柠檬酸对 CHO 细胞生长和代谢的影响

研究了重组中国仓鼠卵巢细胞(CHO)批培养过程中柠檬酸对细胞生长和代谢的影响。结果表明:柠檬酸明显抑制了细胞的生长。与对照组相比,添加12 mm o l/L柠檬酸的处理组细胞的葡萄糖比消耗速率(QG lc)降低了37.5%,渗透压提高了10.0%,乳酸生成量与葡萄糖消耗量的比值增加了27.0%,氨生成量与谷氨酰胺消耗量的比值也增加了。在谷氨酰胺代谢过程中,更多的谷氨酰胺经谷草转氨酶途径生成α-酮戊二酸,参与能量代谢。柠檬酸促使细胞更多地被捕获在G 1期,阻碍细胞的DNA合成,抑制细胞增殖,并促进蛋白的表达。     

Reductive carboxylation supports growth in tumour cells with defective mitochondria

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.     

Functional genomics reveal that the serine synthesis pathway is essential in breast cancer

Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of α-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.     

Deregulated MYC expression induces dependence upon AMPK-related kinase 5

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.     

Progress in the research of GSH in cells

Glutathione (GSH), ??-Glu-Cys-Gly, is one of the most abundant small non-protein thiol molecules in mammalian tissues, particularly in the liver. Although glutathione is present in thiol-reduced (GSH) and disulfide oxidized (GSSG) forms, the predominant form is GSH and its content can exceed 10 mmol/L in liver cells. As an important intracellular reductant, GSH has many biological functions in cells. Its major function is as an anti-oxidant as it can protect proteins from oxidation by reversible posttranslational modification (glutathionylation) and decrease reactive oxygen species-mediated damage. However, it does have numerous other functions, including to chelate metal irons; enhance the absorption of iron, selenium and calcium; participate in lipid and insulin metabolism; regulate cellular events such as gene expression, DNA and protein synthesis, cell proliferation and apoptosis, redox-dependent signal transduction pathways, cytokine production and the immune response; and control protein glutathionylation. Therefore, GSH plays important roles in cell survival and health, and an imbalance in the GSH level can lead to many diseases. In this review, we provide an overview of the function of GSH in mammalian cells and discuss future research of GSH.     

大鼠再生肝8种细胞的葡萄糖代谢基因转录谱预示的代谢活动

为了解大鼠肝再生中8种肝脏细胞的葡萄糖代谢基因转录谱及其预示的葡萄糖代谢活动,用percoll密度梯度离心结合免疫磁珠分选方法分离大鼠再生肝的8种细胞,用RatGenome2302.0芯片等检测上述细胞的葡萄糖代谢基因在大鼠肝再生中的表达变化,用H-Cluster软件分析基因表达模式,用生物信息学和系统生物学等方法分析基因表达变化预示的生理活动.结果表明,48个葡萄糖代谢基因在大鼠肝再生中发生了有意义的表达变化,其中上调、下调和上/下调的基因数分别为20、14、14,上述细胞的相应基因数为14、8和0,11、6和0,6、4和0,7、11和0,11、6和2,6、5和1,12、5和0,9、9和1,呈现21种表达相关性.上述葡萄糖代谢基因转录谱预示,肝细胞和库普弗细胞的3-磷酸甘油醛合成增多,肝细胞和胆管上皮细胞的烯醇式丙酮酸合成增多,肝星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞和树突状细胞的通过三羧酸循环产生ATP活动增强.结论:大鼠肝再生与葡萄糖代谢密切相关.     

1,6-二磷酸果糖对缺氧缺血性心、脑损害的保护作用(综述)

1，6-二磷酸果糖(FDP)是存在于一切活细胞内的糖代谢中间产物。细胞缺氧时，外源性FDP可透过细胞膜进入到细胞内直接供能，并可阻止Ca^2 内流，减少氧自由基的产生、稳定细胞膜，及减少细胞凋亡。多数研究认为FDP对缺氧缺血引起的心、脑损伤具有保护作用，但也有不同的观点。因此有必要进一步临床验证，为FDP在这方面的应用提供有价值的理论依据。     

An oxygen-regulated switch in the protein synthesis machinery

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m(7)-GpppG) 5'?cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α-RBM4-eIF4E2 complex captures the 5'?cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.     

谷氨酰胺对杂交瘤细胞生长代谢的影响

研究了不同浓度的谷氨酰胺对分泌抗小细胞肺癌单克隆抗体的2F7杂交瘤细胞生长、葡萄糖消耗、乳酸及氨的生成等代谢过程的影响。在细胞培养箱中,用含不同浓度谷氨酰胺的培养液培养2F7杂交瘤细胞,培养条件为pH 7.2～7.4,温度37℃,5%CO_2、95%空气。实验表明,适宜于2F7杂交瘤细胞生长的谷氨酰胺浓度为2.5 mmol/L,谷氨酰胺浓度与细胞的葡萄糖消耗呈反比,与乳酸生成浓度呈反比,与氨的生成浓度呈正比。     

生物反应器培养CHO细胞的生长与代谢研究

 在搅拌式生物反应器中,应用批培养、流加培养和灌流培养3种方法对CHO-k1细胞进行了培养.3种方法最大活细胞密度分别达到了2.0×10⁶,2.7×10⁶和9.6×10⁶mL^-1.对培养过程中葡萄糖和氨基酸及代谢产物氨和乳酸的动态变化进行了检测和分析.结果表明,CHO细胞对葡萄糖和谷氨酰胺的摄取以及氨和乳酸的产生与培养液中葡萄糖和谷氨酰胺的浓度正相关,培养过程中控制葡萄糖和谷氨酰胺的加入量能够明显降低氨和乳酸的产生,提高代谢效率,增加细胞产量.     

The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth

Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.     

α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation

Parkinson's disease is the second most common neurodegenerative disorder. Growing evidence indicates a causative role of misfolded forms of the protein α-synuclein in the pathogenesis of Parkinson's disease. Intraneuronal aggregates of α-synuclein occur in Lewy bodies and Lewy neurites, the cytopathological hallmarks of Parkinson's disease and related disorders called synucleinopathies. α-Synuclein has long been defined as a 'natively unfolded' monomer of about 14?kDa (ref. 6) that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous α-synuclein isolated and analysed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of about 58?kDa. Several methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vitro cell crosslinking confirmed the occurrence of the tetramer. Native, cell-derived α-synuclein showed α-helical structure without lipid addition and had much greater lipid-binding capacity than the recombinant α-synuclein studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. On the basis of these findings, we propose that destabilization of the helically folded tetramer precedes α-synuclein misfolding and aggregation in Parkinson's disease and other human synucleinopathies, and that small molecules that stabilize the physiological tetramer could reduce α-synuclein pathogenicity.     

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. Here we show, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells.     

Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes

Little is known about the factors which regulate the growth and development of the mammalian brain. Although proliferation of neuronal cells ceases relatively early in development, certain types of glial cells proliferate and differentiate mainly perinatally. In the perinatal period, the ability of acetylcholine to stimulate phosphoinositide (PI) hydrolysis in brain reaches peak levels, and indeed the stable acetylcholine analogue carbachol can stimulate PI hydrolysis of primary neonatal astroglial cells. As PI hydrolysis is thought to be important in the regulation of cell proliferation, we investigated whether cellular DNA synthesis can be induced by carbachol. Our results show that carbachol stimulates DNA synthesis via muscarinic acetylcholine receptors (mAChRs), in primary astrocytes derived from perinatal rat brain, in an age-dependent fashion. Carbachol is also mitogenic in certain brain-derived astrocytoma and neuroblastoma cell lines, as well as in chinese hamster ovary (CHO) cells expressing recombinant muscarinic receptors. DNA synthesis is strongly activated by carbachol in those brain-derived cell lines and transfected CHO cells that express mAChR subtypes which activate PI hydrolysis efficiently, and poorly activated in cells expressing mAChR subtypes which only weakly activate PI hydrolysis. These results strongly support a role for acetylcholine in regulating astroglial cell growth in the developing brain, and indicate that the specificity of acetylcholine-induced cell proliferation may be determined by the expression of those mAChR subtypes which activate PI hydrolysis.     

谷氨酸能神经传递系统研究进展

谷氨酸广布于大脑皮质等处,以(-酮戊二酸、谷氨酰胺或鸟氨酸等为前体进行合成,通过快突触传递或慢突触传递与突触后膜上的三种类型的受体相结合,参与学习记忆、突触可塑性、细胞凋亡、自主运动神经活动及神经毒性作用等生理和病理功能。     

氧气的适应性机制及低氧预适应研究进展

 氧气是有氧代谢和能量产生的必要条件,对于大多数生物来说都是必不可少的。过多或过少的氧气都可能危害生命,因此研究生物快速响应变化的氧气水平的机制至关重要。当氧气需求超过氧气供应时,细胞就会变得低氧。介绍了氧气的特性以及生理、病理条件下低氧的适应机制,论述了低氧作为的积极方面--低氧预适应的历史和研究进展,展望了低氧预适应在高空、高原等极端环境中以及在卒中等相关临床疾病中的治疗前景。     

SIRT1与糖脂代谢及在运动医学领域的研究展望

SIRT1(silent mating type information regulation 2 homolog1),哺乳动物sir2同系物,一种NAD+依赖的组蛋白脱乙酰化酶。越来越多的证据表明SIRT1通过其脱乙酰化酶活性参与糖脂代谢,其对胰岛β细胞功能和胰岛素信号通路起着正性调节作用,对改善胰岛素的敏感性和维持糖脂代谢稳态具有重要作用。运动疗法是防治2型糖尿病、非酒精性脂肪肝和动脉粥样硬化等慢性疾病主要方法之一,但运动改善慢性疾病的机理还未完全阐明。已有研究表明运动训练能够诱导大鼠骨骼肌和心肌组织中SIRT1表达。运动改善慢性疾病机理是否与SIRT1有关尚不确定。在总结SIRT1与糖脂代谢关系的基础上,结合运动与SIRT1的研究现状,展望SIRT1在运动医学领域的研究前景。     

Functional genomic screen reveals genes involved in lipid-droplet formation and utilization

Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets enclosed in a monolayer of phospholipids and associated proteins. These dynamic organelles serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage.     

高压氧疗对脑损伤治疗作用机制的研究进展

脑损伤可引起脑组织的缺血缺氧。脑供血减少,会引起脑缺氧,但脑缺氧也可单独发生。若脑组织缺血缺氧的状态得不到及时纠正,氧参与的细胞内物质能量代谢将发生异常,如生成ATP的三羧酸循环、离子的转运等基本的生命活动都将受到影响,甚至停止。而蛋白质、核酸、脂质等物质的运输及代谢也将受到影响。这种状况持续数分钟,大脑的生理功能将受到影响,时间愈久这种变化愈难逆转。近年来,有研究表明,高压氧疗(HBOT)能改善损伤引起的脑组织缺血缺氧的状态,并对损伤的脑组织有修复的作用。其可能的途径如下:HBOT通过减少内皮素的生成,改善损伤后的脑血管痉挛状态、平衡脑血流以及降低升高的颅内压等,改善脑组织的缺血缺氧状态;并能通过改变MBP、caspases基因家族、Bcl-2/Bcl-xL以及TSPO、Nogo-A及其受体Ng-R等的表达,起到保护受损脑细胞、减少脑细胞凋亡以及促进神经细胞再生的作用;HBOT还可能通过增加胎盘生长因子(PLGF)的生成,起到促进血管生成及损伤脑组织再生的作用;此外,HBOT还能引起受损脑组织内多种炎症因子的改变,这也在一定程度上影响着脑损伤的预后。HBOT引起多种分子表达的改变,起到对损伤脑组织的修复作用...     

铅离子导致神经胶质细胞毒性的代谢组学机制

以神经胶质细胞为细胞模型,观察铅离子暴露细胞形态变化、细胞存活率和凋亡,并采用UPLC QTOF液质联用技术测定铅离子暴露的神经胶质细胞和正常细胞代谢物水平。结果显示：10 μmol/L 铅离子可引起神经胶质细胞的形态发生显著变化,细胞存活率降低,凋亡比例上升;多达973个潜在代谢物水平变化大于1.5倍（在P<0.05时）;主成分分析显示铅离子暴露组和正常对照组细胞具有显著不同的聚类趋势;差异代谢物的通路富集分析显示铅离子显著改变神经胶质细胞中谷氨酰胺和谷氨酸盐代谢、抗坏血酸/醛酸代谢、丁酸盐代谢以及谷胱甘肽代谢等多个涉及氧化还原等功能的重要代谢通路。铅离子生物毒性作用机制研究可为缓解铅毒性药物研发提供实验基础和理论参考。