Article pubs.acs.org/JAFC
Impact of Dietary Protein Level and Source of Polyunsaturated Fatty Acids on Lipid Metabolism-Related Protein Expression and Fatty Acid Concentrations in Porcine Tissues Dirk Dannenberger,*,† Karin Nuernberg,† Gerd Nuernberg,† and Antje Priepke‡ †
Institute of Muscle Biology and Growth and Institute of Genetics and Biometry, Leibniz Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany ‡ Institute of Animal Production, State Institute for Agriculture and Fishing Research, 18196 Dummerstorf, Germany ABSTRACT: The study assessed the effects of reduced protein (RPD) vs high protein diet (HPD) in combination with n-3/n-6 PUFA-containing plant oils [linseed oil (LO)/sunflower seed oil (SO)] supplementation on lipid metabolism-related protein expression and fatty acid concentrations in porcine tissues. Forty male Landrace pigs (castrates) were allocated into four groups fed diets different in dietary protein and PUFA level and one control group. SCD-1 protein expression in pig muscle, back fat, and liver was not affected by diet. The protein expression of precursor (pSREBP-1c) and active nuclear form of SREBP-1c (mSREBP-1c) in muscle and back fat was affected by diet, however not in liver of pigs. In contrast, the expression of ACC and FAS expression was significantly affected by diet only in the liver. The fatty acid concentrations in muscle, liver, and back fat resulted in higher n-3 PUFA concentrations of LO groups compared to the SO groups. KEYWORDS: pig, muscle, adipose tissue, lipid metabolism, PUFA, reduced protein diet, plant oils
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INTRODUCTION Manipulation of the fatty acid composition of farm animal muscle and adipose tissues has been of great interest in recent years, which is related to an increasing demand on production of meat with desirable nutritional quality for the consumer. Pork is one of the most produced meats in the European Union.1 At the present time the main strategies for altering the nutritional profile of meat are genetic selection and/or dietary manipulations. Dietary strategies used to customize fatty acid composition of pig fat have been proven to be very effective because dietary fatty acids can be incorporated into pig muscle and adipose tissues with little modifications.2−6 The intramuscular fat (IMF) content of the pork is one important trait that influences meat quality characteristics such as meat tenderness, juiciness, flavor, and taste.7 Several studies have suggested a favorable relationship between IMF and meat tenderness and juiciness and recommended that a minimum level of IMF is needed to optimize meat tenderness and consequently increase the consumer acceptance.1,8 Besides the IMF amounts, the muscle fatty acid profile is of increasing interest in regard to public health concerns. It is generally known that selected monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) in the human diet have a number of health benefits.9,10 The majority of the health benefits have been associated with n-3 PUFA. Nevertheless, there is actually a discussion of the disagreement of scientific results with regard to red meat consumption, intake of saturated fatty acids (SFA) and PUFA, and the recommendations of advisory nutrition committees.11−13 The fatty acid composition of adipose and muscle tissues in pigs can be affected by factors such as diet, species, fattiness, age/weight, depot site, gender, breed, and season and hormone levels. It has been shown that the fatty acid profile differs between various © 2014 American Chemical Society
tissues and different fat depots, and reduced energy and protein levels in growing pigs significantly increased IMF, while having only a minor effect on the amount of subcutaneous adipose tissue.14−18 The concept of the reduced protein diet (RPD) intervention is the restriction of muscle growth; however the mechanisms involved in the potential increasing of IMF and mechanisms regulating de novo fatty acid synthesis and fat partitioning in pig muscle and different adipose tissues are not fully understood.17,18 There is an increasing amount of evidence that the fat depots, the back fat and the IMF in pigs, can be regulated by independent mechanisms. IMF is the last fat depot to develop, and it may respond to dietary manipulations in a different manner when compared to other adipose tissues depots.19,20 The tissue-specific increase of expression of lipogenic enzymes could be one reason for higher de novo synthesis of fatty acids fed reduced protein levels. It is known that the regulation of lipogenesis and fat deposition is a complex process, and a range of transcription factorsSREBP1, peroxisome proliferator-activated receptor γ (PPARg)play a key role and regulate different lipogenic key enzymes.21,22 The main enzymes involved in de novo synthesis of SFA are ACC and FAS; the key enzyme in MUFA biosynthesis is SCD1; and the key enzymes in PUFA biosynthesis are Δ6desaturase (Δ6d) and Δ5-desaturase (Δ5d).20 Until now, RPD intervention in combination with n-3/n-6 PUFA-containing plant oils to increase the IMF and n-3/n-6 PUFA contents in pigs has not been investigated and should provide deeper insights into the mechanisms regulating fatty Received: Revised: Accepted: Published: 12453
September 29, 2014 November 25, 2014 November 27, 2014 November 27, 2014 dx.doi.org/10.1021/jf504699a | J. Agric. Food Chem. 2014, 62, 12453−12461
Journal of Agricultural and Food Chemistry
Article
Table 1. Experiment Design and Chemical and Fatty Acid Composition of the Diet Groups (Based on Original Matter)23 LSMSEM (n = 8)a HPD-SO start wt (kg) live wt at slaughter (kg)
68.841.60 120.560.75 ab
dry matter crude protein crude fat crude fiber crude ash starch ME (MJ/kg)
90.0 19.6 5.2 4.7 4.9 36.9 13.7
lysine methionine cysteine threonine
0.93 0.26 0.31 0.60
16:0 18:0 18:1cis-9 18:2n-6 18:3n-3
10.30 3.38 22.81 52.94 6.66
HPD-LO 69.691.60 121.380.75 a Chemical Compositionc 88.7 19.4 5.7 4.6 4.9 36.2 13.7 Amino Acidsc 0.95 0.27 0.31 0.61 Fatty Acids (%)d 10.03 3.84 17.29 29.43 36.11
RPD-SO
RPD-LO
CON
69.361.60 123.190.75 a
69.011.60 120.620.75 a
69.241.60 117.800.75 b
89.8 15.7 5.9 5.3 4.8 40.0 13.5
89.6 15.0 6.3 5.5 5.0 40.7 13.5
89.3 17.4 3.4 4.2 4.8 40.1 13.2
0.82 0.22 0.26 0.48
0.82 0.22 0.27 0.45
0.96 0.24 0.30 0.56
9.89 3.43 22.96 51.69 8.10
9.52 3.75 18.01 31.77 33.80
12.44 1.84 32.46 39.07 6.74
a
LSM: Least-squares mean. SEM: Standard error of the LSM. HPD-SO: High protein diet with sunflower oil. HPD-LO: High protein diet with linseed oil. RPD-SO: Reduced protein diet with sunflower oil. RPD-LO: Reduced protein diet with linseed oil. CON: Control. ba, b: significant effect of diet. cPercent based on original matter. dProportion of total fatty acids. animal. The pigs were weighed during the diet experiment once a week. The pig experiment was carried out at the facilities of University of Rostock (Germany), Faculty of Agricultural and Environmental Sciences. All experiments were conducted simultaneously to avoid seasonal effects between the different feeding groups. All animals were slaughtered at an average live weight of 120 kg in the abattoir of the Leibniz Institute for Farm Animal Biology in Dummerstorf (Germany). The slaughter and dressing procedures were in accordance with EU specifications. Immediately after slaughtering, tissue samples were collected from the right side of the carcass. Longissimus muscles and back fat, directly located over the muscle, were taken from the 13th/14th rib, snap-frozen, and stored at −80 °C until analysis. The liver samples were taken from the top of the Spigelian lobe, snap-frozen, and stored at −80 °C until analysis. Protein Expression Analysis via Western Blotting. Proteins were extracted by homogenizing muscle, liver, and back fat samples in ice-cold lysis buffer (1 g/L Triton X-100, 50 mmol/L HEPES/Tris, 4 mmol/L EGTA, 10 mmol/L EDTA, 100 mmol/L β-glycerophosphate, 15 mmol/L tetrasodium pyrophosphate, 5 mmol/L sodium orthovanadate, 2.5 mmol/L sodium fluoride with protease and phosphatase inhibitors, pH 7.4) using TissueRuptor (Quiagen, Hilden, Germany). After centrifugation (1000g, 10 min, 4 °C), clear protein supernatant aliquots (10 μg) were collected, analyzed for protein concentration (Bio-Rad protein assay cat. no. 500-0006, Munich, Germany) by using microtiter plates and a plate reader (SynergyMx, Biotek, Germany), and stored at −20 °C until further analysis. Post a denaturing protein sample pretreatment [thawing of protein samples, adding blue loading buffer (Cell Signaling Technology, Boston, USA), at 94 °C 4 min], proteins (10 μg/lane) were separated by using selfprepared SDS−PAGE Maxi Gels (Maxi gel casting module; Roth, Karlsruhe, Germany) (7.5% Tris-HCl, 1.0 mm, 43 well) in a Maxi Buffer tank with gel running buffer (cat. no. 3060.1; Roth, Karlsruhe, Germany) with electrophoresis constant power supply (Consort, Turnhout, Belgium). Subsequently, proteins were transferred (90 min, 1.0 mA/cm2) to a PVDF membrane (Roth, Karlsruhe, Germany) with a semidry blotting unit (Hoefer, San Francisco, CA, USA). Nonspecific binding sites were blocked with skim milk powder in TBST (w/v = 5%). For the recognition of pSREBP-1c (125 kDa) the PVDF
acid biosynthesis and fat partitioning. Recently, our research group described the effect of RPD in combination with n-3/n-6 PUFA-containing plant oils, linseed oil (lipids high in n-3 PUFA), and sunflower seed oil (lipids high in n-6 PUFA) on carcass traits, meat quality, and fatty acid profile in porcine muscle of Landrace pigs.23 We hypothesize that RPD in combination with n-3/n-6 PUFA-containing plant oil supplementation led to an increase of IMF contents including higher accumulation of precursor and de novo synthesized long-chain PUFA in muscle associated with minor effect on back fat tissue of pigs. The focus of the present study was to investigate protein expression levels of transcription factors and lipogenic enzymes related to the contents of fatty acid products in muscle, back fat, and liver of pigs fed RPD/high protein diet (HPD) supplemented with plant oils (linseed oil/sunflower seed oil).
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MATERIALS AND METHODS
Experiment. In total 40 male Landrace pigs (castrates) were used in the diet experiment. The animals were individually housed and allocated into five feeding groups (each n = 8) at a live weight of approximately 60 kg (Table 1). The pigs were fed ad libitum from 60 to 100 kg live weight and restricted to 2.8 kg/day until 120 kg. The animals in the experimental groups (1−4, Table 1) were fed with two different levels of protein and two different types of vegetable oil. The animals in the control group were fed with a normal pig diet without plant oil supplementation. The chemical and fatty acid composition of the diet of the five groups (HPD-SO, HPD-LO, RPD-SO, RPD-LO, and CON) are presented in Table 1 (Hohburg Mineralfutter, Lossatal, Germany). The diet experiment was previously described in detail.23 Briefly, the HPD was formulated to contain 19.6% crude protein, and the RPD was formulated to contain 15.5% crude protein. Sunflower oil was used in the diet groups HPD-SO and RPD-SO and linseed oil in diet groups HPD-LO and RPD-LO as fat sources. The diets contained the same level of metabolizable energy (ME) of approximately 13.6 MJ/kg. The feed intake acquisition data was performed for each single 12454
dx.doi.org/10.1021/jf504699a | J. Agric. Food Chem. 2014, 62, 12453−12461
Journal of Agricultural and Food Chemistry
Article
membrane was incubated with specific primary antibody (sc-367, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and for mSREBP1c (68 kDa) the specific primary antibody (sc-366, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used, overnight at 6 °C. After washing, the PVDF membrane was probed with the secondary antibody goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) during 90 min incubation at room temperature, followed by washing three times with TBST and water. For visualization, membranes were developed with SuperSignal West FEMTO chemiluminescent agent (ThermoScientific, Schwerte, Germany). Membranes were scanned with a Chemiluminescence Imager (Intas Science Imaging Instruments, Goettingen, Germany) and band intensities were densitometrically evaluated using LabImage 1D L340 Electrophoresis Software (Kapelan Bio-Imaging, Leipzig, Germany). For recognition of ACC, the primary antibody (ab72046, abcam, Cambridge, U.K.) for FAS (SC-20140, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and for SCD-1 (PAB9094, Abnova, Taipei, Taiwan) was used. For each protein, the same procedure with the same secondary antibody (Rabbit TrueBlot antiRabbit IgG HRP, 18-8816, eBioscience, San Diego, CA, USA) was performed as for the SREBP-1c. Western blot analyses were performed in triplicate, applying all 40 individual protein samples per tissue of all five diet groups per gel. To exclude chemiluminescent protein signal intensity differences between the three repeated blots as factors influencing protein expression data, individual protein expression values were normalized to tubulin intensity for all investigated protein expression values per blot. Statistical evaluation was performed as described in the statistical analysis section. Lipid Extraction and Fatty Acid Analysis. Samples of muscle (approximately 2.0 g), liver (approximately 2.0 g), and back fat (approximately 0.5 g) were thawed at 4 °C. The detailed sample preparation procedure has been previously described by Nuernberg et al.3 Briefly, after homogenization (Ultra Turrax, IKA, Staufen, Germany; T25, 3 × 15 s, 12,000 rpm) and the addition of the fatty acid C19:0 as an internal standard, the total lipids were extracted in duplicate using chloroform/methanol (2:1, v/v) at room temperature. All of the solvents contained 0.005% (w/v) of tert-butylhydroxytoluene (BHT) to prevent the oxidation of PUFAs. The extraction mixture was stored at 5 °C for 18 h in the dark and subsequently washed with 0.02% aqueous CaCl2. The organic phase was dried with Na2SO4 and K2CO3 (10:1, w/w), and the solvent was subsequently removed under nitrogen at room temperature. The lipid extracts were redissolved in 300 μL of toluene, and a 25 mg aliquot was used for methyl ester preparation. Next, 2 mL of 0.5 M sodium methoxide in methanol was added to the samples, which were shaken in a 60 °C water bath for 10 min. Subsequently, 1 mL of 14% boron trifluoride (BF3) in methanol was added to the mixture, which was then shaken for an additional 10 min at 60 °C. Saturated NaHCO3 (2 mL) was added, and the fatty acid methyl esters (FAMEs) were extracted three times in 2 mL of n-hexane. The solvent containing the FAMEs was reduced to dryness under an oxygen-free nitrogen stream, and the FAMEs were resuspended in 100 μL of n-hexane and stored at −18 °C until used for gas chromatography (GC) analysis. The FAMEs were evaporated under an oxygen-free nitrogen stream and dissolved in nheptane for GC analysis. The fatty acid analysis of the muscle lipids was performed using capillary GC with a CP-Sil 88 CB column (100 m × 0.25 mm, Chrompack-Varian, Lake Forest, CA, United States) that was installed in a PerkinElmer gas chromatograph Autosys XL with a flame ionization detector and split injection (PerkinElmer Instruments, Shelton, CT, United States). The detailed GC conditions were recently described by Shen et al.24 Briefly, the initial oven temperature was 150 °C, which was held for 5 min; subsequently, the temperature was increased to 175 °C and then to 200 °C at a rate of 2 °C min−1 and held for 10 min. Finally, the temperature was increased to 225 °C at a rate of 1.5 °C min−1 and held for 25 min. Hydrogen was used as the carrier gas at a flow rate of 1 mL min−1. The split ratio was 1:20, and the injector and detector were set at 260 and 280 °C, respectively. Statistics. The effects of the RPD in combination with vegetable oils on protein expression and fatty acid concentrations were estimated by one-way analysis of variance with fixed factor group (groups 1 to 5)
using the GLM procedure of the SAS software system (SAS Systems, Release 9.2, SAS Institute Inc., Cary, NC). The least-squares means (LSMs) and the standard errors (SEM) of the LSMs are given in the tables. All post hoc tests were performed at a significance level of p ≤ 0.05 using the Tukey−Kramer correction for multiple tests.
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RESULTS Protein Expression. Protein expression of the transcription factor SREBP-1c precursor (pSREBP-1c) in muscle of the present study was not affected by diet; however the active nuclear form of SREBP-1c (mSREBP-1c) was significantly increased in muscle of HPD-SO-fed pigs compared to the pigs of the other diet groups (Figure 1). For SCD-1, the protein expression in longissimus muscle of pigs was not affected by different dietary protein level or linseed/sunflower seed oil supplementation. The expression of the cytosolic enzymes in pig muscle, ACC and FAS, was low, and no abundances were detected in the total muscle extract. In back fat, the expression
Figure 1. Protein expression of SCD (A) and pSREBP-1 and mSREBP-1 (B) in longissimus muscle of pigs fed different diets (normalized to tubulin); a, b: significant effect of diet,
