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
