Deuterium protected linoleic acid

This is a fascinating paper which has distracted me from my thought train on uncoupling, mitochondrial membrane potential and ROS generation because it has aspects involving all three while not being particularly intuitive as to what is going on. I picked it up via comments from Tucker and Raphi on twitter.

Deuterium-reinforced polyunsaturated fatty acids protect agains atherosclerosis by lowering lipid peroxidation and hypercholesterolemia

First aside: Mouse model. This current model, the APOE*3-Leiden mouse, is a model. It's not as totally useless as an APOE total knockout or an LDL receptor knockout model but it's still nothing like a real mouse or like a real human. Mouse lipoprotein management is not like human lipoprotein management. They do not have a cholesterol ester transfer protein. The Leiden mouse has this human gene engineered in. It also has a selective APOE*3 knockout to give a mild elevation of LDL. The end result is a model which has numbers on a lipid panel which look a bit more like a human with metabolic syndrome than the average extreme knockout mouse model.

But it's not a human with metabolic syndrome. It's an APOE*3-Leiden mouse and if you found a cure for its "atherosclerosis" you would have a great tool for helping APOE*3-Leiden mice. Would it translate to helping humans with metabolic syndrome? Hahahahahahahahah bonk. End aside.

Second aside: Does LDL cause atherosclerosis? Hahahahahahahah, bonk. To anyone with any sense atherosclerosis is a response to injury where IGF-1 delivered by platelets attaching to the injured endothelium causes media hypertrophy to reinforce the site of injury. This is accelerated if systemic hyperinsulinaemia also acts as an agonist on those IGF-1 receptors. It can almost certainly be enhanced by delivering lipid peroxides such as 4-HNE, 13-HODE and 9-HODE, though their effects are very, very complex. I'm perfectly willing to believe that any genetic engineered tweak in to a mouse which increases the persistence of linoleic acid containing lipoproteins in the plasma allows time for that LA to spontaneously oxidise and accelerate what looks a bit like atherosclerosis, in the model. Just my view. End second aside.

OK. On to the paper:

The APOE*3-Leiden mice were reared on non specific chow. At 12 weeks of age (Time -4) they were put on to something derived from the AIN-93M diet. All lipids were all supplied as methyl esters of fatty acids, not triglycerides. It contained 1.2% of calories as LA and 9% of calories as sucrose. The intervention group had exactly half of the 1.2% of LA calories supplied in the form of deuterium stabilised, ROS peroxidation resistant D2-linoleic acid.

They were fed these diets for four weeks. At that point (Time zero) 0.15% by weight of reagent grade cholesterol was added to both diets (otherwise the model doesn't work to get the essential-for-funding lipid lesions, no sniggering at the back. It's a model). This "western" diet, which only differed from the run-in diet by the added 0.15% reagent grade cholesterol, caused/allowed some weight gain over the following 12 weeks but less in the D2-LA supplemented group than the normal LA group:

No weight gain on either of the run-in diets, followed by lots of gain in the "normal" LA diet but not the D2-LA diet once the cholesterol was added.

Why?

Why should adding 0.15% of cholesterol produce such diverging weight gains?

Even more exciting is if you look at lean mass vs adipose mass:

Adding just 0.15% cholesterol produced a marked fat mass gain in the normal LA mice and a trend downward in fat mass for the ROS protected D2-LA mice.

On top of that the D2-LA mice started eating extra during the period of fat loss. A lot extra:

Soooooooo. What is going on?

Well, the first thing to realise is that during the four week run-in period after the replacement of chow by the AIN-93M derived diets there were already changes which didn't show in total body weights (graphs A and B). The mice, with or without deuterium stabilised LA, all lost muscle mass and all gained fat mass during those weeks, just under a gram of each. So, even without the reagent grade cholesterol, changes were already on going from a "normal" mouse phenotype on chow towards a "skinny-fat" phenotype on an AIN-93M-like diet. That's clear in graphs C and D. Possibly from the sucrose but there's no way of telling that from the paper.

The changes were on-going before the diets were "westernised" by the addition of 0.15% of reagent grade cholesterol. I suspect that the addition of the cholesterol is a red herring.

Let's go on to look at food intakes.

The mice on the deuterated LA eventually began eating more than those on the standard LA. This became statistically significant at about week five.

Any mouse which is eating extra and losing adipose tissue is either showing malabsorption or uncoupling.

I'll buy the uncoupling, but then I would.

Why the delay to the onset of starting to eat extra? Is there a delay in uncoupling onset? Not necessarily. A normal mouse uses a significant percentage of its caloric intake to generate heat in its brown adipose tissue. There is no need to increase food intake while ever the purported uncoupling from deuterated D2-LA is generating less heat than is needed to maintain body temperature. As heat production increases over time it begins to exceed this essential minimum and so comes to represent a "calories-out" in excess of what is merely needed to keep warm. At this point an increase in food intake becomes necessary to balance the heat lost by supra-physiological uncoupling.

If this is correct, and that's a big if, there is clearly an on-going progressive increase in uncoupling with time. The logical explanation is that there is a progressive increase of deuterated D2-LA in tissues and/or being used for beta oxidation.

How might deuterated, ROS resistant LA, facilitate uncoupling?

I don't know, so it's time for some routine wild speculation. If we could answer that one question the whole scenario becomes straight forward. Sadly it is not at all obvious why D2-LA should facilitate uncoupling. Here's my current best shot. If I think of something more plausible I'll post again:

Let's assume that linoleic acid, whether deuterated or native, allows excess calories in to a cell. This is the doodle from a few posts ago:

which then leads to this doodle:

and this doodle:

This begs the question as to how much damage (signalling?) is done by the stray electrons, how much by superoxide, by hydrogen peroxide or how much is actually mediated by the lipid peroxides generated from linoleic acid per se. Which is the most important mediator?

Let me suggest that D2-LA allows the excessive delta psi, which facilitates both reverse electron transport and pathological ROS generation. As in the previous post the high delta psi eventually allows D2-LA to drive RET at the cost of, via high delta psi, allowing electrons to be lost from the ETC to oxygen at abnormal sites, forming superoxide. Under D2-LA this superoxide has very limited ability to contact oxidisable native linoleic acid.

So now we look more like this with ;

There is a surfeit of ATP, high delta psi and availability of either LA and/or D2-LA to facilitate uncoupling combined with minimal damage to the ETC. You do have to have a source of 4-HNE or a related "damage marker" to facilitate uncoupling but it doesn't need much.

I can't see any more straightforward technique for D2-LA to uncouple. If there is one and it's clear how it works, that would be great. Currently this is the best I can do.

Summary: D2-LA allows uncoupling. That explains everything, but the mechanism for the uncoupling is obscure and I'm guessing.

NB I was also trying to explain to myself why the control group got fat. I don't think they did. A total weight gain of 5g over 12 weeks to give a final weight of 25g sounds like a normal mouse to me. It's the slim mice eating extra food to maintain that slim bodyweight that are abnormal.

Ultimately the paper poses the question: What determines whether a cell deals with excess calories by sequestration to storage vs uncoupling. Obviously this is insulin signalling. But is it an oxidation product of linoleic acid which controls insulin signalling? Are we simply looking at a situation of absolutely suppressed insulin signalling, due to D2-LA being "too" stable?

Peter

Final addendum/aside. There is a claim on 'tinternet, un referenced, that low dose oral deuterium oxide in mammals causes weight loss, rather than the death which is the normal result of high dose exposure. Could D2O trigger uncoupling irrespective of LA type and the catabolism of D2-LA be a simple source of deuterated water by oxidation of the D2-LA? I doubt this but the idea is still a potential explanation. I have hunted support/refutation for this without success.

Protons (70) Uncoupling does suppress insulin signalling

The premise of the last post is that mild mitochondrial uncoupling is protective against fatty liver because it disables insulin signalling and generates heat.

There is a considerable literature looking at "energy stress" in cells induced by marked uncoupling, usually using dinitrophenol (DNP) to profoundly reduce ATP generation from oxidative phosphorylation. The DNP concentration in cell culture to achieve this would usually be 1mM. Oral dosing can transiently achieve plasma levels in mice of around 0.5mM and is probably higher in the liver because it receives the portal blood flow from the site of absorption in the gut, so maybe this has some application to real life. Maybe. These studies are peripherally interesting as dropping ATP this aggressively triggers AMPK activation which translocates GLUT4 to the cell surface to maintain cell viability using ATP from glycolysis. The translocation is independent of any markers of insulin signalling.

An example from 1988

Evidence for two independent pathways of insulin-receptor internalization in hepatocytes and hepatoma cells

from 1995

Phosphatidylinositol3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action

and from 2009

Regulation of glucose transporter 4 traffic by energy deprivation from mitochondrial compromise

The basic premise is that ATP depletion is the activator of AMPK mediated translocation of GLT4s to the plasma membrane. However AMPK is also activated by both fasting and ketogenic diets, neither of which produces an acute ATP deficit. Years ago I suggested that a major activator of AMPK is acute loss of insulin exposure and/or its signalling, independent of ATP status.

So DNP at 1mM (1000μM) in cell culture does indeed deplete ATP and AMPK does indeed translocate GLUT4 under these circumstances. Is the mechanism of action the acute suppression of insulin signalling (secondary to the loss of mitochondrial membrane potential) rather than, or in addition to, the fall in ATP generation per se?

Happily it is quite easy to measure insulin signalling nowadays. It's also possible to use either live mice taking non-lethal doses of DNP or cell culture using therapeutic concentrations of DNP. 

This next paper used DNP in live mice at non lethal dose rates and in cortical neuronal cell culture at 10-40μM concentration as opposed to 1000μM.

The Mitochondrial Uncoupler DNP Triggers Brain Cell mTOR Signaling Network Reprogramming and CREB Pathway Upregulation

Bottom line: Mild uncoupling using a therapeutic concentration of DNP suppresses insulin signalling. In this case they are looking at whole cerebral cortex in their live (until euthanasia for brain removal) mouse model or cortical neuronal cell culture.

"The protein levels of AKT, p-AKT (Thr308), ERK, and p-ERK were examined by immunoblotting which showed that the activated (phosphorylated) forms of these kinases (p-AKT and p-ERK 42/44) were reduced in the cerebral cortex at 24 and 72 h after DNP treatment (Fig. 3c–e). Collectively, these results suggest that insulin receptor signaling is suppressed in cerebral cortical cells in response to mild mitochondrial uncoupling."

That seriously confirms my biases.

I'm perfectly willing to extrapolate from mouse neuronal cells to mouse hepatocytes because this is a basic principle of how I view energy physiology working. I might be wrong, or not.

Low dose DNP and BAM15 will both treat metabolic syndrome in humans.

Conceptually what is happening in metabolic syndrome at the most basic level is that linoleic acid is allowing too many calories in to a cell and this leads to both storage and pathological ROS generation to side-step and/or limit the process. It is a coping mechanism for the failure of LA to signal physiological insulin resistance cf that provided by palmitate.

Uncoupling suppresses insulin signalling. If there are stored calories within the cell they are then made accessible. These calories are used for ox-phos to make up the deficit caused by the uncoupling. At normal weight (ie without excess insulin signalling due to diet) the suppression of insulin signalling will be accommodated by AMPK activation.

Stuff makes sense.

Peter

Protons (69) BAM15

Completely at random Tucker emailed me this paper using the classical model of Bl/6 mice fed an unspecified 60% fat diet to become obese. He thought I might find it interesting from the Protons perspective. He is correct.

Mitochondrial uncoupling attenuates sarcopenic obesity by enhancing skeletal muscle mitophagy and quality control

The background to the mitochondrial uncoupler BAM15 is in this paper

Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane

and there is another group working with the same drug here

Mitochondrial uncoupler BAM15 reverses diet-induced obesity and insulin resistance in mice

BAM15 has the potential to be a blockbuster so I think we should be very, very cautious in how we view these reports of marvellous efficacy. They're probably correct, but caution.

The papers above fit nicely in to the venerable work I've recently been reading relating to DNP, which I'll come back to in a post or two.

Today I just wanted to point out a glaring problem within the BAM15 papers for anyone viewing the work from the Protons perspective.

BAM15 reverses the insulin resistance of diet induced mouse obesity models. As in the last ref:

"Collectively, these data demonstrate that pharmacologic mitochondrial uncoupling with BAM15 has powerful anti-obesity and insulin sensitizing effects without compromising lean mass or affecting food intake." My italics.

I maintain that insulin sensitising makes you fat and that uncoupling should make you thin by suppressing insulin signalling, assisted by a "calories out" component of heat generation.

Let's look at the explanation of how we square this circle.

We need Figure 1 plus the methods section. An oral gavage of BAM15 at 10mg/kg achieves a therapeutic concentration and shows an elimination half life of around 1.7 hours in mice.

 Plasma concentration appears to be sub-therapeutic by four hours max, probably drops too low by two hours. The rest of Figure 1 shows us that BAM15 is essentially liver specific. To demonstrate that the uncoupling is liver specific you have to give that higher dose of BAM15 (50mg/kg) and measure tissue specific oxygen consumption at peak effect,  ie one hour post gavage.

We can also see that by four hours after a therapeutic gavage that liver concentration is also sub therapeutic following a 50mg/kg oral dose:

Then we have the methods section where we can find that the euglycaemic hyperinsulinaemic clamp was performed, as you might expect, after a fast of five hours. The drug is in the food.

By five hours of drug withdrawal the clamp is actually looking at the behaviour of the phenotype induced by the drug, not at the mechanism of action of the drug itself which was used to achieved that phenotype.

If we consider NAFLD as the pathological storage of lipid in the liver secondary to "mopping up" of FFAs released from overly distended adipocytes (with overactive basal lipolysis) then we are in a position to see why suppressing insulin signalling in hepatocytes releases fatty acids from hepatic triglycerides (aka fatty liver). There is no need for export of those released fatty acids in the form of VLDLs because each uncoupled hepatocyte is already in caloric deficit secondary to that uncoupling, which is what is suppressing insulin signalling while simultaneously providing a "calories out" route as heat.

Resisting insulin -> diminishing fat storage.

Continuous disposal of FFAs being stored in the liver from over distended adipocytes, without recycling those FFAs back to adipocytes as VLDLs (aka high fasting triglycerides) produces a slim, insulin sensitive phenotype. Because adipocytes become small, with associated low basal lipolysis.

Just one feature of BAM15, uncoupling, produces both specific hepatic insulin resistance (reducing hepatic triglyceride storage) combined with unstoppable drug-induced hepatic lipid oxidation as a "calories out" route.

Both from that single action of uncoupling. Neither effect is present once the drug wears off.

Insulin sensitivity/resistance being "good" or "bad" is absolutely context specific.

There is no glimmer that any of the above cited papers have an insight as to this being the mechanism of action of BAM15.

But that's how it works.

Support comes from the DNP papers.

Peter

Protons (68) Pathological ROS (1)

This is another non-referenced, thinking out loud post which is the precursor to more normal technical posts. Here we go.

The whole underpinning of the Protons concept is that inadequate generation of ROS secondary to the presence of double bonds in fatty acid fuels causes pathological insulin sensitivity.

Too few ROS.

This post is about how generating too few ROS in mitochondria generates excess ROS in those said mitochondria and what physiology does about this.

Here's the stripped down doodle of the electron transport chain I'm going to use

I've left out complex II, cytochrome C etc to keep it very simple. It's not complete, it's a minimal mental "model". You have been warned.

Next is the normal electron flow from NADH and FADH2 to oxygen:

Electrons passing through complexes I, III and IV pump protons out of the mitochondria to produce an electrical and pH difference between inside and out, the mitochondrial membrane potential, delta psi

and of course delta psi is used to generate ATP by ATP synthase, much as a rotating water mill uses hydrostatic pressure to generate usable energy.

One crucial necessity to allow ATP synthase to function is a supply of ADP. If all of a cell's supply of phosphorylated adenine is in the form of ATP there is minimal ADP as substrate for ATP-synthase to act on, so delta psi will become larger as pumped protons accumulate on the outside of the mitochondrial membrane.

If ATP predominates the cell is replete and the correct response is to limit further caloric ingress. As ETFdh tries to transfer electrons on to the CoQ couple and subsequently to complexes III and IV it becomes progressively harder to pump protons against a rising delta psi. At some point it becomes easier to divert electrons from ETFdh through complex I as reverse electron transport (RET) which generates a very specific, localised ROS signal which is designed to limit insulin signalling and be quenched using superoxide dismutase without doing damage. Saturated fats produce this signal very well because their lack of double bonds maximises the input of FADH2 to facilitate RET:

In this mental model palmitate limits caloric ingress, stops excessive proton pumping and maintains delta psi within physiological limits.

Next we have the situation under linoleic acid oxidation. Here there is a reduced input of FADH2 from ETFdh so it is more difficult to generate the RET needed to limit caloric ingress when ATP is replete and ATP-synthase is no longer consuming delta psi. Protons continue to be pumped and delta psi rises to supra-physiological levels

As delta psi rises the ability to generate RET through complex I also rises until eventually even the relatively low FADH2 input from linoleate can produce RET. However this high delta psi will also allow the generation of ROS at multiple sites in the ETC in addition to that at complex I. Complex IV seems to be a minimal site for this but complex III will produce ROS from sites facing both the mitochondrial matrix and the cytoplasm while complex I appears to only generate on the matrix side, probably from multiple sites under very high delta psi. ETDdh (and mtG-3-Pdh and complex II) can also generate ROS under high delta psi conditions:

This is both good and bad.

Good because it finally hits the signalling pathways needed to limit caloric ingress. Bad because it hits lots of other components of the cell structure in addition.

Summary: consuming linoleic acid will cause your mitochondria to explode.

Except that's preposterous, they don't.

The simplest protective measure is the diversion of calories within the cell in to storage. Those calories are only present in the cell secondary to excess insulin signalling. Because diversion to storage is a classical function of insulin signalling, this will drive obesity whilst also providing some protection from pathological ROS generation.

Additional protection comes from uncoupling.

The core mechanism for the generation of excessive ROS under unmitigated LA oxidation is high delta psi.

Uncoupling lowers delta psi. Doing this is all that is necessary to protect against pathological ROS generation.

But wait.

If linoleic acid allows excess caloric ingress due to a deficit in physiological ROS generation, surely non-specific suppression of ROS generation should increase caloric ingress, increase delta psi to overcome the degree of uncoupling present and re establish pathological ROS generation? Alternatively might uncoupling go on to allow even more excess calorie storage?

Neither happens.

I'll run through some of the papers I've been looking at over the last few months and post about them next.

Peter

Random COVID discussions

 Gotcha Eric.

Linoleic acid panel discussion

Here's the link to the panel discussion about PUFA and obesity, hosted by David Gornoski.

My Big Fat Panel: How Seed Oils Cause Obesity - A Neighbor's Choice by David Gornoski

It was an interesting discussion but I'm not really sure we achieved any consensus as to how you would convince a mainstream scientist that we are correct...

Peter

Chatting post

 Here we go

Peter

Ioannidis

I have great respect John Ioannidis

 
Saving Democracy From the Pandemic

I'm also coming to accept that prison or sectioning for the primary malfeasants is not going to be the best or even a practical solution. Lessons still have to be learned and systems questioned.

Peter

How's it going, Pfi$rael?

There may be some readers who genuinely believe the pfvacine really does protect agains severe illness and even death from covid. That's fine by me.

Using the crudest of datasets it looks like Israel's omicron wave peaked on January 26th 2022 by positive test result.

Quite how long the delay will be for the death toll to peak is a little hard to guess with omicron, but if it's three weeks then the hypervaccinated Israelis are in for a hard time over the next two weeks:

It's worth bearing in mind that it is currently almost impossible to die in a hospital in the UK without testing positive for SARS-CoV-2. I presume Israel is the same, so some of these fatalities may be incidentally positive test results, in which case the mortality peak will follow sooner than expected after that of "cases".

I have an elderly relative approaching end of life care for cancer who was hospitalised about a week ago and tested positive four days after admission. Incidentally she is unvaccinated and completely asymptomatic. Sadly she has now fallen and broken her hip, with an urgent repair planned for about now. 

She is, undoubtedly, a covid hospitalisation statistic. The likely outcome seems uncertain.

Peter

More covid chit chat

Here you go, time flies!

Peter

So you want some DHA? Chickens in Norway

Again via twitter, Tucker retweeted a thread which included the link to this paper

Increased EPA levels in serum phospholipids of humans after four weeks daily ingestion of one portion chicken fed linseed and rapeseed oil

Over the years I keep coming back to this plot

which comes from another paper:

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids

It shows, very clearly, that in rats both linoleic acid and alpha linolenic acids are equally efficacious at suppressing the conversion of ALA to DHA. Given 1% of energy in the diet as ALA then copious amounts of DHA are produced, just so long as the LA proportion of calories is less than 2% of the total. Never mind the ratio. Avoiding PUFA will generate DHA from ALA provided a basic minute minimum of ALA is present in the diet, somewhere around 1% of calories. Over at the right hand side of the plot we can see that even drinking 12% of your energy intake as ALA will not generate significant DHA if you are up in the cardiological nirvana of 16% of energy as LA.

That is in rats.

Chickens superficially appear to be somewhat different.

Switching from soybean oil to a rapeseed/linseed oil mix in the diet increases DHA in the breast meat. Soybean oil gives 14% of lipid as DHA, rapeseed/linseed gives 21%. Of not very much fat in breast meat so the amounts are small in total, but still highly statistically significant.

So ALA in food bumps up DHA in muscle. Of chickens.

This is what the chicken diet looked like, with annotation to give the fat percentage of total calories and the LA and ALA percentages of total calories in the diet, crudely:

We can overlay the chicken diet very approximately, on the rat plot to get this:

which, not surprisingly, suggests that the DHA production in the "high" ALA diet (blue) is actually more influenced by the reduction in LA. At 4% LA we could equally have had ALA at 1% of calories and still got a lower value for DHA than we did by having LA down at 2% of calories.

Might this work for humans too? Could adding ALA to our diet improve DHA availability, with all of what that entails for improved brain development and cognitive function. Just by avoiding LA and maybe drinking a little (traditional) varnish? The study didn't ask this. Instead they fed the above chicken to some humans. Either omega 3 enriched or omega 6 enriched.

This actually dropped DHA in the participants' plasma phospholipids in both groups. Admittedly not by much, so the change was neither statistically nor biologically significant. But it dropped. I would hazard a guess that the chicken simply displaced a richer source of ready-formed DHA from the diet, the tiny amount in the breast meat would do nothing per se. I believe Norwegians consume a certain amount of fish, unless they're given free chicken to eat. 

The rise in EPA must have made the authors happy that something positive came from all of that work. But I don't think you can build a brain out of EPA, DHA looks to be the molecule for that.

Ultimately pre-formed DHA does not look to be necessary for brain development if you have a modest supply of ALA from (grass fed, possibly large) animals and avoid consuming significant amounts of LA. This appears to hold true for rats, chickens and I expect for humans.

Peter

Covid playground

This is just a "post" to allow comments between people interested in the current COVID saga more easily. Comments on all posts older than two weeks are to be moderated by myself, just to keep the spam under control. So here are two weeks of unmoderated commenting scope if people want to exchange comments if I'm off working or weekending with the kids.

There you go Eric. Good idea.

Peter

Rimonabant and adipocytes

Okay. I hope everyone has heard this podcast with Raphi chatting to Tucker and Amber:

I feel Amber articulated the adipo-centric view rather well. The whole podcast reminded me of a few ideas I've had kicking around for ages and it might now be time to do a bit more posting.

I was taken back to good old Rimonabant by some of Tucker's points. It got a passing mention in an old blog post back in 2010. I may not have been terribly impressed at the time.

Rimonabant and hemopressin

For those of us with an adipocentric/insulin based outlook on life Rimonabant is interesting. It takes about 30 seconds on PubMed to pull out

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

CB1 agonists make adipocytes insulin sensitive. Rimonabant blocks this effect, which made me feel good about Protons/insulin/obesity and I left the subject alone in a nice glow of confirmation bias for a few months.

But these are isolated cells, does anything like this happen in real life? I would expect Rimonabant -> adipocytes -> reduced insulin signalling   -> release of free fatty acids -> weight loss -> reduced appetite. 

In that order.

Central to this is that the brain senses energy availability (Amber cited work I'd never heard of, my own ideas are just ideas. I felt it was self evident. You could say I just made it up) and reduces appetite when energy availability is good. Rimonabant frees up calories from adipocytes. But it is nasty stuff in the brain.

Developing drugs which do not pass the blood brain barrier is old hat in anaesthesia. Doing the same for CB1 receptor blockers seems pretty simple too. Just imagine, all the weight loss, none of the suicidal ideation. There are several under development.

But peripheral CB1 blocking drugs hit all sorts of targets ranging from the gut through the liver to the vagus nerve. And adipocytes.

What would an adipo-centrist look for?

Obviously we want an adipocyte specific CB1 receptor knockout mouse. It just has to dawn on you that that is what you want. Which took a while in my case.

So another 30 seconds on PubMed gave me this one:

Adipocyte cannabinoid CB1 receptor deficiency alleviates high fat diet induced memory deficit, depressive-like behavior, neuroinflammation and impairment in adult neurogenesis

and a little wander to the Place-which-shall-not-be-named gets you the full text.

They built a mouse model with a tamoxifen trigger-able deletion of the CB1 gene, specifically in adipocytes. How the hell they do that I can't follow but it's in the methods with links (not followed by me this time I'm afraid). I just have to accept that they can do it and that the technique is very, very clever. Then they fed a German high fat, high linoleic acid diet (around 10% calories from LA, ballpark) to make the mice fat over several months, leading to the start point at week 17 of graph E below. Then they injected tamoxifen daily for 10 days to induce permanent deletion of the CB1 receptor gene in adipocytes only. Here's what happened to the weights.

Black squares are fat mice which keep their CB1 receptors. Open squares are fat mice which lose them. D Both diamonds are controls:

Nothing changes in the gut. Nothing changes in the liver. Nothing changes in the brain. The hypothalamic Reward™ dopaminergic neurons are left untouched. All that happens is that adipocytes lose (at least) the insulin sensitising effect of CB1 receptor activation. Weight completely normalises in less than a month. It's also worth noting that in control mice adipocyte CB1 gene deletion does nothing.

Ultimately the adipo-centric view has phenomenal explanatory power when backed up by the insulin ROS concept. Everything else is higher level signalling and unexciting to me.

There are other interesting things in the paper relating to food intake and uncoupling (ie the rapid weight normalisation occurred without reduction in food intake) but I'll call it a day for now. Except to mention that in human victims of Rimonabant it is well recognised weight loss is greater than can be accounted for by reduction in food intake. Fascinating.

At some time I'll drag myself back to working out the F:N ratio of mixed fats. Two different butters, two lards, one plus 5% soybean oil, done so far. Still got coconut oil and fully hydrogenated coconut oil to go. Losing the will to live.

Perhaps I should ask my son to write me a bit of software to do this!

Peter

Protons (67) a formula revised for butter oil

A couple of months ago Tucker emailed me this study

Docosahexaenoic acid lowers cardiac mitochondrial enzyme activity by replacing linoleic acid in the phospholipidome

The study itself, while interesting, is not the point. The point is that the diet used contained the infamous 42% of calories from fat and was obesogenic. In common with a number of other studies I have been mulling over, the fat was butter oil. This is the butter oil composition

which has 2.3% linoleic acid in 42% of calories giving just over 1% of calories as LA. All I am interested in here is the comparison between 14 weeks on control low fat diet (CD) vs 14 weeks on a low linoleic acid Western Diet (WD). Here are the fat mass data (bodyweight mirrored this)

and here are the IPGTT results. We're just comparing the black control (CON) with the red western diet (WD) lines

So I think we can say that 14 weeks on butter oil is obesogenic and has the appropriate insulin resistance as we expect from any obesity with its increased basal lipolysis of large adipocytes coupled with Protons effects on the non adipose tissues.

Aside:

The diet looks like this:

If anyone thinks this looks like fudge, here is the recipe for a kilo of the stuff

Take 340g of table sugar, add 200g of anhydrous butter fat, bulk it up with 150g of corn starch to help it set and throw in some casein if you feel like it. Mix and extrude (you could cut in in to cubes for human consumption).

Fudge.

Looks yummy. Rewarding. Don't snigger!

End aside.

Butter oil is one of those features of study designs which produce obesity without linoleic acid.

Again, prompted by Tucker, I re-drafted the F:N ratios of MUFA and PUFA in the last post to account for the consumption of one NADH to provide an NADPH for rearranging each double bond during beta oxidation and we can add these revised ratios and some for selected saturate values to the composition table of butter oil like this:

In red are linoleic acid and the short chain fatty acids of an equal or lower F:N ratio cf LA. I threw in oleic acid and C8 caprylic (blue numbers) too to point out that caprylic acid, though higher than LA, is now lower than oleic (I view oleic acid as the mammalian default for "normal" insulin sensitivity) and so might be obesogenic.

So, from the F:N ratio Protons perspective, we have a modest supply of short chain fatty acids of potentially greater insulin sensitising ability (hence obesity promoting) than linoleic acid itself.

The effect of butyrate as a dietary supplement on obesity is controversial and reviewed here

Butyrate: A Double-Edged Sword for Health?

Conclusions totally depend on how you set your study up, what you consider good and what you consider bad. Bear in mind that butyrate is the darling of fibre-philes so consider publication bias too. Conversely it is to a large extent consumed by the colonic epithelium, so not a lot gets through to the systemic circulation. But some clearly does. The snippet of Figure 2 which caught my eye was this section:

I would just point out that anything which decreases lipolysis and increases adipocyte glucose uptake is NOT going to make you skinny. It might make you insulin sensitive (bravo), at least until you get fat enough to leak excess FFAs via augmented basal lipolysis.

You could of course just say butter oil fudge is highly Rewarding, so makes rats and mice fat. How can you tell it's Rewarding? Because it makes rats and mice fat. Except corn oil is also very Rewarding but ad-lib preferential consumption fails to induce obesity. For obesity there is the absolute necessity of calories to enter adipocytes and then stay there. Long term. Dopamine release in the brain might make you choose to eat something over something else but without pathological energy storage... Shrug.

My own concept of how butter oil/sucrose causes obesity is limited by the clear fact that there is no way of simply saying a given F:N ratio will always produce obesity. Too many variables for this to be set in concrete, which allows the hypothesis to side step conflicting evidence. You have been warned.

Random thought: Sucrose (when it doesn't produce a slim insulin sensitive phenotype) usually produces hyperinsulinaemia and insulin resistance (often "skinny fat"). The higher the insulin levels the more effective insulin sensitising dietary components (linoleic acid and now possibly SCFAs) are at allowing that high level of insulin to generate obesity. Probably why the cornstarch is added to the fudge, to augment the insulin response.

As always, alternative explanations welcome.

My thanks to Basti in the comments after the last post which crystalised a lot of this current post. And to Tucker twice over.

Peter

Protons (53) a formula revised

Back in Protons (53) a formula I wrote down how to work out the F:N ratio of (even chain) fatty acids with varying double bonds:

F/N = (n-1-db)/(2n-1)

where n is the length of the carbon chain and db is the number of double bonds.

Oleic C18 is 18-1-1 divided by 36-1, ie 16/35 = 0.457

Linoleic 18-1-2 divided by 36-1, ie 15/35 = 0.423

This is fine up to C18 but C20 and above are targeted to peroxisomes rather than mitochondria so the need for an F:N ratio fades. Peroxisomes have their own signalling systems but research on them is in its infancy.

Anyhoo, Tucker mentioned off blog that during the multistep processing of double bonds there is a step which consumes NADPH. This will have to be re-reduced from the resultant NADP+ by the Krebs Cycle where NADH producing steps have iso enzymes capable of generating NADPH instead of NADH. That reduces the NADH supply to the electron transport chain by 1 NADH per double bond requiring NADPH, so complicates the formula.

The formula ends up as:

F/N = (n-1-db)/(2n-1-db)

It makes a relatively small change to the ratio as the denominator is a much larger number than the numerator.

Oleic acid, originally 0.457 becomes

18-1-1 divided by 36-1-1, ie 16/34 = 0.471

and linoleic acid, originally 0.423 becomes

18-1-2 divided by 36-1-2, ie 15/33 = 0.455

The latter is interesting as it moves linoleic acid upwards towards MUFA and the saturates because the denominator drops.

The value for caprylic (shortest saturate in common consumption) is 0.467 and with the new LA now at 0.455, they are getting closer. Also caprylic is now at lower F:N ratio than oleic. I just wonder if this is part of the explanation of the coconut based diets used by Surwit to induce obesity with LA still limited to 4% of calories...

Thanks to Tucker for the NADPH requirement insight.

Peter

!Kung Bushmen and mongongo nuts yet again

Well, I got that wrong about conjugated linoleic acid (CLA) from mongongo nuts.

The !Kung people eat their mongongo nuts and the large amount of alpha-eleostearic acid converts to 9cis, 11trans CLA:

Alpha-eleostearic acid (9Z11E13E-18:3) is quickly converted to conjugated linoleic acid (9Z11E-18:2) in rats

This 9c, 11t CLA is exactly the same isomer as rumenic acid, the primary CLA of ruminant meat/dairy fats.

It's not a lipolytic agent. Not from monongo nuts, not from ruminants.

For lipolysis you want 10trans, 12cis CLA.

Manufacturing a bulk supply of CLA for marketing as a fat loss drug uses a process of treating ordinary linoleic acid with a catalytic industrial process which isomerises the LA in to roughly a 50:50 mix of 9c, 11t CLA and 10t, 12c CLA plus some odds and sods

The 10t, 12c isomer is a lipolytic agent of some potency. There's a nice review here:

Adipocyte response to conjugated linoleic acid

However it does not appear to be found as a normal component of any biological system as far as I know, though I'm open to someone finding a source. At the moment it looks like it is a drug, manufactured from linoleic acid by an industrial process. The chemical formula might be identical to rumenic acid but on a "shape", charge distribution and metabolism basis (the location and orientation of the double bonds really matters to enzymes) the two have nothing what so ever in common.

In addition to weight loss 10t, 12c CLA can also trigger adipocyte apoptosis. A little apoptosis might be fine if you have adipose tissue hyperplasia (too many adipocytes, rather than too distended adipocytes) but if you have normal levels of overly large adipocytes it will place the burden of accepting excess insulin mediated lipid for storage in to the remaining, already overly large, adipocytes. Or your liver.

This is essentially a lipodystrophy, certainly if taken far enough. As in congenital and acquired lipodystrophies, this will be associated with glucose intolerance, insulin resistance and functional type 2 diabetes. In rodent models you can drive this process somewhat further than you can in human clinical trials. So this study uses mice:

Conjugated Linoleic Acid Supplementation Reduces Adipose Tissue by Apoptosis and Develops Lipodystrophy in Mice

Bear in mind this is a model and has been set up to produce an extreme black/white result and it delivers.

Oral glucose tolerance test and intraperitoneal insulin tolerance tests:

Here are the weights of various organs of interest (check the liver):

Note that the model was set up so there was no weight loss with the 10t, 12c CLA treated group. There is massive adipose tissue loss, adipocyte number loss but no weight loss (it's a model, people are clever). All of the lipid which should be in adipocytes ends up in the liver. What does a 4.44g liver look like in an adipocyte depleted mouse? Like this:

This is the diet:

"The semipurified diet was a low-fat diet and on a calorie basis contained 63% carbohydrate, 11% safflower oil, and 26% protein. Safflower oil was used as a source of fat. Safflower oil (high-oleic type) contained 46% oleic acid (18:1 n-9) and 45% linoleic acid (18:2 n-6) from total fatty acids. CLA was prepared as a free fatty acid at Rinoru Oil Mills (Nagoya, Japan) and stored frozen in plastic bottles blanketed with nitrogen. Linoleic acid was isomerized to CLA with isomers (34% c9, t11/t9, and c11; 36% t10 and c12; 3% c9, c11/c10, and c12; 2% t9, t11/t10, and t12 from total fatty acids). In the CLA-fed group, to keep fat intake constant in the 2 groups, 25% of the safflower oil was replaced with CLA"

That's a quarter of 11% of calories as mixed isomer CLA, the sort you might take as a supplement, ie around 3% of calories. About a third of this is the active 10t, 12c CLA, ie around one percent of calories.

If a human consumes 2000kcal/d then that's 20kcal or 2g of 10t, 12c CLA per day. In this now rather well thumbed study

Comparison of dietary conjugated linoleic acid with safflower oil on body composition in obese postmenopausal women with type 2 diabetes mellitus

they were using 6.4g/d mixed CLA isomers. That will be around 3g/d 10t, 12c CLA. That's exactly the ball park used to produce lipodystrophy and diabetes in mice. The same phenomenon occurs in pigs where after slaughter back fat can be extracted, weighted and processed to detect apoptosis:

Supplementation with conjugated linoeic acid decreases pig back fat deposition by inducing adipocyte apoptosis

Comparable studies would be difficult in humans but least pigs aren't mice.

Where does this leave the !Kung and their mongongo nuts? Well, they certainly never see any 10t, 12c CLA, our liver only converts alpha-eleostearic acid to rumenic acid (assuming we're like rats). This latter is either a weak or non lipolytic/apoptosis agent. Does that leave the !Kung as inexplicable?

No.

It turns out that alpha-eleostearic acid is a rather potent lipolytic agent in its own right, it also induces apoptosis in fat cells in a similar manner to 10t, 12c CLA. Bitter melon seed oil is another, quite well studied source of alpha-eleostearic acid. This gives the flavour:

Altered White Adipose Tissue Protein Profile in C57BL/6J Mice Displaying Delipidative, Inflammatory, and Browning Characteristics after Bitter Melon Seed Oil Treatment

Mongongo nuts are lipolytic until their alpha-eleostearic acid content is detoxified to rumenic acid. To me, this suggests that living on mongongo nuts may carry weight control benefits at some risk of generating a degree of lipodystrophy, however small. I doubt anyone has gone studying adipocytes from the !Kung for markers of apoptosis. It looks like there will be a trade off between degree of lipolysis, giving small, low basal lipolysis adipocytes vs lost adipocytes giving larger, more basally lipolytic remaining adipocytes. I suspect the dose makes the poison.

I think it's probably unimportant to go in to detail about how alpha-eleostearic acid and 10t, 12c CLA induce lipolysis/apoptosis but, not surprisingly, it involves the generation of ROS for both.

Peter

Are you on clenbuterol? (3)

More from Risérus

Trans fatty acids and insulin resistance

"This is especially true [inducing insulin resistance] for conjugated TFA, i.e. conjugated linoleic acid (CLA), which clearly impairs insulin sensitivity."

I think is reasonable to assume that Risérus expects ordinary trans fatty acids to impair insulin sensitivity too, though not quite as effectively as CLA does. He just needs a big enough intervention study to prove it.

Of course he is wrong in this. He's also correct.

There is a saying that the dose makes the poison. CLA warrants a post or two on its own but it's enough to say for now that there is a toxicity syndrome, reliably induced in rodents, because it's ability to induce lipolysis can be frankly too effective. Including death of adipocytes.

Trans fatty acids are the little brother to CLA as far as lipolysis is concerned.

From the Protons point of view oxidising fats, any fats, will be better than glucose, even with insulin, at inducing reverse electron transport through complex I.

Weight loss, ie fat loss, necessitates the oxidation of lost fat. The better the lipolytic agent, the more fat to oxidise and the more insulin resistance.

Extended fasting is classically a state of profoundly increased fatty acid release from adipocytes and the oxidation of those fatty acids, with insulin resistance being intrinsic to this state. And essential for survival. Protons.

So it is impossible to lose fat without the development of some degree of fat oxidation induced insulin resistance.

CLA is good at lipolysis, trans fats less so but still better than a poke in the eye with a sharp stick.

The thought train which goes on from here is that lipolytic agents should acutely reduce insulin sensitivity directly related to the degree of fat loss. In the long term a lipolytic agent which enforces sustained fat loss will provide the low rate of basal lipolysis intrinsic to small adipocytes and so increase insulin sensitivity, especially if the lipolytic agent is not currently active.

I'm going to talk about clenbuterol next but the other agent of interest is metformin. From the Protons view metformin simply blocks the glycerophosphate shuttle, drops the FADH2 input to the electron transport chain so blunts insulin signalling which needs some degree of ROS generation to happen. Blunting insulin signalling allows lipolysis and suppresses hunger in proportion to these fat loss calories. Once adipocytes are small enough from this blunted insulin signalling we are back in to small adipocytes with low basal lipolysis so increased insulin sensitivity, especially if the metformin has worn off... In humans metformin takes a few weeks to "work". I doubt the degree of fat loss needs to be gross, just enough to reduce basal lipolysis a little.

Back to clenbuterol. Calves this time (at least it's not Bl/6 mice!).

Clenbuterol-Induced Insulin Resistance in Calves Measured by Hyperinsulinemic, Euglycemic Clamp Technique

Basically it's looking at acute treatment with a lipolytic agent. Here are the glucose infusion rates under an hyperinsulinaemic clamp:

The black squares are the infusion rates after clenbuterol, the open squares before injection. 

It's clear from the bottom graph, while the drug is active, that the treated  calves are insulin resistant, requiring significantly less glucose during the hyperinsulinaemic clamp compared to before treatment.

The upper graph shows no effect if you wait 16-25 hours before the clamp, ie until the clenbuterol has worn off. Interestingly the square colours are reversed in this upper graph. Even if the rates are ns different, we still have the calves showing as more insulin sensitive in the aftermath of a period of lipolysis. You can't force lipolysis without shrinking adipocytes. Shrunken adipocytes will always have lower basal lipolysis compared to larger adipocytes. This should show as less insulin resistance. There is a suggestion of that here.

Here are the results tabulated

I was going to go on to talk about chronic clenbuterol and the enhanced insulin sensitivity it provides. Undoubtedly chronic, high dose clenbuterol induces low adipocyte size, muscle hypertrophy and markedly improved insulin sensitivity. But the mechanism becomes complex and convoluted. I spent a little time on this fascinating paper which is comprehensible from the Protons point of view but horribly convoluted by beta receptor down regulation leading to blunted adrenaline signalling. Which affects insulin sensitivity directly.

Clenbuterol prevents epinephrine from antagonizing insulin-stimulated muscle glucose uptake

Fascinating but I'll leave that can of worms alone. It does leave me wondering a little about the acute effects of clenbuterol on fully active beta receptors and their interaction with insulin signalling. Messy. I'll leave the above post unchanged but bear in mind a lot is going on when you take an adrenergic agonist drug, in addition to lipolysis!

Peter

Are you lino-philic? (2)

Why do Risérus, Willet and Hu get it so wrong? Apart from habit of course. Out by a Ferguson is their usual standard.

Dietary fats and prevention of type 2 diabetes

Just to regurgitate:

"Taken together, the evidence suggests that replacing saturated fats and trans fatty acids with unsaturated (polyunsaturated and/or monounsaturated) fats has beneficial effects on insulin sensitivity and is likely to reduce risk of type 2 diabetes. Among polyunsaturated fats, linoleic acid from the n-6 series improves insulin sensitivity."

Looking at this study is informative:

Substituting dietary saturated fat with polyunsaturated fat changes abdominal fat distribution and improves insulin sensitivity

Here are the intervention diets

The intervention does exactly what it says on the can. Two five week periods with crossover. The subjects were rock steady for bodyweight throughout the study. Clearly it could not be blinded and the authors speculate that caloric intake might be under reported on the high PUFA arm because there are decades of indoctrination that the PUFA period was "healthy" eating (my phraseology!). I would add that they might even have subconsciously "accidentally" genuinely under eaten rather than under reported. It was only five weeks after all.

Here are the clamp results:

I think it's worth noting that at 120 minutes (Stage of clamp 6) that the glucose infusion rate per unit plasma insulin was still rising in the PUFA period but in the sat fat period the increase had stopped. From the Protons perspective this is the onset of insulin induced insulin resistance, apparently lacking in the PUFA rich period. Not commented on by the researchers but I have the eye of faith. Nice.

Converting the above graph to actual numbers here we have the results table here:

This is all classic Protons.

Protons says insulin signalling makes you fat. Improving insulin sensitivity, ie signalling, will ergo make you fat. Linoleic acid does this better than anything else, pax glitazones. Eventually insulin resistance will occur but only when adipocytes get big enough. This takes longer than five weeks, especially if you succeed in transiently limiting calories to less than those needed to replace calories lost in to adipocytes.

Back to Risérus, Willet and Hu.

To them life appears simple. Skinny people are insulin sensitive. Fat people are insulin resistant. If you could make fat people have the insulin sensitivity of thin people they would become thin. Or at least not diabetic.

Hahahahahahahahahahahaha! Bonk.

They really need Protons.

Peter