An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets

Mycobacterium tuberculosis and other pathogenic mycobacteria export abundant quantities of proteins into their extracellular milieu when growing either axenically or within phagosomes of host cells. One major extracellular protein, the enzyme glutamine synthetase, is of particular interest because of its link to pathogenicity. Pathogenic mycobacteria, but not nonpathogenic mycobacteria, export large amounts of this protein. Interestingly, export of the enzyme is associated with the presence of a poly-L-glutamate/glutamine structure in the mycobacterial cell wall. In this study, we investigated the influence of glutamine synthetase inhibitors on the growth of pathogenic and nonpathogenic mycobacteria and on the poly-L-glutamate/glutamine cell wall structure. The inhibitor L-methionine-S-sulfoximine rapidly inactivated purified M. tuberculosis glutamine synthetase, which was 100-fold more sensitive to this inhibitor than a representative mammalian glutamine synthetase. Added to cultures of pathogenic mycobacteria, L-methionine- S-sulfoximine rapidly inhibited extracellular glutamine synthetase in a concentration-dependent manner but had only a minimal effect on cellular glutamine synthetase, a finding consistent with failure of the drug to cross the mycobacterial cell wall. Remarkably, the inhibitor selectively blocked the growth of pathogenic mycobacteria, all of which release glutamine synthetase extracellularly, but had no effect on nonpathogenic mycobacteria or nonmycobacterial microorganisms, none of which release glutamine synthetase extracellularly. The inhibitor was also bacteriostatic for M. tuberculosis in human mononuclear phagocytes (THP-1 cells), the pathogen's primary host cells. Paralleling and perhaps underlying its bacteriostatic effect, the inhibitor markedly reduced the amount of poly-L-glutamate/glutamine cell wall structure in M. tuberculosis. Although it is possible that glutamine synthetase inhibitors interact with additional extracellular proteins or structures, our findings support the concept that extracellular proteins of M. tuberculosis and other pathogenic mycobacteria are worthy targets for new antibiotics. Such proteins constitute readily accessible targets of these relatively impermeable organisms, which are rapidly developing resistance to conventional antibiotics.     

The glutamine story: where are we now?

PURPOSE OF REVIEW: A recent editorial proclaimed, 'Glutamine, a life saving nutrient, but why?' This review will assess if recent data support glutamine as a life-saving nutrient in critical illness, and, if so, utilize new understanding of gene-nutrient interactions to address potential mechanisms by which glutamine may be 'life-saving'. RECENT FINDINGS: Updated meta-analysis data reveal that glutamine appears to exert a beneficial effect on mortality in critical illness. The questions remaining to be answered regard in what settings and via what method of administration does this phamaconutrient show optimal benefit? It is likely that examination of molecular mechanisms by which glutamine functions will lead to an understanding of how best to utilize glutamine as a pharmacologic agent. Recent laboratory data reveal that these mechanisms include tissue protection, attenuation of inflammation, improved tissue metabolic function, and attenuation of oxidant stress. SUMMARY: Glutamine may be potentially 'life-saving' in critical illness, particularly when administered in doses greater then 0.3 g/kg/day. Present data indicate that glutamine functions as a 'stress signaling molecule' following illness/injury and thus, needs to be given as a pharmacologic agent, rather then as nutritional replacement. Presently, multicenter clinical trials utilizing glutamine as a drug, independent of nutritional needs, are indicated.     

聚乙烯表面的菌膜抑制及其表征

背景:研究表明造成高分子导管相关感染的原因是介入材料在体内停留一段时间容易引起病原菌在其表面吸附、定殖甚至形成菌膜,成为潜在的感染源.抑制菌膜的形成可显著改善高分子导管相关型感染的发生.目的:观察抗感染聚乙烯材料的抗菌效果及其对表面菌膜形成的抑制作用.设计、时间及地点:单一样本观察,于2008-06/2009-05在中科院理化所工程塑料国家工程研究中心完成,其中抗菌检测于2009-03在解放军总参谋部总医院完成.材料:有机抗菌剂为2,4,4'-三氯-2'-羟基二苯醚;无机抗菌剂为IONPURE~(R)系抗菌剂;低密聚乙烯.方法:低密度聚乙烯与有机和无机两种抗菌剂混合后先挤出造粒,然后注塑成片.主要观察指标:①用活菌平板计数法测定材料的抗菌性能.②平板超声波法及扫描电镜判断材料表面菌膜的形成情况.结果:①两种改性抗菌材料对大肠杆菌和金黄色葡萄球菌都有良好的抗菌效果,抗菌率达到99.9%.②细菌菌膜(菌膜)的形成主要包括黏附、繁殖和成熟3个阶段,添加有机抗菌剂的试样在菌膜形成前就可以显著抑制细菌在其表面生长,从而抑制菌膜形成,扫描电镜结果表明其表面未见菌膜形成.空白试样和无机抗菌试样表面均有大量细菌和细胞外基质黏附.结论:有机抗菌聚乙烯对大肠杆菌和金黄色葡萄球菌有良好的抑菌作用,可抑制细菌在材料表面形成菌膜,为防治医用导管相关感染提供了新的途径.     

Renal ammonia production from the nitrogens of glutamine in intact acidotic dogs before and after bicarbonate infusions

Renal ammonia is produced from the amide nitrogen of glutamine, approximately 33-50%. The remainder derives from the amino nitrogen of glutamine and other non amide sources, probably the amino nitrogens of other amino acids. We investigated the acute effects of acid-base perturbations on ammonia production from amide and non amide nitrogen sources to determine how they interrelate. Infusions of glutamine were given to some intact dogs to vary the renal load. Following an acute alkali challenge to dogs in metabolic acidosis, ammoniagenesis from the amide nitrogens decreased significantly when the presentation of glutamine to the kidney was normal or relatively low, but changed less or even increased when the glutamine load was relatively high. In contrast, ammonia from the non amide sources consistently decreased during acute alkalotic challenge at any glutamine load-high or low. Since decreased glutamine deamination leading to glutamate accumulation is generally associated with decreased deamidation in dogs with normal plasma glutamine concentrations, we explain the discrepancy of deamidation at high glutamine loads to an unmasking of a separate effect on the glutaminase (phosphate-dependent) pathway by the acute acid-base changes. Accordingly, our results indicate more than one influence from acute acid-base changes in vivo on renal ammonia formation, one stimulatory and other inhibitory. Nevertheless, the influence of glutamate removal predominates over the other effect on the phosphate-dependent glutaminase pathway at physiological concentrations of glutamine in the intact dog.     

Glutamine and cancer: cell biology,physiology, and clinical opportunities

Glutamine is an abundant and versatile nutrient that participates in energy formation, redox homeostasis, macromolecular synthesis, and signaling in cancer cells. These characteristics make glutamine metabolism an appealing target for new clinical strategies to detect, monitor, and treat cancer. Here we review the metabolic functions of glutamine as a super nutrient and the surprising roles of glutamine in supporting the biological hallmarks of malignancy. We also review recent efforts in imaging and therapeutics to exploit tumor cell glutamine dependence, discuss some of the challenges in this arena, and suggest a disease-focused paradigm to deploy these emerging approaches.     

The role of L-alanyl-L-glutamine in the immune response in vitro

INTRODUCTION: The amino acid glutamine plays an important role in the immune system by providing energy and precursors for biosynthetic processes. For lack of stability it could not so far be generally supplied in total parenteral nutrition. The development of dipeptides consisting of glutamine and a second amino acid offers a solution to this problem. METHODS: In vitro effects of the dipeptide L-alanyl-L-glutamine on different cells of the immune system are assessed and compared to those of glutamine on its own. RESULTS: T-lymphocyte proliferation stimulated with mitogens and alloantigens increased significantly and dose-dependently after addition of L-alanyl-L-glutamine or glutamine. Maximal effects were observed with a concentration of 2 mmol/l of either substance. The stimulatory effects were partly attributed to enhanced cytokine production following glutamine or L-alanyl-L-glutamine treatment. In contrast, the activity of natural killer and cytotoxic T-cells was not influenced by neither amino acid at concentrations of 0.2 and 2 mmol/l, and suppressed at 20 mmol/l. In all experiments, early addition of the amino acids to the cultures proved crucial. CONCLUSION: In this series of in vitro experiments the dipeptide L-alanyl-L-glutamine exerted almost identical immunostimulatory activities to glutamine alone. Its provision in parenteral nutrition appears commendable.     

Inhibition of muscle glutamine formation in hypercatabolic patients.

Glutamine is synthesized primarily in skeletal muscle, and enables transfer of nitrogen to the liver, as well as serving other functions. There is increasing evidence for beneficial clinical effects of glutamine supplementation in critically ill patients. However, the response of endogenous glutamine formation to severe stress is poorly understood. The rates of net protein balance, leucine oxidative decarboxylation, and alanine and glutamine synthesis de novo were determined in leg skeletal muscle of 20 severely burned patients and 19 normal controls in the post-absorptive state. Patients were studied at 14+/-5 days post-burn, and their mean burn size was 66+/-18% of total body surface area. Methods were based on the leg arteriovenous balance technique in combination with biopsies of the vastus lateralis muscle. In the post-absorptive state, patients with severe burns, as compared with healthy control subjects, exhibited accelerated muscle loss (+150%) (i.e. proteolysis minus synthesis) and leucine oxidative decarboxylation (+117%), and depletion of the intramuscular free glutamine pool (-63%). The average rate of glutamine synthesis de novo was decreased by 48%, whereas net alanine synthesis de novo was increased by 174%, in skeletal muscle of burned patients. In conclusion, in severely hypercatabolic burned patients, muscle glutamine formation was suppressed, whereas alanine was the major vehicle for inter-organ nitrogen transport. These changes account for a decreased glutamine availability during prolonged severe stress.     

Effect of inhibition of gluconeogenesis on ammonia production in the perfused rat kidney.

1. Gluconeogenesis from lactate or from glutamine is inhibited by 90-100% by sodium quinolinate (1 mmol/l) or 3-mercaptopicolinate (150 nmol/l) in the perfused rat kidney. L-Tryptophan is not metabolized and is without effect. 2. Lactate uptake and glucose production are inhibited to the same degree by 3-mercaptopicolinate in the kidneys of well-fed or starved rats. 3. Inhibition of gluconeogenesis from glutamine (1 mmol/l) by 3-mercaptopicolinate is accompanied by 50% inhibition of ammonia production, and 34% inhibition of glutamine uptake, in the kidneys of acidotic rats. Ammonia production from glutamine was not inhibited in kidneys from non-acidotic rats. 4. It is concluded that the increased rate of gluconeogenesis from glutamine which occurs in acidotic rats is an essential and primary event regulating all of the increase in ammonia formation.     

Increased glutamine catabolism mediates bone anabolism in response to WNT signaling

WNT signaling stimulates bone formation by increasing both the number of osteoblasts and their protein-synthesis activity. It is not clear how WNT augments the capacity of osteoblast progenitors to meet the increased energetic and synthetic needs associated with mature osteoblasts. Here, in cultured osteoblast progenitors, we determined that WNT stimulates glutamine catabolism through the tricarboxylic acid (TCA) cycle and consequently lowers intracellular glutamine levels. The WNT-induced reduction of glutamine concentration triggered a general control nonderepressible 2–mediated (GCN2-mediated) integrated stress response (ISR) that stimulated expression of genes responsible for amino acid supply, transfer RNA (tRNA) aminoacylation, and protein folding. WNT-induced glutamine catabolism and ISR were β-catenin independent, but required mammalian target of rapamycin complex 1 (mTORC1) activation. In a hyperactive WNT signaling mouse model of human osteosclerosis, inhibition of glutamine catabolism or Gcn2 deletion suppressed excessive bone formation. Together, our data indicate that glutamine is both an energy source and a protein-translation rheostat that is responsive to WNT and suggest that manipulation of the glutamine/GCN2 signaling axis may provide a valuable approach for normalizing deranged protein anabolism associated with human diseases.     

The nutritional requirements for the propagation of poliomyelitis virus by the HeLa cell

Only minimal amounts of poliomyelitis virus were formed by HeLa cells placed in a medium free from glucose and glutamine, even if the medium contained an otherwise full complement of essential and non-essential amino acids, purines, pyrimidines, NB₄⁺, and serum protein. Conversely, within one log of the optimal yield of virus was formed by HeLa cells in a medium containing only glucose, glutamine, and salts, even if the cells had been starved in this medium for 12 hours prior to their inoculation. The presence of glucose alone caused an average 170-fold increase in viral output beyond the amounts formed by the glucose- and glutamine-depleted cells. The addition of glutamine alone caused an average 2000-fold increase; and the addition of both increased the viral formation 40,000-fold. Qualitatively similar results were obtained with unstarved cells, not previously depleted of glucose and glutamine. It follows that only a small proportion of HeLa cells are capable of forming virus unless either glucose or glutamine, or both, are present in the medium. The elaboration of virus was significantly delayed in media containing glucose but no glutamine. The absence of glucose and glutamine did not prevent the fixation of poliomyelitis virus by the cell. When these compounds were added to previously depleted cells even 6 hours after inoculation, and after the excess free virus had been removed by washing and by the addition of specific antiserum, normal amounts of virus were formed despite the degenerative changes caused by the previous glucose and glutamine deprivation. Possible functions of glucose and glutamine in the elaboration of virus are discussed in the text. Such factors other than glucose, glutamine, or salts (e.g. amino acids, purines, pyrimidines, vitamins, protein, or NH₄⁺) as may be needed by HeLa cells for the propagation of poliomyelitis virus, need not be present in the medium and cannot be easily washed out of the cell. Even 12 hours' total deprivation of the cells in salt solution prior to inoculation only slightly decreased their virus-synthesizing capacity in a similarly deficient medium, provided only that adequate amounts of glucose and glutamine were retained.     

Effects of Parathyroid Hormone on Skeletal Muscle Protein and Amino Acid Metabolism in the Rat

Because prominent skeletal muscle dysfunction and muscle wasting are seen in both chronic uremia and in primary hyperparathyroidism, and because markedly elevated parathyroid hormone levels occur in both disorders, potential effects of parathyroid hormone on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism were studied in vitro using isolated intact rat epitrochlearis skeletal muscle preparations. Intact bovine parathyroid hormone and the synthetic 1-34 fragment of this hormone stimulated the release of alanine and glutamine from muscle of control but not from chronically uremic animals. This stimulation was dependent upon the concentration of parathyroid hormone added: At 10⁵ ng/ml parathyroid hormone increased alanine release 84% and glutamine release 75%. Intracellular levels of alanine and glutamine were not altered by parathyroid hormone. Increasing concentrations of the 1-34 polypeptide decreased [³H]leucine incorporation into protein of muscles from both control and uremic animals. Using muscles from animals given a pulse-chase label of [guanido-¹⁴C]arginine in vivo, parathyroid hormone increased the rate of loss of ¹⁴C label from acid-precipitable protein during incubation and correspondingly increased the rate of appearance of this label in the incubation media. Parathyroid hormone increased muscle cAMP levels by 140% and cGMP levels by 185%, but had no effect on skeletal muscle cyclic nucleotide phosphodiesterase activities as assayed in vitro. Adenylyl cyclase activity in membrane preparations from control but not uremic rats was stimulated by parathyroid hormone in a concentration-dependent fashion. However, no stimulation of guanylyl cyclase activity was noted by parathyroid hormone, although stimulation by sodium azide was present. Incubation of muscles with added parathyroid hormone produced a diminished responsiveness towards epinephrine or serotonin regulation of amino acid release and cAMP formation in the presence compared to the absence of parathyroid hormone. In the absence of parathyroid hormone, detectable inhibition of alanine and glutamine release was produced by 10⁻⁹ M epinephrine, whereas in the presence of parathyroid hormone (1,000 ng/ml) inhibition of alanine and glutamine release required 10⁻⁶ M or greater epinephrine. Resistance to cyclic AMP action as well as inhibition of cyclic AMP formation by parathyroid hormone was found. Preincubation of rat sarcolemma with 1-34 parathyroid hormone produced a decreased activity of the isoproterenol-stimulable adenylyl cyclase activity but there was no apparent change in the concentration of isoproterenol required for one-half maximal and maximal stimulation of the enzyme.     

Effects of chronic uraemia on the formation of glucose and urea plus ammonia from L-alanine, L-glutamine and L-serine in isolated rat hepatocytes

The effects of chronic uraemia on glucose production and nitrogen release (urea plus ammonia formation) from alanine, glutamine or serine in isolated rat hepatocytes were studied. Uraemia increased the rate of formation of urea plus ammonia from all three amino acids by 38-93% when they were present at a final concentration of 10 mmol/l. At lower concentrations (2 mmol/l) the rate of nitrogen release was not significantly increased. Hepatocytes from normal rats whose food intake had been restricted to the level of that of uraemic rats did not show the increased rates of nitrogen release. The increased rates of nitrogen release with hepatocytes from uraemic rats were not accompanied by increased rates of glucose synthesis. Instead, accumulation of metabolic intermediates occurred: lactate and pyruvate (alanine or serine as substrates) and glutamate (glutamine as substrate). Livers of uraemic rats had increased activities of glutaminase (30%) and serine dehydratase (100%). Hepatocytes from normal rats treated with phlorhizin to increase the plasma glucagon/insulin ratio behaved in a similar manner to hepatocytes from uraemic rats. They had increased serine dehydratase activity, and increased rates of utilization of serine or glutamine. The possible implications of these findings for human uraemia are discussed.     

A neuronal microtubule-interacting agent, NAPVSIPQ, reduces tau pathology and enhances cognitive function in a mouse model of Alzheimer's disease

Neurofibrillary tangles composed of aggregated, hyperphosphorylated tau in an abnormal conformation represent one of the major pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. However, recent data suggest that the pathogenic processes leading to cognitive impairment occur before the formation of classic tangles. In the earliest stages of tauopathy, tau detaches from microtubules and accumulates in the cytosol of the somatodendritic compartment of cells. Either as a cause or an effect, tau becomes hyperphosphorylated and aggregates into paired helical filaments that comprise the tangles. To assess whether an agent that modulates microtubule function can inhibit the pathogenic process and prevent cognitive deficits in a transgenic mouse model with AD-relevant tau pathology, we administered the neuronal tubulin-preferring agent, NAPVSIPQ (NAP). Three months of treatment with NAP at an early-to-moderate stage of tauopathy reduced the levels of hyperphosphorylated soluble and insoluble tau. A 6-month course of treatment improved cognitive function. Although nonspecific tubulin-interacting agents commonly used for cancer therapy are associated with adverse effects due to their anti-mitotic activity, no adverse effects were found after 6 months of exposure to NAP. Our results suggest that neuronal microtubule interacting agents such as NAP may be useful therapeutic agents for the treatment or prevention of tauopathies.     

Ammonia and glutamine metabolism of the intestine. The effect of lactulose and neomycin

The present work is directed to distinguish between ammonia production by the mucosa and by the intestinal flora, as well as to evaluate the influence of neomycin and lactulose. In vitro studies using rat intestine show that mucosa cells produce ammonia alanine and glutamic acid when incubated with glutamine, whose process can be impaired by neomycin or lactulose. Since the release of the above solutes is virtually the same in germ-free rats, the influence of the bacterial flora might be negligible under the experimental conditions used. Elimination of the aerobic microorganisms results in a minute decrease of ammonia concentration in portal blood in contrast to elimination of the anaerobic flora, which leads to an excessive reduction of ammonia formation. In germ-free rats colonisation with anaerobic microorganisms results in an increment in portal ammonia concentration, whose value, however, is still below levels observed in normal animals. Colonisation with aerobic bacteria has no effect on portal ammonia concentration. Neomycin and lactulose affect ammonia production in the gut by interfering with glutamine uptake in the mucosa cell, thus the influence upon ammonia formation apparently can not be exclusively explained by alterations of the intestinal flora. Possible reasons for the considerable increase in arterial glutamine levels in normal rats are discussed.     

Deficiency in peripheral glutamine production in pediatric patients with burns

Plasma glutamine levels decrease in association with severe injury, which suggests that the consumption of glutamine exceeds the production of glutamine or possibly represents a deficit in the release of glutamine from skeletal muscle. The goal of this study was to assess the peripheral glutamine kinetic response to prolonged stress in children with critical injuries. To accomplish this purpose, we quantitated peripheral glutamine kinetics in vivo with the use of 5N15 glutamine in 5 children with severe burns (total body surface area, 74%+/-14%; mean +/- SEM) and 3 children who underwent elective scar reconstruction. In the children with severe burns, leg blood flow was significantly elevated (16.2+/-2.1 vs 7.5 +/-0.3 mL/min/100 mL leg volume, P < .02) and the arterial concentration of glutamine was significantly reduced (0.31+/-0.04 vs 0.84+/-0.05 mmol/L, P < .001). The rate of glutamine turnover within the leg was significantly reduced in the patients with acute burns, whereas the net efflux of glutamine was similar between the 2 groups. These findings suggest that plasma glutamine concentrations decrease during severe stress as a result of a deficit in peripheral glutamine release in conjunction with an increased central consumption. This preliminary study supports the notion that exogenous glutamine supplementation in pediatric patients with severe injuries may be needed because of this inadequate skeletal muscle response.     

Efficacy and Future of the Aerosoltherapy in the Treatment of Cystic Fibrosis Patients Infected by Pseudomonas aeruginosa

Pseudomonas aeruginosa is considered as the most redoubtable pathogenic agent at cystic fibrosis patients. Its eradication is a priority to avoid the passage to chronic infection, the real turning point of the disease. For this, a wide therapeutic panel of intravenous antibiotics exists, and for some years, the research teams concentrate more and more on the inhaled way. The synthesis of the literature data presented herein focuses on both already experienced molecules (colistin and tobramycin), and on new therapeutics. This review aims at loosening advantages and inconveniences of each of these therapeutic options, while bringing to light the necessity of follow-up studies in order to prove the therapeutic interests of molecules in development.     

霍乱毒素检测方法的研究

霍乱毒素是霍乱弧菌分泌的一种不耐热肠毒素，是霍乱弧菌的主要致病因子。本文介绍了霍乱毒素和霍乱弧菌产毒株检测技术的研究进展。     

Skeletal muscle glutamine production in thermally injured rats

1. The effect of thermal injury (33-35% of body surface area) on the regulation of glutamine metabolism was studied in skeletal muscles of rats 7 days after injury. 2. Injury increased the rates of glutamine production in muscle, skin and adipose tissue preparations, with muscle production accounting for over 90% of total glutamine produced by the hindlimb. 3. Injury produced decreases in the concentrations of skeletal muscle glutamine (36%, P less than 0.001), glutamate (39%, P less than 0.001), alanine (24%, P less than 0.001), pyruvate (35%, P less than 0.001), 2-oxoglutarate (51%, P less than 0.001) and adenosine 5'-triphosphate (38%, P less than 0.001). The concentrations of ammonia (42%, P less than 0.001) and inosine 5'-phosphate (430%, P less than 0.001) were increased. 4. The maximal activity of glutamine synthetase was increased (22-40%, P less than 0.001) in muscles of injured rats, whereas that of glutaminase was unchanged. 5. Hindlimb blood flow decreased by approximately 15% in injured rats, which was accompanied by an enhanced net release of glutamine (80%, P less than 0.001) and alanine (44%, P less than 0.001). 6. It is concluded that there is an enhanced rate of release of both glutamine and alanine from skeletal muscle of thermally injured rats. This may be due to changes in efflux and/or increased intracellular formation of glutamine and alanine.     

Skeletal Muscle Protein and Amino Acid Metabolism in Experimental Chronic Uremia in the Rat: ACCELERATED ALANINE AND GLUTAMINE FORMATION AND RELEASE

The kinetics and factors regulating alanine and glutamine formation and release were investigated in skeletal muscle preparations from control and experimentally uremic rats. These preparations maintained phosphocreatine and ATP levels in vitro which closely approximated levels found in vivo. Alanine and glutamine release from uremic muscle were increased 45.8 and 36.0%, respectively, but tissue levels were unaltered. The increased release of alanine by uremic muscle was not accounted for by decreased rates of medium alanine reutilization via oxidation to CO₂ or incorporation into muscle protein. The maximal capacity of added amino acids such as aspartate, cysteine, leucine, and valine to stimulate net alanine and glutamine formation was the same in uremic and control muscle. Epitrochlearis preparations were partially labeled in vivo with [guanido-¹⁴C]-arginine. On incubation, preparations from uremic animals showed a 54.6% increase in the rate of loss of ¹⁴C-label in acid precipitable protein. Correspondingly, these same uremic preparations showed a 62.7% increase in ¹⁴C-label appearance in the acid-soluble fraction of muscle and in the incubation media. Insulin decreased alanine and glutamine release to an extent threefold greater in uremic than in control preparations, and increased muscle glucose uptake approximately threefold in all preparations. Although basal rates of [4,5-³H]leucine incorporation into protein were decreased 25% in uremic muscles as compared with control muscles, insulin stimulated [³H]leucine incorporation nearly equally in both preparations.     

Effects in vivo of decreased plasma and intracellular muscle glutamine concentration on whole-body and hindquarter protein kinetics in rats.

Glutamine is considered to be a 'conditionally' essential amino acid. During situations of severe stress like sepsis or after trauma there is a fall in plasma glutamine levels, enhanced glutamine turnover and intracellular muscle glutamine depletion. Under these conditions, decreased intramuscular glutamine concentration correlates with reduced rates of protein synthesis. It has therefore been hypothesized that intracellular muscle glutamine levels have a regulatory role in muscle protein turnover rates. Administration of the glutamine synthetase inhibitor methionine sulphoximine (MSO) was used to decrease glutamine levels in male Wistar rats. Immediately after the MSO treatment (t=0 h), and at t=6 h and t=12 h, rats received intraperitoneal injections (10 ml/100 g body weight) with glutamine (200 mM) to test whether this attenuated the fall in plasma and intracellular muscle glutamine. Control animals received alanine and saline after MSO treatment, while saline was also given to a group of normal rats. At t=18 h rats received a primed constant infusion of L-[2,6-3H]phenylalanine. A three-pool compartment tracer model was used to measure whole-body protein turnover and muscle protein kinetics. Administration of MSO resulted in a 40% decrease in plasma glutamine and a 60% decrease in intracellular muscle glutamine, both of which were successfully attenuated by glutamine infusions. The decreased intracellular muscle glutamine levels had no effect on whole-body protein turnover or muscle protein kinetics. Also, glutamine supplementation did not alter these parameters. Alanine supplementation increased both hindquarter protein synthesis and breakdown but the net balance of phenylalanine remained unchanged. In conclusion, our results show that decreased plasma and muscle glutamine levels have no effect on whole-body protein turnover or muscle protein kinetics. Therefore, it is unlikely that, in vivo, the intracellular muscle concentration of glutamine is a major regulating factor in muscle protein kinetics.