Thursday, March 30, 2017

Amgen share price and PCSK9 inhibition with Repatha (2)

I had an email from a PR company representing Amgen, re Repatha and all cause mortality. Here's the bit of interest:


I respect your opinion, but did want to share some additional information with you regarding the 2-year length of the study.

FOURIER was an event-driven study and was to conclude when least 1,630 hard major adverse cardiovascular event (MACE) events were accumulated. Amgen expected the study to run for 43 months with a 2 percent annual event rate in the placebo arm. However, the annual event rate in the placebo arm exceeded 3 percent and led to a faster accumulation of hard MACE events. Since the relative risk reduction in the hard MACE composite endpoint grew from 16 percent in the first year to 25 percent beyond 12 months, Amgen anticipates that a longer duration trial would have led to further relative risk reduction.

Would you please consider correcting this sentence of your post?

“The study was stopped early, presumably to stop the hard end points of dead patients from becoming too obvious.”


You can see how Amgen made their decision. Am I incorrect in my presumption about why the study was terminated early?

Well, technically yes. The protocol is laid out. That's unarguable. So they have a point and have designed the study well, from their point of view.

The fact that 444 people died in the treatment arm vs 426 in the placebo arm was not statistically significant, despite representing a 4.2% increased relative risk of death over the study duration.

What seems to concern Amgen is the implication that all cause mortality had any influence on the decision to terminate the study early. Obviously I cannot know whether this is the case and Amgen are certain that my presumption is incorrect. So maybe some compromise:

If we go with this I can reword the sentence to:

“The study was stopped early due to an unexpected excess of combined cardiac adverse end points in the placebo arm. At this time point the 4.2% increase in relative risk of all cause mortality in the treatment arm was not statistically significant”.

I don't think these facts are arguable with.

Well, that's been interesting. I feel somewhat honoured to have been contacted by a company representing Amgen to correct my presumptions!


Sunday, March 26, 2017

The pathology of evolution

Aaron posted the link to this paper via Facebook:

Selection in Europeans on Fatty Acid Desaturases Associated with Dietary Changes

As the authors comment in the discussion:

"Agricultural diets would have led to a higher consumption of grains and other plant-derived foods, relative to huntergatherer populations. Alleles that increase the rate of conversion of SC-PUFAs to LC-PUFAs would therefore have been favored".

Or to rephrase it slightly, from the legend of Fig 6:

"The adoption of an agricultural diet would have increased LA and decreased ARA and EPA consumption, potentially causing a deficiency in LC-PUFAs".

This is something I have thought about, in more general terms, for some time.

At the time of the switch from hunting animals for their fat to growing grains for their starch the paper suggests that there was a population-wide potential deficiency of the longer chain PUFA, arachidonic acid, EPA and DHA.

This applied a selection pressure to the population. Within the population there was a random distribution of the ability to elongate and desaturate linoleic and alpha linolenic acids to their longer chain derivatives.

People who had this ability in generous amounts did well. Those without, didn't.

What happened to those people who were "without" the lucky gene snps to survive well without animal derived lipids? They didn't "develop" the genes, no individual suddenly develops a better gene. Their intrinsic inability means they didn't reproduce as successfully.

Their genes are currently under represented in the gene pool today.

It has always struck me that the process of getting poorly adapted genes out of the gene pool is what we describe as pathology, illness. Trying to patch it up is what we call medicine. Individuals don't adapt. They either do well or badly. The population "adapts" through the illnesses of those whose genes are not appropriate to the new environment.

The adaptation of our species to the novel situation of agriculture is far from complete. On-going adaptation of a species to a new environment is via the suffering of the individuals with genes more appropriate to the previous long term stable environment. The default for a person with on-going pathology might be to step back 10,000 years rather than continuing to assist evolution of the species via personal pathology. A lot of pathology will be needed.

Miki Ben-Dor has a nice post along these lines this on his blog.


Of course the adaptation to sucrose and bulk seed oils has only just begun. LOTS of pathology needed to adapt the species to those two! Juvenile onset type 2 diabetes is what we call the process.

Saturday, March 25, 2017

Amgen share price and PCSK9 inhibition with Repatha

Just a one-liner to bookmark the death of Repatha.

PCSK9 inhibitors have bombed and the cardiology community is in complete denial. Now this is nothing new, it happens on a regular basis. Repatha produced a massive drop in LDL cholesterol and a small drop in cardiac end points. It also produced a small (ns) rise in both total mortality and cardiovascular mortality.

I have altered the sentence which used to be here in response to a request from a PR company representing Amgen!!!!!! There's a post about it here.

No-one should ever listen to the cardiovascular community on a cholesterol lowering drug. Instead, just look at the share price of Amgen:

The red arrow indicates the release date of the FOURIER study data on March 17th 2017. It's a acute adverse event signalling the failure of a blockbuster drug. The trend in share price also indicates the conversion of an upward trend in price to a downward trend at the time of this adverse event.

As always, the cholesterol hypothesis is dead. It keeps on being killed but, obviously, it never lies down!

Amgen have invested an unimaginable amount of money in their PCSK9 inhibitor. This is a gross failure of basic research. A nerd with smart phone could have told them they were gambling on a very long shot.

They lost.


Friday, March 24, 2017

Will palmitic acid give you cancer or fuel metastasis?

Again thanks to Mike Eades for the full text of this paper and to Marco for poking me about it.

Targeting metastasis-initiating cells through the fatty acid receptor CD36

The executive summary: Both feeding a high fat diet to mice then implanting a certain type of cancer cells or feeding palmitic acid to that certain type of cancer cells pre-implantation makes the cancer much more aggressive once implanted. Up-regulating CD36 (described as a fatty acid transporter) has the same effect.

So. The question is: Should we all abandon high fat diets because fat, particularly palmitic acid, appears to be a promoter of aggressive metastasis?

I have thee things I'd just like to discuss.

I suppose the first is CD36. This is long accepted as a fatty acid transporter which facilitates the entry of FFAs in to those cells which express it on their surface. As far as I was aware this was all it did. My bad. The authors do mention that it promotes the uptake of other substances, including oxLDL, as an aside (they didn't look at this) and they do cite Hale's study using glioblastoms, which is rather more explicit about what CD36 really is:

Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression.

"We confirmed oxidized phospholipids, ligands of CD36, were present in GBM [glioblastomas] and found that the proliferation of CSCs [cancer stem cells], but not non-CSCs, increased with exposure to oxidized low-density lipoprotein".

CD36 is a scaveneger receptor which promotes the uptake of all sorts of lipids and oxidised phospholipids. Of course you can't help but think of 13-HODE and all of the other oxidised omega 6 PUFA derivatives which might or might not have been available to be taken up using extra CD36 receptors. This was not the point of the study, the study was aimed at nailing palmitic acid, to which I will return.

The second point relates to the mice fed the high fat diet.

The mice were fed TD.06414, essentially the same as D12492. Scroll to the bottom of the page to see the metabolic effects!

Lard and sucrose/maltodextrin, designed to produce obesity, hyperglycaemia, hyperinsulinaemia and hyperleptinaemia. No one measured the linoleic acid content of the diet so we can assume, very safely, that the approximate 16% of PUFA in the fat suggested by the manufacturer, is a gross under estimate. No one would expect a diet like this to be anything other than cancer promoting. Throwing in a few extra CD36s will make it worse. Is palmitate the problem in these "high fat" fed mice or is it 13-HODE, other PUFA oxidation products, insulin or leptin?

Point three is the one I'm currently interested in.

Pre incubation of the CD36+ cancer cells with 400micromolar unadulterated palmitic acid, for just 48 hours pre-implantation, promotes markedly increased metastasis when they are injected in to the mouse model. No PUFA, no 13-HODE, no hyperinsulinaemia. Just palmitic acid.

This is undoubtedly the money shot for the research group.

Now, what is going on here? From the focus of my blogging at the moment it's clear that palmitic acid is the highest driver of FADH2 input in to the ETC short of stearic acid. What will 48 hours of high level, uncontrolled FADH2 drive do to reverse electron transport (RET) and the structural integrity of complex I?

This is a model. A concentration of 400micromol palmitate, with no other FFAs, just never happens in real life. This model of extreme palmitate induced RET will force mitochondria to disassemble a pathological amount of their complex I. That's pretty obvious from the work of Guarás. The function of complex I is to reduce the NADH:NAD+ ratio and so disassembling complex I will do the inverse and raise NADH per unit NAD+. I went through the relevance of changes in this ratio, specifically for the generation of aggressive metastatic cancer phenotypes, in 2013 when I posted about Hoffer and B3 therapy for cancer prophylaxis and the modern versions using all of the clever stuff we do nowadays.

Of course you have to wonder about point two above; how much of the cancer promoting effect of obesity might be from the pathology of concurrently elevated fatty acids (reducing complex I availability so NAD+ generation) combined with elevated glucose (supplying the maximum amount of NADH) acting via the NADH:NAD+ ratio, never mind 13-HODE etc. A double whammy.

Personally I'm not about to give up eating butter on the basis of this paper. But that's just me I guess.


BTW, will blocking CD36 be an anti-cancer adjunct? Quite possibly, especially if it blocks 13-HODE entry in to the cell. Or even if it blocks FFA entry when people can't be ars*d to avoid hyperglycaemia while ever they have chronically elevated FFAs.

Thursday, March 23, 2017

Just a little on complex I and models

OK, just a brief summary of the parts I like most from the excellent

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

We should all have a picture of the ETC looking a bit like this, omitting mtG3Pdh and ignoring supercomplex formation:

Guarás et al set up a model, a proof of principle extreme. They blocked the ETC completely at either complex III, or at cytochrome C or at complex IV. They even discussed short term oxygen deprivation as a generator of ROS, equivalent of blocking the extreme end point of the ETC. If you completely block the ETC in this way then any input through FADH2 containing enzymes to CoQ will always generate a massively reduced CoQ pool, one prerequisite for reverse electron transport (RET),

leading to the near complete disassembly of complex I. It's a model, an extreme version of reality. Fascinating in its own right, as was the ability of the fungal CoQH2 oxidase (AOX) to protect complex I completely from any of the engineered ETC defects:

AOX may not pump any protons but it does preserve complex I by reducing the extreme levels of CoQH2 which drive RET.

They subsequently went on to look at more physiological ways to generate RET and came to the conclusion that, as the balance of inputs from NADH vs FADH2 shifted, then the amount of complex I relative to complex III would need to be altered. RET is the physiological signal to balance complexes I and III against NADH and FADH2 supply.

Elegant is not the word.

They even went on to ascertain which cysteine residues were preferentially oxidised to disassemble the complex. They group around the FMN and the CoQ docking area, surprise surprise... OK. If you insist, here is the ribbon diagram from fig 5:

There's a lot of explanation in the text of what the colours and the asterisks mean.

All I really wanted to lay down with this post is that there is a physiological process where FADH2 inputs control complex I abundance. That is how it should be.

When you want a pathological model of complex I destruction, pathological levels of FADH2 input will deliver.


Thursday, March 16, 2017

Protons: More from Dr Speijer

Mike Eades forwarded this paper to me, by Dr Speijer:

Being right on Q: shaping eukaryotic evolution

How good is it?

He covers a vast field including loads on

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.


Supercomplex assembly determines electron flux in the mitochondrial electron transport chain

and even

Mitochondrial fatty acid oxidation and oxidative stress: lack of reverse electron transfer-associated production of reactive oxygen species (another gift from Mike Eades).

I've far from finished reading the paper, these are just some of the gems. At some point I really will get round to a bit more on complex I and RET to regulate susbstrate processing but there is rather a lot happening at home and there might be something of a delay. To say the least.


Stearic acid, FADH2, complex I and cancer

Just a quick aside:

George cited this study in the comments of the last but one post:

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

While I think it is possible that metformin might inhibit mtG3Pdh at levels below those which inhibit complex I, the complex I effect may well still be equally real.

He goes on to say:

"Remodelling the ETC - I like that. The idea of the ETC as a modular assembly that will be reconfigured as the substrate balance shifts. Directly by the effects of the substrates on its outputs".

Remodelling the ETC using RET from FADH2 inputs might be antineoplastic too ie, less complex I would then be available for whatever ox phos the cancer is capable of…

Now: What normal food will generate the highest FADH2 input to the ETC per unit NADH? Correct, stearic acid will.

So what does stearic acid do to breast cancer cells in culture?

Stearate preferentially induces apoptosis in human breast cancer cells

Does it work in a rodent model?

Dietary stearate reduces human breast cancer metastasis burden in athymic nude mice

Do people with breast cancer have low stearate levels in their cell membranes?

Erythrocyte membrane fatty acid composition in cancer patients

Does stearate-driven RET down regulate complex I availability in cancer cells which are partially dependent of glucose derived NADH oxidation via said complex I? And so kill them?



That poor old C57Bl/6 mouse

There is a is a link within the Guarás et al CoQ paper I put up in the last post (which I'll probably go back to in some detail in future posts). It's to a paper by Lapuente-Brun et al:

Supercomplex Assembly Determines Electron Flux in the Mitochondrial Electron Transport Chain

Here is my doodle, butchered from elsewhere, of the  Supercomplex (SC), also known as the Respirasome:

The things to note are the assembly of complexes I, III and IV in to one unit and that there are enclosed molecules of both CoQ and Cytochrome C within the SC. These electron transporters are isolated from their respective general membrane pools so as to ensure maximal efficiency of electron transfer within the integrated SC.

The SC doesn't just happen. It's glued together by specific assembly proteins. In particular C III and C IV are joined by a protein called Cox7A2l (to be renamed supercomplex assembly factor I, SCAFI).

Unless you are a C57Bl/6 mouse.

If you are a C57/Bl/6 mouse your SCAFI  is 4 amino acids too short. It doesn't work.

The whole, excellent, paper by Lapuente-Brun et al is really about supercomplex formation and preferential assembly of the basic complexes. But because it uses the broken C57Bl/6 as an example of defective respiration it does bring home how irrelevant this particular mouse might be to more humans with a more normal metabolism.

Quite how this defect of SC assembly might make that the C57Bl/6 mouse in to the strange metabolic item which it is is not clear.

But, at the core of normal respiratory supercomplex formation, the Bl/6 mouse broken. That's an awful lot of mouse research which is broken. You could almost feel sorry for obesity researchers. But not quite.

Just sayin'.


Thursday, March 09, 2017

Protons: The destruction of complex I

It is quite difficult to express how exciting this paper is to a Protons thread True Believer:

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

The paper covers essential everything I've worked at over the past few years about the ratio of NADH to FADH2 as inputs to the electron transport chain. They even give Dr Speijer an honourable mention. I like these people.

Look at the Graphical Abstract, it's pure Protons:

And these are their highlights:

But, as hinted in the highlights, they take it to another level, beyond where I've gotten to in Protons:

The destruction of complex I by eating saturated fat. Or by generating a few ketones.

Fantastic stuff.


Saturday, March 04, 2017

Trans fats vs linoleic acid

TLDR: Trans fats may not be as bad as they are made out to be.

This paper is comparing a high linoleate diet (using lard) to a soya oil derived (Primex) diet where much of the linoleate has been industrially hydrogenated in to trans fats and fully saturated fats.

A comparison of effects of lard and hydrogenated vegetable shortening on the development of high-fat diet-induced obesity in rats

The thing I like best about it is that, wait for it, they measured the fatty acid composition for their diets! HPLC and all that. Then, they put the results in the paper! On the down side their concepts about energy balance are pure CICO and rats have lingual receptors for fat which link to "hedonistic" centres in the brain. So they understand nothing, but we can forgive them for that.

Here are their results:

I think this might suggest that linoleic acid is obesogenic. Not that I've ever mentioned that before. Now obesity is obesity. What about health? Here are the numbers which matter, obviously insulin is the one we want to look at:

So, clearly, eating 11% of your calories as linoleic acid makes you fat and ill. The fat in the HVF (and the NF control) diet is made of:

"Diet compositions are presented in Supplementary Table 1. Primex pure vegetable shortening, a mixture of partially hydrogenated soybean and palm oil, was used by Dyets Inc. (Bethlehem, PA, USA) to formulate NF and HVF experimental diets".

Compositions panned out as :

Now these are measured, nothing is accepted from USDA food tables etc. So..... If we look at health outcomes (Table 3) in conjunction with diet composition (Table 1) it become pretty evident that, in these rats, trans fatty acid (TFA) feeding at 15% of total calories is positively health generating compared to linoleic acid feeding at 11% of calories. I did not expect this.

Aside: If any diet trial does not have its fatty acid composition measured you have no idea how much linoleic acid it contains. Usually more than you think. Also, joke of the century so far:

Question: If you are in a position of power over innocent folks who are trying to eat healthy food, which fat would you ban?

Answer: The wrong one!

End aside.

I was on a PubMed search looking for trans fat toxicity. In this current study trans fats not only fail to cause insulin resistance, they render insulin-glucose parameters identical to the NF fed rats, despite the TFA fed rats carrying an extra 100g of adipose tissue.

That is very interesting. You can't answer the whys and wherefores from this paper. The missing piece of information is probably FFAs.

Trans fats are very odd. On acute exposure to TFAs isolated adipocytes release stored FFAs. This is what Cromer et al have to say in their study:

Replacing Cis Octadecenoic [Oleic] Acid with Trans Isomers in Media Containing Rat Adipocytes Stimulates Lipolysis and Inhibits Glucose utilization

"Overall, results of this study clearly show that conversion of octadecenoic acid from the cis isomer [oleic acid] to the trans isomer [elaidic acid] in adipocyte media will substantially increase lipolysis and inhibit glucose oxidation and conversion to cell lipid".

Just to clarify: Acute exposure of freshly isolated adipocytes to trans-oleic acid causes fat release, decreased glucose uptake and decreased glucose incorporation in to lipids. Sounds like a weight loss drug to me.

I was expecting this lipolysis to show up as elevated fasting FFAs and elevated fasting insulin. But there isn't any insulin resistance under fasting conditions visible in Table 3 so I think it is reasonable to assume there is no elevation of FFAs at this time either......

The only explanation I can come up with is that the adipocytes of the TFA fed rats are not as "full" as they should be, due to trans fatty acid induced lipolysis. Giving some "space" within an adipocyte allows the insulin sensitising effect of PUFA oxidation to show, certainly while fasting and lipolysis is the correct state to be in. Hence the fasting insulin levels are normal despite the 100g of extra bodyweight.

Or you could theorise that excess lipolysis from the trans fats is being almost exactly matched by decreased lipolysis from the insulin sensitising effects of linoleic acid. The combination just happens to pan out close to normal, provided the rats carry 100g of excess adipose tissue.

Some degree of post prandial hyperinsulinaemia/insulin signalling seems essential just to maintain those extra 100g of accumulated fat, but it's clearly not visible in the post absorptive phase.

If anyone can come up with a better explanation, I'm all ears.

BTW, this obviously relates to Axen and Axen's work. Their hyper-obesogenic diet was based on something Crisco-ish from the 1990's:

"The hydrogenated vegetable fat contained ∼25% long-chain saturated, ∼44% monounsaturated and ∼28% PUFA, with ∼17% of total fat as trans fatty acids (manufacturer’s communication)".

With fat making up 60% of the calories in the diet, and that fat being 28% PUFA, this is somewhere around 17% of total calories as linoleic acid. That is a LOT of linoleic acid. At the time I thought that the trans fatty acids would be to blame. Nowadays I'm not so sure.

How much of the bad rap that trans fats have received is from the PUFA which travelled with them at the time? Without mentioning the amount of fructose in the biscuits. Modern Primex is much more hydrogenated, so lower in PUFA, than whatever Axen and Axen used. It's far less obesogenic too.


Other odd final thought: People who are obese and insulin sensitive: Are they the folks who eat most trans fats along with their hearthealthypolyunsaturated linoleic acid???????

Friday, March 03, 2017

Mitochondria in cancer cells

TLDR: This is just a speculative post about a paper which is interesting from the Protons point of view but is not really related to anything else.

This is the paper by Catalina-Rodriguez et al:

The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis

It is interesting on many levels. At the most basic, it gives you the actual concentration of glucose used to grow the cancer cells. This is pretty unusual.

I picked the paper up while looking at what Lisanti has published recently on the fuelling of cancer growth using ketones derived from glucose via cancer associated fibroblasts. He appears to have been asked to write a commentary on the Catalina-Rodriguez paper. Here it is:

Genetic Induction of the Warburg Effect Inhibits Tumor Growth

The basic idea of the commentary, partly agreed with by Catalina-Rodriguez, is that genetically inducing the Warburg Effect in cancer cells kills them. This is obviously very compatible with Lisanti's ideas, that cancer cells generate ATP via ox phos, and is the diametric opposite of the Warburg Effect.

But the paper by Catalina-Rodriguez, while it does discuss the Warburg Effect, also provides enough information that other explanations are tenable beyond those supported by Lisanti. Anyone looking at Lisanti's work without considering other explanations needs to be more circumspect. The paper is all about the mitochondrial citrate transporter, CIC.

We had better put CIC in to context first. It exports citrate from the mitochondria to liberate mitochondrial derived acetyl-CoA to the cytoplasm for anabolic processes.

It's an antiporter, it exchanges citric acid for malate. Citrate out, acetyl-CoA released, residual oxaloacetate reduced to malate via NADH, malate re enters the TCA.

We can add this to the TCA like this:

So CIC exports citrate and imports malate. Because citrate carries 3 negative charges and malate only two, a proton is carried out with the citrate which maintains electrical neutrality but generates a pH gradient. It's also clear that a cytoplasmic NADH is consumed converting oxaloacetate to malate and this is regenerated within the mitochondria by the TCA as malate reconverts back to oxaloacetate.

So we can look at this as a cycle:

It bypasses all of the energetic processes in the rest of the TCA so delivers very little to the ETC. The normal function of this pathway appears to be a safety valve for the cell when the mitochondria are presented with far too much acetyl-CoA.

In some cancer cells things go very awry.

Two hallmarks of cancer cells appear to be abnormal electron transport function and free radical leakage from the ETC. The free radical leakage is at levels which cause proliferation rather than apoptosis.

Complex I primarily reduces NADH to NAD+. If we have a dysfunctional complex I (either directly or by poor function downstream in the ETC) there is going to be a great deal of NADH per unit NAD+ within the matrix. High levels of NADH inhibit isocitrate dehydrogenase and so drive citrate export in to the aberrant CIC cycle. The cost is limited to one imported cytoplasmic NADH, the benefits are reduced generation of TCA derived NADH and FADH2... Plus cytosolic citrate is an inhibitor of glycolysis, so works as a brake on the excessive supply of pyruvate derived acetyl-CoA.

So some degree of poor ETC function can be accommodated by exporting acetyl-CoA rather than turning the TCA. The red cycle in the above doodle predominates and the TCA is almost inactive.

Under these conditions the mitochondria are largely protected from their dysfunctional electron transport chains. There are ROS (mostly superoxide) being generated but at levels which are not fatal to the cell although they can drive mutagenesis, in both mitochondrial and nuclear genomes.

What happens if you inhibit CIC in a cancer cell which is using CIC to both limit ROS production and to deliver cytoplasmic acetyl-CoA for anabolism? The whole extra-mitochondrial cycle should shut down, meaning acetyl-CoA has nowhere to go other than around the TCA. Less citrate can be exported, so cytosolic citrate levels fall. Falling cytoplasmic citrate allows increased glycolysis and this can feed even more acetyl-CoA in to the TCA. Complex I is still dysfunctional so we are then in to a massively elevated NADH:NAD+ ratio. We will also increase FADH2 driven CoQ reduction via complex II as the TCA turns. A high NADH:NAD+ is the prerequisite to getting an electron on to an oxygen atom to generate superoxide in complex I, back in Protons (22) I speculated this might be at FeS N-1a. A reduced CoQ couple also drives this via reverse electron transport.

Putting an electron on to oxygen aborts all of its downstream proton pumping. What might happen if a very high percentage of electrons jumped ship at complex I due to a grossly elevated NADH:NAD ratio and a reduced CoQ couple?

Increased superoxide, more oxygen consumption (though not necessarily at complex IV), much more ROS. Might the ROS release cytochrome C by oxidation of its cardiolipins, so giving collapse of the mitochondrial membrane potential and triggering apoptosis?

This is not the "genetic induction of the Warburg Effect", however Lisanti describes it. This is over driving a dysfunctional electron transport chain to the point of mitochondrial destruction. The most telling results section graph is this one:

These are the death rates for cancer cell cultures. On the left are three control cultures with functional CIC, on the right are those treated with BTA, a CIC inhibitor. NAC is n-acetyl cysteine. Which is the most lethal treatment? I think we must all agree with the devastating effect of pyruvate alone as being the most lethal treatment. Which gets zero mention in the text anywhere that I can find...

My idea would be that pyruvate is toxic because it bypasses glycolysis so cannot be shut down by the glycolytic inhibitory effect of raised cytoplasmic citrate. There is a spike of acetyl-CoA which cannot be avoided and which CIC levels are not set up to deal with. More acetyl-CoA enters the TCA, more NADH, more superoxide from complex I... The authors focus instead on the fifth column where pyruvate rescues cells from BTA toxicity. I can see no logic to this and have no explanation for that particular result. At least I do actually mention the results which don't fit my hypothesis!

The next very interesting result is the effect of uncoupling. CCCP is an uncoupler which reduces the MMP. This leads to mitophagy +/- apoptosis. A functional CIC cycle saves cells from CCCP damage. You could argue that CIC is just a Saviour of Distressed Cells. But on a more interesting level: Uncoupling normally markedly increases electron flow down the ETC. In normal mitochondria this reduces ROS generation. But if the ETC is leaking electrons to generate ROS under even normal flow it's easy to see why uncoupling can allow massive ROS generation. CIC is protective because it keeps electrons out of the ETC.

They also found that CCCP induces CIC production. My assumption would be that CIC is induced by ROS and CCCP generates ROS. The same thing happens with rotenone by the opposite process. Rotenone does not uncouple, it blocks the ETC at the exit of complex I. This gives reduced conversion of NADH to NAD+, markedly increase the NADH:NAD+ ratio, electrons jump to O2 to N-1a to form superoxide... CIC induction is the solution to limit this.

There are some very interesting results here.

I have absolutely nothing against looking at CIC inhibition as a management for certain subgroups of cancers. It may well help, but thinking about what is actually happening might give more insight.