AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ((M) over dot O(2)). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions ( > 200 mu M O(2)) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in (M) over dot O(2). At physiologically relevant [O(2)] (< 40 mu M O(2)), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular (M) over dot O(2) with no change in sensitivity to [O(2)] as assessed by estimates of cellular P50 and P90 values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on (M) over dot O(2) in normoxia. Treatment of hepatocytes with <= 200 mu M A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular (M) over dot O(2). These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.