Introduction

Post-weaning diarrhea is a major problem in pork industrial production (Wang et al. 2009a, b; Tang et al. 2005, 2009; Deng et al. 2009). Reducing dietary crude protein (CP) level, as a strategy to solve the problem, has been found to limit the frequency and the severity of digestive problems in piglets (Ball and Aherne 1982; Kong et al. 2007). Recent work with the piglet, an excellent animal model for studying the nutrition of human infants (Deng et al. 2007a, b; Elango et al. 2009; Suryawan et al. 2009; Yin et al. 2009), has shown that reducing dietary intake of protein can improve gut function after weaning (Lalles et al. 2007). However, this method results in inadequate provision of amino acids for supporting tissue protein synthesis (Baker 2009; Deng et al. 2009). A strategy to overcome such a nutritional problem may be to supplement free amino acids to low-protein diets (Deng et al. 2007a, b; Hansen et al. 1993; Rotz 2004; Wu et al. 2009). However, little work has been conducted in this area of neonatal nutrition, despite a previous attempt to supplement histidine, isoleucine, and valine to low-protein diets for gilts (Figueroa et al. 2003). Identifying an amino acid that can stimulate protein synthesis in weanling piglets fed a reduced-CP diet is of enormous nutritional importance.

Leucine, as one of the essential amino acids, is of interest for addition to low-protein diets to regulate protein synthesis and animal growth because its biochemical actions include increasing the secretion of growth hormones (Wu 2009), regulating gene expression (Jobgen et al. 2009; Li et al. 2009a; Palii et al. 2009; Stipanuk et al. 2009), and repairing muscles (Dardevet et al. 2000; Tischler et al. 1982). Many studies have demonstrated that leucine can stimulate protein synthesis and inhibit protein degradation in skeletal muscle (Dardevet et al. 2000, 2002; Escobar et al. 2005; Tischler et al. 1982). Leucine has also been implicated to play a signaling role in enhancing the availability of specific eukaryotic initiation factors (Anthony et al. 2000), as well as augmenting the activity of proteins involved in mRNA translation (Davis and Fiorotto 2009; Wu et al. 2010a). This stimulatory effect of leucine is thought to be mediated partly via a mammalian target of rapamycin (mTOR)-dependent process that involves the phosphorylation of S6K1, 4E-BP1 and eIF4E assembly (Kimball and Jefferson 2006). However, little evidence is available regarding a beneficial effect on weaned pigs of supplementing leucine to a low-protein diet. Therefore, the primary objective of this work was to investigate the effect of leucine on growth performance and protein synthesis in multiple organs of weaned pigs fed a low-protein diet.

Materials and methods

Animals and diets

We conducted the experiment in accordance with the Chinese guidelines for animal welfare and it was approved by the animal welfare committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. Eighteen healthy pigs (Landrace × Yorkshire; 8.24 ± 0.67 kg body weight) were weaned at 21 days of age and housed individually in stainless steel metabolism pens (0.8 × 1.8 m) in environmentally controlled facilities (25°C) (Tan et al. 2009a, b). Immediately after removal from sows, piglets were assigned randomly into one of three corn and soybean meal-based diets supplemented with 0, 0.27, or 0.55% l-leucine (Table 1). There were six pigs per diet. The basal diet contained 16.9% crude protein (CP) which was 29% lower than that recommended by NRC (1998), and 1.34% leucine which just met NRC requirement. Thus, the total contents of leucine in the diets supplemented with 0.0, 0.27 and 0.55% leucine were 1.34, 1.61 and 1.88%, respectively. The piglets had free access to their respective diets. On each day, water was added to the diet in a 2:1 ratio. Additional water was available from a nipple drinker (Li et al. 2007; Ruan et al. 2007; Wu et al. 2008). At 32 days of age, all of the pigs were surgically inserted with a jugular catheter for blood sampling and isotope infusion, according to the technique of Li et al. (2008). Feed intake and body weight gain of the animal were recorded weekly (Yin et al. 2008).

Table 1 The composition and nutrient content of experimental diets
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Measurement of protein synthesis

Tissue protein synthesis rates were measured in vivo using a modification of the flooding-dose technique (Frank et al. 2007; Fan et al. 2006). Three days after the catheter insertion and at 1 h after the last meal, all piglets were injected, through the jugular catheter, with a flooding dose of l-phenylalanine (1.50 mmol/kg BW) containing l-[ring-2H5] phenylalanine at 40 mol% (0.60 mmol/kg BW) in sterile saline. The intravenous administration of phenylalanine was completed in 5–10 s. Venous blood samples (1 mL) were taken before injection and 15, 30, and 45 min after injection to measure the specific radioactivity of the extracellular free pool of phenylalanine. Blood samples were centrifuged at 3,000×g for 10 min to obtain plasma (Kong et al. 2009; Li et al. 2009c; Wu et al. 2010b, c). Immediately after the last blood sample was collected, pigs were humanely killed (Deng et al. 2009, 2010). The entire intestine was then rapidly removed and dissected free of mesenteric attachments and placed on a smooth, cold surface tray. The duodenum, jejunum and ileum were separated. The duodenum was the first 30-cm small intestine beginning at the pyloric sphincter. An ileal portion of samples (the 30-cm distal portion of the small intestine ending at the ileocecocolic junction) was also collected (Chen et al. 2007, 2009). Approximately 30 cm from the middle of the small intestine was taken as the jejunal tissue. Their luminal contents were removed by manually stripping out the contents followed by rinsing the lumen with saline (Haynes et al. 2009; Yin et al. 2001). At the same time, longissimus dorsi muscle (spanning the last five ribs on the right side), jejunum, pancreas, kidneys and liver were quickly removed and snap-frozen in liquid nitrogen. The exact time (min) of tracer labeling was recorded from the end of the intravenous injection of phenylalanine to the time of placing tissue samples in liquid nitrogen (Deng et al. 2009).

The isotopic enrichment of l-[2H5]phenylalanine in the tissue free pool was measured according to the procedures of Wang et al. (2007) except that the n-propyl hepta-fluorobutyrate derivative of phenylalanine analyzed using a model 6890 GC linked to a 5973N quadruple MS set on Electron Ionization mode (MacKenzie 1987; Culea and Hachey 1995). Ions with mass-to-charge ratios of 91 and 96 were monitored and converted to percentage of molar enrichment (mol%) using calibration curves. Fractional rates of protein synthesis (FSR) for each tissue were calculated as: FSR (%/day) = (S a × 1,440 × 100%)/(S b × t) where FSR is the percentage of protein renewed in a day, S a is the isotopic enrichment (mol%) of l-[ring-2H5] phenylalanine in the protein-bound pool of tissue at time t, S b is the observed isotopic enrichment (mol%) of l-[ring-2H5]phenylalanine in the free pool of tissue at 15, 30, and 45 min, and t is the exact time (min) of tracer labeling. The isotopic enrichment of l-[2H5]phenylalanine in the free pool of tissue is in equilibrium with that in the aminoacyl-tRNA pool and is therefore an appropriate measure of fractional synthesis rate (Bregendahl et al. 2004).

Protein immunoblot analysis

Antibodies against total 4E-BP1, phosphorylated 4E-BP1 (Thr70), total S6K1, phosphorylated S6K1 (Thr389), total mTOR, and phosphorylated mTOR (Ser2448) were purchased from Cell Signaling (Danvers, MA, USA). Frozen samples were powdered under liquid nitrogen using a mortar and pestle. The powdered tissue was homogenized in seven volumes of buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM NaF, 100 mM KCl, 0.2 mM EDTA, 50 mM β-glycerophosphate, 1 mM dithiothreitol, 0.1 mM phenylmethylsulphonylfluoride, 1 mM benzamidine, and 0.5 mM sodium vanadate) with a Polytron homogenizer and centrifuged at 10,000×g for 10 min at 4°C. The supernatant fluid (0.5 mL) was aliquoted into microcentrifuge tubes containing 0.5 ml of 2 × sodiumdodecyl sulfate (SDS) sample buffer (2 mL of 0.5 M Tris, pH 6.8, 2 mL glycerol, 2 mL of 10% SDS, 0.2 mL of β-mercaptoethanol, 0.4 mL of a 4% solution of bromphenol blue, and 1.4 mL water to a final volume of 8 mL). The samples were boiled for 5 min and cooled on ice before being used for Western blot analysis. The protein samples were separated by electrophoresis on a 7.5% (S6K1), 15% (4E-BP1), or 6% (mTOR) polyacrylamide gel (Kang et al. 2010; Tan et al. 2010). Proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were incubated with the respective primary polyclonal antibodies and a secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Cell Signaling; 1:10,000 dilution in 1% milk) (Hou et al. 2010). Photographs of the membranes were taken using the Kodak Image Station 440, and densitometry was performed with Kodak 1D Network software (Eastman Kodak Company, New Haven, CT, USA).

Hormone and substrate assays in plasma

Plasma glucose was analyzed using a Beckman CX4 and reagents from Shanghai Biotechnologies Ltd. (Shanghai, China) (He et al. 2009). Plasma insulin was measured using a porcine insulin RIA kit that used porcine insulin antibody and human insulin standards (Shanghai Biotechnologies Ltd., Shanghai, China) (Tan et al. 2009b). Plasma amino acids were determined using ion-exchange chromatography, as we previously described (Li et al. 2009b; Yin et al. 2009).

Statistical analysis

Values are expressed as mean ± SEM. Statistical analysis of the data was performed using the GLM procedure of SAS (SAS, 2002), followed by the Student–Neuman–Keuls test to determine treatment effects. Differences between the groups were considered significant when P ≤ 0.05.

Results

Feed intake, performance, and tissue weights

Dietary supplementation with 0.27 and 0.55% leucine had no effect (P > 0.05) on feed intake (Table 2). Daily weight gains were 61 and 41% greater (P < 0.05) in weanling pigs supplemented with 0.55% leucine, respectively, compared with those supplemented with 0.0 and 0.27% leucine. Dietary supplementation with 0.27 and 0.55% leucine increased (P < 0.05) pancreas weight by 40 and 46%, respectively. Dietary supplementation with 0.27% leucine also enhanced (P < 0.05) the weights of stomach and liver.

Table 2 Growth and organ weights of weaning piglets fed different levels of dietary leucine
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Plasma insulin, glucose, and amino acids

Concentrations of insulin and glucose in plasma did not differ (P > 0.05) among either different time points after the intravenous administration of l-[ring-2H5]phenylalanine or dietary treatments (Table 3). Concentrations of phenylalanine in plasma increased (P < 0.001) after the infusion of phenylalanine. At time 15 (baseline values), concentrations of plasma leucine were greater (P < 0.05) in leucine-supplemented pigs compared with the control group (Table 4). Opposite results were obtained for valine and isoleucine at times 30 and 45 min. Leucine supplementation has no effect (P > 0.05) on concentrations of other amino acids in plasma within 45 min after phenylalanine infusion except for threonine, lysine, alanine, glycine, and serine at time 30 min (Table 4).

Table 3 Concentrations of insulin and glucose in the plasma of weanling piglets fed different levels of dietary leucine at 15, 30 and 45 min after intravenous administration of [ring-2H5]phenylalanine
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Table 4 Concentrations of amino acids in the plasma of weanling piglets fed different levels of dietary leucine at 15, 30 and 45 min after intravenous administration of [ring-2H5]phenylalanine
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Protein synthesis

Compared with the 0 and 0.27% leucine groups, dietary supplementation with 0.55% leucine increased (P < 0.05) protein synthesis in the heart, kidney, liver, skeletal muscle, pancreas, spleen, and stomach (Table 5). Dietary supplementation with 0.27% leucine enhanced (P < 0.05) protein synthesis in proximal small intestine, kidney and pancreas, compared with the control group (Table 4).

Table 5 Fractional rates of protein synthesis (%/day) in tissues of weanling piglets fed different levels of dietary leucine
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Phosphorylation of 4E-BP1

Phosphorylated levels of 4E-BP1 (Thr70) and the γ-isoform of 4E-BP1, the repressor protein of eIF4E, are shown in Fig. 1. The 4E-BP1 protein, upon phosphorylation, can be resolved into α-, β-, and γ-forms. The γ-form is the predominant phosphorylated form of the protein and does not bind eIF4E (Romanelli et al. 2002). Compared with the control group, dietary supplementation with 0.55% leucine enhanced (P < 0.05) the phosphorylation state of 4E-BP1 in both skeletal muscle and liver (Fig. 1) while dietary supplementation with 0.27% leucine had no effect (P > 0.05).

Fig. 1
figure 1

Abundance of phosphorylated 4E-BP1 (Thr70) in longissimus muscle and liver of weanling piglets fed different levels of dietary leucine. Data are mean ± SEM for n = 6. Values not sharing the same letter differ (P < 0.05)

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Phosphorylation of S6K1

Dietary supplementation with 0.55% leucine increased (P < 0.05) the phosphorylation level of S6K1(Thr389) in both skeletal muscle and liver, compared with the control group (Fig. 2). Dietary supplementation with 0.27% leucine modestly increased (P < 0.05) the phosphorylation level of S6K1 in the liver but had no effect (P > 0.05) on skeletal muscle, compared with the control group (Fig. 2).

Fig. 2
figure 2

Abundance of phosphorylated S6K1 (Thr389) in longissimus muscle and liver of weanling piglets fed different levels of dietary leucine. Data are mean ± SEM for n = 6. Data are mean ± SEM for n = 6. Values not sharing the same letter differ (P < 0.05)

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Phosphorylation of mTOR

Total mTOR levels in skeletal muscle and liver (Fig. 3), as well as phosphorylated mTOR levels in skeletal muscle (Fig. 4) did not differ (P > 0.05) among pigs fed the different levels of dietary leucine (Fig. 3). However, the phosphorylated level of mTOR (Ser2448) was higher (P < 0.05) in the liver of pigs supplemented with 0.55% leucine compared with the control group (Fig. 4).

Fig. 3
figure 3

The abundance of total mTOR in longissimus muscle and liver of weanling piglets fed different levels of dietary leucine. Data are mean ± SEM for n = 6

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Fig. 4
figure 4

Abundance of phosphorylated mTOR (Ser2448) in longissimus muscle and liver of weanling piglets fed different levels of dietary leucine. Data are mean ± SEM for n = 6. Values not sharing the same letter differ (P < 0.05)

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Discussion

Edmonds and Baker (1987) reported that supplementing 1, 2 and 4% l-leucine to a corn and soybean meal-based diet (20% CP) for 16 days did not affect growth performance in weanling pigs, whereas dietary supplementation with 6% leucine reduced weight gain and food intake. This failure of detecting a beneficial effect of leucine on young pigs was likely due to a high CP content and an imbalance among branched-chain amino acids in the basal diet. Here, we reported for the first time that supplementing 0.55% l-leucine to a low-protein diet (16.9% CP) for 14 days increased weight gain of weanling pigs (Table 2). Additionally, the leucine treatment enhanced protein synthesis (Table 5) and mTOR signaling (Fig. 1) in both skeletal muscle and liver of weanling pigs. These results confirmed and extended the previous in vivo findings that an increase in circulating levels of leucine within physiological ranges stimulated the phosphorylation of both 4E-BP1 and S6K1 in skeletal muscle, liver, and intestine (Escobar et al. 2005; Rhoads and Wu 2009).

Leucine has long been known to stimulate protein synthesis and inhibit protein degradation in incubated skeletal muscle (Tischler et al. 1982). Intensive in vivo studies have extended these in vitro seminal findings and identified that elevated concentrations of leucine in plasma resulting from oral administration or dietary supplementation also increased muscle protein synthesis in young rats (Anthony et al. 2000) and neonatal pigs (Escobar et al. 2005) under physiological conditions. It is now clear that leucine increases muscle protein synthesis by activating the mTOR signaling pathway (Meijer and Dubbelhuis 2004). The results of this study indicated that chronic dietary supplementation with leucine promoted the growth of not only skeletal muscle but also the liver and gastrointestinal tract (Table 2) in young pigs.

Evidence from studies with cultured cells suggests that mTOR is an upstream kinase responsible for phosphorylating both 4E-BP1 and S6K1 (a threonine/serine kinase) in cultured cells (Asnaghi et al. 2004; Um et al. 2004). Phosphorylation of mTOR on Ser2448 is positively related to the activity of mTOR (Lynch et al. 2002; Yonezawa et al. 2004). S6K1 phosphorylates ribosomal protein S6 and promotes the translation of mRNA containing a TOP motif (Um et al. 2004). Additionally, phosphorylation of 4E-BP1 can partly regulate the dissociation of 4E-BP1 with eIF4E, thereby releasing eIF4E for initiation of protein synthesis (Gingras et al. 1999; Raught and Gingras 1999, 2001; Vary and Lynch 2005). Consistent with this notion, dietary supplementation with leucine stimulated the phosphorylation of mTOR, 4E-BP1 and S6K1 proteins in the muscle and liver of young pigs (Figs. 1, 2, and 4). Of particular interest, a novel and important finding from the present study is that the leucine treatment enhanced the phosphorylation of both 4E-BP1 and S6K1 in skeletal muscle despite a lack of change in the phosphorylation of mTOR (Fig. 4). Thus, under physiological conditions in young pigs, 4E-BP1 and S6K1 may be activated independent of changes in mTOR phosphorylation. l-Leucine may directly stimulate 4E-BP1 and S6K1 phosphorylation in cells. It is unlikely that the action of leucine in vivo is mediated by insulin, because plasma levels of this hormone did not differ between control and leucine-supplemented piglets (Table 3). Notably, leucine can be transaminated extensively in skeletal muscle to produce α-keto-isocaproate, glutamate, glutamine, and alanine (Wu and Thompson 1988; Wu et al. 1989). Some of these products may directly phosphorylate both 4E-BP1 and S6K1 in muscle.

Sow milk provides large amounts of branched-chain amino acids to suckling piglets (Kim and Wu 2009). However, when the neonates are removed from their mothers, they respond to the physiological stress with a reduction in food intake (Flynn et al. 2009) and impaired growth of skeletal muscle (Han et al. 2009). Although feeding a low-protein diet appears to be effective in ameliorating the intestinal dysfunction, a reduced provision of dietary amino acids limits tissue protein synthesis (Wu 2009). The observations of the present study and those of others (Escobar et al. 2005) clearly indicate that leucine has potential for activating the machinery for protein synthesis in the skeletal muscle of young pigs. However, due to antagonism among branched-chain amino acids and an imbalance between leucine and threonine in the diet (Dien et al. 1954; Gatnau et al. 1995; Harper et al. 1954), dietary supplementation with leucine alone reduced plasma concentrations of isoleucine, valine, and threonine in piglets (Table 4). Interestingly, the extent of reduction in these three essential amino acids did not appear to limit the effect of leucine on stimulating protein synthesis in skeletal muscle, liver and distal small intestine. Because protein synthesis in tissues of young pigs is very sensitive to circulating levels of threonine (Wang et al. 2007), chronic dietary supplementation with leucine plus isoleucine, valine and threonine to a low-protein diet may hold great promise for enhancing their growth performance. Future studies involving a large number of young pigs are necessary to test this novel hypothesis.

In conclusion, dietary supplementation with 0.55% l-leucine increased the phosphorylation of 4E-BP1 and S6K1 in skeletal muscle and liver, as well as the growth performance of young pigs fed a low-protein diet. Our results also indicate that supplemental l-leucine exerted these effects without affecting mTOR phosphorylation in skeletal muscle. These novel findings provide a molecular basis for designing effective nutritional means to increase the efficiency of nutrient utilization for muscle and whole-body growth in neonates.