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Hepatic Cytochrome P450 4A Expression Level in a Rat Model of Microvesicular Steatosis AMS Abdul Majid1,2 1. School of Pharmaceutical Sciences, University Science Malaysia, Penang, Malaysia, 2. School of Medical Sciences, University of New South Wales, Sydney, Australia Keywords: Cytochrome P450; Orotic acid; Peroxisome proliferator-activated receptor ABSTRACT Microvesicular steatosis in general plays an important role in the pathogenesis of drug, disease and alcohol induced liver damage. Such an event is manifested by the accumulation of lipid within the hepatocytes. Initial experiments conducted previously showed that short-term intake of diets containing Orotic acid (OA) produced rapid and extensive steatosis that follows a microvesicular distribution (Su, Sefton et al. 1999). The present study evaluated the hepatic cytochrome P450 4A expression level in a rat model of microvesicular steatosis that was marked by the intake of diets containing 1% of the Orotic acid for a time period of 21 days. The animals were sacrificed on the 22 nd day and the hepatic microsomal P450 A4 protein level was quantitated using immunoblot analysis. The result of the investigation showed a significant induction of CYP 4A expression level in groups that were fed with diets containing the 1% OA. The CYP4A in the test group livers was found to have been up regulated by 53% when compared to the control group. 1 INTRODUCTION Steatosis is a disease state that occurs due to accumulation of lipid within hepatocytes. This is often due to the intake of alcohol and drugs such as corticosteroids, tetracycline and some non-steroidal anti-inflammatory agents. Factors such as inherited metabolic disorders and diseases with hepatic involvement can also cause such a state (Farrell 1994; Burt, Mutton et al. 1998). The advent of steatosis by OA is thought to be mediated via mitochondrial injury, resulting in the impaired β-oxidation of fatty acids (Berson, De Beco et al. 1998; Burt, Mutton et al. 1998), however the detailed effects of lipid accumulation on hepatic gene expression are still unclear. In the rat, OA has been shown to cause severe fatty infiltration of the liver when administered in concentrations of 0.2% or higher in a purine-deficient diet (Standerfer and Handler 1955). The accumulation of lipids (mainly triglycerides) is due to inhibition of lipoprotein synthesis. The lipoprotein precursors, i.e. the apoprotein and lipid moieties, are synthesised, but the apoprotein is deficient in N-acetylglucosamine, D galactose and N-acetyl neuraminic acid (Martin, Biol et al. 1982) and the conjugation of the two moieties in the liver is impeded (Roheim, Switzer et al. 1965), resulting in a progressive increase in liver fat with a concomitant reduction in serum concentration of very low density lipoproteins and triglycerides (Hay, Fleming et al. 1988). The mechanism however, is not yet fully understood. Previous study showed a significant increase in cholesterol, triglycerides and phospholipids levels in rat liver after feeding of experimental diets containing OA for 21 days (Su, Sefton et al. 1999). Fatty acid overload is accompanied by an increased 2 capacity for oxidation of fatty acids by the mitochondrial peroxisomal and microsomal pathways of liver and heart (Mannaerts and Van Veldhoven 1992). The peroxisomal βoxidation is only a minor pathway for fatty acid oxidation relative to the mitochondrial counterpart, its role is more important for the β-oxidation of very long-chain fatty acids (Osmundsen, Bremer et al. 1991). Diets containing long chain fatty acids have been shown to induce peroxisomal and microsomal oxidation enzymes and also the expression of the cytoplasmic liver-type fatty acid-binding protein (L-FAB) (Issemann, Prince et al. 1992; Reddy and Mannaerts 1994). The L-FAB has been shown to have marked affinity for long-chain fatty acids and is considered to have a significant role in mediating the cellular uptake and intracellular targeting of long-chain fatty acids (Glatz and van der Vusse 1996). Therefore, in the metabolism and transport of long-chain fatty acids during fatty acid overload, the activity of peroxisomal, microsomal enzymes and the expression of LFAB are of great importance. The peroxisome proliferator activated receptors (PPARs; NR1C) belong to the steroid/thyroid/retinoid receptor superfamily. They are nuclear lipid-activatable receptors that control a variety of genes in several pathways of lipid metabolism, including fatty acid transport, uptake by the cells, intracellular binding and activation, as well as catabolism (β -oxidation and ω-oxidation) or storage. Three related isotypes of PPAR have been identified in rat and human, they are PPAR α (NR1C1), PPAR β (NR1C2) and PPAR γ (NR1C3) (Issemann and Green 1990; Dreyer, Krey et al. 1992; Gottlicher, Widmark et al. 1992; Schmidt, Endo et al. 1992; Chen, Law et al. 1993; Sher, Yi et al. 1993; Zhu, Alvares et al. 1993; Kliewer, Forman et al. 1994; Amri, Bonino et al. 1995; Aperlo, Pognonec et al. 1995; Greene, Blumberg 3 et al. 1995; Xing, Zhang et al. 1995). PPARs belong to the TR/RAR subfamily that recognize preferentially the core hexanucleotide motif AGGTCA in the up stream region of the target genes and are also characterized by the ability to form heterodimers with the 9-cis-retinoic acid receptor, RXR (NR2B). PPARs bind to DNAs heterodimerically with RXR as their binding partner. The PPAR: RXR heterodimeric protein binds to the PPAR response elements (PPRE), a direct repeat of two core recognition motifs AGGTCA spaced by one nucleotide (also known as DR1)(Kliewer, Umesono et al. 1992). The PPRE was first located in the promoter region of the acylCoA oxidase gene (Dreyer, Krey et al. 1992; Tugwood, Issemann et al. 1992). PPAR interacts with the upstream-extended core hexamer of the DR1, whereas RXR occupies the downstream motif (Ijpenberg, Jeannin et al. 1997). It has been demonstrated that the carboxy-terminal extension (CTE) regions of both receptors is indeed responsible for the recognition of the 5'-flank of the DR1 in PPREs (Hsu, Palmer et al. 1998). Interestingly, it was observed that the expression of RXR abolished PPARα stimulation of the PRL promoter in pituitary GH4C1 cells (Tolon, Castillo et al. 1998) which suggests that stimulation of the PRL promoter by PPARα was mediated by protein-protein interaction rather than binding of PPAR:RXR to the promoter. It has been proposed that the mechanism of this phenomenon is a ligand-dependent association of PPARα with the transcription factor GHF-1 that stimulates transcription (Tolon, Castillo et al. 1998). This also implies that PPARα would act similarly to a coactivator. It is postulated that in the event of over-expression of RXR, the stimulatory effect of PPARα can be suppressed as RXR might compete for its association with GHF-1. 4 EXPERIMENTAL PROCEDURE Materials The Hyperfilm-MP and reagents for enhanced chemiluminescence were acquired from Amersham Australia (North Ryde, NSW, Australia). Retinal, retinyl acetate and αtocopheryl acetate were purchased from Sigma Chemical Co. (St. Louis, MO). Chemicals used for SDS-polyacrylamide gel electrophoresis were obtained from BioRad laboratories (Richmond, CA). Constituents for the rat experimental diets were purchased from ICN Biochemicals (Seven Hills, NSW, Australia). Other biochemicals were procured from Sigma Chemical Co. (Castle Hill, NSW, Australia) The rabbit antiCYP4A1 IgG was a gift from Prof. G.G. Gibson, University of Surrey. Animal Treatments The research work was carried out according to the guidelines endorsed by the Australian National Health and Medical Research Council and was approved by the University of New South Wales and Central Area Health Service Animal Care and Ethics Committees. 12 male Wistar rats of approximately 3 weeks old with weight range of 237-282 g were divided equally into 2 groups with 3 rats per cage. Each group received basal diets of a high sucrose-containing semi-purified (SP) diet with each kilogram consisting of sucrose (600g), casein (200 g), cellulose (110 g), corn oil (40 g), ICN salt mixture 4179 (40 g), ICN vitamin diet fortification mixture (10 g), αtocopherol (20 mg), and retinyl acetate (8.7 mg). The rat group that had 1% Orotic Acid in their diet was labelled as OA+ group and the group without any Orotic Acid (the control group) was labelled as OA- . The rats were held on the diet for 21 days and had 5 free access to water. All the rats were individually labelled and their weights recorded throughout the 21 days and all the animals were sacrificed on the 22nd day under anaesthesia. Microsome preparation The rat livers harvested were perfused with cold saline solution followed by snap freezing in liquid N2 before storage at -70o C. The hepatic microsomes were prepared according to methods established earlier (Murray, Zaluzny et al. 1986). The liver sections from storage were thawed on ice in microsome preparation buffer (pH 7.4) containing 0.01M K2HPO4, 1mM EDTA and 0.25 M sucrose. The samples were homogenised and centrifuged at 10 000g for 25 minutes at 4o C under vacuum. The pallets were discarded and the supernatant was subjected to ultracentrifugation under vacuum at 35 000rpm for 1 hour 10 minutes at 4 oC. The supernatant was transferred into another tube and stored at -70 oC and the remaining pellet was covered with preparation buffer and homogenised for 5 passes followed by further ultracentrifugation at 4 oC at 35 000rpm for 35 minutes under vacuum. All the supernatant was discarded and the final microsomal pellet was resuspended in 50mM potassium phosphate buffer (pH 7) containing 1 mM EDTA and 20% glycerol and homogenised for 5 passes before being snapped frozen in liquid N2 and stored at -70ºC. Immunoblotting for CYP 450 4A Apoproteins in Rat Hepatic Microsomes. The rat hepatic microsomes protein concentration was determined and standardised using Lowry Protein quantification assay prior to Western Blot analysis. 5 mg / lane of each sample was subjected to electrophoresis on 7.5% acrylamide running gel and 3% polyacrylamide stacking gel (Towbin, Staehelin et al. 1979; Murray, Zaluzny et al. 6 1986). Proteins were transferred to nitrocellulose paper via electrophoresis gel transfer operation. The nitrocellulose paper was left to incubate in anti-rabbit IgG 4A antibody followed by anti-sheep IgG antibody and the immunoreactive proteins were detected by enhanced chemiluminescence on Hyperfilm-MP, and the resultant signals were analyzed by densitometry (Bio-Rad, Richmond, CA). RESULTS: Table 1: Change in rat body mass in during 21 days of dietary conditioning. RAT BODY MASS (g) DAY 1 3 5 7 9 11 13 15 17 19 21 GROUP OA-1 240 226 245 261 274 287 306 322 330 343 351 OA-2 264 253 273 289 299 307 322 332 342 350 359 OA-3 261 243 264 279 289 302 312 325 334 342 352 OA-4 278 269 284 298 308 319 331 342 354 365 375 OA-5 267 256 281 295 309 317 329 336 345 357 370 OA-6 253 241 258 272 284 294 300 316 320 330 332 OA+1 261 249 262 272 277 283 289 296 294 304 317 OA+2 283 271 276 285 289 290 291 301 310 305 311 OA+3 258 251 259 267 267 269 272 280 279 285 295 OA+4 252 241 257 254 261 261 265 267 271 274 278 OA+5 237 230 240 247 252 251 264 264 270 279 289 OA+6 252 243 255 262 265 274 277 282 284 284 287 Table 2 : Food consumption during the 21 days period MASS OF DIET CONSUMED (g) DAY 1 2 CAGE 1 (-OA 1,2,3) CAGE 2 (-OA 4,5,6) CAGE 3(+OA 1,2,3) CAGE 4(+OA 4,5,6) 48 48 48 48 48 48 48 48 3 4 100 100 100 100 100 80 100 87 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 83 90 63 63 86 87 74 73 82 90 85 65 68 70 65 38 77 79 82 61 77 67 85 65 81 70 88 62 82 76 92 58 89 78 91 78 85 73 87 81 91 83 98 100 84 77 99 82 97 83 99 93 100 86 99 81 91 90 82 97 91 83 99 71 92 82 98 99 Total (Kg) 1752 1660 1762 1546 7 Figure 1: Cyp4A immunoreactive protein in rat liver microsomes by Western analysis. 103 76 49 33 +1 Marker MW (kD) +2 +3 +4 +5 OA+ Group +6 -1 -2 -3 -4 -5 -6 OA- Group Figure 2: Cyp4A level between OA+ group and OA-group. Chart showing the Cyp450 Protein level in OA- and OA+ group Densitometer unit 2 1.5 1 0.5 OA+ OA- 0 8 Effects of intake of Orotic Acid containing diets on rat body weight Table 1 shows that the group of rats that were fed with diets containing Orotic acid (OA+) had lower body weights when compared with control (OA-). Student's t-test indicated that the mean values of the two population body mass were significantly different from each other (P< 0.05). The rats also had a lower rate of weight gain when compared to the control, but the total food consumed per cage over the 21 days period was not different as shown by Table 2. The OA- group consumed 1706+65 g and the OA+ group consumed 1655+152g of rat diet. On Day 21, the OA- group had a mean body mass of 357+15 g and the OA+ group had a mean mass of 296+14g. The chart in Figure 2 is a graphical representation of the results shown in Figure 1. The data represents the total amount of Cyp4A in liver microsomes from OA+ group and OA- rats in densitometry units. The OA+ group had a value of 1.560+0.232 unit and the OA- group had a value of 1.0173+0.2463 unit. The student's t-test showed a significant difference between the values of the two groups (P<0.05). This showed that the Cyp4A level was approximately 53+35 % in the OA+ group when compared to the control. DISCUSSION The results of this experiment clearly indicated the up regulation of CyP450 4A proteins in the rat group that was fed with diet containing Orotic acid. Thus, extensive fatty infiltration of the liver was evident. The state of steatosis was observed in the OA+ group liver sample under microscope but no micrographs were taken. However similar 9 findings were made during previous studies (Su, Sefton et al. 1999). The Cyp4A level in the test group was approximately 53+35 % higher than the control group. Previous investigation showed no significant difference between the body weights of the rat group that was fed with diet containing 1% OA and the control group (Su, Sefton et al. 1999). The current investigation however showed otherwise. The body weight of the control group was approximately 20 % greater than the test group although the animals consumed the same amount of diet. Throughout the study, it was observed that the rats which were fed with Orotic acid containing diet appeared to be less physically active compared to the control group. Previous experiments showed that the Orotic acid containing diet resulted in an increase in cholesterol, triglyceride and phospholipids levels in rat liver after feeding of the experimental diets for 21 days (Su, Sefton et al. 1999). It was proposed that lipid deposition could influence CYP function (Murray, Cantrill et al. 1992). This was clearly observed in the previous experiment where Orotic acid containing diet resulted in a decrease in the testosterone hydroxylation activity of CYP2C11, CYP3A and CYP2A1. The expression level of the isoenzymes was also significantly reduced (Su, Sefton et al. 1999). In this experiment the expression level of the CYP4A was dramatically increased in steatotic rats. The CYP4A expression level could be influenced by fatty acids acting as ligands of the PPAR receptors which are known to influence CYP4A regulation. PPARs are capable of specifically interacting with ligands including certain natural compounds such as fatty acids. However the receptors have several peculiarities. Firstly, in contrast to a number of related receptors such as the thyroid hormone receptor, PPARs accommodate several types of ligands and most of the known 10 activators are bona fide ligands (Willson and Wahli 1997). Secondly most of the molecules that specifically bind to PPAR do so with a rather low affinity as compared with the affinity of classical hormones for their cognate receptors. Thirdly, it is found that there is some overlap in ligand recognition by different PPAR isotypes (PPAR α, β and γ). The role of fatty acids has changed from a passive energy-providing molecule to metabolic regulators. The discovery that fatty acids could act as PPAR ligands indicated that at least part of the PPAR-dependent transcriptional activity of fatty acids results from a direct interaction of the nuclear receptor with these molecules. These fatty acids bind to all three PPARs α, β and γ with PPAR α exhibiting the highest affinity, at concentrations that are in agreement with their circulating blood levels. Compared to the unsaturated fatty acids, saturated fatty acids are poor PPAR ligands in general (Forman, Chen et al. 1997; Kliewer, Sundseth et al. 1997; Krey, Braissant et al. 1997), whereas phytanic acid, a dietary branched-chain, isoprenoid-derived fatty acids, binds to PPAR α efficiently (Ellinghaus, Wolfrum et al. 1999). Other PPAR ligands include dicarboxylic fatty acids, eicosanoids and a range of synthetic ligands such as hypolipidermic agents, Prostaglandin 12 analogues and Leukotriene B4 analogues. Alternative Pathways for PPAR activation PPAR α and γ are phosphoproteins. Similar to several other nuclear hormone receptors, PPARs can also be regulated by phophorylation in addition to liganddependent activation. The phophorylation of PPAR α and γ can be influenced by insulin (Shalev, Siegrist-Kaiser et al. 1996). The effect of insulin occurs through the 11 phosphorylation of two microtubule-associated protein (MAP) kinase sites located in the A/B domain of hPPAR α (Juge-Aubry, Hammar et al. 1999). Zhang et al (Zhang, Berger et al. 1996) showed a synergistic effect between insulin treatment and PPAR γ ligand dependent activation on the expression of the target gene aP2. However contrasting results were obtained when exploring the effects of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in modulating PPAR activity (Hu, Kim et al. 1996; Camp and Tafuri 1997). These growth factors were found to decrease the transcriptional activity of PPAR γ while increasing PPAR phosphorylation through MAP kinase signalling. It remains unclear how the nature of triggering signals such as insulin and growth factors can have quite opposite effects on PPAR α signalling. It is possible that their activity involves either specific pleiotropic actions or use of different kinase pathways such as the use of Ras/MAP kinase pathway by insulin to mediate the upregulation of phosphophenol pyruvate carboxykinase gene expression and the inhibition of PPAR α activity by growth hormone through the Janus kinase 2-signal transducer and activator of transcription 5b (JAK2-STAT5b) pathway. The PPAR:RXR heterodimer can alternatively be activated through ligand binding to RXR. Either partner of the PPAR:RXR heterodimer can regulate the transcriptional activity of the DNA-bound complex by interacting with their respective cognate ligand activity of the DNA-bound complex by interacting with their cognate ligand individually or simultaneously. This was observed when RXR ligands such as 9cis-RA and LG 1069 activated the PPRE-driven reporter gene in PPAR:RXR-dependent manner (Kliewer, Umesono et al. 1992; Gearing, Gottlicher et al. 1993; Keller, Dreyer et al. 1993). 12 PPAR-mediated transactivation properties PPAR-mediated transactivation results from the combination of PPAR:RXR binding to PPRE ligand activation of the complex formed by the two species. It is possible that along with the complex is a repressor molecule(s) that renders the PPAR:RXR heterodimer inactive (Horwitz, Jackson et al. 1996; Glass, Rose et al. 1997). Ligand binding (or other activation processes e.g. Phosphorylation) to the PPAR or RXR molecule may change the conformational structure, thus resulting to the dissociation of the repressor molecule(s) and activating the heterodimer complex. This would form a new protein-protein interaction surface that would allow specific contacts with co-activator(s). This subsequently transduces regulatory action to the basal transcriptional machinery. The activated PPAR:RXR complex binds to the PPRE resulting to a change in chromatin structure causing the release of histone molecules. The PPRE-bound PPAR:RXR complex triggers the activation of a co-activator-acetyltransferase complex. This enzyme acetylates the histone tails of the promoter chromatin at the transcription initiation region thereby producing a transcriptionally competent structure. This permits additional transcription factors and the basal transcriptional machinery, including the RNA Pol II initiation complex to assemble with the promoter thus initiating transcription of the particular mRNA. PPAR α tends to be expressed in late development stages in mouse and rat. High levels of the PPAR α mRNA are in brown fat, liver, kidney and the mucosa of stomach and duodenum of adult rat. Other organs that express significant amounts of PPAR α mRNA include the retina, adrenal gland, skeletal muscle and pancreatic islets (Braissant, Foufelle et al. 1996; Lemberger, Braissant et al. 1996). In summary, the expression of PPAR α correlates with high mitochondrial and peroxisomal β-oxidation 13 activities in tissues that primarily use fatty acids as an energy source such as cells of kidney proximal tubules and cardiomyocytes. It has been shown by several investigators that PPAR α expression in rat liver is subject to differential regulation by insulin and glucocorticoids and therefore situations that can induce the levels of plasma glucocorticoids such as stress or fasting can have an effect in the expression of PPAR α (Lemberger, Staels et al. 1994; Steineger, Sorensen et al. 1994). Growth Hormone in contrast has been shown to decrease the PPAR α mRNA levels by 50 % in primary culture of rat hepatocytes (Yamada, Sugiyama et al. 1995). Down-regulation of PPAR α gene expression was also observed in chronic alcoholic liver disease in rat (Wan, Morimoto et al. 1995). The CYP4A enzymes belong to the cytochrome mono-oxygenase that plays a central role in the oxidation of a wide variety of endogenous as well as exogenous compounds. The CYP4A enzymes have been shown to catalyse the ω-hydroxylation of fatty acids and eicosanoids. The rat CYP4A gene family consists of four members that are expressed in the kidney, liver, intestine, heart, colon, lungs, brain and prostate (Kusunose, Ogita et al. 1981; Kimura, Hardwick et al. 1989; Stromstedt, Hayashi et al. 1990; Hardwick 1991). Three isoenzymes of CYP4A have been identified in rat livers that include CYP4A1, CYP4A2 and CYP4A3. CYP4A1 has been found to contain PPRE in their promoter sequence and was shown to respond to PPAR α activators in vivo and cell culture (Muerhoff, Griffin et al. 1992; Krey, Keller et al. 1993; Aldridge, Tugwood et al. 1995). The CYP4A are distinct from other CYP enzymes with less than 30 % homology (Aoyama, Hardwick et al. 1990). All of the three members of the rat CYP4 enzymes have been shown to be induced in the liver by hypolipidemic drugs that includes CYP4A1, CYP4A2 and CYP4A3 (Hardwick, Song et al. 1987; Kimura, 14 Hardwick et al. 1989). Their cDNA sequences show great homology of more than 95 % (Kimura, Hanioka et al. 1989; Kimura, Hardwick et al. 1989). The Cyp4A1 is one of the most active isoenzymes to hydroxylate palmitate, laurate and arachiodonate and this occurs mainly at the ω position (Gibson 1989; Aoyama, Hardwick et al. 1990). Peroxisome proliferators have been shown to induce the expression of several P450 4A enzymes in rat (Kimura, Hardwick et al. 1989). P450 4A enzymes represent a distinct evolutionary branch of the cytochrome P450 superfamily and are distinguished from other mammalian cytochrome P450 enzymes by their ability to catalyse the ωhydroxylation of long and medium chain fatty acids and prostaglandins. The other mammalian P450 iso-enzymes also oxidise fatty acids but their degree of specificity for the terminal, primary C-H bonds of the fatty acid is low when compared to the CYP450 4A sub-type. The significance of ω-hydroxylation as a pathway for the metabolism of saturated fatty acids is still poorly understood. It is however likely that it contributes to the formation of dicarboxylic acids through the subsequent oxidation of the product ωalcohols by cytosolic enzymes. Dicarboxylic fatty acids are substrates for peroxisomal or mitochondrial β-oxidation and it appears that the peroxisomal pathways to be more efficient for the β-oxidation of dicarboxylic acids than the mitochondrial pathway (Okita 1994; Tontonoz, Hu et al. 1994). This difference suggests that microsomal ωhydroxylation would shunt fatty acids to peroxisomes for degradation, which in turn would affect energy conservation from fatty acid oxidation, as the initial step of the peroxisomal pathway is not coupled to ATP production, as it is in the mitochondrial pathways (Tontonoz, Hu et al. 1994). In certain situations, β-oxidation may be incomplete leading to the production of dicarboxylic fatty acids of intermediate length, C6-C10. The levels of C6-C10 dicarboxylic acids are generally elevated in tissues, 15 plasma and urine during fasting and diabetic states where the concentrations of circulating fatty acids are increased due to lypolysis in adipocytes (Mortensen 1992). It was shown that under such conditions, the hepatic concentrations of P450 4A enzymes are also elevated (Imaoka, Shimojo et al. 1988; Barnett, Gibson et al. 1990; Ferguson, Donahue et al. 1993). However, when peroxisome proliferator such as clofibrate was administered (which induces P450 4A) no increase of such dicarboxylic acids were observed (Mortensen 1992). This may reflect a more complete shortening of the dicarboxylic acid by peroxisomal β-oxidation, which is also induced by clofibrate (Okita 1994). Other factors that could contribute to the increase of hepatic free fatty acids level could also be due to competition for metabolic pathways and binding proteins by peroxisome proliferators. This has led to the speculation that fatty acids mediate the response of PPARα to peroxisome proliferators (Lock, Mitchell et al. 1989; Issemann, Prince et al. 1992) and fatty acids have been shown to activate PPARα (Gottlicher, Widmark et al. 1992; Issemann, Prince et al. 1992; Keller, Dreyer et al. 1993). Furthermore, selective inactivation of P450 4A enzymes has been shown to decrease the induction of the peroxisomal fatty acyl-CoA oxidase of rat hepatocytes, suggesting that the production of dicarboxylic acids by P450 4A enzymes contributes to the response to peroxisome proliferators (Kaikaus, Chan et al. 1993). Studies conducted by Kaikaus et al (Kaikaus, Chan et al. 1993) demonstrated that induction of peroxisomal β-oxidation and L-FABP by peroxisome proliferators is mediated via the cytochrome P450 4A ω-hydroxylation pathways. It is suggested that fatty acids metabolised by the enzyme forms into dicarboxylic acid products that may activate the soluble PPAR receptor of the L-FAB and peroxisomal enzymes thus 16 induces them pretranslationally. Oxidised fatty acids may activate the receptors by acting as ligands. The other possibility may include activation of PPAR by protein kinase C-mediated phosphorylation. By comparing PPAR α-deficient and wild type mice, it was shown that starvation and insulin dependent diabetes result in a strong induction of the hepatic CYP4A genes and other lipid-metabolising enzymes such as Acetyl-Coenzyme A through activation of PPAR α provides a negative feedback on the intracellular levels of an endogenous ligand (Devchand, Keller et al. 1996). Secondly it can also be concluded that a pathophysiological state can induce cellular changes that could lead to the activation of PPAR α (Kroetz, Yook et al. 1998). Finally, it can also be assumed that PPAR α may have an important role in the detoxification of some xenobiotics. A more detailed analysis conducted by this laboratory that evaluated the impact of lipid-deficient OA-diet on CYP4A expression and PPARα activation in rodent liver found that the CYP4A protein and laurate ω-hydroxylation activity were elevated in rat hepatocytes after 21 days (Su et al. 2005). CYP4A1 and CYP4A2 mRNAs were found to have been induced more than 2 fold of the control with no apparent effect in corresponding mRNAs level of CYP4A3 and peroxisomal acyl-CoA oxidase. Coadministration of clofibric acid and the OA-diet was found to have prevented hepatic lipid accumulation and caused significant upregulation of CYP4A1-3 and acyl-CoA oxidase mRNAs. Hepatic PPARα protein and retinoid X-receptor-α (RXRα) level was also found to have decreased by the S/OA-diet. The administration of the OA containing diet to control and PPARα-null mice led to hepatic lipid deposition and the microsomal CYP4A protein upregulation in the wild-type but not PPARα-null mice. The results of this study concluded PPARα involvement in the induction of CYP4A in 17 rodent liver by the lipid-devoid OA-diet and suggested that the decreased availability of hepatic PPARα and RXRα after intake of the diet may add to the selective upregulation of hepatic CYP4A1 and CYP4A2 in this model of steotosis. Conclusion The findings from the present study that CYP4A protein expression is up regulated in hepatic steatosis indicate that pathophysiological accumulation of fatty acids may be responsible. Activation of PPARα-mediated gene transcription in fatty liver may contribute to the disordered homeostasis of lipid oxidation that occurs in genetic disease and altered nutrition. 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