Defining the short-term effects of pharmacological 5′-AMP activated kinase modulators on mitochondrial polarization, morphology and heterogeneity
Authors: Kodiha Mohamed , Flamant Etienne , Wang Yi Meng , Stochaj Ursula 
Published: August 2018

Background
Under aerobic growth conditions, mitochondria are the major producers of cellular ATP and crucial for the proper performance of organs and tissues. This applies especially to cells with high energy demand, such as the renal proximal tubule epithelium. Mitochondrial dysfunction contributes to the pathology of human health conditions, including various kidney diseases. The improvement of mitochondrial function ameliorates some of these pathologies. This can potentially be achieved with pharmacological compounds. For example, long-term treatment with activators of 5′-AMP activated kinase (AMPK) enhances mitochondrial biogenesis. However, pharmacological damage control during acute cell injury requires that the short-term effects of these compounds and the impact on healthy cells are also understood. It was our objective to define the changes elicited by established modulators of AMPK activity in healthy renal proximal tubule cells.

Methods
Our work combines confocal microscopy with quantitative image analysis, 3D image reconstruction and Western blotting to provide novel insights into the biology of mitochondria. Specifically, we evaluated the effects of pharmacological AMPK modulators (compound C, AICAR, phenformin, resveratrol) on mitochondrial polarization, morphology and heterogeneity. Microscopic studies generated information at the single cell and subcellular levels. Our research focused on LLC-PK1 cells that are derived from the renal proximal tubule. Mitochondrial heterogeneity was also examined in MCF7 breast cancer cells.

Results
Pharmacological agents that affect AMPK activity in renal proximal tubule cells can alter mitochondrial organization and the electrochemical potential across the inner mitochondrial membrane. These changes were compound-specific. Short-term incubation with the AMPK inhibitor compound C caused mitochondrial hyperpolarization. This was accompanied by mitochondrial fragmentation. By contrast, AMPK activators AICAR, phenformin and resveratrol had little impact. We further show that the biological properties of mitochondria are determined by their subcellular location. Mitochondria at the cell periphery displayed higher MitoTracker/Tom70 values as compared to organelles located in the vicinity of the nucleus. This was not limited to renal proximal tubule cells, but also observed in MCF7 cells. Pharmacological AMPK modulators altered these location-dependent properties in a compound-specific fashion. While the region-dependent differences were enhanced with phenformin, they were ameliorated by resveratrol.

Discussion
We evaluated the rapid changes in mitochondrial characteristics that are induced by pharmacological AMPK modulators. Our research supports the concept that pharmacological agents that target AMPK can rearrange mitochondrial networks at the single cell level. Collectively, these insights are relevant to the development of proper strategies for the short-term adjustment of mitochondrial performance.