A study on features and markers of cellular metabolic rewiring

Abstract

Mammalian cells regulate energy metabolism through molecular responses linked to the microenvironment and nutrient supply. Context dependent abnormalities of energy metabolism are important in disorders such as cancer, diabetes, cardiovascular diseases and neurodegeneration. However, these mechanisms are diverse and often not fully understood. The aim of this study was to investigate cellular mechanisms of metabolic modulation and how it relates to cellular (patho) physiology. We particularly focused on the mitochondria, as these organelles have a critical role in processes of metabolic adaptation. This project investigated metabolic adaptation from two different angles. In one part, we explored metabolic reprogramming as an integral driving force in epithelial to mesenchymal transition (EMT). This is a physiological process that involves important changes in cellular traits and functions. In the other part, we investigated mechanisms and markers of metabolic adaptations occurring when cells and rats were treated with a hypolipidemic modified fatty acids. Although there are conceptual differences between these two contexts, the mechanisms of mitochondrial regulation may be connected. Therefore they may disclose new knowledge that could have applicative value in research on new therapeutic targets and treatment strategies. In the first part, we analysed gene expression in human breast tumours and found that EMT, which facilitates cancer cell migration and possibly metastasis, was associated with reduced mRNA levels of succinate dehydrogenase subunit C (SDHC) and a consequent reduction in mitochondrial activity. Further, we showed that suppression of mitochondrial SDH activity through SDHC CRISPR/Cas9 knockdown or by using the enzymatic inhibitor malonate induced EMT in cell cultures. Similar mitochondrial effects occurred when we modified breast epithelial cells to overexpress EMT-linked transcription factors (TWIST, SNAI2). In these cells, we also observed changes in mitochondrial morphology consistent with loss of functional capacity. These findings describe how altered function of a single mitochondrial metabolic enzyme may have extensive impact on fundamental regulatory programs in a cell. In part two of our studies, we focused on adaptability of fatty acid oxidation, which represent a central fuelling pathway for mitochondrial energy metabolism. Treatment of cells and rats with tetradecylthioacetate (TTA) was used as a strategy to increase the activity of fatty acid oxidation, in order to investigate consequent adaptations in the metabolic machinery. In this work, we also applied targeted modulation of nutrient- and energy sensitive signalling factors such as AMPK, PPARs and mTOR, to investigate mechanisms involved. Our results lead to the characterisation of pyruvate dehydrogenase kinase 4 (PDK4) as a sensitive and robust marker of increased fatty acid oxidation in various contexts of metabolic adaptation. In a separate study, rats were treated with 2-(tridec-12-yn-1-ylthio) acetic acid (1-triple TTA), a derivative of TTA with a carbon-carbon triple bond in omega-1 position. Similar to TTA, 1-triple TTA was found to increase hepatic mitochondrial fatty acid oxidation and reduce plasma lipid level in rats. Further studies are required to determine how the triple bond in 1-triple TTA affects mechanism of action, e.g. through altered bioavailability, stability and ligand-interactions. Through the work of this project, we also validated the use of significantly smaller reaction volumes for analysis of gene expression using quantitative PCR. This was described in a short paper where we reduced the single reaction volume from 10 μl to 1 μl without compromising data quality. Such downscaling facilitates significant savings in the use of chemicals and sample material upon clinical and biomedical applications. In conclusion, this study illuminates important aspects of context-dependent metabolic adaptations through mitochondrial regulation. Shifts in cell metabolism may reflect and interact with mechanisms decisive for cellular (patho) physiology. This may be partly through effects on cellular plasticity, such as the processes of EMT. Hence, implementation of pharmacological approaches to control metabolism may represent an attractive therapeutic strategy in various diseases, including cancer, metabolic diseases and neurodegeneration. This work provided new knowledge on features and markers of metabolic rewiring, and may eventually contribute to future developments of biomedical and clinical applications.