Saturday, July 02, 2022

Brand on 4-HNE facilitated uncoupling

This is one of those papers with strong confirmation bias reinforcing properties, so care is needed. I do indeed have some difficulties with the paper, some of which I'll point out as we go along.

Brand again.

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

So. They wanted to look at how mitochondrial membrane potential controls uncoupling. They also wanted to differentiate between "endogenous" uncoupling and 4-HNE facilitated uncoupling. This is not easy. Certain steps need to be taken. Each step moves you away from reality and closer to answering the question you have in mind. Which may or may not reflect reality.

They used adenine nucleotide translocase (ANT), the enzyme which exchanges ADP for ATP across the inner mitochondrial membrane because a) it uncouples and is closely related to standard UCPs and b) there is a ton of the stuff on all mitochondrial membranes which makes quantitative measurements possible. They also have an inhibitor which allows more information to be extracted.

Here's the setup:

They needed to eliminate FFA induced uncoupling (and energy supply) so added BSA to the medium to soak up endogenous FFAs.

They needed to actively control energisation of the mitochondrial membrane so blocked complex I completely with high dose rotenone to stop oxidation of NADH from endogenous malate or glutamate catabolism.

When they wanted to activate energy supply they used succinate alone working through complex II, which is still functional.

Obviously, to look at uncoupling, they had to block ATP synthase with oligomycin.

Next they equilibrated the proton concentration across the inner membrane using the H+/K+ exchanger nigericin to allow a them to use a voltage only sensitive probe to measure membrane potential.

The degree of mitochondrial activation from succinate was controlled by either titrating with malonate (complex II inhibitor) or cyanide (complex IV inhibitor) to give a stable measurable membrane voltage.

Finally, apparently 4-HNE is active at around 1μM but you need higher concentrations to get an easily measurable effect, especially in the presence of de-fatted BSA, so here they used 35μM.

Having done many (but not all) of the above things they end up with this graph in Figure 1:

which makes everything clear.

This is my biggest problem with the paper. From graph A they extract the information in bar chart B and bar chart C which give details of the calculated proton leaks at 2.5 minutes and 5.0 minutes in to activation with succinate. Which demonstrate quite clearly that HNE facilitated uncoupling via ANT has kicked in by 2.5 minutes and is marked by 5 minutes. This is chart B, extracted from the grey lines in graph A at around 137mV, both time points:

And chart C is from the dark lines in the lower right hand end of graph A at 175mV, only at time 2.5 minutes: 

My problem is that, after many hours trying to curve fit and calculate in Powerpoint, I can never quite extract the numbers from graph A to fit charts B and C.

If we assume that the research group have the raw data are getting their calculations correct we can have this as the main summing up:

"In liver, time at high membrane potential in the presence of HNE results in a striking increase in proton conductance, which may be interpreted in two ways. First, HNE may slowly form covalent adducts with ANT, progressively converting ANT into a form with high proton conductance, or secondly, HNE metabolism may progressively form fatty acids [41], which then activate proton conductance [28]."

The authors consider option two to be unlikely.

If this paper really does hold water we have a situation where high membrane voltage, working through 4-HNE, produces long acting uncoupling in proportion to the time spent at high membrane voltage.

Hence you can run a mitochondrial preparation with supra maximal substrate supply and, rather than exploding/imploding, the correct physiological "decision" is to uncouple in proportion to the excess membrane potential and stabilise it at something close to functionality.

High delta psi -> ROS -> 4-HNE -> ANT/UCP -> damage limitation.

That's how things should work.

Of course that's not always how things pan out.



lapis_exilis said...

“Which explains everything.”HAHAHAHAHAHAHA bonk. 🙃

(Also, the fact that cyanide is a complex 4 inhibitor would probably work great as a party trivia thing! )

lapis_exilis said...

Awwwww it didn’t link my “Now Zoidberg is the popular one” image.
Well. You’ll just have to imagine it! 🙃

cavenewt said...

I'm sorry, lapis_exilis, but Leela is the popular one.

lapis_exilis said...

(In Zoidberg voice) - Awwwww….
Hahaha! Of course Leela is the popular one! I mean…. How cool can she get? 🥰🥰🥰

karl said...

If they are not sharing the raw data, is it still science? (Is this another case of "holding back data in-house" to keep others from making progress? Some authors will respond to a request for data.. )

In the mean time, I've been reading more historical bits about cancer. I already was aware that CAD was considered rare until 1920s-1930s. What I just learned was there was considerable concern appearing well before 1900 about the increases in cancer rates. This is well before concentrated seed oil made it's way into the common diet.

Primitive cultures had extremely few cancer cases - western MD's were puzzled when they examined the native populations. The issue of increasing cancer rates had become a hot issue between the world-wars.

I think T2D is related to cancer, but it seems that there has to be more that changed with the modern diet. Could it be that exposure to metals which so often catalyze biological reactions?

So what was changing in the environment that put cancer on stage? What has changed more recently to cause the T2D pandemic?

That a number of people recognized (way back in time), that most cancers rely on glucose to out grow the immune system - and yet, that narrative died - only to be revived again in the last decades - alarms me that science can really go backwards.

Today, they are ignoring the effect of lab strains on telemer length - tainting a huge amount of cancer research. What happens to cancer rates in wild strains of mice that get exposed to organic-lead? (Most all lab strains die of cancer)..

Metals were used to catalyze LA into plastic -


Blissfully unaware of the heath risks of LA in the diet:

Passthecream said...

Karl, there's this little thing known as the industrial revolution which should more likely be called the pollution revolution! All sorts of noxious chemicals polluted the air in the cities where industrial scale processes were taking place.

For one example it is a cruel sort of twist that in comparison to the gleaming beauty of grandma's porcelain tea service, the life of workers and their families in the towns such as Staffordshire with dozens or hundreds of coal fired bottle kilns was short and ugly and smothered by thick louds of noxious smoke. It was still pretty fugly there up to the 50s. I had a friend who migrated here from there who passed away with emphysema at about 55 y.o. due to growing up in a town like that.

The first recorded type of occupational cancer was coal related:


Not to mention the famous London Smog

Passthecream said...

( ...towns such as those in Staffordshire... , ... clouds ... ) plus other typos at no extra charge.

Passthecream said...

Peter, speculating --- imagine a group of hunter gatherers who have wandered into a very cold region where there is a fabulous resource of sea creatures to hunt, most with high levels of W3 pufa, possibly other pufa capable of staying liquid at low temperatures. It seems as though uncoupling driven by these fats and their by-products could provide useful warmth??

karl said...


Yes - but it just isn't that simple. Read what Ames wrote about this - nature has been waging chemical warfare between plant and animal kingdoms for a long time - many of the 'natural' bits we eat cause mutations.

Besides coal-tar and tobacco, the answers to what causes cancer are totally confounded. There is good evidence that hyper-nutrition is more important than industrial exposure. ( These effects are so large that it was easy to show). There are so many other 'correlations' that are claimed to be causation - but other correlation that show the opposite - more junk science than real science. Scientism - cargo-cult crap.

The fact of the matter - that primitive cultures have/had extremely low cancer rates is still not understood. Was it lack of high carbohydrate diets? Was it exposure to things missing in the modern world? Is it related to photo exposure? Just the pollution? Which pollution matters most? There is nothing here that should give one the warm fuzzy feeling that we know what is going on.

Currently, I'm reading Ravenous: Otto Warburg, the Nazis, and the Search for the Cancer-Diet Connection which I think does give a good background on the enigma - I don't think we 'know' nearly as much as people assume we do. Lots of bits about how the ETC was teased out - how personalities interfered with the progress of science.

There are three things that increased - cancer, CAD, and T2D (not at the same time) - it is possible that there is a connection - some common cause - or not.

Cancer rates were seen to be increasing first - sometime in the 1900 - 1920s - there was an increase in CAD - now starting in the 1960's T2D pandemic sprang forth.

I have my hunches - what I think is likely true - but to say we know? Not even close.

One other bit to consider: we know that lab mice ended up getting bred to have short telomeres which results in they most likely die from tumors - (This puts a huge amount of cancer research in a category of needing to be repeated.. don't hold your breath). Is it possible that there is evolutionary pressure on human telomere length?

Many more questions than answers -- there are all sorts of narratives - more ego trips than dispassionate science.

Simple health diets go back a long ways - vegetarian 'natural' diets have been big, but the data is not kind. Crop selection changed the foods we eat - it is not possible to eat the diet that my great-grandmother had. Some of the pollution looks to be important - but it hardly explains all the changes we have seen.

Eric said...

Another gratuitous threadjack from me here. This may merit its own thread:

or as a free 14 day link:

So, apparently, a study of more than 25,000 participants has shown that taking 2,000 IU of Vitamin D has no effect on cancer rates, cardiovascular disease or bone fracture, and an Australian study has shown it has no effect on longetivity. The NYT article does no explictly mention susceptibility to infection.

From the article, it does not seem like the typical supplement study that was designed to show that supplements don't work. In fact, the lead author said she expected to find a benefit.

So what do we make of it? Was 2,000 units too low? What of Dr. Holick's charts of cancer rates vs. latitude? Could it be that Vit D pills are just another case of reductionim, and it is something in sunlight that cannot be put in bottle? Should Dr. Holick's charts be redrawn with current data now that so many people at low latitudes are hiding from the sun or lathering up with SPF30+? Or is it time to resign and throw out the pills or drops and go hide?

Eric said...

And another off topic but fascinating arctile:

Seems that the mutation to keep making the enzyme to break down lactose was first found 6,600 years ago but remained rare until 4,000 years ago. People have been keeping cattle for at least 10,000 years, though milk production went through ups and downs both regionally and in time, as proven by milk fat residue in clay pot shards. So people were consuming milk even if it caused gas and cramps. Only when famine or epidemics hit did it matter that you had the mutation to survive.

Interestingly, modern Enlish people who do not have the mutation do not seem to show any ill effect. The article does not say explicitly whether they consume milk, though.

Eric said...

And for good measure, I found this also pretty interesting: