Kenneth B. 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.

Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress

SUMMARY The mitogen-activated protein kinase (MAPK) superfamily consists of three main protein kinase families: the extracellular signal-regulated protein kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 family of kinases. Each is proving to have major roles in the regulation of intracellular metabolism and gene expression and integral actions in many areas including growth and development, disease, apoptosis and cellular responses to external stresses. To date, this cellular signal transduction network has received relatively little attention from comparative biochemists, despite the high probability that MAPKs have critical roles in the adaptive responses to thermal, osmotic and oxygen stresses. The present article reviews the current understanding of the roles and regulation of ERKs, JNKs and p38, summarizes what is known to date about MAPK roles in animal models of anoxia tolerance, freeze tolerance and osmoregulation, and highlights the potential that studies of MAPK pathways have for advancing our understanding of the mechanisms of biochemical adaptation.

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.

Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation

Commonly used spectrophotometric methods for determining the extent of lipid peroxidation in animal tissue extracts, such as measurements of diene conjugation and thiobarbituric acid reactive substances (TBARS), have been criticized for their lack of specificity. This study shows that lipid hydroperoxides can be effectively quantified in animal tissue extracts using an assay based on the formation of a Fe(III)xylenol orange complex. Addition of H2O2, cumene hydroperoxides, or methanolic tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)xylenol orange complex absorbance peak at 560-580 nm with a corresponding decrease in the xylenol orange peak at 440 nm. Complex formation measured at 580 nm was saturable with both xylenol orange and Fe (II) concentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) caused a saturable reduction of the Fe(III)xylenol orange complex. Formation of the Fe(III)xylenol orange complex was linear with the amount of tissue extract added. A significant correlation (r = 0.88, p < 0.005) existed between the xylenol orange method of estimating lipid peroxidation and the conventional TBARS assay in a series of animal tissues tested. The time course of increase in A580nm in tests using tissue extracts was typical of a free radical reaction; a lag phase was followed by a log phase. No increase in A580nm was observed up to 24 h when highly peroxidizable arachidonic acid was assayed. These results indicate that the formation of the Fe(III)xylenol orange complex reflects a chemical amplification of the original level of lipid hydroperoxides present in tissue extracts and that peroxidizable lipids do not influence the assay. The potential usefulness of the xylenol orange assay for comparative biochemical and toxicological studies of oxidative stress is discussed.

Bound and determined : a computer program for making buffers of defined ion concentrations

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.

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).

Functional metabolism : regulation and adaptation

In the post-genome era, questions concerning gene products, enzymes, and metabolism are returning to the forefront of life science research. Genetic information on its own does not fully account for an enzyme’s kinetic and regulatory properties or for the behavior of the enzymes within its particular cellular milieu. Unanswered questions about the regulation, integration, and adaptation of metabolism have led to a resurgence of interest in protein, enzymological, and metabolic research for understanding biological processes. Functional Metabolism: Regulation and Adaptation provides a comprehensive survey of metabolism. It includes an in-depth examination of the regulation of carbohydrates, lipids, and amino acids, and approaches to the study of enzyme regulation, signal transduction, and control of transcription and translation. The contributors–an internationally recognized group of researchers–also cover: The metabolic basis of diabetes, obesity, and blood disorders Oxidative stress and antioxidant defenses in health and disease Novel perspectives on biochemical adaptation to environmental stress Application of metabolic knowledge to developments in organ preservation and understanding the origin of life From the basics of metabolic regulation and adaptation to the latest relevant advances in the genetic, proteomic, and enzymatic basis of how cells regulate their functions, Functional Metabolism: Regulation and Adaptation offers the most exhaustive treatment of the subject currently available. It is an essential text for students and practitioners in biochemistry, cell and molecular biology, and biomedicine (Publisher summary).

Life in the slow lane: molecular mechanisms of estivation ☆

Estivation is a state of aerobic hypometabolism used by organisms to endure seasonally arid conditions, often in desert environments. Estivating species are often active for only a few weeks each year to feed and breed and then retreat to estivate in sheltered sites, often underground. In general, estivation includes a strong reduction in metabolic rate, a primary reliance on lipid oxidation to fuel metabolism, and methods of water retention, both physical (e.g. cocoons) and metabolic (e.g. urea accumulation). The present review focuses on several aspects of metabolic adaptation during estivation including changes in the activities of enzymes of intermediary metabolism and antioxidant defenses, the effects of urea on estivator enzymes, enzyme regulation by reversible protein phosphorylation, protein kinases and phosphatases involved in signal transduction mechanisms, and the role of gene expression in estivation. The focus is on two species: the spadefoot toad, Scaphiopus couchii, from the Arizona desert; and the land snail, Otala lactea, a native of the Mediterranean region. The mechanisms of metabolic depression in estivators are similar to those seen in hibernation and anaerobiosis, and contribute to the development of a unified set of biochemical principles for the control of metabolic arrest in nature.

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.