The pharmacokinetics of mycophenolic acid in rats with orotic acid induced nonalcoholic fatty liver disease.

Post-transplantation nonalcoholic fatty liver disease (NAFLD) is common in liver transplant recipients. Changes in the expression levels and activities of drug-metabolizing enzymes and drug transporters have been reported in patients with NAFLD and relevant rodent models. Here, we evaluated whether the pharmacokinetics of mycophenolic acid (MPA), an immunosuppressant, would be altered in rats with NAFLD. NAFLD was induced by feeding a diet containing 1% (w/w) orotic acid for 20 days. The extent of hepatic glucuronidation of MPA to a major metabolite, mycophenolic acid-7-O-glucuronide (MPAG), did not differ between rats with NAFLD and controls. The expression levels of hepatic multidrug resistance-associated protein 2, responsible for biliary excretion of MPAG, were comparable in rats with NAFLD and controls; the biliary excretion of MPAG was also similar in the two groups. Compared with control rats, rats with NAFLD did not exhibit significant changes in the areas under the plasma concentration - time curves of MPA or MPAG after intravenous (5 mg/kg) or oral (10 mg/kg) administration of MPA. However, delayed oral absorption of MPA was observed in rats with NAFLD compared with controls; the MPA and MPAG peak plasma concentrations fell significantly and the times to achieve them were prolonged following oral administration of MPA.

Regulation effects of rosemary (Rosmarinus officinalis Linn.) on hepatic lipid metabolism in OA induced NAFLD rats.

Rosmarinus officinalis Linn. is a kind of medicinal and edible homologous plant, which is popular in the Mediterranean region with a significant effect on mind tranquilization, anti-oxidation, and metabolic improvement. However, the hypolipidemic effects and mechanism of rosemary ethanol extract (RO) and their metabolites are less known. In this study, the hypolipidemic effects of RO and its active compounds were clarified. The results showed that RO, rosmarinic acid (RA) and carnosic acid (CA) significantly reduced the contents of liver triglyceride (TG), total cholesterol (TC), free fatty acids (FFA) and improved cell hypertrophy, vacuolation, and cell necrosis in the liver of orotic acid induced non-alcoholic fatty liver disease (NAFLD) model rats. The mechanism and related pathways of RO and its main metabolites against lipid disorder were related to the up-regulation of the phosphorylation of adenosine 5'-monophosphate(AMP)-activated protein kinase (AMPK) and the inhibition of the sterol regulatory element binding protein-1c (SREBP-1c) cracking into the nucleus, following the down-regulation of fatty acid synthesis. In conclusion, our study demonstrates that RA and CA are active substances of RO, and provides scientific evidence to support functional food product development for improving NAFLD.

Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats.

We investigated whether fatty liver preceded insulin resistance or vice versa using a long-term orotic acid (OA)-induced nonalcoholic fatty liver disease (NAFLD) model without the confounding effects of obesity and hyperlipidemia and explored the role of the liver in insulin resistance. Male Wistar rats were fed with or without OA supplementation for 30, 60, and 90 days. The NAFLD group showed increased liver lipid at 30, 60, and 90 days; glucose intolerance was noted at 60 and 90 days. Furthermore, partial liver proteins and gene expressions related to upstream signaling of insulin were decreased. However, the liver glycogen content was elevated, and gluconeogenesis genes expressions were obviously decreased at 90 days. The occurrence of fatty liver preceded insulin resistance in OA-induced NAFLD without the interference of obesity and hyperlipidemia, and hepatic insulin resistance may not play a conclusive role in insulin resistance in this model.

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola.

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5'-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgE(in)) regained the susceptibility to 5-FOA. Finally, FlgE(in) was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE(out)) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE(out) was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE(out) has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.

Orotic acid induces hypertension associated with impaired endothelial nitric oxide synthesis.

Orotic acid (OA) is an intermediate of pyrimidine nucleotide biosynthesis. Hereditary deficiencies in some enzymes associated with pyrimidine synthesis or the urea cycle induce OA accumulation, resulting in orotic aciduria. A link between patients with orotic aciduria and hypertension has been reported; however, the molecular mechanisms remain elusive. In this study, to elucidate the role of OA in vascular insulin resistance, we investigated whether OA induced endothelial dysfunction and hypertension. OA inhibited insulin- or metformin-stimulated nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation in human umbilical vein endothelial cells. A decreased insulin response by OA was mediated by impairment of the insulin-stimulated phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling pathway in cells overexpressing the p110-PI3K catalytic subunit. Impaired effects of metformin on eNOS phosphorylation and NO production were reversed in cells transfected with constitutively active AMP-activated protein kinase. Moreover, experimental induction of orotic aciduria in rats caused insulin resistance, measured as a 125% increase in the homeostasis model assessment, and hypertension, measured as a 25% increase in systolic blood pressure. OA increased the plasma concentration of endothelin-1 by 201% and significantly inhibited insulin- or metformin-induced vasodilation. A compromised insulin or metformin response on the Akt/eNOS and AMP-activated protein kinase/eNOS pathway was observed in aortic rings of OA-fed rats. Taken together, we showed that OA induces endothelial dysfunction by contributing to vascular and systemic insulin resistance that affects insulin- or metformin-induced NO production, leading to the development of hypertension.

Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system.

Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10ΔpyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.

Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR.

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous ß-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.

Proliferating effect of orotic acid through mTORC1 activation mediated by negative regulation of AMPK in SK-Hep1 hepatocellular carcinoma cells.

Orotic acid (OA) is a tumor promoter of experimental liver carcinogenesis initiated by DNA reactive carcinogens, the molecular mechanisms of which have not been fully elucidated. OA increases cell proliferation and decreases apoptosis in serum-starved SK-Hep1 hepatocellular carcinoma cells, which may ascribe to the inhibition of AMP-activated protein kinase (AMPK) phosphorylation and thus activation of mammalian target of rapamycin complex 1 (mTORC1). The effects of OA on mTORC1 activation, cell proliferation, and cell-cycle progression to S and G2/M phases were completely reversed by rapamycin. Activation of AMPK by a constitutively active mutant or aminoimidazole carboxamide ribonucleotide (AICAR) rescued the effects of OA. In conclusion, OA increases the proliferation and decreases the starvation-induced apoptosis of SK-Hep1 cells via mTORC1 activation mediated by negative regulation of AMPK.

Study on possible mechanism of orotic acid-induced fatty liver in rats.

OBJECTIVE: The aim of this study was to investigate the possible mechanism of orotic acid-induced fatty liver in rats. METHODS: Rats were randomly divided into two groups and fed an AIN-93 diet with 1% orotic acid or without orotic acid for 10 d. Hepatic lipid concentrations, such as triacylglycerol, total cholesterol, and phospholipids, were examined. To clarify the mechanism of orotic acid-induced fatty liver, hepatic enzyme activities and mRNA levels of key enzymes related in lipid metabolism and hepatic gene expression of transcription factors were determined. RESULTS: Orotic acid administration significantly increased hepatic triacylglycerol concentration. The activity and mRNA level of fatty acid synthase were obviously upregulated by orotic acid treatment, whereas the activities and mRNA concentrations of carnitine palmitoyl transferase and microsomal triacylglycerol transfer protein were significantly depressed. Furthermore, orotic acid stimulated the mRNA expression of sterol regulatory element binding protein-1c but did not alter the mRNA concentration of peroxisome proliferator-activated receptor-α in the liver. CONCLUSION: The stimulation of triacylglycerol synthesis induced by orotic acid is mainly caused by enhancement of sterol regulatory element binding protein-1c and its target gene involved in fatty acid biosynthesis. In contrast, the inhibition of fatty acid ß-oxidation and very-low-density lipoprotein secretion were related to the observed lipid accumulation.

Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant.

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Δpta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

Use of the pyrG gene as a food-grade selection marker in Monascus.

Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.

Comparative in vitro toxicity of seven zinc-salts towards neuronal PC12 cells.

Currently much attention has been given to the neurotoxicity of zinc, yet little is known about the influence of the counterions present. Therefore, we investigated the influence of different Zn(2+)-salts (concentrations range 0.05-0.3 mM) on cell viability, ATP and glutathione concentration and caspase activation in differentiated PC12 cells as a model for neuronal cells. Generally, at concentrations of 0.05 mM most Zn(2+)-salts were not cytotoxic except for zinc-citrate. At concentrations between 0.1 and 0.3 mM Zn(2+) a significant decrease in GSH and ATP levels preceded cell death induced by all salts, except of zinc-histidinate. Zinc-citrate and zinc-sulphate turned out to be the most toxic salts particularly at low concentrations. Analyses of caspase 3/7 activity showed that dependent on the concentration and the type of the salt used cell death may show more or less signs of both, necrosis and apoptosis. Interestingly, the uptake of Zn(2+) from zinc-sulphate and zinc-citrate was significantly higher than that of other salts, implicating a correlation between uptake and toxicity. In conclusion, Zn(2+)-salts could be divided into three categories with high (zinc-citrate, zinc-sulphate), moderate (zinc-orotate, zinc-acetate, zinc-chloride(,) zinc-gluconate) and low cytotoxicity (zinc-histidinate).

Fenofibrate prevents orotic acid--induced hepatic steatosis in rats.

The experiments performed in this report were designed to investigate the mechanisms involved in the metabolic alterations associated with orotic acid-induced hepatic steatosis and the effect of fenofibrate, a stimulant of peroxisome proliferators-activated receptor alpha (PPARalpha), on these alterations. Male Wistar rats were divided into three experimental groups: 1) fed a balanced diet (C); 2) fed a balanced diet supplemented with 1% orotic acid (OA); 3) fed OA diet containing 100 mg.kg(-1) bw.day(-1) fenofibrate (OA+F), for 9 days. Administration of OA to rats induced significant increase in the hepatic total lipids content, marked microvesicular steatosis and decrease in plasma lipids concentrations compared to control group. Fenofibrate treatment prevented fatty liver induction, caused an additional reduction on plasma lipids concentrations and caused a 40% decrease in the lipogenic rate in adipose tissue. The results also showed a 40% increase in lipoprotein lipase (LPL) activity in adipose tissue from OA treated group and fenofibrate administration induced a 50% decrease in LPL activity. The liver mRNA expression of PPARalpha and ACO (acyl CoA oxidase) were 85% and 68% decreased in OA group when compared to control, respectively. Fenofibrate treatment increased the PPARalpha and ACO expressions whereas the CPT-1 (carnitine palmitoyl transferase-1) expression was not altered. Our results have shown that fenofibrate treatment decreases the hepatic lipid content induced by OA which is mediated by an important increase in fatty acid oxidation consequent to an increase in hepatic mRNA expression of PPARalpha and ACO.

Integration of genomic and metabonomic data in systems biology--are we 'there' yet?

The measurement of genes, proteins and metabolites has gained increasing acceptance as a means by which to study the response of an organism to stimuli, whether they are environmental, genetic, pharmacological, toxicological, etc. Typically referred to as genomics, proteomics, and metabonomics or metabolomics, respectively, these methods as independent entities have undoubtedly provided new biological insight that was not attainable a decade ago. Not surprisingly, scientists continue to push the boundaries to extract knowledge from data, and it is currently recognized that the full realization of these technologies is limited by a lack of tools to enable data integration. Integration of these 'omic datasets, or integromics, is desirable as it links the individual biological elements together to provide a more complete understanding of dynamic biological processes. Accordingly, in addition to developing new data analysis methods to extract further details from each of the high-content datasets individually, effort is also being expended to create or improve statistical methods, databases, annotations and pathway mapping to maximize our learning. There are several recent examples, in both mammalian and non-mammalian systems, in which genes, proteins and/or metabolites have been integrated using either biology- or data-driven strategies. Herein, key findings are reviewed, gaps in our current tools and technologies are identified and illustrated, and perspective is provided on the potential of integromics in biological research.

Dietary phosphatidylcholine alleviates fatty liver induced by orotic acid.

OBJECTIVE: We compared the effect of dietary phosphatidylcholine (PC) with that of triacylglycerol (TG), both with the same fatty acid profiles, on fatty infiltration in orotic acid (OA)-induced fatty liver of Sprague-Dawley rats. METHODS: Rats were fed an OA-supplemented diets containing TG (TG+OA group) or PC (20% of dietary lipid, PC+OA group) for 10 d. Rats fed the TG diet without OA supplementation served as the basal group. RESULTS: Administering OA significantly increased the weights and TG accumulation in livers of the TG+OA group compared with the basal group. These changes were attributed to significant increases in the activities of fatty acid synthase, malic enzyme, and glucose-6-phosphate dehydrogenase, which are fatty acid synthetic enzymes, and phosphatidate phosphohydrolase, a rate-limiting enzyme of TG synthesis. However, the PC+OA group did not show TG accumulation and OA-induced increases of these enzyme activities. Further, a significant increase in the activity of carnitine palmitoyl transferase, a rate-limiting enzyme of fatty acid beta-oxidation, was found in the PC+OA group. CONCLUSIONS: Dietary PC appears to alleviate the OA-induced hepatic steatosis and hepatomegaly, mainly through the attenuation of hepatic TG synthesis and enhancement of fatty acid beta-oxidation in Sprague-Dawley rats.

Alleviation of fatty liver by alpha-linolenic acid.

We compared the efficacy of alpha-linolenic acid (alpha-LNA, n-3) and linoleic acid (LA, n-6) on orotic acid (OA)-induced fatty liver in Sprague-Dawley rats. Rats were fed semi-synthetic diets containing either LA or alpha-LNA with or without 1% OA for 2 wk. OA supplementation lowered serum lipids in LA+OA groups. In addition to the decline of serum lipids in alpha-LNA groups compared to LA groups, a further decrease was found in alpha-LNA+OA groups compared to LA+OA groups. OA-containing diets significantly increased the liver weights and triacylglycerol (TG) accumulations compared with the OA-free diets. These results were attributed to the significant increases in the activities of phosphatidate phosphohydrolase (PAP), a rate-limiting enzyme of TG synthesis, and glucose-6-phosphate dehydrogenase, a fatty acid synthesis-related enzyme. However, the increase of PAP activity was significantly less in the alpha-LNA+OA group as compared with the LA+OA group. These results suggest that dietary alpha-LNA alleviates OA-induced hepatic TG accumulation through the attenuation of hepatic TG synthesis in rats.

Effects of oral administration of orotic acid on hepatic morphologic characteristics and serum biochemical variables in cats.

OBJECTIVE: To evaluate orotic acid (OA) as a possible etiologic factor in cats with idiopathic hepatic lipidosis (HL). ANIMALS: 20 clinically normal adult female cats. PROCEDURE: Cats were fed a control diet or a diet containing less protein. On day 1 of the control period, blood, urine, and liver biopsy specimens were obtained. Each cat was given an oral dose of water daily. On days 8, 15, and 22, blood and urine specimens were collected as on day 1. On day 29, liver, blood, and urine samples were obtained as on day 1. After a resting period of 30 to 60 days, cats were treated with orotic acid. Serum biochemical analyses, urinary OA-to-creatinine ratios, and liver biopsy specimens were evaluated. Cats were given OA orally (suspension or capsules) for 29 days. Sample collection and data obtained were identical to those described for the control period. RESULTS: Urinary OA-to-creatinine ratios were significantly higher in all treated cats, but ratios were significantly higher in those receiving OA in capsules than in those receiving OA in suspension. Diet or treatment did not alter hepatic biochemical or histologic variables significantly. However, 7 cats given the highest dose of OA in capsules developed azotemia, urolithiasis, and renal changes. CONCLUSIONS: Most concentrations of OA used in this study did not induce HL in cats during a 29-day period, but the highest dosage used did result in renal disease. CLINICAL RELEVANCE: Orotic acid does not appear to be involved in the genesis of HL in cats.

Prevention of orotic-acid-induced fatty liver in male rats by dehydroepiandrosterone and/or phenobarbital.

Dehydroepiandrosterone (DHEA) is a steroid hormone which induces the peroxisome proliferation in rodents. The fatty liver induced by orotic acid and a high sucrose diet in male rats was prevented by the administration of DHEA and/or phenobarbital (PB). A significant increase in the liver weight was induced in the DHEA group (relative weight) and the DHEA + PB group (absolute and relative weight). The liver weight increased more conspicuously in the DHEA + PB group than in the DHEA group. The increase in the liver weight was caused by an increase in the cell size and peroxisome number. In addition, the administration of DHEA alone and the combination of DHEA and PB prevented the lipid droplet accumulation in hepatocytes. The administration of PB alone also prevented the accumulation of lipid droplets without any increase in the liver weight.

Association between hepatic triacylglycerol accumulation induced by administering orotic acid and enhanced phosphatidate phosphohydrolase activity in rats.

Orotic acid is known to cause fatty liver, but it is unclear whether this is caused partly by stimulation of the enzymes for triacylglycerol (TG) synthesis. To understand the change of hepatic TG metabolism in fatty liver induced by orotic acid, we determined the liver tissue TG level and phosphatidate phosphohydrolase (PAP) activity over time in rats fed on a diet containing orotic acid (OA). A dietary lipid content of 10% was achieved by using n-6 fatty acid-rich corn oil in experiment 1, and n-6 fatty acid-rich safflower oil (SO) and n-3 fatty acid-rich fish oil (FO) with the same polyunsaturated fatty acid/monounsaturated fatty acid/saturated fatty acid (P/M/S) ratio in experiment 2. In experiment 1, an increase in the hepatic TG level due to OA intake was observed from day 5 onwards, the level rising approximately 6-fold by day 10. The activity of hepatic microsomal PAP, the rate-limiting enzyme in TG synthesis, increased markedly from day 5 onwards, concurrent with the liver diacylglycerol concentration. A strong correlation (r = 0.974) was observed between the hepatic TG level and microsome-bound PAP activity. In experiment 2, we investigated the effects of dietary fatty acid on OA-induced fatty liver. Compared with the n-6 fatty acid-rich vegetable oil diet, the relative increase in hepatic TG was smaller with the n-3 fatty acid-rich FO diet, and hepatic PAP activity fell markedly to the level for an OA-free diet. In addition, the hepatic TG accumulation and serum TG concentration were lower in the FO group than in the SO group. Nevertheless, because the hepatic TG level was low, it seems that the inhibition of liver PAP activity by FO possibly had a strong influence on the accumulation of TG in the liver. In conclusion, enhanced TG synthesis mediated by changes in liver PAP activity was involved in the hepatic TG accumulation induced by OA administration, this change being markedly suppressed by dietary n-3 fatty acids.