Departments of 1Pharmacology and 2Pathology, Institute of Physiological Sciences, University of Lund, S-221 84 Lund, Sweden; and 3Department of Medical Cell Biology, University of Uppsala, S-75123 Uppsala, Sweden
Submitted 14 November 2002 ; accepted in final form 20 June 2003
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ABSTRACT |
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calcium ion; carbon monoxide
It is easy to envisage that gap junctions are important for coordinating the rhythmicity of the -cells within an islet (35, 36, 47). However, it has also been reported that a rise of [Ca2+]i, evoked by mechanical stimulation of RINm5F cells (7) and of
-cells from rats (5) and ob/ob mice (20), propagates to neighboring cells lacking physical contact. In accord with the idea that diffusible factors aid in the synchronization of the [Ca2+]i signal required for pulsatile release of insulin, the transients of [Ca2+]i often appear in synchrony in
-cells separated by distances up to 80 µm (14). Testing various transmitters of NANC neurons, nitric oxide (NO) has been found to fulfill the criteria for a diffusible synchronizer of the
-cells (11, 14).
Like NO, carbon monoxide (CO) is produced by the islets and acts as an activator of cGMP production (2326). Two main isoforms of the CO-producing enzyme heme oxygenase (HO) have been found in different tissues, i.e., the constitutive isoform HO-2 and the inducible isoform HO-1 (33). We have previously reported that pancreatic islets display an unusually high HO-2 activity and that HO-2 is expressed in all four types of islet endocrine cells (1, 23, 25). In glucose-stimulated isolated islets, the HO substrate hemin was found to stimulate, whereas the HO inhibitor Zn-protoporphyrin suppressed the release of insulin in parallel with the rate of CO production (23, 25). Hence it was proposed that the HO-2-derived CO is a novel, stimulatory regulator of glucose-induced insulin release (2326). On the other hand, the inducible isoform HO-1 has mainly been considered to be expressed as a major part of the defense against different noxious agents and oxidative stress within pancreatic islets (26, 49) as well as in other tissues (33). We now demonstrate that glucose-stimulated -cells from ob/ob mice differ from those in lean controls by combining extensive production of CO, derived from both HO-2 and HO-1, with increased stimulation of insulin release and firing of [Ca2+]i transients. With study of how isolated
-cells are affected by exogenous CO and modifiers of its production, evidence will be provided that CO is a messenger propagating [Ca2+] signals between
-cells.
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MATERIALS AND METHODS |
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Animals. The experiments were performed with adult obese-hyperglycemic mice (ob/ob) and lean (/) C57BL/6J mice. Female 4- to 6-mo-old mice were taken for immunocytochemical demonstration of HO-2 and Western blot measurements of islet activities of HO, NOS, and insulin release. Because of the absence of sex differences, both female and male ob/ob mice were used in the studies of the [Ca2+]i transients. All animals were killed by cervical dislocation. The animal experiments were approved by a local animal welfare committee and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Immunocytochemistry. Pancreatic glands were dissected, divided into pieces, and processed for immunocytochemical demonstration of HO-2, as previously described (1, 23, 25). Cryostat sections, cut at a thickness of 8 µm, were incubated overnight with rabbit antiserum to HO-2 (1:1,000). After being rinsed, the sections were incubated for 90 min with FITC or Texas Red-conjugated donkey anti-rabbit IgG, rinsed, and mounted. The HO-2 antiserum was diluted with PBS. In control experiments, no immunoreactivity could be detected in sections incubated in the absence of HO-2 anti-serum or with antiserum absorbed with an excess of the HO-2-immunizing antigen (100 µg/ml). Epi-illumination and appropriate filter settings for Texas Red and FITC immunofluorescence were used in the microscopic examination of the sections (37).
Western blot analysis. Approximately 150 freshly isolated islets were collected in Hanks' buffer (100 µl) and sonicated on ice (3 x 10 s). Homogenate samples, representing 10 µg of the islet protein, were then run on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrotransfer (1015 V, 60 min; Semi-Dry Transfer Cell; Bio-Rad, Richmond, CA). The membranes were blocked in 9 mM Tris · HCl (pH 7.4) containing 5% nonfat milk powder for 40 min at 37°C. Immunoblotting with rabbit anti-mouse HO-1 (OSA 100; 1:500) and HO-2 (OSA 200; 1:1,000) was performed for 16 h at room temperature. The membranes were washed two times and then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000; Sigma) for 90 min. Antibody binding to HO-2 and HO-1 was detected using 0.25 mM CDP-Star (Tropix, Bedford, MA) for 5 min at room temperature. The chemiluminescence signal was visualized by exposing the membranes to Dupont Cronex X-ray films for 15 min. Appropriate standards, i.e., molecular mass markers, were run in all analyses.
Assay of islet HO activity. Islets were isolated with the aid of collagenase and used for gas chromatographic measurements of the CO production essentially as described by Henningsson and colleagues (23, 25). About 300 islets were collected in 200 µl ice-cold 0.1 M phosphate buffer (pH 7.4) and frozen immediately at 20°C. When assayed, the islets were sonicated on ice, and 30 µl methemalbumin, 100 µl -NADPH (4 mg dissolved in 1 ml 0.1 M phosphate buffer), and 2 mg Hb were added with phosphate buffer up to a final volume of 1 ml. Hb was included in the assay mixture to trap the liberated CO. Methemalbumin solution (substrate) was prepared by dissolving 25 mg hemin, 82.5 mg NaCl, and 12.1 mg Tris base in 5 ml of 0.1 M NaOH, followed by the addition of 5 ml albumin solution (20 g/l) and 5 ml distilled water. The homogenate was then incubated in a water bath at 37°C while protected from light. Aliquots (330 µl) were taken after 6 min of incubation, which was terminated by placing the tubes on ice. The samples were then injected in reaction tubes containing ferricyanide-citric acid (100 µl). Nitrogen was used as a carrier gas and to purge the reaction vessels for 4 min before the samples were injected in them. After a reaction time of 4 min, the liberated CO was brought to a nickel catalyst and allowed to react with H2 to give methane, which was brought further to the detector. CO (99.9%) was used as a standard. The amount of CO produced was calculated from the area under the curve. Protein was measured in samples from the original homogenate as described by Bradford (6).
Assay of islet NOS activity. Isolated islets were collected in ice-cold buffer containing 2 mM HEPES, 0.5 mM EDTA, and 1 mM dithiothreitol, pH 7.2, and immediately frozen at 20°C. On the day of assay, the islets were sonicated on ice, and the solution was supplemented to contain 0.45 mM CaCl2, 2 mM NADPH, 25 units of calmodulin, and 0.2 mM L-arginine in a total volume of 1 ml (26, 41). The homogenate was then incubated at 37°C under constant bubbling with 1.0 ml air/min for 3 h. It was ascertained that the reaction velocity was linear for at least 6 h. Aliquots of the incubated homogenate (200 µl) were passed through an HPLC analysis, and the amount of L-citrulline formed was measured in a Hitachi F-1000 fluorescence spectrophotometer (24, 41). NO and L-citrulline are produced in equimolar amounts. Because NO is highly reactive, the stable L-citrulline is preferably measured. The methodology has been described in detail (24, 41). Protein was determined according to Bradford (6).
Insulin secretion. Freshly isolated islets were preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 10 mM HEPES, 0.1% BSA, and 1 mM glucose as previously described (41). Each incubation vial was gassed with 95% O2 and 5% CO2 to obtain constant pH and oxygenation. After preincubation, the buffer was changed to a medium containing Zn-protoporphyrin and glucose at different concentrations (as specified), and the islets (5 islets/ml medium) were incubated in a volume of 1 ml at 37°C in a metabolic shaker (30 cycles/min). After 60 min of incubation, aliquots of the medium were removed for RIA of insulin (23).
Measurements of cytoplasmic Ca2+ transients. Single cells and small aggregates (<10 cells) were prepared by shaking collagenase-isolated islets in a Ca2+-deficient medium. After suspension in RPMI 1640 medium supplemented with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 30 µg/ml gentamicin, the cells were attached to circular cover glasses during culture for 25 days at 37°C in an atmosphere of 5% CO2 in humidified air. The selection of -cells for analyses was based on their large size and a low nuclear-to-cytoplasmic ratio compared with the islet cells secreting glucagon and somatostatin (4).
The subsequent experimental handling was performed with a basal medium containing 0.5 mg/ml BSA and (in mM) 125 NaCl, 4 KCl, 1.2 MgCl2, 2.6 CaCl2, and 25 HEPES, with pH adjusted to 7.40 with NaOH. After being rinsed in the presence of 3 mM glucose, the cells were loaded with 0.5 µM fura 2-AM during 3040 min incubation at 37°C. The cover glasses with the attached cells were then washed and used as exchangeable bottoms of an open chamber designed for microscopic work (46). The chamber wall was a broad silicon rubber ring (9 mm inner diameter) pressed on the coverslip by the threaded chamber mount and a stainless steel ring. Cannulas fixed to this ring were connected to a two-channel peristaltic pump, allowing steady superfusion of a 2.5-mm medium layer at a rate of 0.75 ml/min. The chamber was placed on the stage of an inverted microscope (Nikon Diaphot) within a climate box maintained at 37°C. The microscope was equipped for epifluorescence fluorometry with a 400-nm dichroic mirror and a x40 Fluor oil immersion objective.
A 75-W xenon arc lamp, combined with 10- to 13-nm half-bandwidth interference filters, was used for excitation. Images were collected through a 30-nm half-bandwidth filter at 510 nm with an intensified charge-coupled device camera (Extended ISIS-M; Phototonic Science, Robertsbridge, UK). The excitation filter was part of a Magiscan image analysis system (Applied Imaging, Gateshead, UK). The amplification of the intensified video camera was set to optimally utilize the dynamic range of the analog-to-digital converter at either wavelength and any [Ca2+]i encountered. The specimens were illuminated only during image capture, and photodamage was minimized with neutral-density filters. Control experiments revealed that exposure to light was particularly harmful when evaluating the effects of porphyrins and mesoporphyrins. Pairs of 340- and 380-nm images, consisting of 16 accumulated video frames divided by 8, were captured, resulting in 1-s delay between images. Ratio frames were calculated after background subtraction, and [Ca2+]i was estimated as previously described (15, 17). The time between successive ratio frames was 6.5 s.
With the use of a previously established protocol (11, 14), it was tested how various substances affect the frequency and synchronization of [Ca2+]i transients during superfusion with medium supplemented with 20 mM glucose, 20 nM glucagon, and 50 µM methoxyverapamil. Glucagon was included in the superfusion medium to counteract the depletion of cAMP known to occur when -cells are separated from the glucagon-producing
-cells (43). The reason for performing the studies in the presence of methoxyverapamil is that blockage of the voltage-dependent Ca2+ channels removes the background disturbances of slow oscillations (13, 32). The effects of gaseous CO were evaluated during periods with three bolus additions of l00-µl medium together with 60-s arrest of the pump at intervals of
4 min. The dilution of the stock solution of CO with the accompanying addition to the chamber was completed within 15 s.
Presentation of data. In the measurements of [Ca2+]i, each experiment refers to analyses of 612 cells (or aggregates) attached to a coverslip. Sudden increases of [Ca2+]i >50 nM were recognized as transients and were considered to be synchronized when occurring within 19.5 s. The proportion of transients appearing in synchrony is referred to as the synchronization index. The synchronization indexes obtained should be compared with a value of 0.13 ± 0.02 (n = 10), which is the degree of synchronization occurring by chance when cells are compared on different coverslips (14). Effects on [Ca2+]i transients were statistically evaluated from paired comparisons using Student's t-test. The effects of Zn-protoporphyrin on the release of insulin from islets of obese and lean mice were compared by the two-way ANOVA test. The results are presented as means ± SE.
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RESULTS |
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Localization of HO-2 in pancreas. The HO-2 immunoreactivity was distributed in a similar way in the pancreas from obese and lean mice. A strong immunoreactivity was seen in the cytoplasm of polygonal ganglionic cell bodies occurring isolated or in groups. Clusters of HO-2-immunoreactive ganglion cells were found also in the periphery of the islets (Fig. 1). Within the islets, almost all endocrine cells displayed HO-2 immunoreactivity, which was weaker than that observed in the ganglionic cell bodies. There was no immunoreactivity for HO-2 in the exocrine cells or in nerve trunks and terminals.
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Islet production of CO and NO. Gas chromatography revealed a high rate of CO production in islet cells. The amounts of CO produced were approximately six times higher in islets from ob/ob mice than in those from the lean controls (Table 1). NO was produced in similar amounts in the islets from the two types of mice at a rate equivalent to 1% of the CO production seen in the ob/ob mice.
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Expression of HO-1 and HO-2. Western blot indicated a more pronounced expression of HO-2 in islets from ob/ob than from lean mice (Fig. 2A). Moreover, there was an exclusive expression of HO-1 in islets from ob/ob mice.
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Effect of the HO inhibitor Zn-protoporphyrin on glucose-stimulated insulin release from isolated islets. To evaluate the role of the HO-CO pathway in glucose-stimulated insulin release, isolated islets were incubated in the absence and presence of the HO inhibitor Zn-protoporphyrin (2, 23, 25). It was found that exposure to 10 µM Zn-protoporphyrin resulted in a prominent inhibition of glucose-stimulated insulin release from ob/ob mouse islets (62%) but only in a modest reduction (24%) in lean control islets (Fig. 2B). A two-way ANOVA test confirmed that islet secretion of insulin in ob/ob mice was particularly sensitive to the suppressive effect of 10 µM Zn-protoporphyrin (P < 0.005).
Effect of CO on the generation of -cell transients of cytoplasmic Ca2+. The obese-hyperglycemic syndrome was characterized by increased firing of [Ca2+]i transients in the
-cells. The number of transients generated during a 10-min period was 16 times higher in
-cells from ob/ob mice (1.78 ± 0.10; n = 88) than in those from the lean controls (0.11 ± 0.04; n = 20).
The effect of exposing -cells from ob/ob-mice to 1 µM hemin, the natural substrate for HO-induced CO production, is shown in Fig. 3. The addition of hemin resulted in an increased generation of [Ca2+]i transients. Table 2 summarizes the results obtained when exposing the
-cells to different concentrations of hemin. It is evident that 0.1 µM is sufficient to stimulate the generation of the transients. In the presence of 10 µM, there was no longer any increase in the number of transients, and 100 µM hemin had a suppressive action. The ability of CO to induce transients of [Ca2+]i was evaluated also by exposing the
-cells to the native gas (Fig. 4). Bolus additions of CO resulted in retarded responses with single transients similar to those occurring spontaneously. The number of transients obtained during a period with repeated additions of CO to final concentrations of 1100 µM was higher than that seen during a preceding control period (Table 3).
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The significance of CO for inducing transients was evaluated also by inhibiting its formation (Figs. 5 and 6). Table 4 summarizes the results of such experiments by using 10 µM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin. Both metalloporphyrins were effective in suppressing the generation of the transients, an effect not seen when the porphyrins lacked the metal component. Actually, mesoporphyrin had the opposite action by increasing the number of [Ca2+]i transients.
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Effects of CO on the synchronization of -cell transients of cytoplasmic Ca2+. It was investigated whether the alterations in the frequency of the [Ca2+]i transients had counterparts in their synchronization (Figs. 5 and 6). This was found to be the case for Zn-protoporphyrin and Cr-mesoporphyrin but not for hemin, protoporphyrin, or mesoporphyrin (Table 5). When the synchronization indexes were compared with those obtained by chance (0.13 ± 0.02), it was evident that the transients will no longer appear in synchrony when the
-cells are exposed to 10 µM of the metalloporphyrins.
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DISCUSSION |
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CO has several properties in common with NO (34), and both gases have been reported to serve as coneurotransmitters in the gastrointestinal tract (3). A further reason for exploring whether CO aids in the generation of -cell transients of [Ca2+]i is that the
-cells are known to contain both constitutive NO synthase (1, 24, 26, 29, 38, 39, 41, 42) and the constitutive isoform of the CO-producing HO (HO-2; see Refs. 2326). We have previously shown that both islet production of CO and glucose-stimulated insulin release are dose dependently suppressed by the HO inhibitor Zn-protoporphyrin but unaffected by protoporphyrin (23, 25). The latter compound has been reported to lack inhibitory effects on HO (33). Moreover, glucose itself and the HO substrate hemin are potent stimulators of islet production of CO (23), and the resulting enhancement of insulin release is reduced significantly by the guanylate cyclase inhibitor ODQ (23). These data suggest that islet production of CO has a regulatory role in glucose-stimulated insulin release and acts, at least in part, through the guanylate cyclase-cGMP system. In support for this idea, addition of gaseous CO was found to potentiate glucose-stimulated insulin release (23).
Both hemin and Zn-protoporphyrin are normal plasma constituents (33), implying that physiological interactions with HO are conceivable. In the present study, it was found that a low concentration (10 µM) of Zn-protoporphyrin is a potent inhibitor (62%) of the excessive insulin release from glucose-stimulated ob/ob mouse islets. In contrast, there was only a modest inhibitory effect (24%) on insulin release from lean mouse islets, similar to what is seen with islets from NMRI mice (25) and Wistar rats (23). Hence the excessive glucose stimulation of insulin release in ob/ob mice may, at least in part, depend on the great CO production in their islets.
CO is not only a stimulator of insulin release but is also a trigger of the transients of [Ca2+]i assumed to coordinate the secretory activity of the -cells. We now observe that hemin increases the frequency of the [Ca2+]i transients and that metalloporphyrins (Zn-protoporphyrin and Cr-mesoporphyrin), at a concentration known to inhibit HO without affecting NO synthase and soluble guanylate cyclase (2), have a suppressive effect. Further evidence that CO is important for the generation of [Ca2+]i transients supposed to coordinate the
-cell rhythmicity was obtained from the lowered synchronization index seen when HO was inhibited by the metalloporphyrins. The observation that alterations of the HO activity affect the firing of [Ca2+]i transients is not surprising when we take into account that CO, like NO, is a stimulator of the cGMP generation by binding to the heme prosthetic group of guanylate cyclase (33, 34). Previous studies have indicated that the guanylate cyclase inhibitor ODQ (9) suppresses the
-cell generation of [Ca2+]i transients (11). We now report an increased firing of transients in the presence of mesoporphyrin, which is known to activate soluble guanylate cyclase (27). The available data do not allow definite conclusions regarding the mechanisms for how cGMP precipitates transients of [Ca2+]i in the pancreatic
-cells. One alternative is that cGMP increases the apparent affinity of the IP3 receptors, as seen in hepatocytes (16).
In the present study, there was a strong immunoreactivity for HO-2 in ganglion cells located in the periphery of mouse islets. The demonstration of a putative neural production of CO close to the -cells and that this gas induces [Ca2+]i transients sufficiently pronounced to interrupt the
-cell electrical activity gives rise to the question whether CO is an NANC neurotransmitter coordinating the rhythmicity of the islets in the pancreas. Evaluating this possibility, it should be kept in mind that CO is less reactive and has a much longer half-life than NO (3, 34). Whereas bolus addition of gaseous NO is known to result in a prompt firing of single transients (11), the corresponding effect of native CO was comparatively slow.
Confocal microscopy in combination with double immunostaining has revealed that a majority of the -cells in NMRI mice display immunoreactivity for HO-2 (23). Observing immunoreactivity for HO-2 in almost all islet cells, we now demonstrate that the
-cells from obese (ob/ob) and lean (/) C57BL/6J mice also have their own production of CO. The islets from the ob/ob mice generated almost 1 nmol CO · min1 · mg protein1, which is sixfold the rate noted in the lean controls. Although ob/ob mice differ from lean mice with regard to the proportion of different cell types in the islets (19), there is no doubt that the obese-hyperglycemic syndrome is associated with a pronounced increase of the
-cell generation of CO. This increase may well explain why the
-cells from the ob/ob mice are particularly active in firing [Ca2+]i transients. As suggested from Western blots, the increased CO production was evoked not only from an enhanced HO-2 expression but also from a marked expression of the HO-1 enzyme. HO-1 is known to be expressed in response to noxious and stressful stimuli such as endotoxin, heavy metals, and oxidative stress (26, 33, 34, 49). HO-1 induction is then thought to protect cells through the activity of HO to metabolize heme to biliverdin, which has antioxidant properties (33, 34, 49). Because ob/ob mice are hyperglycemic, it is conceivable that their
-cells respond to the resulting "glucotoxicity" with induction of HO-1 and an increased HO-2 expression. It was recently observed that islets from hyperglycemic rats express HO-1 1 mo after partial pancreatectomy (30). Because these rats had normal plasma insulin levels, it is likely that hyperglycemia rather than hyperinsulinemia is responsible for the induction of HO-1 in the ob/ob mouse islets. Moreover, it was recently reported in abstract form that it is possible to induce HO-1 in normal rat islets by culture in a high-glucose medium (28).
The islet production of CO exceeds that usually observed (50100 pmol · min1 · mg protein1) in tissues rich in HO-2 (2326, 33, 48). The rate of CO generation in the islets is also considerably higher than the production of NO, which is 10 pmol · min1 · mg islet protein1 in both ob/ob and lean mice (Table 1). The presence of a pronounced CO production in the
-cells may be important for a neural induction of [Ca2+]i transients synchronizing the slow [Ca2+]i rhythmicity. It is known that CO can suppress the activity of constitutive NO synthase in various types of cells, including insulin-releasing cells (23, 25, 26). However, the two gases have been reported to interact also in a synergistic or compensatory way, depending on the microenvironment (18).
The present studies reinforce previous observations that spontaneously occurring [Ca2+]i transients often appear in synchrony in -cells lacking physical contact (11, 14). The presence of such a synchronization in preparations devoid of contaminating nerves and capillaries indicates that
-cells not only receive but also propagate extracellular signals. Being produced in large amounts in the
-cells and generating the type of [Ca2+]i transients known to affect the electrical activity (8), CO may propagate the neurally induced Ca2+ signal from one
-cell to another. In support for this idea, we now demonstrate that imposed alterations of the
-cell production of CO are reflected in their firing of [Ca2+]i transients and that the inhibitory effect of metalloporphyrins has its counterpart in a suppressed synchronization of the transients.
In summary, we have shown that the hyperinsulinemia in ob/ob mice is associated with increased -cell production of CO mediated both by constitutive and inducible HO. Imposed alterations in the production of CO made it possible to propose that this gaseous messenger is not only a positive modulator of glucose-stimulated insulin release but also promotes the generation of [Ca2+]i transients supposed to coordinate the secretory activity of the
-cells.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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