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

Zhu XJ, Dai JQ, Tan X, Zhao Y, Yang WJ - BMC Dev. Biol. (2009)

Bottom Line: However, the intrinsic mechanisms that regulate this process are unclear.Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, PR China. xiao_jingzhu@hotmail.com

ABSTRACT

Background: Cysts 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.

Results: Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha 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 AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha 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.

Conclusion: Together, 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.

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Cellular ATP, ADP, and AMP were determined by HPLC analysis. A. HPLC chromatogram of ATP, ADP, and AMP from Artemia diapause embryos (a) and embryos at three representative developing stages (0, 4, and 8 h, b-d). Arrows indicate peaks of ATP, ADP, and AMP. B. Concentrations of cellular ATP, ADP, and AMP in embryos at different developmental stages were determined by an external standard method. dp, diapause embryos. C. Changes of ADP:ATP (white columns) and AMP:ATP (black columns) ratios in developing embryos.
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Figure 4: Cellular ATP, ADP, and AMP were determined by HPLC analysis. A. HPLC chromatogram of ATP, ADP, and AMP from Artemia diapause embryos (a) and embryos at three representative developing stages (0, 4, and 8 h, b-d). Arrows indicate peaks of ATP, ADP, and AMP. B. Concentrations of cellular ATP, ADP, and AMP in embryos at different developmental stages were determined by an external standard method. dp, diapause embryos. C. Changes of ADP:ATP (white columns) and AMP:ATP (black columns) ratios in developing embryos.

Mentions: Cellular ATP, ADP, and AMP were separated by HPLC analysis in embryos at different developmental stages (Figure 4A). Concentrations of cellular adenylates were calculated by the external standard method (Additional file 2). The results showed that diapause embryos contained large amounts of AMP, while ATP and ADP were merely detectable (Figure 4A, a; Figure 4B, dp). In contrast, ATP remained at a high level in embryos during development (Figure 4B, 0–12 h). The level of ATP increased from initiation of the development until it reached its maximal value at the 8th hour of development (Figure 4B, ATP, 0–8 h). The ATP concentration decreased in the next 4 hours, but still remained higher than at the initiation of development (Figure 4B, ATP, 8–12 h). During development, the AMP:ATP ratios were between 0.1 and 0.3 (Figure 4C). Interestingly, AMPK was not activated in the first 4 hours in which the AMP:ATP ratio was higher than the later stages.


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

Zhu XJ, Dai JQ, Tan X, Zhao Y, Yang WJ - BMC Dev. Biol. (2009)

Cellular ATP, ADP, and AMP were determined by HPLC analysis. A. HPLC chromatogram of ATP, ADP, and AMP from Artemia diapause embryos (a) and embryos at three representative developing stages (0, 4, and 8 h, b-d). Arrows indicate peaks of ATP, ADP, and AMP. B. Concentrations of cellular ATP, ADP, and AMP in embryos at different developmental stages were determined by an external standard method. dp, diapause embryos. C. Changes of ADP:ATP (white columns) and AMP:ATP (black columns) ratios in developing embryos.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667496&req=5

Figure 4: Cellular ATP, ADP, and AMP were determined by HPLC analysis. A. HPLC chromatogram of ATP, ADP, and AMP from Artemia diapause embryos (a) and embryos at three representative developing stages (0, 4, and 8 h, b-d). Arrows indicate peaks of ATP, ADP, and AMP. B. Concentrations of cellular ATP, ADP, and AMP in embryos at different developmental stages were determined by an external standard method. dp, diapause embryos. C. Changes of ADP:ATP (white columns) and AMP:ATP (black columns) ratios in developing embryos.
Mentions: Cellular ATP, ADP, and AMP were separated by HPLC analysis in embryos at different developmental stages (Figure 4A). Concentrations of cellular adenylates were calculated by the external standard method (Additional file 2). The results showed that diapause embryos contained large amounts of AMP, while ATP and ADP were merely detectable (Figure 4A, a; Figure 4B, dp). In contrast, ATP remained at a high level in embryos during development (Figure 4B, 0–12 h). The level of ATP increased from initiation of the development until it reached its maximal value at the 8th hour of development (Figure 4B, ATP, 0–8 h). The ATP concentration decreased in the next 4 hours, but still remained higher than at the initiation of development (Figure 4B, ATP, 8–12 h). During development, the AMP:ATP ratios were between 0.1 and 0.3 (Figure 4C). Interestingly, AMPK was not activated in the first 4 hours in which the AMP:ATP ratio was higher than the later stages.

Bottom Line: However, the intrinsic mechanisms that regulate this process are unclear.Using a phospho-AMPKalpha antibody, AMPK was shown to be phosphorylated in the post-diapause developmental process.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Cell Biology and Genetics, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, PR China. xiao_jingzhu@hotmail.com

ABSTRACT

Background: Cysts 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.

Results: Herein we report the specific activation of an AMP-activated protein kinase (AMPK) in the post-diapause developmental process of Artemia. Using a phospho-AMPKalpha 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 AMPKalpha localized to the nuclei and this location was not affected by intracellular pH. Confocal microscopy analysis of immunofluorescent stained cyst nuclei further showed that AMPKalpha 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.

Conclusion: Together, 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.

Show MeSH
Related in: MedlinePlus