DOI: 10.21767/2572-5459.100012 Relationship Between Liver .

Research ArticleiMedPub JournalsJournal of Animal Research and Nutrition2016ISSN 2572-5459Vol.1 No.2:12http://www.imedpub.com/DOI: 10.21767/2572-5459.100012The Relationship between Liver Lipid Accumulation and Changes inPlasma Amino Acids in Mice Challenged with Carbon TetrachlorideShinya Takagi1, Daichi Oikawa2, Hiromi Ikeda1, Nozomi Tateiwa3, Kazunori Koba3,Vishwajit S. Chowdhury4, Shinobu Yasuo1 and Mitsuhiro Furuse1,*1Laboratory2Facultyof Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japanof Education, Nagasaki University, Nagasaki 852-8521, Japan3Department4Divisionof Nutritional Science, University of Nagasaki, Siebold Nagasaki 851-2195, Japanfor Experimental Natural Science, Faculty of Arts and Science, Kyushu University, Fukuoka 819-0395, Japan*Corresponding author: Mitsuhiro Furuse, Laboratory of Regulation in Metabolism and Behavior, Faculty of Agriculture, Kyushu University,Fukuoka 812-8581, Japan, E-mail: furuse@brs.kyushu-u.ac.jpRec date: Jan 20, 2016, Acc date: Feb 12, 2016, Pub date: Feb 18, 2016Copyright: 2016 Takagi S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.AbstractAcute liver damage induced by the administration ofcarbon tetrachloride is characterized by lipidaccumulation in the liver and changes in the plasma freeamino acid profile. The major objective of the presentstudy was to clarify the relationships between liver lipidlevels and plasma free amino acids, and to estimate liverlipid contents via the concentrations of plasma free aminoacids. Mice were administered with carbon tetrachlorideor olive oil. Liver and plasma samples were obtainedbefore (initial value) and after 1, 3 and 7 days ofadministration. Liver triacylglycerol increased at 1 day andtotal cholesterol was raised at 1 and 3 days post injectionof carbon tetrachloride, and thereafter both returned totheir initial values. L-Phenylalanine, L-serine and L-alaninewere significantly increased, but L-arginine wassignificantly decreased at 1 day. Liver triacylglycerol andtotal cholesterol levels were linearly, but weakly,correlated with several amino acids. Thus, stepwiseregression analysis was applied and we found that theliver triacylglycerol level could be estimated by the levelsof free plasma L-histidine, L-methionine and Lphenylalnine, and that the liver total cholesterol could beestimated by the levels of L-glutamine, L-valine and Lphenylalnine. In conclusion, the increase in plasma free Lphenylalanine plays an important factor in theaccumulation of liver lipid induced by acute treatmentwith carbon tetrachloride in mice.Keywords:Carbon tetrachloride; Liver; Plasma aminoacids; Total cholesterol; TriacylglycerolIntroductionThe liver acts as a vital organ for different metabolicactivities, such as carbohydrate, lipid and protein metabolism,which are interrelated with one another. The interrelationshipsamong these metabolic processes can be confirmed whenthere are injuries to the liver and/or abnormalities in it, whensimultaneous disturbance in functions occurs. Liver cirrhosismost commonly occurs as a result of alcoholism, but somepoisons such as carbon tetrachloride and viral diseases canalso induce liver cirrhosis. Among poisons, carbontetrachloride is widely used for experimental cirrhosis.It is well known that acute administration of carbontetrachloride modifies lipid metabolism, resulting in a fattyliver. Weber et al. [1] summarized an overview of the effects ofcarbon tetrachloride on lipid metabolism in the liver. Carbontetrachloride increases: 1) the synthesis of fatty acids; 2) thesynthesis of triacylglycerols from acetate; 3) the rate of lipidesterification; and 4) the synthesis of cholesterol; but lowers 1)β-oxidation of fatty acids and 2) hydrolysis of triacylglycerols.In the case of protein metabolism, as a result of hepaticinsufficiency due to carbon tetrachloride the plasma freeamino acid levels were distinctly abnormal. Most of the aminoacids except the branched chain amino acids (BCAAs) (i.e.valine, leucine and isoleucine) are metabolized in the liver. TheBCAAs are mainly metabolized in the muscle. Among theamino acids metabolized in the liver, the increased plasmalevel of aromatic acids (AAA) such as phenylalanine andtyrosine is referred to as an indicator of liver disease [2].Fischer et al. [3] proposed the ratio of the sum of the plasmaBCAA levels divided by the sum of the plasma AAA levels, andthis ratio was recognized as Fischer’s ratio. This ratio showedan excellent correlation with a grade of encephalopathy.According to Muratsubaki and Yamaki [4], not only plasma AAAbut also most of the plasma amino acids other than argininewere significantly increased in rats through carbontetrachloride treatment.As mentioned above, carbon tetrachloride stronglyinfluences lipid and amino acid metabolism in the liver.However, the relationships between accumulated levels oflipids in the liver and plasma free amino acid concentration Copyright iMedPub This article is available from: http://animalnutrition.imedpub.com1

Journal of Animal Research and NutritionISSN 2572-5459have not yet been clarified. To clarify this issue, the correlationbetween liver lipid levels and plasma free amino acid levels inmice was determined after challenging the mice with carbontetrachloride.Materials and MethodsAnimals and treatmentsSix-week-old male ICR mice (Japan SLC, Shizuoka, Japan)were housed in conditions including room temperature at22-23 C, humidity at 50-60% and a 12-h light-dark cycle(8:00-20:00); they were housed individually, and had freeaccess to a commercial diet (MF, Oriental Yeast Co. Ltd., Tokyo,Japan) and water.2016Vol.1 No.2:12min, 20–40% B over 3.6 min, 40% B for 1.2 min, 40–60% B over3.8 min, 60% B for 1 min, and 60–10% B over 0.01 min. Justbefore the analysis by UPLC, each sample (10 µl) wastransferred to a UPLC tube, and NAC/OPA (20 µl) and a boratebuffer (70 µl) were added; then it was left for 2 min in a darkroom. The same method was used for the standard solutionscontaining L- and D-amino acids (aspartic acid, glutamic acid,asparagine, serine, glutamine, histidine, threonine, phan,phenylalanine, isoleucine, and leucine), glycine, taurine and γamino butyric acid (GABA).Liver triacylglycerol and total cholesterolanalysisMice were given free access to the commercial diet. After 8days of acclimatization, the mice were divided into sevengroups (n 7) based on similar body weight. One group wasassigned as the initial group without any treatments. Threegroups were exposed to a carbon tetrachloride solution (2ml/kg) and the other three groups were exposed to olive oil (2ml/kg). The carbon tetrachloride solution consisted of thesame volume of carbon tetrachloride and olive oil. The carbontetrachloride solution and the olive oil were orallyadministered once on the day of treatment. Sampling of theinitial group was done on the day of treatment of the other sixgroups. The treated groups were sampled at 1, 3 and 7 dayspost injection. The mice were killed by cervical dislocation anddecapitation. Experimental procedures followed the guidelinesfor animal experiments of the University of Nagasaki (No.26-15).All of the lipids in the liver were extracted using the methodof Folch et al. [5], with some modification. A chloroform/methanol (1:1, vol/vol) solution (12 ml) was added to the liversample, followed by homogenization. Chloroform (6 ml) wasagain added, followed by homogenization. The mixture wasleft for 30 min at 40 C, and filtered. A chloroform/methanol(2:1, vol/vol) solution (2 ml) and distilled water (3.6 ml) wereadded to the filtrate, and the mixture was stirred. The mixturewas left for one day at 4 C in order to separate it into twodistinct phases, and the lower phase was collected. The lowerphase was dried with a nitrogen gas-evaporator, and the driedextract was diluted with 5mL isopropanol, followed by assayusing triacylglycerol and cholesterol determination kits,respectively (Triglyceride E-test wako and Cholesterol E-testwako, Wako Pure Chemical Industries, Ltd., Osaka, Japan).These absorbances were analyzed by Epoch (BioTekInstruments Inc. Vermont, USA).Sample collectionStatistical analysisBlood was collected from the carotid artery into tubescontaining heparin sodium (AY Pharma, Tokyo, Japan) once themice had been decapitated after cervical dislocation. To obtaina plasma sample, blood was centrifuged for 20 min at 850 g.The liver was then removed and weighed. The plasma and liversamples were stored at -80 C until the analysis of lipids andfree amino acids took place.Data from the same treatment were analysed by one-wayanalysis of variance, including the initial value. Whensignificant effects were found, the values were compared bythe Tukey–Kramer test. Statistical significance was set at P 0.05. The results are shown as means S.E.M. Using the dataof all the mice, simple regression analysis was carried outbetween the liver triacylglycerol or total cholesterol contentsand the plasma free amino acid concentration, after whichstepwise regression analysis was also applied.Plasma amino acid assayEach sample of plasma was filtered through an Amicon Ultra centrifugal filter (0.5 ml 3K, Merck Millipore, USA) andcentrifuged at 14,000 g for 15 min. Supernatants of 10 µl wereused. Both the L- and the D-amino acid contents weremeasured by a UPLC system (the AcquityTM UPLC system wascomprised of Waters Binary Solvent Manager, Water SampleManager and Waters FLR Detector) with an ACCQ-TAGTMULTRA C18 1.7 µm 2.1 100 mm column (Waters Corporation,USA). The excitation and emission wavelengths for fluorescentdetection of amino acids were 350 nm and 450 nm,respectively. The system was operated with a flow rate of 0.25ml/min at 30 C.The UPLC gradient system (A 50 mM sodium acetate (pH5.9), B methanol) was 10–20% B over 3.2 min, 20% B for 12ResultsAs shown in Figure 1, liver total cholesterol ((F 3, 24) 8.681, P 0.001) and triacylglycerol ((F 3, 24) 21.995, P 0.0001) contents were significantly modified by carbontetrachloride.In the case of liver total cholesterol, the value sharplyincreased at 1 day after carbon tetrachloride administrationand remained high for up to 3 days, and returned to the initialvalue at 7 days. One the other hand, liver triacylglycerolquickly increased at 1 day and returned to the initial value at 3days.This article is available from:http://animalnutrition.imedpub.com

Journal of Animal Research and NutritionISSN 2572-54592016Vol.1 No.2:12Figure 1: Changes in liver total cholesterol and triacylglycerol after administration of carbon tetrachloride in mice. Open andclosed circles indicate the control and the carbon tetrachloride–treated mice. Different letters for the same treatment indicatesignificant difference at P 0.05.In the present study, we determined 16 amino acids of bothL- and D-forms along with glycine, taurine and GABA, asmentioned above. However, only a few amino acids weresignificantly changed after being challenged with carbontetrachloride (Figure 2). In the case of plasma L-serine ((F 3,24) 5.555, P 0.005), the values significantly increased at 1day and remained high for up to 3 days, but returned to theinitial level at 7 days. Plasma L-phenylalanine ((F 3, 24) Copyright iMedPub12.006, P 0.0001), L-tyrosine ((F 3, 24) 3.510, P 0.05) andL-alanine ((F 3, 24) 5.196, P 0.01) significantly increased ortended to increase at 1 day and returned to the initial value at3 days, but plasma L-arginine ((F 3, 24) 10.245, P 0.001)disappeared at 1 day and showed the initial value at 3 days.Other amino acids were not influenced by carbontetrachloride.3