Fish oil feeding alters liver gene expressions to defend against PPARalpha activation and ROS production

Mayumi Takahashi1, Nobuyo Tsuboyama-Kasaoka1, Teruyo Nakatani1, Masami Ishii2, Shuichi Tsutsumi2, Hiroyuki Aburatani2, and Osamu Ezaki1

1 Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8636; and 2 Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fish oil rich in n-3 polyunsaturated fatty acids has been shown to reduce the risk of cardiovascular diseases partly by reduction of blood triglyceride concentration. This favorable effect mainly results from the combined effects of inhibition of lipogenesis by decrease of SREBP-1 and stimulation of fatty acid oxidation by activation of peroxisome proliferator-activated receptor-alpha (PPARalpha ) in liver. However, because fish oil is easily peroxidized to form hydroperoxides and increases oxidative stress, some defense mechanism(s) against oxidative stress might occur. To understand these complex effects of fish oil diet, the gene expression profile of mice liver was analyzed using high-density oligonucleotide arrays. High-fat diet (60% of total energy intake) as either safflower oil or fish oil (tuna) was given to mice. After 6 mo of feeding, expression levels of a total of 6,521 genes were analyzed. In fish oil diet compared with safflower oil diet, immune reaction-related genes, antioxidant genes (several glutathione transferases, uncoupling protein 2, and Mn-superoxide dismutase), and lipid catabolism-related genes upregulated, whereas cholesterol and fatty acid synthesis-related genes and 17-alpha hydroxylase/C17-20 lyase and sulfotransferases related to production of endogenous PPARalpha ligands and reactive oxygen species (ROS) downregulated markedly. Because upregulation of these antioxidant genes and downregulation of sulfotransferases were also observed in mice administered fenofibrate, altered gene expression related to antioxidant system observed in fish oil feeding was mediated directly and indirectly by PPARalpha activation. However, downregulation of 17-alpha hydroxylase/C17-20 lyase was not due to PPARalpha activation. These data indicate that fish oil feeding downregulated the endogenous PPARalpha -activation system and increased antioxidant gene expressions to protect against ROS excess.

n-3 fatty acids; fibrate; sulfotransferase; glutathione transferase; dehydroepiandrosterone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN RODENTS, FISH OIL FEEDING showed less obesity and lower blood triglyceride levels relative to other dietary oils (4, 13). In humans, increased intake of fish oil showed protective effects against cardiovascular disease (27), but concern remains that increased intake of fish oil may lead to increased lipid peroxidation (23). Contained in fish oil are n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are taken into the body, are mostly delivered to liver (29), and are easily peroxidized to form hydroperoxides and their secondary degradation products (9). These are considered to be deleterious to tissues (37). Thus liver is the primary target organ for oxidative stress in fish oil feeding. Fish oil administration and fibrate, which are peroxisome proliferator-activated receptor-alpha (PPARalpha ) activators, manifest substantial increase in expression of H2O2-generating peroxisomal fatty acyl CoA oxidase, the first enzyme of the classic peroxisomal fatty acid beta -oxidation system, and of microsomal Cyp 4A1 and 4A3 genes (41). Disproportionate increases in H2O2-generating enzymes and H2O2-degrading enzyme catalase and reductions in glutathione peroxidase activity by peroxisome proliferators lead to increased oxidative stress in liver. However, the results of fish oil feeding to animals showed slight increases in the tissue level of thiobarbituric acid reactive substances (33). Thus fish oil feeding may enhance the hepatic antioxidant defense with several mechanism(s).

To identify genes responsible for metabolic alterations and antioxidant systems of fish oil feeding, the gene expression profile of 6,521 genes using oligonucleotide arrays in high-carbohydrate diet, high-safflower oil diet, and high-fish oil diet-fed mice was examined in mice liver. In addition, to examine whether the altered gene expression of fish oil feeding is via PPARalpha activation, mRNA levels of liver from mice administered fenofibrate were examined by Northern blotting.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Diet experiment. Female C57BL/6 mice were obtained from Tokyo Laboratory Animals Science (Tokyo, Japan) at 7 wk of age and fed a normal laboratory diet for 1 wk to stabilize the metabolic conditions. Mice were exposed to 12:12-h light-dark cycle and maintained at a constant temperature of 22°C.

For gene-chip analysis, mice were divided into three groups (n = 5-6 in each group). The first group was given a high-carbohydrate diet that, on a calorie basis, contained 63% carbohydrate, 11% fat, and 26% protein. In the high-carbohydrate diet, safflower oil was used as source of fat. The second group was given a high-safflower oil-rich diet containing 14% carbohydrate, 60% safflower oil, and 26% protein. Safflower oil used here was high-oleic type, containing 46% oleic acid (18:1n-9) and 45% linoleic acid (18:2n-6) from total fatty acids. The third group was given a high-fish oil diet containing 14% carbohydrate, 60% fish oil, mainly from tuna, and 26% protein. Fish oil contained 7% EPA (20:5n-3) and 24% DHA (22:6n-3) and was kindly provided by NOF (Tokyo, Japan). In this study, to elucidate the effects of fish oil feeding, we used a very high-fat diet (60% of total energy intake). The materials and methods of preparation and fatty acid composition of diet were the same as those used in our previous studies (15, 38). Mice were fed each diet for 6 mo. At the end of the experiments, animals were anesthetized at about 10:00 AM by intraperitoneal injection of pentobarbital sodium (0.08 mg/g body wt, Nembutal; Abbot, North Chicago, IL). Liver was isolated immediately, weighed, and homogenized in guanidine-thiocyanate, and RNA was prepared by the method described by Chirgwin et al. (3). RNAs were used for gene-chip analysis and Northern blotting.

To confirm the results of gene-chip analysis and to examine whether up- and downregulation of these genes by 6 mo fish oil feeding were also observed in a short-term feeding period and were due to activation of PPARalpha , Northern blots from the four groups of mice were made. Three groups were the same used in gene-chip analysis, and the fourth group was treated with a direct PPARalpha ligand fenofibrate (Sigma, St. Louis, MO) mixed in high-carbohydrate diet. Because mouse consumed ~1.5-2.0 g chow/day, doses of 0.5% (wt/wt) mixed in diet correspond to 410-550 mg · kg body wt-1 · day-1. Mice were fed each diet for 1 wk and killed similar to gene-chip experiments to obtain RNA. We chose a 1-wk feeding period as short-term feeding, because upregulation of target genes of PPARalpha such as acyl-CoA synthetase (ACS) and lipoprotein lipase (LPL) were observed in a 1-wk period of fish oil feeding or fenofibrate administration (data not shown).

Gene-chip analysis of gene expression. Poly (A)+ RNA was prepared from pooled total RNA from five animals of each group by using an mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ). Double-stranded cDNA (Superscript Choice System, GIBCO BRL) was made from 1 µg of mRNA with a T7-(dT)24 primer containing T7 RNA polymerase promoter site (Amersham Pharmacia). Biotinylated complementary RNA was made from 1 µg of cDNA using BioArray high-yield RNA transcript labeling kit (Enzo) and then fragmented to ~100-200 nucleotides. Ten micrograms of these in vitro transcripts were hybridized to Affymetrix Mu 6500 microarray for 16 h at 45°C with constant rotation at 60 rpm (21). Chips were washed and stained with streptavidin-phycoerythrin (10 µg/ml, Molecular Probes) and biotinylated goat antistreptavidin (3 µg/ml, Vector Laboratories) using tEukGE-WS2 protocol on Affymetrix fluidics station. Chips were scanned using Hewlett-Packard confocal laser scanner and visualized using Affymetrix Gene Chip 3.1 software (Affymetrix). Fluorescence intensity from the safflower oil- and fish oil-fed groups was normalized to that from the carbohydrate-fed group by equating the overall fluorescence intensity for the entire chip of each group. The average values were scaled to 100 so that all chips could be directly compared, and the data were imported into File Maker Pro (File Maker).

In order of fold-change levels in gene expression in liver from fish oil-fed mice relative to safflower oil-fed mice, genes increased more than twofold and decreased less than twofold are listed and examined. Difference call, which defines genes increased or decreased, is derived from this software and is classified in five stages: transcript has increased, decreased, marginally increased, marginally decreased, or has exhibited no change in expression level. Fold-change calculation was carried out as an indication of the relative change of each transcript represented on the probe array. Differentially expressed genes were identified using the following criteria. Absolute call is present, and average difference was >150. Absolute call, which was calculated by this software using several markers, is an indicator of the presence or absence of each gene transcript. The average difference value is a marker of abundance of each gene obtained by comparing the intensity of hybridization to 20 sets of perfectly matched 25-mer oligonucleotides relative to 20 sets of mismatched oligonucleotides using Affymetrix Gene Chip 3.1 software.

Preparation of cDNA probe for Northern blot. The cDNA fragments for cytochrome P-450 17-alpha hydroxylase/c17-20 lyase, sulfotransferases, kappa-immunoglobulin (constant region), gelsolin, and glutathione transferase theta-class type 2 were obtained by PCR from first strand cDNA using mouse liver total RNA. First strand cDNA was prepared using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech) primed with oligo(dT). The PCR primers used were as follows: cytochrome P-450 17-alpha hydroxylase/c 17-20 lyase, 5' primer, 5'-CTACACCTGGCTGCCATGT-3', and 3' primer, 5'-GCCTGATACGAAGCACTTCT-3'; hydroxysteroid sulfotransferase, 5' primer, 5'-AGGAACGAACTGGCTGAATG-3', and 3' primer, 5'-CTTGGGCTACTGTGAAGTGA-3'; phenol/aryl form sulfotransferase, 5' primer, 5'-CCACATTGCAAAGCCTACAC-3', and 3' primer, 5'-CATTAGCTTGGCATAGTGGG-3'; kappa-immunoglobulin (constant region), 5' primer, 5'-GATGCTGCACCAACTGTATC-3', and 3' primer, 5'-AACAGTGGTAGGTCGCTTGT-3'; gelsolin, 5' primer, 5'-GCACTATGGTGGTGGAGCA-3', and 3' primer, 5'-CGTTGGCAATGTGGCTGGA-3'; glutathione transferase theta-class type 2 primer, 5'-GCTTGCTGTTATCGAACGCA-3', and 3' primer, 5'-TGGTCAGACCACTCAAGGAA-3'. PCR was performed with Taq DNA polymerase (Takara, Shiga, Japan). Amplification was made by using the following program: segment 1: 94°C, 1 min; segment 2: 30 cycles of 98°C for 20 s, 68°C for 2 min; and segment 3: 72°C for 10 min. The amplified products were subcloned into pGEM-T Easy vector (Promega, Madison, WI) and verified by sequencing. The cDNA probes for rat glutathione transferase Ya subunit were kindly provided by Dr. Nakagawa at Kitasato University, and rat Mn-SOD was provided by Dr. Ookawara at Hyogo Medical University. These cDNA were used as probes for Northern blotting.

Northern blotting. A portion of RNA (15 µg/lane) was denatured with glyoxal and dimethyl sulfoxide and analyzed by electrophoresis in 1% agarose gels. After transfer to Nylon membranes (New England Nuclear, Boston, MA) and ultraviolet cross-linking, RNA blots were stained with methylene blue to locate 28S and 18S rRNAs and to ascertain the amount of loaded RNAs (35). The blots were hybridized overnight at 42°C with cDNAs that had been labeled with [32P]dCTP (New England Nuclear) by a random prime labeling kit (Amersham Pharmacia Biotech). The filters were washed several times with 1× SSC, 0.1% SDS at room temperature, washed twice at 50°C, and then exposed to X-ray film at -80°C.

Statistics. Statistical comparisons of the groups were made by one-way ANOVA, and when they were significant, each group was compared with the others by Fisher's protected least-significant differences test (Statview 5.0 Abacus Concept, Berkeley, CA). Statistical significance is defined as P < 0.05. Values are means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Phenotypic comparison of mice fed three different diets for 6 mo. The difference of body and tissue weights, blood lipid concentrations, and other metabolic parameters after 5-6 mo feeding are described in our previous studies (13, 15, 38). Briefly, compared with carbohydrate diet, safflower oil diet resulted in two- to threefold increase of wet parametrial white adipose tissue weight with concomitant 40% increase of body weight, but fish oil feeding did not increase white adipose tissue and body weight. The average energy intake of mice fed each diet was not significantly different; energy intakes of mice fed carbohydrate diet, safflower oil diet, and fish oil diet were 7.4 ± 0.5, 7.7 ± 0.9, 7.9 ± 0.5 kcal · day-1 · mouse-1, respectively, when they were measured at 12-13 wk feeding. Liver weight from fish oil-fed mice was 60% greater than that from carbohydrate-fed mice, possibly through peroxisomal proliferation. Fish oil feeding decreased liver triglyceride and cholesterol concentration by 62% and 35%, respectively, compared with safflower oil feeding.

Altered gene numbers among three different diets. Table 1 shows the number of liver genes whose expression levels were altered after a 6-mo feeding period. Of the 6,521 genes, including 3' expressed sequence tags (ESTs), analyzed, 1,669; 1,925; and 1,928 were expressed at significant levels (absolute call is present), and 975; 1,039; and 992 were expressed at substantial levels (absolute call is present, and average difference is >150) in carbohydrate-, safflower oil-, and fish oil-fed female mice, respectively. Of these, in safflower oil- and fish oil-fed mice, compared with carbohydrate-fed mice, 132 (13%), and 117 (12%) genes, respectively, significantly and substantially upregulated (difference call is increased, and its average difference is >150), whereas those of 14 (1%) and 48 (5%) genes, respectively, downregulated significantly (difference call is decreased, and average difference of carbohydrate-fed mice is >150). Within fat diet-fed groups, numbers of up- and downregulated genes in fish oil feeding compared with safflower oil feeding were 63 (6%) and 102 (10%), respectively.

                              
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Table 1.   Number of genes altered in high-fat diet

Upregulated genes of fish oil feeding. Of 63 genes that upregulated in fish oil feeding compared with safflower oil feeding, 24 genes showed increase greater than twofold. In order of the fold increase, they are listed in Table 2 with their average differences (corresponding to expression level) and putative function. When they are classified into subgroups according to their functions, genes related to immune reaction, fat oxidation, and antioxidant emerge (Table 3). Enzymes related to fatty acid oxidation, such as fatty acid transport protein, Cyp 4a-10, long-chain fatty ACS, and carnitine palmitoyltransferase II, upregulated in fish oil-fed mice. Compared with carbohydrate-fed mice, Cyp 4a-10, long-chain fatty ACS also upregulated in safflower oil-fed mice, but their increase in safflower oil-fed mice was smaller than those in fish oil-fed mice. The different expression levels of these genes among three groups might be to the levels of PPARalpha activation. In fish oil-fed mice, testosterone 16-alpha-hydroxylase related to steroid degradation also upregulated by 4.3-fold. Upregulation of these genes is mostly mediated by PPARalpha activation (17). In other genes, alpha 2 type IV collagen, beta-tubulin, insulin-like growth factor-binding protein 2, and biglycan related to cell proliferation and formation of connective tissues also upregulated. However, their physiological roles of upregulation in fish oil feeding have not been elucidated.

                              
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Table 2.   Fish oil diet-induced increases in gene expression in liver


                              
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Table 3.   Altered gene expression by fish oil feeding

Fish oil feeding upregulates immune-reacted genes in a long-term feeding period. Largest increases were observed in immune-reacted genes (Table 2). Components of immunoglobin such as immunoglobin kappa chain and immunoglobin heavy-chain constant region µ (b) markedly upregulated 37- and 24-fold, respectively. Because µ-type heavy chain is found in IgM, an increase of IgM is anticipated. Indeed, in rats, compared with safflower oil-fed group, fish oil feeding enhanced the serum IgM level (12). In relation to immune response, gelsolin involved in phagocytosis of neutrophils and macrophages (36) also upregulated by 10-fold. Leukocyte elastase inhibitor that inactivates enzymes related to immune reaction (34) also upregulated by ninefold. These immunological reactions observed in fish oil-fed mice were not due to hepatocyte injury, because there were no increases of serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) concentrations (data not shown).

To examine whether upregulation of these genes by 6 mo, fish oil feeding was also observed in a short-term feeding period and was related to activation of PPARalpha , expression levels of immunoglobulin kappa chain, and gelsolin in fish oil-fed mice, and mice administered fenofibrate were examined by Northern blotting (Fig. 1). However, fish oil feeding did not upregulate these genes in 1 wk of feeding but markedly upregulated them in 6 mo of feeding. In addition, there was a large variation of immunoglobulin kappa chain expression levels of individual mice in fish oil-fed mice. Gene-chip analysis did not detect this individual variation, because it used a pooled sample. One week of fenofibrate administration did not upregulate these genes but rather downregulated gelsolin expression. Thus upregulation of these immune-related genes is not due to direct PPARalpha activation and may be related to chronic activation of neutrophils against oxidative stress.


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Fig. 1.   Northern blotting for kappa-immunoglobulin (A, B) and gelsolin (C, D) from livers from carbohydrate (Carb.)-, fenofibrate-administered carbohydrate (Fib.)-, and safflower oil (Saf.)- and fish oil-fed mice (Fish) for 1 wk (A, C) and 6 mo (B, D). Total RNA was isolated from livers of carbohydrate diet, fenofibrate-administered carbohydrate diet (for 1 wk only), and safflower oil diet- and fish oil diet-fed mice at 1 wk feeding (A, C) and 6 mo feeding (B, D). Fifteen-microgram aliquots of total RNA were subjected to electrophoresis and transferred to Nylon membranes. The membranes were hybridized with 32P-labeled probe for kappa-immunoglobulin (A, B) and gelsolin (C, D). In autoradiogram, each line represents a sample from an individual mouse. The radioactivity in each band was quantified using an image analyzer. The data for each band are shown in values relative to the mRNA level of carbohydrate diet group mice. In A and B, a typical autoradiogram of kappa-immunoglobulin (24-h exposure) and its relative levels are shown. In C and D, a typical autoradiogram of gelsolin (24-h exposure) and its relative levels are shown. Each value represents a mean ± SE of 5 mice. Statistical differences are shown: *P < 0.05 and **P < 0.01.

Fish oil or fenofibrate administration upregulates antioxidant genes. In this gene-chip analysis, genes having antioxidant activities, such as uncoupling protein-2 (UCP-2), theta-class glutathione transferase type 2, glutathione transferase, glutathione transferase Ya subunit, and manganese superoxide dismutase (Mn-SOD) upregulated by two- to fourfold (Tables 2 and 3). UCP-2 and Mn-SOD, which are located in mitochondria, reduced superoxide formation (6, 26). A recent study of UCP-2 knockout mice revealed an important role of UCP-2 in macrophage-mediated immunity and ROS generation (1). Glutathione transferases in cytosol promoted conjugation of toxic electrophilic xenobiotics to glutathione (22). However, expression levels of other genes related to antioxidant systems, such as Cu-Zn SOD (M60798), cellular (AA123700) and plasma (U13705) glutathione peroxidase, and catalase (L25069), did not alter in fish oil-fed mice (data not shown).

To examine whether upregulation of these genes by 6-mo fish oil feeding was also observed in a 1-wk feeding period and was related to activation of PPARalpha , expression levels of these genes in fish oil-fed mice and fenofibrate-administered mice were examined by Northern blotting. Because it has been previously reported that fish oil and fenofibrate upregulate UCP-2 mRNA (38), glutathione transferase Ya subunit and Mn-SOD (4k and 1k) mRNAs were presented (Fig. 2). In agreement with gene-chip analysis, compared with safflower oil feeding, fish oil feeding also increased these enzyme expressions by 3.8-fold (P < 0.001), 1.3-fold (4k, P < 0.001), and 1.9-fold (1k, P < 0.001), respectively. Administration of 0.5% (wt/wt) fenofibrate for 1 wk increased glutathione transferase Ya subunit and Mn-SOD mRNAs expression by 2.2-fold (P < 0.01), 1.4-fold (4k, P < 0.001) and 2.0-fold (1k, P < 0.001), respectively. In addition, 1-wk administration of fenofibrate also upregulated gene expression of glutathione transferase theta-class type 2 (data not shown). Thus upregulation of glutathione transferases and Mn-SOD by fish oil feeding is also mediated by PPARalpha activation.


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Fig. 2.   Northern blotting for glutathione transferase Ya subunit (A) and Mn-superoxide dismutase (B) from livers from carbohydrate-, fenofibrate-administered carbohydrate-, and safflower oil- and fish oil-fed mice for 1 wk. Total RNA was isolated from livers of carbohydrate diet, fenofibrate-administered carbohydrate diet, and safflower oil diet- and fish oil diet-fed mice at 1 wk feeding. Fifteen-microgram aliquots of total RNA were subjected to electrophoresis and transferred to Nylon membranes. The membranes were hybridized with 32P-labeled probe for glutathione transferase Ya subunit and Mn-superoxide dismutase. In autoradiogram, each line represents a sample from an individual mouse. The radioactivity in each band was quantified using an image analyzer. The data for each band are shown in values relative to the mRNA level of carbohydrate diet group mice. In A, a typical autoradiogram glutathione transferase Ya subunit (24-h exposure) and its relative levels are shown. In B, a typical autoradiogram of Mn-superoxide dismutase (24-h exposure) and their relative levels (4 and 1 kb) are shown. Each value represents a mean ± SE of 5 mice. Statistical differences are shown: *P < 0.05, **P < 0.01, and ***P < 0.001.

Downregulated genes in fish oil feeding. Of 102 genes that downregulated in fish oil feeding compared with safflower oil feeding, 25 genes showed a decrease less than twofold. In order of fold decrease, they are listed in Table 4 with their average differences and putative function. Of 25 genes, 6 genes were related to cholesterol and fatty acid synthesis. They were stearoyl-CoA desaturase, ATP citrate-lyase, fatty acid synthase, squalene epoxidase, farnesyl pyrophosphate synthetase, and low-density lipoprotein receptor. In agreement with our previous findings (15), parallel with decreases of these enzymes, a marked reduction of SREBP-1c may be responsible for downregulation of cholesterol and fatty acid synthesis.

                              
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Table 4.   Fish oil diet-induced decreases in gene expression in liver

In addition, fish oil feeding downregulates several transcription factors, silent mating-type information regulation 2 (Sir 2), ATF-4, P22, and Id2 against safflower oil feeding. However, Sir 2, ATF-4, and P22 in fish oil diet did not differ against carbohydrate diet; i.e., these transcription factors upregulated in safflower oil diet relative to carbohydrate diet. Sir2 encodes a protein that promotes compact chromatin structure, thereby preventing or silencing gene transcription at selected levels. Recently, calorie restriction in yeast failed to extend lifespan in strains mutant for Sir2 (20). Thus the increased longevity-induced calorie restriction requires Sir2 protein. An increase of Sir2 in safflower oil feeding might increase lifespan of hepatocytes. Activation transcription factor 4 interacts with many transcription factors and coactivators such as TATA-binding protein, TFIIB, RAP 30 subunit of TFIIF, and CREB-binding protein (19). Id2 (inhibitor of DNA binding) has recently been shown to bind SREBP-1c and inhibit transcription of fatty acid synthase (24). Thus reduction of Id2 might be an adaptive response against a marked decrease of SREBP-1c mRNA. Further studies are necessary to clarify physiological roles of these transcription factors in high-fat diet.

Fish oil or fenofibrate administration downregulates genes related to induction of endogenous PPARalpha activator. Fish oil feeding downregulated the expression of cytochrome P-450 17-alpha hydroxylase/C17-20 lyase and sulfotransferases (Tables 3 and 4). Cytochrome P-450 17-alpha hydroxylase/C17-20 lyase catalyzes formation of dehydroepiandrosterone (DHEA) from pregnenolone (25). Because the sulfated form of DHEA by hydroxysteroid sulfotransferase is a peroxisomal proliferator (39), marked reduction of both 17-alpha hydroxylase/C17-20 lyase and hydroxysteroid sulfotransferase may be an adaptive response to decrease production of endogenous PPARalpha activators. In addition, hydroxysteroid and phenol/aryl form sulfotransferases produce ROS (10). Sulfation by sulfotransferase is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation. However, the sulfate group is electron withdrawing and may be cleaved off heterolytically in some molecules, leading to electrophilic cation (10).

The mechanism(s) of downregulation of 17-alpha hydroxylase/C17-20 lyase and sulfotransferases were different. Fenofibrate administration did not downregulate 17-alpha hydroxylase/C17-20 lyase mRNA but downregulated hydroxysteroid and phenol sulfotransferases mRNAs by 88% (P < 0.05) and 83% (P < 0.001), respectively, whereas fish oil feeding for 1 wk downregulated all these enzymes (Fig. 3). Thus downregulation of hydroxysteroid and phenol sulfotransferases by fish oil feeding is also mediated through PPARalpha activation, but 17-alpha hydroxylase/C17-20 lyase is not. Because 17-alpha hydroxylase/C17-20 lyase is an enzyme for steroid catabolism, it may be downregulated by a decrease of mature form of SREBP-1c (15).


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Fig. 3.   Northern blotting for cytochrome P-450 17-alpha hydroxylase/C17-20 lyase (A), hydroxysteroid sulfotransferase (B), and phenol/aryl-form sulfotransferase (C) from livers from carbohydrate-, fenofibrate-administered carbohydrate-, and safflower oil- and fish oil diet-fed mice for 1 wk. Total RNA was isolated from livers of carbohydrate diet-, fenofibrate-administered carbohydrate diet-, and safflower oil- and fish oil diet-fed mice at 1 wk feeding. Fifteen-microgram aliquots of total RNA were subjected to electrophoresis and transferred to Nylon membranes. The membranes were hybridized with 32P-labeled probe for cytochrome P-450 17-alpha hydroxylase/C17-20 lyase, hydroxysteroid and phenol/aryl form sulfotransferases mRNAs. In autoradiogram, each line represents a sample from an individual mouse. The radioactivity in each band was quantified using an image analyzer. The data for each band are shown in values relative to the mRNA level of carbohydrate diet group mice. In A, typical autoradiogram of cytochrome P-450 17-alpha hydroxylase/C17-20 lyase (24-h exposure) and its relative levels are shown. In B, typical autoradiogram of hydroxysteroid sulfotransferase mRNAs (48-h exposure) and its relative levels are shown. In C, a typical autoradiogram of phenol/aryl-form sulfotransferase mRNAs (48-h exposure) and its relative levels are shown. Each value represents the mean ± SE of 5 mice. Statistical differences are shown: *P < 0.05, **P < 0.01, and ***P < 0.001.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

By using gene-chip analysis, we have studied genes that were markedly altered in expression levels by fish oil feeding. Although we have selected some genes in limited number of genes, we could show not only that the well-known genes related to fatty acid oxidation and triglyceride synthesis, but also new classes of genes related to PPARalpha activation and ROS production are markedly altered by fish oil feeding.

Fatty acids, particularly eicosanoids and their metabolites (42), constitute one class of endogenous PPARalpha activators, whereas steroid DHEA and its metabolites constitute another class of naturally occurring PPARalpha activators. When DHEA is administrated to rodents, this androgen precursor acts as a strong peroxisome proliferator (7, 40) and inducer of liver peroxisomal enzyme expression and Cyp 4A gene transcription (31). However, DHEA is effective in inducing each gene of these mRNAs in rat liver in vivo (31), but this steroid has no effect in cultured cells (32). This suggests that DHEA per se is not an active inducer but that this metabolite is more active inducer. In rat primary hepatocytes, DHEA-3beta sulfate (DHEA-S), a DHEA metabolite produced by steroid sulfation (14), increases in peroxisomal enzyme and Cyp 4A expression levels at physiologically relevant concentrations of DHEA (10 µM) (32). In addition, because DHEA-S is unable to induce a liver peroxisome proliferative response in vivo when administrated to PPARalpha gene knockout mice (30), it is evident that peroxisome proliferative response by DHEA-S is mediated by PPARalpha . Our finding that fish oil feeding resulted in marked reductions of two important enzymes for DHEA-S formation, namely 17-alpha hydroxylase/C17-20 lyase that catalyzes the conversion of pregnenolone to DHEA and hydroxysteroid sulfotransferase that catalyzes the conversion of DHEA to DHEA-S, indicates an operation of a negative-feedback system to reduce endogenous ligand formation for PPARalpha activation against increased activation of PPARalpha caused by fish oil feeding (Fig. 4). Thus fish oil-mediated PPARalpha activation may inhibit steroid-mediated PPARalpha activation to prevent some PPARalpha -mediated deteriorative effects such as ROS production and cancer formation (41). However, to prove this hypothesis, it is necessary to identify endogenous ligands for PPARalpha activation and to show that fish oil feeding results in a reduction of PPARalpha ligands derived from the steroid pathway.


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Fig. 4.   A proposed model for the negative feedback of peroxisomal proliferator-activated receptor alpha  (PPARalpha ) activation. Fatty acids, in particular, eicosanoids and their metabolites, constitute 1 class of endogenous PPARalpha activators, whereas adrenal steroid dehydroepiandrosterone (DHEA) and its metabolites constitute another class of naturally occurring PPARalpha activators. Cytochrome P-450 17-alpha hydroxylase/C17-20 lyase (P450c17) catalyzes formation of DHEA. Because sulfated form of DHEA (DHEA-S) by hydroxysteroid sulfotransferase (SULT) is a peroxisomal proliferator (30), marked reduction of both P450c17 and SULT by fish oil feeding may be an adaptive response against fish oil-induced PPARalpha activation. Decreased mRNA of SULT is PPARalpha dependent, whereas that of P450c17 is not.

Sulfotransferase is not only related to PPARalpha activation but also to ROS production. Sulfotransferases are phase II drug-metabolizing enzymes that sulfoconjugate a variety of endogenous and exogenous compounds such as biogenic amines, steroid hormones, bile acids, drug, and carcinogens (11, 16). However, the sulfate group is electron withdrawing and may be cleaved off heterolytically in some molecules, leading to an electrophilic cation (10). Fish oil feeding and fenofibrate administration downregulate hydroxysteroid and phenol/aryl form sulfotransferase. Both types of sulfotransferases are related to ROS production. Thus downregulated hydroxysteroid and phenol/aryl form sulfotransferase by fish oil feeding may contribute to a decrease of ROS production.

In this line of evidence, fish oil feeding and fibrate administration upregulated antioxidant genes such as UCP-2, glutathione transferase, and Mn-SOD. Glutathione S-transferases (GST) are a multigene family; four classes of protein, alpha , µ, pi , and theta , have been demonstrated (2). In mouse, alpha -class GST consists of two subunits Ya and Yc. Glutathione transferase (L06047) in Table 2 corresponds to alpha 4-type, which abundantly expresses in lung tissues (43). Ya subunit gene expression is induced by planar aromatic compounds and electrophiles (8), and antioxidant response element is found in the 5'-flanking region of this gene (28). Fish oil feeding upregulated Ya, alpha 4-, and theta -types of GST to quench ROS in cytosol, whereas it upregulated UCP-2 and Mn-SOD to quench ROS in mitochondria (Fig. 5).


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Fig. 5.   A proposed model of antioxidant system in fish oil diet. Long-chain unsaturated fatty acids from fish oils are initially metabolized to acetyl-CoA and medium-chain acyl-CoA (Med acyl-CoA) in peroxisome by beta -oxidation. Reactive oxygen species (ROS) and peroxidized lipid (LOOH) generated in this process are quenched glutathione S-transferase (GST) in cytosol. Acetyl-CoA and Med acyl-CoA are transferred to mitochondria by carnitine acetyltransferase (CAT). Med acyl-CoA is metabolized into acetyl-CoA, and acetyl-CoA is used in the tricarboxylic acid cycle. ROS produced in respiratory chain is reduced by uncoupling protein 2 (UCP-2) through a reduction proton gradient and also quenched by Mn-superoxide dismutase (Mn-SOD). In addition, sulfation of bile acids and steroid hormones by SULT generates ROS. A reduction of SULT may also contribute to a reduction of ROS formation.

Because fish oil-mediated alterations of gene expression related to ROS production were also observed in fenofibrate-administered mice, it is conceivable that alterations of these genes are mediated by PPARalpha activation. However, altered gene expressions observed in fenofibrate administration are due to the direct effects mediated by peroxisome proliferator responsive elements or the indirect effects of other cis-elements by PPARalpha -activated gene products or PPARalpha -unrelated effects. Most of the fenofibrate effects are considered to be mediated directly and indirectly by PPARalpha (17, 18), but it is not ruled out that in case of some genes, similar alterations of gene expression between fenofibrate administration and fish oil feeding were merely coincident and were not related to PPARalpha activation.

In this study, to clarify the effects of fish oil feeding, we used a very high-fat diet (60% of total energy intake). We have observed that there was a linear, dose-dependent effect of fish oil on upregulation of PPARalpha -activated genes such as ACS, LPL, and UCP-2 mRNAs; 10% of fish oil showed a significant increase of ACS, LPL, and UCP-2 mRNAs (data not shown). Thus we expected that alterations of gene expression observed in 60% oil diet would also be observed in the range of daily intake of fish.

Another finding of gene-chip analysis is that immunological reaction was observed in fish oil feeding, but this effect was not observed in PPARalpha activator. Because lipid peroxidation products, which are specific to fish oil feeding, are a potent chemostatic factor for neutrophils (5), it is possible that fish oil feeding increases lipid peroxidation products and induces neutrophil migration and activation in liver tissues. Thus increased immunological reaction as well as induction of antioxidant genes might be an adaptive reaction against increased oxidative stress.

In summary, gene-chip analysis revealed that activators of PPARalpha , such as fish oil and fenofibrate, altered gene expression profiles to defend against excess PPARalpha activation and ROS production.


    ACKNOWLEDGEMENTS

We are grateful to NOF for the supply of fish oil and to Dr. Nakagawa at Kitasato University for the supply of rat GST Ya subunit cDNA and to Dr. Ookawara at Hyogo Medical University for the supply of rat Mn-SOD cDNA.


    FOOTNOTES

This work was supported in part by Special Coordination Funds for Promoting Science and Technology from the Japanese Science and Technology Agency (Tokyo) and by research grants from the Japanese Ministry of Health, Labor, and Welfare (Tokyo) and the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Tokyo).

Address for reprint requests and other correspondence: O. Ezaki, Division of Clinical Nutrition, National Institute of Health and Nutrition, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8636, Japan (E-mail: ezaki{at}nih.go.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00376.2001

Received 24 August 2001; accepted in final form 19 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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