Investigation of the change in BMAL1 levels in response to mitochondrial uncoupling in HEPG2 cells

A study in 2016 demonstrated that the rhythmicity in BMAL1 expression was lost and the expression level of BMAL1 significantly decreased in HepG2 cells with depolarized membrane potential. However, the pathway leading to these changes in BMAL1 levels has not been elucidated. In this study, the molecular mechanism behind the changes in BMAL1 levels was investigated. First, the circadian expression of BMAL1 in serum shock- synchronized HepG2 cells was demonstrated by western blot assay. In order to detect the changes in BMAL1 expression more precisely, the timepoint at which BMAL1 levels peak was determined by Cosinor analysis. Mitochondrial membrane potential was measured in trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP)-treated cells to observe if FCCP-induced uncoupling of oxidative phosphorylation led to mitochondrial membrane depolarization. A 79 percent decrease was detected in mitochondrial membrane potential of FCCP-treated cells as a result of confocal laser scanning microscopy analysis of Rhodamine123- and MitoTracker Green FM-stained cells. Next, a specific SIRT inhibitor, EX527 was applied to cells to investigate if there was a link between SIRT1 activity and BMAL1 downregulation in FCCP-treated cells. After confirming SIRT1 inhibition by detecting significant increases in total protein acetylation in EX527-treated cells, BMAL1 expression was investigated by western blot assay. In accordance with the findings in the literature, BMAL1 was downregulated by 63 percent in FCCP-treated cells with respect to control. However, cells which were treated with both FCCP and EX527 were found to have BMAL1 levels similar to the control which indicates SIRT1 inhibition restores BMAL1 downregulation mediated by FCCP. Consequently, SIRT1 activity was considered to be involved in the pathway leading to BMAL1 downregulation in HepG2 cells in response to FCCP-induced mitochondrial membrane depolarization.