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First published online February 13, 2009
Journal of Experimental Biology 212, 722-730 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.021998
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AMP-activated protein kinase (AMPK) in the rock crab, Cancer irroratus: an early indicator of temperature stress

Markus Frederich*, Michaela R. O'Rourke, Nathan B. Furey and Jennifer A. Jost

Department of Biological Sciences, University of New England, Biddeford, MA 04005, USA


Figure 1
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  		Fig. 1. Model of the AMP-activated protein kinase (AMPK) cascade. Stressors such as hypoxia, exercise or temperature lead to a decrease in cellular ATP and an increase in cellular AMP. This activates AMPK either directly, or indirectly through an upstream AMPK kinase. Once AMPK is activated, it phosphorylates multiple downstream targets, mainly rate-limiting enzymes of all energy metabolism pathways. The effect of this phosphorylation, in summary, leads to an acceleration of all ATP-producing pathways and a deceleration of all ATP-consuming pathways. Therefore, AMPK activation preserves the cellular ATP concentration.

 

Figure 2
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  		Fig. 2. Alignment of AMPK{alpha} amino acid sequences for vertebrate and invertebrate species. The sequence for Cancer irroratus is from this study. The remaining sequences were obtained from GenBank (Aedes aegypti AAX20150, Artemia franciscana ABI13783, Rattus norvegicus NM_023991). Sequence conservation is indicated as: black, no conservation; blue, some conservation; and red, complete conservation among the compared species. More than 60% of the obtained Cancer irroratus sequence (453 amino acids) is conserved in this comparison. A large region of conservation is found in the area flanking the T172 region that activates the AMPK protein. T172 lines up in this sequence comparison at position 176 because it was identified and named in the rat sequence, but shifts slightly when compared with other species.

 

Figure 3
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  		Fig. 3. (A) Reaction time after experimental stimulation in Cancer irroratus at increasing temperatures. Animals slowed down slightly in their response above 18°C, and became significantly slower at 28°C (N=15, *P<0.05, repeated measures ANOVA). The percentage of animals not responding at all is shown in B: all animals righted themselves between 12 and 24°C, no animal was able to turn at 30°C. (C) Heart rate of not experimentally stimulated Cancer irroratus increased with a Q10 of 2.2 between 12 and 26°C and leveled off at 153±27 beats min–1 at 26°C before decreasing again above 30°C (N=6). (D) Lactate concentration in the heart tissue increased significantly above 26°C (N=6, *P<0.05, ANOVA). The vertical dashed line indicates the critical temperature (Tc), the vertical dotted line indicates the pejus temperature (Tp).

 

Figure 4
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  		Fig. 4. Representative western blots and the respective quantification for phosphorylated and therefore activated AMPK (p-AMPK), heat shock protein 70 (HSP70 inducible and total) and the loading control actin, for heart tissue of Cancer irroratus at temperatures between 12 and 28 or 30°C during a fast and progressive temperature increase. (A) AMPK activity remained constant between 12 and 18°C, increased above 18°C and reached significance at 26°C. Two significantly different linear regressions (dashed lines, for equations see text) were fitted using a Q-BASIC program to identify critical points (Yeager and Ultsch, 1989Go). The two regressions intersect at 18.5°C. (B) Total AMPK{alpha} protein remained constant during the fast progressive temperature increase. (C) Total AMPK{alpha} mRNA remained constant during the fast progressive temperature increase. (D) Total HSP70 protein did not show any significant changes during the temperature stress. (E,F) Inducible HSP70 protein and mRNA did not show any significant changes during the temperature stress. However, the slight increase at 28°C might indicate the onset of the heat shock response. For all figures: error bars show ±1 s.e.m., N=4–6 per data point, *P<0.05 vs 12°C, ANOVA. The vertical dashed line indicates the critical temperature (Tc), the vertical dotted line indicates the pejus temperature (Tp).

 

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  		Fig. 5. (A) Keeping Cancer irroratus for 6 h at 26°C led to a constantly high heart rate of 160.9±11.9 beats min–1. (B) Lactate in the heart peaked after 4 h and remained elevated at 6 h. (C) AMPK remained activated throughout the temperature stress. (D) AMPK{alpha} mRNA levels showed an upward trend over the 6 h but reached statistical significance only at the P<0.1 level (ANOVA). (E–G) HSP70 protein (total and inducible) remained constant while HSP70 mRNA levels increased slowly and reached significance at 2, 4 and 6 h. For all figures, the very first data point in each graph represents the value at 12°C for each respective parameter before the temperature increase. N=5–6 per data point, *P<0.05 vs 12°C, ANOVA.

 

Figure 6
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  		Fig. 6. Water temperatures in Casco Bay, where the crabs of this study were caught (buoy GoMOOS CO2 at a depth of 2 m, black, and 20 m, grey). Adapted from Shelford's law of tolerance and the adaptation by Frederich and Pörtner (Frederich and Pörtner, 2000Go); we indicate the optimum range with a maximum scope for exercise, limited by an upper pejus temperature, Tp. When the animals are exposed to temperatures above Tp they enter the pejus range with a limited scope for exercise and AMPK activity increases to ensure an adequate cellular ATP concentration. Further temperature increase leads to the critical temperature, Tc, indicated by the onset of anaerobic metabolism, lactate accumulation and HSP70 expression. Survival time in this pessimum range is limited. Therefore, the first measured marker for cellular stress through temperature is increased AMPK activity. For details see text.

 

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