Janet M. Storey

Metabolic Rate Depression and Biochemical Adaptation in Anaerobiosis, Hibernation and Estivation

For many animals, the best defense against harsh environmental conditions is an escape to a hypometabolic or dormant state. Facultative metabolic rate depression is the common adaptive strategy of anaerobiosis, hibernation, and estivation, as well as a number of other arrested states. By reducing metabolic rate by a factor ranging from 5 to 100 fold or more, animals gain a comparable extension of survival time that can support months or even years of dormancy. The present review focuses on the molecular control mechanisms that regulate and coordinate cellular metabolism for the transition into dormancy. These include reversible control over the activity state of enzymes via protein phosphorylation or dephosphorylation reactions, pathway regulation via the association or dissociation of particle-bound enzyme complexes, and fructose-2, 6-bisphosphate regulation of the use of carbohydrate reserves for biosynthetic purposes. These mechanisms, their interactions, and the regulatory signals (e.g., second messenger molecules, pH) that coordinate them form a common molecular basis for metabolic depression in anoxia-tolerant vertebrates (goldfish, turtles) and invertebrates (marine molluscs), hibernation in small mammals, and estivation in land snails and terrestrial toads.

Metabolic rate depression in animals: transcriptional and translational controls

Metabolic rate depression is an important survival strategy for many animal species and a common element of hibernation, torpor, aestivation, anaerobiosis, diapause, and anhydrobiosis. Studies of the biochemical mechanisms that regulate reversible transitions to and from hypometabolic states are identifying principles of regulatory control that are conserved across phylogenetic lines and that are broadly applied to the control of multiple cell functions. One such mechanism is reversible protein phosphorylation which is now known to contribute to the regulation of fuel metabolism, to ion channel arrest, and to the suppression of protein synthesis during hypometabolism. The present review focuses on two new areas of research in hypometabolism:(1) the role of differential gene expression in supplying protein products that adjust metabolism or protect cell functions for long‐term survival, and (2) the mechanisms of protein life extension in hypometabolism involving inhibitory controls of transcription, translation and protein degradation. Control of translation examines reversible phosphorylation regulation of ribosomal initiation and elongation factors, the dissociation of polysomes and storage of mRNA transcripts during hypometabolism, and control over the translation of different mRNA types by differential sequestering of mRNA into polysome versus monosome fractions. The analysis draws primarily from current research on two animal models, hibernating mammals and anoxia‐tolerant molluscs, with selected examples from multiple other sources.

Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails

The roles of enzymatic antioxidant defenses in the natural tolerance of environmental stresses that impose changes in oxygen availability and oxygen consumption on animals is discussed with a particular focus on the biochemistry of estivation and metabolic depression in pulmonate land snails. Despite reduced oxygen consumption and PO2 during estivation, which should also mean reduced production of oxyradicals, the activities of antioxidant enzymes, such as superoxide dismutase and catalase, increased in 30 day-estivating snails. This appears to be an adaptation that allows the snails to deal with oxidative stress that takes place during arousal when PO2 and oxygen consumption rise rapidly. Indeed, oxidative stress was indicated by increased levels of lipid peroxidation damage products accumulating in hepatopancreas within minutes after arousal was initiated. The various metabolic sites responsible for free radical generation during arousal are still unknown but it seems unlikely that the enzyme xanthine oxidase plays any substantial role in this despite being implicated in oxidative stress in mammalian models of ischemia/reperfusion. We propose that the activation of antioxidant defenses in the organs of Otala lactea during estivation is a preparative mechanism against oxidative stress during arousal. Increased activities of antioxidant enzymes have also observed under other stress situations in which the actual production of oxyradicals should decrease. For example, antioxidant defenses are enhanced during anoxia exposure in garter snakes Thamnophis sirtalis parietalis (10 h at 5 degrees C) and leopard frogs Rana pipiens (30 h at 5 degrees C) and during freezing exposure (an ischemic condition due to plasma freezing) in T. sirtalis parietalis and wood frogs Rana sylvatica. It seems that enhancement of antioxidant enzymes during either anoxia or freezing is used as a preparatory mechanism to deal with a physiological oxidative stress that occurs rapidly within the early minutes of recovery during reoxygenation or thawing. Thus, a wide range of stress tolerant animals display coordinated changes in antioxidant defenses that allow them to deal with oxidative stress that occurs as part of natural cycles of stress/recovery that alter oxygen levels in tissues. The molecular mechanisms that trigger and regulate changes in antioxidant enzyme activities in these species are still unknown but could prove to have key relevance for the development of new intervention strategies in the treatment of cardiovascular ischemia/reperfusion injuries in humans.

Biochemistry of Cryoprotectants

The role of polyhydric alcohols in cryoprotection is probably the most extensively studied feature of insect cold hardiness. The importance of glycerol as a cryoprotectant was first recognized by R. W. Salt after he and others linked the presence of high levels of glycerol with winter hibernation, diapause, or freezing survival (Salt, 1957, 1959, 1961; Wyatt and Kalf, 1957; Chino, 1957). Over the last 30 years, literally hundreds of publications have described the occurrence of glycerol or other polyols in both freeze-tolerant and freeze-avoiding insects (for reviews, see Salt, 1961; Hansen, 1980; Ring, 1980; Somme, 1982; Miller, 1982; Duman et al., 1982; Baust et al., 1982; Zachariassen, 1985; Lee et al., 1986; Storey and Storey, 1988). Glycerol is by far the most common cryoprotectant, but sorbitol, mannitol, ribitol, erythritol, threitol, and ethylene glycol also occur along with a selection of sugars, including trehalose, sucrose, glucose, and fructose (see Fig. 4.1) (Miller and Smith, 1975; Hayakawa and Chino, 1981; Somme, 1982; Gehrken, 1984; Zachariassen, 1985; Hamilton et al., 1985; Storey and Storey, 1988). Glycerol contents that range as high as 25% of the fresh weight of the animal have been reported with polyol concentrations in excess of 2 M in the body fluids of many species (Salt, 1961; Ring, 1981; Zachariassen, 1985; Storey and Storey, 1988). The majority of species produce only a single polyol, but dual or even multiple component systems also occur, glycerol plus sorbitol being the most common pairing (Storey and Storey, 1988).

Tribute to P. L. Lutz: putting life on 'pause'--molecular regulation of hypometabolism.

SUMMARY Entry into a hypometabolic state is an important survival strategy for many organisms when challenged by environmental stress, including low oxygen, cold temperatures and lack of food or water. The molecular mechanisms that regulate transitions to and from hypometabolic states, and stabilize long-term viability during dormancy, are proving to be highly conserved across phylogenic lines. A number of these mechanisms were identified and explored using anoxia-tolerant turtles as the model system, particularly from the research contributions made by Dr Peter L. Lutz in his explorations of the mechanisms of neuronal suppression in anoxic brain. Here we review some recent advances in understanding the biochemical mechanisms of metabolic arrest with a focus on ideas such as the strategies used to reorganize metabolic priorities for ATP expenditure, molecular controls that suppress cell functions (e.g. ion pumping, transcription, translation, cell cycle arrest), changes in gene expression that support hypometabolism, and enhancement of defense mechanisms (e.g. antioxidants, chaperone proteins, protease inhibitors) that stabilize macromolecules and promote long-term viability in the hypometabolic state.

Intermediary metabolism during low temperature acclimation in the overwintering gall fly larva,Eurosta solidaginis

Summary1.The levels of glycogen, lipid, protein, polyols (glycerol and sorbitol), sugars, amino acids, adenylates, and other intermediary metabolites were measured in the overwintering, third instar larvae of the gall fly,Eurosta solidaginis, sampled at specified temperatures during a controlled (1°C per day decrease) low temperature acclimation of the larvae from 15° to − 30°C.2.Glycogen reserves were depleted as temperature was decreased, the decrease in glycogen fully accounting for the observed increases in glycerol, sorbitol, glucose, and trehalose in the larvae at low temperatures. Protein and total glyceride reserves of the larvae, however, were not altered during low temperature acclimation.3.Temperature specific patterns of glycerol and sorbitol accumulation were found. Glycerol concentrations, which were 65% of maximum at 15°C, reached a plateau in concentration of 235 μmol/g wet wt. between 5 and 0°C. Sorbitol first appeared in larvae at 5° C and then increased in concentration rapidly as temperature decreased further to reach a plateau level of 145 μmol/g wet wt. by −10°C.4.The free amino acid pool increased in size by 50% during acclimation from 15 to −5°C, this increase due largely to a 24 μmol/g wet wt. increase in proline concentration and a smaller 4.4 μmol/g wet wt. increase in alanine.5.Arginine phosphate and ATP levels, as well as energy charge and the ratio [ATP]/[ADP]·[Pi], remained high and constant in larvae acclimated to temperatures as low as −5°C but in larvae acclimated to −30°C phosphagen and ATP levels had declined by 54 and 29% respectively and energy charge had decreased from 0.92 to 0.82.6.The data suggest that aerobic metabolism with continued polyol synthesis is fully active in these larvae at temperatures as low as −10°C. However, below −10°C, the temperature at which hemolymph freezing takes place, mitochondria appear to be metabolically inactive. Evidence for this includes the cessation of polyol, sugar, and amino acid accumulation by this temperature and the drop in arginine phosphate, ATP, and energy charge and build-up of lactate at −30°C.7.The regulation of metabolism inE. solidaginis larvae during low temperature acclimation is discussed with particular emphasis on the possible metabolic ‘switches’ regulating the flow of carbon to glycerol versus sorbitol synthesis.

Biochemical adaption for freezing tolerance in the wood frog, Rana sylvatica

Summary1.The wood frog,Rana sylvatica, can survice extracellular freezing during overwintering. Under laboratory conditions freezing occurred at about −2°C and animals survived several days at −6°C.2.Frogs accumulated glucose as a cryoprotectant; glycerol, sorbitol and other sugars were not produced. Average levels of glucose in frozen antimals were 185±40 μmol/ml in blood and 387.8±44.8, 198.3±27.3, 120.8±14.1 and 26.5±2.7 μmol/g wet weight in liver, heart, kidney and leg muscle, respectively.3.Two methods of cold acclimation, 11 weeks at 3°C or a 1°C per day decrease in temperature from 23°C to 0°C, failed to stimulate an anticipatory rise in glucose levels. Only direct exposure to subzero temperature between 0°C and −2°C stimulated synthesis.4.Glucose synthesis appeared to be confined to liver with glucose distributed through the blood to other tissues. Freezing exposure resulted in a decrease in liver glycogen content of over 700 μmol/g wet weight while glycogen content of other tissues was not affected.5.Activities of 17 enzymes in liver and leg muscle were monitored in control and freezing exposed frogs. Freezing exposure increased liver phosphorylase activity by 520% from 3 to 18.6 units/g wet weight and increased phosphorylasea content from 37 to 80%. Freezing also elevated glucose-6-phosphatase activity by 140%. Activities of most other enzymes in liver increased by 30–90% with freezing exposure. Activities of phosphorylase and glucose-6-phosphatase increased in leg muscle with freezing exposure although phosphorylasea content remained at 35%.6.Freezing exposure resulted in the accumulation of lactate in all tissues. Both total adenylates and adenylate energy charge decreased in liver during freezing. In leg muscle adenylates were unaffected but creatine phosphate reserves were depleted.

Low toxic herbicide Roundup induces mild oxidative stress in goldfish tissues

The formulation of Roundup consists of the herbicide glyphosate as the active ingredient with polyethoxylene amine added as a surfactant. The acute toxicity of Roundup (particularly of glyphosate) to animals is considered to be low according to the World Health Organization, but the extensive use of Roundup may still cause environmental problems with negative impact on wildlife, particularly in an aquatic environment where chemicals may persist for a long time. Therefore, we studied the effects of Roundup on markers of oxidative stress and antioxidant defense in goldfish, Carassius auratus. The fish were given 96 h exposure to Roundup at concentrations of 2.5-20 mg L(-1). Exposure to Roundup did not affect levels of lipid peroxides (LOOH) in goldfish brain or liver, and in kidney only the 10 mg L(-1) treatment elevated LOOH by 3.2-fold. Herbicide exposure also had no effect on the concentrations of protein thiols or low molecular mass thiols in kidney, but selective suppression of low molecular mass thiols by 26-29% occurred at some treatment levels in brain and liver. Roundup exposure generally suppressed the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione reductase and glucose-6-phosphate dehydrogenase in fish tissues. For example, SOD activities were reduced by 51-68% in brain, 58-67% in liver and 33-53% in kidney of Roundup treated fish. GST activity decreased by 29-34% in liver. However, catalase activity increased in both liver and kidney of herbicide-exposed fish. To our knowledge this is the first study to demonstrate a systematic response by the antioxidant systems of fish to Roundup exposure.

Freeze tolerance and intolerance as strategies of winter survival in terrestrially-hibernating amphibians

The ability to tolerate extracellular freezing as an adaptation for winter survival was tested in seven species of terrestrially-hibernating amphibians found in eastern Canada. All species had only moderate supercooling abilities, with whole animal supercooling points of -1.5 to -3 degrees C. Two salamander species, Plethodon cinereus and Ambystoma laterale, and the toad, Bufo americanus, were freezing intolerant and were killed when frozen for 24 hr at temperatures just below their supercooling points. The major winter strategy of these animals appears to behavioural avoidance of subzero temperatures. Four species of frogs Rana sylvatica, Hyla versicolor, Hyla crucifer and Pseudacris triseriata, survived extracellular freezing at moderate subzero temperatures (-2 to -4 degrees C) for periods of time ranging up to 2 weeks. All four frog species accumulated low molecular weight carbohydrates as cryoprotectants, glycerol being the major cryoprotectant in adult H. versicolor, while immature adults of this species as well as the other three species all produced high levels of glucose as the cryoprotectant.

Regulation of cryoprotectant metabolism in the overwintering gall fly larva,Eurosta solidaginis: Temperature control of glycerol and sorbitol levels

SummaryAbrupt temperature change, from 23 to 13 °C, 13 to 3 °C or vice versa, was used to study the metabolic events associated with cryoprotectant polyol synthesis and the reversibility of polyol accumulations in the overwintering, freezing tolerant larvae of the gall fly,Eurosta solidaginis.Sorbitol synthesis was induced when larvae acclimated to 13 °C were abruptly moved to 3 °C. A precursor-product relationship between glucose-6-P, glucose and sorbitol was apparent with elevated levels of the compounds in the larvae first detected after 1, 2 and 24 h at 3 °C, respectively. A negative cross-over (increase in fructose-6-P, decrease in fructose-1,6-P2 levels) at phosphofructokinase at 3 °C demonstrated that inhibition at this locus was responsible for the diversion of carbon flow into sorbitol synthesis.Glycerol synthesis was stimulated when larvae acclimated to 23 °C were chilled to 13 °C, with increased glycerol levels first apparent after 2 days at 13 °C. Synthesis was accomplished via an activation of glycogenolysis coupled with a facilitation of flux through the phosphofructokinase locus and an inhibition (negative cross-over) of flux at the pyruvate kinase reaction resulting in a diversion of triose phosphates into the pathway of glycerol synthesis.Warming of the larvae resulted in a rapid catabolism of sorbitol, with a restoration of glycogen reserves, when larvae were switched from 3 to 13 °C. Glycerol content of the larvae, however, did not respond to warming and remained constant when larvae were moved from 13 to 23 °C.The two cyoprotectants appear to have different roles in the overwintering larvae. Glycerol, once synthesized, provides a constant and permanent cryoprotection throughout the winter. Accumulation of this polyol also appears to be anticipatory occurring in response to chilling at relatively high temperatures, well above those at which cryoprotection is needed. Sorbitol, however, is produced only in direct response to cold when freezing temperatures are imminent. Sorbitol provides a variable cryoprotection, levels of the polyol responding to increases or decreases in ambient temperature.