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Promega CorporationPromega Corporation
New tools bring greater understanding
to cellular metabolism research
Mourad Ferhat, Ph.D,
7 Juin 2017
FDSS Users Meeting, Hamamatsu
mourad.ferhat@promega.com
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Glucose uptake
Glucose consumption
Glutamine/Glutamate
Lactate
Today’s talk : focus on new cell-based assays in Metabolism research
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Metabolic shift in cells
NAD+
ADP
ATP
Oxidative
Phosphorylation
Glucose
Pyruvate
Glycolysis
TCA
Glutamine
Lactate
ADP
ATP
a-ketoglutarate
Building
Blocks
NADP+
NADPH
NADP+NAD+
NADH
NAD+ROS
GSH
Anabolic Metabolism
GSSG
NAD/NADH-Glo™
Assay
NADP/NADPH-Glo™
Assay
ROS-Glo™
H2O2 Assay
GSH/GSSG-Glo™
Assay
pGL4 & pNL
Pathway Vectors
pNL Fusion
Vectors
• Metabolic shifts occur to encourage biosynthesis for mitosis.
• NAD, etc –Glo’s
• The shift is accompanied by an increase in ROS and the cell must respond to control ROS.
• ROS-Glo Assay
• GSH/GSSG-Glo Assays
• Signaling pathways are usurped in cancer to maintain the proliferative metabolism.
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The Needs of Quiescent and Proliferating Cells are Different
Quiescent cell – efficiently converts nutrients into energy
Response to internal and external signals
Proliferating cell – rewire metabolism to support growth
Glucose
Glycolysis
TCA
Fatty acids
Glutamine
Glutaminolysis
ATP
Glucose
Glycolysis
TCA
Glutamine
Glutaminolysis
ATP
Nucleotides
Proteins
Lipids
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external & internal signals
Oxidative Phosphorylation
Glucosein media/extracellular
Lactatesecreted
Efficient conversion of glucose into energy
Glucose
Glycolysis
Pyruvate
TCAAcetyl CoALactate
ATP
Highly Glycolytic
Glucosein media/extracellular
Glucose
Glycolysis
Pyruvate
TCAAcetyl CoALactate
Lactatesecreted
Metabolism rewiredlactate/glucose
Glutamine
GlutaminolysisGlutamate
Glutamate
ATP
“Warburg Effect”
“Glutamine Addiction”
Cancer Cells Rewire their Metabolism to Meet Proliferation Needs
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Glutamine Metabolism and Ovarian Cancer Cells
Ovarian cancer glutamine metabolism and cancer aggressiveness
Yang et al. 2016 Cell Metabolism 24:685
Targeting Stromal Glutamine Synthetase in Tumors Disrupts Tumor Microenvironment-
Regulated Cancer Cell Growth
Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in
ovarian cancerYang et al. Mol Syst Biol. (2014) 10:728
Low-Invasive
High-Invasive
Cell lines OVCAR-3 SKOV-3
GlutamineDependence
No Yes
Glutamine Synthetase
Yes No
Glutamate Secretion
Lower Higher
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Metabolite Detection Technology
Metabolite Assays use dehydrogenases selective for each metabolite The concentration of NAD(P)H produced is proportional to the concentration
of metabolite present Dehydrogenases used: Lactate DH, Glucose DH and Glutamate DH To measure Glutamine, there is an additional enzymatic reaction to first
convert glutamine to glutamate
LactateGlucose
GlutamateGlutamine
In Reformulated, Liquid Format,
Luciferin Detection Solution
Our publication in Special Collection Issue: Cancer MetabolismBioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites. D. Leippe, M. Sobol, G. Vidugiris, J.J. Cali, J. Vidugiriene. SLAS Discovery, Vol.22, 366-377 (2017).
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Advantages of Bioluminescent Approach
Simplified protocols
Broad linearity
Wide assay window
Intracellular levels measured in cells cultured in 96-well plates:
• No cell collection• No centrifugation steps
The assay is linear for > 3 logs (from 0.1M to 100M):• Flexibility and convenience when
comparing samples at different metabolite concentrations or following changes over the time
The assay window is >100-500 fold:• 1M is detected with signal >2.5 fold over the background • Maximum signal-to background >200-500• Better discrimination of small changes between samples
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Product Overview
Adenine Dinucleotide AssaysNAD, NADP, NADH, NADPH
Glucose Uptake AssayGlycolysis AssaysGlucose and Lactate
Lipid Metabolism
New
In Development New
GlutaminolysisGlutamine and Glutamate
Oxidative stressROS (H202), GSH/GSSG
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Metabolite Assays are Applicable to Different Samples
Cell Culture:Intracellular
Enzyme Activity
Fluids: Blood, CSF
Tissues
3D, MicrotissuesCell Culture:
Extracellular/Media
Cell Culture:Total =
Cells + Media
Different Samples
Different Preparation
One Assay Chemistry
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Assays Performance
ASSAY LOD Linearity S/B max
Lactate 100nM (5pmol/50l) Up to 200M 240
Glucose 5nM (0.25pmol/50l) Up to 50M >1000
Glutamine 5nM (0.25pmol/50l) Up to 50M > 1000
Glutamate 5nM (0.25pmol/5l) Up to 50M > 500
Incubate 1h
50l Metabolite in PBS
50l Metabolite Detection Reagent
Read luminescence
+
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Assay Protocol
Steps:1. Prepare Detection Reagent2. Prepare Sample: several sample types3. Mix 1:1 in 96 or 384-well plate4. Incubate for 1 hr at RT5. Read luminescence
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Monitoring Changes in Media Using A549 Cells
A549 cells were plated in DMEM media containing 5mM glucose, 2mM glutamine and 10% dialyzed serum
Media samples were removed and changes were measured at 8, 24, 48 and 72 hours
Note: We recommend using dialyzed serum and low glucose media (5 – 10mM)
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Metabolites in Medium: Convenient analysis of multiple metabolites
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Metabolite in Tissues: Schematic
~10 mg tissue sliceIn Inactivation Solution
target ~3 mg/ml
Only a small amount of tissue is needed
Homogenize (mechanical) in acidic Inactivation Solution
Sample = Tissue
Neutralization Solution
Neutralization Solutionʹ
Homogenate ready to be assayed*Multiple metabolites can be measured from a single tissue sample
Glutamate
Protein Assay
GlutamineGlutamateLactate Glucose
*No additional centrifugation or deproteinization steps
are required
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Tissues: Brain and Liver
Mouse Brain Mouse Liver
Good recovery of controls spiked into tissue samples before homogenization
Frozen MouseTissues
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Cellular Dynamics International (CDI) –glucose uptake data : iCell skeletal myoblasts
Raw Data
Control +Insulin +Cyto B0
210 0 5
410 0 5
610 0 5
810 0 5
****
****
RL
UNormalized Data
Control +Insulin0.0
0.5
1.0
1.5
2.0
2.5
****
2-D
G U
pta
ke
(Fo
ld C
ha
ng
e)
Dose-Response Curve
0.01 0.1 1 10 100 1000
1.0
1.5
2.0
2.5
EC50 = 3.2 nM
[Insulin] (nM)
2-D
G U
pta
ke
(Fo
ld C
ha
ng
e)
A B C
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Summary
Cells rewire metabolic pathways to adjust to changing requirements
Bioluminescent Tools for monitoring such changes
Glycolysis
Glutaminolysis
Fatty acid/lipid biosynthesis
TCA Cycle
Pentose Phosphate Shunt
Bioluminescent assays are designed for quantitative metabolite detection in media, cell lysates and tissues: Advantages of bioluminescent detection: sensitive detection (1-5pmole/sample) with broad linear range (0.1 -100µM) and wide dynamic range (max S/B >100 fold)
Recommended