Protocol

Sirtuins

Volume 1077 of the series Methods in Molecular Biology pp 241-258

Date:

Oxygen Flux Analysis to Understand the Biological Function of Sirtuins

  • Dongning WangAffiliated withSarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center
  • , Michelle F. GreenAffiliated withSarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center
  • , Eoin McDonnellAffiliated withSarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center
  • , Matthew D. HirscheyAffiliated withSarah W. Stedman Nutrition and Metabolism Center, Duke University Medical CenterDepartment of Pharmacology and Cancer Biology, Duke University Medical CenterDepartment of Medicine, Duke University Medical Center

* Final gross prices may vary according to local VAT.

Get Access

Abstract

The sirtuins are a family of highly conserved NAD+-dependent lysine deacylases with important roles in metabolic regulation. Of the seven mammalian sirtuins, three localize to the mitochondria: SIRT3, SIRT4, and SIRT5. Mitochondrial sirtuins are crucial regulators of the metabolic network that controls energy homeostasis and impacts cancer, obesity, diabetes, mitochondrial diseases, metabolic disorders, and many other human diseases of aging. To best study the mitochondrial function of the sirtuins, we have employed an oxygen flux analyzer as a tool to track and record the extracellular oxygen consumption rate and acidification rate that reflects mitochondrial respiration and glycolysis, respectfully. Here we described the methods using this assay to study the substrate utilization and mitochondrial function in a human hepatocellular carcinoma cell line, Huh7. Additionally, we have generated a stable SIRT4 knocked-down Huh7 cell line. With this cell line, we evaluated how the absence of SIRT4 affects mitochondrial function, glucose utilization, glutamine oxidation, and fatty acid oxidation in these cells.

Key words

Mitochondrial sirtuins Seahorse XF extracellular flux analyzer Oxygen consumption rate Seahorse assay Substrate utilization Mitochondrial function SIRT4