Investigation of glucose catabolism in hypoxic Mcf 7 breast cancer culture

Hypoxia plays an important role in tumor phenotype and progression and alters glycolysis, with changes in signaling pathways that develop in response to hypoxia. In this study, the effects of oxygen (normoxia/hypoxia) and of glucose levels on the glucose metabolism was investigated in MCF-7 cancer cells. Under either normoxia or hypoxia conditions, the cells were exposed to glucose at different concentrations (0, 5.5, 15 or 55 mM) for either 3, 6, 12, 24 or 48 h. In all groups, cell viability, levels of key enzymes reflecting glycolytic metabolism in cell lysates, glucose consumed in the medium and extracellular lactate levels and wound closure percentages were determined. In hypoxic cells, intracellular consumption of glucose, and extracellular lactate levels due to increased glucose concentration were observed to be higher (compared to normoxia) and as a result of prolonged exposure to hypoxia, cells were observed to develop resistance to the prolonged exposure to hypoxia. The number of glycolytic enzymes obtained at different levels proved that cells had different potential capacities and changing mechanisms for the metabolic needs of the cell depending on the glucose amount in the medium and time in adapting to the oxygen tension. This study showed that there was an important interaction between hypoxia and glucose metabolism in general, and it was concluded that metabolic processes activated by hypoxia could offer new therapeutic targets.     

Chronic hypoxia-induced alterations of key enzymes of glucose oxidative metabolism in developing mouse liver are mTOR dependent

Hypoxia is a potent regulator of gene expression and cellular energy metabolism and known to interfere with post-natal growth and development. Although hypoxia can induce adaptive changes in the developing liver, the mechanisms underlying these changes are poorly understood. To elucidate some of the adaptive changes chronic hypoxia induces in the developing liver, we studied the expression of the genes of mammalian target of rapamycin (mTOR) signaling and glucose metabolism, undertook proteomic examination with 2D gel-MS/MS of electron transport chain, and determined activities and protein expression of several key regulatory enzymes of glucose oxidative metabolism. To gain insight into the molecular mechanism underlying hypoxia-induced liver metabolic adaptation, we treated a subset of mice with rapamycin (0.5 mg/kg/day) to inhibit mTOR postnatally. Rapamycin-treated mice showed lower birth weight, lower body weight, and liver growth retardation in a pattern similar to that observed in the hypoxic mice at P30. Rapamycin treatment led to differential impact on the cytoplasmic and mitochondrial pathways of glucose metabolism. Our results suggest a decrease in mTOR activity as part of the mechanisms underlying hypoxia-induced changes in the activities of glycolytic and TCA cycle enzymes in liver. Chronic postnatal hypoxia induces mTOR-dependent differential effects on liver glycolytic and TCA cycle enzymes and as such should be studied further as they have pathophysiological implications in hepatic diseases and conditions in which hypoxia plays a role.     

ChREBP和Mlx在调控葡萄糖反应基因表达中的作用

近年的研究结果显示,葡萄糖能在转录水平调控糖酵解和生脂酶基因的表达,对肝糖类和脂类动态平衡起协同调控作用.其重要转录因子是糖反应元件结合蛋白（ChREBP）和Max样蛋白X(Mlx),葡萄糖通过ChREBP.Mlx异二聚体调控葡萄糖反应基因的转录.本文主要综述转录因子ChREBP和Mlx的结构与功能,调控葡萄糖反应基因表达的机制,以及影响转录因子表达的因素.     

不同类型神经细胞对低氧的敏感性研究*

Objective: In order to illustrate the hypoxia-induced changes of neural cells in inflammatory response, oxidative stress, and energy metabolism process and to compare the sensitivity of neural cells’ responses to hypoxia. Methods: Different types of neural cells (BV2, N9, Gl261, HT22) were treated with hypoxia (0.1% O₂, 5% CO₂) for 0-24 hours. Cell proliferation was detected by Cell Counting Kit-8 method and cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay. Total RNA was extracted by Trizol reagent, and the inflammation, oxidative stress, and energy metabolism-related genes expression were measured by quantitative real-time PCR and Western blot. The ROS production was detected by flow cytometer with fluorescence probe. Results: Hypoxia stimulation decreased cell proliferation and cell viability. The hypoxia-induced changes of microglial cells (BV2 and N9) were mainly involved in inflammatory response and glucose metabolism process. The changes of astrocytes Gl261 and neural cell HT22 were mainly involved in glucose metabolism process. Hypoxia stimulation significantly increased oxidative stress in microglia and astrocytes. Conclusion: Different types of neural cells have different degrees of sensitivity in response to hypoxic stimulation. In terms of energy metabolism and inflammatory response, microglia are more sensitive to hypoxia treatment, which is manifested as a significant up-regulation of glycolytic enzymes and inflammation genes, whereas microglia and astrocytes are more sensitive to hypoxia treatment in terms of oxidative stress, which is indicated by their quick response and significant increase of ROS production.     

Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production

Thioredoxin-interacting protein (Txnip) has been recently described as a possible link between cellular redox state and metabolism; Txnip binds thioredoxin and inhibits its disulfide reductase activity in vitro, while a naturally occurring strain of Txnip-deficient mice has hyperlipidemia, hypoglycemia, and ketosis exacerbated by fasting. We generated Txnip-null mice to investigate the role of Txnip in glucose homeostasis. Txnip-null mice were hypoglycemic, hypoinsulinemic, and had blunted glucose production following a glucagon challenge, consistent with a central liver glucose-handling defect. Glucose release from isolated Txnip-null hepatocytes was 2-fold lower than wild-type hepatocytes, whereas beta-hydroxybutyrate release was increased 2-fold, supporting an intrinsic defect in hepatocyte glucose metabolism. While hepatocyte-specific gene deletion of Txnip did not alter glucose clearance compared with littermate controls, Txnip expression in the liver was required for maintaining normal fasting glycemia and glucose production. In addition, hepatic overexpression of a Txnip transgene in wild-type mice resulted in elevated serum glucose levels and decreased ketone levels. Liver homogenates from Txnip-null mice had no significant differences in the glutathione oxidation state or in the amount of available thioredoxin. However, overexpression of wild-type Txnip in Txnip-null hepatocytes rescued cellular glucose production, whereas overexpression of a C247S mutant Txnip, which does not bind thioredoxin, had no effect. These data demonstrate that Txnip is required for normal glucose homeostasis in the liver. While available thioredoxin is not changed in Txnip-null mice, the effects of Txnip on glucose homeostasis are abolished by a single cysteine mutation that inhibits binding to thioredoxin.     

低氧环境对三阴性乳腺癌细胞糖代谢途径的重编程

代谢改变是癌细胞的特征之一。研究表明,低氧会使癌细胞的糖代谢发生改变,但是更详细的分子机制仍有待进一步研究。本研究利用转录物组测序技术(RNA-sequencing,RNA-seq)和生物信息学分析发现,低氧导致BT549细胞中334个基因和MDA-MB-231细胞中215个基因在转录水平的表达改变。这些表达变化的基因多与糖代谢相关。进一步分析RNA-seq数据并应用Western 印迹、酶活性检测和代谢产物定量测定的结果显示,低氧通过升高BT549细胞中葡萄糖转运蛋白1(GLUT1)和MDA-MB-231细胞中GLUT1和GLUT3的表达以增加葡萄糖的摄入;低氧使催化糖的无氧氧化途径几乎全部反应的酶都至少有一种同工酶或酶蛋白亚基,以及调节酶6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶3(PFKFB3)和4(PFKFB4)同工酶的表达增加来促进了糖的无氧氧化;低氧还通过增加调节丙酮酸脱氢酶激酶1(PDK1)和3(PDK3)同工酶基因的表达,以及降低关键酶异柠檬酸脱氢酶3(IDH3)同工酶、琥珀酸脱氢酶B亚基和D亚基的表达来减少糖的有氧氧化途径进行;低氧可能还增加磷酸戊糖途径的关键酶葡糖-6-磷酸脱氢酶、糖原合成途径的关键酶糖原合酶GYS1同工酶的表达以促进这2条途径的进行,而对糖异生和糖原分解代谢途径酶基因的表达影响较小。生物信息学分析乳腺癌组织样本在线数据库中糖代谢途径酶基因在转录水平表达结果与细胞研究结果基本一致。总之,该文系统分析了低氧对糖代谢6条代谢途径中全部酶以及2种重要调节酶的影响,可见低氧会通过改变这些酶的同工酶或亚基的基因表达使糖代谢途径进行重编程,这对进一步认识低氧环境下癌细胞糖代谢的分子机制具有一定的意义。