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Metabolic responses to freezing in freeze tolerant reptiles
- Reptile freeze tolerance: Metabolism and gene expression

Natural cold hardiness, including both freeze avoidance and freeze tolerance strategies, typically involves various metabolic adaptations. Two of the common ones are the proliferation of proteins that help the organism to manage ice and the accumulation of high levels of low molecular weight osmolytes that provide colligative antifreeze action to the whole animal in the case of freeze avoidance or to the intracellular milieu in the case of freeze tolerance. What is the status of these in cold hardy reptiles?

Ice management is provided by either antifreeze proteins in freeze avoiding species or ice nucleating proteins (or sometimes nonspecific nucleators) in freeze tolerant species. As mentioned previously, supercooling capacity and resistance to inoculative freezing by C. picta hatchlings increases seasonally and with cold acclimation (as does freeze tolerance) [21], [24] and [91]. The seasonal enhancement of supercooling capacity involves elimination of endogenous ice nucleating agents in the feces (nucleators may have been derived originally from ingestion of nesting soil) [20]. However, tests for thermal hysteresis activity in plasma, which would be indicative of the production of specific antifreeze proteins, were negative for both C. picta and L. vivipara [84] and [89] suggesting that reptiles do not invest in these proteins that are commonly found in coldwater marine fish and terrestrial invertebrates [97]. The plasma of C. picta exhibited considerable ice nucleating capacity that maintained its potency up to a dilution of 1:500 [84]. Both low pH and heat treatments destroyed the activity suggesting that it is proteinaceous. Hence, specific ice nucleating proteins may be present in this species. The dichotomy between an enhancement of resistance to inoculative freezing and the elimination of gut nucleators (both strategies for supercooling) versus the presence of proteinaceous plasma ice nucleators (a strategy for freeze tolerance) is more apparent than real. A freeze tolerant species (or a species that uses either strategy depending on circumstances) benefits from controlling the freezing process, particularly the propagation of ice through its microvasculature. Hence, a reduction or elimination of modes of nonspecific nucleation is not at odds with the use of specific nucleators in selected extracellular compartments to initiate and manage crystal growth in a noninjurious manner.

The other common adaptation for cold hardiness that occurs across phylogeny is the seasonal or freeze-responsive accumulation of high concentrations of low molecular weight metabolites that provide colligative action to resist whole body (freeze avoidance) or intracellular (freeze tolerance) freezing. For example, plasma levels of glucose can rise to 150–300 mM in freeze tolerant wood frogs (control values are 1–5 mM) [81] whereas glycerol levels can reach 2 M or more in freeze avoiding insects. The search for colligative protectants in cold-hardy reptiles has been unimpressive. Polyhydric alcohols such as glycerol are typically insignificant in reptiles ([21], and the total amino acid pool of plasma is low (2–10 mM) [12] and [24]. Glucose and lactate levels do rise in plasma and tissues of both supercooled and frozen reptiles but the levels are low overall and not unlike the values seen when the same species are challenged by hypoxia or anoxia [18], [30], [55] and [67]. Indeed, anoxia exposure at 4 °C resulted in plasma lactate levels in hatchling turtles that were actually 3- to 6-fold higher than in the same species frozen at −2.5 °C [30] which argues against a purposeful synthesis of lactate as a cryoprotectant. Glucose levels do not reach values that would provide substantial colligative action. For example, our studies of painted turtle hatchlings found plasma and organ levels of glucose of only about 15 mM in frozen animals [10], [12] and [85] whereas Costanzo et al. [21] reported midwinter net increases in plasma glucose ranging from 2 to 40 mM depending on year and geographic location of nests sampled. Blood glucose levels rose to 25 mM in freeze exposed L. vivipara [89] and to not, vert, similar20 mM in box turtles [83] but species with poor freeze tolerance, garter snakes and T. s. elegans hatchlings, showed a much small increase to only 3.2–3.5 mM glucose in liver [11] and [13]. Blood and organ lactate reached 5–38 mM in C. p. marginata hatchlings frozen in the laboratory [10] and [85] and plasma lactate was 2–6 mM in hatchlings sampled from nests in midwinter [21]. Plasma lactate was 16–28 mM in freeze tolerant hatchlings of C. picta, E. blandingii, and M. terrapin after 3–7 days frozen at −2.5 °C [30] and rose only as high as 7 mM in organs of 24 h frozen box turtles and 10 mM in 4 h frozen T. s. elegans organs [13] and [83]. Lactate also accumulated in supercooled painted turtle and map turtle hatchlings [2], [18] and [42], apparently as a result of oxygen limitation at subzero temperatures; supercooled painted turtle hatchlings showed a decline in cardiac activity below 0 °C with heart beat ceasing at about −9 °C [4]. Overall, these amounts of glucose and lactate are not impressive for providing colligative resistance to cell volume reduction during freezing and there is no evidence that the production of these metabolites is been specifically enhanced to provide cryoprotection in response to freezing as is the case for glucose production in freeze tolerant frogs [81]. Furthermore, plasma osmolality changes very little with cold acclimation, freezing or over the winter months in reptiles (except under dry conditions where a loss of body water occurs) [21], [83] and [88] so it is apparent that there are no unidentified osmolytes present that could act as cryoprotectants.

However, the relatively low level of cryoprotection offered by colligative osmolytes in hatchling turtles might be augmented by a noncolligative method of cellular water retention. Microscopy of liver slices from C. picta hatchlings that were frozen at −4 °C on a directional stage showed a lower relative amount of extracellular ice (36%) than did comparable slices from skeletal muscle or heart (both 61%) [70]. Instead, micrographs of frozen liver slices showed large amounts of water remaining inside hepatocytes, most of it appearing as spherical droplets around dark granules. These granules appeared to be glycogen which is known to be a highly hydrated macromolecule. Hence, it may be that the high glycogen content of hepatocytes is exploited to achieve significant noncolligative resistance to cell water loss during freezing. Although glycogen granules were not prominent in the muscle tissues, significant intracellular water appeared to remain in muscles along the myofibrils which is where glycogen is known to be localized. Hence, this putative noncolligative mechanism of water retention may represent a significant but previously unappreciated mechanism of natural cryoprotection. Indeed, image analysis of the micrographs showed considerably less ice in all three tissues than was predicted from mathematical calculations based on the osmolality of turtle body fluids [70].

Intermediary energy metabolism during freezing has been examined in garter snakes (T. sirtalis) and hatchling C. picta and T. scripta [9], [10] and [13]. Changes in the levels of glycolytic intermediates in liver were consistent with the activation of liver glycogen breakdown as the carbon source for the glucose and lactate accumulated. The amount of glycogen phosphorylase in the active a form rose sharply in liver in response to freezing in painted turtles and box turtles to catalyze glycogenolysis and supply glucose for both endogenous needs and for export [12] and [83]. Phosphorylase is undoubtedly activated via a β-adrenergic pathway (as also occurs in freeze tolerant frogs) [81] since C. picta liver plasma membranes showed substantial levels of β2-adrenergic receptor binding but virtually no α1-adrenergic receptors [44]. Changes in the substrate (fructose-6-phosphate) and product (fructose-1,6-bisphosphate) levels of phosphofructokinase (PFK) in liver from frozen animals were consistent with inhibitory control placed on glycolysis at this locus [10]. Similar responses by glycolytic intermediates (elevation of hexose phosphates, little or no change in other intermediates) were seen in liver of T. s. elegans hatchlings and garter snakes [9] and [13]. Inhibition of PFK is commonly seen in vertebrate liver under conditions where glucose is being produced for export (such as in response to hypoxia/anoxia) and is prominent in wood frog liver during freeze-stimulated glucose synthesis. Inhibition of PFK is also a component of anoxia-induced metabolic rate depression which serves to adjust the rate of glycolytic ATP output in anoxia to match the greatly reduced rates of ATP-consuming processes to achieve a strong net reduction in metabolic rate [73]. For example, adult C. picta and T. s. elegans submerged in cold water (10 °C) show a very strong drop in metabolic rate to not, vert, similar10% of the corresponding aerobic rate and this is a key factor in their long term survival during winter hibernation under water [46]. Although it has never been directly measured, a comparable freeze-induced suppression of metabolic rate (responding to the anoxia of the frozen state) is strongly suspected to be an important factor in long term organ viability in the frozen state for all freeze tolerant animals [75].

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