O2 O2 O2 O2 H+ H+ H +

O2 O2

O2
O2 O2

Using bioenergetics to reveal metabolic adaptations of Age-related diseases

David Ferrick, PhD Chief Scientific Officer
Seahorse Bioscience

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

CO 2

CO 2 CO2

CO 2 CO2

Glycolysis ECAR
Extracellular Acidification Rate

Mitochondrial Respiration OCR

Oxygen 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 Test

300 250
OCR (% baseline)
0 5 10 20 M M M M HNE HNE HNE HNE

FCCP Antimycin A Oligo

200 150 100 50 0

HNE

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 Thompson
Memorial 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 Glutaminase
Citrate

AOA
Glutamate 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 growth

c-Myc, HIF1a, PTEN RAS
PI3K, AKT LKB1, mTOR

eIF4E, S6K1, 4EBP1

Myc H-Ras, Raf, MEK ERK, p53, PTEN

Immunology

IMMUNOLOGY

Spare Capacity Regulates Memory T Cell Development & Survival

Nature 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 Nodes

Immunity 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 Nodes

Immunity 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 Nodes

Immunity 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

XF-PMP permeabilizes PM
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C2C12 Myoblasts
From 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 = Rosiglitazone
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.

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 therapies

Neurodegeneration: Parkinsons, Huntingtons, Alzheimers

Clinical trials Phase I-IV

2000-2005

2005-2011

Mitochondrial targets Metabolic targets

10 350

105
Cardiovascular disease:

5,425

hepatic failure
diabetes mellitus autoimmunity GVHD Renal disease:

Mitochondrial drugs are going to revolutionize Western medicine

Doug 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 Lausanne

Rat 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 Research

Stem 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 Worldwide

2012: MUSC now has 8 XF Analyzers

XFe - The Worlds Most Advanced Cell Metabolism Analyzer

State-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+

OUT
Q

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+ O2
ADP
ATP

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 Survival
OXPHOS Inhibition (Oligomycin) GLYCOLYSIS Inhibition (2-DG)

A549

H460

CANCER

Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Fast = 25mM Glucose 6mM Glutamine

Slow = 5.5mM Glucose 2mM Glutamine

CANCER

Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Fast = 25mM Glucose 6mM Glutamine

Slow = 5.5mM Glucose 2mM Glutamine

CANCER

Enhanced Metabolic Capacity and Substrate Fluxes in SF188 Human Glioblastoma Cells
Fast = 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 growth

PI3K, AKT

Myc

STEM CELL DIFFERENTIATION

Metabolic switch to glycolytic metabolism may be deterministic for early stem cell development
R 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 Microplate

Sensor 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 Microplate

The 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 Respiration
Spare Capacity
Maximal 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 Kit

10mM

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 Cells

Cell adhesive: Cell-Tak (BD Biosciences).
A sticky protein solution that is used to coat the wells to immobilize cells.