UC Berkeley

Fatty acid entry into peripheral tissues, particularly saturated fatty acids such as the long chain fatty acid (LCFA) palmitic acid (PA), are linked to tissue damage characterized by organ dysfunction, increased inflammation, and cellular death. Tissue damage of peripheral organs contributes to several metabolic disorders, including non-alcoholic fatty liver disease and Type 2 diabetes mellitus (T2DM). While lipotoxicity has been studied at the organismal level, the mechanisms of lipotoxicity at the cellular, organellar, and molecular levels are not fully understood. Mitochondria are master regulators of cellular energy metabolism and serve as signaling hubs to integrate information from within and outside of the cell to maintain cellular homeostasis. Mitochondria are also the main organelles that conduct beta-oxidation of short, medium, and long-chain fatty acids, and are themselves regulated via lipid-protein interactions at the inner and outer mitochondrial membranes. Mitochondria are unique among organelles in that they harbor a distinct polyploid genome, mitochondrial DNA (mtDNA), that is separate from nuclear DNA, encoding respiratory chain components required for energy production. MtDNA molecules are packaged into nucleoids by the replication and transcription regulating protein TFAM, and nucleoids are located near the inner mitochondrial membrane. Therefore, changes in mitochondrial function and morphology due to lipid stress could impact mtDNA integrity. Although the effects of lipotoxicity on mtDNA maintenance are poorly understood, due to insubstantial investigation, there are some reporting on the possible effects of PA treatment on the mitochondrial genome. For example, mtDNA mutation and depletion have been linked to obesity and metabolic syndrome in humans and mtDNA mutation has resulted from lipotoxic oxidative stress. Despite these findings, the relationship between mtDNA and cellular lipid metabolism remain to be characterized. Both mtDNA synthesis and lipid droplet biogenesis are coordinated by proteins enriched at ER-mitochondrial contact sites. I used fluorescence microscopy, molecular techniques, and lipidomic analysis, to probe whether lipid droplet biogenesis and mtDNA replication are spatially linked and coordinated. I used Huh7 cells as the model system for this investigation given the relevancy of hepatocytes in lipid metabolism and the development of metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD). I then induced saturated lipid stress by exposing Huh-7 cells to low-level chronic palmitic acid (PA) exposure. We found that upon PA treatment, mtDNA replication significantly decreases and is not spatially coupled to ER-mitochondrial contacts needed for lipid droplet biogenesis. Instead, saturated lipid stress caused increased ceramide production, mild ER stress, and reduced mitochondrial function. This could be due to the rerouting of fatty acid trafficking during cellular fatty acid influx, which differed between PA and oleic acid (OA) treatment. Here we propose a model in which saturated lipid stress promotes the production of toxic lipid byproducts that remodels organelles involved in the mtDNA synthesis pathway, whereas OA promotes the neutralization and storage of fatty acids to combat lipotoxicity. By studying the mitochondrial response to saturated lipid excess, this work provides new insight on how mitochondria manages two major functions during stress. Our work demonstrates that mitochondria “shut-off” mtDNA synthesis as a response to nutrient stress during lipid excess. Prior to this work, it was known that lipotoxicity causes organelle dysfunction that coincides with apoptosis and inflammation. Much of this work was studied to understand the development of metabolic disorders associated with obesity. Investigating the relationship between lipid metabolism and mtDNA maintenance is a highly under-investigated area of research in the field of metabolic biology. Our work begins to characterize the effect of saturated lipid stress on mtDNA replication and organelle remodeling, with the use of the unsaturated fatty acid oleic acid co-treatment as a rescue strategy for lipotoxic insult. However, further studies are required to create a more comprehensive molecular model that connects lipid flux with changes in mtDNA synthesis. These studies would include the use of more defined and dynamic imaging tools to measure contact sites between mitochondria and lipid metabolizing organelles, such as the ER, as well as imaging techniques to assess changes in mitochondria membrane composition that are in close proximity to mtDNA replication sites.