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Mechanisms of cell survival in hypoxia and hypothermia.

Most animals experience some degree of hypoxia and hypothermia during the course of their natural life history either as a consequence of ambient 'exposure' per se or through metabolic, respiratory and/or circulatory insufficiency. A prevailing experimental approach has been to probe tissues from natural models of hypoxia-tolerant and cold-tolerant vertebrates to look for common mechanisms of defence against O(2) lack and hypothermia. The ability to sustain vital cellular functions in severe cases of either condition varies widely amongst the vertebrates. Like humans, the vast majority of mammals are unable to survive prolonged periods of hypothermia or O(2) deprivation owing to irreversible membrane damage and loss of cellular ion homeostasis in vital organs such as the brain and heart. However, numerous hibernating endotherms, neonatal and diving mammals as well as many ectotherms can tolerate prolonged periods that would, in clinical terms, be called asphyxia or deep hypothermia. The key to their survival under such conditions lies in an inherent ability to downregulate their cellular metabolic rate to new hypometabolic steady states in a way that balances the ATP demand and ATP supply pathways.

Adaptation, Physiological↗

Neither desferrioxamine nor lipoic acid enhances the cold ischaemic tolerance of epigastric free flaps in rats.

This study was designed to test the ability of pre- and postischaemic administration of desferrioxamine (DFX), a combination of a preischaemic single passage perfusion with hypertonic citrate solution (HCA) and postischaemic administration of DFX, and pre- and postischaemic administration of lipoic acid (LA) to enhance the cold (0 degree C) ischaemia tolerance in epigastric free flaps in inbred rats. The ischaemic intervals varied between 0 and 120 hr. The area of flap survival 2 weeks after revascularisation as a percentage of original flap size was recorded as an exponent of ischaemic tolerance. None of the treatment regimens was able to increase flap area survival. Histologically, there were no major differences between the different treatment regimens.

Adipose Tissue↗

Swimming training prevents generation of suppressor macrophages during acute cold stress.

PURPOSE: Acute cold stress induces suppressor macrophages expressing large numbers of receptors to Fc portion of immunoglobulin G (MAC-1+ Fc gammaRII/IIIbright cells), resulting in suppression of splenocyte mitogenesis. The generation of MAC-1+ Fc gammaRII/IIIbright cells is partly mediated by increased glucocorticoid levels during acute cold stress. The aim of the current study was to investigate the effect of swimming training on the generation of the MAC-1+ Fc gammaRII/IIIbright suppressor macrophages by acute cold stress. METHODS: The trained mice underwent a 6-wk endurance swimming training (5 times/wk) in water at 35-36 degrees C for 90 min. The swimming training significantly increased brown adipose tissue mass, suggesting improved cold tolerance. Actually, when the swimming-trained mice were exposed to 5 degrees C for 3 h (acute cold stress), the rectal temperature was not decreased. The proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cells from swimming-trained mice was significantly lower than that from control mice. In addition, the proportion of MAC-1+ Fc gammaRII/IIIbright cells in peritoneal exudate cell population from swimming-trained mice was unaffected by the acute cold stress. The swimming training significantly attenuated the increases in serum corticosterone levels in response to acute cold stress. These results suggested that swimming training not only improves cold tolerance but also inhibits the generation of suppressor macrophages under acute cold stress as well as under normal conditions.

Acclimatization↗

Varietal differences in protein phosphorylation during cold treatment of rice leaves.

The effects of cold stress on protein phosphorylation in leaves of rice (Oryza sativa) seedlings were investigated. Two-week-old rice seedlings were exposed to low temperature (5 degrees) for 6 hr at a relative humidity of 100%. The leaf protein extracts were phosphorylated in vitro. Cold stress stimulated the phosphorylation of a 60 kDa protein in the cold-sensitive rice variety, IR36. In the cold-tolerant rice variety, Kitaibuki, this protein had already been phosphorylated. For greater clarificating of these changes, the effects of cold on rice varieties differing in cold susceptibility were examined. Cold-sensitive rice varieties showed similar the protein phosphorylation in contrast to the cold-tolerant rice varieties.

Cold Temperature↗

Transcriptome analysis of brown adipose tissue in Brandt's vole treated with tannic acid under cold exposure.

BACKGROUND: Tannic acid (TA) is a hydrolysable plant secondary metabolite known to influence multiple physiological processes in animals; however, its role in regulating brown adipose tissue (BAT) thermogenesis remains poorly understood. Notably, the overwinter food caches of Brandt's voles predominantly consist of Artemisia species, which are rich in TA. This study aimed to determine whether TA contributes to cold tolerance in Brandt's voles by activating BAT thermogenesis. Adult male voles were administered TA, after which the masses of BAT and inguinal white adipose tissue (iWAT) were measured, and temperature changes in BAT, the body surface, and the rectum were recorded following exposure to - 20 °C. In addition, transcriptomic analyses of BAT were performed, and the expression and protein levels of key thermogenic markers were assessed. RESULTS: The results showed that TA reduced iWAT mass while exerting minimal effects on BAT mass. TA-treated voles exhibited significantly elevated temperatures in BAT, the body surface, and the rectum after cold exposure. Histological analyses revealed that TA treatment reduced adipocyte area in iWAT while increasing the number of nuclei in brown adipocytes in BAT. In BAT, differentially expressed genes (DEGs) in voles receiving a low TA dose were significantly enriched in pathways related to fat digestion and absorption and peroxisome proliferator-activated receptor (PPAR) signaling. In contrast, DEGs in voles administered a high TA dose were predominantly associated with brown adipocyte differentiation and the upregulation of cold-induced thermogenesis. Moreover, TA administration increased the expression of FFAR4 and UCP1, as well as the protein levels of PGC-1α, PPARγ, and UCP1 following cold exposure. CONCLUSIONS: Collectively, these findings demonstrate that TA enhances cold tolerance in Brandt's voles by promoting thermogenic gene expression and stimulating brown adipocyte differentiation in BAT, providing novel insights into the role of plant secondary metabolites in mammalian cold adaptation and herbivore-plant interactions.

Animals↗

Climatic variability and the evolution of insect freeze tolerance.

Insects may survive subzero temperatures by two general strategies: Freeze-tolerant insects withstand the formation of internal ice, while freeze-avoiding insects die upon freezing. While it is widely recognized that these represent alternative strategies to survive low temperatures, and mechanistic understanding of the physical and molecular process of cold tolerance are becoming well elucidated, the reasons why one strategy or the other is adopted remain unclear. Freeze avoidance is clearly basal within the arthropod lineages, and it seems that freeze tolerance has evolved convergently at least six times among the insects (in the Blattaria, Orthoptera, Coleoptera, Hymenoptera, Diptera and Lepidoptera). Of the pterygote insect species whose cold-tolerance strategy has been reported in the literature, 29% (69 of 241 species studied) of those in the Northern Hemisphere, whereas 85 % (11 of 13 species) in the Southern Hemisphere exhibit freeze tolerance. A randomization test indicates that this predominance of freeze tolerance in the Southern Hemisphere is too great to be due to chance, and there is no evidence of a recent publication bias in favour of new reports of freeze-tolerant species. We conclude from this that the specific nature of cold insect habitats in the Southern Hemisphere, which are characterized by oceanic influence and climate variability must lead to strong selection in favour of freeze tolerance in this hemisphere. We envisage two main scenarios where it would prove advantageous for insects to be freeze tolerant. In the first, characteristic of cold continental habitats of the Northern Hemisphere, freeze tolerance allows insects to survive very low temperatures for long periods of time, and to avoid desiccation. These responses tend to be strongly seasonal, and insects in these habitats are only freeze tolerant for the overwintering period. By contrast, in mild and unpredictable environments, characteristic of habitats influenced by the Southern Ocean, freeze tolerance allows insects which habitually have ice nucleators in their guts to survive summer cold snaps, and to take advantage of mild winter periods without the need for extensive seasonal cold hardening. Thus, we conclude that the climates of the two hemispheres have led to the parallel evolution of freeze tolerance for very different reasons, and that this hemispheric difference is symptomatic of many wide-scale disparities in Northern and Southern ecological processes.

Acclimatization↗

Effects of temperature on contractile properties of skinned muscle fibers from three toad species.

Single fast fibers were isolated from the iliofibularis muscles of three species of toad with different thermal minima for active locomotion: 8 degrees C, American toad, Bufo americanus; 15 degrees C, Rocky Mountain toad, Bufo woodhousei woodhousei; 22 degrees C, Cane toad, Bufo marinus. All experiments were carried out during the summer. Fibers were chemically skinned and maximum isometric tension and unloaded contraction velocity were determined at a series of temperatures between 0 and 35 degrees C. At 25-30 degrees C, isometric tension development has a low temperature dependence (R10 = 1.1-1.3) and is in the range of 210-260 kN X m-2 for each of the three toads. However, at 0-10 degrees C, absolute values of tension increase in the series (B. americanus greater than B. woodhousei greater than B. marinus; i.e., with increasing cold tolerance), while thermal sensitivity between 0 and 10 degrees C is inversely related to cold tolerance. For example, at 0 degree C, maximum isometric tension (Po) for the most northerly distributed species is three times higher than for the subtropical to tropical species (P less than 0.001). R10 for Po (0-10 degrees C) is 1.7 for B. marinus, 1.3 for B. w. woodhousei, and 1.0 for B. americanus. In contrast, unloaded shortening speeds were similar at any given temperature for the three species. It is concluded that adaptations in Bufo myosin for activity at low temperatures largely involves changes in force production.

Adenosine Triphosphatases↗

Rapid cold hardening in the predatory mite Euseius (Amblyseius) finlandicus (Acari: Phytoseiidae).

A rapid cold hardening response was studied in diapause and non-diapause females of the predatory mite Euseius finlandicus. When laboratory reared diapause and non-diapause females were transferred and maintained from the rearing temperature of 20 degrees C for 2 h to -11.5 degrees C and -10 degrees C, 10 to 20% survived respectively. However, conditioning of diapause females for 4 h at a range of temperatures from 0 to 10 degrees C before their exposure for 2 h to -11.5 degrees C, increased survival to approximately 90%. Similarly, conditioning of non-diapause females for 4 h at 5 degrees C before their exposure for 2 h to -10 degrees C increased survival to 90%. A similar rapid cold hardening response in both diapause and non-diapause females was also induced through gradual cooling of the mites, at a rate of approximately 0.4 degrees C per min. The rapid increase in cold tolerance after prior conditioning of the mites to low temperatures, was rapidly lost when they returned to a higher temperature of 20 degrees C. Rapid cold hardening extended the survival time of diapause and non-diapause females at sub-zero temperatures. The cost of rapid cold hardening in reproductive potential after diapause termination was negligible. In non-diapause females, however, the increase in cold tolerance gained through gradual cooling could not prevent cold shock injuries, as both fecundity and survival were reduced.

Journal Article↗

Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster.

Naturally occurring diurnal variations in temperature are sufficient to induce a rapid cold hardening (RCH) response in insects. RCH can increase cold tolerance by 1-2 degrees C and extend the temperature interval at which insects can remain active. While the benefits of RCH are well established, the underlying physiological mechanisms remain unresolved. In this study we investigated the role of RCH on expression of heat shock proteins (Hsp70) after a cold shock, and the effect of RCH on the composition of phospholipid fatty acids (PLFAs) in membranes of Drosophila melanogaster. These experiments were performed on both "control" flies and flies selected for cold resistance in order to additionally examine a possible target for selection for cold tolerance. RCH improved survival following cold shock at -4, -6 and -8 degrees C. No induction of Hsp70 was found following cold shock irrespective of the pre-treatment. In contrast, a 5h RCH treatment was sufficient to induce small, but significant, changes in the composition of PLFAs. Here, the polyunsaturated linoleic acid, 18:2(n-6), increased while monounsaturated (18:1) and saturated (14:0) PLFAs decreased in abundance. These changes were observed in both selection groups and caused a significant increase in the overall degree of unsaturation. This response is consistent with the membrane response typically found during cold acclimation in ectothermic animals and it is likely adaptive to maintain membrane function during cold. Cold selection resulted in PLFA changes (decrease of 18:0 and 18:1 and increase of 14:0 and 16:1), which may improve the ability to harden during RCH.

Acclimatization↗

Tolerance of estrogen-treated rats to acute cold exposure.

Female rats treated chronically with ethynylestradiol (36 micrograms/kg per day) alone, and in combination with the progestational agent, norethynodrel (253 micrograms/kg per day), cooled significantly faster than controls when lightly restrained and exposed to air at 5 degrees C. Rate of cooling of rats given only norethynodrel was similar to that of the control group. In other studies, rate of oxygen consumption was determined for all groups during acute exposure to cold (14 degrees C). All estrogen-treated groups achieved the same maximal rate of oxygen consumption as control and norethynodrel-treated groups during cold exposure, but cooled significantly faster. Two groups of female rats were treated chronically with ethynylestradiol at two separate doses (36 and 61 micrograms/kg per day). An untreated group served as controls. Rate of oxygen consumption of all animals were measured during restraint and exposure to cold (18 degrees C). The estrogen-treated groups again achieved the same maximal rate of oxygen consumption as the control group, but also cooled significantly faster despite the fact that the cold stress was less severe than in the previous experiment. That estrogen-treated rats cooled faster than controls in both studies despite achieving a maximal rate of heat production which did not differ from controls suggests that reduced cold tolerance of estrogen-treated rats may be related to increased heat loss.

Animals↗

Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase.

Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance.

Abscisic Acid↗

Rapid cold-hardening in Culicoides variipennis sonorensis (Diptera: Ceratopogonidae).

Rapid cold-hardening was studied in the adult stage of the biting midge Culicoides variipennis sonorensis Wirth & Jones from a laboratory colony. No individuals could withstand a direct transfer from 20 degrees C (rearing temperature) to -10 degrees C for 2 h; however, an acclimation period of 1 h at 5 degrees C immediately before exposure to -10 degrees C yielded 96% survival. The cold-hardening response was very rapid; increased cold tolerance peaked after only a 1-h exposure to 5 degrees C. No fly withstood 2 h at -20 degrees C, demonstrating the limitations of the cold-hardening response. Varying degrees of cold tolerance also were induced by a 2-h exposure to 37 degrees C. The ability to rapidly cold-harden varied with the age of the adult insect. Rapid cold-hardening in adult C. v. sonorensis may be of ecological and epizootiological significance because it is a mechanism by which a nondiapausing life stage quickly can enhance its tolerance to subzero temperatures.

Analysis of Variance↗

Metabolic cold acclimation after repetitive intermittent cold exposure in rat.

Repetitive intermittent cold exposure (5 degrees C, 6 h/day, 4 weeks) (ICE) resulted in the same cold adaptability as assessed by an enhanced cold tolerance (less drop of colonic temperature at -5 degrees C) and nonshivering thermogenesis (NST) (greater noradrenaline-induced heat production) as that elicited by continuous cold exposure (5 degrees C, 4 weeks) (CA) in rats. Although shorter intermittent (5 degrees C, 2 h/day, 4 weeks) (ICE-2 hr) as well as shorter continuous (5 degrees C, 1 week) (CA-1 wk) cold exposure effected an improved cold adaptability, the magnitude of cold tolerance and NST was smaller as compared with that in CA and ICE. The cold deacclimation process as reflected on the decreased NST did not differ between CA and ICE. Food intake was less in ICE than CA, while increase in body weight during the acclimation period was greater in the former. Increase in adrenal weight was greater in CA than ICE, but plasma corticosterone level did not differ among warm controls (WC), CA, and ICE in resting state (after 18-20 h at warm control temperature of 25 degrees C). Weights of interscapular and dorsocervical brown adipose tissue (BAT) increased to the same degree in CA and ICE. Plasma glucagon level in resting state did not differ among groups, while BAT glucagon levels significantly increased in CA and ICE, but they were higher in dorsocervical site than interscapular site in all acclimated states. Acute cold exposure (-5 degrees C, 15 min) caused increases in plasma corticosterone, glucagon levels, and in BAT glucagon levels in all acclimated groups. The extent of increase was significantly less for plasma glucagon in CA, while plasma corticosterone increased similarly in all groups. These results indicate that repetitive short-term cold exposure could elicit the same cold adaptability as that induced by continuous exposure, but requiring only one-fourth of the time of continuous cold exposure. Moreover, it is suggested that glucagon is involved in both CA and ICE, but the same extent of cold adaptability can be obtained in the less energy-requiring and less stressful state in ICE.

Acclimatization↗

Expression of the antifreeze protein gene in transgenic goldfish (Carassius auratus) and its implication in cold adaptation.

Ocean pout (Macrozoarces americanus) antifreeze protein (AFP) genes (approximately 10(6) copies) were microinjected into the oocytes of goldfish (Carassius auratus). Out of a total of 303 oocytes injected, 235 (77.6%) were normally fertilized after in vitro maturation and insemination, and 136 (57.9%) of the fertilized eggs were hatched. Dot blot analysis of genomic DNA from 2-month-old fish indicated that 30 of the fish (22%) were positive for the AFP transgene. The numbers of integrated AFP genes ranged from a single copy to multiple copies per cell. Two P1 male founders, Y45 and GV16, were crossed separately with a control female, and the inheritance of the AFP transgene was analyzed. Results from Southern blotting and polymerase chain reaction were found to be 7% (5 out of 82) and 58.8% (20 out of 34), respectively, indicating that these two founders are mosaic. The percentage of positive F2 generated from Y45F1 crossed with a control female was 56%, consistent with a Mendelian inheritance. Mature AFP detected by immunoblotting was expressed in both F1 and F2 offspring. These studies indicate that the antifreeze protein gene is successfully transferred and expressed in goldfish. Transgenic goldfish are significantly more cold tolerant than controls when challenged with low temperatures. These studies suggest that the AFP gene may have application in providing cold tolerance in addition to freeze resistance for a variety of fish species.

Acclimatization↗

Poly-beta-hydroxybutyrate metabolism is affected by changes in respiratory enzymatic activities due to cold stress in two psychrotrophic strains of Rhizobium.

Cold stress resulted in a decrease in the poly-beta-hydroxybutyrate (PHB) content of non-cold-acclimated Rhizobium DDSS69 cultures. Analysis of the specific activity of beta-ketothiolase and beta-hydroxybutyrate dehydrogenase revealed that decrease in PHB levels was a result of the inhibition of synthesis of PHB rather than an increase in its breakdown. Rhizobium ATR1, a cold-acclimated strain, revealed the presence of a stable PHB metabolism that did not show any significant differences either in PHB levels or in the activity of enzymes of the PHB metabolism under cold stress, suggesting that PHB is not involved in cold tolerance. Analysis of specific activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway showed the upward regulation of alternate pathways of carbohydrate metabolism under cold stress to rapidly generate energy to overcome the stress. There is diversity in the switching mechanisms of carbon metabolism among cold-acclimated and non-cold-acclimated Rhizobium isolates. Upward regulation of malate dehydrogenase in both isolates suggests that it is a critical input for cold tolerance.

Carbohydrate Metabolism↗

Effect of food restriction on cold adaptability of rats.

To determine the role of the nutritional state in nonshivering thermogenesis during cold adaptation, cold adaptability was compared between cold-adapted (5 degrees C for 4-5 weeks) rats fed ad libitum and cold-adapted rats pair fed with warm controls having the same food intake. Cold-adapted pair-fed rats suffered a significant loss in body weight during cold exposure. However, brown adipose tissue (BAT) in both cold-adapted ad libitum fed and cold-adapted pair-fed rats was enlarged to the same extent as compared with that in control rats. Fat-free dry matter in BAT also increased in cold-adapted ad libitum fed and cold-adapted pair-fed rats to the same extent. Cold tolerance as assessed by the change in the colonic temperature at -5 degrees C was improved relative to control rats and was the same for cold-adapted ad libitum fed and cold-adapted pair-fed rats. Nonshivering thermogenesis as estimated by the noradrenaline-induced increase in oxygen consumption was significantly greater in the cold-exposed rats and there was no significant difference between cold-adapted ad libitum fed and cold-adapted pair-fed rats. These results suggest that an improved cold tolerance by means of nonshivering thermogenesis in brown adipose tissue is closely related to the low temperature itself but not the increased food intake which occurred in the cold.

Adaptation, Physiological↗

Cold-induced thermogenesis in rats nutritionally deprived early in life.

Preweaning nutritional deprivation with or without postweaning partial energy restriction produces animals of small size with defective nonshivering thermogenesis. This is also associated with their inability to tolerate cold exposure to 5 degrees C. Adequate nutritional rehabilitation for a reasonable length of time of 10 days abolishes the defect in cold induced thermogenesis (CIT) although the deficits in body weight or body size are not corrected. This may indicate that the defect may be possibly due to the non availability of enough substrates rather than to a change in the functioning of thermogenic organs such as brown adipose tissue. Sucrose feeding which enhances caloric intake and hence sympathetic activity can reverse the defect in CIT only in older rats suggesting the possibility of delayed maturation of thermoregulatory functions in young rats which are energy deprived. The results of this study possibly indicate that there may be a temporary reduction of the sympathetic nervous system activity during the period of energy restriction which compromises cold tolerance and which is reactivated rapidly following nutritional rehabilitation.

Animals↗