Metabolomics 6 - Metabolomic Fluxes Flashcards
Rationale Behind Cancer Metabolomics: Cancer a metabolic disease?
Warburg 1926: “Stoffwechsel der Tumoren (1926)”
1924: “Cancer cells metabolise glucose in a manner that Is distinct from normal tissues. They tend to ferment glucose into lactate even in the presence of sufficient oxygen to support mitochondrial oxydative phosphorylation”.
1931: Nobelpreis für Physiologie: Entdeckung der Natur und der Funktion des Atmungsferments“
Warburg’s Hypothesis
Used manometric technique to measure
* O2 consumption
C6H12O6 +6O2 ->6CO2 +6H2O
* CO2 production
= equivalent to lactate production in bicarbonate buffers CH2CHOHCOOH + HCO3- -> CH3CHOHCOO- + H2O + CO2
* Used Flexner-Jobling rat carcinoma: Showed that it takes up more O2 than liver and produced lactic acid, even in the presence of O2 (Warburg effect)
* Lactic acid production was 2x that in normal tissue
* Meyerhof quotient O2 consumed/lactic acid produced
Warburg, O. Über den heutigen Stand des Carcinomproblems
The origin of cancer lies in the anaerobic metabolic component of normal growing cells, which is more resistant to damage than is the respiratory component. Damage to the organism favours this anaerobic component and, therefore, engenders cancer.
The Warburg Effect
The utility of anaerobic glycolysis:
- ATP production from glycolysis is approximately 100X faster than from OxPhos.
- Eg when a muscle cell quickly needs large amounts of energy
OXIDATIVE PHOSPHORYLATION
- differentiated tissue
- +O2
- Glucose
- Pyruvate
- Lactate
- CO2
-> Oxidative phosphorylation (-36 mol ATP/mol glucose)
ANAEROBIC GLYCOLYSIS
- differentiated tissue
- -O2
- Glucose
- Pyruvate
- Lactate
-> Anaerobic glycolysis (2 mol ATP/mol glucose)
AEROBIC GLYCOLYSIS (WARBURG EFFECT)
- proliferative tissue or tumor or +/- O2
- Glucose
- pyruvate
- lactate
- CO2
- aerobic glycolysis (Warburg effect) (-4 mol ATP/mol glucose)
The Warburg Effect
-> Role of oxygen
-In the presence of oxygen, non-proliferating (differentiated) tissues metabolize glucose to pyruvate
via glycolysis and then oxidize pyruvate to CO2 via oxidative phosphorylation
in the mitochondria (Krebs cycle)
- Limited oxygen: cells redirect pyruvate away from the Krebs cycle by generating lactate (anaerobic glycolysis).
- Generation of lactate allows glycolysis to continue (by cycling NADH back to NAD+), but results in minimal ATP production compared with the Krebs cycle.
Metabolic Reprogramming in Cancer
Metabolic alterations in cancer:
1. Enhanced glycolysis causing increased lactate production
2. Enhanced glucose import
3. Alterations in glycogen storage
4. IDH1/2 mutations causing reductive carboxylation
5. Enhanced glutaminolysis
6. Mutations in succinate dehydrogenase
7. Mutations in fumarate hydratase
Principle of Metabolic Flux Analysis
Cell culture -> Metabolite extraction -> Sample preparation -> NMR Acquisition -> Data processing -> Metabolic analysis
Tracer based metabolomics and fluxomics
Analytical methods
-> Mass Isotopomer Distribution Analysis (MIDA)
* m+1,2… signals in MS
* ->percentage of each isomer
* No positional information
* Can cover symmetrical molecules such as fumarate and succinate
-> NMR analysis
* 13C-NMR -> direct observation
* insensitive, overlapping signals
* 1H-NMR -> use the ratio of center peaks vs satellites
* Overlappingsignals!
* 1H-1H-TOCSY spectra
* 13C-HSQC
* Direct observation of intensity changes for CH
* Additional information from 13C-couplings -> not limited to CH, also covers COOs
* Large number of increments needed to resolve couplings -> NUS
* 13C-JRES
NMR was used to describe glutaminolysis in cancer cells
- Here we used 13C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis.
- Surprinsingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapud enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anapldrotic oxalacetate was derived from glutamine.
- Rather, glutamine metabolism provides a carbon source that facilitates the cells ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.
An experiment to sort out PC vs PDH activity
-> Zitronensäurezyklus
- PDH would transfer label to C-1 of acetyl-CoA and eventually C-5 of glutamate
- PC would transfer label to C-2 of OAA and eventually C-3 of glutamate.
- Labeling of C-2 of OAA would also result in labeling of C-3 of OAA and ultimately C-2 of glutamate.
- C-3:C-4 = 1:1 ⟹ No PC activity, no anaplerosis
-> Tracer-based NMR can distinguish between PC and PDH product
Sorting out lipid synthesis using elegant NMR experiments
Fatty acid spectra
- Fatty acid synthesis occurs via sequential addition of acetyl-CoA to an elongating chain.
- The methyl and ω-1 carbons are derived from one two-carbon group
- and the ω-2 and ω-3 carbons are derived from another two-carbon group
- The likelihood that both the ω-1 and ω-2 carbons are 13C-labeled is thus determined by the fraction of the acetyl-CoA pool that is 13C-labeled.
Fatty acid spectra
Labelling with [U-13C]glucose
Both, ω1 and ω2 showed a triplet (t) and a double (d)
Coupling pattern at w1: R-CH2-CH2-CH2-CH2-CH3
Triplet at w1: Two coupling 13Cs needed
R-13CH2-13CH2-13CH3
=> Two 13C-labelled AcCoA were used
Doublet at w1: One coupling 13C
R-12CH2-13CH2-13CH3
=> One 13C-labelled AcCoA was used
Same for w2: R-CH2-CH2-CH2-CH2-CH3
Triplet: R-13CH2-13CH2-13CH2-13CH3
Doublet: R-13 CH2-13CH2-12CH2-12CH3
Sorting out the role of glutaminolysis in cancer
Design for two-stage perfusion experiment.:
- In the first stage, cells received [3-13C]glutamine and unlabeled glucose as a bolus and then as a continuous feed.
- In the second stage, cells received [3- 13C]glutamine and [1,6-13C2]glucose.
Phase 1: [3-13C]glutamine labelling
- [3-13C]-α-KG formation
- [2-13C]Asp and [3-13C]Asp (2 and 3 become equal when passing through succinate and fumarate
- [2 and 3-13C]lactate by oxidation of [2/3- 13C]malate by malic enzyme – produces NADPH needed for fatty acid synth
- An equal amount of NADPH came from oxidative PPP (glucose-6 phosphate dehydrogenase (G6PDH) (calculated from C-2/C-3 ratio of lactate)
Phase 2: Co-labelling of [3-13C]glutamine and [1,6-13C]glucose
- Labeling of glutamate at C-4
- Condensation of [2-13C]OAA from [3-13C]glutamine with [2-13C]AcCoA will yield doubly labelled [3,4-13C]glutamine
Quant. Analysis:
- 45% of the labelled glutamate pool was doubly labelled
- glutamate was the major source of anaplerotic flux
- Low amount of cycled glutamate, [2-13C]OAA arises from gln, leading to equal amounts of [2-13C]OAA and [3-13C]OAA
Sorting out the role of glutaminolysis -> Summary
- the cells synthesized fatty acids and lipids primarily with carbon from glucose.
- The cells converted 60% of the glutamine metabolized to lactate.
- This implied a malic enzyme flux high enough to supply the NADPH needed for fatty acid synthesis.
- A large fraction of nitrogen generated during glutamine metabolism was also released from the cell.
- Like many cancer cell lines, these cells exhibited a high rate of glucose consumption and anaerobic metabolism of pyruvate.
- Summing the production of lactate and alanine,
=> >90% of total glucose metabolism. - Therefore, all remaining glucose-dependent activities (glycosylation, fatty acid synthesis, glycerogenesis, nucleotide biosynthesis, pyruvate oxidation, etc.) accounted for <10% of total glucose utilization
Pyruvate carboxylase is critical for non-small lung cancer proliferation
- Infused patients with early-stage non–small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity
- stable isotope–resolved metabolomic (SIRM) analysis to paired freshly resected CA and NC lung tissue slices in culture using either [U-13C] glucose or [U-13C,15N] glutamine as tracers.
- 94% of NSCLC tumours showed overexpressed PC 73% showed elevated PC activity
13C6-glucose was infused into NSLC patients
2.5h hours after infusion the tumour was removed by surgery;
13C3-Asp was determined by GC-MS:
- Enrichment of 13C3 -Asp was on average 117% higher in cancer compared to control tissues
- Possible error by 2nd cycle which produces again 13C3-Asp
- Measuring 13C4-citrate as control: lower => disregard 2nd cycle
Increased PC expression and activity in NSCLC tumors
In vivo PC activity was enhanced in CA tissue compared with that in paired NC lung tissues (n = 34) resected from the same human patients given 13C6 glucose 2.5-3 hours before tumor resection. PC activity was inferred from the enrichment of 13C3-citrate (Cit+3), 13C5-Cit (Cit+5), 13C3-malate (Mal+3), and 13C3-aspartate (Asp+3) as determined by GC-MS,
Further experiments with perfused ex vivo tissue slices
Thin slices of tissue were cultured in 13C6-glucose or 13C5,15N2-glutamine for 24 hours
13C5,15N2-glutamine: Labelled glutamate confirms active glutaminase
Label incorporations point towards increased PC and PDH activity in cancer tissues:
- enrichment in glycolytic 13C3-lactate
- higher fractions of 13C4-, 13C5-, and 13C6-citrate
- the increase in the percentage of enrichment of 13C3-, 13C4-, and 13C5-glutamate
Requires combined PC and PDH activity
Role of glutaminolysis probed by 13C5, 15N2-glutamine treatment of tissue slices for 24 h
No significant differences in the label pattern of glutamate or other Krebs cycle intermediates from incubation of labeled glutamine, suggesting that glutaminase is not more active in cancerous than in paired non- cancerous tissue
Anaplerotic input via GLS did not compensate for the loss of PC activity
Treatment of PC-knockdown cells with 13C5, 15N2 glutamine revealed that anapldrotic input via GLS did not compensate for the loss of PC activity, since GLS activity was attenuated, as interfered from the activity markers. Decarboxylation of glutamine-derived malate by magic enzyme (ME) and reentry of glutamine-derived pyruvate into the Kreby cycle via PC or PDH were also attenuated.
PC Knockdown has significant metabolic effects
Knock-down by shRNA:
- inhibition of cell proliferation by PC suppression is accompanied by a decrease in anaplerotic input into the Krebs cycle
- Reduction of nucleotide and lipid biosynthesis
- Also depletes the GSH pool by blocking de novo glutathione synthesis
- anaplerotic glutaminolysis could not compensate for lost PC-based anaplerosis
These results make PC a clear drug target for NSCLC lung cancers
Reductive carboxylation was proven using metabolic fluxes
Using labelling from [1-13C]glutamine as a readout, we measured a significant and robust decrease in reductive carboxylation when IDH1 messenger RNA (mRNA) was targeted using short hairpin RNAs (shRNAs).
Significant increase of reductive carboxylation under hypoxia
Cells preferentially used glucose carbon for palmitate synthesis under normoxic conditions; however, fatty acids produced under hypoxia were primarily synthesized from glutamine carbon through the reductive pathway.
- Reductive carboxylation of glutamine-derived ɑ-KG accounted for approximately 80% of the carbon used for de novo lipogenesis in A549 cells growing under hypoxia (c).
- Concomitant decrease in the contribution of [U-13C6]glucose to fatty-acid synthesis under this condition.
- The net flux of reductive glutamine metabolism to palmitate synthesis was significantly increased in hypoxic cultures (d).
- Metabolic reprograming of mammalian cells by hypoxia or VHL loss to use reductive glutamine metabolism for lipogenesis.
- HIF stabilization drives transcription of PDK1, which decreases PDH activity and subsequently intracellular citrate levels.
- IDH1 and ACO1 reductively generate lipogenic citrate from glutamine-derived α-KG.
- DCA can inhibit PDKs, forcing increased glucose oxidation in hypoxic cells.
Target: Mutated Isocitrate dehydrogenases (IDH1&2)
In glioblastoma and in acute myeloid leukaemia (AML) - Mutant IDH -> Generate 2-HG from aKG
- 2HG accumulates in mM levels
- 2HG inhibits aKG dependent dioxygenases, involved in histone and DNA demethylation
- => hypermethylation of histones and CpG islands in DNA
- proposed to promote oncogenesis by preventing normal cellular differentiation
- Therapeutic approach:
- Target mutant IDH1 or IDH2
- Several inhibitors now available:
- AGI-5198 for IDH1, suppresses growth of IDH1-mutant human glioma cells in a xenograft model
- AG-221 (enasidenib): survival benefit in a mouse model of IDH2-mutant AML, FDA approved in 2017
Cysteine and Folate Metabolism, and a new metabolic workflow
- Cell lines from primary colon adenocarcinoma (SW480),
- its lymph node metastasis (SW620)
- and a liver metastatic derivative (SW620-LiM2)
- Flux map of central metabolism (13C MFA)
- Recin2 constrained by central metabolism flux map
- Cell line specific genome-scale flux maps
- Cell line specific metabolic targets
Predicted Fluxes through HEX1 (hexokinase), LDH-L (lactate dehydrogenase), PDH (pyruvate dehydrogenase), CS (citrate synthase) and GLS (glutaminase
The cell line-specific flux maps were used to systematically simulate genes’ KO using the minimization of metabolic adjustment (MOMA) algorithm and identify single or target pairs that could selectively inhibit growth in the metastatic cell lines. Overall, 10 single target and 237 target combinations were predicted to impair the proliferation of SW620 and LiM2 (Table S2).
Genome-scale Modelling predicts drug targets
metastatic cells are dependent on cystine uptake and vulnerable to system xCT and glutathione reductase inhibition
Genome-scale Modelling predicted drug targets confirmed by redeployed drugs
Sulfasalazine, a drug approved for the treatment of rheumatoid arthritis, erastin, a recently developed inhibitor of the system xCT
Amersham DNP Experiment
- > 10,000 increase in SNR
- Combines DNP enhancement with temperature factor
- Applicable to single-scan ND NMR applications
- Applicable to 13C, 15N, 31P not easily for 1H
- Addition of trityl radicals to the sample
Polarisation cup
- max 200 µL
- sample
- radical
- solvents to form glass state
Challenges of DNP-NMR and MRI
The entire flux experiment must be carried out during the T1 of the polarised molecule
- Use long-lived atoms with slow relaxation, typically quaternary carbons
- The apparent T1 of pyruvic acid is around 40sec
- The master molecule for in vivo applications is now 1-13C-pyruvic acid (label on the CO)
The radical needs to be removed and the sample needs to be sterilised before injection
- All this has been automatied in GE Healthcare Instrumentation
- There are several clinical trials under way to test this setup
Hyperpolarized 13C MRI: Path to clinical translation in oncology
Clinical grade HP probe fluid path preparation
-> GE SPINlab polarizer + QC system
-> MRI scanner
-> MR Detector Hardware
-> Fast 13C pulse sequence
-> post processing
-> pre clinical studies
-> FDA regulatory approval
-> patient research
Fates of pyruvate
- can be changed into lactate and/or alanine
