Ph. D. Thesis Abstracts

Roles of Akt signaling and its downstream pathways in wood frog freeze tolerance

Abstract:

Wood frogs, Rana sylvatica, are one of only a few vertebrate species that survive prolonged whole body freezing during the winter. Multiple adaptations of physiology and biochemistry that support freeze tolerance have been identified including accumulation of extreme levels of glucose as a cryoprotectant and entry into a hypometabolic state that reduces the energy needs of the animal while frozen. To date, the stress responsive signal transduction networks that trigger and regulate these adaptations have received little attention. The current thesis addressed this subject by exploring responses and regulation of a major intracellular signaling pathway (the Akt pathway) that is centrally involved in mediating cellular growth and proliferation responses, typically responding to extracellular insulin signals. Analysis of four organs (liver, kidney, heart and skeletal muscle) showed activation of the Akt pathway in liver but signs of inhibition occurred in other tissues in response to freezing. Activation of Akt-dependent anti-apoptosis mechanisms in liver was also indicated to support cell survival in the frozen, anoxic state. However, analysis of multiple protein components of the cell cycle and TORC1-dependent protein synthesis showed strong suppression of these in all tissues, although with lesser inhibition in liver. This demonstrates the importance of suppressing energy-expensive cell processes under stress conditions. The data show that, during whole body freezing of wood frogs, (1) ATP expensive cellular events such as the cell cycle and protein synthesis were suppressed; (2) liver remains more metabolically active than other tissues tested; and (3) freeze responsive Akt activation in liver does not universally activate all of its downstream pathways but rather selectively triggers specific targets, particularly those important to glucose production as a cryoprotectant and to cell preservation. The thesis also investigated freeze-responsive antioxidant defenses in wood frog liver and muscle with a focus on mechanisms regulated by the Nrf2 transcription factor and showed that Nrf2 is glucose-responsive. Furthermore, glucose appears capable of differentially affecting gene expression and posttranslational modifications of proteins in liver. Overall, this thesis showed the central importance of the Akt pathway in freeze tolerance and demonstrated tissue- and environment-specific responses by the pathway and its downstream processes.

Cell cycle regulation by post-translational and post-transcriptional mechanisms in an anaerobic extremist – The anoxic tolerant turtle, Trachemys scripta elegans

Abstract:

As a model for vertebrate long-term survival in oxygen restricted environments, the red-eared slider turtle (T. s. elegans) can adapt at the biochemical level to deal with hibernation occurring in oxygen-free (anoxic) cold water (<10°C). In this thesis I hypothesized that the mechanisms which suppress ATP-expensive cell cycle activity, would contribute to establishing an hypometabolic state. To explore this possibility, this thesis studied the post-transcriptional and post-translational mechanisms of cell cycle arrest during anoxic stress in the freshwater turtle.

Results indicated a general regulation of critical cell cycle components, in addition to the possible regulation by signaling cascades (Akt/GSK-3β and ATR/Chk2) that are known to regulate G1/G0 phases of the cell cycle. Importantly, there is extensive regulation of Cyclin D1 protein by (1) Akt/GSK-3β signaling, (2) post-translational modification, (3) an AU-rich region, and (4) microRNA-induced translational suppression. This study also identified a phase-specific cell cycle arrest mechanism involving the Rb/E2F DNA-binding complex in both anoxic liver and kidney tissues. A novel DNA-binding complex ELISA technique was able to identify that both kidney and liver establish an Rb/E2F1 mediated G1 arrest complex by 5 and 20 h anoxia, repectively. By 20 h anoxia, kidney tissue established a reversible state of G0, characterized by the prescence of a p130/E2F4 DNA-bound complex.

Overall, results from this thesis indicate that both kidney and liver enter into a G1 arrest during anoxia. By contrast, the cell cycle in white skeletal muscle was found to be minimally regulated during anoxia and this finding is likely a reflection of its overall post-mitotic nature. Interestingly, kidney established a state of G1 arrest within 5 h anoxia and subsequently transitioned to a sustainable G0 arrest by 20 h anoxia. However, it appears that liver G1 arrest was not established until 20 h anoxia. Future studies will need to explore the regulation of the cell cycle in liver after longer periods of anaerobiosis to determine whether hepatocytes are also able to transition into G0 arrest in a manner similar to kidney tissue.

A study of the metabolic adaptations of marine gastropod molluscs to environmental anoxia stress

Abstract:

Catalytic and regulatory properties of alanopine dehydrogenase (ADH) and pyruvate kinase (PK) from tissues of anoxia tolerant marine gastropods were studied. Particular attention was given to those properties of the enzymes which could help explain their potential role(s) in anaerobic energy metabolism. The physical and kinetic properties of the terminal glycolytic dehydrogenase, ADH, purified to homogeneity from foot muscle of the common periwinkle, Littorina littorea, were examined. The kinetic properties of ADH favor enzyme function in cytoplasmic redox balance during the recovery period following long-term environmental anoxia. Tissue specific isozymes of ADH were found in another marine gastropod, the channelled whelk,Busycotypus canaliculatum. Three isozymic forms, specific for muscle, gill/kidney and hepatopancreas were identified. The three tissue specific isozymes of ADH were purified to homogeneity from foot muscle, gill and hepatopancreas and their kinetic and physical properties were studied. Muscle ADH showed properties which appear to gear this isozyme for alanopine synthesis as an end product of glycolysis. The hepatopancreas isozyme appears suited for a role in alanopine oxidation in vivo. The properties of gill ADH are intermediate between those of the other two forms. Tissue specific forms of PK were also found in B. canaliculatum. Three isozymic forms, specific for red muscle, white muscle and soft tissues, were identified. Furthermore, each PK isozyme was modified in animals subjected to 21 h of anoxic stress such that several physical and kinetic characteristics were altered. Aerobic and anoxic forms of red muscle PK (RPK-AER and RPK-ANX) were purified to homogeneity from radular retractor tissue of B canaliculatum and the physical and kinetic properties of the enzyme were extensively studied. The differences in kinetic properties between RPK-AER and RPK-ANX indicates that red muscle PK activity is probably greatly depressed in vivo during long-term anoxic stress. The anoxia-dependent, in vivo, covalent incorporation of injected (’32)P orthophosphate into RPK-ANX demonstrated that the enzyme is a phosphoprotein. Evidence for the reversibility of this phosphorylation was provided by the kinetic similarities between purified RPK-AER and homogenous alkaline phosphatase treated RPK-ANX.

Metabolic biochemistry of freeze tolerance in vertebrates

Abstract:

A unique group of vertebrate animals has developed complex metabolic adaptations that enable them to survive freezing. This thesis investigates: i) cryoprotectant synthesis in freeze tolerant frogs, ii) freeze tolerance in a newly identified freeze tolerant vertebrate, the garter snake, and iii) metabolic responses elicited by other stresses (anoxia and dehydration) in the garter snake and two frog species. Investigation of cryoprotectant synthesis in spring frogs,Pseudacris crucifer, revealed large amounts of glucose produced during freezing; approximately 0.1 molar. Changes in the levels of glycolytic intermediates indicated that an activation of glycogen phosphorylase and phosphofructokinase (PFK) directed glycolytic flux to cryoprotectant synthesis. Tissue glucose distribution was much lower than in fall animals. These results suggested seasonal variation in glucose transport mechanisms. A similar investigation of cryoprotectant synthesis in spring Hyla versicolorshowed a maintenance of regulatory enzyme controls at glycogen phosphorylase and PFK directing glycogen carbon to cryoprotectant. Only glucose was synthesized as cryoprotectant; quantities of glycerol (the major cryoprotectant of winter H. versicolor) showed no increase. The amount of cryoprotectant produced was directed correlated to glycogen content in the liver. Investigation of freeze tolerance in garter snakes revealed that these snakes were only partially freeze tolerant. Survival of brief freezing exposures (5-10 h at -2.5°C; 30-50% ice) was possible. Two amino acids, glutamate and taurine, were implicated as possible cryoprotective agents. Comparison of the metabolic responses (adenylate levels, anaerobic end products, glycolytic flux) to freezing in garter snakes were similar to those elicited by anoxia. Dehydration timecourses were investigated in two freeze tolerant frog species, Rana sylvatica andPseudacris crucifer. Even though whole body water contents dropped by 50-60 %, individual tissues exhibited little or no change in water content. There were many similarities between metabolic responses to dehydration and those to freezing. The most remarkable similarity between freezing and dehydration was the accumulation of glucose, presumably acting as a cellular protectant; quantities in liver rose to 127 and 220 micromole per gram in R. sylvatica and P. crucifer, respectively.

Phosphofructokinase from white skeletal muscle and liver of rainbow trout (Oncorhynchus mykiss): isolation, characterization and study of enzymatic regulation

Abstract:

Phosphofructokinase (PFK) isozymes from white skeletal muscle and liver of rainbow trout Oncorhynchus mykiss were purified to electrophoretic homogeneity. Muscle PFK was purified 175-fold using phosphocellulose, hydroxylapatite, and ATP-agarose affinity chromatography whereas liver PFK was purified 13,400-fold using acetone precipitation, heat treatment, ammonium sulfate fractionation, and ATP-agarose chromatography. Muscle PFK was a homohexamer having a native molecular mass of 478,000. The enzyme was regulated by the levels of fructose-6-phosphate (F6P), ATP, pH, and allosteric effectors including activators (NH₄⁺, inorganic phosphate, AMP, ADP, and fructose-2,6-bisphosphate [F2,6P₂]) and inhibitors (citrate, phosphoenolpyruvate [PEP], and ATP). Activators increased the enzyme affinity for F6P and released the inhibition by ATP or citrate. Citrate inhibited the enzyme synergistically with ATP. Arrhenius plots of the enzyme activity showed discontinuity at 15 to 16°C, presumably due to conformational alterations in the enzyme. The kinetic behavior of muscle PFK was significantly altered by protein kinase – mediated phosphorylation. The high-phosphate form of the enzyme showed higher activity with increased affinity for F6P and less inhibition by ATP. Protein concentration affected enzyme activity as assessed by two different methods. PFK showed a higher V_max, lower S_0.5 F6P and higher I₅₀ values for ATP as enzyme concentration increased. The association of PFK with myofibrils of trout muscle was affected by pH, ionic strength, protein concentration, and the levels of metabolites or the effectors of the enzyme with binding favored by lower pH values and increased protein concentration. During exercise, muscle PFK is probably activated by increases in the levels of enzyme activators and enzyme phosphorylation state, and enhanced PFK association with myofibrils. Trout liver PFK was also regulated by the levels of F6P, ATP, NH₄⁺, inorganic phosphate, AMP, and F2,6P₂. However, the liver enzyme was not sensitive to citrate inhibition. Contrary to its muscle counterpart, liver PFK was inhibited by protein phosphorylation catalyzed by the catalytic subunit of cAMP-dependent protein kinase and activated by the removal of phosphate through acid phosphatase. The high-phosphate form of liver PFK exhibited a lower V_max, an increased S_0.5 F6P, and higher I₅₀ values for ATP.

Regulation of glycogen metabolism by protein phosphorylation during environmental stress

Abstract:

The enzymes involved in the phosphorylation controlled glycogen catabolic pathway were studied in two different model systems involving anoxia: functional anoxia in exercised fish and environmental anoxia in turtle. Glycogen phosphorylase b from rainbow trout,Oncorhynchus mykiss, white skeletal muscle was purified to near homogeneity. Glucose and ATP inhibited the enzyme; glucose inhibition decreased at lower pH values. Michaelis constants for glycogen, phosphate, and AMP were 128 micromolar, 31 millimolar, and 142 micromolar respectively, at pH 7.2; maximum enzyme activity was obtained at pH 7.5 and 25°C Exhaustive swimming exercise altered tissue glycogen phosphorylase kinase (GPK) and protein kinase A (PKA), GPK activity increasing by 60% in liver and 40% in white muscle of exercised fish. The amount of active PKA rose from 12% to 21% in liver and from 32% to 57% in white muscle after exhaustive swimming coupled with 50% and 70% increases in cellular cyclic AMP levels, respectively. Three forms of alpha-glucosidase were identified in trout liver. Two forms showed acid pH optima, hydrolyzed glycogen, maltose and 4-methylumbelliferyl alpha-glucoside (MUalphaG), and were associated with lysosomes whereas the third was microsomal, had a neutral pH optimum and did not hydrolyze glycogen. Properties of acid alpha-glucosidase type I changed significantly during exercise; maximal activity increased by 80% and K_m values for glycogen and maltose dropped by 50% in exercised, versus control, fish. Exposure of turtles, Trachemys scripta elegans, to submergence anoxia at 7°C, elevated activities of phosphorolytic and glucosidic enzymes in some organs. Phosphorylase a in liver and heart increased significantly after 5 h of anoxia. PKA activity increased 2.3-fold in liver within 1 h of anoxia accompanied by a 60% increase in cAMP levels; however, with longer anoxia active PKA was suppressed to 2.1-3.7% of the total. Protein phosphatase-1 (PP-1) activity in liver decreased to 63% of controls within 1 h and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity also fell in anoxic red muscle and decreased transiently in brain. Within one hour of anoxia, 40% of protein kinase C beta isomer (PKC-beta) and over 80% of PKC-alpha were translocated from cytosol to the membrane fraction. Activity of acid alpha-glucosidase also increased in liver of anoxic turtles. PKA, PP-1, PKC-alpha, and PKC-beta from control turtle liver were purified to homogeneity; physical and kinetic properties of these are presented.

Signal transduction and the function of second messengers and protein kinases in the control of glycogenolysis and cryoprotectant production in freeze tolerant vertebrates

Abstract:

A unique group of lower vertebrates, termed freeze tolerant, have developed complex biochemical mechanisms that allow them to survive whole body freezing. Perhaps the most important of these mechanisms is the production of cryoprotectant, usually in the form of glucose, via an activation of glycogenolysis as a response to freezing. This thesis investigates: (1) whether protein kinase enzymes are responsible for the early activation of glycolysis that stimulates cryoprotectant production in freeze tolerant vertebrates and/or the subsequent inhibition of the pathway in the later stages of a freezing episode, (2) the comparative aspects of the responses of protein kinases elicited by the environmental stresses of dehydration and anoxia, to those of freezing, and (3) the role of the free catalytic subunit of cyclic AMP-dependent protein kinase (PKAc) and protein kinase C (PKC) in cryoprotectant production in Rana sylvatica liver. Short freezing episodes caused a dramatic increase in the percentage of free catalytic PKA (PKAc). This response diminished with increased time frozen in tissues of freeze tolerant vertebrates. The measurement of second messengers over a freezing time course revealed significant increases in adenosine 3′,5′- cyclic monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP₃) in liver and muscle of 4 freeze tolerant animals. An activation of PKA and PKC by these molecules, in response to freezing, is likely. Dehydration stress. not anoxia stress, elicited a remarkably similar protein kinase response to that seen during freezing. Levels of PKAc and its second messenger cAMP, as well as the second messenger for PKC, IP₃, rose in dehydrated liver tissue whereas anoxia did not activate PKAc and only increased liver IP₃. Purified R. sylvatica liver PKAc was not inhibited by high concentrations of salt or glucose and showed an increased affinity for substrates at low temperatures. Additional evidence showed that a freezing stress did not initiate a translocation of PKC to the plasma membrane in R. sylvatica liver and brain tissues. Freeze tolerant vertebrates possess an adaptive strategy to combat freezing which links second messengers and protein kinases to the post-translational modification of glycolytic enzymes conducting the biosynthesis of cryoprotectant.

Metabolism during overwintering in two species of cold-hardy goldenrod gall insects

Abstract:

Metabolism was examined in the larvae of two cold-hardy goldenrod gall insects, the freeze tolerant fly Eurosta solidaginis (Fitch) (Diptera, Tephritidae) and the freeze avoiding moth Epiblema scudderiana (Clemens) (Lepidoptera, Olethreutidae) during overwintering. Acclimatization resulted in specific changes in enzyme activities and cellular metabolites. Enzyme activity data suggested metabolic shifts concurrent with the requirements of both species. Larvae shifted metabolism towards cryoprotectant synthesis in the fall, as witnessed by increased activities of glycerol and sorbitol producing enzymes, and towards the removal of these polyols in the spring with increased activities of catabolic enzymes. Measured increases in lipid unsaturation in the fall helped to maintain membrane fluidity during cold-exposure. Decreased activities of enzymes of the tricarboxylic acid cycle paralleled the reduced demands on oxidative metabolism over the winter. In Eurosta solidaginis, decreased oxidative metabolism and the anoxic state of the animal when frozen further resulted in the lack of lipid reserve depletion over the winter, whereas freeze avoiding Epiblema scudderiana larvae appeared to use lipid reserves to support basal metabolism over the winter. Decreased activities of antioxidant enzymes in Eurosta solidaginisover the winter suggested that these larvae do not experience increased reactive oxygen species formation during freezing and thawing. Increasing antioxidant enzyme activities in Epiblema scudderianaover the winter suggested that these larvae may be subject to oxidative stress, perhaps due to the maintenance of aerobic metabolism during cold exposure.

Antioxidant defenses during estivation in the spadefoot toad, Scaphiopus couchii

Abstract:

Antioxidant enzymes and lipid peroxidation damage were analyzed in an estivating species, to determine whether estivation (a dormancy induced by hot, dry environmental conditions) represented an oxidative stress and to determine whether changes in organ antioxidant systems were made in response to estivation. The model animal used was Scaphiopus couchii, the spadefoot toad, that estivates for about 10 months per year in the Arizona desert during which it loses about 50% of its body water and builds up high levels of electrolytes and osmolytes in body fluids. Activities of six antioxidant enzymes (AOE) and xanthine oxidase/xanthine dehydrogenase as well as the small molecular weight free radical scavenger glutathione were measured in liver, lung, heart, kidney, gut and leg muscle of control and dormant toads. Damage to membranes was assessed by measurement of extent of lipid peroxidation by three different methods. The glutathione ratio, (GSSG) /(GSH), rose significantly in several tissues during estivation. In addition, activities of key AOE were reduced in several tissues, perhaps indicating that the metabolic cost of keeping antioxidant defenses high during estivation was greater than the cost of maintaining AOE at their previous level. Three antioxidant enzymes were further examined: Catalase (CAT) and glutathione reductase (GR) were purified and characterized from liver and glutathione S-transferase (GST) from liver and muscle of awake and estivated toads. CAT activity was lower in liver during estivation but the kinetic properties of catalase were the same in estivated and awake toads. Catalase from toad liver was an active dimer, which has not been demonstrated previously in a eukaryote, to our knowledge. GR and GST of toad liver had altered kinetic properties during estivation, indicating pre- or post-translational modification of the enzyme in the dormant animal. However, GST in muscle was the same for awake and estivated toads. GST is a multiclass family of enzymes which generally render lipid-soluble material soluble in the cytosol, via conjugation to glutathione. Three class of GST, Mu Pi and Alpha, have different substrate specificities; these parameters were used to classify frog and toad GSTs. GST, GR and CAT were relatively insensitive to physiological urea concentrations but were strongly modulated by KCl, suggesting that urea buildup during estivation may serve to modulate passive KCl accumulation as a result of dehydration.

Molecular adaptation to anoxia and recovery from anoxia in the freshwater turtle Trachemys scripta elegans

Abstract:

The responses to stresses associated with oxygen extremes, namely anoxia and oxidative stress associated with recovery from anoxia, were studied in an organism that must tolerate both extremes. The model organism used was the turtle Trachemys scripta elegans that can survive complete anoxia for up to 18 weeks at lower temperatures. Metabolic adaptations that support survival of oxygen extremes in this organism that were examined included: (a) tissue-specific changes in the maximal activities of enzymes of intermediary metabolism, (b) an assessment of the metabolite and enzymatic antioxidant defenses utilized to combat the oxidative stress encountered upon recovery from anoxia, (c) adaptations in specific enzymatic antioxidant defenses and (d) adaptations to anoxia and recovery from anoxia of a specific protease. Of the key enzymes of intermediary metabolism, 14 instances of reduced and only 3 instances of increased enzyme activities were observed in tissues during anoxia as compared to controls. Brain had changes related to both metabolic rate depression and neurotransmitter release. Maximal activities which increased during anoxia included G6PDH in heart, MDH in white muscle and CPT in kidney. Anoxia stress and aerobic recovery produced relatively few changes in organ antioxidant enzyme activities and levels of lipid peroxidation products. Changes included tissue specific decreases in antioxidant enzymatic activities during anoxia, particularly SOD and CAT. The levels of enzymatic antioxidant activities were high in turtles in comparison with other vertebrates and non-vertebrates. Turtles maintained high levels of total glutathione in tissues in comparison to other ectotherms. Tissue-specific changes in the maximal activities of the glutathione-related enzymes occurred, the most dramatic being a decrease in gamma-GTPase during anoxia to 2% of control values. Turtle liver contained one homodimeric alpha class and a unique heterodimeric alpha-like GST. Turtle liver GR revealed a high affinity for GSSG. No new isoforms of either GST or GR were formed during anoxia. MPC displayed an increase in peptidylglutamyl-peptide bond hydrolyzing activity during recovery from anoxia, possibly as a result of oxidative damage to proteins in this tissue. Overall, T. S. elegans maintains continually high defenses against the stresses of anoxia and oxidative stress upon recovery from anoxia.