Survival under stress: molecular mechanisms of metabolic rate depression in animals

For many species, survival under harsh environmental conditions includes metabolic rate depression, an escape into a hypometabolic or dormant state. Studies in my laboratory are analysing the molecular mechanisms and regulatory events that underlie transitions to and from hypometabolic states In systems including anoxia-tolerant turtles and molluscs, estivating snails and toads, hibernating small mammals, and freeze tolerant frogs and insects. Our newest research targets two areas: the role of protein kinases in regulating metabolic adjustments and the role of stress-induced gene expression in producing specific adaptive proteins. Protein kinases A, C and G are all linked to stress-induced signal transduction in various systems, and new studies also show tissue-specific activation of mitogen-activated protein kinases (ERK, JNK, see list of abbreviations p38). Protein adaptations supporting stress tolerance are being sought using cDNA library screening, differential display PCR and Northern blotting to analyse gene expression. These techniques offer new insights into the types of cellular targets that must be coordinated to achieve metabolic suppression and facilitate easy analysis of organ-, time-, and stress-specific gene expression. For example, freeze-induced gene expression in frog liver includes upregulation of genes for subunits of fibrinogen and ADP/ATP translocase, whereas mitochondrial genes coding for subunits of NADH-ubiquinone oxidoreductase subunit 5 and cytochrome C oxidase subunit 1 were upregulated during anoxia in turtle heart

Forever young: Mechanisms of natural anoxia tolerance and potential links to longevity

While mammals cannot survive oxygen deprivation for more than a few minutes without sustaining severe organ damage, some animals have mastered anaerobic life. Freshwater turtles belonging to the Trachemys and Chrysemys genera are the champion facultative anaerobes of the vertebrate world, often surviving without oxygen for many weeks at a time. The physiological and biochemical mechanisms that underlie anoxia tolerance in turtles include profound metabolic rate depression, post-translational modification of proteins, strong antioxidant defenses, activation of specific stress-responsive transcription factors, and enhanced expression of cyto-protective proteins. Turtles are also known for their incredible longevity and display characteristics of “negligible senescence.” We propose that the robust stress-tolerance mechanisms that permit long term anaerobiosis by turtles may also support the longevity of these animals. Many of the mechanisms involved in natural anoxia tolerance, such as hypometabolism or the induction of various protective proteins/pathways, have been shown to play important roles in mammalian oxygen-related diseases and improved understanding of how cells survive without oxygen could aid in the understanding and treatment of various pathological conditions that involve hypoxia or oxidative stress. In the present review we discuss the recent advances made in understanding the molecular nature of anoxia tolerance in turtles and the potential links between this tolerance and longevity

Living without Oxygen: Anoxia-Responsive Gene Expression and Regulation

Many species of marine mollusks demonstrate exceptional capacities for long term survival without oxygen. Analysis of gene expression under anoxic conditions, including the subsequent translational responses, allows examination of the functional mechanisms that support and regulate natural anaerobiosis and permit noninjurious transitions between aerobic and anoxic states. Identification of stress-specific gene expression can provide important insights into the metabolic adaptations that are needed for anoxia tolerance, with potential applications to anoxia-intolerant systems. Various methods are available to do this, including high throughput microarray screening and construction and screening of cDNA libraries. Anoxia-responsive genes have been identified in mollusks; some have known functions in other organisms but were not previously linked with anoxia survival. In other cases, completely novel anoxia-responsive genes have been discovered, some that show known motifs or domains that hint at function. Selected genes are expressed at different times over an anoxia-recovery time course with their transcription and translation being actively regulated to ensure protein expression at the optimal time. An examination of transcript status over the course of anoxia exposure and subsequent aerobic recovery identifies genes, and the proteins that they encode, that enhance cell survival under oxygen-limited conditions. Analysis of data generated from non-mainstream model systems allows for insight into the response by cells to anoxia stress

Glucose-6-Phosphate Dehydrogenase Regulation in Anoxia Tolerance of the Freshwater Crayfish Orconectes virilis

Glucose-6-phosphate dehydrogenase (G6PDH), the enzyme which catalyzes the rate determining step of the pentose phosphate pathway (PPP), controls the production of nucleotide precursor molecules (R5P) and powerful reducing molecules (NADPH) that support multiple biosynthetic functions, including antioxidant defense. G6PDH from hepatopancreas of the freshwater crayfish (Orconectes virilis) showed distinct kinetic changes in response to 20 h anoxic exposure. Km values for both substrates decreased significantly in anoxic crayfish; Km NADP+ dropped from 0.015 ± 0.008 mM to 0.012 ± 0.008 mM, and Km G6P decreased from 0.13 ± 0.02 mM to 0.08 ± 0.007 mM. Two lines of evidence indicate that the mechanism involved is reversible phosphorylation. In vitro incubations that stimulated protein kinase or protein phosphatase action mimicked the effects on anoxia on Km values, whereas DEAE-Sephadex chromatography showed the presence of two enzyme forms (low- and high-phosphate) whose proportions changed during anoxia. Incubation studies implicated protein kinase A and G in mediating the anoxia-responsive changes in G6PDH kinetic properties. In addition, the amount of G6PDH protein (measured by immunoblotting) increased by ∼60% in anoxic hepatopancreas. Anoxia-induced phosphorylation of G6PDH could contribute to modifying carbon flow through the PPP under anoxic conditions, potentially maintaining NADPH supply for antioxidant defense during prolonged anoxia-induced hypometabolism

An Overview of Stress Response and Hypometabolic Strategies in Caenorhabditis elegans: Conserved and Contrasting Signals with the Mammalian System

Studies of the molecular mechanisms that are involved in stress responses (environmental or physiological) have long been used to make links to disease states in humans. The nematode model organism, Caenorhabditis elegans, undergoes a state of hypometabolism called the 'dauer' stage. This period of developmental arrest is characterized by a significant reduction in metabolic rate, triggered by ambient temperature increase and restricted oxygen/ nutrients. C. elegans employs a number of signal transduction cascades in order to adapt to these unfavourable conditions and survive for long times with severely reduced energy production. The suppression of cellular metabolism, providing energetic homeostasis, is critical to the survival of nematodes through the dauer period. This transition displays molecular mechanisms that are fundamental to control of hypometabolism across the animal kingdom. In general, mammalian systems are highly inelastic to environmental stresses (such as extreme temperatures and low oxygen), however, there is a great deal of conservation between the signal transduction pathways of nematodes and mammals. Along with conserving many of the protein targets in the stress response, many of the critical regulatory mechanisms are maintained, and often differ only in their level of expression. Hence, the C. elegans model outlines a framework of critical molecular mechanisms that may be employed in the future as therapeutic targets for addressing disease states

Perspectives in Cell Cycle Regulation: Lessons from an Anoxic Vertebrate

The ability of an animal, normally dependent on aerobic respiration, to suspend breathing and enter an anoxic state for long term survival is clearly a fascinating feat, and has been the focus of numerous biochemical studies. When anoxia tolerant turtles are faced with periods of oxygen deprivation, numerous physiological and biochemical alterations take place in order to facilitate vital reductions in ATP consumption. Such strategies include reversible post-translational modifications as well as the implementation of translation and transcription controls facilitating metabolic depression. Although it is clear that anoxic survival relies on the suppression of ATP consuming processes, the state of the cell cycle in anoxia tolerant vertebrates remain elusive. Several anoxia tolerant invertebrate and embryonic vertebrate models display cell cycle arrest when presented with anoxic stress. Despite this, the cell cycle has not yet been characterized for anoxia tolerant turtles. Understanding how vertebrates respond to anoxia can have important clinical implications. Uncontrollable cellular proliferation and hypoxic tumor progression are inescapably linked in vertebrate tissues. Consequentially, the molecular mechanisms controlling these processes have profound clinical consequences. This review article will discuss the theory of cell cycle arrest in anoxic vertebrates and more specifically, the control of the retinoblastoma pathway, the molecular markers of cell cycle arrest, the activation of checkpoint kinases, and the possibility of translational controls implemented by microRNAs

Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica

<p>Abstract</p> <p>Background</p> <p>The wood frog, <it>Rana sylvatica</it>, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle.</p> <p>Results</p> <p>Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (<it>S</it>_0.5AMP was reduced, Hill coefficient fell to ~1.0) and the transition to the frozen state led to a 3-fold increase in <it>S</it>_0.5AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low <it>S</it>_0.5AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high <it>S</it>_0.5AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH₄Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. <it>S</it>_0.5AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing.</p> <p>Conclusion</p> <p>Maintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.</p

Myostatin levels in skeletal muscle of hibernating ground squirrels

Myostatin, a negative regulator of muscle mass, is elevated during disuse and starvation. Mammalian hibernation presents a unique scenario, where animals are hypocaloric and in torpor, but the extent of muscle protein loss is minimized. We hypothesized that myostatin expression, which is usually increased early in disuse and under hypocaloric conditions, could be suppressed in this unique model. Skeletal muscle was collected from thirteen-lined ground squirrels, Spermophilus tridecemlineatus, at six time points during hibernation: control euthermic (CON); entrance into hibernation (ENT), body temperature (Tb) falling; early hibernation (EHib), stable Tb in torpor for 24 h; late hibernation (LHib), stable Tb in torpor for 3 days; early arousal (EAr), Tb rising; and arousal (AR), Tb restored to 34-37&deg;C for about 18 h. There was no significant increase of myostatin during ENT, EHib or LHib. Unexpectedly, there were approximately sixfold increases in myostatin protein levels as squirrels arose from torpor. The elevation during EAr remained high in AR, which represented an interbout time period. Mechanisms that could release the suppression or promote increased levels of myostatin were assessed. SMAD2 and phosphorylated SMAD2 were increased during EHib, but only the phosphorylated SMAD2 during AR mirrored increases in myostatin. Follistatin, a negative regulator of myostatin, did not follow the same time course as myostatin or its signaling pathway, indicating more control of myostatin at the signaling level. However, SMAD7, an inhibitory SMAD, did not appear to play a significant role during deep hibernation. Hibernation is an excellent natural model to study factors involved in the endogenous intracellular mechanisms controlling myostatin

Angiogenic signaling in the lungs of a metabolically suppressed hibernating mammal (Ictidomys tridecemlineatus)

To conserve energy in times of limited resource availability, particularly during cold winters, hibernators suppress even the most basic of physiologic processes. Breathing rates decrease from 40 breaths/minute to less than 1 breath/min as they decrease body temperature from 37oC to ambient. Nevertheless, after months of hibernation, these incredible mammals emerge from torpor unscathed. This study was conducted to better understand the protective and possibly anti-inflammatory adaptations that hibernator lungs may use to prevent damage associated with entering and emerging from natural torpor. We postulated that the differential protein expression of soluble protein receptors (decoy receptors that sequester soluble ligands to inhibit signal transduction) would help identify inhibited inflammatory signaling pathways in metabolically suppressed lungs. Instead, the only two soluble receptors that responded to torpor were sVEGFR1 and sVEGFR2, two receptors whose full-length forms are bound by VEGF-A to regulate endothelial cell function and angiogenesis. Decreased sVEGFR1/2 correlated with increased total VEGFR2 protein levels. Maintained or increased levels of key ã-secretase subunits suggested that decreased sVEGFR1/2 protein levels were not due to decreased levels of intramembrane cleavage complex subunits. VEGF-A protein levels did not change, suggesting that hibernators may regulate VEGFR1/2 signaling at thes level of the receptor instead of increasing relative ligand

Aestivation: Signaling and hypometabolism

Aestivation is a survival strategy used by many vertebrates and invertebrates to endure arid environmental conditions. Key features of aestivation include strong metabolic rate suppression, strategies to retain body water, conservation of energy and body fuel reserves, altered nitrogen metabolism, and mechanisms to preserve and stabilize organs, cells and macromolecules over many weeks or months of dormancy. Cell signaling is crucial to achieving both a hypometabolic state and reorganizing multiple metabolic pathways to optimize long-term viability during aestivation. This commentary examines the current knowledge about cell signaling pathways that participate in regulating aestivation, including signaling cascades mediated by the AMPactivated kina