Saturday, March 30, 2024

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.


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.


Which makes me happy.



@raphaels7 (twitter) said...

i'm starting to understand how the in vitro "SFAs are bad" trick is pulled

step 1) use supraphysiological doses of ROS
step 2) add SFAs in a context in which it'd never operate physiologically
step 3) notice SFAs make things worse in this 'abberant' context
step 4) add LA, which in the supraphysiological context, causes less damage (or no added damage)
step 5) notice LA is 'better than SFAs' in this supraphysiological context
step 6) erroneously extrapolate behavior of FAs in supraphysiological contexts to physiological ones
step 7) get funded to bash SFAs more and climb the career ladder

karl said...


In the mean time - dosages that people actually get:

There is something happening in the awareness. More people are noticing that the food supply is not safe.

If people eat food that screws up their livers - inducing T2D - or worse - my hunch is that the damage is permanent.

Once people have T2D - they are exposed to super high levels of insulin. It is assumed that glucagon has the opposite effect of insulin - but I'm not so sure as insulin has MANY effects - I doubt that glucagon is simply the opposite of insulin.

There is also Amylin, Glucagon-like peptide-1, Glucagon-like peptide-2, Incretins, etc that are involved in controlling these effects. This hyper-complex web of nonliear feedback loops is not something mere human mortals can model in our heads - though many will claim they understand. What is better is to look at the low level first.

All of this falls back to the proton switch which I think is evolutionary ancient - and should be seen as the substrate that these hormonal control loops were built on top off as evolutionary history progressed. I'm using the term 'proton switch' to indicate that it is normally in one mode or the other - a true switch of state. (it is possible that the evolution of hormonal actors was selected to amplify the effect of the primitive switch).

I want to make a list of what happens in the two states (and what direction)..
Insulin sensitivity
Mitochondria population
ATP production rates

How long is this list? It might help us think about how all of this works.

Terms can mislead - misframe:

T2D is defined as BG dis-regulation - but that definition seems quite wrong to me, it seems that insulin tolerance is how it should be defined. (We define hyperthyroid as a over production of thyroid hormone - so wouldn't it be that an over production of insulin would be defined as hyperinsulinemia? The naming changes the framing of how we think of these diseases).

Anyway - the ADA is still pushing carbs on the T1D public - and has resisted for decades to test the advice in a proper trial. Why is this? It is noticeable that most of their funding comes from food companies that sell food-like-substances that increase insulin. My hunch is if reducing carbs came first - and medications second - most T1D would add years to their lives.

What are the odds that eating 'novel foods' (things we did not evolve with to eat) would be safe long term - even if there are no signs of immediate acute toxic effects? I'm quite sure I don't know ... Seems prudent to avoid novel foods.

Something in the food eaten is causing wide spread illness - I have my hunches which are the biggest factors - but the proper research hasn't been done - and we now have over 5,000 food additives.

One last note - Just finished Gary Taubes' Rethinking Diabetes: What Science Reveals About Diet, Insulin, and Successful Treatments It is good long term and rather wide history of how the anti-fat narrative evolved. (He even mentioned my father's endocrinology professor, Dr. Gordon, - his 1963 diet featured in JAMA inspired Atkins - and I suppose Gordon inspired me as well via the stories my father told me.)

What is disappointing in the book is the lack of awareness of the effect of plant oils on the liver - via T2D. Other than the oxLDL re CVD he seems to have missed the boat once again.

Gyan said...

I am trying to learn more about Sayfried's cancer protocol but, at least from my search, there is nothing after 2019. Updates at his website close at March 2019.
I wonder if people are aware about latest work by Sayfried particularly and metabolic management of cancer generally.

cavenewt said...

Gyan—maybe start here? Perhaps it's because you're doing searches with his name misspelled?

karl said...


Look up ketogenic + DON (blocks glutamine).

I think it is likely that keto reduces cancer rates. Well known that cancers grow faster with high BG - enables out growing the immune system?

Of course high BG also speeds both viral and bacterial replication. Fructose is used in cancer cell cultures..

I don't have the source, but if you rank countries by the amount of grain consumed per person - in matches up with the cancer rates. I figure that is likely true - wish I had the source.

Gyan said...

Does grain consumption vary inversely by human development index. For their is a study

"The estimated global lifetime risk of cancer from birth to death was 25.10% (95% confidence interval (CI): 25.08%-25.11%) in 2020; the risk was 26.27% (95% CI: 26.24%-26.30%) in men and 23.96% (95% CI: 23.93%-23.98%) in women. Significant differences were observed in the risks between countries/regions within world areas and by the human development level. The lifetime risk of cancer was 38.48%, 25.38%, 11.36%, and 10.34% in countries/regions with very high, high, medium, and low Human Development Index, respectively"

Carbs do not cause cancer, and they do not cause diabetes. Restricting carbs may be a good strategy in managing cancer and diabetes however.
I came back to the importance of dietary fat to carb ratio (F:C). Traditionally grain-consuming countries had low fat consumption. As they grow richer, they consume more fats esp more unsaturated fats. Richer a country, more fats it consumes and more PUFA it consumes.
And more hyperinsulinmia it develops.

Gyan said...

Found a paper (not Seyfried's) from 2023--Cell Metabolism

"Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia"

Ketogenic diet delays tumor growth but accelerates cachexia and shortens survival.
In the tumor, increased lipid peroxidation causes ferroptotic death of cancer cells.
In the host, redox imbalance and NADPH depletion cause corticosterone deficiency.
Dexamethasone plus ketogenic diet delays cachexia and preserves delayed tumor growth.

Google scholar gives a Seyfried paper in 2021. A clinical trial in Tehran.

"Effects of Ketogenic metabolic therapy on patients with breast cancer: A randomized controlled clinical trial"
Clinical Nutrition
Volume 40, Issue 3, March 2021, Pages 751-758

KD lead to a reduction in tumor size in the KD compared to the control (27 vs 6 mm, P = 0.01). Stage decreased significantly in patients with locally advanced disease in the KD group after 12 weeks (P < 0.01). No significant differences in response rate were observed in patients with metastatic disease.

Bob Kaplan said...

@Gyan: Tom and his colleagues have completed a manuscript on a protocol that will hopefully be published soon.

Also, Tom sends an information kit and papers to those interested in learning more about metabolic therapies. You can email him at or if you email me ( I can forward you the info.

Bob Kaplan said...

@Gyan wrote: "I wonder if people are aware about latest work by Sayfried particularly and metabolic management of cancer generally."

For a general perspective, see: Press-pulse: a novel therapeutic strategy for the metabolic management of cancer.

Bob Kaplan said...

For more recent preclinical work, see:

Mukherjee et al., 2023: Ketogenic diet as a metabolic vehicle for enhancing the therapeutic efficacy of mebendazole and devimistat in preclinical pediatric glioma.

Mukherjee et al., 2019: Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma.

There's also a recent review by Christos Chinopoulos, one of Tom's colleagues, discussing the role of Complex I activity - how it can support mitochondrial substrate-level phosphorylation in anoxic mitochondria (see Ravasz et al., 2024) or when OXPHOS is compromised - leading to the idea that lower levels of Complex I inhibition could refine treatment strategies down the road: Chinopoulos, 2024: Complex I activity in hypoxia: implications for oncometabolism.

@raphaels7 (twitter) said...

thanks @bobkaplan, i'll follow up on those refs

karl said...

@Gyan who said "Carbs do not cause cancer, and they do not cause diabetes. Restricting carbs may be a good strategy in managing cancer and diabetes however."

A couple of things here - a case can be made that the 'dose makes the poison'. Even a healthy person that eats a mess of carbs will elevate their BS - which is known to speed cancer growth(and viral and bacterial replication - which in turn have the potential to cause cancers). If proto cancers grow rapidly - they can out grow the immune system. So the carbs may not 'cause' the cancer, but may well aid in their survival. I don't think we know this definitively but it appears a promising theory.

I am SURE that I don't know what causes T2D, but I do have my hunches. Seed oils may damage the liver - cause T2D, but then is it that simple? My cat eats almost all industrial chicken - full of LA - his is quite healthy for his age - no metabolic disease. So is LA sufficient on it's own to cause T2D? - Could it be that it is the combination of a high-carb-diet + LA that does the damage?

Also, carbs are not a single thing. While starch is mostly chains of glucose - other carbs, the fructose in sucrose in particular, impact the liver as well. I think the correlation of grains and cancer is real - correlation is not causation, but it is possible that what ever pattern that does cause cancer may well include grains.

I wish I knew for sure what causes T2D - it appears to me to be permanent damage. It is surreal that the huge amount of research money for public health is mostly misspent on junk science when T2D continues to kill and reduce quality of life for so many people. (On the other hand - billions are available for bio-weapons work).

My hunch is that some combination of seed oil, sugars, 24/hr elevated insulin ends up doing damage (the liver?) - and we are left with low-carb as the best tool to prevent further damage.

I have another hunch that chronically elevated insulin causes CVD (much stronger correlation than to any lipoprotein) - and may also be involved with cancer. If we look at the narrative that cancer is first a metabolic disease - and the mutations we see are downstream from metabolic disease, then what ever causes the metabolic disease is the distal cause of cancers.

mct4health said...

I once wrote a post about the causes of T2D, it's not just PUFAs, but also a lack of short and medium fats, i.e. also antibiotics and intestinal microbiome, the state of fat cells, etc.

And I wrote about the interaction of carbohydrates, fats and proteins in the last post. It looks like DNL in high fat environment can change TCA cycle and some food components (e.g. SCFA) can switch these changes back via AMPK and ACC1 phosphorylation. I understand that my layman's observations may not be of interest to anyone, but I think the links to the studies I used are interesting.

Are carbohydrates toxic?

karl said...


The list of what is actually causative of T2D is long - but I don't think anyone has a slam-dunk theory - could be a combination of causes. I just wish people would stop claiming they 'know' when at best we have a tiny lump of jello nailed to the wall - but sagging - ready to fall. Being certain stops discovery of better ideas.

Lets start with defining the tissue that gets damaged: Is it in the liver? brain? pancreas? adipose tissue? muscle?

I think it is likely liver tissue but I'm far from certain. I speculate that PUFAs are involved - but is there a co-causative factor? I think it is likely PERMANENT damage (seems it can be worked around via low-carb - but not cured). How is it, that the disease of our time - leading distal-cause-of-death - with all the research, technology available - and we don't even know for sure which tissue gets damaged?

Here is a thought - someone that has T2D - ends up with a liver transplant - are there cases where the T2D went away?

All sorts of people claim they 'know' - and we could do simple to test many of these hypotheses. Doing good science is really hard - but not impossible.

Healthy people have healthy body-weight set points. Seems that insulin is intimately involved - and thus the proton-switch - but just what controls this set point? How does it get broken? Why doesn't it heal?

Anyway - sorry for the rant - I'm just tired of people saying they 'know' when their hypotheses are not well tested. A good scientist has to put away hubris - be quite humble.

The first job of a scientist, theoretician - is the prove your idea wrong. “The first principle is that you must not fool yourself and you are the easiest person to fool.”
― Richard P. Feynman

So lets hear some theories of which tissue gets damaged - why is it so? What are the flaws in YOUR theory? How could it be tested?

If 4HNE does permanent damage to the liver - what exactly gets damaged?

If people eat some magic combination of fatty acids - is the damage reversed?

It appears that the causative agent(s) are in ultra processed foods - if we looked at which of these products has the highest rates of T2D we could get a list of prime suspects. Instead of papers doing this - there is just hand waving and papers that assume their take is the only right one.

Anyway, I think researchers need to go back to school - learn the scientific method in depth - study epistemology - and actually practice the philosophy. ( there was a reason that PhDs were called philosophers - seems to have been replaced with something to do with grant proposals/grifting, )

Sorry for the rant - I think I read one to many junk papers this week.. how so much garbage gets in journals is mind blowing.

Gyan said...

Been looking at your posts. There is a question I have regarding the 150 g minimum protein plus carb recommendation of the Perfect Health Diet. As I understand, the value comes from the carbohydrate requirement of glycome i.e structural sugars. I have read Jaminet post about when some Optimal Dieters were found with stomach cancers and he speculated that carb plus protein restriction of Optimal Diet had to do with lack of proper mucosa and thus cancer.

However, I have not seen this consideration elsewhere. And this number 150 g is naturally rather ad-hoc.

You have given considerations for upper limit of carb 150 g and upper limit of protein 150 g each on another basis. Do you hold with 150 g P+C requirement of Jaminet?

Gyan said...

I think that following Unger which is called glucagon-centric view of diabetes, there is no damage to liver, but the critical thing is impaired communication between alpha and beta cells of pancreas leading to hyperglucagonemia and excessive production of hepatic glucose.

mct4health said...

As I read Jaminet, he states max 600 kcal of carbohydrates and max 600 kcal of proteins as safe with no extra physical activity. And yes, I think he recommend some minimum carbs for mucus creation. I don't remember him to recommend maximum as the sum. And each of us has an individual limit, it's recommendation.

"Carbohydrate intake:

Carbohydrates should account for 20 to 35% of total energy intake, except for those on a therapeutic ketogenic diet (slightly less) or athletes in training (slightly more).

About 85 % of carbohydrates should be in the form of starches, only a maximum of 15 % of carbohydrates should be fructose, starches should be preferred to sugars.

Starches should be consumed with fat, vinegar and vegetables to minimize their hyperglycemic toxicity. Starches are food, not snacks! " citation from the book.

And about hepatic glucose production, it paradoxically goes up with de novo lipogenesis. That's logical because liver have to dispose glucose somehow using both processes to prevent glycogen storage overflow.