Excessive dietary L-tryptophan regulated amino acids metabolism and serotonin signaling in the colon of weaning piglets with acetate-induced gut inflammation.

L-Tryptophan (Trp) was shown to improve the gut barrier and growth of weaning piglets. However, whether excessive dietary Trp regulates amino acids (AAs) metabolism and gut serotonin (5-HT) homeostasis in piglets with gut inflammation is not clear yet. We hypothesize that excessive dietary Trp alleviates acetate-induced colonic inflammation and gut barrier damage in weaning piglets partially through the regulation of colonic AAs metabolism and 5-HT signaling. Fifty-four 21-day-old weaned piglets were divided into six groups: control, acetate, 0.2%Trp, 0.2%Trp + acetate, 0.4% Trp, and 0.4%Trp + acetate. Piglets were fed a basal diet supplemented with 0%, 0.2%, or 0.4% of Trp throughout the 12-day experiment. During days 0-7, all piglets had free access to diet and drinking water. On day 8, piglets were intrarectal administered with 10 mL of 10% acetate saline solution or 0.9% saline. During days 8-12, all piglets were pair-fed the same amount of feed per kg bodyweight. Results showed that excessive dietary Trp alleviated acetate-induced reductions in daily weight gain and increase in feed/gain ratio. Trp restored (P < 0.05) acetate-induced increase in concentrations of free aspartate, glutamate/glutamine, glycine, 5-HT, and 3-methylindole in the colon, downregulation of zonula occludens-1 and 5-HT reuptake transporter (SERT) expression and upregulation of IL-1ß, IL-8, TLR4, and 5-HT receptor 2A (HTR2A) expression, and the increase in ratios of p-STAT3/ STAT3 and p-p65/p65 in the colon. The above findings suggested that excessive dietary Trp in the proper amount regulated colonic AAs metabolism, 5-HT homeostasis, and signaling that may contribute as important regulators of gut inflammation during the weaning transition.

L-leucine supplementation reduces growth performance accompanied by changed profiles of plasma amino acids and expression of jejunal amino acid transporters in breast-fed intra-uterine growth-retarded piglets.

Previously, we provided an evidence that L-leucine supplementation facilitates growth performance in suckling piglets with normal birth weight. However, it remains hitherto obscure weather breast-fed piglets displaying intrauterine growth restriction (IUGR) show a similar effect in response to L-leucine provision. In this study, seven-day-old sow-reared IUGR piglets were orally administrated with L-leucine (0, 0.7 1.4, 2.1 g/kg BW) twice daily for two weeks. Increasing leucine levels hampered the growth performance of suckling IUGR piglets. The average daily gain of IUGR piglets was significantly reduced in 1.4 g/kg BW and 2.1 g/kg BW L-leucine supplementation groups (P < 0.05). Except for ornithine and glutamine, the plasma concentrations of other amino acids were abated as L-leucine levels increased (P < 0.05). Leucine supplementation led to reduction in the levels of urea, blood ammonia, blood glucose, triglyceride, and total cholesterol, as well as an elevation in the level of low density lipoprotein cholesterol in suckling IUGR piglets (P < 0.05). In addition, 1.4g/kg BW of L-leucine enhanced the mRNA expression of ATB 0,+ , whereas decreased the mRNA abundances of CAT1, y+LAT1, ASCT2 and b 0,+ AT in the jejunum (P < 0.05). Concomitantly, the jejunum of IUGR piglets in L-leucine group contains more ATB0,+ and less SNAT2 protein than in the control (P < 0.05). Collectively, L-leucine supplementation impairs growth performance in breast-fed IUGR piglets, which may be associated with depressed nutritional conditions and alterations in the uptake of amino acids and the expression of amino acid transporters in the small intestine.

Glycine regulates mucosal immunity and the intestinal microbial composition in weaned piglets.

Glycine is an amino acid with a diverse array of health benefits regarding metabolism, immunity, and development. The aim of this study was to test the hypothesis that glycine supplementation alters the intestinal microbial composition and improves the intestinal mucosal immunity of weaned piglets. One hundred and twenty-eight weaned piglets divided into 4 groups were fed with a corn- and soybean meal-based diet supplemented with 0 (control), 0.5, 1, or 2% glycine for 7 days. The intestinal microbiota and tissue samples from the control and the 2% glycine-supplemented piglets were collected for determination of the composition of microbial community and the intestinal mucosal barrier function. Piglets fed with diet containing 2% glycine, instead of 0.5% or 1% glycine, presented elevated average daily gain and feed conversion ratio, as compared with the control. 2% glycine enhanced the abundance of mucins in the jejunum and ileum and mRNA level of porcine ß-defensin (pBD) 2 and pBD-3, as well as the protein level of secretory immunoglobulin A (sIgA) in the jejunum. The mRNA expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, and the protein level of phosphorylated p38 mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), nuclear factor (NF)-κB p65, and claudin-2 in the jejunum were lower in the 2% glycine group than that in the control. In addition, an elevated ratio of CD4+/CD8+ T lymphocytes was observed in the jejunum of piglets receiving diet supplemented with 2% glycine. The colon content of piglets fed with 2% glycine exhibited a reduction in abundance of pathogenic bacteria (Escherichia-Shigella, Clostridium, and Burkholderiales) and an increase in short-chain fatty acid-producing bacteria (Blautia, Lachnospiraceae, Anaerostipes, and Prevotella) in comparison with the control. We conclude that dietary supplementation with 2% glycine improves the intestinal immunological barrier function and the microbial composition, therefore, contributing to the growth performance of weaned piglets.

Glycine Attenuates Citrobacter rodentium-Induced Colitis by Regulating ATF6-Mediated Endoplasmic Reticulum Stress in Mice.

SCOPE: Inflammatory bowel disease (IBD) is an inflammatory gastrointestinal disorder in which endoplasmic reticulum (ER) stress and dysbiosis of the intestinal microbiota are implicated. Glycine supplementation is reported to reduce inflammatory responses in experimental colitis. However, the underlying mechanisms responsible for the beneficial effects remain unclear. METHODS AND RESULTS: Female C57BL/6 mice are orally administered with glycine (3.5 or 5.2 g kg-1 body weight) for 14 continuous days. On day 8 post-glycine supplementation, the mice are orally inoculated with 2 × 109 CFU Citrobacter rodentium (C. rodentium). The results show that glycine alleviates C. rodentium-induced body weight loss, increased disease activity index and spleen weight, colon length shortening, and colonic hyperplasia. Glycine suppresses the activation and infiltration of inflammatory cells, and secretion of pro-inflammatory cytokines in the colon tissues. The apoptosis of colon epithelial cells is also abrogated by glycine, which is associated with the inactivation of activating transcription factor 6α (ATF6α)-C/EBP homologous protein (CHOP) signaling. In addition, glycine administration increases α diversity, restores ß diversity, and abolishes the reduction in Lactobacillus, Bifidobacterium, Alistipes, Turicibacter, and Alloprevotella in the colon. CONCLUSIONS: Glycine supplementation is a nutritional strategy that may ameliorate C. rodentium-induced colitis by regulating ATF6α-CHOP-mediated ER stress and enhancing the abundance of Lactobacillus.

Dietary supplementation with Yucca schidigera extract alleviated heat stress-induced unfolded protein response and oxidative stress in the intestine of Nile tilapia (Oreochromis niloticus).

Heat stress due to global warming exerts deleterious effects on both humans and animals. However, nutritional strategies to reduce heat stress-induced intestinal mucosal barrier dysfunction and the underlying mechanisms remain largely unknown. In the present study, 240 tilapia were distributed into four treatment groups that were fed a basal diet supplemented with or without 0.1% Yucca schidigera extract under normal (28 °C) temperature or heat stress (36 °C) conditions for 2 weeks. Our results showed that tilapia exposed to heat stress resulted in growth arrest, intestinal dysfunction, oxidative damage, endoplasmic reticulum stress, and pro-inflammatory response, which were significantly relieved by yucca supplementation. The alleviative effect of Yucca schidigera extract was related to the down-regulation of mRNA expression of ubiquitin-proteasome system (Polyubiquitin, Proteasome 26S, Proteasome α5, Proteasome ß3, and Ubiquitin-like 3) and inflammatory factors (tumor necrosis factor α, interleukin 1ß, and interleukin 8), as well as the improved histological structure and activation of Hsp70, nuclear factor erythroid 2-related factor 2 signaling, interleukin 10, lysozyme, complement 3, and acid phosphatase in the intestine of tilapia. Collectively, these results indicated that heat stress-induced growth arrest, intestinal dysfunction, and oxidative damage were alleviated by dietary supplementation with Yucca schidigera extract. This offers a nutritional way of improving the growth and intestinal health of tilapia exposed to a hot environment.

Protective Effects of Cinnamaldehyde on the Inflammatory Response, Oxidative Stress, and Apoptosis in Liver of Salmonella typhimurium-Challenged Mice.

Salmonella typhimurium infection is associated with gastrointestinal disorder and cellular injury in the liver of both humans and animals. Cinnamaldehyde, the main component of essential oil from cinnamon, has been reported to have anti-inflammatory, anti-oxidative, and anti-apoptotic effects. However, it remains unknown whether cinnamaldehyde can alleviate Salmonella typhimurium infection-induced liver injury in mice. In the present study, we found that cinnamaldehyde attenuated Salmonella typhimurium-induced body weight loss, the increase of organ (liver and spleen) indexes, hepatocyte apoptosis, and the mortality rate in mice. Further study showed that cinnamaldehyde significantly alleviated Salmonella typhimurium-induced liver injury as shown by activities of alanine transaminase, aspartate transaminase, and myeloperoxidase, as well as malondialdehyde. The increased mRNA level of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IFN-γ) and chemokines (CCL2 and CCL3) induced by Salmonella typhimurium were significantly abolished by cinnamaldehyde supplementation. These alterations were associated with a regulatory effect of cinnamaldehyde on TLR2, TLR4, and MyD88. 16S rDNA sequence analysis showed that Salmonella typhimurium infection led to upregulation of the abundances of genera Akkermansia, Bacteroides, Alistipes, Muribaculum, and Prevotellaceae UCG-001, and downregulation of the abundances of genera Lactobacillus, Enterorhabdus, and Eggerthellaceae (unclassified). These alterations were reversed by cinnamaldehyde supplementation. In conclusion, cinnamaldehyde attenuated the inflammatory response, oxidative stress, and apoptosis in the liver of Salmonella typhimurium-infected mice. Supplementation of cinnamaldehyde might be a preventive strategy to alleviate liver injury caused by Salmonella typhimurium infection in humans and animals.

Dietary Supplementation with Glycine Enhances Intestinal Mucosal Integrity and Ameliorates Inflammation in C57BL/6J Mice with High-Fat Diet-Induced Obesity.

BACKGROUND: Obesity, a major public health problem worldwide, is associated with dysfunction of the intestinal barrier. Glycine (Gly) has been reported to enhance the expression of tight-junction proteins in porcine enterocytes. It is unknown whether Gly can improve intestinal barrier integrity in obese mice. OBJECTIVES: This study tested the hypothesis that Gly enhances the intestinal epithelial barrier by regulating endoplasmic reticulum (ER) stress-related signaling and mitigating inflammation in high-fat diet (HFD)-induced obese mice. METHODS: Five-week-old male C57BL/6J mice were fed a normal-fat diet (ND; fat = 10% energy) or an HFD (fat = 60% energy) and received drinking water supplemented with 2% Gly or 2.37% l-alanine (Ala; isonitrogenous control) daily for 12 wk. Body weight gain and tissue weights, glucose tolerance and the activation of immune cells, as well as the abundances of tight-junction proteins, ER stress proteins, and apoptosis-related proteins in the jejunum and colon were determined. In addition, the body weights of naïve ND and HFD groups (nND and nHFD, respectively) were also recorded for comparison. Differences were analyzed statistically by ANOVA followed by the Duncan multiple-comparison test using SAS software. RESULTS: Compared with ND-Ala, HFD-feeding resulted in enhanced macrophage (CD11b+ and F4/80+) infiltration and immune cell activation by 1.9- to 5.4-fold (P < 0.05), as well as the upregulation of ER stress sensor proteins (including phospho-inositol-requiring enzyme 1α and binding immunoglobulin protein) by 2.5- to 4.5-fold, the induction of apoptotic proteins by 1.5- to 3.2-fold, and decreased abundances of tight-junction proteins by 35%-65% (P < 0.05) in the intestine. These HFD-induced abnormalities were significantly ameliorated by Gly supplementation in the HFD-Gly group (P < 0.05). Importantly, Gly supplementation also significantly enhanced glucose tolerance (P < 0.05) by 1.5-fold without affecting the fat accumulation of HFD-induced obese mice. CONCLUSIONS: Gly supplementation enhanced the intestinal barrier and ameliorated inflammation and insulin resistance in HFD-fed mice. These effects of Gly were associated with reduced ER stress-related apoptosis in the intestine of obese mice.

Dietary L-arginine supplementation reduces lipid accretion by regulating fatty acid metabolism in Nile tilapia (Oreochromis niloticus).

BACKGROUND: Excessive white fat accumulation in humans and other animals is associated with the development of multiple metabolic diseases. It is unknown whether dietary L-arginine supplementation reduces lipid deposition in high fat diet-fed Nile tilapia (Oreochromis niloticus). RESULTS: In the present study, we found that dietary supplementation with 1% or 2% arginine decreased the deposition and concentration of fats in the liver; the concentrations of triglycerides, low-density lipoprotein, total cholesterol, and high-density lipoprotein in the serum; and the diameter of adipocytes in intraperitoneal adipose tissue. Compared with the un-supplementation control group, the hepatic activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase, and hepatic concentration of malondialdehyde were reduced but these for catalase and superoxide dismutase were enhanced by dietary supplementation with 2% arginine. Arginine supplementation reduced the total amounts of monounsaturated fatty acids, while increasing the total amounts of n-3 and n-6 polyunsaturated fatty acids in the liver. These effects of arginine were associated with reductions in mRNA levels for genes related to lipogenesis (sterol regulatory element-binding protein-1, acetyl-CoA carboxylase α, stearoyl-CoA desaturase, and fatty acid synthase) but increases in mRNA levels for genes involved in fatty acid ß-oxidation (carnitine palmitoyltransferase 1α and peroxisome proliferator-activated receptor α). In addition, hepatic mRNA levels for Δ4 fatty acyl desaturase 2 and elongase 5 of very long-chain fatty acids were enhanced by arginine supplementation. CONCLUSION: These results revealed that dietary L-arginine supplementation to tilapia reduced high fat diet-induced fat deposition and fatty acid composition in the liver by regulating the expression of genes for lipid metabolism.

Dietary L-Tryptophan Regulates Colonic Serotonin Homeostasis in Mice with Dextran Sodium Sulfate-Induced Colitis.

BACKGROUND: L-tryptophan (Trp) has been reported to regulate gut immune responses during inflammation. However, the underlying mechanisms are largely unknown. OBJECTIVE: We investigated the role of Trp supplementation on the serotonin receptor (HTR)-mediated immune response in the colon of mice with dextran sodium sulfate (DSS)-induced colitis. METHODS: In Experiment 1, male C57BL/6 mice were randomly assigned to 1 of 4 groups: Control (Con) or L-Trp supplementation [0.1 mg/(g body weight·d) in drinking water] (Trp) with (+DSS) or without 2% DSS in drinking water from days 8 to 14 of the 17-d study. In Experiments 2 and 3, Trp + DSS (Expt. 2) or DSS (Expt. 3) mice were treated as described above and subcutaneously administered with HTR1A or HTR4 antagonists (or their combination) or an HTR2 agonist from days 8 to 14 of the 15-d study. Changes in immune cell phenotypes, inflammatory mediators, and related cell signaling molecules were assessed by flow cytometry, real-time PCR, or Western blot. The mRNA abundances of Trp hydroxylase (Tph1), serotonin reuptake transporter (Slc6a4), and Htr in the colon were also assessed. RESULTS: Trp supplementation before DSS treatment upregulated the expression of colonic Slc6a4 (0.49 compared with 0.30), Htr1a (1.14 compared with 0.65), and Htr4 (1.08 compared with 0.70), downregulated the expression of Htr2a (1.54 compared with 1.89), and decreased the colonic serotonin concentration (11.5 compared with 14.8 nmol/g tissue) (P < 0.01). Trp regulated the DSS-induced immune response partly through attenuating the activation of toll-like receptor 4 (TLR4)-STAT3 signaling and nucleus p-65. Either an HTR2 agonist or HTR1A and HTR4 antagonists reversed the effects of Trp. CONCLUSIONS: In mice treated with DSS, Trp supplementation before DSS administration improved colonic immune responses partly by reducing colonic serotonin and subsequent interactions with HTR1A and HTR4, which are known to be present on neutrophils and macrophages.

Effects of maternal L-proline supplementation on inflammatory cytokines at the placenta and fetus interface of mice.

Dietary L-proline (proline) supplementation during gestation enhances fetal survival and placental development in mice. The objective of the present study was to test the hypothesis that this beneficial effect of proline was associated with alterations in inflammatory response at the placenta and fetus interface. Populations of immune cells present in peripheral blood mononuclear cells (PBMC) were determined by flow cytometry analysis. The concentrations of immunoglobulins in plasma, and the concentrations of cytokines in plasma, uterus, placenta, and amniotic fluid were measured using a bead-based immunoassay. The data showed that proline supplementation led to higher (P < 0.05) populations of B lymphocytes (CD3-CD19+), natural killer (NK) cells (CD3-NK1.1+), and dendritic cells (DCs, CD11c+MHCII+) in peripheral blood, as compared with the controls. Conversely, mice fed a proline-supplemented diet had a lower population of neutrophils (CD11b+F4/80-). Further study showed that proline supplementation decreased (P < 0.05) the concentrations of (1) interleukin (IL)-23, IL-1α, and IL-6 in plasma; (2) IL-6 in the uterus; and (3) tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein (MCP)-1, and IL-17 in the placenta; and (4) interferon (IFN)-γ in amniotic fluid, compared with controls. Conversely, proline supplementation resulted in higher (P < 0.05) concentrations of (1) IL-10, IL-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in plasma; (2) IL-10 and IL-1α in the uterus; and (3) IL-1α, IL-1ß, IL-10, IL-27, and IFN-ß in amniotic fluid, compared with controls. Moreover, concentrations of immunoglobulin (Ig) G2b and IgM were enhanced (P < 0.05) by proline administration. Taken together, our results reveal a regulatory effect of proline in the immunological response at the maternal-fetal interface, which is critical for embryonic development and fetal survival.

Glycine Attenuates LPS-Induced Apoptosis and Inflammatory Cell Infiltration in Mouse Liver.

BACKGROUND: Liver dysfunction impairs immunological homeostasis. Glycine (Gly) has been reported to have antioxidative and anti-inflammatory effects and to regulate apoptosis in various models. OBJECTIVES: The aim of the present study was to determine whether Gly could attenuate LPS-induced liver injury. METHODS: In Experiment 1, 48 6-week-old male C57BL/6 mice were randomly assigned into one of 4 groups: CON (control), GLY [orally administered Gly, 5 g · kg body weight (BW)-1 · d-1 for 6 d], LPS (5 mg/kg BW, intraperitoneally administered), and GLY + LPS (Gly supplementation, and on day 7 LPS treatment). In Experiment 2, mice were untreated, pretreated with Gly as above, or pretreated with Gly + l-buthionine sulfoximine (BSO) (0.5 g/kg BW, intraperitoneally administered every other day) for 6 d. On day 7, mice were injected with LPS as above. Histological alterations, activities of antioxidative enzymes, apoptosis, and immune cell infiltration were analyzed. RESULTS: In Experiment 1, compared with CON, LPS administration resulted in increased karyolysis and karyopyknosis in the liver by 8- to 10-fold, enhanced serum activities of alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH) by 1- to 1.8-fold, and increased hepatic apoptosis by 5.5-fold. Furthermore, LPS exposure resulted in increased infiltration of macrophages and neutrophils in the liver by 3.2- to 7.5-fold, elevated hepatic concentrations of malondialdehyde and hydrogen peroxide (H2O2), and elevated myeloperoxidase (MPO) activity by 1.5- to 6.3-fold. In Experiment 2, compared with the LPS group, mice in the GLY + LPS group had fewer histological alterations (68.5%-75.9%); lower serum ALT, AST, and LDH activities (24.3%-64.7%); and lower hepatic malondialdehyde and H2O2 concentrations (46.1%-80.2%), lower MPO activity (39.2%), immune cell infiltration (52.3%-85.3%), and apoptosis (69.6%), which were abrogated by BSO. Compared with the GLY + LPS group, mice in the GLY + BSO + LPS group had lower hepatic activities of catalase, superoxide dismutase, and glutathione peroxidase by 33.5%-48.5%; increased activation of NF-κB by 2.3-fold; and impaired nuclear factor (erythroid-derived 2)-like 2 signaling by 38.9%. CONCLUSIONS: Gly is a functional amino acid with an ability to protect the liver against LPS-induced injury in mice.

l-Glutamine Represses the Unfolded Protein Response in the Small Intestine of Weanling Piglets.

BACKGROUND: Dysfunction of the endoplasmic reticulum (ER) results in apoptosis, inflammation, and enhanced proteolysis in the small intestine of humans and animals. l-Glutamine (Gln) is required for intestinal mucosal homeostasis in piglets. However, a functional role of the ER in the enterocytes of weanling piglets and its contribution to intestinal mucosal integrity remain largely unknown. OBJECTIVE: This study was conducted to test the hypothesis that preweaning administration of Gln alleviates the activation of unfolded protein response (UPR) in the small intestine of weanling piglets. METHODS: Eighteen sow-reared piglets aged 7 d from 3 litters (6 piglets/litter) were assigned randomly into 1 of 3 treatment groups. Piglets were reared by sows until age 24 d, or were reared by sows and orally administered either l-alanine [1.84 g · kg body weight (BW)-1 · d-1] or Gln (1.52 g · kg BW-1 · d-1) twice daily between 7 and 21 d of age, and then weaned to a corn- and soybean meal-based diet. The small-intestinal samples were collected at 24 d of age for analyses of abundance of proteins related to ER stress and apoptosis, concentrations of inflammatory cytokines, and mRNA abundance for genes implicated in protein degradation. RESULTS: Compared with age-matched suckling piglets, weaning stress increased apoptosis and decreased cell proliferation in the jejunum. The abundance of proteins related to ER stress [binding immunoglobulin protein, activating transcription factor 6α, phosphorylated (p)-inositol-requiring kinase 1α, and p-eukaryotic initiation factor 2α] was elevated by 200% to 320%, and that of apoptotic proteins (CCAAT/enhancer-binding protein homologous protein, p-Jun-N-terminal kinase, caspase-12, cleaved caspase-3, and Bcl-2-associated X) was augmented by 100% to 350% in the jejunum of weanling piglets. The protein abundance for IL-1ß, TNF-α, and IL-8 was increased by 100% to 230% in the jejunum of weanling piglets. These alterations in gene and protein expression were markedly abrogated by Gln supplementation. The mRNA concentration of F-Box protein 32 in the jejunum of weanling piglets was increased by 70%, compared with the control group, and was not affected by Gln supplementation. CONCLUSION: Our results indicate that preweaning administration of Gln to nursing piglets alleviates the weaning-activated UPR.

Maternal L-proline supplementation during gestation alters amino acid and polyamine metabolism in the first generation female offspring of C57BL/6J mice.

We recently reported that dietary supplementation with L-proline (proline) during gestation improved embryonic survival in C57BL/6J mice. The objective of the present study was to test the hypothesis that the effect of maternal proline supplementation on embryonic survival can be carried forward to the first generation female offspring. In the F0 generation, pregnant dams were fed a purified diet supplemented with 0 (control) or 5 g proline/kg diet. The F1 female adult offsprings were bred to fertile males. Fetal survival at embryonic day (E)12.5 and reproductive outcomes at term birth were recorded. The concentrations of amino acids, ammonia, and urea in plasma and amniotic fluid, as well as concentrations of polyamines in placental tissues and amniotic fluid at E12.5 were determined. Results showed that the F1 generation female offspring from proline-supplemented dams had higher (P < 0.05) concentrations of glutamate and taurine in plasma; of putrescine and spermidine in placental tissues; and of glycine, taurine, and spermidine in amniotic fluid at E12.5, as compared with F1 generation female offsprings from dams without proline supplementation. Concentration of proline in the plasma of offspring mice from proline-supplemented dams were lower (P < 0.05), as compared with the control group. No differences in fetal survival, reproductive outcomes, or concentrations of ammonia and urea in plasma and amniotic fluid were observed between the two groups of F1 female offspring. Collectively, our results indicate that the benefits of maternal proline supplementation during gestation on improving embryonic survival and fetal growth in F0 females are not transmitted to their F1 generation females.

Maternal L-proline supplementation enhances fetal survival, placental development, and nutrient transport in mice†.

L-Proline (proline) in amniotic fluid was markedly increased during pregnancy in both pigs and sheep. However, in vivo data to support a beneficial effect of proline on fetal survival are not available. In this study, pregnant C57BL/6J mice were fed a purified diet supplemented with or without 0.50% proline from embryonic day 0.5 (E0.5) to E12.5 or term. Results indicated that dietary supplementation with proline to gestating mice enhanced fetal survival, reproductive performance, the concentrations of proline, arginine, aspartic acid, and tryptophan in plasma and amniotic fluid, while decreasing the concentrations of ammonia and urea in plasma and amniotic fluid. Placental mRNA levels for amino acid transporters, including Slc36a4, Slc38a2, Slc38a4, Slc6a14, and Na+/K+ ATPase subunit-1α (Atp1a1), fatty acid transporter Slc27a4, and glucose transporters Slc2a1 and Slc2a3, were augmented in proline-supplemented mice, compared with the control group. Histological analysis showed that proline supplementation enhanced labyrinth zone in the placenta of mice at E12.5, mRNA levels for Vegf, Vegfr, Nos2, and Nos3, compared with the controls. Western blot analysis showed that proline supplementation increased protein abundances of phosphorylated (p)-mTORC1, p-ribosomal protein S6 kinase (p70S6K), and p-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), as well as the protein level of GCN2 (a negative regulator of mTORC1 signaling). Collectively, our results indicate a novel functional role of proline in improving placental development and fetal survival by enhancing placental nutrient transport, angiogenesis, and protein synthesis.

Glycine supplementation to breast-fed piglets attenuates post-weaning jejunal epithelial apoptosis: a functional role of CHOP signaling.

This study was conducted to test the hypothesis that preweaning  glycine supplementation to breast-fed piglets alleviated the post-weaning  apoptosis of jejunal epithelium through CHOP signaling. Seven-day-old sow-reared piglets were orally administrated with 0, 50, 100, or 200% of glycine intake from sow's milk twice daily for 14 days and then were weaned at 21 days of age. Tissue samples were collected at 28 days of age for determining intestinal morphology, serum diamine oxidase (DAO) activity, abundances of proteins involved in ER stress and apoptosis. Glycine (100-200%) administration increased villus height, the ratio of villus height to crypt depth in the jejunum. Glycine supplementation (200%) enhanced average daily weight gain during the first 2 weeks post-weaning. Serum DAO activity and jejunal epithelium apoptosis were decreased, but the number of goblet cells in the jejunum was increased. Western blot analysis showed that 100-200% glycine enhanced the protein levels of occludin, claudin-1, and zonula occludens (ZO)-1 without affecting those of claudin-3, ZO-2, and ZO-3. Further studies showed that protein abundances of glucose-regulated protein 78 (BiP/GRP78) and p-IRE1α, instead of ATF6α, were reduced by glycine. Among the proteins related to apoptosis, abundances of CHOP and p53 were reduced, whereas those of Bcl-2 and Bcl-xL were enhanced in the jejunum of 100-200% glycine-supplemented piglets. Collectively, our results indicated that preweaning glycine supplementation improved the intestinal development of post-weaning piglets. The beneficial effect of glycine was associated with improved intestinal mucosal barrier and reduced apoptosis of enterocytes through CHOP signaling.

Dietary l-Tryptophan Supplementation Enhances the Intestinal Mucosal Barrier Function in Weaned Piglets: Implication of Tryptophan-Metabolizing Microbiota.

l-Tryptophan (Trp) is known to play an important role in the health of the large intestine. However, a role of dietary Trp in the small-intestinal mucosal barrier and microbiota remains poorly understood. The present study was conducted with weaned piglets to address this issue. Postweaning piglets were fed for 4 weeks a corn- and soybean meal-based diet supplemented with 0 (Control), 0.1, 0.2, or 0.4% Trp. The small-intestinal microbiota and serum amino acids were analyzed by bacterial 16S rRNA gene-based high-throughput sequencing methods and high-performance liquid chromatography, respectively. The mRNA levels for genes involved in host defense and the abundances of tight-junction proteins in jejunum and duodenum were measured by real time-PCR and Western blot techniques, respectively. The concentrations of Trp in the serum of Trp-supplemented piglets increased in a dose-dependent manner. Compared with the control group, dietary supplementation with 0.2⁻0.4% Trp reduced the abundances of Clostridium sensu stricto and Streptococcus in the jejunum, increased the abundances of Lactobacillus and Clostridium XI (two species of bacteria that can metabolize Trp) in the jejunum, and augmented the concentrations of secretory immunoglobulin A (sIgA) as well as mRNA levels for porcine ß-defensins 2 and 3 in jejunal tissues. Moreover, dietary Trp supplementation activated the mammalian target of rapamycin signaling and increased the abundances of tight-junction proteins (zonula occludens (ZO)-1, ZO-3, and claudin-1) in jejunum and duodenum. We suggested that Trp-metabolizing bacteria in the small intestine of weaned pigs primarily mediated the beneficial effects of dietary Trp on its mucosal integrity, health, and function.

Hydroxyproline Attenuates Dextran Sulfate Sodium-Induced Colitis in Mice: Involvment of the NF-κB Signaling and Oxidative Stress.

SCOPE: Inflammatory bowel disease (IBD) is a chronic disease of gastrointestinal tract in which oxidative stress and overactivation of inflammatory response are implicated. The aim of the present study is to test the hypothesis that hydroxyproline (Hyp), an amino acid with an antioxidative property, attenuates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS AND RESULTS: Male C57BL/6 mice supplemented with or without 1% Hyp are subjected to 2.5% DSS in drinking water to induce colitis. Hyp attenuates the severity of colitis as evidenced by reduced disease activity index scores, decreased myeloperoxidase activity, histological damage, and apoptosis. Furthermore, DSS-induced increases in reactive oxygen species accumulation, TNF-α and IL-6 secretion, and malonyldialdehyde activity and a decrease in reduced glutathione in the colon are ameliorated by Hyp. The enhanced phosphorylation of STAT3 and NF-κB following DSS administration is mitigated by Hyp, which is also observed in LPS-treated RAW264.7 macrophages. Moreover, the inhibitory effect of Hyp on IL-6 expression is mainly mediated by the NF-κB signaling, because the induction of STAT3 and IL-6 by LPS is markedly reversed by Bay11-7085, a specific inhibitor NF-κB. CONCLUSION: In summary, Hyp is a critical nutrient with an ability to attenuate DSS-induced colonic damage in mice. This beneficial effect of Hyp is partially mediated by inhibiting the NF-κB/IL-6 signaling and the restoration of redox homeostasis.

Maternal L-glutamine supplementation during late gestation alleviates intrauterine growth restriction-induced intestinal dysfunction in piglets.

Maternal dietary supplementation with L-glutamine (Gln) has been considered as an option to improve fetal growth and to prevent the occurrence of intrauterine growth restriction (IUGR). This study investigated whether maternal Gln supplementation could improve fetal growth as well as the intestinal development during late pregnancy. Sixty pregnant Landrace × Large White multiparous sows were assigned to two groups, either the group fed the control diet or the group with the diet supplemented with 1% Gln from d 85 of gestation until farrowing. One normal body weight piglet and one IUGR piglet were obtained from six litters in each group. Reproductive performance, plasma concentrations of free amino acids and related metabolites as well as piglet growth and tissue indexes were determined. Maternal Gln supplementation during late gestation increased the average birth weight, while decreasing the within-litter variation of newborn piglets. The concentrations of Gln in plasma were lower in IUGR piglets than in normal piglets. Glutamine supplementation enhanced Gln concentrations in maternal and piglet plasma and the piglet jejunum, compared with the Control group. Supplementing Gln suppressed intestinal miR-29a levels, and increased the abundance of extracellular matrix (ECM) and tight junction (TJ) proteins, resulting in increased intestinal weight and improved morphologies of the piglets. Collectively, Gln supplementation to the sow's diet increased fetal growth, decreased the within-litter variation of newborn piglets, and alleviated the IUGR-induced intestinal impairment. These findings suggest the possibility of maternal glutamine supplementation in the prevention and treatment of IUGR in animal production and human medicine.

Glycine enhances expression of adiponectin and IL-10 in 3T3-L1 adipocytes without affecting adipogenesis and lipolysis.

Glycine supplementation has been reported to enhance white-fat loss and improve sensitivity to insulin in animals with obesity or type 2 diabetes. However, the underlying mechanisms responsible for the beneficial effects of glycine remain largely unknown. The purpose of this study was to test the hypothesis that glycine regulates adipocyte differentiation, adipogenesis, and lipolysis, therefore, contributing to white-fat reduction. 3T3-L1 pre-adipocytes were induced to differentiate into adipocytes in the presence of glycine (0, 0.25, 1.0, and 2.0 mmol/L) or resveratrol (50 or 100 µmol/L, served as a positive control) during the differentiation process. Hela and HepG2 cells cultured with oleic acid to induce lipid accumulation in the presence of glycine (0, 1.0, and 2.0 mmol/L) or 10 µmol/L isoproterenol (served as a positive control) for 24 h. Intracellular lipid accumulation, intracellular triglycerides, lipid droplets' diameters of mature adipocytes, mRNA, and protein levels of genes involved in the adipogenesis and lipolysis were analyzed. Isobutylxanthine-dexamethasone-insulin (MDI)-induced adipogenesis in 3T3-L1 cells were blocked by resveratrol, but not by glycine, as shown by decreased lipid contents, reduced diameters of lipid droplets, decreased protein abundances for peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), as well as increased protein abundance of peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), critical transcriptional factors that regulates adipogenesis. However, the mRNA levels of adiponectin and interleukin-10 (IL-10), two adipose-derived adipocytokines with anti-inflammatory effects, were greatly enhanced (P < 0.05) by 2 mmol/L glycine. Compared with non-treated controls, 10 µmol/L isoproterenol significantly decreased (P < 0.05) the intracellular lipid and triglyceride contents induced by oleic acid in Hela and HepG2 cells. mRNA level of fatty acid synthase (FASN), a gene involved in fatty acid synthesis, was significantly reduced (P < 0.05), while that for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase), genes involved in lipolysis were significantly enhanced (P < 0.05) by isoproterenol. However, oleic acid induced the accumulation of intracellular triglyceride and lipid contents were not affected by glycine. In conclusion, glycine exposure enhanced the mRNA levels of adipose-derived adiponectin and IL-10 without affecting adipogenesis and lipolysis in 3T3-L1 adipocytes. These findings provide a possible explanation for the anti-obesity and anti-diabetic effects of glycine that were previously reported in animal models. More studies are needed to uncover the underlying mechanisms responsible for this regulatory effect of glycine on anti-inflammatory adipocytokines expression in both in vitro and in vivo models.

Analysis of Glutathione in Biological Samples by HPLC Involving Pre-Column Derivatization with o-Phthalaldehyde.

Glutathione (GSH) forms conjugates with polyamines in prokaryotes and eukaryotes. There is also evidence suggesting cross-talk between GSH and polyamines to regulate cellular homeostasis and function, particularly under the conditions of oxidative stress. Because of its versatile roles in cell metabolism and function, a number of high performance liquid chromatography (HPLC) methods have been developed for glutathione analysis. Here, we describe our rapid and sensitive method for the analysis of GSH and the oxidized form of glutathione (GS-SG) in animal tissues and cells by HPLC involving pre-column derivatization with o-phthalaldehyde (OPA). OPA reacts very rapidly (within 1 min) with S-carboxymethyl-glutathione at room temperatures (e.g., 20-25 °C) in an autosampler, and their derivatives are immediately injected into the HPLC column without any need for extraction. This method requires two simple steps (a total of 15 min) before samples are loaded into the autosampler: (a) the conversion of GS-SG into GSH by 2-mercaptoethanol; and (b) the oxidation of GSH by iodoacetic acid to yield S-carboxymethyl-glutathione. The autosampler is programmed to mix S-carboxymethyl-glutathione with OPA for 1 min to generate a highly fluorescent derivative for HPLC separation and detection (excitation wavelength 340 nm and emission wavelength 450 nm). The detection limit for GSH and GS-SG is 15 pmol/ml or 375 fmol/injection. The total time for chromatographic separation (including column regeneration) is 16 min for each sample. Our routine HPLC technique is applicable for analyses of cysteine and cystine, as well as polyamines and GSH-polyamine conjugates in biological samples.