Bryan Luu, Ph. D. Biology 2018

Molecular responses to whole-body dehydration in a sequenced vertebrate, Xenopus laevis: Regulation of antioxidants and metabolism by the Sirtuin protein deacetylases



Whole-body dehydration in the African clawed frog, Xenopus laevis, increases hematocrit and blood viscosity, which restrains oxygen delivery. This causes the resting heart rate, differences in arterio-venous blood oxygen contents, and whole-animal lactate to increase.
I hypothesized that dehydration involves changes in cellular signaling through alterations of protein posttranslational acetylation, which can increase antioxidants and regulate metabolism. Seven Sirtuin (Sirt) protein deacetylases were profiled at the mRNA level with RT-qPCR in 6 tissues (liver, muscle, heart, kidney, brain, and lung) of X. laevis under control versus dehydration conditions. At least some sirt transcripts increased in all tissues except for kidney and brain. Similarly, global Sirt activity assays found that Sirt deacetylase activity increased in liver, muscle, heart, and lung. Western blots revealed the relative levels of Ac-SOD2. Results showed that acetylated SOD2 decreased with whole-body dehydration in the lung, heart, and kidney, suggesting that Sirt3 deacetylase activity is triggered by dehydration to activate antioxidant activity in these tissues.
Sirt/PGC-1α/FoxO-mediated upregulation of antioxidants was investigated in lung and brain of X. laevis. Results showed upregulations of these three controllers of antioxidants in lung (but not brain) during dehydration, as evidenced by analyses at the mRNA, protein, and phospho-protein levels. Results suggested that dehydration-induced antioxidant upregulation in X. laevis was mediated by Sirts, in addition to PGC-1a and the FoxO1/3 transcription factors in a tissue-specific manner. Antioxidant capacity assays showed that lung sustained a decrease in antioxidant capacity during dehydration, which suggests that the Sirt/PGC-1α/FoxO response may be a compensatory one to restore antioxidants levels.
In the liver, muscle, and heart, PGC-1α and Hif-1α were assessed for their roles in activating ureagenesis, angiogenesis, and remodelling of the metabolism. MEF2-mediated PGC-1α upregulation occurred in the liver, but not the muscle or heart, whereas Hif-1α increased in all 3 tissues with dehydration. Relative mRNA levels of genes related to glucose metabolism, angiogenesis, ureagenesis and β-oxidation were found to be differentially regulated in response to dehydration. Together, the results suggest that PGC-1α and Hif-1α are modulating gene expression during dehydration to suppress β-oxidation in favour of glycolysis, while ureagenesis and angiogenesis are promoted in liver.