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Glutamine Metabolism (glutamine + metabolism)

Distribution by Scientific Domains

Life Sciences	80%

Selected Abstracts

Improving Glucose and Glutamine Metabolism of Human HEK 293 and Trichoplusiani Insect Cells Engineered To Express a Cytosolic Pyruvate Carboxylase Enzyme

BIOTECHNOLOGY PROGRESS, Issue 1 2003
Cynthia B. Elias
Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusiani High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed. [source]

Glutamine metabolism: Role in acid-base balance,

BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2004
Lynn Taylor
Abstract The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization. The periportal hepatocytes catabolize glutamine and convert ammonium and bicarbonate ions to urea. In contrast, the perivenous hepatocytes are capable of synthesizing glutamine. During metabolic acidosis, the kidney becomes the major site of glutamine extraction and catabolism. This process generates ammonium ions that are excreted in the urine to facilitate the excretion of acids and bicarbonate ions that are transported to the blood to partially compensate the acidosis. The increased renal extraction of glutamine is balanced by an increased release from muscle and liver and by a decreased utilization in the intestine. During chronic acidosis, this adaptation is sustained, in part, by increased renal expression of genes that encode various transport proteins and key enzymes of glutamine metabolism. The increased levels of phosphoenolpyruvate carboxykinase result from increased transcription, while the increase in glutaminase and glutamate dehydrogenase activities result from stabilization of their respective mRNAs. Where feasible, this review draws upon data obtained from studies in humans. Studies conducted in model animals are discussed where available data from humans is either lacking or not firmly established. Because there are quantitative differences in tissue utilization and synthesis of glutamine in different mammals, the review will focus more on common principles than on quantification. [source]

Inhibition of glutamine transport into mitochondria protects astrocytes from ammonia toxicity

GLIA, Issue 8 2007
V. B. R. Pichili
Abstract Hepatic encephalopathy (HE) is a major neurological complication that occurs in the setting of severe liver failure. Ammonia is a key neurotoxin implicated in this condition, and astrocytes are the principal neural cells histopathologically and functionally affected. Although the mechanism by which ammonia causes astrocyte dysfunction is incompletely understood, glutamine, a by-product of ammonia metabolism, has been strongly implicated in many of the deleterious effects of ammonia on astrocytes. Inhibiting mitochondrial glutamine hydrolysis in astrocytes mitigates many of the toxic effects of ammonia, suggesting the involvement of mitochondrial glutamine metabolism in the mechanism of ammonia neurotoxicity. To determine whether mitochondriaare indeed the organelle where glutamine exerts its toxic effects, we examined the effect of L -histidine, an inhibitor of mitochondrial glutamine transport, on ammonia-mediated astrocyte defects. Treatment of cultured astrocytes with L -histidine completely blocked or significantly attenuated ammonia-induced reactive oxygen species production, cell swelling, mitochondrial permeability transition, and loss of ATP. These findings implicate mitochondrial glutamine transport in the mechanism of ammonia neurotoxicity. © 2007 Wiley-Liss, Inc. [source]

Intestinal and hepatic metabolism of glutamine and citrulline in humans

THE JOURNAL OF PHYSIOLOGY, Issue 2 2007
Marcel C. G. Van De Poll
Glutamine plays an important role in nitrogen homeostasis and intestinal substrate supply. It has been suggested that glutamine is a precursor for arginine through an intestinal,renal pathway involving inter-organ transport of citrulline. The importance of intestinal glutamine metabolism for endogenous arginine synthesis in humans, however, has remained unaddressed. The aim of this study was to investigate the intestinal conversion of glutamine to citrulline and the effect of the liver on splanchnic citrulline metabolism in humans. Eight patients undergoing upper gastrointestinal surgery received a primed continuous intravenous infusion of [2- 15N]glutamine and [ureido- 13C,2H2]citrulline. Arterial, portal venous and hepatic venous blood were sampled and portal and hepatic blood flows were measured. Organ specific amino acid uptake (disposal), production and net balance, as well as whole body rates of plasma appearance were calculated according to established methods. The intestines consumed glutamine at a rate that was dependent on glutamine supply. Approximately 13% of glutamine taken up by the intestines was converted to citrulline. Quantitatively glutamine was the only important precursor for intestinal citrulline release. Both glutamine and citrulline were consumed and produced by the liver, but net hepatic flux of both amino acids was not significantly different from zero. Plasma glutamine was the precursor of 80% of plasma citrulline and plasma citrulline in turn was the precursor of 10% of plasma arginine. In conclusion, glutamine is an important precursor for the synthesis of arginine after intestinal conversion to citrulline in humans. [source]

Glutamine metabolism: Role in acid-base balance,

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate-based assays

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010
R. Janke
Abstract Sensitive microplate-based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4,nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2,pmol and a limit of quantitation of 24.8,pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary-phase MDCK cells grown in serum-containing GMEM medium as typically used in influenza vaccine production. Biotechnol. Bioeng. 2010;107: 566,581. © 2010 Wiley Periodicals, Inc. [source]

Effects of glucose and insulin on HepG2-C3A cell metabolism

BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010
Vidya V. Iyer
Abstract HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2-C3A cells were cultured under varying levels of glucose (high, low, and glucose-free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose-free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose-free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2-C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose-free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose-free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2-C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2-C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347,356. © 2010 Wiley Periodicals, Inc. [source]

Metabolism of PER.C6TM cells cultivated under fed-batch conditions at low glucose and glutamine levels

BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006
Luis Maranga
Abstract This is the first study to examine PER.C6TM cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6TM cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced qGln and q by approximately 30%, qAla 85%, and qNEAA 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced qGlc about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y of approximately 6 mol/mol. However, despite no increase in qGln, an increased ammonium ion accumulation and Y were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6TM cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The qGln value was again reduced and, despite an increased q compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6TM cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6TM cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism. © 2006 Wiley Periodicals, Inc. [source]

Effect of dexamethasone on neutrophil metabolism

CELL BIOCHEMISTRY AND FUNCTION, Issue 2 2003
Carolina Garcia
Abstract The effect of dexamethasone on glucose and glutamine metabolism was investigated. The consumption and oxidation of glucose and glutamine, and the production of glutamate and lactate were determined in neutrophils cultured for 3,h in the presence of dexamethasone. The activities and expression of glucose-6-phosphate dehydrogenase (G6PDH) and phosphate-dependent glutaminase were also determined under the same conditions. Addition of dexamethasone to the culture medium caused a significant increase of glucose consumption at 0.5,,m (123.9%) and 1.0,,m (78.3%) concentrations. In spite of this, however, glucose oxidation remained unchanged. The glucocorticoid did not change glutamine consumption but caused a significant increase of glutamate production and did not alter glutamine oxidation. Dexamethasone-treated neutrophils had a significant decrease of G6PDH activity and expression in particular at 1.0,,m concentration. Phosphate- dependent glutaminase activity was also decreased (about 34%) by dexamethasone treatment. A similar effect was observed on glutaminase expression as indicated by RT-PCR analysis. Thus, the effect of dexamethasone on neutrophil metabolism was particularly noticeable with respect to G6PDH and glutaminase activities where a decrease in the respective mRNA levels was demonstrated. Copyright © 2002 John Wiley & Sons, Ltd. [source]

Evidence that glutamine is involved in neutrophil function

CELL BIOCHEMISTRY AND FUNCTION, Issue 2 2002
Tania C. Pithon-Curi
Abstract Phosphate-dependent glutaminase (PDG) activity, a key enzyme of glutamine metabolism, was determined in neutrophils obtained from the intra-peritoneal cavity (PC) or bronchoalveolar space (BAS) after administration of 1,ml or 100,,l, respectively of saline, glycogen solution (1%) or lipopolysaccharide (LPS 0.1,mg (100,,l),1). Neutrophils were obtained by lavage of both sites with 20,ml saline 24,h after the administration of the stimuli. Glycogen and LPS, depending on the site the cells were obtained from, differently modulated PDG activity. Cells from BAS stimulated by glycogen or LPS had raised PDG activity to 30.5,±,5.2 and 42.7,±,12.1,nmol,min,1,mg,1 protein, respectively, when compared with saline (9.1,±,0.9,nmol,min,1,mg,1 protein); mean,±,SEM. On the other hand, cells from PC showed different PDG activity: 52.0,±,12.6,nmol,min,1,mg,1 for saline, 36.5,±,9.5,nmol,min,1,mg,1 for glycogen, and 76.6,±,11.2,nmol,min,1,mg,1 for LPS; mean,±,SEM. Therefore, PDG activity varies with the site from which neutrophils are obtained and the stimulus imposed. The effect of glutamine on nitric oxide (NO) and tumour necrosis factor (TNF) production by peritoneal neutrophils, obtained after glycogen administration, cultured in the presence of LPS (0.5,,g,ml,1) was also examined. The addition of glutamine at concentrations varying from 2 to 20,mM did not markedly affect NO production. Glutamine alone at 2,mM did not modify the production of TNF but in the presence of LPS caused a significant decrease. So, glutamine may preserve the function of neutrophils during infections and injuries. Copyright © 2001 John Wiley & Sons, Ltd. [source]