Protons (75) Tucker; Speakman; Astrup and linoleic acid. And insulin sensitivity

Tucker has a podcast episode in which he chats to John Speakman about obesity. It's one of the more interesting podcasts I've listened to in many years.

Ep. 22: John Speakman—What Causes Obesity?

A very large part of the core discussion is contained within this paper, a massive collaboration, with Speakman as first author:

Total daily energy expenditure has declined over the last 3 decades due to declining basal expenditure not reduced activity expenditure

Basically total daily energy expenditure in the studied populations is down slightly over the last 30 years, despite daily activity energy expenditure going up. This means that basal metabolic rate must have dropped.

Which, of course, begs the question of what might cause basal metabolic rate to fall.

The answer is not obesity.

There are certain groups of people who *do* have a decreased BMR, the most obvious of whom are the post-obese.

The post-obese, like the pre-obese, come with a cluster of abnormalities the two most prominent of which are an enhanced insulin sensitivity and a defect in fat oxidation. And sometimes a depressed metabolic rate, especially BMR. 

To me, the enhance insulin sensitivity is causal, the impaired fat oxidation is secondary. The decreased metabolic rate is simply a longer term downstream effect of chronic under supply of calories to metabolism.

Aside: I haven't discussed it yet but, obviously, pathological insulin sensitivity should also show as an exaggerated ability to over-store fat under peak insulin effect. This shows rather nicely under an hyperinsulinaemic euglycaemic clamp in Astup's lab. See top panel of Fig 2. But currently I'm mostly thinking about fasting conditions. End aside.

So. The core feature of pre or post obesity following on from the pathological insulin sensitivity is a decreased ability to oxidise lipid and a facilitated ability to oxidise carbohydrate. The RQ should rise.

What would happen if you took eight apparently healthy men and fed them, for a week, a complete diet providing 2% PUFA then switched them to a 10% PUFA diet for another week, as a crossover study?

This is the paper, from 1988:

Polyunsaturated:Saturated Ratio of Diet Fat Influences Energy Substrate Utilization in the Human

You can clearly alter the RQ under fasting conditions, on a fixed food quotient diet, simply by altering the dietary fat from 2% of calories as PUFA to 10% PUFA, switching palmitate in or out to balance the PUFA, which was mostly linoleic acid. MUFA were kept constant, as were all other macros.

Within seven days this happened to the fasting RQ values.

Obviously there are three interesting subjects. One showed a decrease in RQ, suggesting enhanced lipid oxidation under linoleic acid. That's unusual. It is normal for linoleic acid to augment the thermic effect of food because it is preferentially oxidised but that is finished well before an overnight fast is finished. Hard to say what was going on with that subject. It wasn't a hospitalised study but all food was provided by the investigators. File it under odd.

The rise in RQ, signifying a change away from lipid and towards carbohydrate oxidation while fasting, was (pax the exception) ubiquitous across all other subjects, but in two subjects there was such a rise in RQ that the investigators seriously considered that there might be a problem with their measurement system. There wasn't. Their comment:

"Although a fasting RQ of 0.9 is unusual, reanalysis of the calibration parameters of the respiratory gas exchange system obtained prior to tests on these subjects revealed no abnormality in analyzer response. No reason for rejection of these RQ values could be determined."

Clearly 10% of LA in the diet moves almost all subjects towards a "pre-obese" phenotype. In two of the eight this move was dramatic. It seems very, very likely to me that these two individuals are at serious risk of obesity in an omega-6 rich environment. Follow up weights over the years would have been lovely but was not remotely the purpose of the study.

You can, within seven days, convert normal people in to pre-obese people, as viewed from metabolic substrate oxidation perspective.

All you have to do is make sure they are eating 10% of their calories from linoleic acid.

Some people will get bitten by this feature of linoleic acid more rapidly than others.

Eventually the whole population will.

Thank your cardiologist.

Peter

Addendum. The world is full of U shaped curves. Adding linoleic acid to the diet causes an initial excess insulin sensitivity. This distends adipocytes. As adipocytes distend they increase their basal lipolysis and release FFAs which cannot be suppressed by insulin. This, at some point, appears to normalise fasting insulin sensitivity at the cost of distended adipocytes, ie obesity, and chronically elevated FFAs. On a starch based diet the high level of post prandial insulin needed to overcome the still (unsupressable) FFA induced insulin resistance at peak absortption will sequester more lipid in to adipocytes, from where they will again leak, via basal lipolysis, leading to frank insulin resistance, hyperinsulinaemia and metabolic syndrome.

Under fasting conditions the pathological insulin sensitivity activates malonyl-CoA formation and the subsequent inhibition of CPT1 mediated entry of fatty acids in to mitochondria. This would, if it occurred in isolation, simply lead to hypometabolism unless enough glucose alone was available to run metabolism. However, it doesn't happen in isolation. It happens combined with obesity, which increases the supply of FFAs irrespective of insulin sensitivity. All that is needed is to elevate FFAs high enough to get adequate substrate in to mitochondria (there is not 100% inhibition of CPT1) and enough lipid derived ROS can then inhibit insulin, reactivate CPT1 and restore metabolism. Hence obese people have high metabolic rates.

The crux comes with conventional dieting. As adipocytes shrink the supply of FFAs from basal lipolysis drops, insulin sensitivity is restored and people get right back to where linoleic acid takes them: obtunded fat oxidation, carbohydrate dependency and hypometabolism. The classical post-diet hungry person.

Why is BMR falling in the developed world despite obesity being rampant? Because everyone is being drugged with linoleic acid to become obese and no one wants to be fat. The more you resist obesity, the more your caloric restriction shows as decreased BMR. The BMR is falling in response to Weight Watchers, Slimming World etc. People are not as fat as linoleic acid "wants" them to be.

Ultimately obesity "fixes" the pathological insulin sensitivity from linoleic acid on both fronts, at the cost of weight gain. But it's not a real fix, it's a sticking plaster and we call it metabolic syndrome.

End.

Foie gras (9) Adipocyte ROS

Time to look at the Vaughan mouse weight gains and actual diet compositions. Here is a recap of the weight gains:

and here is Table 1 with the percentages of energy from linoleic acid added in red by myself:

It's clear that the saturated fat group were only fed 6% of their calories as insulin sensitising linoleic acid. In addition to this relatively low amount of LA, the stearic acid in the saturates has an high F:N ratio and will in part offset the low F:N ratio of the LA component.

If adipocytes resist insulin they stay small.

The oleic acid group were fed 7% of calories as LA but with only 12% of calories as saturated fat there is nothing to oppose the insulin sensitisation effect of LA so they gained a significant amount of weight.

The high (35% of calories) LA diet is uncoupling, produced low levels of ROS as a consequence, and so limited weight gain. Given long enough it would normalise bodyweight.

The high omega 3 diet contained an obesogenic level of LA coupled with an uncoupling level of alpha linolenic acid, though putting a number to the level of ALA needed to uncouple is difficult, but it is lower than the level of LA needed. This is another combination which I expect, given long enough, would normalise body weights. 

I feel that some explanation is needed as to why the LA/ALA 18:3n3 diet mice, and to some extent the high LA diet mice, weigh more than the saturated fat diet mice, to the point that the 18:3n3 diet looks obesogenic.

There are, initially, two effects of fatty acids with multiple double bonds. The first is the reduction in RET through complex I which itself has two effects. Under peak insulin action there is reduced negative feedback so adipocytes distend more than they should. Second is that during fasting, when fat is the primary fuel, RET should occur to limit glucose utilisation in order to spare glucose for the brain/hypothalamus and so limit hunger. With the blunted RET from LA/ALA more glucose is used by muscle etc so more food must be eaten, so breaking the fast, in order to keep glucose levels adequate to limit hunger. Much of this extra food then gets stored.

These two together put fat in to adipocytes and demand more food intake. This is the classic situation under D12492.

This is also likely to be the initial situation when using 35% LA or a mix of 11% LA with 23% ALA in the period before uncoupling becomes established.

The second effect is via uncoupling.

There will be weight loss in these latter two diets, but only once UCP-1 is activated in white adipocytes to lower delta psi and so reduce insulin signalling. At that point adipocyte FFAs can either be oxidised to release heat in situ (beiging of WAT) or transferred to BAT where high levels of UCP-1 can oxidise them to release heat in bulk.

This concept suggests that, by 14 days, the mice on the "18:2n6" diet would be in weight loss and should have low ROS generation due to uncoupling, after an initial weight gain.

The effect should be more marked in the "18:3n3" group, ie an higher initial weight gain, then incipient weight loss by 14 days.

This is why I like the Schwartz data, daily resolution of food intakes and fat mass changes allow you look at things more mechanistically. It would have been nice to have these data from the current pro-linoleic acid study but thats not what the study was all about.

Here are the mRNA data for inflammatory gene expression in adipocytes in vivo (or immediately post euthanasia!), which I am taking to be a surrogate for ROS generation:

 

From the ROS perspective the SFA adipocytes are generating ROS by RET, so are limiting insulin signalling-induced lipid droplet distension. The mice are slim. And healthy.

The 18:2n6 mice are uncoupled, have low ROS due to this and are actively losing weight after an initial gain. Same for the 18:3n3 mice.

The HF fed mice (9.6% LA, obesogenic, failing to limit insulin signalling) have low ROS because they are failing to generate enough of them via RET to limit caloric ingress, ie have "pathological" insulin sensitivity. "Healthy" insulin sensitivity, through healthy ROS, is shown by the SFA group. The HF group are simply sequestering calories in to lipid droplets without oxidising them. Here weight gain is on-going but there is no issue with high ROS because they are effective at sequestering calories. Except...

Now the HF fed mice are really interesting. They have levels of inflammatory gene mRNA expression comparable to all of the other groups, including the SFA group (p>0.05 for the comparison) but look at their MPO activity, an indicator of active inflammation. I've rearranged chart G so all columns are on the same scale:

All groups of mice have comparable levels of inflammatory gene mRNA expression (pax the SFA fed group) but only the HF group have actively inflamed adipose tissue.

Why?

We can say that generating mRNA from pro-inflammatory genes alone is not sufficient to activate the inflammatory cascade to the extent of activating the myeloperoxidase system.

I have to ask myself what, exactly, is the function of these genes we are looking at, within physiology, at the most basic level.

I would suggest that they might be to deal with normal ROS generated from normal metabolism. The SFA diet induces high levels of healthy ROS via RET. It generates a large, effective response in ROS mitigating genes. All other groups, at the 14 day mark of the study, have low levels of RET derived ROS, so low levels of mRNA from inflammatory (or rather mitigating) genes. Including the HF diet group.

What is different about the HF diet group is that they are, through Protons, unable to limit caloric ingress. As much of the excess calories as possible will be rendered in to harmless stored triglycerides but all that is needed to generate frank inflammation is the generation of a delta psi in excess of 170mV. This leads to ROS which are only in a small part derived from RET, ie are mostly pathologically derived.

I think the HF diet fed mouse adipocytes are doing this. There is tissue damage occurring and lipid peroxides are produced at levels which signal danger of serious injury and so macrophages move in to sort out the damage. Probably making incorrect assumptions about the source of the damage, leading to pathology. This appears, in this study, to be independent of the expression of what are considered, in this study, to be pro-inflammatory genes.

Activation of the myeloperoxidase system, as observed in the current study, is not a simple consequence of activating mRNA generation of inflammatory genes.

It just strikes me that expressing a gene and using its product may be greatly influenced by factors this study doesn't address.

So there are at least three descriptions possible for the state of ROS generation in the adipocytes of these mice. There are no simple linear relationships. You need some sort of framework to understand what is going on.

Protons.

Which makes me happy.

Peter

So you want some DHA? Chickens in Norway

Again via twitter, Tucker retweeted a thread which included the link to this paper

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil

Over the years I keep coming back to this plot

which comes from another paper:

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

It shows, very clearly, that in rats both linoleic acid and alpha linolenic acids are equally efficacious at suppressing the conversion of ALA to DHA. Given 1% of energy in the diet as ALA then copious amounts of DHA are produced, just so long as the LA proportion of calories is less than 2% of the total. Never mind the ratio. Avoiding PUFA will generate DHA from ALA provided a basic minute minimum of ALA is present in the diet, somewhere around 1% of calories. Over at the right hand side of the plot we can see that even drinking 12% of your energy intake as ALA will not generate significant DHA if you are up in the cardiological nirvana of 16% of energy as LA.

That is in rats.

Chickens superficially appear to be somewhat different.

Switching from soybean oil to a rapeseed/linseed oil mix in the diet increases DHA in the breast meat. Soybean oil gives 14% of lipid as DHA, rapeseed/linseed gives 21%. Of not very much fat in breast meat so the amounts are small in total, but still highly statistically significant.

So ALA in food bumps up DHA in muscle. Of chickens.

This is what the chicken diet looked like, with annotation to give the fat percentage of total calories and the LA and ALA percentages of total calories in the diet, crudely:

We can overlay the chicken diet very approximately, on the rat plot to get this:

which, not surprisingly, suggests that the DHA production in the "high" ALA diet (blue) is actually more influenced by the reduction in LA. At 4% LA we could equally have had ALA at 1% of calories and still got a lower value for DHA than we did by having LA down at 2% of calories.

Might this work for humans too? Could adding ALA to our diet improve DHA availability, with all of what that entails for improved brain development and cognitive function. Just by avoiding LA and maybe drinking a little (traditional) varnish? The study didn't ask this. Instead they fed the above chicken to some humans. Either omega 3 enriched or omega 6 enriched.

This actually dropped DHA in the participants' plasma phospholipids in both groups. Admittedly not by much, so the change was neither statistically nor biologically significant. But it dropped. I would hazard a guess that the chicken simply displaced a richer source of ready-formed DHA from the diet, the tiny amount in the breast meat would do nothing per se. I believe Norwegians consume a certain amount of fish, unless they're given free chicken to eat. 

The rise in EPA must have made the authors happy that something positive came from all of that work. But I don't think you can build a brain out of EPA, DHA looks to be the molecule for that.

Ultimately pre-formed DHA does not look to be necessary for brain development if you have a modest supply of ALA from (grass fed, possibly large) animals and avoid consuming significant amounts of LA. This appears to hold true for rats, chickens and I expect for humans.

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...

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 (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 (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!

The miracle of safflower oil (3)

TLDR: PUFA in a mixed diet are obesogenic. PUFA under hypoinsulinaemic conditions are not. I doubt they get a free pass long term.



There have been some interesting snippets on Twitter recently, triggered by Diet Doctor's discussion about vegetable oils here. Perhaps the most controversial quote is this one:

"Disclaimer: Vegetable oils are routinely recommended as “heart healthy.” There is high-quality evidence demonstrating that replacing saturated fat with vegetable oils reduces LDL cholesterol levels. But at this point, there is inconsistent evidence whether this translates into fewer heart events or lower rates of cardiovascular mortality".

This is absolutely incorrect for people with pre existing cardiovascular disease as it was found, in a randomised control trial using safflower oil, that increasing vegetable oil for bulk calories will increase all cause mortality (p = 0.05), cardiovascular disease mortality (p = 0.04) and coronary heart disease mortality (p = 0.04). Mortality is an utterly hard end point and particularly the all cause mortality is an end point which cannot be argued with.

Let's rephrase that: in the context of a mixed diet in people with established heart disease vegetable oil (from safflower seeds) is going to increase you risk of death, especially from cardiovascular disease.

The main issue is to ask whether this still applies under low carbohydrate eating conditions. Given the role of insulin in CVD this is far from certain. But context will be crucial here and who would like to be the guinea pig?

The interesting twitter conversation goes like this:

Dr Westman: "In 20 yrs of clinical research and practice using LCHF/keto, I’ve never even mentioned reducing omega 6s, and it works wonderfully. Just cutting carbs gets the job done!"

Tucker: "I disagree, but @drericwestman is an excellent physician who does great work. This is more about determining ultimate causation so we can address people who can't just go low-carb, which is most of the planet".

I think both people are correct. I came to LC because it works. Over decades I've read studies where it works pretty much invariably on a group basis and studies from the mainstream usually advise progressively increasing carbs if they want to knock low carb and secure future funding. You have to pay the mortgage.

I am perfectly willing to accept that consuming carbohydrate in a rapidly absorbable form will overwhelm the liver's ability to protect the systemic circulation from hyperglycaemia so will require systemic hyperinsulinaemia to control that systemic hyperglycaemia. In particular hyperinsulinaemia comes with its attendant problems (ie most of medicine) but obesity only occurs when hyperinsulaemia is marked enough to overcome insulin-induced insulin resistance. I have no doubt this can occur without PUFA but I think it is massively easier in the presence of PUFA, which delay normal insulin-induced insulin resistance in the immediate post prandial period.

The role of polyunsaturated fatty acids is to stop adipocytes developing insulin resistance by limiting ROS generation. Combining hyperinsulinaemia with hypersensitive somatic cells is a recipe for maximising lipid storage in adipocytes and simultaneous packing lipid in to muscles, pancreas and anywhere else you care to imagine that sprouts an insulin receptor (most brain cells excepted).

Eating a low carbohydrate diet side-steps the problem by reducing absolute levels of systemic insulin. Down a set of unrelated rabbit holes I'm looking at what might control hunger under LC eating and PUFA may have some influence on this, but it is clearly a small effect when compared to the same dose of PUFA combined with an insulogenic diet.

Ultimately at low levels of insulin it doesn't matter how well or badly adipocytes respond to/resist insulin. There is so little insulin about that FFAs and ketones are able supply the body's energy needs, given some excess fat (especially visceral fat) available to be utilised.

Back to long term speculation: Do PUFA matter for non-insulin reasons on a low carb diet? Recall that López-Domínguez et al used a low calorie semi-starvation model (which is a partial mimic of low carb eating) to look at longevity in rodents (post is here). It certainly matters under their study conditions but the effect is small enough that I doubt it would show in any way for someone at 40 years of age under a year or two's exposure to a high PUFA but low carbohydrate diet. For those of us in this for the long haul it's much easier not to be the test case and PUFA avoidance seems prudent to me.

And I am undoubtedly still a low carb eater.

Peter

Stearic acid: Skinny-skinny vs skinny-fat

This paper came up in comments to the last post:

Dietary Stearic Acid Leads to a Reduction of Visceral Adipose Tissue in Athymic Nude Mice

I think we can say that, at least in athymic nude mice (which do not seem to be derived from the C57Bl/6 strain), omega 6 PUFA do not cause obesity when compared to either a low fat or high stearic acid synthetic diet (ie the low fat arm is equally synthetic, not more "food-like" ie not chow). At least when you look at total body weights:

So omega 6 PUFA appear to get a free pass here. The actual composition of the diets is in Table 1 of this previous paper and all four contain generous amounts of starch and equal amounts of sucrose:

Dietary Stearate Reduces Human Breast Cancer Metastasis Burden in Athymic Nude Mice

However if you dexa scan the mice you find that the low fat, corn oil and safflower oil groups all have reduced lean mass (probably muscle) and increased visceral fat mass compared to the stearate group. A picture is worth a thousand words so here are some postmortem images with the size of the inguinal fat pads outline by the authors of the paper (no need for me to doodle on this one!). Fig 3:

I really like these images.

Now, cavenewt questioned the relevance of weight/fat alterations from stearate compared to other potential health effects, particularly its affect on cancer metabolism.

The third paper from this same group is

Prevention of carcinogenesis and inhibition of breast cancer tumor burden by dietary stearate

I've been through all three papers and searched on "insulin". The group appears to have no concept that insulin has anything to do with adipocyte size or cancer progression.

A slight handicap when it comes to insight.

In the stearate-visceral fat paper there is a single measurement made of plasma insulin/glucose. Insulin does not vary between diet groups but glucose is significantly lower in the stearate group. I have been unable to work out if the measures were fasting or fed, or even what time of day the samples were taken (ie when the mice were killed). I think that with glucose values in to 200-250mg/dl range these were probably "fed" glucose and insulin levels. The paper does not give us the measured insulin levels, merely that there was no statistically significant difference between groups. But insulin levels come with such huge standard deviations that getting a p value below 0.05 with small group sizes is not going to happen. A ns result does not automatically mean that there were no differences.

Of course a single insulin measurement at one terminal time point tells us nothing about the long term 24h exposure to insulin of the mice, of their adipocytes or of their cancer cells.

So we have to, once again, look at the significance of the changes in fat distribution to attempt to gain insight in to overall insulin exposure. I spent quite some time looking at visceral fat and its significance early last year in this post:

On phosphorylation of AKT in real, live humans. They're just like mice!

and on how stearate might avoid systemic hyperinsulinaemia here:

Dairy and diabetes

Visceral fat is a surrogate for chronic hyperinsulinaemia, particularly fasting hyperinsulinaemia. While I consider non-inflamed visceral fat to be completely benign, or even beneficial for controlling the hunger of fasting, the insulin which maintains that visceral lipid storage is not benign. Chronically elevated insulin (or, more accurately, insulin signalling) should drive both visceral fat storage and xenograft tumour growth in the mice. Probably in humans too.

Happy Solstice and assorted mid-Winter celebrations. If you live in the northern hemisphere that is. Not that I envy those with a Solstice-on-the-beach-without-wooly-hats-and-gloves situation!

Peter

More on drinking varnish

This paper is a gem.

Reducing the Dietary Omega-6:Omega-3 Utilizing α-Linolenic Acid; Not a Sufficient Therapy for Attenuating High-Fat-Diet-Induced Obesity Development Nor Related Detrimental Metabolic and Adipose Tissue Inflammatory Outcomes

What did they do? They fed rats chow or they fed them on one of four other diets enriched in PUFA. The extra PUFA were based around various mixtures of linoleic acid with alpha-linolenic acid, some  were mostly corn oil, some were slanted towards varnish (flax/linseed oil). Total 18-C PUFA made up 9.4% of calories, ie was obesogenic, and this was identical for all of the high fat diets. Overall macros were identical in all of the high fat diets too. There was no sucrose. The rats were fed ad lib.

Here is the link to Table 1 which lists the compositions, it's too big for putting it up as a jpeg. Just look at how utterly fair the composition of the high fat diets were. Even if the absolute amount of linoleic acid in the lard is not accurate, there will be a consistent error across the diets and the results stay plausible. My only complaint is that there was no group where the omega-3 lipids predominated in the diet PUFA, a 50:50 mix was the maximum. Whereas the maximum omega-6 fed group got essentially all of their PUFA from omega-6 PUFA.

The second excellent feature is that the rats were neither semi-starved nor forcibly overfed. Rats are not people. They cannot be verbally asked to overeat to maintain a stable bodyweight nor to calorie restrict to lose weight. They will simply eat until they are no longer feeling hungry. If that happens while they are svelte or not until they are morbidly obese, the rats don't care.

What happened?

Almost nothing. The chow fed rats, with around 3.5% of calories as PUFA, stayed at a reasonable weight. The obesogenic high fat diets (ie nearly 10% of total calories as PUFA) each caused almost exactly the same progression of obesity:

Why almost?

Can you see that the open squares group gain weight slightly more slowly than the other PUFA diet groups? This shows between week six and week 17. The two hashtags mark out a couple of time points where this achieved statistical significance. This slightly less obese group of rats is the group which ate the least alpha-linolenic acid, the most linoleic acid. This suggests that omega-6 PUFA are less fattening than omega-3 PUFA. I like that. Protons likes that.

The effect was fairly small and only shows as an early facilitation of weight gain. By the end of the study the rats and their adipocytes were all about as fat as they were going to get on 9.4% of calories from any family of PUFA.

You can easily hide this effect by under feeding (pair feeding to the same calories as a chow fed group or arbitrarily reducing overall caloric availability) or overfeeding (paid humans or intragastric cannula over-fed rats). If you are an omega-3 lover this can be necessary. But, given a decent study, it shows.

Consuming the 18-C omega-3 rich linseed oil/flax oil/varnish may not make you terribly much fatter than corn oil will eventually make you, but it should get you there quicker. The situation for EPA and DHA is different. Oxidising these will increase the cytoplasmic NADH:NAD+ ratio via peroxisomal oxidation (bad) and give reasonable mitochondrial function from oxidising the residual saturated caprylic acid C-8 (good), which is the normal fate of very long chain fatty acids of any ilk.

Executive summary: Omega-3 18-C fatty acids are more obesogenic than omega-6 18-C fatty acids. The effect is small but real, it might show better if all of the PUFA were alpha-linolenic acid rather than to 50:50 mixture used. It still makes me happy.

Peter




The Protons view (skip this if you're fed up with hearing it over and over again)...

I consider that the mitochondrial oxidation of PUFA will always show as increased peak insulin sensitivity. The cost of that increased insulin sensitivity is fat gain. The fat gain eventually eliminates any benefit from the initial increase in insulin sensitivity. Forced manipulations of the food intake downwards will preserve the intrinsic insulin sensitivity at the cost of chronic hunger. So when high PUFA-fed lab-rats are "pair fed with the chow group" the PUFA rats will look really good, metabolically. The converse, encouragement to overeat, based on avoiding "accidental" weight loss (weight loss is a huge confounder in studies of hepatic lipid accumulation from almost any intervention, PUFA included) by weekly weighing to maintain weight will mask any benefits from saturated fat induced adipocyte insulin resistance. Stacking the deck is crucial to the result you want to get.

Systemic fructose is important

******************************************************************
TLDR: Be cautious of anyone who tells you fructose metabolism is limited to the liver.
******************************************************************

Fructose uptake by the liver is saturable. Drinking two cans of soda sweetened with high fructose corn syrup produces a peak plasma concentration 17mmol/l. Yes, 17mmol/l. On average.

Direct spectrophotometric determination of serum fructose in pancreatic cancer patients

Unfortunately the methods section makes no sense at all, so we have no idea how much fructose was actually consumed:

"In 3 of these subjects, intravenous access was obtained in an antecubital vein, and additional blood samples were taken at baseline and 15, 30, 45, 60, 90, and 120 minutes after ingestion (93 minutes) of two 75-mL cans of a proprietary soda, for determination of serum glucose and fructose concentration. Each 40-oz can of soda contained 75 g of high-fructose corn syrup, which consisted of 55% fructose and 45% glucose as constituent monosaccharides, equating to 41.25 g fructose and 33.75 g glucose, respectively".

Go figure. Two 40oz cans of soda? Some big cans there, even by USA standards!

Anyway, this is the graph they produced:

This next group seems to have managed to write an interpretable methods section but missed peak fructose levels by only sampling at 60 and 120 minutes.

Consumption of rapeseed honey leads to higher serum fructose levels compared with analogue glucose/fructose solutions

Ingesting 75g of neat fructose, as a solution, gives a blood concentration of 130mg/dl, ie they measured just over 7.0mmol/l in real units, at one hour post ingestion.

So fructose gets past the liver and will be taken up by any cells with GLUT3s on their surface. Whole body.

Like adipocytes.

This is a nice paper covering a lot of bases about how adipocytes deal with the fructose they are flooded with every time you down a couple of cans of soda. Or apple juice or.......

Metabolic fate of fructose in human adipocytes: a targeted 13C tracer fate association study

What do adipocytes do with fructose?

They don't oxidise much of it.

They don't convert much to lactate.

They do convert most of it to palmitate and a little to oleate.

They store the oleate.

They release the palmitate as FFAs.

You can't tell from the study how much this palmitate raises systemic FFAs because the study was being performed on "adipocyte-like" cells in cell culture. But, assuming that in most cases fructose would be co-ingested with glucose, you have here the classical situation of elevated free fatty acids, in combination with elevated glucose, in combination with elevated insulin.

This is my definition of metabolic syndrome. The hyperinsulinaemia will, until you become diabetic, eventually control the hyperglycaemia. It may well suppress the elevated FFAs. The glucose and FFAs will be pushed* in to any cell which will respond to insulin.

*Nothing is actually "pushed". Insulin facilitates diffusion (GLUT4s) and maintains a diffusion gradient by removing glucose to glycogen and FFAs to triglycerides.

The liver will be right in the frontline for accepting these FFAs, which should be in adipocytes, and experiencing sustained high levels of insulin (to control glycaemia) will make the hepatocytes hang on to those fatty acids. This is in addition to any intrahepatic trigycerides from fructose-driven DNL. Overall we end up with massively calorically overloaded liver cells. This is the prerequisite to hepatic steatosis and all that is then needed for the generation of inflammatory changes is a source of omega six PUFA. There is a desperate need for liver to say "no" to any more calories. It does by resisting insulin. Which it does by generating ROS. If those ROS meet linoleic acid, it's welcome to 13-HODE, 4-NHE and any other peroxide you care to dig up. These PUFA derivatives do cause insulin resistance per se (as well as 13-HODE stimulating cancer growth), but to me they are just an amplification system derived from what is already happening at the "front end" of the mitochondria... ROS generation by RET, essential to limit grossly excessive caloric ingress.

Peter

Follow on to Tucker's post on PUFA in rats

Tucker posted an excellent discussion of this paper on his blog. Go read it:

Fat Quality Influences the Obesogenic Effect of High Fat Diets

The basic conclusion is that feeding rats a high fat diet makes them fat. If it is PUFA based, including a generous amount of omega 3 alpha linolenic acid, it will cook their liver (figuratively speaking... in actuallity it converts their liver to being full of peroxidised PUFA, en-route to cirrhosis). I have an anecdote-type post on the problems of being married to a cardiologist if you happen to be alcohol addicted somewhere. I really ought to dig it out and hit post.

So. The problems with the paper:

The rats on the PUFA diet, with the gross fatty livers, were less obese than the lard fed rats, had better lean body mass percentage and much better brown adipose tissue hypertrophy and fat oxidation.

The bottom line: If you want look slim and well muscled in your coffin then a safflower oil diet with a heavy dash of varnish might be a good choice...

How come?

The paper was not looking at insulin levels or insulin signalling so it doesn't provide the data we need to come to any conclusions but it has resonances to the comment Zoran made on the previous post.

The Protons Credo (believe if you so wish!) for the situation:

PUFA, of a carbon chain length which targets them for mitochondrial oxidation, input less FADH2 at mitochondrial electron transporting flavoprotein dehydrogenase (mtETFdh) than do saturated fats or MUFA. This lack of FADH2 input limits the ability to reduce the CoQ couple and facilitates electron flow down the electron transport chain (ETC) and so limits the generation of reverse electron transport through complex I. This damped RET limits the ROS generation (superoxide and H2O2) necessary to initiate insulin signalling under fasting and to limit excessive insulin signalling in the fed state.

So on a whole body basis PUFA maintain insulin sensitivity. Insulin acts, rather well, under PUFA compared to under saturated fat, in the fed state. It works less well in the fasted state.

A fed, insulin sensitive animal will do two things of interest on a medium carbohydrate, generous fat diet. It will utilise glucose easily in muscles to burn calories and it will continue to use glucose in adipocytes to esterify FFAs with glycolysis-derived glycerol, to store fat.

So the Protons thread expects insulin sensitivity to cause fat accumulation because of maintained insulin sensitivity in adipocytes at high levels of insulin signalling. The cost of this insulin sensitivity is obesity.

PUFA = obesity, soybean oil is the best, they used safflower here.

Slight aside: The insulin resistance associated with obesity is nothing to do with insulin per se. It is triggered by the fact that very large adipocytes leak free fatty acids irrespective of insulin levels. At elevated FFA levels more insulin is needed to translocate GLUT4s than at low FFA levels.

Back to the rats.

The lard fed rats are the most obese. The PUFA fed rats the least obese.

The lard fed rats are on about 10% of their calories as PUFA in their diet. They are probably almost as fat as a 10% PUFA diet would like them to be, ie their adipocytes are almost as distended as a 10% PUFA diet dictates. The rats are almost as fat as they need to be. They are doing this on 380kJ per day. Because the rats are only allowed a total of 380kJ of energy per day. Did you pick that up in the methods?

The PUFA fed rats want to be truely, grossly obese, much more so than the lard fed rats do, because they are on somewhere between 50% and 60% of their energy intake as PUFA. But there, in the hopper, is that same old 380kJ per rat per day. It doesn't matter how much your adipocytes are crying out for more fat, how empty they feel, how hungry they tell your brain to feel. There, in the hopper, is 380kJ.

These rats are intensely insulin sensitive because their adipocytes are "empty" compared to how thery would like to be. They are "starving" compared to how they would like to be. Their muscles respond to insulin's anabolic effect and I'd be willing to bet their growth hormone levels are through the roof and IGF-1 through the floor (another post there, GH, IGF-1 and starvation). Insulin is going to be low because any glucose released from the liver is easily utilised in the fed state. In the fasting state insulin fails to act effectively so that, while FFAs may be the same as in the lard fed rats, we know (from Figure 2) that lipids are being oxidised much more rapidly on a 24h basis.

PUFA sensitise adipocytes to insulin. Given the choice the animal will eat until obese and become insulin resistant due to adipocyte distension. Combine PUFA with starvation and insulin sensitivity will be maintained. Or enhanced.

Just the Protons view. Any other explanations welcome.

Peter

Of course people should ask how the action of PUFA compares to the action of metformin. They are superficially similar. That might need more doodles I'm afraid!

Linoleic acid and Tuberous Sclerosis

**********************************************************
TLDR: I don't like linoleic acid much.
**********************************************************

Tuberous Sclerosis (TS) is a genetic disease which affects mTOR signalling and predisposes to many problems, one of which is early onset kidney tumours. People with TS also have a tendency to suffer from intractable epilepsy so, almost by accident, a number of them have had their tumours closely monitored while eating a deeply ketogenic diet for the epilepsy. Does the KD help slow tumour growth?

No.

To look at this in more detail a group in Poland has used a TS rat model and tried to manage the disease with a KD. This is the study:

Long-term High Fat Ketogenic Diet Promotes Renal Tumor Growth in a Rat Model of Tuberous Sclerosis

It is a very interesting paper. All rats were euthanased at 14 months of age, having spent differing periods of time eating a close derivative of the F3666 ketogenic diet. The longer the rats had spent on this diet, the more aggressive the renal tumour progression was found to be, especially in the rats which ate it for eight months (the longest duration).

Other than this there were some additional interesting findings. Insulin and glucose did (almost) exactly what you would expect:

   

What is more interesting is that growth hormone increased progressively with duration of time spent on the KD:

This was noted by the authors and was considered as one of the potential explanations of the increase in tumour burden after 8 months on KD:

"Likewise, it has been shown that the growth hormone activates the MAPK pathway, thus its overproduction in the ketogenic groups may also boost ERK1/2 phosphorylation. We believe that HFKD induced the ERK1/2 activation results as a cumulative effect of the renal oleic acid accumulation and the systemic growth hormone overproduction".

I'm not convinced by the oleic acid idea but no one would argue against identifying elevated growth hormone as a stimulant for tumour growth.



Overall we have progressively falling levels of both glucose and insulin, with a progressively rising growth hormone concentration, over the eight months of a ketogenic diet. I asked myself if there might be an explanation for the nature of these reciprocal changes, before thinking about the tumour growth.

Looking at the insulin-glucose levels we can say that insulin sensitivity increased with time on the ketogenic diet, using the surrogate HOMA score based on the product of insulin and glucose. That's despite a concurrent tripling of GH levels, which should induce insulin resistance.

The obvious concept is that GH was being used to maintain normoglycaemia in a set of rats which were developing progressively increasing (pathological) insulin sensitivity and might, theoretically, have become hypoglycaemic on a very low carbohydrate, very low protein diet. That is, despite having been on a high fat diet, they failed to maintain adequate (ie physiological) insulin resistance to spare glucose for the brain.

Does GH sound like a metabolic solution for the problem of pathologically increasing insulin sensitivity? Pathological insulin sensitivity: Is anyone thinking linoleic acid? Well, I am (now there's a surprise).

The rats were raised on a standard chow of un-stated composition before switching to their KD. It seems a reasonable assumption that the chow was relatively low in fat. Exactly how much linoleic acid was supplied is unknown but other rodent chows I've seen described or analysed tend to provide about 2% of calories as linoleic acid.

The F3666 derived diet looks, depending on the lard composition, to be in the region of 18% omega 6 PUFA. That's high (yes, another rodent study has shown that mice develop NASH on this diet, no surprise there).

The rats were raised initially on a starch based diet so their juvenile adipose tissue would probably be composed of DNL derived saturated and monounsaturated fats, supplemented by a little PUFA from the diet. Transition to F3666, which provides approximately 18% of calories in the form of linoleic acid, generates a metabolism much more dependent on linoleic acid. The younger the rats were when they switched to F3666, the less "normal" adipose tissue they would have had available and the more rapidly they would end up with adipose tissue (and plasma) high in linoleic acid.

Rats on true ketogenic diets do not become obese, even on F3666. So we have slim, omega 6 fed rats. Small adipocytes, no excess FFA release, no insulin sensitivity differential between adipocytes and the rest of the body. There is nothing to over-ride the insulin sensitising effect of linoleic acid. Both adipocytes and the rest of the body become progressively more insulin sensitive mediated through linoleic acid. They don't become obese because insulin stays so low due to the lack of carbohydrate and protein. The excess insulin sensitivity only kicks in gradually because their pre-stored, chow derived adipose tissue provides a supply of physiological FFAs which can act as a buffer to the sensitising effect of 18% linoleic acid for a while.

Glucose falls progressively due to the development of progressively increasing pathological insulin sensitivity, linoleic acid induced. GH may well be a stress response to maintain normoglycaemia under these conditions. The GH may or may not be acting as a tumour promoter, but we cannot ignore the role of linoleic acid in its elevation.


Now the tumours.

We all remember Sauer's rats with their xenografts which grew like wildfire as soon as he starved them? Yes. The tumours grew because they were exposed to linoleic acid released from their adipocytes under starvation. Linoleic acid is a precursor for 13-hydroxyoctadecadienoic acid, better known as 13-HODE. Sauer demonstrated that this was the problem very neatly, at the cost of extensive vivisection. I doubt anyone would be allowed to replicate his work today.

We have no idea of either the linoleic acid or the 13-HODE concentration in the plasma of the F3666 fed TS rats. It would be interesting to know. It might matter...

I particularly think it might matter because F3666 is going to be the "off the shelf" KD that a lot of researchers are going to use.....



At the end of the last post I mentioned that fact that any person who is currently obese through following conventional advice to replace healthy saturated fats with 13-HODE generating linoleic acid is probably carting around kilos of a tumour growth-promoting precursor. In Sauer's study all that was needed to release the linoleic acid was starvation. I would suggest that ketogenic eating might do the same, especially if it is based around saturophobic stupidity (think of kids in the USA with tuberous sclerosis on a "medical" ketogenic diet, or the rats in the above study). There is also anecdote on tinternet that patients of Dr Atkins did fine if they had CVD but those with cancer did badly. I find this plausible. They were obese because they were loaded with linoleic acid and they may well have followed an Atkins diet high in hearthealthypolyunsaturates. That's a good way to grow a cancer.

Sauer found a solution in the form of fish oil to limit tumour growth in his rats, most especially EPA. The very long chain omega 3 PUFAs activate g-protein coupled receptors to reduce lipolysis from adipocytes and activate fatty acid oxidation from the diet. VLC omega-3 fatty acids do not promote excessive insulin sensitivity via the Protons based FADH2:NADH ratio concept because they are specifically oxidised in peroxisomes, not mitochonria. The peroxisomes shorten them to C8 length and then pass this to mitochondria as caprylic acid which has a "palmitate-like" FADH2:NADH ratio of 0.47 which is fine for maintaining physiological insulin resistance.

You do have to wonder whether the benefits of fish/oil in a population loaded with linoleic acid might stem largely from this effect of limiting adipocyte release of that linoleic acid. An interesting idea.

I still find it breathtaking how much the lipid hypothesis of heart disease might have done to injure individual people exposed to its recommendations. Which includes much of the world.

Peter

Boiled mashed potatoes for miracle satiety?

The effects of potatoes and other carbohydrate side dishes consumed with meat on food intake, glycemia and satiety response in children.

With thanks to Mike Eades for the full text.

This is an interesting study. Given a meal of meatballs plus a choice of five different carbohydrate sources, a group of children ate a great deal less (in calories) of boiled mashed potatoes than of pasta, rice or either of two types of chips.

"The five treatment sessions consisted of ad libitum servings of (i) rice, (ii) pasta, (iii) boiled and mashed potato (BMP), (iv) baked French fries (BFF) and (v) fried French fries (FFF) with a fixed amount (100 g) of meatballs".

What did they find?

"... children consumed 30–40% less calories at meals with BMP (p less than 0.0001) compared with all other treatments, which were similar".

That's a LOT less calories! Potatoes seem to have some sort of magical satiety property. If you believe in magic. Table 1 gives an inkling of the problems with the study:

As you read through the cooking description you realise (red box) that the carbohydrates had very different amounts of added fat per unit carbohydrate and that some had butter (+/- added milk) while others had canola oil in varying doses. So when we look at Table 3 we have to realise that "CHO amount (g)" means an assorted mix of various fats and carbs:

We have to work back using Table 1 to find out what amounts of carbohydrate and fat were actually eaten and read the cooking details to find out what the fats were in each dish. Some arithmetic gives us this for what was actually eaten:

To my mind the trial here splits in to two. We have BMP, boiled mashed potatoes with 3g of carbohydrate per gram of butter, which is fairly well matched with FFF, chips deep fried in canola oil, with 2g of carbohydrate per gram of canola oil. Both are potatoes. Both provide a roughly similar ratio of calories/grams from glucose and fat. Both are relatively low carbohydrate per unit fat (compared to the other three meals, ie just in this study).

From the Protons point of view the relatively low carb BMP and FFF are supplying glucose from potatoes to drive complex I. However butter also supplies FADH2 at ETFdh, so generates a resistance within adipocytes (and elsewhere) to an excessive insulin facilitated calorie ingress during the period of maximal blood nutrient levels. When calories stop falling in to adipocytes, satiety kicks in. Using FADH2 this happens after eating 508 kcal. With FFF based on canola oil, ie potatoes steeped in 18 carbon omega 3 and 6 PUFA, the beta oxidation generates a much lower input at ETFdh (one less FADH2 per double bond) and so insulin sensitivity at peak nutrient uptake is maintained for longer, fat pours in to adipocytes for longer and almost twice as many calories are consumed (912 kcal) before satiety kicks in. I expect satiety to rise as blood nutrients rise. Not sequestering them in to adipocytes seems the best way to do this. More physiological insulin resistance. I'm guessing the brain does the actual sensing of both glucose and FFAs.

I like that. You can say what you like about the hypothalamus. I prefer to think about the adipocytes and their mitochondria as determining what gets done with food and hunger. There is some input from leptin of course, but that's another post.


The other three carbohydrate dishes are essentially lowish fat foods with between 7g and 10g of carbohydrate per gram of butter or canola oil.

In these lower fat preparations it takes three or four teaspoons of butter to generate satiety vs just under 6 teaspoons of canola oil, roughly twice as much fat is needed when carried with a similar amount of starch. A reasonable fit with a Protons point of view, though not as pleasing as the BMP vs FFF comparison.

How the study was developed is fascinating to think about.

What decisions were made at the planning stage? Obviously, someone had worked out, well before any grant application was submitted, that higher saturated fat with lower carb meals are by far the most satiating. Or maybe they are dumb and they were just lucky to get a result? Personally, I can't see how you engineer a study like this unless you are pretty clever and well informed, not at the mitochondrial level of course, but certainly at the butter level. Mashed potatoes, which already have something of a reputation as a miracle weight loss food, getting a helping hand... From a dollop of butter. It makes sense.

BTW this is Canada. I can't see how such a study would ever have gotten past any ethics review committee in the US of A. Imagine trying to feed BUTTER to American children. Immoral. Plus they might not eat up their carbs!

Peter

High fat fed mice on stearic acid

The concept of finding anything positive about palmitic acid is still tantamount to research suicide. However, stearic acid is a rather different matter. It's lipid "neutral" for those poor folks who still bow their heads and kneel before the altar of the lipid hypothesis. So you can publish good stuff about stearic acid with relative impunity.

Raymond sent me the PhD thesis of Valerie Reeves, Kentucky University.

Reeves, Valerie Lynn, "A DIET ENRICHED IN STEARIC ACID PROTECTS AGAINST THE PROGRESSION OF TYPE 2 DIABETES IN LEPTIN RECEPTOR DEFICIENT MICE (DB/DB)" (2012).Theses and Dissertations--Physiology. Paper 3.

Before we think about leptin receptor defective mice (another day), we can ask questions about the control groups. Such as:

What happens if you feed a fairly typical C57Bl/6 mouse 40% of its calories from fat, based on fully saturated stearic acid?

They stay significantly slimmer than they do on CIAB (chow) and probably slimmer than when fed on 40% oleic acid (olive oil w/o the PUFA).

(EDIT As Tucker pointed out in comments: You might be able to explain the relative weight gains in terms of omega 6 PUFA. Chow was about 13% of energy as PUFA, stearic acid diet about 5% PUFA and the oleic acid diet about 14% PUFA. The correlation of PUFA with fat gain isn’t perfect but it’s quite close… END EDIT)

Now this is clearly impossible, as anyone who has read anything about Bl/6 mice and fat will be very aware. So the poor girl did it again:

This time we have p values sprouting all over the graph like mould in a Winter bathroom. For mice, chow makes you fat. Olive oil makes you fat. Stearic acid doesn't. Impossible I know, but that's twice it has happened. For fat mass the p values never make pay dirt but the writing is on the wall for oleic acid and fat gain too:

The wild type control mice were so nice in this PhD thesis that I thought I'd just put up these few figures before we consider what might happen if (gasp) you put an obese, diabetic db-/- mouse on a highly saturated stearic acid based diet.

I think palmitic acid would do exactly the same as stearic acid did for these mice. But who would risk their career with a finding like that? The corollary is that when you see a C57Bl/6 mouse get fat on a high fat diet, you know there are lots of double bonds in that fat........

Peter

On drinking varnish

Dietary linoleic acid elevates the endocannabinoids 2-AG and anandamide and promotes weight gain in mice fed a low fat diet.

Raphi sent me this link early in the New Year. It’s nice. It demonstrates, at some level of complexity, that omega 6 PUFA at 8% of calories are obesogenic in mice, even if they are fed otherwise fat free CIAB. It’s all about endocannabinoid ligands and receptor activation. Potentially useful when folks get round to starting class actions against the cardiological community and any other health advisors warning against saturated fat. If you limit fat to 30% of calories and saturated fat to 10% you still have 20% PUFA/MUFA in your diet. That’s easily obesogenic. Your cardiologist made you fat. Sue now.

But all of this endocannabinoid stuff is what I call high level signalling. At the core mitochondrial level we know that omega 6 PUFA fail to limit insulin activity under situations where a saturated fat would shut down insulin mediated calorie ingress. In an adipocyte this means that, during oxidation of omega 6 PUFA, insulin continues to signal and fatty acids (and glucose) fall in to the adipocytes, stay there, and you get really hungry. Modified chemicals derived from this system of omega six fatty acids are overlaid on top of the core mitochondrial signalling. A modified derivative of arachidonic acid becomes an endocannabinoid ligand and makes you hungry and fat. The system takes something basic and develops an overlay of enormous complexity, this is what I call higher level signalling.

I hate higher level signalling. Give me the core process anyday.

On this front people may realise I have issues with omega 3 PUFA fats. From the ETC perspective they are worse than omega 6 PUFA and should be more obesogenic. But, in general they’re not. In fact there is a massive industry showing us how good they are for us. But there are suggestions that the core process which makes omega 6 PUFA obesogenic really do apply to the omega 3s. Bear in mind that we are only talking about linoleic and alpha linolenic acids here. Longer fatty acids go to peroxisomes for oxidation and have little influence on core mitochondrial processes, though they do perform a great deal of high level signalling. Here we go:

Sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.

Notice the obesogenic effect of fish oil only shows when sucrose is present in the diet. Replacing sucrose with protein eliminates the effect. Fructose is an unstoppable source of cellular energy intake which needs insulin resistance to limit insulin signalling facilitated ingress of glucose. As insulin continues to act, fat cells sequester calories. Fish oil combined with sucrose is the worst, corn oil is intermediate and, without sucrose, none of the fats are obesogenic.

This makes me happy. I can see the core process at work, never mind what EPA and DHA say to g-protein coupled receptors.

There is another paper which shows a similar effect and I like it rather a lot because the cognitive dissonance, which shines through every word of the text, is rather entertaining. How can you get a life-sustaining source of funding if your data show that omega 3 PUFA are grossly obesogenic? They improve insulin signalling exactly as the ETC effects would predict. The cost of improved insulin responsiveness in adipocytes is obesity. Here we go again:

Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n-3 polyunsaturated fatty acids.

The values to look at begin with the weight gain. All we have to do is to subtract weight at the start of the study period from weight at the end (perhaps the authors don't do arithmetic?). Low fat group gained a gram, added saturated fat group gained 0.6 g, added omega 6 group lost* 2.4g and omega 3 group gained 10.4g.

Ten point four grams.

These are db/db mice which lack a functional leptin receptor. They are diabetic and I feel their chronic hyperglycaemia represents a similar drive to obesity as the fructose loading in the last study, ie an unregulated source of calories which drop in to adipocytes and which require insulin resistance to shut down whatever further caloric ingress it can practically do. Free fatty acids, a reasonable surrogate for the action of unmeasured insulin, are low so this suggests adipocyte sensitivity to insulin is high, hence the weight gain.

Weight gain in the alpha linolenic acid group was over 17 times that of the saturated fat group and 10 times that of the low fat group. Notice saturated fat protected (admittedly ns) against the weight gain seen on the low fat diet. The logic is obvious. What do the authors say? Well, I can find no mention in the discussion of this massive weight gain in the omega 3 group. Zilch. This is the quote from the only mention it gets, in the results section:

"Body weight at the end of the study was somewhat higher in db/db mice fed HF/3 compared with HF/S (Table 1)".

My emphasis.

There is no other mention of the hard fact that omega 3 fats are obesogenic. Also note that in relatively normal, non hyperglycaemic db/+ mice, the omega 3s are not obesogenic. Much the same as for non-fructose fed mice in the previous study.

Now look at the * I put in above. The omega 6 diabetic group LOST 2.4g. Ouch, at the core mitochondrial function level! How can this be? This needs no mention at all in the paper because p is greater than 0.05 (in the twisted stats used by the authors). But brownie points if you have noted the oddity about this particular group of mice.

Well done! Yes, in a group of 5 animals the standard deviation at the end of omega 6 feeding is 8.6. No other group had a standard deviation greater than 3 at any time. How do you get a standard deviation of 8.6? These are diabetic mice. Four gained weight, one became ill and this one lost a lot of weight. That's my guess, just trying to reverse engineer information out of the data supplied by a group of dissonant thinkers...

So, I went to an on-line standard deviation calculator and fed in various options where 4 mice gained some weight and one mouse lost a tonne of weight. Using a 2g gain for 4 possibly healthy mice and a 20g loss for the fifth poorly mouse we get four mice at 44g and one at 22g. This gives a mean weight at the end of the study of 39.5g to with an SD of just over 9. I think something like this is what happened. Would this group notice one skinny mouse in with four fat ones? Hahahahaha!

Summary: When PUFA are being oxidised in the mitochondria of adipocytes, those adipocytes are unable to resist the signal from insulin to distend with fat. The more double bonds in the PUFA has, the greater the effect. Linseed oil should be used for making varnish.

Peter