Hanane

Jing Zhang, Ph.D. Biology, 2013

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.

Kyle Biggar, Ph.D. Biology, 2013

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.

Tamara English, M.Sc. Biology, 1995

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals

 

Abstract:

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than a non-hibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 mM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37°C, while the rat liver fatty acid binding protein was affected with the Kd at 37°C being about half (0.8 mM) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for both proteins at 5°C and 37°C. At the lower temperature, ground squirrel protein bound long chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matches requirements for these fatty acids in the diet. The most common long chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5°C than at 37°C with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal’s body temperature.

Daniel Miller, M.Sc. Biology, 1983

Gas-Liquid Chromatography and Enzymatic Determination of Alanopine and Strombine in Tissues of Marine Invertebrates

 

Abstract:

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, t -strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2’ iminodipropionic acid) were chromatographed as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 microg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Litterina littorea, and the strombine/ alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mM hydrazine buffer, pH 9.0, 5 mM NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.

Roy D. Cole, M.Sc. Biology, 1984

Temperature effect on bivalve anaerobic metabolism.

 

Abstract:

The response of anaerobic metabolism in Mercenaria mercenaria to long term and acute temperature stress revealed qualitative and quantitative changes in metabolic flux. (A) The rate of anaerobic metabolism was sensitive to both long term and acute temperature change. (B) The choice of anaerobic end product was affected by environmental temperature. (C)Long term exposure to high temperature results in acclimation of anaerobic metabolism not seen during acute exposure to high temperature.

 

David Kelly, M.Sc. Biology 1988

Organ-specific control of glycolysis in anoxic turtles

 

Abstract:

Control of glycolysis during anoxia was investigated in five organs (heart, brain, liver, and red and white skeletal muscles) of the freshwater turtle, Pseudemys scripta, after 1 or 5 h of submergence in N2-bubbled water. Lactate was produced as the metabolic end product, with distinct organ differences in the amount (net lactate accumulation was 2.4-fold higher in brain than white muscle) and rate (lactate production in liver dropped 16-fold after the 1st h) of lactate accumulation. ATP and total adenylate contents of all organs were reduced (by 15-32%) after 1 h of submergence, but energy charge was maintained, after 5 h, adenylate contents had fully recovered. Changes in the levels of hexose and triose phosphate intermediates of glycolysis indicated an activation of glycolysis within the 1st h of anoxia exposure in brain, heart, and skeletal muscles. By 5 h, however, these were reversed, and a glycolytic rate depression was indicated, consistent with the overall metabolic rate depression accompanying long-term anaerobiosis in the turtle. Crossover analysis indicated glycolytic control at the pyruvate kinase reaction in all organs during both glycolytic activation and metabolic depression; regulatory control at the phosphofructokinase locus was primarily important only during glycolytic activation in heart and red muscle. The same analysis indicated a very rapid glycolytic inhibition in liver occurring within the 1st h of anoxia exposure; this allows glycogenolysis to be directed toward glucose export yielding the fermentative fuel used by other organs during anoxia.

Ross Whitwam, M.Sc. Biology 1988

Pyruvate kinase from the land snail Otala lactea: regulation by reversible phosphorylation during estivation and anoxia

 

Abstract:

Pyruvate kinase (PK) from tissues of the desert snail Otala lactea (Muller) undergoes a stable modification of its kinetic properties during estivation or in response to anoxia stress. In foot muscle and mantle, the kinetic changes induced by either state were virtually identical and were consistent with a less active enzyme form in estivation or anoxia: S0.5 PEP increased, and I50 values for Mg-ATP and L-alanine decreased, compared to the enzyme in control (aroused) snails. Estivation and anoxia also changed the properties of PK from hepatopancreas; some changes were consistent with a more active enzyme form (S0.5 PEP decreased, I50 values for Mg-ATP and L-alanine increased) but the enzyme lost all sensitivity to the potent activator fructose-1,6-bisphosphate. A time course of changes in I50 Mg-ATP for foot PK and S0.5 PEP for hepatopancreas PK revealed that estivation-induced changes in enzyme properties occurred between 12 and 48 h after snails were deprived of access to food and water, whereas the reversal of these changes occurred within as little as 10 min in foot muscle after arousal was initiated. The molecular basis of the stable modification of PK kinetics appears to be reversible protein phosphorylation. The action of added cyclic-AMP-dependent protein kinase on foot or hepatopancreas PK from control (aroused) snails changed PK kinetic parameters to those characteristic of the enzyme form in estivating animals; the addition of stimulators of endogenous cyclic-GMP-dependent protein kinase or protein kinase C had the same effect. Conversely, treatment with added phosphatases reconverted the properties of foot muscle PK from estivating snails to those characteristic of the control enzyme. The data suggest that reversible phosphorylation control over the activity state of regulatory enzymes of glycolysis is one mechanism contributing to the overall metabolic rate depression of the estivating state.

Clark Holden, M.Sc. Biology 1991

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

 

Abstract:

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE+ ion exchange and 3′-5′-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3+0.74 and 2.7+0.87 micromol glucose-1-P/min/g wet weight, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units/mg protein, respectively. Both enzymes were dimers with native molecular weights of 2l5 000 + l8 000 for glycogen phosphorylase a and 209 000+15 000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87 000+2 000. Both enzymes showed pH optima of 7.5 at 22 °C and a break in the Arrhenius relationship with a two- to fourfold increase in activation energy below 10 °C. Michaelis constant values for glycogen at 22 °C were 0.12+0.004 mg/ml for glycogen phosphorylase a and 0.87+0.034 mg/ml for glycogen phosphorylase b, the Michaelis constant for inorganic phosphate was 6.5 + 0.07 mmol/L for glycogen phosphorylase a and 23.6 mmol/L for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a Ka of 0.176+0.004 mmol/L. Michaelis constant and Ka values decreased by two- to fivefold at 5°C compared with 22 °C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol/L) but was inhibitory to both enzyme forms at high concentrations (2 mol/L). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glyeogen phosphorylase b.

Aleixo Muise, M.Sc. Chemistry 1993

Reversible phosphorylation of fructose 1,6-bisphosphatase mediates enzyme role in glycerol metabolism in the freeze-avoiding gall moth Epiblema scudderiana

 

Abstract:

Fructose-1,6-bisphosphatse (FBPase) from larvae of the freeze-avoiding gall moth Epiblema scudderiana occurs in two forms which are interconverted by reversible phosphorylation and separable by CM-cellulose column chromatography. The phosphoenzyme has properties that would make it the more active form in vivo. Compared with the dephosphorylated form, the phosphoenzyme had 3-fold lower values for Km fructose-1,6-bisphosphate and Ka Mg2+ and lower sensitivities to allosteric inhibitors (I50 values for fructose-2,6-bisphosphate and AMP were 50 % and 10-fold higher, respectively). The proportions of the two enzyme forms in the larvae changed with the seasons and with acclimation to warm (15°C) versus cold (4°C) temperatures. The phosphorylated enzyme predominated (70% of total activity) in early autumn and during the spring, as well as in warm acclimated larvae, all situations where gluconeogenesis via FBPase would be favoured. During the autumn cold-hardening period when the larvae are actively synthesizing the antifreeze, glycerol, the ratio of the two enzyme forms changed to about 50:50. This, plus allosteric inhibition and low temperature effects on enzyme kinetics, would effectively suppress FBPase activity and prevent futile recycling of glycerol carbon back into glycogen during the winter months when the 2 M pool of polyol must be sustained for antifreeze protection. Acclimation studies suggested that low temperature itself might be the signal that triggers enzyme dephosphorylation and this could integrate control over FBPase with the well known phosphorylation-mediated activation of glycogen phosphorylase by low temperature in cold-hardy insects.

Peggy L. Schade, M.Sc. Chemistry, 1996

Glycogen degradation by alpha-glucosidase from a hibernating ground squirrel, Spermophilus lateralis, and a freeze tolerant frog, Rana sylvatica

 

Abstract:

Glycogen degradation in cells can proceed via two routes, the well-known phosphorolytic pathway via glycogen phosphorylase (GP) and the glucosidic pathway involving alpha-glucosidase. The present study analyzes alpha-glucosidase and its potential for net contributions to organ glucose production in two species that exhibit unusual glucose requirements while overwintering at low body temperatures: the hibernating golden-mantled ground squirrel, Spermophilus lateralis, and the freeze-tolerant wood frog, Rana sylvatica. Mammalian liver (ground squirrel, rat) possessed two neutral isozymes of alpha-glucosidase and one acidic form (pH optima 7.0 and 4.0) whereas amphibian liver (wood frog, leopard frog) had one neutral and one acidic enzyme. Tissue distribution of acidic and neutral forms revealed highest activities in liver and kidney and significant changes in activity during hibernation or freezing in selected tissues. Compared with the amount of active GP, acid alpha-glucosidase could make a substantial contribution to net glucose production in several wood frog organs, notably heart. Liver isozymes were separated by isoelectrofocusing prior to characterization of substrate specificity and affinity, inhibitor effects, and thermal denaturation. Neutral isozymes of all species were similar showing limited use of glycogen, strong thermal denaturation at 45°C and above, and generally reduced substrate affinity at the low temperatures characteristic of the hibernating state. Acidic isozymes used glycogen, showed good thermal stability and substrate affinities were largely temperature-independent. Inhibition of acidic alpha-glucosidase by salts and carbohydrates was reduced for the wood frog enzyme, compared with the leopard frog, a factor that may improve enzyme function in the frozen state.