The effects of troglitazone and PMA on AMPK in HepG2 cells
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Type 2 diabetes, as well as other metabolic diseases, is an increasing global health concern and many of the mechanisms of both the disease and its current drug treatments have not been fully described. It has been shown that the anti-diabetic class of drugs, the thiazolidinediones, work via both a known PPARγ-dependent, and a lesser known PPARγ-independent mechanism of action. This PPARγ-independent mechanism likely involves the metabolic regulatory molecule AMPK, which has a newly elucidated inhibitory site of phosphorylation at Ser485/Ser491. In this study we sought to determine if the thiazolidinedione troglitazone affects AMPK in HepG2 liver cells via phosphorylation at both the known Thr172 site as well as the letter understood Ser485 site. We also looked for potential upstream kinases of the Ser485 site by comparing our results to recently proposed mechanisms of phosphorylation here. HepG2 cells were cultured in the lab and treated with troglitazone to determine time- and dose- dependent effects on AMPK. We also treated cultured HepG2 cells with PMA as well as troglitazone and PMA in order to compare mechanisms of action of troglitazone on AMPK. Results were analyzed using common western blot techniques and statistical analysis. Our data found that troglitazone increased AMPK activity by increasing phosphorylation at Thr172 in a time- and dose- dependent manner. The inhibitory site Ser485 was also increasingly phosphorylated with troglitazone treatments, although the net result of troglitazone treatment remained AMPK activation. The recently elucidated results from our laboratory showing the mechanism of p-AMPK Ser485 phosphorylation via PKD after PMA treatment also occurred in HepG2 cells, although this did not appear to be the mechanism by which troglitazone phosphorylated AMPK at Ser485. These data support the current research that there is an AMPK mediated PPARγ-independent mechanism of troglitazone treatment for type 2 diabetes and other metabolic diseases. The results do however bring into question the full effects of the drug on AMPK at a molecular level and leaves room for new research in this area, specifically the exact mechanism by which troglitazone phosphorylates AMPK at Ser485. Our data also brings up new questions as to the simultaneous phosphorylation of AMPK at both Thr172 and Ser485 and what this means for the activity of the molecule as a whole, a current area of critical research. Lastly our data support the newly elucidated mechanism of AMPK phosphorylation at Ser485 via PKD1, an exciting and novel discovery and potential target for therapeutic intervention.