DOI: 10.1111/jpn.12197
ORIGINAL ARTICLE
Effects of soyabean meal- or whey-based diets on lipid metabolism in weaned piglets G. Theodorou1, G. Papadomichelakis2, E. Tsiplakou2, A. D. Lampidonis1, S. Chadio3, G. Zervas2 and I. Politis1 1 Department of Animal Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece 2 Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece, and 3 Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
Summary The present study aimed to test the hypothesis that dietary protein source influences lipid metabolism-related parameters weaned piglets. The effects of soyabean meal (SB) and whey proteins (WP) on gene expression of several genes involved in the lipogenic process in liver, visceral (VAT) and subcutaneous (SAT) adipose tissues, plasma insulin concentration and fatty acid (FA) profile were investigated in 18 weaned piglets. Weaned piglets were fed one of two diets containing either SB or WP as the main protein source. Following a 10-h fasting period, plasma insulin concentration and FA profile were assessed at 56 and 72 days of age, whereas gene expression in liver, VAT and SAT was assessed at 72 days of age. Plasma insulin concentration was not affected by diet, although it was 40% lower in SB fed pigs. The SB pigs had lower 14:0 (p < 0.01) and higher 18:3n-3 (p < 0.001) levels in plasma in comparison with WP pigs. However, these changes were attributed to background differences in the dietary FA profile and not to a direct protein source effect. Gene expression of sterol regulatory elementbinding protein 1 (SREBP-1) in liver and VAT were lower (p < 0.01 and p < 0.05, respectively) in SB compared to WP fed piglets, but no differences occurred in SAT. No changes were observed in sterol regulatory elementbinding protein 2, liver X receptor, peroxisome proliferator-activated receptors a and c and plasminogen activator inhibitor 1 mRNA levels, either in liver or in adipose tissues. In conclusion, dietary protein source, accompanied likely by side alterations in the dietary composition, affects lipid metabolism in pigs through the downregulation of SREBP-1, which is a crucial determinant of lipogenic process. Keywords soyabean meal, whey proteins, lipid metabolism, pigs Correspondence Georgios Theodorou, 75 Iera Odos Str., 118 55 Athens, Greece. Tel: +302105294450; Fax: +30210529442; E-mail: [email protected] Received: 10 November 2013; accepted: 2 April 2014
Dietary interventions that affect lipid metabolism in pigs have received considerable attention, because they represent a useful tool to modulate body fat deposition and improve meat quality (Wood et al., 1996; Gondret and Lebret, 2002). In addition to the agricultural importance, the investigation of such dietary treatments in pigs could serve as a valuable model for studying the intermediary lipid metabolism in humans (Carey, 1997). In this sense, numerous studies have focused in investigating the mechanisms underlying the influence of nutritional treatments on the lipogenic process. Sterol regulatory element-binding protein 1 (SREBP-1) is one of the key transcription factors affected by dietary fat source or crude protein level in pigs. Supplementing diets with very long polyunsaturated (PUFA) fatty acids (Duran-Montg e
et al., 2009) or increasing crude protein content (Zhao et al., 2010) downregulates the expression of SREBP1, which in turn suppresses the gene expression of lipogenic enzymes involved in fatty acid synthesis (acetyl-CoA carboxylase, fatty acid synthase, stearoylCoA desaturase) in liver or adipose tissue. Recent studies in rats and mice have indicated that the source of dietary protein has been shown to affect lipid metabolism. Soy protein when compared to casein reduces the gene expression of lipogenic enzymes in liver (Horton et al., 2002; Ascencio et al., 2004; Tovar et al., 2005) by downregulating SREBP-1 directly (Ascencio et al., 2004) or indirectly, via the liver X receptor (LXR) (Hegarty et al., 2005). This effect is thought to be mediated by the decreased insulin to glucagon ratio, which has been attributed to the lower lysine to arginine ratio and the higher glycine content of soy proteins (Tovar et al., 2002).
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Introduction
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Concomitantly, soy proteins may stimulate proliferator-activated receptor a (PPAR-a) in liver and PPAR-c in adipose tissue increasing, thus, fatty acid oxidation (Tovar et al., 2005) and fatty acid uptake from plasma (Farmer, 2005) respectively. Plasminogen activator inhibitor 1 (PAI-1), apart from its well-known role in maintaining normal haemostasis by regulating the fibrinolytic system, has been also implicated in many other physiological processes, such as the adipose tissue development (Lijnen et al., 2005). To the best of our knowledge, the effects of protein source on the expression of the transcription factors controlling lipogenesis, such as SREBP-1, SREBP-2, LXR and PPAR-a in liver and SREBP-1, SREBP-2, PAI-1 and PPAR-c in adipose tissue, have not been investigated in pigs so far. Soyabean meal and milk products, such as dried whey concentrates, are two protein sources widely used in pig feeding (Mahan, 1993; Yun et al., 2005; Yang et al., 2007); hence, they are suitable for the purpose of this study. The objective of the present study was to compare the effects of using soyabean meal or whey proteins as the main dietary protein source on the gene expression of SREBP-1, SREBP-2, LXR and PPAR-a in the liver and SREBP-1, SREBP-2, PAI-1 and PPAR-c in visceral and subcutaneous adipose tissue of weaned piglets. Plasma insulin concentration and fatty acid profile were analysed in to evaluate possible relationships with gene expression. Materials and methods Animals and diets
Handling and care of the experimental animals conformed to the guidelines of the Faculty of Animal Science and Aquaculture of Agricultural University of Athens. Eighteen castrated male Large White 9 Duroc 9 Landrace piglets weaned at 29 2 days of age were selected from a commercial farm located near the city of Athens. Upon arrival at the experimental facilities, they were allowed an adaptation period of 4 days and were then allotted into two groups (9 pigs/group) balanced for body weight (average BW of 8.4 0.68 kg; mean SD). Piglets were kept in individual cages (1.2 9 0.5 m) and fed ad libitum the following two diets, over a period of 38 days: diet SB, which was formulated to meet the nutrient requirements of piglets (NRC, 1998) using soyabean meal (SB) as the main crude protein (CP) source and diet WP, where SB was totally replaced with a mixture of whey proteins [WP, 70% WheyPro65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg); Hellenic Proteins S.A., Veria, Greece], on equal digestible energy (DE) and CP 2
basis. The mixture of whey proteins was designed to have a content of 660 g CP/kg. The ingredient and chemical composition of diets are summarized in Table 1. Two blood samplings were performed at 56 and 72 days of age (i.e. 21 and 42 days after the start of the experiment, respectively). A 10-h fasting period was applied prior to sampling so as to avoid fluctuations of insulin levels, due to differences in feed intake time. Blood samples were collected in heparinized tubes, and plasma was separated by centrifugation (2000 g, 15 min, 4 °C) and stored at 20 °C for insulin and fatty acid (FA) determination. At the end of the experiment (72 days of age), piglets were stunned and killed by exsanguination. Immediately after, the left lobe of liver and samples of visceral (VAT) and subcutaneous (SAT, neck backfat) adipose tissue were excised, immersed in liquid nitrogen until frozen and subsequently kept at 80 °C pending RT-PCR analyses. Chemical analyses
Feed samples were milled through 1-mm screen prior to analyses. Feed DM was assessed in 5 g samples by oven drying at 105 °C overnight. Routine procedures of Association of Official Analytical Chemists (AOAC) (1984) were used for ether extract (EE; 7.063). Crude protein (CP) was determined as 6.25 9 Kjeldahl nitrogen, using a Kjeltec autoanalyzer unit (Foss, Sweden). All analyses were performed in duplicate. Plasma insulin determination
Plasma insulin concentrations were measured using a double antibody radioimmunoassay kit (rat insulin, Millipore, St. Charles, Missouri, USA), which has 100% specificity for porcine insulin. The sensitivity of the method was 0.081 ng/ml, and the intra- and interassay coefficients of variation were 4.6 and 9.4% respectively. Fatty acid methylesters synthesis and determination
The FA of diets was hydrolysed (with methanolic KOH) and methylated (sulphuric acid catalysis) directly, according to O’Fallon et al. (2007) in duplicate 1 g ground samples. The plasma FA analysis was carried out by the method of Bondia-Pons et al. (2004). Duplicate plasma samples (100 ll) were hydrolysed (with sodium methylate) and subsequently esterified (boron trifluoride-methanol, BF3). In both methods, FA methylesters (FAME) were extracted Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Table 1 Ingredient and chemical composition of the experimental diets
Table 1 (Continued)
Diet* SB Ingredients (g/kg) Maize 621.0 Soyabean meal 342.0 (440 g CP/kg) Whey proteins† – (660 g CP/kg) L-Lysine 80% 2.0 DL-Methionine 99% 1.0 Sodium chloride 5.0 Calcium carbonate 13.0 Monocalcium phosphate 13.0 Mineral–vitamin pre-mix‡ 3.0 Analysed chemical composition (g/kg DM) Dry matter (g/kg) 888.0 Crude protein 232.0 Ether extract 33.1 Total weights of FA 31.4 (mg/100 g DM) Individual FA (% of total FA) 14:0 0.08 16:0 14.40 cis9-16:1 0.16 18:0 2.63 cis9-18:1 21.71 18:2n-6 54.28 18:3n-3 3.35 20:0 0.33 20:1n-9 0.22 22:2 0.60 Calculated chemical composition (g/kg DM)§ Digestible energy 15.5 (MJ/kg DM) Net energy (MJ/kg DM) 9.0 SID¶ crude protein 166 Total methionine + cystine 8.9 SID¶ methionine + cystine 6.9 Lysine 14.0 SID¶ lysine 11.2 Arginine 16.4 SID¶ arginine 12.1 Total lysine/total 0.85 arginine ratio Threonine 8.9 SID¶ threonine 7.2 Glycine 9.7 Calcium 9.2
Diet* WP
754.0 – 210.0 2.0 1.0 4.0 14.0 12.0 3.0 903.0 228.0 40.4 38.7
2.91 17.87 0.40 4.42 24.27 41.42 1.32 0.34 0.20 0.77 15.4 9.6 177 10.4 8.6 17.3 14.2 8.2 5.4 2.11 13.2 11.2 6.1 9.1
SB Total phosphorus Sodium
7.4 2.4
WP 8.4 2.4
*SB, soyabean meal as protein source; WP, whey proteins as protein source. †Mixture of whey proteins (Hellenic Proteins S.A.); 70% WheyPro 65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg) designed to have a content of 660 g CP/kg. ‡Mineral–vitamin pre-mix (Nuevo S.A., N. Artaki, Greece) provided per kg of diet: 15 000 IU vitamin A (retinyl acetate), 2000 IU vitamin D3 (cholecalciferol), 100 mg vitamin E (DL-a-tocopheryl acetate), 3.5 mg menadione (vitamin K3), 2.5 mg vitamin B1, 6 mg vitamin B2, 3 mg vitamin B6, 25 lg cyanocobalamin, 25 mg nicotinic acid, 20 mg pantothenic acid, 2 mg folic acid, 250 lg biotin, 2 mg Co, 4 mg I, 600 lg Se, 300 mg Fe, 100 mg Mn, 100 mg Mg, 320 mg Cu and 240 mg Zn. §Digestible and net energy, macro-element, total amino acid and standardized ileal digestible (SID) protein and amino acid values for maize, soyabean meal and whey proteins were adapted from tabulated data (FEDNA, 2003; NRC, 1988). ¶Standardized ileal digestible nutrients. No data were available on glycine SID values.
Autosystem XL GC equipped with a 30 m 9 0.25 mm i.d. 9 0.25 lm film thickness ΗΡ-Innowax capillary column (Agilent Technologies, Santa Clara, CA, USA, J&W GC columns) and a flame ionisation detector (FID). The oven temperature was programmed for 1 min at 140 °C, raised by 2.5 °C/min to 200 °C, then to 230 °C by 1 °C/min and held for 1 min, and finally to 240 °C by 4 °C/min and held for 10 min. Helium was the carrier gas at a constant pressure of 18 psi, and the temperature of both the injector and FID was set at 250 °C. Fatty acids were identified by comparison with FAME 37 Component Mix (Supelco, Bellefonte, PA, USA), and quantification was achieved using the internal standard (13:0) added prior to hydrolysis. Total weights of FA (mg/100 g) in diets were calculated as the sum of areas for all FA peaks compared to area for 0.5 mg internal standard. Individual FA were expressed as% by weight of total FA. RNA extraction and RT-PCR analysis
with clear n-hexane and transferred into gas chromatograph (GC) vials. The FAME were subsequently analysed in a temperature-programmed run using a Perkin Elmer
Tissue samples were ground while frozen with a mortar and pestle using liquid nitrogen. Total RNA was extracted using TriReagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The quality and quantity of the RNA extracted were confirmed by spectrophotometry as well as gel electrophoresis. Relative levels of mRNA were quantified with real-time, quantitative RT-PCR using SYBR
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Green chemistry. A pair of primers for each of the genes used in this study was constructed using PERLprimer software (Marshall, 2004). All primer pairs are presented in Table S1. The amount of sample RNA was normalized using b-actin and RNA polymerase II (RNA-PII) as reference genes. Equal amounts of total RNA were reverse transcribed with the PrimeScript First Strand cDNA Synthesis Kit (Takara, Otsu, Shiga, Japan), according to the manufacturer’s instructions using a mix of random hexamers and oligo-dT primers. The real-time PCR was performed in the 7500 Real Time system (Applied Biosystems, Carlsbad, CA, USA) using the KAPA SYBR® FAST qPCR Kit (Kapa biosystems, Woburn, MA, USA) according to the manufacturer’s protocol. Each reaction (10 ll) contained 12.5 ng RNA equivalents as well as 150– 300 nM of forward and reverse primers for each gene. The reactions were incubated at 95 °C for 30 s followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. This was followed by a melt curve analysis to determine the reaction specificity. Each sample was measured in duplicates. A modified version of the Pfaffl (2001) normalization method was used for normalization against the two reference genes (Hellemans et al., 2007). Additional information regarding the quantitative RT-PCR assays is provided in Table S2. Statistical analysis
Data were analysed using the SPSS statistical package (version 17.0; IBM, Armonk, NY, USA). Plasma insulin and FA were analysed using a general linear model (GLM) for repeated measures with diet and age as fixed effects, and their interactions and data are presented as least squares (LS) means SEM. For all relative expression analyses, WP was set to 1 and the expression of the SB group is presented as x-fold expression to the WP group, Comparisons between diets were conducted by one-way ANOVA, and data are given as means SEM. Additionally, comparisons between tissues in the expression of SREBP-1 oneway ANOVA were conducted, after setting the mean liver expression arbitrarily to 1. Significance was set at 0.05. The relationship between blood insulin levels and SREBP-1 expression in liver was determined by Pearson’s linear correlation analysis.
fed pigs. Also, age has not been shown to significantly affect insulin levels (Table 2). Plasma 14:0 percentage was lower (p < 0.01), while 18:3n-3 and 20:5n-3 were higher (p < 0.001 and p < 0.01, respectively) for SB compared to WP fed piglets. Decreased 14:0 (p < 0.01), 18:2n-6 (p < 0.001), 18:3n-3 (p < 0.001), 20:5n-3 (p < 0.01) and increased 18:0 (p < 0.001) and 20:4n-6 (p < 0.01) were observed with increasing age. These differences resulted in a proportional increase in plasma SFA (p < 0.001) and a decrease in PUFA (p < 0.05) with age (Table 2). Relative transcription factor mRNA levels in the different tissues
The relative SREBP-1 mRNA level (as SREBP-1/bactin and RNA-PII ratio) in liver homogenates was approximately 45% lower (p < 0.01) in SB compared to WP pigs (Fig. 1). On the other hand, the relative mRNA levels of SREBP-2, LXR and PPAR-a (Fig. 1) were not affected by diet. In VAT, differences similar to those found in liver were observed. The relative SREBP-1 mRNA level was lower (p < 0.05) in SB compared to WP pigs (Fig. 1). No differences were detected in SAT, and moreover, dietary treatments did not affect the expression of SREBP-2, PAI-1 and PPAR-c. The comparison between tissues showed that SREBP-1 expression was lower (p < 0.05) in VAT with regard to liver and SAT (Fig. 2). No correlation was found between plasma levels of insulin and liver SREBP-1 expression (R2 = 0.0018. p = 0.85). Discussion
No statistically significant differences in insulin levels were detected between WB and SB groups, although levels were 1.7 times higher for WP compared to SB
The present work was based on the hypothesis that dietary protein source affects the relative mRNA expression of the transcription factors involved in lipid metabolism in pigs. However, in practical pig feeding, a change in the protein source is inevitably accompanied by side alterations in other nutrients, in an attempt to comply with nutrient recommendations. In addition to the protein source, diets SB and WP differed in the corn percentage (and hence starch content), the net energy content and FA composition (Table 1), the potential impact of which is discussed. Transcription factor SREBP-1 is expressed in the liver and adipose tissues in pigs and rodents, where it regulates the expression of several lipogenic enzymes (Ascencio et al., 2004; Hsu et al., 2004). Its expression is reduced in rats and mice fed soy proteins (Torres et al., 2006), which have lower lysine to arginine ratio compared to casein (Sanchez and Hubbard, 1991). The present work showed that the expression of
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Results Plasma insulin and fatty acid profile
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Table 2 Plasma insulin concentration (ng/ml) and fatty acid (FA) composition (% of total FA) in piglets fed diets with soyabean meal or whey proteins (LS means SEM) Diet (D)*
Insulin 14:0 16:0 18:0 cis9-18:1 18:2n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3 SFA§ MUFA¶ PUFA**
Age (A)
Significance‡
SB
WP
SEM†
56 days
77 days
SEM†
D
A
D9A
191.9 0.54 20.26 17.10 15.49 18.97 0.48 0.56 9.21 0.42 1.13 2.22 39.46 19.91 34.22
318.1 0.67 21.08 17.20 14.79 18.51 0.20 0.57 9.93 0.28 1.15 2.26 40.44 18.70 34.23
55.95 0.031 0.351 0.449 0.403 0.669 0.015 0.031 0.423 0.032 0.097 0.124 0.365 0.500 0.556
279.8 0.67 20.96 15.51 14.73 20.32 0.40 0.57 8.41 0.40 1.15 2.68 39.13 19.16 35.14
230.3 0.54 20.38 18.79 15.55 17.16 0.28 0.53 10.73 0.29 1.13 1.80 40.87 19.45 33.31
48.30 0.029 0.329 0.415 0.380 0.553 0.015 0.030 0.411 0.027 0.078 0.114 0.309 0.470 0.539
ns ** ns ns ns ns *** ns ns ** ns ns ns ns ns
ns ** ns *** ns *** *** ns ** ** ns *** *** ns *
ns ns ns ns ns ns * ns ns t ns ns * ns ns
*SB, soyabean meal as protein source; WP, mixture of whey proteins [WP, 70% WheyPro65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg); Hellenic Proteins S.A.] as protein source. †SEM, pooled standard error of means (n = 9 individual piglets per diet). ‡ns, not significant; t, 0.10 < p < 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. §SFA, total saturated FA, including individual FA which are not presented (12:0 + 14:0 + 15:0 + 16:0 + 17:0 + 18:0). ¶MUFA, total monounsaturated FA, including individual FA which are not presented (cis9-16:1 + cis7-16:1 + cis9-18:1 + cis11-18:1). **PUFA, total polyunsaturated FA, including individual FA which are not presented (18:2n-6 + 18:3n-6 + 18:3n-3 + 20:2 + 20:3n-6 + 20:4n-6 + 20:5n-3 + 22:4n-6 + 22:5n-3 + 22:6n-3).
Fig. 1 Gene expression of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2), liver X receptor (LXR) and peroxisome proliferator-activated receptor a (PPAR-a) in liver, and of SREBP-1, SREBP-2, plasminogen activator inhibitor-1 (PAI-1) and peroxisome proliferator-activated receptor c (PPAR-c) in visceral (VAT) and subcutaneous (SAT) adipose tissues in 72-day-old pigs fed diets with soyabean meal (SB) or whey proteins (WP) (means SEM). mRNA levels were normalized to b-actin and RNA polymerase II (RNA-PII). The mean value of the WP group within each gene and tissue was set to 1. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01) in the expression between diets.
SREBP-1 was significantly lower in the SB, compared to WP fed pigs, both in liver and in VAT. The result was similar to that observed in rats and mice fed soy proteins (Ascencio et al., 2004). The understanding of the mechanism by which SB proteins and their amino acid profile exert their effects on SREBP-1 is not complete. In rodents, several studies led to the conclusion that the regulation of SREBP-1 is mediated mainly by
insulin; it is decreased in rats and mice fed soy proteins concentrates in contrast to those fed casein (Tovar et al., 2002) and in turn downregulates SREBP-1 (Ascencio et al., 2004; Torres et al., 2006). However, earlier studies in pigs have failed to establish a clear relationship between dietary amino acid profile and insulin secretion (Zijlstra et al., 1996) or between insulin and expression of SREBP-1 (Zhao et al.,
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2010). In the current study, the 40% lower insulin in SB compared to WP fed pigs (Table 2) may imply a pattern similar to that seen with rodents (i.e. linkage between protein source, insulin and SREBP-1). Those numerically lower values could be partially explained by the lower starch content in SB diets (lower maize). However, the statistically insignificant differences preclude from arriving to any valid conclusion. Furthermore, no correlation was found between plasma insulin levels and SREBP-1 expression. These data would suggest that insulin might not be the only factor regulating SREBP-1 expression in pigs and that other hormones may be involved (Gondret et al., 2001) in a concerted or not action with insulin. Another issue not to be dismissed herein is the dietary net energy (NE) content. Despite the equal dietary digestible energy (DE), the NE content was lower in SB compared to WP diet by 0.6 MJ/kg (calculated values; Table 1). In addition to the aforementioned aspects, the lower NE content could have likely contributed to the lower SREBP-1 expression in SB pigs. This hypothesis has not been considered by Zhao et al. (2010) and Tovar et al. (2005) and should be further investigated. Regardless to the mechanisms involved, the downregulation of SREBP-1 by SB is an important finding emerging from the present work, as it is a major determinant of tissue lipogenic capacity in pigs (Gondret et al., 2001). In contrast to SREBP-1, no effects of dietary protein source on the expression of SREBP-2 and LXR in liver or SREBP-2 in adipose tissues were observed in pigs. The SREBP-2 preferentially binds to promoters of genes involved in cholesterol metabolism, such as hydroxymethyl glutaryl-CoA (HGM-CoA) reductase and the low-density lipoprotein receptor (LDLr)
(Amemiya-Kudo et al., 2002). In soy protein fed rats, the expression of SREBP-2 increases as a result of low cholesterol content in the liver, with respect to those fed a casein diet (Tovar et al., 2005), and increases the expression of HMG-CoA reductase and LDLr (Ascencio et al., 2004). These decreased cholesterol and oxysterol (intermediates of cholesterol metabolism) concentrations (Torres et al., 2006) in turn inhibit the transcription of LXR in liver, which additionally reduces SREBP-1 expression (Steffensen and Gustafsson, 2004). However, the respective impact of LXR on SREBP-1 is expected to be quite low (Hegarty et al., 2005). The present data suggest firstly that cholesterol metabolism was not affected by diets, as indicated by SREBP-2 and LXR in liver and SREBP-2 in adipose tissue, and secondly, both SREBP-2 and LXR played a minor and/or no role in regulating SREBP-1 expression in liver. The lack of difference in the expression of PPAR-a in liver between SB and WP fed pigs indicated that diet composition could have not affected FA oxidative metabolism in contrast to reports in rats (Tovar et al., 2005). The transcription factors PPAR-c and PAI-1 have a functional role in differentiation and development of adipose tissue. The PPAR-c stimulates FA uptake and esterification into triglycerides in a concerted action with SREBP-1 that regulates lipogenesis to fill the lipid droplet (Farmer, 2005). Recent studies in rats showed that PPAR-c is activated in adipose tissue by soy proteins, resulting in an upregulation of adipogenesis and probably FA uptake from plasma in contrast to casein (Tovar et al., 2005). High expression of PAI-1 may be associated with lower body and fat pad weight and reduced adipocyte size, but its specific role in adipose tissue is not clear (Lijnen et al., 2005). Therefore, dietary protein source did not appear to affect FA uptake, transport and oxidative metabolism in the present study. An important issue to be addressed in the present study was the potential effect of dietary FA on the transcription factors, and particularly SREBP-1. Diet SB had relatively lower saturated FA (SFA; mainly 14:0, 16:0 and 18:0) and higher polyunsaturated FA (PUFA; mainly 18:2n-6 and 18:3n-3) percentages than diet WP (Table 1). Polyunsaturates have strong inhibitory effects on SREBP-1 (Deckelbaum et al., 2006) and LXR expression (Pawar et al., 2003) and can bind directly to PPAR-a upregulating its expression (Desvergne and Wahli, 1999; Sampath and Ntambi, 2005) in the liver of rats. However, previous studies in pigs have showed that only very long-chain dietary n-3 PUFA, such as those of fish or DHA oil (rich in 22:6n-3), have the capacity of inhibiting the SREBP-1, but not PPAR-a, expression in liver,
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Fig. 2 Differences in the relative expression of sterol regulatory element-binding protein 1 (SREBP-1) between liver, visceral (VAT) and subcutaneous (SAT) adipose tissue in 72-day-old pigs (means SEM). mRNA levels were normalized to b-actin and RNA polymerase II (RNA-PII). Mean liver expression was arbitrarily set to 1. Different letters indicate significant differences in gene expression between tissues (p < 0.05).
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whereas no effects have been observed in adipose tissue (Hsu et al., 2004; Liu et al., 2005; Duran-Montge et al., 2009). On the other hand, the effects of tallow, sunflower or linseed oil (rich in SFA, 18:2n-6 and 18:3n-3, respectively) on transcriptional factors in liver and adipose tissue were insignificant (DuranMontge et al., 2009). Thus, it appears unlikely that the rather small differences observed in the dietary FA profile herein could have produced background effects on SREBP-1 mRNA expression, either in liver or in adipose tissues. The differences in plasma (Table 2) FA profile between SB and WP pigs mainly depicted the incorporation of dietary FA into plasma lipids, that is lower 14:0 and higher 18:3n-3 in SB fed pigs and not a protein source effect. Any other differences in FA observed in plasma between groups were more likely due to endogenous FA metabolism. As SREBP-1 was the major transcription factor downregulated by SB diets, it was compared between tissues. The expression of SREBP-1 was found to be higher in liver and SAT than in VAT, but did not differ between liver and SAT (Fig. 2), in contrast to the findings of Gondret et al. (2001), who reported higher SREBP-1 expression in white adipose tissue than in liver. A possible explanation is that Gondret et al. (2001) determined SREBP-1 expression in 16-weekold pigs in the fed state, while in this study pigs aged 10 weeks (72 days) and after a short-time fasting. However, as previously reported (Hsu et al., 2004), a short-time feed deprivation does not affect SREBP-1 expression in pigs. Despite the difference between the present study and that of Gondret et al. (2001), the present results corroborate their conclusion on the tissue-specific importance of SREBP-1 in pigs. Conclusions The major finding emerging from the present study was that soyabean meal fed to weaned pigs reduced the expression of SREBP-1 in liver and in visceral adipose tissue when compared to whey proteins. The References Amemiya-Kudo, M.; Shimano, H.; Hasty, A. H.; Yahagi, N.; Yoshikawa, T.; Matsuzaka, T.; Okazaki, H.; Tamura, Y.; Iizuka, Y.; Ohashi, K.; Osuga, J.; Harada, K.; Gotoda, T.; Sato, R.; Kimura, S.; Ishibashi, S.; Yamada, N., 2002: Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes. Journal of Lipid Research 43, 1220–1235.
SREBP-2, LXR and PPAR-a in liver and SREBP-2, PAI-1 and PPAR-c expression in adipose tissues were not affected. The differences found in plasma FA profile between SB and WP pigs reflected the dietary FA profile and cannot be solely attributed to dietary protein source. Differences in the expression of SREBP-1 were observed between liver and adipose tissues, indicating a tissue-specific effect on the overall lipid metabolism. In conclusion, feeding soyabean meal-based diets to weaned piglets downregulates the expression of SREBP-1, which is a crucial determinant of lipogenic process because it controls the gene transcription of several lipogenic enzymes. Acknowledgements The present study was partially supported by the JSK Communication Management. The authors are grateful to Hellenic Proteins S.A. for providing the whey proteins (WheyPro 65 and WheyPro 80). The authors also acknowledge the assistance of Dr. R. Chronopoulou, Mrs. D. Tsoli and Mrs. E. Tomara during the experimental and analytical procedures. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Sequences and relative positions of forward (F) and reverse (R) primers for liver X receptor (LXR), sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and 2), peroxisome proliferator-activated receptors a and c (PPARa and PPARc), plasminogen activator inhibitor-1 (PAI-1), RNA polymerase II (RPII) and b-actin (ACTb) used in real-time PCR. Table S2. qPCR assays information for liver X receptor (LXR), sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and 2), peroxisome proliferator-activated receptors a and c (PPARa and PPARc), plasminogen activator inhibitor-1 (PAI-1), RNA polymerase II (RPII) and b-actin (ACTb)
Ascencio, C.; Torres, N.; Isoard-Acosta, F.; G omez-P erez, F. J.; Hern andez-Pando, R.; Tovar, A. R., 2004: Soy protein affects serum insulin and hepatic SREBP-1 mRNA and reduces fatty liver in rats. Journal of Nutrition 134, 522–529. Association of Official Analytical Chemists (AOAC), 1984: Official Methods of Analysis, 10th edn. Association of Official Analytical Chemists, Washington, DC, USA. Bondia-Pons, I.; Castellote, A. I.; L opezSabater, M. C., 2004: Comparison of
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conventional and fast gas chromatography in human plasma fatty acid determination. Journal of Chromatography B 809, 339–344. Carey, G. B., 1997: The swine as a model for studying exercise-induced changes in lipid metabolism. Medicine and Science in Sports and Exercise 29, 1437–1443. Deckelbaum, R. J.; Worgall, T. S.; Seo, T., 2006: n-3 Fatty acids and gene expression. American Journal of Clinical Nutrition 83, 1520–1525.
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Desvergne, B.; Wahli, W., 1999: Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocrine Reviews 20, 649–688. Duran-Montg e, P.; Theil, P. K.; Lauridsen, C.; Esteve-Garcia, E., 2009: Dietary fat source affects metabolism of fatty acids in pigs as evaluated by altered expression of lipogenic genes in liver and adipose tissues. Animal 3, 535–542. Farmer, S. R., 2005: Regulation of PPARgamma activity during adipogenesis. International Journal of Obesity and Related Metabolic Disorders 29, 13–16. Fundaci on Espa~ nola para el Desarrollo de la Nutrici on Animal, 2003: Tablas FEDNA de composicion y valor nutritivo de alimentos para la fabricacion de piensos compuestos, 2nd edn. FEDNA, Madrid, Spain. Gondret, F.; Lebret, B., 2002: Feeding intensity and dietary protein level affect adipocyte cellularity and lipogenic capacity of muscle homogenates in growing pigs, without modification of the expression of sterol regulatory element binding protein. Journal of Animal Science 80, 3184–3193. Gondret, F.; Ferr e, P.; Dugail, I., 2001: ADD-1/SREBP-1 is a major determinant of tissue differential lipogenic capacity in mammalian and avian species. Journal of Lipid Research 42, 106–113. Hegarty, B. D.; Bobard, A.; Hainault, I.; Ferre, P.; Bossard, P.; Foufelle, F., 2005: Distinct roles of insulin and liver X receptor in the induction and cleavage of sterol regulatory element-binding protein-1c. Proceedings of the National Academy of Sciences USA 102, 791–796. Hellemans, J.; Mortier, G.; De Paepe, A.; Speleman, F. J.; Vandesompele, J., 2007: qBase relative quantification framework and software for management and automated analysis of realtime quantitative PCR data. Genome Biology 8, R19. Horton, J. D.; Goldstein, J. L.; Brown, M. S., 2002: SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. Journal of Clinical Investigation 109, 1125–1131. Hsu, J. M.; Wang, P. H.; Liu, B. H.; Ding, S. T., 2004: The effect of dietary docosahexaenoic acid on the expression of porcine lipid metabolism-related genes.
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Journal of Animal Science 82, 683–689. Lijnen, H. R.; Alessi, M. C.; Van Hoef, B.; Collen, D.; Juhan-Vague, I., 2005: On the role of plasminogen activator inhibitor-1 in adipose tissue development and insulin resistance in mice. Journal of Thrombosis and Haemostasis 3, 1174– 1179. Liu, B. H.; Wang, Y. C.; Kuo, C. F.; Cheng, W. M.; Shen, T. F.; Ding, S. T., 2005: The effects of docosahexaenoic acid oil and soybean oil on the expression of lipid metabolism related mRNA in pigs. Asian-Australasian Journal of Animal Science 18, 1451–1456. Mahan, D. C., 1993: Evaluating two sources of dried whey and the effects of replacing the corn and dried whey component with corn gluten meal and lactose in the diets of weanling swine. Journal of Animal Science 71, 2860–2866. Marshall, O. J., 2004: PerlPrimer: crossplatform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20, 2471–2472. National Research Council (NRC), 1998: Nutrient Requirements of Swine, 10th revised edn. National Academy Press, Washington DC, USA. O’Fallon, J. V.; Busboom, J. R.; Nelson, M. L.; Gaskins, C. T., 2007: A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs. Journal of Animal Science 85, 1511–1521. Pawar, A.; Botolin, D.; Mangelsdorf, D. J.; Jump, D. B., 2003: The role of liver X receptor-a in the fatty acid regulation of hepatic gene expression. Journal of Biological Chemistry 2278, 40736– 40743. Pfaffl, M. W., 2001: A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Research 29, 2002–2007. Sampath, H.; Ntambi, J. M., 2005: Polyunsaturated fatty acid regulation of genes of lipid metabolism. Annual Review of Nutrition 25, 317–340. Sanchez, A.; Hubbard, R. W., 1991: Plasma amino acids and the insulin/ glucagon ratio as an explanation for the dietary protein modulation of atherosclerosis. Medical Hypotheses 36, 27–32.
Steffensen, K. R.; Gustafsson, J. A., 2004: Putative metabolic effects of the liver X receptor (LXR). Diabetes 53, 36–42. Torres, N.; Torre-Villalvazo, I.; Tovar, A. R., 2006: Regulation of lipid metabolism by soy protein and its implication in diseases mediated by lipid disorders. Journal of Nutritional Biochemistry 17, 365–373. Tovar, A. R.; Ascencio, C.; Torres, N., 2002: Soy protein, casein, and zein regulate histidase gene expression by modulating serum glucagon. American Journal of Physiology Endocrinology and Metabolism 283, 1016–1022. Tovar, A. R.; Torre-Villalvazo, I.; Ochoa, M.; Elias, A. L.; Ortiz, V.; Aguilar-Salinas, C. A.; Torres, N., 2005: Soy protein reduces hepatic lipotoxicity in hyperinsulinemic obese Zucker fa/fa rats. Journal of Lipid Research 46, 1823–1832. Wood, J. D.; Brown, S. N.; Nute, G. R.; Whittington, F. M.; Perry, A. M.; Johnson, S. P.; Enser, M., 1996: Effects of breed, feed level and conditioning time on the tenderness of pork. Meat Science 44, 105–112. Yang, Y. X.; Kim, Y. G.; Lohakare, J. D.; Yun, J. H.; Lee, J. K.; Kwon, M. S.; Park, J. I.; Choi, J. Y.; Chae, B. J., 2007: Comparative efficacy of different soy protein sources on growth performance, nutrient digestibility and intestinal morphology in weaned pigs. Asian-Australasian Journal of Animal Science 20, 775–783. Yun, J. H.; Kwon, I. K.; Lohakare, J. D.; Choi, J. Y.; Yong, J. S.; Zheng, J.; Cho, W. T.; Chae, B. J., 2005: Comparative efficacy of plant and animal protein sources on the growth performance, nutrient digestibility, morphology and caecal microbiology of the early-weaned pigs. Asian-Australasian Journal of Animal Science 18, 1285–1293. Zhao, S.; Wang, J.; Song, X.; Zhang, X.; Ge, C.; Gao, S., 2010: Impact of dietary protein on lipid metabolism related gene expression in porcine adipose tissue. Nutrition and Metabolism 7, 1–13. Zijlstra, R. T.; Mies, A. M.; McCracken, B. A.; Odle, J.; Gaskins, H. R.; Lien, E. L.; Donovan, S. M., 1996: Short-term metabolic responses do not differ between neonatal piglets fed formulas containing hydrolyzed or intact soy proteins. Journal of Nutrition 126, 913–923.
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
ORIGINAL ARTICLE
Effects of soyabean meal- or whey-based diets on lipid metabolism in weaned piglets G. Theodorou1, G. Papadomichelakis2, E. Tsiplakou2, A. D. Lampidonis1, S. Chadio3, G. Zervas2 and I. Politis1 1 Department of Animal Husbandry, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece 2 Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece, and 3 Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
Summary The present study aimed to test the hypothesis that dietary protein source influences lipid metabolism-related parameters weaned piglets. The effects of soyabean meal (SB) and whey proteins (WP) on gene expression of several genes involved in the lipogenic process in liver, visceral (VAT) and subcutaneous (SAT) adipose tissues, plasma insulin concentration and fatty acid (FA) profile were investigated in 18 weaned piglets. Weaned piglets were fed one of two diets containing either SB or WP as the main protein source. Following a 10-h fasting period, plasma insulin concentration and FA profile were assessed at 56 and 72 days of age, whereas gene expression in liver, VAT and SAT was assessed at 72 days of age. Plasma insulin concentration was not affected by diet, although it was 40% lower in SB fed pigs. The SB pigs had lower 14:0 (p < 0.01) and higher 18:3n-3 (p < 0.001) levels in plasma in comparison with WP pigs. However, these changes were attributed to background differences in the dietary FA profile and not to a direct protein source effect. Gene expression of sterol regulatory elementbinding protein 1 (SREBP-1) in liver and VAT were lower (p < 0.01 and p < 0.05, respectively) in SB compared to WP fed piglets, but no differences occurred in SAT. No changes were observed in sterol regulatory elementbinding protein 2, liver X receptor, peroxisome proliferator-activated receptors a and c and plasminogen activator inhibitor 1 mRNA levels, either in liver or in adipose tissues. In conclusion, dietary protein source, accompanied likely by side alterations in the dietary composition, affects lipid metabolism in pigs through the downregulation of SREBP-1, which is a crucial determinant of lipogenic process. Keywords soyabean meal, whey proteins, lipid metabolism, pigs Correspondence Georgios Theodorou, 75 Iera Odos Str., 118 55 Athens, Greece. Tel: +302105294450; Fax: +30210529442; E-mail: [email protected] Received: 10 November 2013; accepted: 2 April 2014
Dietary interventions that affect lipid metabolism in pigs have received considerable attention, because they represent a useful tool to modulate body fat deposition and improve meat quality (Wood et al., 1996; Gondret and Lebret, 2002). In addition to the agricultural importance, the investigation of such dietary treatments in pigs could serve as a valuable model for studying the intermediary lipid metabolism in humans (Carey, 1997). In this sense, numerous studies have focused in investigating the mechanisms underlying the influence of nutritional treatments on the lipogenic process. Sterol regulatory element-binding protein 1 (SREBP-1) is one of the key transcription factors affected by dietary fat source or crude protein level in pigs. Supplementing diets with very long polyunsaturated (PUFA) fatty acids (Duran-Montg e
et al., 2009) or increasing crude protein content (Zhao et al., 2010) downregulates the expression of SREBP1, which in turn suppresses the gene expression of lipogenic enzymes involved in fatty acid synthesis (acetyl-CoA carboxylase, fatty acid synthase, stearoylCoA desaturase) in liver or adipose tissue. Recent studies in rats and mice have indicated that the source of dietary protein has been shown to affect lipid metabolism. Soy protein when compared to casein reduces the gene expression of lipogenic enzymes in liver (Horton et al., 2002; Ascencio et al., 2004; Tovar et al., 2005) by downregulating SREBP-1 directly (Ascencio et al., 2004) or indirectly, via the liver X receptor (LXR) (Hegarty et al., 2005). This effect is thought to be mediated by the decreased insulin to glucagon ratio, which has been attributed to the lower lysine to arginine ratio and the higher glycine content of soy proteins (Tovar et al., 2002).
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Introduction
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Concomitantly, soy proteins may stimulate proliferator-activated receptor a (PPAR-a) in liver and PPAR-c in adipose tissue increasing, thus, fatty acid oxidation (Tovar et al., 2005) and fatty acid uptake from plasma (Farmer, 2005) respectively. Plasminogen activator inhibitor 1 (PAI-1), apart from its well-known role in maintaining normal haemostasis by regulating the fibrinolytic system, has been also implicated in many other physiological processes, such as the adipose tissue development (Lijnen et al., 2005). To the best of our knowledge, the effects of protein source on the expression of the transcription factors controlling lipogenesis, such as SREBP-1, SREBP-2, LXR and PPAR-a in liver and SREBP-1, SREBP-2, PAI-1 and PPAR-c in adipose tissue, have not been investigated in pigs so far. Soyabean meal and milk products, such as dried whey concentrates, are two protein sources widely used in pig feeding (Mahan, 1993; Yun et al., 2005; Yang et al., 2007); hence, they are suitable for the purpose of this study. The objective of the present study was to compare the effects of using soyabean meal or whey proteins as the main dietary protein source on the gene expression of SREBP-1, SREBP-2, LXR and PPAR-a in the liver and SREBP-1, SREBP-2, PAI-1 and PPAR-c in visceral and subcutaneous adipose tissue of weaned piglets. Plasma insulin concentration and fatty acid profile were analysed in to evaluate possible relationships with gene expression. Materials and methods Animals and diets
Handling and care of the experimental animals conformed to the guidelines of the Faculty of Animal Science and Aquaculture of Agricultural University of Athens. Eighteen castrated male Large White 9 Duroc 9 Landrace piglets weaned at 29 2 days of age were selected from a commercial farm located near the city of Athens. Upon arrival at the experimental facilities, they were allowed an adaptation period of 4 days and were then allotted into two groups (9 pigs/group) balanced for body weight (average BW of 8.4 0.68 kg; mean SD). Piglets were kept in individual cages (1.2 9 0.5 m) and fed ad libitum the following two diets, over a period of 38 days: diet SB, which was formulated to meet the nutrient requirements of piglets (NRC, 1998) using soyabean meal (SB) as the main crude protein (CP) source and diet WP, where SB was totally replaced with a mixture of whey proteins [WP, 70% WheyPro65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg); Hellenic Proteins S.A., Veria, Greece], on equal digestible energy (DE) and CP 2
basis. The mixture of whey proteins was designed to have a content of 660 g CP/kg. The ingredient and chemical composition of diets are summarized in Table 1. Two blood samplings were performed at 56 and 72 days of age (i.e. 21 and 42 days after the start of the experiment, respectively). A 10-h fasting period was applied prior to sampling so as to avoid fluctuations of insulin levels, due to differences in feed intake time. Blood samples were collected in heparinized tubes, and plasma was separated by centrifugation (2000 g, 15 min, 4 °C) and stored at 20 °C for insulin and fatty acid (FA) determination. At the end of the experiment (72 days of age), piglets were stunned and killed by exsanguination. Immediately after, the left lobe of liver and samples of visceral (VAT) and subcutaneous (SAT, neck backfat) adipose tissue were excised, immersed in liquid nitrogen until frozen and subsequently kept at 80 °C pending RT-PCR analyses. Chemical analyses
Feed samples were milled through 1-mm screen prior to analyses. Feed DM was assessed in 5 g samples by oven drying at 105 °C overnight. Routine procedures of Association of Official Analytical Chemists (AOAC) (1984) were used for ether extract (EE; 7.063). Crude protein (CP) was determined as 6.25 9 Kjeldahl nitrogen, using a Kjeltec autoanalyzer unit (Foss, Sweden). All analyses were performed in duplicate. Plasma insulin determination
Plasma insulin concentrations were measured using a double antibody radioimmunoassay kit (rat insulin, Millipore, St. Charles, Missouri, USA), which has 100% specificity for porcine insulin. The sensitivity of the method was 0.081 ng/ml, and the intra- and interassay coefficients of variation were 4.6 and 9.4% respectively. Fatty acid methylesters synthesis and determination
The FA of diets was hydrolysed (with methanolic KOH) and methylated (sulphuric acid catalysis) directly, according to O’Fallon et al. (2007) in duplicate 1 g ground samples. The plasma FA analysis was carried out by the method of Bondia-Pons et al. (2004). Duplicate plasma samples (100 ll) were hydrolysed (with sodium methylate) and subsequently esterified (boron trifluoride-methanol, BF3). In both methods, FA methylesters (FAME) were extracted Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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Table 1 Ingredient and chemical composition of the experimental diets
Table 1 (Continued)
Diet* SB Ingredients (g/kg) Maize 621.0 Soyabean meal 342.0 (440 g CP/kg) Whey proteins† – (660 g CP/kg) L-Lysine 80% 2.0 DL-Methionine 99% 1.0 Sodium chloride 5.0 Calcium carbonate 13.0 Monocalcium phosphate 13.0 Mineral–vitamin pre-mix‡ 3.0 Analysed chemical composition (g/kg DM) Dry matter (g/kg) 888.0 Crude protein 232.0 Ether extract 33.1 Total weights of FA 31.4 (mg/100 g DM) Individual FA (% of total FA) 14:0 0.08 16:0 14.40 cis9-16:1 0.16 18:0 2.63 cis9-18:1 21.71 18:2n-6 54.28 18:3n-3 3.35 20:0 0.33 20:1n-9 0.22 22:2 0.60 Calculated chemical composition (g/kg DM)§ Digestible energy 15.5 (MJ/kg DM) Net energy (MJ/kg DM) 9.0 SID¶ crude protein 166 Total methionine + cystine 8.9 SID¶ methionine + cystine 6.9 Lysine 14.0 SID¶ lysine 11.2 Arginine 16.4 SID¶ arginine 12.1 Total lysine/total 0.85 arginine ratio Threonine 8.9 SID¶ threonine 7.2 Glycine 9.7 Calcium 9.2
Diet* WP
754.0 – 210.0 2.0 1.0 4.0 14.0 12.0 3.0 903.0 228.0 40.4 38.7
2.91 17.87 0.40 4.42 24.27 41.42 1.32 0.34 0.20 0.77 15.4 9.6 177 10.4 8.6 17.3 14.2 8.2 5.4 2.11 13.2 11.2 6.1 9.1
SB Total phosphorus Sodium
7.4 2.4
WP 8.4 2.4
*SB, soyabean meal as protein source; WP, whey proteins as protein source. †Mixture of whey proteins (Hellenic Proteins S.A.); 70% WheyPro 65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg) designed to have a content of 660 g CP/kg. ‡Mineral–vitamin pre-mix (Nuevo S.A., N. Artaki, Greece) provided per kg of diet: 15 000 IU vitamin A (retinyl acetate), 2000 IU vitamin D3 (cholecalciferol), 100 mg vitamin E (DL-a-tocopheryl acetate), 3.5 mg menadione (vitamin K3), 2.5 mg vitamin B1, 6 mg vitamin B2, 3 mg vitamin B6, 25 lg cyanocobalamin, 25 mg nicotinic acid, 20 mg pantothenic acid, 2 mg folic acid, 250 lg biotin, 2 mg Co, 4 mg I, 600 lg Se, 300 mg Fe, 100 mg Mn, 100 mg Mg, 320 mg Cu and 240 mg Zn. §Digestible and net energy, macro-element, total amino acid and standardized ileal digestible (SID) protein and amino acid values for maize, soyabean meal and whey proteins were adapted from tabulated data (FEDNA, 2003; NRC, 1988). ¶Standardized ileal digestible nutrients. No data were available on glycine SID values.
Autosystem XL GC equipped with a 30 m 9 0.25 mm i.d. 9 0.25 lm film thickness ΗΡ-Innowax capillary column (Agilent Technologies, Santa Clara, CA, USA, J&W GC columns) and a flame ionisation detector (FID). The oven temperature was programmed for 1 min at 140 °C, raised by 2.5 °C/min to 200 °C, then to 230 °C by 1 °C/min and held for 1 min, and finally to 240 °C by 4 °C/min and held for 10 min. Helium was the carrier gas at a constant pressure of 18 psi, and the temperature of both the injector and FID was set at 250 °C. Fatty acids were identified by comparison with FAME 37 Component Mix (Supelco, Bellefonte, PA, USA), and quantification was achieved using the internal standard (13:0) added prior to hydrolysis. Total weights of FA (mg/100 g) in diets were calculated as the sum of areas for all FA peaks compared to area for 0.5 mg internal standard. Individual FA were expressed as% by weight of total FA. RNA extraction and RT-PCR analysis
with clear n-hexane and transferred into gas chromatograph (GC) vials. The FAME were subsequently analysed in a temperature-programmed run using a Perkin Elmer
Tissue samples were ground while frozen with a mortar and pestle using liquid nitrogen. Total RNA was extracted using TriReagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The quality and quantity of the RNA extracted were confirmed by spectrophotometry as well as gel electrophoresis. Relative levels of mRNA were quantified with real-time, quantitative RT-PCR using SYBR
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Green chemistry. A pair of primers for each of the genes used in this study was constructed using PERLprimer software (Marshall, 2004). All primer pairs are presented in Table S1. The amount of sample RNA was normalized using b-actin and RNA polymerase II (RNA-PII) as reference genes. Equal amounts of total RNA were reverse transcribed with the PrimeScript First Strand cDNA Synthesis Kit (Takara, Otsu, Shiga, Japan), according to the manufacturer’s instructions using a mix of random hexamers and oligo-dT primers. The real-time PCR was performed in the 7500 Real Time system (Applied Biosystems, Carlsbad, CA, USA) using the KAPA SYBR® FAST qPCR Kit (Kapa biosystems, Woburn, MA, USA) according to the manufacturer’s protocol. Each reaction (10 ll) contained 12.5 ng RNA equivalents as well as 150– 300 nM of forward and reverse primers for each gene. The reactions were incubated at 95 °C for 30 s followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. This was followed by a melt curve analysis to determine the reaction specificity. Each sample was measured in duplicates. A modified version of the Pfaffl (2001) normalization method was used for normalization against the two reference genes (Hellemans et al., 2007). Additional information regarding the quantitative RT-PCR assays is provided in Table S2. Statistical analysis
Data were analysed using the SPSS statistical package (version 17.0; IBM, Armonk, NY, USA). Plasma insulin and FA were analysed using a general linear model (GLM) for repeated measures with diet and age as fixed effects, and their interactions and data are presented as least squares (LS) means SEM. For all relative expression analyses, WP was set to 1 and the expression of the SB group is presented as x-fold expression to the WP group, Comparisons between diets were conducted by one-way ANOVA, and data are given as means SEM. Additionally, comparisons between tissues in the expression of SREBP-1 oneway ANOVA were conducted, after setting the mean liver expression arbitrarily to 1. Significance was set at 0.05. The relationship between blood insulin levels and SREBP-1 expression in liver was determined by Pearson’s linear correlation analysis.
fed pigs. Also, age has not been shown to significantly affect insulin levels (Table 2). Plasma 14:0 percentage was lower (p < 0.01), while 18:3n-3 and 20:5n-3 were higher (p < 0.001 and p < 0.01, respectively) for SB compared to WP fed piglets. Decreased 14:0 (p < 0.01), 18:2n-6 (p < 0.001), 18:3n-3 (p < 0.001), 20:5n-3 (p < 0.01) and increased 18:0 (p < 0.001) and 20:4n-6 (p < 0.01) were observed with increasing age. These differences resulted in a proportional increase in plasma SFA (p < 0.001) and a decrease in PUFA (p < 0.05) with age (Table 2). Relative transcription factor mRNA levels in the different tissues
The relative SREBP-1 mRNA level (as SREBP-1/bactin and RNA-PII ratio) in liver homogenates was approximately 45% lower (p < 0.01) in SB compared to WP pigs (Fig. 1). On the other hand, the relative mRNA levels of SREBP-2, LXR and PPAR-a (Fig. 1) were not affected by diet. In VAT, differences similar to those found in liver were observed. The relative SREBP-1 mRNA level was lower (p < 0.05) in SB compared to WP pigs (Fig. 1). No differences were detected in SAT, and moreover, dietary treatments did not affect the expression of SREBP-2, PAI-1 and PPAR-c. The comparison between tissues showed that SREBP-1 expression was lower (p < 0.05) in VAT with regard to liver and SAT (Fig. 2). No correlation was found between plasma levels of insulin and liver SREBP-1 expression (R2 = 0.0018. p = 0.85). Discussion
No statistically significant differences in insulin levels were detected between WB and SB groups, although levels were 1.7 times higher for WP compared to SB
The present work was based on the hypothesis that dietary protein source affects the relative mRNA expression of the transcription factors involved in lipid metabolism in pigs. However, in practical pig feeding, a change in the protein source is inevitably accompanied by side alterations in other nutrients, in an attempt to comply with nutrient recommendations. In addition to the protein source, diets SB and WP differed in the corn percentage (and hence starch content), the net energy content and FA composition (Table 1), the potential impact of which is discussed. Transcription factor SREBP-1 is expressed in the liver and adipose tissues in pigs and rodents, where it regulates the expression of several lipogenic enzymes (Ascencio et al., 2004; Hsu et al., 2004). Its expression is reduced in rats and mice fed soy proteins (Torres et al., 2006), which have lower lysine to arginine ratio compared to casein (Sanchez and Hubbard, 1991). The present work showed that the expression of
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Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Results Plasma insulin and fatty acid profile
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Table 2 Plasma insulin concentration (ng/ml) and fatty acid (FA) composition (% of total FA) in piglets fed diets with soyabean meal or whey proteins (LS means SEM) Diet (D)*
Insulin 14:0 16:0 18:0 cis9-18:1 18:2n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:5n-3 22:6n-3 SFA§ MUFA¶ PUFA**
Age (A)
Significance‡
SB
WP
SEM†
56 days
77 days
SEM†
D
A
D9A
191.9 0.54 20.26 17.10 15.49 18.97 0.48 0.56 9.21 0.42 1.13 2.22 39.46 19.91 34.22
318.1 0.67 21.08 17.20 14.79 18.51 0.20 0.57 9.93 0.28 1.15 2.26 40.44 18.70 34.23
55.95 0.031 0.351 0.449 0.403 0.669 0.015 0.031 0.423 0.032 0.097 0.124 0.365 0.500 0.556
279.8 0.67 20.96 15.51 14.73 20.32 0.40 0.57 8.41 0.40 1.15 2.68 39.13 19.16 35.14
230.3 0.54 20.38 18.79 15.55 17.16 0.28 0.53 10.73 0.29 1.13 1.80 40.87 19.45 33.31
48.30 0.029 0.329 0.415 0.380 0.553 0.015 0.030 0.411 0.027 0.078 0.114 0.309 0.470 0.539
ns ** ns ns ns ns *** ns ns ** ns ns ns ns ns
ns ** ns *** ns *** *** ns ** ** ns *** *** ns *
ns ns ns ns ns ns * ns ns t ns ns * ns ns
*SB, soyabean meal as protein source; WP, mixture of whey proteins [WP, 70% WheyPro65 (650 g CP/kg) + 30% WheyPro 80 (800 g CP/kg); Hellenic Proteins S.A.] as protein source. †SEM, pooled standard error of means (n = 9 individual piglets per diet). ‡ns, not significant; t, 0.10 < p < 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001. §SFA, total saturated FA, including individual FA which are not presented (12:0 + 14:0 + 15:0 + 16:0 + 17:0 + 18:0). ¶MUFA, total monounsaturated FA, including individual FA which are not presented (cis9-16:1 + cis7-16:1 + cis9-18:1 + cis11-18:1). **PUFA, total polyunsaturated FA, including individual FA which are not presented (18:2n-6 + 18:3n-6 + 18:3n-3 + 20:2 + 20:3n-6 + 20:4n-6 + 20:5n-3 + 22:4n-6 + 22:5n-3 + 22:6n-3).
Fig. 1 Gene expression of sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2), liver X receptor (LXR) and peroxisome proliferator-activated receptor a (PPAR-a) in liver, and of SREBP-1, SREBP-2, plasminogen activator inhibitor-1 (PAI-1) and peroxisome proliferator-activated receptor c (PPAR-c) in visceral (VAT) and subcutaneous (SAT) adipose tissues in 72-day-old pigs fed diets with soyabean meal (SB) or whey proteins (WP) (means SEM). mRNA levels were normalized to b-actin and RNA polymerase II (RNA-PII). The mean value of the WP group within each gene and tissue was set to 1. Asterisks indicate significant differences (*, p < 0.05; **, p < 0.01) in the expression between diets.
SREBP-1 was significantly lower in the SB, compared to WP fed pigs, both in liver and in VAT. The result was similar to that observed in rats and mice fed soy proteins (Ascencio et al., 2004). The understanding of the mechanism by which SB proteins and their amino acid profile exert their effects on SREBP-1 is not complete. In rodents, several studies led to the conclusion that the regulation of SREBP-1 is mediated mainly by
insulin; it is decreased in rats and mice fed soy proteins concentrates in contrast to those fed casein (Tovar et al., 2002) and in turn downregulates SREBP-1 (Ascencio et al., 2004; Torres et al., 2006). However, earlier studies in pigs have failed to establish a clear relationship between dietary amino acid profile and insulin secretion (Zijlstra et al., 1996) or between insulin and expression of SREBP-1 (Zhao et al.,
Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
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G. Theodorou et al.
2010). In the current study, the 40% lower insulin in SB compared to WP fed pigs (Table 2) may imply a pattern similar to that seen with rodents (i.e. linkage between protein source, insulin and SREBP-1). Those numerically lower values could be partially explained by the lower starch content in SB diets (lower maize). However, the statistically insignificant differences preclude from arriving to any valid conclusion. Furthermore, no correlation was found between plasma insulin levels and SREBP-1 expression. These data would suggest that insulin might not be the only factor regulating SREBP-1 expression in pigs and that other hormones may be involved (Gondret et al., 2001) in a concerted or not action with insulin. Another issue not to be dismissed herein is the dietary net energy (NE) content. Despite the equal dietary digestible energy (DE), the NE content was lower in SB compared to WP diet by 0.6 MJ/kg (calculated values; Table 1). In addition to the aforementioned aspects, the lower NE content could have likely contributed to the lower SREBP-1 expression in SB pigs. This hypothesis has not been considered by Zhao et al. (2010) and Tovar et al. (2005) and should be further investigated. Regardless to the mechanisms involved, the downregulation of SREBP-1 by SB is an important finding emerging from the present work, as it is a major determinant of tissue lipogenic capacity in pigs (Gondret et al., 2001). In contrast to SREBP-1, no effects of dietary protein source on the expression of SREBP-2 and LXR in liver or SREBP-2 in adipose tissues were observed in pigs. The SREBP-2 preferentially binds to promoters of genes involved in cholesterol metabolism, such as hydroxymethyl glutaryl-CoA (HGM-CoA) reductase and the low-density lipoprotein receptor (LDLr)
(Amemiya-Kudo et al., 2002). In soy protein fed rats, the expression of SREBP-2 increases as a result of low cholesterol content in the liver, with respect to those fed a casein diet (Tovar et al., 2005), and increases the expression of HMG-CoA reductase and LDLr (Ascencio et al., 2004). These decreased cholesterol and oxysterol (intermediates of cholesterol metabolism) concentrations (Torres et al., 2006) in turn inhibit the transcription of LXR in liver, which additionally reduces SREBP-1 expression (Steffensen and Gustafsson, 2004). However, the respective impact of LXR on SREBP-1 is expected to be quite low (Hegarty et al., 2005). The present data suggest firstly that cholesterol metabolism was not affected by diets, as indicated by SREBP-2 and LXR in liver and SREBP-2 in adipose tissue, and secondly, both SREBP-2 and LXR played a minor and/or no role in regulating SREBP-1 expression in liver. The lack of difference in the expression of PPAR-a in liver between SB and WP fed pigs indicated that diet composition could have not affected FA oxidative metabolism in contrast to reports in rats (Tovar et al., 2005). The transcription factors PPAR-c and PAI-1 have a functional role in differentiation and development of adipose tissue. The PPAR-c stimulates FA uptake and esterification into triglycerides in a concerted action with SREBP-1 that regulates lipogenesis to fill the lipid droplet (Farmer, 2005). Recent studies in rats showed that PPAR-c is activated in adipose tissue by soy proteins, resulting in an upregulation of adipogenesis and probably FA uptake from plasma in contrast to casein (Tovar et al., 2005). High expression of PAI-1 may be associated with lower body and fat pad weight and reduced adipocyte size, but its specific role in adipose tissue is not clear (Lijnen et al., 2005). Therefore, dietary protein source did not appear to affect FA uptake, transport and oxidative metabolism in the present study. An important issue to be addressed in the present study was the potential effect of dietary FA on the transcription factors, and particularly SREBP-1. Diet SB had relatively lower saturated FA (SFA; mainly 14:0, 16:0 and 18:0) and higher polyunsaturated FA (PUFA; mainly 18:2n-6 and 18:3n-3) percentages than diet WP (Table 1). Polyunsaturates have strong inhibitory effects on SREBP-1 (Deckelbaum et al., 2006) and LXR expression (Pawar et al., 2003) and can bind directly to PPAR-a upregulating its expression (Desvergne and Wahli, 1999; Sampath and Ntambi, 2005) in the liver of rats. However, previous studies in pigs have showed that only very long-chain dietary n-3 PUFA, such as those of fish or DHA oil (rich in 22:6n-3), have the capacity of inhibiting the SREBP-1, but not PPAR-a, expression in liver,
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Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH
Fig. 2 Differences in the relative expression of sterol regulatory element-binding protein 1 (SREBP-1) between liver, visceral (VAT) and subcutaneous (SAT) adipose tissue in 72-day-old pigs (means SEM). mRNA levels were normalized to b-actin and RNA polymerase II (RNA-PII). Mean liver expression was arbitrarily set to 1. Different letters indicate significant differences in gene expression between tissues (p < 0.05).
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whereas no effects have been observed in adipose tissue (Hsu et al., 2004; Liu et al., 2005; Duran-Montge et al., 2009). On the other hand, the effects of tallow, sunflower or linseed oil (rich in SFA, 18:2n-6 and 18:3n-3, respectively) on transcriptional factors in liver and adipose tissue were insignificant (DuranMontge et al., 2009). Thus, it appears unlikely that the rather small differences observed in the dietary FA profile herein could have produced background effects on SREBP-1 mRNA expression, either in liver or in adipose tissues. The differences in plasma (Table 2) FA profile between SB and WP pigs mainly depicted the incorporation of dietary FA into plasma lipids, that is lower 14:0 and higher 18:3n-3 in SB fed pigs and not a protein source effect. Any other differences in FA observed in plasma between groups were more likely due to endogenous FA metabolism. As SREBP-1 was the major transcription factor downregulated by SB diets, it was compared between tissues. The expression of SREBP-1 was found to be higher in liver and SAT than in VAT, but did not differ between liver and SAT (Fig. 2), in contrast to the findings of Gondret et al. (2001), who reported higher SREBP-1 expression in white adipose tissue than in liver. A possible explanation is that Gondret et al. (2001) determined SREBP-1 expression in 16-weekold pigs in the fed state, while in this study pigs aged 10 weeks (72 days) and after a short-time fasting. However, as previously reported (Hsu et al., 2004), a short-time feed deprivation does not affect SREBP-1 expression in pigs. Despite the difference between the present study and that of Gondret et al. (2001), the present results corroborate their conclusion on the tissue-specific importance of SREBP-1 in pigs. Conclusions The major finding emerging from the present study was that soyabean meal fed to weaned pigs reduced the expression of SREBP-1 in liver and in visceral adipose tissue when compared to whey proteins. The References Amemiya-Kudo, M.; Shimano, H.; Hasty, A. H.; Yahagi, N.; Yoshikawa, T.; Matsuzaka, T.; Okazaki, H.; Tamura, Y.; Iizuka, Y.; Ohashi, K.; Osuga, J.; Harada, K.; Gotoda, T.; Sato, R.; Kimura, S.; Ishibashi, S.; Yamada, N., 2002: Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes. Journal of Lipid Research 43, 1220–1235.
SREBP-2, LXR and PPAR-a in liver and SREBP-2, PAI-1 and PPAR-c expression in adipose tissues were not affected. The differences found in plasma FA profile between SB and WP pigs reflected the dietary FA profile and cannot be solely attributed to dietary protein source. Differences in the expression of SREBP-1 were observed between liver and adipose tissues, indicating a tissue-specific effect on the overall lipid metabolism. In conclusion, feeding soyabean meal-based diets to weaned piglets downregulates the expression of SREBP-1, which is a crucial determinant of lipogenic process because it controls the gene transcription of several lipogenic enzymes. Acknowledgements The present study was partially supported by the JSK Communication Management. The authors are grateful to Hellenic Proteins S.A. for providing the whey proteins (WheyPro 65 and WheyPro 80). The authors also acknowledge the assistance of Dr. R. Chronopoulou, Mrs. D. Tsoli and Mrs. E. Tomara during the experimental and analytical procedures. Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Sequences and relative positions of forward (F) and reverse (R) primers for liver X receptor (LXR), sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and 2), peroxisome proliferator-activated receptors a and c (PPARa and PPARc), plasminogen activator inhibitor-1 (PAI-1), RNA polymerase II (RPII) and b-actin (ACTb) used in real-time PCR. Table S2. qPCR assays information for liver X receptor (LXR), sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and 2), peroxisome proliferator-activated receptors a and c (PPARa and PPARc), plasminogen activator inhibitor-1 (PAI-1), RNA polymerase II (RPII) and b-actin (ACTb)
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