Activation of an AMP-activated protein kinase is involved in post-diapause development of Artemia franciscana encysted embryos

Abstract

BackgroundCysts of Artemia can remain in a dormant state for long periods with a very low metabolic rate, and only resume their development with the approach of favorable conditions. The post-diapause development is a very complicated process involving a variety of metabolic and biochemical events. However, the intrinsic mechanisms that regulate this process are unclear.ResultsHerein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKα antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process. Results of kinase assay analysis showed that this phosphorylation is essential for AMPK activation. Using whole-mount immunohistochemistry, phosphorylated AMPK was shown to be predominantly located in the ectoderm of the early developed embryos in a ring shape; however, the location and shape of the activation region changed as development proceeded. Additionally, Western blotting analysis on different portions of the cyst extracts showed that phosphorylated AMPKα localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKα localized to the nuclei when activated. Moreover, cellular AMP, ADP, and ATP levels in developing cysts were determined by HPLC, and the results showed that the activation of Artemia AMPK may not be associated with cellular AMP:ATP ratios, suggesting other pathways for regulation of Artemia AMPK activity.ConclusionTogether, we report evidence demonstrating the activation of AMPK in Artemia developing cysts and present an argument for its role in the development-related gene expression and energy control in certain cells during post-diapause development of Artemia.

Gene expression, metabolic regulation and stress tolerance during diapause

“…Delegation of phosphorylated (active) AMPK to this narrow timeframe implies promotion of post-diapause development rather than inhibition of metabolism and development in diapause-destined embryos. These results are intriguing, especially because the reported amount of active AMPK in diapause embryos is different from the quantity in embryos immediately post-diapause and yet to initiate development [76]. Setting AMPK in motion at this time, along with restricted distribution of the enzyme mostly to ectoderm nuclei, is a sign AMPK influences gene expression during the differentiation of post-diapause Artemia embryos, rather than exercising metabolic regulation per se.…”

“…Apparently, changes to mitochondria do not explain metabolic rate depression in diapause Artemia embryos, and this leaves the way open for other mechanisms including those mediated by AMPK. Artemia contain AMPK, although it appears not to be phosphorylated in early postdiapause embryos when the AMP:ATP ratio is highest [75,76]. Phosphorylated AMPK is first detected 4 h post-diapause and increases for 12 h, but it is absent from nauplii and adults.…”

Role of AMP-activated protein kinase in metabolic depression in animals

“…Strangely, AMP:ATP ratios were unusually high in both non-diapause and diapause embryos of this insect (Hand et al 2011). AMPK activation was not observed in diapausing A. franciscana embryos, in spite of an elevated AMP:ATP ratio (Zhu et al 2009) and profound metabolic depression (Patil et al 2013). Paradoxically in A. franciscana embryos, AMPK activation was seen rather during post-diapause development when AMP levels dropped dramatically, but the mechanism of AMPK activation is unknown (Zhu et al 2009).…”

“…AMPK activation was not observed in diapausing A. franciscana embryos, in spite of an elevated AMP:ATP ratio (Zhu et al 2009) and profound metabolic depression (Patil et al 2013). Paradoxically in A. franciscana embryos, AMPK activation was seen rather during post-diapause development when AMP levels dropped dramatically, but the mechanism of AMPK activation is unknown (Zhu et al 2009). During this phase of development of Artemia embryos, DNA synthesis and cell division are blocked (Olson and Clegg 1978), which could be mediated by AMPK, and AMPK might also influence gene expression (MacRae 2010).…”

Two p90 Ribosomal S6 Kinase Isoforms Are Involved in the Regulation of Mitotic and Meiotic Arrest in Artemia

“…Diapause embryos are composed of 4000 cells arrested at G 1 /S phase. In these embryos, metabolic activity is greatly reduced and RNA/protein synthesis does not occur (25)(26)(27)(28). Post-diapause embryos, in which the cell cycle is arrested at the G 2 /M phase (29), are obtained by storing diapause embryos at Ϫ20°C for at least 3 months (30).…”