Intra-uterine growth restriction (IUGR) is a major determinant of infant mortality and morbidity in human obstetrics and domestic animal production(Reference Wu, Bazer and Wallace1). In addition to showing a high rates of perinatal mortality, surviving neonates with IUGR display retarded effects on postnatal development, feed utilisation efficiency, and intestinal development and maturity(Reference Wu, Bazer and Cudd2). Moreover, emerging evidence suggests that IUGR contributes to a broad range of metabolic disorders and chronic diseases in adults, including cardiovascular disorder, hypertension, obesity, insulin resistance and diabetes type II(Reference Barker, Eriksson and Forsen3,Reference Park, Stoffers and Nicholls4) . It is generally believed that maternal, placental or fetal origin risk factors adversely affect fetal growth and contribute to the development of IUGR(Reference Faraci, Renda and Monte5). However, nutritional strategies to improve the growth of IUGR piglets are not available due to the lack of knowledge about the mechanisms of IUGR in humans and animals.
Aside from serving as the substrate for protein synthesis, l-Leucine, a nutritionally essential amino acid for both humans and animals(Reference Wu, Bazer and Dai6,Reference Li, Yin and Tan7) , has a signalling role to activate the mechanistic target of rapamycin, thereby stimulating protein synthesis and inhibiting proteolysis in the small intestine(Reference Rhoads and Wu8), skeletal muscle(Reference Columbus, Fiorotto and Davis9–Reference Escobar, Frank and Suryawan12) and mammary gland tissue(Reference Appuhamy, Knoebel and Nayananjalie13,Reference Lei, Feng and Zhang14) . Dietary supplementation with l-Leucine or a provision of leucine-rich meal leads to enhanced intestinal development and increased protein accretion in piglets(Reference Li, Yin and Tan7,Reference Yin, Yao and Liu15,Reference Zhang, Qiao and Ren16) . Despite the fact that sow’s milk contains a high proportion of l-Leucine(Reference Wu and Knabe17,Reference Davis, Nguyen and Garcia-Bravo18) , our previous study showed that l-Leucine supplementation improved intestinal development and whole-body growth in normal suckling piglets(Reference Sun, Wu and Li19). This result indicates that the extra provision of l-Leucine is required for maximal growth in piglets due to the catabolism of l-Leucine by small intestinal epithelial cells and bacteria in pigs(Reference Dai, Zhang and Wu20,Reference Chen, Li and Wang21) .
IUGR piglets are characterised by continuous impairment of intestinal, liver and muscle development(Reference Wang, Chen and Li22–Reference Liu, Lin and Wang24). Using temporal proteomic approaches, Wang et al. demonstrated that alterations in amino acid metabolism might be one of the underlying mechanisms responsible for the impaired fetal development in IUGR fetuses(Reference Wang, Chen and Li22). Further studies show that the concentration of l-Leucine in the umbilical vein(Reference Lin, Liu and Feng25) was lowered in IUGR piglets compared with that of control. The plasma branched-chain amino acid concentrations of umbilical artery and vein were lower in growth-restricted neonates than in normal body weight controls(Reference Cetin, Marconi and Bozzetti26–Reference Wu, Pond and Ott30). Consistently, branched-chain amino acid-supplemented diet partially improved fetal growth restriction caused by maternal undernutrition in mice(Reference Mogami, Yura and Itoh31). Supplementing weaned IUGR piglets with l-leucine improved muscle protein synthesis and aided in weight gain, as shown in a study by Xu et al.(Reference Xu, Bai and He32). These findings imply that l-leucine might be a nutritionally vital factor in the growth and development of IUGR neonates. However, it remains unknown whether supplementation of l-leucine improves the growth of IUGR piglets during the suckling period. Neonatal piglets serve as an excellent model for addressing the scientific problem related to newborn human beings and provide evidence on the regulation role of l-leucine on early growth and metabolism of suckling piglets. Thus, we investigated the impact of l-leucine supplementation on IUGR suckling piglets. Our data indicate that l-leucine administration presents an adverse effect in breast-fed IUGR piglets, with a significant reduction in average daily gain and plasma free amino acid levels. Additionally, we profiled the blood biochemistry and the expression of amino acid transporters in the small intestine.
Materials and methods
Animals and experimental design
All the animal treatment procedures were approved by the Institutional Animal Care and Use Committee of China Agricultural University and conducted strictly following the Guide for the Care and Use of Laboratory Animals of the Chinese Association for Laboratory Animal Science and Use. A total of sixty crossbred (Large White × Landrace × Duroc) female neonatal IUGR piglets (it has been pointed out that female newborns are more prone to develop IUGR(Reference Radulescu, Ferechide and Popa33)) weighing 0·85 (sem 0·02) kg were chosen and randomly divided into five groups assigned to five lactating multiparous sows in parity 3. Twelve IUGR pigs fed by each sow were randomly allocated to four treatment groups (0, 0·7 1·4, 2·1 g/kg BW of l-leucine supplementation) with three piglets per group. Thus, fifteen piglets in total were included in each group. All the piglets and lactating sows were maintained in a farrowing pen with temperature at 19–21°C. The obstetric tables for lactating sows were equipped with steel piglet isolation guardrail and floating guardrails. The isolation guardrails assisted the piglets to more effectively contact the sow’s teats. The floating guardrails function to prevent piglets from being crushed by sows. People who drenched the piglets were not blind to the treatment. To achieve an isonitrogenous feeding, 1·43, 0·95, 0·48 and 0 g/kg BW of L-alanine were mixed into the 10 ml of l-leucine-containing solutions. All the breast-fed IUGR piglets were given orally twice per day at 0800 and 1600 hours with the prepared saline solution containing l-leucine and/or l-alanine for 14 consecutive days (from 7 to 21 d of age) (Fig. 1). To ensure that the isonitrogenous l-leucine/l-alanine supplements were added in equal volume, plastic medical graduated injector syringes were used to quantify the volume of solution. These liquid preparations were injected into the throat of the piglets. The feed formula for lactating sows was designed following the nutrition requirements provided by National Research Council, which was the same as our previous report(Reference Sun, Wu and Li19). Based on intakes of milk and body weight, the amount of leucine supplemented to the piglets was 100 %, 200 % or 300 % of the leucine consumed by 7-d-old piglets. All piglets were afforded free access to teats of sow (the frequency was approximately every 1·5 h) and drinking water, and milk intake was recorded on 14 and 21 d of age using the weigh–suckle–weigh technique(Reference Wu, Flynn and Knabe34). Milk intake did not differ among the four groups of piglets. Body weight was recorded at 0700 hours on 7, 14 and 21 d of age.
Experimental design showing intra-uterine growth restriction (IUGR) piglets supplemented with l-leucine or l-alanine from 14 to 21 d of age. Sixty female IUGR piglets weighing 0·85 (sem 0·02) kg were randomly assigned to five lactating sows in parity 3. Twelve IUGR pigs fed by each sow were randomly allocated to one of four treatment groups, as indicated. Each group contained a total of fifteen piglets. To achieve isonitrogenous feeding, l-alanine was added to 10 ml of l-leucine-containing solutions. All of the breast-fed IUGR piglets were given orally twice a day for 14 consecutive days (from 7 to 21 d of age). l-ala, l-alanine; l-leu, l-leucine.
At the end of the test period (day 21 of age), piglets undergoing the last l-leucine and/or l-alanine supplementation were subjected to blood collection 1 h later. Blood samples obtained from the anterior vena cava of piglets were collected into EDTA anticoagulant tubes. Plasma separation was implemented by centrifugation at 3000 g for 5 min at 4°C and frozen at –80°C before analysis. For sampling, six piglets were randomly selected from the control and the 1·4 g/kg BW l-leucine treatment group. We chose piglets from 1·4 g/kg BW l-leucine owing to this dose of l-leucine showed an obvious effect on growth performance of IUGR piglets. In addition, we have previously reported that 1·4 g/kg BW l-leucine improved intestinal development and growth in normal suckling piglets(Reference Sun, Wu and Li35), so as to make a contrast with the suckling IUGR piglets. The piglets were anaesthetised with Zoletil (10 mg/kg BW) and then euthanised via exsanguination in a slaughterhouse. Thereafter, the heart, liver, spleen, lung and kidney tissues were removed from the body and weighed. Each segment (duodenum, mid-jejunum and mid-ileum) of small intestine was flushed using ice-cold PBS (0·137 M NaCl, 2·7 mM KCl, 0·01 M Na2PO4, 1·8 mM KH2PO4, pH 7·4), followed by drain off excess fluid and weight record. Approximately 1 cm of segments from distal duodenum, mid-jejunum and mid-ileum samples, respectively, was snap frozen in N2 and stored at −80°C for the subsequent assays.
Blood biochemical analysis
Plasma was separated by centrifugation at 3000 g for 5 min at 4°C and stored at −80°C until analysis. Plasma contents of urea, glucose, ammonia, TAG, total cholesterol, HDL-cholesterol and LDL-cholesterol were analysed by colorimetric methods using commercial kits purchased from Nanjing Jiancheng Bioengineering Inc. All the assay methods were carried out in accordance with the protocols provided by the manufacturers. The biochemical reaction principles for urea determination are as follows: Urea is hydrolysed by urease, resulting in the production of ammonia and carbon dioxide. In an alkaline medium, ammonia and phenol chromogenic reagents produce a blue substance. The amount of this substance is proportional to the urea content and can be evaluated colorimetrically at 640 nm. Quantification of blood glucose was performed using a glucose oxidase method. Glucose oxidase catalyses the formation of gluconic acid and hydrogen peroxide (H2O2) from glucose. H2O2 reacts with peroxidase to yield o-tolidine, which leads to the generation of a blue substance. The absorbance of this coloured substance was measured at 630 nm. Determination of blood ammonia was based on the following principle: The rate of enzymatic reaction, that is, the conversion of NAD(P)H to NAD(P)+, was proportional to the concentration of ammonia in the reaction system in the presence of excess α-ketoglutarate, NAD(P)H and sufficient glutamate dehydrogenase. TAG levels were quantified by GPO-PAP method. TAG were hydrolysed into glycerol and NEFA by lipoprotein lipase. Glycerol is converted to 3-phosphoglycerol by ATP and glycerol kinase, which is then oxidised by phosphoglycerol oxidase to produce phosphoric acid. Subsequently, phosphoric acid is converted into dihydroxyacetone and H2O2 by glycerophosphate oxidase. H2O2 together with 4-aminoantipyrine and 4-chlorophenol is catalysed by peroxidase to form red quinone compounds, the absorbance of which at 510 nm is proportional to the concentration of TAG. The content of total cholesterol was determined by COD-PAP method. Cholesteryl ester is hydrolysed to free cholesterol by cholesterol ester hydrolase, which is oxidised to cholestenone by cholesterol oxidase to generate H2O2, followed by a catalysation process by peroxidase in the presence of 4-aminoantipyrine and phenol to generate a red quinoneimine pigment. HDL-cholesterol and LDL-cholesterol were assessed by surfactants treatment methods. HDL-cholesterol forms soluble complexes in the presence of surfactants, allowing HDL-cholesterol to react directly with enzyme reagents to produce H2O2. The red quinone compounds of 4-(p-benzoquinone-monoimino)phenazone are synthesised by oxidase in the presence of H2O2, 4-amino-antipyrazoline and phenol. The absorbance of 4-(p-benzoquinone-monoimino)phenazone at 546 nm was proportional to the content of HDL-cholesterol. Amphoteric surfactants selectively protect LDL-cholesterol, while non-LDL-cholesterol are eliminated by cholesterol enzymatic reagents. Cholesterol is released from LDL-cholesterol and participates in the Trinder reaction, which produces a coloured substance measured at 546 nm.
Analysis of free amino acids in plasma and small intestine
A known value of 100 μl of plasma samples was acidified with 100 μl of 1·5 M HClO4, followed by neutralisation with 50 μl of 2 M K2CO3. The extract was obtained by centrifugation (21 000 g , 10 min) and subjected to derivatisation with o-phthaldialdehyde and analysis of amino acids. For small intestine tissue, ∼40 mg powdered sample was weighed and acidified with 200 μl of 1·5 M HClO4 and then was mixed with 100 μl of 2 M K2CO3. After a derivatisation with o-phthaldialdehyde, free amino acid levels in plasma and small intestinal tissues were detected by reversed-phase HPLC using a HPLC apparatus (Waters Inc.) equipped with an analytical column (C18; 4·6 mm × 15 cm, 3 μm) protected by a guard column (C18; 4·6 mm × 5 cm, 20–40 μm) and a Model 2475 Multi λ fluorescence detector as previously described(Reference Wu and Meininger36).
Detection of mRNA expression of transporters for amino acids and peptides
Jejunal tissues (∼40 mg per sample) were pulverised in N2, and total RNA extraction from the tissues was performed by using a commercial Trizol reagent (CWBIO). After a quality evaluation, the total RNA was subjected to reverse transcription by a PrimeScript RT Master Mix kit (TaKaRa) following the instructions provided by manufacturer. Reverse transcription solution system was prepared in a 20 μl final volume containing 2·0 μl of 5 × PrimeScript RT Master Mix, 2·0 μl of RNA (25 ng/μl) and RNA enzyme-free water. The reaction condition was 37°C 15 min, 85°C 5 s, followed by cooling at 4°C. The cDNA products were preserved at −20°C. Real-time quantitative PCR was conducted by using SYBR Green reagent (TaKaRa) executing the procedure as follows: (1) 95°C, 30 s; (2) 95°C, 5 s; 60°C, 34 s; 40 cycles. The reaction system was composed of template cDNA, forward primer (200 nM), reverse primer (200 nM), SYBR Mix, ROX and PCR grade water. The primer sequences used for real-time PCR are listed in Table 1. β-actin, which was stably expressed in the jejunum of piglets exposed to the present treatment condition, was selected as internal reference. Gene expression relative to β-actin was normalised by comparative Ct (2-ΔΔCt) method(Reference Livak and Schmittgen37).
Primers sequences used for quantitative real-time PCR
B0AT1, system B0 neutral AA transporter; ATB 0,+, system B0,+ neutral AA transporter; ASCT2, Na+-neutral AA exchanger; CAT1, cationic amino acid transporter 1; y+LAT1, y+ L amino acid transporter-1; rBAT, basic amino acid transporter; b0,+AT, b0,+ amino acid transporter; PepT1, intestinal peptide transporter.
Determination of protein abundance of amino acid transporters
Approximately 40 mg of frozen jejunum samples was pulverised in N2 and subjected to protein extraction using RIPA buffer (50 mM Tris-HCl (pH 7·4), 150 mM NaCl, 1 % NP-40, 0·1 % SDS, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) added with a protease inhibitors cocktail (Roche Applied Science). Bicinchoninic acid assay was carried out in order to quantify the protein concentration for each samples. Thirty microgram of proteins was electrophoretic separated by 10 % SDS polyacrylamide gels, followed by transfer to a polyvinylidene difluoride membrane (Millipore). After blocking with 5 % non-fat milk at room temperature for 1 h, the membranes were incubated with a primary antibody overnight at 4°C and then were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The following primary and secondary antibodies were used: anti-β-actin (1:2000, Santa Cruz Biotechnology, sc-47778), anti-ATB0,+ (1:500, Santa Cruz Biotechnology, sc-169348), anti-CAT1 (1:500, Santa Cruz Biotechnology, sc-33087), anti-PAT1 (1:500, Santa Cruz Biotechnology, sc-161150), anti-rBAT (1:500, Santa Cruz Biotechnology, sc-32930), anti-SNAT2 (1:500, Santa Cruz Biotechnology, sc-166366), anti-xCT (1:500, Santa Cruz Biotechnology, sc-79359), horseradish peroxidase-AffiniPure goat anti-rabbit IgG (1:5000, Huaxingbio Co., Ltd., HX2031), horseradish peroxidase-AffiniPure goat anti-mouse IgG (1:5000, Huaxingbio Co., Ltd., HX20321) and horseradish peroxidase-AffiniPure rabbit anti-goat IgG (1:5000, Huaxingbio Co., Ltd., HX2030). The protein bands were visualised by using the ImageQuant LAS 4000 mini system (GE Healthcare) and quantitative analysed by Quantity One software (Bio-Rad Laboratories).
Statistical analysis
The statistical unit was the pig because all of the pigs were reared in the same testing room. The sow was not included in the statistical model. Data were analysed by one-way ANOVA followed by Student–Newman–Keuls multiple comparison or the independent-samples t-test procedure within the SPSS statistical software. Values are expressed as mean values with their standard error of the mean. P < 0·05 indicates statistically significant.
Results
Effects of L-leucine on growth performance and organ weight of suckling intra-uterine growth restriction piglets
We observed no obvious stress in all the piglets respond to drenching act. Importantly, a recent publication demonstrates that the act of drenching does not impose additional stress to low birth weight piglets(Reference Van Tichelen, Prims and Ayuso38), providing a support on the handling method used in our study. The effects of l-leucine on the growth performance of IUGR newborn piglets are shown in Table 2. At the end of the trial, the body weight of piglets receiving 0·7, 1·4 and 2·1 g/kg BW of l-leucine supplementation was decreased by 5·0, 11·4 and 5·8 %, respectively, compared with the control group (P < 0·05). The average daily gain of piglets was lower by 19·3, 24·2 and 19·8 %, respectively, with the increased level of l-leucine from day 7 to 21 of age. An increasing dose of l-leucine significantly reduced the average daily gain of IUGR suckling piglets for the entire trial period by 8·6, 18·9 and 11·7 %, respectively (P < 0·01). Although a reduction in growth performance was observed, the organ index (organ weight/body weight) of the heart, liver, kidney, lung, spleen and small intestine of IUGR newborn piglets was not affected by l-leucine supplementation (Fig. 2).
The growth performance of breast-fed intra-uterine growth restriction piglets supplemented with varying levels of l-leucine (Means values with their pooled standard errors of the mean, n 15)*
* Piglets were supplemented with l-leucine twice daily.
a,b,c,dMean values within a column sharing different superscript letters differ (P < 0·05).
d0 represents 7 d of age.
Effect of 1·4 g/kg BW l-leucine on organ index (tissue weight/ body weight) of breast-fed intra-uterine growth restriction piglets. Values are means with their standard error of the mean, n 6. Piglets were supplemented with 1·4 g/kg BW l-leucine or 0·95 g/kg BW l-alanine (the isonitrogenous control) twice daily for 2 weeks. 
, Ctl; 
, Leu.
Effects of l-leucine levels on blood biochemistry in newborn piglets with intra-uterine growth restriction
As shown in Table 3, l-leucine supplementation significantly reduced the contents of urea, blood ammonia, glucose, TAG and total cholesterol in plasma (P < 0·01), with a linear relationship with the increase of l-leucine levels. Concretely, dietary leucine levels reduced the plasma urea in IUGR suckling piglets significantly by 17·6, 24·7 and 26·1 % (P < 0·05), respectively. Ammonia content in plasma was decreased by 5·9, 33·1 and 31·7 % with the increase of l-leucine supplement (P < 0·05). Plasma glucose concentration significantly decreased by 9·3, 11·5 and 8·5 % in leucine-treated group compared with the control (P < 0·05). Piglets subjected to increased l-leucine supplementation displayed declining plasma TAG by 15·8, 25·8 and 30 %, respectively. No differences were observed in regard to plasma HDL for IUGR piglets supplemented with different levels of l-leucine (P > 0·05). The plasma LDL content was increased by 13·6, 18·1 and 22·0 % with the increase of l-leucine supplementation (P < 0·05).
The biochemical parameters of plasma from breast-fed intra-uterine growth restriction piglets at 21 d of age*
(Means values with their pooled standard errors of the mean, n 15)
* Piglets were supplemented with l-leucine twice daily.
a,b,cMean values within a row sharing different superscript letters differ (P < 0·05).
Amino acid profiles in plasma and intestinal tissues in suckling piglets with intra-uterine growth restriction receiving l-leucine
The data on plasma free amino acid concentrations of breast-fed IUGR newborn piglets receiving different levels of l-leucine are shown in Table 4. l-leucine supplementation significantly increased the blood leucine content of 21-d-old newborn piglets compared with that of the control group (P < 0·01). With increasing l-leucine supplementation, the plasma levels of the essential amino acids, including histidine, isoleucine, methionine, phenylalanine, threonine and valine, were decreased significantly (P < 0·01). Similarly, the contents of non-essential amino acids including alanine, arginine, aspartic acid, asparagine, citrulline, glutamine, glutamic acid, glycine, serine, taurine and tyrosine also showed a marked reduction, as the level of leucine supplement increased (P < 0·01). The amino acid profiles in the duodenum, jejunum and ileum tissues of IUGR suckling piglets orally administered with l-leucine are listed in Table 5. l-leucine treatment (1·4 g/kg BW) significantly increased the leucine content in all segments of small intestine including duodenum, jejunum and ileum of IUGR suckling piglets (P < 0·05). The contents of aspartate, glutamic acid, glutamine and glycine in duodenum of IUGR suckling piglets were reduced in response to supplementation with l-leucine (P < 0·05). In the jejunum, glycine and taurine in l-leucine-treated group decreased significantly (P < 0·01) by 20·2 and 23·3 %, respectively. The levels of glycine, citrulline and taurine in ileum were significantly decreased (P < 0·05), whereas the contents of glutamate were significantly increased in piglets supplemented with 1·4 g/kg BW leucine compared with those given 0 g/kg BW leucine (P < 0·05).
Effect of l-leucine supplementation on concentrations of amino acids in the plasma of breast-fed intra-uterine growth restriction piglets at 21 d-old (µM)*
(Means values with their pooled standard errors of the mean, n 15)
* Piglets were supplemented with l-leucine twice daily.
a,b,cMean values within a row sharing different superscript letters differ (P < 0·05).
Effects of l-leucine supplementation on amino acid concentrations of small intestine in intra-uterine growth restriction breast-fed piglets (µmol/g tissue)
(Means values with their pooled standard errors of the mean, n 6)*
* Piglets were supplemented with 1·4 g/kg BW l-leucine twice daily.
Regulation of the expression of transporters for amino acids and peptides by l-leucine in the jejunum of suckling piglets with intra-uterine growth restriction
The effects of 1·4 g/kg BW of l-leucine on the mRNA levels of genes encoding amino acid transporters in the jejunum of IUGR suckling piglets are shown in Fig. 3. In comparison with the control group, l-leucine markedly increased the mRNA level of ATB 0,+ , whereas decreased the mRNA expression of ASCT2, y + LAT1, B 0,+ AT and CAT1 (P < 0·05). Consistently, the mRNA level of oligopeptides transporter PepT1 in l-leucine treatment group was also lower than that of the control (P < 0·05). However, no significant difference in the expression of B 0 AT1 and rBAT genes was observed between the two groups (P > 0·05). The abundances of amino acid transporters at protein level in the jejunum of IUGR suckling piglets were detected by Western blot. As shown in Fig. 4, l-leucine supplementation significantly increased the level of ATB0,+ protein in the jejunum (P < 0·05), whereas decreased the abundance of SNAT2 (P < 0·01), compared with the control. However, l-leucine treatment showed no significant impact on the levels of CAT1, PAT1, rBAT and xCT (P > 0·05).
The mRNA levels of amino acid transporters (ATB 0,+ , CAT1, rBAT, y + LAT1, B 0 AT1, ASCT2 and b 0,+ AT) related to leucine uptake, and small peptide transporter PepT1 in the jejunum of 21-d-old suckling intra-uterine growth restriction (IUGR) piglets. The IUGR piglets were provided with 1·4 g/kg BW l-leucine or 0·95 g/kg BW l-alanine (the isonitrogenous control) for 2 weeks between 7 and 21 d of age. Values are means with their standard error of the mean, n 6. *P < 0·05.
The abundances of ATB0,+, CAT1, PAT1, rBAT, SNAT2 and xCT proteins in the jejunum of 21-d-old suckling piglets born with intra-uterine growth restriction (IUGR). (a) Representative immunoblotting bands. (b) The statistical analysis of protein abundance evaluated by gray value using Image J (NIH). The two groups as shown indicate IUGR piglets orally supplemented with 0·95 g/kg BW l-alanine (the isonitrogenous control) or 1·4 g/kg BW l-leucine, respectively, between 7 and 21 d of age. Values are means with their standard error of the mean, n 6. *P < 0·05.
Discussion
It has been well established that IUGR fetuses present an elevation in the risk of fetal death or perinatal mortality and morbidity compared with normal fetuses(Reference Sharma, Shastri and Sharma39,Reference Malhotra, Allison and Castillo-Melendez40) . Of note, piglets exposed to weaning are highly predisposed to pathogenic infection and disease(Reference Dou, Gadonna-Widehem and Rome41,Reference McLamb, Gibson and Overman42) , particularly metabolically abnormal IUGR piglets. Thus, nutritional reinforcement prior to weaning may contribute to the growth and health of pigs. Interestingly, a comparatively low level of leucine in the umbilical vein of IUGR during late gestation has been noted(Reference Lin, Liu and Feng25). This prompted us to propose a hypothesis that l-leucine supplementation during the suckling period may improve the growth performance of IUGR piglets. Unexpectedly, contrary to the growth promotion effect of leucine on normal suckling piglets, administration of leucine led to decreased production performance in IUGR piglets during suckling period, accompanied by lower plasma amino acid levels, changes in plasma biochemical parameters and the expression of amino acid transporters in the jejunum. Previous studies on weaned IUGR piglets (14–35 d of age) fed a basal diet supplemented with 0·3–0·35 % l-leucine have shown that l-leucine improves growth performance and glycolipid metabolism(Reference Xu, Bai and He32,Reference Zhang, Xu and Han43) . These evidence suggests that l-leucine is beneficial to IUGR piglets in response to weaning stress. Here, the present study on breast-fed IUGR piglets prior to weaning (14–21 d of age) demonstrated that l-leucine (0·7–2·1 g/kg BW) exhibited a negative effect on whole-body growth. IUGR piglets may be difficult to adapt to the extra addition of l-leucine during a normal feeding period of breast milk. Hence, although l-leucine is in favour of supporting the growth of normal suckling piglets and IUGR piglets post-weaning, it is not recommended to be supplied to IUGR piglets during a pre-weaning stage following the doses provided in this study. However, as l-leucine at lower dose exerted a supportive effect on the growth of weaned IUGR piglets and high l-leucine supplementation has been shown to facilitate the catabolism of valine and isoleucine(Reference Xu, Bai and He32,Reference Wessels, Kluge and Hirche44,Reference Bertocchi, Bosi and Luise45) , whether leucine at a lower dose (< 0·7 g/kg BW) or in combination with valine and isoleucine is beneficial to the growth of breast-fed IUGR requires additional study in the future.
Urea is a predominant form of elimination for amino groups deriving from amino acids(Reference Weiner, Mitch and Sands46). A low urea level has been implicated in amino acid malnutrition(Reference Benabe and Martinez-Maldonado47). In this experiment, the plasma urea concentration of IUGR piglets decreased with the increasing l-leucine, which evinces an aggravating amino acid malnutrition. Indeed, l-leucine supplementation reduced amino acid transport in the jejunum of IUGR pigs, resulting in a fall in plasma levels of a variety of amino acids and a corresponding decrease in urea. In parallel, a reduction in plasma glucogenic amino acids, which can be converted to glucose, may engender a decrease in blood glucose levels of breast-fed IUGR piglets provided with l-leucine supplementation. Blood ammonia is a product originating from amino acid deamination(Reference Krebs48). Thereby, the decline in plasma amino acids in response to the increased delivery of l-leucine occurs accompanied by a reduction in ammonia. Additionally, it has been confirmed that IUGR piglets exhibit impaired intestinal morphology and nutrient absorption capacity, as well as decreased intestinal tissue protein abundance and aminopeptidase activity in comparison with normal piglets(Reference Wang, Lin and Liu23,Reference Wang, Huo and Shi49) . The addition of l-leucine in this experiment may exacerbate the intestinal burden of suckling IUGR piglets.
Amino acids serve not only as the basic unit of protein synthesis but also act as precursors for various bioactive molecules and metabolic energy substrates(Reference Wu50). It has been noted in suckling piglets with normal weight that a 2-week l-leucine supplementation increased the levels of lysine and methionine in plasma(Reference Sun, Wu and Li35). In contrast, this study revealed that l-leucine treatment significantly suppressed the plasma contents of lysine and methionine in IUGR suckling piglets. Lysine, as the first limiting amino acid of pigs fed with typical grain-based diet, is essential for the synthesis of muscle proteins, hormones, Ig and Hb as well as the regulation of nutrients metabolism and gene expression(Reference Liao, Wang and Regmi51). A deficiency of dietary lysine has been observed to induce growth retardation, alteration in amino acids metabolism and impairment of immune function(Reference Han, Yin and Wang52,Reference Yin, Han and Li53) . In general, methionine is regarded as the second or third limiting amino acid in typical diets of pig production. Beyond protein synthesis, methionine has been functioned in providing methyl groups and the synthesis of multiple bioactive molecules(Reference Yang, Htoo and Liao54). Given that the two limiting amino acids are indispensable and crucial for the growing development and physiological function of piglets, a reduction in lysine and methionine induced by leucine supplementation undoubtedly results in slow growth in IUGR suckling piglets.
Compared with normal piglets, piglets with IUGR display an abnormal concentration of plasma amino acids(Reference MacKay, Brophy and McBreairty55), which may induce a distinct metabolic response to leucine supplementation. Our previous study with normal newborn piglets indicated that leucine supplementation significantly increased the glycine levels in plasma and ileum(Reference Sun, Wu and Li35). Conversely, administration of leucine, in the present study, reduced the contents of glycine and serine (biosynthetically linked with glycine) in the plasma from suckling IUGR piglets. Glycine is abundant in the plasma of suckling piglets and serves to facilitate glutathione biosynthesis, protein synthesis and maximal growth of piglets(Reference Flynn, Knabe and Mallick56–Reference Wang, Dai and Wu58). Additionally, glycine and serine appear as the donors of one carbon unit and participate in the biosynthesis of purine, pyrimidine and s-adenosylmethionine(Reference Reina-Campos, Diaz-Meco and Moscat59,Reference Wang, Wu and Dai60) . Given the particular importance of glycine as abovementioned, a reduction in glycine levels in plasma and small intestine, as observed in this study, may be part of the reasons for the decreased growth performance of IUGR suckling piglets fed with l-leucine. Furthermore, as shown by our previous study, l-leucine increased the intestinal mucosa levels of glutamine and glutamate which provides energy source and thus promotes the growth of normal suckling piglets(Reference Sun, Wu and Li35). Nevertheless, l-leucine treatment significantly reduced the contents of glutamine and glutamate in intestinal tissues from IUGR suckling piglets. These amino acids are known as the crucial energy substrate for intestinal mucosa metabolism(Reference Blachier, Boutry and Bos61), which may be the underlying reason in part for the slow growth of suckling IUGR piglets in response to the additional l-leucine. It is worth noting that supplementing with l-leucine significantly decreased plasma levels of valine, isoleucine and tryptophan. Evidence has suggested that l-leucine provision dose-dependently accelerates the catabolism of valine and isoleucine and decreases available tryptophan(Reference Wessels, Kluge and Hirche44,Reference Bertocchi, Bosi and Luise45) , which may be an additional essential factor for the depressed performance of breast-fed IUGR piglets in this trial and conduces to clarifying the reason for the reduction of valine levels in the ileum in response to l-leucine supplementation.
The protein and polypeptide are hydrolysed into amino acids in the cavity of small intestine, after which the amino acids are transferred through a series of transporters. Following the observations from IUGR in primate model and humans, a decreased activity of key amino acid transporters was identified in placenta(Reference Pantham, Rosario and Weintraub62,Reference Roos, Jansson and Palmberg63) . It has been recognised that maternal protein malnutrition leads to down-regulation of nutrient transporters and contributes to the development of IUGR(Reference Jansson, Pettersson and Haafiz64). Changes in nutrient absorption capacity of IUGR piglets after birth suggest a potential dependence on the expression of transporters for nutrients, particularly for amino acids(Reference Lin, Wang and Wu65,Reference Ji, Wu and Dai66) . Thus, we determined the impact of leucine on the expression of amino acid transporters in intestinal tissues of suckling IUGR piglets. Individual amino acid can be transported by multiple amino acid transporters. In mammals, l-leucine transporters are mainly located at the apical membrane of small intestinal epithelium(Reference Buddington, Elnif and Puchal-Gardiner67). Na+-dependent neutral amino acid transporter ATB0, + appears as one of the key transporters responsible for l-leucine absorption in the jejunum(Reference Samluk, Czeredys and Skowronek68), and up-regulation of its expression promotes the uptake of l-leucine. In agreement with our results from suckling piglets with normal weight(Reference Sun, Wu and Li35), l-leucine supplementation significantly increased the expression of ATB0, + in the jejunum of suckling IUGR piglets. ASCT2, y+LAT1, B0,+AT and CAT1 belong to the ASC, y+L, b0,+ and y+ system, respectively, and their expression in the jejunum of IUGR suckling piglets was suppressed in response to l-leucine administration. These transporters are known to be involved in the transport of the following amino acids: ASCT2 (glutamine, serine, cysteine, alanine, threonine and valine)(Reference Ni, Yu and Li69), y+LAT1 (arginine, lysine, histidine, glutamine, leucine, alanine, cysteine, methionine)(Reference Cleal, Brownbill and Godfrey70), B0,+AT (neutral amino acids)(Reference Jando, Camargo and Herzog71) and CAT1 (arginine, histidine, lysine, ornithine)(Reference Chen, Yin and Tu72). By comparison with our previous study(Reference Sun, Wu and Li35), despite the fact that down-regulation of y+LAT1 was observed in both IUGR and normal suckling piglets following l-leucine supplementation, the expression of B0AT1 and b0,+AT was dramatically enhanced in normal breast-fed piglets while showing no effect and being significantly lowered in IUGR suckling piglets, respectively. These findings partially shed light on the discrepancy between the responses of normal and IUGR suckling piglets to l-leucine supplementation at the same dose. Na+-coupled neutral amino acid transporters 2 (SNAT2) belongs to system A transporters that extensively distributed in most tissues including small intestine(Reference Broer73). The expression of this transporter is modulated by multiple factors such as nutritional status (amino acids level) and stressors(Reference Mando, Tabano and Pileri74). The present experiment indicated that SNAT2 level was reduced in the jejunum of IUGR suckling piglets supplemented with 1·4 g/kg BW l-leucine, implying SNAT2 may respond to the low plasma amino acid levels induced by leucine. In addition, l-leucine inhibited the expression of PepT1, which is responsible for the transport of amino acids in the form of small peptides(Reference Spanier75), in the jejunum of IUGR piglets during suckling period. Together, the reduction in plasma amino acid concentrations in IUGR suckling piglets supplemented with 1·4 g/kg BW l-leucine is possibly linked to the regulation of intestinal transport function, which is mediated by a diverse array of transporters for amino acids and peptides.
In conclusion, a provision of l-leucine (0·7–2·1 g/kg BW) impairs the growth performance in IUGR suckling piglets along with depressed nutritional status and plasma amino acids levels, as well as changed expression of amino acid transporters in the jejunum. These findings hint that supplementation of leucine presents an adverse impact on breast-fed piglets with IUGR in the conditions described in this study. Since neonatal piglets well mimic human infant features, a range of doses of l-leucine supplementation in low-birth weight infants at suckling period may also present an adverse effect on growth.
Acknowledgements
The authors extend their gratitude to all those who volunteered for this study. This work was supported by the National Natural Science Foundation of China (no. 31625025, 31301979, 32172749, 32202701), the Zhengzhou 1125 Talent Program (no. 2016XT016), the 2115 Talent Development Program of China Agricultural University (no. 00109016) and the ‘111’ Project (B16044).
The authors’ contributions were as follows: Y. S. and Z. W. designed the research; Y. S. conducted the research; Y. S., N. L., H. J., Z. D. and Y. Y. analysed the data; Y. J., H. J. and Z. D. provided methodology. Y. J. drafted the original manuscript. Y. J. and Z. W. revised the manuscript; Z. W. had primary responsibility for the final content and all authors read and approved the final manuscript.
The authors declare no conflict of interests.
