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O2 O2 O2 O2 H+ H+ H ++
O2 O2
O2O2 O2
Using bioenergetics to reveal metabolic adaptations of Age-related diseasesDavid Ferrick, PhD Chief Scientific OfficerSeahorse Bioscience
H+
H+
H
CO2 CO2 CO2 CO 2 CO 2
XFe Analyzers, FluxPaks, Stress Test Kits and Reagents
XFe96 Analyzer
XFe24 Analyzer
XFe96 & XFe24 FluxPaks
XF Stress Test Kits & Reagents
How high can we go?
Rapidly Approaching 500 Publications
O2 O2 O2 O2 O2 O2
O2 O2 O2
H
H + H+H + + + H +H
CO 2
CO 2 CO2
CO 2 CO2
Glycolysis ECARExtracellular Acidification Rate
Mitochondrial Respiration OCROxygen Consumption Rate
Measuring Mitochondrial Respiration and Glycolysis in a Microplate
4 drug injection ports per well Add inhibitors, stimulants, substrates, and compounds Injections are defined by the user
Cell Stress and Degeneration
CARDIOVASCULAR
Spare capacity reveals sub-lethal stress response
XF Cell Mito Stress Test300 250OCR (% baseline)0 5 10 20 M M M M HNE HNE HNE HNE
FCCP Antimycin A Oligo
200 150 100 50 0HNE
0
25
50
75
100
125
150
175
200
Time (min)
Cell type: Neonatal Rat Ventricular Myocytes
Biochem J. 2009 Nov 15;425(1):99-107 Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal Bradford G. Hill, Brian P. Dranka, Luyun Zou, John C. Chatham and Victor M. Darley-Usmar
Cancer
CANCER
Oncogenes drive metabolism to support malignant growth
Otto Warburg 1883 - 1970
Craig ThompsonMemorial Sloan-Kettering Cancer Center
Reuben Shaw, Lew Cantley
CANCER
Prostate Cancer Cells Display Glycolytic Phenotype
Cell type: Prostate Tumor Cells Primary Prostate Epithelial Cells PC-3M PCS
J Cancer Sci Ther 2011, S1 Free Base Lysine Increases Survival and Reduces Metastasis in Prostate Cancer Model Mibrahim-Hashim, et al
CANCER
XF Assay Reveals Glutamine Metabolism is Required for Anchorage-Independent Growth
Acetyl CoA Oxaloacetate
Aminotransferases GlutaminaseCitrate
AOAGlutamate Glutamine
-ketoglutarate Succinate
Dimethyl -ketoglutarate (DMK)
Cell type: HCT116 Colon Cancer Cells
Proc Natl Acad Sci USA. 2010 May 11; 107(19):8788-8793 Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity Weinberg, F. and Chandel, N., Northwestern University
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
ETO
Biochim Biophys Acta. 2010 Nov 16; Epub ahead of print. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells Lisa S. Pike, Amy L. Smift, Nicole J. Croteau, David A. Ferrick, Min Wu
CANCER
Oncogenes drive metabolism to support malignant growthc-Myc, HIF1a, PTEN RASPI3K, AKT LKB1, mTOR
eIF4E, S6K1, 4EBP1
Myc H-Ras, Raf, MEK ERK, p53, PTEN
Immunology
IMMUNOLOGY
Spare Capacity Regulates Memory T Cell Development & SurvivalNature 2009 July 2 Enhancing CD8 T-cell memory by modulating fatty acid metabolism Pearce E et al. University of Pennsylvania
Nave T cells Effector T cells Memory T cells
Cell type: Mouse Lymphocytes Spleen & Lymph NodesImmunity 2011 December 28 Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, and Pearce, EL, Washington School of Medicine
IMMUNOLOGY
Spare Capacity in CD8+ T Cells Is Dependent on Mitochondrial Fatty Acid Oxidation
Cell type: Mouse Lymphocytes Spleen & Lymph NodesImmunity 2011 December 28 Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, and Pearce, EL, Washington School of Medicine
IMMUNOLOGY
Spare Capacity in CD8+ T Cells Is Dependent on Mitochondrial Fatty Acid Oxidation
Cell type: Mouse Lymphocytes Spleen & Lymph NodesImmunity 2011 December 28 Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development van der Windt, JW, and Pearce, EL, Washington School of Medicine
IMMUNOLOGY
Activated and Quiescent T cells Have Distinct Metabolic Phenotypes
Whats the Next Experiment?
Isolated Mitochondria Assays
The effects of known respiratory inhibitors on the pattern of mitochondrial respiration are demonstrated in A) the level of respiratory coupling (the coupling experiment) and B) the sequential determination of complex I, II, III and IV-dependent respiration (the electron flow experiment). .
PLoS One; 2011 Jul 25. (7):e21746. High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria Rogers G, Brand M D, Petrosyan S, Ashok D, Elorza A, Ferrick D and Murphy A N
XF Plasma Membrane Permeabilizer is a cytolysin that selectively permeabilizes the plasma membrane
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C2C12 MyoblastsFrom laboratory of Dr. Anne Murphy: Divakaruni, AS et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. MANUSCRIPT SUBMITTED
A Mitochondrial Mystery, Solved
A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans Bricker, et al. Science 6 July 2012: 96-100. DOI:10.1126/science.1218099 Identification and Functional Expression of the Mitochondrial Pyruvate Carrier Herzig, et al. Science 6 July 2012: 93-96. DOI:10.1126/science.1218530
OBESITY/DIABETES
Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.C2C12 Myoblasts
Pyruvate, malate and DCA in the presence of oligomycin.
Avandia = RosiglitazoneFrom laboratory of Dr. Anne Murphy: Divakaruni, AS et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. MANUSCRIPT IN SUBMITTED
OBESITY/DIABETES
Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.C2C12 Myoblasts
Pyruvate, malate and DCA in the presence of oligomycin. From laboratory of Dr. Anne Murphy: Divakaruni, AS et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. MANUSCRIPT IN SUBMITTED
OBESITY/DIABETES
Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.
From laboratory of Dr. Anne Murphy: Divakaruni, AS et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. MANUSCRIPT IN SUBMITTED
OBESITY/DIABETES
Mitochondrial Pyruvate Carrier May Improve the Glucose handling Characteristics of TZDs and Insulin Sensitizers
From laboratory of Dr. Anne Murphy: Divakaruni, AS et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. MANUSCRIPT IN SUBMITTED
Clinical
Ten-fold increase in mitochondrial therapiesNeurodegeneration: Parkinsons, Huntingtons, Alzheimers
Clinical trials Phase I-IV
2000-2005
2005-2011
Mitochondrial targets Metabolic targets
10 350
105Cardiovascular disease:
5,425
hepatic failurediabetes mellitus autoimmunity GVHD Renal disease:
Mitochondrial drugs are going to revolutionize Western medicineDoug Wallace, Director, Center for Mitochondrial and Epigenomic Medicine, CHOP
proximal tubulopathy
Cancer
DIAGNOSTICS
A Clinical Platform for Bioenergetic Profiling of Human Blood
Summary
Catch the Wave
Understanding Metabolism is Not Easy
However, Measuring Metabolism Can Be Easier
MODEL ORGANISMS XF Technology
Reveals Beyond Cells: in Cells andand Zebrafish Bioenergetics C. elegans Tissues
HepG2 liver cells
Hepatocytes
Cardiomyocytes
Heart mitochondria
Podocytes
Renal Proximal Tubules
MCF7 breast cancer
Epithelial cells
INS1 beta cells
Whole Islets
Lymphocytes
PBMC
Cortical neurons
Synaptosomes
Fibroblasts
Cybrids
Myotubes
L6 myoblasts
Isolated Mitochondria
Adipose tissue
SC adipocytes
Human skin fibroblasts
Data courtesy of Auwerx Lab, cole Polytechnique Fdrale de LausanneRat retinal endothelial
Microvascular endothelial
Hippocampal
Stem Cells
Aortic endothelial
Intact muscle fibers
DISEASES OF AGING
XF Assays Reveal Age-Related Metabolic Phenotypes
Cell 148, 421-433, Feb 3, 2012. Resveratrol Ameliorates Aging-Related Metabolic Phenotypes by Inhibiting cAMP Phosphodiesterases Sung-Jun Park et al. Labortory of Obesity and Aging Research, Genetics and Developmental Biology Ceenter, NIH
MODEL ORGANISMS
Beyond Cells: C.elegans and Zebrafish
FCCP injection
***
basal
FCCP basal FCCP
C.elegans
Zebrafish
Data courtesy of Auwerx Lab cole Polytechnique Fdrale de Lausanne
PLoS One. 2011 6(9):e25652 Bioenergetic profiling of zebrafish embryonic development Stackley KD, Beeson CC, Rahn JJ, Chan SS.
Seahorse Impacts Cellular Bioenergetics ResearchStem Cell Biology Cancer Cardiovascular
Nature. 2010 Dec 2;468(7324):659-63. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Gurumurthy S, Xie SZ, Alagesan B, Kim J, Yusuf RZ, Saez B, Tzatsos A, Ozsolak F, Milos P, Ferrari F, Park PJ, Shirihai OS, Scadden DT, Bardeesy N.
Cancer. 2011 Jun 30; Individualizing antimetabolic treatment strategies for head and neck squamous cell carcinoma based on TP53 mutational status Sandulache VC, Skinner HD, Ow TJ, Zhang A, Xia X, Luchak JM, Wong LJ, Pickering CR, Zhou G, Myers JN.
BBA Molecular Cell Research, 2011 Aug 4 Susceptibility to simvastatin-induced toxicity is partly determined by mitochondrial respiration and phosphorylation state of Akt] Mullen PJ, Zahno A, Lindinger P, Maseneni S, Felser A, Krhenbhl S, Brecht K.
Neurodegeneration
Obesity, Diabetes, Metabolic Disorders
Aging
J Neurochem. 2011 Jul; 118(1):79-92. Mitochondrial aconitase knockdown attenuates paraquat-induced dopaminergic cell death via decreased cellular metabolism and release of iron and H(2) O(2) Cantu D, Fulton RE, Drechsel DA, Patel M.
Invest Ophthalmol Vis Sci. 2011 Oct 6 [Epub ahead of print] High Glucose Induces Mitochondrial Morphology and Metabolic Changes in Retinal Pericytes Trudeau K, Molina AJ, Roy S.
Nature. 2009 May 21; 459(7245):387-92. Bmi1 regulates mitochondrial function and the DNA damage response pathway Liu J, Cao L, Chen J, Song S, Lee IH, Quijano C, Liu H, Keyvanfar K, Chen H, Cao LY, Ahn BH, Kumar NG, Rovira, Ii, Xu XL, Van Lohuizen M, Motoyama N, Deng CX, Finkel T.
Rapid Acceptance of XF Technology Worldwide2012: MUSC now has 8 XF Analyzers
XFe - The Worlds Most Advanced Cell Metabolism AnalyzerState-of-the-art optics for rapid calibration.
Intuitive touchscreen navigation. Similar to a smartphone, easy to learn.
Precision Cartridge Handling Improved Mixing Uniformity Improved temperature regulation throughout assay XFe tells you if you left the lid on the microplate
Independent data acquisition processing. Use the controller while an assay is running.
Mitochondrial Electron Transport Chain
+ H+ H+ H+H+ H H+
OUTQ
Cyt C e- e-
e- e-
Cyt C
e-eH++ ++H 2OH HH
IN
e- e+`H NAD+ HH+` +
O2 OHH+H2O H+ H+ H+ O2ADPATP
NADH
Krebs Cycle
How can we use the OCR measurement to characterize these complexes?
Mutation Research 509 (2002) 127151 Peter S. Rabinowitchafter Mandavilli et al
Physiology/Pathophysiology Is Not A One-Way Street
Information
Function
nucDNA mtDNA
Protein
Environment
CANCER
Simultaneous Measurement of Respiratory and Glycolytic Capacity Reveals Mechanism of Tumor SurvivalOXPHOS Inhibition (Oligomycin) GLYCOLYSIS Inhibition (2-DG)
A549
H460
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma CellsFast = 25mM Glucose 6mM Glutamine
Slow = 5.5mM Glucose 2mM Glutamine
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma CellsFast = 25mM Glucose 6mM Glutamine
Slow = 5.5mM Glucose 2mM Glutamine
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma CellsFast = 25mM Glucose 6mM Glutamine
Slow = 5.5mM Glucose 2mM Glutamine
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Biochim Biophys Acta. 2010 Nov 16; Epub ahead of print. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells Lisa S. Pike, Amy L. Smift, Nicole J. Croteau, David A. Ferrick, Min Wu
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Biochim Biophys Acta. 2010 Nov 16; Epub ahead of print. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells Lisa S. Pike, Amy L. Smift, Nicole J. Croteau, David A. Ferrick, Min Wu
CANCER
Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Biochim Biophys Acta. 2010 Nov 16; Epub ahead of print. Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells Lisa S. Pike, Amy L. Smift, Nicole J. Croteau, David A. Ferrick, Min Wu
CANCER
Oncogenes drive metabolism to support malignant growthPI3K, AKT
Myc
STEM CELL DIFFERENTIATION
Metabolic switch to glycolytic metabolism may be deterministic for early stem cell developmentR e s p i r a t i o n
G l y c o l y s i s
The EMBO Journal (2012), 114. HIF1a induced switch from bivalent to exclusively glycolytic metabolism during ESC-to-EpiSC/hESC Transition Kevin Y. Lee,1 Stephane Gesta,1 Jeremie Boucher,1 Xiaohui L. Wang,1 and C. Ronald Kahn1
You have options
XFe96 Analyzer
Measuring Mitochondrial Respiration and Glycolysis in a MicroplateSensor Probes Fiber Optics
XFe Cell Culture Microplate Assay Medium Solid State Sensor Cell Monolayer
Well 1
Well 2
Measuring Mitochondrial Respiration and Glycolysis in a MicroplateThe sensor The biocartridge is cartridge is A transient raised, bringing raised, bringing microthethe system system back chamber is to back to baseline formed baseline
The transient micro-chamber enables rapid measurement of mitochondrial respiration and glycolysis in minutes
Well 1
Well 2
Automated Drug Injection Ports Enable Kinetic, Functional Data
4 drug injection ports per well Add inhibitors, stimulants, substrates, and compounds Injections are defined by the user
XF Cell Mito Stress Test
Kit measures the key parameters of mitochondrial function: Basal RespirationSpare CapacityMaximal Respiration
ATP Production Maximal and Spare Capacity Proton Leak
ATP Production Basal Respiration
Proton Leak Non-mitochondrial Respiration
XF Cell Mito Stress Test Profile
XF Glycolysis Stress Test Kit10mM
Extracellular Acidification
ECAR (mpH/min) ECAR(mpH/min)
Measures the key parameters of glycolytic function:Glycolytic Reserve Capacity
Non-glycolytic Acidification Glycolysis Glycolytic Capacity Glycolytic Reserve
Glycolytic Capacity Glycolysis Glycolysis Non-glycolytic Acidification
XF Glycolysis Stress Test Profile
XF Palmitate-BSA FAO Reagent
Provides the first fully validated and standardized Palmitate-BSA FAO reagent: No substrate preparation Activity tested Quality controlled
How to Work with Suspension CellsCell adhesive: Cell-Tak (BD Biosciences).A sticky protein solution that is used to coat the wells to immobilize cells.