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Background: Glutamine (Gln) has an important effect on the growth performance and immune function of piglets. However, the effect of Gln on intestinal immunity in piglets through modulating the signaling pathways of the helper T cells 17 (Th17)/regulatory T cells (Treg) immune response has not been reported., Objective: This study aimed to determine the effect of Gln on piglet growth performance and immune stress response and its mechanism in piglets., Methods: Twenty-four weaned piglets were randomly assigned to 4 treatments with 6 replicates each, using a 2 × 2 factorial arrangement: diet (basal diet or 1% Gln diet) and immunological challenge [saline or lipopolysaccharide (LPS)]. After 21 d, half of the piglets on the basal diet and 1% Gln diet received the intraperitoneal injection of LPS and the other half received the same volume of normal saline., Results: The results showed that Gln increased average daily feed intake and average daily weight gain in comparison with the control group (P < 0.05). Dietary Gln increased the villus height, villus height-to-crypt depth ratio, and the abundance of Bacteroidetes, Lactobacillus sp., and Ruminococcus sp. while reducing the abundance of Firmicutes, Clostridium sensu stricto 1 sp., and Terrisporobacter sp. (P < 0.05). Furthermore, Gln increased the concentration of short-chain fatty acids in the colon and the expression of genes of interleukin (IL)-10, transforming growth factor-beta-1, forkhead box P3 while downregulating the expression of genes of IL-6, IL-8, IL-1β, tumor necrosis factor-α, IL-17A, IL-21, signal transducer and activator of transcription 3, and rar-related orphan receptor c in ileum (P < 0.05). Correlation analysis demonstrated a strong association between colonic microbiota, short-chain fatty acids, and ileal inflammatory cytokines., Conclusions: These results suggest that dietary Gln could improve growth performance and attenuate LPS-challenged intestinal inflammation by modulating microbiota and the Th17/Treg immune response signaling pathway in piglets., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Background: Early life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions., Objectives: In this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis., Methods: A hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2'-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids., Results: In comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch., Conclusions: Together our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The intestinal epithelium is one of the fastest self-renewal tissues in the body, and glutamine plays a crucial role in providing carbon and nitrogen for biosynthesis. In intestinal homeostasis, phosphorylation-mediated signaling networks that cause altered cell proliferation, differentiation, and metabolic regulation have been observed. However, our understanding of how glutamine affects protein phosphorylation in the intestinal epithelium is limited, and identifying the essential signaling pathways involved in regulating intestinal epithelial cell growth is particularly challenging., Objectives: This study aimed to identify the essential proteins and signaling pathways involved in glutamine's promotion of porcine intestinal epithelial cell proliferation., Methods: Phosphoproteomics was applied to describe the protein phosphorylation landscape under glutamine treatment. Kinase-substrate enrichment analysis was subjected to predict kinase activity and validated by qRT-PCR and Western blotting. Cell Counting Kit-8, glutamine rescue experiment, chloroquine treatment, and 5-fluoro-2-indolyl deschlorohalopemide inhibition assay revealed the possible underlying mechanism of glutamine promoting porcine intestinal epithelial cell proliferation., Results: In this study, glutamine starvation was found to significantly suppress the proliferation of intestinal epithelial cells and change phosphoproteomic profiles with 575 downregulated sites and 321 upregulated sites. Interestingly, phosphorylation of eukaryotic initiation factor 4E-binding protein 1 at position Threonine70 was decreased, which is a crucial downstream of the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Further studies showed that glutamine supplementation rescued cell proliferation and mTORC1 activity, dependent on lysosomal function and phospholipase D activation., Conclusion: In conclusion, glutamine activates mTORC1 signaling dependent on phospholipase D and a functional lysosome to promote intestinal epithelial cell proliferation. This discovery provides new insight into regulating the homeostasis of the intestinal epithelium, particularly in pig production., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

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-1β, 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., (Copyright © American Society for Nutrition 2019.)

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications., Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model., Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured., Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05., Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.

Background: Programmed cell death plays a fundamental role in intestinal development and mucosal homeostasis. Dysregulation of these processes is associated with an impaired intestinal-mucosal barrier, reduced nutrient absorption, and initiation and progression of intestinal diseases. 4-Hydroxy-2-nonenal (4-HNE), a product of lipid peroxidation, is commonly used to induce oxidative stress in cells. l-Glutamine is known to protect cells from apoptosis. However, the underlying mechanisms are largely unknown., Objective: This study was conducted to test the hypothesis that l-glutamine attenuates 4-HNE-induced apoptosis by modulating glutathione (GSH) and thioredoxin (TXN) antioxidant systems and the expression of genes involved in 4-HNE metabolism in enterocytes., Methods: Intestinal porcine epithelial cell line 1 (IPEC-1) cells were cultured with or without 4-HNE (30 μmol/L) in the presence of 0.05 or 0.25 mmol l-glutamine/L (a physiological concentration in the lumen of the small intestine) for indicated time periods. Cell viability, abundances of apoptotic proteins, mitochondrial membrane depolarization, production of reactive oxygen species (ROS) and GSH, and expression of genes involved in the biosynthesis of GSH, thioredoxin, and 4-HNE metabolism were determined., Results: Compared with basal medium containing 0.05 mmol l-glutamine/L, 4-HNE enhanced apoptosis by 19.6% (P < 0.05) in a caspase-3-dependent manner. This effect was accompanied by elevated intracellular ROS production (39.5% and 85.3% for 2- and 4-h treatment, respectively), increased mitochondrial depolarization by 80%, and decreased intracellular GSH concentrations by 17.7%. These effects of 4-HNE were reduced by 0.25 mmol l-glutamine/L. Further study showed that the protective effect of l-glutamine was associated with the enhanced expression of genes involved in GSH production (including GCLC, GCLM, GSR, CBS, and CTH) by 3.9-14-fold, as well as genes involved in 4-HNE metabolism [e.g., glutathione S-transferase A (GSTA)1 and GSTA4] by 1.9-7.2-fold. The mRNA levels for ADH5, AKR1C1, AKR1A1, and TXNRD1 were enhanced 1.4-8.8-fold by 4-HNE but were not changed in cells co-treated with 4-HNE and l-glutamine., Conclusion: These findings indicate that l-glutamine attenuates 4-HNE-induced apoptosis by regulating GSH-related redox homeostasis and enhancing GSTA-mediated metabolism in enterocytes.

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., (© 2016 American Society for Nutrition.)

Impaired glucagon-like peptide (GLP-1) secretion or response may contribute to ineffective insulin release in type 2 diabetes. The conditionally essential amino acid glutamine stimulates GLP-1 secretion in vitro and in vivo. In a randomized, crossover study, we evaluated the effect of oral glutamine, with or without sitagliptin (SIT), on postprandial glycemia and GLP-1 concentration in 15 type 2 diabetes patients (glycated hemoglobin 6.5 ± 0.6%). Participants ingested a low-fat meal (5% fat) after receiving either water (control), 30 g l-glutamine (Gln-30), 15 g L-glutamine (Gln-15), 100 mg SIT, or 100 mg SIT and 15 g L-glutamine (SIT+Gln-15). Studies were conducted 1-2 wk apart. Blood was collected at baseline and postprandially for 180 min for measurement of circulating glucose, insulin, C-peptide, glucagon, and total and active GLP-1. Gln-30 and SIT+Gln-15 reduced the early (t = 0-60 min) postprandial glycemic response compared with control. All Gln treatments enhanced the postprandial insulin response from t = 60-180 min but had no effect on the C-peptide response compared with control. The postprandial glucagon concentration was increased by Gln-30 and Gln-15 compared with control, but the insulin:glucagon ratio was not affected by any treatment. In contrast to Gln-30, which tended to increase the total GLP-1 AUC, SIT tended to decrease the total GLP-1 AUC relative to control (both P = 0.03). Gln-30 and SIT increased the active GLP-1 AUC compared with control (P = 0.008 and P = 0.01, respectively). In summary, Gln-30 decreased the early postprandial glucose response, enhanced late postprandial insulinemia, and augmented postprandial active GLP-1 responses compared with control. These findings suggest that glutamine may be a novel agent for stimulating GLP-1 concentration and limiting postprandial glycemia in type 2 diabetes.

We investigated the effects of glutamine on the development of colonic fibrosis and on the expression of the major fibrogenic factors in a rat model of experimental colitis. Colitis was induced in one-half of the male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). L-glutamine (25 mg/kg) was administered rectally to one-half of the controls and one-half of the colitic rats. The control, control+glutamine, TNBS, and TNBS+glutamine groups were studied at d 2 and 7 after colitis induction. Glutamine induced a significant decrease in the area of colon fibrosis and in the staining of alpha-smooth muscle actin positive cells within areas of extracellular matrix deposits in the submucosa. Collagen synthesis was stimulated following TNBS administration, with a significant increase in procollagen IV, collagen III, and collagen Ialpha2 mRNA levels in the colon by d 2 after TNBS instillation. Tissue inhibitor of metalloproteinase, connective tissue growth factor, transforming growth factor-beta, platelet-derived growth factor, and phosphorylated Smad3 were overexpressed in the colon of TNBS-treated rats. These effects were significantly abrogated in the colitic rats treated with glutamine. Our findings suggest that glutamine treatment not only attenuates the outcome of TNBS-induced colitis by reducing the inflammatory response but also by downregulating the increased expression of several gene pathways that contribute to the accumulation of matrix proteins. This molecule may be an interesting candidate for reducing the risk of fibrosis and stricture formation in inflammatory bowel disease.

Doxorubicin (DOX), a widely used anticancer drug, has a dose-dependent cardiotoxicity, attributed mainly to free radical formation. The cardiomyocyte oxidative stress occurs rapidly after DOX treatment, resulting in harmful modifications to proteins, lipids, and DNA. Previous data showed that oral l-glutamine (Gln) prevented cardiac lipid peroxidation and maintained normal cardiac glutathione (GSH) levels in DOX-treated rats. Our aim in this study was to examine the effect of Gln on DOX-induced cardiac oxidative stress in a tumor-bearing host. Female Fisher344 rats with implanted MatBIII mammary tumors were randomized into 2 groups: a Gln group that received l-Gln (1 g.kg(-1).d(-1)) (n = 10) via a Gln-enriched diet and/or gavage with 50% Gln suspension during the whole experiment and a control group that was fed the same diet formulation without Gln and/or were gavaged with water. All rats received a single injection of 12 mg/kg DOX and were killed 3 d later. GSH levels of hearts, livers, tumors, and blood, as well as cardiac histological alterations, lipid peroxidation, peroxinitrite levels, and caspase-3 activation were determined. Cardiac physiologic alterations were assessed by ultrasound imaging before and 3 d after DOX administration. The Gln supplementation resulted in lower cardiac lipid peroxidation and peroxintrite levels and elevated cardiac catalase enzyme activity and GSH compared with the controls, without affecting those of the tumors. DOX-induced alterations of the echocardiographic parameters were significantly reduced in the Gln-supplemented rats. These data indicate that Gln is able to reduce the oxidative damage of cardiomyocytes that occurs soon after DOX administration and thus protects the heart of a tumor-bearing host from DOX-induced cardiomyopathy.

Total parenteral nutrition (TPN) results in intestinal mucosal atrophic changes due to an absence of enteral nutrition; however, the mechanisms responsible for this are not fully understood. It has been shown that bone morphogenetic protein (BMP) activation inhibits intestinal epithelial cell (EC) proliferation. Therefore, we hypothesized that the BMP pathway could be upregulated by TPN. To address this, we randomly assigned mice to receive TPN or to be enterally fed (control) for 7 d. Mucosal EC isolates were harvested from the entire length of small intestine for RNA and protein measurements. Full-thickness, mid-small bowel was processed for histological examination. TPN increased the abundance of BMP2, BMP4, and BMP type II receptor at the RNA and protein levels. Phosphorylation of Smad1, Smad5, and Smad8 also was greater in the TPN group than in the control, which helped to confirm activation of this pathway. Interestingly, the TPN and control groups did not differ in the mRNA expression of the extracellular soluble bmp antagonists, noggin, gremlin, chordin, or follistatin. Compared to the control group, the expression of c-Myc (cellular myelocytomatosis) mRNA was lower, whereas the level of p21(WAF1/CIP1) was greater, in the TPN group. Because the BMP family may function through suppression of Wnt-beta-catenin signaling, this pathway was also examined. mRNA expression of Wnt 3, Wnt5a, and the Wnt receptor Lrp5 were lower in the TPN group compared to controls. The results suggest that the BMP signaling pathway may be involved in the development of intestinal mucosal atrophy due to TPN administration.

Glutamine (Gln) 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 study was to test the hypothesis that Gln regulates intercellular junction integrity and TJ proteins through the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Caco-2 cells. Deprivation of exogenous and endogenous glutamine decreased transepithelial electrical resistance (TER) (P < 0.01) and increased permeability (P < 0.01). Both wortmannin and LY294002, PI3K inhibitors, prevented the TER decrease and the permeability increase induced by Gln deprivation (P < 0.001). Gln deprivation also caused decreased TJ protein claudin-1 (P < 0.001). Both wortmannin and LY294002 treatment prevented this effect (P < 0.001). Deprivation of Gln increased phosphor-Akt protein. Gln supplementation reversed this effect. Decreased TER and increased permeability associated with Gln deprivation were not observed in small interfering RNA for p85 transfected Caco-2 cells. In conclusion, Gln regulates intercellular junction integrity and TJ proteins through the PI3-Kinase/Akt pathway.

Dietary supplementation of glutamine prevents intestinal dysfunction and atrophy in weanling piglets, but the underlying mechanism(s) are largely unknown. This study was conducted to test the hypothesis that weaning or glutamine may modulate expression of genes that are crucial for intestinal metabolism and function. In Expt. 1, we obtained small intestine from 28-d-old pigs weaned at 21 d of age and from age-matched suckling piglets. In Expt. 2, piglets were weaned at 21 d of age and then had free access to diets supplemented with 1% L-glutamine (wt:wt) or isonitrogenous L-alanine (control). At d 28, we collected small intestine for biochemical and morphological measurements and microarray analysis of gene expression using the Operon Porcine Genome Oligo set. Early weaning resulted in increased (52-346%) expression of genes related to oxidative stress and immune activation but decreased (35-77%) expression of genes related to macronutrient metabolism and cell proliferation in the gut. Dietary glutamine supplementation increased intestinal expression (120-124%) of genes that are necessary for cell growth and removal of oxidants, while reducing (34-75%) expression of genes that promote oxidative stress and immune activation. Functionally, the glutamine treatment enhanced intestinal oxidative-defense capacity (indicated by a 29% increase in glutathione concentration), prevented jejunal atrophy, and promoted small intestine growth (+12%) and body weight gain (+19%) in weaned piglets. These findings reveal coordinate alterations of gene expression in response to weaning and aid in providing molecular mechanisms for the beneficial effect of dietary glutamine supplementation to improve nutrition status in young mammals.

The development of inflammatory bowel disease may involve immune dysfunction. Because enteral glutamine is the main source of amino acids for the intestinal mucosa and is metabolized at high rates by both enterocytes and immunocytes, the aim of this study was to ascertain the protective role of glutamine supplementation in a DSS-induced model of mild experimental colitis on metabolic, immune, and intestinal variables. Lewis rats were fed diets supplemented with glutamine (glutamine diet, G group) or an isoenergetic isonitrogenous control diet (C group) from postnatal d 21 (weaning) and continuing to d 35. On d 30, half of the rats from both groups were given 0.5% DSS in drinking water (G-DSS and C-DSS groups). Glutamine supplementation increased the plasma concentrations of Thr, Gln, Cit, His, and Arg and enhanced the ratio of essential to nonessential amino acids irrespective of DSS treatment. DSS administration increased the plasma Gln concentration, indicating a reduced utilization of this amino acid by the intestinal tissue. Regarding the gut-associated lymphoid tissue lymphocyte populations, DSS increased the percentages of CD3(+) T lymphocytes from Peyer's patches, NK and B lymphocytes from mesenteric lymph nodes, and NK CD8(-) cells from intraepithelial lymphocytes. The administration of glutamine did not affect the inductive populations nor did it modify T-cell subtypes or the percentage of intraepithelial lymphocytes of gut-associated lymphoid tissue. However, glutamine supplementation reduced the feces water contents in the DSS-treated but not in the untreated rats. These results indicate that glutamine supplementation can improve barrier function in rats with colitis.

This study examined the regulation of glutamine synthetase protein levels, in response to changes in external glutamine concentration, in mouse C2C12 skeletal muscle cells. Glutamine, at concentrations as low as 0.25 mmol/L, downregulated endogenous and exogenous (plasmid encoded) glutamine synthetase with maximal effect at 2 mmol/L. Glutamine appears to act by changing the stability of the glutamine synthetase protein, and the effect was partially blocked by the proteasome inhibitor MG132. The addition of the glutamine structural analog and glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, in the presence or absence of glutamine, also resulted in low glutamine synthetase protein levels. Otherwise, the effect was specific for glutamine, and the only compounds able to mimic the effect of glutamine were amino acids, glutamate, alanine, and ornithine, which can be converted to glutamine. Other amino acids, analogs, and products of glutamine metabolism were without effect. Methionine sulfoximine, an inhibitor of glutamine synthetase, stabilized the protein and prevented the glutamine effect. Thus, in mouse C2C12 skeletal muscle cells, glutamine synthetase protein expression is regulated by glutamine through changes in the rate of degradation of the protein. The effect is specific to glutamine, which acts directly without requiring prior metabolism.

Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Recent studies showed that glutamine regulates protein metabolism and intestinal inflammation among other mechanisms by reducing proinflammatory cytokine release. Because regulation of the inflammatory response was shown to be linked to proteolysis regulation, we hypothesized that glutamine pretreatment could act on IL-8 production in human intestinal epithelial cells through the regulation of inhibitor kappaB (IkappaB) ubiquitination. The HCT-8 cells were pretreated for 24 h with 0.6, 2, or 10 mmol/L glutamine. IL-8 concentration and IkappaB (free and ubiquitinated) expressions were assessed by ELISA and immunoblotting, respectively. A pretreatment with 10 mmol/L glutamine decreased IL-8 production under both basal and proinflammatory conditions (both P < 0.05). In the presence of a proteasome inhibitor (MG132), the ubiquitin-IkappaBalpha complex expression was not significantly modified by glutamine under basal conditions but decreased significantly under proinflammatory conditions (P < 0.05). After the addition of 10 mmol/L of glutamine, the free IkappaBalpha expression increased under basal and stimulated conditions (both P < 0.05). A glutamine pretreatment of 10 mmol/L did not affect ubiquitin expression or proteasome activity. This study indicates that glutamine pretreatment may reduce the intestinal inflammatory response by limiting the proteolysis of IkappaBalpha.

The intestinal epithelium may serve as a nidus for inflammation that can cause local and systemic organ dysfunction. Relative to the adult, the immature intestine is exquisitely sensitive to inflammatory agents. Glutamine (Gln), an amino acid that is rapidly depleted during critical illness, modulates intestinal inflammation in vitro and in vivo. Here we relate Gln status to activation of the inhibitor of kappaB (IkappaB)/nuclear factor (NF)-kappaB signaling pathway in fetal-derived (H4) and adult (Caco-2) enterocytes. In the absence of Gln with or without LPS, H4 cells expressed more interleukin (IL)-8) than Caco-2 cells. Gln supplementation partially prevented the LPS-induced elevation of IL-8 in both cell types. IkappaBalpha was significantly decreased in both H4 and Caco-2 cells with Gln deprivation, and this was followed by an increase in NF-kappaB p65 in the nucleus. DNA binding of NF-kappaB was increased in both H4 and Caco-2 cells with Gln deprivation. IkappaBalpha phosphorylation was not altered by Gln status in either H4 or Caco-2 cells. Proteasomal inhibition after Gln depletion in Caco-2 cells was associated with an increase in the IkappaB-ubiquitin complex, but a decrease in complex formation in H4 cells, indicating that Gln deprivation alters IkappaBalpha through a pathway that differs from Caco-2 cells. We speculate that a reduced capacity of the immature enterocyte (H4) to respond to Gln deprivation with increased synthesis of IkappaBalpha rather than increased proteolysis as seen in the Caco-2 cells is the underlying mechanism.

Epithelial cell apoptosis is an important regulator of normal gut mucosal turnover; however, excessive apoptosis may inhibit mucosal restitution during pathophysiologic states. Apoptosis is induced by oxidative stress and cytokines, but regulation by specific nutrients has been infrequently studied under these conditions. Glutamine (Gln) is an important metabolic fuel for intestinal epithelial cells and a precursor to the antioxidant glutathione (GSH), which has antiapoptotic effects. In cultured intestinal epithelial cells, Gln depletion increases oxidant-induced apoptosis. This study examined whether Gln protects against apoptosis induced by the cytokine tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in the human colon carcinoma cell line, HT-29. TRAIL-induced apoptosis in HT-29 cells was characterized by an increase in the percentage of cells in the sub-G1 fraction by flow cytometry, nuclear condensation and the activation of caspase-8 and caspase-3. TRAIL-induced apoptosis was completely prevented by Gln, but not inhibited by other amino acids, including the GSH constituents, glutamate, cysteine and glycine. Similar antiapoptotic effects of Gln occurred when apoptosis was induced by a combination of tumor necrosis factor-alpha and interferon-gamma. Cellular GSH was oxidized during TRAIL-induced apoptosis. This effect was completely blocked by Gln, however, inhibition of GSH synthesis with buthionine sulfoximine did not alter Gln antiapoptotic effects. Furthermore, glutamate prevented GSH oxidation in response to TRAIL but did not protect against TRAIL-induced apoptosis. These results show that Gln specifically protects intestinal epithelial cells against cytokine-induced apoptosis, and that this occurs by a mechanism that is distinct from the protection against oxidative stress mediated by cellular GSH.

Dietary glutamine (Gln) has been shown to be important for maintenance of the intestinal barrier. To investigate the role of the epithelium in this Gln dependence, Caco-2 cells were raised on semipermeable membranes under conditions that model different regions of the crypt and villus. Gln availability was controlled by addition to the medium and treatment with methionine sulfoximine to inhibit Gln synthetase (GS). Barrier function was assayed by measuring transepithelial electrical resistance and fluxes of [(14)C]mannitol and fluorescein isothiocyanate-dextran. The barrier function of these monolayers was found to require the Gln provided either in the medium at the apical or basal surface or via GS. However, the barrier was no more sensitive to Gln deprivation than it was to accumulation or maintenance of total protein. These results suggest that the in vivo dependence of the gut mucosal barrier on Gln likely involves roles separate from maintenance of the epithelial barrier per se.

The heat shock protein, heme oxygenase-1 (HO-1), contributes to the protection of the intestine. Some experimental models suggest that induction of HO-1 by glutamine may contribute to the preservation of intestinal mucosa. The effect of an enteral infusion of glutamine for 6 h on HO-1 expression in duodenal mucosa was studied in healthy men and women and compared with an isonitrogenous mixture of amino acids. After enteral infusion, endoscopic duodenal biopsies were performed and either fixed in formalin for immunohistochemistry or frozen for HO-1 mRNA analysis by reverse transcriptase-polymerase chain reaction. Histologic examination revealed that HO-1 was constitutively expressed in intestinal epithelial cells (IEC), and that glutamine increased the grade of HO-1 immunostaining (P

After exhaustive exercise, intravenous or oral glutamine promoted skeletal muscle glycogen storage. However, when glutamine was ingested with glucose polymer, whole-body carbohydrate storage was elevated, the most likely site being liver and not muscle, possibly due to increased glucosamine formation. The rate of tricarboxylic acid (TCA) cycle flux and hence oxidative metabolism may be limited by the availability of TCA intermediates. There is some evidence that intramuscular glutamate normally provides alpha-ketoglutarate to the mitochondrion. We hypothesized that glutamine might be a more efficient anaplerotic precursor than endogenous glutamate alone. Indeed, a greater expansion of the sum of muscle citrate, malate, fumarate and succinate concentrations was observed at the start of exercise (70% VO2(max)) after oral glutamine than when placebo or ornithine alpha-ketoglutarate was given. However, neither endurance time nor the extent of phosphocreatine depletion or lactate accumulation during the exercise was altered, suggesting either that TCA intermediates were not limiting for energy production or that the severity of exercise was insufficient for the limitation to be operational. We have also shown that in the perfused working rat heart, there is a substantial fall in intramuscular glutamine and alpha-ketoglutarate, especially after ischemia. Glutamine (but not glutamate, alpha-ketoglutarate or aspartate) was able to rescue the performance of the postischemic heart. This ability appears to be connected to the ability to sustain intracardiac ATP, phosphocreatine and glutathione.

Glutamine is normally considered to be a nonessential amino acid. However, recent studies have provided evidence that glutamine may become "conditionally essential" during inflammatory conditions such as infection and injury. It is now well documented that under appropriate conditions, glutamine is essential for cell proliferation, that it can act as a respiratory fuel and that it can enhance the function of stimulated immune cells. Studies thus far have determined the effect of extracellular glutamine concentration on lymphocyte proliferation and cytokine production, macrophage phagocytic plus secretory activities and neutrophil bacterial killing. Other cells of the immune system remain to be studied. The high rate of glutamine utilization and its importance to the function of lymphocytes, macrophages and neutrophils have raised the question "why glutamine?" because these cells have access to a variety of metabolic fuels both in vivo and in vitro. I have attempted to answer this question in this article. Additionally, knowledge of the rate of utilization and the pathway of metabolism of glutamine by cells of the immune system raises some intriguing questions concerning therapeutic manipulation of utilization of this amino acid such that the proliferative, phagocytic and secretory capacities of cells of the defense system may be beneficially altered. Evidence to support the hypothesis that glutamine is beneficially immunomodulatory in animal models of infection and trauma, as well as trauma in humans, is provided.

Whether on the scale of a single cell, organ or organism, glutamine homeostasis is to a large extent determined by the activities of glutaminase (GA, EC 3.5.1.2) and glutamine synthetase (GS, EC 6.3.1.2), the two enzymes that are the focus of this report. GA and GS each provide examples of regulation of gene expression at many different levels. In the case of GA, two different genes (hepatic- and kidney-type GA) encode isoforms of this enzyme. The expression of hepatic GA mRNA is increased during starvation, diabetes and high protein diet through a mechanism involving increased gene transcription. In contrast, the expression of kidney GA mRNA is increased post-transcriptionally by a mechanism that increases mRNA stability during acidosis. We found recently that several isoforms of rat and human kidney-type GA are formed by tissue-specific alternative RNA splicing. Although the implications of this post-transcriptional processing mechanism for GA activity are not yet clear, it allows for the expression of different GA isoforms in different tissues and may limit the expression of GA activity in muscle tissues by diverting primary RNA transcripts to a spliceform that produces a nonfunctional translation product. The expression of GS enzyme is also regulated by both transcriptional and post-transcriptional mechanisms. For example, the GS gene is transcriptionally activated by glucocorticoid hormones in a tissue-specific fashion. This hormonal response allows GS mRNA levels to increase in selected organs during catabolic states. However, the ultimate level of GS enzyme expression is further governed by a post-transcriptional mechanism regulating GS protein stability. In a unique form of product feedback, GS protein turnover is increased by glutamine. This mechanism appears to provide a means to index the production of glutamine to its intracellular concentration and, therefore, to its systemic demand. Herein, we also provide experimental evidence that GS protein turnover is dependent upon the activity of the 26S proteosome.

With its many proposed metabolic roles, glutamine would seem to have major potential in normal animal production systems as well as during situations involving adverse challenges. In practice, however, responses to glutamine supplementation have been inconsistent. Thus, during lactation and growth studies in ruminants, both positive and null effects on production responses have been reported. Similarly, therapeutic responses to glutamine supplementation during various digestive tract disorders have been inconsistent in both pigs and ruminants. This is despite a proven involvement in the nucleic acid biosynthesis necessary to support cell proliferation. In sheep, at least, glutamine may exert a protective effect against hepatic amino acid (AA) oxidation, particularly for methionine. This may offer anabolic potential because methionine is the first limiting AA in a number of animal feedstuffs. Glutamine is also important in control of metabolic acidosis, but, in contrast to rodents, the main site of production seems to be extra-hepatic. In the immune system, while lymphocyte proliferation is glutamine-dependent, intracellular concentrations are low (in contrast to other tissues, such as muscle and liver). Instead, glutamate is accumulated, but the majority of this (approximately 65%) is derived in vivo from plasma glutamine. In sheep, endotoxin challenge elevates the plasma flux of glutamine, with a corresponding decrease in plasma concentration. At the same time, both the glutamate accumulation and fractional rate of protein synthesis within lymphocytes are enhanced. These lymphocyte responses, however, are not altered by an AA supplement that contains glutamine. Overall, although glutamine obviously plays important metabolic roles within the body, supplementation does not appear to provide consistent beneficial or therapeutic effects, except during certain catabolic situations. Glutamine availability, therefore, does not seem to be a limitation in many challenge situations. Rather, glutamine may signal alterations in nutrient demands among organs and a better understanding of this role may increase understanding of where modulation of glutamine status would be beneficial.

Severe infection causes marked derangements in the flow of glutamine among organs, and these changes are accompanied by significant alterations in regional cell membrane transport and intracellular glutamine metabolism. Skeletal muscle, the major repository of glutamine, exhibits a twofold increase in glutamine release during infection, which is associated with a significant increase in endogenous glutamine biosynthesis. Despite an increase in glutamine synthetase activity in skeletal muscle, the intracellular glutamine pool becomes depleted, indicating that release rates exceed rates of synthesis. Simultaneously, the circulating pool of glutamine does not increase, indicating accelerated uptake by other organs. The liver appears to be the major organ of glutamine uptake in severe infection; studies in endotoxemic rodents have shown net hepatic glutamine uptake to increase by as much as 8- to 10-fold. This increase is due partially to increases in liver blood flow, but also to a three- to fourfold increase in hepatocyte System N activity in the liver. Cytokines and glucocorticoids mediate the increased uptake of glutamine by the liver in septic states as well as other compounds. Sepsis does not appear to induce an increase in System N gene expression, indicating that the increase in hepatic glutamine transport observed during severe infection is probably regulated at the protein level. The bowel displays a decrease in glutamine utilization during sepsis, a response that may be related to the decrease in circulating insulin-like growth factor-1 (IGF-1) levels that is characteristic of sepsis. Recent studies suggest that IGF-1 has a direct effect on stimulating glutamine transport across the gut lumen and thus may represent a therapeutic avenue for improving gut nutrition during severe infection. The cells of the immune system (lymphocytes, macrophages) are also major glutamine consumers during inflammatory states in which cell proliferation is increased. Under these conditions, glutamine availability can become rate limiting for key cell functions, such as phagocytosis and antibody production.

Glutamine is the most abundant free amino acid in the human body; it is essential for the growth of normal and neoplastic cells and for the culture of many cell types. Cancer has been described as a nitrogen trap. The presence of a tumor produces great changes in host glutamine metabolism in such a way that host nitrogen metabolism is accommodated to the tumor-enhanced requirements of glutamine. To be used, glutamine must be transported into tumor mitochondria. Thus, an overview of the role of glutamine in cancer requires not only a discussion of host and tumor glutamine metabolism, but also its circulation and transport. Because glutamine depletion has adverse effects for the host, the effect of glutamine supplementation in the tumor-bearing state should also be studied. This communication reviews the state of knowledge of glutamine and cancer, including potential therapeutic implications.

Numerous studies indicate beneficial effects of glutamine (Gln) in many models of catabolic adult rats. No data were available for aged rats. The effects of oral L-Gln-enriched diet were tested in endotoxemic 24-mo old rats. First, rats received for 7 d (from d0 to d7) an oral diet supplemented with either L-Gln [1g/(kg. d)] or casein (Cas: isonitrogenous supply) prior to lipopolysaccharide (LPS) challenge. The rats were then killed after 24 h food deprivation (from d7 to d8). Endotoxemia induced a catabolic response as shown by muscle glutamine depletion, hyperphenylalaninemia, small bowel atrophy and impaired functionality and bacterial translocation. The Gln-enriched diet did not prevent muscle Gln depletion but significantly (P

Because parenteral feeding is associated with negative N balance and reduced rates of protein synthesis in intestinal mucosa, we hypothesized that luminal exposure to specific amino acids or energy fuels would stimulate intestinal protein synthesis. We studied the acute effects of luminal nutrients on mucosal protein synthesis in the absence of systemic influences. Multiple jejunal segments constructed in piglets deprived of food overnight (n = 6) were randomly assigned to luminal perfusion with saline, 30 mmol/L amino acid mixture with or without 50 mmol/L glucose, or 30 mmol/L glutamine for 90 min. Protein synthesis was then measured by luminal perfusion with L-[2,6-(3)H]-phenylalanine. Energy substrates (glucose, short-chain fatty acids or beta-hydroxybutyrate) had no effect on mucosal protein synthesis. Relative to saline, a 30 mmol/L amino acid mixture or 30 mmol/L glutamine suppressed mucosal protein synthesis by 20-25% (P < 0.05). On the basis of these surprising results, we speculated that a coordinate reduction of proteolytic processes would be required to maintain positive intestinal N balance. Although intestinal protein catabolism cannot be assessed directly, the 30 mmol/L amino acid mixture acutely suppressed mucosal levels of mRNA encoding ubiquitin, 14-kDa ubiquitin conjugating enzyme and the C9 subunit of the proteasome by 20-30% (P < 0.05), demonstrating the sensitivity of components of the ATP-ubiquitin proteolytic pathway to acute regulation by nutrients. The suppression of protein synthesis by luminal amino acids in the absorptive state might lower intestinal utilization of amino acids to ensure efficient allocation of absorbed nutrients to nonintestinal tissues.

To examine the effects of dietary glutamine on lymphocyte function, male mice aged 6 wk were fed for 2 wk one of three isonitrogenous, isocaloric diets, which varied in glutamine concentration. The control diet included 200 g casein/kg, providing 19.6 g glutamine/kg; the glutamine-enriched diet provided 54.8 g glutamine/kg partly at the expense of casein; and the alanine + glycine-enriched diet provided 13.3 g glutamine/kg. The plasma concentrations of a number of amino acids varied because of the diet fed. The plasma glycine concentration was greater in mice fed the alanine + glycine-enriched diet (380 +/- 22 micromol/L) than in mice fed the control (177 +/- 17 micromol/L) or the glutamine-enriched (115 +/- 18 micromol/L) diets. The plasma glutamine concentration was greater in mice fed the glutamine-enriched diet (945 +/- 117 micromol/L) than in those fed the diet enriched with alanine + glycine (561 +/- 127 micromol/L), but was not different from that in mice fed the control diet (791 +/- 35 micromol/L). There was a significant linear relationship between the amount of glutamine in the diet and plasma glutamine concentration (r = 0.655, P = 0.015). Plasma alanine concentration was unaffected by diet. The reason for the lack of effect of increasing the amount of alanine in the diet upon its concentration in the circulation may relate to its use by the liver. Thymidine incorporation (56 +/- 18 kBq/well versus <10 kBq/well), expression of the alpha-subunit of the interleukin-2 receptor (62 versus 30% receptor positive cells) and interleukin-2 production [189 +/- 28 versus 106 +/- 5 (control) or 61 +/- 13 (alanine + glycine enriched) ng/L] were greater for concanavalin A-stimulated spleen lymphocytes from mice fed the glutamine-enriched diet compared to those from mice fed the other two diets. Thus, increasing the amount of glutamine in the murine diet enhances the ability of T lymphocytes to respond to mitogenic stimulation. Taken together, these observations suggest that increasing the oral availability of glutamine could promote the T-cell driven, cell-mediated immune response.

Glutamine supplementation is beneficial for preventing intestinal atrophy and maintaining mucosal functions in metabolically stressed patients. The mechanisms by which glutamine prevents mucosal atrophy remain unclear. In particular, the role of glutamine in the survival of cells under stress is unknown. Intestinal epithelial cells (IEC-6) were cultured in media with or without supplementation of L-glutamine. A low concentration of L-glutamine (1.0 mmol/L) was sufficient to minimize the percentage of floating cells under basal conditions. Heat shock at 43 degrees C for 90 min decreased (P < 0. 001) the number of attached cells, while increasing (P < 0.001) the number of floating cells, which is a measurement of the extent of cell death in these cultures. Glutamine enhanced attached cell count and diminished heat shock-induced cell death in a dose-dependent manner. Of note, 2 mmol/L was suboptimal in both respects, thus indicating that heat-shocked cells require higher concentrations of glutamine for optimal cell survival. Maximal effect was achieved with 8 mmol/L glutamine, which increased (P < 0.001) cell growth (indicated by the number of attached cells) and diminished (P < 0. 001) cell death (indicated by the number of floating cells). Further increase of L-glutamine concentration to 12 or 20 mmol/L did not provide additional benefit in minimizing cell death. Heat shock protein 70 (hsp 70) mRNA was induced by heat shock only in cultures supplemented with L-glutamine, and the induction was more consistent and greater in cultures containing higher concentrations of glutamine. Thus, glutamine supplementation reduced heat shock-induced cell death. This effect, together with the maintenance of cell growth, may play a key role in the prevention of intestinal mucosal atrophy.

Burn patients develop pathophysiological alterations, which include extensive nitrogen loss, malnutrition, markedly increased metabolic rate and immunologic deficiency. This predisposes burn patients to frequent infections, poor wound healing, increased length of hospitalization and increased mortality. The nutritional support requires high protein and high energy diets preferably administered enterally soon after injury. The effects of increased dietary components such as glutamine, arginine and (n-3) fatty acids and related compounds have been evaluated in burn victims. These components, when supplied in quantities two to seven times of those in normal diets of healthy persons, appear to have beneficial pharmacological effects on the pathophysiological alterations associated with burns. However, the efficacy of immune-enhancing diets remains to be convincingly shown.

L-Glutamine is the most abundant free amino acid of the human body and is essential for the culture of many cell types. Clinically, reduction of glutamine by administration of glutaminase or the use of glutamine analogs is a common therapy for patients with acute lymphocytic leukemia. In the current study, we investigated the influence of glutamine concentrations on the human myelomonocytic cell line U937. Decreasing the glutamine concentration evoked a reduction in DNA synthesis (R2 = 0.9885, P < 0.0001), increased cell volume (P < 0.01) and the cytoplasm/nuclear ratio, and enhanced the development of vacuoles but did not influence cell viability. Culturing cells in reduced concentrations of glutamine augmented the percentage of cells expressing CD64 (Fc receptor for IgG/FcgammaRI, P < 0.01), CD11b (complement receptor type 3/CR3, P < 0.001) and CD71 (transferrin receptor, P < 0.05). The percentage of U937 cells expressing CD23 (low affinity receptor for IgE/FcepsilonRII) was increased at low concentrations of glutamine at both the protein (P < 0.01) and mRNA levels. The percentage of U937 cells phagocytizing opsonized E. coli (P < 0.001) or latex particles (P < 0.001) was enhanced by lowering the glutamine concentration. In conclusion, reducing glutamine concentration causes differentiation of the cell line U937 along the monocytic pathway. These effects may indicate a mechanistic basis for prior published evidence that glutaminase and glutamine antagonists are effective anti-tumor agents.

Numerous studies in humans and rats have shown that glutamine supplementation during stressful conditions has favorable outcomes. However, the requirements for glutamine during weaning are unknown. Thus, the effects of glutamine supplementation in healthy and infected weaned pigs were investigated. At 21 d of age, pigs were weaned to an elemental diet supplemented with glutamine (+Gln) or an isonitrogenous diet containing nonessential amino acids (-Gln). At 26 d of age, pigs were intraperitoneally injected with Escherichia coli (+Ecoli) or buffered saline (-Ecoli) and killed at 28 d of age. Infection decreased (P < 0.05) plasma and intramuscular glutamine concentrations, but infected pigs that received +Gln diets had higher intramuscular glutamine levels than those that received -Gln diets. Infected pigs had elevated (P < 0.05) total leukocyte counts, and blood lymphocyte responses ([3H]-thymidine incorporation) to a mixture of phorbol myristate acetate and ionomycin were reduced. White blood cell counts were greater (P < 0.05) in +Gln than -Gln pigs. The peak responses to concanavalin A (Con A) by lymphocytes of +Ecoli+Gln pigs were greater (P < 0.05) than those of +Ecoli-Gln pigs and not different than those of noninfected pigs. Hence, glutamine supplementation maintained muscular glutamine concentrations and normalized lymphocyte function in infected pigs.

The early-weaned pig develops intestinal atrophy and provides a readily accessible animal model for determining the role of dietary supplementation of glutamine (Gln, a major fuel for enterocytes) in preventing intestinal damage. Three experiments were conducted to determine the stability of dietary Gln in the acidic part of the gastrointestinal tract using pigs surgically fitted with a T-cannula in mid-duodenum (Exp. 1), and the effects of dietary Gln supplementation on the villus height and lamina propria depth of duodenum and jejunum (Exp. 2) as well as growth performance (Exp. 3) of pigs weaned at 21 d of age. Postweaning pigs were fed for 14 d corn- and soybean meal-based diets supplemented with 0.0, 0.2, 0.6 or 1.0% free L-Gln. Dietary Gln was not subject to measurable acid hydrolysis in the stomach and upper part of duodenum and was substantially available for the small intestine for metabolic utilization. Glutamine supplementation (1.0%) prevented jejunal atrophy (as indicated by villus height) during the first week postweaning and increased the gain:feed ratio (an indicator of growth performance) by 25% during the second week postweaning. Glutamine supplementation (1.0%) increased plasma concentrations of aspartate, glutamate and alanine and reduced the extent to which plasma taurine concentration fell in postweaning pigs. These results provide an experimental basis for enteral use of Gln in swine production and clinical nutrition to prevent intestinal epithelial damage.

Recent work indicates that cell volume is an important regulator of proteolysis in liver. The antiproteolytic effects of insulin and some amino acids (e.g., glutamine and glycine) are mediated by increases in cell volume. The purpose of the present study was to assess the role of nutritional state in the cell volume-dependent regulation of proteolysis in isolated perfused rat liver. In rats that had been prelabeled by an intraperitoneal injection of L-[4,5-3H]leucine 16-20 h prior to the perfusion experiment, hepatic proteolysis was studied by determination of [3H]label release into effluent perfusate. In separate perfusion experiments [3H]inulin and [14C]urea, acting as markers for extracellular and the sum of extra- plus intracellular space, were employed for determination of effector-induced cell volume changes. Proteolysis in the perfused rat liver was inhibited by insulin-like growth factor-I (IGF-1) and taurocholic acid. Both agonists increased the intracellular water space. The nutritional state of the livers had marked influence on the hormone- and amino acid-dependent regulation of proteolysis. In livers from food-deprived rats for 24 h, the swelling responses to glycine, glutamine and alanine were enhanced, whereas the insulin- and IGF-1-induced increases of cell volume were diminished. A stronger inhibition of proteolysis was observed in livers from food-deprived rats upon addition of the amino acids, whereas the insulin- and IGF-1-mediated inhibition of proteolysis was attenuated. Independent of the nutritional state, a close relationship between the cellular hydration state and the corresponding inhibition of proteolysis was observed, regardless of whether cell volume was modified by amino acids, hormones, hypoosmotic exposure or bile acids. We conclude that the nutritional state markedly modifies the swelling potency of amino acids and hormones in liver and by this means affects proteolysis.

C57BL/6J (B/6J) mice are genetically predisposed to become overweight and develop hyperglycemia if raised on a high fat diet. The purpose of the present study was to explore the effect of dietary supplementation of L-glutamine (Gln), an inhibitor of fatty acid oxidation, on the development of hyperglycemia and excessive weight gain. Groups of 10 age- and weight-matched male B/6J mice were raised on one of four diets: 1) a low fat, low sucrose (LL), studied separately, 2) a high fat, low sucrose (HL) diet alone, 3) high fat, low sucrose supplemented with L-glutamine (HL+Gln) and 4) high fat, low sucrose supplemented with L-alanine (HL+Ala). Energy intake, body weight, plasma glucose and insulin concentrations were monitored over time. We found no difference in energy intake per unit body weight between any groups after the first 2 wk of feeding. However, the mean +/- SEM for body weight (27.1 +/- 0.6 g) of the LL group measured at 16 wk was lower (P < 0.05) than that of the HL group at 37.9 +/- 1.9 g. Also, after 5.5 mo, the mean +/- SEM for plasma glucose and insulin concentrations in the LL group of mice were 6.9 +/- 0.4 mmol/l and 146 +/- 30 pmol/l, which were lower (P < 0.05) than those in the HL group at 10.1 +/- 0.9 mmol/l and 438 +/- 84 pmol/l, respectively. Although both amino acids caused a 10% reduction (P < 0.05) in body weight compared with HL feeding at wk 16, only Gln supplementation resulted in persistent reductions in both plasma glucose and insulin concentrations over 5.5 mo. In another experiment, when Gln was added to the high fat (HL) diet of heavy hyperglycemic animals for 2 mo, body weight gain, hyperglycemia and hyperinsulinemia were attenuated. In conclusion, supplementing glutamine to a high fat diet reduces body weight and attenuated hyperglycemia and hyperinsulinemia in B/6J mice.

The effect of dietary asparagine on rat growth was investigated. Diets were formulated with L-amino acids so as to contain asparagine, aspartic acid, glutamine and/or glutamic acid in all possible combinations and then fed to weanling rats for 3 weeks. Of the four, only asparagine was found to be essential for optimal growth, and it was essential regardless of the presence or absence of any dietary combination of these related amino acids. In selected dietary groups, the unbound asparagine levels were measured in various tissues over an 8-day period. Muscle asparagine levels were reduced for asparagine-deprived animals over the entire period studied; brain levels were decreased only after 7 days of dietary depletion, while hepatic levels were unaffected by dietary asparagine deprivation. In a related series, animals were more drastically depleted of asparagine by combining dietary deprivation with asparaginase treatment, causing a rapid decrease in cellular concentration of asparagine, which affected protein and DNA synthesis for those organs undergoing hyperplastic growth. Thus, asparagine may be rate limiting to protein synthesis for this extreme case as well as during dietary asparagine deprivation, which also decreased intracellular levels of unbound asparagine and led to irreversible deficits in development.

The report of Baliga et al. (1) that the nutritionally essential amino acids promote initiation of protein synthesis by isolated rat liver ribosomes has been reinvestigated. We were unable to observe any particular influence of essential amino acids on the incorporation of leucine into protein. We find instead that glutamine, glutamate, asparagine and aspartate appear to exert a greater influence than the essential amino acids on leucine incorporation by the ribosomes and suggest that the availability of amino acids in cell-free systems primarily affects elongation.