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.


Sunday, March 24, 2024

Foie Gras (8) Vaughan's macrophages

Okay, back to the Vaughan paper

and a look at their macrophages in cell culture. The pattern is consitent so I'll just run through MCP-1 gene mRNA expression. As in the last post I am going to work on the basis that these genes are, in the absence of trauma or infection, going to respond to mitochondrial/NOX ROS production. It's a graph of relative expression, the control represents the response to the ROS being produced by supplying glucose at around 30mmol/l. That will be in response to NOX-2 being activated through calmodulin kinase II as we would expect. Even without insulin, these ROS will activate the phosphorylation of AKT and translocate GLUT4s to the cell membrane. It's the black column:

Adding 50 μmol/l of stearic acid (white column) will generate ROS by reverse electron transport though complex I, via an increase FADH2:NADH ratio, physiologically to limit insulin facilitated glucose ingress. Even if there is no insulin present, it still generates the ROS, which will limit the facilitation provided by NOX-2 derived ROS. Saturated fatty acids do this at low delta psi levels, there is no multi-enzyme/complex ROS generation, they just generate neat and tidy, fully physiological RET. The cell responds appropriately with superoxide dismutase and catalase and a coincidentally extended lifespan. Okay, I can't resist it, here he is again:

Oleic acid is another whole series of posts, so I'll mostly gloss over it. Oleic acid is designed to facilitate insulin signalling as a physiological balance to palmitic acid. It generates some ROS but not enough to significantly resist insulin signalling. There are many concepts stemming from this and here is not the place to discuss them.

Linoleic acid (hatched column) utterly fails to generate enough RET to limit glucose ingress, so glucose continues to enter, while at the same time the LA provides calories in excess of what the cell needs. The problem is that LA cannot shut down glucose ingress because it fails, through its double bonds, to generate the RET facilitated ROS to limit that glucose ingress.

The cell is full of ATP and depleted of ADP so ATP synthase cannot turn. Delta psi rises and above 170mV large numbers of ROS are generated. These come from a combination of complex II, complex III, electron transporting flavoprotein dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase. Oh, and I guess some from RET. All secondary to high delta psi.

All stemming from inadequate RET derived ROS from LA's surfeit of double bonds. A few prophylactic ROS, produced without an high delta psi, stops an awful lot of problems. I think the phrase is that a stitch in time saves nine...

I think here is a good place to point out that ROS generate a response in inflammatory gene mRNA expression, probably at any level of exposure. Low levels will result in normal physiology, high levels will result in frank inflammation,  but also will induce inflammation limiting gene expression, in which I would include multiple uncoupling protein genes. Failure to limit high levels of ROS generation clearly results in apoptosis or, given a catastrophic failure of the ETC secondary to 4-HNE and associates, necrosis.

This paper is quite unique in view of the low dose of fatty acids used. If they had chosen the standard route of 1000μmol/l FFAs in addition to glucose at 25mmol/l (utterly non physiological) then RET would have dominated, giving apoptosis/necrosis levels of ROS, meaning stearic acid would be bad (usually palmitic is used), oleic less so and LA would be almost harmless. There is an infinite supply of such papers to reinforce the stupidity of saturophobia.

Okay, the in vivo mice next.


Foie Gras (7) What do you mean by inflammation?

This paper contains a very superficial and bullet point overview of the inflammatory cascade. It omits swathes of anti inflammatory pathways and many of the boxes on the chart could be massively expanded. The paper is interested in using phytotoxins (mis-termed phytonutrients) as anti inflammatory agents, so all I take from the paper is their doodle

Animal Models of Inflammation for Screening of Anti-inflammatory Drugs: Implications for the Discovery and Development of Phytopharmaceuticals

A legible version of the diagram is in the paper if anyone would like to actually read it.

We can simplify the process slightly with the red arrow. In addition I've circled in blue the component that is being used as a marker of inflammation in the Vaughan paper:

We can now consider the basic process a little more carefully. This massive review (no, I've not read it all) brings to prominence the central role of ROS in inflammation

and has this nice illustration featuring mitochondria as sources of ROS. Superoxide generation is never accidental. It is a tightly controlled and highly specific process to achieve certain ends. Mitochondria and NOXs are not making mistakes or having accidents:

BTW MPO is the myeloperoxidase we've seen recently in Vaughan's paper. HOCl is bleach, it's an inflammatory tool. We use it to kill germs.

So this lets us redraw this doodle of inflammation with a small modification:

Right up at the top of the inflammation doodle is a small red circle labelled PLA2, phospholipase A2. Its job, in the event of tissue injury, signalled by ROS and derivatives, is to release arachidonic acid from lipid membranes which then allows the generation of a raft of inflammatory mediators using cyclooxygenase and lipoxygenase enzymes.

Corticosetroids are potent suppressors of PLA2. They also releases both glucose and FFAs from the liver and adipocytes in to the blood stream. Physiological cortisol does this to deal with the sort of situation where you and a bunch of your mates are about to go and drive an angry mammoth into a bog, to kill it and butcher it. But high FFAs and high glucose together generate metabolic ROS, which are pro inflammatory.

You want raw energy, not to be crippled by an acute inflammatory reaction. So cortisol also down regulates the inflammatory cascade which *should* have been triggered, via ROS from the ETC, while flooding mitochondria with large amounts of substrate in anticipation of incipient need.

So cortisol floods your metabolism with calories, simultaneously controlling inflammation, and your mitochondria convert those calories in to the ability to successfully kill a mammoth, ie obtain food for a month for you and your tribe. Those calories get used successfully. Nothing is overloaded, nothing is damaged. Glucose and FFAs get used up during the hard work involved, facilitated by AMP-kinase rather than insulin. There is no generation of bulk inflammation, and what minor ROS mediated lipid peroxidation does occur has the routine inflammatory response suppressed. 

Evolution sets the levels correctly. It may be blind but it recognises functionality when it sees it.

Now imagine you're an Homo modernus visiting your bank manager in the 1960s to get a mortgage with a borderline adequate income and a just passable deposit, for the house of your dreams. The manager is a local petty Hitler and, even if he intends to grant the mortgage, he's going to really make you suffer, mentally, for it. Because he can.

You're stressed pre interview. So cortisol floods your system. Glucose and FFAs are made plentiful to allow you to fight at your hardest during a necessary mammoth kill. But there are consequences to killing the bank manager and most people, understandably, decide not to do so. Much as they might wish to. Especially if he refuses to grant you the debt you can barely afford. For your safety. There is no energy usage, no AMPK activation, just a serious availability of calories.

If the levels are high enough they will flood your mitochondria and provide substrate well in excess of what is needed for current ATP demand. If ATP synthase refuses to turn because ATP is high and ADP is low, while NADH and FADH2 keep supplying electrons to the ETC at the CoQ couple, delta psi will rise above that safe figure in the region of 170mV and ROS will be produced in large amounts from multiple sites, giving both insulin resistance and tissue damage.

You can use pharmaceuticals to inhibit phospholipase A2 as much as you like to avoid any response to this damage, classically using prednisolone or dexamethasone, but there is still the flood of calories generating ROS at tissue damaging levels. You might feel okay because the whole inflammatory cascade is suppressed, but cells are still dying from high ROS levels. If these happen to be endothelial cells lining your coronary arteries you are in trouble. Some will be.

So I would posit that the increased risk of CVD under corticosteroids could be (in part, there are many other issues) an effect of acute ROS injury. Under corticosteroids the inflammatory response is suppressed but the excess calories are still generating excess ROS. The damage is still done.

Of course the same applies to metabolic syndrome.

I define metabolic syndrome as the inability to shut down FFA availability in the presence of insulin and elevated glucose. FFAs are high from basal lipolysis at the same time as glucose from the diet causes hyperglycaemia. If substrate supply is high enough ROS will be generated and damage will be done. This time there will be an actual inflammatory response, no corticosteroid involved, and people can make statements like "obese people are chronically inflamed". They are producing mitochondria mediated ROS to physiologically resist insulin. If they are forced to continue to accept calories despite resisting insulin, the ROS become simply damaging. Inflammation follows. 

If you do not link this "inflammation" back to ROS then you would, logically, treat it with dexamethasone. Which would be a booboo, as we say in the UK. You would suppress inflammation while supplying even more ETC input, so worsening ROS mediated damage. Those poor coronary artery endothelial cells. In the absence of slaughtering a mammoth of course.

It's also worth thinking about the role of 4-HNE. Just a skim of the abstract of this book chapter is worth a moment:

4-HNE is, in itself, a product of ROS damage. It is also a generator of ROS per se. Much of the inflammatory cascade is self amplifying (which is why we have systems to turn inflammation off as well as on). Taking exogenous 4-HNE has profound effects on ROS mediated signalling in the insulin cascade, at low dose by injection to enhance, at high dose (with other lipid peroxides) orally to inhibit. It matters in the inflammatory cascade as much as in the insulin cascade.

My interest, getting back to Vaughan's obese mice without hepatic inflammation, is rooted in the factors which generate 4-HNE in situ around the ETC and which factors stop this. I'm talking about ROS generation.

Back to those mice and their non-inflamed hepatocytes and adipocytes.


Tuesday, March 19, 2024

Foie Gras (6) inflammatory mRNAs

 Okay, time to look at this study

It presented itself to me while I was looking  for explanations as to why linoleic acid was or wasn't inflammatory in hepatocytes. Being "inflammatory" is determined, here, by the response of treating freshly isolated cells, in this case macrophages, to incubation with linoleic acid at 50 μmol/l for 12 hours, in addition to the normal culture medium. Some ferreting around on the internet suggests that the culture medium used, when fresh, contained glucose at 33 mmol/l. The concentration of LA, 50μM, is low and physiological for a FFA concentration under hyperglycaemia. The macrophages were assessed for expression of pro-inflammatory genes coding for these proteins:

Monocyte Chemoattractant Protein-1 (MCP-1)

Macrophage inflammatory protein-1 (MIP-1)

F4/80 is a gene for the surface marker of macrophages

and good old TNFα and Il6 get thrown around too.

We can ignore the anti-inflammatory gene expression Ym1 and Ym2.

This is what they found for pro-inflammatory gene mRNA relative expression, linoleic acid is tall hatched bars. It's a log scale:

So we can say LA is pro-inflammatory in this model. No one in obesity research wants to find that the cardiological darling and cholesterol lowering PUFA are pro-inflammatory. I told you they were rank amateurs. Anyway, they developed another model, in-vivo this time, which got the correct results.

They fed the mice the diets I mentioned in the last post for four weeks then looked at pro-inflammatory gene expression in both white adipose tissue and liver tissue immediately after euthanasia.

This was much more satisfactory. In both tissues the high PUFA diet was not inflammatory, with a trend towards it being anti-inflammatory in adipose tissue:

Adipose, from Fig 2:

and liver from Fig 3:

However, there is another snippet of information also available in Fig 3. The activity of myeloperoxidase system tells us slightly more than the relative mRNA for inflammatory genes do in the bulk of the figures. Myeloperoxidase tells us whether the WAT cells in Fig2 are actually using the pro inflammatory genes to produce inflammation. This is what they found. The chart is split in two because the HF (high fat diet, something like D12492) was so activating it was an order of magnitude higher than all of the others, so needed it's own scale:

I think we have to be very, very careful about what we mean by "inflammation". In fact I might just take a brief pause here and explain what I think "inflammation" means, before running through what is happening in this study. That may need some cellular physiology rather than mitochondrial physiology.

Here's my conundrum:

CVD is an "inflammatory" condition, sic.

The most potent anti-inflammatory agents available to modern medicine are the corticosteroids.

Corticosteroids make CVD much, much worse.

Somebody, somewhere, has an odd idea about what "inflammation" might be.

With apologies for the hiatus.


Monday, March 18, 2024

Foie Gras (5) An aside on how to stay slim

 Another one-liner:

Now, I like this paper. They found some weird stuff which is fully Protons compliant. That's not today's post. I just wanted to share this graph:

These people are clearly rank amateurs rather than hardcore obesity researcher, they have utterly failed to "improve" the saturated fatty acid (SFA) diet to make it obesogenic. Want to stay slim? Cocoa butter. As good as chow, and who would want to eat chow? Though I'd personally choose beef fat.

The linoleic acid diet is also not very fattening. Hmmmmm.

Even without looking at Table 1 you absolutely know what has been done, both to get the low weight gain on LA and to eliminate the inflammatory changes in the liver (which are there in cell culture).

Hard to decide whether to go through this study or start on the mitochondrial data from the hungry Italian rats. Or maybe go to Winks Meadow to see if the Green Lipped orchids are up and flowering yet. 

The sun is shining.


Foie Gras (4) RER

A quick one-liner from the Italian rats in

Fat Quality Influences the Obesogenic Effect of High Fat Diets

These are the RERs of the rats on the last day of the study. The horizontal lines are the food quotients calculated from the macros of the diets. Under stable conditions 24h RER should equal FQ. These are not stable conditions

It's clear that both groups of rats are catabolising protein for energy in excess of what is present in the diet. More so for the lard diet than the safflower/linseed. They are, undoubtedly, hypocaloric.

The lard based diet rats fail to oxidise lipid despite it being in the diet because they are still sequestering lipid in to adipocytes. Basal lipolysis may be high but hyperinsulinaemia (not measured) from incipient metabolic syndrome is recycling some of that lipid back in to adipocytes. Where it's not being oxidised. The rats are cold and hungry.

The safflower/linseed group have normal fat oxidation, ie equal to FQ, because fat is not being lost in to adipocytes, it's being lost by uncoupling so lipid oxidation on RER is normal. It may provide nothing but heat but the RER looks good. Of course these rats are warm and hungry.

I like it.


Edit, I don't like the rats being hungry! Just the RER data. End edit.

Foie Gras (3) The Japanese mice

So the interesting question about the rats in the Italian study which were fed on the Safflower/linseed oil diet is:

How calorically restricted were they?

In the absence of a control group allowed ad lib high fat intake (or even one fed chow) we will have to look elsewhere to estimate this. Japan is a good start with this paper:

Just give Bl6 mice a free choice to munch chow and/or drink corn oil and they will eat/drink progressively more corn oil over 4 weeks until they are stable at ~75% of their calories from corn oil, 25% from chow. Which gives us in the region of 38% of calories from linoleic acid. They are weight comparable to chow-only fed rats throughout, maybe slightly lighter:

Under stable conditions (weeks six to eight) they feed themselves 35% extra total calories to maintain an identical bodyweight to the chow fed mice:

This is because they were uncoupling, primarily in interscapular brown adipose tissue but (from other papers, a future post) they also do so in white adipose tissue.

Back to

Fat Quality Influences the Obesogenic Effect of High Fat Diets

If we imagine the Italian rats had been offered ad-lib access to the safflower/linseed diet we could expect them to eat somewhere in the region of 380kJ x 135%, so around 500-530kJ/d.

They got fat on just 380kJ/d. Like the lard fed rats. But by two weeks they were a bit less fat (p<0.05).

How does this work?

On day one of the safflower/linseed diet there is a marked increase in insulin sensitivity, by the standard Protons/polyunsaturate mechanism, with an associated hypocaloric episode as calories poured in to adipocytes but no additional food was provided. There is no significant increase in uncoupling immediately.

But now we have an insulin sensitive liver and the standard response of the liver to ingress of excess non-carbohydrate calories is to signal, using FGF21, to BAT to induce uncoupling giving thermogenesis and calorie disposal.

The time scale for onset of uncoupling could be estimated if we had daily food intakes and body weights, but we don't, so let's just guess at around a week.

As uncoupling in white adipocytes kicks in they will become poorly able to respond to insulin with the correct ROS signal, so insulin signalling decreases and they release FFAs. At the same time as this suppressed insulin signalling occurs BAT will be disposing of bulk calories by thermogenesis and the caloric drive for the pancreas to secret insulin also drops, again assisting lipolysis.

By two weeks there is active, on going weight loss from an obese baseline. Lipid is being lost at an excessive rate accentuated by hypocaloric eating and the liver is dealing with this excess, under hypoinsuliaemia, in part via BAT and in part by the peroxisomal mechanism described in the previous post, the cost of which is, in rats (and mice), of hepatic lipid accumulation.

Would the lipid accumulation have occurred if the Italian rats had not been calorically restricted?


Uncoupling made the Japanese rats hungry, they ate extra to stop pathological weight loss. The extra calories include some carbohydrate and would have slowed lipolysis by raising insulin secretion to a level where lipolysis did not overload the liver. There are several papers to cover this in future posts.

Given long term ad-lib access to an uncoupling diet based on PUFA the rats would have eventually and gradually lost weight until they matched bodyweight with their non existent control group. Assuming these mice are anything to go by, who did it over a period of 10 weeks. From here:

I plotted the numbers for the body weights in Table 2, by eye, in PowerPoint. The dashed lines are the ones to follow. Obese on ad-lib lard, back to control (low fat, 35% of calories from sucrose) mouse weight on ad-lib safflower oil based diet giving LA at 35% of calories:

Okay, that will do for today.

I would not, in any way, endorse drinking either varnish or safflower oil, even if they produce weigh normalisation by what are, to me, metabolically convincing/plausible mechanisms.

Better not to make your adipocytes pathologically insulin sensitive, then you wouldn't need to address the obesity with potentially pathological double bonds in your food.

Probably safflower oil and hepatic inflammation next.


Saturday, March 16, 2024

Foie Gras (2) Lard fed rats

There is too much in the Italian rat study for a single post. Here's the easy bit discussing the rats fed the lard based diet.

I guess everyone is familiar with this graph in Figure 1 of this paper from the Schwartz lab:

Obesity is associated with hypothalamic injury in rodents and humans

which provides this gem. These are the *daily* caloric intakes of rats on bog standard lab chow, in grey, or during the sudden onset of feeding lard based D12492, in black:

These are Long-Evans rats weighing 300-350g. I drag this up because Protons suggests that on day 1 of exposure to D12492 the rats immediately sequester approximately 70kcal of energy in to adipocytes (ignoring processing losses) while still maintaining the basal (when chow fed) intake of 75kcal which are needed to run the rat's metabolism.

This immediate significant fat gain on day one (seen in section F of the same figure) raises adipocyte diameter, so increases basal lipolysis, so reduces hunger, such that on day two the extra food intake needed is lower and eventually, by day seven, excess food intake is no longer significantly increased and by day 14 it is normalised. Simple, yes?

Now let's return to the current study of interest discussed by Tucker, which I think of as the "Italian" study:

This used Sprague-Dawley rats weighing 250g, so probably more actively growing than the rats in the Schwartz lab study. They were measured as consuming 90kcal/d (380kJ/d) of chow assessed over the time before the study started. So we can plot the food intake of rats in the Italian study on a modification of graph H of the Schwartz study. I've left the chow fed line from the Schwartz study as an imaginary chow fed control line which was omitted by this group and I've renumbered the y axis to reflect the values of caloric intake actually reported in the Italian study:

We know that the red line for the lard fed group is close to correct from the methods section:

"Rats were divided in two groups with the same mean body weight (250 ± 5 g) and were pair fed with 380 kJ [90kcal] metabolisable energy (ME)/day (corresponding to the spontaneous energy intake of the same rats, that was assessed [on chow] before the start of the experiment) of a lard-based (L) or safflower-linseed oil based (S) diet for two weeks."

The Italian rats, without any access to the luxurious amounts of D12492 provided to the Schwartz rats, still got fat. 

If you view this from my perspective this is not surprising. The Italian rats still lost calories in to adipocytes but, without access to extra food, had an hypocaloric crisis. They ended up with a reduced percentage of protein mass in their carcass, despite an increased fat mass. Obese and sarcopaenic.

However, by two weeks on a diet which sequesters lipid in to adipocytes at the cost of reduced growth, a few of the rats will have achieved a sufficient increase in basal lipolysis to normalise hunger, as per the Schwartz rats, at the "cost" of obesity. It is very simple to multiply 380kJ/d by 14 days and get 5320kJ of offered food over 14 days. The mean of the actual food intake over the study was measured as 5286kJ in total for the lard fed rats. We don't have individual data but any rat still hungry on day 14 will have eaten all of its 380kJ, but no extra, because none was on offer. Adequately obese rats will have, via increased basal lipolysis, not needed to eat all of the 380kJ offered, ie these rats will have slightly reduced the mean total energy intake, by 34kJ, probably in the last few days of the study.

These rats are in a difficult position, they must maintain an adequate fat mass for increased basal lipolysis to offset increased insulin mediated lipid sequestration induced by the linoleic acid component of their diet. As they grow they will have to increase fat mass to maintain adequate basal lipolysis to function.

Running on basal lipolysis derived FFAs at a time when you have access to dietary glucose is the basic definition of metabolic syndrome.

If you keep adipocytes small by forcibly keeping insulin low (ie caloric restriction) you will completely side step increased basal lipolysis, side step insulin resistance, side step or delay many diseases and *increase* the duration of the miserable, hungry existence which will be your extended life.

There is no way in which you can transfer sufficient the FFAs from insulin sensitive but non-adequately distended adipocytes to hepatocytes as is needed to maintain a fatty liver. Caloric restriction is highly protective. Just ask any obesity researcher, the cure for fatty liver is hunger. Oops, I mean weight loss rather than hunger, in obesity doublespeak.

Safflower/linseed oil next.


Wednesday, March 13, 2024

Foie Gras (1) Peroxisomes

Jaromir posted this link on Twitter/X. It's excellent groundwork for further discussions relating to the study cited by Tucker in rats and the hepatic lipidosis in mice mentioned by Bill Lagakos.

Fasting induces hepatic lipid accumulation by stimulating peroxisomal dicarboxylic acid oxidation

which can be summarised in this picture, modified very sightly to remove their drug manipulations. Each step was validated by a set of experiments, it looks to have been a large part of several people's lives over several years:

This looks very, very much like a protective mechanism, put in place within hepatocytes (which are the final port of call for FFAs when the system is overloaded from excess lipolysis, think of ethanol or fructose acting on adipocytes) to protect them from potentially lethal damage from excess beta oxidation derived ROS.

It's executed by peroxisomes. Anyone who is still shamefully unaware of Dave Speijer's ideas of the role of peroxisomes in the protection of LECA (last eukaryote common ancestor) from ROS damage should go and read     

How to deal with oxygen radicals stemming from mitochondrial fatty acid oxidation

Ultimately, when the liver cells are flooded with FFAs the peroxisomes respond* by producing DCAs, di-carboxylic acids. This is basically taking a FFA such as palmitic acid and sticking, by a specific process, a second carboxyl group on to the omega end. This is used a signal to increase peroxisomal oxidation to offload calories without generating the high delta psi which would damage mitochondria. Shortening DCAs ends up with the dicarboxylic acid succinate (HOOC.CH2.CH2.COOH) which is exported to mitochondria where it increases the NADH:NAD+ ratio resulting in the generation of inhibitory metabolites which divert FFAs from beta oxidation to stored triglycerides, fatty liver.

All that is missing from the story is the role of ROS.

*I have absolutely no data on this but, if you expect the signal for peroxisomes to generate dicarboxylic acids by omega oxidation is going to be anything other than mitochondrial derived ROS, then I cannot help you.

Peroxisomal beta oxidation generates hydrogen peroxide (well duh). And we can guesstimate how much H2O2 is being produced in hepatocytes under these circumstances. Enough to replace the missing insulin when a rat is being fasted. Never forget that insulin is merely a superficial overlay over the ROS signal. If you want to get damaging levels of FFAs out of the vicinity of mitochondria you need to divert them to be stored as inactive triglycerides. That requires activation of the "insulin" signalling pathway, mediated by H2O2. Without insulin all you need are the ROS. You can buy hydrogen peroxide in a bottle (here working on adipocytes) which, at an extracellular concentration of ~1mmol, will act to provide what we describe as peak insulin signalling:

Equally, you can evolutionarily generate these insulin equivalent levels of H2O2 from peroxisomes whenever FFAs are too high and insulin is too low. It's a safety net.

Which can go wrong.

It does go wrong, spectacularly so, in cats.

Just imagine a slightly more obese than normal domestic moggy, fed on chicken fat and starch (better known as Crapinabag). It never goes without eating for more than a few hours because a) there is dry "kibble" available at all times and b) the cat is hungry all the time c) whenever it eats, a proportion of its food is lost in to its adipocytes, keeping it hungry. That's why it's fat.

Now drop its level of insulin to basal fasting levels for three days. Maybe a road traffic accident, getting shut in a garden shed, having an acute infection, anything which stops it eating. It becomes hypoinsulinaemic. You can even do it by being too aggressive when putting your porky cat on a weight loss diet, without needing complete fasting.

It will do lipolysis. It has a huge fat mass. It will *really* do lipolysis. The hepatocytes are going to get massively overloaded with FFAs. The peroxisomes will kick in and protect the liver by signalling conversion of dangerous FFAs to harmless inactivated triglycerides. Fatty liver. There are limits to this protection. Evolution has not anticipated a cat with a body condition score of 9/10. The liver becomes so overloaded with lipid that cellular damage takes over and we are in to hepatic lipidosis. You can get the flavour of it here

Basically, after years of continuous enhanced insulin signalling from a largely carbohydrate and LA diet  insulin is suddenly withdrawn and basal lipolysis can take over. With a vengeance. FFAs cannot be recycled to adipocytes because there is too little insulin. Hepatocytes have to take over. They use peroxisomal ROS to signal lipid sequestration in the absence of insulin.

There can be so much hepatic lipid sequestration that severe hepatocellular damage occurs and then it's up to the ICU clinician to try to save the cat's life. This is done by oesophageal tube feeding for anything up to a month. If the cat survives, recovery can be complete. The current advice is to feed protein based foods, the worry being insulogenic carbohydrate food might worsen hepatic lipid storage. Quite why no one has considered acipimox is beyond me, or peripheral low dose insulin infusion to target adipocytes rather than hepatocytes as an anti-lipolytic... But then I'm totally out of ICU work nowadays.


I hope people get the concept that fasting can trigger hepatic lipidosis. Bill Lagakos commented that he has seen chow fed mice develop the mouse equivalent of hepatic lipidosis by extended fasting. I completely believe him.

It puts us in a better position to understand what is happening in Tucker's cited study of safflower oil inducing fatty liver, which I was not expecting. I had been edging towards these concepts for weeks but the information from Jaromir and Bill have been extraordinarily helpful.


Sunday, March 10, 2024

Foie gras from safflower oil hiatus

Tucker discussed a paper in some detail here on his substack. I fell for it hook line and sinker. It's been pulling me around for weeks. How badly? I've had a copy of Gold's book The Deep Hot Biosphere since late February and I'm only on page 10.

Here's the paper:

Fat Quality Influences the Obesogenic Effect of High Fat Diets

I *think* I understand what is going on but am not quite certain enough to hit "publish" of the third version of my blog post about it. I have been back through so many layers of references that some interesting studies have come up and I think I'll write about one of these next while I continue to mull over the fatty livers. I'm not in an "ignore the blog" phase. It's just the study which has me hooked is very, very complicated and, to me, very, very unexpected. So I can't leave it alone.

More when I can.


Wednesday, February 21, 2024

Electrochemistry (2) Superoxide

I keep trying to get back to simple things like weight loss/gain/insulin/LA but the electrochemistry won't leave me alone.

Someone, possibly Jaromir (mct4health) or Brad Marshall, pointed out that the pyruvate dehydrogenase complex (PDC) is a significant source of ROS.

So I was rootling around through papers on PDC and read, in a now-lost paper, that the decarboxylase component of the complex converted pyruvate to acetate but that a similar effect could be achieved by reacting isolated pyruvate with a source of free radicals to give acetate and carbon dioxide. It was probably H2O2 they were working with. The decarboxylation is a lot quicker with the enzyme but a chemical source of ROS will get the job done.

Anyway, the significance only dawned on me weeks later and I hadn't saved the paper. It's gone.

There are, it turns out, many papers looking at pyruvate decarboxylation using H2O2, other ROS or RNS. It's a generic trait. I went through this first paper in some detail:

Here's a simplified version of their Figure 1 which is the basic reaction. An electron from the H2O2 attacks the alpha carbon of the pyruvate to give the completely unstable 2-hydroperoxy-2-hydroxypropanoate which spontaneously degrades to acetate and CO2:

"The reaction of pyruvate and H2O2 produces acetate, carbon dioxide (CO2) and water; its transition intermediate has been recently confirmed..."

Essentially the H2O2 is providing an electron which destabilises the alpha carbonyl group and the molecule then rearranges itself in to the decarboxylation products.

Now look at Nick Lane's slides in the last post. First we need this bit:

in which CO2 accepts a geochemical derived electron to become a bound CO molecule and a bound oxygen anion. This lets us re write this line

(in which the activated CO2 derivatives are highlighted with red ovals) in to the much simpler form of:

Ultimately we can convert an acetyl group and  CO2 to pyruvate using a geochemical electron from the origin of life scenario.

We can do the exact opposite and convert pyruvate to acetate and CO2, again using a donated electron, this time from H2O2.

Pyruvate is stable. You can buy it in tablet form as a metabolic nutritional "supplement". It won't convert to vinegar in the jar. If you were to carbonate a bottle of vinegar it would stay as "fizzy vinegar" long term without converting to pyruvate. Exactly as you could mix H2 and CO2 in aqueous solution and they would remain stable without a hint of formate formation. Until you add an electron.

Then the reaction moves. The change in energy is quite small and you could push the reaction one way or the other way depending on the relative concentrations of acetate, CO2 or pyruvate. The electron is what makes it happen, in either direction.

This is not unique to the reaction of pyruvate with hydrogen peroxide.

A little more grubbing around suggests that peroxinitrite is even better:

"... oxygen consumption studies confirmed that peroxynitrite mediates the decarboxylation of pyruvate to free radical intermediates. Comparing the yields of acetate and free radicals estimated from the oxygen uptake studies, it is concluded that pyruvate is oxidized by both one- and two-electron oxidation pathways..."

No one seems to have looked at superoxide but you can bet your bottom dollar it does the same. In fact, for the similar reaction of alpha ketoglutarate to succinate, superoxide will do the job:

The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist

"The significance of KG [alpha ketoglutarate], a metabolite that can detoxify H2O2 and O2- with the concomitant formation of succinate in this process is also discussed."

So what?

The interconversion of metabolites of the TCA appears to be quite possible mediated by nothing other than the availability of spare electrons and the relative concentrations of the core reactants. From Nick Lane's doodles the essential component for the actual dissipation of energy in the sections of the TCA at the origin of life are actually mediated by the availability of electrons. No enzymes required.

The availability of electrons from geochemical sources is what drove the conversion of COand protons to core metabolites of the essential parts of the TCA. No electrons, no conversion of anything in to anything else.

Today the electrons come from all sorts of places, reduced ferredoxin, NADH, FADH2 etc. But originally, in the beginning, I suspect that the first source of free electrons to replace the geochemical source might have been superoxide.

We know that LUCA used oxygen despite the anoxic conditions of the early Earth. She had superoxide dismutase, catalase and a precursor of haemoglobin which stored (precious) oxygen. So LUCA actively controlled oxygen availability, superoxide dismutation and hydrogen peroxide catabolism to oxygen and water. Presumably for a very specific purpose.

All that is needed to convert pyruvate to acetate is a free electron. Electrons are continuously being placed on to ferredoxin by our prototypical membrane bound hydrogenase. Once the cellular supply of ferredoxin has been largely converted to reduced ferredoxin then a) trying to move more electrons to ferredoxin gets harder and b) there is enough reduced ferredoxin to be worth activating metabolism and growth c) this can be initiated by transferring electrons on to stored oxygen (possibly derived from radiolysis or photolysis of water) and allow the superoxide generated to perform the process of converting one metabolite to another.

Ferredoxin (and even ATP, once evolved) could then be used for the more obscure reactions that might require more complicated metabolite interconversions, possibly not amenable to simple ROS mediated methods.

We are still using superoxide today, from reverse electron transport through complex I (directly comparable to that of the prototypical hydrogenase), as the core control of metabolism (pax NOX enzymes). The above speculative narrative would start with superoxide as the actual catalyst at first, one step removed from the origin of life, rather than as a signal. It will be interesting to see whether the modern production of superoxide has any residual enzymic function per se or whether it is now merely a signal/mediator, working through modification of functional sulphydryl groups on proteins which now perform their essential redox catalysis deep within their active sites.

I find it a fascinating idea.


Addendum.  These papers were formative of the above ideas but are a bit like excess baggage to the core principle. I enjoyed them so here they are:

You can do other interesting things with sources of electrons and core members of the modern TCA. If you would like to decarboxylate oxaloacetate to malonate just add ROS:

Malonate as a ROS product is associated with pyruvate carboxylase activity in acute myeloid leukaemia cells

"We have shown that malonate can be formed from oxaloacetate by chemical conversion under the influence of hydrogen peroxide..."

The ROS in this paper which convert oxaloacetate to malonate appears to come from the pyruvate carboxylase enzyme. This is their previous paper which they cited above:

Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

"However, in vitro treatment of oxaloacetate and pyruvate confirm that these conversions are in fact induced by hydrogen peroxide as shown in Figure S4."

"In addition, previous reports have established that ROS mediate the non-enzymatic conversions including that of α-ketoglutarate into succinate [24]-[26]."

This is the alpha-ketoglucarate paper:

Nonezymatic formation of succinate in mitochondria under oxidative stress

"The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO2 and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H2O2 and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO2 in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress..."

Okay, that will do!

Wednesday, January 31, 2024


Passthecream posted this link in comments which is a nice listen to Nick Lane explaining his ideas to a non biochemistry audience. I enjoyed it.

Youtube recalled that I watched it and suggested this similar item

which also has some core concepts in it. This few minutes really caught my eye as it's something I've been think about for a long time. It's a reiteration of his ideas from pages p133 to 140 of Transformer with all of the doodles merged in to two slides

Although he does specify a negatively charged surface in the book, this doesn't get fitted in to the brief overview he presents in the talk. But this negative charge is fundamental to the chemistry being discussed. I've snapshotted the two slides and added in the supply of electrons needed for each step of the reaction, with a different colour for each electron or pair of electrons, all coming from the charged surface.

Aside. The two red ovals pick out a single, bound, negatively charged oxygen atom. If you're trying to keep the charges balanced it is helpful to realise that they are the same moiety illustrated in two places on different slides. End aside.

This is pure electrochemistry. I picked up a paper years ago which was looking at origin of life reactions driven by an external voltage. You can drive the sort of reactions Nick Lane is describing with a tenth of a 1.5 volt battery's potential. The beauty of vents is that they supply the battery.

A subgroup of industrial chemical engineers is well aware of this phenomenon and they are amazed that electrochemistry for organic carbon based molecular synthesis has never been commercialised. This abstract gives the flavour of their frustration

A Survival Guide for the "Electro-curious"



Sunday, January 28, 2024

Life (40) Proton pumping

Okay. Time to finish the complex I series. Under conditions of a cell surface membrane which is partially permeable to protons and (less so) to hydroxyl ions there can be a proto-metabolism based on the ingress of protons driving both carbon fixation and energy generation, with neutralisation by OHions. This is dependent on having a partially permeable membrane to both of these ions. Subsequently, by using the simultaneous impermeability to (larger, less permeant) Na+ ions, combined with the above ability to neutralise protons with OH-, a Na+/H+ antiporter can establish a Na+ potential to drive a proto-ATP synthase. Koonin's group discussed it here:

Evolutionary primacy of sodium bioenergetics

As the protocell membrane becomes progressively more impermeable to both H+ and  OHthen running a Na+/H+ antiporter becomes progressively more difficult. At the same time this makes proton pumping potentially advantageous. This is how I am guessing that proton pumping may have developed.

If we start from that neat doodle from

looking like this:

we can reverse model it back to a simpler NiFe hydrogenase in a proton semi-permeable membrane and need just four images to sum it up:

This has the ocean at pH 6 protonating acidic amino acids in a channel from the ocean to the FeS cluster. There is also a side chain of acidic amino acids in contact with the NiFe cluster which are non-protonated because they are contiguous with the cytoplasmic fluid of pH10.

A molecule of hydrogen arrives at the NiFe cluster and is split in to a pair of electrons and a pair of protons:


The electrons hop on to the FeS cluster and thence to ferredoxin (accompanied by their ability to do work) to give reduced ferredoxin, Fd2-, while the protons go to the waiting carboxylates of the amino acids on the route to the pH10 cytoplasm:

which then leaves the complex ready for the next hydrogen molecule to come along after the protons on the cytoplasmic route's amino acids have been deprotonated by the pH10 cytoplasm:

The cost of this manoeuvre being a small fall in the intracellular pH, to be neutralised by the same alkaline vent fluid which supplied the molecular hydrogen.

That seems quite simple.

If we consider what might happen if the availability of hydroxyl ions is curtailed by progressively rising impermeability of the cell membrane to both H+ and OH- then the process must halt. With the evolution of soluble hydrogenases, and especially of electron bifurcation, then Fd2- might become more plentiful but molecular hydrogen less so.

We can consider what the immediate advantage might be to a cell to consume Fd2- and regenerate molecular hydrogen by running this complex in reverse.

So now I've set the intracellular pH to pH7 and left the ocean fixed at pH6. It's a big ocean.

In this scenario all of the amino acids in the complex would be protonated:


If we allow a Fd2- molecule to place a pair of electrons on to the FeS cluster:

these can combine with a pair of protons to form molecular hydrogen. These protons should come from the (very slightly) more acidic ocean channel:

This reaction is exothermic and needs no proton gradient. It leaves us with a deficit of protons in the oceanic pH channel:

which you would expect to be replenished from the bulk ocean. But we have a certain amount of free energy available from the high energy Fd2- molecule used to make the molecular hydrogen. All that is needed is an electrostatic/conformational change comparable to the "Doohickey" function of the last several posts and it becomes simple to take two protons from the cytoplasmic influenced amino acids and put then on to the oceanic side using the energy available from Fd2- oxidation:

What might be the immediate advantage of doing this?

The pH7 environment on the cellular side will allow spontaneous re-protonation of the acidic residues in the complex:

which will clearly leave a very small and very localised area of higher pH, here designated as pH8 for illustrative purposes only:

We have now produced a very localised accentuation of the progressively feebler pH gradient resulting from the cell membrane becoming progressively more opaque to OHions.

As cell energetics are highly Na+ dependent, as per the introduction to this post, establishing a small area of accentuated pH gradient will allow the immediate advantage of facilitating the struggling Na+/H+ antiporting process, at the cost of allowing the loss of the newly developed localised area of pH 8 (as shown) back down to pH7 (not shown). Like this:

which is fine except a simple "monogenetic" antiporter is actually pretty useless at low membrane potentials, as in:

So it would be better to have the ancient ancestor of the modern MRP ultra-low proton gradient antiporter instead. Here we have several protons each "kicking" another inward channel to finally 
"kick" a Na+ ion out of the cell:

At this point having MRP snuggle up to a membrane bound hydrogenase to access a better pH gradient is starting to look vaguely like a complex I precursor, but not quite. All we have is a small improved localised pH gradient, no gross expulsion of protons, and the sole use is to generate a Na+ ion gradient. But that would be advantageous, immediately.

Now let's worsen matters still further and drop the intracellular pH to 6.5, where even the mighty MRP antiporter is in trouble. We can get extracellular protons to the half way inward mark, and intracellular Na+ to the half way outward mark but there is insufficient pH drive to complete their respective journeys. Stalemate:

Now if we just think about that energy input from "wasting" a Fd2- molecule we can have a conformational/electrostatic change in the green outlined amino acid (modern day aspartate D72 in the original diagram) like this:

giving a "push" to help the struggling MRP antiporter:

by providing a "kick" which the pH gradient can't manage alone. As it stands there need be no outward proton translocation, just a push to the MRP antiporter. In fact the localised pH gradient would be lost on Na+ antiporting but the cell would have bought a better Na+ gradient for ATP synthase in return:

In this last image I've suggested that blocking off access to the ocean would be an incremental advantage too as it might make a conformational change in the "kicking" acidic amino acid more effective at facilitating MRP antiport completion.

None of this is a proton pump. But as the cell membrane become essentially impermeable to protons there develops an advantage to running MRP in reverse. All you have to do is attach the kicking-complex the wrong way round to MRP and you could kick a Na+ in to the cell and two H+ out of the cell, then start of using protons in ATP synthase. Or completely drop the module which translocates Na+ and just use the "kick" to push two protons outwards. Or, given a power source like the NADH:CoQ couple, kick four protons out wards, as in complex I. Notice the "kicker" is on the opposite end of the MRP antiporter derivative here and the Na+ module has been abandoned/replaced:

Given a less potent power source such as the Fd2-/H+ couple you can just drive out one proton, as Ech does:

You can also, if you're Pyrococcus furiousus living at 100degC, still pump Na+ ions (it's not easy to build a proton tight membrane at 100degC, so Na+ energetics are retained) by flipping one proton channel round, pushing a proton outwards and allowing this proton back inwards to antiport a Na+ ion outwards:

which is a proton-neutral technique to establish a Na+ ion gradient.

All you have to do is to develop a "kicker" for MRP and the world is your oyster. There are many derivatives of this type of pump with various subunits arranged in various orders. It's a molecular Lego set. All that is needed is for each step during its development to be continuously advantageous.

The concept that modern derivatives might be the best guide as to where and how life began fascinates me and has been laid out by Nick Lane's group here:

It makes a lot of sense to me.