Satiety (02) TD.130051

One of the reasons that I'm excited about Shulman's description of Thr¹¹⁶⁰ is that it provides us with a very simple way of assessing or quantifying insulin resistance without all of the problems of hyperinsulinaemic euglycaemic clamps, and there are a lot of problems with those.

It doesn't even seem beyond the realms of possibility that Shulman's lab might develop yet another NMR protocol to track this phosphorylation in vivo. That would be very cool.

On a very simple basis I'd like to make some suggestions about what the phosphorylation of Thr¹¹⁶⁰ might mean from the Protons perspective.

This is the Shulman paper

Short-term overnutrition induces white adipose tissue insulin resistance through sn-1,2-diacylglycerol/PKCε/insulin receptor Thr1160 phosphorylation

Shulman has a pathway from the accumulation of diacyl glycerols (DAGs) in the cell surface membrane to the activation of phosphokinase C ε (PKCε) which, along with many other targets, phosphorylates Thr¹¹⁶⁰ and so induces insulin resistance.

So this particular pathway to insulin resistance looks like a sensor built around the accumulation of the penultimate molecule in triglyceride synthesis and could be viewed as a marker of supply of lipid. My own ideas are rooted in the ability of fatty acids to generate ROS mediated insulin resistance as a physiological function. We are looking at a different level of the same signal. It would be very interesting to look at the ability of differing DAGs to activate PCKε. I would predict that saturated fatty acids are better activators than PUFA, following the ROS pattern the sensor is built around. This would be very cool and could be really useful for "blaming" saturated fats for the "defect" of insulin resistance.

Anyway, I am going to consider insulin resistance, aka phosphorylation of Thr¹¹⁶⁰, under various circumstances

I've taken the Schwartz lab graph yet again, which we now all know,

blotted out the food intakes and put an hypothetical scale of percentage phosphorylation level of Thr¹¹⁶⁰. We can put four clear cut data points on such a chart from Shulman's work

Chow is easy because it shouldn't change much in a week. Fasting is easy too because, if we fasted a human for a week they would clearly develop insulin resistance in order to spare glucose for brain usage, as observed by Shulman in the video. A mouse would die in less than a week of fasting.

We know D12942 produces higher insulin resistance than chow but lower than fasting. The paradox being that the D12492 fed rats are moving towards the fasting value despite having over-eaten for a week.

Before we go on to consider this we need to think about the custom diet made by Harlan for this perviously discussed (not very good) study

A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue

where TD.130051, with 50% of calories as fat, produced zero weight gain in excess of that of mice on chow. So here we have an high fat diet which does not produce "hyperphagia" or, as Shulman phrases it in the title of his paper, "overnutrition". Mice eat enough to grow, no more, no less. At 50% fat.

Where would we pin the donkey tail for TD.130051 on the red line which marks the level of insulin resistance on day seven?

If we ignore D12492 and simply think of the phosphorylation of Thr¹¹⁶⁰ as being a surrogate for how much of the adipocyte (in this study) metabolism is being based on lipid oxidation, and so generating ROS, we can assume it will come somewhere between the value for chow (18% fat) and fasting (virtually 100% of calories from fat derived from adipose tissue). This is very simple, though impossible to specify numerically. I would expect something like this:

For fasting there should be a delay while liver and muscle glycogen is depleted then a rapid rise in insulin resistance to the fasting level.

For TD.130051 there would also be a delay but we are not expecting any need for glycogen depletion, all we need is for the adipocytes to adapt from the level of ROS production associated with 18% fat to that associated with 50% fat. I've assumed this is a couple of days too, pale blue line. Or you could, in Shulman-speak, assume this is the timescale for a 50% fat diet to increase the level of DAGs in the cell membrane so activates PCKε and so generate Thr¹¹⁶⁰ phosphorylation mediated insulin resistance.

Not that Shulman views a rise in insulin resistance as a simple adaptation to an high fat diet, be that exogenous fat or secondary to fasting. It's a defect to be corrected, unless it's from fasting. And I would expect TD.130051 to be *worse* than D12492 in this respect. This is obvious because you have to resist insulin if you want to stay slim. Saturated fat does this. TD.130051 is very high in saturates and only around 6% linoleic acid.

So in the case of TD.130051 there is absolutely no suggestion of "overnutrition" as a Shulman-esque explanation for the insulin resistance. Normal weight, normal calorie intake, normal growth but with 50% fat. I predict it will still lead to insulin resistance. This is to limit glucose ingress to offset the calories from fat so that an adipocyte, and the whole organism, would have an RQ or RER (to use the horrible but correct new terms) which matches the food quotient (FQ). And not get fat.

That's how it works, using ROS/DAGs, whichever you prefer. Although the ROS signal is far more fundamental than the DAG signal, it's probably much more labile too (but also capable of a rapid response) and there is clearly some advantage in smoothing it out with an overlay (slower but more even response). The DAG signal will be a derivative of the ROS signal. It will concur with it. In both cases the insulin resistance is utterly physiological.

D12492 is more complicated.

Peter

Satiety (01) Shulman's gift of threonine 1160

 I have to acknowledge an important gift from Dr Shulman's lab in this paper:

Short-term overnutrition induces white adipose tissue insulin resistance through sn-1,2-diacylglycerol/PKCε/insulin receptor Thr1160 phosphorylation

That gift is the amino acid Threonine¹¹⁶⁰ (Thr¹¹⁶⁰), part of the insulin receptor.

We all know the story, the insulin receptor is always trying to activate itself, via its built in autophosphorylation subunit and this self activation process is kept under control by a complex process using phosphatases, which are under redox control. You know, ROS.

Thr¹¹⁶⁰ is a switch, apparently independent of the above process. If Thr¹¹⁶⁰ is left alone the activation system works perfectly well whenever insulin docks with the receptor.

If Thr¹¹⁶⁰ is phosphorylated, the activation module doesn't work because the shape of the catalytic domain has been carefully modified to stop it functioning. Insulin can dock with the receptor, but nothing happens.

At the level of an individual insulin receptor the ability to respond to insulin is controlled by a simple on/off switch at the Thr¹¹⁶⁰ site.

Shulman's excellent paper goes on to show how D12492 (yes, that D12492) induces phosphorylation of Thr¹¹⁶⁰  within seven days and so induces insulin resistance. It details the mechanism (which is irrelevant to understanding the physiology) in great detail.

Now, there is a paradox.

In this video clip:

at time point 9.15 he describes insulin resistance as a "defect". His term, not mine.

At time point 10.00 he points out that Thr¹¹⁶⁰ is conserved from humans to fruit flies, so insulin resistance must have a serious survival benefit.

At time point 10.35 he observes that this crucial insulin resistance pathway is activated under starvation, to spare glucose for the brain, hence its conservation.

At 11.22 he points out that "overnutrition" activates this pathway, leading to metabolic disease.

The paradox, not explicitly stated as such, is that Thr¹¹⁶⁰ phosphorylation is induced by both starvation and by "overnutrition".

I love this.

I happened on the video quite by chance and it took me some weeks before I went back and pulled out the amino acid involved (and looked up the papers about it) and thought through what Shulman was saying in the video.

I think Thr¹¹⁶⁰ is going to provide a fantastic tool to allow us to consider all sorts of data points. It's difficult to know where to start.

But it won't be from insulin resistance as a "defect"

Peter

Rapeseed oil for weight loss (4): Hypocaloric satiety

This post is about the next anomaly in the paper

A highly saturated fat-rich diet is more obesogenic than diets with lower saturated fat content

and is looking at this graph:

Again, this post is pure speculation. I don't even know if the lab had a janitor.

The red oval highlights a serious "That's odd" moment. With this sort of finding you can

a) Say "That's odd" and think about investigating it based on what probably happened.

b) Report it, accept that you have no idea what it is all about and say so.

c) Pretend it didn't happen, but leave the anomaly in the graph without mentioning it in the results or discussion.

d) Falsify the data.

To their credit the group took option c), as far as I can glean after making myself read the results and skim-read the discussion section. The didn't take option d), to their slight credit.

Why do I find this so interesting?

Because rats, subjected to a 40% reduction (down to 165kJ/d) in available calories from their preferred calorie intake (270kJ/d) quite suddenly, after about 10 days of clearing the food hopper, started to leave some of the full (but small) amount of offered food.

In real terms this means that, suddenly, they weren't hungry. During a 40% calorie restriction.

Just think about that.

Nothing at all is reported to have changed at the time point of the sudden drop in hunger.

Oooooh, now that's a challenge if ever I saw one. The change was transient and the rats were almost back to clearing their hopper at the time they were executed ("euthanised" under CO2 anaesthesia).

Aside: If you have an anaesthetic machine with a CO2 regulator and a cylinder of CO2 you can easily dial a 50:50 mixture of CO2:O2 and try inhaling it. I would suggest that is no fun. No fun at all. I guess the study's ethics committee have never tried this. Maybe they don't have an anaesthetic machine with a CO2 regulator to try it with. The way I used to have. End aside.

Anyhoo. Here's a Powerpoint of lines popping up on diagrams.

TLDW?

The janitor turned up the heating. Day 109.

Peter

PS this wouldn't work in humans, not even transiently. We're too big, we live very close to our thermoneutral point compared to rats.

Links to papers briefly used in the presentation.

Mouse EE graph from https://pubmed.ncbi.nlm.nih.gov/26042200/

Mouse to rat EE conversion from

https://journals.physiology.org/doi/epdf/10.1152/ajpregu.00137.2012

Rapeseed oil for weight loss (3) Canola oil vs butter round two

Now to get down to the nitty gritty of the first anomaly in the paper

A highly saturated fat-rich diet is more obesogenic than diets with lower saturated fat content

The oddity is the blip downwards of weight in canola fed rats, highlighted by the red oval on the graph:

which needs to be read in conjunction with the food intakes in this graph, also highlighted by a red oval:

I've copied the food intake graph and laid it over the weight graph to make it a bit clearer:

Because the red graph is in grams of food I've annotated the important energy intakes next to the related data points. Obviously the switch from chow to high fat diet (fine orange line) massively alters the caloric intake despite the weight eaten being the same on the day before and after the switch.

Interminable aside: if any paper gives you the caloric density of any food used you are assured that the group are idiots, even if they are very, very clever idiots. They believe you can fool a rat's hypothalamus in to over-consuming calories by increasing the caloric density of the food. Or fool it in to under-consuming calories by diluting them down with sawdust or the equivalent (usually cellulose). As if evolution is that gullible. Do not, under any circumstances, accept any of such a group's ideas about satiety, food intake or bodyweight. Ever. End aside.

Anyhoo. On day one the high fat fed rats increased their energy intake from around 227kJ (55kcal) to 355kJ (85kcal), a rise of ~56%. I find this impressive and in the same league as the Schwartz lab rats on D12492, who increased their caloric intake by ~87% on day one in this venerable graph:

In Protons terms the canola oil fed rats still used 227kJ for running their metabolism but "lost" 128kJ in to their adipocytes via the enhanced insulin signalling of linoleic acid (LA) and alpha linolenic acid (ALA) due to their low F:N ratios. The rats needed 227kJ, they lost 128kJ so needed to replace those 128kJ, therefore they ate 227+128 = 355kJ. They stopped at 355kJ because that's when they stopped being hungry. Overall weight did not change because 227kJ of chow weighs the same as 355kJ of the canola oil diet (Hmmm, chance or pilot study????). 128kJ is about 3g of fat gain.

Simple.

What happened next is much more exciting. On day two of the canola oil diet the rats weren't hungry. They only ate 155kJ that day. The weight of food eaten dropped by ~13g, and so did the weight (~10g) of the rats, food is heavy, they ate less, they weighed less. But they still needed those 227kJ to run their metabolism. They ate until they weren't hungry, that's what they do. Why weren't they hungry?

They must have accessed the full 227kJ that they needed, otherwise they would still have been hungry. They obtained the missing 72kJ from their adipocytes. Without "trying", without being hungry. Remember, they always eat to satiety.

The logical explanation is that there was an acute drop in insulin signalling in their adipocytes which released 72kJ worth of FFAs, which meant that there was no need to eat the 72kJ's worth of food needed to reach the full 227kJ to run metabolism. 72kJ is the energy available from about 2g of fat.

From the Protons perspective acute loss of insulin signalling is a standard effect of reduced mitochondrial membrane potential caused by uncoupling. PUFA uncouple. At high intakes this over rides the obesogenic effect of their reduced F:N ratio.

The blip of weight down then back up again mostly reflects food weights with a lesser contribution from adipose weights, but the latter adipose changes are the factor responsible for the hunger changes which drive the food intake changes.

Uncoupling explains the blip. The only thing which surprised me was how rapidly the effect kicked in. I would have expected that about a week would be about the time scale but here it was 48h. Those are the data. I have to revise my views in the light of it. You don't often get given a gift-horse (of daily food intakes and daily body weight changes) to consider examining the mouth of!

The following day the rats increased their food intake. Under steady growth the rats still needed the standard 227kJ/d plus some extra for uncoupling losses. Adipocyte size does not continue to shrink because the uncoupling using biological uncoupling proteins is ATP dependent, low intra mitochondrial ATP reduces the effectiveness of uncoupling proteins. This is distinctly different from chemical uncoupling to which there is no upper limit and so these can give extreme weight loss or even death.

The rats still needed 227kJ/d for metabolism and 62kJ/d for uncoupling, hence 289kJ/d. This happened pretty consistently throughout the rest of the high fat period. They stayed slim but not too slim.  Despite eating around 20% extra in calories compared to the chow fed mice.

Peter

There is another very strange/interesting feature on the food intake graph. I'll see when I can find time to describe it.

Rapeseed oil for weight loss (2) and butter for obesity round one

This is the next paper. These people are good. Really good. There is almost nothing amateurish in this paper:

A highly saturated fat-rich diet is more obesogenic than diets with lower saturated fat content

and here is the graph which sums it up

I'm not saying these people aren't stacking the deck and I'm not for a moment suggesting that they understand anything about obesity. But they deliver in spades to support the current (anti saturated fat) narrative.

I'm going to speculate from here onwards about how they managed it.

The above graph did not materialise out of thin air.

They made their own foods in their own lab. For the high fat diet they "chose" 67% of calories as fat, For the low fat period they "chose" 27% of energy as fat.

Why?

Because, obviously, markedly different values would not have generated the above graph. They could, and I suspect they did, formulate almost any diet macro ratio they wanted to. It's called a pilot study and and I think you should seriously consider that they did one, and kept quiet about it.

Very few people using a rodent diet induced obesity model would consider 27% of calories from fat to be low in fat. Their chow was ~11% of calories as fat and very low in PUFA (Diet 5075, which included beef tallow as a partial fat source. It's not made anymore but you can guess it resembles 5001 but with less PUFA. Probably. But who knows?). My presumption for butter is that medium and short chain fatty acids are, by the Protons hypothesis, insulin sensitising. At low dose rates they are largely diverted from gut to liver and never reach peripheral adipocytes. So they are not obesogenic. They increase hepatic insulin sensitivity which lowers the penetration of both glucose and insulin to the systemic circulation and, certainly in the case of MCT oil, are slimming. My guess is that much less than 27% of calories in the "low fat" period would have produced active weight loss. So you simply do not use less than 27% of calories as fat from butter, if you want obesity in your model. 

If you supply enough insulin sensitising shorter chain fatty acids that they penetrate past the liver to peripheral adipocytes they will continue to work their insulin sensitising effect but this time on adipocytes rather than hepatocytes. With enhanced adipocyte insulin signalling there is enhanced fat gain. This appears to be optimal at 67% of calories from butter. Much more than 67% would tend towards ketogenically low levels of insulin exposure and the insulin sensitising effect would become irrelevant. Again, a guess, but much over or under 67% would have generated less obesity. It's a model, it's designed to make a point.

In the canola oil arm the high fat diet provide ~20% of calories as LA, verging on an uncoupling dose rate. In addition to this there are 10% of calories as ALA, a significantly better activator of UCP1 and UCP2 than LA. This is clearly a factor in the high fat group and there are oddities on the graph and elsewhere in the paper which do suggest that this is the case. Another post.

What would have happened if they had lowered the canola oil to a real low fat level, say 4% of calories from PUFA? LA at under 4% of energy is not usually obesogenic, though it should tend that way via the Protons F:N ratio, absenting uncoupling effects. Some fat storage is beneficial. And ALA would accentuate this effect, so long as levels were high enough for the Protons effect to occur but low enough to avoid uncoupling effects. So very low dose canola oil might well be obesogenic and was sensibly avoided.

You don't write your experimental protocol until you are damned sure it is going to deliver. The required result here is to show that "saturated" fat rich diets are obesogenic and "PUFA" rich diets are not.

They succeeded. As I say, they are good. Here's the start of their discussion:

"The present study tested canola, lard, and butter, respectively, low, moderate, and rich sources of SFA, widely consumed in the human diet, in an animal model of dietary obesity. As predicted, results confirmed the hypothesis that an SFA-rich diet is more obesogenic than diets with lower SFA content."

They obtained the result they predicted. Probably by dint of a lot of hard work.

Peter

Have you thought about electron transporting flavoprotein dehydrogenase and its substrate electron transporting flavoprotein?

This is not really a post. I just want somewhere to place a concept, outside of the original post in which it is embedded, where it is more easily recognisable and findable.

It's worth noting that this paper:

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency

describes the process of remodelling the ETC in great detail, especially down regulation of complex I when there is an high input from other sources to the CoQ couple. It's mediated by reverse electron transport and it happens fast.

The basic TLDR is that if you take fat adapted mitochondria they will be using mtETFdh to generate a significant proportion of their maximal oxygen consumption for ATP generation. This means that complexes I and II will be down regulated, so supplying electrons to these complexes cannot match the oxygen consumption which would be generated if mtETFdh was maximally active. We have no available direct supplies of electron transporting flavoprotein to supply FADH2 in the way that beta oxidation does. "Dysfunction" is actually an artefact of not inputting adequate electrons to the CoQ couple via mtETFdh.

This applied both to studies on high fat diets and studies on fasting. It implies extreme caution if one is to decide that high PUFA diets, when high in overall fat, do actually cause *any* mitochondrial dysfunction, if only tested using inputs from glutamate/malate or succinate.

So this has major implications as a generic "how to read a paper" factor.

The insight is based on the oxygen consumptions in this paper where "disrupted bioenergetics" are claimed.

Rapeseed oil‑rich diet alters hepatic mitochondrial membrane lipid composition and disrupts bioenergetics

I wrote this in the post about the above paper:

There is nothing wrong with these mitochondria. Bioenergetic are *not* disrupted, as suggested by the title of the paper. Let's dig deeper.


What is happening is that the study is taking mitochondria from fat-adapted rats and feeding them on either a complex I input or a complex II input. Fatty acids, even LA, make significant use of electron transporting flavoprotein (ETF) dehydrogenase as their input to the CoQ couple. Mitochondria adapt their electron transport chains to the substrates available. If mitochondria from rats fed 40% of calories from fat are significantly dependent on mtETFdh for input to the CoQ couple, and have down regulated both complexes I and II, then feeding the preparation on substrates specifically aimed at complex I or II will obviously produce sub-maximal oxygen consumption. Which is what happens under either state 3 respiration or FCCP uncoupling.


Under the "tickover" conditions of state 4 respiration the uncoupling from PUFA shows clearly.


Obviously, to restore visibly normal mitochondrial function, what's needed is a supply of reduced ETF to use as a substrate for mtETFdh. As supplied by beta oxidation. Sadly you can't just buy reduced electron transporting flavoprotein from Sigma Aldridge, so you end up with artifactual mitochondrial "dysfunction".

Another aside: that is exactly what is happening here too

Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

discussed here

Will fasting destroy your mitochondria? (No).

It *appears* as if mitochondria adapted to high input from mtETFdh are dysfunctional if you fail to supply them with adequate electron transporting flavoprotein! The study did try to get around this by using octanoyl-carnitine (50μmol/l) as a lipid input to generate reduced ETF but clearly even 50μmol/l of palmitate will provide twice the ETF of 50μmol/l octanoate and the chaps in the study were running total FFAs of up to 3000μmol/l, not 50μmol/l, at the time that the muscle biopsies were taken. Utilising 3000μmol/l of FFAs provides a lot of ETF. So "dysfunction" is really an experimental artefact induced by lack of metabolic substrate for mtETFdh (secondary to using an homeopathic level of octanoate in this case or no mtETFdh substrate at all in most studies).

That is all.

Peter

Rapeseed oil for weight loss (1): Norwitz vs Goodrich (eventually, scroll down if bored by the very long Protons preamble)

Rats fed a diet with 40% of calories from rapeseed oil are slim.

Rapeseed oil‑rich diet alters hepatic mitochondrial membrane lipid composition and disrupts bioenergetics

"Rats assigned to the modified diet showed a slight delay in growth (Fig. S1, left), probably related to a lower food intake (Fig. S1, right)."

Like this

That's correct, feeding a 40% fat diet where the fat is rapeseed oil produces less weight gain compared to standard carpinabag chow. Because the rats had, and I quote, "a lower food intake". This translates, for less mealy mouthed people, as "the rats were less hungry while still eating to satiety". However at week two, when weight was already down, the rats were actually eating *more* calories of the rapeseed oil diet, compared to the chow fed group. Uncoupling. Decreased insulin signalling.

Aside: they also note:

"The only significant [gross pathological] alteration observed in rats submitted to the 20 % rapeseed oil diet was a decrease in the [hepatic] triglyceride content (56 ± 7.1 mg/dL as compared to 137 ± 29.5 mg/dL for control rats) after 33 days of treatment."

Yes, you read that correctly, rapeseed oil is protective against the fatty liver which is routinely induced by chow with 7% of calories as PUFA, mostly LA. That's a huge drop in hepatic trigs. Anyone want to cure NAFLD?

End aside.

Now, from the Protons point of view there are two ways of avoiding obesity (or NAFLD). You can

a) get your ROS in to the range equivalent of treating an adipocyte with H2O2 at around ~3-4mmol/l, ie employ physiological insulin resistance generated by saturated fats during the peak absorptive phase after a meal. My approach. It works.

or

b) reduce your ROS generation to a level too low for peak insulin signalling, say to the equivalent of ~0.01-0.03mmol of H2O2.

That means uncoupling.

PUFA, especially linoleic acid, fail to generate adequate ROS via Dave Speijer's F:N ratio hypothesis which is the basic underpinning of Protons. So they are obesogenic.

However, they are also uncoupling.

Which effect predominates determines whether the PUFA based diet you have chosen to eat is obesogenic or not. How much PUFA, which species, whether they are mitochondrially targeted. Linoleic acid (LA) at 35% of calories is uncoupling and will normalise the weight of a lard fed mouse. Alpha linoleic acid (ALA) will do the same, but studies tend to use high dose rates. People love all-or-nothing models. So simple to secure funding.

Hypolipidemic Activity of Peony Seed Oil Rich in α-Linolenic, is Mediated Through Inhibition of Lipogenesis and Upregulation of Fatty Acid β-Oxidation

More translation: "Mediated Through Inhibition of Lipogenesis and Upregulation of Fatty Acid β-Oxidation" actually translates as "reduced insulin signalling". If you can measure virtually everything but have no hypothesis to hang you facts on, you'll get nowhere.

At what level of intake this effect kicks in is difficult to determine but it will undoubtedly be lower for ALA than for LA.

Now we can look at the current study in those terms.

Do the liver mitochondria uncouple?

Yes, and no.

The numbers are best for experiments using succinate to feed the mitochondrial preparations. With a priming dose of succinate followed by a flood of ADP the mitochondrial run flat out (state 3 respiration).

Here there is a slightly *lower* O2 consumption in the mitochondria from rapeseed oil fed rats, a paradox:

That doesn't look like uncoupling.

We have a large supply of metabolic substrate and a large supply of ADP to allow ATP-synthase to turn but still, in supposedly uncoupled mitochondria, oxygen consumption is reduced. The same happens when feeding with glutamate-malate to input at complex I but something went wrong with these data at the week 22 mark. Overall it doesn't look like these mitochondrial are uncoupled on either substrate.

Next we can look at what happens when the added ADP has run out (state 4 respiration). Now ATP-synthase cannot turn because there is no ADP available, so any oxygen consumption must be via uncoupling. That's exactly what we see. There is now also plenty of intra-mitochondrial ATP to allow uncoupling protein activation. So yes, uncoupling happens in this state:

Equally unexpected for already uncoupled mitochondrial is the effect of full uncoupling with FCCP. Here we again have paradoxically reduced oxygen consumption in the uncoupled mitochondrial preparations from the rapeseed oil fed rats.

So the question is why should supposedly pre-uncoupled mitochondrial have lower oxygen consumptions than either fully uncoupled mitochondria or mitochondria running at full chat? Whether you "feed" your preparation with a complex I input or a complex II input?

There is nothing wrong with these mitochondria. Bioenergetics are *not* disrupted, as suggested by the title of the paper. Let's dig deeper.

What is happening is that the study is taking mitochondria from fat-adapted rats and feeding them on either a complex I input or a complex II input. Fatty acids, even LA, make significant use of electron transporting flavoprotein (ETF) dehydrogenase as their input to the CoQ couple. Mitochondria adapt their electron transport chains to the substrates available. If mitochondria from rats fed 40% of calories from fat are significantly dependent on mtETFdh for input to the CoQ couple, and have down regulated both complexes I and II, then feeding the preparation on substrates specifically aimed at complex I or II will obviously produce sub-maximal oxygen consumption. Which is what happens under either state 3 respiration or FCCP uncoupling.

Under the "tickover" conditions of state 4 respiration the uncoupling from PUFA shows clearly.

Obviously, to restore visibly normal mitochondrial function, what's needed is a supply of reduced ETF to use as a substrate for mtETFdh. As supplied by beta oxidation. Sadly you can't just buy reduced electron transporting flavoprotein from Sigma Aldridge, so you end up with artifactual mitochondrial "dysfunction".

Another aside: that is exactly what is happening here too

Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

discussed here

Will fasting destroy your mitochondria? (No).

It *appears* as if mitochondria adapted to high input from mtETFdh are dysfunctional if you fail to supply them with adequate electron transporting flavoprotein! The study did try to get around this by using octanoyl-carnitine (50μmol/l) as a lipid input to generate reduced ETF but clearly even 50μmol/l of palmitate will provide twice the ETF of 50μmol/l octanoate and the chaps in the study were running total FFAs of up to 3000μmol/l, not 50μmol/l, at the time that the muscle biopsies were taken. Utilising 3000μmol/l of FFAs provides a lot of ETF. So "dysfunction" is really an experimental artefact induced by lack of metabolic substrate for mtETFdh (secondary to using an homeopathic level of octanoate in this case or no mtETFdh substrate at all in most studies). 

End aside.

Okay. Protons says that reducing the mitochondrial membrane potential reduces superoxide production (and its derivative H2O2) to levels which do not promote activation of the insulin signalling cascade.

Delta psi is definitely reduced by the rapeseed oil feeding. These are the values for delta psi of a fully charged membrane using glutamate-malate (succinate is the same) but before any ADP has been added, ie this is as high as the membrane potential can get with the substrate supplied (the graph is deliberately upside down as delta psi is always negative):

The membrane potential drops by less in the mitochondria from rapeseed oil fed rats when ADP is supplied but it is still always lower that for the control rats.

Is this reflected in lower production of H2O2?

Yes:

The only unanswered question is how does a production rate of H2O2 of just over 2000pmol/mg prot/15mins in uncoupled mitochondria fed succinate through complex II compare to just over 3000pmol/mg protein/15mins in the normally coupled mitochondria? And how do these relate to the steady state exposure to H2O2 used in

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

to generate this graph

This we can't answer directly. The fact that the rats fed rapeseed oil were lighter than those fed chow suggests, to me, that there was less insulin signalling going on in the adipocytes of the slim rats.

People may disagree.

That's fine, just it makes sense to me.

But: Is it Good or Bad to run your (slim) metabolism on uncoupling levels of PUFA when compared to running on insulin resisting saturated fat?

Ah, now that's a question. There are suggestions as to the correct answer.

Running your metabolism on a mix of LA and ALA which allows uncoupling to a normal or a slightly slim bodyweight might make you look good. Without having listened to any of the podcasts of Nick Norwitz (he's clearly a bright guy) I can easily accept he's fully able to maintain a slim bodyform, combined with high ketone levels, using PUFA, even if he doesn't mind getting sunburned occasionally as a trade off.

Running your metabolism predominantly on fat while avoiding PUFA also works, looking at Tucker Goodrich here. Normal bodyweight, minimal risk of sunburn.

So here's the decider, and it's not sunburn:

Who is most at risk of pancreatitis? I know, I know, none of us will get pancreatitis. But we might. You can stack the deck. This is Nick's pancreas on PUFA:

Distinctive roles of unsaturated and saturated fatty acids in hyperlipidemic pancreatitis

"At sufficiently high concentrations, unsaturated fatty acids were able to induce acinar cells injury and promote the development of pancreatitis." [Not my typo].

Well, look what happens when you emphasise PUFA for maximum ketosis:

Early- and late-onset complications of the ketogenic diet for intractable epilepsy

I'm sure this won't happen and Nick will be fine. But, if it does happen, pancreatitis is a really fun condition to have (not) and, if you get a good dose of it, you are heading for the ITU for quite a long stay. Will you live or die? That's largely dependent on whether you develop acute (some say adult) respiratory distress syndrome (ARDS). While people have tried treating this with a combined heart-lung transplant, it's better avoided if you want to survive.

You can pretty well predict who will develop ARDS as they come in to the ITU by simply counting the double bonds in their plasma free fatty acids.

An increase in serum C18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome

"The calculated ratios of serum free fatty acids (ie., the ratio of C18 unsaturated fatty acids linoleate and oleate to fully saturated palmitate, C16:0) increased and predicted the development of ARDS in at-risk patients."

Now, I have fundamental ideas about choice of lipid sources for weight normalisation. Those ideas are compatible both with the choices made by Tucker and those made by Nick. Both are correct in their diametrically opposed choices. They are both correct for bodyweight. But there are nuances. I like nuances.

One choice is more likely to lead to the ITU and to the development of ARDS than the other.

My advice is to NEVER need to go to the ITU and DO NOT develop ARDS.

There are some real lifestyle choices which influence these outcomes. Rapeseed oil for weight loss works. It also raises the number of double bonds in pretty much all of your tissues.

Choose wisely.

Peter

Addendum. BTW just to clarify: You don't actually need pancreatitis to develop ARDS. It's a generic route to death in the ITU. Simple severe trauma will do the job. The PUFA are still what influence whether you live or die.

Protons (77) Shulman PUFA and insulin sensitisation. Or not. Or so.

Over in the comments to the last post on metformin and Shulman's lab, Tucker pointed out that Shulman was an author (penultimate, so a senior author) on Nowotny et al's 2013 paper

Mechanisms Underlying the Onset of Oral Lipid–Induced Skeletal Muscle Insulin Resistance in Humans

which starts its discussion with the controversy about whether PUFA, particularly from soybean oil,  induce insulin resistance or insulin sensitivity. The most contradictory paper they cite is Xiao et al from 2006. In my head I think of this as the Hot Chocolate or the Cocoa study, which I discussed here as pure Protons in a cup of hot chocolate:

Differential effects of monounsaturated, polyunsaturated and saturated fat ingestion on glucose-stimulated insulin secretion, sensitivity and clearance in overweight and obese, non-diabetic humans

The two studies have diametrically opposite conclusions and this is an obvious opportunity for insight.

To me, it contradicts Protons, so let's go! Sadly, as Tucker points out, the chance for insight is completely missed by the Shulman group. They settled for PUFA -> insulin resistance. They lack the Protons perspective.

Equally sadly I have no handy mental label for the Nowotny/Shulman paper, probably because it produces no confirmation bias induced dopamine release in my head. My bad.

So let's compared the two.

The Cocoa study fasted obese subjects for 12 hours then fed them almost nothing but fat (with just a little carbohydrate) in small aliquots over 28-30 hours depending on how you read the methods. These subjects, by the time they started their hyperglycaemic clamp, had been either fully fasting or running their metabolism on the study fat, for approaching forty hours. In the control section of the study there were minimal calories supplied throughout, just a little carbohydrate in each drink. We can consider our control section to be, of necessity, in the functional insulin resistance of (very mild) starvation.

They are in a functional, very physiological, insulin resistance which is essential secondary to fasting or a fat based metabolism.

In Nowotny/Shulman their subjects were fasted for 10 hours in the aftermath of three days of a "high carbohydrate" eating period. By the start of the study period the subjects might have been ready for breakfast but they will have had absolutely no need (with a liver still full of glycogen) for the physiological insulin resistance adaptation to an absence of carbohydrate food intake such as would be necessitated by 40 hours of near fasting.

Subjects either drank 900kcal of soybean oil, started a 6h infusion of ~900kcal of intravenous soybean oil (Intralipid) or, for the control group, a 6h infusion containing approximately 54kcal of glycerol, ie mild fasting of 16h in total for the controls. Glycerol is primarily used by the liver for gluconeogensis so it will be a bit like the carbohydrate from the drink in the Cocoa study.

So Nowotny/Shulman  only looked at soybean oil vs nothing on a deliberately carbohydrate based metabolism of their mildly fasted control group. After carb loading to ensure a glucose based metabolism.

And soybean oil triggered insulin resistance. Glycerol didn't. QED, PUFA cause insulin resistance, directly. Diacylglycerols blah blah and all that crap as a mechanism, which is what the study seems to have been all about.

But the soybean oil was only being compared to a fully primed glucose based metabolism. Using an hyperinsulinaemic clamp and glucose supply to normoglycaemia.

In the Cocoa study the various fats were being compared to what was approaching a 40 hour fast. These subjects were already physiologically insulin resistant.

Adding more fat will (at approximately the correct 24h metabolic requirement for calories) either increase or decrease the degree of fasting insulin resistance based on Protons, the F:N ratio and reverse electron transport derived ROS.

Here PUFA cause a decrease in the physiological insulin resistance of 40 hours of fasting. Saturated fat augments it. MUFA is neutral:

You have to be very, very careful about what you are comparing to what.

Without thinking through the methods sections you could easily be forgiven for believing that the Nowotny/Shulman paper shows that PUFA cause insulin resistance. Possibly uniquely.

They clearly do, compared to to glucose. But they are *less* effective than saturated fats in performing the essential metabolic function of resisting insulin when glucose is in short supply. Shulman missed this, despite having Xiao point it out (*sarcasm warning*) in words of one syllable. PUFA are insulin sensitising when compared to whatever FFAs an obese person has available after nearly 40 hours of fasting.

Peter

Afterthought: The Cocoa SFA group required just under 40μmol/kg/min of glucose to maintain 20mmol/l in their plasma. The PUFA group needed about 55μmol/kg/min of glucose.

Given an infinite supply of donuts, which state would result in you eating the most?

Mmmmmm PUFA. The fat that makes you fat. By limiting insulin resistance.

Metformin (16) The LaMoia Shulman review

I first came across Gerard Shulman and his research group at Yale here:

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

and, although they are now looking at other targets for metformin's action, mitochondrial glycerol-3-phosphate dehydrogenase inhibition appears to be adequate to explain most of its clinical features.

I finally looked up who he is because, while looking for papers about certain aspects of metformin, I found this comprehensive review paper:

Cellular and Molecular Mechanisms of Metformin Action

which contains the bias confirming lines:

"Taken together, these studies indicate that metformin’s effect to increase insulin-stimulated peripheral glucose uptake is secondary to improved glycemic control and reversal of glucose toxicity, which can mostly be attributed to metformin’s ability to directly inhibit hepatic gluconeogenesis and HGP."

My own turn of phrase was:

"It [metformin] *appears* to improve insulin sensitivity, lowering the plasma level of insulin and glucose, but this is because it inhibits hepatic gluconeogenesis via inhibiting mtG3Pdh. That drops hepatic glucose output and that is what lowers the insulin level." I'm slightly cautions about the glucotoxicity aspect.

If you want more of an idea about how Shulman works there is a relatively short interview here which gives the flavour.

https://www.youtube.com/watch?v=qXxZ-I9N7Kc

Obviously he needs to take about four more steps backwards up the course of insulin resistance before he reaches perilipins and basal lipolysis. Whether he will ever go a step further beyond that and realise how linoleic acid controls the adipocyte size which controls the perilipins is possibly another order of magnitude further away. He also has zero concept that insulin resistance, which he notes is utterly preserved across all of those metazoan species which use insulin (which is most of us), is a functionally protective mechanism. As in here:

Insulin resistance is a cellular antioxidant defense mechanism

Until you realise insulin resistance is an antioxidant defence mechanism you will keep trying to "cure" it.

Never the less, he's a bright guy.

Tracy LaMoia, who is first author on the above two author review, seems to be a recent addition to the Shulman lab and is deeply steeped in metformin function. To the point where she is first author of this paper in addition to the review:

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis

I've yet to examine how convincing the complex IV part of the study might be, there's a lot to read, but it does pretty convincingly destroy any residual notion that metformin acts clinically by inhibiting complex I.

Or that a one millimolar or higher concentration of metformin is in any way related to clinical usage/efficacy. In fact actually measuring plasma metformin and reporting it in your research appears to be unusual.

This has consequences.

If you read any paper where they are using metformin at 1mM, 5mM or even 20mM to blockade complex I, crash ATP supply and thus activate AMPK, you can absolutely bin all of the cell culture sections of the paper. On diabetes, cancer, ageing etc. All of them. It is always the first thing I check.

Any section of such studies describing in-vivo work, be that mouse, rat or human, will give results that are likely to be believable. Though interpretation of the findings will be unreliable when swathes of the research population still mistakenly believe that metformin is an insulin sensitiser which works by blockade of complex I after being concentrated within mitochondria to 1000 times plasma level.

Which is preposterous. See piericidin A in LaMoia's paper above.

Peter

Protons (76) Those D12492 fed mice (Speakman and Tucker again)

For people who are thinking about re-listening to Tucker's discussion with Prof Speakman, at

Ep. 22: John Speakman—What Causes Obesity?

you could do worse than to check the section from time point of 24 minutes through to 26m 20. Speakman is describing exactly the phenomenon in the graph below, beautifully illustrated from the Schwartz laboratory. I may just have mentioned this many times in multiple blog posts:

He also describes, in brief, the concept of Reward as applied to these data.

To me, the Reward hypothesis has approximately zero explanatory power for the phenomenon in the graph and Speakman eloquently describes this deficit. He and Tucker discuss how an addictive drug drives progressively increasing consumption, but an high fat diet clearly has a decreasing drive to eat until near normal consumption resumes by about a week.

But always with residual obesity and slow, on-going weight gain.

Let's consider a better explanation for the behaviour of the mice in the Schwartz lab.

Linoleic acid in the D12492 is around 18% of total calories, according to a table I downloaded from Research Diets in 2011. This is well above the insulin sensitising dose noted for humans in the last post.

The whole argument from the Protons hypothesis is that linoleic acid has the ability to facilitate insulin signalling to a) increase post-prandial fat storage b) inhibit fasting fatty acid oxidation. That is a recipe for an acute loss of calories in to adipocytes and an hypocaloric crisis.

Which is easily corrected by eating some more. As in the above mice.

Now, before we look at the next paper, some ground rules need to be set out.

Metformin.

This is the most mis-represented drug ever investigated and almost all of the conclusions published about it are incorrect.

Metformin is an inhibitor of insulin signalling which therefore results in a decreased phosphorylation of AKT. Every time. See here here

here and many more places. It *appears* to improve insulin sensitivity, lowering the plasma level of insulin and glucose, but this is because it inhibits hepatic gluconeogenesis via inhibiting mtG3Pdh. That drops hepatic glucose output and that is what lowers the insulin level.

And don't forget SHORT syndrome, discussed here.

Having established that, let's put some ideas in to perspective. Linoleic acid is a pathological insulin sensitiser. Metformin is an insulin desensitiser.

The converse drugs to metformin are the glitazones. In vivo these *increase* the phosphorylation of AKT. What else would you expect? They really are insulin sensitisers. Their standard side effect is a worsening of obesity. Of course.

We are now in a position to explain the "hyperphagia" of mice fed high fat, high linoleic acid diets such as the D12492 used in the Schwartz lab.

We need to look at this paper:

Metformin Reduces Body Weight Gain and Improves Glucose Intolerance in High-Fat Diet-Fed C57BL/6J Mice

The mice were offered something very similar to D12451 (45% fat rather than the 60% fat of D12492)  but we don't know from which company it was purchased or even if the lard included was from Japan or America. No gas chromatography was used this time so a best guess might be around 10-15% of total calories as LA.

In the first hour of access each mouse eats roughly 5.5g of it, ie 28kcal, that's roughly a third of the 70kcal/d that the Schwartz mice would eat in a full 24h period while on a chow diet:

But the really interesting finding is what happens when you either reduce insulin signalling with metformin or increase it with pioglitazone.

Blunting insulin signalling (metformin 300mg/kg p/o) before access to the food decreases the one-hour food consumption by 80%.

That's 80 per cent.

5.5kcal in an hour to 1.0kcal in an hour.

The food is still yummy, it will still light up the endogenous opioid, endocannabinoid and serotonin systems (dopamine too I guess) of the hypothalamus but the hyperphagia essentially disappears. The hyperphagia is made worse by pioglitazone, of course.

It's simply about pathological insulin sensitivity being corrected by an insulin signalling inhibitor.

It is an energy supply problem.

So metformin is a partial rescue drug for LA toxicity. It's not perfect but it illustrates basic physiological principles. Obviously the correct solution to obesity is the reduction of linoleic acid in the diet to around or just below 2% of calories. Ruminant fat. Not metformin. Not a GLP-1 agonist.

Oh, almost forgot. Near normalisation of calorie intake: I've said it before, hyperphagia ameliorates over a week because distended adipocytes increase their basal lipolysis and will raise FFAs high enough to a) induce enough insulin *resistance* to reduce LA's lipid storage effect and b) overcome the blockade of CPT1 from malonyl-CoA. Adequate calories then become available *provided* adipocytes stay distended. Under-eating simply shrinks the adipocytes, reduces basal lipolysis mediated FFA release and re-establishes pathological insulin sensitivity. Because there is now a need to maintain adipocyte size, food intake must trickle along at levels just high enough to maintain adequate obesity for adequate caloric availability from increased basal lipolysis to resist insulin.

Peter

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.

Protons (74) Arne Astrup and the formerly obese

I have a soft spot for Arne Astrup. Back in the days of the depths of the Danish fat taxation stupidity, he was one of the voices of reason speaking out against the tax. It was a near miss for sanity. Academics have since argued that the tax was repealed too soon (and the sugar tax never got started) and that it was actually "working", at least among those who couldn't hop over the border to buy their (Danish?) butter in Germany. Had it been allowed to continue to "work" we might have successfully forced a whole nation to avoid fat, especially saturated fat. Where might that have led? If you wish to compose the answer on a postage stamp it is just three letters long, which will fit neatly on to even the smallest stamp.

Anyhoo. People may have noticed that I like this paper from the Astrup led lab

Fat metabolism in formerly obese women

mostly because Table 3 confirms all of my biases by showing formerly obese women are exquisitely insulin sensitive, which is pure Protons:

The rest of the paper is more difficult.

The formerly obese are, as expected, only deriving around 35% of their energy from fat oxidation at time zero on the graph below while the never-obese controls are deriving just under 80% of their energy needs from lipid oxidation, time zero again. These values are while sitting still on a bicycle ergonometer, after an over night fast. Not quite basal metabolic rate or resting energy expenditure but pretty close:

It is also worth noting that performing exercise at 50% of VO2 max (previously individually measured) completely normalises energy production derived from fat oxidation. Those are time points 15-60min. All we need here is for AMPK to instigate oxidation of the fatty acids available while suppressing their formation. Then the pathological insulin sensitivity is bypassed. There are several posts possible on AMPK but again, here is not the place to explore the control of insulin signalling by AMPK and vice versa. Both happen.

Finally, the really strange thing is that these formerly obese women have modestly *elevated* FFAs, both at rest and throughout exercise, consistently around 300µmol/l greater than controls.  If the formerly obese have all this extra lipid available, why don't they oxidise it?

We can say, quite conclusively, that these FO women have normal electron transport chains. Under exercise they oxidise lipids exactly as well as control women do. My assumption is that there has to be a signalling problem which is inhibiting fatty acid oxidation but can be over-ridden by AMPK activation.

We know, from a mass of rodent and human studies, that when you allow a subject access to carbohydrate food (or an OGTT) after an extended fast, they perform de novo lipogenesis, giving an RER > 1.0, for about an hour. The duration is interesting. Insulin-induced insulin resistance (which is complex and probably involves the glycerophosphate shuttle) usually comes in to effect at around about an hour in many models. This will reduce insulin signalling from its peak action under "hungry" conditions to a more moderate "fed" signal.

So peak insulin signalling after a fast, but before establishment of some physiological limitation, is a potential major driver of de novo lipogenesis with storage as triglyceride and, as we shall see, an effective inhibitor of fat oxidation.

Mechanistically we have to look briefly at the Randle Cycle.

Two of the many actions of insulin are to activate the pyruvate dehydrogenase complex and the acetyl-CoA carboxylase complex. This generates malonyl-CoA which inhibits CPT1 mediated transport of fatty acids in to mitochondria. Hence FFAs are available but not oxidised. But there is no problem with the mitochondrial ETC itself, all that is needed is for the insulin signal to be reduced.

During that initial refeeding period both the RQ of >1.0 and the inability to oxidise lipid can be viewed as manifestations of marked insulin sensitivity. Carbohydrate uptake is enhanced by insulin and the products of this carbohydrate catabolism are diverted, by insulin, to metabolites which inhibit fat oxidation and away from the Krebs Cycle and the electron transport chain.

Aside: My interest is in ROS based control systems so I have tended to ignore such details. But here the downstream effects of excessive insulin signalling on the Randle Cycle do matter. My bad. End aside.

My premise is that obesity is cause by a pathological sensitivity to the hormone insulin, mediated by linoleic acid. If this is correct then we would expect pathological lipid synthesis/storage to be combined with an inhibition of fatty acid oxidation. The normal "one hour" of peak insulin sensitivity is extended or even becomes continuous by using linoleic acid as a significant energy source (pax uncoupling intakes).

Here we have the formerly obese who are, without a doubt, destined to become obese again in the future. We also have people with obese parents who are not yet obese themselves. Both show the accentuated insulin sensitivity in combination with depressed fatty acid oxidation, both at rest and post prandially. All that is required to do this is to allow insulin to continue to act at peak efficacy under conditions where a functional limitation should have been imposed. Linoleic acid replacing palmitate/stearate under the Protons hypothesis provides exactly this.

So, in Astrup's particular group of formerly obese subjects described in the current study, it has proved possible to have inhibited fatty acid oxidation with sufficient severity that it leads to elevated plasma FFAs, because they cannot be used for energy generation. All as a consequence of augmented insulin signalling.

This particular group of FO subjects do appear to be a rather extreme example. Other FO people assessed by Astrup's group in previous studies do not feature the elevated FFA or profoundly depressed fasting insulin aspects, though the inability to oxidise lipid to produce adequate energy is a consistent feature over many studies. I think this current group of women are probably outliers who give an insight in to mechanisms. That's good.

It's also clear that these formerly obese women have a metabolic rate under fasting conditions significantly lower than that of the never obese controls. This is not surprising. Fat oxidation is being largely inhibited by elevated malonyl-CoA and a significant portion of glucose is being diverted from energy production to form that malonyl-CoA and its derived and stored lipid.

The FO women's resting energy expenditure is 3.77kJ/min, ie 0.9kcal/min, 1296kcal/24h. Never obese women expend 4.88kJ/min at rest, 1.2kcal/min, 1728kcal/24h. Except of course the FO women are not oxidising fat, because they are unable to effectively oxidise FFAs. They are using glucose.

So they are 461kcal/24h "hungrier" than never obese controls and are running on limited supplies of glucose from glycogen. The obvious solution is to access more glucose, which insulin has actively locked in to the liver/muscle stores of glycogen.

Traditionally this is solved in the real world by raiding the fridge at 3am. For something sweet. Much of which will be diverted to storage.

Weight gain.

It happens.

Peter