Changes in microRNA expression related to ischemia-reperfusion injury in the kidney of the thirteen-lined ground squirrel during torpor.

During the hibernation season, the thirteen-lined ground squirrel undergoes cyclical torpor and arousal periods. The decrease and restoration of metabolic rate and oxygen delivery during torpor and arousal, respectively, may cause reperfusion-ischemia injury in the kidneys. In order to maintain the structural integrity of the kidneys necessary for renal function resumption during arousal, the thirteen-lined ground squirrel has developed adaptive methods to prevent and repair kidney injury. In this present study, computational methods were used to clean and analyze sequenced kidney RNA samples. Significantly differentially expressed microRNAs and enriched gene sets were also determined. From the gene set analysis, the results showed an increase in ubiquitin-related processes and p53 signaling pathways which suggested the occurrence of kidney damage during torpor. There was also an observed increase in cell cycle processes and the anchoring junction cellular compartment which may lend to the prevention of kidney injury. From the differentially expressed microRNAs, miR-27a (log2FC = 1.639; p-value = 0.023), miR-129 (log2FC = 2.516; p-value = 0.023), miR-let-7b (log2FC = 2.360; p-value = 0.025), miR-let-7c (log2FC = 2.291; p-value = 0.037) and miR-let-7i (log2FC = 1.564; p-value = 0.039) were found to be significantly upregulated. These biochemical adaptations may allow the thirteen-lined ground squirrel to maintain kidney structure and function during hibernation.

Role of MicroRNAs in Extreme Animal Survival Strategies.

The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to play a major role. In this chapter, we discuss the activation, expression, biogenesis, and unique attributes of miRNA regulation required to facilitate profound metabolic rate depression and implement stress-specific metabolic adaptations. We examine the role of miRNA in strategies of biochemical adaptation including mammalian hibernation, freeze tolerance, freeze avoidance, anoxia and hypoxia survival, estivation, and dehydration tolerance. By comparing these seemingly different adaptive programs in traditional and exotic animal models, we highlight both unique and conserved miRNA-meditated mechanisms for survival. Additional topics discussed include transcription factor networks, temperature dependent miRNA-targeting, and novel species-specific and stress-specific miRNAs.

44 Current Challenges in miRNomics.

Mature microRNAs (miRNAs) are short RNA sequences about 18-24 nucleotide long, which provide the recognition key within RISC for the posttranscriptional regulation of target RNAs. Considering the canonical pathway, mature miRNAs are produced via a multistep process. Their transcription (pri-miRNAs) and first processing step via the microprocessor complex (pre-miRNAs) occur in the nucleus. Then they are exported into the cytosol, processed again by Dicer (dsRNA) and finally a single strand (mature miRNA) is incorporated into RISC (miRISC). The sequence of the incorporated miRNA provides the function of RNA target recognition via hybridization. Following binding of the target, the mRNA is either degraded or translation is inhibited, which ultimately leads to less protein production. Conversely, it has been shown that binding within the 5' UTR of the mRNA can lead to an increase in protein product. Regulation of homeostasis is very important for a cell; therefore, all steps in the miRNA-based regulation pathway, from transcription to the incorporation of the mature miRNA into RISC, are under tight control. While much research effort has been exerted in this area, the knowledgebase is not sufficient for accurately modelling miRNA regulation computationally. The computational prediction of miRNAs is, however, necessary because it is not feasible to investigate all possible pairs of a miRNA and its target, let alone miRNAs and their targets. We here point out open challenges important for computational modelling or for our general understanding of miRNA-based regulation and show how their investigation is beneficial. It is our hope that this collection of challenges will lead to their resolution in the near future.

Insights from a vertebrate model organism on the molecular mechanisms of whole-body dehydration tolerance.

Studies on the molecular mechanisms of dehydration tolerance have been largely limited to plants and invertebrates. Currently, research in whole body dehydration of complex animals is limited to cognitive and behavioral effects in humans, leaving the molecular mechanisms of vertebrate dehydration relatively unexplored. The present review summarizes studies to date on the African clawed frog (Xenopus laevis) and examines whole-body dehydration on physiological, cellular and molecular levels. This aquatic frog is exposed to seasonal droughts in its native habitat and can endure a loss of over 30% of its total body water. When coping with dehydration, osmoregulatory processes prioritize water retention in skeletal tissues and vital organs over plasma volume. Although systemic blood circulation is maintained in the vital organs and even elevated in the brain during dehydration, it is done so at the expense of reduced circulation to the skeletal muscles. Increased hemoglobin affinity for oxygen helps to counteract impaired blood circulation and metabolic enzymes show altered kinetic and regulatory parameters that support the use of anaerobic glycolysis. Recent studies with X. laevis also show that pro-survival pathways such as antioxidant defenses and heat shock proteins are activated in an organ-specific manner during dehydration. These pathways are tightly coordinated at the post-transcriptional level by non-coding RNAs, and at the post-translational level by reversible protein phosphorylation. Paired with ongoing research on the X. laevis genome, the African clawed frog is poised to be an ideal animal model with which to investigate the molecular adaptations for dehydration tolerance much more deeply.

Phosphoproteomic Analysis of X enopus laevis Reveals Expression and Phosphorylation of Hypoxia-Inducible PFKFB3 during Dehydration.

Xenopus laevis tolerate dehydration when their environments evaporate during summer months. Protein phosphorylation has previously shown to regulate important adaptations in X. laevis, including the transition to anaerobic metabolism. We therefore performed phosphoproteomic analysis of X. laevis to further elucidate the cellular and metabolic responses to dehydration. Phosphoproteins were enriched in cellular functions and pathways related to glycolysis/gluconeogenesis, the TCA cycle, and protein metabolism, among others. The prominence of phosphoproteins related to glucose metabolism led us to discover that the hypoxia-inducible PFKFB3 enzyme was highly phosphorylated and likely activated during dehydration, a feature of many cancers. Expression of the four transcript variants of the pfkfb3 gene was found all to be upregulated during dehydration, potentially due to the enrichment of hypoxia responsive elements in the pfkfb3 promoter. These results further support the role of anaerobic glycolysis during dehydration in X. laevis and elucidate a potential mechanism for its increased rate.

MicroRNA expression in the heart of Xenopus laevis facilitates metabolic adaptation to dehydration.

Xenopus laevis survive severe dehydration during the summer months in their natural range. MicroRNA regulate translation of target mRNAs and have shown to be differentially expressed in response to dehydration in X. laevis. During dehydration, heart rate is elevated which appears to compensate for the reduced oxygen delivery capability due to increased hematocrit. We hypothesized that microRNAs would be differentially expressed in the heart to modulate gene expression levels in response to dehydration. The present study assessed changes in the microRNAome of X. laevis heart in response to severe dehydration (30% loss of body water) using microRNA-seq. We show that target genes are enriched for RNA, DNA, and transcription factor binding activities, cardiac muscle contraction, and glycolysis/gluconeogenesis. These results suggest that microRNAs contribute to gene expression reorganization in the heart in response to dehydration, putatively supporting the increased physiological demands and ATP production needs by the heart under these conditions.

Advances and applications of environmental stress adaptation research.

Evolution has produced animals that survive extreme fluctuations in environmental conditions including freezing temperatures, anoxia, desiccating conditions, and prolonged periods without food. For example, the wood frog survives whole-body freezing every winter, arresting all gross physiological functions, but recovers functions upon thawing in the spring. Likewise, many small mammals hibernate for months at a time with minimal metabolic activity, organ perfusion, and movement, yet do not suffer significant muscle atrophy upon arousal. These conditions and the biochemical adaptations employed to deal with them can be viewed as Nature's answer to problems that humans wish to answer, particularly in a biomedical context. This review focuses on recent advances in the field of animal environmental stress adaptation, starting with an emphasis on new areas of research such as epigenetics and microRNA. We then examine new and emerging technologies such as genome editing, novel sequencing applications, and single cell analysis and how these can push us closer to a deeper understanding of biochemical adaptation. Next, evaluate the potential contributions of new high-throughput technologies (e.g. next-generation sequencing, mass spectrometry proteomics) to better understanding the adaptations that support these extreme phenotypes. Concluding, we examine some of the human applications that can be gained from understanding the principles of biochemical adaptation including organ preservation and treatments for conditions such as ischemic stroke and muscle disuse atrophy.

DNA methylation and regulation of DNA methyltransferases in a freeze-tolerant vertebrate.

The wood frog is one of the few freeze-tolerance vertebrates. This is accomplished in part by the accumulation of cryoprotectant glucose, metabolic rate depression, and stress response activation. These may be achieved by mechanisms such as DNA methylation, which is typically associated with transcriptional repression. Hyperglycemia is also associated with modifications to epigenetic profiles, indicating an additional role that the high levels of glucose play in freeze tolerance. We sought to determine whether DNA methylation is affected during freezing exposure, and whether this is due to the wood frog's response to hyperglycemia. We examined global DNA methylation and DNA methyltransferases (DNMTs) in the liver and muscle of frozen and glucose-loaded wood frogs. The results showed that levels of 5-methylcytosine (5mC) increased in the muscle, suggesting elevated DNA methylation during freezing. DNMT activities also decreased in muscle during thawing, glucose loading, and in vitro glucose experiments. Liver DNMT activities were similar to muscle; however, a varied response to DNMT levels and a decrease in 5mC highlight the metabolic role the liver plays during freezing. Glucose was also shown to decrease DNMT activity levels in the wood frog, in vitro, elucidating a potentially novel regulatory mechanism. Together these results suggest an interplay between freeze tolerance and hyperglycemic regulation of DNA methylation.

Naked mole rats activate neuroprotective proteins during hypoxia.

Naked mole rats are a long-lived animal model that age much like humans, but that can also withstand oxidative damage, cancer, neurodegenerative diseases, and severe hypoxic conditions, which is of particular interest to this study. The conditions of their underground burrows result in competition for oxygen consumption, yet despite this oxygen deprivation they emerge unscathed. To understand the mechanisms in place to facilitate neuronal preservation during hypoxia, we investigated the protein levels of well-known cell-stress factors. We found that under hypoxic conditions, nearly half of the proteins measured increased expression in brain, while only a few decreased. Under hypoxic conditions there appeared to be a HIF1α-centered response, where HIF1α and its interactors carbonic anhydrase 9, CITED2, p21/CIP1, and NFκB1, among others, were upregulated. Concurrently, a hypoxia-induced decrease of cytochrome c was consistent with decreased mitochondrial function and protection from apoptosis. The picture that emerges is one of neuroprotection, cell-cycle arrest, and the promotion of antiapoptotic functions, all of which are consistent with conserving energy and maintaining neural integrity under low oxygen levels. These results suggest how this species may be poised to face hypoxia and contribute to its remarkable ability to deal with myriad of other damaging factors and sets the stage for future work on the neuroprotective facilitators we identified.

Glucose and urea metabolic enzymes are differentially phosphorylated during freezing, anoxia, and dehydration exposures in a freeze tolerant frog.

Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress.

Transcriptional regulation of metabolism in disease: From transcription factors to epigenetics.

Every cell in an individual has largely the same genomic sequence and yet cells in different tissues can present widely different phenotypes. This variation arises because each cell expresses a specific subset of genomic instructions. Control over which instructions, or genes, are expressed is largely controlled by transcriptional regulatory pathways. Each cell must assimilate a huge amount of environmental input, and thus it is of no surprise that transcription is regulated by many intertwining mechanisms. This large regulatory landscape means there are ample possibilities for problems to arise, which in a medical context means the development of disease states. Metabolism within the cell, and more broadly, affects and is affected by transcriptional regulation. Metabolism can therefore contribute to improper transcriptional programming, or pathogenic metabolism can be the result of transcriptional dysregulation. Here, we discuss the established and emerging mechanisms for controling transcription and how they affect metabolism in the context of pathogenesis. Cis- and trans-regulatory elements, microRNA and epigenetic mechanisms such as DNA and histone methylation, all have input into what genes are transcribed. Each has also been implicated in diseases such as metabolic syndrome, various forms of diabetes, and cancer. In this review, we discuss the current understanding of these areas and highlight some natural models that may inspire future therapeutics.

Roles for lysine acetyltransferases during mammalian hibernation.

The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is a well-known model for studying hibernation. While in a torpid state, these animals globally suppress energy expensive processes, while supporting specialized pathways necessary for survival. Lysine acetyltransferases (KATs) play a crucial role in modulating the expression and activity of a wide-variety of cellular pathways and processes, and therefore, may play a role during hibernation when the cell is shifting to an energy conservative, cytoprotective state. Here we measured protein levels of four KATs (CBP, PCAF, GCN5L2, HAT1), total histone acetyltransferase (HAT) activity, and the levels of acetylation of histone H3 lysine 9 (H3K9ac), in multiple tissues across the torpor-arousal cycle. Our results show a tissue-specific response of KATs, particularly in the adipose tissues where specific KATs (PCAF and GCN5L2), HAT activity, and H3K9ac increased in the metabolically active BAT while HAT1, HAT activity and H3K9ac decreased in WAT. Liver showed significant increases in the KAT PCAF whereas skeletal muscle had decreased CBP and GCN5L2. Both liver and skeletal muscle showed no change in HAT activity and H3K9me3 increased in muscle during torpor. Together, these results suggest KATs may play specialized roles in the different tissues of the ground squirrel to contribute to the hibernator phenotype.

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.

Histone methylation in the freeze-tolerant wood frog (Rana sylvatica).

Freeze-tolerant animals survive sub-zero temperatures and long-term starvation associated with the winter by lowering their metabolic rate using a variety of transcriptional, translational, and post-translational regulatory methods. Histone methylation is one mechanism that is known to regulate gene expression at the transcriptional level. Here, we measured relative protein levels of seven histone methyltransferases (SMYD2, SETD7, ASH2L, RBBP5, SUV39H1, EHMT2, and SET8), four methylated histone H3 residues (H3K4me1, H3K9me3, H3K27me1, and H3K36me2), the methyltransferase activity on H3K4, and methylation of p53 (p53K370me2 and p53K372me1) in the skeletal muscle and liver of the freeze-tolerant wood frog (Rana sylvatica) during the freeze-thaw cycle. Overall, the results reveal a tissue-specific expression of histone methyltransferases and the methylation sites on histone H3 during freezing and thaw. In liver, H3K4me1 significantly decreased during freezing, H3K9me3 remained constant across conditions, H3K27me1 increased only during thaw, and H3K36me2 increased during freezing and then decreased during thaw (p < 0.05, n = 4). In skeletal muscle, H3K4me1 and H3K27me1 both decreased during freezing, whereas H3K9me3 and H3K36me2 were maintained across freezing and thaw (p < 0.05, n = 4). Methylation of p53 was also tissue-specific, where no changes were seen in liver tissue; however, p53 in skeletal muscle was differentially methylated. Overall, these results provide an evidence for the potential role methylation of histones and non-histone proteins play in freezing survival and entrance into a hypometabolic state.

Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.