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INTRODUCTION |
Multicellular organisms have evolved complex homeostatic
mechanisms to sense and respond to a diverse range of exogenous and endogenous signals. One such mechanism appears to require the biochemical events which follow the activation of a peroxisome proliferator-activated receptor
(PPAR).1 PPARs are members of
the nuclear steroid hormone receptor superfamily and function to
transduce a variety of environmental, nutritional, and inflammatory
signals into a defined set of cellular responses (1). Three PPAR
isoforms, 
, 
/
, and 
, have been identified (1-3). Each
exhibits a high degree of sequence and structural homology (2), but
they possess individual patterns of tissue distribution (4, 5).
Activation of a PPAR causes the receptor to heterodimerize with a
9-cis-retinoic acid receptor (6), thereby conferring upon it
the ability to bind DNA and transcriptionally regulate a subset of
genes possessing a peroxisome proliferator response element, consensus
5'-AACTAGGTCAAAGGTCA-3' (7), in their promoter region (1, 8, 9).
While PPARs had long been considered to be orphan receptors, recent
reports indicate that a number of natural and xenobiotic ligands with
specificity for PPARs do indeed exist. The first ligands described were
the insulin-sensitizing thiazoladinediones which are specific
ligands for the PPAR isoform (10). Since that time, a number of
natural endogenous molecules have been found to be capable of
activating PPARs. For example, 15-deoxy-
12,14
prostaglandin J2 represents a natural PPAR ligand (11,
12). Many specific fatty acid species and their derivatives, especially polyunsaturated fatty acids (13-16), the inflammatory mediator leukotriene B4 (17), and the eicosanoid
8(S)-hydroxyeicosatetraenoic acid (18), have now been
demonstrated to be ligands for PPAR. Studies employing the
PPAR-deficient mouse have also revealed that the natural steroid
hormone dehydroepiandrosterone-3-sulfate (DHEAS) is a specific
activator of PPAR (19). Furthermore, a vast array of man-made
compounds are capable of activating PPAR. These include the
synthetic arachidonic acid analog eicosatetrayeinoic acid, the
hypolipidemic agents WY-14,643 and clofibrate, certain non-steroidal
anti-inflammatory drugs, phthalate ester plasticizers, plus a number of
other xenobiotic compounds (1, 20, 21). Based upon their capacity to
elicit cellular responses to a variety of stimuli, the PPARs may
represent a class of molecules which allow the biochemical adaptation
to a diverse range of internal and external signals. These include
nutritional and inflammatory agents as well as a number of potentially
toxic substances. PPAR activation results in the transcriptional
up-regulation of many genes, including those involved in peroxisomal
and mitochondrial fatty acid -oxidation, some lipid binding proteins
and apolipoproteins, certain isozymes of the cytochrome P450 family,
and antioxidant enzymes (1, 17, 22, 23). In addition, activation of
PPARs has been demonstrated to antagonize signaling through an array of
important pathways, including STATs, AP-1, and NF-
B (13, 24-28).
There is strong evidence to suggest that the deleterious changes to the
immune system that occur as an individual or experimental animal ages,
including a reduced capacity to be effectively vaccinated and the
dysregulated production of a number of pleiotropic cytokines, are
associated with a decreased ability to effectively handle oxidative
stress (29-33). That elevated levels of cellular oxidative stress are
present in aged experimental animals is indicated by elevations in
tissue and circulating lipid peroxide levels (32-34) as well as
oxidized proteins (35, 36). The redox-regulated and oxidant
stress-activated transcription factor NF-B has been reported to be
active in the heart, liver, kidney, brain, and cardiac muscle of aged
experimental animals, without alterations in the amounts of NF-B
subunits or of the inhibitory molecule IB present in the cytosol
(37-39). Under resting conditions, NF-B exists in the cytoplasm as
a dimer bound to the inhibitory protein IB. Signaling by various
cell stimuli appear to converge at the generation of increased levels
of intracellular reactive oxygen species (ROS), causing the
phosphorylation and ubiquitination of IB and its release from
NF-B. The NF-B dimer then translocates to the nucleus and binds
to the promoter region of genes possessing a B motif (consensus
GGGRNNT(Y)CC), thereby causing recruitment of transcriptional machinery
and induction of gene transcription (40).
We have recently demonstrated that NF-B is also present in an active
state in the macrophages and lymphocytes which reside in the spleens of
aged mice (34). This active NF-B was demonstrated to correlate with
the expression of the NF-B-regulated genes IL-6, IL-12, macrophage
migration inhibitory factor, cyclooxygenase-2, and tumor necrosis
factor- (34). We found that the administration of specific PPAR
activators or the dietary antioxidant vitamin E to aged rodents
effectively reduced the elevated levels of active NF-B,
reestablished control over proinflammatory cytokine production, and
reduced lipid peroxide levels in various tissues (34). These findings
suggest a role for PPAR in the maintenance of redox balance during
the aging process.
The studies presented herein demonstrate that low dose DHEAS or
WY-14,643 administration to aged animals elicits a number of biologic
changes, which are mediated through a process involving PPAR
activation. We employed a strain of mice bearing a null mutation in
PPAR (PPAR 
/) to experimentally demonstrate that normal
PPAR function is necessary to effectively maintain a balance in
cellular redox state. PPAR / mice were found to express indicators of oxidative stress much earlier in their lifespans than
wild-type mice. Administration of PPAR-specific activators to
wild-type and knockout animals was capable of reducing the age-associated elevations in NF-B activity and constitutive
pro-inflammatory cytokine production in the PPAR wild-type animals
but not their PPAR-deficient counterparts. Vitamin E supplementation
lowered the levels of active NF-B present in the spleens of both
aged wild-type and aged knockout animals. Similar results were obtained using young PPAR +/+ and PPAR / mice rendered
redox-imbalanced at an early age by feeding a vitamin E-deficient diet.
In addition, a decline in cellular PPAR expression was observed to
occur with normal aging. This was accompanied by similarly reduced
levels of acyl-CoA oxidase and catalase mRNA expression, enzymes
that are transcriptionally up-regulated following administration of PPAR activators. Either vitamin E supplementation or treatment of
aged mice with PPAR activators was able to cause elevations in
splenic PPAR, acyl-CoA oxidase, and catalase mRNA to levels typically observed in the spleens of young animals. Our findings indicate that the therapeutic administration of PPAR activators is
able to regulate cellular oxidant/antioxidant balance through a
mechanism that appears to require a functional PPAR.
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EXPERIMENTAL PROCEDURES |
Animals and Diets--
Colonies of PPAR wild type (PPAR
+/+) and homozygous knockout (PPAR /) mice were expanded from
breeding pairs obtained from Dr. F. J. Gonzalez (Metabolism
Branch, National Institutes of Health, Bethesda, MD). The derivation
and phenotypic characteristics of these animals have previously been
reported (41). Female mice were used for all of the experiments
reported herein. Six-week-old and 20-24-month-old female C57BL/6 mice
were purchased from the National Institute on Aging. All mice were
housed in the University of Utah Animal Resource Center, which
routinely monitors for the most prevalent murine pathogens, employs
sentinel animals as a means for early detection of murine hepatitis
virus, and guarantees strict compliance with regulations established by
the Animal Welfare Act. Normal maintenance chow, vitamin E-deficient
(tocopherol-stripped) chow, and vitamin E-containing control chow were
purchased from Harlan Teklad (Madison, WI). Mice were anesthetized with
halothane and sacrificed by cervical dislocation. Any mice with
evidence of gross internal or external pathology at the time of
sacrifice were excluded from the study.
Supplementation Therapy--
DHEAS (5-androsten-3-ol-17-one
3-sulfate) (Sigma), dissolved directly in the drinking water at a
concentration of 100 µg/ml, was prepared fresh on a weekly basis and
was available to the mice ad libitum, resulting in doses of
approximately 300-500 µg/day. WY-14,643
([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid) (Chemsyn
Science Laboratories, Lenexa, KS) was added directly to the chow
resulting in 250 µg/day doses. -Tocopherol (Sigma) was applied to
the chow in order to achieve a supplemental vitamin E dose of 4.6 mg/day. Peroxisome-proliferating doses of DHEA
(5-androsten-3-ol-17-one) (Sigma) and WY-14,643 were provided in the
chow at 0.5% w/w (25 mg/day) and 0.1% w/w (5 mg/day), respectively.
Freshly prepared food was provided every 2-3 days and was continuously
available to the mice during the 2-week supplementation period.
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared from cell suspensions of whole spleens using a modified
protocol of Dignam et al. (42). Briefly, cells were washed
twice in ice-cold phosphate-buffered saline containing 1 mM
phenylmethylsulfonyl fluoride (PMSF) and resuspended in 1 ml of buffer
A (10 mM HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM PMSF, 1 mM
DTT, and 0.5% Nonidet P-40) for 10 min on ice, vortexing every 2 min.
Nuclei were collected by centrifugation at 1000 × g
for 5 min at 4 °C. The nuclear pellet was washed with 1 ml of buffer A without Nonidet P-40. 25 µl of buffer B (10 mM HEPES,
pH 7.9, 420 mM NaCl, 25% glycerol, 5 mM
MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 10 µg/ml aprotinin, 100 µM leupeptin, 1 mM
PMSF, and 1 mM DTT) was added to the pellet and, following
sonication for 10 s, incubated for 30 min on ice. Nuclear debris
was removed by centrifugation at 13,000 × g for 10 min. The supernatant was collected, the protein content of the nuclear
extract determined (43), and an electrophoretic mobility shift assay
(EMSA) was performed. While contamination of the nuclear extracts with
small quantities of cytosolic proteins is possible, cytosolic NF-B
will not bind DNA due to its association with the inhibitory molecule
IB (44).
EMSA--
Equal amounts of nuclear extracts (2 µg of protein)
were incubated with 30,000 cpm of 32P labeled NF-B
specific probe (Promega). Reactions were performed in a 20-µl total
volume containing 2 µg of nuclear extract, 4 µl of 5× gel shift
binding buffer (20 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, and
20% glycerol), 1.5 µg of poly(dI-dC), and 1 µl of probe. For
supershift assays, 2 µl of an appropriate anti-NF-B subunit
antibody (Santa Cruz Biotechnology, Inc.) was added to each reaction.
The reaction was incubated at room temperature for 15 min, loaded on a
4% native polyacrylamide gel, and run in 0.5× TBE buffer. The gel was
dried and subjected to autoradiography. NF-B-specific bands were
confirmed by competition with a 50-fold excess of an unlabeled NF-B
probe, which resulted in no shifted band, or by preparing the reaction
with excess labeled nonspecific probe, which did not reduce the
intensity of the NF-B band.
Cell Culture and ELISA--
Cells obtained from the spleens of
PPAR +/+ or PPAR / animals were cultured under serum-free
conditions. Briefly, mice were anesthetized with halothane and
sacrificed by cervical dislocation. Single cell suspensions were
prepared from the spleens of these animals. The collected splenocytes
were washed three times in Dulbecco's phosphate-buffered saline and
cultured at 107 cells/ml in freshly prepared serum-free
medium consisting of RPMI 1640, 1% Nutridoma-SR (Boehringer Mannheim),
200 mM L-glutamine, antibiotics, and 5 × 10
5 M 2-mercaptoethanol. Cells were incubated
for 24 h at 37 °C in an atmosphere of 5% CO2 in
air. Cell culture supernatants were then collected for quantitative
evaluation of immunoactive IL-6 or IL-12 (p40) by ELISA, as described
previously (45). Monoclonal rat anti-murine cytokine antibodies and
murine recombinant IL-6 and IL-12 cytokine standards were purchased
from PharMingen (San Diego, CA).
Reverse Transcriptase (RT)-PCR--
mRNA was prepared by the
method of Chomczynski (46) and RT-PCR was performed with adaptations
for rapid cycling with the 1605 air thermocycler (Idaho Technology,
Idaho Falls, ID), as described previously (45). PCR conditions were:
denaturation, 94 °C for 1 s, annealing for 1 s, and
elongation, 72 °C for 8 s. 16 cycles were performed for
-actin and 30 cycles were performed for PPAR, acyl-CoA oxidase,
and catalase. Gene-specific sequences were derived from GenBank
submissions. Oligonucleotides and annealing temperatures (AT) used for
these analyses are as follows: -actin (AT = 60 °C): 5'-GGG
TCA GAA GGA CTC CTA TG-3' and 5'-GTA ACA ATG CCA TGT TCA AT-3'. PPAR
(AT = 55 °C): 5'-GTG GCT GCT ATA ATT TGC TGT G-3' and 5'-GAA
GGT GTC ATC TGG ATG GGT G-3'. Acyl-CoA oxidase (AT = 55 °C):
5'-CGA CCT TGT TCG GGC AAG TGA GGC GC-3' and 5'-GGA GCT CAG ACG TGT CCC
AGG G-3'. Catalase (AT = 55 °C): 5'-ATG TCG GAC AGT CGG GAC
C-3' and 5'-CAT GTC AGG ATC CTT CAG G-3'.
Following PCR, the samples were mixed with an equal volume of stop
solution, heated to 95 °C for 5 min, and electrophoresed on a 6%
acrylamide gel. Bands were detected by autoradiographic exposure for
16 h at 70 °C. Sizes of the bands were determined by
comparing to the migration pattern of a 32P-end-labeled
MspI digest of pBR322 (New England Biolabs, Beverly, MA).
Lipid Peroxidation Assay--
Immediately following sacrifice,
mouse livers were perfused with cold 0.9% NaCl via the portal vein
prior to removal. Livers were weighed, and 10% homogenates were
prepared in 1.15% KCl using a Tissue Tearor homogenizer. Supernatants
obtained by centrifugation at 3500 × g of liver
homogenates were either left on ice or subjected to an oxidative stress
in vitro by incubating them for 1 h at 37 °C in the
presence of 10 µM FeSO4, after which time
EDTA was added to a final concentration of 10 mM. Lipid
peroxides were assayed using a modified procedure of Buege and Aust
(47). Briefly, 250 µl of 10% tissue homogenate or working standard
(1,1,3,3-tetramethoxypropane; Sigma) was added to 500 µl of
TCA-TBA-HCl reagent (15% w/w trichloroacetic acid, 0.375% w/v
thiobarbituric acid, 0.25 N hydrochloric acid). The
resulting mixture was vortexed and heated at 95 °C for 15 min. The
mixture was then cooled on ice for 15 min, centrifuged at 3000 rpm for
10 min, and the absorbance of the supernatant at 535 nm was determined
using a Molecular Devices multiwell plate spectrophotometer. The degree
of lipid peroxidation was expressed as concentration of thiobarbituric
acid-reactive substances (TBARS) participating in the reaction per gram
of tissue.
| |
RESULTS |
PPAR / Mice Exhibit an Obese Phenotype--
While expanding
the PPAR +/+ and PPAR / mouse colonies, it became apparent
that the body weights of 14-week-old PPAR / mice were greater
than age-matched PPAR +/+ mice. As has been described previously
(48, 49), an observed 30% increase in body weight persisted throughout
adulthood, and the overweight phenotype was more exaggerated in males.
Gross pathologic examination revealed a markedly increased amount of
intraabdominal adipose tissue in the PPAR / mice (data not
shown). The recent report that PPAR / mice demonstrate reduced
basal levels of mitochondrial fat-metabolizing enzymes may offer an
explanation for these observations (23). An inability to efficiently
catabolize fatty acids would cause them to be redirected for storage in
adipose tissue, thereby accounting for the obese phenotype of PPAR
/ mice. The reported inability of PPAR / mice to
transcriptionally up-regulate enzymes for fatty acid catabolism in
response to administration of PPAR activators may be why these mice
exhibit lipid droplets in their livers following an overnight fast (48,
49), suggesting that endogenous molecules (i.e. fatty acids)
are not capable of activating PPAR, or of being enzymatically
metabolized, in the PPAR / mice.
PPAR / Mice Exhibit an Aged Proinflammatory Phenotype at a
Younger Chronological Age than PPAR +/+ Mice--
When splenocytes
were obtained from 4-month-old PPAR +/+ and PPAR / mice and
analyzed by EMSA for NF-B activity, PPAR +/+ mice did not express
nuclear NF-B activity in their spleens, while age-matched PPAR
/ mice already exhibited high levels of nuclear NF-B activity
(Fig. 1A). In vitro
activation of splenocytes from PPAR +/+ and PPAR / mice with
a low dose of LPS (10 ng/ml) was able to modestly increase nuclear
NF-B activity in both groups (Fig. 1A). The levels of
NF-B activity seen in splenocytes from 4-month-old PPAR +/+ mice
following activation with LPS, however, did not reach the levels seen
in splenocytes from age-matched PPAR / mice in the absence of
exogenous activators (Fig. 1A). The secretion of two
NF-B-driven cytokines, IL-6 and IL-12, by either control or
LPS-activated splenocytes from PPAR +/+ and PPAR / mice was
quantitated by ELISA. PPAR / splenocytes were found to produce
2-3 times more IL-6 and IL-12, both in the absence of exogenous
stimulation (Fig. 1B) and following activation with LPS
(Fig. 1C), than splenocytes from PPAR +/+ mice.
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Fig. 1.
PPAR / mice acquire a physiologically
aged phenotype, indicative of a state of redox imbalance, at a younger
age than PPAR +/+ mice. Splenocytes obtained from 4-month-old
PPAR +/+ (open bars) or PPAR /
(filled bars) mice were cultured at equivalent
cell densities in the absence or presence of 10 ng/ml LPS. After 45 min, cells were harvested, nuclear extracts were prepared, and an
NF-B EMSA was performed (A). Data shown are typical of
mice from each group. Cells treated with 0 ng/ml (B) or 10 ng/ml (C) LPS were cultured for 24 h, and IL-6 and
IL-12 levels (mean ± S.D. of 3 mice/group) in supernatants were
quantitated by ELISA.
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Peroxisome Proliferating Doses, but Not Therapeutic Doses, of DHEAS
or WY-14,643 Cause the Activation of NF-B in Young PPAR +/+
Mice--
We questioned whether the effects that we have previously
reported to occur following the administration to aged mice of natural and xenobiotic agents capable of activating PPAR (45,
50)2 were dependent upon the
presence of a functional PPAR. PPAR +/+ and PPAR / mice at
2 and 15 months of age were compared in these studies. As has been
previously demonstrated with C57BL/6 strain mice (45), both PPAR
+/+, and PPAR / mice at 2 months of age exhibited only minimal
levels of nuclear NF-B activity in their spleens (Fig.
2). When peroxisome-proliferating doses of DHEAS or WY-14,643 were added to the diets of young PPAR +/+ mice
for a period of 2 weeks, a marked elevation in nuclear NF-B activity
was consistently observed in spleen (Fig. 2) and liver tissue (data not
shown). It is not known whether the induction of NF-B represents a
transient response or is long- lasting and contributes to the
hepatocarcinogenic effects of long term and high dose administration of
peroxisome proliferators to rodents (52). Spleens of animals provided
therapeutic doses of DHEAS or WY-14,643 that have previously been
demonstrated by us to cause marked reductions in splenic NF-B
activity, inflammatory cytokine production, and tissue TBARS levels
(34), (approximately 40-fold lower than peroxisome proliferating doses)
showed no nuclear NF-B-inducing activity (Fig. 2). Consistent with
their reported inability to respond to PPAR activators (41), no
nuclear NF-B was observed in young PPAR / mice treated with
DHEAS or WY-14,643 at either the high (peroxisome-proliferating) or low
doses (Fig. 2). These results demonstrate that, while
peroxisome-proliferating doses of PPAR-specific activators are
capable of activating NF-B, lower doses of PPAR activators appear
not to cause deleterious changes in cellular redox balance.
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Fig. 2.
Peroxisome proliferating doses of DHEAS or
WY-14,643 activate NF-B in PPAR +/+ mice but not PPAR /
mice, while therapeutic doses of PPAR-specific activators do not
activate NF-B in either. Two-month-old PPAR +/+ and PPAR
/ mice were fed a control diet or chow containing low (therapeutic)
or high (peroxisome proliferating) doses of DHEA or WY-14,643 for a
period of 2 weeks. Spleens were then harvested, nuclear extracts were
prepared, and an NF-B EMSA was performed. Data are representative of
results from experiments performed twice employing two mice per
group.
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|
Effects Derived from the Therapeutic Administration of DHEAS or
WY-14,643 to Aged Mice Are Dependent upon a Functional
PPAR--
When animals were evaluated at 15 months of age, both
PPAR +/+ and PPAR / mice were found to abnormally express
significant levels of nuclear NF-B in their unstimulated spleens
(Fig. 3A). This observation is
consistent with our recent findings in C57BL/6 strain mice (45). When
15-month-old PPAR +/+ and PPAR / mice were treated with
therapeutic doses (34) of DHEAS or WY-14,643 (300-500 and 250 µg/day, respectively), or with a supplemental dose of the antioxidant
vitamin E (3 IU/day), the PPAR +/+ mice exhibited markedly reduced
nuclear NF-B activity in their spleens following all three
treatments (Fig. 3A). The levels of nuclear NF-B activity
in the treated aged PPAR +/+ mice were comparable to the levels seen
in the young PPAR +/+ mice fed the same diet as aged control animals
(identical composition without the supplements). Analysis of whole cell
extracts of splenocytes from aged and supplemented-aged mice by Western
blotting has revealed similar expression of the NF-B subunit p65
(data not shown). PPAR / mice responded to treatment with
vitamin E by exhibiting a lowering of nuclear NF-B levels (Fig.
3A) and a reduction in constitutive IL-6 (Fig.
3B) and IL-12 production (Fig. 3C). The treatment
of 15-month-old PPAR / mice with DHEAS or WY-14,643, however,
had no modifying influence on splenic NF-B activity (Fig.
3A) or pro-inflammatory cytokine production (Fig. 3,
B and C). Reductions in splenic NF-B activity
and cytokine production in PPAR +/+ mice, but not in PPAR /
mice, were observed following the systemic treatment of aged animals
with DHEAS or WY-14,643 for only 3 days, while both PPAR +/+ and
PPAR / mice responded rapidly to supplementation with additional
vitamin E (data not shown). These results strongly suggest that a
functional PPAR is required to promote the beneficial effects of
DHEAS or WY-14,643 treatment of aged mice. As expected, the capacity of
antioxidant supplementation of aged mice with vitamin E to correct the
age-associated dysregulation in the NF-B system is independent of
PPAR influences, as this treatment was effective in both the PPAR
+/+ and PPAR / mice.
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Fig. 3.
Administration of DHEAS or WY-14,643 lowers
constitutive NF-B activity and pro-inflammatory cytokine production
in 15-month-old PPAR +/+ mice but not PPAR / mice.
Spleens were isolated from 2-month-old, 15-month-old, or treated
15-month-old PPAR +/+ or PPAR / mice. Nuclear extracts were
prepared, and an NF-B EMSA was performed (A). Data are
representative of results from experiments performed on four separate
occasions employing two mice per group. Alternatively, splenocytes were
cultured in serum-free medium for 24 h and ELISAs were performed
on culture supernatants to quantitate IL-6 (B) and IL-12
(C) levels (mean ± S.D. of 3 mice/group).
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Supershift assays of nuclear extracts obtained from aged PPAR +/+
mice revealed the presence of both p50 and p65 subunits, with an
apparent absence of cRel (Fig. 4). The
presence of two supershifted bands upon the addition of the -p50
antibody indicates the presence of both p50/p50 homodimers and, because
of its similar gel mobility to that observed upon the addition of
-p65, p50/p65 heterodimers. This conclusion is similar to the
observations made by Supakar et al. (53) in their studies of
age-associated elevations in liver NF-B activity and is supported by
the finding that the addition of both -p50 and -p65 antibodies
resulted in the clear appearance of two supershifted bands. Similar
results were seen in splenic nuclear extracts obtained from
15-month-old PPAR / mice (data not shown). These findings, in
addition to the observed elevations in IL-6 and IL-12 production, imply
that the NF-B found in the nuclear extracts of splenocytes from aged
mice is indeed capable of transcription-regulating activities.
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Fig. 4.
The nuclear NF-B seen in spleens from aged
mice is composed of p50 and p65 subunits. Nuclear extracts
isolated from spleens of 15 month old PPAR +/+ mice were incubated
with antibodies recognizing the NF-B subunits p50, p65, or cRel,
prior to performing an NF-B EMSA. Similar results were obtained with
splenic nuclear extracts from 15-month-old PPAR / mice.
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The Therapeutic Administration of DHEAS or WY-14,643 Requires a
Functional PPAR to Lower the Levels of NF-B Activity and
Inflammatory Cytokine Production in Redox-imbalanced Adult
Mice--
Dietary supplementation with antioxidants, in particular
vitamin E, has been well documented to have beneficial effects on numerous physiologic parameters both during aging and in a number of
pro-oxidant disease states (54-57). In our hands, dietary
supplementation of aged mice with vitamin E was capable of eliciting
its antioxidant effects independent of a functional PPAR. We
therefore questioned whether a depletion of vitamin E from the diets of
2-month-old PPAR +/+ and PPAR / mice might rapidly promote a
physiologically aged phenotype in a chronologically young animal. Mice
were fed a control diet or a diet deficient in vitamin E for a period
of 4 weeks. These diets were identical, except that a
tocopherol-stripped fat source and vitamin mix containing no added
-tocopherol were substituted for the normal fat and vitamin mix,
respectively. As expected, the PPAR +/+ and PPAR / mice fed
control diets showed no measurable nuclear NF-B activity in their
spleens. Both the 3-month-old PPAR +/+ and PPAR / animals
consuming the vitamin E-deficient diet, however, exhibited nuclear
translocation of NF-B in their spleens (Fig.
5). Supershift assay of splenic nuclear
extracts obtained from these mice showed the presence of NF-B
composed of p50 and p65 subunits, similar to what was found in the aged
mice (data not shown).
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Fig. 5.
Feeding of a vitamin E-deficient diet causes
the activation of NF-B. Groups of five 2-month-old PPAR +/+
and PPAR / mice were maintained on the control diet or a vitamin
E-deficient diet for a period of 4 weeks, after which time splenic
nuclear extracts were prepared and an NF-B EMSA was performed. Data
shown are typical of mice from each group.
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Groups of 2-month-old PPAR +/+ and PPAR / mice were
maintained on vitamin E-deficient diets for a period of 6 weeks. For the last 2 weeks, subgroups of animals from both diet groups were provided with DHEAS, WY-14,643, or vitamin E supplements. Both PPAR
+/+ and PPAR / vitamin E-depleted mice demonstrated reduced levels of nuclear NF-B activity in their spleens following vitamin E
supplementation (Fig. 6A).
Similarly, PPAR +/+ mice provided with DHEAS or WY-14,643
demonstrated levels of nuclear NF-B activity that closely resembled
the levels found in splenocytes from 2-month-old mice fed the control
diet (Fig. 6A). The PPAR / mice, however, did not
show any reduction in splenic nuclear NF-B levels following dietary
supplementation with either DHEAS or WY-14,643 (Fig.
6A).
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Fig. 6.
The levels of nuclear NF-B and
inflammatory cytokine production induced by the feeding of a vitamin
E-deficient diet are lowered by dietary supplementation with DHEAS or
WY-14,643 in PPAR +/+ mice, but not in PPAR / mice.
Beginning at 8 weeks of age, mice were maintained on the normal diet or
were fed the vitamin E-deficient diet for a total of 6 weeks. During
the final 2 weeks, groups of 3 mice were provided with supplemental
vitamin E, WY-14,643, or DHEAS in their diets. Splenocytes were
harvested, nuclear extracts were then prepared, and an NF-B EMSA was
performed (A). Data shown are typical of mice from each
group. Alternatively, splenocytes were cultured in serum-free medium
for 24 h and ELISAs were performed on culture supernatants to
quantitate IL-6 (B) and IL-12 (C) levels
(mean ± S.D. of 3 mice/group).
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The transcription-regulating activity of the NF-B localized to the
nucleus in spleens of mice provided with vitamin E-deficient diets was
confirmed by ELISA to measure the abnormal constitutive production of
the cytokines IL-6 and IL-12. Splenocytes from young PPAR +/+ and
PPAR / mice cultured overnight without additional stimulation
produced only very low levels of IL-6 (Fig. 6B) and IL-12
(Fig. 6C). However, splenocytes from age-matched PPAR +/+ and PPAR / mice maintained on the vitamin E-deficient diet for 6 weeks, which induced the activation of NF-B, demonstrated the
spontaneous production of IL-6 and IL-12. This constitutive pro-inflammatory cytokine production could be effectively reduced in
PPAR +/+ mice and partially lowered in the PPAR / animals following the reintroduction of a supplemental dose of vitamin E into
the diets for a period of 2 weeks. A supplementation with DHEAS or
WY-14,643 to the vitamin E-deficient diets resulted in a reduction of
the spontaneous IL-6 and IL-12 production only in the PPAR +/+ mice.
DHEAS or WY-14,643 administration was completely incapable of lowering
the spontaneous production of IL-6 and IL-12 in PPAR / mice.
These results further implicate that the activation of PPAR is
capable of having an anti-oxidant effect in mice rendered redox
imbalanced either through dietary manipulation or as a normal result of aging.
Activation of PPAR Modulates Tissue Levels of Lipid
Peroxides--
We and others have previously observed elevated levels
of lipid peroxides in the tissues of aged experimental animals and humans (29, 32-34, 58). Vitamin E supplementation has been demonstrated to reduce tissue levels of lipid peroxides (34, 59). In
addition, our laboratory has demonstrated that administration of
vitamin E or specific activators of PPAR is capable of reducing the
age-associated elevations in tissue lipid peroxide levels in the spleen
and liver (34). We therefore determined the levels of lipid
peroxidation in livers from PPAR +/+ and PPAR / mice of
various ages. Furthermore, we questioned whether the capacity of DHEAS
and WY-14,643 to modulate the amount of cellular lipid peroxides is
through the activation of PPAR. The TBARS assay was used to measure
tissue lipid peroxide levels in liver homogenates from PPAR +/+ and
PPAR / mice that were young (3 months old), middle aged (12 months old), aged (24 months old), or aged but provided supplementation
with DHEAS, WY-14,643, or vitamin E. The lipid peroxide levels in liver
homogenates were measured either directly (Fig.
7A) or following in
vitro oxidation in the presence of iron (Fig. 7B). At 3 months of age, both PPAR +/+ and PPAR / mice exhibited
similarly low levels of tissue lipid peroxidation. By 12 months of age,
however, the PPAR / mice exhibited approximately 2.5-fold
elevations in liver TBARS levels than their age-matched PPAR +/+
counterparts. At 24 months of age, both strains of mice exhibited
elevated TBARS levels both under basal conditions or subsequent to
in vitro oxidation. Supplementation of aged PPAR +/+ mice
with DHEAS or WY-14,643 was capable of reducing tissue lipid peroxides
to levels seen in young animals, while similar treatment of aged
PPAR / mice was completely ineffective. As expected,
supplementation with the dietary antioxidant vitamin E was capable of
reducing the levels of tissue TBARS in both PPAR +/+ and PPAR
/ mice, although this treatment appeared to be somewhat more
effective in PPAR +/+ animals. These results suggest that the tissue
lipids of PPAR / mice exhibit indications of oxidative damage
and are more susceptible to oxidative stress at an earlier age than
PPAR +/+ mice. Supplementation with specific activators of PPAR
was only capable of lowering the levels of liver lipid peroxides in
aged PPAR +/+ mice. In addition, liver tissue from aged PPAR +/+
animals supplemented with DHEAS or WY-14,643 exhibited greater
resistance to lipid peroxidation following an in vitro
oxidative stress, suggesting that tissues from these animals have
regained an inherent capacity to more effectively cope with naturally
encountered oxidative stresses than age-matched PPAR /
mice.
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Fig. 7.
Age-associated elevations in tissue lipid
peroxide levels occur at a younger age in PPAR / mice and are
reduced following dietary supplementation with DHEAS or WY-14,643 in
aged PPAR +/+ mice only. Liver homogenates were obtained from
PPAR +/+ (open bars) and PPAR / mice
(filled bars) that were 3, 12, or 24 months old,
or 24 months old treated with a 2-week regimen of DHEAS, WY-14,643, or
vitamin E. TBARS assays were performed either immediately upon
sacrifice (A) or following in vitro oxidation in
the presence of 10 µM iron for 60 min at 37 °C
(B). Data are the mean values ± S.D. of 3 mice/group.
The absence of error bars implies small S.D. values.
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Age-associated Reductions in Splenic Expression of PPAR,
Acyl-CoA Oxidase, and Catalase Are Reversed following DHEAS
Supplementation--
Examination of mRNA obtained from the spleens
of young, aged, and DHEAS-treated aged C57BL/6 strain mice revealed an
age-associated decline in the levels of mRNA encoding PPAR,
which was accompanied by a similar decrease in mRNA for the
peroxisome-associated enzymes acyl-CoA oxidase and catalase (Fig.
8A). The levels of PPAR
mRNA in splenocytes from young mice was determined by densitometric analysis to be between 2.5- and 10-fold higher than in splenocytes from
aged animals (data not shown). Therapeutic treatment of aged C57BL/6
mice with DHEAS for 2 weeks, which we have previously demonstrated to
reduce the spontaneous activation of NF-B and NF-B-driven genes
in the spleen (34) resulted in the up-regulation of PPAR, acyl-CoA
oxidase, and catalase mRNA to levels normally observed in young
mice (Fig. 8A). Furthermore, aged C57BL/6 mice supplemented
for 2 weeks with vitamin E also demonstrated elevated expression of
mRNA for PPAR (Fig. 8B), suggesting that balancing the cellular redox state may provide a level of transcriptional regulation for this gene. The prooxidant state observed in the cells of
aged animals may therefore be a cause of the age-associated reductions
in PPAR gene expression. Reciprocally, reductions in PPAR gene
expression may, in part, contribute to the prooxidant phenotype of
aging through an age-associated deficiency in the efficient modulation
of cellular redox state.
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Fig. 8.
Transcript levels of PPAR are reduced in
aged mice and are elevated following dietary supplementation with DHEAS
or vitamin E. To evaluate the levels of PPAR, acyl-CoA oxidase,
catalase, and -actin, RT-PCR was performed on mRNA extracted
from the splenocytes from C57BL/6 mice that were 2 or 24 months old, or
24 months old treated with a 2-week regimen of DHEAS (A) or
vitamin E (B). -Actin was employed to ensure equal
loading of cDNA. Data are representative of three separate
experiments employing two mice per group.
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DISCUSSION |
Elevated levels of cellular oxidative stress contribute to the
pathophysiology of a number of clinical conditions, disease states, and
to aging. In addition to causing damage to cellular constituents,
recent evidence suggests that reactive oxygen species can alter
cellular function through their ability to affect signal transduction
processes (60). Our laboratory has recently demonstrated that aged
C57BL/6 strain mice express a markedly elevated activity of the
redox-regulated transcription factor NF-B in a number of lymphoid
organs when compared with young adult controls (34). Activities by
dysregulated cytokines and proteins under NF-B control could be
responsible for changes in immune competence and may contribute to
other diseases that accompany aging (61-66). Supplementing the diets
of aged mice with modest doses of chemical agents capable of activating
PPAR reduced the nuclear activity of NF-B to levels seen in young
animals. This was paralleled by a correction of the dysregulated
constitutive expression of a number of NF-B-regulated cytokines and
other proteins (see Ref. 34, and references therein). The experiments
described herein were designed to question whether the in
vivo NF-B-correcting effects resulting from treatment of aged
and redox-imbalanced mice with PPAR activators is mediated through
mechanisms that require a functional PPAR.
Activation of PPAR in vivo causes an up-regulation of the
mRNA and protein levels of a number of peroxisome- and
non-peroxisome-associated enzymes, and structural proteins. Included
among these induced genes are enzymes responsible for fatty acid
-and 
-oxidation, bile acid and glycerolipid synthesis, amino acid
metabolism, certain apolipoproteins and fatty acid-binding proteins,
lipoprotein lipase, the antioxidant enzymes catalase and
Cu,Zn-superoxide dismutase, and mediators of the glutathione pathway
(17, 22). While the -oxidative enzymes induced by PPAR activators
are themselves capable of generating hydrogen peroxide, induction of
the antioxidant enzyme catalase appears to be able to effectively
counterbalance this effect (67, 68). Following administration of agents
capable of activating PPAR, the catalase activity within the cytosol of liver and kidney cells was found to increase greater than 5-fold (69). Indices of cellular oxidative stress are not demonstrated to be
altered following the administration of modest doses of PPAR
activators, indicative of the enzymatic antioxidant capacity of the
induced catalase (70).
Activators of PPARs have now been implicated in the protection against
pro-oxidant-mediated damage induced by a number of systems. These
include protection against transition metal-induced oxidation of
LDL-associated lipids, CCl4-mediated hepatotoxicity, and
others (71-76). Recent evidence also suggests that PPAR is capable
of regulating plasma levels of lipid peroxides (77), indices of oxygen
radical-mediated damage, and that administration of the PPAR
activator bezafibrate reduces circulating lipid peroxide levels (78).
PPAR activation appears to be capable of modulating the duration of
an inflammatory response, probably through the transcriptional
up-regulation of enzymes necessary for the metabolic clearance of
leukotrienes as PPAR / mice were found to elicit an extended
response to inflammatory stimuli (17).
The cellular activation of PPAR has been demonstrated to antagonize
activities by a number of transcription factors, including NF-B,
AP-1, and STATs (24, 25). These effects were possibly achieved through
a sequestration of essential transcription coactivators such as p300,
Src-1, or CBP, or via direct protein-protein or protein-DNA
interactions involving PPAR itself. PPAR also forms stable
interactions with other transcription factor complexes, thereby
exerting an inhibitory influence on cellular signaling processes (13,
26, 27). Recently, it was demonstrated, in human aortic smooth muscle
cells cultured in vitro, that specific activators of PPAR
are capable of inhibiting IL-1-induced IL-6 and prostaglandin
production. PPAR activation was also able to inhibit
cyclooxygenase-2 expression, primarily through the repression of
NF-B transactivation (28). For any single or combination of the
aforementioned reasons, providing aged animals with specific activators
of PPAR results in reductions in NF-B activity and, subsequently,
causes declines in the expression of NF-B-regulated genes.
The observed age-associated declines in PPAR expression may occur
for a number of reasons. The gene encoding PPAR is under the
transcriptional control of glucocorticoids (GCS) (79). As such, PPAR
levels fluctuate in rhythm with circulating GCS concentrations and the
levels of PPAR are also up-regulated during times of stress, which
are accompanied by increased GCS secretion by the adrenal glands (80).
Interestingly, the ability to respond to PPAR activators is enhanced
in starved mice, a condition that increases circulating GCS
concentrations and that has been employed, in the form of caloric
restriction, as a therapeutic intervention to prevent a number of
pathologic outcomes in aging (81, 82). However, age-associated declines
in tissue responsiveness to the anti-inflammatory effects of GCS have
been demonstrated in aged experimental animals despite elevated levels
of GCS in the circulation (83-85).
Seven putative binding sites for the basal transcription factor Sp1
have been identified in the promoter region of the PPAR gene (86).
The DNA binding activity, and therefore transcription regulating
capacity, of a number of transcription factors including Sp1 and the
glucocorticoid receptor are susceptible to alterations in cell redox
state (87). Sp1 has been demonstrated to possess reduced DNA binding
efficiency in nuclear extracts obtained from the tissues of aged rats
(87, 88). An increase in the intracellular oxidative state in aged
animals and the lowering of oxidative state following administration of
PPAR activators or -tocopherol suggest that the ability of Sp1 or
glucocorticoid receptor to act as a transcriptional regulators may be
affected in our model.
The NF-B-driven cytokines tumor necrosis factor-, IL-1, and
IL-6 have been demonstrated to cause a reduction in the expression of
PPAR (89, 90). These same cytokines have been found to be expressed
at higher levels by cells from aged animals and, in the case of IL-6,
can be demonstrated in the serum of aged humans and mice (61, 62, 66).
Whether the effects of these cytokines are mediated through the
generation of intracellular ROS, or through another defined
cell-signaling mechanism, is currently being studied in our laboratory.
Whatever the reason(s) behind the reductions in PPAR expression, our
data offer a possible explanation for observations that it is more
difficult to induce responses following administration of PPAR
activators to aged rodents (91).
A number of the consequences of the age-associated decline in PPAR
expression may contribute to the molecular alterations observed in
aging. The decreased expression of acyl-CoA oxidase may contribute to
the age-associated accumulation of lipid stores and an inability to
efficiently metabolize very long chain and polyunsaturated fatty acids,
outcomes evidenced by increases in membrane rigidity and oxidative
damage to lipids (91-93). It has been demonstrated, employing the
acyl-CoA knockout mouse (94), that elevated levels of hydrogen peroxide
are present in liver tissue at 4 months of age (95). This is followed
by a lowering of liver H2O2 levels as the
animals age, during which time they undergo liver regeneration as a
result of spontaneous PPAR activation from endogenous agonists (95).
Our results demonstrate that oxidative damage to tissue lipids is
reduced following the administration of PPAR-specific activators.
The age-associated decrease in catalase mRNA expression may have an
enormous impact on the overall cellular oxidative state because of its
capacity to detoxify hydrogen peroxide, the reactive oxygen species
which itself has been demonstrated to be sufficient for the activation
of NF-B (51, 60, 96-98). The ability of dietary supplementation
with PPAR activators to facilitate the transcriptional up-regulation
of acyl-CoA oxidase and catalase may, in turn, increase the turnover of
damaged and chain-propagating lipids and other pro-inflammatory fatty
acid derivatives, as well as lowering cellular levels of hydrogen
peroxide and other ROS. This would, in turn, cause reductions in the
level of NF-B activity as well as lower the production of
NF-B-driven gene products.
It remains to be established whether the administration of
PPAR-specific activators to mice with an intact PPAR gene elicits its beneficial effects directly within the spleen or in another organ(s) with higher levels of PPAR expression, such as in the liver
(3, 5). Either of these primary sites of PPAR-ligand interaction
could allow for the re-establishment of an appropriate redox balance in
certain individual cells within a particular organ and possibly in the
whole animal. The cells which are beneficially affected by PPAR
activators are likely to be those expressing PPAR, as well as those
cells in the immediate vicinity of PPAR-expressing cells. Recent
work by our laboratory and others has provided convincing evidence that
consideration of the roles of PPAR, its activators, and
PPAR-regulated genes may have important clinical applications toward
maintaining redox balance during aging and re-establishing redox
balance caused by pro-inflammatory or oxidant stress-related disease
states. If similar age-associated changes are observed in humans, it
might offer an explanation for the increased incidence of a number of
disease states with aging.
Activation
Modulates Cellular Redox Status, Represses Nuclear Factor-
B
Signaling, and Reduces Inflammatory Cytokine Production in Aging*
§ and
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Abstract
Introduction
