Satiety (05) Threonine/alanine and the fasting insulin resistance

Shulman had a "That's interesting" moment in his 2016 paper which unfortunately got filed under "everything else was as we expected", and placed on the penultimate page of the supplementary data.

Here's the moment:

"Basal and clamp plasma insulin levels were subtly increased in [chow fed] InsrT1150A mice (Supplemental Table 1)."

Terminology note and clarifications: InsrT1150A are the Thr¹¹⁵⁰A mutants discussed the previous posts, ie mice with an un-phosphorylatable alanine in the place of the Thr¹¹⁵⁰ of their insulin receptor. The during-clamp high insulin looks like a non related phenomenon, more a feature of altered insulin catabolism under clamp conditions. Which might be interesting, but not for today's discussion. Oh, and this didn't show up after a six hour fast (Supplemental Table 2) because there was still a glucose based metabolism at that time. I think the anomaly will be intrinsic to markedly fat based metabolism.

Here's Supplemental Table 1

The change in fasting insulin is in the wrong direction and it's the only statistically significant change, excepting the clamp insulin level.

Shulman is perfectly entitled to use the term "subtly" because the difference between 4.5μU/ml and 6.2μU/ml is unlikely to be of any physiological significance. It's like feeling uranium minerals are slightly warm to the touch, noting it and ignoring it. Whereas the actual follow on from warm uranium was Hiroshima.

These mice which have been engineered to invariably fail to phosphorylate the Thr¹¹⁵⁰ location on their insulin receptor are more insulin resistant, not less.

So, when assessed using a genuine fasting insulin level, these mice *do* resist insulin more than control mice do, despite their absolute lack of a phosphorylatable Thr¹¹⁵⁰.

Which brings us back to ROS.

I hope everyone recalls this paper:

Electron Transport Chain-dependent and -independent Mechanisms of Mitochondrial H2O2 Emission during Long-chain Fatty Acid Oxidation

discussed here, featuring this graph

Genuine fasting metabolism is based on fatty acids. These, irrespective of the mitochondrial membrane potential, will generate ROS and mediate redox dependent insulin resistance. No cellular caloric overload needed, fatty acid oxidation simply resists insulin signalling.

A layer on top of this is the enzymic phosphorylation of Thr¹¹⁵⁰ which will reduce insulin facilitated glucose ingress, and oxidation, in addition to the ROS signal, so you need less of an ROS signal.

The ROS signal is still there, you can't oxidise fatty acids without generating ROS. I think you can reduce the need for ROS using the supplementary Thr¹¹⁵⁰ system to also resist insulin. So, for a given level of ROS, the enzymic mechanism enhances glucose restriction and keeps tighter control over the inner mitochondrial membrane potential. Obviously if delta psi is high ROS are generated by any substrate oxidation at similar levels to those from fatty acid oxidation, as in the above graph.

So the ROS insulin signal is higher in the Thr¹¹⁵⁰/A mice which gives a mild increase in fasting insulin resistance. The ROS signal is higher either because there is more lipid oxidation occurring or (more likley) some glucose oxidation is occurring when the tissues are already energy replete from FAO. This will raise delta psi and increase ROS production from this source in addition to FAO, providing extra redox mediated insulin resistance.

Obviously once insulin acts on adipocytes to suppress FFA release during a clamp then the FFAs fall, so does the ROS signal and all behaves as normal and Shulman is happy.

That is where I was mentally sitting while thinking about the Thr¹¹⁵⁰/A mice before Tucker emailed me this paper, explicitly invoking ROS mediated insulin resistance:

In adipose tissue, increased mitochondrial emission of reactive oxygen species is important for short-term high-fat diet-induced insulin resistance in mice

Which is rather nice. The ROS are real.

Sadly the paper a bit like the curate's egg, good in places. I think it's worth a post in its own right.

Peter

Satiety (04) D12942 and insulin resistance(s)

These are just some of the illustrations I doodled out for the last post while thinking about insulin sensitivity/resistance in D12942 fed mice. I hope they make it clearer what Shulman was looking at in 2016 and what he moved on to look at in 2021, comparing D12942 feeding to control mice versus to his mouse model with the Thr¹¹⁵⁰ to alanine (Thr¹¹⁵⁰A) switch.

I've left various possible routes for the development of insulin resistance dashed for D12942 because that still needs a significant amount of discussion, see below. I've also added in yellow a line for the state of phosphorylation of the Thr¹¹⁵⁰A substituted mouse mutant. What the 30 minute clamp is looking at is the residual physiological insulin resistance at a time when hunger on D12942 has almost normalised and the level of phosphorylation of Thr¹¹⁵⁰ is approaching what it should be if D12942 was a physiological high fat diet.

This clamp induced change will be a generic pattern, here are all of the scenarios I imagined two posts ago, with the Thr¹¹⁵⁰A mutant added, just to reiterate the colour scheme:

and here's what I expect happens when you measure insulin sensitivity by glucose infusion rate during a mild insulin clamp at 30 minutes:

If I am correct it suggests that all of these cells are physiologically normal under normal clamp conditions. This is not "pathological" insulin resistance.

The next question is why cells under all of these conditions have differing levels of insulin resistance before the clamp started.

The obvious explanation is that the phosphorylation of Thr¹¹⁵⁰ is a surrogate for the activity of PKCε, which is a surrogate for cell membrane DAGs, which are a surrogate for free fatty acid availability. High fat diets provide high fatty acid availability, so high levels of phosphorylation of Thr¹¹⁵⁰. If you are oxidising fat it is essential that you reduce the oxidation of glucose by an appropriate amount.

The phosphorylation of Thr¹¹⁵⁰ is an enzymic mechanism of inducing insulin resistance, in insulin sensitive cells, in proportion to the availability of free fatty acids.

This is completely separate from the redox induced insulin resistance posited by the Protons hypothesis.

It has its uses. It has its limitations.

The first use is that it allows fatty acid induced insulin resistance without the need for those redox changes intrinsic in fatty acid oxidation. If you want a safety mechanism to limit ROS generation to tightly controlled levels this could be one method of achieving it. A fine tuning mechanism.

The second is as a safety net for when the redox signal from fatty acid oxidation fails, as it does under the beta oxidation of linoleic acid.

This latter is what I want to look at in more detail today.

When we change a mouse from chow to D12942 there is a sudden switch from a metabolic substrate mixture with a normal redox balance to one with reduced ability to generate ROS. Because PUFA are preferentially oxidised, the effect should occur rapidly.

The insulin secreted in response to a meal acts *too* effectively and activates both the pyruvate dehydrogenase complex and the acetyl-CoA carboxylase complex to allow the generation of malonyl-CoA in the cytoplasm, which inhibits fatty acid entry in to the mitochondria. This marked fall in FAO results in a marked fall in ROS generation and reduces ROS derived insulin resistance still further.

This is very straight forward from the ROS perspective. Insulin over-acts, fatty acid oxidation plummets, there is an hypocaloric state which cannot be corrected by fatty acid oxidation and so carbohydrate oxidation predominates. D12942 only provides 20% of calories as carbohydrate so the mice need to eat an awful lot of it to stay alive. Here's the acute ROS mediated pathological insulin sensitivity and associated hunger. The unusable fat is mostly (but not completely) dumped in to adipocytes by the same insulin sensitivity.

Over the following days the free fatty acid levels in plasma rise because they are not being oxidised (see Astrup 1998, discussed here) and this allows the progressive accumulation of DAGs in the plasma membrane where they activate PKCε, which phosphorylates Thr¹¹⁵⁰ *irrespective* of the levels of ROS being generated within a given cell. If D12942 behaved like a physiological high fat diet we should get this, just from phosphorylation of Thr¹¹⁵⁰:

But failed fatty acid oxidation derived insulin resistance has dropped the baseline from which Thr¹¹⁵⁰  insulin resistance has to start. Combining the immediate intrinsic ROS signal change with the slower rise in FFA/DAG mediated insulin resistance gives this pattern:

As Thr¹¹⁵⁰ mediated insulin resistance rises, irrespective of the failed ROS signal, it progressively limits the pathological *excess* insulin signalling and allows progressively more fatty acid oxidation to become possible. The hunger of the first day gradually reduces until by day seven, while hunger is still slightly above that of chow fed mice, it is no longer statistically significantly so.

The next situation we need to look at is the mouse with the Thr¹¹⁵⁰A engineered mutation when fed D12942 but I've written enough for today. Oh, and I think we also need to consider the situation once *genuine* pathological insulin resistance is established in control and Thr¹¹⁵⁰A mice.

Peter

Satiety (03) 30 minutes vs 140 minutes

The most serious tool available to the Shulman lab in both the 2016 and 2021 studies was a mouse strain in which they had replaced the Thr¹¹⁵⁰ (the mouse equivalent of the human Thr¹¹⁶⁰) of the insulin receptor with an alanine. Alanine is a threonine with the hydroxyl group removed and the side chain shortened by one carbon atom. There is nowhere to place a phosphate group on an alanine, so these mice are unable to develop pThr¹¹⁵⁰ induced insulin resistance. Nowhere to place an appropriate phosphate = no pThr¹¹⁵⁰ insulin resistance.

With zero pThr¹¹⁵⁰ derived insulin resistance they had a rock steady standard of high insulin sensitivity against which to compare other less insulin sensitive states, using basic tools such as an hyperinsulinaemic euglycaemic clamp, glucose uptake, pAKT etc.

It turned out to be easy to demonstrate that these alanine substituted mice were perfectly capable of maintaining hepatic insulin sensitivity despite increased hepatic triglyceride/diglyceride content when fed D12492, much as you would expect in the absence of a phosphorylatable Thr¹¹⁵⁰.

The problem was that the group was unable to demonstrate any adipocyte insulin resistance at all after a week of feeding D12942 to their control mice. You would have thought that a week of "overnutrition" would, if you were Shulman, make adipocytes insulin resistant.

So they tweaked the hyperinsulinaemic euglycaemic clamp conditions to allow them to look for more subtle signs of preserved insulin sensitivity in the Thr¹¹⁵⁰/alanine mice or, more accurately, of active insulin resistance in the control mice. As they state:

"In our previous study, we did not observe any protective effects on WAT insulin action in HFD-fed InsrT^1150A mice (6). However, these assessments of WAT metabolism were performed during the final stages of a 140-minute HEC with an insulin infusion rate at 2.5 mU/(kg•min). Suppression of WAT lipolysis occurs rapidly after the onset of hyperinsulinemia (16), and the degree of WAT insulin resistance after just several days of HFD feeding is subtle and can be surmounted with high plasma insulin concentrations. Thus, any differences in WAT lipolysis may have been obscured in our previous studies involving the InsrT^1150A mice.

In order to address this possibility, we performed a much shorter 30-minute HEC study with a lower-dose insulin infusion rate (2.0 mU/[kg-min]) to evaluate insulin action in WAT in InsrT^1150A mice subjected to 7-day HFD."

They did two things at the same time, one was to reduce the dose of insulin used in the clamp and the second was to shorten the duration of the clamp from 140 minutes to 30 minutes. 

They ran in to problems doing this. The biggest is that, if you slog through supplementary data to the first paper in 2016, you get this chart:

which gives you an insulin concentration in plasma of around 30μU/ml, or, as we say in the rest of the world, 210pmol/l, ie a modest fed-state value was used as the clamp level. That's a very low insulin level for a clamp, they're already looking for subtleties. This the *high* infusion rate value from 2016. Once they had dropped the insulin infusion rate and changed the measurement point to 30 minutes rather than 140 minutes, they got this as the clamp level of insulin in 2021, again from the supplementary data:

Here we have a clamp level of insulin, with the *reduced* infusion rate, which is *higher* than in their initial 2016 study, this time we have ~38μU/ml, ie 270pmol/l vs the 200pmol/l of the earlier study. I very much doubt that the level is actually genuinely significantly higher, statistically or biologically. It's just unchanged.

But it is certainly not lower.

Ouch.

This is actually completely plausible as the insulin level achieved with a given infusion rate will have a lot of variables which affect the end result and Shulman's group were probably simply unlucky.

So they ignored it. At least publicly.

Ouch. 

So the difference in adipocyte insulin resistance between the 2016 study and the 2021 study is that, at essentially the same insulin level, things are very different depending on whether you open Schrodinger's mouse box at 30 minutes vs opening it at 140 minutes.

Let's make this absolutely clear: 

Thr¹¹⁵⁰/alanine mice are never insulin resistant. Full stop.

D12942 fed mice are insulin resistant, with significantly phosphorylated Thr¹¹⁵⁰ while eating an HFD, but lose their insulin resistance at some time point between 30 minutes and 140 minutes of exposure to mildly elevated insulin in combination with normoglycaemia. This insulin resistance is present at 30 minutes. It's gone by 140 minutes. Phosphates come off of Thr¹¹⁵⁰ somewhere within this time window.

I might rephrase that in the future but it's good enough for today.

These are the core findings about adipocyte insulin resistance from the two Shulman studies. That's it.

I have come to loathe both of these studies, but, when you extract the facts from the doublespeak, they do describe reality as I see it.

Pause. Deep breath.

How well does reality comply with Shulman's data? We have this study

Resistance to symptomatic insulin reactions after fasting

As Shulman tells us, fasting phosphorylates human Thr¹¹⁶⁰. The subjects of the above study had an insulin tolerance test before fasting and then fasted for 60 days. This is likely to have phosphorylated the Thr¹¹⁶⁰ of their insulin receptors to the maximum physiological level possible. Under these extreme fasting conditions the insulin tolerance test was repeated. Despite the weight loss the insulin concentrations in plasma were remarkably consistent between the two tolerance tests. Kudos to whoever calculated the individualised insulin boluses.

Shulman's data tell us that we can expect, with insulin and a little glucose, to de-phosphorylate Thr¹¹⁶⁰ to achieve a minimally insulin resistant state a time point somewhere between 30 minutes and 140 minutes after insulin exposure, pretty much independent of the insulin concentration used. This is what happens to the plasma glucose levels, the fall of which represents insulin's action, after an insulin bolus at time point zero:

In the "before fast" state there is only a modest percentage of insulin receptors with phosphorylation of Thr¹¹⁶⁰, so insulin acts rapidly to give a glucose nadir at 30 minutes.

In the "after fast" state Thr¹¹⁶⁰ of the insulin receptors are predominantly phosphorylated and it takes time to de-phosphorylate them, so the glucose nadir occurs at 60 minutes rather than at 30 minutes. Exactly in the window specified by Shulman's data but, obviously, never explicitly stated.

When I find two utterly non related studies both of which tell me the same story, I have a tendency to believe them.

Shulman actually uses this phrase (the term  InsrT1150A means the Thr¹¹⁵⁰/alanine modified mouse which we have been discussing and Insr is the insulin receptor):

"Mutation of this residue from a threonine to an alanine (i.e., InsrT1150A) shields Insr from this pathogenic phosphorylation and preserves hepatic insulin signaling and hepatic insulin sensitivity in HFD-fed mice... "

The same applies to adipocytes. My italics and my red colouration to the text for emphasis. Shulman's lab is very, very good at doing things. Understanding their own data and deriving how life works from them, not so much so.

There is no pathology here, excepting what we'll come to look at in terms of failed physiological insulin resistance under D12942.

Peter

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.