Hanane

Ryan Bell.  M.Sc. Biology 2008.

Regulation of glutamate dehydrogenase in hypometabolic states

 

Abstract:

Glutamate dehydrogenase (GDH) is a key enzyme in nitrogen metabolism and has significant roles in amino acid catabolism and urea biosynthesis. The role of this enzyme in important metabolic processes suggests that it may be regulated in animals that survive in unforgiving environments. Such animals typically respond to harsh environmental conditions with strong overall metabolic rate suppression as well as species- and tissue specific metabolic alterations. To better understand the role of amino acid metabolism in hypometabolism, GDH was investigated in hibernating Richardson’s ground squirrels (Spermophilus richardsonii), anoxia-tolerant freshwater turtles (Trachemys scripta elegans), and estivating land snails (Otala lactea). Studies analyzed GDH substrate affinities, effects of metabolite activators and inhibitors, pH and/or temperature effects, and the actions of protein kinases and protein phosphatases in modifying enzyme properties. Liver GDH from ground squirrels was regulated by reversible protein phosphorylation in a manner that could activate the oxidation of glutamate to assist in energy production and contribute to gluconeogenesis during hibernation. Similarly, foot muscle GDH from land snails was regulated by reversible phosphorylation with the subsequent activation of the glutamate-oxidizing reaction to aid in energy production and urea biosynthesis in the estivating state. Conversely, under conditions of oxygen deprivation, the freshwater turtle utilizes reversible phosphorylation to inactivate both the glutamate-oxidizing and glutamate-synthesizing reactions of GDH. Thus, it appears that GDH and its effects on amino acid metabolism play an important role in animals during hypometabolism.

 

Neal Dawson, M. Sc. Biology, 2009

Regulation of tail muscle energetics during anoxia in the freshwater crayfish, Orconectes virilis

 

Abstract:

Metabolic rate depression is vital to the survival of many organisms in the face of low oxygen levels. This is achieved by a coordinated suppression of both ATP-consuming and ATP-producing metabolic pathways. The role of reversible protein phosphorylation in metabolic rate depression during anoxia was explored in the tail muscle of the anoxia-tolerant freshwater crayfish, Orconectes virilis. This study investigated glutamate dehydrogenase (GDH), the enzymatic bridge between amino acid and carbohydrate metabolism, arginine kinase (AK), an important enzyme involved in regulation of phosphagen reserves, and hexokinase (HK), the enzyme at the forefront of carbohydrate metabolism. The data obtained showed that GDH and AK are regulated by reversible phosphorylation during anoxia, resulting in less phosphorylated, less active forms of these enzymes. Experiments were performed under normoxic and anoxic conditions, and protein expression levels, susceptibility to urea denaturation, structural stability, response to specific protein kinase and phosphatase incubations as well as elution profiles from an ion-exchange column were explored. The data from GDH suggests that amino acid metabolism is left largely separate from carbohydrate metabolism by the reduction of this vital bridge point. AK results suggest that precious ATP is not involved in the regeneration of phosphagen reserves during anoxia. HK was also explored using similar experiments, and it seems that HK protein levels increase during anoxia, and reversible phosphorylation seems to increase protein stability and affect cellular localization. Overall, these studies suggest that reversible phosphorylation plays a key role in the regulation of muscle energetics in the freshwater crayfish, O. virilis.

Oscar A. Aguilar, M. Sc. Biology, 2009

Regulation of the MEF-2 and the SMAD family of transcription factors in the freeze tolerant wood frog, Rana sylvatica

 

Abstract:

The wood frog, Rana sylvatica is a native North America capable of withstanding full body freezing when ambient temperatures drop below 0°C. During the freeze exposure, approximately 65-70% of the extracellular fluid gets converted into ice. Anoxia, ischaemia, osmotic and oxidative stress are some of the consequences which result from a freezing cycle. The dynamic nature of cells allows them to adapt to a wide array of stress conditions at different organizational levels. Transcription factors are key regulators of gene expression responsible for adaptation. In the present study, the MEF2 and SMAD family of transcription factors are demonstrated to have importance in the wood frog during freezing. The proteins were initially associated with developmental controls, however recent studies have found them to be involved in stress responses. Western blots were used to assess the expression and phosphorylation levels of MEF2A, MEF2C, SMAD1, SMAD2, SMAD3, and SMAD4 during torpor. It was generally found that MEF2A, MEF2C, and SMAD3 were post-translationally (phosphorylated) at Thr312, thr300, ser425 sites, respectively during 24h and 8h thawing. RT-PCR analysis of MEF2 and SMAD target genes (calreticulin, glucose transporter-4, creatine kinase (brain and muscle) and serpine1, myostatin, tsc22d3, respectively) revealed a modest up-regulation during 24h freezing in wood frog brain, heart, skeletal muscle, liver and kidney in selected transcripts. These results show that the two families of transcription factors are transcriptionally active during freezing, which comes as no surprise given the signals which regulate these proteins as well as the functions of the genes they activate.

Marcus Allan, M.Sc. Biology, 2010

Nuclear factor (NF)-kappaB regulation in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:

When environmental conditions become unfavorable, such as during winter, many small mammals are able to enter into a state of dormancy known as hibernation in order to conserve energy. Energy conservation is accomplished via a drastic decline in metabolic and physiological activity in association with a decrease in body temperature, which is periodically interspersed with brief bouts of arousal back to their euthermic values. These drastic changes in oxygen consumption and concentration, perfusion of tissues and energy consumption results in an elevated susceptibility to oxidative stress which can cause severe tissue damage. Hibernators are able to mitigate this damage using antioxidants and their associated pathways in a coordinated response. In the present study, the role of the redox sensitive transcription factor NF-κB was investigated to gain insight into its regulation during hibernation. NF-κB is an essential transcription factor which is known to regulate many targets including antioxidant, antiapoptotic/pro-survival and pro-inflammatory genes. The extent and duration of the NF-κBs response depends on its interactions with its multiple upstream effectors. During hibernation it was found that NF-κB and its signaling components have different expression patterns which are tissue dependant and change along the torpor–arousal cycle. Overall, NF-κB was found to be maximally activated during entrance into torpor, with its cytoprotective downstream genes being upregulated in time for next subsequent arousal in both liver and skeletal muscle tissue. Therefore, these results suggest that antioxidant defenses are upregulated throughout torpor-arousal and that NF-κB may help mediate such protective responses.

Shannon N. Tessier, M.Sc. Biology, 2010

Molecular adaptations of skeletal muscle and cardiac muscle in the hibernating thirteen-lined ground squirrel, Spermophilus tridecemlineatus.

 

Abstract:

Many small mammals face severe problems during the winter – little or no food supply and yet huge energy costs for keeping their bodies warm. To escape these problems, they hibernate, entering states of deep torpor where metabolic rate falls to just 2-4% of normal and body temperature falls to near 0°C. Remarkably, skeletal muscle sustains cell size and strength despite extended periods of disuse during torpor whereas cardiac muscle actually promotes cell growth (hypertrophy) to support the stronger cardiac contractions needed in the cold. Despite overall suppression of transcription and translation during hibernation, the present research identified and analyzed selected muscle genes and their products that were up-regulated during torpor in striated muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus). These changes in myocyte enhancer factor-2 (MEF2a, MEF2c) transcription factor levels as well as altered expression of selected downstream targets (e.g. glucose transporter 4, myogenic differentiation protein) aid skeletal and cardiac muscle in meeting metabolic challenges associated with hibernation. MEF2 transcription factors were significantly elevated at various points in the torpor-arousal cycle suggesting a significant role for MEF2-mediated gene transcription in the selective adjustment of striated muscle proteins. Muscle plasticity in the hibernator was also evidenced by torpor-responsive changes in the levels of important contractile (troponin I, α/β-tropomyosin), sarcomeric (myomesin) and cytoskeleton proteins (desmin, andvimentin). These data provides new insights into muscle remodeling during hibernation and the role of selected genes/proteins in balancing programs of atrophy, stasis andmyogenesis.

 

Helen Alyx Holden, M.Sc. Biology, 2011

Anuran adaptations to climatic stress: Immune responses and the SMAD family in the wood frog, Rana sylvatica, and the African clawed frog, Xenopus laevis

 

Abstract:

The wood frog, Rana sylvatica, survives freezing over winter. The African clawed frog,Xenopus laevis, withstands substantial dehydration seasonally. The effects of environment on these frogs‟ immunity were investigated with a focus on antimicrobial peptides. Expression of brevinin-1SY was analyzed during freezing, dehydration, anoxia, and development in R. sylvatica. Brevinin-1SY responded differently to each stress, suggesting environmentally regulated expression. Upregulation of hepcidin mRNA was demonstrated during dehydration in X. laevis liver, as were hepcidin agonists, STAT 3 andcMYC. Alternatively, hepcidin antagonizing TGF-β-mediated SMADs weredownregulated and the BMP-mediated SMADs, promoters of hepcidin expression, did not change. Molecular controls of X. laevis skeletal muscle growth were also explored during dehydration. Myostatin, a muscle growth antagonizer, was downregulated during dehydration, whereas cMYC, a muscle growth agonizer, and GLUT 4, a glucose transporter, were upregulated; differential control of SMADs was documented. The data suggest that, during estivation, muscle growth signals are promoted.

William C. Plaxton, Ph.D. Biology, 1984

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.

Thomas A. Churchill, Ph.D. Biology 1992

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.

Yanjing Su, Ph.D. Chemistry, 1992

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 Vmax, lower S0.5 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 Vmax, an increased S0.5 F6P, and higher I50 values for ATP.

 

Hossein Mehrani, Ph.D. Chemistry, 1994

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 Km 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.