Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay

Brittany A. Stork; Adam Dean; Brian York

doi:10.14440/jbm.2022.374

Authors

Brittany A. Stork Baylor College of Medicine
Adam Dean Baylor College of Medicine
Brian York Baylor College of Medicine

DOI:

https://doi.org/10.14440/jbm.2022.374

Keywords:

peroxisome, seahorse assay, fatty acid metabolism

Abstract

The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse® system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.

Author Biography

Brian York, Baylor College of Medicine

Assistant Professor

Department of Molecular and Cellular Biology

Our research is focused on understanding the physiological contributions of transcriptional coregulators and their upstream signaling inputs. The majority of our research is geared toward unraveling the metabolic and inflammatory pathways involved in liver homeostasis and disease. Over the past decade, we have defined the Steroid Receptor Coactivator (SRC) family as master gatekeepers of systems metabolism. We utilize a variety of in vitro and in vivo models to elucidate novel functions of these potent transcriptional regulators.

In search of the upstream signaling inputs to the SRCs, we have recently identified the calcium kinase signaling cascade as a major player. These studies have evolved into a new research arm of the laboratory that is dedicated to understanding perturbations in calcium signaling as it pertains to hepatic metabolism, inflammation and angiogenesis. Importantly, this triad of processes are central to the development of metabolically induced pathologies beginning with Non Alcoholic Fatty Liver Disease (NAFLD) that progresses to Non Alcoholic Steatohepatitis (NASH) and ultimately to Hepatocellular Carcinoma (HCC).

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