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The Peroxisome Proliferator-Activated Receptor α (PPARα) Regulates the Plasma Thiobarbituric Acid-Reactive Substance (TBARS) Level
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
237, 606–610 (1997)
RC977196
The Peroxisome Proliferator-Activated Receptor a (PPARa) Regulates the Plasma Thiobarbituric Acid-Reactive Substance (TBARS) Level Ikuo Inoue,1 Satoru Noji, Man-zhen Shen, Keiichi Takahashi, and Shigehiro Katayama The Fourth Department of Medicine, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-04, Japan
Received May 30, 1997
We investigated whether liver expression of the peroxisome proliferator-activated receptor a (PPARa) gene is related to the plasma thiobarbituric acid-reactive substance (TBARS) level, as well as to plasma cholesterol (TC) level and plasma triglyceride (TG) level in rats fed a high fat chow containing a variety of fatty acids. Only the plasma TBARS level showed a significant negative correlation with the liver PPARa mRNA level (TC, RÅ0.001, pÅ0.9967; TG, RÅ0.248, pÅ0.1276; TBARS, RÅ0.439, pÅ0.0046). Although futher studies are needed to clarify whether the increase of the liver PPARa mRNA level confers a reduction in plasma TBARS levels, it is likely that PPARa activity plays a regulatory role in the pathogenesis of hyperlipidemia and atherosclerosis. q 1997 Academic Press
The peroxisome proliferator-activated receptors (PPAR) is a member of the steroid hormone receptor superfamily (1). Three types of PPARs have been described in rodents and humans: PPARa, Nuc1 (also called PPARb or PPARd), and PPARg. In the adult rat liver, PPARa is the most predominat isoform. PPARa is activated by a variety of hypolipidemic fibrates such as bezafibrate, clofibrate, fenofibrate, and gemfibrozil. Moreover, PPARa is acivated by medium, long, and very long chain fatty acids. Recently, Lermann et al. reported that PPARg expressed in adipose tissue is also acivated by the thiazolidinediones, i.e. troglitazone, pioglitazone, and BRL42346c (2). PPARs are ligand-activated transcription factors that control gene expression by interacting with specific respose elements (PPREs) located upstream of responsive genes (3). The genes containig PPRE motifs 1 All correspondence should be addressed to Ikuo Inoue, M.D., The Fourth Department of Internal Medicine, Saitama Medical School. 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-04, Japan. Fax: 81492-94-9752.
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include acyl-CoA oxidase (3), peroxisomal bifunctional enzyme (4), liver fatty acid binding protein (5), and microsomal CYP4A6, cytochrome P450 fatty acid v-hydoxylase (6). In addition, the genes coding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (7) the apoproteins A-I (8), A-II (9), and C-III (10) are also regulated by PPARa, resulting in the association of PPARa with lipid regulation. Recently, we reported that troglitazone and bezafibrate altered b-oxidation in peroxisomes (11) as well as in mitochondoria (12). In addition, we have reported that troglitazone (13) exert antioxidation effects, resulting in the reduction of the plasma thiobarbituric acid reactive substances (TBARS) levels. A high-fat diet containing a variety of fatty acids is known to change the plasma cholesterol and triglycerides levels. The dietary v-6 and v-3 fatty acids suppress lipogenesis in the liver, whereas saturated and monounsaturated fatty acids do not (14). In addition, the composition of dietary fatty acid has been reported to alter the susceptibility of low-density lipoprotein (LDL) to oxidation, and may cause a change in the plasma TBARS level (15). In this study, we investigated whether plasma concentrations of TBARS are altered in rats fed a high fat chow containing a variety of fatty acids. We also investigated whether expression of the PPARa gene in the liver is related to the plasma TBARS level, as well as plasma lipid levels. MATERIAL AND METHODS Animal treatment. Male Sprague-Dawley rats were obtained from Charles River Japan at the age of 4 weeks. They were housed three per cage and allowed free access to tap water and standard laboratory rat chow (Clea Japan Inc., Tokyo, Japan). Rats were housed in a temperature controlled room (227C) under a 12-h lightdark (8 PM to 8 AM) cycle. Three weeks later, the animals were divided into five groups and fed one of the following diets for 5 weeks. 1) Normal rat chow (lipid 16%, milk casein 20%, starch 54%, vitamins 1%, minerals 4%, cellulose powder 5%, w/w; Soy group, nÅ9). The
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lipid in the diet of the Soy group consisted of soy bean fat, which is rich in polyunsaturated fatty acid. 2) Olive diet, in which the lipid of the normal rat chow was replaced by olive oil. This regimen was rich in monounsaturated fatty acid (Olive group, nÅ9). 3) Palm diet, in which the lipid of the normal rat chow was replaced by palm oil containing a lot of saturated fatty acids (Palm group, nÅ9). 4) Caproic diet, in which the lipid of the normal rat chow was relaced by caproic acid, a medium chain fatty acid (Caproic group, nÅ9). 5) Normal rat chow in combination with eicosapentaenoic acid (EPA; 1000 mg/kg/ day) (EPA group, nÅ9). An additional study was performed by feeding rats one of the following diets for 10 days: 1) normal rat chow, which was similar to the Soy group described above (nÅ9), 2) normal rat chow in combination with troglitazone (200 mg/kg/day; Tro group, nÅ9), 3) normal rat chow in combination with bezafibrate (80 mg/kg/day; Beza group, nÅ9). Drugs were dissolved in polyethylene glycol/water (194:12:20 wt/ wt) and administered orally by gavage to the EPA group, Tro group, or Beza group. Preparation of lipoproteins. At the end of the feeding period, rats were killed after fasting overnight. Body and liver weights were measured, and tissue samples were collected. Blood samples were collected immediately into tubes containing EDTA (0.1% final concentration). After separation of the plasma, the lipoproteins were isolated by sequential density ultracentrifugation.
sities of the bands were evaluated using a UV-light box imaging system (Atto, Tokyo). Some of the PCR products were also identified by the capillary electrophresis (18). Statistical analysis. Parametric data are expressed as the mean { SD. Non-parametric data are expressed as the median and quartiles. Differences between groups were evaluated by Scheffe´’s F test.
RESULTS Although the body and liver weights in the Caproic group were significantly lower than those of the other groups, the differences in the body and liver weights among the other groups were not significant (data not shown). There were no difference in liver enzymes such as glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, g-glutamyl transferase, lactic dehydrogenase, total bilirubin between the four groups. All laboratory data were within normal ranges in agematched rats (data not shown). Although plasma TC, TG, and HDL-C levels were not affected by the different
Laboratory analyses. Plasma cholesterol (TC) and triglyceride (TG) levels were determined by enzymatic methods. Plasma glucose levels were determined by a glucose oxidase method and insulin levels by a radioimmunoassay using rat insulin as the standard. The plasma TBARS levels were determined by the method of Yagi (16). Determination of fatty acid composition of lipoproteins. The fatty acids LDL, high density lipoprotein (HDL) and very low density lipoprotein (VLDL), were extracted from rat plasma according to the method of Folch et al. (17). The fatty acids were methylated and the resulting fatty acid methyl esters were identified using a GC17A gas chromatograph (Shimadzu Corp., Kyoto, Japan) equipped with an Omegawax 30 m 1 0.25 mm capillary column (Supelco, Inc., Bellefonte, PA). Reverse transcriptase polymerase chain reaction (RT-PCR). Total tissue RNA was isolated from liver homogenate using a commercial kit (Isogen, Nippon Gene Co. Ltd., Toyama, Japan). Preliminary experiments had shown that PPARa mRNA expression was low and that accurate measurements would not be possible by Northern analysis. We therefore performed a RT-PCR procedure to examine PPARa expression levels. Total RNA (0.3 mg) was used as a template for DNA synthesis using oligo(dT) primer and a DNA cycle kit (GeneAmp RNA PCR Kit, Perkin Elmer, New Jersey, USA) according to the manufacture’s instructions. The RT reaction was performed twice at 427C for 15 min to maximize cDNA synthesis and was terminated by heating at 997C for 5 min. The resulting cDNA was used as the template for PCR. Oligonucleotide primers for PPARa RT-PCR were designed to amplify partial cDNA sequences. The synthetic oligonucreotides were obtained from Nippon Flour Mills Co., Ltd. (Kanagawa Japan). The primers used for PPARa were 5*-CCT TTT TGT GGC TGC TAT-3* for the forward primer and 5*-TCC CTG CTC TCC TGT ATG-3* for the reverse primer, giving a 356 bp fragment. The primers used for glyceraldehyde3-phosphate-dehydrogenase (GAPDH) were 5*-ACC ACA GTC CAT GCC ATC AC-3* for the forward primer and 5*-TCC ACC ACC CTG TTG CTG TA-3* for the reverse primer, giving a 452 bp fragment. PCR reactions were performed using the same temperatures as for denaturation (947C, 30s) and extension (727C, 90s), but the annealing temperature was 567C for 50 s. The number of amplification cycles was 33. The PCR products were analyzed by polyacrylamide gel electrophoresis on a 7.5% gel (NPU - 7.5 type, Atto Corporation, Tokyo, Japan), which was run for 2 h at 20 mA. DNA was visualized by ethidium bromide staining at a concentration of 10 mg/ml. The inten-
FIG. 1. Serum fatty acids level in the Soy group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), EPA group (nÅ9), and Caproic group (nÅ9). Three pools of three freshly prepared plasma for each group were determined.
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FIG. 2. Serum TBARS level in the Caproic group (nÅ9), EPA group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), and Soy group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median.
dietary lipids, the administration of EPA, which is a polyunsaturated fatty acid (v-3), reduced the circulation lipid levels, but the difference was not significant (data not shown). Fatty acid profiles of plasma in all groups are shown in Figure 1. In Palm group, the plasma levels of saturated fatty acid, i.e. lauric acid (C12:0) and myristic acid (C14:0), increased as compared with those of the other groups. In the Olive group, oleic acid (C18:1, v-9) was the major component. In the Soy group, polyunsaturated fatty acid, i.e. linoleic acid (C18:2, v-6), linolenic acid (C18:3, v-3), and eicosadienoic acid (C20:2, v-6), increased as compared with the other groups. EPA adminitration increased the plasma EPA (C20:5, v-3) and docosapentaenoic acid (C22:5, v-3) levels 10-fold and 2-fold, respectively, as compared with the Soy group (data not shown), but the docosahexaenoic acid (C22:6, v-3) level was not changed by EPA adminitration (data not shown). In all rats, the plasma fatty acids level reflected the levels of VLDL, LDL, and HDL (data not shown).
FIG. 3. Example of the detection of liver peroxisome proliferatoractivated receptor a PPARa and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the Caproic group (lane 1), EPA group (lane 2), Olive group (lane 3), Soy group (lane 4), and Palm group (lane 5). S: Standard maker.
FIG. 4. Liver PPARa mRNA level in the Caproic group (nÅ9), EPA group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), and Soy group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median. *põ0.05.
The plasma lipid peroxide levels were determined by measuring the TBARS levels. Although the plasma TBARS levels in the EPA group were reduced significantly in comparison with those of the Soy group, there were not the significant difference among the the TBARS levels of the others groups (Figure 2). Figure 3 indicates the typical level of PPARa mRNA expression in all groups. Figure 4 shows the densitometer analysis of the PPARa mRNA levels in all groups. The PPARa mRNA levels in the EPA and Caproic groups were significantly increased. Although there were no significant difference among the other groups, the PPARa mRNA levels were ranked as follows: Olive group ú Soy goup ú Palm group. The length of the PCR products determined by capillary electrophoresis was 356 { 0.05 bp (mean { SD), and was used to quantify the amount of the PPARa mRNA. Figure 5 shows the relationship between the logarithm of the PPARa mRNA level and the plasma TC, TG, and TBARS levels. Only the plasma TBARS level showed a significant negative correlation with the PPARa mRNA level in all groups (TC, RÅ0.001, pÅ0.9967; TG, RÅ0.248, pÅ0.1276; TBARS, RÅ0.439, pÅ0.0046). As shown in Figure 6, bezafibrate administration significantly increased the expression of PPARa mRNA, whereas troglitazone administration did not increase the expression of PPARa mRNA. DISCUSSION It is well known that a high-fat diet induces peroxisomal proliferation (19). Several studies have demon-
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with the fact that fatty acids have similar physiological actions as peroxisomal proliferators like bezafibrate. Dietary v-6 and v-3 fatty acid suppress lipogenesis in the liver, whereas saturated fatty acid and monounsaturated fatty acid do not have that effect (14). In addition, the composition of dietary fatty acids affects the susceptibility of LDL to oxidation, suggesting that the plasma TBARS level would be altered by a variety of dietary fatty acid. Recently, we (21) and others (22) have also reported that HDL, which is the main cholesterol-carrying particle in rodents (23), can be oxidized. Monounsaturated oleic acid makes lipoprotein more resistant to in vitro oxidation, whereas polyunsaturated fatty acids are less resistent (24,25). In fact, the lag phases of oxidation of LDL and HDL in the EPA group and Soy group were shorter than those of the Palm group and Olive groups (15). Interestingly, the plasma TBARS level in the EPA group had decreased (Figure 2). On the other hand, the PPARa mRNA level had increased in the EPA group. Moreover, as shown in Figure 5, the plasma TBARS level was the factor with the strongest correlation to the increase the the PPARa mRNA level. It is likely that EPA activates PPARa (26), resulting in a fall in the plasma TBARS level. By contrast, the plasma TBARS level was also reduced by administration of troglitazone, which is a ligand of PPARg, without activating liver PPARa Troglitazone is structurally similar to vitamin E, which is an antioxidant, and causes a ruduction of the plasma TBARS level (13). The mechanism of reduction of the plasma TBARS level by the activation of PPARa is not clear. Recently, it has been reported that the PPARa affected the duration of inflammation induced by leukotriene B4/arachidonic acid (27). Reactive oxygen-induced free radicals play an important role as mediators of tissue injury associated with many pathological conditions, such as inflammatory and ischemic states. The reactive oxygen FIG. 5. The relationship between the liver PPARa mRNA level and plasma TC, TG, and TBARS level.
strated that all fatty acids can activate PPAR to various extents. Steineger et al. have indicated that increasing the number of double bonds in fatty acids augments PPAR mRNA expression (20). As shown in Figure 3, our in vivo data indicate that the liver PPARa mRNA levels of the EPA and Caproic groups had increased as compared with those of the other groups. Based on our finding that the plasma palmitoleic (C16:1), eicosatrienoic (C20:3, v-9), and docosahexaenoic (C22:6, v-3) acid level in the Caproic group had increased (data not shown), the rise in the PPARa mRNA level in the Caproic group might be due to the increased plasma monounsaturated and polyunsaturated fatty acid levels. The activation of PPAR by fatty acid is in agreement
FIG. 6. Liver PPARa mRNA level in the Tro group (nÅ9) and Beza group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median. *põ0.05.
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species, such as superoxide and hydroxyl radicals, can act on membranous lipids or lipids in lipoprotein particle, resulting in increased plasma TBARS level. When linked to the activation of function of peroxisome, H2O2 is generated. Although excessive H2O2 production may damage the genes and protein, catalase scavenges H2O2 . Catalase, which is the main enzyme contained in peroxisomes, may also scavenge reactive oxygen species, such as alkoxyl radical, hydroxyl radical, and peroxide radical, induced by Fenton reactions in addition to the reaction with H2O2 . If the activation of PPARa induces catalase activity, reactive oxygen species would be scavenged in peroxisomes. In fact, it has been reported that administration of a high fat diet (28), bezafibrate (29), or EPA (30) increases the amount of peroxisomal catalase activity. As discussed above, the activation of peroxisome might cause a reduction of plasma TBARS level. In fact, administration of bezafibrate (31), or EPA (32), prevents cardiovascular disease in spite of a mild reduction of serum cholesterol level. In the present study, we have shown that the level of expression of the PPARa gene in the liver is negatively correlated with the plasma TBARS level. Although futher studies are needed to clarify whether the increase of the liver PPARa mRNA level confers some benefit in preventing vascular complications, it is likely that PPARa activity plays a regulatory role in the pathogenesis of hyperlipidemia and atherosclerosis, in addition to its role in the inflammatory diseases, rheumatoid arthritis, lupus, and psoriasis (27). ACKNOWLEDGMENTS We are grateful to Dr. Keiichi Sano and Ms. Rie Yanagisawa (Jokoh Co. Ltd., Tokyo, Japan) for performing the capillary electrophoresis and to Ms. Atuko Neuchi (Saitama Medical School) for technical assistance. This work was supported by a grant-in-aid from the annual meeting for circulating regulatory factors.
REFERENCES 1. Issemann, I., and Green, S. (1990) Nature 347, 645–650. 2. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A. (1995) J. Biol. Chem. 270, 12953–12956. 3. Tugwood, J. D., Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L., and Green, S. (1992) EMBO J. 11, 433–439. 4. Zhang, B., Marcus, S. L., Miyata, K. S., Subramani, S., Capone, J. P., and Rachubinski, R. A. (1993) J. Biol. Chem. 268, 12939– 12945. 5. Issemann, I., Prince, R., Tugwood, J., and Green, S. (1992) Biochem. Soc. Trans. 20, 824–827.
6. Muerhoff, A. S., Griffin, K. J., Johnson, E. F. (1992) J. Biol. Chem. 267, 19051–19053. 7. Rodriguez, J. C., Gil-Gomez, G., Hegardt, F. G., and Haro, D. (1994) J. Biol. Chem. 269, 18767–18772. 8. Vu-Dac, N., Schoonjans, K., Laine, B., Fruchart, J. C., Auwerx, J., and Staels, B. (1994) J. Biol. Chem. 269, 31012–31018. 9. Vu-Dac, N., Schoonjans, K., Kosykh, V., Dallongeville, J., Fruchart, J. C., Staels, B., and Auwerx, J. (1995) J. Clin. Invest. 96, 741–750. 10. Hertz, R., Bishara-Shieban, J., and Bar-Tana, J. (1995) J. Biol. Chem. 270, 13470–13475. 11. Inoue, I., Takahashi, K., Katayama, S, Harada, Y., Negishi, K., Itabashi, A., and Ishii, J. (1995) Metabolism 44, 1626–1630. 12. Mannaerts, G. P., Debeer, L. J., Thomas, J., and De-Schepper, P. J. (1979) J. Biol. Chem. 254, 4585–4595. 13. Inoue, I., Katayama, S., Takahashi, K., Negishi, K., Sonoda, M., and Komoda, T. Biochem. Biophys. Res. Commum. 235, 113– 116. 14. Clarke, S. D., and Jump, D. B. (1996) Lipids 31(Suppl), S7-S11. 15. Takahashi, K., Shen, M.-Z., Noji, S., Inoue, I, Wakabayashi, T. Negishi, K., and Katayama, S. (1997) Diabetes 31(Suppl). [Abstract] 16. Yagi, K. (1976) Biochem. Med. 15, 212–216. 17. Folch, L. (1957) J. Biol. Chem. 226, 497–509. 18. Ulfelder, K. J., Schwartz, H. E., Hall, J. M., and Sunzeri, F. J. (1992) Anal. Biochem. 200, 260–267. 19. Neat, C. E., Thomassen, M. S., and Osmundsen, H. (1980) Biochem. J. 186, 369–371. 20. Steineger, H. H., Sorensen, H. N., Tugwood, J. D., Skrede, S., Spydevold, O., and Gautvik, K. M. (1994) Eur. J. Biochem. 225, 967–974. 21. Nakajima, T., Sakagishi, Y., Katahira, T., Nagata, A., Kuwae, T., Nakamura, H., Inoue, I., Takahashi, K., Katayama, S., and Komoda, T. (1995) Biochem. Biophys. Res. Commun. 217, 407– 411. 22. Hurtado, I., Fiol, C., Gracia, V., and Caldu´, P. (1996) Atherosclerosis 125, 39–46. 23. Suckling, K. E., and Jackson, B. (1993) Prog. Lipid Res. 32, 1– 24. 24. Berry, E. M., Eisenberg, S., Friedlander, Y., Harats, D., Kaufmann, N. A., Norman, Y., and Stein, Y. (1992) Am. J. Clin. Nutr. 56, 394–403. 25. Scaccini, C., Nardini, M., D’Aquino, M., Gentili, V., Di-Felice, M., and Tomassi, G. (1992) J. Lipid Res. 33, 627–633. 26. Go¨ttlicher, M., Demonz, A. Swensson, D., Tollet, P., Berge, R., ˚ . (1993) Biochem. Pharmacol. 46, 2177– and Gustafsson, J.-A 2184. 27. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39–43. 28. Krahling, J. B., and Tolbert, N. E. (1982) Ann. NY Acad. Sci. 386, 433–435. 29. Stegmeier, K., and Schmidt, F. H. (1982) Ann. NY Acad. Sci. 386, 449–452. 30. Yamazaki, R. K., Shen, T., and Schade, B. (1987) Biochim. Biophys. Acta 920, 62–67. 31. Ericsson, C.-E., Hamsten, A., Nilsson, J., Grip, L., Svane, B., and de Faire, U. (1996) Lancet 347, 849–853. 32. Harris, W. S. (1989) J. Lipid Res. 30, 785–807.
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237, 606–610 (1997)
RC977196
The Peroxisome Proliferator-Activated Receptor a (PPARa) Regulates the Plasma Thiobarbituric Acid-Reactive Substance (TBARS) Level Ikuo Inoue,1 Satoru Noji, Man-zhen Shen, Keiichi Takahashi, and Shigehiro Katayama The Fourth Department of Medicine, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-04, Japan
Received May 30, 1997
We investigated whether liver expression of the peroxisome proliferator-activated receptor a (PPARa) gene is related to the plasma thiobarbituric acid-reactive substance (TBARS) level, as well as to plasma cholesterol (TC) level and plasma triglyceride (TG) level in rats fed a high fat chow containing a variety of fatty acids. Only the plasma TBARS level showed a significant negative correlation with the liver PPARa mRNA level (TC, RÅ0.001, pÅ0.9967; TG, RÅ0.248, pÅ0.1276; TBARS, RÅ0.439, pÅ0.0046). Although futher studies are needed to clarify whether the increase of the liver PPARa mRNA level confers a reduction in plasma TBARS levels, it is likely that PPARa activity plays a regulatory role in the pathogenesis of hyperlipidemia and atherosclerosis. q 1997 Academic Press
The peroxisome proliferator-activated receptors (PPAR) is a member of the steroid hormone receptor superfamily (1). Three types of PPARs have been described in rodents and humans: PPARa, Nuc1 (also called PPARb or PPARd), and PPARg. In the adult rat liver, PPARa is the most predominat isoform. PPARa is activated by a variety of hypolipidemic fibrates such as bezafibrate, clofibrate, fenofibrate, and gemfibrozil. Moreover, PPARa is acivated by medium, long, and very long chain fatty acids. Recently, Lermann et al. reported that PPARg expressed in adipose tissue is also acivated by the thiazolidinediones, i.e. troglitazone, pioglitazone, and BRL42346c (2). PPARs are ligand-activated transcription factors that control gene expression by interacting with specific respose elements (PPREs) located upstream of responsive genes (3). The genes containig PPRE motifs 1 All correspondence should be addressed to Ikuo Inoue, M.D., The Fourth Department of Internal Medicine, Saitama Medical School. 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-04, Japan. Fax: 81492-94-9752.
0006-291X/97 $25.00
include acyl-CoA oxidase (3), peroxisomal bifunctional enzyme (4), liver fatty acid binding protein (5), and microsomal CYP4A6, cytochrome P450 fatty acid v-hydoxylase (6). In addition, the genes coding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (7) the apoproteins A-I (8), A-II (9), and C-III (10) are also regulated by PPARa, resulting in the association of PPARa with lipid regulation. Recently, we reported that troglitazone and bezafibrate altered b-oxidation in peroxisomes (11) as well as in mitochondoria (12). In addition, we have reported that troglitazone (13) exert antioxidation effects, resulting in the reduction of the plasma thiobarbituric acid reactive substances (TBARS) levels. A high-fat diet containing a variety of fatty acids is known to change the plasma cholesterol and triglycerides levels. The dietary v-6 and v-3 fatty acids suppress lipogenesis in the liver, whereas saturated and monounsaturated fatty acids do not (14). In addition, the composition of dietary fatty acid has been reported to alter the susceptibility of low-density lipoprotein (LDL) to oxidation, and may cause a change in the plasma TBARS level (15). In this study, we investigated whether plasma concentrations of TBARS are altered in rats fed a high fat chow containing a variety of fatty acids. We also investigated whether expression of the PPARa gene in the liver is related to the plasma TBARS level, as well as plasma lipid levels. MATERIAL AND METHODS Animal treatment. Male Sprague-Dawley rats were obtained from Charles River Japan at the age of 4 weeks. They were housed three per cage and allowed free access to tap water and standard laboratory rat chow (Clea Japan Inc., Tokyo, Japan). Rats were housed in a temperature controlled room (227C) under a 12-h lightdark (8 PM to 8 AM) cycle. Three weeks later, the animals were divided into five groups and fed one of the following diets for 5 weeks. 1) Normal rat chow (lipid 16%, milk casein 20%, starch 54%, vitamins 1%, minerals 4%, cellulose powder 5%, w/w; Soy group, nÅ9). The
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lipid in the diet of the Soy group consisted of soy bean fat, which is rich in polyunsaturated fatty acid. 2) Olive diet, in which the lipid of the normal rat chow was replaced by olive oil. This regimen was rich in monounsaturated fatty acid (Olive group, nÅ9). 3) Palm diet, in which the lipid of the normal rat chow was replaced by palm oil containing a lot of saturated fatty acids (Palm group, nÅ9). 4) Caproic diet, in which the lipid of the normal rat chow was relaced by caproic acid, a medium chain fatty acid (Caproic group, nÅ9). 5) Normal rat chow in combination with eicosapentaenoic acid (EPA; 1000 mg/kg/ day) (EPA group, nÅ9). An additional study was performed by feeding rats one of the following diets for 10 days: 1) normal rat chow, which was similar to the Soy group described above (nÅ9), 2) normal rat chow in combination with troglitazone (200 mg/kg/day; Tro group, nÅ9), 3) normal rat chow in combination with bezafibrate (80 mg/kg/day; Beza group, nÅ9). Drugs were dissolved in polyethylene glycol/water (194:12:20 wt/ wt) and administered orally by gavage to the EPA group, Tro group, or Beza group. Preparation of lipoproteins. At the end of the feeding period, rats were killed after fasting overnight. Body and liver weights were measured, and tissue samples were collected. Blood samples were collected immediately into tubes containing EDTA (0.1% final concentration). After separation of the plasma, the lipoproteins were isolated by sequential density ultracentrifugation.
sities of the bands were evaluated using a UV-light box imaging system (Atto, Tokyo). Some of the PCR products were also identified by the capillary electrophresis (18). Statistical analysis. Parametric data are expressed as the mean { SD. Non-parametric data are expressed as the median and quartiles. Differences between groups were evaluated by Scheffe´’s F test.
RESULTS Although the body and liver weights in the Caproic group were significantly lower than those of the other groups, the differences in the body and liver weights among the other groups were not significant (data not shown). There were no difference in liver enzymes such as glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, g-glutamyl transferase, lactic dehydrogenase, total bilirubin between the four groups. All laboratory data were within normal ranges in agematched rats (data not shown). Although plasma TC, TG, and HDL-C levels were not affected by the different
Laboratory analyses. Plasma cholesterol (TC) and triglyceride (TG) levels were determined by enzymatic methods. Plasma glucose levels were determined by a glucose oxidase method and insulin levels by a radioimmunoassay using rat insulin as the standard. The plasma TBARS levels were determined by the method of Yagi (16). Determination of fatty acid composition of lipoproteins. The fatty acids LDL, high density lipoprotein (HDL) and very low density lipoprotein (VLDL), were extracted from rat plasma according to the method of Folch et al. (17). The fatty acids were methylated and the resulting fatty acid methyl esters were identified using a GC17A gas chromatograph (Shimadzu Corp., Kyoto, Japan) equipped with an Omegawax 30 m 1 0.25 mm capillary column (Supelco, Inc., Bellefonte, PA). Reverse transcriptase polymerase chain reaction (RT-PCR). Total tissue RNA was isolated from liver homogenate using a commercial kit (Isogen, Nippon Gene Co. Ltd., Toyama, Japan). Preliminary experiments had shown that PPARa mRNA expression was low and that accurate measurements would not be possible by Northern analysis. We therefore performed a RT-PCR procedure to examine PPARa expression levels. Total RNA (0.3 mg) was used as a template for DNA synthesis using oligo(dT) primer and a DNA cycle kit (GeneAmp RNA PCR Kit, Perkin Elmer, New Jersey, USA) according to the manufacture’s instructions. The RT reaction was performed twice at 427C for 15 min to maximize cDNA synthesis and was terminated by heating at 997C for 5 min. The resulting cDNA was used as the template for PCR. Oligonucleotide primers for PPARa RT-PCR were designed to amplify partial cDNA sequences. The synthetic oligonucreotides were obtained from Nippon Flour Mills Co., Ltd. (Kanagawa Japan). The primers used for PPARa were 5*-CCT TTT TGT GGC TGC TAT-3* for the forward primer and 5*-TCC CTG CTC TCC TGT ATG-3* for the reverse primer, giving a 356 bp fragment. The primers used for glyceraldehyde3-phosphate-dehydrogenase (GAPDH) were 5*-ACC ACA GTC CAT GCC ATC AC-3* for the forward primer and 5*-TCC ACC ACC CTG TTG CTG TA-3* for the reverse primer, giving a 452 bp fragment. PCR reactions were performed using the same temperatures as for denaturation (947C, 30s) and extension (727C, 90s), but the annealing temperature was 567C for 50 s. The number of amplification cycles was 33. The PCR products were analyzed by polyacrylamide gel electrophoresis on a 7.5% gel (NPU - 7.5 type, Atto Corporation, Tokyo, Japan), which was run for 2 h at 20 mA. DNA was visualized by ethidium bromide staining at a concentration of 10 mg/ml. The inten-
FIG. 1. Serum fatty acids level in the Soy group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), EPA group (nÅ9), and Caproic group (nÅ9). Three pools of three freshly prepared plasma for each group were determined.
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FIG. 2. Serum TBARS level in the Caproic group (nÅ9), EPA group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), and Soy group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median.
dietary lipids, the administration of EPA, which is a polyunsaturated fatty acid (v-3), reduced the circulation lipid levels, but the difference was not significant (data not shown). Fatty acid profiles of plasma in all groups are shown in Figure 1. In Palm group, the plasma levels of saturated fatty acid, i.e. lauric acid (C12:0) and myristic acid (C14:0), increased as compared with those of the other groups. In the Olive group, oleic acid (C18:1, v-9) was the major component. In the Soy group, polyunsaturated fatty acid, i.e. linoleic acid (C18:2, v-6), linolenic acid (C18:3, v-3), and eicosadienoic acid (C20:2, v-6), increased as compared with the other groups. EPA adminitration increased the plasma EPA (C20:5, v-3) and docosapentaenoic acid (C22:5, v-3) levels 10-fold and 2-fold, respectively, as compared with the Soy group (data not shown), but the docosahexaenoic acid (C22:6, v-3) level was not changed by EPA adminitration (data not shown). In all rats, the plasma fatty acids level reflected the levels of VLDL, LDL, and HDL (data not shown).
FIG. 3. Example of the detection of liver peroxisome proliferatoractivated receptor a PPARa and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) in the Caproic group (lane 1), EPA group (lane 2), Olive group (lane 3), Soy group (lane 4), and Palm group (lane 5). S: Standard maker.
FIG. 4. Liver PPARa mRNA level in the Caproic group (nÅ9), EPA group (nÅ9), Olive group (nÅ9), Palm group (nÅ9), and Soy group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median. *põ0.05.
The plasma lipid peroxide levels were determined by measuring the TBARS levels. Although the plasma TBARS levels in the EPA group were reduced significantly in comparison with those of the Soy group, there were not the significant difference among the the TBARS levels of the others groups (Figure 2). Figure 3 indicates the typical level of PPARa mRNA expression in all groups. Figure 4 shows the densitometer analysis of the PPARa mRNA levels in all groups. The PPARa mRNA levels in the EPA and Caproic groups were significantly increased. Although there were no significant difference among the other groups, the PPARa mRNA levels were ranked as follows: Olive group ú Soy goup ú Palm group. The length of the PCR products determined by capillary electrophoresis was 356 { 0.05 bp (mean { SD), and was used to quantify the amount of the PPARa mRNA. Figure 5 shows the relationship between the logarithm of the PPARa mRNA level and the plasma TC, TG, and TBARS levels. Only the plasma TBARS level showed a significant negative correlation with the PPARa mRNA level in all groups (TC, RÅ0.001, pÅ0.9967; TG, RÅ0.248, pÅ0.1276; TBARS, RÅ0.439, pÅ0.0046). As shown in Figure 6, bezafibrate administration significantly increased the expression of PPARa mRNA, whereas troglitazone administration did not increase the expression of PPARa mRNA. DISCUSSION It is well known that a high-fat diet induces peroxisomal proliferation (19). Several studies have demon-
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with the fact that fatty acids have similar physiological actions as peroxisomal proliferators like bezafibrate. Dietary v-6 and v-3 fatty acid suppress lipogenesis in the liver, whereas saturated fatty acid and monounsaturated fatty acid do not have that effect (14). In addition, the composition of dietary fatty acids affects the susceptibility of LDL to oxidation, suggesting that the plasma TBARS level would be altered by a variety of dietary fatty acid. Recently, we (21) and others (22) have also reported that HDL, which is the main cholesterol-carrying particle in rodents (23), can be oxidized. Monounsaturated oleic acid makes lipoprotein more resistant to in vitro oxidation, whereas polyunsaturated fatty acids are less resistent (24,25). In fact, the lag phases of oxidation of LDL and HDL in the EPA group and Soy group were shorter than those of the Palm group and Olive groups (15). Interestingly, the plasma TBARS level in the EPA group had decreased (Figure 2). On the other hand, the PPARa mRNA level had increased in the EPA group. Moreover, as shown in Figure 5, the plasma TBARS level was the factor with the strongest correlation to the increase the the PPARa mRNA level. It is likely that EPA activates PPARa (26), resulting in a fall in the plasma TBARS level. By contrast, the plasma TBARS level was also reduced by administration of troglitazone, which is a ligand of PPARg, without activating liver PPARa Troglitazone is structurally similar to vitamin E, which is an antioxidant, and causes a ruduction of the plasma TBARS level (13). The mechanism of reduction of the plasma TBARS level by the activation of PPARa is not clear. Recently, it has been reported that the PPARa affected the duration of inflammation induced by leukotriene B4/arachidonic acid (27). Reactive oxygen-induced free radicals play an important role as mediators of tissue injury associated with many pathological conditions, such as inflammatory and ischemic states. The reactive oxygen FIG. 5. The relationship between the liver PPARa mRNA level and plasma TC, TG, and TBARS level.
strated that all fatty acids can activate PPAR to various extents. Steineger et al. have indicated that increasing the number of double bonds in fatty acids augments PPAR mRNA expression (20). As shown in Figure 3, our in vivo data indicate that the liver PPARa mRNA levels of the EPA and Caproic groups had increased as compared with those of the other groups. Based on our finding that the plasma palmitoleic (C16:1), eicosatrienoic (C20:3, v-9), and docosahexaenoic (C22:6, v-3) acid level in the Caproic group had increased (data not shown), the rise in the PPARa mRNA level in the Caproic group might be due to the increased plasma monounsaturated and polyunsaturated fatty acid levels. The activation of PPAR by fatty acid is in agreement
FIG. 6. Liver PPARa mRNA level in the Tro group (nÅ9) and Beza group (nÅ9). Upper and lower lines indicate the 10th and 90th percentiles, boxes indicate the 25th and 75th percentiles. The line through each box indicates the median. *põ0.05.
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species, such as superoxide and hydroxyl radicals, can act on membranous lipids or lipids in lipoprotein particle, resulting in increased plasma TBARS level. When linked to the activation of function of peroxisome, H2O2 is generated. Although excessive H2O2 production may damage the genes and protein, catalase scavenges H2O2 . Catalase, which is the main enzyme contained in peroxisomes, may also scavenge reactive oxygen species, such as alkoxyl radical, hydroxyl radical, and peroxide radical, induced by Fenton reactions in addition to the reaction with H2O2 . If the activation of PPARa induces catalase activity, reactive oxygen species would be scavenged in peroxisomes. In fact, it has been reported that administration of a high fat diet (28), bezafibrate (29), or EPA (30) increases the amount of peroxisomal catalase activity. As discussed above, the activation of peroxisome might cause a reduction of plasma TBARS level. In fact, administration of bezafibrate (31), or EPA (32), prevents cardiovascular disease in spite of a mild reduction of serum cholesterol level. In the present study, we have shown that the level of expression of the PPARa gene in the liver is negatively correlated with the plasma TBARS level. Although futher studies are needed to clarify whether the increase of the liver PPARa mRNA level confers some benefit in preventing vascular complications, it is likely that PPARa activity plays a regulatory role in the pathogenesis of hyperlipidemia and atherosclerosis, in addition to its role in the inflammatory diseases, rheumatoid arthritis, lupus, and psoriasis (27). ACKNOWLEDGMENTS We are grateful to Dr. Keiichi Sano and Ms. Rie Yanagisawa (Jokoh Co. Ltd., Tokyo, Japan) for performing the capillary electrophoresis and to Ms. Atuko Neuchi (Saitama Medical School) for technical assistance. This work was supported by a grant-in-aid from the annual meeting for circulating regulatory factors.
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