Oxygen--A Key Regulatory Metabolite in Metabolic Defense Against Hypoxia

Two main defense strategies against hypoxia tolerant animalshave been identified in earlier studies: (i) reduction in energyturnover and (ii) improved energetic efficiency of those metabolicprocesses that remain. Two model systems were developed fromthe highly anoxia tolerant aquatic turtle—(i) tissue slicesof brain cortex (to probe cell level electrophysiological responsesto oxygen limitation) and (ii) isolated liver hepatocytes (toprobe signalling and defense). In the latter, a series of mechanismsunderpinning hypoxia defense is initiated with an oxygen sensor(probably a heme protein) and a message transduction pathwayleading to the specific activation of some genes (increasedexpression of several proteins) and to specific down regulationof other genes (decreased expression of several other proteins).The pathway seems similar to oxygen regulated schemes in othercells. The main roles for the oxygen sensing and signal transductionsystem appear to include coordinate down regulation of energydemand and energy supply pathways in metabolism. By this means,hypoxia tolerant cells stay in energy balance as they down regulateto extremely low levels of ATP turnover. The main ATP demandpathways in normoxia (protein synthesis, protein degradation,glucose synthesis, urea synthesis, and maintenance of electrochemicalgradients) are all depressed to variable degree during anoxiaor extreme hypoxia. However, Na⁺ K⁺ ATPase is the main energysink in anoxia—despite significant reductions in cellmembrane permeability ("channel arrest"). Turtle brain corticalcells also show lower permeability than do homologous hypoxiasensitive cells, but in this case under acute anoxia, thereis no further change in cell membrane conductivity. These twomodels may supply guidelines for further studies of estuarineanimals on how normoxic maintenance ATP turnover rates can bedown regulated by an order of magnitude or more—to newhypometabolic steady states prerequisite for surviving prolongedhypoxia or anoxia     

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis (PGC-1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1α, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.     

Surviving hypoxia without really dying

In cases of severe O(2) limitation, most excitable cells of mammals cannot continue to meet the energy demands of active ion transporting systems, leading to catastrophic membrane failure and cell death. However, in certain lower vertebrates, hypoxia-induced membrane destabilisation of the kind seen in mammals is either slow to develop or does not occur at all owing to adaptive decreases in membrane permeability (i.e. ion 'channel arrest'), that dramatically reduce the energetic costs of ion-balancing ATPases. Mammalian cells do, however, exhibit a whole host of adaptive responses to less severe shortages of oxygen, which include energy-balanced metabolic suppression, ionic-induced activation of O(2) receptors and the upregulation of certain genes, all of which enhance the systemic delivery of oxygen and promote energy conservation. Accumulating evidence suggests that the mechanisms underlying these protective effects are orchestrated into action by putative members of an O(2)-sensing pathway that most if not all cells share in common. In this review we address three major questions: (i) how do cells detect shortages of oxygen and subsequently set in motion adaptive mechanisms of either energy production or energy conservation; (ii) how do these mechanisms restructure cellular pathways of ATP supply and demand to ensure that ion-motive ATPases are given priority over other cell functions to preserve membrane integrity in energy-limited states; and (iii) what mechanisms of molecular and metabolic defence against acute and long-term shortages of oxygen set hypoxia-tolerant systems apart from their hypoxia-sensitive counterparts?     

Metabolic depression: a response of cancer cells to hypoxia?

Hypoxic tumours have the worst prognosis because they are the most aggressive and the most likely to metastasize. This may be because these aggressive cancers have a hypoxic core which generates signals that activate angiogenesis which enables the supply of nutrients and oxygen to a rapidly growing outer oxidative shell. The hypoxic core is a crucial element of this hypothesis, as is the fact that the cells in the hypoxic core are inherently adapted to survive hypoxia. We reasoned therefore that cancer cells exposed to hypoxia/anoxia should show the hallmarks of adaptation to hypoxia/anoxia, i.e. a down-regulation of protein synthesis and a reverse Pasteur effect. We tested this hypothesis in transformed (MCF-7) and normal (HME) human mammary epithelial cells, by exposing both cell types to a range of oxygen concentrations, including anoxia. We find that indeed protein synthesis is down-regulated in the MCF-7, but not in the HME cells in response to anoxia. The data on glycolysis are not as clear-cut, but in the light of similar previous measurements on hypoxia-tolerant animals, is still consistent with the hypothesis.     

Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss)</Emphasis>

In many vertebrates, a short episode of oxygen lack protects against myocardial necrosis during a subsequent, longer period of oxygen deprivation. This protective effect, termed preconditioning, also improves the functional recovery. Improved functional recovery has been reported for hypoxia-sensitive, in situ perfused rainbow trout hearts, but appears absent in another strain of rainbow trout that has a more hypoxia-tolerant heart. The results for the hypoxia-tolerant rainbow trout heart, however, might have occurred because the preconditioning stimuli were insufficient in either intensity or type to elicit cardioprotective effects. In the present study, we attempted to induce preconditioning in in situ perfused hearts from hypoxia-tolerant rainbow trout (Oncorhynchus mykiss), acclimated and tested at 10 °C, by either doubling the anoxic preconditioning stimulus (PO₂ of the perfusate <0.5 kPa) relative to earlier studies or by using short exposures to high concentrations of adrenaline. In addition, anoxic-preconditioning experiments were conducted at an acutely elevated temperature (15 °C) to increase myocardial sensitivity to oxygen lack. The effect of preconditioning stimuli was assessed by measuring cardiac performance before and after exposure to a 20-min anoxic challenge. In addition, myocardial condition was evaluated at the termination of the experiment by measuring myocardial concentrations of glycogen, high energy phosphates and lactate, as well as activities of pyruvate kinase and lactate dehydrogenase. Maximal cardiac performance in oxygenated control hearts was unchanged by the 2-h experimental protocol, whereas inclusion of a 20-min period of anoxia led to 25 and 35% reductions in maximal cardiac performance at 10 and 15 °C, respectively. Reduced contractility, however, could not be ascribed to myocardial necrosis, as the biochemical and energy state of the hearts was unaffected. Hence, anoxic exposure merely stunned the myocardium. At 10 °C, neither the anoxic nor adrenergic preconditioning protocols improved post-anoxic cardiac performance. Further, the preconditioning protocols did not reduce post-anoxic myocardial dysfunction at 15 °C, despite the increased cardiac sensitivity to anoxia at this temperature. Thus, despite using strong and different preconditioning stimuli compared with earlier studies, the cardio-protective effect of preconditioning seems to be absent in rainbow trout hearts that are inherently more hypoxia-tolerant.     

An in vivo study of common carp (Cyprinus carpio L.) liver during prolonged hypoxia

Hypoxia induced apoptosis has been studied extensively in many mammalian cell lines but there are only a few studies using whole animal models. We investigated the response of the intact liver to hypoxia in a hypoxia tolerant fish, the carp (Cyprinus carpio, L). We exposed carp to hypoxia for up to 42 days, using oxygen level (0.5 mgO₂/L) that were slightly higher than the critical oxygen level of carp. There was extensive DNA damage in liver cells, especially during the first week of exposure, indicated by a massive TUNEL signal. However there was no change in cell proliferation, cell number or size, no increase in caspase-3 activity, no increase in single stranded DNA and this, combined with a number of other observations, led us to conclude there was no increase in apoptosis in the liver during hypoxia. There was up-regulation of some anti-apoptotic genes and proteins (Bcl-2, HSP70, p27) and down-regulation of some pro-apoptotic genes (Tetraspanin 5 and Cell death activator). The cells appeared to enter cell cycle arrest, presumably to allow repair of damaged DNA. As there was no change in cell proliferation and cell number, the damaged cells were not entering apoptosis and must have recovered during prolonged hypoxia.     

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.     

Tolerance of isolated rat hearts to low-flow ischemia and hypoxia of increasing duration: Protective role of down-regulation and ATP during ischemia

We tested the hypothesis that down-regulated hearts, as observed during low-flow ischemia, adapt better to low O₂ supply than non-down-regulated, or hypoxic, hearts. To address the link between down-regulation and endogenous ischemic protection, we compared myocardial tolerance to ischemia and hypoxia of increasing duration. To that end, we exposed buffer-perfused rat hearts to either low-flow ischemia or hypoxia (same O₂ shortage) for 20, 40 or 60 min (n = 8/group), followed by reperfusion or reoxygenation (20 min, full O₂ supply). At the end of the O₂ shortage, the rate·pressure product was less in ischemic than hypoxic hearts (p < 0.0001). The recovery of the rate·pressure product after reperfusion or reoxygenation was not different for t = 20 min, but was better in ischemic than hypoxic hearts for t = 40 and 60 min (p < 0.02 and p < 0.0002, respectively). The end-diastolic pressure remained unchanged during low-flow ischemia (0.024 ± 0.013 mmHg·min^–1), but increased significantly during hypoxia (0.334 ± 0.079 mmHg·min^–1). We conclude that, while the duration of hypoxia progressively impaired the rate·pressure product and the end-diastolic pressure, hearts were insensitive of the duration of low-flow ischemia, thereby providing evidence that myocardial down-regulation protects hearts from injury. Excessive ATP catabolism during ischemia in non-down-regulated hearts impaired myocardial recovery regardless of vascular, blood-related and neuro-hormonal factors. These observations support the view that protection is mediated by the maintenance of the ATP pool.     

A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (<Emphasis Type="Italic">Hemiscyllum ocellatum</Emphasis>)

Hypoxia and warm ischemia are primary concerns in ischemic heart disease and transplant and trauma. Hypoxia impacts tissue ATP supply and can induce mitochondrial dysfunction that elevates reactive species release. The epaulette shark, Hemiscyllum ocellatum, is remarkably tolerant of severe hypoxia at temperatures up to 34°C, and therefore provides a valuable model to study warm hypoxia tolerance. Mitochondrial function was tested in saponin permeabilised ventricle fibres using high-resolution respirometry coupled with purpose-built fluorospectrometers. Ventricular mitochondrial function, stability and reactive species production of the epaulette shark was compared with that of the hypoxia-sensitive shovelnose ray, Aptychotrema rostrata. Fibres were prepared from each species acclimated to normoxic water conditions, or following a 2 h, acute hypoxic exposure at levels representing 40% of each species’ critical oxygen tension. Although mitochondrial respiratory fluxes for normoxia-acclimated animals were similar for both species, reactive species production in the epaulette shark was approximately half that of the shovelnose ray under normoxic conditions, even when normalised to tissue oxidative phosphorylation flux. The hypoxia-sensitive shovelnose ray halved oxidative phosphorylation flux and cytochrome c oxidase flux was depressed by 34% following hypoxic stress. In contrast, oxidative phosphorylation flux of the epaulette shark ventricular fibres isolated from acute hypoxia exposed the animals remained similar to those from normoxia-acclimated animals. However, uncoupling of respiration revealed depressed electron transport systems in both species following hypoxia exposure. Overall, the epaulette shark ventricular mitochondria showed greater oxidative phosphorylation stability and lower reactive species outputs with hypoxic exposure, and this may protect cardiac bioenergetic function in hypoxic tropical waters.     

动物抗低氧胁迫相关基因的表达调控机制

体内氧浓度的稳定是机体维持自身功能的一个必要条件。在低氧条件下,机体内部在低氧信号的刺激下形成一个强大的防御体系以保护自己的组织。在采取防御的过程中,低氧诱导因子-1 (hypoxia inducible factor-1,HIF-1)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、促红细胞生成素(erythropoietin, EPO)、核因子-κB (nuclear factor-κB, NF-κB)等基因表达上调。HIF-1是一个与低氧胁迫相关的转录因子,它的激活与体内氧浓度相关。VEGF是HIF-1下游的一个靶基因,它是至今发现的一个在促血管新生方面起着最关键作用的因子。NF-κB能够抑制由低氧引起的细胞凋亡。以上这些基因在动物抗低氧胁迫方面起着重要作用,综述了低氧条件下HIF-1、VEGF、EPO、NF-κB的功能、表达特性以及调控机制。