From the Department of Anesthesiology, University of
Illinois, Chicago, Illinois, 60612,
Veterans Affairs
Chicago Health Care System West Side Division, Chicago, Illinois,
60680, ** Departmento de Bioquimica y Biologia Molecular,
Universidad de Salamanca, Salamanca 37007, Spain, and
§ Institute of Pharmacology, Catholic University Medical
School, Rome 00168, Italy
Received for publication, August 8, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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Activation of peroxisome
proliferator-activated receptors (PPARs) can regulate brain physiology
and provide protection in models of neurological disease; however,
neither their exact targets nor mechanisms of action in brain are
known. In many cells, PPAR The peroxisome proliferator-activated receptors
(PPARs)1 are nuclear hormone
receptors that can bind to PPAR elements (AGGTCA n AGGTCA) to
activate transcription (1-3) normally as heterodimers with the
retinoic acid receptor (4-6). Three major subtypes have been
identified ( Recently, it has been shown that PPAR Studies carried out in liver, adipose tissue (34-37), and muscle
(38-40) showed that in periphery, PPAR To test this hypothesis we examined the effects of TZDs on glucose
uptake and metabolism in primary enriched cultures of rat cortical
astrocytes. Incubation with these drugs increased glucose utilization
(assessed as loss of glucose from the incubation media), 2-deoxy-glucose uptake, and lactate production. These effects did not
appear to be mediated by activation of PPAR Materials--
Cell culture reagents (DMEM and antibiotics) and
LPS (Salmonella typhimurium) were from Sigma. Fetal
calf serum (FCS) was from Invitrogen. Pio was from Takeda
Pharmaceuticals North America (Lincolnshire, IL). Rosi was from
GlaxoSmithKline. Trog was from Parke-Davis. GW347845 was a gift of Tim
Willson (GlaxoSmithKline). Polyclonal antibodies directed against
GLUT-1 (AB-1340, which recognizes the carboxyl terminus of rat GLUT-1)
and GLUT-3 (AB-1344, which recognizes the carboxyl terminus of the
mouse GLUT-3) were from Chemicon International (Temecula, CA).
Horseradish peroxidase-conjugated rabbit secondary antibodies were from
Vector Laboratories (Burlingame, CA). Enhanced chemiluminescence
reagents were from Pierce. Taq polymerase and cDNA
synthesis reagents were from Promega. Cell viability was assessed by
measurement of released lactate dehydrogenase according to the
manufacturer's procedures, using the CytoTox-96 kit from Promega.
Cells--
Primary astrocytes were from cerebral cortices of
postnatal day 1-2 Harlan Sprague-Dawley rats as described previously
(48). Media were changed every 3 days. After 2 weeks growth in DMEM containing 25 mM glucose, 10% FCS, and antibiotics
(penicillin and streptomycin) the cultures consisted of 95-98%
astrocytes and 2-3% microglia.
Glucose and Lactate Assays--
Glucose and lactate
concentrations in the incubation media was assessed by a quantitative
enzymatic colorimetric kit, according the manufacturer's instructions
with some modifications. Briefly, 5-µl aliquots of media were
incubated with 100 µl of Sigma Diagnostic Glucose Trinder reagent for
10 min at 37 °C, and the absorbance was read at 492 nm. For lactate,
samples were incubated with 90 µl of Sigma Diagnostic Lactate reagent
for 20 min at room temperature, and absorbance was read at 550 nm. In
each assay, a standard curve was prepared in the range of 0 to 100 mg/100 ml of D-glucose or 0 to 50 mg/100 ml of
L-lactate in DMEM. Levels in each sample were calculated by
interpolation from standard curves.
2-Deoxy-Glucose Uptake--
For glucose uptake experiments,
astrocytes were pre-incubated in low glucose (5 mM instead
of 25 mM) DMEM with 1% FCS containing different
concentrations of Pio. During pre-incubation, aliquots were collected
at different time points for subsequent glucose and lactate
assessments. After 6 h of pre-incubation, the medium was replaced
by 250 µl of glucose-free DMEM to which was added increasing
concentrations of D-glucose (0.05-1 mM) and 1 µCi/ml of 2-deoxy-D-[2,6-3H]glucose (2-DG;
Amersham Biosciences), final concentration 24 nM, and cells
were further incubated for 10-90 min. At the end of the uptake
experiment the medium was removed, and the cells were washed three
times with 1 ml of ice-cold phosphate-buffered saline and then lysed by
adding 250 µl of 200 mM NaOH. An aliquot of 200 µl was
assayed for 3H by liquid scintillation counting. Protein
content was measured in the same samples by Bradford's method in 50 µl of the remaining lysates. [3H]2-DG uptake was
expressed in pmol/mg of protein or nmol of glucose/mg protein.
NOS2 Induction and Activity Measurements--
Cells were grown
to 90-95% confluency, the growth media were removed, the cells were
washed in serum-free media, and incubations were carried out in fresh
DMEM containing antibiotics and 1% FCS. NOS2 was induced by incubation
with bacterial endotoxin LPS (1 µg/ml). Induction of NOS2 was
assessed indirectly by nitrite production in the cell culture media. An
aliquot of the cell culture media (80 µl) was mixed with one-half
volume of Griess reagent (49), and the absorption was measured at 550 nm. Solutions of NaNO2 dissolved in DMEM/1% FCS served as standards.
mRNA Analysis--
Total cytoplasmic RNA was prepared from
cells using TRIZOL reagent (Invitrogen), aliquots were converted to
cDNA using random hexamer primers, and mRNA levels were
estimated by real time, quantitative RT-PCR (50). The primers used for
GLUT-1 detection were 939F (5'-TGA AAG AAG AGG GTC GGC AGA TAA-3'),
corresponding to bases 939-960, and 1301R (5' AGA TGG CCA CGA TGC TCA
GAT AGG-3'), complementary to bases 1278-1301 of the rat GLUT-1
cDNA sequence, which yield a 363-bp product. The primers used for
GLUT-3 were 978F (5'-GGA GCG GGC AGG AAG GAG AA-3') and 1305R (5'-AGA
AAA CGA GGA AGG CAG CGA AGA-3'), which correspond to bases 978-998 and
1281-1305, respectively, and which yield a 328-bp product. The primers
used for glyceraldehyde-3-phosphate dehydrogenase detection were
796F (5' GCC AAG TAT GAT GAC ATC AAG AAG) and 1059R (5' TCC AGG GGT TTC
TTA CTC CTT GGA), which yield a 264-bp product. The primers used for
PPAR Western Blot Analysis--
Astrocytes were grown until confluent
and then incubated in DMEM containing 5 mM glucose and 30 µM Pio or the equivalent amount of
Me2SO for 6 or 24 h. Cells were washed twice
with ice-cold phosphate-buffered saline, harvested by scraping,
collected by centrifugation (700 × g for 10 min), and
placed on ice. Subcellular fractionation was carried out as described
(51, 52). Briefly, cells were resuspended in lysis buffer (250 mM sucrose, 5 mM NaN3, 2 mM EGTA, 1/100 protease inhibitor mixture (P8320; Sigma),
and 20 mM HEPES, pH 7.4) and then homogenized using a
Dounce homogenizer. The homogenate was centrifuged at 760 × g for 5 min to remove unbroken cells and nuclei; the
supernatant was collected and centrifuged at 31,000 × g for 60 min to pellet the crude plasma membrane and collect
the supernatant as cytoplasmic fraction. Protein content was determined
by Bradford's method using bovine serum albumin as standard. Ten µg
of protein of each sample were mixed 1:3 with 3× gel sample buffer
(150 mM Tris-HCl, pH 6.8, 7.5% SDS, 45% glycerol, 7.5%
bromphenol blue, 15% cAMP Assay--
Cells were incubated in 1% FCS in DMEM in the
presence of indicated drugs. An initial time course (10-90 min) was
carried out in the presence of 10 µM Pio, and a 30-min
incubation time was chosen for the experiments. After incubation, cells
were washed three times with physiological saline (Ca2+-
and Mg2+-free) and then flooded with 100 µl of 5%
trichloroacetic acid. After 5 min, the supernatant was removed, and
cells were washed again with 100 µl of water. Trichloroacetic acid
supernatant and wash water were combined and extracted with ether. cAMP
was assessed using an enzymatic immunoassay kit purchased from
Biomedical Technologies Inc. (Staughton, MA) according to the
manufacturer's instructions. Sample concentrations were calculated
using a standard curve constructed by plotting relative binding values
(B/Bo = cAMP bound/cAMP total) versus log of
concentration (pg/ml).
Isolation and Assay of Brain Mitochondria--
Rat brain
mitochondria were isolated as described (54). Briefly, brains were
homogenized in isolation buffer using 10 ml per gram of brain, in a
50-ml centrifuge tube with a tight fitting pestle. The homogenate was
centrifuged at 5,000 × g for 10 min, the supernatant
was removed, and the pellet was re-extracted with isolation buffer. The
two supernatants were combined and centrifuged at 16,700 × g. The upper half from this was discarded, and the lower
portion combined 1:1 with 15% Percoll and mixed gently. The resulting
material was layered onto a Percoll step gradient (23% on top of 40%)
and centrifuged at 30,700 × g. The fraction accumulating at the interface was collected, slowly diluted 1:4 with
isolation buffer, and centrifuged at 16,700 × g for 10 min to yield the final mitochondrial pellet.
Mitochondrial respiration was carried out at 30 °C and monitored as
described previously (55). Respiratory control ratios measured as the
ratio of state III (ADP-supported) to state IV (resting) were
consistently above ten. Respiration was initiated by the addition of
substrate (pyruvate, 10 mM final concentration). Calculations of respiratory rates, in units of ng atoms oxygen per min
per mg, were derived from the slopes of oxygen consumption in the
reaction chamber, which were linear for over 10 min.
Measurement of Mitochondrial Membrane Potential--
Primary rat
astrocytes were grown in 25-cm2 Petri dishes at a density
of about 200,000 cells/cm2 in DMEM containing 1% FCS. When
confluent, cells were treated with 10 µM of Pio or Trog
or the corresponding amount of vehicle (Me2SO). After
6 h, the medium was removed, and cells were trypsinized and used
for measurements of JC-1 aggregate fluorescence as described (56). For
each sample, the measurements were made three times, and the S.D.
values were within 10% of the means. The ratios of FL2 (fluorescence
because of JC-1 aggregation) to FL1 fluorescence (because of monomeric
JC-1) were determined and normalized to the values obtained for
incubation in vehicle alone. Complete mitochondrial depolarization was
obtained by addition of carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (5 µM for 5 min) to the cells, and that value was assigned as 0%.
In Vivo Experiments--
Female Sprague-Dawley rats (Charles
River Laboratories, Wilmington MA), weighing 250-300 g were housed in
groups of four at 22 °C and in a 12-h light/dark cycle with free
access to food and water. Pio was added to Purina chow 5002 at 0 or 100 ppm. Rats were provided free access to diets for different days as indicated. On the day of the experiment, the animals were killed by
decapitation. The brains were quickly removed from the skull, and the
cortices were dissected under aseptic conditions. Coronal slices were
made across the cortex to produce sections with two cut surfaces and
having approximately the same weight, as assessed by protein
measurement at the end of each experiment. All slices from the same
animal were collected in a 35-mm Petri dish containing 7 ml of DMEM
containing 5 mM glucose and 1% FCS and incubated at
37 °C in a humidified atmosphere containing 5% CO2.
After 45 min of pre-incubation, during which the incubation medium was replaced twice, the slices were transferred to a 48-well plate containing 500 µl/well of low glucose DMEM and further incubated for
4 h. Aliquots of 5 µl were collected after different times to
assess glucose levels. At the end of each experiment measurements of
lactate dehydrogenase showed no differences in lactate dehydrogenase released between control and Pio-treated slices.
Data Analysis--
Experiments were done at least in triplicate
unless noted otherwise. Data were analyzed by one- or two-way ANOVA
followed by Dunnett's multiple comparison or Bonferroni post hoc
tests, and p values < 0.05 were considered significant.
Rat Astrocytes Express PPAR Pioglitazone Increases Glucose Utilization--
Cortical rat
astrocytes were incubated in low glucose (5 mM) containing
medium, and glucose consumption, assessed as loss of glucose from the
incubation media, was measured after different times up to 24 h in
the presence of increasing amounts of Pio (0-30 µM).
Control cells used glucose from the media (8.1 ± 1.0% per h),
most likely because of maintenance of basal energetic needs. Pio time-
and dose-dependently increased the rate of glucose utilization (9.0 ± 0.9 and 12.8 ± 0.5% per h, at 10 and 30 µM, respectively; see Fig.
2), and this effect was significant at 30 µM Pio as soon as 4 h of incubation. After 24 h, Pio-treated cells continued to show an increased amount of glucose
utilization (control, 80.0 ± 1.6% compared with 30 µM Pio with almost 100% of the available glucose used)
(data not shown).
Pioglitazone Increases 2-Deoxy-glucose Uptake--
In control
astrocytes, uptake of 2-DG was linear during 90 min of incubation (Fig.
3A). Pre-treatment with Pio
(30 µM for 6 h) increased subsequent 2-DG uptake by
~33% (from 151 ± 9 to 196 ± 3 fmol per min per mg of
protein; p < 0.0001, two-way ANOVA). To characterize
which glucose transporter(s) were involved in this increase, a similar
experiment was performed in the presence of different amounts of cold
D-glucose, and the amount of 2-DG taken up was measured
after 45 min (Fig. 3B). The stimulatory effect of Pio was
observed primarily at the higher doses of cold D-glucose
(0.5-1 mM), suggesting an increased glucose flux through GLUT-1, which has a lower affinity for glucose than does GLUT-3 (53).
Effect of Pioglitazone on Glucose Transporter Expression--
The
effects of Pio on astrocyte glucose utilization measured after 6 h
were not significantly reduced by co-incubation with either a
transcriptional (5 µg/ml actinomycin D) or translational (10 µg/ml
cycloheximide) inhibitor (Fig. 4),
suggesting that effects on gene transcription or protein translation
were not involved in this process. Consistent with this, we found that
Pio treatment (6 h with 30 µM) did not modify the steady
state mRNA levels of either GLUT-1, which is mainly expressed in
glial and endothelial cells, or GLUT-3, which is mainly expressed in
neurons (Fig. 5, A and
B). Similar results were obtained after longer incubation periods (up to 24 h) (data not shown) suggesting that increases in
GLUT mRNA levels were not responsible for increased glucose uptake.
Analysis of GLUT-1 and GLUT-3 protein levels revealed that incubation
with Pio for 6 h did not significantly increase either membrane or
cytosolic levels of GLUT-1 (Fig. 6,
left panels) although a slight increase (~20% increase in
two experiments) in cytosolic levels was observed. However, after
24 h of incubation, we observed a significant increase (greater
than 2-fold) in cytosolic levels and a slight increase (roughly 25%)
in membrane levels of GLUT-1 in Pio-treated versus control
astrocytes (Fig. 6, right panels). These results suggest
that increased glucose utilization observed up to 6 h of
incubation is not because of an increase in GLUT-1 levels but more
likely because of an increase in glucose flux through pre-existing
GLUT-1 protein.
Pioglitazone Increases Lactate Production--
Rat cortical
astrocytes were incubated in DMEM containing 5 mM glucose,
and lactate levels in the incubation media were measured after
different times (Fig. 7). Treatment with
Pio significantly increased lactate production (p < 0.0001, two-way ANOVA), suggesting that Pio influences glucose
metabolism, as well as its uptake.
TZD Effects Are Not Mediated by PPAR TZD Effects Involve the cAMP/Protein Kinase A
Signaling Pathway--
To begin to characterize possible mechanisms of
action of Pio on astrocyte glucose metabolism, we tested inhibitors of
several signaling pathways. The ability of Pio to increase lactate
production was not modified by co-incubation with either an ATPase
inhibitor (ouabain) or with a phosphatidylinositol 3-kinase inhibitor
(wortmannin) (data not shown). However, incubation with a highly
selective protein kinase A inhibitor (KT-5720; 100 nM)
attenuated the effects of Pio (Fig.
9A). Consistent with
this, we found that incubation with Trog significantly increased
intracellular cAMP levels after 30 min. Treatment with Pio (10 µM) showed a tendency to increase cAMP levels at this
time point, although the values were not significant (p = 0.07, unpaired t test) compared with vehicle (Fig.
9B).
TZDs Influence Mitochondrial Function--
Because effects of TZDs
on glucose metabolism and lactate production could be secondary
consequences of effects on mitochondrial function, we tested whether
TZDs directly modified mitochondrial respiration (Fig.
10). In freshly isolated rat brain
mitochondria, incubation with Pio or Trog reduced the pyruvate-driven
state III respiration, measured by loss of oxygen from the incubation media (Fig. 10A). The consequences of TZD inhibition of
mitochondrial respiration was examined in whole astrocytes (Fig.
10B). Mitochondrial membrane potentials ( Pioglitazone Reduces Astrocyte Cell Death--
To test whether
incubation with TZDs influenced cell viability, we tested the effects
of Pio on astrocyte cell death because of glucose deprivation (Fig.
11). Astrocytes maintained for 24 h in either high (25 mM) or low (5 mM) glucose
containing media showed some cell death (5 to 10%, assessed by lactate
dehydrogenase release), which was not significantly reduced by
co-incubation with Pio (30 µM). In contrast, overnight
incubation in the absence of glucose increased lactate dehydrogenase
release 4-fold over control values (to 18 ± 2%), and this was
reduced to control values by Pio (p < 0.001, unpaired
t test). These data suggest that the effects of TZDs on
astrocytes can provide protection against hypoglycemia-induced injury.
TZD Effects on Glucose Metabolism in Vivo--
To establish
whether TZDs could influence brain glucose metabolism in animals, we
tested the effects of acute and of chronic TZD treatment on brain
sections prepared from Pio-fed adult rats (Fig.
12). Incubation of cortical sections
prepared from control rats with TZDs significantly increased glucose
loss from the media, similar to what we observed in primary astrocyte
cultures (data not shown). We then compared glucose utilization in
sections prepared from rats that were provided oral Pio (100 ppm) for 1, 3, or 10 days. The glucose utilization in sections from 1 day Pio-fed rats was not significantly different from that of control
rats. However, after 3 days of treatment, the glucose utilization was
~50% greater in sections prepared from Pio-fed animals than control
animals. However, this potentiating effect of orally provided Pio was
transient, because after a longer period (10 days) the sections from
Pio-fed animals showed a similar glucose use as did control sections. Although we do not yet know the cellular source of glucose use in these
sections, these results demonstrate that oral treatment with Pio can
influence brain glucose metabolism.
Our data demonstrate for the first time that PPAR It has been shown that astrocytes are the main source of energy for
neuronal activity (43). Therefore, in this study we examined the effect
of Pio on glucose metabolism using primary enriched cultures of rat
cortical astrocytes. In this model Pio increased glucose utilization as
assessed by loss of glucose from the culture media and glucose uptake
as measured by influx of radiolabeled 2-DG. Because the delivery of
glucose within the brain is mediated primarily by GLUT-1, present in
the blood-brain barrier, as well as in glial cells, and to a smaller
extent by GLUT-3 presents in neurons (42), we tested whether Pio
modified GLUT-1 or GLUT-3 expression. Measurements of
2-deoxy-glucose uptake suggested an increased flux through
GLUT-1, because significant increases due to Pio occurred at the higher
but not the lower glucose concentrations tested (53). In other systems,
the regulation of GLUT-1 expression is under both transcriptional and
post-transcriptional control (61, 62). However, in our studies the
increase in glucose utilization observed at 6 h was not reduced by
co-treatment with cycloheximide or actinomycin D, and measurements of
GLUT-1 and GLUT-3 mRNA levels did not reveal significant effects
due to Pio treatment at either early (6 h) or later (24 h) time points, again suggesting that glucose-modifying effects are mediated by non-genomic mechanisms.
In insulin-sensitive cells, about 50% of the GLUT-1 and most of the
GLUT-4 is located in intracellular vesicles, and both are translocated
to the plasma membrane in response to insulin (63). Because TZDs can
increase GLUT-4 translocation from the intracellular pool to the plasma
membrane (51) we assessed whether Pio modified either the cytosolic or
membrane content of GLUT-1. However, no significant differences were
observed after 6 h of incubation, whereas a slight increase in
both cytoplasmic and plasma membrane content of GLUT-1 was observed
after 24 h of incubation. We conclude that the effects of Pio on
glucose uptake at early time points (within 6 h) are not
associated with significant changes in glucose transporter expression.
However, at later times, an increase in GLUT-1 content could contribute
to or account for the increased rate of metabolism within the cells.
Our data also suggest that the effects of TZDs on astrocyte metabolism
are not mediated by PPAR Several studies (64-66) have now shown that TZDs can trigger specific
effects within a relatively short time range, consistent with the
existence of transcription-independent mechanisms of action. Incubation
with 15-deoxy- In contrast, we found that inhibition of protein kinase A with the
selective inhibitor KT-5720 (100 nM) significantly reversed the metabolic effects of Pio. Consistent with this, we also found that
astrocyte cAMP levels were increased (albeit slightly) after 30 min of
incubation with Pio and significantly increased upon incubation with
Trog. To our knowledge these results are the first demonstration of an
effect of TZDs on intracellular cAMP levels. The greater effect due to
incubation with Trog is consistent with our findings that Trog more
potently increased glucose utilization and lactate production than did
Pio. However, further studies are needed to better characterize the
effects of TZDs on the kinetics and magnitude of intracellular cAMP
levels. There are numerous pathways linking changes in cAMP to effects
on glucose metabolism. For example chronic treatment with cAMP
analogues increased glucose utilization by up-regulating GLUT-1
expression in human choriocarcinoma cells (68), although opposite
results were observed in primary mouse placental cells (69). In
insulin-sensitive tissues, several studies have shown that glucose
uptake was enhanced by regulation of GLUT-1 expression through the
cAMP/protein kinase A pathway. However, in myoblasts these effects were
observed only after 24 h (70) and only at the higher
concentrations (within the mM range) of cAMP analogues
(71). Similarly, cAMP increased glucose uptake in T3T-L1 adipocytes by
increasing GLUT-1 protein content in the plasma membrane after 8 (72)
or 16 h (73). Interestingly, in brown adipocytes norepinephrine
enhanced glucose transport across the membrane by a mechanism involving
cAMP and increased GLUT-1 affinity for its substrate (63). Taken
together, these reports indicate that glucose uptake can be regulated
by cAMP via several distinct mechanisms. Finally, and in light of the fact that TZDs directly inhibit mitochondrial respiration, reports demonstrating inhibition of the pyruvate dehydrogenase complex by
cAMP-dependent phosphorylation of pyruvate dehydrogenase
kinase (74, 75) suggest an additional mechanism to account for
suppression of pyruvate-driven respiration.
It has been shown that the glutamate released upon neuronal activity is
taken up by astrocytes, causes an increase in astroglial glycolysis,
and subsequently increases the uptake of glucose from the incubation
media and lactate release into the media, thereby coupling in this way
neuronal activity with energy metabolism (43). We therefore
hypothesized that increased glucose uptake because of Pio might also
increase lactate production by astrocytes. We observed that Pio induced
a rapid increase in lactate production that was evident as soon as
after 2 h of treatment, suggesting that the effect of Pio is to
increase glucose metabolism and subsequently cause uptake from
extracellular pools. A similar effect has been demonstrated in rat
muscle cells in which inhibition of insulin-stimulated mitochondrial
fuel oxidation by Trog leads to an increase of anaerobic glycolysis and
glucose transport (76).
Our current findings demonstrate that TZDs also directly inhibit the
ability of acutely isolated brain mitochondria to catalyze pyruvate-driven state III respiration, consistent with previous findings that TZDs directly inhibit mitochondrial fatty acid metabolism (64, 76). Although inhibitory effects on mitochondrial respiration are
difficult to reconcile with subsequent protective actions, it has been
hypothesized previously (56, 60) that the increase in anaerobically
derived ATP due to mitochondrial impairment by nitric oxide
subsequently contributes to sustained mitochondrial membrane potential,
and we observe a similar phenomenon after 6 h of incubation with
TZDs. Furthermore, mitochondrial hyperpolarization has been shown to
protect cells against cytotoxic damage, and in our experiments we find
that treatment with Pio reduced astrocyte cell death due to hypoglycemia.
Based upon the above considerations, we propose the following scheme to
explain the ability of TZDs to increase astroglial glucose metabolism.
A similar scheme has been proposed for the ability of low doses of
nitric oxide to inhibit initial oxidative respiration but that
eventually lead to higher intracellular ATP levels and protective
effects. We suggest that after entry into the cell, TZDs modulate
(either directly or possibly by a mechanism involving cAMP) enzyme
activities present within the mitochondrial membrane causing inhibition
of ongoing pyruvate-driven respiration. Whether this is a selective
effect on pyruvate oxidation (i.e. by inhibition of pyruvate
dehydrogenase) or is a more general inhibition of mitochondrial
substrate utilization is not yet known. Increased cytosolic pyruvate
results in greater lactate production, observed as an increase in
extracellular lactate levels. In astrocytes, this inhibitory effect on
mitochondrial function is compensated by an increase in anaerobic
glycolysis allowing for continued ATP production, as well as further
pyruvate and lactate synthesis. Eventually, the reduced intracellular
glucose levels are replenished by glucose transport through the GLUT-1
because of mass action through the transporter and without significant
change in GLUT-1 levels (at least initially).
At later times, mitochondrial respiration recovers, because the actions
of TZDs are neither toxic nor irreversible. Accumulated ATP can be
translocated via the adenine nucleotide translocase into the
mitochondria, where hydrolysis to ADP releases protons that contribute
to maintenance and increase of the membrane potential. Because
hyperpolarization of the mitochondrial membrane is postulated to be
protective, for example by conferring resistance to depolarization and
release of pro-apoptotic factors, the net result of TZD treatment, at
least in astrocytes, is protective and allows cells to withstand subsequent noxious stimuli.
It has been shown recently, both in vitro and in
vivo, that PPAR agonists increase glucose uptake and
metabolism. Because astrocytes store glucose and provide lactate to
neurons on demand, we tested effects of PPAR
agonists on astroglial
glucose metabolism. Incubation of cortical astrocytes with the PPAR
thiazolidinedione (TZD) agonist pioglitazone (Pio) significantly
increased glucose consumption in a time- and dose-dependent
manner, with maximal increase of 36% observed after 4 h in 30 µM Pio. Pio increased 2-deoxy-glucose uptake
because of increased flux through the type 1 glucose
transporter. However, at this time point Pio did not increase
type 1 glucose transporter expression, nor were its effects blocked by
transcriptional or translational inhibitors. Pio also increased
astrocyte lactate production as soon as 3 h after incubation.
These effects were replicated by other TZDs; however, the order of
efficacy (troglitazone > pioglitazone > rosiglitazone)
suggests that effects were not mediated via PPAR
activation. TZDs
increased astrocyte cAMP levels, and their glucose modifying effects
were reduced by protein kinase A inhibitors. TZDs inhibited state III
respiration in isolated brain mitochondria, whereas in astrocytes they
caused mitochondrial membrane hyperpolarization. Pio protected
astrocytes against hypoglycemia-induced cell death. Finally, glucose
uptake was modified in brain sections prepared from Pio-fed rats. These
results demonstrate that TZDs modify astrocyte metabolism and
mitochondrial function, which could be beneficial in neurological
conditions where glucose availability is reduced.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
(or
), and
) with distinct actions on cell
physiology and different specificity in their ligand-binding properties
(7). PPAR
is widely expressed in the adipose tissue where it
controls adipocyte differentiation and lipid metabolism (8). The
agonists of PPAR
include fatty acids, non-steroidal anti-inflammatory drugs like ibuprofen (9), the natural ligand 15-deoxy-
-12,14-prostaglandin J2 (10), thiazolidinediones (TZDs) such as troglitazone (Trog), pioglitazone (Pio), and rosiglitazone (Rosi) (11), and high affinity tyrosine-based agonists like GW347845
(7). TZDs were originally designed as antidiabetic drugs because of
their insulin-sensitizing effects, and several are in clinical use
(12-14).
is involved in the regulation
of inflammatory responses (15-18). Thus, PPAR
activation contributes to reduce the secretion of proinflammatory cytokines and
neurotoxic substances from activated monocytes (19-21) and microglia
(22). Our previous studies showed that PPAR
agonists are able to
down-regulate neuronal inducible nitric-oxide synthase expression and
to reduce cerebellar granule cell death in vitro (23) and
in vivo (24). More recently, we demonstrated that PPAR
agonists reduced the development of clinical symptoms in experimental
autoimmune encephalomyelitis, a commonly used animal model for multiple
sclerosis (25). These findings have led to the possibility that PPAR
agonists could provide protection in other neurodegenerative diseases,
such as Alzheimer's disease (26), stroke (27), Parkinson's disease
(28), and multiple sclerosis (25, 29-31). PPAR
activation may
regulate several functions in brain, in addition to inflammation,
including promoting neurite extension (32) and inducing myelin gene
expression (33). However, neither the exact targets nor the mechanisms
of protection due to PPAR
activation in brain are well known.
regulates glucose metabolism by increasing glucose uptake through facilitative glucose transporter proteins (GLUTs). Delivery of glucose in the central nervous system is
mainly mediated by GLUTs present on the brain-blood barrier, on glial
cells, and on neurons (41, 42). However the primary energy source in
brain is provided by astrocytes. These cells are able to store glucose
in the form of glycogen and provide lactate to neurons on demand (43).
Because glucose metabolism could be adversely affected in some
neurodegenerative disorders (44-47), we hypothesized that one
mechanism of PPAR
agonists protection during disease could be
related to an increase of cerebral metabolism of glucose.
or to changes in gene
expression but instead because of a rapid effect on other cellular
signaling systems possibly related to changes in mitochondrial respiration. Our results demonstrate that TZDs can increase astroglial glucose metabolism, suggesting that this class of drugs may be therapeutically useful in conditions in which brain glucose levels or
availability are limited.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were 642F (5'-GCC TTG CTG TGG GGA TGT CTG-3') and 995R (5'-CGA
AAC TGG CAC CCT TGA AAA ATG-3') from the rat PPAR
sequence
NM_013124.1, and they yield a 354-bp product. Cycling conditions
were 35 cycles of denaturation at 94 °C for 10 s, annealing at
61 °C for 15 s, and extension at 72 °C for 20 s,
followed by 2 min at 72 °C, in the presence of SYBR Green (1:10,000
dilution of stock solution from Molecular Probes, Eugene, OR) carried
out in a 20-µl reaction in a Corbett Rotor-Gene (Corbett Research,
Sydney, Australia). Relative mRNA concentrations were calculated
from the relative take-off point of the PCR reactions using the
manufacturer's software included in the unit. Correct product
synthesis was confirmed by melting curve analysis, which yielded a
single melting point, and by electrophoresis through 2% agarose gels
containing ethidium bromide of the PCR products.
-mercaptoethanol), denatured at 70 °C for 5 min, and separated through SDS-10% acrylamide gels. Denaturation at
70 °C versus boiling reduces aggregation of GLUT-1 in
gels.2 Apparent
molecular weights were estimated by comparison to
BenchMarkTM prestained protein ladder standards
(Invitrogen). After electrophoresis, proteins were transferred to
polyvinylidene difluoride membranes by semi-dry electrophoretic
transfer. The membranes were blocked with 10% (w/v) low-fat milk in
TBST (10 mM Tris, 150 mM NaCl, 0.1% (w/v)
Tween 20, pH 7.6) for 1 h and incubated in the presence of
antibody (anti-GLUT-1 at 1:5000 dilution, anti-GLUT-3 at 1:1000 dilution) overnight with gentle shaking at 4 °C (53). The primary antibody was removed, and the membranes were washed four times in TBST
and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody diluted 1:40,000 for GLUT-1 and 1:8,000 for GLUT-3. After four washes in TBST,
bands were visualized by incubation in enhanced chemiluminescence reagents for 1 min and exposure to x-ray film for 1 min (for membranes) or 5-10 min (for cytosolic). Band intensities were determined using
ImageJ software (National Institutes of Health) from autoradiographs obtained from at least two different exposure times, and background intensities (determined from an equal-sized area of the film
immediately below the band of interest) were subtracted.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
PCR analysis showed that the
mRNA corresponding to PPAR
is present in unstimulated confluent
primary cultures of rat cortical astrocytes (Fig.
1). Incubation with the PPAR
agonist
Pio (30 µM) did not significantly modify steady state
PPAR
mRNA levels measured after 24 h. Inflammatory
activation with bacterial endotoxin LPS significantly reduced (by about
50%) PPAR
steady state mRNA levels measured after 24 h,
and this decrease was not significantly affected by co-incubation with
Pio. These results demonstrate that as reported previously (57),
rat astrocytes express PPAR
mRNA and that its levels, although
responsive to inflammatory stimuli, are not significantly altered upon
incubation with a selective PPAR
agonist.
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Fig. 1.
Rat astrocytes express
PPAR . Total RNA was prepared from primary
cortical astrocytes after 24 h of incubation in media alone, with
30 µM Pio, with 1 µg/ml LPS, or with Pio and LPS. Real
time RT-PCR analysis for levels of the PPAR
mRNA was carried out
described under "Experimental Procedures." The top panel
shows a representative gel image of PCR products obtained at the end of
the analysis from two different RNA samples for each condition. The
bottom panel shows the real time quantitative results and is
the mean ± S.D. of two different samples for each group, each
measured in triplicate. The experiment was carried out two other times
with similar results. Incubation with LPS significantly reduced PPAR
mRNA levels (p = 0.0011, two-way ANOVA).
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Fig. 2.
Pio dose- and time-dependently
increases glucose utilization. Primary cortical astrocytes were
incubated in DMEM containing 5 mM glucose and 1% FCS,
together with 0, 10, or 30 µM Pio. Glucose levels in the
culture media were measured at indicated times. Data are means ± S.E. of n = 4 replicates and are expressed as % of
glucose consumed, determined as loss of glucose from the incubation
media. *, p < 0.05; ***, p < 0.001 versus control (two-way ANOVA, Bonferroni multiple post hoc
comparison).
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Fig. 3.
Pio increases 2-deoxy glucose uptake.
A, astrocytes were pre-incubated in DMEM with 5 mM glucose containing 0 or 30 µM Pio. After
6 h, cells were incubated in DMEM containing 0.5 mM
D-glucose and 1 µCi/ml 2-DG (24 nM) for
indicated times. Data are means ± S.E. of n = 3-6 replicates and are expressed as pmol of 2-DG per mg of protein.
***, p < 0.001 versus control (two-way
ANOVA, Bonferroni multiple post hoc comparison). B,
astrocytes were treated as in A, after which 2-DG uptake was
measured in the presence of different concentrations of cold
D-glucose. Data are means ± S.E. and are expressed as
nmol of total glucose taken up per mg of protein. *, p < 0.05; ***, p < 0.001 versus control
(two-way ANOVA, Bonferroni multiple post hoc comparison).
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Fig. 4.
Transcriptional and translational inhibitors
do not prevent Pio effects. Cortical rat astrocytes were
coincubated with a transcriptional (5 µg/ml actinomycin D) (Act
D) or translational (10 µg/ml cycloheximide (CHX)
inhibitors in the presence of DMEM containing 5 mM glucose
and 0 or 30 µM Pio. Glucose utilization was assessed
after 6 h as loss of glucose from the media. The data are
means ± S.E. of n = 4 samples and are expressed
as % loss of starting glucose levels. ***, p < 0.001 versus no Pio (one-way ANOVA followed by Newman-Keuls
multiple comparison test).
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Fig. 5.
Effect of Pio on glucose transporter mRNA
expression. Astrocytes were incubated in DMEM containing 5 mM glucose in the presence of 0 or 30 µM Pio.
After 6 h, total cytosolic RNA was prepared and used for RT-PCR
analysis of Glut-1, Glut-3, and glyceraldehyde-3-phosphate
dehydrogenase (GDH) mRNA levels. A,
representative agarose gel electrophoresis showing levels of GLUT-1 and
GLUT-3 PCR products from control and Pio-treated samples (results for
two different samples from each group, each done in duplicate, are
shown). B, results of real-time PCR analysis showing the
mean ± S.E. of the calculated cycle number take-off values
(n = 4 replicates per group).
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Fig. 6.
Effect of Pio on GLUT-1 expression.
Astrocytes were incubated 6 (left panels) or 24 h
(right panels) in complete media containing 0 or 30 µM Pio. Whole cell lysates were prepared and used to
isolate membrane and cytoplasmic fractions, and equal aliquots were
used for Western blot analysis of GLUT-1. In each panel,
membrane and cytoplasmic samples from two different preparation are
shown (C1 and P1). The bottom bar
graphs show calculated band densities, and the error
bars represent analytical error in the densitometric analysis for
each sample. Note that film exposure times were 1 min for membrane
samples and 5 or 10 min for cytoplasmic samples, and therefore the
relative intensities cannot be compared directly.
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Fig. 7.
Effect of Pio on lactate production.
Astrocytes were incubated in 5 mM glucose in the presence
of 0 or 30 µM Pio. Lactate levels were assessed at the
indicated times. Data are means ± S.E. of n = 4 replicates, and there was a significant effect of Pio on lactate
production (p < 0.001, two-way ANOVA). ***,
p < 0.001 versus control (Bonferroni post
hoc comparison).
Activation--
Although
Pio can bind to and activate PPAR
, it can elicit PPAR
-independent
effects, as well. We therefore examined the effects of several
different TZDs on astrocyte lactate production (Fig. 8). The order of efficacy for lactate
production was found to be Trog > Pio > Rosi, which is the
inverse order of their reported efficacies for activating the PPAR
.
Furthermore, co-incubation with GW347845, a high affinity, non-TZD
PPAR
agonist (7), failed to increase lactate production (Fig.
8A). Indeed, even at a concentration of 10 µM,
at which dose Pio shows only a slight increase in glucose utilization,
Trog significantly increased glucose utilization (Fig. 8B)
whereas GW347485 had no effects. Together, these results suggest
that the effects of Pio (and other TZDs) on astrocyte lactate and
glucose metabolism could be mediated by a mechanism of action not
involving PPAR
activation, in contrast to their more well
characterized anti-inflammatory actions (20, 58). In contrast,
inflammatory activation of astrocytes by LPS, as assessed by expression
of NOS2 and nitrite production (see Fig. 8C), was inhibited
by TZDs in approximately the same order of potency as their ability
to activate PPAR
(GW347845 > Rosi > Pio > Trog).
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Fig. 8.
TZDs metabolic effects are not mediated by
PPAR activation. A, astrocytes
were incubated in presence of 10 µM of different PPAR
agonists, and lactate production was measured at indicated times. Data
are means ± S.E. of n = 4 replicates, ***,
p < 0.001; **, p < 0.01; and *,
p < 0.05 Pio-treated versus control,
one-way ANOVA. B, at the end of the experiment, glucose
levels were assessed in the incubation media. Data are expressed as % of glucose consumed and are analyzed by one-way ANOVA, followed by
Dunnett's multiple comparison test. *, p < 0.05 versus control. GW, GW347845. C,
astrocytes were incubated with 1 µg/ml of endotoxin LPS in the
presence of indicated PPAR
agonists (all at 10 µM) and
nitrite levels after indicated times as a marker of NOS2 induction.
Data are nmol of nitrite per well and are means ± S.E. of
n = 4 replicates. Data are analyzed by two-way
ANOVA.
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Fig. 9.
Pio affects glucose metabolism via the
cAMP/protein kinase A pathway. A, the stimulatory
effect of Pio (30 µM) on lactate was reversed by a
selective protein kinase A inhibitor (KT-5720) used at 100 nM (Ki = 56 nM). Data were
expressed as mg/100 ml of lactate and were analyzed by one-way ANOVA,
followed by Newman-Keuls multiple comparison test. **,
p < 0.01 versus control. B,
astrocytes were incubated in low glucose DMEM for 30 min in the
presence of 10 µM Pio or Trog, or vehicle alone, after
which intracellular cAMP levels were determined. Trog (10 µM) increased cAMP levels (1.86 ± 0.5 versus 0.08 ± 0.02 pg of cAMP per ml of Trog and
Me2SO, respectively, p < 0.05, unpaired
t test); Pio (10 µM) induced a slight but
non-significant increase (to 0.29 ± 0.08, p = 0.07, unpaired t test).
) were
measured by using flow cytometry with the fluorescent day JC-1 (56).
Incubation with TZDs significantly increased the ratio of aggregate to
monomer JC-1 fluorescence in astrocytes (30% increase by Pio, 43%
increase by Trog), indicating a hyperpolarization of mitochondria
. The ability of an initial inhibition of respiration to cause
subsequent mitochondrial hyperpolarization has been described
previously (59, 60) and may involve intramitochondrial hydrolysis of
anaerobically derived ATP (see "Discussion").
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Fig. 10.
TZDs modify mitochondrial function.
A, freshly isolated rat brain mitochondria was incubated in
the presence of the indicated concentrations of Pio or Trog or the
appropriate amount of Me2SO vehicle. State III respiration
was initiated by addition of pyruvate, and oxygen consumption was
measured over the next 10 min. The data are means ± S.E. of
three-four experiments. B, astrocytes were incubated for
6 h in 10 µM Pio or Trog, or vehicle only. At that
time the mitochondrial membrane potential was determined by
fluorescence measurement of JC-1 aggregation state. The data are
means ± S.D. of n = 3 experiments. **,
p < 0.05; ***, p < 0.01 versus
vehicle, unpaired t test.
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Fig. 11.
Pio reduces hypoglycemia-induced cell
death. Astrocytes were incubated in DMEM containing 1% FCS and
either 0, low (5 mM), or high (25 mM) glucose
and in the presence or absence of Pio (30 µM). After
24 h, lactate dehydrogenase release and total lactate
dehydrogenase levels were determined. Data are means ± S.E. of
three-four measurements. ***, p < 0.001, unpaired
t test.
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Fig. 12.
Pio increases glucose utilization in rat
cortical slices. Cortical slices were prepared from adult rats
kept on a diet containing Pio for 1, 3, or 10 days and further
incubated in vitro. Glucose levels in the incubation media
were measured after 4 h. Data are expressed as % of glucose
utilization and are means ± S.E. of n = 4 replicates per group. The same experiment was carried out twice. Data
are analyzed by unpaired t test. *, p < 0.05 versus control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
agonists of
the TZD family can modify glucose metabolism in brain glial cells,
despite the fact that the regulation of glucose metabolism in brain is
primarily insulin-independent. TZDs increased astrocyte glucose uptake
from the media and lactate production and release from the cells. The
mechanism underlying this increase in glucose metabolism is not yet
clear; however, in contrast to other studies, our results do not
support an effect of TZDs, at least at early time points, on glucose
transporter expression but instead point to rapid events occurring at
the mitochondrial level. Furthermore, our results suggest that the
effects of TZDs on astrocyte metabolism are not mediated by PPAR
activation, because the most effective TZD was troglitazone, which of
the TZDs tested has the lowest efficacy of PPAR
activation (7), and
because the highly selective PPAR
agonist GW347485 was ineffective.
Together, our data suggest the existence of a novel rapid and
non-genomic action of TZDs upon astroglial cell physiology, which may
involve changes in intracellular cAMP. These findings raise the
possibility that TZDs could be used therapeutically to increase
astroglial energy stores, as well as their ability to provide lactate
to neurons when needed.
activation but instead by rapid activation
of an intracellular signaling system, possibly leading to modification
of mitochondrial function. Measurements of lactate production show that
the order of efficacy for increasing production was Trog > Pio > Rosi, inverse of their reported affinities for binding and
activating PPAR
. We also found that Trog (at 10 µM)
was the only TZD able to increase glucose utilization and moreover that
the high affinity PPAR
agonist GW347845 had no effect on glucose
metabolism. In contrast, in the same cells the TZDs reduced
LPS-dependent nitrite production (presumably because of
increased NOS2 expression) with efficacies similar to their ability to
activate PPAR
, indicating the presence of functional PPAR
on
these cells.
-12,14-prostaglandin J2 (as well as Pio and Trog)
induces a rapid and transient activation of the mitogen-activated
protein kinase/extracellular signal-regulated kinase pathway in
vascular smooth muscle cells by an activation of phosphatidylinositol
3-kinase (65) suggesting that some actions of TZDs might be mediated
via this protein kinase. However, incubation of astrocytes with a
phosphatidylinositol 3-kinase inhibitor (wortmannin) had no significant
effect on the Pio-dependent increases in glucose uptake or
lactate production. Because glucose uptake and the rate of glycolytic
metabolism are increased by activation of
Na+K+ ATPase in astrocytes (67) we also
tested the effect of a Na+K+ ATPase inhibitor
(ouabain). However, under the conditions used, ouabain did not reverse
Pio effects.
activation can exert anti-inflammatory effects
(25), regulate myelin gene expression (33), and increase neurite
extension (32) in brain cells. For these reasons, PPAR
agonists,
including TZDs like Pio and Rosi, are being considered as potential
neuroprotective agents in neurodegenerative disorders, including
Alzheimer's disease, Parkinson's disease, stroke, and multiple
sclerosis. It is possible that increases in intracellular glucose could
contribute to the anti-inflammatory effects by increasing
expression of stress proteins, such as inhibitory I
B proteins, which
would reduce glial inflammation. However, because glucose metabolism is
perturbed in the early stages of Alzheimer's disease (77-79) and in
multiple sclerosis (80-82), the effects described herein suggest an
additional mechanism, i.e. maintenance of brain energy
supplies, by which TZD treatment could provide benefit.
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ACKNOWLEDGEMENTS |
---|
We thank Anthony Sharp and Patricia Murphy for excellent technical assistance with cell culture and animal procedures, Chanannait Paisansathan and Richard Ripper for help with mitochondrial preparations, and Dr. Jonathan Art for help with immunocytochemical staining of astrocyte cultures.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Multiple Sclerosis Society, Takeda Pharmaceuticals North America, and GlaxoSmithKline.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.
¶ To whom correspondence should be addressed: 1819 West Polk St., MC519, Rm. 540, Chicago, IL 60612. Tel.: 312-996-8377; Fax: 312-996-9680; E-mail: cdr@uic.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M208132200
2 C. Dello Russo and D. L. Feinstein, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; 2-DG, 2-deoxy-glucose; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GLUT, glucose transporter; LPS, lipopolysaccharide; NOS, nitric-oxide synthase; Pio, pioglitazone; Rosi, rosiglitazone; Trog, troglitazone; TZD, thiazolidinedione; RT, reverse transcriptase; ANOVA, analysis of variance.
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