Cocaine- and Amphetamine-regulated Transcript (CART) Is Expressed in Several Islet Cell Types During Rat Development
Departments of Physiological Sciences (NW,BON,EE,FS) and Cell and Molecular Biology (HM), Lund University, Lund, Sweden, and Yerkes National Primate Research Center (MK), Emory University, Atlanta, Georgia
Correspondence to: Nils Wierup, Lund University, Dept. of Physiological Sciences, Sect. of Neuroendocrine Cell Biology, BMC F10, 22 184 Lund, Sweden. E-mail: nils.wierup{at}mphy.lu.se
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Summary |
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(J Histochem Cytochem 52:169177, 2004)
Key Words: CART cocaine- amphetamine- regulated transcript pancreatic nerves islet development INS-1 (832/13)-cells ghrelin gastrin VIP islet hormones neuropeptides
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Introduction |
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The developing islets are a rich source of bioactive peptides. Gastrin, peptide YY (PYY), and neuropeptide Y (NPY) are expressed in islet endocrine cells and display marked plasticity in expression levels and distribution patterns during development (Larsson et al. 1976; for reviews see MyrsenAxcrona et al. 1997
; Mulder et al. 1998
). Recently, we found the gastric hormone ghrelin to be produced by a unique endocrine islet cell type that is more common pre- and neonatally than in islets of adults as studied in humans (Wierup et al. 2002b
) and rat (Wierup et al. 2002a
). We therefore examined CART peptide and mRNA expression during pre- and postnatal islet development in the rat. We also studied the relationship of CART expression to that of islet hormones and markers of islet development. In addition, the possibility of proliferative effects of CART was examined using clonal insulin cells (INS-1 832/13) (Hohmeier et al. 2000
). In view of the wide expression of CART in neuronal tissue of the gut, we also examined its expression in pancreatic nerves, using markers for nerve identity.
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Materials and Methods |
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Immunocytochemistry
Antibodies were diluted in PBS, pH 7.2, containing 0.25% bovine serum albumin and 0.25% Triton X-100. Sections were incubated with primary antibodies (Table 1) overnight at 4C, followed by rinsing in PBS with Triton X-100 twice for 10 min. Thereafter, secondary antibodies with specificity for rabbit, guinea pig, sheep, or mouse IgG, coupled to either fluorescein isothiocyanate (FITC) (DAKO, Copenhagen, Denmark; Jackson, West Grove, PA; Sigma, St Louis, MO), Texas Red (Jackson), or 7-amino-4-methyl coumarin-3-acetic acid (AMCA) (Jackson) were applied to the sections. Incubation was for 1 hr at room temperature (RT). Sections were again rinsed in Triton X-100-enriched PBS twice for 10 min and then mounted in PBS:glycerol 1:1. In addition, ghrelin antibodies, directly conjugated with 5-(and 6)-carboxyfluorescein (FAM), were used (Table 1). The specificity of immunostaining was tested using primary antisera preabsorbed with an excess amount of homologous antigen (100 µg of peptide/ml antiserum in working dilution) or by omission of primary antibodies. Double or triple immunofluorescence was also used, with combinations of primary antibodies (rabbit, guinea pig, sheep, or monoclonal antibodies). The two or three primary antibodies were incubated simultaneously overnight at 4C, followed by rinsing in PBS with Triton X-100 twice for 10 min. Then the two or three secondary antibodies were incubated simultaneously for 1 hr at RT. In these studies, the controls included tests for inappropriate binding of the secondary antibodies. When directly conjugated ghrelin antibodies in combination with conventional indirect immunostaining were used, the incubations were in sequence, ending with the directly conjugated antibody.
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Image Analysis and Morphometry
Immunofluorescence was examined in an epifluorescence microscope (Olympus BX60). By changing filters, the localization of the different secondary antibodies in double and triple staining was determined. Images were captured with a digital camera (Olympus DP50). Islets (n=58/animal) were randomly selected from different parts of the sections from five individuals. To quantify cells co-expressing CART and islet hormones [insulin, glucagon, somatostatin, and pancreatic polypeptide (PP)], immunoreactive cells were counted and the mean percentage of the various islet cells also expressing CART was calculated. ISH radiolabeling was examined in bright- or darkfield. To quantify the density of labeling for CART mRNA in islets, areas of ISH radiolabeling were calculated. Islets were selected as above. The labeled area, i.e., grain density within an islet and total islet area, was measured, using NIH image software, and the density of labeling was expressed as percentage of the total islet area (Mulder et al. 1995). All sections used were hybridized simultaneously and under identical conditions. Density of CART mRNA labeling is shown as means ± SEM. Data were analyzed by a one-way ANOVA followed by Bonferroni's correction. Differences with a value of p<0.05 were considered significant.
Cell Culture and Proliferation Assay
INS-1 (832/13) cells were seeded and grown to confluence in 5-cm dishes in RPMI cell culture medium with 11.1 mM glucose supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM ß-mercaptoethanol. All incubations were at 37C in a humidified atmosphere containing 5% CO2. Proliferation was determined by measuring the incorporation of radiolabeled thymidine into DNA. The cells were incubated with or without CART 55-102 (American Peptide; Sunnyvale, CA) at different concentrations for 24 hr. During the last hour of incubation [methyl-3H]-thymidine (10 µCi) was present. Incubation was stopped by placing the dishes on ice. Then the cells were washed with PBS, trypsinized, and centrifuged at 10,000 rpm for 2 min at 4C. Next, the cells were sonicated in 5 mM NaOH twice for 10 sec. Aliquots of the homogenate were precipitated with 5% trichloroacetic acid (TCA) and centrifuged as above. The pellet was washed twice with 5% TCA, again centrifuged, and then dissolved in Soluene 350 at RT for 2 hr. A liquid scintillation cocktail was added and the radioactivity measured in a scintillation counter (Beckman LS6500; Beckman Instruments, Fullerton, CA). Radioactivity was expressed as dpm and was normalized to the total protein concentration. Protein was determined with a Bio-Rad protein assay kit (Bio-Rad; Hercules, CA) based on the Lowry method (Lowry et al. 1951).
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Results |
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Discussion |
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Previous studies (Bouwens et al. 1994) suggest that islet neogenesis is dependent on differentiation from ductal precursor cells rather than on proliferation of pre-existing islet cells. The widespread co-expression of CART and the duct cell/islet cell precursor marker CK20 suggests a role for CART in islet neogenesis from duct cells to islet cells.
The present finding that CART is expressed in a subpopulation of proliferating cells (expressing Ki67) further supports a possible role for CART in islet development and/or maturation. The co-expression of CART and gastrin in the islets during the neonatal period is in analogy with the rat antral G-cells, in which CART and gastrin also are co-expressed (Ekblad et al. 2003). Gastrin is a hormone with documented trophic effects on islet cells (Larsson et al. 1976
; Wang et al. 1993
), and the co-expression of CART and gastrin further suggests a regulatory role for CART in islet development. Although CART is co-expressed with a trophic hormone and a proliferation marker during islet development, we were unable to detect any effects of CART on cell proliferation in INS-1 cells. However, cultured INS-1 cells, which continuously divide, clearly exhibit proliferation dynamics that are different from those of islet cells in vivo. Another possibility is that CART plays a role in islet cell differentiation that may not be detected in a proliferation assay. Supporting this hypothesis, CART has been reported to inhibit apoptosis and to promote survival and differentiation of various neurons in vitro (Louis 1996
). Interestingly, whereas normal adult insulin cells lack CART expression, recently presented data indicate that CART is expressed in the insulin cells in several rat type 2 diabetes models (Wierup et al. 2003
).
We could also demonstrate CART in pancreatic nerve fibers and in nerve cell bodies of pancreatic ganglia. This is in analogy with the rat gastrointestinal tract, in which CART is widely expressed in the enteric nervous system (Couceyro et al. 1998; Ekblad et al. 2003
). CART IR was seen in pancreatic CGRP-containing and VIP-containing nerve fibers and in VIP-containing cell bodies of pancreatic ganglia. The latter is reminiscent of the GI tract, in which CART is co-expressed with VIP in nerve fibers, and in submucous and myenteric nerve cell bodies (Ekblad et al. 2003
). Conceivably, the CART-containing fibers storing VIP emanate from local ganglia, whereas the CART-containing fibers storing CGRP are sensory fibers emanating from spinal ganglia (Koylu et al. 1998
), and/or the nodose ganglion (Broberger et al. 1999
). Our data do not support expression of CART in adrenergic neurons; see also Dun et al. (2000b)
. A recent study reported that CART stimulates rat pancreatic exocrine secretion and that the effect was reduced after blocking parasympathetic activity with atropine and was abolished after vagotomy (Cowles et al. 2001
). These findings may suggest an action of CART on parasympathetic nerve transmission in the pancreas but do not tell whether this action is exerted centrally or peripherally. One function of CART in pancreatic neurons may be to modulate neuron-mediated exocrine pancreatic secretion.
In summary, this study shows for the first time that CART is transiently expressed in a majority of islet cells during development and that it is expressed in pancreatic neurons. Although CART is highly expressed and co-expressed with proliferation markers during islet development, it does not affect proliferation in INS-1 cells. However, a role in cell survival and/or differentiation cannot be excluded. Islet CART represents a considerable source of the peptide during development that may affect systemic levels. Further studies are needed to clarify the specific role of CART during islet development.
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Acknowledgments |
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We thank Eva Hansson, Karin Jansner, Ann-Christin Lindh, and Doris Persson for expert technical assistance.
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Footnotes |
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