Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus

The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling.


Introduction
The mucosal barrier of intestine is the first defense line against the luminal hostile environment [1]. The maintenance of intestinal mucosa function mainly depends on the mucosal barrier of gastrointestinal tract that consists of non-specific barrier mechanisms, specific immunological responses and intestinal microecology [2][3][4][5]. The mucin-type glycoproteins or mucins that are mainly synthesized and secreted by the goblet cells in the intestinal mucosa are the important component of non-specific barrier mechanisms [6].
Rotavirus is a kind of double-stranded RNA icosahedral RNA virus [7]. It is a major pathogen inducing severe gastroenteritis and diarrhea in children and the other young animals [7,8]. This could be due to rotavirus damaging the mucosal barrier of the proximal small intestine, including the decrease of the mucin production and the goblet cell number [9][10][11][12].
Mucins, a kind of glycoproteins with molecular weights ranging from 0.5 to 20 MDa, consist of the oligosaccharide chains and the protein core [13,14]. The protein core of small-intestinal mucins in pigs contains a lot of threonine that represents 28-35% of the total amino acid residues [15]. Recent studies have shown that dietary leucine supplementation may decrease serum threonine concentration of pigs [16], and do not affect the total tract apparent threonine digestibility in pigs [17]. Additionally, as a functional amino acid, leucine may regulate protein metabolism in intestines of pigs through the mTOR signaling pathway [16,18,19], and stimulate the expression of some specific proteins in tissues and cells [20][21][22][23]. Leucine treatment could also inhibit the autophagy via activating the mTOR signaling pathway, which affects the cellular survival and function [24][25]. Thus, it is possible that dietary leucine supplementation stimulated the mucin synthesis in the intestinal mucosa of pigs, and attenuated the effects of rotavirus infection on the mucin production and the goblet cell number of intestinal mucosa. However, these have not been determined.
Therefore, the present study was conducted to test the hypothesis that dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of the weaned pigs infected by porcine rotavirus.

Animals and diets
The animal protocol for this study was approved by the Animal Care and Use Committee of Sichuan Agricultural University. A total of 24 crossbred (Duroc × Large White × Landrace) barrows, which were weaned at 21 d of age, were housed individually in the metabolism cage (1.5 m × 0.7 m × 1.0 m). The lighting of room was natural, and temperature was maintained at 25-28°C. The pigs were provided with the diets 4 times daily at 0800, 1200, 1600 and 2000 h, and had free access to drinking water. At 0800 h of d 1, 11 and 18, the body weight and feed intake of all pigs were measured, which were used to calculate average daily weight gain (ADG), average daily feed intake (ADFI) and feed conversion. During the experiment, the health of all pigs was monitored every day, and there were not any unexpected deaths.

Experimental design and sample collection
After 3 d of acclimatization, based on the initial body weights and origin of litters, twenty four piglets were weighed (7.41 ± 0.77) and assigned randomly to 1 of 2 diets supplemented with 1.00% (w/w) L-leucine or 0.68% (w/w) L-alanine (n = 12) for 17 d. On day 11, following orally infused with 5 mL of the sterile 100 mM sodium bicarbonate solution in all piglets, half of the piglets on each diet were orally infused with 4 mL (10 6 Tissue culture infective dose 50 (TCID 50 )/mL) of procine rotavirus (PRV) dissolved in the essential medium, while the other half were orally infused with the same amount of the sterile essential medium. After PRV infusion, the diarrhea of all pigs was observed. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid. The mean cumulative score of diarrhea was calculated as [(∑ fecal scores for 1 week PRV infusion)/n] [27].
On d 18, following weighing, all piglets received their normal diets. At 1.5 h after feeding, the blood samples of 20 mL were obtained by the jugular vein, and centrifuged at 3500 × g for 10 min. The serum was stored at -80°C until analysis. Following the blood sampling, the piglets were killed by an intracardial injection of Na pentobarbital (50 mg/kg body weight) and jugular exsanguinations. Then, the small intestine was removed, and the jejunum (proximal half of the small intestine) was quickly isolated and flushed with ice-cold saline. The segment of the jejunum was collected and fixed in 10% neutral-buffered formalin for the analysis of histomorphology and goblet cells. The tissue of jejunal mucosa was collected through scraping the intestinal wall with a glass microscope slide, frozen in liquid nitrogen, and stored at -80°C until ELISA and Western Blot analysis.
Porcine rotavirus preparation and virus titre determination PRV used in this study was a tissue culture-adapted Ohio State University (OSU) strain (ATCC #VR-893). The virus was propagated in the IPEC-J2 cell line, a generous gift from Professor Per Torp Sangild (Copenhagen University, Danmark), as described previously [28]. Briefly, after the pre-activation with 5 μg/mL trypsin (type IX, Sigma) for 30 min at 37°C, the PRV was used to inoculate the IPEC-J2 cell. Following 1 h of incubation at 37°C, the inoculation were removed. IPEC-J2 cells were washed three times with sterile PBS, then incubated at 37°C in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12 medium). When the extensive cytopathic effect (cell destruction) was observed with microscope, the cultures were frozen and thawed three times, and centrifuged at 3000 × g for 10 min. The supernatants containing the PRV were stored at -80°C. The virus titre (TCID 50 value) was determined as described previously [29]. Briefly, IPEC-J2 cells were grown to 80-90% confluence in ninety-six-well plates, and then infected with 50 μL aliquots of 1:10 serial dilutions (in DMEM/F12 medium) of PRV samples (8 wells/ dilution). Following the incubation for 4 d at 37°C in 5% CO 2 , the cytopathic effect was visualized by staining the remaining viable cells with crystal violet. The virus titres were calculated with the Speaman method (Speaman 1908) and expressed as log 10 (TCID 50 ) [30].

Chemical analyses
The crude protein of diets was determined according to the Association of Official Analytical Chemists (AOAC 1995) method (AOAC Method 988.05) [31]. The concentrations of amino acids in diets were determined following hydrolyzing in 6 N HCl at 110°C for 24 h (AOAC method 994.12) [31]. Amino-acid analyses were performed by using an L-8800 Amino Acid Analyzer (Hitachi, Tokyo, Japan). The concentrations of plasma free amino acids were also determined using an L-8800 Amino Acid Analyzer (Hitachi, Tokyo, Japan), as previously described by Mao et al. (2014) [32]. Serum urea was measured by an assay kit from Nanjing Jiancheng Biochemistry (Nanjing, China) according to the manufacturer's instructions.
Analysis of the rotavirus antibody concentration of the serum and the mucin1 and 2 concentrations of the jejunum Serum rotavirus antibody concentration was measured by using the commercially available pig enzyme-linked immunosorbent assay (ELISA) kit from TSZ ELISA (Framingham, MA) according to the manufacturer's instructions. The mucin 1 and 2 in the jejunal mucosa were determined using the commercially available pig mucin 1 and 2 ELISA kits from CUSABIO Biotech Co. Ltd. (China) according to the manufacturer's instructions. The serum rotavirus antibody and the mucin 1 and 2 in the jejunal mucosa were quantified by using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT), and absorbance was measured at 450 nm.

Analysis of the histomorphology and goblet cells in the jejunum
The histomorphology and the count of goblet cells in the jejunum was determined as described previously [33,34]. Briefly, following the fixing, the segment of the jejunum was embedded in paraffin. Consecutive sections (5 μm) were stained with hematoxylin-eosin and Periodic Acid Schiff-Alcian Blue (PAS-AB) for histomorphological examination and counting goblet cells, respectively. The villus height and crypt depth of the jejunal mucosa was measured at 40 × magnification with an Olympus CK 40 microscope (Olympus Optical Company). The number of positively stained goblet cells was counted within 10 randomly selected villi using Image-Pro Plus software, version 6.0 (Media Cybernetics).

Western Blot analysis
The antibody against phosphorylated mTOR (Ser 2448 ) was purchased from Cell Signaling (Davers, MA). The antibody against β-actin was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Protein levels for the phosphorylated mTOR and β-actin in the jejunal mucosa were determined by Western Blot analysis as described previously [22,23].

Statistical analysis
All data were expressed as mean ± standard error. Data for the growth performance of pigs during the first 10 d of trial were analyzed using the unpaired t test. The other data were analyzed as a 2 × 2 factorial with the general linear model procedures of the SAS (Version 8.1; SAS Institute, Gary, NC). The factors of models included the main effects of leucine treatment (supplemented or unsupplemented with leucine in the diet) and PRV challenge (PRV or sterile essential medium) as well as their interaction. All analyses were performed using SAS (Version 8.1; SAS Institute, Gary, NC). P<0.05 was considered to indicate statistical significance, and P<0.10 was considered to indicate statistical tendency.

Results
The effect of dietary leucine supplementation and/or PRV infusion on the growth performance and diarrhea in the weaned pigs During the first 10 d of trial, supplementing 1.0% leucine in the diet could improve the feed efficiency (P = 0.09), and had no significant effect on the ADG and ADFI of the weaned pigs ( Table 2). After PRV infusion, dietary leucine supplementation also improved the feed efficiency (P = 0.05), and decreased the mean cumulative score of diarrhea (P<0.05) in the weaned pigs (Table 2). However, PRV infusion impaired the ADG and feed efficiency (P<0.05), and increased the mean cumulative score of diarrhea (P<0.05) in the weaned pigs (Table 2). In addition, dietary leucine supplementation attenuated the effect of PRV infusion on the feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09) in the weaned pigs ( Table 2).
The effect of dietary leucine supplementation and/or PRV infusion on the serum concentrations of free amino acids and urea in the weaned pigs Dietary 1% leucine supplementation could increase the serum leucine concentration (P<0.05), and decrease the serum concentrations of threonine, valine, isoleucine, methionine, phenylalanine, lysine, histidine and urea (P<0.05) of the piglets (Table 3) (Table 3). In addition, dietary leucine supplementation might attenuate the effect of PRV infusion on the serum concentrations of threonine (P = 0.09), phenylalaine (P = 0.07), lysine (P = 0.08), histidine (P = 0.09) and urea (P = 0.08) in the weaned pigs ( Table 3).
The effect of dietary leucine supplementation and/or PRV infusion on the serum rotavirus antibody in the weaned pigs PRV infusion increased the serum rotavirus antibody (P<0.05), but dietary leucine supplementation had no significant effect on the serum rotavirus antibody in the weaned pigs (Table 4). Furthermore, there was no interaction between dietary leucine supplementation and PRV infusion with regard to the serum rotavirus antibody in the weaned pigs (Table 4).    (Table 5). However, PRV infusion increased the crypt depth (P<0.05), and decreased the villus height: crypt depth (P = 0.07) of the jejunal mucosa in the weaned pigs (Table 5). In addition, dietary leucine supplementation might attenuate the effect of PRV infusion on the villus height (P = 0.07) and villus height: crypt depth (P = 0.06) of the jejunal mucosa in the weaned pigs ( Table 5).

The effect of dietary leucine supplementation and/or PRV infusion on the number of goblet cells and the concentrations of mucin 1 and 2 in the jejunal mucosa of the weaned pigs
Dietary 1% leucine supplementation could increase the number of goblet cells (P<0.05, Table 5) and the mucin 1 and 2 concentrations (P<0.05, Table 6) in the jejunal mucosa of the weaned pigs. However, PRV infusion decreased the number of goblet cells (P<0.05, Table 5) and the mucin 1 and 2 concentrations (P<0.05, Table 6) in the jejunal mucosa of the weaned pigs. In addition, dietary leucine supplementation attenuated the effect of PRV infusion on the number of goblet cells (P < 0.05, Table 5) and the mucin 1 (P = 0.08, Table 6) and 2 (P = 0.07, Table 6) concentrations in the jejunal mucosa of the weaned pigs. The effect of dietary leucine supplementation and/or PRV infusion on the phosphorylated mTOR level of the jejunal mucosa in the weaned pigs Supplementing 1% leucine in the diet could increase the phosphorylated mTOR level of the jejunal mucosa in the weaned pigs (P<0.05, Fig 1). However, the phosphorylated mTOR level of the jejunal mucosa was decreased by PRV infusion in the weaned pigs (P<0.05, Fig 1). In addition, dietary leucine supplementation attenuated the effect of PRV infusion on the phosphorylated mTOR level of the jejunal mucosa in the weaned pigs (P = 0.08, Fig 1).

Discussion
Rotavirus is one of the main pathogens that lead to the symptomatic gastroenteritis in the young animals, as well as in the young children [7,8]. Recent study of our lab has shown that PRV challenge decreased the growth performance, impaired the intestinal morphology, and increased the diarrhea in the pigs [35], as well rotavirus could decrease the mucin levels and goblet cell numbers in the intestinal mucosa of the mice [9], which was consistent with the present study (Tables 2, 5 and 6). In addition, this study showed that PRV challenge increased the serum rotavirus antibody in the weaned pigs (Table 4). These results indicated that PRV challenge model was successful. It has previously been shown that moderate levels of leucine in diets can improve the growth performance of the weaned pigs, but the excess of dietary leucine depress the feed intake and limit growth [16,18,36,37]. In this study, dietary 1% leucine supplementation could improve the feed efficiency of weaned pigs under the normal and PRV challenge conditions (Table 2). Thus, it is clear that the effect of the similar leucine supplementing level on the growth performance of weaned pigs is different among these studies, which could be derived from the differences in the content of leucine and/or protein of diets.
The morphology is important to maintain the normal intestinal function, especially digestive and absorptive function [38]. This study showed that dietary leucine supplementation could improve the morphology, and attenuate the effect of PRV challenge on the morphology in the jejunum (Table 5). These determined that, under normal and PRV challenge conditions, leucine treatment increased the digestion and absorption of nutrients in the intestine of pigs, which could be an important reason that supplementing 1% leucine in diets improved the growth performance and decreased the diarrhea.
The blood concentrations of free amino acids and urea could indirectly indicate the amount of amino acids that are available for tissue growth [39]. This study also showed that PRV challenge increased the serum concentrations of free amino acids and urea in the weaned pigs (Table 3), which determined that PRV challenge could impair the protein metabolism of whole body. It is possible that PRV inducing the decrease of growth performance partially derived from the protein metabolic disturbance. However, recent studies have shown that oral leucine administration may significantly affect the serum or plasma levels of the free amino acids in rats, mice and pigs [16,18,23,[40][41][42][43], which is similar with the present study (Table 3). In this study, dietary leucine supplementation also attenuated, to some extent, the effect of PRV challenge on the blood concentrations of free amino acids and urea in the weaned pigs (Table 3), which could determine that supplementing leucine in diets attenuated the protein metabolic disturbance induced by PRV challenge, and then possibly improved the growth performance. Some studies with humans and pigs have shown that 20-70% of the first-pass metabolism of essential amino acids in diets is consumed by the portal-drained viscera (PDV), and a large amount of dietary threonine (40-60%) were extracted by the PDV (dominated by the intestinal mucosa) in the first pass metabolism [44,45]. In addition, the peptide backbone of small-intestinal mucins in pigs contains a lot of threonine that represents 28-35% of the total amino acid residues [46]. The present study also showed that, under normal and PRV challenge conditions, dietary leucine supplementation decreased serum threonine concentration of pigs at 1.5 h after feeding (Table 3), which is consistent with the result of the previous studies [16,23,[40][41][42][43]. However, this study showed that supplementing 1% leucine in diets could increase the mucin 1 and 2 concentrations of mucosa (Table 6). Mucins mainly synthesized and secreted by the goblet cells are the important component of non-specific barrier mechanisms in the intestinal mucosa, which will protect animals and humans against enteric pathogens [6]. Recent studies have shown that rotavirus damages the mucosal barrier function of the proximal small intestine, including decreasing the mucin levels via destroying the goblet cells, which is one of the reasons that rotavirus leads to the diarrhea [9][10][11][12]. Thus, in this study, dietary 1% leucine supplementation attenuate the effect of PRV challenge on the mucin 1 and 2 concentration and the goblet cell number in the jejunal mucosa of weaned pigs (Tables 2, 5 and 6), which could be the reason that leucine treatment decreased the diarrhea induced by the PRV infection.
In the cell or tissue, the mTOR signaling is one of the most important pathways regulating the protein metabolism and the autophagy that may directly affect the cellular function and survival through exerting both beneficial and aggravating effects on the progression of disease [47,48]. In the present study, PRV challenge decreased the phosphorylated mTOR level in the jejunal mucosa of weaned pigs (Fig 1), which could impair the mucin synthesis and the survival of the goblet cells. Thus, it is possible that rotavirus decreasing the mucin production was relative with inhibition of the mTOR signaling pathway.
Recent studies have shown that dietary leucine supplementation may stimulate the mTOR signaling and the protein synthesis in the intestine of piglets [16,18], and enhance the production of specific proteins in various tissues and cells through stimulating the mTOR signaling [20][21][22][23]. Moreover, oral leucine administration can decrease the protein degradation in human duodenum, and increase the mucosal cell proliferation via activating the PI3K/Akt/mTOR signaling [19]. In this study, dietary 1% leucine supplementation also stimulated the phosphorylated mTOR level in the intestinal mucosa of pigs, and even attenuated, to some extent, the effect of PRV challenge on it (Fig 1). Therefore, these indicated that supplementing 1% leucine in diets efficiently alleviated the effect of PRV challenge on the mucin production and the goblet cell number of the jejunal mucosa possibly via stimulating the phosphorylated mTOR level in the goblet cells.

Conclusion
Rotavirus infection impaired the growth performance of the weaned pigs via the protein metabolic disturbance of whole body and the diarrhea derived from the jejunal mucosa dysfunction. However, dietary 1% leucine supplementation alleviated the effect of rotavirus infection on growth performance and diarrhea in weaned pigs, which could be due that leucine treatment could improve the protein metabolism, the digestive and absorptive function of intestine, and non-specific barrier mechanism of intestinal mucosa. These findings provide a basis for researching the different functions of leucine in animals and humans.