The Effects of Glucagon-like Peptide-2 on the Tight Junction and Barrier Function in IPEC-J2 Cells through Phosphatidylinositol 3-kinase–Protein Kinase B–Mammalian Target of Rapamycin Signaling Pathway

Glucagon-like peptide-2 (GLP-2) is important for intestinal barrier function and regulation of tight junction (TJ) proteins, but the intracellular mechanisms of action remain undefined. The purpose of this research was to determine the protective effect of GLP-2 mediated TJ and transepithelial electrical resistance (TER) in lipopolysaccharide (LPS) stressed IPEC-J2 cells and to test the hypothesis that GLP-2 regulate TJ and TER through the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in IPEC-J2 cells. Wortmannin and LY294002 are specific inhibitors of PI3K. The results showed that 100 μg/mL LPS stress decreased TER and TJ proteins occludin, claudin-1 and zonula occludens protein 1 (ZO-1) mRNA, proteins expressions (p<0.01) respectively. GLP-2 (100 nmol/L) promote TER and TJ proteins occludin, claudin-1, and zo-1 mRNA, proteins expressions in LPS stressed and normal IPEC-J2 cells (p<0.01) respectively. In normal cells, both wortmannin and LY294002, PI3K inhibitors, prevented the mRNA and protein expressions of Akt and mTOR increase induced by GLP-2 (p<0.01) following with the significant decreasing of occludin, claudin-1, ZO-1 mRNA and proteins expressions and TER (p<0.01). In conclusion, these results indicated that GLP-2 can promote TJ’s expression and TER in LPS stressed and normal IPEC-J2 cells and GLP-2 could regulate TJ and TER through the PI3K/Akt/mTOR pathway

Construction and expression of two-copy engineered yeast of feruloyl esterase

Background: Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results: The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZ\u3b1A-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZ\u3b1FA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50\ub0C, and was stable at a pH range of 4.0\u20136.5 and at below 45\ub0C. Its activity was not significantly affected by K+, Ca2+, Mg2+, Cu2+, Zn2+, Mn2+, Na+ and EDTA, but activated by Fe2+. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions: The two-copy strain GSKZ\u3b1FA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris

Dietary licorice flavonoids powder improves serum antioxidant capacity and immune organ inflammatory responses in weaned piglets

Weaning often induces oxidative stress and inflammatory response in piglets. This study investigated the effects of dietary licorice flavonoids powder (LFP) supplementation on antioxidant capacity and immunity in weaned piglets. Notably, 96 Landrace × Yorkshire × Duroc (DLY) weaned piglets were randomly allocated to four treatments with 6 replicates (4 animals per replicate) and fed with diet supplementation with 0, 50, 150, and 250 mg/kg LFP, respectively. The trial lasted for 5 weeks. The results showed that dietary LFP supplementation effectively increased the liver index (P &lt; 0.05). In addition, dietary LFP supplementation reduced serum aspartate aminotransferase activity (P &lt; 0.01). Piglets fed with 50 mg/kg LFP decreased total cholesterol and HDL-C content in serum (P &lt; 0.05) and increased serum alkaline phosphatase activity (P &lt; 0.01). Similarly, supplementation with 150 mg/kg LFP elevated the activity of total antioxidant capability (T-AOC) in serum (P &lt; 0.01) and dietary with 150 and 250 mg/kg LFP increased T-AOC activity in spleen (P &lt; 0.01). Moreover, dietary with 150 mg/kg LFP addition enhanced (P &lt; 0.05) the serum IgG content of piglets. Additionally, compared with the control group, dietary 250 mg/kg LFP supplementation upregulated (P &lt; 0.05) the mRNA abundance of Interleukin (IL)-1β and monocyte chemoattractant protein 1 (MCP-1) in the spleen. Meanwhile, dietary 150 and 250 mg/kg LFP supplementation downregulated (P &lt; 0.05) mRNA abundance of IL-10, and MCP-1 and 250 mg/kg LFP upregulated (P &lt; 0.05) the expression of intercellular adhesion molecule 1 (ICAM-1), IL-1β, IL-6, and tumor necrosis factor α (TNF-α) in the thymus. In conclusion, LFP supplementation improved the immune function of piglets by regulating the activity of serum biochemical enzymes, improving the antioxidant capacity, and alleviating inflammation of immune organs. This study indicated that LFP is potential alternative protection against early weaned stress in piglets

L-theanine improves intestinal barrier functions by increasing tight junction protein expression and attenuating inflammatory reaction in weaned piglets

L-theanine, a characteristic non-protein amino acid first extracted from green tea leaves, has multiple biological functions, including antioxidant, immune and anti-inflammatory functions. However, the effect of L-theanine on intestinal barrier function of piglets remains unclear. This study aimed to investigate the role and underlying molecular mechanism of L-theanine in tight junction protein expression and intestinal mucosa inflammation in weaned piglets. Our data showed that L-theanine increased the expression of zonula occludens 1, claudin 1 and occludin in jejunum and ileum of piglets. In addition, L-theanine inhibited the mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), IL-6 and Toll-like receptor 4 (TLR4) and the protein expressions of TNF-α, phospho-p38 MAPK (mitogen-activated protein kinase), phospho-IĸB and nuclear NF-ĸB (nuclear factor-ĸB) p65. Taken together, our results indicated that L-theanine could improve intestinal barrier function in weaned piglets, and the effect might be mediated through inhibition of the TLR4/p38 MAPK/NF-κB signaling pathway

Effects of spermine supplementation on the morphology, digestive enzyme activities, and antioxidant capacity of intestine in weaning rats

The main objective of this study was to investigate the effects of different doses of spermine and its extended supplementation on the morphology, digestive enzyme activities, and intestinal antioxidant capacity in weaning rats. Nineteen-day-old male rats received intragastric spermine at doses of 0.2 and 0.4 μmol/g BW for 3 or 7 d, whereas control rats received similar doses of saline. The results are as follows: 1) In the jejunum, the seven-day supplementation with both doses of spermine significantly increased crypt depth (P < 0.05) compared with the control group; the supplementation extension of the high spermine dose increased villus height and crypt depth (P < 0.05); in the ileum, the low spermine dose significantly increased villus height and crypt depth compared with the control group for 7 days (P < 0.05). 2) The 3-day supplementation with high spermine dose increased alkaline phosphatase activity in the jejunum (P < 0.05). 3) In the jejunum, the anti-hydroxyl radical (AHR), total superoxide dismutase (T-SOD), catalase (CAT), and total antioxidant capacity (T-AOC) activities were increased (P < 0.05); however, the malondialdehyde (MDA) content was reduced (P < 0.05) in groups supplemented with the high spermine dose relative to those in the control groups after 3 and 7 d; moreover, the anti-superoxide anion (ASA) and glutathione (GSH) contents increased with the high spermine dose that lasted for 3 days (P < 0.05). Furthermore, the T-SOD and CAT activities (after 3 and 7 d), ASA (after 3 d), and AHR (after 7 d) increased with the high spermine dose compared with those of the low spermine dose (P < 0.05). Extending the supplementation duration (7 d) of the high spermine dose decreased the MDA content and ASA and T-AOC activities (P < 0.05). These results suggested that spermine supplementation can modulate gut development and enhance the antioxidant status of the jejunum in weaning rats, and a dosage of 0.4 μmol spermine/g BW had better effects than the dosage of 0.2 μmol spermine/g BW on accelerating gut development and increasing antioxidant capacity

Tissue Distribution of Porcine FTO and Its Effect on Porcine Intramuscular Preadipocytes Proliferation and Differentiation.

The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation

Hydroxy Selenomethionine Improves Meat Quality through Optimal Skeletal Metabolism and Functions of Selenoproteins of Pigs under Chronic Heat Stress

Chronic heat stress (CHS) induces metabolic changes in skeletal muscle from growth to maintenance that jeopardizes growth performance, carcass traits, and meat quality of pigs. We investigated the protective effect of dietary organic selenium (hydroxy-4-methylselenobutanoic acid, OH-SeMet) on CHS-induced skeletal muscle damages of growing pigs, and the corresponding responses of selenoproteins. A total of 40 ((Landrace ×Yorkshire) × Duroc) pigs with an average live weight of 49.64 ± 2.48 kg were used in this 4-week trial. Pigs were randomly allotted to 5 groups: The control group was raised on a basal diet in a thermoneutral environment (22 ± 2 °C); and four CHS groups were raised on a basal diet and supplemented with Se 0.0, 0.2, 0.4, and 0.6 mg/kg as OH-SeMet, respectively, in hyperthermal condition (33 ± 2 °C). CHS resulted in significant decrease of growth performance, carcass traits, and meat quality, which were associated with reduced (p &lt; 0.05) serum alkaline phosphatase (ALP) and total superoxide dismutase (T-SOD) and increased (p &lt; 0.05) serum creatine (CK), sarcous heat shock protein 70 (HSP70), glucokinase (GCK), phosphoenolpyruvate carboxykinase (PEPCK), and malondialdehyde (MDA) contents. Meanwhile, four metabolism-related genes and seven selenoprotein encoding genes were abnormally expressed in skeletal muscle. Dietary OH-SeMet addition partially alleviated the negative impact of CHS on carcass traits and improved meat quality. These improvements were accompanied by the increase in Se deposition, the anti-oxidative capacity of serum and muscle, and protein abundance of GPX1, GPX3, GPX4, and SELENOP. Supplementation with 0.6 mg Se/kg (OH-SeMet) restored the sarcous PEPCK, and 0.4 and 0.6 mg Se/kg (OH-SeMet) restored all abnormally expressed metabolism-related and selenoprotein encoding genes. In summary, dietary supplementation with OH-SeMet beyond Se requirement mitigated CHS-induced depression of carcass traits and meat quality of pigs associated with optimal skeletal metabolism, enhanced antioxidant capacity, and regulation of selenoproteins in skeletal muscle of pigs

Urinary Metabolomic Approach Provides New Insights into Distinct Metabolic Profiles of Glutamine and N-Carbamylglutamate Supplementation in Rats

Glutamine and N-carbamylglutamate can enhance growth performance and health in animals, but the underlying mechanisms are not yet elucidated. This study aimed to investigate the effect of glutamine and N-carbamylglutamate supplementation in rat metabolism. Thirty rats were fed a control, glutamine, or N-carbamylglutamate diet for four weeks. Urine samples were analyzed by nuclear magnetic resonance (NMR)-based metabolomics, specifically high-resolution 1H NMR metabolic profiling combined with multivariate data analysis. Glutamine significantly increased the urine levels of acetamide, acetate, citrulline, creatinine, and methymalonate, and decreased the urine levels of ethanol and formate (p &lt; 0.05). Moreover, N-carbamylglutamate significantly increased the urine levels of creatinine, ethanol, indoxyl sulfate, lactate, methymalonate, acetoacetate, m-hydroxyphenylacetate, and sarcosine, and decreased the urine levels of acetamide, acetate, citrulline, creatine, glycine, hippurate, homogentisate, N-acetylglutamate, phenylacetyglycine, acetone, and p-hydroxyphenylacetate (p &lt; 0.05). Results suggested that glutamine and N-carbamylglutamate could modify urinary metabolome related to nitrogen metabolism and gut microbiota metabolism. Moreover, N-carbamylglutamate could alter energy and lipid metabolism. These findings indicate that different arginine precursors may lead to differences in the biofluid profile in rats