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in Rat Liver1
Institut für Ernährungswissenschaften, Martin-Luther-Universität Halle-Wittenberg, D-06108 Halle/Saale, Germany
2To whom correspondence should be addressed. E-mail: eder{at}landw.uni-halle.de.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol/kg (FF25, FF250 rats) or a fat heated at 50°C for 38 d, with vitamin E concentrations of 25 or 250 mg -tocopherol/kg (OF25, OF250 rats) for 63 d. Differences in gene expression were considered to be significant at a ratio of at least 1.4. In the OF25 rats, the expression of 47 genes was altered; in the OF250 rats, the expression of 37 genes was altered, and in the FF250 rats, the expression of 21 genes was altered compared with FF25 rats. In both OF25 and OF250 rats, a series of target genes of the peroxisome proliferator-activated receptor (PPAR) was upregulated. Determination of gene expression of acyl CoA oxidase and activity of catalase confirmed that oxidized fats caused peroxisome proliferation in the liver. In OF25 and OF250 rats, there was also upregulation of 12 and 5 genes involved in xenobiotic metabolism and stress response, of 7 and 7 genes involved in protein metabolism, of 5 and 2 genes encoding intracellular effectors or modulators and of 5 and 6 genes, respectively, encoding activators or repressors of transcription or translation. In conclusion, this study provides indirect evidence that dietary oxidized fats cause an activation of the PPAR, irrespective of the dietary vitamin E concentration. Identification of several other differentially regulated genes may be helpful to understand the effects of oxidized fats on animal metabolism.
KEY WORDS: • rats • oxidized fats • vitamin E • cDNA array • gene expression
The typical Western diet contains large quantities of PUFA that are heated or processed to varying degrees. In fast food restaurants, fat is heated in fryers for up to 18 h daily, at temperatures close to 180°C. For cost effectiveness, heated fats are used for up to 1 wk before they are discarded and replaced with fresh fats. Such fats have high concentrations of lipid peroxides (1). Several studies with animals showed that the consumption of oxidized fats affects the metabolism in several ways (2–5). Phenotypic changes in the metabolism are due to altered regulation of a large number of genes. Most studies performed with animals fed oxidized fats considered only a few genes or the proteins encoded by these genes. The current study was undertaken to identify and examine a comprehensive collection of genes whose expression was altered by dietary oxidized fats. Many of the effects caused by oxidized fats are mediated by oxidative stress (6,7). In this case, a high intake of antioxidants ought to prevent these effects. Therefore, we conducted a bifactorial experiment with growing rats that were fed diets with either a fresh or an oxidized fat and varying concentrations of vitamin E, which is the most effective protector against lipid peroxidation (8). The lower vitamin E concentration (25 mg -tocopherol equivalents/kg) meets the requirement for vitamin E (9), whereas the higher concentration (250 mg -tocopherol equivalents/kg) provided an excess supply of vitamin E. The concentrations of various lipid peroxidation products in heated fats depend on their thermal treatment (10). In a recent study, we found that an oxidized fat heated at a relatively low temperature over a long period containing high concentrations of primary lipid peroxidation products affected the lipid metabolism of rats more than an oxidized fat heated at a high temperature for a shorter period (11). Therefore, we planned to use a dietary fat treated at a moderate temperature (50°C) over a long period (38 d) in this study. To investigate the effects of long-term intake of the oxidized fat, we fed the experimental diets for 9 wk, thereby encompassing almost the entire growth period of the rats.
Recent developments in molecular techniques have made it possible to monitor changes in the gene expression of multiple transcripts, and thereby generate large gene expression profiles. One of these newly established methods is cDNA array technology, which is a powerful tool in a variety of research fields (12–14). To our knowledge, there has been no array-based investigation of the effects of oxidized fat in animals. The identification of gene expression patterns induced by oxidized fat might provide useful insights into the molecular mechanism underlying their diverse actions. In the current study, we used a cDNA array containing 1176 genes involved in the metabolism of lipids, carbohydrates, amino acids, xenobiotics, and vitamins as well as genes encoding receptors, hormones, and various other proteins. We decided to evaluate the gene expression profile of the liver because this tissue is the major target of oxidized lipids that are absorbed from the intestine.
It was shown recently that dietary oxidized fats (15) as well as cyclic fatty acid monomers (CFAM)3 (16), components of heated fats, lead to activation of the peroxisome proliferator-activated receptor (PPAR) and cause an increased expression of its target genes. The evaluation of our arrays in this study also showed that rats fed oxidized fats have an increased expression of various genes encoding proteins that are involved in the mitochondrial, microsomal, and peroxisomal oxidation of fatty acids. Some of these are target genes of the PPAR. We therefore also investigated the hypothesis that oxidized fat activates this receptor and causes proliferation of peroxisomes. To confirm this hypothesis, we determined the expression of acyl CoA oxidase (ACO) and the activity of catalase. Both are marker enzymes of peroxisomes; it was shown that peroxisome proliferation leads to upregulation of ACO and catalase (17). Upregulation of genes encoding proteins involved in the oxidation of fatty acids is expected to reduce concentrations of lipids in liver and plasma. Upregulation of genes encoding proteins involved in peroxisomal ß-oxidation is expected to reduce the concentrations of highly unsaturated (n-3) PUFA, which are the main substrates of these proteins. Reduced concentrations of (n-3) PUFA in membrane lipids might influence the gene expression of fatty acid desaturases, which are located in the microsomal membrane and are involved in the maintenance of membrane fluidity (18). To investigate a potential relationship between oxidized fat and alterations in lipid metabolism caused by peroxisome proliferation and increased oxidation of fatty acids, we determined also the concentrations of various lipids in liver and plasma, the fatty acid composition of liver phospholipids, and the gene expression of fatty acid desaturases.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol equivalents/kg). The 4 diets were: fresh fat, 25 mg -tocopherol equivalents/kg (FF25); fresh fat, 250 mg -tocopherol equivalents/kg (FF250); oxidized fat, 25 mg -tocopherol equivalents/kg (OF25); and oxidized fat, 250 mg -tocopherol equivalents/kg (OF250). The oxidized fat was a mixture of sunflower oil and lard (1:1), which was heated for 38 d at a temperature of 50°C. The fresh fat was a mixture of sunflower oil and lard in a ratio of 31:69. This ratio was chosen to equalize the fatty acid composition of the fresh fat with that of the oxidized fat. The fatty acid composition of both fats was similar but the oxidized fats had much higher concentrations of peroxides (918 vs. 5 mEq O2/kg), TBARS (22 vs. 0.01 mmol/kg), and conjugated dienes (277 vs. 14 µmol/kg) than the fresh fat. The major fatty acids in the fresh fat and the oxidized fat were (g/100 g total fatty acids): myristic acid (14:0), 1.1 vs. 0.9; palmitic acid (16:0), 19.2 vs. 17.4; stearic acid (18:0), 11.5 vs. 9.8; oleic acid [18:1(n-9)], 35.6 vs. 36.9; linoleic acid [18:2(n-6)], 26.1 vs. 26.9; and -linolenic acid [18:3(n-3)], 0.6 vs. 0.3. Other fatty acids, including trans-fatty acids, were present only in traces (<0.5 g/100 g fatty acids).
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-tocopherol equivalents/kg by individual supplementation of all-rac--tocopheryl acetate after determination of the basal tocopherol concentrations of the diets (the biopotency of all-rac--tocopheryl acetate was considered to be 67% of that of -tocopherol). To standardize food intake, the diets were administered in restricted amounts. The amount of food offered each day was increased continuously during the experiment from 8.3 to 17.4 g. Thus, all of the rats within 1 experiment consumed identical amounts of food. Water was freely available from nipple drinkers. The experimental diets were fed for 63 d. The rats were then killed by decapitation under light anesthesia with diethyl ether. Blood was collected into glass tubes. The liver was excised, divided, and frozen immediately in liquid nitrogen. All experimental procedures described followed established guidelines for the care and handling of laboratory animals and were approved by the council of Saxony-Anhalt.
Gene expression profile.
cDNA array analysis was performed using the Hybond Atlas Rat 1.2 array (Clontech) containing 1176 unique cDNAs spotted on a nylon membrane. Probing of cDNA array was performed according to the manufacturer’s directions. Briefly, total RNA was prepared from livers using RNAzol B reagent (Wako Chemie Neuss). The quality and quantity of RNA samples were assessed by measuring the optical density of each sample at 260 and 280 nm. Equal amounts of the RNA of 3 rats of each group were mixed into 1 pool of 50 µg. Therefore, 3 RNA pools per treatment group were used for a separate hybridization. A pooled set of primers complementary to the genes represented on the array was used for the reverse transcription probe synthesis, which was labeled with [33P]-dATP (NEN, Perkin-Elmer). Hybridization was allowed to proceed for 18 h at 63°C. The array membrane was washed and exposed to a phosphorus imaging screen for 24 h. The array images were visualized using the Bio-Image analyzer Fujifilm BAS 1500 and TINA 2.0 software (Raytest). Gene-specific signal intensities on the arrays were quantified using AtlasImage 2.01 software (Clontech) and corrected for background. Normalization of the signal intensities was done by relating them to the mean of the signal intensities of all genes. In our prestudies, this method of normalization yielded a higher reproducibility than normalization by various housekeeping genes. The background was the mean signal intensity of the unspotted fields on the array. Genes whose signal intensities were at least 1.5-fold the background level were considered. Of the 3 hybridizations done for each treatment group, the mean signal intensity for each gene was calculated. Mean signal intensities of the FF250, OF25, and OF250 groups were compared with those of the FF25 group. A difference in gene expression at a ratio threshold of 1.4 or more was considered to be potentially relevant. To verify the reproducibility of this cDNA array analysis, we analyzed 3 separate arrays of a pooled rat liver sample. The mean CV of the intensities of the detected signals was 22%.
ACO and 
9-, 6-, and 5-desaturase gene expression.
mRNA analysis was done as RT-PCR with real-time detection (Rotorgen 2000, Corbett Research using Sybr Green I). For quantification, a standard curve was generated with purified PCR products (extraction of cut ethidium bromide-stained bands following 2% agarose gel electrophoresis by MinElute Gel Extraction Kit (Qiagen) in concentrations of 105–108 copies (PicoGreen DNA Quantitation Kit, Molecular Probes). For normalization purposes, the copy number of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an independent internal control.
For cDNA synthesis, 2.3 µg total RNA was used (Omniscript RT Kit, Qiagen, Mastercycler Personal, Eppendorf). PCR was carried out using 100 µL Rotorgene PCR tubes with a final volume of 15 µL reaction mixture containing 3.5 mmol/L MgCl2, 125 µmol/L dNTP (Roth), 5 U Taq DNA polymerase, 1.5 µL 10X buffer (all from Promega), 0.38 µL 5X Sybr Green I (Sigma-Aldrich), 2 µL of first-strand cDNA or standard, and 2 µL of the primer mix. The following forward (F) and reverse (R) primers were selected using Primer Pairs software (Applied Biosystems): ACO (EC 1.3.3.6, EMBL ID: RNACO1) F: 5' CGG CGG GCA CGG CTA TTC T 3' R 5' GCT GTG GCT GGA TCC GCT GAC TC 3', primer mix (F, R): 2.5 pmol/µL (Roth).
9-Desaturase (EC 1.14.99.5; EMBL ID: RNSCD) F: 5' CCG TGG CTT TTT CTT CTC TCA 3', R: 5'CTT TCC GCC CTT CTC TTT GA 3', primer mix F: 3.5 pmol/µL, R: 7.5 pmol/µL (Roth). 6-Desaturase (EC 1.14.99; EMBL ID: AB621980), F: 5'CTT TCT CCT CCT GTC CCA CAT 3', R: 5'CAT TGC CGA AGT ACG AGA GGA 3' primer mix (F, R): 7.5 pmol/µL. 5-Desaturase (EC 1.14.99, EMBL ID: AF320509), F: 5'CCC CAT GCA CAT TGA TCA TG 3', R: 5' AAC GTT GCA GGT TGC CTG TAG 3'7,5 primer mix (F, R): 7,5 pmol/µL. GAPDH (EC 1.2.1.12; EMBL ID: RNGADPHR), F: 5'GCA TGG CCT TCC GTG TTC C 3', R: 5'GGG TGG TCC AGG GTT TCT TAC TC 3' primer mix 1.5 pmol/µL (MWG). Reactions were run in triplicate in separate tubes starting with a single 120-s denaturation step at 95°C. The DNA of ACO and desaturases was amplified in 25 cycles of 20 s denaturation at 95°C, 20 s/30 s annealing at 58°C/60°C and 20 s/40 s elongation at 72°C, respectively. Fluorescence was measured at 85°C/72°C. A final melting curve guaranteed the authenticity of the target product. Data were analyzed using the Rotorgene 2000 Detection System. The threshold cycle of standards was used to calculate the copy number of the target gene.
-Tocopherol concentration.
Concentrations of individual tocopherols in liver, adipose tissue, plasma and fats were determined by HPLC (19).
Plasma and liver lipids. Total lipids from the liver were extracted with a mixture of n-hexane and isopropanol (3:2, v:v) (20). Phospholipids of the extract were separated by normal phase HPLC using a Supelco-Si column (25 cm x 0.46 cm, LC-Si 5 µm, 13 nm, Supelco) and an elution system consisting of various mixtures of chloroform and methanol (21). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were collected using a fraction collector and methylated with trimethylsulfonium hydroxide (22). The FAME were separated by GC (23). For quantification of the phospholipids, individual phospholipids (cardiolipin, PE, phosphatidylinositol, PC, phosphatidylserine, sphingomyelin) were separated by HPLC using a Kromasil Si (60 5 µm, 12.5 cm x 4 mm, CS Langerwehe) column, an elution system consisting of chloroform, methanol, ammonia (30%), and water and analyzed by light scattering detection (24). For determination of the concentrations of triglycerides and cholesterol in the liver, an aliquot of the lipid extract was dried and the lipids were dissolved in a small volume of Triton X-100 (25). Concentrations of triglycerides in liver, plasma, and VLDL were determined using an enzymatic reagent kit (Ecoline 25, catalog-no. 1.14856.001, Merck).
Hepatic catalase activity. Catalase activity was determined in liver homogenate at 25°C using hydrogen peroxide as a substrate according to the method of Aebi (26). One unit of catalase activity is defined as the amount consuming 1 µmol hydrogen peroxide/min.
Statistics. Results were tested by two-way ANOVA (fat type, vitamin E concentration, and their interaction). Differences in treatment effects were considered significant at P < 0.05. When the interaction was significant, means were compared by Tukey’s post-hoc analysis.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-tocopherol in the liver did not differ between OF25 rats and FF25 rats but was lower in OF250 rats than in FF250 rats. The -tocopherol concentrations in adipose tissue and plasma were lower in OF25 rats than in FF25 rats, and they were lower in OF250 rats than in FF250 rats.
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400 yielded signal intensities above background levels. The correlation analysis of the gene expression levels relative to the FF25 group is shown in Fig 1. In OF25 rats, the expression of 47 genes was altered (39 upregulated, 8 downregulated); in OF250 rats, the expression of 37 genes was altered (27 upregulated, 10 downregulated) and in the FF250 rats, the expression of 21 genes was altered (18 upregulated, 3 downregulated) compared with FF25 rats. OF25 rats had an increased expression of 3 genes in the liver encoding proteins of mitochondrial ß-oxidation, of 2 genes encoding proteins of peroxisomal ß-oxidation, and of 4 genes encoding proteins involved in microsomal 
- and -1 hydroxylation of lauric acid (Table 3). In OF250 rats, the same genes were increased as was that of carnitine palmitoyl transferase II. Some genes of peroxisomal ß-oxidation and microsomal hydroxylation were also increased in FF250 rats. In OF25 rats, 4 other genes involved in the lipid metabolism were expressed differentially in FF25 rats; 3 were upregulated and 1 was downregulated (Table 4). In OF250 rats, 2 other genes involved in lipid metabolism were changed, and in FF250 rats, 1 other gene involved in lipid metabolism was changed (Table 4). OF25 rats also had upregulation of 7 genes involved in protein metabolism. The same genes were upregulated in OF250 rats; in the FF250 rats, 5 of these 7 genes were upregulated. Compared with FF25 rats, OF25 rats showed upregulation of 13 genes involved in xenobiotic metabolism and the stress response. In OF250 and FF250 rats, 6 of these 13 genes were upregulated. In OF25 rats, 5 genes encoding intracellular effectors and modulators were upregulated; in OF250 rats, 2 of them were upregulated. Among the genes encoding activators or repressors of transcription or translation, 5 were downregulated in OF25 rats and 6 were downregulated in OF250 rats. In FF250 rats, none of the genes encoding intracellular effectors and modulators or activators or repressors of transcription or translation differed from expression in FF25 rats. In all 3 groups of rats (OF25, OF250, FF250), the gene encoding ATP synthase was downregulated compared with FF25 rats, and the gene encoding dC-stretch binding protein was upregulated. In OF25 rats, the gene encoding cofilin was downregulated and that encoding Na+, K+, ATPase was upregulated, compared with FF25 rats. In OF250 and FF250 rats, the gene encoding microglobulin was downregulated compared with FF25 rats.
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6-desaturase, and 5-desaturase and increased activity of catalase in the liver than FF25 or FF250 rats (Table 5). The mRNA concentration of 9-desaturase did not differ between rats fed oxidized fat and those fed fresh fat. The dietary vitamin E concentration did not influence relative mRNA or activities of those enzymes.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1.6 can underestimate the number of genes that are differentially regulated by a dietary treatment (27,28). The cDNA array method is less sensitive and less reliable than other methods to quantify mRNA levels such as real-time detection PCR or Northern blotting (28,29). This may explain the different results of cDNA array and real-time detection PCR regarding gene expression of acyl CoA oxidase in the FF250 group compared with the FF25 group.
The oxidized fat used in this study, prepared by heating at a relatively low temperature over a long period, had high concentrations of primary lipid peroxidation products such as peroxides and hydroperoxides. Compared with most other studies of the effects of thermoxidized fats (2–4,30), feeding the diet containing the oxidized fat had less effect on the growth of the rats. This indicates that the oxidized fat used in this study was moderately oxidized compared with other studies. The fact that body weight development was only slightly lower in rats fed the oxidized fat than in those fed the fresh fat is advantageous from a methodological viewpoint because the effects of oxidized fats were not confounded by secondary effects of reduced growth. Because the control and oxidized fats were also equalized for their fatty acid composition, the effects observed in rats fed oxidized fats can be attributed predominantly to lipid peroxidation products present in the oxidized fats. The observation that rats fed oxidized fats had lower concentrations of -tocopherol in the liver than rats fed the fresh fat confirms recent studies showing that oxidized dietary lipids enhance tocopherol turnover in animal tissues (6,7,31).
The evaluation of the cDNA array showed that dietary oxidized fats led to an upregulation of some key genes encoding proteins involved in mitochondrial and peroxisomal ß-oxidation of fatty acids and - and -1 hydroxylation of fatty acids. Some of the upregulated genes such as carnitine palmitoyl transferase I, medium chain acyl CoA dehydrogenase, acyl CoA oxidase, 3-keto acyl CoA thiolase, or cytochrome P450 (CYP) 4A1 are target genes of PPAR. Increased levels of mRNA concentrations of ACO as determined by real-time PCR and an increased activity of catalase, both marker enzymes of peroxisomes, suggest that the oxidized fat caused peroxisome proliferation in the liver. An increased concentration of phospholipids in the liver, which was observed in rats treated with peroxisome proliferators (32), is an indicator of peroxisome proliferation in rats fed the oxidized fat. Our work, therefore, indirectly confirms a recent study showing that oxidized fats activate PPAR and cause increased gene expression or activities of some downstream genes such as ACO or CYP4A1 (15). The oxidized fat used in our study contained a large number of different lipid peroxidation products. Therefore, it is unknown which compounds were responsible for activating PPAR. In a recent study (16), feeding diets containing CFAM, which are components of heated fats, caused effects in rats similar to those observed in our study. Although we did not analyze the concentrations of CFAM in the fats we used, we assume that CFAMs were present and might have contributed to the effects observed in the rats fed the oxidized fat. Consideration of genes monitored by the cDNA array as well as the activity of catalase and gene expression of ACO determined by real-time PCR shows that raising the vitamin E concentration of the diet from 25 to 250 mg -tocopherol/kg did not prevent activation of PPAR and the increased expression of genes involved in fatty acid degradation caused by dietary oxidized fats.
We (33) and others (34) observed previously that oxidized dietary fats reduce lipid concentrations in liver and plasma. The current study clearly indicates that this effect might be due at least in part to enhanced fatty acid oxidation in the liver. Lower concentrations of highly unsaturated (n-3) PUFA in liver phospholipids, which were also reported previously in rats fed a diet containing CFAM (16), might be caused by an increased rate of peroxisomal ß-oxidation. Highly unsaturated (n-3) PUFA are the main substrate of peroxisomal ß-oxidation (35). Our study also shows that oxidized dietary lipids upregulate 5- and 6-desaturase in the liver. We suspect that this effect may be caused by reduced concentrations of highly unsaturated fatty acids in liver membranes of rats fed an oxidized fat. Highly unsaturated fatty acids such as 22:5(n-3) and 22:6(n-3) as components of membrane phospholipids increase the fluidity of membranes (36). Gene expression of 5- and 6-desaturases is controlled by the fluidity of the microsomal membrane (18). Upregulation of 5- and 6-desaturases could be a compensatory means of maintaining membrane fluidity in animals fed an oxidized fat.
The current study shows that dietary oxidized fat leads to an increased expression of genes encoding proteins that are involved in the microsomal biotransformation of xenobiotics. This finding agrees with a previous study that observed increased activities of various detoxifying microsomal enzymes in rats and guinea pigs fed oxidized fats (31,37). Upregulation of genes encoding proteins involved in the stress response such as glutathione-S-transferase or phospholipid hydroperoxide glutathione peroxidase in rats fed the oxidized fat may be caused at least in part by increased formation of reactive oxygen species (ROS) in the liver due to the oxidized fat. Activation of mitochondrial, microsomal, and peroxisomal enzymes leads to an increased generation of ROS (17) and may therefore be 1 important source of ROS in the liver of rats fed the oxidized fat. On the assumption that stress response genes were upregulated by increased formation of ROS, it is plausible that an improvement in the vitamin E status of the rats by increasing the dietary vitamin E concentration might have reduced the expression of some of those genes in rats fed the oxidized fat.
In addition to genes involved in lipid metabolism or xenobiotic metabolism and stress response, the expression of a number of genes encoding proteins involved in several other metabolic pathways were upregulated in rats fed the oxidized fat. The ubiquitine-proteasome pathway is the primary proteolytic pathway of eukaryotic cells. It controls the levels of numerous proteins involved in gene regulation, cell division, and surface expression as well as the stress response and inflammation. The proteasome system is now considered to be a cellular defense mechanism because it also removes irregular or damaged proteins generated by mutations, translational errors, or oxidative stress (38). Consumption of oxidized fat may induce increased damage of proteins in the liver through enhanced generation of ROS. Upregulation of activators of proteasomal proteins could be a compensatory mechanism to protect against cell damage by oxidized proteins.
Id proteins constitute a family of helix-loop-helix (HLH) transcription factors that are important regulators of cellular differentiation and proliferation (39). Id proteins lack a basic DNA binding region and are capable of inhibiting gene expression by forming inactive heterodimers with basic HLH transcription factors (40). The reduced expression of inhibitors of the DNA-binding proteins Id-2 and Id-3 suggests that dietary oxidized fats may stimulate cellular differentiation and proliferation. G-proteins are involved in many signal transduction pathways. Upregulation of various genes encoding G-proteins also suggests that oxidized fats affect signal transduction in hepatocytes in several ways. Alterations in the expression of several other genes, which cannot be discussed in detail, show that the oxidized fat affected several metabolic pathways. The finding that most of them were independent of the dietary vitamin E concentration suggests that the alterations were due only in part to oxidative stress. We therefore assume that the expression of most genes was altered by specific lipid peroxidation products present in the oxidized fat.
The comparison of FF250 with FF25 rats in our study shows that increasing the dietary vitamin E concentration leads to overexpression of several genes encoding peroxisomal or microsomal proteins. This finding is in close agreement with a recent study showing that oral treatment with -tocopherol increases the concentrations and activities of various microsomal enzymes such as CYP and the catalytic activities of various microsomal enzymes such as CYP1A1, CYP1A2, CYP2B1, CYP2C, and NADPH-cytochrome-P450 reductase in the liver of male rats (41).
Advances in array technology have allowed us to investigate genes differentially expressed in rat liver in response to a dietary oxidized fat and to different dietary vitamin E concentrations. Overall, in this study, the oxidized fat induced genes encoding proteins involved in the metabolism of lipids, proteins, and xenobiotics as well as genes encoding various intracellular effectors, activators, and repressors. This confirms that dietary oxidized fat influences many biochemical pathways in animals.
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FOOTNOTES |
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3 Abbreviations used: ACO, acyl CoA oxidase; CFAM, cyclic fatty acid monomer; CYP, cytochrome P450; FF25, diet containing fresh fat with 25 mg -tocopherol/kg; FF250, diet containing fresh fat with 250 mg -tocopherol/kg; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLH, helix-loop-helix; MUFA, monounsaturated fatty acids; OF25, diet containing oxidized fat with 25 mg -tocopherol/kg; OF250, diet containing oxidized fat with 250 mg -tocopherol/kg; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PPAR, peroxisome proliferator-activated receptor ; ROS, reactive oxygen species.
Manuscript received 2 December 2003. Initial review completed 7 January 2004. Revision accepted 19 March 2004.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) at least 1.4-fold in rats fed FF250, OF25, or OF250 compared with rats fed the FF25 diet
) at least 1.4-fold in rats fed FF250, OF25, or OF250 compared with rats fed the FF25 diet