RESUMO
Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.
Assuntos
Aclimatação , Desidratação/enzimologia , Enzimas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Tardígrados/enzimologia , Animais , Desidratação/genética , Dessecação , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Conformação Proteica , Interferência de RNA , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Tardígrados/genética , Regulação para Cima , VitrificaçãoRESUMO
Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.
Assuntos
Soroalbumina Bovina , Trealose , Aclimatação , Animais , Membrana Celular/metabolismo , Crioprotetores/farmacologia , Ficoll , Congelamento , Insetos/metabolismo , Larva/metabolismo , Prolina/metabolismoRESUMO
Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing-induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport, and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.
Assuntos
Gelo , Perciformes , Animais , Congelamento , Multiômica , Vertebrados , Adaptação Fisiológica/fisiologia , Aclimatação/genéticaRESUMO
Cryopreservation presents a critical challenge due to cryo-damage, such as crystallization and osmotic imbalances that compromise the integrity of biological tissues and cells. In contrast, various organisms in nature exhibit remarkable freezing tolerance, leveraging complex molecular mechanisms to survive extreme cold. This review explores the adaptive strategies of freeze-tolerant species, including the regulation of specific genes, proteins, and metabolic pathways, to enhance survival in low-temperature environments. We then discuss recent advancements in cryopreservation technologies that aim to mimic these natural phenomena to preserve cellular and tissue integrity. Special focus is given to the roles of glucose metabolism, microRNA expression, and cryoprotective protein modulation in improving cryopreservation outcomes. The insights gained from studying natural antifreeze mechanisms offer promising directions for advancing cryopreservation techniques, with potential applications in medical, agricultural, and conservation fields. Future research should aim to further elucidate these molecular mechanisms to develop more effective and reliable cryopreservation methods.
RESUMO
For the breadth of the winter, Dryophytes versicolor can survive full body freezing utilizing a phenomenon known as metabolic rate depression (MRD). Epigenetic transcriptional control on gene expression, such as histone methylation and acetylation, can aid in implementing a balance between permissive and restricted chromatin required to endure this stress. As such, this study explores the interplay between histone lysine methyl and acetyl transferases (HKMTs, HATs), as well as the abundance of various acetyl-lysine and methyl-lysine moieties on histone H3 and H4. Results showing that overexpression of transcriptionally repressive marks, and under expression of active ones, suggest a negative effect on overall gene transcription in skeletal muscle tissue.
Assuntos
Epigênese Genética , Histonas , Lisina , Músculo Esquelético , Histonas/metabolismo , Músculo Esquelético/metabolismo , Lisina/metabolismo , Acetilação , Metilação , Animais , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , CongelamentoRESUMO
Rana sylvatica (also known as Boreorana sylvatica) is one of the few vertebrates that spend extreme winters showing no physiological signs of life. Up to 70% of the total body water of the wood frog freezes as extracellular ice. Survival in extreme conditions requires regulation at transcriptional and translational levels to activate prosurvival pathways. N6-methyladenosine (m6A) methylation is one of the most common RNA modifications, regulating transcript processing and translation by executing important functions that affect regulatory pathways in stress conditions. In the study, regulation of m6A-related proteins in the liver of R. sylvatica was analyzed during 24 h frozen and 8 h thaw conditions. Decreases in the activity of demethylases of 28.44 ± 0.4% and 24.1 ± 0.9% of control values in frozen and thaw tissues, respectively, were observed. Total protein levels of m6A methyltransferase complex components methyltransferase-like 14 and Wilm's tumor associated protein were increased by 1.28-fold and 1.42-fold, respectively, during freezing. Demethylase fat mass and obesity, however, showed a decreasing trend, with a significant decrease in abundance during recovery from frozen conditions. Levels of mRNA degraders YTHDF2 and YTHDC2 also decreased under stress. Overall, increased levels of m6A methylation complex components, and suppressed levels of readers/erasers, provide evidence for the potential role of RNA methylation in freezing survival and its regulation in a hypometabolic state.
Assuntos
Metiltransferases , Ranidae , Animais , Congelamento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metilação , Ranidae/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Fígado/metabolismoRESUMO
The freeze-tolerant anuran Dryophytes chrysoscelis, Cope's gray treefrog, mobilizes a complex cryoprotectant system that includes glycerol, glucose, and urea to minimize damage induced by freezing and thawing of up to 65% of body water. In this species' eastern Northern American temperate habitat, oscillations of temperature above and below freezing are common; however, the effects of repeated freezing and thawing in this species are unstudied. The biochemical and physiological effects of repeated freeze-thaw cycles were therefore evaluated and compared with cold acclimation and single freeze-thaw episodes. Glycerol was elevated in plasma, liver, and skeletal muscle of both singly and repeatedly frozen and thawed animals compared with cold-acclimated frogs. In contrast, urea was unchanged by freezing and thawing, whereas glucose was elevated in singly frozen and thawed animals but was reduced toward cold acclimation levels after repeated bouts of freezing. Overall, the cryoprotectant system was maintained, but not further elevated, in all tissues assayed in repeatedly frozen and thawed animals. For repeated freeze-thaw only, hepatic glycogen was depleted and plasma hemoglobin, indicative of erythrocyte hemolysis, increased. Postfreeze recovery of locomotor function, including limb and whole body movement, was delayed with repeated freeze-thaw and was associated with glycerol accumulation and glycogen depletion. Individuals that resumed locomotor function more quickly also accumulated greater cryoinjury. Integrated analyses of cryoprotectant and cryoinjury accumulation suggest that winter survival of D. chrysoscelis may be vulnerable to climate change, limited by carbohydrate stores, cellular repair mechanisms, and plasticity of the cryoprotectant system.
Assuntos
Crioprotetores , Glicerol , Animais , Congelamento , Anuros/fisiologia , Glucose , UreiaRESUMO
Populations from different climates often show unique growth responses to temperature, reflecting temperature adaptation. Yet, whether populations from different climates differ in physiological temperature acclimation remains unclear. Here, we test whether populations from differing thermal environments exhibit different growth responses to temperature and differences in temperature acclimation of leaf respiration. We grew tropical and subtropical populations of two mangrove species (Avicennia germinans and Rhizophora mangle) under ambient and experimentally warmed conditions in a common garden at the species' northern range limit. We quantified growth and temperature responses of leaf respiration (R) at seven time points over ~10 months. Warming increased productivity of tropical populations more than subtropical populations, reflecting a higher temperature optimum for growth. In both species, R measured at 25 °C declined as seasonal temperatures increased, demonstrating thermal acclimation. Contrary to our expectations, acclimation of R was consistent across populations and temperature treatments. However, populations differed in adjusting the temperature sensitivity of R (Q10) to seasonal temperatures. Following a freeze event, tropical Avicennia showed greater freeze damage than subtropical Avicennia, while both Rhizophora populations appeared equally susceptible. We found evidence of temperature adaptation at the whole-plant scale but little evidence for population differences in thermal acclimation of leaf physiology. Studies that examine potential costs and benefits of thermal acclimation in an evolutionary context may provide new insights into limits of thermal acclimation.
Assuntos
Aclimatação , Clima , Aclimatação/fisiologia , Temperatura , Respiração , Folhas de Planta/fisiologiaRESUMO
The great complexity of extracellular freezing stress, involving mechanical, osmotic, dehydration and chemical perturbations of the cellular milieu, hampers progress in understanding the nature of freezing injury and the mechanisms to cope with it in naturally freeze-tolerant insects. Here, we show that nuclear DNA fragmentation begins to occur in larval haemocytes of two fly species, Chymomyza costata and Drosophila melanogaster, before or at the same time as the sub-zero temperature is reached that causes irreparable freezing injury and mortality in freeze-sensitive larval phenotypes. However, when larvae of the freeze-tolerant phenotype (diapausing-cold acclimated-hyperprolinemic) of C. costata were subjected to severe freezing stress in liquid nitrogen, no DNA damage was observed. Artificially increasing the proline concentration in freeze-sensitive larvae of both species by feeding them a proline-enriched diet resulted in a decrease in the proportion of nuclei with fragmented DNA during freezing stress. Our results suggest that proline accumulated in diapausing C. costata larvae during cold acclimation may contribute to the protection of nuclear DNA against fragmentation associated with freezing stress.
Assuntos
Drosophila melanogaster , Insetos , Animais , Congelamento , Larva , Temperatura Baixa , Aclimatação , ProlinaRESUMO
The wood frog (Rana Sylvatica) can endure the sub-zero temperatures of winter by freezing up to 65% of total body water as extracellular ice and retreating into a prolonged hypometabolic state. Freeze survival requires the coordination of various adaptations, including a global suppression of metabolic functions and select activation of pro-survival genes. Transcription factors playing roles in metabolism, stress tolerance, and cell proliferation may assist in making survival in a frozen state possible. In this study, the role of Forkhead box 'other' (FOXO) transcription factors in freeze tolerance, and related changes to the insulin pathway, are investigated. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frogs subjected to freezing for 24 h and thawed recovery for 8 h. Levels of active FOXO3 increased in brain, kidney, and liver during freezing and thawing, suggesting a need to maintain or enhance antioxidant defenses under these stresses. Results implicate FOXO involvement in the metabolic regulation of natural freeze tolerance.
Assuntos
Criopreservação , Fatores de Transcrição , Animais , Congelamento , Fatores de Transcrição/metabolismo , Criopreservação/métodos , Aclimatação , Ranidae/metabolismoRESUMO
The wood frog, Rana sylvatica (aka Lithobates sylvaticus) is the main model for studies of natural freeze tolerance among amphibians living in seasonally cold climates. During freezing, â¼65% of total body water can be converted to extracellular ice and this imposes both dehydration and hypoxia/anoxia stresses on cells. The current study analyzed the responses of the alpha subunit of the hypoxia-inducible transcription factor (HIF-1), a crucial oxygen-sensitive regulator of gene expression, to freezing, anoxia or dehydration stresses, examining six tissues of wood frogs (liver, skeletal muscle, brain, heart, kidney, skin). RT-PCR revealed a rapid elevation hif-1α transcript levels within 2 h of freeze initiation in both liver and brain and elevated levels of both mRNA and protein in liver and muscle after 24 h frozen. However, both transcript and protein levels reverted to control values after thawing except for HIF-1 protein in liver that dropped to â¼60% of control. Independent exposures of wood frogs to anoxia or dehydration stresses (two components of freezing) also triggered upregulation of hif-1α transcripts and/or HIF-1α protein in liver and kidney with variable responses in other tissues. The results show active modulation of HIF-1 in response to freezing, anoxia and dehydration stresses and implicate this transcription factor as a contributor to the regulation of metabolic adaptations needed for long term survival of wood frogs in the ischemic frozen state.
Assuntos
Criopreservação , Desidratação , Animais , Congelamento , Desidratação/metabolismo , Criopreservação/métodos , Hipóxia/metabolismo , Ranidae/metabolismo , Músculo Esquelético/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The goldenrod gall fly (Eurosta solidaginis) is a well-studied model of insect freeze tolerance. In situations of prolonged winter subzero temperatures, larvae of E. solidaginis accept ice penetration throughout extracellular spaces while protecting the intracellular environment by producing extreme amounts of glycerol and sorbitol as cryoprotectants. Hypometabolism (diapause) is implemented, and energy use is reprioritized to essential pathways. Gene transcription is one energy-expensive process likely suppressed over the winter, in part, due to epigenetic controls. The present study profiled the prevalence of 24 histone H3/H4 modifications of E. solidaginis larvae after 3-week acclimations to decreasing environmental temperatures (5 °C, -5 °C and -15 °C). Using immunoblotting, the data show freeze-mediated reductions (p < 0.05) in seven permissive histone modifications (H3K27me1, H4K20me1, H3K9ac, H3K14ac, H3K27ac, H4K8ac, H3R26me2a). Along with the maintenance of various repressive marks, the data are indicative of a suppressed transcriptional state at subzero temperatures. Elevated nuclear levels of histone H4, but not histone H3, were also observed in response to both cold and freeze acclimation. Together, the present study provides evidence for epigenetic-mediated transcriptional suppression in support of the winter diapause state and freeze tolerance of E. solidaginis.
Assuntos
Histonas , Tephritidae , Animais , Histonas/genética , Histonas/metabolismo , Congelamento , Temperatura Baixa , Tephritidae/metabolismo , Crioprotetores/farmacologia , Crioprotetores/metabolismo , Larva/metabolismoRESUMO
Glycerol and aquaporin 9 (aquaglyceroporin) are known to be involved in freeze tolerance in the Japanese tree frog Hyla japonica. However, the regulatory mechanisms of freeze tolerance in this species have not been fully elucidated. In the present study, we focused on the inter- and intracellular dynamics of glucose to analyze the role of glucose and glucose-related proteins such as transporter and metabolic enzymes in freeze tolerance. Serum glucose concentrations were compared among the frogs that were nonhibernating, hibernating, and thawed after freezing at -4°C for 6 hr. Serum concentrations of glucose in thawed frogs were significantly higher than those in hibernating and nonhibernating, active frogs. Periodic acid-Schiff staining showed that the accumulation of glycogen in the hepatocytes increased before hibernation and decreased after freezing and thawing. Quantitative RT-PCR analysis using the liver showed that, compared with active frogs, the type 2 glucose transporter gene (glut2) was upregulated in frozen frogs, the liver glycogen phosphorylase gene (pygl) was upregulated in frozen or thawed frogs, and the type 2 glycogen synthase gene (gys2) was upregulated in hibernating frogs. Immunohistochemistry of liver sections showed that, compared with nonhibernating frogs, Glut2 proteins were clearly increased most likely on the plasma membrane of hepatocytes in hibernating frogs and further increased by freezing, then decreased after thawing. These results suggest the possibility that glucose acts as a cryoprotectant in H. japonica.
Assuntos
Anuros , Glucose , Animais , Anuros/metabolismo , Crioprotetores/metabolismo , Congelamento , Glucose/metabolismo , FígadoRESUMO
Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on the mechanisms of individual CP interactions, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here, we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, which survives long-term cryopreservation in liquid nitrogen. The CPs proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine and sarcosine accumulate in hemolymph in a ratio of 313:108:55:26:6:4:2.9:0.5â mmolâ l-1. Using calorimetry, we show that artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it probably forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.
Assuntos
Gelo , Trealose , Animais , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores , Congelamento , Larva , Nitrogênio , ProlinaRESUMO
The wood frog (Rana sylvatica) undergoes numerous changes to its physiology and metabolic processes to survive the winter months, including adaptations that let them endure whole-body freezing. The regulation of key enzymes of central carbohydrate metabolism in the liver plays a crucial role in mediating the synthesis and maintenance of high concentrations of glucose as a cryoprotectant during freezing as well as glucose reconversion to glycogen after thawing. The present study characterized the regulation of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) from wood frog liver during freezing, FBPase being a crucial enzyme regulating gluconeogenesis. Liver FBPase was purified to homogeneity from control and frozen wood frogs by a one-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and demonstrated a significant decrease in sensitivity to its substrate fructose-1,6-bisphosphate in the liver of frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant decrease in serine phosphorylation (by 53%) for FBPase from frozen frogs. Taken together, these results suggest that FBPase is suppressed, and gluconeogenesis is inhibited during freezing. This response acts as an important component of the metabolic survival strategy of the wood frog.
Assuntos
Frutose , Ranidae , Animais , Congelamento , Glucose/metabolismo , Fígado/metabolismo , Ranidae/metabolismoRESUMO
Subzero temperatures are among the most significant factors defining the distribution of organisms, yet, certain taxa have evolved to overcome this barrier. The microscopic tardigrades are among the most freeze-tolerant animals, with selected species reported to survive milli-Kelvin temperatures. Here, we estimate survival of fully hydrated eutardigrades of the species Ramazzottius varieornatus following exposures to -20 °C and -80 °C as well as -196 °C with or without initial cooling to -80 °C. The tardigrades easily survive these temperatures, yet with a significant decrease in viability following rapid cooling by direct exposure to -196 °C. Hence, post-freeze recovery of R. varieornatus seems to rely on cooling rate and thus controlled ice formation. Cryophilic organisms are renowned for having cold-active enzymes that secure appropriate reaction rates at low temperatures. Hence, extreme freeze-tolerance in R. varieornatus could potentially involve syntheses of cryoprotectants and de novo transcription. We therefore generated a reference transcriptome for this cryophilic R. varieornatus population and explored for differential gene expression patterns following cooling to -80 °C as compared to active 5 °C controls. Specifically, we tested for fast transcription potentially occurring within 25 min of cooling from room temperature to a supercooling point of ca. -20 °C, at which the tardigrades presumably freeze and enter into the ametabolic state of cryobiosis. Our analyses revealed no evidence for differential gene expression. We, therefore, conclude that extreme freeze-tolerance in R. varieornatus relies on controlled extracellular freezing with any freeze-tolerance related genes being constitutively expressed.
Assuntos
Gelo , Tardígrados , Animais , Temperatura Baixa , Congelamento , Tardígrados/genética , TemperaturaRESUMO
Wood frogs, Rana sylvatica, endure the freezing of â¼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.
Assuntos
Antioxidantes , Ranidae , Animais , Antioxidantes/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Congelamento , Hipóxia/genética , Músculo Esquelético/metabolismo , Ranidae/fisiologiaRESUMO
Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5â°C-acclimated) and frozen (-15â°C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.
Assuntos
Piruvato Quinase/química , Piruvato Quinase/metabolismo , Tephritidae/enzimologia , Aclimatação , Animais , Temperatura Baixa , Congelamento , Larva/enzimologia , Fosforilação/fisiologia , Tephritidae/crescimento & desenvolvimento , Tephritidae/metabolismoRESUMO
Cryoprotectants play an essential role in the survival of some amphibians in response to different stress conditions such as freezing, anoxia, and dehydration. Glucose is one of the cryoprotectants important for freeze-tolerant frogs. The aim of the present study was to investigate the survival strategies of Anatolian mountain frogs (Rana macrocnemis and Rana holtzi), which are terrestrial hibernators, by examining the changes in glucose and water content in some tissues at subzero temperatures. In the current study, animals were exposed to freezing (-2.5 °C), anoxia, and dehydration treatments. During these treatments, all frogs survived. The glucose levels in the plasma, liver, and skeletal muscle and the water content of the tissues were measured during the freezing, anoxia, and dehydration. Changes in body weight were also recorded in both species. During the freezing, a 3.3-fold increase was seen in the blood glucose level of R. macrocnemis (1.35 ± 0.25 to 4.45 ± 0.51 µmol mL-1), whereas the blood glucose level of R. holtzi exhibited a 4.5-fold increase (1.90 ± 0.25 to 8.67 ± 2.22 µmol mL-1). In the liver, a 6.7-fold increase was seen in the glucose level of R. macrocnemis (5.66 ± 0.15 to 38.27 ± 8.53 µmol g-1) and the increase in R. holtzi was approximately 6.0-fold (2.25 ± 0.46 to 13.36 ± 1.32 µmol g-1) during freezing. The liver glucose levels of both species also increased significantly in response to the anoxia and dehydration. In both species, the glucose levels of the skeletal muscle were found to be higher in dehydration than with freezing and anoxia. In conclusion, our results suggest that glucose may be identified as an important cryoprotectant that plays an important role in the survival of Anatolian mountain frogs during extreme conditions.
Assuntos
Desidratação , Glucose , Animais , Criopreservação/métodos , Congelamento , Hipóxia , RanidaeRESUMO
In recent years there have been several advancements in organ preservation that have yet to see widespread clinical translation. While static cold storage (SCS) at 2⯰C-4⯰C continues to be the state-of-the-art strategy, it contributes to the current shortage of transplantable organs due to the limited preservation times it affords combined with the limited ability of marginal grafts to tolerate SCS. The era of optimizing storage solutions to minimize SCS-induced hypothermic injury has largely plateaued in its improvements, resulting in a shift towards the use of machine perfusion systems to provide continuous metabolic support, or the use of sub-zero storage temperatures to leverage the protection brought forth by a reduction in metabolic demand. Many of the rigors that organs are subjected to at low sub-zero temperatures (-80⯰C to -196⯰C) commonly used for mammalian cell preservation have yet to be surmounted, and therefore the focus of this article lies on an intermediate range of storage temperatures (0⯰C to -20⯰C) where much success has been seen in the past two decades. Numerous mechanisms leveraged by organisms capable of withstanding prolonged periods at these temperatures through either avoiding or tolerating the formation of ice has provided a foundation for some of the more promising efforts, and thus we aim to contextualize the translation of these nature-derived strategies to mammalian organ preservation.