Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%–370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%–30%). Neither -ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%–40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%–21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

Metabolic modulation induced by chronic hypoxia in rats using a comparative proteomic analysis of skeletal muscle tissue

Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure.     

A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension

Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.     

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence:

at first, TCA cycle enzymes are activated;

secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Q_o2 and a considerable one of O₂ consumption in cyanide-resistant respiration pathway;

thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

It is supposed, that coordination of carbon and energy metabolism in B. flavum depends on intracellular ATP concentration or energy charge value.     

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.     

Comparative metabolic flux profiling of melanoma cell lines: beyond the Warburg effect

Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the "reverse" (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma.     

缺氧应激对肝癌细胞代谢信号通路的调节作用

通过实验阐明在缺氧条件下糖酵解相关基因表达的变化规律及对肿瘤细胞和正常细胞增殖的影响,并探索活性氧(ROS)介导肝癌细胞代谢途径及对相关基因表达和酶活性的调节作用.以SMMC-7721人肝癌细胞和L02正常肝细胞作为研究对象,分别在单纯缺氧及加葡萄糖缺氧条件下,观察细胞生长,并检测糖代谢关键酶:丙酮酸激酶(pyruvate-kinase,PK)、己糖激酶(hexokinase,HK)、琥珀酸脱氢酶(succinic dehydrogenase,SDH)、异柠檬酸脱氢酶(isocitric dehydrogenase,IDH)mRNA表达水平和乳酸脱氢酶(lactate dehydrogenase,LDH)活性.还检测了pkb基因及缺氧诱导因子hif-1的表达.实验结果说明:a.肿瘤细胞较正常细胞具有更强的缺氧耐受性;b.缺氧条件下,糖酵解途径的增强是保证肿瘤细胞能快速增殖的机制之一;c.ROS通过HIF-1介导了糖代谢通路相关酶的基因表达,参与肝癌细胞缺氧信号通路调节,用抗氧化剂干预可以降低肿瘤细胞的缺氧耐受能力.     

Metabolic changes in the glucose-induced apoptotic blastocyst suggest alterations in mitochondrial physiology

Mammalian preimplantation embryos experience a critical switch from an oxidative to a predominantly glycolytic metabolism. In this study, the change in nutrient metabolism between the 2-cell and blastocyst stages was followed by measuring single embryo concentrations of tricarboxylic acid (TCA) cycle and glycolytic metabolites with microfluorometric enzymatic cycling assays. When the normal values were established, further changes that occur as a result of the induction of apoptosis by exposure to high-glucose conditions were examined. From the 2-cell to the blastocyst stage, the embryos experienced an increase in TCA metabolites and a dramatic increase in fructose 1,6-bisphosphate (FBP). The high TCA metabolites may result from accumulation of substrate due to a slowing of TCA cycle metabolism as glycolysis predominates. Embryos exposed to elevated glucose conditions experienced significantly lower FBP, suggesting decreased glycolysis, significantly higher pyruvate, suggesting increased pyruvate uptake by the embryos in response to decreased glycolysis, and increased TCA metabolites, suggesting an inability to oxidize the pyruvate and a slowing of the TCA cycle. We speculate that the glycolytic changes lead to dysfunction of the outer mitochondrial membrane that results in the abnormal TCA metabolite pattern and triggers the apoptotic event.     

Gene expression profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish

Low oxygen levels (hypoxia) play a role in clinical conditions such as stroke, chronic ischemia, and cancer. To better understand these diseases, it is crucial to study the responses of vertebrates to hypoxia. Among vertebrates, some teleosts have developed the ability to adapt to extremely low oxygen levels. We have studied long-term adaptive responses to hypoxia in adult zebrafish. We used zebrafish that survived severe hypoxic conditions for 3 wk and showed adaptive behavioral and phenotypic changes. We used cDNA microarrays to investigate hypoxia-induced changes in expression of 15,532 genes in the respiratory organs (the gills). We have identified 367 differentially expressed genes of which 117 showed hypoxia-induced and 250 hypoxia-reduced expressions. Metabolic depression was indicated by repression of genes in the TCA cycle in the electron transport chain and of genes involved in protein biosynthesis. We observed enhanced expression of the monocarboxylate transporter and of the oxygen transporter myoglobin. The hypoxia-induced group further included the genes for Niemann-Pick C disease and for Wolman disease [lysosomal acid lipase (LAL)]. Both diseases lead to a similar intra- and extracellular accumulation of cholesterol and glycolipids. The Niemann-Pick C protein binds to cholesterol from internal lysosomal membranes and is involved in cholesterol trafficking. LAL is responsible for lysosomal cholesterol degradation. Our data suggest a novel adaptive mechanism to hypoxia, the induction of genes for lysosomal lipid trafficking and degradation. Studying physiological responses to hypoxia in species tolerant for extremely low oxygen levels can help identify novel regulatory genes, which may have important clinical implications.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.     

Fumarate reductase activity maintains an energized membrane in anaerobic Mycobacterium tuberculosis

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD(+). This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO(2) incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope ((13)C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from (13)C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.     

Spatial Reorganization of Saccharomyces cerevisiae Enolase To Alter Carbon Metabolism under Hypoxia

Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-¹³C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.     

GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.