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Turkish Journal of Fisheries and Aquatic Sciences 17: 405-415 (2017)

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www.trjfas.org ISSN 1303-2712 DOI: 10.4194/1303-2712-v17_2_20 RESEARCH PAPER

Fatty Acid Composition, Lipogenic Enzyme Activities and Mrna Expression of Genes Involved in The Lipid Metabolism of Nile Tilapia Fed with Palm Oil Christian Larbi Ayisi 1, Jin-Liang Zhao1,* 1

Shanghai Ocean University, Key Laboratory of Freshwater Fishery Germplasm Resources, Ministry of Agriculture, P. R. China.

* Corresponding Author: Tel.: +86 2161900435 ; Fax: +86 2161900405; E-mail: [email protected]

Received 20 September 2016 Accepted 21 October 2016

Abstract This study was aimed at elucidating the effects of replacing fish oil (FO) with palm oil (PO) on tissue fatty acid composition, lipogenic enzyme activities and mRNA expression of genes related to lipid metabolism in Nile tilapia, Oreochromis niloticus (6.72± 0.14g). An eight week feeding trial was conducted using five isonitrogenous and isolipidic diets containing 0% PO, 25% PO, 50% PO, 75% PO and 100% PO. PO supplementation led to a significant increase in total saturated fatty acid (SFA), total mono unsaturated fatty acids (MUFA) and 18: 2n-6, whiles DHA, EPA, total n-3 as well as 20: 2n-6 were reduced significantly in the liver (P＜0.05). With the exception of glycerol-3-phosphate acyltransferase (GPAT) enzyme activity, supplementing tilapia diet with PO significantly increased fatty acid synthesase (FAS), acetyl-CoA carboxylase (ACC), steroyl-CoA desaturase 1 (SCD1), ATP citrate lyase (ACYL), carnitine palmitoyltransferase Ia (CPTIa) and carnitine palmitoyltransferase Ib (CPT Ib) (P＜0.05). In addition, significant/positive correlations were observed among dietary PO and/or liver tissue FA with FAS, ACC, SCD1 and ACYL mRNA expression while a negative correlation was recorded for CPTI mRNA expression. Generally, inclusion of PO in tilapia diets resulted in lipid accumulation in the liver and altered the key gene expression of lipid metabolism. Keywords: Stearoyl-CoA desaturate 1, Fatty acid synthesase, Carnitine palmitoyltransferase (CPT) I, Acetyl-CoA Carboxylase, ATP Citrate Lyase, Lipid metabolism, Oreochromis niloticus, Palm oil.

Introduction Fish oils are considered as the main source of lipid in aquaculture feeds to promote growth and development of farmed species by providing essential polyunsaturated fatty acids (PUFAs), especially high unsaturated fatty acids (HUFAs) (Sargent et al., 2002). However, as a result of increase in aquaculture activities as well as the limited availability of fish oil and fish meal, there are calls for the use of alternative lipid and protein sources to develop fish farming practices that are sustainable (Kazemi et al., 2016). Plant oils rich in C18 polyunsaturated fatty acids (PUFA) are potential and suitable candidates to replace fish oils in aquaculture feeds (Hafezieh et al., 2010). Palm oil is currently the most abundant vegetable oil in the world (Ochang et al., 2007a) with the global production of palm oil projected to increase by over 30% by 2020 due to the continuous supply by developing countries (Ayisi and Zhao, 2014). At present, many preliminary studies have been conducted to determine the effects of replacing fish oil with palm oil in tilapia (Ochang et al., 2007b; Ng

and Wang, 2010), catfish (Ochang et al., 2007a), large yellow croaker (Duan et al., 2014) and Juvenile Chu‟s Croaker, Nibea coibor (Huang et al., 2016). Changes in dietary fatty acid composition can have effects on the regulation of fatty acid oxidation through a variety of genomic and non-genomic mechanisms (Morash et al., 2009). Recent studies indicated that replacing fish oil with palm oil could act as a modifier in lipid metabolism hence increasing lipid accumulation in fish tissue and whole body. For instance, replacing FO with PO led to an increase in liver lipid deposition in Juvenile Chu‟s Croaker, Nibea coibor (Huang et al., 2016). Similarly, replacing FO with other VO resulted in increased lipid levels in liver in tilapia (Peng et al., 2015) and rainbow trout (Guler and Yildiz, 2011). Lipid accumulation results from the balance between synthesis of fatty acids (lipogenesis) and fat catabolism via β-oxidation (lipolysis), and many key enzymes and transcriptional factors are involved in these metabolic processes (Chen et al., 2015). These enzymes include lipogenic enzymes

© Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey in cooperation with Japan International Cooperation Agency (JICA), Japan

C. L. Avisi and J. L. Zhao / Turk. J. Fish. Aquat. Sci. 17: 405-415 (2017) 406 (such as fatty acid synthase (FAS), and acetyl-CoA lipogenic enzyme activities as well as lipid carboxylase (ACC) , and lipolytic enzymes (such metabolism related gene expression. Fatty acid as carnitinepalmitoyltransferase I (CPT I), composition of the liver and muscle were analyzed. hormone-sensitive lipase (HSL) and adipose Also enzyme activities of Fatty acid synthesase triacylglyceride lipase (ATGL) (Elliott and Elliott, (FAS), Acetyl-CoA Carboxylase (ACC), Steroyl-CoA 2009). desaturase 1 (SCD1), ATP Citrate Lyase (ACYL), Acetyl-CoA carboxylase (ACC) is not only a Glycerol-3-phosphate acyltransferase (GPAT), key enzyme in fatty acid synthesis via the ACCa, Carnitine Palmitoyltransferase I were examined in the but also plays an important role by regulating fatty liver. Finally, mRNA expression of SCD1, ACC, acid oxidation via the ACCb. ACC is a biotinFAS, ACYL, CPTI a and CPTI b as well as GPAT dependent enzyme that catalyzes the irreversible were studied. carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid biosynthetic pathway Materials and Methods (Cheng et al., 2011). FAS is a key enzyme that regulates the de novo Feed and Feeding Trial biosynthesis of long-chain fatty acids from acetylCoA and malonyl-CoA in the presence of NADPH Five isonitrogenous (33% crude protein) and (Dong, et al., 2014). isolipidic diets (10%) were prepared for the eight Carnitine palmitoyltransferase (CPT) I is weeks feeding trial. The variation was made to have considered as the main regulatory enzyme in effects on fish oil and palm oil as shown in Table 1. mitochondrial fatty acid oxidation because it catalyses Fish meal, soybean meal and rapeseed meal were used the conversion of fatty acyl-CoAs into fatty acylas the protein sources, while palm oil and fish oil carnitines for entry into the mitochondrial matrix were used as the sources of lipid. All dry ingredients (Kerner and Hoppel, 2000). Various nutrients (defatted fish meal, soybean meal, rapeseed meal, modulate the mechanisms involved in CPT I wheat meal, mineral mix and vitamin mix) were regulation and consequently mitochondrial βmixed using the progressive enlargement method. The oxidation of fatty acids. For example, CPT I experimental diets were prepared by mixing the dry expression was increased in the red muscle, adipose ingredients with palm oil, fish oil and distilled water tissue and liver between three and seven fold in in a Hobart mixer, and the resulting moist dough rainbow trout (Oncorhynchus mykiss) fed high PUFA pelleted using a meat mincer through a 1-mm die. The diet compared to the mixed fatty acid control diet 1-mm diameter pelleted diets were wet extruded, air (Morash et al., 2009). Also, PUFAs can also act on dried, broken up and sieved into proper pellet size. All CPT I activity indirectly via changes in the experimental diets were stored at -20 C until time of mitochondrial membrane composition. The fatty acid feeding. The fatty acid composition of the test diets composition of the outer mitochondrial membrane has are given in Table 2. been shown to be of particular importance to the regulation of CPT I because it can affect membrane Experimental Procedures properties and the binding affinity of the allosteric regulator M-CoA to the enzyme (Morash et al., 2009). Nile tilapia fingerlings (6.72±0.14, initial Fatty acid can serve as a substrate for β-oxidation to weight) were obtained from Tilapia Germplasm provide energy. β-oxidation is the major process by Station of Shanghai Ocean University, China. Prior to which fatty acids are oxidized, by sequential removal the start of the experiment, fish were transported to of two carbon units from the acyl chain. CPT I is aquarium facilities at Shanghai Ocean University and frequently described as the „rate-limiting enzyme‟ of acclimated for two weeks. Fish were fed commercial β-oxidation flux in liver, heart and skeletal muscle diets obtained from Shanghai Jin Yuan Trade (Eaton, 2002). containing 30% crude protein twice daily to apparent SCD are known for their roles in synthesizing satiation. Fish were starved for 24 hours before the unsaturated fatty acids (Ardiyanti et al., 2012). feeding trial, weighed, and randomly distributed into Stearoyl-CoA desaturase (SCD) synthesizes oleate 15 rectangular fiber glass tanks (150× 60× 40 cm) at necessary for the biosynthesis of triglycerides and 40 fish per tank with water maintained at 210 litres. other lipids (Miyazaki et al., 2004). Although studies Dissolved oxygen (DO) concentration, pH and water have investigated the replacement of fish oil with temperature were monitored on daily basis using YSI palm oil on growth and lipid deposition in fish, the 556 instrument (YSI, Yellow Springs, Ohio). underlying molecular processes involved in fatty acid Ammonia-N and Nitrite-N were analyzed metabolism and the change of lipid deposition as a spectrophotometrically on a weekly basis following response to dietary fish oil replacement by palm oil standard methods (APHA, 1998). Each diet was are seldom known. randomly offered to a tank and its replicates to sum The aim of the present study was to investigate up to 15 experimental tanks. During experimental the underlying mechanisms of dietary fish oil period of eight weeks, fish were offered the replacement with palm oil on fatty acid metabolism, experimental diets to apparent satiation twice daily at

C. L. Avisi and J. L. Zhao / Turk. J. Fish. Aquat. Sci. 17: 405-415 (2017)

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Table 1. Formulation and proximate composition of experimental diets (g/100 g in dry matter)

Ingredient Fish meal* Soybean meal* Wheat meal* Rapeseed meal* Fish oil* Palm oil* Soybean phospholipid* Mineral mix** Vitamin mix*** Ca(H2PO4) Choline chloride Inositol Total Proximate composition (%) Moisture Protein Lipid Ash

PO 0 6.00 30.00 22.50 30.00 6.00 0.00 2.50 0.55 0.40 1.50 0.50 0.05 100

Dietary PO replacement level (%) PO 25 PO50 6.00 6.00 30.00 30.00 22.50 22.50 30.00 30.00 4.50 3.00 1.50 3.00 2.50 2.50 0.55 0.55 0.40 0.40 1.50 1.50 0.50 0.50 0.05 0.05 100 100

PO75 6.00 30.00 22.50 30.00 1.50 4.50 2.50 0.55 0.40 1.50 0.50 0.05 100

PO100 6.00 30.00 25.50 30.00 0.00 6.00 2.50 0.55 0.40 1.50 0.50 0.05 100

10.30 33.18 9.82 5.25

10.53 33.15 9.81 5.60

10.70 33.23 9.88 5.40

10.50 33.17 9.85 5.50

10.82 33.09 9.87 5.53

*Fish meal, Soybean meal, Wheat meal, Soybean phospholipase, Palm oil, Vitamin premix, Mineral mix and Ca (H2PO4) were supplied by Nonghao Feed Company (Shanghai, China). **Mineral mix (mg kg-1 dry diet): Cu (CuSO4), 2.0; Zn (ZnSO4), 34.4; Mn (MnSO4), 6.2; Fe (FeSO4), 21.1; I (Ca (IO3)2), 1.63; Se (Na2SeO3), 0.18; Co (CoCl2), 0.24; Mg (MgSO4.H2O), 52.7. *** Vitamin premix (IU or mg kg-1 diet ): vitamin A, 16000 IU; vitamin D, 8000 IU; vitamin K, 14.72; thiamin, 17.8; riboflavin, 48; pyridoxine, 29.52; cynocobalamine, 0.24, tocopherols acetate, 160; ascorbic acid (35%), 800; niacinamide, 79.2; calcium-Dpantothenate,73.6; folic acid, 6.4; biotin, 0.64; inositol, 320; choline chloride, 1500; L-carnitine, 100.

Table 2. Main fatty acid composition (% of total fatty acids) of experimental diets FATTY ACID(S) PO 0 0.15±0.00 5.54±0.21 23.88±0.49 5.31±0.04 34.88±0.46 6.02±0.34 23.56±0.68 29.58±0.85 20.65±0.77 0.56±0.05 21.21±0.22 5.52±0.23 0.32±0.01 4.25±0.14 5.66±0.35 15.75±0.19 1.33±0.05 34.96±0.18 0.87

PO 25 0.14±0.01 4.52±0.04 25.90±0.08 5.76±0.06 36.32±0.83 5.23±0.59 25.42±0.09 30.65±0.47 21.57±0.05 0.47±0.03 22.04±0.89 3.96±0.15 0.33±0.00 3.19±0.01 4.15±0.04 11.63±0.57 1.30±0.21 33.67±0.34 1.07

Dietary PO replacement level (%) PO 50 PO 75 PO 100 0.12±0.00 0.12±0.01 0.11±0.00 3.20±0.05 2.25±0.09 1.27±0.1 27.00±0.39 28.06±0.37 29.19±0.27 5.43±0.12 5.30±0.13 5.15±0.05 35.75±0.19 35.73±0.33 35.72±0.72 3.15±0.04 2.06±0.15 0.89±0.01 28.40±0.16 31.16±0.20 33.77±0.16 31.55±0.77 33.22±0.43 34.66±0.32 23.07±0.22 23.62±0.32 24.33±0.03 0.41±0.00 0.25±0.05 0.20±0.02 23.48±0.33 23.87±0.18 24.53±0.62 3.98±0.04 3.79±0.09 3.63±0.03 0.32±0.01 0.30±0.00 0.29±0.01 2.20±0.06 1.41±0.04 0.62±0.00 2.85±0.16 1.69±0.02 0.58±0.02 9.35±0.04 7.19±0.39 5.12±0.31 1.29±0.33 1.19±0.43 0.93±0.05 32.83±0.18 31.06±0.09 29.65±0.14 1.08 1.15 1.20

12:0 14:0 16:0 18:0 Total SFA‟s 16:1(n-7) 18:1(n-9) TOTAL MUFAs 18:2(n-6) 20:4(n-6)ARA Total n-6 18:3(n-3) 18:4(n-3) 20:5(n-3)EPA 22:6(n-3)DHA Total n-3 DHA/EPA Total PUFAs Total SFA/total PUFA n-3:n-6 0.74 0.52 0.39 0.30 0.20 ARA= Arachidonic acid; EPA= Eicosapentanoic acid; DHA= Decosahexanoic acid; SFA= saturated fatty acids; MUFA= mono unsaturated fatty acid; PUFA= polyunsaturated fatty acid.

08:00 and 16:00. Sample Collection Fish were starved 24 hours prior to harvest after

completion of the trial period. Seventy-five (five per tank) fish at the end of the trial were randomly sampled, euthanized with an overdose of tricaine methane sulfonate (MS-222 at 200mg/L in culture water), liver and muscle samples taken, pooled and

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C. L. Avisi and J. L. Zhao / Turk. J. Fish. Aquat. Sci. 17: 405-415 (2017)

stored at -80°C for subsequent determination of fatty acid composition, lipogenic enzyme activity and mRNA expression.

Before the assay was performed, tissues (liver and muscle) and feed were grinded to powder individually. (1) Total lipid (TL) was extracted from freeze – dried samples with chloroform–methanol (2:1, V/V), according to the method of (Folch et al., 1957). (2) Fatty acid methyl esters (FAME) were prepared by transesterification with 0.4 M KOHmethanol, and then detected by gas chromatograph (GC-7890A, USA) using methyl heneicosanoate (C21:0) as the internal standard. (3) Fatty acid content was determined using the normalization method. All measurements were performed in triplicates and the fatty acids content expressed as % total FA.

reaction contained 1-µl cDNA sample, 10 µl SYBR green I Master Mix (TaKaRa), 0.5µl of each primer and 8 µlH2O. PCR amplification was performed in triplicate wells using the following protocol: 3 min at 95C, 45 cycles consisting of 10s at 95 C, 15s at 63 C and 25 s at 72C. A melting curve analysis was performed to confirm that a single PCR product had been amplified. Approximately, there was an equal amplification efficiency for all genes ranging between 95.1% and 99.3%. β-actin gene was used as the reference to quantify the target genes relatively. In order to quantify the transcripts of lipid metabolism related genes, the normalized gene expression of the group fed the control diet (0% PO) was set to 1. The expression of the target genes were expressed relating them to the control group. At the end of the reaction, the fluorescent data were converted into Ct values. Each transcript level was normalized to b-actin using the 2 -DDCT Method (Livak and Schmittgen, 2001).

Lipogenic Enzyme Activities

Statistical Analysis

Enzyme activities of Fas, Acc, Acyl, CPTI, Gpat and Scd1 were measured using enzyme linked immunosorbent assay (ELISA). A total of 0.5-1.0 g of liver were homogenized using a ground glass homogenizer on ice. The homogenates were centrifuged (20,000 rpm, 50 mins at 4 C), and the clear phase between the top layer and the pellets used for the analysis. Samples were analyzed in a 96-well plates by ELISA (Shanghai MLBIO Biotechnology Co. Ltd, China). Optical Density (OD) was measured in an ELISA microplate reader (Bio Tek Synergy, USA) at 450nm. A standard curve was generated according to the manufacturer‟s instruction, and the standard diversity calculated with Excel 2003. Enzyme activity units (IU), defined as moles of substrate converted to product per minute at assay temperature, were expressed per mg of hepatic soluble protein specific activity or per gram of liver tissue wet weight.

All data were analyzed by one-way analysis of variance and Turkey‟s multiple test to compare treatment means. Differences were considered significant at 0.05 probability level for all data. All analysis was performed using the Graph Pad prism V.5.03 and results presented as mean ± standard error of the mean (SEM).

Fatty Acid Composition of Feed, Liver and Muscle

RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (RT-Qpcr) Expression analysis of FAS, ACC, SCD 1, ACYL, CPTIa and CPTIb was performed using realtime PCR with beta-actin as house-keeping gene (Oku et al. 2006). Total RNA was isolated from the samples using Trizol Reagent (Invitrogen). RNA concentration was determined by conventional agarose electrophoresis and through absorbance measurements (ratio 260/280 ≥2). The genomic DNA contaminating the RNA samples was digested by RNase-free DNase I (TaKaRa) incubation for 15 min at 37 C. Next, 2ug of RNA was transcribed into cDNA using M-MLV reverse transcriptase. All cDNA samples were stored at –20C until analysis. Real-time PCR was conducted on a Mini Option Real-time PCR machine (Bio-Rad). The 20-µl

Results Fatty Acid Compositions in Liver and Muscle The fatty acid composition of liver and muscle following 56 days of feeding experimental diets are presented in tables 4 and 5, respectively. Replacing FO with PO had significant influence on the fatty acid composition of the liver and muscle. Whereas total SFA, total MUFA and 18:2n-6 (LA) increased significantly (P