Dihydroartemisinin ameliorates innate inflammatory response induced by Streptococcussuis-derived muramidase-released protein via inactivation of TLR4-dependent NF-κB signaling.

Muramidase-released protein (MRP) is now being recognized as a critical indicator of the virulence and pathogenicity of Streptococcus suis (S. suis). However, the identification of viable therapeutics for S. suis infection was hindered by the absence of an explicit mechanism for MRP-actuated inflammation. Dihydroartemisinin (DhA) is an artemisinin derivative with potential anti-inflammatory activity. The modulatory effect of DhA on the inflammatory response mediated by the virulence factor MRP remains obscure. This research aimed to identify the signaling mechanism by which MRP triggers the innate immune response in mouse spleen and cultured macrophages. With the candidate mechanism in mind, we investigated DhA for its ability to dampen the pro-inflammatory response induced by MRP. The innate immune response in mice was drastically triggered by MRP, manifesting as splenic and systemic inflammation with splenomegaly, immune cell infiltration, and an elevation in pro-inflammatory cytokines. A crucial role for Toll-like receptor 4 (TLR4) in coordinating the MRP-mediated inflammatory response via nuclear factor-kappa B (NF-κB) activation was revealed by TLR4 blockade. In addition, NF-κB-dependent transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinases (MAPKs) activation was required for the inflammatory signal transduction engendered by MRP. Intriguingly, we observed an alleviation effect of DhA on the MRP-induced immune response, which referred to the suppression of TLR4-mediated actuation of NF-κB-STAT3/MAPK cascades. The inflammatory response elicited by MRP is relevant to TLR4-dependent NF-κB activation, followed by an increase in the activity of STAT3 or MAPKs. DhA mitigates the inflammation process induced by MRP via blocking the TLR4 cascade, highlighting the therapeutic potential of DhA in targeting S. suis infection diseases.

Intersex is required for female sexual development in Hermetia illucens.

Sex-determination pathways are extremely diverse. Understanding the mechanism of sex determination in insects is important for genetic manipulation of the pest population and for breeding of economically valuable insects. Although sex determination has been well characterized in the model species Drosophila melanogaster, little is known about this pathway in Stratiomyidae. In the present study, we first identified the Drosophila intersex (ix) homolog in Hermetia illucens, also known as the black soldier fly, which belongs to the Stratiomyidae family and which is an important insect for the conversion of various organic wastes. Phylogenetic analyses and multiple sequence alignment revealed that Hiix is conserved compared with Drosophila. We showed that Hiix is highly expressed in internal genitalia. Disruption of the Hiix gene using CRISPR/Cas9 resulted in female-specific defects in external genitalia and abnormal and undersized ovaries. Taken together, our study furthers our understanding of sex determination in insects and could facilitate breeding of H. illucens.

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.

S100A4 protects mice from high-fat diet-induced obesity and inflammation.

As a member from S100 calcium-binding protein family, S100A4 is ubiquitous and elevated in tumor progression and metastasis, but its role in regulating obesity has not been well characterized. In this study, we showed that S100A4 was mainly expressed by stromal cells in adipose tissue and the S100A4 level in adipose tissue was decreased after high-fat diet (HFD). S100A4 deficient mice exhibited aggravated symptoms of obesity and suppressed insulin signaling after 12 weeks of HFD. Aggravated obesity in S100A4 deficient mice were found to be positively correlated with higher inflammatory status of the liver. Then, we found that extracellular S100A4 or overexpressed S100A4 inhibited adipogenesis and decreased mRNA levels of inflammation gene in 3T3-L1 adipocytes in vitro; whereas small interfering RNA (siRNA)-mediated suppression of S100A4 displayed the opposite results. Additionally, the protective effect induced by S100A4 during HFD-induced obesity was tightly related with activation of Akt signaling in adipose tissues, as well as livers and muscles. Taken together, we demonstrate that S100A4 is an inhibitory factor for obesity and attenuates the inflammatory reaction, while activating the Akt signaling, which suggest that S100A4 is a potential candidate for the treatment of diet-induced obesity and its complications.

S100A4 promotes lung tumor development through ß-catenin pathway-mediated autophagy inhibition.

Autophagy has emerged as a critical pathway in tumor development. S100A4 plays important roles in tumor metastasis, but its role in regulating autophagy has not been well characterized. In this study, we found that S100A4 was significantly upregulated in lung adenocarcinoma tissues. Clinical investigation demonstrated that high expression level of S100A4 was associated with tumor size and advanced tumor grades of lung adenocarcinoma patients. Moreover, our results revealed that extracellular S100A4 or overexpression of S100A4 inhibited starvation-induced autophagy and promoted cell proliferation in lung cancer cells in vitro; whereas small interfering RNA (siRNA)-mediated suppression of S100A4 increased autophagy and reduced cell viability in both A549 and LLC cells. Additionally, S100A4 inhibited starvation-induced autophagy to promote tumor cell viability via the Wnt pathway. Increased expression of ß-catenin consistently led to a decreased LC3-II protein abundance. Further, the inhibitory effect of S100A4 on autophagy and its promotion role in cell proliferation was abolished in A549 and LLC cells using the receptor for advanced glycation end products (RAGE)-specific inhibitor (FPS-ZM1). S100A4-deficient mice showed retarded tumor development. This effect was well correlated with increased expression of autophagy markers. Our findings demonstrate that S100A4 promotes lung tumor development through inhibiting autophagy in a ß-catenin signaling and S100A4 receptor RAGE-dependent manner, which provides a novel mechanism of S100A4-associated promotion of tumor development.

Intestinal Epithelial Cell Endoplasmic Reticulum Stress and Inflammatory Bowel Disease Pathogenesis: An Update Review.

The intestinal epithelial cells serve essential roles in maintaining intestinal homeostasis, which relies on appropriate endoplasmic reticulum (ER) function for proper protein folding, modification, and secretion. Exogenous or endogenous risk factors with an ability to disturb the ER function can impair the intestinal barrier function and activate inflammatory responses in the host. The last decade has witnessed considerable progress in the understanding of the functional role of ER stress and unfolded protein response (UPR) in the gut homeostasis and its significant contribution to the pathogenesis of inflammatory bowel disease (IBD). Herein, we review recent evidence supporting the viewpoint that deregulation of ER stress and UPR signaling in the intestinal epithelium, including the absorptive cells, Paneth cells, goblet cells, and enteroendocrine cells, mediates the action of genetic or environmental factors driving colitis in experimental animals and IBD patients. In addition, we highlight pharmacologic application of chaperones or small molecules that enhance protein folding and modification capacity or improve the function of the ER. These molecules represent potential therapeutic strategies in the prevention or treatment of IBD through restoring ER homeostasis in intestinal epithelial cells.

Interactions between Intestinal Microbiota and Host Immune Response in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Although the etiology and pathogenesis of IBD remain unclear, both genetic susceptibility and environmental factors are implicated in the initiation and progression of IBD. Recent studies with experimental animal models and clinical patients indicated that the intestinal microbiota is one of the critical environmental factors that influence nutrient metabolism, immune responses, and the health of the host in various intestinal diseases, including ulcerative colitis and Crohn's disease. The objective of this review is to highlight the crosstalk between gut microbiota and host immune response and the contribution of this interaction to the pathogenesis of IBD. In addition, potential therapeutic strategies targeting the intestinal micro-ecosystem in IBD are discussed.

Cinnamicaldehyde regulates the expression of tight junction proteins and amino acid transporters in intestinal porcine epithelial cells.

BACKGROUND: Cinnamicaldehyde (CA) is a key flavor compound in cinnamon essential oil possessing various bioactivities. Tight junction (TJ) proteins are vital for the maintenance of intestinal epithelial barrier function, transport, absorption and utilization of dietary amino acids and other nutrients. In this study, we tested the hypothesis that CA may regulate the expression of TJ proteins and amino acid transporters in intestinal porcine epithelial cells (IPEC-1) isolated from neonatal pigs. RESULTS: Compared with the control, cells incubated with 25 µmol/L CA had increased transepithelial electrical resistance (TEER) and decreased paracellular intestinal permeability. The beneficial effect of CA on mucosal barrier function was associated with enhanced protein abundance for claudin-4, zonula occludens (ZO)-1, ZO-2, and ZO-3. Immunofluorescence staining showed that 25 µmol/L CA promoted the localization of claudin-1 and claudin-3 to the plasma membrane without affecting the localization of other TJ proteins, including claudin-4, occludin, ZO-1, ZO-2, and ZO-3, compared with the control cells. Moreover, protein abundances for rBAT, xCT and LAT2 in IPEC-1 cells were enhanced by 25 µmol/L CA, while that for EAAT3 was not affected. CONCLUSIONS: CA improves  intestinal mucosal barrier function by regulating the distribution of claudin-1 and claudin-3 in enterocytes, as well as enhancing protein abundance for amino acid transporters rBAT, xCT and LAT2 in enterocytes. Supplementation with CA may provide an effective nutritional strategy to improve intestinal integrity and amino acid transport and absorption in piglets.

Glycine Regulates Protein Turnover by Activating Protein Kinase B/Mammalian Target of Rapamycin and by Inhibiting MuRF1 and Atrogin-1 Gene Expression in C2C12 Myoblasts.

BACKGROUND: The regulation of protein turnover in skeletal muscle is essential for the maintenance of integrity, growth, and function of this tissue. We recently reported that glycine enhances skeletal muscle growth in young pigs. However, the underlying mechanisms remain unknown. OBJECTIVE: This study was conducted with a mouse myoblast cell line, C2C12, to test the hypothesis that glycine activates protein kinase B/mammalian target of rapamycin (Akt/mTOR), as well as inhibits 5'-adenosine monophosphate-activated protein kinase (AMPK) and the expression of genes for proteolysis. METHODS: C2C12 myoblasts were cultured with 0, 0.25 (physiologic concentration in mouse plasma), 0.5, or 1.0 mmol glycine/L. Cell proliferation, activation of mammalian target of rapamycin complex 1 (mTORC1), AMPK signaling, mRNA levels of atrogin-1 and muscle-specific ring finger protein 1 (MuRF1), and protein synthesis and degradation were measured in the absence or presence of an Akt inhibitor, LY294002, or an AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR). RESULTS: Compared with control cells, 0.25-1.0 mmol glycine/L enhanced cell growth (by 12-15%) after 24 h (P < 0.05). Glycine treatment led to increased DNA replication (by 70-80%) while enhancing mTORC1 activation by upregulating Akt and inhibiting AMPK signaling (P < 0.05). Accordingly, glycine exposure increased (P < 0.05) the rate of protein synthesis (by 20-80%) and inhibited (P < 0.05) the rate of protein degradation (by 15-30%) in a concentration-dependent manner in C2C12 cells. These observations were validated by the use of an Akt inhibitor, LY294002, or an AMPK activator, AICAR. Moreover, glycine addition resulted in decreased mRNA levels for atrogin-1 and MuRF1 (by 20-40% and 30-50%, respectively; P < 0.05). The repressing effect of glycine on the expression of MuRF1, instead of atrogin-1, was abolished by LY294002 (P < 0.05). CONCLUSIONS: These findings indicate that glycine plays a previously unrecognized role in enhancing protein synthesis and inhibiting protein degradation in C2C12 cells. Glycine regulates protein turnover by activating mTORC1 and by inhibiting the expression of genes for proteolysis. Our results indicate that glycine is a functional amino acid that improves muscle cell growth.

Glycine Regulates Expression and Distribution of Claudin-7 and ZO-3 Proteins in Intestinal Porcine Epithelial Cells.

BACKGROUND: Glycine traditionally is classified as a nutritionally nonessential amino acid in humans and animals. Because of its abundance in the body and its extensive use via multiple pathways, requirements for glycine are particularly high in neonates. Our recent studies show that dietary glycine supplementation is needed for optimal intestinal development in piglets. Importantly, reduced concentrations of glycine in the lumen of the small intestine are associated with gut dysfunction in low-birth-weight piglets. However, the mechanisms responsible for the beneficial effects of glycine on the intestinal mucosal barrier are largely unknown. OBJECTIVE: This study tested the hypothesis that glycine may regulate the expression and distribution of tight junction (TJ) proteins, thereby contributing to intestinal mucosal barrier function. METHODS: Enterocytes isolated from the jejunum of a healthy newborn pig were propagated to establish a stable cell line. The cells were cultured with 0.05 mmol glycine/L (control; concentration in the small intestinal lumen of low-birth-weight piglets) or 0.25 or 1.0 mmol glycine/L for the indicated periods of time. Epithelial barrier integrity and expression and localization of TJ proteins were analyzed by using monolayer transepithelial electrical resistance (TEER) and paracellular permeability, Western blot, and immunofluorescence imaging. RESULTS: Compared with controls, cells cultured with 0.25 or 1.0 mmol glycine/L increased TEER (P < 0.05) by 46-53% and 80-111%, respectively, at 60-72 h. Correspondingly, paracellular permeability was reduced (P < 0.05) by 6-21% and 18-27% for 0.25 or 1.0 mmol glycine/L treatment, respectively, at 36-72 h. Compared with controls, protein abundances for claudin-3, claudin-7, and zonula occludens (ZO) 3 were enhanced (25-33%, P < 0.05) by 0.25 and 1.0 mmol glycine/L at 8 h, whereas those for occludin, claudin-1, claudin-4, and ZO-2 were not affected. Compared with controls, 1.0 mmol glycine/L reduced the protein abundance of ZO-1 by 20% at 8 h (P < 0.05), but 0.25 mmol glycine/L had no effect. A glycine concentration of 0.25 mmol/L sustained the localization of claudin-7 and ZO-3 to the interface between enterocytes. Interestingly, 1 mmol glycine/L promoted the distribution of claudin-4 and claudin-7 to the cytosol and nucleus, and the localization of ZO-3 to the plasma membranes, while decreasing the distribution of ZO-1 at cell-cell contact sites, compared with control cells. CONCLUSION: Physiologic concentrations of glycine support intestinal mucosal barrier function by regulating the abundance and distribution of claudin-7 and ZO-3 in enterocytes. Supplementation with glycine may provide an effective nutritional strategy to improve intestinal integrity in piglets.

L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells.

BACKGROUND: The tight junctions (TJs) are essential for maintenance of the intestinal mucosal barrier integrity. Results of our recent work show that dietary l-glutamine (Gln) supplementation enhances the protein abundance of TJ proteins in the small intestine of piglets. However, the underlying mechanisms remain largely unknown. OBJECTIVE: This study was conducted to test the hypothesis that Gln regulates TJ integrity through calcium/calmodulin-dependent kinase 2 (CaMKK2)-AMP-activated protein kinase (AMPK) signaling which, in turn, contributes to improved intestinal mucosal barrier function. METHODS: Jejunal enterocytes isolated from a newborn pig were cultured in the presence of 0-2.0 mmol Gln/L for indicated time points. Cell proliferation, monolayer transepithelial electrical resistance (TEER), paracellular permeability, expression and distribution of TJ proteins, and phosphorylated AMPK were determined. RESULTS: Compared with 0 mmol Gln/L, 2.0 mmol Gln/L enhanced (P < 0.05) cell growth (by 31.9% at 48 h and 11.1% at 60 h). Cells treated with 2 mmol Gln/L increased TEER by 32.2% at 60 h, and decreased (P < 0.05) TJ permeability by 20.3-40.0% at 36-60 h. In addition, 2.0 mmol Gln/L increased (P < 0.05) the abundance of transmembrane proteins, such as occludin, claudin-4, junction adhesion molecule (JAM)-A, and the plaque proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 by 1.8-6 times. In contrast, 0.5 mmol Gln/L had a moderate effect on TJ protein abundance (20.2-70.5%; P < 0.05) of occludin, claudin-3, claudin-4, JAM-A, and ZO-1. 2.0 mmol Gln/L treatment led to a greater distribution of claudin-1, claudin-4, and ZO-1 at plasma membranes compared with 0 mmol Gln/L. This effect of Gln was mediated by the activation of CaMKK2-AMPK signaling, because either depletion of calcium from the medium or the presence of an inhibitor of CaMKK2 abrogated the effect of Gln on epithelial integrity. CONCLUSION: Our findings indicate that activation of CaMKK2-AMPK signaling by Gln is associated with improved intestinal mucosal barrier function through enhancing the abundance of TJ proteins and altering their intracellular localization in intestinal porcine epithelial cells.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Nutritional epigenetics with a focus on amino acids: implications for the development and treatment of metabolic syndrome.

Recent findings from human and animal studies indicate that maternal undernutrition or overnutrition affects covalent modifications of the fetal genome and its associated histones that can be carried forward to subsequent generations. An adverse outcome of maternal malnutrition is the development of metabolic syndrome, which is defined as a cluster of disorders including obesity, hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertension and insulin resistance. The transgenerational impacts of maternal nutrition are known as fetal programming, which is mediated by stable and heritable alterations of gene expression through covalent modifications of DNA and histones without changes in DNA sequences (namely, epigenetics). The underlying mechanisms include chromatin remodeling, DNA methylation (occurring at the 5'-position of cytosine residues within CpG dinucleotides), histone modifications (acetylation, methylation, phosphorylation, ubiquitination and sumoylation) and expression and activity of small noncoding RNAs. The enzymes catalyzing these reactions include S-adenosylmethionine-dependent DNA and protein methyltransferases, DNA demethylases, histone acetylase (lysine acetyltransferase), general control nonderepressible 5 (GCN5)-related N-acetyltransferase (a superfamily of acetyltransferase) and histone deacetylase. Amino acids (e.g., glycine, histidine, methionine and serine) and vitamins (B6, B12 and folate) play key roles in provision of methyl donors for DNA and protein methylation. Therefore, these nutrients and related metabolic pathways are of interest in dietary treatment of metabolic syndrome. Intervention strategies include targeting epigenetically disturbed metabolic pathways through dietary supplementation with nutrients (particularly functional amino acids and vitamins) to regulate one-carbon-unit metabolism, antioxidative reactions and gene expression, as well as protein methylation and acetylation. These mechanism-based approaches may effectively improve health and well-being of affected offspring.

Intimacy and a deadly feud: the interplay of autophagy and apoptosis mediated by amino acids.

Autophagy (i.e., "self-eating") and apoptosis (i.e., type I programmed cell death) are essential and intimately involved in molecular, cellular, and whole-body homeostasis in humans and animals. Autophagy has been categorized as a mechanism of intracellular degradation, recycling, defense, and survival. To date, three types of autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy. Recent discoveries strongly suggest that macroautophagy also modulates type II programmed cell death under specific circumstances. Autophagy and apoptosis are fundamentally distinct processes, but are interconnected by common stress initiators and intermediate regulators. During the past two decades, the role of amino acid metabolism and signaling in the regulation of apoptosis and autophagy has been intensively studied. In this review, we summarize recent advances in our understanding of the molecular mechanisms that regulate both autophagy and apoptosis in the context of amino acid signaling.

Dietary L-leucine supplementation enhances intestinal development in suckling piglets.

L-Leucine is a signaling amino acid in animal metabolism. It is unknown whether supplementing L-leucine to breast-fed neonates may enhance their small-intestinal development. This hypothesis was tested with a piglet model. Seven-day-old sow-reared pigs with an average birth weight of 1.45 kg were assigned randomly to the control or leucine group (n = 30/group). Piglets in the leucine group were orally administrated with 1.4 g L-leucine/kg body weight, whereas piglets in the control group received isonitrogenous L-alanine, twice daily for 14 days. The supplemental L-leucine amounted to 200 % of L-leucine intake from sow's milk by 7-day-old pigs. At the end of the 2-week experiment, tissue samples were collected for determining intestinal morphology, expression of genes for intestinal leucine transporters (real-time RT-PCR and western blot analysis), and plasma metabolites and hormones. L-leucine administration increased (P < 0.05) villus height in the duodenum, an elevated ratio of villus height to crypt depth in the duodenum and ileum, plasma concentrations of leucine, glutamine and asparagine, as well as body-weight gains. mRNA levels for L-leucine transporters (SLC6A14, SLC6A19 and SLC7A9) and the abundance of the ATB(0,+) protein were increased (P < 0.05) but those for SLC7A7 mRNA and the LAT2 protein were decreased (P < 0.05) in the jejunum of leucine-supplemented piglets, compared with the control. Plasma concentrations of ammonia, urea, triglycerides, and growth-related hormones did not differ between the control and leucine groups. Collectively, these results indicate that L-leucine supplementation improves intestinal development and whole-body growth in suckling piglets with a normal birth weight.

l-Tryptophan Activates Mammalian Target of Rapamycin and Enhances Expression of Tight Junction Proteins in Intestinal Porcine Epithelial Cells.

BACKGROUND: Besides serving as a substrate for protein synthesis, L-tryptophan (L-Trp) is used via serotonin-, kynurenine-, and niacin-synthetic pathways to produce bioactive compounds crucial for whole-body homeostasis. It is unknown whether L-Trp itself can regulate metabolic pathways in animal cells. OBJECTIVE: This study tested the hypothesis that L-Trp may activate mammalian target of rapamycin (mTOR) complex 1 and enhance expression of tight junction (TJ) proteins in intestinal porcine epithelial cells. METHODS: Jejunal enterocytes, intestinal porcine epithelial cell line 1 (IPEC-1) isolated from newborn pigs, were cultured in customized Dulbecco's modified Eagle medium (DMEM) supplemented with or without L-Trp for the indicated time periods. Cell proliferation, L-Trp metabolism, protein turnover, mRNA abundance for L-Trp transporters [solute carrier family 3 member 1 (SLC3A1), solute carrier family 6 member 14 (SLC6A14), solute carrier family 6 member 19 (SLC6A19), and Na(+)/K(+) ATPase subunit-α1 (ATP1A1)], abundance of proteins involved in mTOR signaling, and TJ proteins were determined. RESULTS: L-Trp was not degraded in IPEC-1 cells. Compared with basal medium containing 0.04 mmol/L L-Trp, 0.4 and 0.8 mmol/L L-Trp enhanced (P < 0.05) protein synthesis by 45-52% and cell growth by 17% and 25% on day 1 and 72% and 51% on day 2, respectively, while reducing (P < 0.05) protein degradation by 12% and 22%, respectively. These effects of L-Trp were associated with mTOR activation and increased (P < 0.05) mRNA abundance for L-Trp transporters (SLC6A19, SLC6A14, and SLC3A1) by 1.5-2.7 fold and ATP1A1 by 3 fold. L-Trp also upregulated (P < 0.05) the abundance of occludin, claudin-4, zonula occludens (ZO) 1 and 2 by 0.5-2 fold but did not affect expression of claudin-1 or ZO-3 in IPEC-1 cells. CONCLUSION: L-Trp is not catabolized by pig small intestinal epithelial cells but can regulate intracellular protein turnover and expression of TJ proteins in these cells.