derived from the human fetal pancreas are regulated in vitro by specific growth factors including activin A, ß-cellulin (1), hepatocyte growth factor/scatter factor (2), vitamin derivatives such as nicotinamide (3), and agents that induce blockade of phosphatidylinositol 3-kinase (4).
Exendin 4, a long-lasting analog of the intestinal hormone glucagon-like peptide 1 (GLP-1), has been reported to stimulate both the differentiation of ß-cells from ductal progenitor cells and the proliferation of ß-cells when given to rats (5). Moreover, we also previously showed that GLP-1 reconstituted a biphasic insulin release in human fetal pancreatic cells exposed to the peptide (6). More recently, also in rodents, GLP-1 has been shown to enhance the pancreatic expression of the homeodomain transcription factor PDX 1 and the capacity of the pancreas to generate new ß-cells (7). In this report we have investigated the role of exendin 4 in the maturation of human fetal pancreatic endocrine precursor cells toward insulin production both in vitro and in vivo after transplantation into nude rats.
Research Design and Methods
Human tissue
Human fetal pancreases used in these experiments were provided by Advanced Bioscience Resources (Oakland, CA) and Central Laboratory for Human Embryology, University of Washington (Seattle, WA) after the termination of pregnancy by dilatation and extraction between 16 and 24 wk of gestation.
Informed consent for tissue donation was obtained by the procurement center. In addition, our own Institutional Review Board reviewed and approved use of fetal tissue for these studies.
Tissue preparation
Tissue was processed as described previously (8) by digestion with collagenase type 11 (Sigma, St. Louis, MO). Three to four fetal pancreases were used for each set of experiments. The islet-like cell clusters (ICCs) formed from each of the digests were cultured for 3–5 d in RPMI-1640 in the presence of 10% human serum. As previously characterized (2, 9), the ICCs derived from fetal pancreases consist of about 5% endocrine cells and mostly undifferentiated epithelial cells.
Exendin 4 treatment
Exendin 4 (Sigma) at the dose of 10 nM was added to the culture media on the day of tissue processing. Exendin 4 treatment was applied for 4 d. One or two of the culture dishes were used as control in each set of experiments.
Measurement of insulin and DNA content
For insulin extraction, the ICCs were hand-picked using direct vision under a stereoscope, homogenized in distilled water, and sonicated. Aliquots of the sonicates were extracted with acid/ethanol for insulin RIA using a solid-phase assay kit (Diagnostic Products, Los Angeles, CA) as described previously (2) or dried for DNA quantitation by a fluorometric technique (10).
Transplantation experiments
Animals used in this study were Rowlett athymic nude rats obtained from Charles River Breeding Laboratories (Charles River, MA). They were housed in microisolater cages in a semisterile room. Animals were maintained according to the NIH Guide for the Care and Use of Laboratory Animals.
ICCs (1000/animal) were transplanted into nude rats under the kidney capsule using a positive displacement pipette as described previously (11). A total of 13 animals received the transplants.
Exendin 4 treatment
Forty-eight hours after transplantation, rats were treated with exendin 4 or saline solution for 10 consecutive days. Exendin 4 was administrated by ip injection, once a day, at the dose of 1 nmol/kg body weight. Every day before injection, rats were weighed and their blood glucose was measured, using a glucometer (One Touch; Lifescan Inc., Milpitas, CA) on a blood sample collected from the tail vein.
Functional response
Eight weeks after transplantation, fasted rats were given glucose 3 g/kg ip. Blood was withdrawn from the tail vein before (T0) and 30 min after (T30) glucose administration. Samples were immediately centrifuged, and plasma was stored at -20 C until the measurement of human C-peptide levels using a RIA that does not cross-react with rat C-peptide (DPC).
Grafts were removed 24 h after glucose challenge and processed for histological analysis. For this study, four rats were used in each experimental group.
Immunohistochemistry and morphometry
For the in vitro study, ICCs cultured in the presence or absence of exendin 4 were harvested, washed with PBS, and fixed in 4% paraformaldehyde for 30 min at 4 C. After fixation, the ICCs were pelleted and embedded in agarose, dehydrated, and embedded again in paraffin.
Double-immune fluorescence labeling was performed on 5-µm-thick paraffin sections of ICCs using sheep IgG antihuman insulin (Binding Site, Birmingham, UK) and rabbit IgG antimouse PDX 1 (kind gift from Dr. Joel Habener). The secondary antibodies used were lissamine rhodamine conjugated donkey antisheep IgG and fluorescein isothiocyanate-conjugated donkey antirabbit IgG (both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). In each experimental group, the percentage of PDX 1-positive cells to the total number of cells in the ICCs was calculated.
For the in vivo study, kidneys bearing the transplanted fetal ICCs were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sequentially sectioned. Eight to 10 sections per graft were stained for insulin using the immunoalkaline phosphatase technique as described previously (8). A guinea pig antiporcine insulin (Chemicon, Temecula, CA) was used as the primary antibody. Sections were then counterstained with hematoxylin. Additionally, graft sections from both groups were stained for glucagon and somatostatin. For these, staining antiglucagon and antisomatostatin antibodies (ICN Pharmaceutical, Orsay, France), both raised in rabbit, and a peroxidase-conjugated goat antirabbit IgG (KPL; Dynatech Corp., St. Quentin en Yevelines, France) were used. Sections from rat pancreases in both experimental groups were also immunostained for insulin.
Quantitative evaluation of total ß-cell area as well as 
- and 
-cell area was performed using a computer-assisted image analysis procedure based on a BX 40 microscope (Olympus Corp., Melville, NY) connected via a video camera to a PC computer and using the Visiolab 1000 software (Biocom, Les Ulis, France). The area of insulin-positive cells as well as those of total graft area was evaluated in each stained section. The relative ß-cell area in the graft was determined by stereological morphometric methods, calculating the ratio between the area occupied by insulin-positive cells and that occupied by the noninsulin-positive cells present in the graft. Within the sections, the graft area could be easily distinguished from the kidney tissue by morphological criteria. To determine the percentage of ß-cells in the rat pancreas, we calculated the ratio between the area occupied by insulin-positive cells and that occupied by the total pancreatic tissue. The number of animals studied for ß-cell area determination in the grafts was four in the exendin 4-treated group and five in the control group.
Statistical analysis
Statistical analysis of observed differences was analyzed by t test.
Results
In vitro study
To determine the capacity of exendin 4 to alter ß-cell differentiation in fetal ICCs, we measured the amount of insulin within the ICCs treated with exendin 4, compared with controls. After 4 d of exendin 4 treatment, the measurement of insulin content per DNA showed no significant differences between the two groups (Fig. 1
).
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).
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, A and B). Indeed, in a minimum of 1200 total cells counted in ICC sections from separate experiments, 39% of cells exhibited nuclear staining for PDX 1 in the exendin 4-treated ICCs, but in the control group, only 16% of cells showed strong nuclear staining for PDX 1. The comparison of sections immunostained for the PDX 1 in exendin 4-treated ICCs and control ICCs showed that, in the majority of ICCs treated with exendin 4, most of the cells expressed high levels of nuclear PDX 1 as judged by the intensity of the fluorescence (Fig. 2, A and B). In control ICCs we consistently found that the expression of PDX 1 was mainly restricted to insulin-positive cells, and, therefore, a much smaller number of cells exhibited PDX 1 staining in this group, compared with the exendin 4-treated group.
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Follow-up of transplantation of human fetal ICCs.
Chronic exendin 4 treatment has been reported to reduce food intake and weight gain in Zucker rats (12). Because the food intake and the possible consequent variations in the blood glucose may modify the hormonal balance that could in turn interfere with our study in ß-cell mass and function, we studied the effect of exendin 4 treatment in the evolution of body weight and blood glucose concentration in nude rats during the course of the treatment. These two parameters were measured every day during the treatment and once a week during the following 6 wk. We did not find any significant decrease in the body weight of exendin 4-treated rats, compared with controls. The glycemia in both groups ranged between 3.6 and 4.4 mM and was indistinguishable between control and exendin 4-treated animals (Fig. 3).
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To distinguish between endogenous rat insulin and that secreted by human ß-cells in the transplant, human C-peptide was measured using an RIA that does not cross-react with rat C-peptide (8). The basal levels of human C-peptide were similar at T0 (before glucose administration) in blood samples from rats treated with exendin 4 and the controls but by 30 min after administration of glucose, the levels of human C-peptide significantly rose in exendin 4-treated rats (Fig. 4). This indicates that transplanted tissue, originally consisting of mainly undifferentiated precursor cells, had developed into functionally mature ß-cells. In contrast, the untreated animals had no increase in the levels of human C-peptide, reflecting that the ß-cells in the transplant of control rats were not responsive to glucose.
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, C–F) paralleled the functional data as evidenced by the analysis of insulin-containing cells. The area of insulin-positive cells in grafts from exendin 4-treated animals contained three times more ß-cells than the ones from control animals. We also expressed this parameter as the ratio of ß-cell area to the whole graft area to eliminate any bias introduced by an eventual difference in the original number of ICCs transplanted in rats. The percentage of ß-cell area per graft area was significantly increased (P < 0.05) in exendin 4-treated rats, compared with control rats (Fig. 5). Moreover, to confirm that the increase in the surface area occupied by ß-cells was due to an increase in differentiation rather than a loss of undifferentiated tissue originally transplanted, we also evaluated the total surface area of the grafts, which appeared to be similar in both groups (not shown).
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- plus -cells to the total graft area was not significantly different between the two groups. The values were 0.8 ± 0.03% in the exendin 4-treated group and 1.3 ± 0.02% in the control group. To evaluate the effect of exendin 4 treatment in the native islets in rats, we measured the ß-cell mass within the pancreas of rats receiving transplants in both exendin 4 and control groups. We found no significant increase in the ß-cell area per pancreas in the exendin 4-treated rats, compared with controls. The ratio of ß-cell area to total pancreatic tissue area was 0.32 ± 0.07% in exendin 4-treated rats vs. 0.25 ± 0.03% in controls.
Discussion
One of the major limitations to cell-based insulin replacement therapies in type 1 diabetes is the scarcity of human islets for transplantation. Human fetal pancreatic endocrine cells or their precursors are a potential source of cells for clinical transplantation (11). However, transplanting fetal cells or tissue could be useful only if the endocrine cells and their precursors were able to grow and differentiate into mature insulin-producing cells that can meet the metabolic demands in insulin-deficient states. Therefore, the identification of factors that influence the growth and differentiation of endocrine precursor cells has important implications in the treatment of diabetes.
Studies from our laboratory previously identified specific growth factors that promote ß-cell differentiation in cultured ICCs (1, 2, 3). In the study reported here, we investigated the capacity of exendin 4 to stimulate precursor cell differentiation and ß-cell neoformation in ICCs derived from the human fetal pancreas. Exendin 4, a long-lasting analog of the intestinal hormone GLP-1, is a 39-amino acid peptide produced in the salivary gland of the Gila monster lizard (Heloderma suspectum) (13). It shares a 53% amino acid sequence with mammalian GLP-1, and it is an agonist at GLP-1 receptor (14). GLP-1 and exendin 4 reduce appetite and fat deposition, improve parameters associated with glucose intolerance in Zucker rats (15), and improve glucose control in db/db mice (16).
In addition to these metabolic effects, several studies show potent regulatory effects of GLP-1 and exendin 4 on ß-cell growth. In diabetic mice, exendin 4 promotes ß-cell neogenesis (7). The peptide also stimulates the expression of the transcription factor PDX 1 (7) and increases endocrine cell mass in the pancreas of old glucose-intolerant rats (17) and diabetic rats (5). Here, we first evaluated the expression of insulin and the transcription factor PDX 1 in cultured human ICCs exposed to exendin 4. Our results show that exendin 4 treatment induces the expression of PDX 1 protein as indicated by the increase in the number of cells within the ICCs exhibiting strong nuclear staining for PDX 1. Exendin 4 could act directly by signaling through GLP-1 receptor in ICCs to stimulate PDX 1 expression. The presence of GLP-1 receptor mRNA in human fetal ICCs has been shown by RT-PCR (Itkin-Ansari, P., personal communication).
Other studies related to the effect of GLP-1 in cells in vitro had shown a stimulatory effect of GLP-1 on insulin gene expression in a rat cell line RIN (18) and an increase in cell proliferation in ß-cell line INS 1 (19). In this study we found no increase in the insulin content of ICCs treated with exendin 4 and no increased number of insulin-positive cells within the ICCs. The reason that PDX 1 expression is not followed by an increase in insulin gene expression is not clear. PDX 1 is a key factor for pancreas development in mice and human (20, 21). PDX 1 is also an important transcription factor regulating the glucose-stimulated insulin gene expression (22, 23, 24). In animal models of pancreas regeneration, the increase of ß-cell mass during regeneration is accompanied by the expression of PDX 1 in ductal cells (25, 26). However, a study in human ß-cell lines had shown that, despite the introduction of PDX 1 into TRM-6 cell line derived from human fetal pancreas, the cells did not differentiate into insulin-producing cells (27). This observation is in agreement with our present results, suggesting that PDX 1 expression is necessary in the pathway toward differentiation of precursor cells into insulin-producing ß-cells, but the presence of other key components is required for ß-cell differentiation. It is likely that other transcription factors involved in the eventual maturation of fully differentiated ß-cells, such as Beta2/Neuro D, Nkx 6.1, and Nkx 2.2 (28, 29), are not regulated by exendin 4. Therefore, despite the initiation of ß-cell differentiation as demonstrated by PDX 1 expression, the precursor cells failed to proceed through the differentiation process to achieve the terminal phenotype of mature insulin-producing ß-cells.
We also investigated the effect of exendin 4 administrated in vivo on the differentiation and functional maturation of ß-cells in human ICCs transplanted into athymic nude rats.
These results are in keeping with other reported stimulatory effects of exendin 4 and GLP-1 in ß cell differentiation in the rodent pancreas (5, 7, 17, 30). Eight weeks after transplantation, the graft showed a significant increased ß-cell surface area in those animal treated with the peptide. The increase suggests either a rather indirect mechanism for the activation of the pathways leading to ß-cell differentiation or activation of the transcriptional cascade up to a point at which PDX 1 alone cannot induce insulin expression in the absence of other activators of gene expression provided in vivo. We previously reported that the mechanisms activated during the acquisition of an ultimate ß-cell mass after fetal pancreatic cell transplantation are those causing differentiation of precursors into endocrine cells rather than proliferation of preexisting ß-cells (31). The data in the present study also suggest that the mechanism for the increased ß-cell mass resulting from the in vivo exendin 4 treatment is more dependent on cell differentiation because it is to be expected that most of the PDX 1-positive cells obtained in vitro, became insulin cells in vivo. However, some contribution to the ß-cell mass from proliferation cannot be dismissed because exendin 4 may also stimulate insulin secretion from the native rat islets that could act as growth-stimulating factor in the transplanted ICCs. Moreover, it is known that in addition to its effect on insulin secretion, GLP-1 has also a regulatory effect on extrapancreatic tissues such as liver, muscle, and adipose tissue (32). Although the effect of GLP-1 and its analog in stimulation of growth factor production by the liver has not been documented, we can hypothesize that the expression of growth factors such as IGF1 and hepatocyte growth factor by the liver could be enhanced by exendin 4, which in turn could stimulate ß-cell growth and differentiation in the transplanted ICCs.
This study also shows that ß-cells in the transplanted ICCs from exendin 4-treated animals exhibit functional maturation 8 wk after transplantation; in fact, the levels of circulating human C-peptide in exendin 4-treated rats in response to glucose were significantly increased, but the control group failed to adjust their ß-cell response to the secretagogue. This indicated that not only are there more ß-cells in the graft from the rats treated with exendin 4 but also that the newly formed ß-cells are functionally mature and responsive to glucose. The disparity between the in vivo and in vitro results raises interesting questions. The in vivo system most certainly provides an additional stimulus that allows the cells to proceed toward a well-defined terminal phenotype. The in vitro system could then represent a valuable tool for testing the effect of various growth factors added to the cells along with exendin 4, which could reproduce the beneficial effect of exendin 4 administrated in vivo. Such experiments could help elucidate the mechanism of action of exendin 4 on ß-cell differentiation in human fetal pancreatic cells.
In summary, we have shown that in the human fetal pancreas, the proliferation and differentiation of endocrine precursor cells into insulin-producing ß-cells can be positively regulated by exendin 4. Moreover, the peptide accelerates the functional maturation of fetal ß-cells as indicated by the stimulated insulin secretion in response to glucose. These observations are, therefore, pertinent to potential clinical application of exendin 4 in the treatment of diabetes.
Acknowledgments
Footnotes
This work was supported by Juvenile Diabetes Research Foundation Grant 1-1999-526 (to A.H.). J.M. was a recipient of a postdoctoral fellowship from the Association de Langue Française pour l’Etude du Diabète et des Maladies Métaboliques.
Abbreviations: GLP-1, Glucagon-like peptide 1; ICC, islet-like cell cluster.
Received January 31, 2002.
Accepted July 2, 2002.
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