Akt signaling and freezing survival in the wood frog, Rana sylvatica.

BACKGROUND The wood frog (Rana sylvatica) exhibits well-developed natural freeze tolerance supported by multiple mechanisms of biochemical adaptation. The present study investigated the role and regulation of the Akt signaling pathway in wood frog tissues (with a focus on liver) responding to freezing stress. METHODS Immunoblotting was used to assess total and phospho-Akt levels, total and phospho-PDK1, PTEN protein level, as well as total and phospho-FOXO1 levels. RT-PCR was used to investigate transcript levels of PTEN and microRNAs. RESULTS Akt was inhibited in skeletal muscle, kidney and heart after 24h freezing exposure with a reversal after thawing. The responses of the main kinase (PDK-1) and phosphatase (PTEN) that regulate Akt were consistent with freeze activation of Akt in liver; freezing exposure activated PDK-1 via enhanced Ser-241 phosphorylation whereas PTEN protein levels were reduced. Levels of three microRNAs (miR-26a, miR-126 and miR-217) that regulate pten expression were elevated in liver during freezing. One well-known role of Akt is in anti-apoptosis, mediated in part by Akt phosphorylation of Ser-256 on FOXO1. Freezing triggered an increase in liver phospho-FOXO1 Ser-256 content, suggesting that an important action of Akt may be apoptosis inhibition. CONCLUSIONS Akt activation in wood frog is stress and tissue specific, with multi-facet regulations (posttranslational and posttranscriptional) involved in supporting this specific signal transduction response. GENERAL SIGNIFICANCE This study implicates the Akt pathway in the metabolic reorganization of cellular metabolism in support of freezing survival.

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