Lecture 5: glycolysis Flashcards
What’s the Warburg effect in cancer? (don’t learn by heart)
A form of modified cellular metabolism, which tend to favor a fermentation over the aerobic respiration pathway that most other cells of the body prefer. It’s characterized by an increase in glucose uptake and the production of lactate.
What’s the Pasteur effect and what is the effect of the Pasteur effect on glucose consumption? (don’t learn by heart)
The inhibiting effect of (the presence of) oxygen on the fermentation process. It is a sudden change from anaerobic to aerobic. Glucose consumption is reduced in the presence of oxygen.
Two metastatic breast cancer cell lines, one cell line being more aggressive than the other, are compared based on their glucose consumption rate.
Consider the Warburg and Pasteur effect: what similarities are there between these two cell lines under normoxic and hypoxic conditions and what differences are there? (don’t learn by heart)
- Similarities: both cell lines have a higher glucose consumption rate under hypoxic conditions (Warburg effect) and a lower glucose consuption rate under normoxic conditions (Pasteur effect).
- Differences: the glucose consumption rate of the aggressive cell line under normoxic and hypoxic conditions is much higher compared to the non-invasive cell line.
This phenomenon of differences and similarities between the aggressive and non-invasive cell line due to the Warburg and Pasteur effect are consistent with PET scans. What do these PET scans show? (don’t learn by heart)
That a higher glucose uptake correlates with more aggressive phenotypes and poorer clinical outcomes.
What is shown by the fact that these cancer have two different phenotypes (with the biggest difference → glucose consumption rate), but have the same genotype?
Bistability
What do yeast cells produce during fermation?
Ethanol
Glycolysis is divided into two pathways: upper and lower glycolysis. Based on what can you divide these two parts of glycolysis?
Based on ATP use or production → in upper glycolysis, ATP is used and in lower glycolysis ATP is produced.
The trehalose cycle is an important process in prokaryotes like yeast cells. What kind of molecule is trehalose and what is the function of trehalose?
Trehalose is a sugar composed of two glucose molecules. It is an important player in sensing stress signals and helping the cell adapt to these stress signals. It helps proteins to stay folded and to keep the integrity of cells during osmotic stress. (This is one of the reasons e.g. bacteria can live under harsh conditions.)
What happens in yeast cells when they are grown on glucose in regard to glycolysis?
Yeast cells will reduce/stop their production of trehalose.
What happens if the first enzyme tps1 that converts glucose (G6P) to trehalose is knocked down in yeast mutants?
The cells cannot grow on glucose anymore. Some metabolites of glucose become constant/reach a steady state over time, but only a few metabolites increase in concentration. The metabolites that increase in concentration, are the ones stuck between the upper and lower glycolysis. So it seems there’s an imbalance between the upper and lower part of glycolysis.
What metabolite typically increases in concentration when Tps1 is knocked down?
Fru16bP (FbP)
So what happens if Tps1 is knocked down? What kind of imbalance is created in the upper and lower part of glycolysis?
- The upper part of glycolysis goes too fast → because of the Tps1 knockdown, glucose-6-phosphate (G6P) cannot be converted to trehalose-6-phoshpate (T6P). T6P cannot inhibit hexokinase, a kinase that converts glucose to G6P. So the upper part is overstimulated to produce lots of G6P. G6P is converted to F6P.
- The lower part goes too slow → as a consequence of G6P not being converted to T6P, now there’s no phosphate group to remove from T6P. The upper part doesn’t produce any phosphate groups anymore, which are needed in the lower part of glycolysis. So that’s why the lower part moves too slow.
What key enzyme is important in ATP production during glycolysis? What does it produce and to what does it respond?
PFK (Phosphofructokinase), the enzyme responsible for the production of Fru16bP. It responds to the energy state of the cell by sensing the amount of AMP. If AMP is low, ATP is high and vice versa.
So if ATP is needed (thus AMP is high), Fru16bP is converted so that it can be used in lower glycolysis for ATP production.
Is it normal for cell products/metabolites to accumulate in a cel?
No, there should be a molecule/process that inhibits the production of this metabolite once it increases in concentration too much. Also called negative feedback.
Why can you consider this model as bistable?
Because with the use of two different phosphate concentrations, you get two different phenotypes of glycolysis.
Explain why the Tps1 mechanism/Trehalose cycle is so important.
To prevent imbalanced glycolysis, the upper and lower part of glycolysis should be in balance.
The trehalose cycle prevents that the upper part of glycolysis is overstimulated (by inhibiting hexokinase that converts glucose ito G6P). Not only does the trehalose cycle prevent overstimulation of the upper part, but it also ensures that there’s enough phosphate for the lower part of glycolysis (by inhibiting the upper part). Thus, the trehalose cycle is important for balanced glycolysis.
The lecturer tells about his PhD: he made a computer model of glycolysis. What did he see in a graph that made him realise he forget something in his model?
The importance of phosphate. He assumed that there’s a set level of phosphate in cells (10 mM), but when he peformed glycolysis he saw that at the end there was much more phosphate than only 10 mM (>100 mM phosphate). (So there must be an extra reservoir somewhere in the cell).
So what did the lecturer do when he realised phosphate concentrations are much more dynamical than he first realized?
He realized that there would be a phosphate source somewhere and he figured out that there’s vacuolar yeast with lots of phosphate in it. So he changed his model (see picture).
Based on this model, what did the lecturer see in the computed graphs of cellular concentrations of metabolites of glycolysis, like ATP G6P and F16bP?
Imbalance state of glycolysis, where now all the metabolites are in a steady state, except F16bP that is accumulating.
If this model of glycolysis is bistable and there’s a balanced and imbalanced state of this system, what does it mean when glycolysis is imbalanced? And what needs to occur to balance this system?
F16bP accumulates, due to having no/low Tps1. Phosphate (P) is low, because P is also used to form F16bP. The intracellular concentration of P is now lower than in the vacuole of the cell and P will diffuse to try to increase the intracellular P concentration (i.e. to restore equilibrium).
What happens to the imbalance if the intracellular concentration of phosphate is increased to 30 mM?
It will result in a global steady state, the upper and lower part of glycolysis are balanced. Where also the concentration of F16bP is balanced.
Examine this picture and see if you can understand it. What is the line called that separates the imbalanced state from the steady state?
The line is called the separatrix, it literally separates the two states.
There were different combination of yeast cells and the medium they grew on:
- Tps1 deficient + glucose
- Wildtype + glucose
- Tps1 deficient + galactose
- Wildtype + galactose
What was the result of the pH measurement of these cells?
- The Tps1 mutant growing on glucose slowly acidifies, so this mutant produces very low ATP levels on glucose.
- The WT growing on glucose has a small drop in pH (metabolic imbalance), but quickly adapts itself.
What conclusion can be made based on this picture, especially when looking at the yeast clones on the plate with galactose and glucose?
You can see yeast cell growing next to each other, where one is yellow (high pH) and one is blue (low ph). So it support the fact that there should be two subpopulations.
From this, the steady and imbalanced state were computed and converted into graphs. Explain what happens when glucose is added and the cells goes in the imbalanced state.
When glucose is added, the upper part of glycolysis is highly stimulated and FBP accumulates. FBP is used as a substrate in the lower part of glycolysis and also needs phosphate as a substrate. Only, this phosphate is used to produce FBP. So while FBP is increasing, phosphate is descreasing. This creates a very unstable state.
So what can be concluded based on this core model?
When yeast cells notice that [P] is decreasing, they use a positive feedback mechanism in the first 10 minutes to produce enough ATP in the lower part of glycolysis. This ATP is then used in the upper part of glycolysis to produce trehalose from G6P and T6P. Thus, the reason why yeast cell produce less trehalose is due to the fact that trehalose is only needed in the first 10 minutes when an imbalance is created due to the increase of FBP and decrease of phosphate. You can see this in graph E.
The conclusion that yeast cells restore imbalance by positive feedback and the use of small concentrations of trehalose, are all based on computer models. So how would we measure if this is really the case intracellularly?
By measuring the flux (rate of reaction) by observing the metabolite. This is done by the use of a molecular ink, here 13C is used. 13C glucose is added to cells and the labeling in different pools of metabolism is measured over time. With a mass spectrometer and calculations, you can get an estimate for the flux/rate of reaction. This is depicted in the picture (blue is metabolite concentration in WT growing on glucose, red is how the label is distributed).
This data can be used to see how much flux is directed to tps1 relative to hexokinase. What is seen?
What is seen is that almost 30% of all glucose goes towards the Tps1 cycle in the first 5 minutes. After these 5 minutes, the system goes back to baseline.
So if there’s a balanced and imbalanced state, also depicted in this picture: what is the function of trehalose cycling?
Trehalose cycling prevents the ball going into the imbalanced state. Once in the imbalanced state, trehalose cycling can be used to go back into the steady state.
