Exposure of marine invertebrates to high temperatures leads to a switch from aerobic to anaerobic metabolism, a drop in the cellular ATP concentration ([ATP]), and subsequent death. In mammals, AMP-activated protein kinase (AMPK) is a major regulator of cellular [ATP] and activates ATP-producing pathways, while inhibiting ATP-consuming pathways. We hypothesized that temperature stress in marine invertebrates activates AMPK to provide adequate concentrations of ATP at increased but sublethal temperatures and that AMPK consequently can serve as a stress indicator (similar to heat shock proteins, HSPs). We tested these hypotheses through two experiments with the rock crab, Cancer irroratus. First, crabs were exposed to a progressive temperature increase (6 degrees C h(-1)) from 12 to 30 degrees C. AMPK activity, total AMPK protein and HSP70 levels, reaction time, heart rate and lactate accumulation were measured in hearts at 2 degrees C increments. AMPK activity remained constant between 12 and 18 degrees C, but increased up to 9.1(+/-1.5)-fold between 18 and 30 degrees C. The crabs' reaction time also decreased above 18 degrees C. By contrast, HSP70 (total and inducible) and total AMPK protein expression levels did not vary significantly over this temperature range. Second, crabs were exposed for up to 6 h to the sublethal temperature of 26 degrees C. This prolonged exposure led to a constant elevation of AMPK activity and levels of HSP70 mRNA. AMPK mRNA continuously increased, indicating an additional response in gene expression. We conclude that AMPK is an earlier indicator of temperature stress in rock crabs than HSP70, especially during the initial response to high temperatures. We discuss the temperature-dependent increase in AMPK activity in the context of Shelford's law of tolerance. Specifically, we describe AMPK activity as a cellular marker that indicates a thermal threshold, called the pejus temperature, T(p). At T(p) the animals leave their optimum range and enter a temperature range with a limited aerobic scope for exercise. This T(p) is reached periodically during annual temperature fluctuations and has higher biological significance than earlier described critical temperatures, at which the animals switch to anaerobic metabolism and HSP expression is induced.
