Anesth Analg 124(6): 1857-1871

Subcellular Energetics and Metabolism: A Cross-Species Framework

Introduction

As organisms that depend mostly on aerobic metabolic pathways for the provision of adenosine triphosphate (ATP), it is not surprising that humans consider oxygen to be an essential component of life. The most ancient forms of life, however, neither consumed nor produced oxygen, utilizing anaerobic anoxygenic photosynthesis (AnAnP) to generate ATP.¹ It was not until the development of oxygenic (oxygen-producing) photosynthesis 2.4-3 billion years ago that the atmosphere began to be “contaminated” with oxygen.¹,2 The introduction of oxygen to the environment led to the development of primitive mechanisms to protect against oxidative stress, some of which eventually morphed into subcellular pathways capable of consuming this dangerous metabolic byproduct and in doing so producing energy.² The tradeoff between oxidative stress and the production of energy (known as the “Respiration Paradox”³), which still exists today, was thus established billions of years ago.

Viruses, Bacteria, and Archaea can survive in completely anoxic environments,⁴-7 and more recently, Metazoan (kingdom Animalia) species that can tolerate anoxic environments have also been identified.⁸ The questions then arise – if modern, multicellular organisms can survive in completely anoxic environments, are these alternative metabolic pathways utilized by humans, can they be co-opted and used to our advantage, and what can they teach the scientific community about cell death and, potentially, survival in the face of anoxic stress? The purpose of this review is to explore the ability of cells (e.g. cancer) and organisms (e.g. pre-implantation embryos) to survive, and in some cases thrive, in ostensibly hostile environments, and identify common themes that suggest therapeutic applications for humans subjected to hypoxic or oxidative stress.

Human Embroygenesis

Mammalian embryos are a useful starting point for understanding how organisms can adapt to hypoxia because the development and growth of the neurologic system precedes the development of the cardiovascular system. In the adult, hypoxia-induced ischemia appears related to the balance between oxygen delivery and consumption, with the most metabolically active organs (e.g. brain) most susceptible to injury.⁹,10 It is therefore interesting to note that while the adult brain is relatively intolerant of hypoxia, the developing brain requires hypoxia to properly form. This counterintuitive phenomenon was demonstrated convincingly by Morriss and New in 1979, who subjected rat embryos to oxygen levels ranging from 5-40% and found that higher oxygen levels prevented neural tube closure [Figure 1].¹¹

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Figure 1

Thirteen-somite embryo cultured at 40% O₂, demonstrating broad opening of the forebrain (abnormal cranial fold) as compared to embryos cultured at 5% O₂.

The dependence of developing organs on subatmospheric oxygen concentrations is unsurprising when one considers that the partial pressure of oxygen (pO₂) ranges from 0.5-30 mm Hg (1-5%) in the intrauterine environment.¹² In contrast, the intracellular oxygen concentrations in most adult cells ranges from 2-9% although exceptions such as the renal medulla safely exist at concentrations as low as 1%.¹³ Studies utilizing the hypoxia marker pimonidazole, which binds protein and DNA at oxygen levels below 2%, demonstrate extensive hypoxia in both the neural tube and developing heart of murine embryos during organogenesis.¹⁴

In fact, during embryogenesis oxygen acts more like a signal and less like an electron acceptor, guiding both stem cell and endothelial progenitor differentiation.¹³,15 Prior to the development of the circulatory system, fetal oxygen levels typically do not exceed 3%, making achievement of “normal” post-gestational levels of oxygen almost impossible.¹⁶,17 Because oxygen travels by diffusion, an oxygen gradient exists across the developing animal, with higher oxygen levels in areas proximal to maternal spiral arteries, and lower levels dorsally [Figure 2].¹³ The fate of individual stem cells is affected by local levels of oxygen. For example, trophoblastic stem cells exposed to high levels of oxygen become giant cells, whereas those exposed to lower levels of oxygen become spongiotrophoblasts.¹⁸ Even in adults, animal data suggests that regional hypoxia helps maintain stem cells in their pluripotent state.¹⁹ The ontogenic properties of molecular oxygen are not limited to mammals and are shared across species (e.g. in Drosophila melagnogaster, oxygen levels strongly influence the development of the trachea).²⁰

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Figure 2

During development, cells are exposed to varying levels of O₂, which influences their differentiation.

The link between varying oxygen levels and organogenesis is formed by hypoxia inducible factor 1α (HIF-1α), which is expressed almost ubiquitously in the developing embryo.² Both HIF-1α and vascular endothelial growth factor (VEGF) bind to pimonidazole, suggesting that hypoxia induces adaptive processes such as HIF-1α upregulation and angiogenesis.¹³,15 Mice deficient in HIF-1 are marked by defective neural tube development and angiogenesis (particularly in neurologic tissue), and do not survive past embryonic day 10.5.²¹,22 It is not clear whether HIFs arose initially to protect against hypoxia (or oxidative stress) and were co-opted to support organogenesis, or vice versa, but it is clear that this class of transcription factors is tightly coupled to oxygen availability and that without it, development and survival are not possible. Because hypoxia is an essential precondition for proper development, and metabolically active organs can develop and grow in low oxygen environments, the question then arises – how is it possible for organ systems (e.g. neurologic system) to thrive in hypoxic environments that would be fatal in the fully developed organism?

Several general strategies have evolved for adapting to hypoxia. Many are shared across organisms, and some are used during development. First, alternative pathways for producing energy may be utilized (e.g. anaerobic glycolysis). Indeed, in the very earliest stages of development (pre-implantation embryo), very little oxygen is consumed by developing cells, and mitochondria demonstrate relatively few foldings, also suggesting minimal aerobic respiration.²³-25 Pyruvate is the preferred energy source of the pre-implantation embryo, as it can be used to produce ATP (albeit inefficiently) without the need for oxygen.²⁵-27

Second, in low oxygen environments, oxygen delivery can be increased via strategies such as augmenting cardiac output (immediate) or HIF-1α–mediated angiogenesis (delayed).¹² During embryological development, an increase in cardiac output is not possible until the development of the cardiovascular system is complete (in human embryos, the primitive left ventricle is not formed until approximately ED 30). Angiogenesis is rampant during embryogenesis, primarily driven by hypoxia-responsive factors such as HIF-1α, HIF-2α, and VEGF.¹³,15

Third, when oxygen availability is scarce, energetic needs can be attenuated through a variety of mechanisms, including reductions in adipogensis, ion-channel activity and protein synthesis (also see Hibernation, below).¹³,28 In the developing embryo, which grows at exponential rates, this is a challenge. By the time the blastocyst stage is achieved (post-implantation), oxygen consumption increases markedly and glucose becomes the preferred energy source, with 85% of it being metabolized through aerobic respiration.²⁵,27

Fourth, accumulating data suggest that cellular damage associated with hypoxia may not result only from energetic failure, but also from the production of reactive oxygen species (ROS) that occurs in response to a hypoxic insult followed by re-establishment of normal oxygen levels. This phenomena is also known as ischemia-reperfusion injury (see Ischemia-Reperfusion and Reactive Oxygen Species, below), and may depend on the organism's ability to defend against oxidative stress.²⁹

Lastly, the developing brain may also be resistant to intracellular calcium overload. Calcium accumulation is central to the development of neurologic injury following ischemia. In the classic model of hypoxia-induced neurologic damage, lack of oxygen decreases the activity of ATP-dependent processes such as maintenance of transmembrane ion potentials and neurotransmitter reuptake. The resulting membrane depolarization leads to release of glutamate, dopamine, and other neurotransmitters. These excitotoxins bind to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors, increasing the concentration of intracellular calcium and ultimately causing cellular damage and death.³⁰,31 Limiting calcium accumulation during hypoxia may explain the relative resistance of mammalian neonatal neuronal tissues to hypoxic stress, and inactivation of NDMA receptors has been observed after hypoxia. ³¹,32 Interestingly, elevations in intracellular calcium levels may also facilitate HIF-1 expression (see below).³³ [Table 1]

Table 1

Strategies for Adapting to Hypoxia

Strategies for Adapting to Hypoxia
Alternative energetic pathways
Increased oxygen delivery
Decreased energy needs
Defense against oxidative stress
Attenuation of calcium accumulation

Hypoxia Inducible Factors and Mammalian Adaption to Hypoxia

Evolution has produced mechanisms for responding to hypoxia, all capable of either activating transcription or inhibiting translation to protect oxygen-dependent organisms. These mechanisms include the hypoxia-inducible-factors and mammalian target of rapamycin (mTOR) kinase, the unfolded protein response, and NF-κB.³⁴-36 Of these, the HIFs are the best known and understood. In the presence of oxygen, the alpha subunits of the HIF proteins (HIF-1α, HIF-2α, HIF-3α) are degraded by HIF prolyl hydroxylases (PHDs).³,37,38 When intracellular oxygen drops below a critical value (approximately 10 mm Hg³⁹), PHDs become inactive and HIFs begin to accumulate. This increase in HIFs affects as many as 1,500 target genes through the cis-acting hypoxia response element (HRE), which contains the sequence 5’-RCGTG-3’ (where R = A or G).³⁷ With over a thousand target genes, describing the impact of HIFs in the context of a review article is impossible, so we will therefore focus on those most relevant to hypoxia and oxidative stress.

HIF-1 activates several angiogenic and arteriogenic growth factors including VEGF, angiopoietin 2 (ANGPT2), stromal derived factor 1 (SDF1), platelet-derived growth factor B (PDGFB), placental growth factor (PGF), and stem cell factor (SCF). Collectively, these factors increase the delivery of oxygenated hemoglobin to chronically hypoxic tissues.⁴⁰ HIF-2 activates the transcription of genes for erythropoietin (EPO) and proteins needed to absorb and deliver iron, thereby increasing the carrying capacity of blood.³⁷,41-43 While these responses can have profound effects over prolonged periods, they do little to ameliorate the effects of hypoxia in the short term.

More immediately, cells deprived of oxygen can adapt by shifting energy production away from oxidative phosphorylation. Oxidative phosphorylation, in which NADH and FADH₂ donate electrons to the electron transport chain (ETC) to drive ATP synthase using oxygen as the terminal electron acceptor, is highly efficient, producing more ATP per molecule of substrate than any other known form of energy production. However, the coordinated passage of electrons across the cytochromes and dehydrogenases that make up the electron transport chain is a finely tuned process, and oxygen tensions that deviate in either direction from normality lead to the loss of electrons and production of free radicals which not only reduce the amount of ATP generated but can lead to extensive cellular damage.³⁷ To promote the use of alternative metabolic pathways in the setting of hypoxia, HIF-1 upregulates pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK-1) and consequently pyruvate dehydrogenase (PDH), which prevents the conversion of pyruvate to Acetyl-CoA for use in oxidative phosphorylation.³⁷,44

HIF-1 also suppresses fatty acid β-oxidation (another source of Acetyl CoA) through inhibition of acyl-CoA dehydrogenases.⁴⁵ Both of these activities deprive the TCA cycle of acetyl-CoA, attenuate the ability of NADH and FADH₂ to donate electrons to the ETC, and decrease oxygen dependent ATP production. HIF-1 also inhibits the TCA cycle through indirect inhibition of aconitase)⁴⁶ and in doing so modifies oxidative phosphorylation directly. Because the mitochondria serve as a receptacle for a molecule (O₂) necessary to preserve continuous oxidative phosphorylation, the activities of HIF-1 on PDK-1 and PDH help maintain cell function in the face of decreased O₂ supply.⁴⁷

Shutdown of the TCA cycle and oxidative phosphorylation would be fatal unless energy were available from other pathway(s). Towards that end, HIF-1 also activates lactate dehydrogenase-A (LDHA), promoting the conversion of pyruvate to lactate, shunting pyruvate away from the TCA cycle (the “Pasteur effect”), and allowing energy production to continue.³⁷ Because this pathway is less efficient than oxidative phosphorylation, greater supplies of glucose are necessary to maintain stable levels of ATP. Fortunately, HIFs upregulate genes encoding glucose transporters.⁴⁸ HIF-1 also facilitates a subunit switch in cytochrome c oxidase (COX) that increases the efficiency of electron transfer and attenuates the production of ROS (H₂O₂) at lower oxygen levels.⁴⁹,50 This subunit switch is important because hypoxia does not shut down oxidative phosphorylation completely and ROS are harmful. HIF-1 also upregulates the micro-RNA miR-210, which inhibits ETC Complex 1 and reduces ROS production .⁴⁶ Lastly, HIF-1 can induce mitochondrial autophagy, which decreases oxidative phosphorylation by reducing the supply of mitochondria through its interactions with BNIP3 and BNIP3L.⁵¹ Hypoxia inducible factor HIF-2α plays a similar role in blocking the conversion of pyruvate to acetyl-CoA and thereby depriving the TCA cycle of a crucial intermediary.³ In mice deficient in the conversion enzyme PHD-1, HIF-2α levels are elevated when compared to wild-type mice.⁵²

Knockdown of PHD-1 leads to an upregulation of peroxisome proliferator-activated receptor alpha (PPARα), a “master regulator” of metabolism that can reduce glucose oxidation through its effects on pyruvate dehydrogenase lipoamide kinase isozyme 4 (Pdk4).⁵² PPARα-mediated activation of PDK-4 inhibits PDH.⁵³ Skeletal muscles from PHD-1 deficient mice consume less oxygen and tolerate ischemia better than their wild-type counterparts,⁵² likely due to a combination of decreased oxidative phosphorylation, reduced ROS production, and increased glycolysis (also mediated by PPARα).⁵² Additionally, in the setting of ischemia, PHD-1 deficient mice preserve high energy phosphates better than wild-type mice.⁵² [Figure 4] HIFs also exert their hypoxia-moderating effects through EPO. In addition to increasing oxygen carrying capacity, which is a long term approach, EPO also activates anti-apoptosis proteins bcl-2 and bcl-XL, inhibits brain derived neurotrophic factor, caspases, and decreases the development of inflammation and excitotoxicity.⁵⁴-56 These short term effects are particularly relevant in protecting the central nervous system from hypoxia,⁵⁴-56 although human trials to date have been negative.⁵⁷,58

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Figure 4

HIF-1 and HIF-2 activate thousands of genes, many of which impact metabolism and are protective in the setting of hypoxia.

Physiologic adjustments to hypoxia are also mediated through non-HIF pathways. Because protein synthesis is so energetically intense, hypoxic stresses such as changes in redox state, glucose availability, glycosylation, and energy loss lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum.³⁵ These proteins then activate an unfolded protein response (UPR), a system of signal cascades including activation of PKR-like kinase [PERK], activating transcription factor 6α [ATF6α], and inositol requiring 1α [IRE1α]. These responses ultimately reduce RNA translation, protein production, and therefore energy expenditure.³⁵,59 Similarly, hypoxia downregulates the activity of the a serine/threonine kinase mTOR, a protein that modifies protein synthesis in response to stress, nutrient deprivation, and hypoxia.⁶⁰ Because mTOR drives energy consuming processes such as protein synthesis and cell growth, effects of hypoxia on mTOR lead to decreased cell growth.³⁵ Importantly, the ability of hypoxia to stimulate mTOR is independent of HIF.⁶⁰-62

Hypoxia also activates NF-κβ,⁶³-65 which regulates apoptosis, inflammation, both the innate and adaptive immune responses, the cellular stress response, cell adhesion and proliferation, and tissue remodeling.³⁴ Normal NF-κβ function is essential for survival, perturbations in NF-κβ activity are linked to cancer development, and abnormalities in NF-κβ function have been documented in autoimmune diseases and sepsis.⁶⁶,67 Although NF-κβ activation is a HIF-independent response to hypoxia, both the HIF and the NF-κβ hypoxia responses are mediated by prolyl hydroxylases.⁶³

Ischemia-Reperfusion and Reactive Oxygen Species

In 1960, Jennings et al. first suggested that reperfusion of previously unperfused tissue beds may contribute to injury.⁶⁸ In 1977, Bulkely and Hutchins observed that the majority of perioperative infarctions occurring in patients undergoing coronary artery bypass grafting (CABG) surgery occurred in well-perfused, grafted territories of the myocardium.⁶⁹ A decade later, Parks and Granger demonstrated that histologic injury was worse after three hours of ischemia followed by one hour of reperfusion than after four hours of ischemia with no reperfusion in a feline intestinal ischemia model.⁷⁰ Taken together, these landmark findings raised the possibility that restoring oxygen delivery to previously hypoxic tissues may itself be harmful. At the level of the cell, injury caused by ischemia and reperfusion is multifactorial and incompletely understood, but includes alterations in ion pump function (and resultant ionic disturbances), expression of pro-inflammatory genes, repression of “protective” genes, activation of apoptosis, autophagy, and necrosis, and activation of both the innate and adaptive immune systems (autoimmunity).⁷¹,72

A common source of injury following reperfusion is the combination of accumulated reactive oxygen species (ROS) combined with reduced anti-oxidant capacity.⁷³ During normal aerobic respiration, an estimated 1-20% of the electrons destined to flow through the ETC chain are ejected prematurely, leading to the creation of superoxide (•O₂^{) which subsequently interacts with a variety of molecules to produce both ROS and reactive nitrogenous species (RNS).}⁷⁴-78 While the quantity and sources of these electron “leaks” are not completely understood, both complexes I and III appear to be major contributors.⁷⁹-84 Supranormal levels of ROS have been demonstrated following ischemia-reperfusion in a variety of animal and human models.⁸⁵-87 While the source of ROS is not completely understood, ROS begins to accumulate even during the ischemic process when tissue oxygen levels are lower. .⁴⁷,82,86,88 This accumulation is then compounded by an additional ROS “burst” that occurs with re-introduction of O₂ to ischemic cells.⁸⁹-92 ROS and RNS are highly reactive species which, after overwhelming the body's endogenous antioxidant systems, disrupt membrane integrity, and contribute to necrosis and cell death by lipid membrane peroxidation, and alteration of cellular proteins and ribonucleic acids.⁷²,93

The theory that ROS/RNS play a prominent role in injury following ischemia and reperfusion is indirectly supported by several findings. First, free radical scavengers attenuate ischemia-reperfusion injury.⁹⁴-96 Second, when compared to wild-type cells, HIF-1α knockouts cultured in a hypoxic environment (1%) die prematurely, despite higher intracellular levels of ATP.⁴⁸ This suggests that hypoxia-induced decreases in oxidative phosphorylation may not be the primary cause of death after hypoxic insult. HIF-1α knockouts cells subjected to hypoxia also produce lethal levels of ROS.⁸⁸ Current data suggest that mitochondrial ROS lead to HIF-1 stabilization⁹⁷-100 and that ROS scavengers inhibit HIF-1 stabilization.⁴⁷,98,99,101 Third, skeletal muscle of mice deficient in PHD1 consumes less oxygen than muscle from wild-type mice and displays less necrosis and reduced ROS production with limb ischemia.⁵² While ROS and RNS are not the only sources of injury following ischemia and reperfusion (see above), understanding the impact of ROS/RNS formation is important because it is potentially modifiable and may present clinicians with an interventional opportunity.

Hibernation

Several animal species periodically enter controlled, photoperiodically determined states of hypometabolism known as hibernation.¹⁰² A universal component of hibernation is a decrease in core body temperature, which decreases the metabolic rate and the need for oxygen. In small animals, decreased body temperature is accompanied by reductions in cardiac output, ventilation, and in some cases increased peripheral vasoconstriction.¹⁰³-106 Collectively, these macro-physiologic changes decrease VO₂ to approximately 5% of euthermic baseline levels.¹⁰² Complementing this decreased need for oxygen are decreases in immunological and hematologic function, and increased antioxidant activity.³⁰

For reasons that are unclear, many hibernating animals (rodents in particular) undergo brief periods of arousal during the hibernating state, which increases VO₂ and subjects them to the risk of oxygen supply:demand mismatching. During these periods, PaO₂ as low as 10 mm Hg has been recorded in some animals.¹⁰²,104 Despite such arousals, these animals exhibit normal levels of plasma lactate and brain iNOS expression, suggesting they experience only moderate oxidative stress. This ability to tolerate what would be considered severe oxygen supply:demand mismatching is thought to be mediated by increased reliance on anaerobic and non-carbohydrate metabolic pathways,¹⁰⁷,108 ion channel arrest,¹⁰⁹,110 and preconditioning-like phenomena.¹⁰⁴,111 Interestingly, both preconditioning and hibernation upregulate a similar set of genes involving metabolism, immune responsiveness, and ion channel activity.¹¹²,113

Some have suggested that hibernating animals constantly subject themselves to oxidative stress during euthermia to precondition themselves in preparation for hibernation and arousal/rewarming.¹⁰⁴ The artic ground squirrel, for instance, maximally expresses HIF-1 and iNOS during a euthermic state (when PaO₂ ~ 60 mm Hg), despite decreases in PaO₂ to approximately 10 mm Hg during arousal periods.¹¹⁴ Arctic ground squirrels (AGS) thus experience mild, chronic hypoxia during euthermy, which increases HIF-1 and iNOS levels and provides protection during arousal/rewarming.¹¹⁴,115 While ROS may also trigger beneficial effects downstream, they are potentially harmful and mitigated by concomitant antioxidant production (e.g. ascorbate) and redistribution.¹⁰³

Even in their euthermic state, hibernating animals (e.g. ground squirrels, hamsters, and bats) tolerate hypoxia better than their homeothermic counterparts (e.g. rats, guinea pigs).¹⁰⁴,111,116,117 AGS, for instance, can tolerate 8-10 minutes of asphyxia leading to cardiac arrest with almost no neurologic damage. ¹⁰⁸,116,117 Potential mechanisms for this resiliency include improved hypothermia tolerance (which occurs naturally when oxygen supplies are low and the metabolic rate is regulated downward), higher plasma levels of β-hydroxybutyrate (which can be utilized as an alternative energy source), stronger antioxidant defense systems, and alteration of voltage-sensitive calcium channels (which prevents calcium accumulation and improves cerebral ischemia tolerance).¹⁰⁴,109,118,119

Of particular interest is the ability of the AGS to prevent ischemic depolarization (ID) and subsequent intracellular calcium accumulation. During the induction stage of ischemia, electron transport is inhibited, intracelluar ATP stores decrease, intracellular acidosis ensues, and the excitatory neurotransmitter glutamate is released, causing intracellular calcium accumulation. Additionally, genes controlling free radical production are unregulated. These changes conspire to activate at least five independently damaging events, called “perpetrators.” Both glutamate excitotoxicity and calcium accumulation are critical components of the transition from reversible cellular stress to irreversible cell death.¹²⁰ As described above (Human Embryogenesis), neonatal rat pups are relatively tolerant to hypoxia in part because of the ability of neuronal tissue to prevent hypoxia-induced calcium influx and glutamate accumulation.³²

Similarly, the AGS survive both oxygen and glucose deprivation despite significant depletion of ATP, suggesting that the ability of these animals to tolerate ischemia is due not only to reduced oxygen consumption but also the ability to cope with depleted intracellular energy stores.¹¹⁷ Inhibition of the epsilon protein kinase C (ePKC) protein, which inhibits both the Na/K-ATPase and voltage-gated Na channels, severely attenuates the AGS’ tolerance for hypoxia and shortens the time to ischemic depolarization (ID), providing further evidence that AGS and other hibernating animals tolerate loss of high energy substrates by preventing membrane depolarization and the subsequent electrolyte disarray that ultimately leads to cell death.¹¹⁶

Even more surprising, when AGS finally succumb to oxygen supply:demand mismatching and ischemic depolarization does occur, glutamate efflux is less harmful to these animals.¹²¹,122 This finding is likely due to decreased expression of NMDA receptors and lower levels of glutamate in the AGS.¹²³ AGS also activate mitogen activated protein kinase (MAPK) stress pathways during euthermia and arousal.¹¹⁵ MAPK activation correlates with HIF-1 expression, can be triggered by small increases in intracellular calcium,¹²¹ and has been associated with attenuation of calcium accumulation in an in vitro preconditioning model (rat hippocampal slices).¹²⁴

Diving Adaptations

While humans can typically only tolerate several minutes of apnea, some species of diving whales (Physeter catodon) and seals (Mirounga leonina, Cystophora cristata) can remain under water for much longer periods of time – in some cases, over an hour.¹⁰² These species have developed several macro-physiologic adaptations that allow them to store large amounts of oxygen, including higher hemoglobin levels (up to 25 g/dL) and myoglobin (which also behaves as an antioxidant), high tidal volumes when surfacing, increased tissue capillary density, the ability to cool during submergence, and redistribution of blood flow during periods of apnea.¹⁰²

Despite these macro-physiologic adaptations, diving animals eventually deplete most of their oxygen stores. Seals may achieve PaO₂ levels as low as 12 mm Hg.¹²⁵ Remarkably, seals can maintain cerebral integrity (measured using electroencephalography) to PaO₂ levels as low as 7-10 mm Hg,³⁰ which are substantially lower than the “critical” value of 25-40 mm Hg that affects ATP production in non-diving mammals.¹²⁶ Clearly, other adaptations exist to maintain organ function and promote survival. Some diving animals have an increased ability to utilize anaerobic pathways. Because such pathways are inefficient compared to oxidative phosphorylation, increased substrate availability is needed for this mechanism and increased glycogen stores, increased glycolytic capacity, and an enhanced ability to metabolize lactic acid have been documented in these animals.¹²⁷-129

Additionally, diving mammals may be able to transiently “switch off” the metabolic activity of certain tissue beds. In particular, in vitro studies have demonstrated that hepatic and renal tissue can transiently reduce their ATP consumption to 1% of resting of resting rates. These organs may also reduce ion permeability through channel arrest, which is particularly important as maintenance of ionic gradients is highly energy-intensive.¹²⁹-131 Interestingly, myocardial and hepatic tissue in non-diving mammals may also reduce ATP demand and utilization during hypoxia, likely through inhibition of cytochrome c oxidase and mitochondrial function.¹³²,133

Tissue from diving mammals subjected to oxidative stress produces more superoxide than non-diving animals, suggesting that diving mammals have adapted mechanisms to tolerate, rather than prevent, the formation of ROS.¹³⁴ Diving seals, for instance, express large amounts of antioxidant enzymes¹³⁵ and exhibit higher levels of superoxide in several tissue beds than the shorter/shallower diving dolphins, despite experiencing less oxidative stress.¹³⁶ Such behavior may be mediated by HIF-1, as its expression has been identified in seals and is associated with lower levels of protein oxidation.¹³⁷,138 Neuroglobin, a recently discovered protein located in the central nervous system, ¹³⁹ may play a role in attenuating ROS damage in diving animals, although its exact function in controversial.¹³⁴,140

Cancer

The study of cancerous cells is useful to investigators interested in subcellular energetics for two reasons. First, cancer is the second leading cause of death in the United States¹⁴¹, and understanding how cancerous cells survive, thrive, and spread in austere environments may lead to potentially therapeutic interventions. Second, the survival strategies utilized by cancerous cells may be applicable to other cell types and have implications for non-cancerous disease states (sepsis) and various biological threats (hypoxia).

Non-cancerous cells have narrow tolerances for pH, oxygenation, and nutrient supply. In contrast, cancerous cells survive in unfavorable conditions without consistent access to the bloodstream and at lower pH (typically 6.2-6.8, as opposed to 7.2-7.4 for normal cells).³⁵,142,143 Oxygen diffusion occurs at distance no more than 200 um (up to 20 cell layers) and the partial pressure of oxygen, which varies cyclically, may be as low as 5 mm Hg in some cancerous cells.³⁹,144 Thus in order for tumors to grow, neovascularization, hypoxia tolerance, or both are required.³⁵ Hypoxia in cancerous cells leads to HIF-1 mediated angiogenesis via VEGF and angiopoeitin 2.³⁹ VEGF-mediated angiogenesis is essential for cancerous cells to grow beyond a few millimeters in size and is thought to force metastatic cells out of dormancy.¹⁴⁵,146 [Figure 5]

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Figure 5

cancerous cells often live in a hypoxic environment. Their ability to survive, and in some cases thrive, is due to upregulation of a variety of HIF-1 mediated genes that modify metabolism, inhibit apoptosis, induce angiogenesis, and promote invasion and/or metastasis.

Rapidly-dividing cells consume increased amounts of oxygen, worsening local hypoxia.¹⁴⁷ Because cancerous cells often have less access to oxygen (especially prior to VEGF-mediated angiogenesis), alternate metabolic pathways such as anaerobic metabolism are required. Interestingly, cancerous cells often preferentially convert glucose to lactate even under normoxic conditions, a behavior known as the Warburg effect.¹⁴⁸ Because anaerobic metabolism is significantly less efficient than oxidative phosphorylation, cancerous cell thus have unusually high glucose requirements (forming the basis for [^{F]-deoxyglucose positron-emission tomography as a tumor identification tool).}¹⁴⁸ Thus, in cancerous cells, HIF-1 stabilization also leads to upregulation of GLUT1 and GLUT3 glucose transporters, further facilitating the use of inefficient anaerobic metabolic pathways.¹⁴⁹

In addition to its effects on angiogenesis and inducing a shift from oxidative phosphorylation to glycolytic energy production, HIF-1 also reduces the mitochondrial supply by inhibiting mitochondrial biogenesis through a C-MYC dependent mechanism. HIF-1 thus protects the growing tumor from oxidative stress, and impacts genes that influence metastasis.³⁹,148,150 Hypoxia also induces autophagy via BNIP3 and BNIP3L, thus further promoting tumor progression.⁵¹

Both hypoxia and ROS activate HIF-1.⁴⁷,97-101 HIF-1 stability is particularly affected by both hydrogen peroxide (H₂O₂) and nitric oxide (NO).¹⁵¹-157 Many forms of cancer over-express NO synthase (NOS) which increases endogenous levels of NO, stabilizes the HIF family of transcription factors, and initiates the pro-survival metabolic changes described above even when oxygen levels are normal (see Hypoxia Inducible Factors and Mammalian Adaption to Hypoxia).¹⁵⁸,159 Coincidentally, hypoxia, which induces a pro-survival response in cancerous cells, also confers resistance to both radiation therapy and chemotherapy through a variety of mechanisms.¹⁴⁴,160 Oxygen increases DNA damage and higher levels of oxygen thus increase the efficacy of radiation therapy (and some chemotherapeutic agents).¹⁴⁴ While radiation therapy leads to increased local levels of oxygen due to destruction of susceptible cells and reduced oxygen consumption, HIF-1 is up-regulated following radiation therapy, likely due to ROS production.¹⁶¹ Similarly, fewer cells cycle (and thus susceptible to chemotherapy) in low oxygen environments.³⁹ Additionally, the same limited blood supply that delivers oxygen also delivers lower levels of chemotherapy, making drug delivery a challenge.¹⁴⁴

Hypoxia tolerance by cancer cells, while generally pro-survival, may also lead to oncologic treatment opportunities. As compared to hypoxia-sensitive human glioblastoma multiforme cells, hypoxia tolerant human glioblastoma multiforme cells maintain ATP concentrations and stable mitochondrial membrane potentials in the setting of hypoxia. While the mechanism(s) responsible for this preservation have yet to be clarified, the development of hypoxia tolerance is accompanied by increased sensitivity to various mitochondrial inhibitors, a potentially useful characteristic in the development of chemotherapeutic agents.¹⁶² Other treatment strategies based on the hypoxic behavior of cancer cells includes the use of prodrugs which are activated by hypoxia (thus leaving normal cells alone), HIF-1 targeting, hypoxia-selective gene therapy, and even recombinant anaerobic bacteria (for a excellent review on the subject, see Semenza¹⁴⁹ and also Brown and Wilson¹⁴⁴).

Sepsis

Globally, uncontrolled infection (sepsis) is responsible for 721,000 to 2.1 million deaths/year.¹⁶³-166 As in many disease processes, oxygen supply and demand and/or subcellular derangements are thought to play a prominent role in the manifestation of sepsis. Specifically, septic pathology may be thought of in terms of three distinct strata, all of which may be abnormal - the macrovasculature (blood pressure, cardiac output, hemoglobin concentration, arterial oxygen saturation, mixed venous oxygen saturation), the microvasculature (small vessels which bring red blood cells into close proximity to tissues that need oxygen and are deranged during sepsis), and the subcellular apparatus (mitochondria and electron transport chain). [Figure 6] Therapies designed to improve macrovascular function have largely been a failure.¹⁶⁷-169 Trials targeting microcirculatory dysfunction have yielded conflicting results.¹⁷⁰

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Figure 6

Sepsis may be best conceptualized in terms of three distinct strata - the macrovasculature (blood pressure, cardiac output, hemoglobin concentration, arterial oxygen saturation, mixed venous oxygen saturation), the microvasculature, and the subcellular apparatus (mitochondria and electron transport chain, reactive species).

Targeting subcellular processes in sepsis has been challenging. Mitochondrial dysfunction plays a prominent role in sepsis.¹⁷¹,172 Total detectable cytochrome levels fall during the initiation of sepsis,¹⁷¹ and cytochrome aa₃ transitions from an oxidized to a reduced state as sepsis progresses in a variety of sepsis models,¹⁷³-176 despite the maintenance of adequate oxygen delivery. Animal studies of experimental sepsis (lipopolysaccharide [LPS] infusion) have demonstrated mitochondrial swelling, respiratory dysfunction, and partial uncoupling of oxidative phosphorylation.¹⁷⁷

Several investigators have attempted to better understand oxygenation at the level of the cell, using tissue oxygen tension measurements. These studies, which have been conducted in septic rat models (LPS and cecal ligation and puncture [CLP]), pigs (LPS), and humans, have not produced consistent results. ¹⁷⁹,182,186,190,196,201-206 Hotchkiss examined the impact of CLP in rodents using the cellular hypoxia marker [18F]fluoromisonidazole, and found no evidence of cellular hypoxia during sepsis.¹⁹⁵ Of note, these studies examined intracellular oxygenation at varying time points, ranging from 0-42 hours after initiation of experimental sepsis.

Rather than focus on tissue oxygen tension, some investigators have attempted to measure high energy phosphate levels. As with the tissue oxygen tension experiments, initial animal work utilizing P₃₁ nuclear magnetic resonance (NMR) to measure high energy phosphates (e.g. ATP) have also produced conflicting results.¹⁸⁹,193,194,197,198,200 These early studies utilized inconsistent, single time points, and did not discriminate between survivors and non-survivors. A more recent animal study demonstrated nitric oxide overproduction and mitochondrial dysfunction (exhibited by both ATP depletion and complex I inhibition) that generally worsened over time and with increasing sepsis severity.²⁰⁸ Singer and Brealey synthesized data from multiple animal models of varying severity and time points, and found that mitochochondrial dysfunction does occur over longer periods of time and with higher severity.²⁰⁹

Early human studies reiterated these longer-term animal findings. Muscle ATP levels are lower in septic patients than in those undergoing elective surgical procedures.²¹⁰,211 More recently, Brealey et al. analyzed vastus lateralis muscle samples in septic humans and found that non-survivors had lower tissue ATP, less complex I (NADH dehydrogenase) activity, and complex IV (aa₃; c oxidase) activity than sepsis survivors and non-septic controls.²⁰⁷ Similarly, Fredriksson et al. found lower ATP levels in the leg muscles of septic patients as compared to healthy controls undergoing elective surgery. Additionally, this group found reductions in complex I and IV activity in intercostal and leg muscles, respectively, as well as evidence of increased mitochondrial oxidative stress.²¹² The growing appreciation that decreased oxygen consumption is due to defects in cellular respiration, rather than abnormal oxygen delivery has led to the development of the term “cytopathic hypoxia.”²¹³ It is not clear whether this mitochondria-driven reduction in oxygen consumption and accompanying “metabolic shutdown” is harmful or an attempt at organ preservation by down-regulating organ function and thus the requirement for high energy phosphates, or some combination of the two. Key elements of this metabolic shutdown include mitochondrial inhibition, reduced mitochondrial biogenesis, and decreased hormonal stimulation.²¹⁴ Sepsis-induced organ quiescence is reversible if the organism survives.²⁰⁸,214-216

Interestingly, the increase in inflammatory mediators that accompanies sepsis (as well as ischemia-reperfusion injury, see Ischemia-Reperfusion and Reactive Oxygen Species above) results in overproduction of ROS and RNS, both of which induce mitochondrial dysfunction. HIF-1, which is protective in ischemia-reperfusion injury⁸⁸,217-220 and preconditioning,¹⁹,20 exerts control over mitochondrial metabolism,⁸⁸ and appears to be upregulated in the setting of sepsis.²²¹ The complex link between HIF-1 and ROS/RNS (ROS stabilize HIF-1,⁹⁷-100 and ROS scavengers have been shown to destabilize HIF-1,⁴⁷,98,99,101 as described above) may explain why, despite some promising small pre-clinical trials, a phase III trial of NOS inhibition in humans with severe sepsis increased mortality in the treatment group.²²² Further strengthening the theory that hypoxia, HIF-1, and inflammatory disarray (e.g. infection, ischemia) are related is the connection between hypoxia and the immune system - hypoxia increases the expression of Toll-like receptors TLR2 and TLR6, and HIF-1 enhances the bactericidal activity of phagocytes and enhances innate immunity in a variety of cell types.²²³-225

Conclusions

While oxygen is often considered an essential component of life, the natural world provides multiple examples of complex organisms that survive, and in some cases thrive, in hypoxic or anoxic environments. These include, but are not limited to all mammalian embryos, as well as some hibernating and diving mammals. Additionally, the development of hypoxia tolerance improves the survival of ischemic myocardium (through preconditioning) and increases cancer metastasis. HIF-1 is central to hypoxic adaptation, activating over 1500 target genes and leads to angiogenesis, a shift towards anaerobic metabolism, and electron transport chain modifications which increase electron transfer efficiency, enable the activation of iNOS, and control the balance of ROS. ROS are both essential (vasodilation, decreased platelet and neutrophil adhesion) and potentially harmful (oxidative stress), and the ability of organisms to manage ROS is relevant to both preconditioning and the response to uncontrolled infection and inflammation. Greater understanding of the similarities between various pathologies and evolutionary adaptations, especially regarding the electron transport chain, production and attenuation of reactive species, and alternate metabolic pathways, may allow for the development of novel treatment modalities (discussed in the second part of this review).

University of Virginia

Robert H. Thiele, M.D.

Role: This author wrote the manuscript

Conflicts: Robert H. Thiele reported no conflicts of interest

Attestation: Robert H. Thiele approved the final manuscript

Corresponding Author: Robert H. Thiele, M.D., University of Virginia, Department of Anesthesiology PO Box 800710, Charlottesville, VA 22908-0710, Phone: 434-243-9412, FAX: 434-982-0019, ude.ainigriv@w7thr

Abstract

While it is generally believed that oxidative phosphorylation and adequate oxygenation are essential for life, human development occurs in a profoundly hypoxic environment and “normal” levels of oxygen during embryogenesis are even harmful. The ability of embryos to not only survive, but thrive in such an environment is made possible by adaptations related to metabolic pathways. Similarly, cancerous cells are able to not only survive, but grow and spread in environments that would typically be fatal for healthy adult cells. Many biological states, both normal and pathological, share underlying similarities related to metabolism, the electron transport chain, and reactive species. The purpose of Part I of this review is to review the similarities between embryogenesis, mammalian adaptions to hypoxia (primarily driven by hypoxia inducible factor-1), ischemia-reperfusion injury (and its relationship with reactive oxygen species), hibernation, diving animals, cancer, and sepsis, with a particular focus on the common characteristics that allow cells and organisms to survive in these states.

Abstract

Footnotes

Funding: NIGMS (1K08GM115861-01A1)

Information for LWW regarding depositing manuscript into PubMed Central: This paper does not need to be deposited in PubMed Central.

Footnotes

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