Department of Physiology, University of Missouri-Columbia, Columbia, Missouri 65212
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ABSTRACT |
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We used
-escin-permeabilized pig cerebral microvessels (PCMV) to study the
organization of carbohydrate metabolism in the cytoplasm of vascular
smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG
and Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262,
1999) that intact PCMV metabolize the glycolytic intermediate
[1-13C]fructose 1,6-bisphosphate (FBP) to
[1-13C]glucose with negligible production of
[3-13C]lactate, while simultaneously
metabolizing [2-13C]glucose to
[2-13C]lactate. Thus gluconeogenic and
glycolytic intermediates do not mix freely in intact VSM cells
(compartmentation). Permeabilized PCMV retained the ability to
metabolize [2-13C]glucose to
[2-13C]lactate and to metabolize
[1-13C]FBP to
[1-13C]glucose. The continued existence of
glycolytic and gluconeogenic activity in permeabilized cells suggests
that the intermediates of these pathways are channeled (directly
transferred) between enzymes. Both glycolytic and gluconeogenic flux in
permeabilized PCMV were sensitive to the presence of exogenous ATP and
NAD. It was most interesting that a major product of
[1-13C]FBP metabolism in permeabilized PCMV was
[3-13C]lactate, in direct contrast to our
previous findings in intact PCMV. Thus disruption of the plasma
membrane altered the distribution of substrates between the glycolytic
and gluconeogenic pathways. These data suggest that organization of the
plasma membrane into distinct microdomains plays an important role in
sorting intermediates between the glycolytic and gluconeogenic pathways
in intact cells.
-escin; glycolysis; gluconeogenesis; permeabilization; channeling; vascular smooth muscle; caveolae
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INTRODUCTION |
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IN THE TEXTBOOK VIEW of the cell, the intermediates and
enzymes of carbohydrate metabolism are freely diffusible components of
the cytoplasm. However, it is unlikely that this is the case in living
cells. Most glycolytic enzymes probably exist in both free and bound
states (for example, see Ref. 26). The interactions of glycolytic
enzymes with the F-actin cytoskeleton are well-documented (2), and an
actin-binding region has been found in the glycolytic enzyme aldolase
(23). Associations of glycolytic enzymes with microtubules have also
been demonstrated repeatedly (36) and a glycolytic enzyme-binding
domain has recently been identified on -tubulin (35). Thus
glycolytic enzymes are probably not freely diffusible in intact cells.
The enzymes of the glycolytic pathway may engage in metabolite channeling (31). Metabolite channeling occurs when an intermediate is transferred directly from one enzyme to another, or when an intermediate is present at locally high concentrations that are out of equilibrium with the bulk of the cytoplasm (24). Localization of enzymes to structures such as actin filaments or microtubules would facilitate this process. The intermediates of gluconeogenesis may be similarly channeled.
The concentrations of glycolytic enzymes are similar to the concentrations of glycolytic intermediates within the cell (32). Therefore, if the intermediates of glycolysis and gluconeogenesis are channeled, the access of exogenous substrates to the pathways will be limited because most enzyme active sites will be occupied by substrates (32). Likewise, once a particular substrate molecule has entered a pathway, it is unlikely to diffuse away. Thus each intermediate remains within the pathway, making it unavailable for use by other pathways in which it is also an intermediate (compartmentation). Compartmentation of glycolytic, gluconeogenic, and glycogenolytic intermediates has been shown in previous studies in our laboratory (9, 11-13) and others (1, 15).
We have recently found that vascular smooth muscle of isolated pig cerebral microvessels (PCMV) utilizes [1-13C]fructose 1,6-bisphosphate ([1-13C]FBP; a glycolytic intermediate) for gluconeogenesis, while simultaneously utilizing [2-13C]glucose for glycolysis. Thus exogenous [1-13C]FBP does not mix with the [2-13C]FBP derived from glucose breakdown, and this tissue exhibits a compartmentation of glycolysis and gluconeogenesis (18). Because glycolytic enzymes are known to associate with microtubules, we examined the role of microtubules in compartmentation of glycolysis and gluconeogenesis. Our data suggested that glycolytic rate is partially regulated by the availability of binding sites for glycolytic enzymes on tubulin (18). However, microtubules did not appear to be involved in the regulation of gluconeogenic flux. In addition, associations of glycolytic enzymes with microtubules did not appear to be the basis of the compartmentation of metabolism we observed. Based on these results, we hypothesized that gluconeogenic enzymes are localized elsewhere within the cell, and that a portion of the glycolytic pathway is also localized to structural elements other than microtubules.
Recently, a number of studies have demonstrated that the plasma
membrane is organized into microdomains (such as caveolae) in which
proteins of related functions are concentrated (22). Glycolytic enzymes
are known to associate with the plasma membrane (34) and the enzyme
phosphofructokinase has recently been identified in caveolae (29). Thus
we hypothesized that the plasma membrane could be the site of
gluconeogenic enzyme localization, as well as the location of a portion
of the glycolytic pathway. We disrupted the plasma membrane of vascular
smooth muscle (VSM) of PCMV using -escin to examine the role of the
plasma membrane in the regulation and compartmentation of carbohydrate
metabolism in this tissue. The results of these studies provide
important new information about the organization of metabolism in
living cells.
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MATERIALS AND METHODS |
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Tissue collection. Pig brains were obtained at a local abattoir within 30 min of slaughter. Brains were placed in ice-cold physiological saline solution (PSS) for transport to the laboratory. PSS consisted of the following (in mM): 116 NaCl, 4.6 KCl, 1.16 KH2PO4, 25.3 NaHCO3, 2.5 CaCl2, 1.16 MgSO4, and 5 glucose, pH 7.4. PSS was oxygen- and pH-equilibrated before use by gassing with 95% O2/5% CO2. To prevent microbial contamination, 303 mg/l penicillin G and 100 mg/L streptomycin sulfate were added to PSS. PSS was also filtered through a 0.22-µm filter before use (Micron Separations, Westboro, MA). Brains were stored in fresh PSS at 4°C until use.
Microvessel isolation. Microvessels were isolated as previously described (18) by a modification of the method of Sussman et al. (33). Microvessels were isolated from three brains for each experiment. The brains were placed in HEPES-buffered PSS (HBPSS). HBPSS contained 118 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgSO4, 28 mM HEPES, 1.0 mM NaH2PO4, 0.2% (wt/vol) BSA, 1 U/ml heparin, and 10 µM isoproterenol, pH 7.4. HBPSS was supplemented with antibiotics and filtered as described for PSS. The outer layers of the brain were removed and the cerebral cortex was dispersed by aspiration into a plastic vacuum flask. The aspirated material was homogenized by five strokes in a stainless steel Dounce-type homogenizer (Dura-Grind, Thomas Scientific, Swedesboro, NJ). Microvessels were collected by pouring the brain homogenate over nylon meshes, which trapped the vessels while allowing smaller pieces of tissue to pass through. A 210-µm nylon mesh (Small Parts, Miami Lakes, FL) was used first to remove large vessels from the homogenate. These vessels were discarded. The material that passed through the 210-µm mesh was filtered over a 105-µm mesh, which trapped vessels of intermediate size. Smaller vessels and debris, which passed through this mesh, were discarded. The vessels adhering to the 105-µm mesh were rinsed with HBPSS, then collected by inverting the mesh and rinsing the vessels into a clean container. PCMV isolated in this manner are largely composed of VSM cells (18).
Permeabilization of microvessels.
Microvessels were permeabilized by incubation in Buffer B containing 50 µM -escin for 30 min at 21°C. Buffer B contained (in mM) 150 sucrose, 35 potassium acetate, 5 MgSO4, 5 NaH2PO4, 40 HEPES, and 35 KCl, pH 7.55 (4).
Permeabilization conditions and solutions were adapted from Iizuka et
al. (14) and from previous studies (3, 4, 7). After permeabilization,
microvessels were rinsed with fresh Buffer B.
Metabolic studies of permeabilized microvessels. Permeabilized microvessels were resuspended in 9 ml of Buffer B containing 5 mM [1-13C]FBP (Omicron Biochemicals, South Bend, IN) and 5 mM [2-13C]glucose (pH 7.55; Cambridge Isotope Laboratories, Andover, MA). Buffer B also contained the cofactors ATP (1 mM) and NAD (1 mM), as well as an ATP-regenerating system composed of phosphocreatine (PCr, 10 mM), creatine (Cr, 10 mM), and creatine phosphokinase (CPK, 2.5 U/mL). The suspension was mixed to ensure even distribution of microvessels, and 8 ml was pipetted into a 25-cm2 polystyrene cell culture flask (Corning Costar, Cambridge, MA). The flask was incubated for 3 h at 37°C in a shaking bath. At the conclusion of the incubation, a 5.5-ml sample of the suspension was withdrawn from the flask. The sample was centrifuged (Marathon 6K, Fisher Scientific) at 1,000 g for 5 min to pellet the microvessels. For NMR analysis, 4 ml of the resulting supernatant were frozen in liquid nitrogen. A 4-ml sample of the starting solution containing labeled substrates was also saved for NMR analysis.
Metabolic studies of intact microvessels.
Metabolism in intact microvessels was examined largely as described
above, except that microvessels were not permeabilized with -escin,
the incubations were performed in HBPSS rather than Buffer B, and no
additional cofactors were supplied.
Effects of exogenous ATP on metabolism in permeabilized vessels. To determine the effect of exogenous ATP concentration on metabolism in permeabilized vessels, microvessels were isolated from three brains and permeabilized as described above. The vessels were then split into two aliquots. One aliquot was incubated in Buffer B containing labeled substrates and additional cofactors as described above. The second aliquot was incubated in an identical solution, except that no ATP was provided.
Effects of exogenous NAD on metabolism in permeabilized microvessels. Metabolism in permeabilized vessels was examined at several concentrations of exogenous NAD to determine how the presence of this cofactor affected glycolytic and gluconeogenic rate. Microvessels were isolated and permeabilized as described above, then split into two aliquots. One aliquot was incubated as described above, in incubation medium containing 1 mM NAD. The second aliquot was incubated in an identical solution, except that the concentration of NAD was changed to 0, 0.2, 2, or 4 mM.
Effect of variations in phosphorylation potential on metabolism in permeabilized microvessels. We also examined metabolism in permeabilized vessels at varying ATP-to-ADP ratios ([ATP/ADP]) to determine whether phosphorylation potential modified glycolytic rate. Microvessels were isolated and permeabilized as described above, then split into two aliquots. One aliquot was incubated in the standard solution described above containing labeled substrates, 1 mM ATP, 1 mM NAD, 10 mM PCr, 10 mM Cr, and 2.5 U/mL CPK. Because K' = [ATP][Cr]/[ADP][PCr] = 100 (20), [ADP] for this solution = 0.01 mM, and [ATP/ADP] = 100. The second aliquot was incubated in the same solution, except that the concentrations of PCr and Cr were changed to either 10 mM PCr and 1 mM Cr ([ATP/ADP] = 1,000) or 1 mM PCr and 10 mM Cr ([ATP/ADP] = 10). Thus [ATP/ADP] was varied over two orders of magnitude in these experiments. Data obtained at [ATP/ADP] = 10 and [ATP/ADP] = 1,000 were normalized to the values obtained at [ATP/ADP] = 100.
NMR spectroscopy. Supernatant solutions from metabolic experiments (and the starting solution for each experiment) were lyophilized to powder in a Speed Vac (Savant Instruments, Farmingdale, NY). Dry samples were resuspended in 800 µl of 99.9% D2O (Cambridge Isotope Laboratories, Andover, MA) containing 25 mM 3-(trimethylsilyl)-1-propanesulfonic acid (TMSPS) as a chemical shift reference. A 650-µl aliquot of this solution was transferred to a 5-mm NMR tube for NMR spectroscopy.
13C-NMR was performed using a Bruker DRX 500 spectrometer. One thousand two hundred scans were acquired after sixty-four dummy scans using a 30° pulse angle at 125.77 MHz, 33,333-Hz sweep width, and 1-s predelay. A total of 32,768 points were acquired and processed with line broadening of 1 Hz before Fourier transform of the data. All spectra were broad-band proton decoupled. All peak positions and intensities were normalized to the signal of TMSPS, set at 0 ppm. Peak intensity was calculated using Bruker software. No corrections for nuclear Overhauser effects were made because these effects were expected to be the same for all experiments. Supernatants from intact PCMV were examined as above, except that the number of scans was 300.Statistical analysis. Significant differences in the metabolism of intact and permeabilized microvessels were detected by comparing 13C-NMR peak intensities of interest using two-tailed t-tests for two samples assuming unequal variances. Significant differences between 13C-NMR peak intensities of microvessels incubated at 0 and 1 mM ATP were detected using a two-tailed t-test for paired samples. Significant differences between 13C-NMR peak intensities of microvessels incubated at [ATP/ADP] = 10 and 100 and [ATP/ADP] = 100 and 1,000 were detected using two-tailed t-tests for paired samples. Values of P < 0.05 were considered significant. All statistical calculations were performed using Microsoft Excel 97 software.
Reagents. Na2HPO4 and NaH2PO4 were purchased from Aldrich Chemical, Milwaukee, WI. All other chemicals (except where otherwise stated) were obtained from Sigma Chemical, St. Louis, MO.
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RESULTS |
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PCMV are capable of both glycolysis and gluconeogenesis.
-Escin-permeabilized PCMV incubated with labeled substrates in the
presence of 1 mM ATP, an ATP-regenerating system, and 1 mM NAD retained
their glycolytic ability, metabolizing
[2-13C]glucose to
[2-13C]lactate and
[1-13C]FBP to
[3-13C]lactate. Permeabilized PCMV also
metabolized [1-13C]FBP to
[1-13C]glucose, demonstrating that the
gluconeogenic pathway remained active in permeabilized cells (Fig.
1). Thus PCMV retain metabolic activity,
despite extensive disruption of the plasma membrane and free access of
cytoplasmic components to the extracellular solution. These results
suggest that both glycolytic and gluconeogenic intermediates are
channeled in VSM of PCMV because these small compounds would otherwise
diffuse out of cells and be diluted by the extracellular solution,
halting metabolism (see DISCUSSION).
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Permeabilization alters metabolic flux and the distribution of
substrates between glycolysis and gluconeogenesis.
As discussed above, both the glycolytic pathway and the gluconeogenic
pathway remained active in PCMV after -escin treatment. However,
considerable alterations in both metabolic pathway flux and the
distribution of substrates between the two pathways were observed in
permeabilized PCMV relative to intact PCMV (Fig.
2). In the presence of 1 mM NAD, 1 mM ATP,
and an ATP-regenerating system, permeabilization significantly
(P < 0.0001) reduced flux of
[2-13C]glucose to
[2-13C]lactate, to 20.8% of the flux measured
in intact PCMV. The flux of [1-13C]FBP to
[1-13C]glucose in permeabilized PCMV was also
significantly (P < 0.0001) reduced, to 27.5% of the flux in
intact PCMV.
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Exogenous ATP is required for maximal glycolysis in permeabilized
PCMV.
Permeabilized PCMV that were incubated with labeled substrates in the
presence of 1 mM NAD and an ATP-regenerating system (but no ATP) had
minimal glycolytic flux (Fig. 3).
Production of [3-13C]lactate from
[1-13C]FBP was significantly enhanced in the
presence of 1 mM ATP (P < 0.001). In the presence of ATP,
there was a corresponding decrease in the production of
[1-13C]glucose from
[1-13C]FBP (P < 0.01). Production of
[2-13C]lactate from
[2-13C]glucose was almost undetectable in the
absence of ATP, and was significantly increased in its presence
(P < 0.05). Thus exogenous ATP is required for maximal
glycolytic activity in permeabilized cells. These data demonstrate that
the plasma membrane is permeable to molecules at least as large as
glycolytic intermediates (the largest of which is FBP, molecular weight
406.1) because ATP (molecular weight 551.1) is able to enter cells
freely. These data also suggest that exogenous FBP has a higher
affinity for the glycolytic pathway than the gluconeogenic pathway
because gluconeogenesis from FBP was markedly decreased in the presence
of ATP.
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Exogenous NAD is required for maximal glycolytic rate in
permeabilized PCMV.
Permeabilized PCMV incubated with labeled substrates, ATP, and an
ATP-regenerating system (but no NAD) produced very little [3-13C]lactate from
[1-13C]FBP (Fig.
4). When metabolism was examined over a
range of NAD concentrations (0.2, 1, 2, and 4 mM) a clear relationship
between NAD concentration and [3-13C]lactate
production was observed, with half-maximal
[3-13C]lactate production at 0.68 mM NAD.
[1-13C]glucose production showed an inverse
relationship to NAD concentration, declining as [NAD]
increased. Thus when appropriate cofactors are available,
[1-13C]FBP is preferentially metabolized by the
glycolytic pathway.
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Glycolysis in permeabilized PCMV is not sensitive to variations in
phosphorylation potential.
We also examined the role of phosphorylation potential in metabolism in
permeabilized PCMV. The ratio of ATP-to-ADP ([ATP/ADP]) in
the incubation solution was varied from 10 to 1,000 by varying the
concentrations of Cr, PCr, and ATP. All data were normalized to the
values obtained at [ATP/ADP] = 100. No significant
differences in lactate production from either
[1-13C]FBP or
[2-13C]glucose were observed at either high
([ATP/ADP] = 100) or low ([ATP/ADP] = 10) phosphorylation potentials, relative to lactate production at
[ATP/ADP] = 100 (Fig.
6). Therefore, phosphorylation potential
did not affect lactate production from either
[2-13C]glucose or
[1-13C]FBP over a 100-fold range of
[ATP/ADP] values.
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DISCUSSION |
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We have previously demonstrated that a compartmentation of carbohydrate metabolism exists in vascular smooth muscle of PCMV (18). Intact PCMV metabolized the glycolytic intermediate [1-13C]FBP almost entirely to [1-13C]glucose (gluconeogenesis), rather than to [3-13C]lactate (glycolysis). Simultaneously, intact PCMV metabolized [2-13C]glucose to [2-13C]lactate via glycolysis. Thus the intermediates of glycolysis and the intermediates of gluconeogenesis do not mix freely in the cytoplasm of VSM cells. We are currently investigating the structural basis of this compartmentation.
The intermediates of glycolysis and gluconeogenesis are channeled. Metabolite channeling may be one aspect of the structural basis of compartmentation. Generally, metabolic intermediates are considered to be channeled if they are "transferred from one enzyme to another without complete equilibration with the surrounding medium" (24). This transfer can occur directly, via enzyme-enzyme associations. Alternatively, locally high concentrations of intermediates that are kept out of equilibrium with the bulk solution of the cell can facilitate the interaction of enzyme with substrate (24). Channeling has been demonstrated in a variety of biochemical pathways, including phosphatidylcholine biosynthesis (6) and DNA replication (27). It has been suggested that the intermediates of glycolysis and other metabolic pathways are channeled as well (for a recent review, see Ref. 25). Glycolytic enzymes and intermediates are present at similar concentrations in the cell, suggesting that most enzyme active sites are occupied by substrates in vivo (32). Thus, if glycolytic intermediates are channeled directly between enzymes, exogenous intermediates will have limited access to the pathway because there will be few free active sites. This model is consistent with our previous results in PCMV showing that access of exogenous FBP to the glycolytic pathway is restricted (18).
In this study, we examined metabolism in VSM cells permeabilized withGlycolysis and gluconeogenesis are modulated by cofactors in
permeabilized cells.
We found that both glycolytic rate and gluconeogenic rate were
modulated by ATP and NAD in permeabilized PCMV. Therefore, -escin
permeabilization effectively removed the ability of the plasma membrane
to serve as a diffusive barrier to small molecules. Although
restrictions to diffusion of small molecules within the cytoplasm may
still exist after plasma membrane permeabilization, these intracellular
diffusion barriers are unlikely to be significant over the long (3 h)
incubation times used in these experiments. Maximal glycolysis was
observed when 1 mM NAD, 1 mM ATP, and an ATP-regenerating system were
supplied in addition to the labeled substrates. In the absence of NAD,
glycolysis from either [1-13C]FBP or
[2-13C]glucose was low, indicating that most of
the cytoplasmic NAD had diffused out into the incubation medium.
Exogenous ATP also modulated both glycolysis and gluconeogenesis.
Lactate production was low in the absence of added ATP, again
demonstrating diffusion into the extracellular medium. Addition of 1 mM
ATP to the incubation medium increased lactate production from both
[1-13C]FBP and
[2-13C]glucose, while decreasing
[1-13C]glucose production from
[1-13C]FBP. Conversion of
[1-13C]FBP to
[1-13C]glucose was highest when glycolysis was
inhibited in the absence of NAD and ATP, and declined as
[NAD], [ATP], and glycolytic rate increased.
Metabolism of [1-13C]FBP to
[3-13C]lactate is energetically more favorable
than metabolism of [1-13C]FBP to
[1-13C]glucose. Thus in the presence of
sufficient cofactors (NAD and ATP) and given free access of
[1-13C]FBP to the glycolytic pathway (as is
provided by permeabilization), it would be expected that the major
product of [1-13C]FBP metabolism would be
[3-13C]lactate. The decreased production of
[1-13C]glucose that we observed in the presence
of ATP and NAD may therefore simply reflect the partitioning of
[1-13C]FBP between more and less energetically
favorable pathways, with metabolism via glycolysis favored when
conditions are appropriate.
Role of plasma membrane in pathway sorting. Our results in permeabilized cells can be accounted for by the existence of metabolite channeling. Either enzyme-enzyme interactions or localized enzyme systems (or both) are necessary for channeling to occur. Because glycolysis and gluconeogenesis appear to occur in separate metabolic compartments in intact cells, we hypothesized that the enzymes of glycolysis must be spatially separated from the enzymes of gluconeogenesis. We have investigated the potential role of enzyme associations to microtubules as one structural basis of this phenomenon. However, although associations of glycolytic enzymes with microtubules appeared to regulate glycolytic pathway flux, microtubules did not appear to be required for compartmentation of metabolism to exist (18).
The
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ACKNOWLEDGEMENTS |
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The technical assistance of Tina Roberts is appreciated.
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FOOTNOTES |
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This work was supported by an Established Investigator Grant from the American Heart Association (C. D. Hardin), National Heart, Lung, and Blood Institute Training Grant HL-07094 (support to P. G. Lloyd), American Heart Association (Heartland Affiliate) Predoctoral Fellowship 9910198Z (P. G. Lloyd), and National Science Foundation instrumentation Grant CHE-89-08304. Pig brains were provided by Excel, Marshall, MO.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. D. Hardin, Dept. of Physiology, MA415 Medical Sciences Bldg., Univ. of Missouri-Columbia, Columbia, MO 65212 (E-mail: HardinC{at}health.missouri.edu).
Received 16 June 1999; accepted in final form 29 October 1999.
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