Wednesday, September 09, 2020

Protons (60) 4-hydroxy-2-nonenal

I've been re-reading Dave Speijer's

Being right on Q: shaping eukaryotic evolution

I cannot over emphasise how both broad and detailed this work is. This current post came from following a single link in the section on uncoupling.

Back in 2000 people were bulk manufacturing human uncoupling proteins using E. coli and assembling them within the membranes of synthetic lipid vesicles. They had problems getting the UCP1 to function correctly but eventually, by dint of an enormous amount of hard work, they found this requirement (the title says it all):

Coenzyme Q is an obligatory cofactor for uncoupling protein function

The UCP1 derived from E.coli could be activated by the addition of coenzyme Q, more specifically in its oxidised form CoQ. This is slightly counterintuitive as you might expect CoQH2 to be more of a signal that "excess" electrons were present in the ETC and that uncoupling to reduce the mitochondrial proton gradient might be a good idea.


The next snippet was provided by Brand's group

Superoxide activates mitochondrial uncoupling proteins

who used the oxidation of xanthine by xanthine oxidase to generate superoxide in-situ, to demonstrate that superoxide was, or could generate, the necessary co factor to allow UCP3 (in this case) to function. This is much more understandable because excess input to the ETC in the absence of a need for ATP is the classical situation for ROS generation and so ROS are more plausible as a signal to institute uncoupling compared to the oxidised version of CoQ.

Then comes this paper, again from Brand et al (which is the one I picked up from Dr Speijer's work):

Synergy of fatty acid and reactive alkenal activation of proton conductance through uncoupling protein 1 in mitochondria

The evil molecule 4-hydroxy-2-nonenal (4-HNE) is synergistic with fatty acids in activating UCP1. Physiological uncoupling is generally thought of as a Good Thing. 4-HNE as a Bad Thing. Perhaps we should be careful about making value judgements about molecules.

From an evolutionary perspective there is no obvious reason (to me) why UCPs might not be activated directly by superoxide itself but in this case the preferred solution appears to have been to allow superoxide to modify linoleic acid within/around the mitochondrial inner membrane into 4-HNE, which can then act as a cofactor to UCP1 to synergise, in this experiment, with free palmitic acid to dissipate the membrane potential and so to limit excess ROS production.

So UCPs in general appear to respond to an inappropriately high level of ROS generation by activating the safety valve of uncoupling the mitochondrial membrane potential. Linoleic acid derived 4-HNE is key to this process.

I have argued that the normal mechanism for limiting calorie ingress into a replete cell is for ROS to disable insulin signalling. And that PUFA fail to generate the appropriate ROS needed because they fail to deliver an appropriate supply of FADH2 to ETFdh and subsequent reduction of the CoQ couple. So PUFA allow an excessive, poorly controlled calorie supply. Eventually enough energy will be supplied that an excess of ATP combined with a paucity of ADP limits the activity of complex V, so membrane voltage will finally rise, the flow of electrons will back up and lots of ROS will finally be generated. At this stage there is still too much input, too little demand and a problem looking for a solution.

Uncoupling is one solution. Electrons can be allowed to continue to pass down the ETC and to pump protons but these protons are allowed back through the UCP, generating heat rather than ATP and reducing the membrane potential. Which will limit the ROS generation which might otherwise become too high.

Now, if you accept that PUFA are the cause of the situation and that uncoupling is the solution, which fatty acids would you expect to be the best activators of UCPs when uncoupling proves to be needed?

Correct. PUFA are the most effective protonophores when used by UCPs to reduce the inner mitochondrial membrane potential. As in:

Polyunsaturated fatty acids activate human uncoupling proteins 1 and 2 in planar lipid bilayers

Aside: It's worth reading the methods section of this paper. It gives insight in to a) how phenomenally difficult it is to set up models to look at individual protein functions in isolation and b) how far from physiological such models are. Difficult, extreme, necessary. But interpret with caution. And think about any requirement for 4-HNE. End aside.

Let's go up a level from the ETC to the cell plasma membrane and insulin signalling. If you are a cell and you are swamped with incoming calories but can only signal using ROS by the time that ongoing incoming calories are continuously too high, what other strategies might you apply?

How about augmenting the PUFA-inadequate insulin resistance by using 4-HNE to generate a few of the necessary extra ROS? As in:

The lipid peroxidation by-product 4-hydroxy-2-nonenal (4-HNE) induces insulin resistance in skeletal muscle through both carbonyl and oxidative stress

Additional cellular insulin resistance, supplied by 4-HNE, is a logical solution to a situation where insulin resistance is needed but is not happening appropriately. The role of 4-HNE can be viewed as being protective by uncoupling at the level of the mitochondrial membrane and also protective by augmenting insulin resistance at the cell surface membrane.

And to re-iterate again: Insulin resistance in adipocytes is synonymous with decreased fat storage and/or increased lipolysis.

I think it is very reasonable to assume that our physiology knows all about PUFA and how to deal with them. The end result may not always be what we want, but it will be adaptive. I think the context in which we are exposed to them is very important, especially the level of insulin, the rate of beta oxidation (which beaks down 4-HNE and related molecules) and the total quantity of linoleic acid in the diet. Getting 1% from mammoth fat is perfectly oaky. Getting much more on a ketogenic diet can be dealt with. Margarine on your baked potato might be a no-no.

I also think that bulk ingesting aged corn oil from a deep fat fryer might not provide a particularly physiological supply of 4-HNE.

But clearly, given the correct experimental set up, we can arrange that diets based around safflower oil can be less obesogenic than those based around lard, despite the very much higher linoleic acid content of the safflower oil. It provides a tool to understand papers like this one:

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice

(HT to Amber O'Hearn for resurfacing the paper which has been on my "think about it" list for a long time)

which uses these diets

How the authors describe the diets is unimportant, all that matters is the PUFA content. Here are the weight graphs:

The minimum weight gains are the LF_PO at 1% PUFA (low total fat), over lain by the HF_CB (high total fat) but just over 1% of calories as PUFA. HF_PO is worst due to 4.5% of calories as PUFA. HF_OO diet is almost as bad with the same PUFA percentage.

Yet another aside: I would never argue that there is no influence of the lipid species available to be incorporated in to adipocyte triglycerides. All-palmitate would turn adipose to candle wax, all-linoleic acid into a liquid. So there are decisions made re storage vs oxidation taken at several layers above the ETC with are not unimportant but are not my forte. End aside.

But the safflower oil based diet, despite over 35% of calories as PUFA, is almost as weight gain limiting as the two low PUFA diets.

If you wanted to explain findings like this you would need to look at the level of heat generation, the level of 4-HNE production, the rate of oxygen consumption and possibly the level of insulin signalling in the post prandial period. But there are mechanisms to support a possible explanation.

Is linoleic acid a potential adjunct to weight loss? Mostly "no" is the short answer. But it appears to depend on how carefully you set up your study and what result you would like to get. Possibly how long you run the study for. Not that there any biases involved. It might also rather depend on how close you want to get to eating F3666 high PUFA ketogenic rodent food. And how many double bonds you might be willing to accept into your inner mitochondrial membrane lipids.

Personally, no thanks.



In the first paper CoQ probably works by generating the 4-HNE needed by UCP1 while CoQH2 doesn't. I'd speculate that because CoQ is an electron acceptor, which normally accepts electrons from the terminal FeS cluster of complex I, it might be looking to accept electrons from other sources in a lipid bilayer preparation. In the synthetic lipid by bilayer there are molecules of linoleic acid. Under conditions of available oxygen I see no reason why CoQ might not accept/steal a pair of electrons from a double bond in linoleic acid which would leave behind a reactive lipid radical which is a good candidate for combining with oxygen and eventually forming the 4-HNE needed by UCP1 to work efficiently. Just a guess.


GarlicPudding said...

Off topic comment but I wanted to thank you for your blog! I always make sure to read every post. Although I don't know what you are talking about at least 85% of the time, and have some trouble connecting the dots at times on how it relates in practical terms (which doesn't seem to be the purpose of the blog directly), I do understand that PUFA equals no bueno.

Passthecream said...

Just tying together a couple of speculative ideas - interesting that the Brand work used xanthine oxidase ( XOR is actually an oxioreductase, a two faced enzyme). It is a widely distributed enzyme that seems to easily pump out free radicals whenever it bumps into any one of a number of substrates. Cuppa coffee anyone? So I wonder if caffeine generates a nice clean ( dirty!!!) ROS signal? Anyone who suffers gout spends a little time contemplating this enzyme's effects.

Relevant to the capacity to handle dietary LA, I was reading about desaturases and their co-workers, elongases. Vertebrates lack one which can produce LA from stearate/palmitate. Those cold waxed up outermost cell types you mention would usually be cutting their fully saturated triglycerides with oleic acid for which we do have a desaturase, hence neatsfoot oil. When you see yaks standing out in the snow it is mufa which keeps their legs from turning into candles. Bovine antifreeze. But not LA. ( Which led me to wondering how eg chickens might develop significant levels of LA in their flesh, must be in the corn.)

Wrt a hypothetical carbaholic diet, only just enough lipid in it to stop you gagging, and with the lack of the desaturase to produce linoleic acid, I wonder what your stored fat profile would become?

Passthecream said...

Peter: "Let's go up a level from the ETC to the cell plasma membrane and insulin signalling. " --- had me searching for those papers from the NZ group about cell surface oxygen consumption. Another rabbit hole.

Passthecream said...

You might assume that mammoth legs would have been a rich source of mufa given their habitat.

(Those which were outstanding in their field did not have the full leg wax.)

Tucker Goodrich said...

Nice post. I have nothing to add to my comment on your previous discussion of Speijer's "Q" paper (replying to @Kenneth Strain):

Where I noted Speijer's observation:

"Here, palmitate (saturated C-16) and an oxidized (!) FA (4-hydroxy-2-nonenal) enhanced UCP proton transport."

I commented:

"4-hydroxy-2-nonenal is 4-HNE, and so the breakdown of TLCL is a fundamental part of mitochrondial function and regulation.

"The problem occurs, based on my reading, when TLCL overwhelms the regulatory/reactive systems. One sees this is occurring when glutathione (GSH) is diminished, and HNE is escaping the mitochondria unbound to GSH to wreak havoc on surrounding structures, like DNA. The presence of HNE bound to various other things is a marker for every part of the MetS, broadly defined, which includes cardiovascular disease and Alzheimer's. It's everywhere, along with the other N-6 peroxides."

I don't have anything to add to that, as I think it's spot on, even after three years of learning.

It's why my post (in response to Peter & Dr. Eades's discussions) explaining the fundamental problem in these processes contains the word "EXCESS":

"The Cause of Metabolic Syndrome: Excess Omega-6 Fats (Linoleic Acid) in Your Mitochondria"

Peter observes above:

"So UCPs in general appear to respond to an inappropriately high level of ROS generation by activating the safety valve of uncoupling the mitochondrial membrane potential. Linoleic acid derived 4-HNE is key to this process."

And yes, you can certainly figure out ways to tweak the inputs to alter the outputs. But these are generally non-physiological tweaks, and are not without their own hazards. So you can use "F3666 high PUFA ketogenic rodent food", but you will still kill the liver, as I noted in this post:

"Hello, Can We Have Your Liver?"": Understanding a High-PUFA Diet.

Where a high PUFA diet produces a better outcome, in some respects: "tl;dr: A diet high in omega-6 and omega-3 polyunsaturated fatty acids has some positive effects on the body: lower weight gain, better preservation of lean mass..."

But, as with Maratos-Flier's keto mouse experiments, it still kills the liver via steatosis, even when weight gain is not part of the outcome.

As Peter also notes, excess n-6 is not something you want. HNE does loads of other fun things, too, as it damages ~24% of the proteins in the cell. Just check out what it does to ATP!

"Omega-6 HNE breaks ATP synthase, hence mitochondria, early in Alzheimer’s."

Peter said...

Hi Tucker, agree. Overall I have a fascination with papers which disprove the Protons hypothesis that the number of double bonds being oxidised in mitochondria are drivers of energy storage. Papers like the one discussed are deeply interesting from their ability to prove me wrong... As you comment, no one in their right mind wants to saturate their most crucial lipid membranes/structures with LA. Beyond what is needed for functionality.


That’s interesting. Caffeine, giving ROS and increased lipolysis is an archetypal weight loss drug. I wonder why Atkins induction banned methyl xanthines? The headache on top of keto adaptation was no fun at the time. I was a serious coffee drinker in those days.

As insulin controls desaturases I’d guess mono unsaturated would be prevalent…

Did you pick up that mammoths are argued to be semi hibernating (from the paper which cites the single elephant!), ie through the worst of the winter they stood still and slept standing up? Yep, lots of outstanding mammoths, maybe that’s why ALA might have been high too (if it ever was)? Of course this might vary with time of year. Do other species vary their leg waxing for winter conditions?


Unknown said...

I have heard that caribou accumulate high concentrations of PUFA in leg joint tissue, presumably to prevent joint stiffness from reduced temperature.

Abraham said...


Abraham said...

Passthecream said...

According to this study, oleic acid is the major fatty acid in human adipose tissue at 50%, in bottom fat anyway. Stearic is disappointingly low approx 6% with palmitic near 24% and linoleic approx 13%.

But then you'd need to consider what these people were eating (?) and also to notice that these are autopsied samples so could in fact represent the epitome of bad lipid profiles.

Those vertebrates which do have higher LA content such as chickens probably get that from eating invertebrates such as snails, slugs and bugs, and various plants -- many small vertebrates consume a high quantity of insects and/or plant matter.

Passthecream said...

I feel compelled to speculate whether mammoths had not only the typical elongases but also a full array of trunkases?

Peter said...

Hmmm, 1979. There was already a differential between older people (less LA and younger people (more LA). This is on the cusp of the massive population injury inflicted by the cardiologists. Another 30 years of avoiding saturated fat and mainlining sugar might/will have made matters even worse!

It would be interesting to unpack the trunkases...


Passthecream said...

It could be a fascinating tusk.

Mammoths hibernating while standing up would have been an easy target for restless hunter gatherers.

Peter said...

Hadn't thought of that!