Scopinaro and biliarypancreatic diversion

The late Nicola Scopinaro was an interesting chap. I came across him while reading about the use of the biliarypancreatic diversion (BPD) operation for the management of obesity and diabetes. He developed the operation in the 1970s and produced a string of publications about it over around 40 years. He died in 2020.

I can appreciate his practical abilities. In an obituary a friend describes how, during a parachute malfunction in the 1970s, Scopinaro spent his time during the descent in working out how to best position himself on impact to minimise the probability of any of the 13 fractures he sustained leading to a penetrating injury of his abdominal or thoracic viscera, or brain. He survived, hitting the ground at ~100kph. So he can work things out. An impressively pragmatic person.

His operation works.

If anyone wants the details there is always Scopinaro's comprehensive (and possibly mildly biased) review from the early days here:

Biliopancreatic diversion

but the core is that it pretty well always works and while there can be catastrophic problems these can be relatively simply managed. Inject B vitamins sooner rather than later if your patient's brain malfunctions and perform revision surgery to increase the protein absorption section if they develop protein malnutrition. And a few others. All in the paper.

Here's what the operation does.

If that's not clear we can analyse it in a little more detail. Most of the small intestine is separated from the stomach and is simply left in place to act as a conduit for bile salts and pancreatic juices to be transferred to the far end of the small intestine. We can remove this conduit from the diagram and replace it with the large red arrow like this:

The last 250cm of the small intestine is plumbed directly to a truncated stomach and functions to absorb glucose and sucrose (using the brush border sucrase enzyme), highlighted in blue below:

The conduit provided by the rest of the small intestine delivers the bile salts and pancreatic secretions to the last 50cm of small intestine. This 50cm section is the only section of the gut which is able to digest starch, fat and protein, that's the region highlighted in red:

Under these condition it is impossible to overfeed using anything containing starch, fat or protein. People with this alteration to their digestive system usually eat around 3000kcal/d, with just under half of the food eaten going down the loo.

If you make them over-eat to a total of ~5000kcal/d by adding an extra 2000kcal of fat/starch there is absolutely no change to their weight over 15 days. I prefer not to think about the resulting changes to their already execrable lower bowel function during this period.

Here are the weight loss data from a case series who had a milder version of the above procedure. Roughly 70% loss initial excess weight (IEW) maintained for longer than 18 years:

The full operation as described above gives more like an 80% permanent loss of IEW.

You can develop all sorts of ideas about how this operation works physiologically, what bypassing the bulk of the small intestine does to GPL-1, GIP, vagal sectioning, endocananbinoids etc etc but the bottom line is that Scopinaro was a pragmatic surgeon and what he means by satiety and appetite may not be quite the same as I do.

Which puts us in a position to think about Tataranni's paper comparing BPD patients with normal weight people as regards insulin sensitivity and RQ. And maybe basal metabolic rate.

Peter

Synchronicity and the origins of Protons (2)

This is the paper which Amber mentioned in her podcast conversation, primarily in the context that low carbohydrate, high fat diets markedly reduce hunger in diabetic rats. I wasn't looking at that aspect, what had caught my attention was the caloric intakes of the non diabetic rats on different levels of linoleic acid inatke and I had this post pretty well complete. Which looked pretty uninteresting unless you have a Protons perspective. Here's the post very much as was:

                                        *****************

I happened on this paper by Edens and Friedman many years ago:

Response of Normal and Diabetic Rats to Increasing Dietary Medium-Chain Triglyceride Content

and this is the core quote:

"On the other hand, LCT-fed [corn oil, 55% linoleate] normal rats overate for several days when they were given the higher fat diet."

Notice the word "overate" and that this was transient, then look at Figure 5, from which I've removed section B because that is just about the diabetic rats which are irrelevant to the current discussion:

I think it is not unreasonable to draw a straight line through the calorie intakes, provided we ignore the upper trace of the corn oil fed rats (filled dots) in the section circled in blue, which are the ones we are interested in:

NB the line trends downwards because the rats are slowing their growth rate so need fewer calories per day as the weeks go by.

Next we can look at the blue circled area and add in, by eye on Powerpoint, a smoothed line for the calorie intake during this period. Which looks like this, again in blue:

The 25% fat by weight diet supplied around 43% of calories as corn oil which gives around 24% of calories as linoleic acid.

We've seen something similar before of course, from the Schwartz lab:

on to which we can draw a similar set of lines:

I think exactly the same phenomenon is happening in both diets, one from 1984, the other from modern day D12942. The effect is much smaller and goes on for half the time period but it's there. These differences give us some insight in to what has been tweaked over the decades to improve the obesogneic nature of diets leading to the development D12942 and D12451.

Aside: The reason why the effect is small and the effect of MCT oils is minimal is another whole discussion. On the to-do list. End aside.

Is the 25% fat diet from 1984 going to be obesogenic when the rats only "overate" for a few days? Of course it is. Rats on D12942 only over eat for seven days before food intake drops to statistically indistinguishable from chow fed rats, but they still get slowly fatter over the weeks. So too would the rats in this venerable study, had they eaten it for long enough. IMNSVHO.

The discussion section is interesting because the authors are continuously trying to tease metabolic effects apart from "palatability" effects. That's good but the lack of concepts that insulin signalling is a redox based system and that the generation of superoxide/H2O2 is controlled by the relative proportion of FADH2 and NADH produced by a given metabolic substrate means that the conclusions must, necessarily, be far from complete.

So it lacks the Protons hypothesis and cannot tease out why a jump in linoleic acid intake causes a brief period of "overeating". And, of course, if you considered these few days of significantly increased caloric intake to be the only effect of the high fat corn oil diet you might be forgiven for concluding that polyunsaturated fats are non obesogenic The authors published in 1984 so cannot be criticised for being unaware that the redox state controls caloric ingress in to individual cells, as falls out from the appreciation of the ratio of FADH2 to NADH from fats vs carbohydrate as they affect the function of the ETC, RET and superoxide generation. Especially the effect of sub-physiological production of FADH2 per unit NADH as it features in the beta oxidation of linoleic acid.

Where as the difference in redox signalling generated by linoleate vs stearate (and to a lesser extent palmitate) has good explanatory power.

Peter

Synchronicity and the origins of Protons

Amber O'Hearn has a podcast conversation up on Spotify with LowCarbLogic here. Very early on in the discussion she mentions this particular study from Mark Friedman. He has championed the concept that obesity is a result of the sequestration of lipid in to adipocytes, fat trapping. This is mediated by insulin, hence the role of low carbohydrate diets, ie low insulin diets, in the management of obesity.

This idea, promoted by Gary Taubes, has profoundly shaped my thoughts on obesity, in common with those of Amber. It is difficult to over emphasise how important this is to any sort of understanding. It's that mind-bending concept that obesity is *causal* to over eating.

Your adipocytes steal your calories, so you have to eat extra calories to have adequate substrate for energy generation to run your metabolism. 

Getting fat makes you hungry.

Of course there are a number of major problems with this simple but incomplete hypothesis. The two most spectacular of which are the observation that countries such as Japan and China, whose populations ate the vast majority of their caloric intake as rice, historically had no obesity and, conversely, that of rodents fed on an high fat, low carbohydrate diet become grossly, grossly obese. D12942 supplies only 20% of calories as carbohydrate but is the gold standard for generating diet induced obesity in rats and mice.

What separates fat trapping from obesity is the failure to limit insulin signalling to the appropriate physiological level. Correctly functioning insulin signalling does not cause obesity.

You cannot talk about what controls obesity without talking about what controls insulin signalling. Oddly enough there is more to insulin signalling than the level of insulin in the plasma.

Understanding insulin signalling is impossible without appreciating that the system is intrinsically related to the actions of reactive oxygen species, as elegantly demonstrated by Czech back in 1974. You can activate all of the functions of insulin on adipocytes in cell culture by exposure to hydrogen peroxide. Low concentrations activate the insulin signalling cascade, higher concentrations inhibit it.

Of course there is no point in talking about the generation of ROS without understanding the work of David Speijer. There is a comprehensive description of his ideas, from 2019, and his thinking goes back to 2011. Probably further. I would also say that Skulachev's work on ROS and membrane potential is fairly essential too.

All I have done in the Protons thread is to throw together the ideas from Czech and Speijer and spend nearly 14 years thinking about them. With a few other inputs from evolutionary biology and the origin of life, largely from Nick Lane. And especially thinking about the paradoxes.

Oh, the trigger for this post was that I have already written a separate post about that study published by Mark Friedman in his early days, back in 1984. It's the same study which Amber cited at the start of the podcast. Having her discuss the same study which I'd already written about but not quite published struck me as profound synchronicity. Got me thinking. Anyway, this post is too long and philosophical to go on to doodle lines all over graphs so I'll update the original post to include Amber's mention and get it published separately fairly soon.

Peter

Addendum. There has been some posting recently on X about the role of the Randle cycle by which the conversion of glucose to malonyl-CoA inhibits the uptake of long chain fatty acids in to mitochondria, leading to failure of fatty acid oxidation and preferential oxidation of glucose derivatives. This is absolutely correct. The massive hole in citing the "Randle cycle" to explain obesity is in failing to ask what limits this from happening. Obviously it is limited by limiting insulin signalling. What limits insulin signalling? Well duh, ROS limit insulin signalling. Presenting a cell with a mix of glucose and long chain saturated fats means that the oxidation of these fatty acids correctly limits insulin's ability to activate both the PDH complex and the ACC complex. So the concentration of malonyl-CoA is kept at a functional level.

Saturated fats limit insulin signalling to allow co-oxidation, in the same cell, of both glucose and lipid substrates. Hence the generation of whole organism respiratory exchange ratios that indicate both fatty acid and glucose oxidation are occurring concurrently. As they do.

Unless that fatty acid FAILS to generate adequate ROS to apply this essential limitation system.

Linoleic acid's low ROS signal (compared to stearate) allows excess insulin signalling to facilitate malonyl-CoA generation and the immediate inhibition of fat oxidation, even within the first hour of ingestion of D12451 (kid brother of D12942). I hope you have all read Matsui et al's paper where an oral dose of metformin 300mg/kg, 30 minutes before food access after an overnight fast, completely normalised caloric intake of D12451 when it is eventually re-supplied. And Chung et al's paper where they tracked the RER daily during the three day transition from low fat to D12451. This 45% fat diet *raises* the RER, despite increasing fat provision and decreasing carbohydrate provision. All just Protons.

It's quite straight forward.

Of course, without Protons, you're lost.

I'll stop now.

P.

Satiety (06) The MCAT mice

Tucker emailed me this paper while I was writing the last post:

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

I was particularly excited by it because they report actual H2O2 production in pmol min-1 [mg wet weight]-1. Or, in olden-speak, pmol/min/mg tissue. Shrug.

This section of Figure 5 superficially appears to confirm all of my biases. Respiration supported by lipid oxidation generates more ROS from D12942 fed mice than chow fed mice.

Except that's not a question I would have asked. These are cells in very specific cell culture conditions and the white and black bars are both from cells being "fed" on exactly the same concentration of palmitoyl-CoA. Theoretically the ROS production should be the same, given the same fatty acid. Plenty from palmitate, a little less from linoleate.

But here the question they asked is about cells extracted from a mouse exposed to D12942 for a week versus those from a mouse exposed to chow for a week and now both are currently being exposed to an identical level of palmitate.

It's very probable that the D12942 derived cells have more mtETFdh complexes than chow fed and that these complexes generate more ROS than cells from mice fed chow. We'll never know because they measured levels of complexes I, II, III and V, but not the complex delivering the primary lipid-derived FADH2 input to the ETC, mtETFdh. Go figure.

However.

When you read the methods section you realise they are not looking at physiology at all. These are not cells fed on simple substrates and then the ROS production is being measured. They are cells whose ETC chain is fully inhibited at the ATP synthase level by oligomycin at 6.7μg/ml. That is a solid inhibitory level. So, essentially, they are "pressurising" the ETC to a high membrane potential using succinate (10mM, also not a low level) and then looking at the ROS leakage under these conditions as the test substrate is applied, in addition to the succinate. The mtETFdh complex is famous for ROS generation under high membrane potential in far-from-physiological preparations.

This is interesting but has absolutely nothing to do with physiology and certainly gives no information about fatty acid mediated ROS generation at low physiological mitochondrial membrane potential. The whole point of ROS generation from fatty acid oxidation is that it does NOT require an elevated membrane potential.

Aside: Red flag warning. They also looked at ROS generation under state 3 respiration conditions, in which the cell preparation is flooded with ADP, which allows ATP synthase to dissipate membrane potential while forming ATP, so reduces ROS generation, which it did. Here outlined in red from section C of Figure 5:

But you can't dissipate membrane potential through complex V using ADP when that complex V is 100% blockaded by 6.7μg/ml of oligomycin. ADP only consumes membrane potential when ATP synthase is active. Flooding a cell preparation with a generous supply of ADP when ATP synthase is inhibited by oligomycin *should* generate zero uncoupling, so there will still be an high membrane potential and high ROS production. Yet ADP clearly reduces ROS generation in this model. I cannot see why this should be the case. It shouldn't.

I think the word is "dodgy". End aside.

So I'm not exactly overwhelmed by their permeabilised cell ROS generation model. It tells us nothing about in-vivo physiology.

The MCAT part of the study is even worse, in it's own way. These are real live mice which have been engineered to produce human catalase in their mitochondrial matrix (in addition to their own catalase) and they do show a marked preservation of insulin sensitivity when fed on D12942.

This is reported as a Good Thing.

They are tinkering with the second core physiological messenger, H2O2, (the first messenger is superoxide and this should be unaltered by catalase) within the mitochondrial matrix, and you would expect this to cause detectable physiological (or pathological) changes.

On chow it doesn't. They used a 10% of energy from fat chow and, not surprisingly, the reduction in mitochondrial matrix H2O2 had no measurable effect on insulin sensitivity. There is no need for grossly detectable physiological insulin resistance when only 10% of dietary calories are coming from fat. Insulin signalling preservation is an essential net benefit here.

The knock-in mice demonstrated some effects in some adipose depots under D12942 feeding, rendering them apparently less resistant to insulin's ability to phosphorylate AKT than they should be, ie more like chow fed mice. I have a 10 slide PowerPoint deck dissecting the problems with their data presentation, which I will spare you. But ultimately the effect is much smaller than presented but it is still present. Mostly.

What we actually want to know is whether the decrease of the normal ROS signal from FAO in the MCAT knock-in mice makes them behave as if they have an inadequate ROS signal, as mice fed 18% linoleate do. Do they get *extra* obese on D12942?

We don't know.

MCAT mice on low fat chow don't have any change to their insulin sensitivity compared to wild type. They have a normal, very carbohydrate based metabolism which requires minimal insulin resistance at low mitochondrial membrane potential.

Wild type mice on a 60% fat diet should, excepting the linoleate effect (which is initially huge), resist insulin and stay slim, they should be soourcing 60% of their calories from fatty acid oxidation and limiting the rump of their calorie generation from the small amounts of carbohydrate and protein in D12942. MCAT mice, like linoleate fed mice, shouldn't.

It would be nice if MCAT mice on D12942 became "hyperphagic" as wild type mice do for a few days, but then kept it up for the full week and beyond, until their adipocytes simply became so full that they were damaged and releasing those inflammatory mediators that long term D12942 fed WT mouse adipocytes eventually produce. You might even pick this up in weight gain changes at one week, the MCAT mice on D12942 should be fatter than the wild type mice on D12942.

I guess the alternative is that the overlay of PKCε and Thr¹¹⁵⁰ might kick in to rescue matters, which would limit the excessive insulin sensitivity of the MCAT mice. And would be boring. Hard to say, but of PKCε is under redox control then this route to salvation won't happen.

Sadly we'll never know about the weight gains. Despite the mice being weighed at the one week mark their weights are not reported.

As far as I can find no one else has fed MCAT mice on D12942 or its equivalent. They do very well long term on an unspecified chow diet but that is both expected and very, very different to feeding D12942.

Overall the paper turned out to be hugely disappointing. C’est la vie.

Peter

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