New tools bring greater understanding to cellular metabolism research

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1 New tools bring greater understanding to cellular metabolism research Mourad Ferhat, Ph.D, 7 Juin 2017 FDSS Users Meeting, Hamamatsu mourad.ferhat@promega.com

2 Today s talk : focus on new cell-based assays in Metabolism research Glucose uptake Glucose consumption Glutamine/Glutamate Lactate 2

Metabolic shift in cells Metabolic shifts occur to encourage biosynthesis for

NAD, etc Glo s The shift is accompanied by an increase in ROS and the cell

ROS-Glo Assay GSH/GSSG-Glo Assays Signaling pathways are usurped in cancer to

NAD/NADH-Glo Assay pgl4 & pnl Pathway Vectors Glucose ADP Lactate GSH/GSSG-Glo

3 Metabolic shift in cells 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. NAD/NADH-Glo Assay pgl4 & pnl Pathway Vectors Glucose ADP Lactate GSH/GSSG-Glo Assay pnl Fusion Vectors ATP ATP Oxidative Phosphorylation ADP Glutamine NAD+ NADH NAD+ Glycolysis NAD+ Pyruvate GSH TCA a-ketoglutarate NADP+ NADPH NADP/NADPH-Glo Assay Building Blocks GSSG ROS NADP+ ROS-Glo H 2 O 2 Assay Anabolic Metabolism 3

4 The Needs of Quiescent and Proliferating Cells are Different Glucose Glucose Fatty acids Glycolysis Response to internal and external signals Glycolysis Nucleotides Proteins TCA ATP ATP TCA Lipids Glutaminolysis Glutaminolysis Glutamine Glutamine Quiescent cell efficiently converts nutrients into energy Proliferating cell rewire metabolism to support growth 4

5 Cancer Cells Rewire their Metabolism to Meet Proliferation Needs Oxidative Phosphorylation Glucose in media/extracellular external & internal signals Highly Glycolytic Glucose in media/extracellular Glucose Glycolysis Glutamine Addiction Glucose Warburg Effect Glycolysis Pyruvate ATP Pyruvate ATP Lactate Acetyl CoA TCA Lactate Acetyl CoA TCA Lactate secreted Lactate secreted Glutamate Glutaminolysis Glutamine Glutamate Efficient conversion of glucose into energy Metabolism rewired lactate/glucose 5

Glutamine Metabolism and Ovarian Cancer Cells Ovarian cancer glutamine metabolism and cancer aggressiveness Low- Invasive High- Invasive Cell lines OVCAR-3 SKOV-3 Glutamine Dependence Glutamine

6 Glutamine Metabolism and Ovarian Cancer Cells Ovarian cancer glutamine metabolism and cancer aggressiveness Low- Invasive High- Invasive Cell lines OVCAR-3 SKOV-3 Glutamine Dependence Glutamine Synthetase Glutamate Secretion No Yes Lower Yes No Higher 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 cancer Yang et al. Mol Syst Biol. (2014) 10:728 Yang et al Cell Metabolism 24:685 6

Metabolite Detection Technology In Reformulated, Liquid Format, Luciferin Detection Solution Lactate Glucose Glutamate Glutamine Metabolite Assays use dehydrogenases selective for each metabolite The

7 Metabolite Detection Technology In Reformulated, Liquid Format, Luciferin Detection Solution Lactate Glucose Glutamate Glutamine 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 Our publication in Special Collection Issue: Cancer Metabolism Bioluminescent 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, (2017). 7

8 Advantages of Bioluminescent Approach The assay is linear for > 3 logs (from 0.1 M to 100 M): Flexibility and convenience when comparing samples at different metabolite concentrations or following changes over the time Broad linearity Simplified protocols Intracellular levels measured in cells cultured in 96-well plates: No cell collection No centrifugation steps Wide assay window The assay window is > fold: 1 M is detected with signal >2.5 fold over the background Maximum signal-to background > Better discrimination of small changes between samples 8

Product Overview Oxidative stress ROS (H 2 0 2 ), GSH/GSSG Adenine Dinucleotide Assays NAD, NADP, NADH, NADPH Glucose Uptake

9 Product Overview Oxidative stress ROS (H ), GSH/GSSG Adenine Dinucleotide Assays NAD, NADP, NADH, NADPH Glucose Uptake Assay New Glycolysis Assays Glucose and Lactate In Development Lipid Metabolism New Glutaminolysis Glutamine and Glutamate 9

10 Metabolite Assays are Applicable to Different Samples Cell Culture: Intracellular Tissues Cell Culture: Extracellular/ Media Cell Culture: Total = Cells + Media Different Samples Different Preparation One Assay Chemistry Enzyme Activity Fluids: Blood, CSF 3D, Microtissues 10

11 Assays Performance 50 l Metabolite in PBS + 50 l Metabolite Detection Reagent Incubate 1h Read luminescence ASSAY LOD Linearity S/B max Lactate 100nM (5pmol/50 l) Up to 200 M 240 Glucose 5nM (0.25pmol/50 l) Up to 50 M >1000 Glutamine 5nM (0.25pmol/50 l) Up to 50 M > 1000 Glutamate 5nM (0.25pmol/5 l) Up to 50 M >

12 Assay Protocol Steps: 1. Prepare Detection Reagent 2. Prepare Sample: several sample types 3. Mix 1:1 in 96 or 384-well plate 4. Incubate for 1 hr at RT 5. Read luminescence 12

13 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) 13

14 Metabolites in Medium: Convenient analysis of multiple metabolites 14

15 Metabolite in Tissues: Schematic ~10 mg tissue slice In Inactivation Solution target ~3 mg/ml Only a small amount of tissue is needed Sample = Tissue Homogenize (mechanical) in acidic Inactivation Solution Neutralization Solution Neutralization Solutionʹ *No additional centrifugation or deproteinization steps are required Homogenate ready to be assayed* Multiple metabolites can be measured from a single tissue sample Lactate Glucose Glutamate Glutamine Glutamate Protein Assay 15

16 Tissues: Brain and Liver Mouse Brain Mouse Liver Good recovery of controls spiked into tissue samples before homogenization Frozen Mouse Tissues 16

Cellular Dynamics International (CDI) glucose uptake data : icell skeletal myoblasts A Raw Data B Normalized Data C 8 10 05 2.

17 Cellular Dynamics International (CDI) glucose uptake data : icell skeletal myoblasts A Raw Data B Normalized Data C **** **** RLU **** 2-DG Uptake (Fold Change) DG Uptake (Fold Change) Do 0 Control +Insulin +Cyto B 0.0 Control +Insulin

Summary Cells rewire metabolic pathways to adjust to changing requirements Glycolysis Bioluminescent Tools for monitoring such changes Glutaminolysis Fatty acid/lipid biosynthesis TCA Cycle Pentose

18 Summary Cells rewire metabolic pathways to adjust to changing requirements Glycolysis Bioluminescent Tools for monitoring such changes 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 ( µM) and wide dynamic range (max S/B >100 fold) 18

How to Integrate Cellular Metabolism Assays Into Your Research: Considerations and Challenges

How to Integrate Cellular Metabolism Assays Into Your Research: Considerations and Challenges Donna Leippe Sr Research Scientist Outline for Today s Webinar Introduction to Cell Metabolism Metabolite Detection