HomeLarge Type Edition
HOME ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:B997-B1006 (2004)
© 2004 The Gerontological Society of America

Modulation of PPAR in Aging, Inflammation, and Calorie Restriction

Bokyung Sung1, Seongjoon Park1, Byung Pal Yu2 and Hae Young Chung1,

1 College of Pharmacy, Aging Tissue Bank, Pusan National University, Busan, Korea.
2 Department of Physiology, The University of Texas Health Science Center at San Antonio.

Address correspondence to Hae Young Chung, PhD, College of Pharmacy, Pusan National University, Jangjeon-dong, Geumjeong-ku, Busan, 609-735, Korea. E-mail: hyjung{at}pusan.ac.kr


   Abstract
Top
 Abstract
Experimental Procedures
 Results
 Discussion
 References
 
Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily of transcription factors, are key regulators in various pathophysiological processes related to energy metabolism including lipid, carbohydrate metabolism, and inflammation. At present, little information is on the effect of age and calorie restriction (CR) on PPARs. In the present study, we investigated how age and CR (60% of the ad libitum intake) modulate PPARs in kidneys obtained from Fischer 344 rats, ages 13 and 25 months. Results showed that nuclear protein, mRNA level, and DNA binding activity of PPARs decreased with age, while CR blunted the reduction. Our findings were verified in separate experiments in which rats were injected with lipopolysaccharide, with the result of increased susceptibility to inflammation. Based on these findings, we conclude that the altered expression of PPARs may be due to increased oxidative stress with age, and that CR prevents these decreases through its antioxidative action.

PEROXISOME proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear hormone receptor superfamily (1). PPARs play major roles in a broad spectrum of biological processes, including cell proliferation, differentiation, glucose homeostasis, eicosanoid signaling, insulin sensitivity, glucose and lipid metabolism, bone formation, and tissue remodeling. Recent studies showed that PPAR activation can regulate inflammatory responses (1,2). PPARs modulate gene expression by binding with their heterodimeric partner retinoid X receptor (RXR) to specific PPAR-response elements (PPREs).

The PPAR subfamily consists of three isotypes: PPAR{alpha}, PPARß/{delta}, and PPAR{gamma}. Intensive study of PPARs during the last few years has revealed their importance in both normal physiology and in the pathology of various tissues. PPAR{alpha} and PPAR{gamma} are the two main categories of these receptors and are well characterized. PPAR{alpha} is present in the liver, heart, and, to a lesser extent, skeletal muscle. When activated, PPAR{alpha} promotes fatty acid oxidation, ketone body synthesis, and glucose sparing. PPAR{gamma} is expressed in adipose tissue, lower intestine, and cells involved in immunity. Activation of PPAR{gamma} induces the differentiation of adipocytes and stimulates triglyceride storage. Thus, PPAR{gamma} activation improves insulin sensitivity by inducing insulin-sensitizing hormones such as adiponectin, all of which promote glucose utilization (3).

More recently, PPAR{alpha} and PPAR{gamma} have been implicated as regulators of inflammatory responses (2). A role for PPAR{gamma} in inflammation also has been suggested in the regulation of monocyte/macrophages (4), where the ligands of this receptor inhibit the activation of macrophages and the production of inflammatory cytokines. Several studies have demonstrated that PPAR{alpha} and PPAR{gamma} inhibit the expression of inflammatory genes, such as cytokines, metalloproteases, and acute phase proteins (5–8). Interestingly, all available data indicate that the PPAR activity mediates, through oxidative stress-sensitive pathways, redox-responsive nuclear factor-{kappa}B (NF-{kappa}B), activator protein-1 (AP-1), and signal transducers and activators of transcription (STAT) (9). These findings suggest an important role for PPARs in controlling the inflammatory process, which could lead to potential therapeutic approaches for age-related inflammatory diseases.

Aging is a complex and multifactorial process, molecularly characterized by progressive, deleterious alterations in various transcription factors. From the standpoint of cell signal transduction pathways, age-related functional alterations can be elicited from an organism's loss in the ability to maintain the cellular signal pathways, partly due to increased oxidative status during aging (10).

Recent research provides ample evidence for most of the transduction factors involved in the cellular signaling activity being extremely sensitive to oxidative stress and the redox state of the cell (11). The current oxidative stress hypothesis of aging offers mechanistic explanations for increased oxidative stress and redox imbalance during aging (12). This hypothesis predicts that the age-related redox imbalance results from elevated oxidative status due to unregulated reactive species (RS) and an accompanying weakened, counter-acting, antioxidative defense force that causes a disruption in normal cellular functions. A recent report from this laboratory on the modulation of redox-sensitive transcription factors during aging highlighted the essential nature of the redox status in the regulation of transcription factors (10).

Our recent proposal on the age-related inflammatory process presented evidence that the oxidative process of the inflammatory process may be a major link bridging normal aging and many age-related degenerative diseases (13,14). Molecular explorations revealed that redox-sensitive NF-{kappa}B regulated proinflammatory molecules such as tumor necrosis factor (TNF{alpha} and TNFß), interleukins (IL-1ß, IL-2, and IL-6), chemokines (IL-8 and RANTES), adhesion molecules (ICAM-1, VCAM-1, and E-selectin), and that enzymes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) increased with age (13).

Calorie restriction (CR), a potent experimental paradigm for the retardation of aging, has been shown to exhibit broad and effective antioxidative properties (12). Recent research provides strong evidence that CR may exert diverse anti-aging benefits by its ability to modulate age-related oxidative stress and by its antiinflammatory effect (14,15). Several studies reported that CR attenuates age-related increases of proinflammatory cytokines (IL-1ß, IL-6, and TNF-{alpha}) in myocardial ischemia-reperfusion injury and other diseases (16–18). Based on the data from these studies and others, the beneficial effects of CR can be viewed as an outcome of the proper maintenance of cellular signal transduction activities through the modulation of various redox-sensitive transcription factors. Supporting this notion, our laboratory recently reported that the binding activity of redox-sensitive transcription factors, AP-1, NF-{kappa}B, and HIF-1, which DNA-increased during the aging process, was effectively suppressed by CR (10).

Much is known about the activation of PPARs via PPREs and various PPRE participations, but far less information exists for the mode of PPAR modulation during aging. Although, the aging research literature does show that the activity and metabolism of peroxisomes decline (19,20) and that expression of PPAR{alpha} is reduced during the aging process (21,22), to date, no data are reported on the molecular regulation of PPAR{gamma} and PPAR{alpha} by CR during the aging process.

In the present study, we attempted to document age-related changes in the expression and activity of PPAR{alpha} and PPAR{gamma} and the extent of their modulation by CR in aged rat kidney. In addition, we present data on lipopolysaccharide (LPS)-induced inflammatory reaction to further characterize the modulation of PPAR{alpha} and PPAR{gamma} under acute inflammatory conditions.


   EXPERIMENTAL PROCEDURES
Top
 Abstract
 Experimental Procedures
Results
 Discussion
 References
 
Animals and LPS Treatment Procedure
Animal.-- Rat maintenance procedures for specific pathogen-free (SPF) status and the dietary compositions for chow and CR regimens have been previously reported (23). This study complies with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication No. 85–23) and was approved by an Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.

Briefly, male SPF Fischer 344 rats were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% {alpha}-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix, and 3% Solka-Floc. The ad libitum (AL)-fed group had free access to both food and water. The animals designated as CR were fed 60% of the food intake of their AL-fed littermates, beginning at 6 weeks of age. Rats at 16 and 25 months of age were killed by decapitation, and the kidneys were quickly removed and rinsed in ice-cold buffer (100 mM Tris, 1 mM EDTA, 0.2 mM phenylmethyl-sulfonylfluoride [PMSF], 1 µM pepstatin, 2 µM leupeptin, 80 mg/L trypsin inhibitor, 20 mM ß-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate [pH 7.4]). Tissues were immediately frozen in liquid nitrogen and stored at –80°C.

LPS treatment.-- SPF male Fischer 344 rats at 13 and 31 months of age were separated into young (n = 6) and old (n = 6) groups, respectively. To investigate the effects of inflammation on the aging process, two additional groups were used for the injection of bacterial LPS. LPS was injected intraperitoneally with 5 mg/kg body weight doses in young (n = 6) and old (n = 6) rats. After 5 hours, rats were killed by decapitation and the kidneys quickly removed. The tissue was immediately immersed in liquid nitrogen and stored at –80°C (24).

Primers.-- Primers for reverse transcriptase-polymerase chain reaction (RT-PCR) were synthesized by Bioneer (Daejeon, Korea) and had the following sequences (the expected sizes are given in parentheses). rat PPAR{gamma}: sense, 5'-GGTGTGATCTTAACTGTCGG-3'; antisense, 5'-CATGGACACCATACTTGAGC-3' (530 bp), rat PPAR{alpha}: sense, 5'-GATGACCTGGAAAGTCCCTT-3'; antisense, 5'-AGGTAGGCTTCGTGGATTCT-3' (595 bp) and rat GAPDH: sense, 5'-GGGTGATGCTGGTGCTGAGTATGT-3'; antisense 5'-AAGAATGGGAGTTGCTGTTGAAGTC-3' (617 bp).

Preparation of nuclear extracts.-- All solutions, tubes, and centrifuges were maintained at 0°–4°C. For each nuclear extract preparation, three rat kidney tissues were pooled. The preparation of nuclear extracts was based on previous methods (25). All buffers contained 2 µg/ml of each protease inhibitor: aprotinin and leupeptin and 0.5 mM PMSF. Two grams of rat kidney harvested from young and old rats, were minced in phosphate-buffered saline. Minced tissue was incubated for two 15-minute intervals in relaxation buffer 1 (RBI; 100 mM KCl, 5 mM MgCl2, 5 mM EGTA, 5 mM sodium pyrophosphate, pH 6.8), followed by two 10-minute washes in RBII (50 mM KCl, 5 mM MgCl2, 1 mM EGTA, 1 mM sodium pyrophosphate, pH 6.8). The tissue was then washed with homogenization buffer A (0.3 M sucrose, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 15 mM HEPES, pH 7.5, 0.5 mM EGTA, 2 mM EDTA, 10 mg/ml bovine serum albumin, 1 mM dithiothreitol [DTT]), followed by homogenization in fresh homogenization buffer A. Kidney tissue was disrupted with homogenizer. Kidney homogenates were then centrifuged at 1000 g for 10 minutes in a Beckman JA-25-50 rotor (Beckman, Fullerton, CA), and the pellets were solubilized in homogenization buffer B (0.3 M sucrose, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 15 mM HEPES, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 10 mg/ml bovine serum albumin, 1 mM DTT) containing 0.5% (v/v) Triton X-100. The solubilized pellet was homogenized, followed by homogenization with a hand-held Teflon glass Dounce homogenizer. The nuclear pellet was solubilized in 10 ml of lysis buffer (10 mM HEPES, pH 7.5, 100 mM KCl, 3 mM MgCl2, 0.1 mM EDTA, 10% [w/v] glycerol). Nuclei were lysed by drop-wise addition of 3 M NH4SO4 (pH 7.9) to a final concentration of 0.4 M, and the resulting extract was gently shaken for 30 minutes at 4°C. The lysate was ultracentrifuged at 126,000 g for 1 hour, and the supernatant was collected. Solid NH4SO4 (0.3 g/ml) was added slowly to the supernatant, and nuclear proteins were allowed to precipitate on ice for 30 minutes. Precipitated proteins were pelleted by ultracentrifugation at 126,000 g for 30 minutes and resuspended in 500 µl of dialysis buffer (25 mM HEPES, pH 7.6, 40 mM KCl, 0.1 mM EDTA, 10% [w/v] glycerol, 1 mM DTT). The muscle nuclear protein extract was dialyzed twice for 1 hour each time against dialysis buffer, and aliquots were quick frozen and stored at –80°C in aliquots until the electrophoretic mobility shift assay (EMSA, see below) was done. Once thawed, nuclear protein extracts were not refrozen for use. The total protein concentration in samples was measured with a Sigma (St. Louis, MO) protein assay reagent kit containing bicinchoninic acid.

Electrophoretic mobility shift assay.-- The EMSA method was used to characterize the binding activities of PPAR in nuclear extracts (26). PPRE oligonucleotide was 5'-CAAAACTAGGTCAAAGGTCA-3'. Protein-DNA binding assays were performed with 10 µg of nuclear protein. To minimize the effect of salt on binding, the concentration of salt was adjusted to the same level in all samples. Nonspecific binding was blocked by using 1 µg of poly(dI-dC) · poly(dI-dC). The binding medium contained 5% glycerol, 1 mM MgCl2, 50 mM NaCl, 0.5 mM EDTA, 2 mM DTT, 1.0% Nonidet P40 (NP40), and 10 mM Tris/HCl, pH 7.5. In each reaction, 20,000 cpm of a radio-labelled probe was included. Samples were incubated at room temperature for 20 minutes, and the nuclear protein-32P-labelled oligonucleotide complex was separated from free 32P-labelled oligonucleotide by electrophoresis through a 5% native polyacrylamide gel in a running buffer containing 50 mM Tris, pH 8.0, 45 mM borate, and 0.5 mM EDTA. After separation was achieved, the gel was vacuum-dried for autoradiography and was exposed to Fuji X-ray film (Seoul, Korea) for 1–2 days at –80°C.

To determine the specificity of the nuclear protein binding, competitions were carried out under the same conditions using a 100-fold molar excess of the unlabeled PPRE oligonucleotide probe.

Western blotting.-- Western blotting was carried out as described previously (27). Homogenized samples were boiled for 5 minutes with a gel-loading buffer (0.125 M Tris-Cl, 4% SDS, 10% 2-mercaptoethanol, pH 6.8, 0.2% bromphenol blue) at a ratio of 1:1. Total protein-equivalent samples were separated by SDS-polyacrylamide gel as described by Laemmli (28), and transferred to poly(vinylidene fluoride) membrane at 15 V for 1 hour in a semidry transfer system. The membrane was immediately placed into blocking buffer (5% nonfat milk) in TBS-T buffer (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20). The blot was allowed to block at room temperature for 1 hour. The membrane was incubated with anti-PPAR{gamma} and anti-PPAR{alpha} for 1 hour at 25°C, followed by an antirabbit immunoglobin G (IgG)-horseradish peroxidase-conjugated antibody for 1 hour at 25°C. Antibody labeling was detected using enhanced chemiluminescence per the manufacturer's instructions. Prestained protein markers were used for molecular weight determinations.

Isolation of RNA.-- Total RNA was isolated using TRIZOL reagent (Gibco-BRL, Gaithersburg, MD), as per the manufacturer's instructions. Briefly, tissue samples were homogenized in the presence of Trizol (2 ml per 100 mg tissue) with several strokes in a tissue homogenizer. Aliquots of 0.2 ml chloroform per 1 ml homogenate were added. The samples were shaken vigorously for 15 seconds, and kept in ice for 5 minutes. The suspension was centrifuged at 12,000 g for 15 minutes at 4°C. The aqueous phase was transferred to a fresh tube, to which an equal volume of isopropanol was added, and the samples were kept at 4°C for 15 minutes. Samples were again centrifuged at 12,000 g for 15 minutes at 4°C. The supernatant was removed and the RNA pellet was washed once with 75% ethanol by vortexing and subsequently centrifuged at 7500 g for 8 minutes at 4°C. The pellet was dried for 10–15 minutes. The RNA pellet was dissolved in diethylpyrocarbonate (DEPC)-treated water.

Assay of RT-PCR.-- Two micrograms of total RNA were used to generate a cDNA template for RT-PCR. The first strand synthesis was performed using random hexamers (Promega, Madison, WI) and Superscript II reverse-transcriptase (Invitrogen, Carlsbad, CA). The first-strand cDNA products were further diluted and used as the PCR template. The PCR reaction in a total volume of 25 µl, containing 0.25 units of Taq polymerase, 0.25 mM dNTPs, PCR buffer (PE Applied Biosystems, Foster City, CA), and 50 ng of the relevant oligonucleotide primers. Parallel amplifications (20, 23, 26, and 29 cycles) of a given cDNA were used to determine the optimum number of cycles. For each gene under study, a readily detectable signal within the linear range was observed after 29 cycles. For the actual analysis, samples were heated for 5 minutes at 94°C, and then 23 cycles were carried out, each consisting of 1 minute at 94°C, 30 seconds at 55°C, and 1 minute at 72°C. This was followed by a final 10-minute extension at 72°C. In this study, GAPDH was used as the expression control. PCR reaction products were separated on a 1.2% agarose gel and visualized under ultraviolet light after ethidium bromide staining.

Statistical analysis.-- For Western blotting and RT-PCR, data from three independent experiments were collected. The statistical significance of the differences between the treatments (age and diet) was determined by the analysis of variance (ANOVA) method. One-factor ANOVA was conducted to analyze significant differences among all possible age and diet pairs. Differences among the mean value of individual groups were assessed by the Fischer's Protected Least Significant Difference (LSD) post hoc test. Values of p <.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Experimental Procedures
 Results
Discussion
 References
 
Effect of Age and CR on PPAR{alpha} and PPAR{gamma} mRNA Levels
To determine gene expression alterations in PPAR{alpha} and PPAR{gamma} during aging, the RT-PCR assay was carried out to measure the abundance of PPARs mRNAs in young (13 months) and old (25 months) AL and CR rats. As shown in Figure 1, we observed a slight decrease in PPAR{gamma} (Figure 1A) and PPAR{alpha} (Figure 1B) levels in mRNA. Compared with young AL rats, old AL rats showed decreased PPAR{alpha} and PPAR{gamma} mRNA levels of 30% and 46%, respectively. However, old CR rats showed higher levels of both PPAR{alpha} and PPAR{gamma} than their AL counterparts.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 1. Effect of age and calorie restriction (CR) on peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) and PPAR{gamma} mRNA levels. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of renal PPAR{gamma} (A) and PPAR{alpha} (B) were performed from RNA isolated from ad libitum (AL)-fed and CR rats. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used in separate PCR reactions to control for the efficiency of cDNA synthesis in each sample. A representative result is presented from three experiments. Densitometric measurements showed age-related decreases of PPAR{gamma} and PPAR{alpha} expressions in rat kidneys (n = 3 per group). Statistical significance: *p <.05; **p <.01 versus young AL rats; #p <.05; ##p <.01 CR versus the age-matched AL rats

 
Effect of Age and CR on Nuclear PPAR{alpha} and PPAR{gamma} Protein Levels
To verify the possibility that a reduction of PPAR{alpha} and PPAR{gamma} levels in mRNA causes a decline in PPAR{alpha} and PPAR{gamma} protein levels during the aging process, we examined nuclear protein levels by Western blot analysis using PPAR{alpha}- and PPAR{gamma}-specific antibody. The results in Figure 2 show that nuclear levels of PPAR{gamma} (Figure 2A) decreased with age in AL rats (84% decrease) and that CR (26% decrease) ameliorated this change. Likewise, aging markedly decreased nuclear levels of PPAR{alpha} (Figure 2B). Compared with young AL rats, we observed a 53% decrease in old AL rats; however, the decrease (23%) was deterred in old CR rats.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Effect of age and calorie restriction (CR) on peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) and PPAR{gamma} nuclear protein levels. Western blot analysis of renal nuclear PPAR{gamma} (A) and PPAR{alpha} (B) were performed from RNA isolated from ad libitum (AL)-fed and CR rats. A representative result is presented from three experiments. Densitometric measurements showed an age-related decrease of PPAR{gamma} and PPAR{alpha} expression in rat kidneys (n = 3 per group). Statistical significance: **p <.01; ***p <.001 versus young AL rats; ##p <.01; ###p <.001 CR versus the age-matched AL rats

 
Changes in PPAR Binding Activity by Aging and CR
We performed EMSA on rat kidney PPAR-DNA binding to examine whether a change in renal PPARs expression affects PPAR-DNA binding to PPRE, which is in the PPAR-binding domain. Figure 3A shows the film from the EMSA of PPAR-DNA binding in the AL and CR rats. Using a PPRE oligonucleotide, PPAR-DNA binding activity was significantly lower in old (25 months) compared with young (13 months) rats in the AL groups. We found that PPAR-DNA binding activity decreased by 64% in the old AL groups compared with the young (Figure 3B). However, old CR rats showed higher (19% decreased) activity levels compared with their age-matched AL group. These data clearly showed that PPAR activation declines with age and that CR attenuates this age-related decline.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 3. Calorie restriction (CR) suppresses age-related decreases in peroxisome proliferator-activated receptor (PPAR) binding activity. A, The electrophoretic mobility shift assay (EMSA) method was used to compare nuclear PPAR binding activities in kidney nuclear protein from ad libitum (AL)-fed and CR rats. Lane 1: probe without nuclear protein sample (BL); Lane 2–5: kidney nuclear protein samples of AL and CR rats, ages 13 and 25 months, respectively. Lane 6: competition assay using 100-fold excess of unlabeled PPAR-response elements (PPRE) oligonucleotide. A representative result is presented from three experiments. B, Densitometric measurements showed age-related PPAR-DNA binding activity in rat kidneys (n = 3 per group). Statistical significance: *p <.05; ***p <.001 versus young AL rats; #p <.05; ###p <.001 CR versus the age-matched AL rats

 
PPAR{alpha} and PPAR{gamma} Expression Changes During Aging and LPS Challenge
To duplicate our findings and to further understand the association between alterations of PPARs and the inflammatory response during aging, groups of young and old rats were challenged with a potent inflammatory stimulus, LPS. First, we used RT-PCR to measure mRNA levels in LPS-treated young and old rats. The results shown in Figure 4 indicate decreased concentrations of PPAR{gamma} (Figure 4A) and PPAR{alpha} (Figure 4B) in old (31-month-old) rats compared with young (13-month-old) rats, not only in the control group but also in the LPS-treated group, particularly, the old LPS-treated rats, which showed the lowest amount of PPARs among all groups.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 4. Changes in peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) and PPAR{gamma} expression during aging and lipopolysaccharide (LPS) challenge. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of renal PPAR{gamma} (A) and PPAR{alpha} (B) were performed from RNA isolated from control and LPS-treated rats. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used in separate PCR reactions to control for the efficiency of cDNA synthesis in each sample. C, Western blot analysis was performed to investigate renal nuclear PPAR{gamma} protein levels in nuclear extracts from control and LPS-treated rats (n = 6 per group). A representative result is presented from three experiments. Densitometric measurements showed age-related decrease of PPAR{gamma} and PPAR{alpha} expression in rat kidneys. The data shown are presented as young LPS untreated rat. Statistical significance: *p <.05; **p <.01; ***p <.001 versus young untreated rats; #p <.05; ##p <.01 LPS-treated rats versus the age-matched untreated rats

 
Effect of Age and LPS-Induced Inflammation on Nuclear PPAR{gamma} Protein Level
Previous studies report a decrease in PPAR{alpha} with age and inflammatory stimuli in various tissues (21,22,29). In this study, we investigated alterations of PPAR{gamma} with age. To determine whether aging and LPS-induced inflammation influence PPAR{gamma} expression, we performed Western blot analysis using a PPAR{gamma}-specific antibody. We observed the level of nuclear PPAR{gamma} protein to decrease with age and to be altered after LPS treatment (Figure 4C). When comparing old and young rats in the control group, we found that the nuclear PPAR{gamma} protein level was significantly reduced (49%) in old rats compared with young rats. However, in LPS-treated groups, old rats showed an even greater reduction of nuclear PPAR{gamma} level compared with LPS-treated young rats. The old LPS-treated rats expressed the lowest amount of nuclear PPAR{gamma} proteins among all groups.

DNA Binding Activity of PPRE During Aging and LPS
To investigate the binding of PPAR to the PPRE consensus sequence during aging and inflammation, we used EMSA with nuclear extracts prepared from aged rat kidney to confirm PPAR DNA binding activity. EMSA data (Figure 5) showed that the PPAR binding activity decreased in old rats compared with young rats. Moreover, this age-related down-regulation was further decreased by the LPS challenge, indicating an increased susceptibility to inflammatory stimuli with age. This result corresponded with the decreases in nuclear PPARs and gene expression.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 5. Effects of age and lipopolysaccharide (LPS) on the PPAR-response elements (PPRE) DNA binding activity. A, The electrophoretic mobility shift assay (EMSA) method was used to compare nuclear peroxisome proliferator-activated receptor (PPAR) binding activities in kidney nuclear protein. Lane 1: probe without nuclear protein sample (BL); Lane 2–5: kidney nuclear protein samples of control (CTL) and LPS-treated (LPS) rats (n = 6 per group). Lane 6: competition assay using 100-fold excess of unlabeled PPRE oligonucleotide. A representative result is presented from three experiments. B, Densitometric measurements showed age and LPS-effected PPAR-DNA binding activity in rat kidneys. The data shown are presented as young LPS untreated rat. Statistical significance: *p <.05; **p <.01 versus young untreated rats; #p <.05; ##p <.01 LPS-treated rats versus the age-matched untreated rats

 

   DISCUSSION
Top
 Abstract
 Experimental Procedures
 Results
 Discussion
References
 
In a previous study, our group showed that age-related inflammation and oxidative stress enhanced various mRNA levels and protein expression in aged rat kidney (13). Although many studies have shown a close association between redox-regulated transcription factors and aging and/or inflammation (10), the effects of age on the alterations of PPARs are not well explored. Recent molecular data reveal that the susceptibility of aged animals to inflammatory insults may be due to an increased oxidative state and a disrupted redox state in these animals (13,24).

To better understand the alterations of PPARs during aging and their modulation at the molecular level, we assessed the extent of PPAR changes in mRNA and protein levels with age. In addition, we injected inflammatory LPS into young and old rats to examine a possibly altered PPAR status by prooxidative inflammation. Results revealed an age-related reduction of PPAR{alpha} and PPAR{gamma} in both mRNA and nuclear protein levels, which was detected in LPS-treated rats. Activation of PPARs is indicated by DNA-binding to PPRE, the PPAR binding domain of the transcriptional regulating region. Our data provided the first direct evidence of altered PPARs with age and decreased PPAR DNA binding by inflammation. More interestingly, our results showed for the first time that the antioxidative effects of CR blunt age-induced decreases in PPAR{alpha} and PPAR{gamma} mRNA levels and protein expression. Data further revealed that CR improved the age-induced decrease in PPAR DNA binding activity to PPRE.

The down-regulation of PPARs by inflammation and oxidative stress was reported previously (22,30). Considering the close relation between oxidative stress and aging, the possible influence of aging on PPARs alterations can be expected. Among the altered expressions of inflammatory mediators, NF-{kappa}B and its related genes are of importance (13). Chung and colleagues recently proposed the molecular inflammatory hypothesis of aging to emphasize the organism's susceptibility to incessant oxidative stresses and a disrupted redox balance (at submicroscopic and molecular levels), which gradually lead toward a proinflammatory stage during aging, which may develop into a full-blown pathologic condition during senescence (13).

So far, there are just a few studies reporting the age effect on PPAR expression, which focused mainly on PPAR{alpha}. For instance, Chao and colleagues (31) reported that aging had no effect on PPAR{alpha} expression but that RXR changed in liver. However, these authors' findings conflict with those by Iemitsu and colleagues (21) and Carrera and colleagues (32), who showed that age causes a marked reduction in the expression and activity of PPAR{alpha} in old rat liver and heart. Our current data obtained from the kidney agree with what was found about old rat liver and heart.

Recently, a report (22) on an animal model of aging showed that the supplementation of a PPAR{alpha} agonist, Wy 14,643, and dehydroepiandrosterone (DHEAS) corrected the age-induced up-regulation of NF-{kappa}B activity and the expression of several NF-{kappa}B-regulated genes. In addition, PPAR{alpha}-deficient mice are shown to have higher oxidative stress at an earlier age than wild-type mice and have an exacerbated inflammatory response to LPS stimulation (33). Taken together, these results give a strong implication to PPAR{alpha} in the inflammatory process and oxidative stress.

PPAR{gamma}, another member of the PPAR subfamily, may also be involved in modulating inflammation. PPAR{gamma} activation through its agonists could interfere with the NF-{kappa}B and AP-1 pathways, and down-regulate NF-{kappa}B and AP-1-dependent genes, including COX-2, iNOS, and cytokines. However, the molecular mechanisms by which PPAR{gamma} and PPAR{alpha} regulate inflammatory response genes are not fully understood. A recent paper reports the activation of PPAR{alpha}-induced I{kappa}B{alpha} mRNA and protein expression and reduced NF-{kappa}B DNA binding activity, although it did not affect p65 nuclear translocation (34). PPAR{gamma} inhibits NF-{kappa}B-driven transcription by physically interacting with both p65 and p50 (35).

Much of the accumulated evidence strongly implicates oxidative stress as a major factor contributing to the aging process and age-related diseases, such as neurodegenerative diseases, arteriosclerosis, diabetes, and inflammatory diseases (10). Recent molecular studies show that the induction of oxidative stress is associated with the down-regulation of PPARs (36–38) and that activation of PPAR{alpha} leads to a neuroprotective effect (39). In addition, the activation of PPAR{gamma} is shown to reduce oxidative stress and nitrative stress (40).

Our findings on age-induced inflammation of PPARs were further tested in LPS-challenged rats. As expected, we observed that aged animals showed a much greater responsiveness to the LPS challenge than the young, implying an enhanced susceptibility and sensitivity to the inflammatory stimulus with age. Thus, the LPS challenge exacerbated the already existing oxidative stress (41,42), which may lead to further decreases in PPARs.

The possible inverse association between age-related changes in PPARs levels and oxidative stress was further supported by the antioxidative action of CR. Recent studies on CR provide clear evidence that CR likely exerts its diverse benefits through its antioxidative ability to suppress age-related oxidative stress while upholding the antioxidant defense system, including major ROS/RNS scavengers during aging (43). In the present study, CR blunted the down-regulation of PPARs in aged rat kidneys, which could be explained by CR's antioxidative and antiinflammation action.

Conclusion
Our current data showed that the down-regulation of PPARs by the aging process might be correlated with age-related oxidative stress, which was counteracted by the antioxidative action of CR. Based on these and other data, we propose that the prevention of the age-related decline in PPARs may be partly responsible for the suppression of the increased inflammatory process during aging. Molecular insights into the modulation of PPARs during aging and CR's ability to prevent decreases in PPARs will provide a better understanding of the role of PPARs in the aging process and the mechanisms underlying antiinflammatory action of CR.


   Acknowledgments
 
This research was supported by a grant (PF0320801-00) from the Plant Diversity Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Korean government.


   Footnotes
 
Decision Editor: Arlan Richardson, PhD

Received March 19, 2004

Accepted June 19, 2004


   References
Top
 Abstract
 Experimental Procedures
 Results
 Discussion
 References
 

  1. Auwerx J. PPAR{gamma}, the ultimate thrifty gene. Diabetologia. 1999;42:1033-1049.[Medline]
  2. Guan Y, Zhang Y, Breyer MD. The Role of PPARs in the Transcriptional Control of Cellular Processes. Drug News Perspect. 2002;15:147-154.[Medline]
  3. Ferre P. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes. 2004;53:S43-50.[Abstract/Free Full Text]
  4. Chawla A, Barak Y, Nagy L, et al. PPAR-gamma dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation. Nat Med. 2001;7:48-52.[Medline]
  5. Ricote M, Huang J, Fajas L, et al. Expression of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc Natl Acad Sci U S A. 1998;95:7614-7619.[Abstract/Free Full Text]
  6. Jiang C, Ting AT, Seed B. PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998;39:82-86.
  7. Law RE, Goetze S, Xi XP, et al. Expression and function of PPAR{gamma} in rat and human vascular smooth muscle cells. Circulation. 2000;101:1311-1318.[Abstract/Free Full Text]
  8. Okada M, Yan SF, Pinsky DJ. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) activation suppresses ischemic induction of Egr-1 and its inflammatory gene targets. FASEB J. 2002;16:1861-1868.[Abstract/Free Full Text]
  9. Barbier O, Torra IP, Duguay Y, et al. Pleiotropic actions of peroxisome proliferator-activated receptors in lipid metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol. 2002;22:717-726.[Abstract/Free Full Text]
  10. Kim HJ, Jung KJ, Yu BP, et al. Modulation of redox-sensitive transcription factors by calorie restriction during aging. Mech Ageing Dev. 2002;123:1589-1595.[Medline]
  11. Lavrovsky Y, Chatterjee B, Clark RA, et al. Role of redox-regulated transcription factors in inflammation, aging and age-related diseases. Exp Gerontol. 2000;35:521-532.[Medline]
  12. Yu BP. Aging and oxidative stress: modulation by dietary restriction. Free Radic Bio. Med. 1996;21:651-668.
  13. Chung HY, Kim HJ, Kim KW, et al. Molecular inflammation hypothesis of aging based on the anti-aging mechanism of calorie restriction. Microsc Res Tech. 2002;59:264-272.[Medline]
  14. Chung HY, Kim HJ, Kim JW, et al. The inflammation hypothesis of aging: molecular modulation by calorie restriction. Ann N Y Acad Sci. 2001;928:327-335.[Medline]
  15. Yu BP, Chung HY. Stress resistance by caloric restriction for longevity. Ann N Y Acad Sci. 2001;928:39-47.[Medline]
  16. Chandrasekar B, Nelson JF, Colston JT, et al. Calorie restriction attenuates inflammatory responses to myocardial ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol. 2001;280:H2094-H2102.[Abstract/Free Full Text]
  17. Muthukumar AR, Jolly CA, Zaman K, et al. Calorie restriction decreases proinflammatory cytokines and polymeric Ig receptor expression in the submandibular glands of autoimmune prone (NZB x NZW)F1 mice. J Clin Immunol. 2000;20:354-361.[Medline]
  18. Chandrasekar B, McGuff HS, Aufdermorte TB, et al. Effects of calorie restriction on transforming growth factor beta 1 and proinflammatory cytokines in murine Sjogren's syndrome. Clin Immunol Immunopathol. 1995;76:291-296.[Medline]
  19. Youssef J, Badr M. Biology of senescent liver peroxisomes: role in hepatocellular aging and disease. Environ Health Perspect. 1999;107:791-797.[Medline]
  20. Beier K, Volkl A, Fahimi HD. The impact of aging on enzyme proteins of rat liver peroxisomes: quantitative analysis by immunoblotting and immunoelectron microscopy. Virchows Arch B Cell Pathol Incl Mol Pathol. 1993;63:139-146.[Medline]
  21. Iemitsu M, Miyauchi T, Maeda S, et al. Aging-induced decrease in the PPAR-alpha level in hearts is improved by exercise training. Am J Physiol Heart Circ Physiol. 2002;283:H1750-H1760.[Abstract/Free Full Text]
  22. Poynter ME, Daynes RA. Peroxisome proliferator-activated receptor{alpha} activation modulates cellular redox status, represses nuclear factor-{kappa}B signaling, and reduces inflammatory cytokine production in aging. J Biol Chem. 1998;273:32833-32841.[Abstract/Free Full Text]
  23. Bertrand HA, Herlihy JT, Ikeno Y, et al. Dietary restriction. In: Yu BP, ed. Methods in Aging Research. Boca Raton, FL: CRC Press; 1999: 271–300.
  24. Kwon HJ, Sung BK, Kim JW, et al. The effect of lipopolysaccharide of enhanced inflammatory process with age: modulation of NF-{kappa}B. J Am Aging Assoc. 2001;24:163-172.
  25. Hattori M, Tugores A, Veloz, L, et al. A simplified method for the preparation of transcriptionally active liver nuclear extracts. DNA Cell Biol. 1990;9:777-781.[Medline]
  26. Kerr LD. Electrophoretic mobility shift assay. Meth Enzymol. 1995;254:619-632.[Medline]
  27. Habib A, Creminon C, Frobert Y, et al. Demonstration of an inducible cyclooxygenase in human endothelial cells using antibodies raised against the carboxyl-terminal region of the cyclooxygenase-2. J Biol Chem. 1993;268:23448-23454.[Abstract/Free Full Text]
  28. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685.[Medline]
  29. Sanguino E, Ramon M, Michalik L, et al. Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha. Biochem Pharmacol. 2004;67:157-166.[Medline]
  30. Mueller C, Weaver V, Vanden Heuvel JP, et al. Peroxisome proliferator-activated receptor {gamma} ligands attenuate immunological symptoms of experimental allergic asthma. Arch Biochem Biophys. 2003;418:186-196.[Medline]
  31. Chao C, Youssef J, Rezaiekhaleigh M, et al. Senescence-associated decline in hepatic peroxisomal enzyme activities corresponds with diminished levels of retinoid X receptor alpha, but not peroxisome proliferator-activated receptor alpha. Mech Ageing Dev. 2002;123:1469-1476.[Medline]
  32. Carrera M, Laguna JC. Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha. Biochem Pharmacol. 2004;67:157-166.
  33. Delerive P, De Bosscher K, Besnard S, et al. Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1. J Biol Chem. 1999;274:32048-32054.[Abstract/Free Full Text]
  34. Delerive P, Gervois P, Fruchart JC, et al. Induction of I{kappa}B{alpha} expression as a mechanism contributing to the anti-inflammatory activities of PPAR{alpha} activators. J Biol Chem. 2000;275:36703-36707.[Abstract/Free Full Text]
  35. Chung SW, Kang BY, Kim SH, et al. Oxidized low lipoprotein inhibits interleukin-12 production in lipopolysaccharide-activated mouse macrophages via direct interactions between PPAR{gamma} and NF-{kappa}B. J Biol Chem. 2000;276:32681-32687.
  36. Bagi Z, Koller A, Kaley G. PPAR{gamma} activation, by reducing oxidative stress, increases NO bioavailability in coronary arterioles of mice with Type 2 diabetes. Am J Physiol Heart Circ Physiol. 2004;286:H742-H748.[Abstract/Free Full Text]
  37. Meerarani P, Reiterer G, Toborek M, et al. Zinc modulates PPARgamma signaling and activation of porcine endothelial cells. J Nutr. 2003;133:3058-3064.[Abstract/Free Full Text]
  38. Xu J, Fu Y, Chen A. Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth. Am J Physiol Gastrointest Liver Physiol. 2003;285:G20-G30.[Abstract/Free Full Text]
  39. Deplanque D, Gele P, Petrault O, et al. Peroxisome proliferator-activated receptor-alpha activation as a mechanism of preventive neuroprotection induced by chronic fenofibrate treatment. J Neurosci. 2003:;23;:6264-6271.
  40. Tao L, Liu HR, Gao E, et al. Antioxidative, antinitrative, and vasculoprotective effects of a peroxisome proliferator-activated receptor-gamma agonist in hypercholesterolemia. Circulation. 2003;108:2805-2811.[Abstract/Free Full Text]
  41. de Rochemonteix-Galve B, Marchat-Amoruso B, Dayer JM, et al. Tumor necrosis factor and interleukin-1 activities in free lung cells after single and repeated inhalation of bacterial endotoxin. Infect Immun. 1991;59:3646-3650.[Abstract/Free Full Text]
  42. Hsieh V, Amoruso-Marchat B, Rylander R, et al. Oxygen metabolites from lavage and interstitial lung cells after inhalation of endotoxin in guinea pigs. Int Arch Allergy Immunol. 1994;104:42-47.[Medline]
  43. Espinosa-Heidmann DG, Suner I, Hernandez EP, et al. Age as an independent risk factor for severity of experimental choroidal neovascularization. Invest Ophthalmol Vis Sci. 2002;43:1567-1573.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Services
Right arrow Download to citation manager
PubMed
Right arrow PubMed Citation

HOME ARCHIVE SEARCH TABLE OF CONTENTS