New tools bring greater understanding to cellular metabolism research

Promega CorporationPromega Corporation

New tools bring greater understanding

to cellular metabolism research

Mourad Ferhat, Ph.D,

7 Juin 2017

FDSS Users Meeting, Hamamatsu

[email protected]

2Promega CorporationPromega Corporation

Glucose uptake

Glucose consumption

Glutamine/Glutamate

Lactate

Today’s talk : focus on new cell-based assays in Metabolism research


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.


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


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


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


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).


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


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


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


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

+


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


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)


Metabolites in Medium: Convenient analysis of multiple metabolites


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


Tissues: Brain and Liver

Mouse Brain Mouse Liver

Good recovery of controls spiked into tissue samples before homogenization

Frozen MouseTissues


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


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)

Science

New tools bring greater understanding to cellular metabolism research