Desaturase in Slow- or Fast-Growing Rabbit Geno - ACS Publications

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Activity, Expression and Substrate Preference of the #6Desaturase in Slow- or Fast-growing Rabbit Genotypes Cesare Castellini, Alessandro Dal Bosco, Simona Mattioli, Magdalena Davidescu, Lanfranco Corazzi, Lara Macchioni, S Rimoldi, and Genciana Terova J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05425 • Publication Date (Web): 08 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Journal of Agricultural and Food Chemistry

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Activity, Expression and Substrate Preference of the ∆6Desaturase in Slow- or Fast-growing Rabbit Genotypes Cesare Castellini⊥, Alessandro Dal Bosco⊥, Simona Mattioli⊥, Magdalena Davidescuψ, Lanfranco Corazziψ, Lara Macchioniψ, Simona Rimoldi±, Genciana Terova±§ ⊥

Dept.of Agricultural, Food and Environmental Science, University of Perugia – Borgo 20 Giugno, 74. 06100 Perugia, Italy. ψ Dept. of Experimental Medicine, University of Perugia, Piazza Gambuli 1, 06123 Perugia, Italy ± Dept. of Biotechnology and Life Sciences (DBSV), University of Insubria, Via J.H. Dunant, 3. 21100 Varese, Italy. § Inter-University Centre for Research in Protein Biotechnologies, “The Protein Factory,” Polytechnic University of Milan and University of Insubria, Varese, Italy. 1

Corresponding author: Prof Alessandro Dal Bosco. Email: [email protected]

1

ACS Paragon Plus Environment


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Abstract.

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In the present paper liver fatty acid ∆6 desaturation (fads2) activity was analyzed in two rabbit

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strains with slow- (S, 27.5 g/d) or fast-growing (F, 48.5 g/d) rate. The fatty acid profile of the

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liver showed a different PUFA profile in the two strains with lower n-6/n-3 ratio in the S

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rabbits. The expression of fads2 was two folds higher in S than in F rabbits, whereas enzyme

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activity resulted higher in F and more oriented toward the desaturation of linoleic acid (90%).

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In contrast, S showed a higher preference for linolenic acid (38.9% vs. 10%). This study

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identified a single difference in the fads2 amino acid sequence between these two strains.

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Such difference consists in the substitution of Gly104 to Ser104 in the sequence of F fads2.

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These results indicate for the first time that genetic selection for performance may affect the

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preference for PUFA toward desaturation of linoleic/linolenic acid.

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Keywords: rabbit, linoleic acid, competition, long-chain polyunsaturated fatty acids, liver

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metabolism

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INTRODUCTION

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In Western countries the human diets are considered unbalanced in terms of PUFA with a

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striking increase in the n-6 as well as a decrease in n-3 intake 1. During the last 100 years, the

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n-6 to n-3 ratio has increased to 10-20:1, far from the optimal dietary intakes, which should be

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about 1-4:1 2.

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On the other hand, humans are quite inefficient long chain polyunsaturated fatty acid

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(LCPUFA) producers, being largely dependent on dietary intake 3. LCPUFAs are particularly

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abundant in the brain and retina and have relevant roles in many physiological pathways and

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pathological disorders such as cardiovascular diseases, reproductive dysfunctions, depression,

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and immune response 4,5.

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Currently, fish which is the main supply of n-3 LCPUFA 6, is becoming progressively scarce

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and less sustainable due to the high pressure on global fish stocks, and aquaculture is unlikely

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to be a sustainable source because the feed used contains large amounts of wild fish 7.

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Therefore, the capacity of terrestrial animals to elongate and desaturate essential fatty acids

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(EFA) has been widely explored

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production of LCPUFA of the n-6 and n-3 series requires as a substrate linoleic acid (LA;

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C18:2n-6) and α-linolenic acid (ALA; C18:3n-3) 9. This process is catalyzed by an elongase

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and two desaturase enzymes, ∆5- and ∆6- desaturase (fads1 and fads2), which introduce

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double bonds into the EFA 10. The fads2 acts twice, once on the 18-carbon PUFA and again

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after they are converted to 24-carbon derivatives for that reason it is considered as one of the

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limiting factors for the conversion to the longer-chain homologs 11.

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Terrestrial animals have a low capacity of conversion for EFA precursors into LCPUFA

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owever, such a poor efficiency is modulated by exogenous factors such as feed, species,

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gender and breed

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more n-6 fatty acids than in the past

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in an attempt to find other sources of n-3 LCPUFA. The

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.

13-15

. The current rearing systems seem to produce meat with less n-3 and 16,17

due to use of feedstuffs rich in n-6 and poor in n-3 3



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and/or a possible secondary effect arising from intensive selection of animals for productive

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traits. Indeed, selection can modify the expression of genes coding for enzymes involved in

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LCPUFA synthesis as well as the relative enzymatic activity 18.

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Thus, in the present study we analyzed the ∆6-desaturase activity with different substrate

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precursors (LA or ALA) on hepatic microsomes isolated from rabbit of two genetic strains

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with different rate of growth. The fads2 gene expression in liver of both rabbit strains was

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also quantified by a cutting edge analytical method (droplet digital PCR). Other genetic

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factors that could explain the difference in fads2 gene expression and the relative activity

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were studied too.

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MATERIAL AND METHODS

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Reagents

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For this study, [1-14C]-18:2n-6 and [1-14C]-18:3n-3 (58.2 and 51.7 mCi/mmol, respectively)

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were purchased from PerkinElmer (Boston, USA). ATP, CoASH, NADPH, MgCl2, NaF,

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glutathione, BF3, unlabeled LA and ALA, linolenic acid methyl ester, and organic solvents

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were obtained from Sigma Aldrich (Italy). Stearidonic acid methyl ester (C18:4n-3) was

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obtained from Cayman Chemical (Michigan 48108, USA). Silica gel plates impregnated with

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10% AgNO3 were purchased from StepBio (Bologna, Italy).

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Animals and diets

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Forty male rabbits were raised from weaning to 80 days of age in the experimental farm of the

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Department of Agricultural, Food and Environmental Science of Perugia University

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according to the International Guiding Principles for Biomedical Research Involving Animals

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National Rules

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The rabbits were housed in individual cages under a constant photoperiod of 16 hours of light

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and according to guidelines for rabbit experimental breeding purposes

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.

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per day, at temperatures ranging from 15 to 28 °C and at a relative humidity of 60 to 75%,

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and were fed ad libitum with a commercial diet formulated according to standard

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requirements 21 (Table 1). Water was also administered ad libitum.

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The two rabbit strains compared were: a meat-type (F), and a slow-growing (S). Fast-growing

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animals were selected from a New Zealand White origin for litter size and weight at weaning

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and at 80d for at least 16 generation. On the contrary, the slow-growing breed was not

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submitted to genetic selection for productive traits 22.

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Productive performance

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Daily gain, feed consumption and feed to gain ratio were analyzed. Body weight (BW) and

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feed intake (FI) was measured weekly, weight gain (WG) and feed conversion ratio (FCR)

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were then calculated.

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At 80 days of age, 10 rabbits per group with weights close to the average of the group (±

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10%) were selected and slaughtered in the departmental processing plant 12 hours after feed

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withdrawal under the supervision of veterinarians from the University of Perugia; none of the

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animals underwent any form of transportation. The rabbits were sacrificed by severing the

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carotid arteries and jugular veins following electro-stunning and the carcasses were prepared

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according to the methods described by Blasco and Ouhayoun

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immediately removed and transported to laboratory of Department.

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and the livers were

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Analytical determinations

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Chemical analysis

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The chemical composition of the feed was determined according to the AOAC 24.

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The fatty acid profiles of the feed and the livers were determined by gas chromatography after

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lipid extraction according to Folch et al. 25. In particular, 1 mL of lipid extract was evaporated

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under a stream of nitrogen, and the residue was derivatized by the addition of 3 mL of sulfuric 5



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acid (3% in methyl alcohol). After incubating at 80°C for 1 h, the methyl esters were

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extracted with petroleum ether, and 1 µl was injected into the gas chromatograph (Mega 2 -

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model HRGC; Carlo Erba, Milan Italy), which was equipped with a flame ionization detector.

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Separation of the fatty acid methyl esters (FAMEs) was performed using an Agilent (J&W)

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capillary column (30 m × 0.25 mm I.D; CPS Analitica, Milan, Italy) coated with a DB-wax

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stationary phase (film thickness of 0.25 mm). The operating conditions for column injection

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of the 1 µl sample volumes were as follows: the temperatures of the injector and detector were

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set at 270 °C and 280 °C, respectively, and the detector gas flows were H2 at 50 mL min−1 and

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air at 100 mL min−1. The oven temperature was programmed to provide a good peak

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separation, as follows: the initial oven temperature was set at 130 °C, this temperature

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increased at a rate of 4.0 °C min−1 to 180 °C and was held for 5 min, the temperature was then

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increased at a rate of 5.0 °C min−1 to 230 °C, and the oven was held at the final temperature

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for 5 min. Helium was used as a carrier gas at a constant flow rate of 1.1 mL min−1. Individual

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fatty acid methyl esters were identified by referencing the retention times of the FAME

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authentic standards. The relative proportion of each fatty acid was expressed as a percentage.

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The mean value of each fatty acid was used to calculate the sum of the saturated (SFA),

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mono-unsaturated (MUFA) and polyunsaturated (PUFA) fatty acids and LCPUFA of different

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series (n-3 and n-6). Microsomal lipids were extracted with trichloromethane: methyl alcohol

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(2:1, v/v) according to Folch et al. 25. Endogenous free fatty acids were analyzed by TLC on

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silica gel plates and developed with trichloromethane: methyl alcohol: 15% ammonia

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(65:25:4, v/v). The plates were air dried and exposed to iodine vapors, followed by

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densitometric quantification. The endogenous amount of free LA and ALA in the liver

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microsomes was calculated according to the FA profile of the liver.

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Molecular analysis 6


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Total RNA extraction

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Total RNA was extracted from rabbit liver; the tissue lysis and homogenization were carried

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out in a closed system using gentle MACS Dissociator and single use gentle MACS M tubes

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(MiltenyiBiotec). The total RNA isolation was performed by an automated purification

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process using the Maxwell® 16 Instrument and Maxwell® 16 Tissue LEV total RNA

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purification Kit (Promega, Italy). The quantity and purity of RNA was assessed

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spectrophotometrically by using NanoDrop (Thermo Scientific). The total RNA integrity was

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checked by electrophoresis on agarose gel (1%) stained with ethidium bromide.

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Analysis of rabbit fatty acid desaturase (fads2) coding sequence

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The RNAs (3 µg) extracted from liver of 10 rabbits/strain, were reverse transcribed into

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cDNAs using SuperScript® III Reverse Transcriptase (Life Technologies) and Oligo d(T)16,

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following the manufacturer’s protocol. To perform PCR, 100 ng of each resulting cDNA were

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amplified with Advantage 2 Polymerase Mix (Clontech), which is an optimized blend of PCR

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enzymes that includes a proofreading DNA polymerase, and the designed primers in a final

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volume of 50 µl. Two sets of primers (FADS2 sense 1 + antisense 1 and FDAS2 sense 2 +

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antisense 2; Table 2) were designed on the basis of Oryctolagus cuniculus fads2 gene

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sequence published in Genebank database (Accession number XM_002721017.1; Figure 1,).

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Each set of primers amplified for a partial cDNA sequence of about 800 base pairs (bp)

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covering the complete fads2 open reading frame (ORF) of 1335 bp. The PCR run parameters

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were the followings: initial denaturation step at 95 °C for 1 min followed by 32 cycles of

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denaturation for 30 s at 95 °C, annealing for 30 sec at 53 °C, and extension for 1.30 min at 68

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°C. A final extension step at 70°C for 10 min was performed at the end of amplification

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cycles. The PCR products were loaded on 1% agarose gel, excided, and cloned in pGEM®T-

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Easy vector (Promega, Italy) to be then sequenced in both directions (T7 and SP6).

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Gene expression analysis by Digital PCR (dPCR) 7



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The quantification of hepatic fads2 gene expression of 10 rabbits/strain was performed by

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means of The QX200 Droplet Digital PCR (ddPCR™) System.

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The concept of digital PCR was first described by Sykes et al.

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combination of limiting dilution, end-point PCR, and Poisson statistics could yield an

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absolute measure of nucleic acid concentration. Digital PCR consists on traditional PCR

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amplification and fluorescent-probe–based detection methods without the need for standard

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curves.

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Briefly, the QX200 Droplet Generator (BIO-RAD) partitioned samples (20 µl into 20,000

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nanoliter-sized droplets) for PCR amplification. Following amplification using a T100TM

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thermal cycler (BIO-RAD), droplets from each sample were analyzed individually on the

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QX200 Droplet Reader (BIO-RAD), where PCR-positive (with florescence) and PCR-

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negative droplets (without florescence) are counted to provide absolute quantification of

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target DNA in digital form.

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Poisson statistical analysis of the numbers of positive and negative droplets yields absolute

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quantitation of the target sequence. The PCR reactions were made on 50 ng of total RNA

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from each liver sample (10 rabbits/strain), using One-Step RT-ddPCR Kit for Probes (BIO-

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RAD) and a TaqMan® MGB Probe assay for rabbit fads2 gene (Life Biotechnologies). The

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amplification conditions were the following: a reverse transcription step at 55 °C for 30 min,

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enzyme inactivation at 95 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30

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sec, annealing/extension at 60°C for 1 min and a final step at 98°C for 10 min to heat

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inactivate the enzyme.

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Bisulfite specific PCR

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The CpG-rich regions of the fads2 promoter were identified by in silico analysis of rabbit

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fads2 gene sequence published in GeneBank database (sequence ID: NW_003159343.1)

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using MethPrimer (plus CpG Island Prediction) 27. The methylation of CpG sites within the

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, who recognized that the

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fads2 promoter were quantified by bisulfite specific PCR. Genomic DNA was extracted from

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liver of 10 rabbits/strains using the DNeasy® Blood and Tissue Kit (Qiagen) including RNase

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I treatment.

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Genomic DNA samples (1 µg) were acid-catalyzed converted with bisulfite using the

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EpiTect® Fast Bisulfite Kit (Qiagen) and stored at -20 °C until further analysis. Two

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fragments, one of 192 bp and the other of 282 bp, corresponding to regions of the fads2

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promoter between -272 and -463, and between -590 and -871, respectively (adenine of ATG

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was numbered +1) (Figure 1), were amplified by PCR from 100 ng of bisulfite-treated DNA.

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The PCR reactions were performed with Taq DNA Polymerase (Qiagen) supplied with Q-

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solution and primer sets: BSF_FADS2_fw1 + BSF_FADS2_rv1 and BSF_FADS2_fw2 +

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BSF_FADS2_rv2 (Table 2). Thermocycling conditions were: initial denaturation at 94 °C for

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3 min, followed by 40 cycles at 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min and a final

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extension step of 10 min at 72 °C. PCR products were then separated on 2% agarose gel,

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purified and cloned in pGEM®T-Easy vector (Promega, Italy) and then sequenced.

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Preparation of hepatic microsomes

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Microsomes were isolated from fresh rabbit liver (approximately 2 g) by standard methods.

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Livers were excised and homogenized in 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, and 0.1

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M potassium phosphate (pH 7.2). The homogenate was centrifuged at 1500 x g for 10 min to

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remove cell nuclei, unbroken cells, and debris. The supernatant was centrifuged at 8000 x g

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for 20 min to eliminate the crude mitochondrial fraction The resulting supernatant was

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centrifuged at 105,000 x g for 60 min; microsomes were resuspended in an appropriate

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amount of 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, and 0.1 M potassium phosphate (pH

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7.2); and aliquots were dispensed into vials for storage at -80 °C until use. The microsomal

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protein concentration was determined by Bradford assay 28 with bovine serum albumin as the

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standard.

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Determination of ∆6-desaturase enzymatic activity

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The ∆6-desaturase activity was determined by measuring the amount of [1-14C]-18:3n-6

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produced from [1-14C]-18:2n-6 and [1-14C]-18:4n-3 produced from [1-14C]-18:3n-3.

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The reaction medium, in a total volume of 0.5 ml, contained the following: 4 mM ATP, 0.5

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mM CoASH, 1.25 mM NADH, 2 mM MgCl2, 12 mM NaF, 1.5 mM glutathione, 0.5 M

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potassium phosphate, pH 7.2, 40 nmol 1-14C-labeled fatty acids (AS ≅ 3 nCi/nmol), and 5 mg

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microsomal proteins. Considering the amount of free LA and ALA in microsomes, in LA

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incubations a total of about 70 nmol (F) and 64 nmol (S) LA was present, whereas

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endogenous ALA was almost negligible (about 4 nmol in F and 8 nmol in S, see Table 5). In

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ALA incubations, the activity was determined in the presence of about 30 nmol (F) and 24

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nmol (S) LA (Table 5).

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In vitro assays were also performed to study the competition of ALA vs LA and viceversa on

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the ∆6-desaturase activity. ∆6-desaturase activity was determined by incubating microsomes

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with 40 nmol of radiolabeled LA or ALA (AS ≅ 3 nCi/nmol) in the presence of increased

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amounts of ALA or LA (from 20 to 160 nmol), respectively.

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The reaction was stopped by adding 1 ml of 10% (w/v) KOH in methyl alcohol. Lipids were

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saponified by heating for 1 h at 80 °C and acidified with 8 M HCl, with the fatty acids being

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extracted with n-hexane in a 3-step extraction. BF3/ methyl alcohol (14%) was added and

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heated at 100°C for 2 min to yield fatty acids methyl ester (FAME). After the 3-step

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extraction with hexane, the distribution of radioactivity among the substrates and products of

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∆6 desaturase activity were determined by TLC with silica gel plates impregnated with 10%

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AgNO3. Plates were developed in n-hexane/diethyl ether (85:15, v/v) for the separation of

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labeled trienes (C18:3n-6) from labeled dienes (C18:2n-6) and in n-hexane/diethyl ether (2:3, 10


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v/v) for the separation of labeled tetraenes (C18:4n-3) from labeled trienes (C18:3n-3). The

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spots were made visible under U.V. by spraying with 2′,7′-dichlorofluorescein (0.2% w/v in

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ethyl alcohol), scraped into scintillation vials and counted for radioactivity, using a liquid

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scintillation analyzer (Tri-carb Packard, model 1600 CA). The enzyme activity was expressed

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as pmol of 18:3n-6 or 18:4n-3 produced from 18:2n-6 or 18:3n-3, respectively in 30 min per

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mg proteins.

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Statistical analyses

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Data were assessed using the Generalized Linear Model (GLM) procedure of STATA

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Duncan multiple comparison test (p

Desaturase in Slow- or Fast-Growing Rabbit Geno - ACS Publications

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