Pro- and anti-apoptotic microRNAs are differentially regulated during estivation in Xenopus laevis.

Xenopus laevis, the African clawed frog, undergoes seasonal estivation to survive periods of drought when its lake-bed habitats dry up. The frog can lose ∼30% of its total body water, leading to conditions of impaired blood flow and ischemia which risk cellular survival under these harsh conditions. MicroRNAs are short, noncoding, single-stranded RNAs 21-24 nt long that have been widely implicated in hypometabolic responses, and serve functions including apoptosis survival. The levels of three pro-apoptotic and four anti-apoptotic miRNAs were measured in liver and skeletal muscle of estivating X. laevis, and bioinformatic analysis was performed to verify potential mRNA targets of these miRNAs. Members of pro-apoptotic miRNAs miR-15a, miR-16, and miR-101 showed upregulation as a result of dehydration stress, while anti-apoptotic miRNAs miR-19b, miR-21, miR-92a, and miR-155 showed differential regulation between the two tissues. Together, these miRNAs act in a more diverse fashion than arbitrarily pro- or anti-apoptotic, and encompass functions ranging from the inhibition of cell proliferation through cell cycle arrest to the prevention of skeletal muscle atrophy.

Increased transcript levels and kinetic function of pyruvate kinase during severe dehydration in aestivating African clawed frogs, Xenopus laevis.

The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

Identification of a novel dehydration responsive gene, drp10, from the African clawed frog, Xenopus laevis.

During periods of environmental stress a number of different anuran species employ adaptive strategies to promote survival. Our study found that in response to dehydration (i.e., loss of total body water content), the African clawed frog (Xenopus laevis) increased the expression of a novel gene (drp10) that encodes a structural homolog of the freeze-responsive FR10 protein found in wood frogs. Similar to FR10, the DRP10 protein was found to also contain a highly conserved N-terminal cleavable signal peptide. Furthermore, DRP10 was found to have high structural homology to the available crystal structures of type A and E apolipoproteins in Homo sapiens, and a type IV LS-12 anti-freeze protein in the longhorn sculpin, Myoxocephalus octodecemspinosis. In response to dehydration, the transcript expression of drp10 was found to increase 1.52 ± 0.16-fold and 1.97 ± 0.11-fold in response to medium (15%) and high (30%) dehydration stresses in the liver tissue of X. laevis, respectively, while drp10 expression increased 2.12 ± 0.12-fold and 1.46 ± 0.16-fold in kidney tissue. Although the molecular function of both dehydration-responsive DRP10 and the freeze-responsive FR10 have just begun to be elucidated, it is likely that both are frog-specific proteins that likely share a similar purpose during water-related stresses.

Global DNA modifications suppress transcription in brown adipose tissue during hibernation.

Hibernation is crucial to winter survival for many small mammals and is characterized by prolonged periods of torpor during which strong global controls are applied to suppress energy-expensive cellular processes. We hypothesized that one strategy of energy conservation is a global reduction in gene transcription imparted by reversible modifications to DNA and to proteins involved in chromatin packing. Transcriptional regulation during hibernation was examined over euthermic control groups and five stages of the torpor/arousal cycle in brown adipose tissue of thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Brown adipose is crucial to hibernation success because it is responsible for the non-shivering thermogenesis that rewarms animals during arousal. A direct modification of DNA during torpor was revealed by a 1.7-fold increase in global DNA methylation during long term torpor as compared with euthermic controls. Acetylation of histone H3 (on Lys23) was reduced by about 50% when squirrels entered torpor, which would result in increased chromatin packing (and transcriptional repression). This was accompanied by strong increases in histone deacetylase protein levels during torpor; e.g. HDAC1 and HDAC4 levels rose by 1.5- and 6-fold, respectively. Protein levels of two co-repressors of transcription, MBD1 and HP1, also increased by 1.9- and 1.5-fold, respectively, in long-term torpor and remained high during early arousal. MBD1, HP1 and HDACs all returned to near control values during interbout indicating a reversal of their inhibitory actions. Overall, the data presents strong evidence for a global suppression of transcription during torpor via the action of epigenetic regulatory mechanisms in brown adipose tissue of hibernating thirteen-lined ground squirrels.