Ameliorative Potential of L-Alanyl L-Glutamine Dipeptide in Colon Cancer Patients Receiving Modified FOLFOX-6 Regarding the Incidence of Diarrhea, the Treatment Response, and Patients' Survival: A Randomized Controlled Trial.

Background and Objectives: Diarrhea induced by chemotherapy may represent a life-threatening adverse effect in cancer patients receiving chemotherapy. FOLFOX, an effective treatment for colon cancer, has been associated with diarrhea with high severity, particularly with higher doses. Management of diarrhea is crucial to increase the survival of cancer patients and to improve the quality of life. Glutamine is an abundant protein peptide found in blood and has a crucial role in boosting immunity, increasing protein anabolism, and decreasing the inflammatory effects of chemotherapy on the mucosal membranes, including diarrhea. This study aimed to provide evidence that parenteral L-alanyl L-glutamine dipeptide may have a positive influence on the incidence of diarrhea, treatment response, and the overall survival in colon cancer patients treated with modified FOLFOX-6 (mFOLFOX-6). Materials and Methods: Forty-four stage II and III colon cancer patients were included in this study where they were treated with the standard colon cancer chemotherapy mFOLFOX-6 and were randomly allocated into glutamine group and placebo group, each of 22 patients. Results: L-alanyl L-glutamine dipeptide was found to be significantly effective in decreasing the frequency and severity of diarrhea when compared to the placebo group, particularly after four and six cycles of mFOLFOX-6. There was no significant difference between the studied groups regarding to the overall survival. Conclusion: L-alanyl L-glutamine dipeptide can be considered as an add-on with chemotherapy to improve the quality of life and the overall survival of colon cancer patients.

Production of L-alanyl-L-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase.

BACKGROUND: L-Alanyl-L-glutamine (Ala-Gln) represents the great application potential in clinic due to the unique physicochemical properties. A new approach was developed to synthesize Ala-Gln by recombinant Escherichia coli OPA, which could overcome the disadvantages of traditional chemical synthesis. Although satisfactory results had been obtained with recombinant E. coli OPA, endotoxin and the use of multiple antibiotics along with toxic inducer brought the potential biosafety hazard for the clinical application of Ala-Gln. RESULTS: In this study, the safer host Pichia pastoris was applied as an alternative to E. coli. A recombinant P. pastoris (named GPA) with the original gene of α-amino acid ester acyltransferase (SsAet) from Sphingobacterium siyangensis SY1, was constructed to produce Ala-Gln. To improve the expression efficiency of SsAet in P. pastoris, codon optimization was conducted to obtain the strain GPAp. Here, we report that Ala-Gln production by GPAp was approximately 2.5-fold more than that of GPA. The optimal induction conditions (cultivated for 3 days at 26 °C with a daily 1.5% of methanol supplement), the optimum reaction conditions (28 °C and pH 8.5), and the suitable substrate conditions (AlaOMe/Gln = 1.5/1) were also achieved for GPAp. Although most of the metal ions had no effects, the catalytic activity of GPAp showed a slight decrease in the presence of Fe3+ and an obvious increase when cysteine or PMSF were added. Under the optimum conditions, the Ala-Gln generation by GPAp realized the maximum molar yield of 63.5% and the catalytic activity of GPAp by agar embedding maintained extremely stable after 10 cycles. CONCLUSIONS: Characterized by economy, efficiency and practicability, production of Ala-Gln by recycling immobilized GPAp (whole-cell biocatalyst) is represents a green and promising way in industrial.

Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise.

We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1ß, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1ß, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation.

Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids.

L-glutamine (Gln) is a key metabolic fuel for intestinal epithelial cell proliferation and survival and may be conditionally essential for gut homeostasis during catabolic states. We show that L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide, protects mice against jejunal crypt depletion in the setting of dietary protein and fat deficiency. Separately, we show that murine crypt cultures (enteroids) derived from the jejunum require Gln or Ala-Gln for maximal expansion. Once expanded, enteroids deprived of Gln display a gradual atrophy of cryptlike domains, with decreased epithelial proliferation, but stable proportions of Paneth and goblet cell differentiation, at 24 h. Replenishment of enteroid medium with Gln selectively activates mammalian target of rapamycin (mTOR) signaling pathways, rescues proliferation, and promotes crypt regeneration. Gln deprivation beyond 48 h leads to destabilization of enteroids but persistence of EGFP-Lgr5-positive intestinal stem cells with the capacity to regenerate enteroids upon Gln rescue. Collectively, these findings indicate that Gln deprivation induces a reversible quiescence of intestinal stem cells and provides new insights into nutritional regulation of intestinal epithelial homeostasis.

Formulation and characterization of ethylcellulose microparticles containing .L-alanyl- L-glutamine peptide.

CONTEXT: The L-alanyl-L-glutamine peptide (AGP) has been effective to promote acute glycemia recovery during long-term insulin-induced hypoglycemia (IIH), and the oral administration of AGP is suggested to prevent prolonged hypoglycemia, such as nocturnal hypoglycemia. OBJECTIVE: Considering the ability of AGP on glycemia recovery and AGP's fast metabolism, the aim of current study was to obtain and characterize ethylcellulose microparticles to deliver the drug for a prolonged time. MATERIALS AND METHODS: Microparticles were prepared by simple and double emulsification/hardening method and characterized by scanning electron microscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), Fourier transform infra-red (FTIR) and FT-Raman spectroscopy and in vitro release. RESULTS AND DISCUSSION: Spherical structures with a mean diameter between 9.30 µm and 13.19 µm were formed. TG analysis showed that the thermal stability of AGP was even more increased by encapsulation with ethylcellulose. In addition, TG, DSC, FTIR and FT-Raman analyses proved that AGP was encapsulated in a molecular way. Higher values of encapsulation efficiency were observed for the microparticles prepared by double emulsification (57.83-83.67%) than for those prepared by simple emulsification (18.37%). However, the last ones could release the peptide in a quicker and more extensive manner than those prepared by double emulsification. CONCLUSION: For the first time, microparticles containing AGP were developed and exhibited prolonged in vitro release as well as protection to the drug, and it could be considered as a dosage form for patients who suffer from insulin-induced hypoglycemia and/or nocturnal hypoglycemia.

[Immobilizing engineered Escherichia coli cells into zeolitic imidazolate framework 8 for efficient biosynthesis of Ala-Gln].

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.

Clean Production of l-Alanyl-l-glutamine by an Efficient Yeast Biocatalyst Expressing α-Amino Acid Ester Acyltransferase without N-Glycosylation.

l-Alanyl-l-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the N-glycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g·(L·min)-1, respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.

Structural diversity and concentration dependence of pyrazine formation: Exogenous amino substrates and reaction parameters during thermal processing of l-alanyl-l-glutamine Amadori compound.

The Amadori compound of glucose and l-alanyl-l-glutamine (Ala-Gln-ARP) was prepared and characterized by UPLC-MS/MS and NMR. There were no pyrazines produced by heated Ala-Gln-ARP alone due to the asynchronicity of regenerated l-alanyl-l-glutamine and α-dicarbonyl compounds. High temperature (130 °C) and long reaction time could facilitate the 2,5-dimethylpyrazine formation at a small concentration (33.4 ± 3.47 µg/L). The exogenous amino substrates would lower the formation temperature of pyrazines and make it to be generated effectively. Extra supplied l-alanyl-l-glutamine could generate 2,5-dimethylpyrazine at 110 °C, while higher temperature of 140 °C could strengthen the formation of 2,5-dimethylpyrazine (793 ± 119 µg/L) and stimulate the generation of other pyrazines, including methylpyrazine and 2,6-dimethylpyrazine. The exogenous alanine, glutamic acid, and glutamine was also beneficial to enhance the pyrazines formation, especially the glutamic acid. Furthermore, alkaline pH of thermal reaction made pyrazines increase significantly than in neutral medium and further enriched their species such as unsubstituted pyrazine and trimethylpyrazine.

Adenylation domains of nonribosomal peptide synthetase: A potential biocatalyst for synthesis of dipeptides and their derivatives.

Dipeptides and their derivatives are important functional compounds that can be applied to fields such as medicine and food. As biological macromolecules, the adenylation domains of nonribosomal peptide synthetase (NRPS) can recognize and activate various building blocks, such as amino acids, for the biosynthesis of nonribosomal peptides. In this way, the amide bond formation can be achieved through a nucleophilic reaction where the adenylation domain serves as a biocatalyst and is further used to conduct dipeptide synthesis. In this study, the adenylation domains (BAA2, BBA2, and BCA4) of bacitracin synthetase were predicted and expressed. The substrate evaluation results showed that adenylation domains displayed broad substrate selectivity for amino acids in vitro. Furthermore, the use of dipeptide synthesis in adenylation domains suggested that the polarity of amino acids could have an influence on nucleophilic reactions. Finally, L-alanyl-L-glutamine and aspartame were successfully synthesized through catalysis by the adenylation domains BAA2 and BCA4, respectively. This study expands on approaches to the synthesis of functional dipeptides and their derivatives based on the chemoenzymatic process.

Peritoneal Dialysis Fluid Supplementation with Alanyl-Glutamine Attenuates Conventional Dialysis Fluid-Mediated Endothelial Cell Injury by Restoring Perturbed Cytoprotective Responses.

Long-term clinical outcome of peritoneal dialysis (PD) depends on adequate removal of small solutes and water. The peritoneal endothelium represents the key barrier and peritoneal transport dysfunction is associated with vascular changes. Alanyl-glutamine (AlaGln) has been shown to counteract PD-induced deteriorations but the effect on vascular changes has not yet been elucidated. Using multiplexed proteomic and bioinformatic analyses we investigated the molecular mechanisms of vascular pathology in-vitro (primary human umbilical vein endothelial cells, HUVEC) and ex-vivo (arterioles of patients undergoing PD) following exposure to PD-fluid. An overlap of 1813 proteins (40%) of over 3100 proteins was identified in both sample types. PD-fluid treatment significantly altered 378 in endothelial cells and 192 in arterioles. The HUVEC proteome resembles the arteriolar proteome with expected sample specific differences of mainly immune system processes only present in arterioles and extracellular region proteins primarily found in HUVEC. AlaGln-addition to PD-fluid revealed 359 differentially abundant proteins and restored the molecular process landscape altered by PD fluid. This study provides evidence on validity and inherent limitations of studying endothelial pathomechanisms in-vitro compared to vascular ex-vivo findings. AlaGln could reduce PD-associated vasculopathy by reducing endothelial cellular damage, restoring perturbed abundances of pathologically important proteins and enriching protective processes.

Retinoic acid receptor gamma is targeted by microRNA-124 and inhibits neurite outgrowth.

During brain development, neurite outgrowth is required for brain development and is regulated by many factors. All-trans retinoic acid (RA) is an important regulator of cell growth and differentiation. MicroRNA-124 (miR-124), a brain-specific microRNA, has been implicated in stimulating neurite growth. In this study, we found that retinoic acid receptor gamma (RARG) expression was decreased, whereas miR-124 expression was increased during neural differentiation in mouse Neuroblastoma (N2a) Cells, P19 embryonal carcinoma (P19) cells, and mouse brain, as detected by immunoblotting or RT-qPCR. And we proved that miR-124 inhibited RARG expression by binding to the 3' UTR of RARG with a luciferase reporter assay. Upregulation of miR-124 (using miR-124 overexpressing plasmid and miR-124 mimic) led to a significant decrease in RARG protein in N2a cells and primary neurons. Therefore, we asked whether and how the miR-124/RARG axis regulates neuronal outgrowth, which is poorly understood. Strikingly, RARG knockdown by shRNA stimulated neurite growth in N2a cells and primary neurons, whereas RARG overexpression (without 3' UTR) inhibited neurite growth in N2a cells, P19 cells, and primary neurons. Furthermore, RARG knockdown could partially eliminate neurite outgrowth defects caused by the inhibitor of miR-124, while RARG overexpression could reverse the neurite outgrowth enhancing effect of the upregulation of miR-124. Collectively, the data reveal that miR-124/RARG axis is critical for neurite outgrowth. RARG emerges as a new target regulated by miR-124 that modulates neurite outgrowth, providing a novel context in which these two molecules function.

Effects of glutamine and alanine supplementation on muscle fatigue parameters of rats submitted to resistance training.

OBJECTIVE: Although glutamine and alanine have properties that could delay fatigue, recent evidence showed that these amino acids impaired central fatigue markers. Nevertheless, the effect of this intervention on muscle fatigue is unknown. The aim of this study was to investigate the effects of glutamine and alanine supplementation on muscle fatigue parameters in rats submitted to resistance training (RT). METHODS: Wistar rats were distributed into the following groups: sedentary (SED), exercised (CON), exercised and supplemented with alanine (ALA), glutamine and alanine in their free form (G+A) or l-alanyl-l-glutamine (DIP). Trained groups underwent a ladder-climbing exercise for 8 wk. In the last 3 wk of RT, supplementations were offered in water with a 4% concentration. RESULTS: G+A and DIP supplementation increased the muscle content of glutamine and glutamate. DIP administration increased glycogen and lactate dehydrogenase (LDH) concentrations in muscle, whereas ALA and G+A supplementation reduced plasma LDH and creatine kinase levels. All trained groups presented higher levels of muscle glutathione (GSH) than SED. There was no difference between groups in lactate, xanthine, hypoxanthine, thiobarbituric acid reactive substances, 8-isoprostane and GSH in plasma; adenosine monophosphate deaminase, citrate synthase and monocarboxylate transporters 1 and 4 in muscle; and glycogen and GSH in the liver. Moreover, physical performance did not differ between groups. CONCLUSION: Glutamine and alanine supplementation improved muscle fatigue markers without affecting exercise performance.

Effects of Glutamine and Alanine Supplementation on Adiposity, Plasma Lipid Profile, and Adipokines of Rats Submitted to Resistance Training.

Glutamine and alanine are lipogenic and could prevent the effects of resistance training (RT) in reducing adiposity and modulating lipid profile. Thus, we aimed to investigate the effects of RT and glutamine and alanine supplementation, in their free or conjugated form, on relative epididymal adipose tissue (EAT) and brown adipose tissue (BAT) weight, plasma lipid profile, and adipokines in EAT. Thirty Wistar rats, aged two months, were distributed into five groups: control (CTRL), trained (TRN), trained and supplemented with alanine (ALA), glutamine and alanine in their free form (GLN+ALA), or L-alanyl-L-glutamine (DIP). Trained groups underwent a ladder-climbing exercise for eight weeks, with progressive load increase. Supplementations were offered in a solution with a concentration of 4% in the last 21 days of training. Food consumption and body weight gain were decreased in the TRN group compared with CTRL. RT also reduced relative EAT and BAT weight, while supplementations, especially with ALA, increased adipose tissue mass. RT reduced total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) (TRN vs. CTRL), whereas glutamine and alanine supplementation increased TC and LDL-c, impairing lipid profile modulation by physical exercise. RT did not affect the concentrations of adipokines in EAT, but DIP supplementation increased interleukin- (IL-) 6 and IL-10. In conclusion, RT reduced adiposity and modulated lipid profile, whereas glutamine and alanine supplementation increased adiposity and impaired lipid profile but increased the concentration of the anti-inflammatory cytokines IL-6 and IL-10 in EAT.

[Expression, purification, characterization and application of α-amino acid ester acyltransferase from recombinant Escherichia coli].

α-Amino acid ester acyltransferase (Aet) catalyzes the L-alanyl-L-glutamine forming reaction from L-alaine methylester hydrochloride and L-glutamine. In this study, the recombinant Escherichia coli saet-QC01 was used to express the α-amino acid acyltransferase, and its expression conditions were optimized. The recombinant protein was separated and purified by Ni-NTA affinity chromatography, and its enzymatic properties and catalytic applications were studied. The induction conditions suitable for enzyme production optimized were as follows: The temperature was 20 ℃, the induction stage (OD600=2.0-2.5), IPTG concentration was 0.6 mmol/L, induction time was 12 h. The optimal reaction conditions of α-amino acid acyltransferase were 27 ℃, pH 8.5, it was most stable between pH 7.0 and 8.0 and relatively stable in an acidic environment, and low concentration of Co²âº or EDTA could promote the enzyme activity. Under optimal reaction conditions, 600 mmol/L of L-alaine methylester hydrochloride and 480 mmol/L of L-glutamine, the yield of L-alanyl-L-glutamine reached 78.2 g/L and productivity of 1.955 g/L/min, the conversion rate reached 75.0%. α-Amino acid ester acyltransferase has excellent acid-basei resistance, high catalytic efficiency. These characteristics suggest its application prospects in the industrial production.

Targeted Metabolomic Profiling of Peritoneal Dialysis Effluents Shows Anti-oxidative Capacity of Alanyl-Glutamine.

Readily available peritoneal dialysis (PD) effluents from PD patients in the course of renal replacement therapy are a potentially rich source for molecular markers for predicting clinical outcome, monitoring the therapy, and therapeutic interventions. The complex clinical phenotype of PD patients might be reflected in the PD effluent metabolome. Metabolomic analysis of PD effluent might allow quantitative detection and assessment of candidate PD biomarkers for prognostication and therapeutic monitoring. We therefore subjected peritoneal equilibration test effluents from 20 stable PD patients, obtained in a randomized controlled trial (RCT) to evaluate cytoprotective effects of standard PD solution (3.86% glucose) supplemented with 8 mM alanyl-glutamine (AlaGln) to targeted metabolomics analysis. One hundred eighty eight pre-defined metabolites, including free amino acids, acylcarnitines, and glycerophospholipids, as well as custom metabolic indicators calculated from these metabolites were surveyed in a high-throughput assay requiring only 10 µl of PD effluent. Metabolite profiles of effluents from the cross-over trial were analyzed with respect to AlaGln status and clinical parameters such as duration of PD therapy and history of previous episodes of peritonitis. This targeted approach detected and quantified 184 small molecules in PD effluent, a larger number of detected metabolites than in all previous metabolomic studies in PD effluent combined. Metabolites were clustered within substance classes regarding concentrations after a 4-h dwell. PD effluent metabolic profiles were differentiated according to PD patient sub-populations, revealing novel changes in small molecule abundance during PD therapy. AlaGln supplementation of PD fluid altered levels of specific metabolites, including increases in alanine and glutamine but not glutamate, and reduced levels of small molecule indicators of oxidative stress, such as methionine sulfoxide. Our study represents the first application of targeted metabolomics to PD effluents. The observed metabolomic changes in PD effluent associated with AlaGln-supplementation during therapy suggested an anti-oxidant effect, and were consistent with the restoration of important stress and immune processes previously noted in the RCT. High-throughput detection of PD effluent metabolomic signatures and their alterations by therapeutic interventions offers new opportunities for metabolome-clinical correlation in PD and for prescription of personalized PD therapy.

Production of l-alanyl-l-glutamine by recycling E. coli expressing α-amino acid ester acyltransferase.

In this study, the optimum induction and reaction conditions, and fermentation process of producing Ala-Gln by E. coli Origami 2 overexpressing α-amino acid ester acyltransferase (OPA) were investigated. Besides, the Ala-Gln synthesis by OPA achieved the maximum molar yield of 94.7% and productivity of 1.89g/L/min due to the extremely high enzyme activity. On this basis, repeated-cycle batch fermentation to produce Ala-Gln indicated that OPA could maintain high Ala-Gln yields and enzyme stabilities after several cell recycling. Consequently, the cost-efficient and environmentally friendly approach for Ala-Gln production by recycling OPA makes a great contribution to further industrial-scale applications.

l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

Effects of glutamine and alanine supplementation, in their free form or as dipeptide, on fatigue parameters of rats submitted to resistance training / Efeitos da suplementação com glutamina e alanina, na forma livre ou como dipeptídeo, sobre parâmetros de fadiga de ratos submetidos ao treinamento resistido

Fatigue is defined as the inability to maintain muscle power and strength, impairing performance. Nutritional interventions have been used to delay this phenomenon, such as glutamine and alanine supplementation. These amino acids might attenuate several causes of fatigue, since they are important energy substrates, transport ammonia avoiding the accumulation of this toxic metabolite and attenuate muscle damage and oxidative stress. Thus, the aim of this study was to evaluate the effects of glutamine and alanine supplementation on central and muscle fatigue parameters of rats submitted to resistance training (RT). Forty adult Wistar rats (60 days) were distributed into five groups: SED (sedentary, receiving water), CON (trained, receiving water), ALA, G+A and DIP (trained and supplemented with alanine, glutamine and alanine in their free form, and Lalanyl-L-glutamine, respectively). Trained groups underwent a ladder-climbing exercise, with progressive loads, for eight weeks. Supplements were diluted in water to a 4% concentration and offered ad libitum during the last 21 days of experiment. RT increased plasma glucose, the muscle concentrations of ammonia and glutathione (GSH) and the muscle damage parameters - plasma creatine kinase (CK) and lactate dehydrogenase (LDH), whereas decreased muscle glycogen. G+A supplementation prevented the increase of muscle ammonia by RT, while ALA and G+A administration reduced plasma CK and LDH, and DIP supplementation increased the muscle content of glycogen and LDH. Contrary to expectations, DIP administration increased central fatigue parameters, such as plasma concentration of free fatty acids (FFA), hypothalamic content of serotonin and serotonin/dopamine ratio. Despite these results, there was no difference between groups in the maximum carrying capacity (MCC) tests. In conclusion, supplementation with glutamine and alanine improves some fatigue parameters, but does not affect physical performance of rats submitted to RT

O termo fadiga é definido como a incapacidade de manutenção da força e da potência musculares, prejudicando a performance. Intervenções nutricionais têm sido utilizadas para retardar este fenômeno, como a suplementação com glutamina e alanina. Estes aminoácidos poderiam atenuar diversas causas de fadiga, pois são importantes substratos energéticos, carreiam amônia evitando o acúmulo deste metabólito tóxico e atenuam a lesão muscular e o estresse oxidativo. Logo, o objetivo deste estudo foi avaliar os efeitos da suplementação com glutamina e alanina sobre parâmetros de fadiga central e muscular em ratos submetidos ao treinamento resistido (TR). Foram utilizados 40 ratos Wistar adultos (60 dias de idade), distribuídos nos grupos: SED (não treinados, recebendo água), CON (treinados, recebendo água), ALA, G+A e DIP (treinados e suplementados com alanina, glutamina e alanina livres, e L-alanil-L-glutamina, respectivamente). Os grupos treinados realizaram um exercício de escalada em escada, com aumento progressivo de carga, durante oito semanas. A suplementação foi diluída a 4% em água e ofertada via oral, ad libitum, durante os últimos 21 dias de experimento. O TR aumentou a glicemia, as concentrações musculares de amônia e de glutationa (GSH) e os parâmetros de lesão muscular - creatina quinase (CK) e lactato desidrogenase (LDH) no plasma, enquanto reduziu o glicogênio no músculo. A suplementação com G+A preveniu o aumento de amônia muscular promovido pelo TR, enquanto a administração de ALA e G+A reduziu as concentrações de CK e LDH no plasma, e a suplementação com DIP aumentou o conteúdo muscular de glicogênio e de LDH. Ao contrário do esperado, a administração de DIP aumentou parâmetros de fadiga central, como as concentrações plasmáticas de ácidos graxos livres, o conteúdo hipotalâmico de serotonina e a razão serotonina/dopamina. Apesar disso, não houve diferença entre os grupos nos testes de carga máxima. Em conclusão, a suplementação com glutamina e alanina melhora alguns parâmetros de fadiga, mas não afeta o desempenho físico em ratos submetidos ao TR

Efeito da suplementação com L-glutamina e L-alanina, livres ou como dipeptídeo, sobre a lesão, inflamação e citoproteção em modelos de estresse in vivo e in vitro / Effects of supplementation with L-glutamine and L-alanine, in their free form or as dipeptide, on muscle damage, inflammation and cytoprotection of in vivo and in vitro stress models

Subprojeto 1: Determinação do efeito anti-inflamatório e citoprotetor da suplementação com L-glutamina e L-alanina, ou com L-alanil-L-glutamina (DIP) em ratos submetidos a treinamento resistido. Exercícios intensos reduzem a disponibilidade de glutamina, comprometendo a função imune e a recuperação de atletas. O objetivo do estudo foi avaliar os efeitos da suplementação oral crônica com L-glutamina e L-alanina, nas formas livres ou como dipeptídeo (DIP), sobre parâmetros de lesão, inflamação e citoproteção em ratos Wistar adultos submetidos a treinamento resistido (TR). Neste estudo, o TR reduziu a concentração de glutamina no plasma e no músculo EDL. No entanto, este efeito foi atenuado pelos suplementos contendo L-glutamina, os quais aumentaram os conteúdos da proteína de resposta ao estresse (HSP70) em células do sistema imune (PBMC) e no EDL, concomitantemente à redução da ativação do NF-kB e a da concentração de citocinas no EDL. O efeito protetor das suplementações também foi evidenciado pela atenuação de marcadores de lesão (CK e LDH) e inflamação (TNF-α e IL-1ß), bem como pelo aumento nas concentrações de marcadores anti-inflamatórios (IL-6, IL-10 e MCP-1) no plasma. Nossos resultados sugerem que a suplementação oral crônica com L-glutamina (administrada com L-alanina livre ou como DIP) promoveu efeitos citoprotetores mediados pela HSP70 em resposta à lesão e inflamação induzidas pelo TR. Subprojeto 2: Efeitos da L-alanil-L-glutamina sobre as vias de sinalização da insulina e da mTOR/S6K, e citoproteção em células musculoesqueléticas C2C12. O dipeptídeo L-alanil-L-glutamina é conhecido por modular o metabolismo e a viabilidade celular. Contudo, os efeitos sobre os componentes clássicos das vias de sinalização da insulina e da mTOR/S6K, bem como o efeito citoprotetor em células musculares, são pouco esclarecidos. O objetivo deste estudo foi investigar o efeito do DIP sobre as vias de sinalização da insulina e da mTOR/S6K em miotubos C2C12, em condições normais ou resistentes à insulina. A exposição crônica à insulina (24h) promoveu resistência à insulina, reduzindo os conteúdos totais do receptor beta (IR-ß) e do substrato do receptor de insulina (IRS-1), e diminuindo a fosforilação de IRS-1, AKT e P44/42 MAPK. Adicionalmente, houve redução na expressão do transportador de glicose (GLUT4) e HSP70, redução da viabilidade celular e menor fosforilação de p70S6k e S6, proteínas relacionadas à síntese proteica. Em contraste, a suplementação com DIP aumentou os conteúdos totais de IR-ß e IRS-1 e a fosforilação de IRS-1 e AKT. A glicólise anaeróbia e a capacidade glicolítica, além da fosforilação de p70S6k e S6, foram aumentadas pelo DIP em condições normais e na resistência à insulina. Nestas condições experimentais, nossos resultados sugerem que a suplementação com DIP melhorou as vias de sinalizações da insulina e da mTOR/S6K, aumentou a captação e metabolização da glicose, independente da estimulação com insulina e, finalmente, promoveu citoproteção resgatando parcialmente as células de um estado resistente à insulina, por meio do aumento de HSP70 e ativação das etapas finais da via mTOR/S6K.

Subproject 1: Determination of the anti-inflammatory and cytoprotective effects of supplementation with L-glutamine and L-alanine, or with L-alanyl-L-glutamine in rats submitted to resistance training. Intense exercise reduces glutamine availability, compromising immune function and recovery of athletes. The objective of the study was to evaluate the effects of chronic oral supplementation with L-glutamine and L-alanine, in their free form or as dipeptide (DIP), on muscle damage, inflammation and cytoprotection in adult Wistar rats submitted to resistance training (RT). In this study, RT reduced glutamine concentration in plasma and EDL muscle. However, this effect was attenuated by supplements containing L-glutamine, which increased the contents of the stress response protein (HSP70) in immune system cells (PBMC) and EDL, concomitantly with the reduction of NF-kB activation and the concentration of cytokines in EDL. The protective effect of supplementation was also evidenced by attenuation of lesion markers (CK and LDH) and inflammation (TNF-α and IL-1ß), as well as by the increase in anti-inflammatory plasma markers (IL-6, IL-10 and MCP-1). Our results suggest that chronic oral supplementation with L-glutamine (administered along with free L-alanine or as DIP) promoted HSP70-mediated cytoprotective effects in response to RT-induced injury and inflammation. Subproject 2: Effects of L-alanyl-L-glutamine on the components of insulin and mTOR/ S6K signaling pathways and cytoprotection in C2C12 musculoskeletal cells. The dipeptide L-alanyl-L-glutamine is known to modulate metabolism and cell viability. However, the effects on the classical components of insulin and mTOR/ S6K signaling pathways, as well as the cytoprotective effect on muscle cells, are poorly understood. The aim of this study was to investigate the effect of DIP on insulin and mTOR/ S6K signaling pathways in C2C12 myotubes, under normal or insulin resistant conditions. Chronic insulin exposure (24h) promoted insulin resistance, reducing the total contents of the insulin receptor (IR-ß) and the insulin receptor substrate (IRS-1), and decreasing the phosphorylation of IRS-1, AKT and P44/ 42 MAPK. In addition, there was a reduction in the expression of glucose transporter (GLUT4) and HSP70, reduction of cell viability and defective phosphorylation of p70S6k and S6, which are related to protein synthesis. On the other hand, DIP supplementation increased the total contents of IR-ß and IRS-1 and the phosphorylation of IRS-1 and AKT. Anaerobic glycolysis and glycolytic capacity, in addition to phosphorylation of p70S6k and S6, were increased by DIP under normal conditions and in insulin resistance. In our experimental conditions, our results suggest that DIP supplementation improved the signaling pathways of insulin and mTOR/ S6K, increased glucose uptake and metabolism, independent of insulin stimulation, and finally promoted cytoprotection by partially rescuing the cells of an insulin resistant state, by increasing HSP70 and activating the final stages of the mTOR/ S6K pathway.