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 ($$ \dot{M}_{{{\text{O}}_{ 2} }} $$). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O2) 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 $$ \dot{M}_{{{\text{O}}_{ 2} }} $$. At physiologically relevant [O2] (<40 μM O2), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular $$ \dot{M}_{{{\text{O}}_{ 2} }} $$ with no change in sensitivity to [O2] 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 $$ \dot{M}_{{{\text{O}}_{ 2} }} $$ in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular $$ \dot{M}_{{{\text{O}}_{ 2} }} $$. 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.