谷氨酰胺在肠道的代谢及其对肠粘膜的保护作用

谷氨酰胺是一种十分重要的具有特殊营养作用的条件必需氨基酸。肠道是消耗谷氨酰胺的主要器官。肠粘膜细胞既可利用从肠腔食糜中摄取的谷氨酰胺,也利用从血液中摄取的谷氨酰胺。谷氨酰胺是肠粘膜细胞的主要能源物质,提供氮质参与细胞核酸和蛋白质合成代谢,促进粘膜细胞更新再生。机体谷氨酰胺缺乏,可导致肠粘膜萎缩。在严重创伤、烧伤、感染、恶性肿瘤等病理状态下,补充外源性谷氨酰胺,可维持和恢复肠粘膜的代谢、结构和功能,防止肠道细菌和毒素移位,减轻应激代谢反应。     

腺病毒E1B-19K基因过表达对HEK293细胞生长和代谢的影响

细胞凋亡是动物细胞大规模培养中影响活细胞密度和目的产品质量的重要因素,过表达抗凋亡基因是目前常用的提高工程细胞凋亡抗性的一种策略。拟在HEK293细胞中过表达腺病毒E1B-19K基因,挑取了不同E1B-19K表达水平的单克隆细胞,考察在不同培养条件下细胞的凋亡水平和代谢情况。E1B-19K的过表达可显著增强细胞在低葡萄糖、低血清和无谷氨酰胺3种培养条件下的抗凋亡能力,使凋亡细胞比例降低60%~80%;E1B-19K的过表达可使批次培养HEK293细胞的衰退期延迟2天,而对细胞的葡萄糖、乳酸和谷氨酰胺等的代谢无显著影响。结果表明,过表达E1B-19K是一种有效减缓HEK293细胞在培养过程中凋亡的策略。     

谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响

微囊化技术是一种有发展潜力的生物技术,在细胞移植和药物控释等方面具有广泛的应用。然而由于目前微囊化细胞规模化培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。为了了解微囊化重组CHO细胞的生长代谢特性为今后规模化培养优化提供技术参考,考察了主要氮源物质谷氨酰胺对微囊化重组CHO细胞生长代谢及内皮抑素表达的影响。结果显示:当谷氨酰胺起始浓度从2.69mmolL增加到9.05mmolL时最大活细胞密度并没有增高,细胞增殖没有显著差异。当谷氨酰胺起始浓度较低(2.69mmolL)时,葡萄糖的比消耗速率较大;当谷氨酰胺起始浓度增高时(7.91mmolL～9.05mmolL)葡萄糖和谷氨酰胺的比消耗速率增大,但细胞对葡萄糖和谷氨酰胺的利用率降低。谷氨酰胺对产物表达有显著影响,起始浓度为4.97mmolL时的内皮抑素累积浓度最高,达546.36ngmL,过低和过高谷氨酰胺起始浓度下内皮抑素的累积浓度均较低。     

大鼠谷氨酰胺转运蛋白SNAT2-EGFP融合蛋白的表达与鉴定

谷氨酰胺转运蛋白是中枢神经系统中一种重要的中性氨基酸转运蛋白,对谷氨酰胺的跨膜转运十分重要。为了更方便地研究大鼠谷氨酰胺转运蛋白2(SNAT2)在细胞膜上的表达与定位,利用亚克隆技术将增强型绿色荧光蛋白(EGFP)构建于SNAT2的C端,通过菌液PCR、酶切和DNA测序鉴定重组真核表达质粒;将测序正确的重组质粒瞬时转染人胚胎肾细胞(HEK293T cells),用Western blot和激光共聚焦电子显微镜荧光检测技术鉴定SNAT2-EGFP的表达与亚细胞定位。结果表明,SNAT2-EGFP融合蛋白重组质粒在细胞中表达并正确定位于细胞膜上。SNAT2-EGFP融合蛋白重组质粒的成功构建为今后深入研究SNAT2的结构和功能提供了一个有效的工具。     

L-谷氨酰胺对PC12细胞中grp75的调控作用

条件必需氨基酸谷胺酰胺可上调细胞中热激蛋白(hsp)的表达,为观察谷氨酰胺是否对hsp 家族成员grp75的表达具有调控作用,以PC12细胞为模型用免疫组化、蛋白质印迹法和RT-PCR 等方法检测谷胺酰胺对grp75基因的表达的影响;并以MTT法观察谷氨酰胺对PC12的细胞和grp75低表达的PC12细胞缺糖损伤的保护作用。结果表明谷氨酰胺可以上调grp75的表达,特别是对缺糖细胞的上调作用更显著;但这种上调作用与谷氨酰胺的作用浓度和作用时间并未显示出有明显的关系。MTT检测显示,谷氨酰胺使细胞在缺糖条件下的存活率明显上升;grp75低表达细胞与未转染的细胞相比这种保护效应明显降低,说明谷氨酰胺通过调节grp75的表达对缺糖损伤起到保护作用     

谷氨酰胺在杂交瘤细胞培养中的降解与代谢

谷氨酰胺(Glutamine,Gln)是动物细胞培养中一种很特殊的必需氨基酸,常用培养基中其浓度在0.7～5 mmol/L之间,可作为细胞生长的主要能源和氮源,并参与合成嘌呤、嘧啶、蛋白质和多肽.但谷氨酰胺的水解又是培养体系中主要毒性副产物氨的重要来源,其水解途径有二:(1)非酶水解即化学降解,生成氨和吡咯烷酮羧酸,这是个一级反应,其反应常数和温度、酸碱度、血清浓度有关;(2)被谷氨酰胺酶所水解,生成氨和谷氨酸.     

植物转谷氨酰胺酶

植物转谷氨酰胺酶廖祥儒,朱新产,赵军（西北农业大学,陕西杨陵７１２１００）（中国科学院西北水土保持研究所）关键词转谷氨酰胺酶,蛋白质交联蛋白质交联,作为转译后蛋白质的一种修饰方式,与细胞结构形成有关,对稳定组织结构和细胞骨架有重要意义。现已发现,和Ｎ...     

L-谷氨酰胺对PC12细胞中grp75的调控作用

条件必需氨基酸谷胺酰胺可上调细胞中热激蛋白（hsp）的表达,为观察谷氨酰胺是否对hsp家族成员grp75的表达具有调控作用,以PC12细胞为模型用免疫组化、蛋白质印迹法和RT—PCR等方法检测谷胺酰胺对grp75基因的表达的影响：并以MTT法观察谷氨酰胺对PC12的细胞和grp75低表达的PC12细胞缺糖损伤的保护作用。结果表明谷氨酰胺可以上调grp75的表达．特别是对缺糖细胞的上调作用更显著;但这种上调作用与谷氨酰胺的作用浓度和作用时间并未显示出有明显的关系。MTT检测显示,谷氨酰胺使细胞在缺糖条件下的存活率明显上升：grp75低表达细胞与未转染的细胞相比这种保护效应明显降低,说明谷氨酰胺通过调节grp75的表达对缺糖损伤起到保护作用。     

L-谷氨酰胺生产及分离纯化研究进展

谷氨酰胺是一种条件性必需氨基酸,具有重要的生理作用,是一种极有开发前途的新药.本文对谷氨酰胺的生理作用、应用、生产及分离纯化工艺进行了综述.     

胰腺癌通过调控非经典谷氨酰胺代谢维持氧化还原稳态

胰腺导管腺癌是一种高度恶性的肿瘤,近年来胰腺癌的治疗并未取得突破性进展。从代谢的角度干预胰腺癌发生发展成为具有重要应用前景的治疗策略。胰腺癌细胞的增殖高度依赖于葡萄糖和谷氨酰胺。胰腺癌细胞并不依赖葡萄糖维持细胞内还原力NADPH的生成,而通过非经典谷氨酰胺代谢途径产生还原力,进而维持细胞的氧化还原稳态。蛋白精氨酸甲基转移酶可作为氧化压力的感受器,通过调控苹果酸脱氢酶的甲基化水平将非经典谷氨酰胺代谢通路和氧化还原稳态偶联起来。     

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. IEC-18; NIH/3T3; glutaminase; 6-diazo-5-oxo-L-norleucine; glutathione     

Glutamine synthesis in the developing porcine placenta

Glutamine plays a vital role in fetal carbon and nitrogen metabolism and exhibits the highest fetal:maternal plasma ratio among all amino acids in pigs. Such disparate glutamine levels between mother and fetus suggest that glutamine may be actively synthesized and released into the fetal circulation by the porcine placenta. We hypothesized that branched-chain amino acid (BCAA) metabolism in the placenta plays an important role in placental glutamine synthesis. This hypothesis was tested by studying conceptuses from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day). Placental tissue was analyzed for amino acid concentrations, BCAA transport, BCAA degradation, and glutamine synthesis as well as the activities of related enzymes (including BCAA transaminase, branched-chain alpha-ketoacid dehydrogenase, glutamine synthetase, glutamate-pyruvate transaminase, and glutaminase). On all days of gestation, rates of BCAA transamination were much greater than rates of branched-chain alpha-ketoacid decarboxylation. The glutamate generated from BCAA transamination was primarily directed to glutamine synthesis and, to a much lesser extent, alanine production. Placental BCAA transport, BCAA transamination, glutamine synthesis, and activities of related enzymes increased markedly between Days 20 and 40 of gestation, as did glutamine in fetal allantoic fluid. Accordingly, placental BCAA levels decreased after Day 20 of gestation in association with a marked increase in BCAA catabolism and concentrations of glutamine. There was no detectable catabolism of glutamine in pig placenta throughout pregnancy, which would ensure maximum output of glutamine by this tissue. These novel results demonstrate glutamine synthesis from BCAAs in pig placentae, aid in explaining the abundance of glutamine in the fetus, and provide valuable insight into the dynamic role of the placenta in fetal metabolism and nutrition.     

Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity.

The amino acid concentrations in the phototrophic bacterium Rhodospirillum rubrum were measured during growth under nif-repressing and nif-derepressing conditions. The effects of ammonium, glutamine, darkness, phenazine methosulfate, and the inhibitors methionine sulfoximine and azaserine on amino acid levels of cells were tested. The changes were compared to changes in whole-cell nitrogenase activity and ADP-ribosylation of dinitrogenase reductase. Glutamate was the dominant amino acid under every growth condition. Glutamine levels were equivalent when cells were grown on high-ammonia (nif-repressing) medium or glutamate (nif-derepressing) medium. Thus, glutamine is not the solitary agent that controls nif expression. No other amino acid correlated with nif expression. Glutamine concentrations rose sharply when either glutamate-grown or N-starved cells were treated with ammonia, glutamine, or azaserine. Glutamine levels showed little change upon treatment of the cells with darkness or ammonium plus methionine sulfoximine. Treatment with phenazine methosulfate resulted in a decrease in glutamine concentration. The glutamine concentration varied independently of dinitrogenase reductase ADP-ribosylation, and it is concluded that an increase in glutamine concentration is neither necessary nor sufficient to initiate the modification of dinitrogenase reductase. No other amino acid exhibited changes in concentration that correlated consistently with modification. Glutamine synthetase activity and nitrogenase activity were not coregulated under all conditions, and thus the two regulatory cascades perceive different signal(s) under at least some conditions.     

Enzymology of Glutamine Metabolism Related to Senescence and Seed Development in the Pea (Pisum sativum L.)

The metabolism of glutamine in the leaf and subtended fruit of the aging pea (Pisum sativum L. cv. Burpeeana) has been studied in relation to changes in the protein, chlorophyll, and free amino acid content of each organ during ontogenesis. Glutamine synthetase [EC 6.3.1.2] activity was measured during development and senescence in each organ. Glutamate synthetase [EC 2.6.1.53] activity was followed in the pod and cotyledon during development and maturation. Maximal glutamine synthetase activity and free amino acid accumulation occurred together in the young leaf. Glutamine synthetase (in vitro) in leaf extracts greatly exceeded the requirement (in vivo) for reduced N in the organ. Glutamine synthetase activity, although declining in the senescing leaf, was sufficient (in vitro) to produce glutamine from all of the N released during protein hydrolysis (in vivo). Maximal glutamine synthetase activity in the pod was recorded 6 days after the peak accumulation of the free amino acids in this organ.

In the young pod, free amino acids accumulated as glutamate synthetase activity increased. Maximal pod glutamate synthetase activity occurred simultaneously with maximal leaf glutamine synthetase activity, but 6 days prior to the corresponding maximum of glutamine synthetase in the pod. Cotyledonary glutamate synthetase activity increased during the assimilatory phase of embryo growth which coincided with the loss of protein and free amino acids from the leaf and pod; maximal activity was recorded simultaneously with maximal pod glutamine synthetase.

We suggest that the activity of glutamine synthetase in the supply organs (leaf, pod) furnishes the translocated amide necessary for the N nutrition of the cotyledon. The subsequent activity of glutamate synthetase could provide a mechanism for the transfer of imported amide N to alpha amino N subsequently used in protein synthesis. In vitro measurements of enzyme activity indicate there was sufficient catalytic potential in vivo to accomplish these proposed roles.

     

Glutamine homeostasis and mitochondrial dynamics

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.     

The relation between glutamine and the immunodepression observed in exercise

Summary. Glutamine is the most abundant amino acid in the body. It is an important fuel for some key cells of the immune system. Both the plasma concentration of glutamine and the functional ability of immune cells in the blood are decreased after prolonged, exhaustive exercise. Glutamine feeding has had beneficial effects in clinical situations, and the provision of glutamine after intensive exercise has decreased the incidence of infections, particularly of upper respiratory tract infections. However, the precise effect of glutamine on immunodepression in this situation is not yet established. Received January 2, 2000 / Accepted February 1, 2000     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

The role of glutamine in the immune system and in intestinal function in catabolic states

Summary Glutamine is designated a non-essential amino acid: however, evidence is accumulating that glutamine becomes essential when catabolic conditions prevail.It has been established that glutamine is an important fuel for lymphocytes and macrophages, even when resting. Plasma and muscle glutamine concentrations are decreased after trauma such as burns, major surgery, and in sepsis. The effectiveness of the immune system is decreased after trauma: this may be due, in part, to the decrease in plasma glutamine concentrations.Most studies on sepsis in humans have shown plasma glutamine concentrations to bedecreased: this may be due to an increased rate of utilization of glutamine by lymphocytes and macrophages during proliferation or phagocytosis. In contrast, several studies on rats showincreased plasma glutamine levels in sepsis. A species difference in the way in which glutamine is metabolised could be the main reason for the conflicting results. Other contributory factors could be diurnal variation and timing of sample collection.A substantial amount of dietary glutamine is taken up by intestinal cells. When the supply of glutamine via the diet is decreased, glutamine is taken up from the circulation by the intestine. In total parenteral nutrition (TPN) sepsis can sometimes occur because the gut is rested, leading to villous atrophy and increased gut mucosal barrier permeability. There is now a move towards the use of enteral nutrition in preference to TPN. Provision of exogenous glutamine has had beneficial effects in humans and animals, particularly in improving intestinal function. The safety and efficacy of glutamine administration to humans is discussed in detail.     

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the?highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to?produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or?a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by?glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.     

Amino acid transport in a human neuroblastoma cell line is regulated by the type I insulin-like growth factor receptor

Insulin-like growth factor I (IGF-I) and IGF-II stimulate cancer cell proliferation via interaction with the type I IGF receptor (IGF-IR). We put forward the hypothesis that IGF-IR mediates cancer cell growth by regulating amino acid transport, both when sufficient nutrients are present and when key nutrients such as glutamine are in limited supply. We examined the effects of alphaIR3, the monoclonal antibody recognizing IGF-IR, on cell growth and amino acid transport across the cell membrane in a human neuroblastoma cell line, SK-N-SH. In the presence of alphaIR3 (2 micro/ml), cell proliferation was significantly attenuated in both control (2 mM glutamine) and glutamine-deprived (0 mM glutamine) groups. Glutamine deprivation resulted in significantly increased glutamate (system X(AG)(-)), MeAIB (system A), and leucine (system L) transport, which was blocked by alphaIR3. Glutamine (system ASC) and MeAIB transport was significantly decreased by alphaIR3 in the control group. Addition of alphaIR3 significantly decreased DNA and protein biosynthesis in both groups. Glutamine deprivation increased the IGF-IR protein on the cell surface. Our results suggest that activation of IGF-IR promotes neuroblastoma cell proliferation by regulating trans-membrane amino acid transport.