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The role of AMP-activated protein kinase in initiating metabolic rate suppression in goldfish hepatocytes Lau, Gigi Yik Chee


The ability to undergo metabolic rate suppression (MRS) markedly improves chances of survival during aquatic hypoxia. In this thesis, I specifically tested the hypothesis that AMP-activated protein kinase (AMPK) initiates MRS in hepatocytes from the common goldfish (Carassius auratus). My first goal was to investigate the responses of isolated hepatocytes to changes in O₂. Goldfish hepatocytes showed a gradual decrease in cellular oxygen consumption rate (MO₂) as O₂ was decreased from normoxia (~310 µM O₂) down to the apparent P₉₀ of 13 µM, below which there was a steep decline in MO₂. The apparent P₉₀ for hepatocyte respiration matched published measurements of venous [O₂], which suggests that hepatocyte MO₂ in vivo may be regulated by O₂. To address the relationship between AMPK and MRS, several drugs were used to manipulate AMPK activity. I was able to activate AMPK with 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) under normoxic conditions, which caused a reduction in MO₂; this decrease was mediated through a decrease in protein synthesis rate via eukaryotic elongation factor 2 (eEF2) phosphorylation. Specifically, a maximal 7.5-fold activation of AMPK resulted in a 24% reduction in MO2, thus supporting the notion that AMPK activation initiates MRS. We then used compound C, a general protein kinase inhibitor, in an attempt to reverse the AICAR effects on AMPK activation, but compound C did not reverse the effects of AICAR. A recently discovered specific AMPK activator, A769662, was also used to manipulate AMPK activity. However, at all doses, A769662 failed to activate AMPK. Nevertheless, whenever I was able to activate AMPK via AICAR incubation, there was a consistent lowering of metabolic rate. Thus I have provided evidence to support the hypothesis that AMPK is important in the initiation of MRS in goldfish hepatocytes.

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