Olena Doran

Effect of dietary fatty acids on expression of lipogenic enzymes and fatty acid profile in tissues of bulls

This study investigated the effects of dietary linolenic acid (C18:3n-3) v. linoleic acid (C18:2n-6) on fatty acid composition and protein expression of key lipogenic enzymes, acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase (SCD) and delta 6 desaturase (Δ6d) in longissimus muscle and subcutaneous adipose tissue of bulls. Supplementation of the diet with C18:3n-3 was accompanied by an increased level of n-3 fatty acids in muscle which resulted in decrease of n-6/n-3 ratio. The diet enriched with n-3 polyunsaturated fatty acids (PUFAs) significantly inhibited SCD protein expression in muscle and subcutaneous adipose tissue, and reduced the Δ6d expression in muscle. There was no significant effect of the diet on ACC protein expression. Inhibition of the Δ6d expression was associated with a decrease in n-6 PUFA level in muscles, whereas repression of SCD protein was related to a lower oleic acid (C18:1 cis-9) content in the adipose tissue. Expression of ACC, SCD and Δ6d proteins was found to be relatively higher in subcutaneous adipose tissue when compared with longissimus muscle. It is suggested that dietary manipulation of fatty acid composition in ruminants is mediated, at least partially, through the regulation of lipogenic enzymes expression and that regulation of the bovine lipogenic enzymes expression is tissue specific.

Differential effects of reduced protein diets on fatty acid composition and gene expression in muscle and subcutaneous adipose tissue of Alentejana purebred and Large White × Landrace × Pietrain crossbred pigs.

The present study assessed the effect of pig genotype (fatty v. lean) and dietary protein and lysine (Lys) levels (normal v. reduced) on intramuscular fat (IMF) content, subcutaneous adipose tissue (SAT) deposition, fatty acid composition and mRNA levels of genes controlling lipid metabolism. The experiment was conducted on sixty intact male pigs (thirty Alentejana purebred and thirty Large White × Landrace × Pietrain crossbred), from 60 to 93 kg of live weight. Animals were divided into three groups fed with the following diets: control diet equilibrated for Lys (17·5 % crude protein (CP) and 0·7 % Lys), reduced protein diet (RPD) equilibrated for Lys (13·2 % CP and 0·6 % Lys) and RPD not equilibrated for Lys (13·1 % CP and 0·4 % Lys). It was shown that the RPD increased fat deposition in the longissimus lumborum muscle in the lean but not in the fatty pig genotype. It is strongly suggested that the effect of RPD on the longissimus lumborum muscle of crossbred pigs is mediated via Lys restriction. The increase in IMF content under the RPD was accompanied by increased stearoyl-CoA desaturase (SCD) and PPARG mRNA levels. RPD did not alter backfat thickness, but increased the total fatty acid content in both lean and fatty pig genotype. The higher amount of SAT in fatty pigs, when compared with the lean ones, was associated with the higher expression levels of ACACA, CEBPA, FASN and SCD genes. Taken together, the data indicate that the mechanisms regulating fat deposition in pigs are genotype and tissue specific, and are associated with the expression regulation of the key lipogenic genes.

Combined effects of dietary arginine, leucine and protein levels on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of crossbred pigs

The cumulative effects of dietary arginine, leucine and protein levels on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig longissimus lumborum muscle and subcutaneous adipose tissue (SAT) were investigated. The experiment was performed on fifty-four intact male pigs (Duroc × Pietrain × Large White × Landrace crossbred), with a live weight ranging from 59 to 92 kg. The pigs were randomly assigned to one of six experimental treatments (n 9). The treatments followed a 2 × 3 factorial arrangement, with two levels of arginine supplementation (0 v. 1 %) and three levels of a basal diet (normal protein diet, NPD; reduced protein diet, RPD; reduced protein diet to achieve 2 % of leucine, RPDL). The results showed that dietary arginine supplementation did not affect the intramuscular fat (IMF) content and back fat thickness, but increased the total fat in SAT. This effect was associated with an increase in fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) mRNA levels in SAT, which suggests that arginine might be involved in the differential regulation of some key lipogenic genes in pig muscle and SAT. The increase in IMF content under the RPD, with or without leucine supplementation, was accompanied by increased FASN and SCD mRNA levels. Arginine supplementation did not influence the percentage of main fatty acids, while the RPD had a significant effect on fatty acid composition in both tissues. Leucine supplementation of RPD did not change IMF, total fat of SAT and back fat thickness, but increased 16 : 0 and 18 : 1cis-9 and decreased 18 : 2n-6 in muscle.

Effect of pig breed and dietary protein level on selected fatty acids and stearoyl-coenzyme A desaturase protein expression in longissimus muscle and subcutaneous fat

The aims of the study were 1) to investigate effects of a low protein diet on fatty acids content and composition of the LM and subcutaneous adipose tissue in 3 genetically diverse breeds, Large White × Landrace, Alentejano, and Bizaro, and 2) to determine whether the effect of the low protein diet of fatty acid composition is associated with dietary modulation of stearoyl-CoA desaturase (SCD) protein expression. The experiments were conducted on 12 Large White × Landrace, 12 Alentejano, and 10 Bízaro female and male pigs. The average animal BW at the beginning of experiments was 40.8, 40.7, and 38.3 kg for Large White × Landrace, Alentejano, and Bízaro, respectively, and the BW of animals at slaughter was 90 kg. The diets contained 202 or 169 g/kg DM of CP (high and low protein diets, respectively) and were balanced in essential AA. The diets were fed until the animals reached 90 kg BW (approximately 73 d). It was established that Large White × Landrace pigs had a less (P = 0.001) total fatty acid content in subcutaneous adipose tissue when compared with Alentejano and Bízaro and less (P < 0.001) intramuscular fat (IMF) content when compared with Alentejano. There was a positive relationship between SCD protein expression in the LM and MUFA content (r = 0.627, P = 0.029) and SCD protein expression and total muscle fatty acids content (r = 0.725, P = 0.008) in Large White × Landrace but not in Alentejano and Bizaro breeds. It has been suggested that SCD protein expression is associated with regulation of fat deposition only in breeds with genetic predisposition to a low IMF content.

Fatty acid composition and lipogenic enzyme protein expression in subcutaneous adipose tissue of male pigs vaccinated against boar taint, barrows, and entire boars.

Objectives of this study were to compare fatty acid composition of subcutaneous adipose tissue of entire boars, barrows, and male pigs vaccinated against boar taint with a vaccine containing a GnRH analogue-protein conjugate (Improvac, Pfizer Animal Health) and to investigate the association between fatty acid composition and protein expression of key lipogenic enzymes in entire boars, barrows, and vaccinated pigs. Differences between groups were observed in the content of total SFA (P≤0.001), MUFA (P=0.035), and n-6 PUFA (P≤0.001) but not n-3 PUFA (P=0.373). Total SFA were greater (P<0.001) in barrows and vaccinated pigs compared with entire animals. This was accompanied by an increase (P<0.05) in the protein expression of the lipogenic enzyme fatty acid synthase in barrows and vaccinated pigs. Total MUFA content was increased (P<0.001) in barrows compared with entire and vaccinated pigs. This was not accompanied (P>0.05) by an increase in expression of stearoyl-CoA desaturase protein, the enzyme catalyzing MUFA biosyntheses. Total n-6 PUFA content did not differ (P<0.001) between entire and vaccinated pigs but was lower in barrows. Expression of Δ6-desaturase protein, one of the key enzymes of PUFA biosynthesis, was greater (P<0.05) in vaccinated pigs than in barrows but did not differ significantly between vaccinated and entire animals. We conclude that fatty acid profile of animals vaccinated against boar taint is similar to that of entire male pigs and that the effect of physical castration and vaccination on fatty acid composition involves changes in lipogenic enzyme protein expression.

Investigation of Testosterone, Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.

Influence of oilseed supplement ranging in n-6/n-3 ratio on fatty acid composition and Δ5-, Δ6-desaturase protein expression in steer muscles

This study investigated effects of roasted or extruded oilseed supplementation ranging in n-6/n-3 ratios from 0.3 to 5.0 on the fatty acid composition and expression of delta-5 desaturase (Δ5d) and Δ6-desaturase (Δ6d) protein in commercial steer cheek (m. masseter) and diaphragm (pars costalis diaphragmatis) muscles. In general, the n-6/n-3 ratio of the diet had a subsequent effect on the muscle n-6/n-3 ratio (P < 0.05), with muscle 18:2n-6 and 18:3n-3 content relating to proportion of dietary soya bean and linseed (P < 0.01). Compared with canola, pure linseed and soya bean diets reduced 14:1c-9 and 16:1c-9 (P < 0.05) but increased 18:1t-11 and c-9,t-11 conjugated linoleic acid (CLA) content (P < 0.01). Oilseed processing had a minor influence but extruded oilseeds increase 18:1t-11 and c-9,t-11 CLA compared with roasted (P < 0.05). Polar lipid 18:3n-3 and n-3 long-chain polyunsaturated fatty acid (LC, ⩾20 carbons PUFA) derivative content increased in relation to dietary linseed supplementation in the diaphragm (P < 0.01), whereas only 18:3n-3 was increased in the cheek (P < 0.01). Protein expression did not differ between diets; however, in each muscle the Δ5d protein expression had a stronger association with the desaturase products rather than the precursors. The relationship between Δ5d protein expression and the muscle LC n-6/n-3 ratio was negative in both muscles (P < 0.05). The relationship between Δ6d protein expression and the LC n-6/n-3 ratio was positive in the cheek (P < 0.001) and negative in the diaphragm (P < 0.05). In conclusion, diet n-6/n-3 ratio affected muscle 18:2n-6 and 18:3n-3 deposition, whereas the Δ5d and Δ6d protein expression had some influence on the polar lipid LC-PUFA profile. Results reaffirm that processed oilseeds can be used to increase the proportion of fatty acids potentially beneficial for human health, by influencing the formation of LC-PUFA and reducing the n-6/n-3 ratio.

Metabolism of Androstenone, 17β-Estradiol and Dihydrotestosterone in Primary Cultured Pig Hepatocytes and the Role of 3β-Hydroxysteroid Dehydrogenase in This Process

Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD) in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS). 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5∶1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

Influence of betaine and arginine supplementation of reduced protein diets on fatty acid composition and gene expression in the muscle and subcutaneous adipose tissue of cross-bred pigs

The isolated or combined effects of betaine and arginine supplementation of reduced protein diets (RPD) on fat content, fatty acid composition and mRNA levels of genes controlling lipid metabolism in pig m. longissimus lumborum and subcutaneous adipose tissue (SAT) were assessed. The experiment was performed on forty intact male pigs (Duroc×Large White×Landrace cross-breed) with initial and final live weights of 60 and 93 kg, respectively. Pigs were randomly assigned to one of the following five diets (n 8): 16·0 % of crude protein (control), 13·0 % of crude protein (RPD), RPD supplemented with 0·33 % of betaine, RPD supplemented with 1·5 % of arginine and RPD supplemented with 0·33 % of betaine and 1·5 % of arginine. Data confirmed that RPD increase intramuscular fat (IMF) content and total fat content in SAT. The increased total fat content in SAT was accompanied by higher GLUT type 4, lipoprotein lipase and stearoyl-CoA desaturase mRNA expression levels. In addition, the supplementation of RPD with betaine and/or arginine did not affect either IMF or total fat in SAT. However, dietary betaine supplementation slightly affected fatty acid composition in both muscle and SAT. This effect was associated with an increase of carnitine O-acetyltransferase mRNA levels in SAT but not in muscle, which suggests that betaine might be involved in the differential regulation of some key genes of lipid metabolism in pig muscle and SAT. Although the arginine-supplemented diet decreased the mRNA expression level of PPARG in muscle and SAT, it did not influence fat content or fatty acid composition in any of these pig tissues.

Effect of Breed on Fatty Acid Composition and Lipogenic Enzyme Abundance in the Subcutaneous Adipose Tissue of Pigs

UNLABELLED This study investigated the role of lipogenic enzyme expression in breed-specific fat deposition in pigs. OBJECTIVES (i) determine effect of breed on the relative abundance of the key lipogenic enzymes stearoyl-CoA desaturase (SCD), delta-6 desaturase (Δ6D), and fatty acid synthase (FAS) in pig subcutaneous adipose tissue. (ii) Investigate breed-specific relationships between lipogenic enzyme abundance and fatty acid composition. Large White × Piétrain, Piétrain, and Duroc × Piétrain pigs were used. Expression of SCD, Δ6D, and FAS was analyzed by Western blotting. Fatty acid composition was determined by gas chromatography. FAS protein in Large White × Piétrain pigs was similar to the Piétrain breed, but was significantly higher than Duroc × Piétrain. A positive relationship was found between FAS abundance and the saturated fatty acids (SFAs), for Large White × Piétrain pigs, but not for the other breeds. Δ6D was significantly higher in Large White × Piétrain compared with Duroc × Piétrain and Piétrain. This was accompanied by significantly higher total n-3 poly-unsaturated fatty acids (PUFAs) in the Large White × Piétrain when compared to the other breeds. CONCLUSIONS (i) increased subcutaneous adipose tissue SFA content in Large White × Piétrain pigs (but not Piétrain and Duroc × Piétrain) is related to increased abundance of FAS protein; (ii) high n-3 PUFA content in Large White × Piétrain pigs is related to activation of Δ6D protein synthesis; (iii) SCD and Δ6D abundance does not contribute to between-breed differences in MUFA and n-6 PUFA content of pig subcutaneous adipose tissue.