Protons (81) Crown like structures

Hyperinsulinaemia, while being unable to suppress basal lipolysis, is still able to facilitate uptake and storage of fatty acids within adipocytes. In this study the rats which were fed an high polyunsaturate diet were fatter and more hyperinsulinaemic than those fed higher saturate diet, all other aspects of the diets being constant.

Diet fat composition alters membrane phospholipid composition, insulin binding, and glucose metabolism in adipocytes from control and diabetic animals

But when you take adipocytes out of the intact rat and ask how well they processed glucose, the high PUFA group adipocytes were *more* insulin sensitive.

The metabolic milieux renders the live rats insulin resistant (elevated FFAs from increased basal lipolysis) while the individual adipocytes extracted from the rats are pathologically insulin sensitive. So the rats become fat and insulin resistant at the macroscopic level but retain insulin sensitivity at the adipocyte level when supplied FFAs are lowered to the tissue culture levels used.

Aside: There is now an on-line calculator which converts ng/ml of insulin to pmol/l insulin. Anyone who has had to convert grams to moles and then get the decimal point correct when converting to picomoles will understand.  Happy happy happy. 100ng/ml is 225pmol/l, a mild post prandial value. 1000ng/ml is 2250pmol/l, aggressive hyperinsulinaemic clamp levels. Most "ordinary" high insulin clamps use the highest post prandial levels of around 1000pmol/l, ie a bit less than half  highest values on the graph above. The above graph is close to physiology. End aside with happy dance.

To me this sets the scene that elevated plasma insulin, combined with enhanced insulin sensitivity at the adipocyte level, can successfully repackage lipids from basal lipolysis back in to adipocytes. That's Carpentier's idea in this paper, as in the last posts.

Increased postprandial nonesterified fatty acid efflux from adipose tissue in prediabetes is offset by enhanced dietary fatty acid adipose trapping

It is also quite possible to break adipocytes by doing this. We have two opposing processes. Enhanced translocation of glucose and fatty acids in to adipocytes under the failure to correctly resist insulin's storage signal, due to inadequate (but far from zero) ROS generation by linoleic acid. The second is the ability of distended adipocytes to release FFAs irrespective of insulin's action by enhanced basal lipolysis. This limits adipocyte size and supplies a competing substrate for insulin sensitive cells which requires the rejection of a certain amounts of glucose (ie insulin resistance) in proportion to the FFAs available from basal lipolysis.

Obviously excess storage, mediated via linoleic acid, wins. Otherwise there would be no linoleic acid mediated obesity. So when an adipocyte is at maximum size and there is a sudden surge in insulin/glucose/FFA availability then the adipocyte will attempt to get bigger. At some point it will fail.

Which leads me on to this D12492 mouse paper:

Adipocyte Death, Adipose Tissue Remodeling, and Obesity Complications

The photomicrographs are very pretty. The red arrows (placed by the authors) indicate "crown like structures" (CLSs). This is a simple H&E stained image of adipose tissue from a mouse after eating D12492 for eight weeks:

The CLSs appear to be lipid droplets with thickened material surrounding them which looks a lot like cytoplasm. If you go on to use immunohistochemistry to label perilipin A, which labels the protein surrounding the lipid droplet in functional adipocytes, there isn't any. The numbers indicate individual CLSs. Golden brown indicates perilipin A, clearly stained in the (un-numbered) living adipocytes:

If you stain the same section with F4/80, which picks out macrophages, you get this:

which shows that what, on H&E, looks like s thick surround of adipocyte cytoplasm, is in fact a population of macrophages surrounding the remains of a dead adipocyte. Big Eaters. They are clearing up debris

This is the image from a mouse sacrificed after 12 weeks of eating D12942:

and by week 16 we have this

There are no adipocytes visible in this image. It's all crown-like structures. The authors have not placed arrows because they would need to be everywhere. By 16 weeks of the mice eating D12942 their adipose tissue is in crisis, many adipocytes are dead and there is a marked inflammatory response which is clearing up the debris.

By 20 weeks there is significant recovery of adipose architecture, presumably from a supply of stem cells/preadipocytes, but the formation of CLSs continues, a consequence of the continued feeding of D12942. This is the view at week 20 when the study ended:

There is nothing tidy about the death of adipocytes during the formation of CLSs under D12942. The process is known as pyroptosis. I'm not sure how real pyroptosis might be, after all ferroptosis is a well recognised and well researched process which seems to be little more that linoleic acid intoxication. But assuming pyroptosis is real it is considered to be part of the innate immune system by which cells, when they have certain types of overwhelming infection, kill themselves. The process is messy.

Macrophages don't like mess. They get in there to sort it out. They also signal to the rest of the body that something is very wrong and it's time to optimise metabolic conditions to maximally enhance immune function.

Here's what it looks like if you immunostain the macrophages of CLSs for TNF-α or Il-6

Of course both of these cytokines will cause insulin resistance. Which is adaptive (another post there, you think the innate immune system does stupid things?). Not only in the surrounding adipocytes but also systemically. Which leaves a few open questions.

There are thinkers who surmise that adipose tissue inflammation is causal of insulin resistance and even that this insulin resistance, which generates hyperinsulinaemia, might be the actual cause of obesity. I know it sounds strange, but who knows? Everyone is welcome to their opinion.

Or we could hypothesis, as I do, that linoleic acid is the cause of insulin *sensitivity* which enhances insulin's storage signal (without inflammation) to the point where adipocytes die in association with over distension and there is then a massive inflammatory response as a secondary consequence.

There's a lot more to say about unhappy adipocytes and cytokines but I'll leave this post now by suggesting that the residual insulin resistance seen in IGT when FFAs are normalised by acipimox, as in here:

Overnight Lowering of Free Fatty Acids With Acipimox Improves Insulin Resistance and Glucose Tolerance in Obese Diabetic and Nondiabetic Subjects

might be mediated by TNF-α, IL-6 and their kindred signaling molecules from CLSs.

Sadly even this may not be quite as simple as it sounds.

Peter

Protons (80) The Carpentier Paradox (Carpentier III)

Preamble.

Direct quotes from Carpentier:

"Raglycerol, a marker of total AT lipolytic rate..."

"Plasma glycerol appearance was lower in IGT..."

"Postprandial palmitate appearance (Rapalmitate) was higher in IGT..."

If we combine the second two statements we can re write the findings as:

In people with IGT the rate of lipolysis is decreased (glycerol release) and simultaneously increased (FFA release) in the post prandial period.

A paradox. Oooh exciting! It would have made a great title for the paper.

So I wrote this post.

Just a one liner based on Tucker's link:

Obesity and metabolic perturbations after loss of aquaporin 7, the adipose glycerol transporter

discussing this paper

Aquaporin 7 deficiency is associated with development of obesity through activation of adipose glycerol kinase

If you knock out the glycerol/water transporter aquaporin 7 you get an obese mouse model, late onset.

This KO increases the glycerol content of adipocytes and, in all probability, drives the reaction

glycerol + ATP <-> glycerol-3-P + ADP

to the right, on the basis of increased glycerol concentration. The enzyme is glycerokinase.

This using a sledge hammer to move reaction kinetics and I doubt it has much to do with generic obesity.

But it does demonstrate that if you drive glycerol-3-phosphate formation you can drive obesity. Then comes this little snippet from the discussion:

"Lazar and coworkers demonstrated that thiazolidinediones markedly increased Gyk [Glycerokinase] mRNA level in adipocytes, resulting in triglyceride accumulation through enhancement of the conversion of glycerol into glycerol-3-P (21)."

This is ref 21 

A futile metabolic cycle activated in adipocytes by antidiabetic agents

The glitazones allow "futile" cycling of FFAs from triglycerides back in to triglycerides WITHOUT releasing the glycerol from the cell. Like aquaporin 7 KO mice but without all of that complicated genetic engineering.

Aside: "Futile" cycling is anathema to evolution. You either have an unavoidable thermogenic effect of an essential process, like protein catabolism, or you have a useful thermic effect like thermogenic uncoupling. The latter is derived from essential uncoupling to avoid damaging elevations of delta psi in mitochondria, wastefull but essential. Futile cycling without fulfilling a need or without an essential underlying process wastes energy which should be used to make babies. Survival of the fecundest is how it goes. "Futile" cycling is pathology. End aside.

So you cannot use glycerol release as an index of total lipolysis if subjects are taking glitazones to become fat. Oops, I mean to become insulin sensitive. Ah, is there any difference?

Which brings us right back to Carpentier's failure to discuss the *fall* in glycerol release from adipocytes concurrent with the *rise* in FFA release in the post prandial period.

Of course Carpentier's subjects weren't taking pharmaceutical activators of PPARγ.

But they were Canadians who had managed to eat sufficient linoleic acid to get themselves in to prediabetes.

Which begs the question: Is linoleic acid a glitazone mimetic? Well, no. But it generates functional PPARγ activators which *are* glitazone mimetics. You know, 9-HODE, 13-HODE and, of course, 4-HNE. All of which, at the correct concentration, would activate PPARγ and allow "futile" cycling of intra-adipocyte FFAs back to triglycerides without releasing their glycerol.

I'm embarrassed that I was unaware of this.

Carpentier is being paid a group leader's salary to be unaware of it. Also, who the hell scrutineered the paper?

Oops. And oops.

Peter

Protons (80) Carpentier II

I've been wanting to write about this paper for some time. But it annoys me. A lot.

Increased postprandial nonesterified fatty acid efflux from adipose tissue in prediabetes is offset by enhanced dietary fatty acid adipose trapping

I only realised yesterday that it is from Carpentier's group. Clearly Carpentier is asking questions about subjects which I am interested in. So it's time to say something.

First comes the title. From my point of view it absolutely concurs with what I would expect. If we accept that people have impaired glucose tolerance because they have accentuated lipid release from adipocytes (due to increased lipid droplet size necessitating elevated basal lipolysis), then storing lipid after a meal *should* increase FFA efflux from adipocytes. Make them big, they then "leak" (in a very controlled manner).

Carpentier used a very comprehensive tracer study to show that this effect is real and does occur (though they didn't look at, and clearly don't have, an hypothetical mechanism). The other finding they report is that this rise in efflux is not from chylomicrons spilling FFAs when they dock with extracellular lipoprotein lipase. The excess FFA efflux comes from adipocyte intracellular lipolysis.

This is consolidated in the first sentence of the abstract:

"The mechanism of increased postprandial nonesterified fatty acid (NEFA) appearance in the circulation in impaired glucose tolerance (IGT) is due to increased adipose tissue lipolysis..."

Both of which confirm my biases. Which makes me want to like the paper.

Here's the fly in the ointment, also from the abstract:

"Plasma glycerol appearance was lower in IGT (P = 0.01), driven down by insulin resistance and increased insulin secretion."

So.

The group is saying that they have documented elevated postprandial FFA efflux from adipocyte lipolysis in subjects with IGT. But they have NOT detected a rise in glycerol from that lipolysis. Quite the opposite.

What's it to be? More lipolysis giving elevated FFA efflux, or less lipolysis giving less glycerol efflux?

You can't have both at the same time. In the abstract and the discussion they are claiming that hyperinsulinaemia secondary to insulin resistance is suppressing glycerol release. But not suppressing (accentuated) FFA release.

Go figure.

So I've sat on the paper, because it confirms most of my biases but doesn't make sense.

The paper is important because, if their FFA flux data are believable, what they are saying is that adipocytes of people with IGT are releasing FFAs in the post prandial period, but there is, at the same time, enhance uptake of FFAs in to adipocytes.

In my terms: accentuated basal lipolysis, which is protective of adipocytes from over distention, is being offset by FFA uptake by adipocytes as a consequence of enhance insulin and insulin signalling secondary to linoleic acid's inability to resist it.

It matters because there is a battle over adipocyte size. When excessive insulin signalling wins over basal lipolysis, people get hurt. Especially their adipocytes do.

The downstream effects are not pretty.

Peter

Protons (79) Define insulin resistance

It occurred to me while finishing the Carpentier post that it is a beautiful model of metabolic syndrome.

My definition of insulin resistance is an adaptive response to limit insulin-facilitated metabolic substrate ingress in to a cell when an alternative metabolic substrate is being utilised concurrently. With a few caveats.

This is exactly what Carpentier generated when he infused Intralipid/heparin to supply FFAs continuously during an hyperglycaemic clamp test. Look at the control group (open circles):

With glucose clamped at 20mmol/l from 120min onward insulin eventually rises to ~700pmol/l which suppresses FFA availability toward the end of the clamp to around 0.050mmol/l or lower. At this point the subjects are running their metabolism almost completely on the glucose supplied by the infusion and FFAs are, appropriately, sequestered in to adipocytes.

The filled circles are the same people but this time, still with glucose clamped at 20mmol/l, they cannot suppress FFAs using insulin because the FFAs are being supplied exogenously using Intralipid. Free fatty acid release from adipocytes will still drop to near zero, as in the control situation, but plasma FFAs are artificially maintained exactly at fasting levels by the infusion.

The insulin resistance of fasting is real. This essential insulin resistance is not some "problem" to be "cured". It is the suppression of glucose uptake when fatty acid generated ROS are signalling that glucose is not needed, so insulin mediated glucose uptake is also not currently needed. Conveniently, this leaves glucose free for use by the brain.

Intralipid here supplies almost exactly the FFAs needed to imitate fasting (~0.70mmol/l) at a time when blood glucose is clamped at 20mmol/l and insulin is high. Resisting insulin under these circumstances is NOT pathology. It is purely adaptive. Fatty acids at 0.70mmol/l supply almost all of a subject's metabolic needs outside of the brain. Subjects do not need the glucose uptake which insulin and hyperglycaemia are trying to force on them. So they resist it. I would do the same.

You can "cure" this insulin "resistance" by turning off the lipid infusion. Probably in less than half an hour, extrapolating from Shulman's work.

So what goes wrong in metabolic syndrome?

The issue in metabolic syndrome is that you cannot turn off the supply of free fatty acids by pressing the stop button on an infusion pump full of Intralipid.

In metabolic syndrome the fatty acids are coming from adipocytes which are larger than they should be and as such have elevated basal lipolysis. We've all read this:

Effect of cell size on lipolysis and antilipolytic action of insulin in human fat cells

showing the effect of cell size on basal lipolysis:

and the inability of insulin, even at preposterous dose rates, to suppress this lipolysis:

So the (inappropriate) fatty acid supply to insulin sensitive cells in obesity *requires* insulin resistance. It is derived from large adipocytes, not small adipocytes (which have low rates of basal lipolysis), ie adipose hypertrophy necessitates insulin resistance while adipose hyperplasia does not. At the same fat mass.

Of course it is possible to stop free fatty acid release mediated through basal lipolysis using acipimox. Again, we've all read this one:

Overnight Lowering of Free Fatty Acids With Acipimox Improves Insulin Resistance and Glucose Tolerance in Obese Diabetic and Nondiabetic Subjects

and struggled to make out the numbers from its spectacularly low quality pdf file illustrations. I think I have the scales correct here:

The insulin tolerance is markedly improved, clearly, but is it not normal. Also, if you work through the rest of the paper, the mitochondria are still far from normal and I have absolutely no problem with adducts of 4-HNE and its relatives causing problems in their own right within the electron transport chain. They are, after all, an intrinsic part of both insulin's activation and deactivation pathways. I wouldn't ignore them. It's a whole series of potential posts about how and why they might be formed. Or not.

But to get back to metabolic syndrome. The obvious question is "Why are adipocytes so big as to be spilling FFAs through size-related elevated basal lipolysis in the first place?".

Insulin. Insulin makes small fat cells in to large fat cells. Stearate is the most effective fatty acid at generating the ROS signal which limits this, with palmitate a close second. Failure to limit insulin signalling, as neatly demonstrated by safflower oil in the Cocoa study, is what makes an adipocyte excessively insulin sensitive and subsequently engorged. With insulin resistance following on as a secondary change derived from the size of adipocytes.

Linoleic acid is a dud for limiting insulin signalling. It's the pathology.

Peter

Protons (78) Carpentier

I went to Edinburgh for a CPD meeting and skipped social media for four days. I've come back ready to leave it alone for a while longer and to get back to doing some blogging.

Tucker and I have batted this paper, best known as the Carpentier Study, around by email in the past:

Acute enhancement of insulin secretion by FFA in humans is lost with prolonged FFA elevation

and it surfaced in my memory as no tweeted this on X:

"Considering you can use LA to quickly induce IR ... the answer is complicated."

Yes, it's complicated. Both correct and incorrect.

So here is Carpentier's graph of what happens when you use an hyperglycaemic clamp to 20mmol/l, ie the right hand side of the graph where the necessary infusion rate to achieve this concentration is illustrated:

This is completely clear cut. Infusing Intralipid (~50% linoleic acid) for 48h up to and throughout the hyperglycaemic clamp markedly reduces the amount of glucose needed to maintain 20mmol/l in the blood, which signifies insulin resistance.

There are two fundamental problems here. The first is that the subjects were fed, throughout the 48h of the lead up to the clamp, a tightly controlled diet. The total number of calories is not specified but Tucker suggested from other papers by the same group that it was in the region of 2100kcal/d, designed to maintain weight stability.

This was fed either without the Intralipid or with the Intralipid, which provided an additional 1720kcal/24h, if it was included.

So in the "No Intralipid" arm the subjects were on a diet designed to maintain weight stability.

In the "Intralipid" arm the subjects were receiving 3820kcal/d, ie being calorically overloaded during the 48h leading up to the clamp.

Anyone who has even superficially glanced at

Insulin resistance is a cellular antioxidant defense mechanism

will be aware that caloric overload absolutely *should* induce insulin resistance. Otherwise there would be reductive stress (too many calories entering insulin sensitive cells) leading to an excessively high delta psi and subsequent oxidative stress, ie excessive generation of reactive oxygen species. 

The control situation is very different to the Intralipid situation. They are utterly different on a overall calorie supply basis, which is fundamental to the essential adaptive nature of insulin resistance.

Okay.

The second problem (or beauty, next post) is the continuation of the infusion through the hyperglycaemic clamp. In the control situation the subjects were only receiving, intravenously, glucose at the steady state of the hyperglycaemic clamp. Around 200μmol/kg/min.

In the Intralipid arm they were receiving 40ml/h of Intralipid, ie 80kcal/h in addition to the glucose at ~130μmol/kg/min. It's beyond my willpower to convert the Intralipid supply to μmol/kg/min and we don't know the weights of the subjects anyway.

But Protons says that the calories from fat should cause enough insulin resistance to limit insulin facilitated glucose ingress to cells by an amount of calories equivalent to those supplied by the fat. This will happen with Intralipid or any other lipid emulsion, non of which was used, or was available at the time.

The issue Protons has with Intralipid is that it will not cause *enough* superoxide generation, by reverse electron transfer, to adequately resist insulin by the correct amount. If I smooth out the curves from Carpentier's paper we get this:

and if I add in what I would expect an highly saturated fat infusion to produce, we would get this this:

and if I wanted to be perverse I would predict this to be the effect of adding a safflower oil infusion (70% LA) with an even higher linoleic acid content than soybean oil:

Of course this has not been done. What has been done is the Cocoa Study by Xiao (also with Carpentier as co-author) using oral rather than intravenous fats:

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

which again used an hyperglycaemic clamp to 20mmol/l of glucose, which gives exactly what Protons would predict:

The hypothesis that linoleic acid generates insulin resistance promptly, as a direct effect of the generation of reactive aldenhydes formed from linoleic acid in the bloodstream, is not supported by either of the Carpentier papers discussed here.

Far more plausible is the Protons hypothesis in which linoleic acid fails to generate the ROS signal and so fails to correctly limit insulin signalling.

The same ROS signal generates satiety in the brain stem. And it also limits the insulin mediated increase in the size of adipocytes. Linoleate oxidation absolutely causes insulin resistance. No doubt. Unfortunately it doesn't cause enough insulin resistance when compared to the normal physiological mix of palmitate, stearate and oleate.

"It's complicated" applies.

Peter

Luckily the lipid peroxidation hypothesis generates the same message as the Protons hypothesis, limit linoleic acid intake. Maybe it doesn't matter which is correct, excepting it's nice to have an explanation for Carpentier's work. I have to say that the simple message "Linoleate = badness", while beautifully simple, has limited explanatory power for studies like these. "It's complicated" hits the nail on the head.

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