Protons (64) The miracle of fish oil (6)

Preamble: I started this current series of post about the ability of fatty acids with multiple double bonds to limit weight gain. To me, this is a paradox. Paradoxes are, without a doubt, the most productive sources for the development of an idea. Even as I started this current post I had no idea where it was going to end up and was bit surprised at where the metabolism took me. So be it. Let's begin.

Beta oxidation in peroxisomes does not consume oxygen and does not produce CO2.

The first step of oxidation of saturated fats runs like this:

R-CH2-CH2-COOH + FAD -> R-CH=CH-COOH + FADH2

In peroxisomes this is followed by

FADH2 + O2 -> FAD + H2O2

then

2xH2O2 -> Signalling -> Catalase -> 2xH2O + O2

The energy from FADH2 is released as heat and the oxygen is regenerated.

The NADH from beta oxidation is of no immediate use in a peroxisome and has to be transferred to mitochondria before it can be utilised. I suppose it could be phosphorylated to NADPH for anabolism but I have no data on that. It's not clear how reducing equivalents might be transferred from peroxisomes to mitochondria. There is speculation about something along the lines of the malate-aspartate shuttle used to import cytoplasmic NADH in to mitochondria.

It's also something of a truism that peroxisomes cease beta oxidation at C8 and then export this (by uncertain mechanism) to mitochondria for completion of oxidation. Digging back through the reference trail leads to the origin of this as the finding that isolated peroxisome preparations happily oxidise lauric acid but won't oxidise caprylic acid (much). Clearly oxidising DHA will never produce caprylic acid directly because there are double bonds within the residual eight carbon atoms. What exactly happens to truncated DHA at the C8 length appears to be an unasked question.

And there will be no CO2 production in peroxisomes as they do not have the TCA, that's limited to mitochondria.

So beta oxidation in peroxisomes produces heat, NADH, acetyl-CoA and signalling H2O2. And perhaps some caprylic acid from any saturated fatty acids being oxidised. It requires no oxygen consumption and results in no CO2 production, which gives an unchanged respiratory exchange ratio (RER).

Going back to

Enhanced Peroxisomal beta-Oxidation Is Associated with Prevention of Obesity and Glucose Intolerance by Fish Oil-Enriched Diets

some of these things become clear. We have these measurements of oxygen consumption:

The round symbols are the fish oil fed groups. Average VO2 through 24h is reduced by fish oil from about 3500ml/kg/h to about 3000ml/kg/h, ie that's a just under 15% reduction.

Here are the RER figures, still fish oil as circles. As expected high fat diets show a low RER, low fat diets show the converse. The reduced O2 consumption is exactly balanced by a reduced CO2 production and the RER is still largely set by the dietary carbohydrate-fat ratio.

Clearly, under fish oil, approximately 15% of calories are being used to generate heat and anabolic substrate without consuming oxygen or being transferred to the ETC.  Provided there is enough fish oil to stimulate peroxisomal proliferation the changes are quantitively independent of the absolute amount of fish oil. 

So with fish oil at as low as 10% of calories, not all of which are PUFA, VO2 is dropped by 15% suggesting that the peroxisomes are activated and are oxidising more fatty acids than just the PUFA from the diet. Presumably on the low fat fish oil diet the peroxisomes are also metabolising palmitate and oleate derived from carbohydrate by de novo lipogenesis too.

If we go to this paper:

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

we can see, by clever carbon 13 labelling, that peroxisomal derived acetyl-CoA in cardiac muscle (and I would guess most other extra-hepatic sites) does not enter mitochondria, it all stays in the cytoplasm as malonyl-CoA.

These data are from perfusing hearts with docosanoate, a C24, fully saturated, fully peroxisome targeted fatty acid. We get lots of labelled malonyl-CoA in the cytoplasm, minimal labelled citrate in the mitochondria.

The next fascinating paper (HT to Peter Schmitt for the link) used erucic acid, another peroxisome targeted fatty acid.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver

In the liver peroxisomal oxidation of fatty acids generates acetate but that this is still converted to acetyl-CoA and then malonyl-CoA without entering mitochondria. We know from the Randle cycle that malonyl-CoA is an inhibitor of fatty acid oxidation so it should come as no surprise that erucic acid feeding to peroxisomes inhibits fatty acid oxidation in mitochondria. So we end up with lipid accumulation within the liver, progressing to fatty liver and NASH. I have mention before that in rodent models of alcoholic fatty liver disease fish oil is one of the most effective generators of alcohol induced liver damage...

But perhaps the best line from this last paper is:

"Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio..."

It seems very, very unlikely that fish oil will be any different.

We find ourselves in a situation where peroxisomal oxidation of fatty acids generates benign heat combined with large amounts of anabolic substrate and a high NADH:NAD+ ratio without requiring oxygen while simultaneously inhibiting mitochondrial fatty acid oxidation while shifting metabolism to glucose.

Does that look like a recipe for cancer?

It does to me.

I had no idea that there is a large literature looking at the role of peroxisomes in all sorts of cancer types. Woohoo, they are a drug target! Perhaps avoiding peroxisome activating fatty acids and their derivatives might be a better approach. Apart from accepted Bad Things like drinking erucic acid or 4-HNE (a superb peroxisome activator) we might ask serious questions about drinking bulk fish oil.

Perhaps don't.

Peter

Addendum: I recall this study (observational but not a food frequency questionnaire in sight), which I was fairly uncertain about back in 2013

Plasma phospholipid fatty acids and prostate cancer risk in the SELECT trial

Now I'm more convinced...

Protons (63) 4-hydroxy-2-nonenal (2)

Brief aside for a one-liner-ish post.

This study gives an idea of what happens when you drink 4-HNE:

A high oxidised frying oil content diet is less adipogenic, but induces glucose intolerance in rodents

Here are the diets. The rats on heated soybean oil (HO) ate relatively little so all of the other groups were partially starved to the caloric intake of the HO group.

and here are the weight gains (green circles).

Note especially how thin the 4-HNE fed rats (blue rectangle) were and how the fish oil fed rats (red rectangle), even under caloric restriction were still the fattest, fatter even than the fresh soybean oil fed rats. On the same calories.

This diet had no sucrose, it was starch based. Like the rodent chow in the last 4-HNE post which gave the greatest weight gain on DHA or EPA. Starch diets seem worse than sucrose diets when mixed with fish oil...

Back to 4-HNE. These rats were mildly glucose intolerant too. Glucose was ns different from controls throughout an OGTT but the area under the curve was greater under 4-HNE.

In this case we have, I would speculate, 4-HNE causing insulin resistance within adipocytes, limiting fat storage (ie reducing loss into adipocytes), so limiting hunger by actually reducing fat gain. 

And doing god only knows what other damage along the way to these un-arguably thin rats. As the authors suggest, the HO diet could even be destroying the beta cells of the pancreas...

Peter

Addendum HT to raphi for this link in the comments. Well, that's cool

4-hydroxynonenal causes impairment of human subcutaneous adipogenesis and induction of adipocyte insulin resistance

Protons (62) The miracle of fish oil (5)

Okay, its time to look at this paper:

Enhanced Peroxisomal beta-Oxidation Is Associated with Prevention of Obesity and Glucose Intolerance by Fish Oil-Enriched Diets

We have four diets, two based around low fat (10% fish oil or 10% lard), each with a little soybean oil thrown in. The other two were 60% of calories from fat, either from fish oil or lard, both with generous soybean oil added. Fatty acid composition was measured by gas chromatography. I've replaced the percentage of lipid as linoleic acid with percent of total calories as LA written in red.

Notice the study altered protein levels in addition to switching starch for fat. Talk about failing to control your variables.

Then we just have to feed ad-lib for eight weeks and look at the weights. In fact we can actually look at the fat mass in addition to weight, which is much nicer. We can also look at the energy efficiency, weight gain per unit calories absorbed. Note the columns have changed order, I've again added the linoleate percentages of calories in red, highlighted the energy efficiency in blue and circled the results of interest in green:

I did a rough back-of-the-envelope calculation for the number of mg/kg/d of DHA consumed by the mice fed the 10% fish oil diet. It works out as around 1250mg/kg/d on a semi-purified diet background, so probably in the peroxisome activating level.

Obviously the amount of fat stored roughly follows the LA content of the diet outside the green circle anomaly.

Notice that the "average energy absorbed" is lowest in the fattest group of mice. These animals are not making fat out of nothing. To understand this you have to go back to this image from a long time ago:

The top line is HFD (D12492, fed to Long Evans rats). All of the "hyperphagia" needed due to rapid weight gain occurs during the first 10 days and is only statistically elevated during the first 6 days. If the averaged food intake of the mice in the current study is lower than the brief "hyperphagic" phase this would explain the low overall calorie intake. The effect might be exacerbated in part due to the strain of mouse used, in this case the Swiss mouse, which is not prone to obesity in the way that many rodent strains are.

I started to look at these mice in terms of energy budgets. The mice are fed ad lib so are all going to eat exactly as much food as they need. No more, no less.

The high lard, high LA fed mice need 77.85kJ/d to meet basal metabolic rate, thermogenesis, cage exploration and this will also include a modest loss of calories in to their already distended adipose stores. They can do all of this on 77.85kJ/d. This is what they need.

The high fish oil fed mice should need very slightly less than 77.85kJ/d because they are lighter, so have less tissue to support metabolically, ie have minutely lower BMR and, again being lighter, it takes less calories to move themselves around their cage. They are also barely losing any fat in to their adipocytes. Despite all of these small combined contributions to decreased caloric needs they still have to absorb more calories (83.42kJ/d) than the fat mice to support a significantly lower weight and fat mass.

Clearly they are losing some of those extra calories but in this case the calories end up in peroxisomes rather than in adipocytes. I would predict that these mice will have a normal body temperature (this is tightly controlled in awake mice) but they will be physiologically adapted to dissipate excess waste heat. At 20degC this is easy for a mouse, it normally spends more calories on thermogenesis than it does on BMR at this temperature.

So the thin mice are unable to meet their basic caloric needs without having to "over-consume" food to make up the deficit induced by heat generation. In peroxisomes. They are not overeating and then using heat generation as a technique to stay slim. They are eating enough food to meet their needs but the lipid sources in their food are intrinsically and wastefully heat generating.

Just as the fat mice are fat because they lose calories in to adipocytes due to linoleic acid, so the skinny mice are skinny because they lose heat calories through their skin due to DHA in peroxisomes.

Ad-lib fed mice never over or under consume calories. They eat to meet their needs. Exactly.

Peter

Note that the mice fed 10% of calories fish oil "over-consume" to total 92.51kJ/d. They need more daily calories than the 60% fish oil fed mice because they are losing some calories in to adipocytes. That poses some more questions. As does the low oxygen consumption in the fish oil fed mice. An interesting paper. More to think about.

Protons (61) The miracle of fish oil (4)

There is a huge body of work on the requirement for DHA, its lipid peroxides, its use in the body but almost nothing about its bulk disposal. As in what happens to the excess DHA when you drink 60% of your total daily calories as fish oil, for weeks. As a rat.

I've been chasing tenuous leads as to whether DHA is catabolised in peroxisomes, in mitochondria or in both. I'd like a nice clear cut answer, but you can't always have what you want. It's clear that DHA can only be synthesised in peroxisomes because it requires elongation from ALA to eventually form a 24 carbon PUFA which is then shortened by beta oxidation to the C22 DHA. Only peroxisomes appear to deal with the beta oxidation of C24 fatty acids. For C22 and especially C20 its not quite so clear cut.

On that basis I'm willing to go with peroxisomes as the main site of DHA catabolism, grudgingly and without hard data. Peroxisomal degradation is particularly difficult to justify from the simple FADH2:NADH ratio because DHA has so many double bonds that it's not going to drive reverse electron transfer through complex I. But it might be too fattening of course...

While searching I came across this study:

Enhanced Peroxisomal beta-Oxidation Is Associated with Prevention of Obesity and Glucose Intolerance by Fish Oil-Enriched Diets

which provides a number of points which need discussion, but for today I'm looking at following the reference trail back through DHA and peroxisomes.

The trail is good at level one backwards, with a nice paper on reagent grade DHA gavaged in to rats, but beyond that it drifts off into partially hydrogenated fish oil (goodness only knows what that contains but it undoubtedly induces peroxisome proliferation) and beyond that in to very long chain mono unsaturated fatty acids which do the same thing but neither helps me with DHA/EPA catabolism.

So I'll just start with the DHA gavage paper today

Docosahexaenoic acid shows no triglyceride-lowering effects but increases the peroxisomal fatty acid oxidation in liver of rats

The biggest problem with it is that for a lot of the work they were using group sizes of three rats. You don't do stats with n=3 group sizes, so I see it more of a proof of concept paper.

Rats were ad-lib fed semisynthetic diets (experiment I) +/- added cholesterol (experiment II). They were also gavaged with 500mg/kg, 1000mg/kg or 1500mg/kg of pure DHA daily for 10 days. Controls got nothing or palmitate 1500mg/kg/d. I'll come back to experiment III later.

Control rats grew at normal rat growth rate. DHA at 500mg/kg increased weight gain over the 10 days, 1500mg/kg did not, giving a comparable growth rate to controls. Using 1000mg/kg/d varied in effect but that's probably due to n=3 group sizes. Palmitate at 1500mg/kg/d is not obesogenic (well, whodathunkit?).

These are the numbers, relevant weight gains outlined in red:

Experiment III is even more interesting. Here they fed the rats ad-lib on a then-current 1993 style fairly high quality crapinabag, possibly something a bit like 5001. They gavaged EPA as well as DHA and had palmitate as control, all at 1000mg/kg/d, there was no untreated control group. This time we have n=5 rats. From the blue square palmitate gave 33g weight gain, DHA 52g and EPA 58g over 10 days. I particularly like this as DHA might be peroxisomaly directed but EPA, being shorter, less so. I get the impression this is not "all or nothing". DHA looks as if it might simply go through mitochondria if there is just a little of it around. If there is a lot it around it induces peroxisome proliferation and peroxisomal beta oxidation. Putting double bonds through mitochondria should produce fat gain, through peroxisomes less so. If you have my biases. Of course we have no idea re fat gain vs muscle gain in these rats.

The thing which struck me is how neatly you can control weight gain by choice of dose of DHA and by choice of background diet. I like it. Rats are so like people.


It's also interesting to look at Table 5

The column of interest here is outlined in red again. This is the ability to oxidise palmitoyl-CoA in the presence of potassium cyanide. Because KCN completely blocks the respiratory chain at complex IV any oxidation of palmitate in its presence is exclusively within peroxisomes. Increasing doses of DHA increase peroxisomal palmitate oxidation. Given high enough DHA ingestion peroxisomal activation appears able to over ride the weight gain effect of low dose DHA.

Summary: Low dose DHA causes increased weight gain in growing rats at 500mg/kg/d. At 1500mg/kg/d it doesn't, almost certainly through peroxisomal activation.

Adding DHA or EPA to a particularly healthy low fat/high complex carbohydrate diet might make the weight gain worse. In a rat.

Does this mean anything for humans?

Perhaps if you are going to take fish oil, take lots. Or, better still, none at all.

Peter

Oh, and, as far as I can see, no one has ever taken radio-labelled DHA and fed it to isolated peroxisomes or isolated mitochondria and looked at labelled metabolite or CO2 production. The test fatty acid has always been palmitate. Which is odd.

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 HPLC 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