Friday, January 18, 2019

Cell surface oxygen consumption (1)

Back in the comments thread to the More on insulin and the glycerophosphate shuttle post there has been some discussion as to whether Warburg/Seyfried was/is correct about cancers being glycolysis driven or whether ox-phos is the core processes driving cancer metabolism. Raphi and Altavista have fairly opposite views.

Altavista rather liked this paper, using supra pharmacological concentrations of metformin to block complex I in tissue cultured cancer cells. It is true that this intervention produced a dose dependent decrease in oxygen consumption, but we have no idea of what the absolute oxygen consumption of the cells was, only the relative fall from control cell levels. I have huge problems with this paper. Relative change smells like relative risk, as in cardiology...

So I went looking to find out whether cancer cells do consume oxygen in decent amounts and whether this oxygen is simply metabolised by their mitochondria.

It seems that oxygen metabolism is not the sole prerogative of mitochondria.

This paper is fascinating reading and Table 1 gives us the actual oxygen consumption rates of assorted cancer cell lines in culture.

Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines

Total oxygen consumption varies from as high as almost 28pmol/sec/10^6 cells in the HeLa line to as low as 5pmol/sec/10^6 cells in the P815 cell line.

So there is no doubt that cancer cells in culture do consume oxygen.

Does that imply they are using it for ox-phos? Fascinatingly, the answer is no. Not completely.

A significant proportion of the oxygen consumption is occurring at the cell surface plasma membrane. If you acutely block the ETC of the mitochondria using myxothiazol this plasma membrane oxygen consumption continues and appears to account for around half of the total oxygen consumption, the exact percentage varies.

What is really interesting is what happens if you generate ρ° derivatives of your cell line. These have no functional ETC at all but still consume significant amounts of oxygen, usually in the order of around 90% of the total amount consumed by the parent cell line. They have obviously adapted to their lack of mitochondrial ETC by hugely up regulating cell surface oxygen consumption.

Why? How?

I'll probably put posts up about these questions as we do have some ideas. But the whole reason I went looking was to decide whether cancer cells perform ox-phos. It seems that at least some of them do. Those which don't (major mutations of complex I genes) tend to be very, very unpleasant in patients. Amongst other cancers it's possible that the degree of dysfunction in ox-phos and its replacement by plasma membrane oxygen consumption may correlate with the degree of malignancy of the cancer.

Nothing is black and white. Many cancers respire to various degrees. Not always very well.

You can't tell from simple oxygen consumption if a cancer cell line is respiring using the mitochondrial ETC or performing plasma membrane oxygen consumption. Your Clark electrode can't tell you. That explains a chunk of why people can't agree on whether cancers use ox-phos or not. All consume oxygen, but some use more ox-phos than others.

I just thought it was interesting.

Peter

12 comments:

Marko Peteri said...

Hi Peter,

Thanks for your great investigate work! This stuff is so fascinating, especially due to the wide range of overgeneralized myths still spreading. You are contributing with you deep quality ponderings in immense information-rate per word provided, indicating a great cognitive machinery.

I'd love to hear your thoughts on Grant Genereaux (I find his personal story extremely fascinating) and Garrett Smiths findings/hypotheses possibly stating that there is no such thing as "Vitamin" A, and that the science behind its Vitamin status might be fundamentally bonked. Just a quick look comparing the carotenoid and retinoid molecules and all their other derivatives, they look very similar to PUFAs, which begs the question: are all polyunsaturated aliphatic compounds potentially toxic (in "normal" amounts)?

I'd recommend the following steps:

Step 1. Listen to this podcast from December 2018 with Dr Garrett Smith, which gives some background/context on Grants story and Garrets findings: https://www.bslnutrition.com/episode53/

Step 2. Read following free ebooks authored by the Canadian engineer/geologist Grant Genereux in order: https://ggenereux.blog/my-ebooks/

Step 3. Dr. Garrett Smith has done some work with finding studies in his blog/forum here, which he updates continuously: https://nutritionrestored.com/blog-forum/

raphi said...

@peter

nice paper, i'm going to keep that O2 consumption table handy and see if i can fit mitochondrial ultrastructural data to the O2 consumption rates. UCP2 is over-expressed in these 7 cancer cell lines http://www.ncbi.nlm.nih.gov/pubmed/19480693 (Eugene Fine and Richard Feinman) so i wouldn't be surprised even if cancer cell lines showing lots of mitochondrial O2 aren't aren't coupling much of it to ATP production. as a further control we might want to see how many carbons from glutamine and glucose end up in CO2...

Another one from Fine and Feinman http://www.fasebj.org/content/29/1_Supplement/725.22 seems that the MCF-7 breast cancer cells I used, that were supposedly oxphos competent due to a high rate of O2 consumption, have their growth inhibited by acetoacetate. As do the MDA MB 231 and SW480 cell lines.

with my bias, what i read into the p^0 lines is that they confirm we can get significant O2 use in cancer cells without mitochondria and they can stay alive, strongly suggesting fermentation must be the back-up generator.

what would be the experiment we'd need to absolutely show that at least 1 cell line is totally competently using oxphos to obtain most of its ATP needs?

Passthecream said...

Some time ago when I was looking for more information about the ETC I was surprised to find some similar components apparently residing in outer cell membranes. Old news I suppose:

Transplasma membrane electron transport: enzymes involved and biological function
https://www.tandfonline.com › doi › pdf


This paper is similar to the one you link above, same authors:

https://www.researchgate.net/publication/6630754_Plasma_Membrane_Electron_Transport_A_New_Target_for_Cancer_Drug_Development

Passthecream said...

'Suprised to find' LOL.
I didn't find them, merely read about them!

Peter said...

@pass, also same authors:

https://www.ncbi.nlm.nih.gov/pubmed/15178469

“It is worth noting that the ability to reduce oxygen at the cell surface to support glycolytic metabolism may be a fingerprint of the early endosymbiotic origin of eukaryotic cells” Surface oxygen consumption pumps protons too, but indirectly, by an antiporter... A Na+/H+ antiporter no less! I do wonder if these folks are on to something.

@raphi. I may be underthinking this but wouldn't feeding caner cells on lactate get rid of ATP from glycolysis while providing a perfect substrate for ox-phos? Leaves you with glutamate and a group of other amino acid confounders of course...

@Marko, thanks for the heads up but I've still not finished the podcast where Peter Attia interviewed Seyfried yet! I'll keep the concept in mind .

Peter

Peter said...

@raphi, just making the children lunch when "UCPs" gave a nasty thought about lactate and O2/ATP!

Peter

Passthecream said...

https://www.ncbi.nlm.nih.gov/m/pubmed/24789919/

Peter said...

Nice!

Peter

Passthecream said...

That first link I pasted was borked.
Here's a link to the .pdf


Transplasma membrane electron transport: enzymes involved and biological function:

https://www.tandfonline.com/doi/pdf/10.1179/135100003125001198

It's worth a look.

karl said...

This got my attention:

"It seems that oxygen metabolism is not the sole prerogative of mitochondria."

But think about it - if we think of eukaryotes as cells that expanded the area of cell wall in folded sheets in the mitochondria - we can imagine that the primitive cell that got engulfed and became the mitochondria had to do what it could to survive - and some of that likely lives on in the MT-DNA

I know that one of the ways cells are suspected of being cancers is that they are small - relying on the cell-wall rather than their mitochondria.

My hunch is that 'most' cancers have something wrong with their mitochondria - at least enough wrong that they ignore the autophagy signal that lets them become immortal.

So if a cell had a wall that didn't use much O2 - it would likely not make it in a new career as a cancer cell - so cells that have more active walls would be selected for.

To me the real question is just what goes wrong in a MT to ignore the autophagy signal? (Likely not just one case).

Anyway fascinating.

Passthecream said...

The more I learn about cells the more I wonder how there is any room for just plain outer membrane. There are so many transporters and surface tags etc such that a significant portion of cellular energy is taken up in operating the various transporters. These papers refer to a plasma membrane potential, 15 to 35mV, wikip. has it maxing out near 200mV before arcing over and a capacitance of 2uF per square cm. By a rough calc that means an hepatocyte has a pm capacitance of approx 2nF. In parallel with the very low resistance of electron, proton and ion transporters would suggest that membrane potential changes can happen very quickly ie T = R×C is small.

Karl & Peter, not Macavity but I did see a pic of a maze with Hairy Maclary :)

Passthecream said...

PS in models of insulin resistance my brain takes it literally as (channel) resistance but I find it easier to think about insulin conductance, or lack of.