Are the effects of catch-and-release angling evident in changes to mRNA abundances related to metabolism, acid-base regulation and stress in lake trout (Salvelinus namaycush) gills?

Catch-and-release (C&R) angling is a conservation-oriented practice intended to reduce the impact recreational angling has on fish populations. Even though most recreationally angled fish are released, little is known about how C&R angling impacts fish at the cellular or tissue level. As the first to explore the impacts of C&R angling on mRNA abundances, our study aimed to identify how the stress of angling influenced metabolism, acid-base regulation and cellular stress in the gills of lake trout (Salvelinus namaycush). Because gills are responsible for metabolic gas exchange, are crucial sites of acid-base homeostasis and respond to stressors quickly, we hypothesized that the relative mRNA abundance of genes related to these three physiological processes would be altered after angling. We took gill samples of live lake trout at 0, 2 or 48 h after fish were angled by rod and reel, and then used quantitative PCR (qPCR) to measure the relative abundance of nine candidate mRNA transcripts. Heat shock protein 70 (hsp70) mRNA levels significantly increased over 5-fold 2 h after angling, indicating a potential activation of a cytoprotective response. However, contrary to our hypothesis, we observed no change in the relative mRNA abundance of genes related to metabolism or acid-base regulation in response to C&R angling within a 48-h period. As C&R angling can negatively impact fish populations, further use of transcript-level studies will allow us to understand the impact C&R has on specific tissues and improve our knowledge of how C&R influences overall fish health.

The contributions of parental lactation on offspring development: It's not udder nonsense!

The Developmental Origins of Health and Disease (DOHaD) hypothesis describes how maternal stress exposures experienced during critical periods of perinatal life are linked to altered developmental trajectories in offspring. Perinatal stress also induces changes in lactogenesis, milk volume, maternal care, and the nutritive and non-nutritive components of milk, affecting short and long-term developmental outcomes in offspring. For instance, selective early life stressors shape the contents of milk, including macro/micronutrients, immune components, microbiota, enzymes, hormones, milk-derived extracellular vesicles, and milk microRNAs. In this review, we highlight the contributions of parental lactation to offspring development by examining changes in the composition of breast milk in response to three well-characterized maternal stressors: nutritive stress, immune stress, and psychological stress. We discuss recent findings in human, animal, and in vitro models, their clinical relevance, study limitations, and potential therapeutic significance to improving human health and infant survival. We also discuss the benefits of enrichment methods and support tools that can be used to improve milk quality and volume as well as related developmental outcomes in offspring. Lastly, we use evidence-based primary literature to convey that even though select maternal stressors may modulate lactation biology (by influencing milk composition) depending on the severity and length of exposure, exclusive and/or prolonged milk feeding may attenuate the negative in utero effects of early life stressors and promote healthy developmental trajectories. Overall, scientific evidence supports lactation to be protective against nutritive and immune stressors, but the benefits of lactation in response to psychological stressors need further investigation.

Non-nutritive bioactive components in maternal milk and offspring development: a scoping review.

Lactation is a critical time in mammalian development, where maternal factors shape offspring outcomes. In this scoping review, we discuss current literature concerning maternal factors that influence lactation biology and highlight important associations between changes in milk composition and offspring outcomes. Specifically, we explore maternal nutritional, psychosocial, and environmental exposures that influence non-nutritive bioactive components in milk and their links to offspring growth, development, metabolic, and behavioral outcomes. A comprehensive literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews (PRISMA-ScR) guidelines. Predetermined eligibility criteria were used to analyze 3,275 papers, and the final review included 40 primary research articles. Outcomes of this review identify maternal obesity to be a leading maternal factor influencing the non-nutritive bioactive composition of milk with notable links to offspring outcomes. Offspring growth and development are the most common modes of programming associated with changes in non-nutritive milk composition due to maternal factors in early life. In addition to discussing studies investigating these key associations, we also identify knowledge gaps in the current literature and suggest opportunities and considerations for future studies.

The role of humanin in natural stress tolerance: An underexplored therapeutic avenue.

BACKGROUND: The discovery of humanin (HN/MTRNR2) 20 years ago blazed a trail to identifying mitochondrial derived peptides with biological function. SCOPE: Humanin is associated with pro-survival, cytoprotective, anti-inflammatory, and anti-oxidative properties and may play a role in reducing neurodegenerative and metabolic disease progression. Although the role of humanin in vitro and in vivo laboratory models is well characterized, the regulation of humanin in natural models that encounter lethal cytotoxic and oxidative insults, as part of their natural history, require immediate research. In this review, we discuss the conservation of humanin-homologues across champion hibernators, anoxia and freeze-tolerant vertebrates and postulate on the putative roles of humanin in non-model species. SIGNIFICANCE: We hope characterization of humanin in animals that are naturally immune to cellular insults, that are otherwise lethal for non-tolerant species, will elucidate key biomarkers and cytoprotective pathways with therapeutic potential and help differentiate pro-survival mechanisms from cellular consequences of stress.

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk.

Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.

Maternal effects in mammals: Broadening our understanding of offspring programming.

The perinatal period is a sensitive time in mammalian development that can have long-lasting consequences on offspring phenotype via maternal effects. Maternal effects have been most intensively studied with respect to two major conditions: maternal diet and maternal stress. In this review, we shift the focus by discussing five major additional maternal cues and their influence on offspring phenotype: maternal androgen levels, photoperiod (melatonin), microbiome, immune regulation, and milk composition. We present the key findings for each of these topics in mammals, their mechanisms of action, and how they interact with each other and with the maternal influences of diet and stress. We explore their impacts in the contexts of both predictive adaptive responses and the developmental origins of disease, identify knowledge gaps and research opportunities in the field, and place a particular emphasis on the application and consideration of these effects in non-model species and natural ecological systems.

Oxidative Damage? Not a Problem! The Characterization of Humanin-like Mitochondrial Peptide in Anoxia Tolerant Freshwater Turtles.

Mitochondria was long thought to be an "end function" organelle that regulated the metabolic flux and apoptosis in the cell. However, with the discovery of the mitochondrial peptide (MDP) humanin (HN/MTRNR2), the cytoprotective and pro-survival applications of MDPs have taken the forefront of therapeutic and diagnostic research. However, the regulation of humanin-like MDPs in natural model systems that can tolerate lethal environmental and cytotoxic insults remains to be investigated. Red-eared sliders are champion anaerobes that can withstand three continuous months of anoxia followed by rapid bouts of oxygen reperfusion without incurring cellular damage. Freshwater turtles employ extensive physiological and biochemical strategies to combat anoxia, with metabolic rate depression and a global enhancement of antioxidant and cytoprotective pathways being the two most important contributors. The main aim of this study was to uncover and characterize the humanin-homologue in freshwater turtles as well as investigate the differential regulation of humanin in response to short and long-term oxygen deprivation. In this study we have used de novo and homology-based protein modelling to elucidate the putative structure of humanin in red-eared sliders as well as an ELISA and western immunoblotting to confirm the protein abundance in the turtle brain and six peripheral tissues during control, 5 h, and 20 h anoxia (n = 4/group). We found that a humanin-homologue (TSE-humanin) is present in red-eared sliders and it may play a cytoprotective role against oxidative damage.

Dynamic regulation of histone H3 lysine (K) acetylation and deacetylation during prolonged oxygen deprivation in a champion anaerobe.

Trachemys scripta elegans can survive up to three months of absolute anoxia at 3 °C and recover with minimal cellular damage. Red-eared sliders employ various physiological and biochemical adaptations to survive anoxia with metabolic rate depression (MRD) being the most prominent adaptation. MRD is mediated by epigenetic, transcriptional, post-transcriptional, and post-translational mechanisms aimed at shutting down cellular processes that are not needed for anoxia survival, while reprioritizing ATP towards cell processes that are vital for anaerobiosis. Histone acetylation/deacetylation are epigenetic modifications that maintain a proper balance between permissive chromatin and restricted chromatin, yet very little is known about protein regulation and enzymatic activity of the writers and erasers of acetylation during natural anoxia tolerance. As such, this study explored the interplay between transcriptional activators, histone acetyltransferases (HATs), and transcriptional repressors, sirtuins (SIRTs), along with three prominent acetyl-lysine (K) moieties of histone H3 in the liver of red-eared sliders. Western immunoblotting was used to measure acetylation levels of H3-K14, H3-K18, and H3-K56, as well as protein levels of histone H3-total, HATs, and nuclear SIRTs in the liver in response to 5 h and 20 h anoxia. Global and nuclear enzymatic activity of HATs and enzymatic activity of nuclear SIRTs were also measured. Overall, a strong suppression of HATs-mediated H3 acetylation and SIRT-mediated deacetylation was evident in the liver of red-eared sliders that could play an important role in ATP conservation as part of the overall reduction in metabolic rate.

Multi-tissue profile of NFκB pathway regulation during mammalian hibernation.

Hibernators have evolved effective mechanisms to overcome the challenges of torpor-arousal cycling. This study focuses on the antioxidant and inflammatory defenses under the control of the redox-sensitive and inflammatory-centered NFκB transcription factor in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), a well-established model of mammalian hibernation. While hibernators significantly depress oxygen consumption and overall metabolic rate during torpor, arousal brings with it a rapid increase in respiration that is associated with an influx of reactive oxygen species. As such, hibernators employ a variety of antioxidant defenses to combat oxidative damage. Herein, we used Luminex multiplex technology to examine the expression of key proteins in the NFκB transcriptional network, including NFκB, super-repressor IκBα, upstream activators TNFR1 and FADD, and downstream target c-Myc. Transcription factor DNA-binding ELISAs were also used to measure the relative degree of NFκB binding to DNA during hibernation. Analyses were performed across eight different tissues, cerebral cortex, brainstem, white and brown adipose tissue, heart, liver, kidney, and spleen, during euthermic control and late torpor to highlight tissue-specific NFκB mediated cytoprotective responses against oxidative stress experienced during torpor-arousal. Our findings demonstrated brain-specific NFκB activation during torpor, with elevated levels of upstream activators, inactive-phosphorylated IκBα, active-phosphorylated NFκB, and enhanced NFκB-DNA binding. Protein levels of downstream protein, c-Myc, also increased in the brain and adipose tissues during late torpor. The results show that NFκB regulation might serve a critical neuroprotective and cytoprotective role in hibernating brains and selective peripheral tissue.

Maternal high-fat diet induces sex-specific changes to glucocorticoid and inflammatory signaling in response to corticosterone and lipopolysaccharide challenge in adult rat offspring.

BACKGROUND: Maternal obesity as a result of high levels of saturated fat (HFD) consumption leads to significant negative health outcomes in both mother and exposed offspring. Offspring exposed to maternal HFD show sex-specific alterations in metabolic, behavioral, and endocrine function, as well as increased levels of basal neuroinflammation that persists into adulthood. There is evidence that psychosocial stress or exogenous administration of corticosterone (CORT) potentiate inflammatory gene expression; however, the response to acute CORT or immune challenge in adult offspring exposed to maternal HFD during perinatal life is unknown. We hypothesize that adult rat offspring exposed to maternal HFD would show enhanced pro-inflammatory gene expression in response to acute administration of CORT and lipopolysaccharide (LPS) compared to control animals, as a result of elevated basal pro-inflammatory gene expression. To test this, we examined the effects of acute CORT and/or LPS exposure on pro and anti-inflammatory neural gene expression in adult offspring (male and female) with perinatal exposure to a HFD or a control house-chow diet (CHD). METHODS: Rat dams consumed HFD or CHD for four weeks prior to mating, during gestation, and throughout lactation. All male and female offspring were weaned on to CHD. In adulthood, offspring were 'challenged' with administration of exogenous CORT and/or LPS, and quantitative PCR was used to measure transcript abundance of glucocorticoid receptors and downstream inflammatory markers in the amygdala, hippocampus, and prefrontal cortex. RESULTS: In response to CORT alone, male HFD offspring showed increased levels of anti-inflammatory transcripts, whereas in response to LPS alone, female HFD offspring showed increased levels of pro-inflammatory transcripts. In addition, male HFD offspring showed greater pro-inflammatory gene expression and female HFD offspring exhibited increased anti-inflammatory gene expression in response to simultaneous CORT and LPS administration. CONCLUSIONS: These findings suggest that exposure to maternal HFD leads to sex-specific changes that may alter inflammatory responses in the brain, possibly as an adaptive response to basal neuroinflammation.

Metabolic reorganization in winter: Regulation of pyruvate dehydrogenase (PDH) during long-term freezing and anoxia.

Wood frogs, Rana sylvatica, can undergo prolonged periods of whole body freezing during winter, locking as much as 65-70% of total body water into extracellular ice and imposing both anoxia and dehydration on their cells. Metabolic rate depression (MRD) is an adaptation used by R. sylvatica to survive these environmental stresses, where a finite amount of ATP generated through anaerobic metabolism is directed towards maintaining pro-survival functions, while most ATP-expensive cellular processes are temporarily reduced in function. Pyruvate dehydrogenase (PDH) is a vital metabolic enzyme that links anaerobic glycolysis to the aerobic TCA cycle and is an important regulatory site in MRD. PDH enzymatic activity is regulated via reversible protein phosphorylation in response to energetic demands of cells. This study explored the posttranslational regulation of PDH at three serine sites (S232, S293, S300) on the catalytic E1α subunit along with protein expression of four pyruvate dehydrogenase kinases (PDHK1-4) in response to 24 h Freezing, 8 h Thaw, 24 h Anoxia, and 4 h Recovery in the liver and skeletal muscle of R. sylvatica using Luminex multiplex technology and western immunoblotting. Overall, inhibitory regulation of PDH was evident during 24 h Freezing and 24 h Anoxia, which could indicate a notable reduction in glycoytic flux and carbon entry into the tricarboxylic acid cycle as part of MRD. Furthermore, the expression of PDHK1-4 and phosphorylation of PDH at S232, S293, and S300 were highly tissue and stress-specific, indicative of how different tissues respond differently to stress within the same organism.

Dynamic regulation of six histone H3 lysine (K) methyltransferases in response to prolonged anoxia exposure in a freshwater turtle.

The importance of histone lysine methylation is well established in health, disease, early development, aging, and cancer. However, the potential role of histone H3 methylation in regulating gene expression in response to extended periods of oxygen deprivation (anoxia) in a natural, anoxia-tolerant model system is underexplored. Red-eared sliders (Trachemys scripta elegans) can tolerate and survive three months of absolute anoxia and recover without incurring detrimental cellular damage, mainly by reducing the overall metabolic rate by 90% when compared to normoxia. Stringent regulation of gene expression is a vital aspect of metabolic rate depression in red-eared sliders, and as such we examined the anoxia-responsive regulation of histone lysine methylation in the liver during 5 h and 20 h anoxia exposure. Interestingly, this is the first study to illustrate the existence of histone lysine methyltransferases (HKMTs) and corresponding histone H3 lysine methylation levels in the liver of anoxia-tolerant red-eared sliders. In brief, H3K4me1, a histone mark associated with active transcription, and two corresponding histone lysine methyltransferases that modify H3K4me1 site, significantly increased in response to anoxia. On the contrary, H3K27me1, another transcriptionally active histone mark, significantly decreased during 20 h anoxia, and a transcriptionally repressive histone mark, H3K9me3, and the corresponding KMTs, similarly increased during 20 h anoxia. Overall, the results suggest a dynamic regulation of histone H3 lysine methylation in the liver of red-eared sliders that could theoretically aid in the selective upregulation of genes that are necessary for anoxia survival, while globally suppressing others to conserve energy.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 4. Regulation of pyruvate dehydrogenase complex and metabolic fuel selection.

Mammalian hibernation is characterized by extensive adjustments to metabolism that typically include suppression of carbohydrate catabolism and a switch to triglycerides as the primary fuel during torpor. A crucial locus of control in this process is the pyruvate dehydrogenase complex that gates carbohydrate entry into the tricarboxylic acid cycle. Within the complex, the E1 enzyme pyruvate dehydrogenase (PDH) is the main regulatory site and is subject to inhibitory phosphorylation at three serine residues (S232, S293, S300). To determine if marsupial hibernators show a comparable focus on PDH to regulate fuel metabolism, the current study explored PDH control by site-specific phosphorylation in the South American marsupial, monito del monte (Dromiciops gliroides). Luminex multiplex technology was used to analyze PDH responses in six tissues comparing control and hibernating (4days continuous torpor) animals. Total PDH content did not change significantly during hibernation in any tissue but phospho-PDH content increased in all. Heart PDH showed increased phosphorylation at all three sites by 8.1-, 10.6- and 2.1-fold for S232, S293 and S300, respectively. Liver also showed elevated p-S300 (2.5-fold) and p-S293 (4.7-fold) content. Phosphorylation of S232 and S293 increased significantly in brain and lung but only S232 phosphorylation increased in kidney and skeletal muscle. The results show that PDH suppression via enzyme phosphorylation during torpor is a conserved mechanism for inhibiting carbohydrate catabolism in both marsupial and eutherian mammals, an action that would also promote the switch to fatty acid oxidation instead.

Strategies of biochemical adaptation for hibernation in a South American marsupial Dromiciops gliroides: 1. Mitogen-activated protein kinases and the cell stress response.

Hibernation is a period of torpor and heterothermy that is typically associated with a strong reduction in metabolic rate, global suppression of transcription and translation, and upregulation of various genes/proteins that are central to the cellular stress response such as protein kinases, antioxidants, and heat shock proteins. The current study examined cell signaling cascades in hibernating monito del monte, Dromiciops gliroides, a South American marsupial of the Order Microbiotheria. Responses to hibernation by members of the mitogen-activated protein kinase (MAPK) pathways, and their roles in coordinating hibernator metabolism were examined in liver, kidney, heart and brain of control and versus hibernating (4days continuous torpor) D. gliroides. The targets evaluated included key protein kinases in their activated phosphorylated forms (p-ERK/MAPK 1/2, p-MEK1, p-MSK1, p-p38, p-JNK) and related target proteins (p-CREB 2, p-ATF2, p-c-Jun and p-p53). Liver exhibited a strong coordinated response by MAPK members to hibernation with significant increases in protein phosphorylation levels of p-MEK1, p-ERK/MAPK1/2, p-MSK1, p-JNK and target proteins c-Jun, and p-ATF2, all combining to signify a strong activation of MAPK signaling during hibernation. Kidney also showed activation of MAPK cascades with significant increases in p-MEK1, p-ERK/MAPK1/2, p-p38, and p-c-Jun levels in hibernating animals. By contrast, responses by heart and brain indicated reduced MAPK pathway function during torpor with reduced phosphorylation of targets including p-ERK/MAPK 1/2 in both tissues as well as lower p-p38 and p-JNK content in heart. Overall, the data indicate a vital role for MAPK signaling in regulating the cell stress response during marsupial hibernation.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3ß(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3ß, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3ß (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 3. Activation of pro-survival response pathways.

The South American marsupial, monito del monte (Dromiciops gliroides) uses both daily torpor and multi-day hibernation to survive in its southern Chile native environment. The present study leverages multiplex technology to assess the contributions of key stress-inducible cell cycle regulators and heat shock proteins to hibernation in liver, heart, and brain of monito del monte in a comparison of control versus 4day hibernating conditions. The data indicate that MDM2, a stress-responsive ubiquitin ligase, plays a crucial role in marsupial hibernation since all three tissues showed statistically significant increases in MDM2 levels during torpor (1.6-1.8 fold). MDM2 may have a cytoprotective action to deal with ischemia/reperfusion stress and is also involved in a nutrient sensing pathway where it could help regulate the metabolic switch to fatty acid oxidation during torpor. Elevated levels of stress-sensitive cell cycle regulators including ATR (2.32-3.91 fold), and the phosphorylated forms of p-Chk1 (Ser345) (1.92 fold), p-Chk2 (Thr68) (2.20 fold) and p21 (1.64 fold) were observed in heart and liver during hibernation suggesting that the cell cycle is likely suppressed to conserve energy while animals are in torpor. Upregulation of heat shock proteins also occurred as a cytoprotective strategy with increased levels of hsp27 (2.00 fold) and hsp60 (1.72-2.76 fold) during hibernation. The results suggest that cell cycle control and selective chaperone action are significant components of hibernation in D. gliroides and reveal common molecular responses to those seen in eutherian hibernators.

The regulation of heat shock proteins in response to dehydration in Xenopus laevis.

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45-1.65-fold) in the kidneys and lungs, and HSP40 (1.39-2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43-0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39-1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20-1.36-fold), but decreased in skeletal muscle (0.27-0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39-0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.

Regulation of pyruvate dehydrogenase (PDH) in the hibernating ground squirrel, (Ictidomys tridecemlineatus).

Pyruvate dehydrogenase (PDH) is a vital regulatory enzyme that catalyzes the conversion of pyruvate into acetyl-CoA and connects anaerobic glycolysis to aerobic TCA cycle. Post-translational inhibition of PDH activity via three serine phosphorylation sites (pS232, pS293, and pS300) regulate the metabolic flux through the TCA cycle, decrease glucose utilization, and facilitate lipid metabolism during times of nutrient deprivation. As metabolic readjustment is necessary to survive hibernation, the purpose of this study was to explore the post-translational regulation of pyruvate dehydrogenase and the expression levels of four mitochondrial serine/threonine kinases (PDHKs), during torpor-arousal cycles in liver, heart, and skeletal muscle of 13-lined ground squirrels. A combination of Luminex multiplex technology and western immunoblotting were used to measure the protein expression levels of total PDH, three phosphorylation sites, S232, 293, 300, and the expression levels of the corresponding PDH kinases (PDHK1-4) during euthermic control, entrance, late torpor, and interbout arousal. Liver and heart showed strong inhibitory PDH regulation, indicating a possible decrease in glucose utilization and a possible preference for ß-oxidation of fatty acids during periods of low temperature and starvation. On the contrary, skeletal muscle showed limited PDH regulation via phosphorylation, possibly due to alternate controls. Phosphorylation of PDH may play an important role in regulating aerobic and anaerobic metabolic responses during hibernation in the 13-lined ground squirrel.

MAP kinase signaling and Elk1 transcriptional activity in hibernating thirteen-lined ground squirrels.

BACKGROUND: The thirteen-lined ground squirrel (I. tridecemlineatus) becomes hypometabolic during hibernation; characterized by reductions in energy expensive processes like transcription during periods of low temperature and starvation. We were interested in elucidating the mechanisms of transcriptional control by studying the MAPK pathway and downstream transcription factors in skeletal muscle. We were also interested in how environmental factors, such as body temperature, that fluctuate during hibernation, can affect transcriptional regulation. METHODS: Protein abundance of MAPKs and effectors were quantified using immunoblotting and Luminex® multiplex assays. DNA-protein interaction (DPI)-ELISA was used to quantitatively assess the binding of transcription factors to DNA promoter sequences under environmental conditions which are reflective of those during torpor. RESULTS: JNK2/3 showed increases in protein abundance during early arousal. The transcription factor, Elk1, showed increases in phosphorylation of S383 (which is indicative of increased activity) during arousal that accompany decreases in total protein levels. Further analysis on the relative binding of this transcription factor to its consensus sequence during euthermia and torpor showed that its binding is significantly different when environmental conditions such as temperature, [urea], and [Ca2+] were changed. CONCLUSION: We show evidence of Elk1 regulation during hibernation, and its activity could be influenced by environmental factors such as temperature, [urea] and [Ca2+], as well as post-translational modifications via JNK2/3. GENERAL SIGNIFICANCE: I. tridecemlineatus has natural mechanisms of transcriptional regulation during hibernation through phosphorylation and changes to the cellular environment. We identify regulation of JNK and Elk1 in the skeletal muscle of hibernating I. tridecemlineatus.

Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana sylvatica).

The common wood frog, Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65-70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP) response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46), and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70), Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.