Alanyl-Glutamine Dipeptide Attenuates Non-Alcoholic Fatty Liver Disease Induced by a High-Fat Diet in Mice by Improving Gut Microbiota Dysbiosis.

Non-alcoholic fatty liver disease (NAFLD) manifests as a persistent liver ailment marked by the excessive buildup of lipids within the hepatic organ accompanied by inflammatory responses and oxidative stress. Alanyl-glutamine (AG), a dipeptide comprising alanine and glutamine, is commonly employed as a nutritional supplement in clinical settings. This research aims to evaluate the impact of AG on NAFLD triggered by a high-fat diet (HFD), while concurrently delving into the potential mechanisms underlying its effects. The results presented herein demonstrate a notable reduction in the elevated body weight, liver mass, and liver index induced by a HFD upon AG administration. These alterations coincide with the amelioration of liver injury and the attenuation of hepatic histological advancement. Furthermore, AG treatment manifests a discernible diminution in oil-red-O-stained regions and triglyceride (TG) levels within the liver. Noteworthy alterations encompass lowered plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) concentrations, coupled with elevated high-density lipoprotein cholesterol (HDLC) concentrations. The mitigation of hepatic lipid accumulation resultant from AG administration is aligned with the downregulation of ACC1, SCD1, PPAR-γ, and CD36 expression, in conjunction with the upregulation of FXR and SHP expression. Concomitantly, AG administration leads to a reduction in the accumulation of F4/80-positive macrophages within the liver, likely attributable to the downregulated expression of MCP-1. Furthermore, AG treatment yields a decline in hepatic MDA levels and a concurrent increase in the activities of SOD and GPX. A pivotal observation underscores the effect of AG in rectifying the imbalance of gut microbiota in HFD-fed mice. Consequently, this study sheds light on the protective attributes of AG against HFD-induced NAFLD through the modulation of gut microbiota composition.

Effects of tributyrin and alanyl-glutamine dipeptide on intestinal health of largemouth bass (Micropterus salmoides) fed with high soybean meal diet.

To investigate the effects of dietary tributyrin (TB) and alanyl-glutamine (AGn) on the intestinal health of largemouth bass (Micropterus salmoides) fed with high-level soybean meal (SM) diet, six isonitrogenous (41.36%) and isolipidic (10.25%) diets were formulated and fed to largemouth bass (initial body weight 25.5 ± 0.5g) for 8 weeks. The two control diets contained 34.8% peanut meal (PM) and 41.3% SM, while the other four experimental diets supplemented TB at 0.1% (TB0.1), 0.2% (TB0.2) and AGn at 1% (AGn1), 2% (AGn2) in SM, respectively. The results showed that there were no significant differences in weight gain, survival rate, and hepatosomatic index among all groups (P>0.05), while feed coefficient rate in AGn1, AGn2 and TB0.2 groups was significantly lower than that in SM group (P< 0.05). Compared with the PM group, the intestinal inflammation of largemouth bass in SM group were obvious, accompanied by the damage of intestinal structure, the decrease of digestive enzyme activity, and the up-regulation of proinflammatory cytokines. Compared with the SM group, the activities of intestinal trypsin, lipase and foregut amylase in TB and AGn groups increased significantly (P<0.05), and the gene expression levels of acetyl-CoA carboxylase (ACC), caspase-3, caspase-8, caspase-9, tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß) were down-regulated, while the gene expression levels of target of rapamycin (TOR) and eIF4E-binding protein (4E-BP) were up-regulated in all experimental groups (P<0.05). It can be concluded that supplementation of 1%-2% AGn and 0.1%-0.2% TB can alleviate enteritis caused by high-level soybean meal, and the recommend level is 2% AGn and 0.2% TB.

Alanyl-Glutamine (Ala-Gln) Ameliorates Dextran Sulfate Sodium (DSS)-Induced Acute Colitis by Regulating the Gut Microbiota, PI3K-Akt/NF-κB/STAT3 Signaling, and Associated Pulmonary Injury.

The aim of this study was to investigate the protective effect of alanyl-glutamine (Ala-Gln) on acute colitis complicated by pulmonary injury induced by dextran sulfate sodium (DSS) in C57BL/6 mice. The results showed that Ala-Gln intervention alleviated weight loss, the disease activity index (DAI), colon shortening, and pathological injury and regulated the absolute number of CD4+T-cell subsets in mesenteric lymph nodes (MLNs). In addition, Ala-Gln intervention significantly ameliorated the composition of the gut microbiota in mice with DSS- induced acute colitis, significantly decreasing the relative abundance of Desulfovibrionaceae and increasing the abundances of Gastranaerophilales, Clostridia-vadinBB60, and Alistipes. Moreover, Ala-Gln treatment significantly inhibited the activation of the PI3K-Akt/NF-κB/STAT3 inflammatory signaling pathways in the colon of mice with DSS-induced acute colitis. Notably, Ala-Gln intervention also alleviated the pulmonary injury as well as the imbalance in levels of CD4+T-cell subsets in pulmonary tissue in mice with DSS-induced acute colitis. In conclusion, Ala-Gln alleviates DSS-induced acute colitis by regulating the gut microflora and PI3K-Akt/NF-κB/STAT3 signaling pathways, as well as by alleviating accompanying pulmonary injury.

Alanyl-Glutamine Protects Mice against Methionine- and Choline-Deficient-Diet-Induced Steatohepatitis and Fibrosis by Modulating Oxidative Stress and Inflammation.

Nonalcoholic steatohepatitis (NASH) is a common chronic liver disease with increasing prevalence rates over years and is associated with hepatic lipid accumulation, liver injury, oxidative stress, hepatic inflammation, and liver fibrosis and lack of approved pharmacological therapy. Alanyl-glutamine (Ala-Gln) is a recognized gut-trophic nutrient that has multiple pharmacological effects in the prevention of inflammation- and oxidative-stress-associated diseases. Nevertheless, whether Ala-Gln has a protective effect on NASH still lacks evidence. The aim of this study is to explore the influence of Ala-Gln on NASH and its underlying mechanisms. Here, C57BL/6 mice were fed a methionine- and choline-deficient (MCD) diet to establish the model of NASH, and Ala-Gln at doses of 500 and 1500 mg/kg were intraperitoneally administered to mice along with a MCD diet. The results showed that Ala-Gln treatment significantly attenuated MCD-induced hepatic pathological changes, lowered NAFLD activity score, and reduced plasma alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) levels. Ala-Gln dramatically alleviated lipid accumulation in liver through modulating the expression levels of fatty acid translocase (FAT/CD36) and farnesoid X receptor (FXR). In addition, Ala-Gln exerted an anti-oxidant effect by elevating the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX). Moreover, Ala-Gln exhibited an anti-inflammatory effect via decreasing the accumulation of activated macrophages and suppressing the production of proinflammatory mediators. Notably, Ala-Gln suppressed the development of liver fibrosis in MCD-diet-fed mice, which may be due to the inhibition of hepatic stellate cells activation. In conclusion, these findings revealed that Ala-Gln prevents the progression of NASH through the modulation of oxidative stress and inflammation and provided the proof that Ala-Gln might be an effective pharmacological agent to treat NASH.

Alanyl-Glutamine Protects against Lipopolysaccharide-Induced Liver Injury in Mice via Alleviating Oxidative Stress, Inhibiting Inflammation, and Regulating Autophagy.

Acute liver injury is a worldwide problem with a high rate of morbidity and mortality, and effective pharmacological therapies are still urgently needed. Alanyl-glutamine (Ala-Gln), a dipeptide formed from L-alanine and L-glutamine, is known as a protective compound that is involved in various tissue injuries, but there are limited reports regarding the effects of Ala-Gln in acute liver injury. This present study aimed to investigate the protective effects of Ala-Gln in lipopolysaccharide (LPS)-induced acute liver injury in mice, with a focus on inflammatory responses and oxidative stress. The acute liver injury induced using LPS (50 µg/kg) and D-galactosamine (D-Gal) (400 mg/kg) stimulation in mice was significantly attenuated after Ala-Gln treatment (500 and 1500 mg/kg), as evidenced by reduced plasma alanine transaminase (ALT) (p < 0.01, p < 0.001), aspartate transaminase (AST) (p < 0.05, p < 0.001), and lactate dehydrogenase (LDH) (p < 0.01, p < 0.001) levels, and accompanied by improved histopathological changes. In addition, LPS/D-Gal-induced hepatic apoptosis was also alleviated by Ala-Gln administration, as shown by a greatly decreased ratio of TUNEL-positive hepatocytes, from approximately 10% to 2%, and markedly reduced protein levels of cleaved caspase-3 (p < 0.05, p < 0.001) in liver. Moreover, we found that LPS/D-Gal-triggered oxidative stress was suppressed after Ala-Gln treatment, the effect of which might be dependent on the elevation of SOD and GPX activities, and on GSH levels in liver. Interestingly, we observed that Ala-Gln clearly inhibited LPS/D-Gal exposure-induced macrophage accumulation and the production of proinflammatory factors in the liver. Furthermore, Ala-Gln greatly regulated autophagy in the liver in LPS/D-Gal-treated mice. Using RAW264.7 cells, we confirmed the anti-inflammatory role of Ala-Gln-targeting macrophages.

[Innovations in peritoneal dialysis]. / Innovationen in der Peritonealdialyse.

Peritoneal dialysis used to be a common treatment for acute kidney failure that required dialysis. In favor of continuous, extracorporeal renal replacement procedures, it disappeared from the scene in the western world, whereas it continues to be used in structurally poor countries due to its simplicity and low resource intensity. Recently, the shortages in medical care in the context of the coronavirus disease 2019 (COVID-19) pandemic led to renewed worldwide interest in peritoneal dialysis as a safe option in acute kidney failure requiring dialysis. The introduction of biocompatible solutions 20 years ago was expected to reduce mortality or technical failure. Unfortunately, so far this could only be implied but not confirmed in studies. Immunomodulatory adjuvants are an innovative option which have the potential to improve the local immunocompetence and prevent the loss of peritoneal function. Currently, the vision of a wearable artificial kidney is getting closer. Intensification of dialysis dose also appears achievable with minimal dialysate volumes. In times of global warming, the regeneration of dialysates could not only save relevant amounts of water but also have a favorable impact on the CO2 balance. In summary, peritoneal dialysis is currently enjoying a comeback. This article describes the current and future developments of this procedure.

How to Improve the Biocompatibility of Peritoneal Dialysis Solutions (without Jeopardizing the Patient's Health).

Peritoneal dialysis (PD) is an important, if underprescribed, modality for the treatment of patients with end-stage kidney disease. Among the barriers to its wider use are the deleterious effects of currently commercially available glucose-based PD solutions on the morphological integrity and function of the peritoneal membrane due to fibrosis. This is primarily driven by hyperglycaemia due to its effects, through multiple cytokine and transcription factor signalling-and their metabolic sequelae-on the synthesis of collagen and other extracellular membrane components. In this review, we outline these interactions and explore how novel PD solution formulations are aimed at utilizing this knowledge to minimise the complications associated with fibrosis, while maintaining adequate rates of ultrafiltration across the peritoneal membrane and preservation of patient urinary volumes. We discuss the development of a new generation of reduced-glucose PD solutions that employ a variety of osmotically active constituents and highlight the biochemical rationale underlying optimization of oxidative metabolism within the peritoneal membrane. They are aimed at achieving optimal clinical outcomes and improving the whole-body metabolic profile of patients, particularly those who are glucose-intolerant, insulin-resistant, or diabetic, and for whom daily exposure to high doses of glucose is contraindicated.

An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept.

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell-cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.

Protective effect of glutamine and alanyl-glutamine against zearalenone-induced intestinal epithelial barrier dysfunction in IPEC-J2 cells.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, has a negative effect on porcine intestine. Glutamine (Gln) and alanyl-glutamine (Ala-Gln) are nutrients with potential preservation functions similar to those of the intestinal epithelial barrier. The protective role of Gln and Ala-Gln on ZEN-induced intestinal barrier dysfunction was evaluated in this study. Additionally, the ability of Gln and Ala-Gln to protect the intestinal barrier was investigated. Our results showed that lactate dehydrogenase (LDH) activity, paracellular permeability and reactive oxygen species (ROS) level were increased by ZEN, while the glutathione (GSH) level was decreased by ZEN. Gln and Ala-Gln promoted the proliferation of cells and attenuated the ZEN-induced increase in cytotoxicity, cell apoptosis and paracellular permeability. Gln and Ala-Gln alleviated barrier function damage, which was additionally induced by ZEN by increasing the antioxidant capacity of cells. In addition, Gln and Ala-Gln upregulated intestinal barrier associated gene expressions including pBD-1, pBD-2, MUC-2, ZO-1, occludin and claudin-3. This study revealed that Gln and Ala-Gln had similar effects in protecting intestinal epithelial barrier function against ZEN exposure in IPEC-J2 cells. A new treatment for alleviating ZEN-induced injury to the intestine through nutritional intervention is provided.

The use of dipeptide supplementation as a means of mitigating the negative effects of dietary soybean meal on Zebrafish Danio rerio.

Soybean meal (SBM) inclusion in aquaculture diets has been found to negatively affect growth and induce intestinal inflammation in fish. The objective of this study was to determine the effect of health-promoting dipeptide supplementation into SBM-based feeds on growth performance, intestinal health, and muscle free amino acid composition, an indicator of dietary amino acid availability, in a zebrafish model. There were five treatment groups in this study. The first group ((+) Control) received a fishmeal-based diet. The second group ((-) Control) received SBM-based diet. The last three groups (Ala-Glu, Car, and Ans) were fed SBM-based diets, supplemented with alanyl-glutamine, carnosine, and anserine respectively. The Ala-Glu and Car groups experienced a significantly higher weight gain than the (-) Control group, weighing 35.38% and 33.96% more, respectively at the conclusion of the study. There were no significant differences in gene expression among the groups, but Ala-Glu had the highest expression of both nutrient absorption genes measured, PepT1 and fabp2. Ala-Glu had significantly longer intestinal villi, and a significantly higher villus length-to-width ratio than the (-) Control group. The Car group had a significantly higher post-prandial tissue concentration of lysine, compared to the (-) Control group. The increase in villus surface area and expression of nutrient absorption genes represent an improvement in intestinal absorptive capacity in the Ala-Glu group. The results from this study provide support for the use of alanyl-glutamine and carnosine supplementation as a means of improving growth performance of zebrafish fed with a high level SBM-based diet.

Expert consensus on clinical application of intravenous alanyl-glutamine dipeptide / 中华临床营养杂志

Alanyl-glutamine dipeptide is an important component in parenteral nutrition, which can be decomposed into alanine and L-glutamine in vivo. It plays multiple functions including maintaining intestinal barrier, improving immunity, promoting protein synthesis, and regulating the production and release of inflammatory mediators. Substantial clinical evidences have demonstrated its favorable effectiveness and safety. Rational application of alanyl-glutamine dipeptide can reduce postoperative complications, shorten hospital stay and save medical costs. There are still controversies at home and abroad on the applicable population and dosage of alanyl-glutamine dipeptide. Chinese Society of Parenteral and Enteral Nutrition organized China's experts of related disciplines to compile international standards in accordance with the latest guidelines and consensus, so as to achieve the goal of standardized application and patient benefits.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status / Resposta de choque térmico em perda auditiva induzida por ruído: efeitos da suplementação com dipeptídeos de alanil-glutamina sobre o estado das proteínas de choque térmico

Abstract Introduction: The 72 kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). Objective: We explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. Methods: Female 90 day-old Wistar rats (n = 32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124 dB SPL for 2 h) and 14 days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. Results: We found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. Conclusions: Our data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72 kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Resumo Introdução: A proteína de choque térmico de 72 kDa, HSP72 localizada intracelularmente, tem papéis citoprotetores e anti-inflamatórios cocleares na orelha interna durante situações de ruído estressantes. A expressão dessa proteína pode ser potencializada pela suplementação com dipeptídeo de alanil-glutamina. Por outro lado, essas proteínas atuam como sinais pró-inflamatórios no meio extracelular. Objetivo: Investigar se a perda auditiva induzida por ruído promove alterações tanto das proteínas HSP72 intracelulares quanto extracelulares na resposta de choque térmico e se a suplementação com alanil-glutamina pode modificar a resposta de choque térmico e evitar a perda auditiva. Método: Ratos Wistar fêmeas, com 90 dias de idade (n = 32), foram divididos aleatoriamente em quatro grupos: controle, perda auditiva induzida por ruído, tratados com alanil-glutamina e perda auditiva induzida por ruído mais alanil-glutamina. Os potenciais evocados auditivos do tronco encefálico foram avaliados antes da exposição ao ruído (124 dB NPS por 2 h) e 14 dias após. A cóclea, o complexo nuclear coclear e amostras de plasma foram coletadas para mensuração de HSP72 intra e extracelular com um kit Elisa de alta sensibilidade. Resultados: Houve um aumento nos níveis de HSP72 intra e extracelular no grupo perda auditiva induzida por ruído, que foi minimizado pelo tratamento com alanil-glutamina. Além disso, o índice H das HSP72 (razão HSP72 extracelular/HSP72intracelular plasma/cóclea) aumentou no grupo perda auditiva induzida por ruído, mas foi limitado pelo tratamento com alanil-glutamina, embora o alanil-glutamina não tenha efeito no limiar auditivo. Conclusões: Nossos dados indicam que o dano coclear induzido pela exposição ao ruído é acompanhado por marcadores da resposta de choque térmico locais e sistêmicos. Além disso, alanil-glutamina reduziu os marcadores de estresse, mesmo não tendo efeito sobre a perda auditiva induzida por ruído. Finalmente, os níveis plasmáticos de proteínas de choque térmico de 72 kDa podem ser usados como biomarcador do estresse auditivo, após a exposição ao ruído.

Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid.

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.

Protective Effects Oncorneal Endothelium During Intracameral Irrigation Using N-(2)-l-alanyl-l-Glutamine.

Corneal endothelial disease is a global sight-threatening disease, and corneal transplantation using donor corneas remains the sole therapeutic option. A previous work demonstrated that N (2)-alanyl-glutamine (Ala-Gln) protected against apoptosis and cellular stress, and maintained intestinal tissue integrity. In this pursuit, the present study aimed to examine the effect of Ala-Gln in the protection of the corneal endothelium and expand its range of potential clinical applications. Mice in the control group were intracamerally irrigated with Ringers lactate injection, whereas those in the experimental group were irrigated with Ringers lactate injection containing Ala-Gln. The mean intraocular pressure increased to 44 ± 3.5 mm Hg during intracameral irrigation (normal range 10.2 ± 0.4 mmHg). In vivo confocal microscopy results showed that the addition of Ala-Gln protected the morphology, structure, and density of the corneal endothelial cells. Optical Coherence Tomography (OCT) measurements showed that corneal thickness was not significantly different between the two groups, because of the immediate corneal edema after irrigation, but the addition of Ala-Gln obviously promoted the recovery of the corneal edema. Scanning electron microscopy indicated that the corneal endothelial cells were severely ruptured and exfoliated in the Ringer's group accompanied with cellular edema, when compared with the Ala-Gln group. The intracameral irrigation using Ala-Gln protected the structure and expression of cytoskeleton and Na-K-ATPase, which exhibited a regular distribution and significantly increased expression in comparison to Ringer's group. Furthermore, Ala-Gln maintained the mitochondrial morphology and increased the activity of mitochondria. Moreover, transmission electron microscopy showed that intracameral irrigation of Ala-Gln reversed the ultrastructural changes induced by the acute ocular hypertension in mice. Our study demonstrates that the intracameral irrigation of Ala-Gln effectively maintained the corneal endothelial pump function and barrier function by protecting the mitochondrial function and preventing the rearrangement of cytoskeleton in acute ocular hypertension in mice.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status.

INTRODUCTION: The 72kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). OBJECTIVE: We explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. METHODS: Female 90 day-old Wistar rats (n=32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124dB SPL for 2h) and 14days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. RESULTS: We found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. CONCLUSIONS: Our data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Dietary alanyl-glutamine improves growth performance of weaned piglets through maintaining intestinal morphology and digestion-absorption function.

Alanyl-glutamine (Ala-Gln), a highly soluble and stable glutamine dipeptide, is known to improve gut integrity and function. The aim of this study was to evaluate whether dietary Ala-Gln supplementation could improve growth performance, intestinal development and digestive-absorption function in weaned piglets. A total of 100 purebred Yorkshire piglets weaned at 21 days of age were assigned randomly to four dietary treatment groups and fed a basal diet (control group) or a basal diet containing 0.15%, 0.30% and 0.45% Ala-Gln, respectively. Compared with the control group, piglets fed the Ala-Gln diets had higher average daily gain and lower feed : gain and diarrhea rate (P < 0.05). Moreover, dietary Ala-Gln supplementation increased villous height and villous height : crypt depth ratio in duodenum and jejunum (P < 0.05), as well as the activities of maltase and lysozyme in jejunum mucosa (P < 0.05). In addition, a decrease in serum diamine oxidase activity and crypt depth in duodenum and jejunum was observed in piglets fed the Ala-Gln diets (P < 0.05). Serum cytosolic phospholipase A2 (cPLA2) concentration and gene expression of cPLA2, Na+-dependent glucose transporter 1, glucose transporter 2 and peptide transporter 1 in jejunum were increased by feeding Ala-Gln diets relative to control diet (P < 0.05). These results indicated that feeding Ala-Gln diet has beneficial effects on the growth performance of weaned piglets, which associated with maintaining intestinal morphology and digestive-absorption function.

Alanyl-glutamine ameliorates lipopolysaccharide-induced inflammation and barrier function injury in bovine jejunum epithelial cells.

The aim of this study was to investigate the effects of alanyl-glutamine (Ala-Gln) on the regulation of lipopolysaccharide (LPS)-induced inflammation and barrier function in bovine jejunum epithelial cells (BJECs). BJECs were exposed (or not) to 1 µg/mL LPS for 24 h to generate a pro-inflammatory model. The cells were then treated with different concentrations of Ala-Gln (0.25, 0.5, 1.0, 2.0, or 4.0 mmol/L) to detect any regulatory effects on the inflammation and barrier function of BJECs. LPS decreased cell viability and enhanced the production of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. LPS induced inflammation and damaged the barrier function of BJECs, as evidenced by up-regulated mRNA and protein expression of inflammatory factors and down-regulated expression of tight junction proteins. Conversely, Ala-Gln rescued the decrease in cell viability and prevented the accumulation of ILs after LPS exposure by reducing the mRNA and protein expression levels of inflammatory factors. In addition, Ala-Gln induced the mRNA and protein expression of multiple tight junction proteins, and thus reconstituted the barrier function of BJECs. In conclusion, Ala-Gln attenuates injury from inflammation and repairs damaged intestinal barrier induced with LPS, suggesting its potential as a therapeutic agent against intestinal inflammation in mammals.

Evitar (l-Alanyl-l-Glutamine) Regulates Key Signaling Molecules in the Pathogenesis of Postoperative Tissue Fibrosis.

AIMS: Hypoxia and the resulting oxidative stress play a major role in postoperative tissue fibrosis. The objective of this study was to determine the effect of l-alanyl-l-glutamine (Ala-Gln) on key markers of postoperative tissue fibrosis: hypoxia-inducible factor (HIF) 1α and type I collagen. METHODS: Primary cultures of human normal peritoneal fibroblasts (NPF) established from normal peritoneal tissue were treated with increasing doses of Ala-Gln (0, 1, 2, or 10 mM) with hypoxia ([2% O2] 0-48 hours; continuous hypoxia) or after hypoxia (0.5, 1, 2, 4 hours) and restoration of normoxia (episodic hypoxia) with immediate treatment with Ala-Gln. Hypoxia-inducible factor 1α and type 1 collagen levels were determined by enzyme-linked immunosorbent assay. Data were analyzed with 1-way analysis of variance followed by Tukey tests with Bonferroni correction. RESULTS: Hypoxia-inducible factor 1α and type I collagen levels increased in untreated controls by 3- to 4-fold in response to continuous and episodic hypoxia in human NPF. Under continuous hypoxia, HIF-1α and type I collagen levels were suppressed by Ala-Gln in a dose-dependent manner. l-alanyl-l-glutamine treatment after episodic hypoxia also suppressed HIF-1α and type I collagen levels for up to 24 hours for all doses and up to 48 hours at the highest dose, regardless of exposure time to hypoxia. CONCLUSIONS: l-alanyl-l-glutamine significantly suppressed hypoxia-induced levels of key tissue fibrosis (adhesion) phenotype markers under conditions of continuous as well as episodic hypoxia in vitro. This effect of glutamine on molecular events involved in the cellular response to insult or injury suggests potential therapeutic value for glutamine in the prevention of postoperative tissue fibrosis.

Design of composite microparticle systems based on pectin and waste material of propolis for modified l-alanyl-l-glutamine release and with immunostimulant activity.

Catabolic conditions like acquired immunodeficiency syndrome, cancer, and burn can cause immunosuppression. Amino acids such as alanine and glutamine are essential for the activity of the immune system. Propolis is immunostimulant and the waste of propolis extraction has been reused with technological and therapeutic purposes. Therefore, this study describes the association of propolis byproduct extract (BPE) with pectin to prepare spray-dried microparticles containing the dipeptide l-alanyl-l-glutamine as stimulant systems of neutrophils. The use of a factorial design allowed selecting the best formulation, which was characterized by morphology, size, and entrapment efficiency analyses. In addition, the systems were characterized by thermal and X-ray diffraction analysis, Fourier-transform infrared spectroscopy, in vitro drug release, and in vitro cytotoxicity and stimulation test of neutrophils. Small well-structured microparticles with good entrapment efficiency values were achieved. Thermal stability of formulation was observed, and it was proved that pectin, BPE and l-alanyl-l-glutamine were dispersed throughout the matrix. The drug was released from the microparticles during 24 h governed by swelling and diffusion. The drug-loaded formulations showed a significant stimulating effect on neutrophils. These structures could increase the activity of immune cells, and other in vitro and in vivo studies should be performed in the future.

Oral administration of alanyl-glutamine and glutamine improve random pattern dorsal skin flap survival in rats.

OBJECTIVES: Skin flap necrosis is the most common postoperative side effect in reconstructive surgeries. Glutamine (GLN) has been shown to accelerate wound healing process. The purpose of this study was to evaluate the effects of GLN either in free form or in the dipeptide form along with L- alanyl (Ala-GLN) on random skin flaps survival in rats. MATERIALS AND METHODS: Dorsal skin flaps with caudal bases (8 ×2 cm) were established in 24 adult male Wistar rats. Then, the animals were randomly assigned into 3 groups (n=8). Control, GLN (0.75 g/kg) and Ala-GLN (0.75 g/kg). All groups administrated orally 24 and 6 hr before flap elevation and continued repeatedly daily until 7 days postoperation. The flap survival rate and vascular density using histological analysis were evaluated. Vascular endothelial growth factor (VEGF) by immunohistochemical method was determined. RESULTS: Seven days after surgery, the mean surviving area in the GLN and Ala-GLN groups were significantly greater than in the untreated control group (P<0.001). Furthermore, in comparison with the control group, the number of blood vessels and VEGF-positive cells in treated groups with GLN and Ala-GLN were significantly higher. However, no significant differences were observed between treated groups with GLN and Ala-GLN. CONCLUSION: The findings from this study indicate that oral administration of GLN in free form or in the dipeptide (Ala-GLN) could promote neovascularization and improve skin flap survival in rats.