The role of AMP-activated protein kinase in initiating metabolic rate suppression in goldfish hepatocytes

• Main Author: Lau, Gigi Yik Chee
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/29357

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.