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

  • 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

  • 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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Literature Cited

Literature Cited
   Begriche, K. , Massart, J. , Robin, M. A. , Borgne‐Sanchez, A. , and Fromenty, B. 2011. Drug‐induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver. J. Hepatol. 54:773‐794.
   Bennett, M.J. 2007. Assays of fatty acid beta‐oxidation activity. Methods Cell. Biol. 80:179‐197.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Eaton, S. , Bartlett, K. , and Pourfarzam, M. 1996. Mammalian mitochondrial beta‐oxidation. Biochem. J. 320:345‐357.
   Fillmore, N. and Lopaschuk, G.D. 2013. Targeting mitochondrial oxidative metabolism as an approach to treat heart failure. Biochim. Biophys. Acta 1833:857‐865.
   Fillmore, N. , Mori, J. , and Lopaschuk, G.D. 2013. Mitochondrial fatty acid oxidation alterations in heart failure, ischemic heart disease, and diabetic cardiomyopathy. Br. J. Pharmacol. doi:10.1111/bph. [Epub ahead of print].
   Fogarty, S. and Hardie, D.G. 2010. Development of protein kinase activators: AMPK as a target in metabolic disorders and cancer. Biochim. Biophys. Acta 1804:581‐591.
   Hynes, J. , Marroquin, L.D. , Ogurtsov, V.I. , Christiansen, K.N. , Stevens, G.J. , Papkovsky, D.B. , and Will, Y. 2006. Investigation of drug‐induced mitochondrial toxicity using fluorescence‐based oxygen‐sensitive probes. Toxicol. Sci. 92:186‐200.
   Hynes, J. , Nadanaciva, S. , Swiss, R. , Carey, C. , Kirwan, S. , and Will, Y. 2013. A high‐throughput dual parameter assay for assessing drug‐induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays. Toxicol. In Vitro 27:560‐569.
   Jones, P.M. and Bennett, M.J. 2002. The changing face of newborn screening: Diagnosis of inborn errors of metabolism by tandem mass spectrometry. Clin. Chim. Acta 324:121‐128.
   Lapidus, R.G. and Sokolove, P.M. 1993. Spermine inhibition of the permeability transition of isolated rat liver mitochondria: An investigation of mechanism. Arch. Biochem. Biophys. 306:246‐253.
   Lopaschuk, G.D. and Jaswal, J.S. 2010. Energy metabolic phenotype of the cardiomyocyte during development, differentiation, and postnatal maturation. J. Cardiovasc. Pharmacol. 56:130‐140.
   Lopaschuk, G.D. , Ussher, J.R. , Folmes, C.D. , Jaswal, J.S. , and Stanley, W.C. 2010. Myocardial fatty acid metabolism in health and disease. Physiol. Rev. 90:207‐258.
   McGarry, J.D. and Foster, D.W. 1980. Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem. 49:395‐420.
   Olpin, S.E. , Manning, N.J. , Pollitt, R.J. , and Clarke, S. 1997. Improved detection of long‐chain fatty acid oxidation defects in intact cells using [9,10‐3H]oleic acid. J. Inherit. Metab. Dis. 20:415‐419.
   Palladino, A.A. , Chen, J. , Kallish, S. , Stanley, C.A. , and Bennett, M.J. 2012. Measurement of tissue acyl‐CoAs using flow‐injection tandem mass spectrometry: acyl‐CoA profiles in short‐chain fatty acid oxidation defects. Mol. Genet. Metab. 107:679‐683.
   Rinaldo, P. , Matern, D. , and Bennett, M.J. 2002. Fatty acid oxidation disorders. Annu. Rev. Physiol. 64:477‐502.
   Rogers, G.W. , Brand, M.D. , Petrosyan, S. , Ashok, D. , Elorza, A.A. , Ferrick, D.A. , and Murphy, A.N. 2011. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One 6:e21746.
   Saudubray, J.M. , Coude, F.X. , Demaugre, F. , Johnson, C. , Gibson, K.M. , and Nyhan, W.L. 1982. Oxidation of fatty acids in cultured fibroblasts: A model system for the detection and study of defects in oxidation. Pediatr. Res. 16:877‐881.
   Serra, D. , Mera, P. , Malandrino, M.I. , Mir, J.F. , and Herrero, L. 2013. Mitochondrial fatty acid oxidation in obesity. Antioxid. Redox Signal. 19:269‐284.
   Wu, M. , Neilson, A. , Swift, A.L. , Moran, R. , Tamagnine, J. , Parslow, D. , Armistead, S. , Lemire, K. , Orrell, J. , Teich, J. , Chomicz, S. , and Ferrick, D.A. 2007. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am. J. Physiol. Cell. Physiol. 292:C125‐C136.
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