Increase In Glutamine Concentration Research Articles

Abstract 3972: Novel renal cell carcinoma therapy targeting glutaminolysis

Abstract Objective: Metabolic reprogramming of cancer cells contributes to tumor development and provides a target for cancer therapy ASCT2 is a glutamine transporter on the plasma membrane. Glutamine is converted into the cell to glutamate by glutaminase (GLS) and utilized by the TCA cycle. This glutaminolysis has been implicated in tumor progression in various malignancies, including renal cell carcinoma. In the present study, we investigated whether the unique strategy that effectively targets glutamine addiction would be effective as a treatment for RCC. Methods: Tumor tissue (T) and normal renal tissue (N) from surgical specimens of 66 patients with RCC were collected, and metabolomic analysis using liquid chromatography-mass spectrometry (LC/MS) to calculate the T/N ratio of Gln and Glu, respectively, were performed. In addition, immunohistological (IHC) staining of paraffin-embedded sections of 106 metastatic RCC cases was used to compare GLS and ASCT2 expression in tumor and normal tissue. In vitro, cell viability of the human RCC cell lines 786-O and 769-P was assessed by WST-8 assays. The protein expression was detected by Western blotting. Intracellular concentrations of glutamine and glutamate were analyzed using LC/MS. V9302 was used as the specific ASCT2 inhibitor, and CB839 as the specific GLS inhibitor. As the xenograft model in vivo, athymic nude mice were injected subcutaneously with 786-O cells and given various doses of V9302 and CB839 intraperitoneally. Results: LC/MS assays using surgical specimens showed a notable increase in glutamine concentration in tumor tissue. IHC assays confirmed high expression of ASCT2 in tumor tissue, and patients with high expression of ASCT2 had a poorer prognosis than those with low expression. Treatment of V9302 with 786-O and 769-P resulted in a limited decrease in cell viability. LC/MS analysis confirmed that V9302 treatment reduced glutathione metabolites. V9302 administration was shown to increase intracellular glutamine levels, presumably by compensatory mechanisms, whereas the combination of V9302 and CB839 significantly decreased glutamine levels in 786-O cells. Western blot analysis showed that the expression levels of ASCT2 and GLS1 decreased after the combination therapy. The xenograft study confirmed that the combination of V9302 and CB839 significantly inhibited 786-O tumor growth compared with controls within three weeks after treatment (p <0.001). Conclusion: It has been shown that ASCT2 expression is a marker of poor prognosis in RCC and that suppression of ASCT2 has an antitumor effect by affecting glutathione metabolism. On the other hand, the administration of V9302 raised the issue that a compensatory mechanism increases intracellular glutamine levels. Our results suggest that a novel combination strategy using CB839 in addition to V9302 could overcome the tolerance for the monotherapy and effectively target glutamine addiction in RCC. Citation Format: Yoshinari Muto, Akihito Takeuchi, Kenji Zennami, Eiji Sugihara, Ryoichi Shiroki, Hideyuki Saya, Makoto Sumitomo. Novel renal cell carcinoma therapy targeting glutaminolysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3972.

Glutamine Imaging: A New Avenue for Glioma Management.

The glutamine pathway is emerging as an important marker of cancer prognosis and a target for new treatments. In gliomas, the most common type of brain tumors, metabolic reprogramming leads to abnormal consumption of glutamine as an energy source, and increased glutamine concentrations are associated with treatment resistance and proliferation. A key challenge in the development of glutamine-based biomarkers and therapies is the limited number of in vivo tools to noninvasively assess local glutamine metabolism and monitor its changes. In this review, we describe the importance of glutamine metabolism in gliomas and review the current landscape of translational and emerging imaging techniques to measure glutamine in the brain. These techniques include MRS, PET, SPECT, and preclinical methods such as fluorescence and mass spectrometry imaging. Finally, we discuss the roadblocks that must be overcome before incorporating glutamine into a personalized approach for glioma management.

Longitudinal osmotic and neurometabolic changes in young rats with chronic cholestatic liver disease

Type C hepatic encephalopathy (type C HE) is increasingly suspected in children with chronic liver disease (CLD), and believed to underlie long-term neurocognitive difficulties. The molecular underpinnings of type C HE in both adults and children are incompletely understood. In the present study we combined the experimental advantages of in vivo high field 1H magnetic resonance spectroscopy with immunohistochemistry to follow longitudinally over 8 weeks the neurometabolic changes in the hippocampus of animals having undergone bile duct ligation as pups. Rats who develop CLD early in life displayed pronounced neurometabolic changes in the hippocampus characterized by a progressive increase in glutamine concentration which correlated with plasma ammonia levels and a rapid decrease in brain myo-inositol. Other neurometabolic findings included a decrease in other organic osmolytes (taurine, choline-containing compounds and creatine), ascorbate and glutamate. At the cellular level, we observed an increase in glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4) expression in the hippocampus at 4 weeks post bile duct ligation (BDL), together with astrocytic morphological alterations. These findings differ from observations in the brain of adult rats following BDL, and are in keeping with the commonly accepted theory of age-dependent vulnerability.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp (Marsupenaeus japonicus) acclimated at different salinities.

Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism.

Acute effects of phenylbutyrate on glutamine, branched-chain amino acid and protein metabolism in skeletal muscles of rats.

Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed invivo and invitro experiments to examine the effects of PB on glutamine (GLN), branched-chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5mM). L-[1-14 C] leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched-chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate-aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH4+ and K+ in their effects on the Na+,K+-ATPase and the Na+,K+, 2 Cl- and water transporter NKCC1. Stimulation of the Na+,K+-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na+,K+-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.

Apical stem culturing to enhance cell sap assimilates towards grain sucrose and glutamine metabolism in wheat

Apical stem culturing offers an alternative approach of manipulating cell sap for wheat grain carbon and nitrogen metabolism in a near in vivo conditions. Employing this technique, role of sucrose and glutamine in transport stream on sucrose metabolism, ammonia assimilation and aminotransferase activities were assessed towards starch and protein accumulation in two wheat genotypes PBW 343 (low yield) and PBW 621 (high yield). At mid-milky stage, detached tillers were cultured in complete liquid medium containing varied concentration of glutamine and sucrose for seven days during year 2012–13 and 2013–14. Increasing glutamine concentration from 17 to 25 mM in the culture medium having 117 mM sucrose enhanced activities of nitrate reductase, glutamate synthase, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) at 4 days after culturing (DAC) in correspondence with an increase in soluble protein content. However, at 7 DAC content of soluble protein decreased whereas starch accumulation increased showing, thereby a compensatory effect on carbon and nitrogen metabolism. Apparently, activities of sucrose synthase, soluble acid and neutral invertase significantly decreased. Increasing sucrose concentration from 117 to 125mMled to an increase in transformation of sugars to starch in grain but protein content decreased. PBW621 showed high protein content due to higher activities of GOT, GPT at 4 DAC which subsequently increased carbon skeleton of proteins towards starch synthesis at 7 DAC. Grain filling processes in terms of soluble sugars/starch were strongly correlated to invertase activities whereas proteins to aminotransferases.

Normalizing Glutamine Concentration Causes Mitochondrial Uncoupling in an In Vitro Model of Human Skeletal Muscle

Glutamine has been considered essential for rapidly dividing cells, but its effect on mitochondrial function is unknown. Human myoblasts were isolated from skeletal muscle biopsy samples (n = 9) and exposed for 20 days to 6 different glutamine concentrations (0, 100, 200, 300, 500, and 5000 µM). Cells were trypsinized and manually counted every 5 days. Seven days before the end of exposure, half of these cells were allowed to differentiate to myotubes. Afterward, energy metabolism in both myotubes and myoblasts was assessed by extracellular flux analysis (Seahorse Biosciences, Billerica, MA). The protocol for myoblasts was optimized in preliminary experiments. To account for different mitochondrial density or cell count, data were normalized to citrate synthase activity. Fastest myoblast proliferation was observed at 300 µM glutamine, with a significant reduction at 0 and 100 µM. Glutamine did not influence basal oxygen consumption, anaerobic glycolysis or respiratory chain capacity. Glutamine significantly (P = .015) influenced the leak through the inner mitochondrial membrane. Efficiency of respiratory chain was highest at 200-300 µM glutamine (~90% of oxygen used for adenosine triphosphate synthesis). Increased glutamine concentration to 500 or 5000 µM caused mitochondrial uncoupling in myoblasts and myotubes, decreasing the efficiency of the respiratory chain to ~70%. Glutamine concentrations, consistent with moderate clinical hypoglutaminemia (300 µM), bring about an optimal condition of myoblast proliferation and for efficiency of aerobic phosphorylation in an in vitro model of human skeletal muscle. These data support the hypothesis of hypoglutaminemia as an adaptive phenomenon in conditions leading to bioenergetic failure (eg, critical illness).

Single- and multiple-dose pharmacokinetics of arginase inhibitor Nω-hydroxy-nor-L-arginine, and its effect on plasma amino acids concentrations in Wistar rats

Arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) augments synthesis of nitric oxide (NO) exerting therapeutic effects in rodent models for cardiovascular and airway diseases. This study examined single- and multiple-dose pharmacokinetics and effects of nor-NOHA on plasma amino acids in Wistar rats. Animals were administered 30 mg/kg nor-NOHA in a single bolus intravenous (i.v.) or intraperitoneal (i.p.) injection, or five once-daily i.p. injections at the same dose, or vehicle. Nor-NOHA and amino acids were assayed in blood plasma by high-performance liquid chromatography. After a bolus i.v. injection, the elimination of nor-NOHA was rapid (the mean residence time was 12.5 min). The area under the concentration-time curve and maximum concentration were higher by 17% and 31%, respectively, after the fifth as compared to the first i.p. injection. A shift in arginine utilization towards the synthesis of NO was indicated by elevated citrulline-to-ornithine and citrulline-to-arginine ratios. No changes in plasma arginine were observed. Increased glutamine concentrations might indicate an alternative detoxification pathway for ammonia due to inhibition of hepatic arginase. In conclusion, pharmacokinetic data of the present study can guide rational dosing of nor-NOHA in future studies. Limitations of the strategy of NO modulation via arginase inhibition should be further explored.

Altered Glutamatergic Metabolism Associated with Punctate White Matter Lesions in Preterm Infants

Preterm infants (∼10% of all births) are at high-risk for long-term neurodevelopmental disabilities, most often resulting from white matter injury sustained during the neonatal period. Glutamate excitotoxicity is hypothesized to be a key mechanism in the pathogenesis of white matter injury; however, there has been no in vivo demonstration of glutamate excitotoxicity in preterm infants. Using magnetic resonance spectroscopy (MRS), we tested the hypothesis that glutamate and glutamine, i.e., markers of glutamatergic metabolism, are altered in association with punctate white matter lesions and “diffuse excessive high signal intensity” (DEHSI), the predominant patterns of preterm white matter injury. We reviewed all clinically-indicated MRS studies conducted on preterm infants at a single institution during a six-year period and determined the absolute concentration of glutamate, glutamine, and four other key metabolites in the parietal white matter in 108 of those infants after two investigators independently evaluated the studies for punctate white matter lesions and DEHSI. Punctate white matter lesions were associated with a 29% increase in glutamine concentration (p = 0.002). In contrast, there were no differences in glutamatergic metabolism in association with DEHSI. Severe DEHSI, however, was associated with increased lactate concentration (p = 0.001), a marker of tissue acidosis. Findings from this study support glutamate excitotoxicity in the pathogenesis of punctate white matter lesions, but not necessarily in DEHSI, and suggest that MRS provides a useful biomarker for determining the pathogenesis of white matter injury in preterm infants during a period when neuroprotective agents may be especially effective.

Regulation of amino acid metabolism as a defensive strategy in the brain of three freshwater teleosts in response to high environmental ammonia exposure

Many teleosts have evolved mechanisms to cope with ammonia toxicity in the brain when confronted with high environmental ammonia (HEA). In the present study, the possible role of conversion of accumulated ammonia to glutamine and other free amino acids in the brain of three freshwater teleosts differing in their sensitivities to ammonia was investigated. The detoxification mode of ammonia in brain is suggested to be through amination of glutamate to glutamine by the coupled activities of glutamate dehydrogenase (GDH), transaminase (aspartate aminotransaminase ‘AST’ and alanine aminotransaminase ‘ALT’) and glutamine synthetase (GSase). We investigated the metabolic response of amino acids in the brain of highly sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less sensitive cyprinid Cyprinus carpio (common carp) and the highly resistant cyprinid Carassius auratus (goldfish) when exposed to 1mM ammonia (as NH4HCO3; pH 7.9) for 0h (control), 3h, 12h, 24h, 48h, 84h and 180h. Results show that HEA exposure increased ammonia accumulation significantly in the brain of all the three species from 12h onwards. Unlike in trout, ammonia accumulation in carp and goldfish was restored to control levels (48–84h); which was accompanied with a significant increase in glutamine content as well as GSase activity. In trout, glutamine levels also increased (84–180h) but GSase was not activated. The elevated glutamine level in trout was accompanied by a significant depletion of the glutamate pool in contrast to the stable glutamate levels seen in carp and goldfish. This suggests a simultaneous increase in the rate of glutamate formation to match with the demand of glutamine formation in cyprinids. The activity of GDH was elevated significantly in carp and goldfish but remained unaltered in trout. Also, the transaminase enzymes (AST and ALT) were elevated significantly in exposed carp and goldfish while only ALT was up-regulated in trout. Consequently, in carp and goldfish both aspartate and alanine were utilized under HEA, whereas only alanine was consumed in trout. With ammonia treatment, significant changes in concentrations of other amino acids also occurred. None of the species could detoxify brain ammonia into urea. This study suggests that protective strategies to combat ammonia toxicity in brain are more pronounced in carp and goldfish than in trout.

Expression of glutamine synthetase in Tegillarca granosa (Bivalvia, Arcidae) hemocytes stimulated by Vibrio parahaemolyticus and lipopolysaccharides

The blood cockle, Tegillarca granosa, is a widely consumed clam in the Indo-Pacific region. Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. We identified the GS of T. granosa (Tg-GS) from hemocytes by 3'- and 5'-rapid amplification of cDNA ends (RACE)-PCR. The full-length cDNA consisted of 1762 bp, with a 1104-bp open reading frame encoding 367 amino acids. Sequence comparison showed that Tg-GS has homology to GS of other organisms, with 79.78% identity with GS from the Pacific oyster Crassostrea gigas, 71.98% identity with GS from the zebrafish Danio rerio, and 68.96% identity with human Homo sapiens GS. A C-beta-Grasp domain and an N-catalytic domain were identified in Tg-GS, indicating that Tg-GS should be classified as a new member of the GS family. A quantitative RT-PCR assay was used to detect mRNA expression of Tg-GS in five different tissues. Higher levels of mRNA expression of GS were detected in the tissues of hemocytes and the mantle. Up-regulation of GS by challenge with the bacteria Vibrio parahaemolyticus and with bacterial wall lipopolysaccharides showed that GS plays a role in anti-bacterial immunity. We conclude that pathogen infection significantly induces expression level of Tg- GS, and that activation of GS influences the immune response of T. granosa by increasing glutamine concentration.

Influence of different ammonium, lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10−4, 6.7 × 10−4 and 13.4 × 10−4 M). The liquid medium with 0.1 mg l−1 2,4-d and 6.7 × 10−4 M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10−4 to 6.7 × 10−4 M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10−4 to 13.4 × 10−4 M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l−1 2,4-d and 6.7 × 10−4 M glutamine (118 and 91 mg of proteins g−1 DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g−1 DW for “Jihel” cultivar and 182 mg of sugars g−1 DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l−1 BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l−1, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.

Simultaneous Infusion of Glutamine and Branched‐Chain Amino Acids (BCAA) to Septic Rats Does Not Have More Favorable Effect on Protein Synthesis in Muscle, Liver, and Small Intestine Than Separate Infusions

Glutamine and branched-chain amino acids (BCAA; valine, leucine, and isoleucine) are used as nutrition supplements in the treatment of proteocatabolic illness. We hypothesized that simultaneous administration of BCAA and glutamine affects protein metabolism more significantly than separate administration. In the present study, we evaluated their effect on protein synthesis in skeletal muscle, liver, and jejunum of septic rats. Twenty-four hours after induction of sepsis by subcutaneous injection of turpentine, the rats were infused for 6 hours with 5 mL of 1.75% glutamine, 1.75% BCAA, 1.75% glutamine+BCAA, or saline solution. The control group consisted of intact rats infused with saline. Protein synthesis was measured at the end of infusion by a "flooding method" with [3,4,5-(3)H]phenylalanine. In turpentine-treated animals, we observed a decrease in glutamine concentration in blood plasma and skeletal muscle, a decrease in BCAA concentration in liver and jejunum, and a decrease in protein synthesis in all tissues. Glutamine or glutamine+BCAA infusion increased glutamine concentration in plasma and muscle and stimulated protein synthesis in the liver. The BCAA infusion enhanced concentrations of BCAA in plasma and tissues, but the effect of BCAA on protein synthesis was insignificant. Synergistic effect of simultaneous infusion of glutamine and BCAA on protein synthesis was not observed. We conclude that glutamine infusion to rats with septic injury may significantly improve impaired protein synthesis in the liver and that there is no synergistic effect of glutamine and BCAA infusion on protein synthesis in skeletal muscle, liver, and jejunum.

Use of unusual storage temperatures to improve the amino acid profile of potatoes for novel flavoring applications

To investigate the potential of potato culls as a source of novel flavorings, free amino acid profiles of juice from tubers stored at 0°C, 12°C or 24°C were analysed by gas chromatography/mass spectrometry after separation on cation exchange resin and derivatization with N-methyl- t-butyl-dimethyl-silyl-trifluoroacetamide. Juices were also incubated for 42 h with or without a commercial protease for comparison. Storage at all three temperatures increased glutamine concentration. The relative percentages of asparagine and glutamine decreased while several other amino acids increased in the juice of potatoes stored at 0°C and 24°C. Small shifts in free amino acid profile, increasing alanine, valine, aspartic acid, asparagine and arginine levels, were obtained by incubating juice from potatoes stored at 0°C without added protease while the protease produced substantial increases but with emphasis on leucine and isoleucine and phenylalanine, which increased bitterness. Since storage alone increased the concentration of single amino acids such as alanine, methionine and phenylalanine by several fold, it may be the preferred means of improving potato culls for novel flavor applications where bitterness must be minimized.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Amino acid ratios in plasma and tissues in a rat model of liver cirrhosis before and after exercise

In human liver cirrhosis (LC), the Fischer ratio (branched-chain amino acid (BCAA)/aromatic amino acid (AAA)) is used as a marker of hepatic encephalopathy. Similarly, the plasma BCAA levels decrease while AAA levels increase in LC rats. However the BCAA and AAA ratio and the influence of exercise on this ratio remains to be clarified in various tissues of the LC rat model. Normal and LC rats received 2-weekly injections of olive oil or CCl 4 for 10 weeks, respectively, and half of the rats from each group were subjected to one time exercise on a treadmill. BCAA, AAA, glutamate (GLU) and glutamine (GLN) concentrations were measured in plasma, liver, brain, heart and skeletal muscles. Decreased BCAA and increased AAA concentrations in brain and heart were also observed in LC rats and these changes were significantly amplified by exercise. The Fischer ratio in the LC group with and without exercise was decreased in skeletal muscles, while it remained unchanged in the liver. Decreased GLU and increased GLN concentrations were shown in most LC samples following exercise. In conclusion, specific alterations of the amino acid patterns were observed in various tissues in the LC group. These alterations were further emphasized by exercise.

Cytokine-stimulated nitric oxide production and inducible NO-synthase mRNA level in human intestinal cells: lack of modulation by glutamine

Background & aims: Excess NO production has been reported during intestinal inflammation. Modulation of the inflammatory response with nutrients in critically ill patients has gained increasing interest. Glutamine has beneficial effects on gut mucosa but its effects on human intestinal NO production during an inflammatory response are not known. Methods: Caco-2/TC7 and HCT-8 cells were stimulated with a cytokine mixture (IL-1β, TNFα, IFNγ) and duodenal biopsies from human healthy volunteers in organ culture were stimulated with IL-1β. All cultures were performed in the presence of 2–10 mmol/l glutamine. NO release in culture supernatant and iNOS mRNA level in cultured cells or biopsies were assessed by nitrate reduction and Griess assay and RT-PCR, respectively. Results : In Caco-2, HCT-8 cells and duodenal biopsies, cytokine stimulation increased iNOS mRNA level 1.2-fold (ns), 3.8-fold ( P=0.02), 4.7-fold ( P=0.03) and NO production 1.4-fold (ns), 9.1 ( P=0.01) and 1.7-fold ( P=0.01), respectively. Increasing glutamine concentration had no significant effect on NO production and iNOS mRNA in any type of culture, stimulated or not by cytokines. In various models of human intestinal cells, glutamine does not further increase NO production induced by pro-inflammatory cytokines.