Cells of non-adipose tissues have a limited capacity fortorage of lipids. For a long time, fatty liver or hepaticteatosis was considered to be a benign condition. However,ecent data have indicated a wide spectrum of clinical andathologic manifestations that subjects with non-alcoholicepatic steatosis develop, which together are termed non-lcoholic fatty liver disease. The manifestations of non-lcoholic fatty liver disease are similar to those seen inatients with alcoholic liver disease and range from mildepatic steatosis, steatohepatitis, fibrosis, to cirrhosis [1–3]nd, rarely, to hepatocellular carcinoma [4].
This work was supported by a research grant from the Kieikai Researchoundation.
Evidence from human studies and animal models haveuggested that lipid accumulation in the liver plays an impor-ant role in the pathogenesis of heart failure, obesity, andiabetes [5]. Therefore, the discovery of nutrients that amelio-ate fatty liver is of interest. We previously reported that oroticcid (OA)–induced fatty liver is partly alleviated by treatmentith �-3 polyunsaturated fatty acid–containing fat [6,7]. Thus,ur data and those of others have suggested that some nutrientsould alleviate fatty liver induced by OA [8,9].
Phosphatidylcholine (PC) is a major component of di-tary phospholipids and is absorbed well in humans andnimals when administered orally [10]. Recent reports haveuggested that PC may play a protective role in liver injury.anty and Zeisel [11] and Albright and Ziesel [12] stressed
he importance of choline in dietary PC based on the facthat choline deficiency enhances the level of cytokines, suchs transforming growth factor-�1, and suppresses the ex-
ression of antioxidation enzymes, such as superoxide dis-
utase and catalase, leading to apoptosis. Lieber et al. [13]nd Folch et al. [14] reported that polyunsaturated PC pre-ents fatty liver in alcoholic fatty infiltration in humans andnimals. However there has been no study of whether PCffects the onset of fatty liver induced by OA. In the presenttudy, we evaluated the effects of dietary PC versus triac-lglycerol (TG) on OA-induced fatty liver in rats.
aterials and methods
nimals and diets
All aspects of the experiment were conducted accordingo guidelines provided by the ethical committee of experi-ental animal care at Saga University (Saga, Japan).Male Sprague-Dawley rats ages 4 wk were purchased
rom Seiwa Experimental Animals (Fukuoka, Japan) andoused individually in an air-conditioned room (24°C) with12-h light/dark cycle. After a 1-wk adaptation period, ratsere assigned to one of three groups (five rats each).Basal diets were prepared according to recommendations
f the American Institute of Nutrition (AIN) and containedin weight%) 20 of casein, 10 of fat, 1 of vitamin mixtureAIN-93G), 3.5 of mineral mixture (AIN-93), 0.25 of cho-ine bitartrate, 0.3 of L-cystine, 0.002 of tert-butylhydroqui-one, 5 of cellulose, 10 of sucrose, 13.2 of �-cornstarch, and-cornstarch, to make 100. The OA diets were prepared byupplementation of 1.0% OA to the basal diet at the expensef �-cornstarch. Dietary fats were designed to have a con-tant ratio of polyunsaturated to monounsaturated to satu-ated fatty acids as presented in Table 1. Dietary fat in theG diets was composed of a mixture of several vegetableils (high oleic safflower oil, high linoleic safflower oil, andalm oil). Dietary fat in the PC diet was a mixture of PC andG, with a 2:8 ratio. Palm oil, safflower oils, and egg PCere provided from Kewpie Co. Ltd. (Tokyo, Japan). The
nimals received the diets for 10 d. At the end of the feedingeriod, rats were killed by decapitation after a 9-h starva-ion. Livers were excised immediately, and serum was sep-rated from blood.
nalysis of lipids
Liver lipids were extracted according to the method ofolch et al. [15] and concentrations of TG and phospholipidere measured by the methods of Fletcher [16] and Bartlett
17], respectively. Serum TG, total cholesterol, and phos-holipid were measured using enzyme assay kits fromako Pure Chemicals (Tokyo, Japan) according to theanufacturer’s instructions.
reparation of liver subcellular fractions
A piece of liver was homogenized in 6 vol of a 0.25-M
ucrose solution containing 1 mM of ethylene-diaminetetra-
cetic acid (EDTA) in 10 mM of Tris-HCl buffer (pH 7.4).fter precipitating the nuclei fraction, the supernatant was
entrifuged at 10 000g for 10 min at 4°C to obtain mito-hondria. The resulting supernatant was recentrifuged at25 000g for 60 min to precipitate microsomes, and theemaining supernatant was used as the cytosol fraction. Theicrosomal pellet was resuspended in a 0.25 M of sucrose
olution containing 1 mM of EDTA in 10 mM of Tris-HCluffer (pH 7.4). Protein concentration was determined byhe method of Lowry et al. [18], with bovine serum albuminsed as the standard.
ssays of hepatic enzyme activity
The enzyme activities of malic enzyme (EC 1.1.1.40)ere determined as previously described [19]. The reaction
olution contained 64 mM of triethanolamine hydrochloridepH 7.4), 1.2 mM of malic acid, 1.2 mM of oxidized nico-inamide adenine dinucleotide phosphate, and 4 mM of
nCl2. The reaction was initiated by the addition of 0.6 to.8 mg of a protein source (cytosol) in a final assay volumef 1 mL at 27°C and absorbance was monitored at 340 nmor 2 min.
The enzyme activities of glucose-6-phosphate dehydro-enase (EC1.1.1.49) were determined as previously de-cribed [20]. The reaction solution contained 0.16 M ofris-HCl buffer (pH 7.6), 30 mM of MgCl2, 3.3 mM oflucose-6-phosphate, 1.6 mM of oxidized nicotinamide ad-nine dinucleotide phosphate, and 1 U of 6-phosphoglu-onate dehydrogenase. The reaction was initiated by theddition of 0.3 to 0.4 mg of a protein source (cytosol) in anal assay volume of 1 mL at 30°C and the absorbance wasonitored at 340 nm for 2 min.The enzyme activities of fatty acid synthase (FAS; EC
.3.1.85) were determined as previously described [21]. Theeaction solution contained 0.1 M of phosphate buffer (pH.0), 0.2 M of EDTA, 50 �M of acetyl coenzyme A (CoA)nd 0.3 mM of reduced nicotinamide adenine dinucleotidehosphate. The entire solution was equilibrated at 30°C.he reaction was initiated by the addition of 0.8 to 1.0 mgf a protein source (cytosol) and absorbance was monitoredor 2 min at 340 nm. The malonyl-CoA–dependent rate wasonitored for 3 min.The enzyme activities of carnitine palmitoyl transferase
CPT; EC2.3.1.23) were determined as previously described21]. The reaction solution contained 58 mM of Tris-HCluffer (pH 8.0), 1.25 mM of EDTA, 1.25 mM of L-carnitine,.25 mM of 5,5=-dithiobis-2-nitrobenzoic acid, 37.5 �M ofalmitoyl-CoA, and 0.1% Triton-X. The entire solution wasquilibrated at 27°C. The reaction was initiated by theddition of 0.2 to 0.4 mg of a protein source (mitochondria)nd absorbance was monitored for 5 min at 412 nm. The-carnitine–independent rate was monitored for 5 min. Theifference between with and without L-carnitine gave the-carnitine–dependent rate for formation of CoA-SH.
The enzyme activities of phosphatidic acid phosphohy-
rolase (PAP; EC3.1.3.4) were determined as previouslyescribed [22]. The reaction solution contained 0.05 M ofris-HCl (pH 7.0), 1 mM of L-�-phosphatidate and 1 mM ofC liposomes suspended (prepared by sonicating for 10 min
n 0.9% NaCl with a Branson 250 sonifier; Branson,anbury, CT, USA), 1.25 mM of Na2-EDTA, and 50 to00 �g of a protein source (microsome) in a final assayolume of 0.2 mL. Each assay was performed in the pres-nce of 3.25 mM of MgCl2. The mixture was incubated for5 min at 37°C and the reaction was terminated by theddition of 0.8 mL of a solution containing 0.13% sodiumodecylsulfate, 1.25% ascorbic acid, 0.32% ammonium mo-ybdate-4H2O, and 0.75 N of H2SO4. The phosphomolyb-ate color was developed at 45°C for 20 min and absor-ance was measured at 820 nm. Non-enzymatic phosphateelease was determined by using inactivated enzymes boiledor 1 min without substrate. The enzyme activity was ex-ressed as nanomoles per minute per milligram of protein.
nalysis of mRNA expression
Total RNA was extracted from 300 mg of liver by usingTRIzol Reagent (Invitrogen, Tokyo, Japan). A TaqManniversal PCR Master Mix (Applied Biosystems, Tokyo,
Initial body weight (g) 297 � 6Final body weight (g) 344 � 3Food intake (g/day) 20.6 � 0Feed efficiency (g gain/g intake) 0.196 � 0Liver weight (g/100 g body weight) 4.88 � 0
OA, orotic acid; PC, phosphatidylcholine; TG, triacylglycerolDifferent letters show significant difference at P � 0.05.
* Rats were fed each diet for 10 d. Data are means � standard error of the me
Rn00569117_m1 for FAS, Rn00580702_m1 for CPT-1,s99999901_s1 for 18S RNA; Applied Biosystems) weresed for quantitative real-time reverse transcriptase poly-erase chain reaction analysis of CPT-1, FAS, and 18SNA expression in the liver. The amplification was per-
ormed with a real-time polymerase chain reaction systemABI Prism 7000 Sequence Detection System, Applied Bio-ystems). Results were expressed as a relative value afterormalization to 18S RNA expression.
tatistical analyses
All values are expressed as mean � standard error of theean. Data were analyzed with one-way analysis of vari-
nce, and all differences were inspected by Duncan’s newultiple-range test [23] using SPSS statistical software
SPSS Inc., Chicago, IL, USA). P � 0.05 was consideredtatistically significant.
esults
ffects of dietary PC on body weight gain and livereight in OA-fed rats
Body weight, food intake, feed efficiency, and livereights are presented in Table 2. Feed efficiency was sim-
lar across groups, whereas final body weight was slightlyower in both OA groups. Compared with the OA-freeroup (basal group), the TG � OA group showed a markedncrease in liver weight; the PC � OA group had a loweriver weight compared with the TG � OA group.
ffects of dietary PC on lipid concentration in the livernd serum in OA-fed rats
Rats fed the OA diet had a hepatic TG concentrationhree times higher than that in the basal group (Figure 1).owever, dietary PC attenuated OA-induced TG accumu-
ation by 45% compared with the TG � OA group. BothA groups showed a slight increase in hepatic phospholipid
oncentration. The administration of OA decreased serumG, phospholipid, and total cholesterol concentrations (Fig-
re 2). Compared with the TG � OA group, the PC � OAroup tended to have lower serum TG, phospholipid, andholesterol levels.
ffects of dietary PC on hepatic enzyme activitiesnvolved in lipid metabolism in OA-fed rats
Figure 3 shows the activities of malic enzyme, glucose--phosphate dehydrogenase, FAS, PAP, and CPT in theiver. The addition of OA to the basal diet increased activ-ties of malic enzyme (1.8-fold) and glucose-6-phosphateehydrogenase (2.7-fold) and tended to increase the activi-ies of FAS (1.3-fold) and PAP (1.3-fold). However, addi-ion of PC to the OA diet abolished the OA-induced in-reases in these enzyme activities, actually making themower than those from the basal group. In contrast, CPTctivity was higher (by 1.2-fold) in the PC � OA group,lthough CPT activities were comparable between groupsed the TG and basal diets. It seems likely that PC enhancesatty acid �-oxidation in the fatty infiltration condition in-uced by OA.
IG. 2. Effects of dietary PC on serum lipid concentration in OA-fed rats.G without OA (basal diet) for 10 d. Values are expressed as mean � stan
IG. 1. Effects of dietary PC on hepatic lipid concentration in OA-fed rats.ats were fed OA-supplemented diets containing TG or PC or a dietontaining TG without OA (basal diet) for 10 d. Values are expressed asean � standard error of the mean of five rats. See MATERIALS AND
ETHODS for composition of diets. abDifferent letters indicate significantifferences at P � 0.05. OA, orotic acid; PC, phosphatidylcholine; PL,hospholipid; TG, triacylglycerol.
f diets. abDifferent letters indicate significant differences at P � 0.05. OA, oroti
ffects of dietary PC with OA on FAS and CPT-1 mRNAevels
A significant increase in the hepatic mRNA level of FASas found in the TG � OA group compared with the basalroup (Figure 4). The OA-induced increase in FAS mRNAxpression was abolished by the addition of PC to the OAiet. No significant difference in hepatic mRNA level ofPT-1 was observed between the basal and TG � OAroups (Figure 4), whereas the PC � OA group tended tohow an enhanced expression compared with the TG � OAroup.
iscussion
It is well known that OA administration induces fattyiver [6,24,25]. However, the cause and pathogenesis ofA-induced steatohepatitis remains poorly understood. The
iver plays the central role in lipid metabolism and may benjured as a result of lifestyle-related diseases [26,27].herefore, it is important to discover nutrients that willrotect the liver from these injuries. The present study washe first to demonstrate that dietary PC attenuates the TGccumulation and hepatomegaly induced by OA.
Several human and animal studies have attempted tolleviate liver injury with dietary lipids, including phospho-ipids and other ingredients [6,8,9]. Recently, Navder et al.13] and Lieber et al. [14] reported protective effects ofolyunsaturated PC on alcoholic steatohepatitis in humansnd animals. However, it is unknown whether treatmentith PC alters lipid metabolism in the OA-induced fatty
iver. The present study was undertaken to explore theffects of dietary PC on TG metabolism in OA-inducedatty liver.
Rats fed OA had higher relative liver weights after feed-ng for 10 d as compared with those fed the basal diet asresented in Table 2. The final liver weight, however, wasower in the PC � OA group than in the TG � OA group,
ere fed OA-supplemented diets containing TG or PC or a diet containingror of the mean of five rats. See MATERIALS AND METHODS for composition
Rats wdard er
c acid; PC, phosphatidylcholine; PL, phospholipid; TG, triacylglycerol.
lthough no significant difference in food intake betweenroups was observed.
The same tendency was seen in the concentration ofepatic TG (Figure 1). OA administration resulted in TGccumulation in the liver, consistent with previous reports
IG. 3. Effects of dietary PC on hepatic enzyme activities involved in lipidr PC or a diet containing TG without OA (basal diet) for 10 d. Values areETHODS for composition of diets. abDifferent letters show significant diffe6PDH, glucose-6-phosphate dehydrogenase; ME, malic enzyme; OA, orohospholipid; TG, triacylglycerol.
IG. 4. Effects of dietary PC on FAS and CPT-1 mRNA levels in OA-fedats. Rats were fed OA-supplemented diets containing TG or PC or a dietontaining TG without OA (basal diet) for 10 d. Values are expressed asean � standard error of the mean of five rats. See MATERIALS AND
ETHODS for composition of diets. abDifferent letters indicate significantifferences at P � 0.05. CPT-1, carnitine palmitoyl transferase-1; FAS,atty acid synthase; OA, orotic acid; PC, phosphatidylcholine; PL, phos-
wholipid; TG, triacylglycerol.
6,24,25]. Although the hepatic TG content in the TG � OAroup was three times that in the basal group, partiallyeplacing TG with PC (20% of fat) in the diet attenuated theG accumulation to two-thirds. OA administration tended
o cause a slight increase in hepatic phospholipid content inhe liver, although the extent of the increase was comparableetween the two OA-fed groups. In agreement with previ-us reports [6,24,25], concentrations of serum TG, phos-holipids, and total cholesterol were markedly lower inA-treated groups than in the OA-free basal group, and a
urther decrease occurred when TG was partially replacedith PC in the OA-diet (Figure 2).The excess accumulation of liver lipids can result from
wo mechanisms: 1) increased expression of lipogenic en-ymes and genes combined with the impaired entry of fattycids into the mitochondrial �-oxidation pathway, or 2) thempairment of secretion of very low-density lipoproteinsrom the liver. To investigate the underlying mechanism byhich dietary PC alleviates OA-induced TG accumulation
n the liver, we measured enzyme activities involved in fattycid and TG syntheses and fatty acid oxidation.
The activities of malic enzyme and glucose-6-phosphateehydrogenase, which provide reduced nicotinamide ade-ine dinucleotide phosphate required for FAS (Figure 3),
lism in OA-fed rats. Rats were fed OA-supplemented diets containing TGed as mean � standard error of the mean of five rats. See MATERIALS AND
rane-bound PAP activities also tended to be enhanced byupplementation with OA. However, partial replacement withC abolished these OA-induced increases. FAS and PAP areey enzymes in the regulation of FA and TG de novo synthe-is. Therefore, it is plausible that a synchronized reduction ofatty acid synthesis and TG synthesis is required for the alle-iation of OA-induced fatty liver by dietary PC.
To examine the effect of dietary PC on the transcrip-ional regulation of lipid metabolism after OA administra-ion, we analyzed the hepatic mRNA expression of lipo-enic (FAS) and lipolytic (CPT) genes by real-timeolymerase chain reaction. The mRNA expression of FASended to increase after OA feeding, and the increasedxpression of FAS mRNA was totally abolished by dietaryC. The good correlation between enzyme activity andRNA expression of FAS suggests that the regulation ofAS after feeding with OA and PC occurred at the gene
ranscriptional step. Thus, FAS expression might be impor-ant in the development of OA-induced fatty liver and itsrevention with dietary PC.
Impairment of fatty acid oxidation at the level of CPTeportedly stimulates the esterification of fatty acids to TGhen substrate supply is available. Miyazawa et al. [28]
eported that feeding rats with OA decreased the �-oxida-ion capacity of the liver. However, in the present study, OAdministration did not affect hepatic enzyme activity andNA expression of CPT. The reason for these discrepancies
s unclear, but they may be due to the differences of nutri-ional conditions and animal species. The PC � OA grouphowed a marked increase in CPT activity compared withhe TG � OA group, although the mRNA expression ofPT was not different between groups. Malonyl-CoA is a
egulator of CPT activity [29,30]; the reduced activities ofatty acid synthetic enzymes by dietary PC (Figure 2) wouldesult in decreased malonyl-CoA content and thereafter re-ease the inhibition of CTP activity through post-transcrip-ional regulation. This possible mechanism needs furthertudy under several sets of experimental conditions.
The latter mechanism is supposed in part to be an in-olvement of microsomal triglyceride transfer protein,hich catalyzes the binding of lipids to apolipoprotein-B
31, 32]. This hypothesis is supported by the recent findinghat administering OA inhibits hepatic microsomal triglyc-ride transfer protein activity (T. Yanagita et al., unpub-ished data). However, we have not evaluated the effect ofC on microsomal triglyceride transfer protein activity. Itemains to be determined in a future study.
The PC molecule contains a hydrophilic constituent base,holine, and a hydrophobic fatty acid component. DietaryG and PC used in the present experiment had the same
atty acid composition and content, so the effects of dietaryatty acids could be ignored. Anderson and Holub [33] and
urata et al. [34] reported that supplementation of free-holine or PC had no effects on hepatic and serum lipidrofiles in rats. However, Lieber et al. [14] showed that
olyunsaturated PC supplementation attenuated alcoholic
atty liver diseases in non-human primates. Further, PCupplementation lowered the risk of coronary heart diseases35,36]. In this study, we also observed that PC intake couldlleviate the higher activity of plasma alanine aminotrans-erase, an enzyme involved in liver cell damage, induced byA (data not shown).In conclusion, supplementation with dietary PC sup-
resses OA-induced fatty infiltration by attenuating TG syn-hesis through the downregulation of fatty acid synthesisnd the upregulation of �-oxidation. Findings of the presenttudy also suggest that PC might be useful in the preserva-ion of liver functions.
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