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.

Anyhoo.

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.

Peter

BTW

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.

Ultra processed food (2) Haub vs Hall

I’ve taken this post down. With the information that Haub specifically restricted calories none of it makes any sense, however you discuss around the edges. Apologies to anyone who as been embarrassed, if it’s any consolation it has been much worse for myself!

Peter

Protons (59) The NDI1 guys and gals are good

This little chap:

featured in this paper

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

which I discussed here. Obviously a group which can get the above image in to a Cell Metabolism paper has an admirably relaxed outlook on their own work and probably on science in general. You have to be good to have that mindset.

So now they have given us this review:

Role of Mitochondrial Reverse Electron Transport in ROS Signaling: Potential Roles in Health and Disease

which really summarises, at the most basic level, the nuts and bolts of what is happening to drive RET in the ETC under assorted inputs. The Protons starting point.




Edit: I've just fixed a broken link in Protons (03) Superoxide. Back in 2008 I was just starting to tease out the differences between glucose oxidation and lipid oxidation and the initial paper which started me on superoxide was this one from Muller et al

High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates

Once you twig that palmitate always drives complexes I and II but linoleate doesn't drive complex II so much... The NDI1 people make this soooooo much easier that it was back then. End edit.

They even found DHODH as another input (dihydroorotate dehydrogenase, I had to look it up too). Section A shows high ATP demand, low delta psi, minimal RET. Section C shows what happens when supply of nutrients exceeds ATP demand, delta psi rises and RET increases.

They've also got the TCA and beta oxidation working in parallel, as they do:

and have included the NADH:FADH2 ratios (admittedly upside down but I'm not complaining).

I hope their next move is in to subtleties of chain length and saturation to start to see how fatty acids have different ROS generating potential.

Then to relate ROS to insulin secretion/signalling. Insulin to obesity. Physiological vs pathological insulin resistance. Maybe metformin too.

But it's a great start. These people will go far.

Peter

So you want some DHA?

It seems like a very long time ago (only last year!) that George Henderson posted links in comments to the blog* about the absolutely crucial work done by Gibson and colleagues, documented in this paper

*Ooooh look, I just noticed how to link to comments. I'm so tech savvy!

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

Another aside: Paywalled. If anyone has a few pence looking for a home Alexandra Elbakyan might be a good destination. I didn't say that. End aside.

It is impossible to say how good this work is. It's very good.

I'm no hyper-enthusiast for DHA. It's a tool. It does a job. Saturating yourself with the stuff is very likely to be a Bad Thing. This is perhaps best exemplified by the fierce negative feedback exerted by all of dietary C18 VLCPUFA precursors (omega 6 and omega 3s) on its synthesis (I would assume the same happens for arachidonic acid as well). The conversion of alpha linolenic acid to DHA is, for rats at least (and I would go with for humans too), very, very easily achieved by simply getting close to eliminating linoleic acid from the diet and also keeping ALA low, under 3% of calories. Here's my favourite figure from the paper, already tweeted and blogged by George:

These are the DHA levels in phospholipids, presumably LDL and HDL secreted by the liver, extracted from plasma after three weeks of dietary intervention in Hooded Wistar rats.

"We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA [ie all combined PUFA*] is low (less than 3% of energy)."

*My insert for emphasis.

Does anyone begin to recognise a pattern to PUFA requirements here?

Peter

Random aside. Rats. Have they been scavengers of the small amounts of edible tissue left on mammoth carcasses after humans had finished with them? Are rats evolved to be opportunist high fat, low PUFA adapted facultative carnivores? Now that's an interesting and useless thought but might help explain why they behave exactly as humans do on Surwit diets compared to low PUFA Surwit-like derivatives. Well, the idea entertains me. But then I like rodent studies...

Tweaks are ubiquitous

As so often happens you occasionally stumble over a gem by accident, like this one.

A neutral lipid-enriched diet improves myelination and alleviates peripheral nerve pathology in neuropathic mice

Ignore the TrJ mice, just look at the control mice.

They were being fed crapinabag chow or high sucrose (gasp), high starch (gasp), high anhydrous butter fat (mega gasp) based diets.

If I told you that the anhydrous butter fat diet was not supplemented with soya bean oil (OMG, these poor mice will develop life threatening PUFA deficiency in the six weeks of the study! Assume a sarcasm apology as being provided) would that make your ears prick up?

So the PUFA content of the Ultra Processed (sarcasm apology repeated) sugar/butter diet turned out to be 3.0% of the lipid calories, which makes PUFA under 1.5% of total calories...

Okay. You don't even need to read the results, you already know what the body weights are going to look like. In case you can't be bothered and would just like some confirmation bias, here they are:

So. When researchers add "x" percent of soya bean oil to anhydrous butter fat diets to "prevent PUFA deficiency" you know that they are doing this, absolutely, because without the PUFA their butter based diets will not produce obesity. They know this, or at least the DIO manufacturers know this.

This insight is very, very important.

Over the years it has become clear to me that there are certain things which "everybody does" which are essential for getting the "desired" result. When Surwit specifies adding a PUFA based oil to an hydrogenated coconut oil based diet it is because he knows that it will NOT be obesogenic without it. He won't know why, but he will know that it is essential.

I think this is a general principle. I especially think it will apply to the intra cerebral injection of insulin behaving as a satiety hormone. There will be something which is routinely done which produces this effect and it won't be the insulin, it will be a "tweak", a normal lab procedure done for some plausibly justifiable reason. That's why it cannot be replicated in the hard nosed commercial lab of company which manufactures the insulin in question and which is looking to market the effect of the insulin, not of some dubious tweak (of which they are unaware).

I've no idea what the tweak might be.

But it will be there.

Peter

The miracle of fish oil (4) Adipocere

I learned a new word yesterday.

Adipocere.

Sadly, I find this word very fascinating. Here's why.

This is the first step of beta oxidation of a saturated fatty acid:

FADH2 derived electrons provide a large amount of energy as pumped protons on their route down the electron transport chain to oxygen. That's a standard part of life for any modern mitochondrion or aerobic bacterium.

But think of the old ways. Imagine you are an anaerobic microbe deep in an anaerobic peat bog in northern Siberia and that you would never even contemplate this new fangled oxygen based metabolism thing. Your core metabolic energy molecule might well be a very negative potential reduced ferredoxin molecule. There might be others but I rather like ferredoxins so I'll go with this one. If you want to do anything with reduced ferredoxin you need an electron acceptor. If you live in said peat bog with a dead mammoth you might just find a fatty acid molecule with a double bond. That's the electron acceptor you've been looking for. Bingo.

Obviously you can only do this once with each double bond, and sometimes the fatty acid will break at the time of reduction of the double bond giving a pair of shortened molecules. Eventually everything ends up as pretty much a mess of saturated hydrocarbons which is termed "adipocere": fat-wax. A well recognised post mortem change in wet, anaerobic environments.

Which means that if you dig said mammoth out of the bog 40,000 years later you are not going to get a very pretty picture from your GC output when you go looking for the PUFA content of the mammoth adipose tissue.

So these guys have a problem:

The Fat from Frozen Mammals Reveals Sources of Essential Fatty Acids Suitable for Palaeolithic and Neolithic Humans

(Belated HT to Tucker for this paper, got carried away with adipocere!)

They need to reverse engineer the composition of the adipocere to make a best guess as to what was present in the mammoth while it was alive. Also adipocere formation is random. Sometimes a lot, sometimes a little, even within the same carcass. Tough call.

They used a combination of what they thought the mammoth might have eaten, what modern elephants have as their PUFA ratios and the output from the HPLC machine to do the best they could. I do not envy them in this task.

Here is their main results table after the reverse engineering process

The top line (MY) is the mammoth, the others are horses and bison. The mammoth fat is calculated to have been composed of 7% linoleic acid and 18% alpha linolenic acid before adipocere.

Which looks preposterous to me.

The superscripts to the calculated percentages are the actually measured percentages. That would be 3.6% LA and 0.0% ALA, which were the basis for the modelling.

The superscript c to the MY identifier links to ref 19 which it claims specifies that "grass fed elephants" have similar values for PUFA to the mammoth values presented. Which sounds convincing.

Accumulation of polyunsaturated fatty acids by concentrate selecting ruminants

Until you find it is only one elephant.

And that there is absolutely no information in the paper about whether this one elephant was wild, ie grass fed, or was domesticated, ie concentrate fed. In fact none of the individuals have any information about grass fed, grain fed, hunted or slaughtered. There is no information as to what proportion of those 25% PUFA in the elephant's fat depot were LA vs ALA either. There is no information.

Who will bet it was a domestic working elephant fed on grains?

Me for one.

Especially because I've read this paper:

Molecular characterization of adipose tissue in the African elephant (Loxodonta africana)

All wild animals, culled as part of an elephant management operation.

How do their adipose tissue fatty acids pan out? In the absence of adipocere formation of course.

That looks a bit like around 1% LA and 2% ALA.

I'd eat that.

Peter

Protons (58) When hydrogen peroxide becomes insulin

Preamble: I'm not going to discuss NADPH oxidase 4 or rho zero cells at this stage, not that these are unimportant or boring. For today's post it's just about some of the ROS from mitochondria.

Amber O'Hearn re-tweeted this paper,

Academic urban legends

with "Full disclosure: I didn't check the references" added. Which amused me greatly.

So you have to follow references back and back and back to be certain that the absolute fact that "X" causes "Y" is supported by more than someone's ad hoc hypothesis number 3297 as a one line throw away in a textbook from 1952. Or, worse, that they said the exact opposite! It happens.

I've spent an inordinate amount of time going through very old references in the past few weeks. The idea that hydrogen peroxide is an insulin mimetic turns out to be sound. It's not just an insulin mimetic for control of glucose uptake, it appears to be able to replace all of insulin's actions from initiation of signalling through to inhibition of signalling at high dose rates. The exogenous amounts needed in cell culture are compatible with the amounts generated by mitochondrial preparations under plausible conditions, as far as I am able to understand from the methods sections of isolated mitochondria papers. BTW For anyone who owns a MAGA hat you cannot replace parenteral insulin with parenteral hydrogen peroxide for diabetes management, undesirable effects will occur at the whole organism level.

I started out from this 2005 paper

Insulin Action Is Facilitated by Insulin-Stimulated Reactive Oxygen Species With Multiple Potential Signaling Targets

and went back in time to find out if it was true. This next paper is from 1974 when people were using transition metal ions to generate ROS, giving the realisation you could do the same thing with hydrogen peroxide alone, without the copper (or chromium) ion:

Evidence for Electron Transfer Reactions Involved in the Cu2+-dependent Thiol Activation of Fat Cell Glucose Utilization

This image is the rate of uptake of glucose into adipocytes under the influence of hydrogen peroxide in the culture medium:

The effect was evident at 10micromol, peaked at 1mmol and was obtunded or eliminated by 4mmol. Bear in mind that these are the concentrations in the medium outside the cell. The concentration in the cytoplasm will be lower and within the mitochondria lower still. Catalase don'tchano. In isolated mitochondrial preps generating ROS in-situ we are talking nanomoles rather than micro or millimoles. But the pattern is there, where small amounts of peroxide get glucose in to adipocytes and larger amounts suppress this.

We can also look at the incorporation of glucose in to lipids and activation of the pyruvate dehydrogenase (PDH) complex using this paper, a jump forward to 1979:

The Insulin-like Effect of Hydrogen Peroxide on Pathways of Lipid Synthesis in Rat Adipocytes

where the pattern is repeated in the activation and deactivation by phosphorylation of the PDH complex at low and high hydrogen peroxide exposure (same pattern is seen for incorporation of glucose in to lipid too, graphs are in the paper):

It's worth noting that the effect is present in the absence of glucose but  is enhanced when glucose is present at low levels. High levels of glucose swamp the effect (I didn't follow that particular ref) but I find this plausible because the glycerophosphate shuttle will be better able to generate supplementary ROS given a little glycolysis to work with.

I won't cite any of the many isolated cell culture papers showing that the oxidation of palmitate is good at generating ROS and that linoleic acid is poor at this, I've been through that too many times. They usually use high dose pure palmitate combined with hyperglycaemia and are aghast that cells die under these conditions. Palmitate is the devil incarnate. A deeper view allows more understanding.

Relating insulin signalling to mtG3Pdh activation and/or fatty acid oxidation ties ROS generation to insulin signalling and goes a long way to explaining many phenomena.

Peter

More lactate wars

I have a suspicion that lactate as a portable energy source might be going to become quite interesting. I'll hit publish on this post which has been lying around on the draft list for some time. Here goes.

Some groups of researchers have been interested in lactate as a fuel for oxidative metabolism for a very long time. My own biases rather like this approach, so beware.

Back in 2008 we have this paper:

Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma Cells (CCF-STTG1)

"Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget."

which is clearly preposterous if you are part of Fulghum's group in Kentucky. From 2019:

Mitochondria-associated lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in murine striated muscle

"We find that cardiac mitochondria do not contain LDH ... These results indicate that cytosolic, and not mitochondrial, LDH promotes cardiac lactate oxidation."

"Our findings show negligible levels of lactate oxidation in isolated mitochondria from heart and skeletal muscle in sedentary, acutely exercised, and exercise-adapted conditions."

A finding which was promptly addressed in 2020 by Mailloux, now in Canada:

Lactate dehydrogenase supports lactate oxidation in mitochondria isolated from different mouse tissues

"Using the guide supplied by Passarella et al., we counter the conclusions drawn by Fulghum et al. and demonstrate that mitochondria oxidize lactate."

"Collectively, we can conclude lactate is a good fuel for mitochondrial bioenergetics in mammalian cells."

This is clearly an ongoing battleground and I doubt the exchange of half bricks is finished yet. It certainly brings to mind the astrocyte-neuron lactate shuttle

Lactate: the ultimate cerebral oxidative energy substrate?

which has largely been destroyed by

Control of brain energy supply by astrocytes

which I had a think about in this post. I do wonder if this declaration of destruction might be a little premature too. As was said in the Monty Python sketch: "I'm not dead yet!".

Ultimately, isolated mitochondria are very, very far away from anything physiological. I get the impression that the conditions they are studied under are utterly critical for the results you might like to get, or not get. The models are not useless per se but anything found needs to be considered very carefully, often in the absence of knowledge about what does and doesn't matter within the methods section and which may well have been tweaked to get the result desired. And in the context of what might be published next year.

I think abandoning lactate as a super-fuel might be a little premature. Beware of my biases.

Peter

Ultra processed food

This piece of epidemiological meta-analysis, hot off the press, is doing the rounds at the moment:

Consumption of ultra-processed foods and health status: a systematic review and meta-analysis

It illustrates yet another major error in nutrition research.

There are a few key words which flag a given publication for me as junk. If I see "reward" it signifies that the authors consider that certain foods force re-consumption and that such overeaten food has to be stored as fat. High "reward" overcomes the normal control of metabolism which has existed for millenia. This concept is junk to me.

The second phrase which alerts me is the "caloric density" of food. People really do think that you can trick metabolism in to overconsumption. That people and rats are programmed to (say) eat 100 mouthfuls per day. Put more calories in to each mouthful and you get fat. Another junk concept.

Now we have ultra processed food as the next junk term. Let's play a thought experiment.

Given a saucepan, a cooker, some milk, some rennet and a cheesecloth I think it's quite possible your granny might be able to put together something resembling a casein rich cheese-precursor. Somehow I doubt that she could produce a freeze dried pack of lab grade casein powder, so I think we can consider such a powder to be an ultra processed food component.

Sucrose can be extracted from beets or cane without too much technology but modern sucrose coming out of something resembling the Cantley sugar beet factory in Norfolk might be considered as ultra processed, never mind the smell. So might raw refined corn starch.

If you work at Sigma Aldrich you can take soya bean oil and convert it by an unknown (to me) and undoubtedly very, very clever technique in to tricaprylin, a triplet of octanoic acid molecules attached to a glycerol backbone. I challenge your granny to even extract the soybean oil from the soya beans, let alone convert it to tricaprylin. So I think we can suggest that this interesting oil is more than a little ultra processed.

Mix these components up and supply them to a lab in Japan to feed to some rats. We can merely look at the end weights from this paper:

Effects of Different Fatty Acid Chain Lengths on Fatty Acid Oxidation-Related Protein Expression Levels in Rat Skeletal Muscles

Feed one set of rats on crapinabag, which is about as un-processed as anything fed to a lab-rat ever gets.

Feed the next set on the tricaprylin mix, 60% of calories as this fat with generous casein, sucrose and cornstarch.

A final set can be fed with the same ultra-processed diet as the tricaprylin rats but with the soya bean oil left as soya bean oil.

Which rats get fattest? Okay, soya bean oil it is.

Which rats stay slimmest? Tricky. Whole food crapinabag or ultra-processed synthetic caprylic acid based syntho-food?

Well, I'd hardly be posting this if the ultra-processed food came out badly, now would I?

Here's Table 2

So, "whole food" SC crapinabag fed rats ended up at 239g bodyweight, seriously ultra-processed octanoate based MCFA at 216g, seriously ultra-processed soya bean oil based LCFA at 244g.

It's not the ultra processing. It's the effect on insulin, insulin signalling and the ability to resist insulin signalling when the resistance to that signal is physiologically appropriate. None of which was looked at in the paper, it was about something else.

Of course these are the PUFA levels:

The crapinabag was 11% fat, I think we can assume around just over half of that was linoleic acid, probably with a little alpha linolenic acid thrown in.

The 60% of calories as fat in the ultra processed diets both provided the same ratio of omega 3 to omega 6 but the absolute levels of total PUFA were around 3% for the MCFA fed rats and around 34% PUFA in the LCFA group.

The numbers speak for themselves.

What appears to matter is how capable adipocytes are to say "no" to extra in-coming calories. There are obviously a ton of down stream effects of distended adipocytes. Looking at PUFA combined with insulin shows how they get fat.

I'm the last person to suggest junk made of sucrose and starch are problem free but you have to be very careful of processed vs unprocessed as terminology when applied to foods. It's not likely to be as simple as it looks.

Peter

PS tricaprylin is interesting in its own right as it is weird stuff, but today I'm just looking at processed vs unprocessed. I hope no one would suggest that tricaprylin is an un processed food component.

Protons (57) When glucose becomes palmitate

 I'll just put this up as a brief post, there is a lot of background to it.

We all know that long chain fully saturated fatty acids yield approximately twice as much NADH as FADH2 giving an FADH2:NADH ratio just under 0.5 and that this high rate of FADH2 input at the CoQ couple facilitates superoxide generation by reverse electron transport through complex I.

Equally, we know that glucose oxidation, with five times the generation of NADH as FADH2, gives us a ratio of 0.2 and minimal reverse electron transport

We also know that, in order to balance the cytosolic NAD+:NADH ratio that NADH must be converted back to NAD+ to allow glycolysis to continue. This can be done using the malate-aspartate shuttle, conversion of pyruvate to lactate (both of which are redox neutral) or by using the glycerophosphate shuttle.

The latter is far from redox-neutral from the FADH2 input perspective. A cytoplasmic NADH is converted to an FADH2 within mtG3Pdh. This inputs at the CoQ couple. As far as the mitochondria are concerned that cytoplasmic NADH never existed. It behaves exactly as an FADH2. So, while the glycerophosphate shuttle is active, glucose presents to the mitochondria as two FADH2 and four NADH, giving us a nice, rather neat, FADH2:NADH ratio of 0.5. Slightly higher than palmitate or stearate.

I consider the glycerophosphate shuttle as generating essential ROS for insulin signalling. Small amounts of ROS generation facilitates insulin signalling. Large amounts inhibit it. Glucose, even hyperglycaemia, dose not generate ROS by the RET route. Adding insulin does do so because as the pyruvate dehydrogenase complex becomes more active then so the glycoerophosphate shuttle also becomes more active. The FADH2:NADH from glucose rises from 0.2 towards 0.5 and ROS increase to generate (given enough activation of the PDH complex) insulin resistance.

Insulin induced insulin resistance.

Peter

Protons (56) The miracle of fish oil (3)

I think this one is too important to leave it where George Henderson posted it in comments:

Of mice and men: Factors abrogating the antiobesity effect of omega-3 fatty acids

The group is from Norway. I tend to think they might be biased pro-fish oil. I also think they might be interested in why a paradox has occurred and this has overcome their intrinsic bias. I like their title too.

It appears that the weight loss routinely found in mouse experiments is remarkably difficult to replicate in humans. It can be abrogated (their word) by sugar, refined carbohydrates and omega 6 fatty acids. The refs are in the paper.

This gives the possibility for a given lab to set up a specific experiment to produce the result it wants/requires by manipulating these factors. That's called a pilot study and it doesn't often get mentioned in the paper per se. The mouse weight loss will not be replicated by a human popping three fish oil capsules before a meal of chips fried in sunflower or soya oil with a Big Gulp or two on the side.

George looks at this from the endocannabinoid signalling level within the brain.

I look at it from the adipocyte mitochondrial level control of insulin signalling coupled with the amount of insulin generated. They are both layers of signalling derived from the same process.

Nice.

Peter

Quick edit: Of course if a human removed sugar, refined starch and seed oils from their diet they might lose weight spontaneously with or w/o the fish oil. Maybe it might help, maybe not, but I doubt that has been looked at!

Protons (55) The miracle of fish oil (2)

I have a feed to my email account which has worked out that I am interested in longevity studies and particularly the role of PUFA in the inner mitochondrial membrane. This paper popped out today:

Dietary fatty acids and oxidative stress in the heart mitochondria

The diets were roughly 16% of calories as coconut oil, olive oil or fish oil. Fed to rats for 16 weeks, which is a fair length of time in the life of a rat. They were interested in the effect of unsaturation on the measurable oxidative damage done to mitochondrial proteins and the peroxidation of inner mitochondrial membrane lipids.

TLDR for the paper itself: If I was taking fish oil I would stop.

But of course I'm more interested in the body weights.

Here is the summary of the lipids in the diets, butchered out of Table 1:

And here, in its entirety, is Table 2 giving the weights at the end of 16 weeks. We can ignore the fish oil plus probucol group, except to note that they were even fatter than the fish oil group, don't you love those good old antioxidants:

Obviously none of the weights are significantly different from each other and 10-20 grams on a 500g rat is not a huge difference. Except food supply was limited to a fixed, slowly increasing amount as the rats grew. There is no mention of uneaten food so I think it is reasonable to assume all rats ate all of the food offered. So on a rigidly fixed calorie intake the fish oil fed rats were heaviest. I won't mention Arnie rats or C57Schwartz6 mice after my embarrassment in the comments to the last post.

Very roughly the modest excess weight goes up with the double bond index of the diet. On a rigidly fixed, mildly hypocaloric diet, even if p stays stubbornly above 0.05.

Also distinctly non-significant but appropriately trending are the fasting glucose readings. Those are in Table 3:

Highest in the Coconut oil group, trending down to lowest in the Fish oil group. Fish oil leaves you insulin sensitive.

Insulin signalling in adipocytes makes you fat.

If the rats were allowed to eat ad lib then the calories lost in to adipocytes would be replaced by eating more food. Eating the food would get the blame for the adipocytes being bigger than they ought to be.

Incorrectly.

Peter

Pesky PSCK9 inhibitors (2)

Eric put various links in the comments to the first PSCK9 post leading, eventually, to this study:

Sequence Variations in PCSK9, Low LDL, and Protection against Coronary Heart Disease

which gives us these results:

"Of the 3363 black subjects examined, 2.6 percent had nonsense mutations in PCSK9; these mutations were associated with a 28 percent reduction in mean LDL cholesterol and an 88 percent reduction in the risk of CHD (P = 0.008 for the reduction; hazard ratio, 0.11; 95 percent confidence interval, 0.02 to 0.81; P = 0.03). Of the 9524 white subjects examined, 3.2 percent had a sequence variation in PCSK9 that was associated with a 15 percent reduction in LDL cholesterol and a 47 percent reduction in the risk of CHD (hazard ratio, 0.50; 95 percent confidence interval, 0.32 to 0.79; P = 0.003)."

and the conclusion:

"These data indicate that moderate lifelong reduction in the plasma level of LDL cholesterol is associated with a substantial reduction in the incidence of coronary events, even in populations with a high prevalence of non–lipid-related cardiovascular risk factors."

Well. There we go. Again.

Soooooo. What is the glaring omission from the study results?

That's correct, there is no body count. Presumably the paper was written by cardiologists and/or lipidologists.

Perhaps we should get a body count.

Lets go to UK Biobank and some folk in Denmark. Here we have

Low LDL Cholesterol by PCSK9 Variation Reduces Cardiovascular Mortality

"In causal, genetic analyses, a 0.5-mmol/l (19.4-mg/dl) lower LDL cholesterol was associated with risk ratios for cardiovascular and all-cause mortality of 0.79 (95% confidence interval [CI]: 0.63 to 0.99; p = 0.04) and 1.02 (95% CI: 0.94 to 1.12; p = 0.63) in the Copenhagen studies, 0.79 (95% CI: 0.58 to 1.08; p = 0.14) and 0.98 (95% CI: 0.87 to 1.10; p = 0.75) in the UK Biobank."

and in conclusion:

"Genetically low LDL cholesterol due to PCSK9 variation was causally associated with low risk of cardiovascular mortality, but not with low all-cause mortality in the general population."

Note, particularly that in the UK Biobank data, there was no significant risk reduction for CVD events in addition to the no, zero, zilch, nil reduction of risk in all cause mortality. None. I'm a subject in UK Biobank.

So why would anyone expect PSCK9 inhibitors, certainly in the UK, to do any better than genetic PSCK9 activity reduction?

Perhaps such people have a drug to sell in a broken paradigm.

Peter

Protons (54) The miracle of fish oil

This paper has absolutely nothing to do with obesity:

Feeding into old age: long-term effects of dietary fatty acid supplementation on tissue composition and life span in mice

The researchers fed mice on chow until 450 days of age. For some they then started blending in sunflower oil (omega-6 based) and for others they added in fish oil to the same chow. The composition of the diets was sufficiently similar that there was no effect on lifespan found, either median or maximum. But there was an effect on bodyweight. I bring this up because, while sunflower oil would be reasonably expected to be obesogenic, fish oil certainly would not.

Unless you view it from the Protons perspective of course. Here the mitochondrial oxidation of omega-3 PUFA should be more obesogenic than omega-6, which is almost never the finding in rodent studies and which is why, over the years, I collect any studies which suggest this. To confirm my bias.

Crucially the people running this current study were interested in longevity, not obesity.

Despite this, not only did they weigh the mice weekly (which most studies do) but they also reported those weights in detail (which many don't).

"Mean body weights in all three groups (over the entire experiment) and SEMs were 30.9 ± 0.1, 29.9 ± 0.1 and 28.7 ± 0.09 for n-3 rich, n-6 rich and controls, respectively."

Graphically it looks like this:

If we take the rather crowded data points over in to PowerPoint we can crudely rough in some curves:

The red line is the fish oil group, yellow the sunflower oil and blue the chow.

Fish oil should make you fat. Confirming this bias is remarkably difficult, so you can imagine how I feel about these data points.

Quite how fish oil can be shown to be so beneficial most of the time is beyond me. I think the aphorism goes something like "current medical research reflects current medical bias". Possibly from John Ioannidis?

Peter

Of course the fish oil mice might have looked like Arnie* on steroids. Or they might not.

*Having had the joke explained to me in comments I can't look at this without giggling. C57Schwarz6 mice!

Pesky PSCK9 inhibitors

For a variety of reasons I'm rather ignoring the blog at the moment. But this is too good not to post, HT to Carlos Monteiro for the link:

Serious Adverse Events and Deaths in PCSK9 Inhibitor Trials Reported on ClinicalTrials.gov: A Systematic Review

PCSK9 inhibitors do not work. However much they cost, they're useless.

This confirms (again) that the lipid hypothesis of CVD is bollocks. It was so in the 1950s. Nothing has ever changed that.

Happily only Evolocumab will kill you prematurely (with the data so far).

Peter

EDIT cavenewt emailed me this press release (see her comment for quotes). Permanent alteration of your PSCK9 gene... what could possibly go wrong? END EDIT

ARDS and Tucker Goodrich

Tucker has been very, very busy over the last few weeks.

Does Consumption of Omega-6 Seed Oils Worsen ARDS and COVID-19?

You will need some time. Maybe some coffee.

Peter

Total, utter aside:

"Although we initially also identified monoclonal antibodies that appeared to bind exclusively to 4-HNE-LDL, these were lost during the cloning procedure."

The first ever auto immune disease to be tied to a specific human leucocyte antigen receptor subtype was ankylosing spondylitis, linked to HLA B27. The monoclonal antibody was developed by my wife's PhD supervisor working in Prof Ebringer's lab, as part of her own PhD. She lost it during the cloning procedure. It happens. As Tucker says:

Bummer.

Blood "energy" content

"One-liner" post. This is exciting (picked up on twitter from Mike Eades):

Effects of dietary carbohydrate content on circulating metabolic fuel availability in the postprandial state

It's Ludwig's group. I've skim read but not looked at the detail. I like what I see.

Over the last year or so I've ventured in to the morass of older papers about the CNS response to infusion of various metabolic substrates where you get bogged down in the various neural groups which respond in various ways to high vs low glucose etc. It's messy and it's rare for people to have asked the questions in quite the way I might have phrased them.

Eventually I simply started adding up the energy content of "blood" in various states, especially under extended fasting when hunger becomes blunted. Being me I tended to add them up in terms of how much NADH and FADH2 might be available. I kept getting pushed towards the idea that hunger might be a simple matter of the energy content of the blood supplying the hypothalamus. Clearly that is one core thing that the CNS monitors (using ROS of course).

Could hunger be this simple?

Okay, there is also clearly a neural input (think hepatic FFA infusion via the portal vein suppressing food intake) but ultimately if the brain is being perfused with too few calories, it is going to do anything it can to make you eat. The classic is reactive hypoglycaemia or insulin induced hunger where I suspect the problem is (in myself in pre low carb days) not absolute hypoglycaemia (I could get this at BG around 4.5mmol/l) but the accompanying low FFA availability giving low brain stem energy availability. But of course measuring FFAs is not as simple as measuring glucose...

Anyway, it's fantastic to see some serious researchers looking at the concept of blood energy content. They will have to add the Protons concept eventually, to explain why things happen as they do, but they're on an exciting trajectory.

Peter

Fancy some serology? (3) In Japan

I notice that the COVID-19 state of emergency has been lifted in the last remaining areas of Japan as of last Monday.

I think they lost about 800 people in the pandemic. The seroprevalence in Tokyo is at least 6% in the populace attending a community clinic or two and at least 10% in healthcare workers. Exposure has widespread.

All countries have had their individual approaches to managing the pandemic, some sensible, others less so. What worked and what didn't will probably be lost in the avalanche of lies used to cover the arses of incompetent politicians, certainly here in the UK.

I found this ancient (2014) snippet by accident somewhere on t'internet:

"But one country has managed to keep obesity down with the help of a controversial government policy that probably wouldn't fly in the U.S. That country is Japan, where only about 3.5% of the population is classified as obese, compared to rates as high as 30% or greater in countries like the U.S. And it's not just a generally healthier diet and lifestyle that's kept the Japanese trim.

Citizens must adhere to government-mandated waistline limits or face consequences. The government has established waistline limits for adults ages 40 to 74. Men must maintain a waistline at or below 33.5 inches; for women, the limit is 35.4 inches. The "metabo law" went into effect in 2008, with the goal of reducing the country's overweight population by 25% by 2015. The government's anti-obesity campaign aims to keep "metabolic syndrome" — a number of factors that heighten the risk of developing diabetes and vascular diseases, such as obesity and high blood pressure, glucose and cholesterol levels — in check, thus minimizing the ballooning health care costs of Japan's massive ageing population.

Those who stray beyond the state-mandated waistlines are required to attend counseling and support sessions. Local governments and companies that don't meet specific targets are fined, sometimes quite heavily".


From Snopes (FWIW) it seems this is basically true, assuming the numbers for waistlines are real:

"Japan requires citizens between the ages of 45 and 74 to have their waistlines measured once a year and potentially seek medical attention.

Unlike individuals, however, companies and local governments can be assessed financial penalties if the citizens in their charge do not meet government standards".

I guess that having a national policy to limit metabolic syndrome might or might not have any influence of the course of a pandemic which targets people with metabolic syndrome.

We'll never know...

While the obvious initial advice for mitigating infection with SARS-CoV-2 was to try not to be elderly and to try not to be diabetic it now looks like simply trying not to be diabetic might have been all that mattered.

Peter

Fancy some serology? (2)

I thought I would just take a break from trying to simplify the Protons electron transport chain as regards ultra low fat diets and talk about sensitivity and specificity of serology tests for a break.

People may have noticed I'm quite keen on serology and am rather less than enthusiastic about PCR for test, track and trace in a situation where the SARS-CoV-2 virus is present throughout the country, as it is here in the UK. Stupid is as stupid does.

However, serology is not quite as straight forward as I might like either.

There are a number of serology tests coming on to the market, and many have a 100% sensitivity and 98% specificity. It difficult to express how phenomenally accurate these test are. If I submit a blood sample for some routine analysis I accept that 95% for these sorts of accuracy assessments is pretty good, we're dealing with biological systems, there is room for grey zones.

So presently serology has a 100% sensitivity. That means it will always pick up seropositive people. If you have antibodies, this test will find them. Always. Getting a negative needs some thought.

This is addressed by specificity. A 98% specificity means that a negative on the test will be correct 98 times out of 100. If the test says you don't have antibodies, it is also most likely correct, a one in fifty error rate there.

It is difficult to over emphasise quite how good these values for sensitivity and specificity are for a lab test. They are very, very good.

At detecting antibodies.

If antibodies are found in a healthy person it is, with a test this good, pretty well certain that they have been exposed to the disease and, in the absence of illness, that they are immune. Or at least they were at the time of exposure.

Sadly human immune systems can be recalcitrant in cooperating with serology.

The Royal College of Pathologists short presentation on serology is now up on Youtube

The COVID 19 pandemic: testing – serological diagnostics for COVID 19

and here is a screenshot from just after 18 minutes in:

The dotted red line is the lower limit of the serology assay used. All of the patients have had known, absolutely certain, clinical disease. If you use a serology test which is 100% sensitive and 98% specific, you will pick up everyone over the red dashed line. A negative result will be correct 98% of the time.  That is what a highly sensitive, highly specific test does. To put that in a more visually clear image here is another screenshot:

Again, below the red dotted line you will be classified as seronegative, you are seronegative. That does not mean that you have not been exposed. It doesn't matter how good your test is. The test cannot see below the red line, above the red line the test is phenomenal. This not a problem with the test, the test is not for exposure/recovery from the disease. It is just for antibodies above a certain level. This is the limit of serology testing, it is undermined by the ability to recover from this infection without seroconverting. It happens, it's on the graphs. It's not the test's fault.

To a large extent recovery without seroconversion suggest that the innate immune system is at work (or you are simply unable to become infected) and that would fit nicely with the reports suggesting that hyperglycaemia over 10mmol/l is bad news and hyperglycaemia below 10mmol/l gives a slightly better outlook in severely ill patients. Hyperglycaemia is a good way of suppressing the innate immune system.

Passthecream put an interesting link in the comments of the last serology post which suggests the innate immune system is also adaptive, it remembers, no antibodies needed...

Adaptation in the Innate Immune System and Heterologous Innate Immunity

Sadly, at our rudimentary level of understanding of the immune system, we are in no position to assess whether a given person might be seronegative but still immune.

Having said all of this, it's worth remembering that being seropositive without having being ill suggests you are immune. It's interesting to see the WHO position on this. The WHO currently suggests that there is no evidence that having antibodies confers immunity.

That is interesting and absolutely, currently, technically correct. Thus far seropositive people have never been challenge-tested with virulent virus, so there can be no evidence that antibodies are protective (try getting that one through ethics committee review!). It is theoretically possible that a person could have been exposed to virulent virus, have never been ill, have developed antibodies, and yet is still be susceptible to the virus. You can imagine that this might be the case.

Well, actually, I can't.

So, if you are the head of an ITU in a UK district general hospital in London and you find you are one of those lucky people who are solidly IgG seropositive without ever having been ill, what would you do as regards PPE for yourself?

As the WHO says, there is no evidence that being seropositive is protective, as yet. But for antibodies produced in vivo, by someone who was never unwell, for these antibodies not to be protective would have to be a first of a kind as regards immunology (vaccine induced seropositivity is a whole different ball game).

I love this guy:

COVID-19: ICU care, long-term effects and immunity with Dr Richard Breeze

(Hat tip to Unknown for the link and no, Breeze didn't use any PPE while treating the large wave of COVID-19 patients which passed through Lewisham District General Hospital's upgraded ITU)

Knowledge over protocol. He also strikes me as the sort of person who might look at a patient on a ventilator who was developing barotrauma because "protocol" suggest "Xml/kg" as the "correct" tidal volume setting and who might reach over and reduce (gasp) the tidal volume setting. Just my guess. Or avoid intubated ventilation if at all possible (which was what they did).

You have to contrast this with the hospital managers who discharged SARS-CoV-2 positive patients in to unprotected nursing homes because "it's protocol".

I get the impression that good medics (and there are some excellent ones out there) don't seem to be the sort of people that become the politico-medics who guide the government...

Peter

Aside: I just can't get over Dr Breeze working without PPE. Sort of thing I might have done under the circumstances. I can't believe it was allowed nowadays!

Low fat vs low carb again (2)

For your enjoyment I have simplified this graph from

Hyperinsulinemia Drives Diet-Induced Obesity Independently of Brain Insulin Production

out of Jim Johnson's lab:

down to this graph (at great effort) to show only the mice on standard chow:

These are the insulin responses to an IP glucose tolerance test at a year of age in mice which have been fed on good quality chow all of their lives. The mice in the top curve are phenotypically normal in their insulin response to glucose, the mice in the lower curve have had three out of four of their insulin genes knocked out. They weigh the same.

We all know from the Surwit posts that the normal insulin exposure mice have an 11% decrease in median lifespan compared to low insulin exposed mice, Jim Johnson's lab again.

None of us is in a position to have our insulin genes partially silenced from before birth, but we do have a choice as to how much insulin we expose ourselves to, based on our dietary choices.

What we need to know is what the insulin response to a given meal might be if we were to try to imitate the partial insulin gene knockout mice. Very few studies have provided this sort of information but the current pre print from Hall et al does just this. Here is the graph

The red curve is from a group of people fed a single meal of a mildly ketogenic diet. With insulin peaking between 20 and 30micoU/ml this is quite similar to the value in mice with reduced insulin gene load, those pan out at around the 22microU/ml mark (don't you wish everyone just used picomoles all the time? Well, I do). Or you can eat low fat, plant based and choose to expose yourself to over 100microU/ml of insulin. Doing this you might still lose a little weight (another post, eventually), you might lose a little fat but you also might lose a few years of lifespan as the insulin drives ageing with its associated chronic diseases.

How many years? If the median lifespan for humans is around 70-80 years and we are talking about an 11% reduction that gives us a ballpark of just under a decade lost. As a thought experiment.

PBLF, plant based low fat. ABLC, animal based low carbohydrate.

There is no choice.

Peter

Total aside: I really hope that Hall keeps the title of this paper unchanged in the version which eventually gets published. It's a single sentence of prose which encapsulates what is wrong with nutrition research. It is absolutely, totally factually accurate, while being completely selective in its choice of factual content to give an absolutely misleading impression. As a declaration of bias it is unbeatable. I love it.

Low fat vs low carb again

I guess everyone knows about this pre-print

A plant-based, low-fat diet decreases ad libitum energy intake compared to an animal-based, ketogenic diet: An inpatient randomized controlled trial

There is a wealth of data to enjoy and a lot to say from the Protons and insulin point of view but just a brief look gives us equal weight loss, equal fat loss and the sort of changes in fat free mass you would expect from likely shifts in glycogen and its associated water:



Clearly the extra 600kcal eaten under ketogenic conditions did nothing to blunt fat loss, much as we would expect from the low carb perspective. The extra calories did not evaporate, they were lost through increased energy expenditure, especially during sleep and while sedentary:

These people have uncoupled metabolism during the period of eating the ketogenic diet, they generate heat. As measured within the limits of indirect calorimetry. You could argue about a greater faecal, urinary or breath mediated loss of calories too but that's less important than a measured equivalent weight loss despite higher, extremely accurately measured caloric intake.

That's all pretty boring.

What is really, really interesting is the equivalent spontaneous weight loss under the period of high carbohydrate intake. Over the years I've looked at the carbosis vs ketosis for potential mechanisms and this study may go some way to clarifying what is going on. The very low fat eating certainly does not limit the penetration of either glucose nor insulin past the liver. Both spike systemically after every meal. But still there is spontaneous weight loss due to a suppressed appetite.

Under low fat eating less "waste" heat is generated, metabolism is coupled. Tightly coupled metabolism means people needed less calories. The subjects, under very low fat eating, lost weight without any biochemical markers of inadequate calories. Just as they did under ketogenic eating.

That's really interesting. With data, lots of it including important things like the effect of a typical meal on blood glucose, insulin and lactate. Plenty to work with. Needs a lot of thinking about.

Peter

BTW does this sound like a metabolic advantage to ketogenic eating? Rhetorical question, 24h energy expenditure combined with utterly accurate food intake measurements tells us something...

Fancy some serology?

Just a one liner-ish type post.

I had the privilege of listening-in to one of the weekly Royal College of Pathologist webinars on the SARS-CoV-2 virus, this one on serology testing. These webinars are really fantastic, they are given as a 15 minute presentation by a scientist at the top of their field, in the complete absence of political interference or the sort of financial pressures applied to produce a 100% specific, 98% sensitive serology test to make billions of dollars for a commercial company. They have spent their careers as coronavirus "enthusiasts". The presentations are by pathologists, for pathologists. They are technical and utterly honest (as far as I can tell).

So. There are three types of people in the world. If you have had SARS-CoV-2, confirmed by PCR, have been seriously unwell, hospitalised, needed supplementary oxygen and been considered for a respirator/ITU admission then the chances are good that you will be solidly seropositive for SARS-CoV-2 on a blood sample in recovery. I would suggest that your medical history might be quite a big hint in this direction, which might render the use of the serology test under these circumstances somewhat superfluous.

The second type of person has also had SARS-CoV-2, confirmed by PCR test, been clearly unwell but not so unwell as to need any hospital admission for management. With the best possible testing using multiple different antibodies and different test techniques these people are very, very difficult to detect on a serology basis. Many will be negative on serology within the limits of what we have available now and what will be developed commercially. That is worth thinking about.

The third type of person has never been ill, has never been PCR tested, has no idea whether they have been exposed to SARS-CoV-2 or not. These are the apparently healthy population, the sort of people John Ioannidis sampled in Santa Clara County.

Of the 3300 people Ioannidis tested, 2.5-4.2% turned out to be sero-positive. Listening to the RCPath webinar on the problems of serology testing in mildly unwell people (let alone those apparently never unwell) this implies that the values from Ioannidis might well be the absolute, rock bottom, tip of the iceberg minimum. Exposure has probably been much, much higher in this still healthy population.

I find that rather hopeful.

It is difficult to describe how badly I feel that the COVID-19 pandemic has been managed here in the UK. I don't make political posts on the blog (or anywhere else) but the level of utter incompetence of our current government is breathtaking. I suppose a different administration could have done worse, but that's hard to imagine.

Peter

Surwit diet and derivatives (3) 5LJ5 vs D12330: Chow vs Surwit

TLDR: A "healthy", complex carbohydrate, low glycaemic index diet appears to markedly shorten the median lifespan of mice when compared to a diet of maltodextrin/sucrose with hydrogenated coconut oil, irrespective of obesity or insulin gene dose.

This is the second excellent paper from Jim Johnson's lab:

Reduced Circulating Insulin Enhances Insulin Sensitivity in Old Mice and Extends Lifespan

It is slightly different from the 2012 paper as these mice are full knockout for the Ins1 gene and this time it is the Ins2 gene that is present as a full complement or at half knockout, to adjust the insulin gene dosage.

The study was never intended to compare the two diets, the diets were simply intended to provide a fairly normal insulin environment using a rodent chow against an high insulin environment generated by a Surwit type diet. It was the insulin exposure which was the focus of the study.

But, ultimately, the study did compare the two diets and in some detail.

Just to summarise the diets. Both had 4% of calories as PUFA, primarily linoleic acid. The 5LJ5 chow used a slow release carbohydrate (as uncooked ground wheat) combined with a little extra protein from soybean meal. The D12330 (Surwit type) diet was the usual hydrogenated coconut oil with maltodextrin/sucrose plus casein as the sole protein source.

Maximum individual longevities came out as expected, with the 5LJ5 coming out best and the low insulin gene dose conferring benefit to both diets.

These are the mean lifespans of the four longest lived mice in each group (top decile) as shown by the open circles/squares on the bar chart, taken from the end stage of the survival curves as shown here:

That's relatively unexciting and no one would be surprised by it.

What surprised me was the longevity advantage to the Surwit diet groups when assessed at median life span. Not only did the Surwit groups both do a great deal better than the chow groups at median lifespan but there was only a very small improvement (about 3%) obtained by reducing insulin exposure. In fact the normal gene-dose, obese, high insulin-exposure Surwit diet group (purple) had a longer median lifespan compared to the reduced insulin-exposure group that was on chow. Which was better again than that of the ordinary mice fed on chow.

If we simply ignore the reduced insulin exposure groups we can also suggest, based on these data, that the unmodified Surwit diet produces a median longevity gain in the order of 16% over a top-of-the-range excellently formulated lab animal breeding chow.

If the Surwit diet was a drug it would knock spots off of metformin, rapamycin, ethanol, caffeine or glucosamine for median lifespan extension. These mostly gain around 10% in median life span extension.

I accept that, for the four mice which made it in to extreme old age, there is a small disadvantage to the Surwit diet, but this only becomes apparent at those lifespans at over 750 days of age, out of a max of just over 900 days.

A quick look round the literature shows us that feeding a 60% fat diet where the PUFA content comes out at around 15% of total calories (high PUFA lard as the fat source), combined with Surwit-like levels of maltodextrin/sucrose, is a catastrophe. As in this one using TD.06414.

At the risk of speculating; there may be a host of problems triggered by a wheat/soybean based diet which do not appear to occur with a casein/saturated fat based diet, certainly until extreme old age is achieved. Or there could be some specific advantage to a highly saturated fat based diet which over rides the problems provided by maltodextrin/sucrose. Lots of possibilities, no obvious answers!

Fascinating study.

Peter

Surwit diet and derivatives (2) It's the insulin

EDIT Please ignore any ref to sucrose as the Surwit type diet used here contained none. My apologies. END  EDIT.

I've been spending some time re-reading a couple of papers out of Jim Johnson's lab and I'll start with this one because it has a core message which is absolutely crucial and possibly under appreciated. Sorry if the text is a bit repetitive but the idea is not completely intuitive.

Hyperinsulinemia Drives Diet-Induced Obesity Independently of Brain Insulin Production

The mice in this study were full knockouts for the Ins2 gene and also had either no knockout or half knockout of the Ins1 gene. Reduced insulin gene dose reduces insulin secretion which completely protected them from the obesogenic effect of the Surwit (D12330) diet. You cannot become hyperinsulinaemic in response to Surwit's maltodextrin/sucrose if you have only one out of 4 insulin genes functioning (with two out of four you can). Lack of hyperinsulinaemia normalises fat storage as one effect. Lack of hyperinsulinaemia also eliminates long term insulin-induced insulin resistance as a second, non related effect. Both effects are independently the direct result of reduced insulin exposure. The mice stay slim because they are eu-insulinaemic on a Surwit diet. The mice stay insulin sensitive because they are eu-insulinaemic on a Surwit diet. One cause, two responses. Shared causality tends to give correlated effects. But we all know about correleations and causality...

Despite being insulin sensitive the low Ins2 mice do not become obese because their knockouts stop them making enough insulin to achieve this. They are insulin sensitive but they are genetically unable use their insulin sensitivity. They are beautiful, to me at least. In an abstract sense.

Okay, have some graphs:

Top line in pink, obesogenic diet with normalish insulin phenotype, they get fat. Red line is the obesogenic diet with blunted insulin secretion. They don't get fat.

And insulin resistance: just consider the 52 week values here, these mice are a bit hit and miss re glucose/insulin function very early in life. By a year they show their true phenotype.

Just to reiterate: On the obesogenic diet fasting insulin is high because there has been a year of exposure to high insulin from the maltodextrin/sucrose of the Surwit diet when combined with a fairly normal pancreas, pink circles. The red triangles are the same diet but with blunted insulin exposure due to their Ins1 partial gene knockout. Ergo, low insulin exposure is causative of low insulin resistance at a year of age, even on the Surwit diet.

So. Insulin sensitivity, a surrogate for low insulin exposure, is a Good Thing. Using that insulin sensitivity by increasing insulin exposure will make you fat and insulin resistant as two separate effects from the same change.

I'll take a brief pause here for that to sink in before looking at the next paper from Jim Johnson's lab which translates these findings in to longevity studies. Which are really weird.

Peter