Journals of Gerontology Series A: Biological Sciences and Medical Sciences Large Type Edition
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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:1252-1264 (2005)
© 2005 The Gerontological Society of America

Betaine Suppresses Proinflammatory Signaling During Aging: The Involvement of Nuclear Factor-{kappa}B via Nuclear Factor-Inducing Kinase/I{kappa}B Kinase and Mitogen-Activated Protein Kinases

Eun Kyung Go1, Kyung Jin Jung1, Ji Young Kim1, Byung Pal Yu2,3 and Hae Young Chung1,2,

1 Department of Pharmacy, College of Pharmacy, and 2Longevity Life Science and Technology Institutes Pusan National University, Gumjung-ku, Busan, Republic of Korea.
3 Department of Physiology, The University of Texas Health Science Center at San Antonio.

Address correspondence to Hae-Young Chung, PhD, Department of Pharmacy, College of Pharmacy, Pusan National University, San 30, Jang-jun-dong, Gumjung-ku, Busan, 609-735, Korea. E-mail: hyjung{at}pusan.ac.kr


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Betaine is an important human nutrient obtained from various foods. In the present study, we assessed the anti-inflammatory effect of betaine on nuclear factor-{kappa}B (NF-{kappa}B) during aging. Sprague-Dawley (SD) rats, ages 7 and 21 months, were used in this study. The older rats were fed betaine. To elucidate the effect of betaine on oxidative stress-induced NF-{kappa}B and its signaling pathway at molecular levels, YPEN-1 cells were used. Results showed that betaine suppressed NF-{kappa}B and its related gene expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule-1 (ICAM-1) in aged kidney. Furthermore, betaine attenuated oxidative stress-induced NF-{kappa}B via nuclear factor-inducing kinase/I{kappa}B kinase (NIK/IKK) and mitogen-activated protein kinases (MAPKs) in the YPEN-1 cells. On the basis of these results, we concluded that betaine suppressed the age-related NF-{kappa}B activities associated with upregulated NIK/IKK and MAPKs that were induced by oxidative stress. Thus, betaine might be useful as a preventive agent against the activation of NF-{kappa}B induced during inflammation and aging.

BETAINE is a major water-soluble component of Lycium chinensis that has been used as a traditional remedy in Southeast Asia for hepatic disorders (1). Also, betaine functions as an oxidative metabolite of choline by donating methyl groups, and has the ability to covert homocysteine to methionine. Thus, it is a nutrient that plays an important role in the health of the cardiovascular system. Studies have suggested that betaine helps to reduce potentially toxic levels of homocysteine, a naturally occurring amino acid that can be harmful to blood vessels, thereby contributing to the development of heart disease, stroke, and Alzheimer's disease (2–4). In addition, betaine has been shown to enhance the expression and activity of key participants in vascular inflammation related to deteriorated vessel function, such as hyperosmotic cell shrinkage or hypoosmotic cell swelling in liver and kidney (5,6). In addition, betaine is shown to be beneficial in the treatment of nonalcoholic fatty liver disease (7). These data strongly suggest that betaine regulates inflammatory processes.

Many researchers report that inflammatory reactions combined with the disruption of an organism's physiological control and the persistence of inflammation can lead to a wide range of diseases and a deterioration of cellular function (8). The activation of nuclear factor-{kappa}B (NF-{kappa}B) and its dependent genes has been shown to be significantly associated with the processes of inflammation, atherosclerosis, cancer, and aging and with the pathological functions of these conditions (9,10).

NF-{kappa}B regulates the transcription of proinflammatory molecules such as tumor necrosis factor (TNF), interleukins (ILs), chemokines, adhesion molecules, and inducible enzymes such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The NF-{kappa}B transcription factor, which is formed by the hetero- or homodimerization of proteins from the rel family, is the subject of a recent review. NF-{kappa}B exists in the cytoplasm in inactive form complexed by an inhibitory subunit, I{kappa}B{alpha} (10). The exposure of cells to various extracellular stimuli results in the activation of I{kappa}B kinase (IKK), and the phosphorylated I{kappa}B is further ubiquitinated and degraded by beta-transducin repeat-containing proteins (ß-TrCP) and proteasomes, respectively (11–13).

These stimuli lead to the activation and translocation of NF-{kappa}B into the nucleus, and enhance the binding of NF-{kappa}B to regulatory elements and promoters of DNA. Nuclear factor-inducing kinase (NIK) is a member of the mitogen-activated protein kinase kinase (MKK) family that may either directly or indirectly phosphorylate or activate IKK{alpha}/ß, leading to the phosphorylation and degradation of I{kappa}B{alpha} followed by NF-{kappa}B activation (14). Also, regarding mitogen-activated protein kinases (MAPKs), p38MAPK, extracellular signal-regulated kinase (ERK), and c-jun-N-terminal kinase (JNK) constitute an additional level of gene regulation by the transcription factor NF-{kappa}B (15,16). Each of these MAPK subfamilies is activated by specific upstream MKKs, which dually phosphorylate MAPKs on a threonine and tyrosine residue separated by an intervening amino acid characteristic for each MAPK subfamily (17–19).

According to recent reports, the relation between NIK/IKK, MAPK, and NF-{kappa}B activation has been observed during aging (20–22). NF-{kappa}B is activated by increased oxidative stress and inflammatory status, which are two of the most important occurrences commonly observed during aging (23,24). Aging is characteristically described as a time-dependent functional decline, leading to the cell's incapacity to withstand external and internal challenges (25). Because the most plausible cause of aging is regarded as oxidative stress that the major molecular motives for inflammation, there is close correlation among oxidative stress, inflammation, and aging.

As reports are scarce on the association between proinflammatory factor NF-{kappa}B and betaine in the aging process, we thought that it would be interesting to know about the ability of betaine to regulate NF-{kappa}B during aging. From the present study, we found that it is possible that betaine suppressed the NF-{kappa}B pathway through the activation of NIK/IKK and MAPKs during aging and that betaine led to the modulation of the age-related proinflammatory signaling pathway in the NIK/IKK and MAPK cascade.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were obtained from Samtako (Osan, Korea) and were fed a diet of the following composition: 21% soybean protein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15% a-methionine, 0.2% choline chloride, 5% salt mix, 2% vitamin mix, and 3% Solka-Floc (International Fiber Co., North Tonawanda, NY).

SPF male Sprague-Dawley rats (7 and 21 months old) were used as young and old rats, respectively. To investigate the effects of betaine on NF-{kappa}B regulation, we fed the rats three different doses of betaine. Betaine was mixed and grinded as 0.01%, 0.02%, or 0.04% of the rat chow and was fed to the 21-month-old rats for 10 days. Considering that each animal ate on average 3 mg, 6 mg, or 12 mg of betaine per day, this amount would be equal to 30, 60, or 120 mg/kg.

Rats at 7 and 21 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, trypsin inhibitor at 80 mg/L, 20 mM ß-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate [pH 7.4]). The tissue was immediately frozen in liquid nitrogen and stored at –80°C.

Reagents
Betaine (Figure 1) was purchased from Sigma (Sigma Chemical Co., St. Louis, MO). 2',7'-Dichlorofluorescin diacetate (DCF-DA) was obtained from Molecular Probes, Inc. (Eugene, OR). A horseradish peroxide-conjugated donkey antirabbit antibody was obtained from Amersham (Amersham, Buckinghamshire, U.K.). Polyvinylidene difluoride membranes were obtained from Millipore Corporation (Bedford, MA). The radionucleotide [32P]-adenosine triphosphate (ATP) was obtained from Amersham. Kinase inhibitors were purchased from Calbiochem (Darmstadt, Germany). All other chemicals were available of the highest purity from Sigma.



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  		Figure 1. Chemical structure of betaine

 
Tissue Preparation
One gram of kidney was homogenized with 10 ml of ice-cold buffer (50 mM phosphate buffer containing 1 mM EDTA, 1 µM pepstatin, trypsin inhibitor at 80 mg/L, 20 mM ß-glycerophosphate, 20 mM NaF, 2 mM sodium orthovanadate [pH 7.4]) and centrifuged at 900 g at 4°C for 15 minutes. The supernatants were recentrifuged at 12,000 g at 4°C for 15 minutes to yield sedimented mitochondrial fraction and postmitochondrial supernatant fraction. The supernatant was regarded as a cytosol.

For the nuclear protein, 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 (26). The total protein concentration in samples was measured with a protein assay reagent kit containing bicinchoninic acid (Sigma).

Assessment of Oxidative Stress in Aged Kidney
Reactive species (RS) generation was measured as previously described using a fluorescence probe (27). Briefly, 25 µM DCF-DA was added to homogenates for 250 µl of final volume. Changes in fluorescence intensity were measured every 5 minutes for 30 minutes on a fluorescence plate reader (GENius; Tecan Instruments, Salzburg, Austria) with excitation and emission wavelengths set at 485 and 530 nm, respectively.

ONOO– generation was measured by monitoring the oxidation of dihydrogenrhodamine (DHR) 123 (28). Briefly, 10 µl homogenates were added to the rhodamine solution (50 mM sodium phosphate buffer, 90 mM sodium chloride, 5 mM diethylenetriaminepentaacetate [DTPA], and DHR 123). Changes in fluorescence intensity were measured every 5 minutes for 30 minutes on a fluorescence plate reader (GENius, Tecan Instruments, Salzburg, Austria) with excitation and emission wavelengths set at 485 and 530 nm, respectively.

Cell Culture Conditions and Measurement of Intracellular Oxidative Stress
YPEN-1 cells (rat endothelial cells) were obtained from ATCC (American Type Culture Collection, Rockville, MD). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Nissui Co., Tokyo, Japan) supplemented with 5% heat-inactivated (56°C for 30 minutes) fetal bovine serum (Gibco, Grand Island, NY), glutamine at 233.6 mg/ml, penicillin–streptomycin at 72 mg/ml, and amphotericin B at 0.25 mg/ml, and were adjusted to pH 7.4–7.6 with NaHCO3 in an atmosphere of 5% CO2. The fresh medium was replaced after 1 day to remove nonadherent cells or cell debris.

Cell survival was quantified by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma), which measures mitochondrial activity in viable cells (29). This method is based on the conversion of MTT to MTT-formazan crystal by mitochondrial enzyme. Cells seeded at a density of 3 x 104 per well in a 48-well plate were allowed to adhere overnight, and then the culture medium was replaced with fresh serum-free DMEM. After preincubation for 1 hour with betaine, cells were treated with 15 µM tert-butyl hydroperoxide (t-BHP). For the MTT assay, MTT was freshly prepared at 5 mg/mL, a 0.5 mL aliquot of MTT stock solution was added to each well, and the plate was incubated at 37°C for 3 hours in a humidified 5%, CO2/95% air incubator. Cells were then disrupted with solubilizing solution (dimethyl sulfoxide/absolute ethanol, 1:1). The formazan dye produced by viable cells was quantified in an enzyme-linked immunosorbent assay (ELISA) microplate reader at an absorbance of 560 nm.

For the determination of intracellular RS scavenging activity of YPEN-1 cells, cells were seed in a 96-well plate. After 1 day, the medium was changed to fresh serum-free medium. The cells were treated with or without betaine and incubated for 1 hour. After treatment with t-BHP (20 mM) for 30 minutes, the medium was replaced with fresh serum-free medium, and then DCF-DA (12.5 mM) was added. Total RS were measured with fluorescence intensity of DCF for 1 hour. The fluorescence was used at excitation and emission wavelengths of 485 and 535 nm, respectively.

Preparation of Cytosolic and Nuclear Extracts From Cultured Cells
Betaine- and t-BHP-treated cells were washed with phosphate-buffered saline (PBS), and then 1 mL of ice-cold PBS was added. Pellets were harvested at 970 g at 4°C for 5 minutes. The pellets were then suspended in 10 mM Tris (pH 8.0), with 1.5 mM MgCl2, 1 mM dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors, and incubated on ice for 15 minutes. It was centrifuged at 15,610 g at 4°C for 15 minutes. The supernatants were used as cytosolic fraction, and the pellets were resuspended in 10 mM Tris (pH 8.0), with 50 mM KCl, 100 mM NaCl, and protease inhibitors, incubated on ice for 30 minutes, then were centrifuged at 15,610 g at 4°C for 30 minutes. After that, supernatants were used as nuclear fraction.

Protein Measurement by Western Blot
To investigate changes in expressions of various proteins, we used western blot experiments to examine cytosol and nuclear fractions of kidney. Samples were boiled for 5 minutes with a gel loading buffer (125 mM Tris–HCl, 4% sodium dodecylsulfate, 10% 2-mercaptoethanol, pH 6.8, and 0.2% bromophenol blue) at a ratio of 1:1. Total protein equivalents for each sample were separated on an 8%–15% sodium dodecylsulfate–polyacrylamide mini-gel using a Laemmli buffer system (30). Samples were then transferred to a polyvinylidene difluoride membrane at 100 V for 1.5 hours. The blot was allowed to block using 1%–5% nonfat milk in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween-20 at room temperature for 1 hour. The blot was incubated with specific primary antibody, followed by a secondary antibody (a horseradish peroxidase-conjugated donkey antirabbit antibody) (1:5000; Amersham), at 25°C for 2 hours, respectively. Antibody labeling was detected using enhanced chemiluminescence (ECL) (Amersham) per the manufacturer's instructions, which was exposed to hyperfilm (Amersham).

Assessment of NF-{kappa}B Binding Activity
First, the electrophoretic mobility shift assay (EMSA) method was used to characterize the binding activities of NF-{kappa}B transcription factors in nuclear extracts (31). NF-{kappa}B oligonucleotide was 5'-GAGAGGCAAGGGGATTCCCTTAGTTAGGA-3'. Protein-DNA binding assays were performed with 20 µg of nuclear protein. To minimize salt on binding, the concentration of salt was adjusted to the same level in all samples. Unspecific 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 dithiothreitol, and 10 mM Tris/HCl, pH 7.5. In each reaction, 20,000 cpm of a radiolabeled probe was included.

For the NF-{kappa}B binding assay, 1.0% Nonidet P-40 was included. Samples were incubated at room temperature for 20 minutes, and the nuclear protein with 32P-labeled oligonucleotide complex was separated from free 32P-labeled 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 exposed to Fuji x-ray film for 1–2 days at –80°C.

NF-{kappa}B activity was examined using a luciferase plasmid DNA, pTAL-NF-{kappa}B, that contains a specific binding sequence for NF-{kappa}B (BD Biosciences Clontech, Palo Alto, CA) (32). Transfection was carried out using FuGENE 6 Reagent (Roche, Indianapolis, IN). Briefly, 2 x 104 cells per well were seeded in 48-well plates. When cultured cells reached {approx}50% confluence, cells were treated with 0.1 µg DNA/0.2 µl FuGENE 6 complexes in a total volume of normal medium (5% serum contained) with 500 µl for 48 hours. Subsequently, 0.5 µM t-BHP was treated after the plate was changed with serum-free medium, and treatments of several dose of betaine were performed 1 hour previously. After additional incubation for 6 hours, cells were washed with PBS and added with Steady-Glo Luciferase Assay System (Promega, Madison, WI) to the plate. Luciferase activity was measured by a luminometer (GENius, Tecan Instruments). Raw luciferase activities were normalized by protein concentration per each well.

Statistical Analysis
Results were analyzed statistically by one-factor analysis of variance. Values of p <.05 were considered statistically significant.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cytoprotective Effect of Betaine on t-BHP-Induced Cytotoxicity
The MTT assay showed that t-BHP expressed toxicity in a dose-dependent (5–60 µM) fashion in the YPEN-1 cells for up to 6 hours (Figure 2A) with an 80% survival with a dose of 15 µM, which was used in subsequent experiments. To determine the effect of betaine on t-BHP-induced cytotoxicity, cells were pretreated with betaine at 50 and 200 µM and additionally incubated with 15 µM t-BHP for 6 hours for subsequent experiments. The result of the MTT assay (Figure 2B) showed that betaine had an ability to inhibit cytotoxicity in a dose-dependent manner.



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  		Figure 2. Tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity was protected by betaine treatment in YPEN-1 cells. A, YPEN-1 cells were treated with the indicated concentration of t-BHP for 6 hours. B, YPEN-1 cells were treated with 15 µM t-BHP in the presence of betaine at 50 and 200 µM for 6 hours. Viable cells were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Betaine was added to the medium 1 hour before the t-BHP treatment. Results of one-factor analysis of variance: *p <.05, ***p <.001 vs untreated control; #p <.05 vs 15 µM t-BHP-treated group, respectively

 
Inhibitory Effect of Betaine on t-BHP-Induced Oxidative Stress
To determine oxidative stress, RS generation was detected in t-BHP-treated cells. Thus, we have determined the effects of betaine on t-BHP-induced intracellular RS generation in YPEN-1 cells using DCF-DA, which is oxidized by RS to fluorescence DCF. YPEN-1 cells treated with 15 µM t-BHP displayed fluorescence intensity before incorporation with DCF-DA. Intracellular RS formation resulting from t-BHP treatment was significantly reduced when betaine (10, 50, 100, or 200 µM) was present in the medium (Figure 3). The results showed that betaine has an intracellular capacity to prevent oxidative stress.



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  		Figure 3. Tert-butyl hydroperoxide (t-BHP)-induced reactive species (RS) generation was suppressed by betaine in YPEN-1 cells. A, Quantitative analysis of fluorescence intensity using 2',7'-dichlorofluorescin diacetate (DCF-DA) was detected after treatment with vehicle or 15 µM t-BHP in the absence or presence of 10, 50, 100, or 200 µM betaine for 6 hours. B, Intracellular RS were detected by DCF-DA using a fluorescence microscope. a: untreated control, b: 15 µM t-BHP treated alone, c: 15 µM t-BHP and 32 µM betaine, d: 15 µM t-BHP and 75 µM betaine, e: 15 µM t-BHP and 150 µM betaine. Results of one-factor analysis of variance: **p <.01 vs untreated control; #p <.05 vs 15 µM t-BHP-treated group

 
Suppressive Action of Betaine on Age-Related Oxidative Stress
To elucidate the effect of betaine on RS formation during aging, total RS and ONOO– generation in rat kidney was examined. Total RS and ONOO– generation in old rats fed betaine was lower than that of the old rats not fed betaine (Figure 4, A and B). Our results indicate that betaine suppressed an increase in oxidative stress during aging and that level of oxidative stress remained at a similar level in the young rats' aging process.



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  		Figure 4. Betaine modulated increased oxidative stress during aging. A, The 2',7'-dichlorofluorescin diacetate (DCF-DA) method was used to determine the effect of betaine on reactive species generation in aged kidney homogenate. B, Dihydrogenrhodamine (DHR) 123 method was used to determine the effect of betaine on ONOO– generation in aged kidney homogenate. Results of one-factor analysis of variance: **p <.01, ***p <.001 vs young rats; #p <.05, ##p <.01, ###p <.001 vs old nonbetaine-fed rats, respectively. RFU = Related fluorescence unit

 
Modulation of Age-Related NF-{kappa}B Activation by Betaine
To determine age-related activation of NF-{kappa}B, first we examined nuclear protein levels by western blot using p65-specific and p50-specific polyclonal antibodies. Data in Figure 5A clearly reveal that nuclear translocation of NF-{kappa}B significantly increased in aged rat, whereas old, betaine-fed rats showed lower levels of NF-{kappa}B in a dose-dependent manner compared to their old, nonbetaine-fed counterparts. Second, to verify the DNA binding of NF-{kappa}B, EMSA was carried out with nuclear proteins isolated from young, old, and old rats fed betaine. Figure 5B shows that the binding activity of NF-{kappa}B was upregulated during aging and that betaine suppressed up-regulation of NF-{kappa}B. Third, to verify the effect of betaine on oxidative stress-induced NF-{kappa}B activation in endothelial cells, luciferase activity was detected by t-BHP treatment and betaine treatment after transient transfection of a plasmid containing NF-{kappa}B consensus sequence and luciferase reporter (Figure 5C). NF-{kappa}B luciferase activity increased by twofold compared to transfected cell exposure of 0.25 µM t-BHP for 6 hours, and betaine showed a dose-dependent ability to decrease NF-{kappa}B luciferase activity. Overall, the data indicate that the activation of NF-{kappa}B is implicated in t-BHP-induced oxidative stress, whereas activation of NF-{kappa}B was inhibited by betaine.



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  		Figure 5. Betaine suppressed age-related increase of nuclear factor-{kappa}B (NF-{kappa}B) activity. A, Western blot was performed to detect nuclear p50 and p65 protein levels in renal homogenate (30 µg of protein) from young, old, and old rats fed betaine. One representative blot of p50 and p65 is shown from three experiments that yielded similar results, respectively. Hypoxia-inducible factor-1ß (HIF-1ß) was amplified as a positive control. B, The electrophoretic mobility shift assay method was used to compare NF-{kappa}B binding activities between old rats fed betaine and their counterparts. One representative result is shown from three experiments that yield similar results. Results of one-factor analysis of variance: *p <.05 vs young rats; ##p <.01, ###p <.001 vs old nonbetaine-fed rats, respectively. C, Activity of NF-{kappa}B was determined by luciferase reporter assay. Cells were treated with 0.25 µM tert-butyl hydroperoxide (t-HBP) for 6 hours, and betaine (40, 100, or 250 µM) was given 1 hour previously. Luciferase activity is reported as a percentage of activity compared with that in control cells. Statistical significance of difference between untreated control group and treated groups in luciferase activity of NF-{kappa}B was determined using one-factor analysis of variance: *p <.05 vs nontransfection control; ##p <.01 vs transfected and untreated t-BHP; $$p <.01, $$$p <.001 vs transfected with.25 µM t-BHP, respectively. RLU = Related luminescence unit

 
Effect of Betaine on Cytoplasmic I{kappa}B{alpha} and I{kappa}Bß: Degradation and Phosphorylation
As shown in Figure 6A, the levels of I{kappa}B{alpha} and I{kappa}Bß proteins in cytoplasmic extract decreased in old rats. However, old betaine-fed rats showed higher levels of these proteins than did their old, nonbetaine-fed counterparts. Results showed that I{kappa}B{alpha} phosphorylation increased with age, but that old rats fed betaine showed lower levels than did old nonbetaine-fed rats (Figure 6B). Thus, these findings strongly indicate that NF-{kappa}B activation during aging is likely elicited by age-related increases in I{kappa}B{alpha} and I{kappa}Bß degradation.



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  		Figure 6. Betaine prevented age-related degradations of I{kappa}B{alpha}, I{kappa}Bß, and phosphorylation of I{kappa}B{alpha}. Western blot was performed to detect I{kappa}B{alpha}, I{kappa}Bß (A), and p-I{kappa}B{alpha} (B) protein levels in renal cytoplasmic extracts (100 µg of protein) from young, old, and old rats fed betaine. One representative blot of I{kappa}B{alpha}, I{kappa}Bß, and p-I{kappa}B{alpha} is shown from three experiments that yielded similar results, respectively. ß-actin was amplified as a positive control. Results of one-factor analysis of variance: *p <.05, ***p <.001 vs young rats; #p <.05, ##p <.01, ###p <.001 vs old nonbetaine-fed rats, respectively

 
Inhibition of Expression of NF-{kappa}B-Dependent Genes by Betaine
From the results presented in Figure 5B, NF-{kappa}B binding activity increased in old rat kidney. We also determined NF-{kappa}B-dependent gene expressions, such as iNOS, COX-2, vascular cell adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule-1 (ICAM-1). These genes are shown to have an NF-{kappa}B binding site located in their promoter regions that is controlled by NF-{kappa}B regulation (8–10). Our results showed that protein levels correlated with NF-{kappa}B activity (Figure 7) and that betaine modulated NF-{kappa}B-dependent gene expression.



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  		Figure 7. Betaine suppressed expression of nuclear factor-{kappa}B (NF-{kappa}B)-dependent genes during aging. Western blot was performed to detect cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule-1 (ICAM-1) protein levels in renal cytoplasmic extracts (100 µg of protein) from young, old, and old rats fed betaine. One representative blot of each protein is shown from three experiments that yielded similar results, respectively. ß-actin was amplified as a positive control. Results of one-factor analysis of variance: *p <.05, **p <.01 vs young rats; #p <.05, ##p <.01 vs old nonbetaine-fed rats, respectively

 
Effects of Betaine on COX-2 Expression via IKK/p38/ERK/JNK Inhibitor
COX-2 is an NF-{kappa}B-responsive gene and is regulated by various kinases that cause the translocation of NF-{kappa}B to elevate COX-2 expression (33). To confirm the mechanism of the NF-{kappa}B pathway, cells were pretreated with betaine, IKK inhibitor (BAY11-7085), p38 inhibitor (SB203580), ERK inhibitor (PD098059), and JNK inhibitor (SP600125) for 1 hour. After cells were incubated with 15 µM t-BHP for an additional 6 hours, NF-{kappa}B-responsive gene COX-2 expression was assessed. As shown in Figure 8A, t-BHP-induced COX-2 was suppressed by betaine and kinase inhibitors, which means that betaine possibly modulates COX-2 through these kinases. To verify the results shown in Figure 8A, we tested the effects of betaine on IKK{alpha}/ß, ERK1/2, p38, and JNK expression (Figure 8B). Results showed that the addition of betaine significantly inhibited phosphorylation of IKK{alpha}/ß, ERK1/2, p38, and JNK. Moreover, results showed that the addition of betaine significantly inhibited phosphorylation of NIK, MKK1/2, and MKK3/6 (Figure 8C). Finally, our data suggested that betaine prevents NF-{kappa}B nuclear translocation via NIK/IKK and MAPKs (Figure 8D). Thus, the decreased NF-{kappa}B activity by betaine that we observed indicates the involvement of NIK/IKK and MAPKs in t-BHP-induced NF-{kappa}B activation.



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  		Figure 8. Betaine attenuated nuclear factor-{kappa}B (NF-{kappa}B)-responsive cyclooxygenase-2 (COX-2) via nuclear factor-inducing kinase/I{kappa}B kinase (NIK/IKK) and mitogen-activated protein kinases (MAPKs) induced by oxidative stress. YPEN-1 cells were grown to 80% confluence in 100-mm dishes in Dulbecco's modified Eagle medium. A, Cells were pretreated (1 hour) in betaine (100 µM) inhibitors containing medium. After that, the cells were stimulated with tert-butyl hydroperoxide (t-BHP) (15 µM) in the presence of betaine and each kinase inhibitor. B, After stimulation with t-BHP (20 minutes for phosphorylation of IKK, extracellular signal-regulated kinase [ERK]1/2, p38, and c-jun-N-terminal kinase [JNK]) in the absence (–) or presence (+) of betaine (20 and 100 µM), the cells were washed and lysed. Western blot was performed for each kinase such as p-IKK, p-ERK1/2, p-p38, and p-JNK. C, After stimulation with t-BHP (10 minutes for phosphorylation of NIK, mitogen-activated protein kinase kinase [MKK]1/2, and MKK3/6) in the absence (–) or presence (+) of betaine (20 and 100 µM), the cells were washed and lysed. Western blot was performed for each kinase such as p-NIK, p-MKK1/2, and p-MKK3/6. D, After stimulation with t-BHP (1 hour for NF-{kappa}B) in the absence (–) or presence (+) of betaine (20 and 100 µM) the cells were washed and lysed. ß-actin and hypoxia-inducible factor-1ß (HIF-1ß) were amplified as a positive control. Results of one-factor analysis of variance: *p <.05, **p <.01, ***p <.001 vs untreated control; #p <.05, ##p <.01 vs 15 µM t-BHP-treated group, respectively

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Betaine is an important human dietary nutrient obtained from a variety of foods (34), especially popular as a major component of Lycium chinensis fruit, which has long been used in tea and in traditional medicine to treat hepatic disorders (1). Betaine is a metabolite of choline and is also known as trimethylglycine (TMG). The principal physiologic role of betaine is as an osmolyte and methyl donor (transmethylation). As an osmolyte, betaine protects cells and vessels from hyperosmotic cell shrinkage or hypoosmotic cell swelling (5,6). As a methyl donor, betaine participates in methionine metabolism in the human liver and kidney. Betaine can reduce the elevated serum homocysteine concentrations associated with mild or severe hyperhomocystinuria via the methionine cycle (35). In addition, betaine decreases progression of nonalcoholic fatty liver disease (NAFLD) (7), but it contributes to various diseases, including coronary, cerebral, hepatic, and vascular disease induced by inflammation (36–38).

The interrelation between inflammation and aging highlights the important role of NF-{kappa}B in the proinflammatory state during aging. Recent reviews show that upregulated NF-{kappa}B activity seems to be a widespread biological phenomenon in aged animals and that NF-{kappa}B is a critical transcription factor involved in the pathogenesis of many disorders, including inflammatory diseases (23,24). It is reported that oxidative stress, one plausible cause of aging, follows the same course as inflammation, which is implied intimate association among oxidative stress, inflammation, and aging (39,40).

Activated redox-sensitive transcription factor NF-{kappa}B is translocated from the cytoplasm into the nucleus (23). NF-{kappa}B is retained in the cytoplasm by the binding of a family of inhibitors (I{kappa}B{alpha}, I{kappa}Bß, and I{kappa}B{gamma}). Activation by stimuli requires sequential phosphorylation of I{kappa}B by I{kappa}B-kinase (IKK1 or IKK2), ubiquitination and degradation by the proteasome, which is followed by translocation of NF-{kappa}B/Rel proteins into the nucleus. NF-{kappa}B controls the expression of numerous proinflammatory gene products including cytokines, molecules involved in endothelial cell adhesion and antigen presentation, as well as inflammatory enzymes such as COX-2 and iNOS (41).

There is wide application of t-BHP to investigate the mechanism of oxidative stress-induced cell injury (42) because t-BHP induces oxidative stress that is responsible for lipid peroxidation, DNA adduct formation, and the induction of apoptosis leading to inflammation (43,44). To elucidate the anti-inflammatory effect of betaine, our present study was undertaken to determine the effects of betaine on NF-{kappa}B activity in the presence of oxidative stress in both a cell culture system and in aged rat kidney. Our data show that treatment with betaine inhibited accumulated oxidative stress in aged kidney and that t-BHP induced cellular oxidative status in YPEN-1 cells. Moreover, based on our results from western blot, EMSA, and the reporter assay, we conclude that betaine inhibits proteolytic degradation of I{kappa}B, binding of the p50/p65 heterodimer, and NF-{kappa}B-dependent transcriptional activity during aging. Although the modulation of other transcription factors by betaine was not investigated in the present study, we speculate that betaine might be able to modulate other redox-sensitive transcription factors (as we have seen this in similar work) (23,24).

NF-{kappa}B activation has been suggested to be mediated by two distinct signaling pathways. First, the NIK/IKK pathway is involved in the induction of transcriptional activation of NF-{kappa}B (22). Second, MAPKs regulate NF-{kappa}B activation via multiple mechanisms. One of the most extensively investigated intracellular signaling cascades involved in proinflammatory responses is the MAPK pathway (45–47). In the present study, different inhibitors of kinases were used in YPEN-1 cells to confirm the suppressive action of betaine on oxidative stress in the NF-{kappa}B pathway, and we validated COX-2 expression, which is regulated by NF-{kappa}B recognizing the COX-2 promoter (48,49). To determine if COX-2 expression is blocked by upstream NF-{kappa}B signaling in t-BHP-stimulated YPEN-1 cells, we investigated the inhibitory effects of an IKK inhibitor (BAY11-7805), a p38 inhibitor (SB203580), an ERK inhibitor (PD098059), and a JNK inhibitor (SP600125) (50–53). All inhibitors prevented induced COX-2 expression, suggesting that the NIK/IKK and MAPK signaling pathway has a role in the regulation of COX-2 expression by t-BHP. This finding also suggests an inhibitory mechanism from betaine in the NF-{kappa}B signaling pathway via NIK/IKK and MAPKs in YPEN-1 cells stimulated with t-BHP.

Conclusion
The present study strongly indicates that betaine decreases NF-{kappa}B activation via NIK/IKK and MAPKs, suggesting that betaine may be a useful agent for the suppression of age-related inflammation and may improve deleterious conditions related to chronic age-related diseases.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
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 References
 
This work was supported by a grant from the Korean Research Foundation (KRF-2005-015-C00484). We also are grateful to the Aging Tissue Bank for the supply of the aging tissue.

Eun Kyung Go and Kyung Jin Jung contributed equally to this work.


   Footnotes
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 Abstract
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 Results
 Discussion
 References
 
Decision Editor: James R. Smith, PhD

Received March 26, 2005

Accepted June 6, 2005


   References
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 Abstract
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 Results
 Discussion
 References
 

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