Keqin Yan, M.Sc. Biology, 2005

Glucose regulated protein and heat shock protein expression in hibernating mammals



During mammalian hibernation, most physiological activities are dramatically suppressed, but selected genes are up-regulated to provide protein products that protect cells and organs for long term survival in the hypometabolic, hypothermic state. In this study, the roles of chaperone proteins including glucose regulated proteins and heat shock proteins were assessed in two species: thirteen-lined ground squirrels, Spermophilus tridecemlineatus, and little brown bats, Myotis lucifugus. RT-PCR and Western blot techniques were used to examine gene and protein expression. Compared with euthermic control squirrels and bats, glucose regulated protein 75 (Grp75), Grp94 and Grp170 were elevated in some tissues at the mRNA and/or protein levels with organ-specific patterns of response by grp transcripts and GRP protein. The up-regulation of grp mRNA may be important for rapidly elevating the protein content of these chaperones during hibernation and arousal; elevated GRP protein would then aid in the folding of other proteins that are newly synthesized during hibernation and/or in the renaturation of proteins that become misfolded at low body temperatures. Heat shock proteins (Hsps) including Hsp40, Hsp72, Hsp73 and Hsp90 were elevated in some tissues of hibernating ground squirrels and bats. Analysis of partial amino acid sequences of Grps and Hsps showed very high identities (88-100%) compared with human or mouse sequences which indicates similar structures and functions of Grps and Hsps among mammalian species. The data support the idea that Grps and Hsps are up-regulated during hibernation to function as chaperones to bind non-native polypeptides and suppress protein aggregations caused by low temperature during hibernation.


Jun Du, M.Sc. Chemistry, 2005

Anti-apoptotic and antioxidant defenses in the freeze tolerant wood frog, Rana sylvatica.



Multiple biochemical adaptations support natural freeze tolerance by wood frogs, Rana sylvatica. The present research explored the role of anti-apoptotic and antioxidant defenses in organ survival of freeze/thaw stresses using PCR and Western blotting to analyze the expression of selected genes and proteins. The STAT family of transcription factors mediate both pro- and anti-apoptotic gene responses. Elevated amounts of phosphorylated (active) Stat5 (Tyr694) and/or phospho-Stat3 (Ser727) in selected frog organs during freeze/thaw suggest activation of anti-apoptotic defenses to help organs recover from metabolic insults caused by freezing. However, levels of phospho-Stat1 (Tyr701), a pro-apoptotic signal, also rose in kidney and muscle during thawing. Increased amounts of anti-apoptotic proteins including Bcl-2 and phospho- Bcl-2 (Ser70) in liver and skeletal muscle and Bcl-xL (and phospho-Bcl-2) in kidney could help counteract freeze-induced apoptotic signals that were evidenced by higher levels of Bad protein (liver, muscle, kidney) and phospho-Bad (Ser112) (kidney) and enhanced DNA laddering. Antioxidant defense via glutathione S-transferase (GST) was evaluated by analyzing the expression of GST isozymes. GST Pi protein rose in four organs during freeze/thaw and GST Pi mRNA was freeze up-regulated in liver. GST M1/2, M5, A3 and A5 were freeze- or thaw- responsive in selected organs. Freeze-induced changes in the transcription factors, Nrf2 and MafG, and elevated MafG in the nucleus suggest that these regulate the freeze up-regulation of antioxidant enzymes by targeting the antioxidant response element of genes. Both anti-apoptotic and antioxidant defenses are important aspects of natural freezing survival.





Jiayun Zhou, M.Sc. Chemistry, 2006

Regulation of enzymes of energy metabolism – AMP deaminase and creatine kinase – in an anoxia tolerant turtle.



The red-eared slider turtle (Trachemys scripta elegans) is one of the few vertebrate species that can survive long term oxygen deprivation. Maintenance of viable cellular energetics is key to anoxia survival and, in response to low oxygen, most anoxia tolerant animals show a drop in total adenylate levels while energy charge remains stable. To better understand how turtles regulate their energy metabolism when deprived of oxygen, the present studies focused on the control of two important enzymes in muscle energy metabolism: AMP deaminase (AMPD) and creatine kinase (CK). AMPD activity increased under anoxia in turtle skeletal muscle and the effects of ATP∙Mg and ions indicated that allosteric controls are part of the mechanism of AMPD regulation. In vitro incubations to stimulate the actions of endogenous protein kinases and phosphatases showed that AMPD is a phosphoenzyme and suggested that reversible phosphorylation has a central role in AMPD regulation under aerobic versus anoxic conditions. CK from turtle heart is also a phosphoprotein and anoxia-induced metabolic rate depression was accompanied by a strong increase in the fraction of dephosphorylated CK that showed increased affinity for creatine. Incubation studies implicated selected protein kinases (PKA, PKG, and AMPK) and phosphatases (PP1) as responsible for heart CK regulation. However, anoxia-responsive changes in kinetic properties of skeletal muscle CK did not appear to be caused by a change in phosphorylation state. Regulation of muscle CK under anoxia may be linked with changes in the binding of CK with myofibrils and the effects of binding on enzyme properties.

Lin Xie, M.Sc. Biology, 2007

Antioxidant and anti-apoptotic defenses in the anoxia-tolerant turtle, Trachemys scripta elegans.  



The freshwater turtle, Trachemys scripta elegans, utilizes various biochemical adaptations to survive anoxia-reoxygenation cycles without apparent tissue damage. This thesis focused on changes in the expression and regulation of selected enzymes and proteins involved in antioxidant and anti-apoptotic defense in turtle tissues in response to anoxic submergence and reoxygenation recovery. Western blotting showed that levels of the antioxidant enzyme, manganese superoxide dismutase (Mn SOD), were significantly higher (P<0.05) in heart and skeletal muscle during anoxia. Among four isozymes of glutathione S-transferase (GST), GST K1 expression level was enhanced in kidney, liver and muscle during anoxia, but remained stable in heart. GSTT1, GSTP1 and GSTM3 were elevated in a tissue-specific manner. Anoxia-induced upregulation of the transcription factor, Nrf2, coupled with translocation of Nrf2 into the nucleus in anoxia, indicated that Nrf2 is probably involved in activating downstream antioxidant genes such as GST. Analysis of antiapoptotic proteins (Bcl-XL, Bcl-2 and Mcl-1) also showed enhanced expression in selected tissues during anoxia exposure whereas the pro-apoptotic protein, Bad, was suppressed via phosphorylation during anoxia in muscle. Levels of bcl-xl mRNA were also quantified to assess the relationship between bcl-xl gene and Bcl-XL protein expression under anoxia. The results indicate that enhancement of antioxidant and anti-apoptotic defenses is an important adaptive mechanism for effectively dealing with low oxygen and oxidative stresses over cycles of anoxia/reoxygenation in turtles.

Jacques Niles, M.Sc. Biology, 2007

Freeze tolerant frogs: expression and regulation of transcription factors of the unfolded protein response and the ER-associated degradation.  



Wood frogs (Rana sylvatica) are the primary model used in the study of freeze tolerance in vertebrates and much is known about the adaptations of physiology, biochemistry and gene expression that support winter freezing survival, particularly their natural cryoprotective processes. Freezing and/or its components (anoxia and dehydration) places multiple stresses on cells; one of these is endoplasmic reticulum (ER) stress, a condition caused by accumulation of unfolded or misfolded proteins in the ER. The regulated expression of selected transcription factors, such as ATF4, that trigger genes that protect against ER stress is important for cell survival of freezing. During ER stress, the unfolded protein response (UPR) and the ER-associated degradation (ERAD) pathway are triggered, which can potentially lead to apoptosis.  Western blots were used to evaluate the responses by key protein components of the UPR and the ERAD under freezing, anoxia and dehydration stresses in two major organs of wood frogs (skeletal muscle and liver).  The proteins analyzed included the activating transcription factors (ATF3, ATF4, ATF6), the growth arrest and DNA damage proteins (GADD34, GADD153), and the EDEM and XBP1 proteins.  Stress-induced redistribution of transcription factors between cytoplasmic and nuclear fractions was also evaluated. All three stresses triggered the UPR in both tissues but only freezing of skeletal muscle seemed to trigger the ERAD.  Only anoxic treated skeletal muscle showed metabolic signs of potential apoptosis. It was concluded that wood frog organs activate the UPR as a means of stabilizing cellular proteins and shutting down global protein synthesis in order to survive freezing exposures without irreparable injury.

Ryan Bell.  M.Sc. Biology 2008.

Regulation of glutamate dehydrogenase in hypometabolic states



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



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



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.



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.



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.