Early weaning (14- to 21-d weaning age) is one of the most stressful events that pigs encounter in swine production because weaned pigs must rapidly adapt to the changes in diet, physical environment and social environment(Reference van Beers-Schreurs and Spaeth1,Reference Moeser, Klok and Ryan2) . Weaned piglets have reduced feed intake immediately after weaning, which is highly associated with the negative changes in intestinal architecture and function, such as villous atrophy and impairment of intestinal barrier function, decreased enzymatic activities and increased expression of proinflammatory cytokines(Reference Yang, Wang and Xiong3,Reference Yang, Xiong and Wang4) . The metabolism shifts in the intestinal epithelium play a significant role in maturation of the intestinal epithelium(Reference Kumar, Srivillibhuthur and Joshi5,Reference Gehart and Clevers6) . Our previous study has also reported that weaning influences energy and protein metabolism in the intestinal epithelium, which further impacts the proliferation of intestinal epithelial cells in weaned piglets(Reference Yang, Xiong and Wang4).
Intestinal renewal is critically important in intestinal development, maintenance and recovery from tissue damage; therefore, the intestinal epithelial cells have notably high energy demands due to the rapid renewal(Reference van Beers-Schreurs and Spaeth1,Reference Pinto and Clevers7) . Glutamate, aspartate and glutamine are the major oxidative substrates utilised by the intestinal epithelial cells, whereas glucose and fatty acids are minor oxidative substrates(Reference Stoll, Henry and Reeds8,Reference Windmueller and Spaeth9) . However, our previous study has reported that weaned piglets supplemented with glucose, soya oil or glutamine exhibited different responses. Pigs fed with glucose or soya oil had better growth performance due to enhanced feed intake, whereas pigs supplemented with glutamine have improved immunity(Reference Lv, Xiong and Yang10). Glucose, fatty acids and glutamine have different metabolism pathways; thus, they may have different physiological functions(Reference Mendoza and Van11–Reference Makkink, Negulescu and Qin13). On the basis of previously published research, we hypothesised that the intestinal metabolism may be altered at the organismal level in those pigs. The mammalian target of rapamycin (mTOR) is a crucial signalling node that integrates environmental cues to regulate cell survival, proliferation and metabolism. Lysosomal mTOR complex 1 (mTORC1) modulates the cell response to growth factors and nutrients by increasing protein synthesis and cell growth and repressing the autophagy-lysosomal pathway(Reference Wallroth, Koch and Marat14). Thus, the present study aims to investigate the effects of dietary supplementation of glucose, soya oil or glutamine on protein expression in crypt compartment and the mTORC1 signalling pathway along the jejunal crypt–villus axis in weaned piglets.
Methods
Animals and experimental treatments
The experimental design and procedures used in the present study were approved by the Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. Weaned piglets used in this experiment were bred from Duroc boars to Landrace × Large Yorkshire sows. A total of forty-two piglets were weaned at 21 d of age, grouped by body weight and sex and were randomly assigned to one of the three treatments. Each treatment group had fourteen piglets, including seven gilts and seven barrows. They were fed isoenergetic diets supplemented with either glucose, soya oil or glutamine. Glucose, soya oil and glutamine accounted 2·5 % of metabolic energy in each diet. Glucose and soya oil were edible grade and refined, and glutamine was obtained from Sigma (G3126). The diets were formulated to meet the nutrient requirements for weaned piglets(15). The nutrient composition is depicted in online Supplementary Table S1. The experiment was conducted for 28 d with two phases and 2 weeks in each phase. There were no differences in initial body weight among glucose, soya oil and glutamine treatments (7·36 (sd 0·23) v. 7·39 (sd 0·19) v. 7·43 (sd 0·17) kg; P = 0·968), and there were significant differences in body weight on day 14 (9·84 (sd 0·30) v. 10·03 (sd 0·30) v. 9·08 (sd 0·22) kg, P = 0·046) but not on day 28 (14·77 (sd 0·53) v. 14·73 (sd 0·62) v. 13·50 (sd 0·49) kg, P = 0·208).
Animal management and sample collection
The piglets were housed individually in an environmentally controlled nursery room with hard plastic slatted flooring. All piglets had free access to drinking water. On day 14 or 28, seven piglets (four barrows and three gilts) were randomly selected from each treatment and were euthanised for tissue sampling. The remained pigs were euthanised after the completion of the experiment (day 28). Intestinal segments were collected from the mid-jejunum, rinsed with ice-cold PBS and divided into two sections. One (approximately 1–2 cm) section was fixed with 4 % formaldehyde-phosphate buffer and kept at 4°C for microscopic assessment of the mucosal morphology. The other section was used for sequential isolation of small intestinal epithelial cells.
Sequential isolation of small intestinal epithelial cells along the crypt–villus axis was performed according to our published research(Reference Xiong, Yang and Tan16). In brief, the jejunum was flushed once with PBS and then incubated at 37°C for 30 min in 15 ml of oxygenated PBS. After pre-incubation, the jejunal segments were then filled with 15–30 ml of isolation buffer (5 mm Na2EDTA, 10 mm HEPES, 0·5 mm dithiothreitol, 0·25 % bovine serum albumin, 2·5 mmd-glucose, 2·5 mml-glutamine, 0·5 mmdl-β-hydroxybutyrate sodium salt, oxygenated with an O2–CO2 mixture (19:1, v/v)) for sequential isolation of three epithelial cell fractions (upper villus: F1; middle villus: F2; crypt: F3) from the villus tip to the crypt bottom. Isolated cell fractions were pooled for each piglet. After that, the cell fraction was washed twice with an oxygenated cell suspension buffer (155 mm KCl at pH 7·4) and centrifuged at 400 g for 4 min at 4°C. The washed cells were immediately frozen by liquid N2 and stored at −80°C for further use.
Measurement of jejunal morphology
The cross sections of intestinal samples preserved in 4 % formaldehyde-phosphate buffer were prepared using standard paraffin embedding techniques. The samples were sectioned at 5 μm thickness and were stained with haematoxylin–eosin(Reference Xiong, Yang and Wang17). The villus height and crypt depth were measured in the stained sections under a microscope with 40× combined magnification using an image processing and analysis system (version 1; Leica Imaging Systems Ltd). At least fifteen well-oriented intact villi and the associated crypt depths were measured in each section for each piglet.
Sample preparation and isobaric labelling
Briefly, the crypt cells were resuspended in lysis buffer (7m urea, 2 m thiourea, 4 % w/v 3-((3-cholamidopropyl) dimethylammonio) propanesulfonate, 20 mm tributyl phosphate and 0·2% Bio-lyte (pH 3–10)) containing a protease inhibitor cocktail (Roche Diagnostics). The cells were disrupted with disposable tissue-grinding pestles for 5 min and then treated by ultra sonication (Sonics & Material) for 10 min. Trypsin digestion and iTRAQ labelling were performed according to the manufacturer’s protocol (Applied Biosystems Inc.) and dried under vacuum.
High pH reverse phase separation and low pH nano-HPLC-MS/MS analysis
The iTRAQ-labelled samples were divided into twelve fractions by high pH separation using an Aquity ultra performance liquid chromatography system (Waters Corporation) that was connected to a reverse phase column (BEH C18 column, 2·1 × 150 mm, 1·7 μm) (Waters Corporation) and dried under vacuum. High pH separation was performed using a linear gradient (0–45 %) for 45 min in water-ammonium formate-acetonitrile solvents (pH 10·0).
Each fraction was dissolved in solvent A (5 % acetonitrile, 0·1 % formic acid) and analysed on TripleTOF 5600 systems (AB SCIEX) in an information-dependent mode. Briefly, peptides were separated on reverse-phase column (ZORBAX 300SB-C18 column, 5 μm, 0·1 × 15 mm; Micromass) using an Eksigent 1D PLUS system (AB SCIEX) at an analytical flow rate of 300 nl/min. The peptides were separated with 120 min linear gradient from 5 to 40 % solvent B (0·1 % formic acid/90 % acetonitrile). Survey scans were acquired from 400 to 1500 with up to fifteen precursors selected for MS/MS and dynamic exclusion for 20 s.
Database analysis and quantification
Mascot software (version 2.3.02, Matrix Science) was used to simultaneously identify proteins. The search parameters were as follows: (1) Uniprot pig database; (2) peptide tolerance was set as 25 ppm and MS/MS tolerance was set as 0·2 Da; (3) the fixed modifications of MMTS were set as cysteine; and oxidation of methionine was considered as a variable modification. Scaffold was used to quantify the isobaric tag peptide and for protein identification. For protein identification, the filters were set as follows: significance threshold P < 0·05 (with 95 % confidence) and ion score or expected cut-off <0·05 (with 95 % confidence). For protein quantitation, the filters were set as follows: ‘median’ was chosen for the protein ratio type; the minimum precursor charge was set to 2 and minimum peptides were set to 2; only 2 and >2 unique peptides were used to quantify proteins. The median intensities were set as normalisation, and outliers were removed automatically. The peptide threshold was set as above for identity. Quantitative ratios were log2 normalised for final quantitative testing. The normality of data was checked with the Shapiro–Wilk test and then analysed using t test, Mann–Whitney test and ANOVA (followed by a Tukey–Kramer post hoc test).
Bioinformatics analysis
The differential proteins were blasted against Swiss-Prot human database. The Ingenuity Pathway Analysis (content version: 47547484) was used to classify these identified proteins and enrich networks.
Expression of mammalian target of rapamycin complex 1 signalling pathway-related proteins in weaned piglets
The mTOR signalling pathway, phosphor-mTOR, eukaryotic initiation factor-4E binding protein-1 (4EBP1), phosphor-4EBP1, S6 kinase (S6K), phosphor-S6K, AMP-activated protein kinase and β-actin protein abundances in enterocytes along the crypt–villus axis of the jejunum were determined by Western blotting as previously reported(Reference Xiong, Yang and Tan16). Total proteins were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Biyuntian) containing phenylmethylsulfonyl fluoride. The sample was then centrifuged, and the resulting supernatants were sampled for analyses of their protein concentrations before Western blotting of the target proteins.
The following antibodies were used for Western blot analysis. Antibodies for mTOR, phospho-mTOR (Ser2448), 4EBP1, phospho-4EBP1 (Thr70), S6K, phospho-S6K (Thr421/Ser424, Thr389) and AMP-activated protein kinase and β-actin were purchased from Cell Signaling Technology. β-actin was used to normalise the abundance of the target proteins. Quantification of bands was carried out using Image J software (Bio-Rad).
Statistical analysis
The intestinal morphology and Western blot data were analysed using two-way ANOVA in SAS software (version 9.2; SAS Institute Inc.). Values are mean with their standard errors of the mean. P values <0·05 were used to indicate statistical significance.
Results
Jejunal morphology
There were no significant differences (P > 0·05) in villus height, crypt depth and villus height:crypt depth (V:C) of the jejunum among the three treatments on days 14 and 28 (Table 1; online Supplementary Fig. S1).
Table 1. Effects of dietary supplementation with different energy sources on jejunal morphology of weaned piglets* (Mean values and pooled standard errors)
VH, villus height; CD, crypt depth.
* Seven piglets per treatment.
Differentially expressed proteins in the crypt compartment cells in the glucose, soya oil and glutamine groups
A total of 5200 proteins were identified in the present study using Mascot version 2.3.2. A protein with ≥1·2-fold or ≤0·8-fold difference and a P value ≤0·05 was regarded as being differentially expressed. A total of 137 differentially expressed proteins were identified in the crypt compartment of pigs among the three groups on day 14. The highest number of differentially expressed proteins was between the glucose and glutamine groups on day 14. These 137 differential proteins were classified into twenty-three groups based on major intestinal functions, that is, transcription; metabolism of protein, amyloidosis, processing of RNA, catabolism of protein, degranulation of cells, fatty acid metabolism, repair of DNA, deubiquitination of protein, synthesis of protein, processing of ribosomal RNA, stabilisation of protein, cell spreading, cytolysis, nonsense-mediated mRNA decay, organisation of actin cytoskeleton, replication of viral replicon, replication of flaviviridae, stabilisation of mRNA, metabolism of TAG, oxidation of fatty acid or reorganisation of cytoskeleton and synthesis of amine (Fig. 1(A)).
Fig. 1. (A) The functional categories of differentially expressed proteins in the glucose, soya oil and glutamine groups on day 14. (B) The functional categories of differentially expressed proteins in the glucose, soya oil and glutamine groups on day 28.
A total of fifty-six differentially expressed proteins were identified in the crypt compartment of pigs from the three groups on day 28. The highest number of differential proteins was between the soya oil and glutamine groups. These fifty-six differential proteins were classified into twenty-three groups based on major intestinal function, that is, transcription of DNA, G1 phase, metabolism of membrane lipid derivative, synthesis of carbohydrate, concentration of lipid, deubiquitination of protein, metabolism of DNA, proliferation of lymphocytes, primary adenocarcinoma, apoptosis of thyroid tumour cell lines, cell viability of monocyte-derived dendritic cells, chemotaxis of monocytes, chronic myelomonocytic leukaemia, concentration of TAG, congenital anaemia, differentiation of stem cell lines, differentiation of keratinocytes, fragmentation of DNA, G1 phase of pancreatic cancer cell lines, hydrolysis of TAG or infection of hepatoma cell line and invasion of colorectal cancer cell lines (Fig. 1(B)).
The differential proteins between the soya oil and glutamine groups on day 14 or 28 were also networked on Table 2. These differential proteins mainly took part in few networks, including antimicrobial response, inflammatory response, cell death and survival, digestive system development and function and carbohydrate metabolism.
Table 2. Differentially expressed proteins involved in each network of jejunal crypt cells among the three groups*
* Seven piglets per treatment.
† Gene name.
Mammalian target of rapamycin complex 1 pathway in villus and crypt compartment by Western blotting
On day 14, the protein expression of mTOR and p-mTOR was higher (P < 0·05) in the jejunal villus compartment of pigs supplemented with glucose than pigs in the glutamine group, whereas pigs in the soya oil group had the lowest expression (Fig. 2(A)). 4EBP1 and AMP-activated protein kinase expressions were higher (P < 0·05) in the jejunal crypt compartment of pigs in the glucose and glutamine groups than pigs in the soya oil group (Fig. 2(C)).
Fig. 2. Effects of dietary supplementation of different energy sources on protein expression of the mammalian target of rapamycin complex (mTORC1) signalling pathway in the upper villus (A, B) or crypt cell (C, D) fraction. The relative abundance of proteins in mTOR signalling pathway proteins is based on the expression of these proteins determined by Western blot on day 14 or 28. Data are expressed as mean values with their standard errors. a,b,c Mean values with unlike letters within the same organ are significantly different (P < 0·05). AMPK, AMP-activated protein kinase; mTOR, mammalian target of rapamycin; p-mTOR, phosphor-mTOR; 4EBP1, eukaryotic initiation factor-4E binding protein-1; p-4EBP1, phospho-4EBP1; S6K, S6 kinase; p-S6K, phosphor-S6K. 
, Glucose; 
, soya oil; 
, glutamine.
On day 28, pigs fed with glucose had greater (P < 0·05) protein expression of mTOR than pigs fed with soya oil and had greater (P < 0·05) AMP-activated protein kinase expression than pigs fed with glutamine (Fig. 2(B)). Pigs fed with glutamine had greater (P < 0·05) protein expression of 4EBP1 in the jejunal crypt compartment than pigs in the soya oil group. The protein expression of p-mTOR was the highest (P < 0·05) in the jejunal crypt compartment of pigs fed glucose than pigs fed with soya oil or glutamine (Fig. 2(D)).
Discussion
Weaning stress is characterised by poor growth, decreased feed intake and diarrhoea(Reference Pluske, Hampson and Williams18). The intestine has a high rate of energy expenditure for performing its digestion and absorption processes. Inadequate food intake in weaned piglets may contribute to intestinal dysfunction and morphology atrophy(Reference Pie, Lalles and Blazy19). Three types of substrates (amino acids, glucose and fatty acids) are oxidised by intestinal tissues as the energy source, in which amino acids (i.e. glutamine, glutamate and aspartate) are the major contributors to mucosal oxidative energy generation(Reference Stoll, Henry and Reeds8,Reference Windmueller and Spaeth9) . Our previous study reported that weaned piglets supplemented with glucose or soya oil demonstrated better growth performance possibly due to enhanced feed intake, whereas those supplemented with glutamine may have improved immunity and intestinal oxidative capacity(Reference Lv, Xiong and Yang10). In the present study, we aimed to investigate the effects of these three energy sources on intestinal function, which may attribute to the changes of performance and intestinal health in weaned pigs.
The intestinal villus height:crypt depth ratio is widely adopted to assess the nutrient digestion and absorption capacity of the small intestine(Reference Montagne, Crevieu-Gabriel and Toullec20). Given the better growth rate in pigs fed with glucose and soya oil on day 14, it is reasonable to expect that the intestinal morphology of pigs may be altered. However, there were no significant differences in the intestinal morphology among the three groups on day 14. The reason may be that glutamine, the main respiratory substrate of enterocytes, has a better energy supply capacity in enterocytes than soya oil or glucose and it promoted the recovery of intestinal morphology. Intestinal epithelial cells in the crypt proliferate in weaned piglets in response to weaning(Reference Yang, Xiong and Wang4). Enteral nutrients play an important role in regulating intestinal protein accretion, epithelial cell proliferation and mucosal growth(Reference Yang, Wang and Xiong3). In the present study, we isolated and identified the proteins being expressed in jejunal crypt epithelial cells of the pigs supplemented with glucose, soya oil or glutamine. There were more differential expressed proteins in the crypt compartment of the three groups on day 14 than on day 28. There were more proteins involved in the nutrient metabolism or repair of DNA on day 14 expressed in the three groups. However, the main function of the differentially expressed proteins on day 28 included transcription or metabolism of DNA. These results indicated that the protein metabolism of enterocytes in the crypt compartment was adjusted after a 28-d period among the three groups. In addition, the network analysis of the differentially expressed proteins showed that there were differences in the antimicrobial response between soyabean oil or glucose v. glutamine. Paneth cells are specialised epithelial cells located at the base of small intestinal crypts that act as important effectors of innate immunity through their secretion of antimicrobial peptides(Reference Bevins and Salzman21). Glutamine has been found to increase antimicrobial peptide expression and reduce gut inflammation in animal models of inflammatory bowel diseases(Reference Hu, Deng and Yu22). Thus, we speculated that Gln may affect the expression of antimicrobial peptides through the Paneth cells in the crypt compartment of weaned piglets.
mTOR, as the catalytic subunit of two distinct protein complexes, mTORC1 and mTORC2, is the major regulator of growth in animals and is the key link between the availability of nutrients in the environment and the control of most anabolic and catabolic processes(Reference Laplante and Sabatini23,Reference Sabatini24) . Nutrient signals carried by free amino acids and glucose regulate mTORC1 activation through the Rag guanosine triphosphatases(Reference Castellano, Thelen and Moldavski25). Given the central role of mTORC1 in promoting lipid biosynthesis, dietary lipids may provide feedback on mTORC1 activation. However, whether and how mTORC1 senses dietary lipids in a cell-autonomous manner remains unclear(Reference Dibble and Manning26). The 4EBP1 and the translation regulator S6K are two key downstream targets of mTOR(Reference Inoki, Corradetti and Guan27). Our previous study showed that the mTOR signalling pathway may be involved in regulating intestinal epithelial cell renewal along the crypt–villus axis(Reference Yang, Xiong and Wang4). The present study also showed that glucose, soya oil and glutamine had differential effects on the related protein expression of the mTORC1 pathway in the jejunum of weaned piglets. On day 14 or 28, piglets supplemented with glucose or glutamine had higher protein expression of 4EBP1 in the jejunal crypt compartment compared with the soya oil group. On day 28, piglets supplemented with glucose had higher protein expression of p-mTOR in the jejunal crypt compartment compared with the soya oil group. These results may indicate that dietary glucose or glutamine activated mTORC1 and increased the related protein expression of mTORC1 signalling pathway. Compared with suckling piglets, proteins involved in the tricarboxylic acid and β-oxidation were significantly down-regulated, but glycolysis was significantly up-regulated in crypt cells of the early-weaned piglets(Reference Xiong, Yang and Tan16). Villus cells use luminal nutrients as well as arterial nutrients, while crypt cells use arterial nutrition(Reference Alper28). The suckling-weaning transition coincides with metabolic changes that are probably related to the metabolism difference between villus and crypt cells. Our present results also indicated that enterocytes in crypt compartment may make better use of glucose or glutamine than soya oil.
In conclusion, these results indicated that isoenergetic glucose, soya oil or glutamine had no effect on jejunal morphology of weaned piglets; however, they had different effects on protein metabolism in crypt compartment. Compared with soya oil, glucose or glutamine may be better energy supplies for enterocytes in jejunal crypt compartment. Pig is a good model for human nutrition and medicine, and the pig gastrointestinal tract is much closer to that in humans than that in rodents. Thus, the present finding may be useful for human in stress to develop future therapeutic interventions.
Acknowledgements
This work was supported by National Natural Science Foundation of China (no. 31572420; no. 31872991), Hunan Provincial Natural Science Foundation of China (2018JJ3094); Key project of Hunan Provincial Education Department (16A096) and Research Foundation of Education Bureau of Hunan Province, China (18B476).
The authors’ contributions were as follows: X. X. and Y.-L. Y. designed the study; X. X., D.-H. L. and D.-F. X. performed the experiments and collected samples; X. X. and D.-H. L. analysed the data and wrote the manuscript; and X. X., L.-J. Z., M.-H. S., Y.-H. L. and Y.-L. Y. revised the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0007114519002629
