Abstract
Cocaine- and amphetamine-regulated transcript (CART) is an anorexigenic peptide widely expressed in the central and peripheral, including the enteric, nervous systems. CART is also expressed in pituitary endocrine cells, adrenomedullary cells, islet somatostatin cells, and in rat antral gastrin cells. We used immunocytochemistry (IHC) and in situ hybridization (ISH) to study CART expression in developing rat pancreas. We also examined co-expression of CART and islet hormones and developmental markers and the effect of CART on proliferation using clonal insulin cells (INS-1 832/13). A major portion of each of the islet cell types, except the ghrelin cells, expressed CART during a period before and around birth. Two weeks postnatally, CART expression was restricted to somatostatin cells. Pre- and early postnatally, many of the CART-expressing cells co-expressed cytokeratin 20 (CK20), a marker of duct cells and islet precursor cells, the trophic hormone gastrin, and a smaller subpopulation also harbored the proliferation marker Ki67. CART was also expressed in pancreatic nerve fibers, both sensory and autonomic, and in ganglion nerve cell bodies. Although highly expressed in the developing islets, CART did not affect proliferation of INS-1 cells. We have demonstrated that CART is expressed in several islet cell types during rat development but is restricted to somatostatin cells and neurons in the adult rat.
Keywords
Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered brain neuropeptide (Douglass et al. 1995) with potent anorexigenic properties (Lambert et al. 1997; Kristensen et al. 1998). Although a fragment of ovine CART peptide was sequenced several years ago (Spiess et al. 1981), the sequence of the rat brain CART mRNA was elucidated by Douglass et al. (1995). An anorexigenic role for CART peptide has recently been emphasized by the observations that CART-null mice develop obesity when fed a high-calorie diet (Asnicar et al. 2001) and that humans with mutations in the CART gene develop obesity and exhibit a lower metabolic rate (Challis et al. 2000; del Giudice et al. 2001). Furthermore, CART peptide inhibits gastric acid secretion and gastric emptying (Okumura et al. 2000), stimulates pancreatic exocrine secretion in the rat (Cowles et al. 2001), and has antinociceptive effects (Ohsawa et al. 2000; Damaj et al. 2003). CART peptides have been reported to inhibit apoptosis and promote survival and differentiation of various neurons in vitro (Louis 1996). CART is widely expressed in the central nervous system. CART mRNA is one of the most highly expressed mRNAs in the rat hypothalamus (Gautvik et al. 1996; Koylu et al. 1997;1998). CART is also expressed in the peripheral nervous systems, including sympathetic preganglionic (Dun et al. 2000a), primary sensory (Dun et al. 2000b), and enteric (Couceyro et al. 1998; Ekblad et al. 2003) neurons. CART is also expressed in certain endocrine cells, including a population of pituitary endocrine cells (Couceyro et al. 1997; Koylu et al. 1997), adrenomedullary cells (Couceyro et al. 1997; Koylu et al. 1997; Dun et al. 2000b), islet somatostatin cells (Jensen et al. 1999), and antral gastrin (G)-cells, as studied in the rat (Ekblad et al. 2003). Thus, a collection of data indicates that CART belongs to the already large family of regulatory peptides with a distribution in central and peripheral nerves and in neuroendocrine cells.
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 Myrsen-Axcrona 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.
Materials and Methods
Animals and Tissue Processing
Sprague-Dawley rats, (M&B; Ry, Denmark) were housed under alternate 12-hr periods of light and dark with free access to standard rat food and tapwater. Fetuses at embryonic (E) day 13 (n = 12), 15 (n=28), 17 (n=15), 19 (n=28), and 20 (n = 30), and neonates at postnatal (P) day 0 (n = 5), 1 (n=6), 2 (n=5), 3 (n=5), 5 (n=5), 8 (n=5), 10 (n=5), 12 (n=4), and 30 (n = 4) and adult (10–50-week age) rats (n = 10) were used. The pancreas was dissected out, fixed overnight in Stefanini's solution (2% paraformaldehyde and 0.2% picric acid in 0.1 M PBS, pH 7.2), rinsed thoroughly in Tyrode's solution containing 10% sucrose, and frozen on dry ice. Sections (10 μm thick) were cut and thaw-mounted on slides. The project was approved by the Animal Ethics Committee in Lund and Malmö.
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.
In Situ Hybridization
A mix of two synthetic 30-mer oligodeoxyribonucleotide probes for CART mRNA was used. They were complementary to the sequences 133–162 and 130–159 of rat CART mRNA (GenBank accession number U16826). The sequence specificity of the probes was determined using the GenBank database; no significant similarity with other mammalian mRNAs was found. The CART probes were synthesized by the Biomolecular Resource Facility of Lund University. All probes were 3′-end tailed by [35S]-dATP (NEN; Stockholm, Sweden) (Mulder et al. 1993). The ISH protocol has been described previously (Mulder et al. 1993). In brief, sections were air-dried, fixed in 4% paraformaldehyde for 15 min, washed twice for 5 min in PBS, and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. Then the sections were dehydrated in graded ethanols, treated with chloroform for 5 min, ethanol (95.5%) for 5 min, and again air-dried. Hybridization was carried out in sealed moisturizing chambers at 37C overnight, using CART probe concentration 0.9 pmol ml–1, followed by stringent posthybridization washing (1 × SSC; 0.15 M NaCl; 0.015 M sodium citrate). The slides were dipped in NTB 2 emulsion (Eastman Kodak; Rochester, NY) and stored in light-sealed boxes at 4C for 20–30 days. They were then developed in Kodak D-19, fixed in Kodak polymax, and mounted in Kaiser's glycerol gelatin. For control purposes, hybridization in the presence of a 100-fold excess of unlabeled probe was performed.
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 = 5–8/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.
Details of the antibodies
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 β-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).
Results
Immunocytochemistry
CART immunoreactivity (IR) was seen in many cells in all fetal and neonatal islets studied (Figures 1A and 1B). To identify the CART IR cells, triple immunostainings for CART/insulin/somatostatin, CART/glucagon/somatostatin, and double immunostainings for CART/PP and CART/ghrelin were performed. The stainings revealed co-expression of CART and all islet hormones tested, except ghrelin. CART was co-expressed with insulin from E13 to P10, with glucagon from E13 to P10, with PP from E15 to P10, and with somatostatin from E15 to adult (Figures 1C-1F). Ghrelin cells were few but were regularly seen from E15 to P30, as previously reported (Wierup et al. 2002a). These cells consistently lacked CART IR, as examined at several developmental stages (Figure 3B). To quantify the relative numbers of islet cells co-expressing CART and each of the islet hormones and to study the developmental pattern of co-expression, immunostained cells in sections from different developmental stages were counted. The results are summarized in Figure 2. CART-expressing insulin IR cells peaked at P0 (57% of all insulin IR cells) and gradually declined to 2% at P10. CART-expressing glucagon IR cells peaked at E15 (54%) and decreased to 1% at P10, while CART-expressing PP IR cells peaked at P1 (80%) and declined to 55% at P10. CART-expressing somatostatin IR cells increased from E15 (40%) to P1 (70%) and declined to 40% on P3. After P12, CART was expressed in the somatostatin cells only, and in adult rat (10–50 weeks) 40% of all somatostatin cells expressed CART. To examine islet gastrin expression and its possible relation to CART, double staining for CART and gastrin was performed. At birth, the vast majority (70%) of gastrin IR cells also expressed CART (Figure 3A). After P3, only a few gastrin IR cells co-expressed CART. Gastrin IR cells were undetectable after P10, as previously reported (Ekelund et al. 1985). The duct cell/islet precursor cell marker CK20 has previously been reported as a marker for islet neogenesis (Bouwens et al. 1994) and, in agreement with their observations, we found CK20 expression in many cells in the islet periphery during development. Double staining for CART and CK20 revealed a high frequency of cells with co-expression from E17 to P3 (Figure 3D). At E17–P1, 50–65% of all CART IR cells also expressed CK20. To study whether CART IR cells were proliferating cells, double staining for CART and the proliferation marker Ki67 was performed. A subpopulation of the CART IR cells co-expressed Ki67 (Figure 3C). However, such cells were fewer than those co-expressing CART and CK20.
Immunofluorescence photomicrographs. (
CART IR was also seen in pancreatic nerve fibers (from E17 to adult), predominantly in ganglia and around blood vessels and ducts, more rarely in exocrine parenchyma, and occasionally in islets. Some nerve cell bodies within pancreatic ganglia were CART IR. Double staining for CART and the neuron-specific marker human neuronal protein (HuC/D) revealed a neuronal identity of CART-expressing cells in the ganglia (Figure 4A). In addition, ganglionic cell bodies co-expressing CART and VIP were regularly detected (Figure 4D). To characterize the CART-expressing nerve fibers, we examined the additional presence of CGRP, a marker for sensory neurons, tyrosine hydroxylase (TH), a marker for adrenergic neurons, and VIP. Double staining for CART and CGRP revealed many fibers with co-expression (Figure 4B). Double staining for CART and VIP also revealed fibers with co-expression (Figure 4C). However, such fibers were rarer than those co-expressing CART and CGRP. Double staining for CART and TH revealed a close relationship between CART-containing and TH-containing nerve fibers but no firm evidence of co-existence (Figure 4E).
In Situ Hybridization
Labeling for CART mRNA was found both in the center and at the periphery of the islets from E15 to P10. Subsequently (from P12 to adult), labeling was confined to single or small clusters of cells at the islet periphery (Figure 5). The labeled area in each islet was calculated and expressed as the percentage of the total islet area. CART mRNA labeling covered 25% of the total islet area at E15 and reached its peak between P0 and P2 (45–60%), after which it declined to 7% at P10 (Figure 6).
Relative frequency (mean percentage) of cells of each islet cell type co-expressing CART at different time points around birth. •, PP IR cells; ▴, somatostatin IR cells; ▪, insulin IR cells; ○, glucagon IR cells. For all cell types, the frequency peaks around birth and then declines after P2.
Cell Proliferation Assay
To study whether CART influenced cell proliferation, we treated INS-1 (832/13) cells with 1–10 nM CART for 24 hr. Radiolabeled thymidine incorporation into newly synthesized DNA was measured and radioactivity expressed as dpm/mg protein. CART did not affect thymidine incorporation compared to the controls run in parallel.
Discussion
In this study we demonstrate that CART peptide and CART mRNA are highly expressed in rat pancreatic islets during development. Interestingly, CART is expressed in the majority of all islet cells and in each of the four classical islet cell types (glucagon-, insulin-, somatostatin-, and PP-cells) during a period around birth. Subsequently (at P10 and beyond) CART expression in islet cells becomes restricted to a subpopulation of the somatostatin cells. The expression of CART in the classical islet cell types displayed a similar pattern during the period of development examined, in that the relative number of cells of each cell type expressing CART peaked around birth and decreased 1 week postnatally. We recently reported that ghrelin cells constitute a new islet cell type in human and rat islets (Wierup et al. 2002a,b). The present finding, i.e., that islet ghrelin cells lack CART expression, suggests a developmental phenotype for the ghrelin cells that differs from that of the classical islet cell types. It might be worth mentioning in this context that ghrelin cells in the rat virtually disappear from the islets during the first month postnatally and therefore have a fate that differs from the main islet cells (Wierup et al. 2002a).
Immunofluorescence photomicrographs of neonatal (P1) rat islets. (
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).
Immunofluorescence photomicrographs of pancreatic neuronal tissue of rats (P30), illustrating that CART (red in all images) is expressed in nerve fibers and nerve cell bodies. (
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 sub-mucous 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.
Darkfield images of ISH autoradiograms. Labeling for CART mRNA in islets (within dashed line) at two different stages of development. CART mRNA is expressed throughout the islet at P2 (
Density of labeling (mean percentage ± SEM) for CART mRNA in islets over the developmental period E15-P10. CART mRNA labeling density peaks between P0 and P2. ∗ p<0.05 P0–P2 vs E15 and P0–P2 vs P10.
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.
Footnotes
Acknowledgements
Supported by the Swedish Research Council (Project no. 4499), DA00418, DA10732, the Swedish Diabetes Association, the Novo Nordic Foundation, the Royal Physiographic Society, and the Hedberg, Påhlsson, and Gyllenstiernska Krapperup Foundations.
We thank Eva Hansson, Karin Jansner, Ann-Christin Lindh, and Doris Persson for expert technical assistance.