Assessment of Fatty Acid Beta Oxidation in Cells and Isolated Mitochondria

George W. Rogers1, Sashi Nadanaciva2, Rachel Swiss2, Ajit S. Divakaruni3, Yvonne Will2

1 Seahorse Bioscience, North Billerica, Massachusetts, 2 Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc, Groton, Connecticut, 3 Department of Pharmacology, University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.3
DOI:  10.1002/0471140856.tx2503s60
Online Posting Date:  May, 2014
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Abstract

Fatty acid beta oxidation is a major pathway of energy metabolism and occurs primarily in mitochondria. Drug‐induced modulation of this pathway can cause adverse effects such as liver injury, or be beneficial for treating heart failure, type 2 diabetes, and obesity. Hence, in vitro assays that are able to identify compounds that affect fatty acid oxidation are of value for toxicity assessments, as well as for efficacy assessments. Here, we describe two high‐throughput assays, one for assessing fatty acid oxidation in cells and the other for assessing fatty acid oxidation in isolated rat liver mitochondria. Both assays measure fatty acid‐driven oxygen consumption and can be used for rapid and robust screening of compounds that modulate fatty acid oxidation. Curr. Protoc. Toxicol. 60:25.3.1‐25.3.19. © 2014 by John Wiley & Sons, Inc.

Keywords: fatty acid beta oxidation; oxygen consumption; cells; mitochondria

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Fatty Acid Beta Oxidation in Cells
  • Basic Protocol 2: Assessment of Fatty Acid Oxidation in Isolated Rat Liver Mitochondria
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of Fatty Acid Beta Oxidation in Cells

  Materials
  • Cells to be tested: e.g., C2C12 (ATCC no. CRL‐1772) or HepG2 cells (ATCC no. 77400)
  • Growth medium for C2C12 cells (see recipe) or for HepG2 cells (see recipe)
  • Substrate‐limited medium (see recipe; also see Critical Parameters and Troubleshooting)
  • Oligomycin, FCCP, rotenone, and antimycin A (Seahorse Bioscience, XF Cell Mito Stress Test Kit, cat. no. 101706‐100; alternatively, each of these compounds can be purchased from Sigma)
  • Test compounds
  • Fatty Acid Oxidation (FAO) assay medium (see recipe)
  • 10 mM etomoxir
  • XF Palmitate‐BSA FAO Substrate and BSA control (Seahorse Bioscience, cat. no. 102720‐100)
  • XF96, XFe96, XF24, or XFe24 Fluxpaks (XF cartridges and tissue culture plates; Seahorse Bioscience, cat. no. 102310‐001, 102416‐001, 100850‐001, or 102340‐001, respectively)
  • XF96, XFe96, XF24, or XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, cat. no.100900‐100, 101991‐100, 100736‐100, or 102238‐100, respectively)
  • 96‐well plates for diluting compounds
  • Non‐CO 2 incubator
  • Additional reagents and equipment for tissue culture ( )

Basic Protocol 2: Assessment of Fatty Acid Oxidation in Isolated Rat Liver Mitochondria

  Materials
  • Sprague‐Dawley rats (male, 150 to 300 g; Charles River Laboratories)
  • Buffer I (see recipe)
  • Buffer II (see recipe)
  • BCA (bicinchoninic) Protein Assay Reagent (see )
  • Heavy mineral oil (MP Biomedical, cat. no. 150138)
  • Respiration buffer (see recipe)
  • Glutamate/malate stock solution (see recipe)
  • Palmitoyl CoA/carnitine/malate stock solution (see recipe)
  • MitoXpress probe (Luxcel Biosciences)
  • Test compounds
  • Vehicle: DMSO
  • ADP stock solution (see recipe)
  • Dissecting instruments
  • Glass tissue homogenizer with Teflon pestle (55 ml; Thomas Scientific, cat. no. 3431D94)
  • Power drill (hand‐held or static)
  • Glass beakers
  • Plastic funnel
  • Cheesecloth
  • 50‐ml centrifuge tubes
  • Refrigerated high‐speed centrifuge
  • Disposable transfer pipets (3 ml)
  • Standard clear‐bottom 96‐well plate for BCA protein assay
  • Black‐body clear‐bottom 96‐well plate (Costar 3631 or equivalent) for oxygen‐consumption assay
  • Plate warmer (VWR, cat. no. 12621‐108)
  • 30°C water bath (for warming solutions)
  • Time‐resolved fluorescence plate reader with temperature control and kinetic analysis software
  • Absorbance plate reader
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, ) and BCA protein assay ( )
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Figures

Videos

Literature Cited

Literature Cited
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