Modelling energy intake (2): Corn oil

This post has been waiting around for some time so I thought I'd just put it up before settling down to read

The carbohydrate-insulin model: a physiological perspective on the obesity pandemic

but I'm also looking at

Food intake in diabetic rats: isolation of primary metabolic effects of fat feeding

Hyperphagia in Rats with Experimental Diabetes Mellitus:A Response to a Decreased Supply of Utilizable Fuels

and this one is pure Protons and ruminants

CALCIUM SOAPS OF FATTY ACIDS WITH VARYING UNSATURATION ASFAT SUPPLEMENTS FOR LACTATING COWS 

ditto this

Effect of supplementation with corn oil on postpartum ovarian activity, pregnancy rate, and serum concentration of progesterone and lipid metabolites in F1 (Bos taurus x Bos indicus) cows

and there might be at perhaps two more post on canagliflozin. Maybe.

When on earth I'll get to post on these I have no idea. Working on it!

Anyhoo. Back to today:

I thought it might be interesting to very, very crudely apply Kevin Hall's mathematic model to a much more interesting study. This one came my way via Jacob in comments quite a few weeks ago.

Response of body weight to a low carbohydrate, high fat diet in normal and obese subjects

This graph is an example of one single individual out of a total of five people in 1973, so we are talking about near-anecdotal data, but fascinating never the less.

The diet contained a fixed 168g/d of carbohydrate and 64g/d of protein plus a variable amount of fat over time. This is the weight change curve for subject "1", as far as I can make out:

This person lost around 2kg in the first 10 days on the diet then regained just a small amount over the following 25 days. For comparison if we now go on to look at Table 3 from Hall's

How Strongly Does Appetite Counter Weight Loss? Quantification of the Feedback Control of Human Energy Intake

we can see that a standard "lifestyle intervention" (Weight Watchers and exercise perhaps????) established an enforced caloric deficit of around 700kcal per day, which was eroded by hunger ("appetite") at something like an exponential rate, approaching the re establishment of baseline caloric intake with persistent ongoing hunger:

In the first month or so this caloric deficit triggered something around 2kg of weight loss. So if we took the graph for subject 1 at the top of the post we might reasonably assume an initial deficit of in the region of 700kcal with a rapid onset of hunger which would try to erode this weight loss effort. Something like:

Absolute CI decrease + CO increase = hunger, ie real life.

Except the subject in the graph at the top of the post was not in a weight loss study. He/she was in an overfeeding study. Carbohydrate and protein were kept fixed (and non-ketogenic) and progressively more fat was added to the diet, in steps, every five days. Here is the graph with the calorie intakes illustrated for this particular individual.

Yes, that is just under 6000kcal/d at a stable reduced weight.

Two things come to mind.

First is that it is stated that all subjects consuming over 2700kcal/d of fat felt warm all of the time and sweated easily. Second is that DLW measurement of TEE would certainly pick this up perfectly well and estimate a massive "calories out". Those would be as heat. Reversing-engineering weight loss to estimate changes in food intake is clearly completely out of its depth here. What happened?

The fat was corn oil.

Linoleic acid -> 4HNE -> activates uncoupling to blunt insulin signalling and causes insulin resistance per se -> hot, sweaty weight loss.

It takes a significant amount of linoleic acid to do this, well in excess of that needed to augment fat storage.

This effect appears to apply just as well to humans as it did to those mice in The ginger paradox (3), even when overfeeding is exogenously enforced. Clearly the mice which actively lost weight "effortlessly" (ie mice never do the human "appetite" battle unless they are exogenously semi-starved) on safflower oil used uncoupling to blunt insulin signalling and so increase lipolysis and adipocyte derived calorie supply.

Subject 1, on corn oil, had a peak of around 84% of calories from fat which put the linoleic acid percentage in the region of 40% of 6000kcal, well in to uncoupling levels. Corn oil in the 1970s was suggested to be around 45% LA.

Now, what would be expected to happen if we massively over fed with a lower LA, less uncoupling fat? The estimate for LA in olive oil in the 1970s was 7%. In this next graph the maximum LA percentage was 6% of calories, which is more akin to an obesogenic dose than to an uncoupling dose. Overfeeding olive oil does this:

That is 9kg weight gain in 40 days, and still going.

Now, what might we expect if we tried the same thing with beef fat? My expectations:

Weight gain would be even greater.

Metabolic syndrome would develop rapidly.

It should be much harder to sustain a 6000kcal diet of mostly beef fat than it was from either an uncoupling or  an obesogenic fat source.

The group didn't try overfeeding beef fat, sensibly.

There are a number of studies which I have picked up over the years which suggest that the uncoupling effect of double bonds kicks in at essentially all levels of their metabolism. At low levels the effect is over-ridden by the effect of failing to limit insulin signalling in adipocytes as per the Protons concept, leading to weight gain ie insulin still signals perfectly well and it does so more than is physiologically appropriate, especially in the immediate post prandial period. As uncoupling comes to predominate the ability of a low mitochondrial membrane potential to markedly suppress ROS generation becomes progressively more and more dominant, so insulin signalling becomes profoundly blunted. It will never get to high enough levels where insulin-induced insulin resistance should have kicked in, so the Protons concept becomes irrelevant. Under uncoupling, mitochondrial metabolism is functionally hypoinsulinaemic, it should resemble that of reduced insulin gene dose mice in Jim Johnson's lab where reduced insulin signalling was simply the end result of reduced insulin production, 24/7. It should also resemble ketogenic, hypoinsulinaemic eating.

Whether it is via 4-HNE/UCPs or 2, 4-dinitrophenol, high enough levels of uncoupling will absolutely blunt insulin signalling, with subsequent increase in access to adipocyte calories and consequentially suppressed hunger, leading to adipose tissue loss without increased food intake, with a few small caveats thrown in.

Peter

Modelling energy intake: Canagliflozin

It is quite possible to make a very reasonable estimate of how many calories a given person has consumed over the previous few weeks by estimating their total energy expenditure (TEE) using doubly labelled water (DLW), estimating the calories supplied from fat in adipose stores using the changes measured by DEXA scanning and applying a little arithmetic:

TEE (by DLW) - Fat mass change (by DEXA) = Food derived calories

Nice and simple. And very, very expensive.

Quite a few years ago Kevin Hall's group had the idea that you might be able to reverse engineer the intake of food calories simply from the change in weight over time using a mathematical model. They validated this against a two year conventional diet study where weight, TEE by DLW and fat mass changes by DEXA were repeatedly measured. They produced this paper:

Validation of an inexpensive and accurate mathematical method to measure long-term changes in free-living energy intake

Their model is pretty good within certain limits. You could trip it flat on its face pretty easily but that's not today's post. Just assume it works in the above study and also in this one:

How strongly does appetite counter weight loss? Quantification of the feedback control of human energy intake

The second study piggybacked on a diabetes study using canagliflozin, a sodium glucose co-transporter inhibitor which increases urinary glucose excretion. Canagliflozin produces the loss of around 90g/d of glucose, ie around 400kcal/d. This loss is insensible, other than via counting the number of trips to the bathroom. There was no specification within the study protocol to lose weight or to restrict calories.

Long-term efficacy and safety of canagliflozin monotherapy in patients with type 2 diabetes inadequately controlled with diet and exercise: findings from the 52-week CANTATA-M study

The interesting questions are whether this silent caloric loss produces weight reduction, what does it do to caloric intake and what mechanisms might be at work.

Here are the weight changes:

So. Obviously losing 400kcal/day does produce weight loss. Or is that genuinely obvious? Surely, if the hypothalamus wants to "see" a certain number of calories to run metabolism, shouldn't it immediately increase calories eaten to counter that 400kcal deficit? Yes, it should. Immediately. Except...

Here is what happened to the energy intake. The solid black line is Hall's model which does not include the starting point at time zero with weight change zero. I've added the red curve to include this and roughed in the rest of the data points as well as a curve in powerpoint can manage:

It's quite clear from the data points that there was an initial drop in total energy intake to a nadir, somewhere within the first three weeks. DLW is an averaging technique so the location of the nadir is an unknown but it must have happened, to explain the data points generated where week three is below time zero.  The effect is more marked in the placebo group and probably represents simply being in the trial and tidying up, in both groups, the worst of the normally execrable diabetic diet prior to starting the study.

The effect is blunted in the canagliflozin group, presumably because of those 400kcal/d down from day one and their hypothalamus will have noticed this and have kicked their cortex in to doing something about it (hunger). By 15 weeks the extra calorie intake estimate (around +350kcal/d) is getting pretty close to the urinary calorie loss estimate (around -400kcal/d). 

But for the first 15 weeks calorie intake was estimated to be well below urinary calorie loss. Food was ad libitum. Why any weight loss?

That's interesting.

Also, despite increasing food calories to match urinary losses, weight remained stable at over three kilograms below baseline, with no suggestion of weight regain at the end of a year.

That's interesting too.

Hall goes on to treat the changes in weight as an engineering control system, a bit like a black box, without any attempt at integrating any basic physiology. A quick search of the text shows no mention of insulin in the whole paper. Not surprising, given the stance taken by Hall over the CIM of obesity.

But even the most basic, strawman-facilitating version of the CIM of obesity has no problem explaining the results in some depth. It takes about 30 seconds on PubMed to ascertain what canagliflozin does to the insulin requirement of people with DMT2.

It drops the requirement.

Addition of canagliflozin to insulin improves glycaemic control and reduces insulin dose in patients with type 2 diabetes mellitus: A randomized controlled trial

For patients still using their own pancreas for insulin this seems very likely to simply be reflected in a spontaneous fall in plasma insulin, triggered by the loss of 90g/d of glucose which exits through the bladder rather than requiring insulin to stuff it in to storage within the body.

If we assume insulin drops by a fixed amount in proportion to 90g less of glucose, and stays at this reduced level for as long the canagliflozin is given, there will be an acute rise in lipolysis which will supply adipocyte derived calories to partially make up for the urinary loss.

As the hypothalamus monitors energy status it will see 90g/d of glucose as absent but being replaced by, initially, roughly a kilo of fat from adipocytes over three weeks. More arithmetic:

400kcal glucose x 21 days = 8400kcal deficit from glycosuria.

Weight loss of 1kg over three weeks = 9000kcal of fat from adipocytes.

I would suggest that fat loss comes as a direct response to lowered insulin levels and will easily at least partially replace the glucose loss, certainly initially. The fat loss can be described as "calories-in" without actually eating them. So people with an acutely lowered insulin level eat less than you would expect.

Let's look at this the correct way round. An all-glucose caloric deficit of 400kcal/d was acutely established which directly resulted in rapid drop in plasma insulin levels. Lipolysis was acutely increased which largely offset the glycosuric calorie deficit. Because over several weeks lipolysis gradually slowed to an appropriate level determined by the the new insulin levels, food calories had to increase in proportion, to maintain an adequate energy flux to keep the hypothalamus happy. Eventually extra food-in will equal urinary glucose-out giving stable weight. But with lower insulin levels this will occur at a lower total fat mass.

The weight loss/calorie intake deficit were both caused, directly, by a fall in insulin levels. Utterly simplistic CIM.

Kevin Hall is a great source of data. Of insight?

Not so much.

Peter

Back to a semblance of normality: A couple of conversations

Hi all.

Life is back to a semblance of normality now. I've de-spammed/approved the comments on older posts and will try to read all of the comments as soon as practical.

As a brief update, Brian Sanders and I had a chat which is now up on the Peak Human website. It was fun. Nothing too detailed in the way of biochemistry and lots and lots of "I don't know about....." or "I don't have a framework to integrated that into..." sort of statements.

As life should be.

Peter from Hyperlipid on Are medical professionals giving the absolutely wrong advice?

The other thing which happened just before our vacation was a chat with Amber O'Hearn. She is really interested in sleep and diet, as in

The therapeutic properties of ketogenic diets, slow-wave sleep, and circadian synchrony

and it was a great privilege to throw in some ROS derived ideas which might have been helpful towards her presentation at AHS21

Does dietary mismatch affect us via sleep?

Very interesting. I have previously been sent a paper by a reader (long time ago) where death from sleep deprivation is an ROS phenomenon, largely centred on ROS damage to the gut. But, while fascinated, it didn't make a lot of sense to me until Amber filled in a lot of gaps. It still doesn't completely make sense but time and some thought might help with that!

I have to say, adenosine looks to be a very interesting molecule too, even if not directly ROS controlled...

Peter

COVID-19

Brief one-liner:

There was a question in comments about what tweaks people might apply to themselves to minimise the risk of severe COVID-19 when they get around to being exposed to the SARS-CoV-2 virus, as we all will.

For subtleties anyone could do a great deal worse than follow George Henderson on Twitter.

For myself I rather like his tweet related to the Virta Health intervention:

which has been reinforced by this AI facilitated mining operation of the morass of published "risk factors" for severe COVID-19, with thanks to James for the link:

A Machine-Generated View of the Role of Blood Glucose Levels in the Severity of COVID-19

Clearly in 2021 DMT2 is currently due to either a lifestyle choice or to a lack of (accurate) information.

So for COVID-19 my specific medical advice to minimise serious illness is still the same.

Try not to be elderly. Try not to be diabetic.

Peter

Update

Lots of posts part written but currently I'm getting camping gear ready for our family holiday with kayaks, hills and tents. At the same time the essential big car is in the garage getting it's rear differential fixed/replaced and I'm not sure we would all fit into the MX5...

Normal service will be resumed when I get some time!

Peter

Nourish Balance Thrive Podcast

I had a chat with Megan Hall of Nourish Balance Thrive. I feel it went quite well and I got most of the core ideas of the Protons/ROS hypothesis over in a relatively concise manner. The microphone continues to work:

Here it is on Apple Podcasts

The True Cause of Insulin Resistance and Obesity (and What To Do Instead)

and on the NBT website

The True Cause of Insulin Resistance and Obesity (and What To Do Instead)

Peter

Jay Bhattacharya in conversation with Lord Sumption

This came to me via Ivor and Facebook. I keep struggling with the worry that the current pandemic might be the beginning of the end of western liberal democracy. The interview is not encouraging and Lord Sumption does encapsulate exactly where this feeling I have might be coming from.

A Conversation with Lord Sumption

If anyone is hopeful that we are getting out of this mess anytime soon then they had better not watch it.

Peter

More time wasted on vaccines

My thanks to Jonathan Engler for the tweet. This is HMS Queen Elizabeth.

She has a complement of 1,600 when fully staffed (dirtied my hands in Wikipedia to check that) so 1,400 on board sounds very plausible.  All are fully vaccinated and work under navy orders specifying social distancing, masks and track-n-trace. Those 1,400 people service a set of warplanes with armaments which you would not want to be on the receiving end of.

There are 100 COVID-19 cases so far, no deaths. I wonder if the case numbers might not have peaked yet.

This is the Diamond Princess.

She had a crew of 1045, looking after a passenger list of 2,666 whose demographic included 14 people sufficiently elderly (and I presume diabetic enough) that they died of COVID-19.

So the crew, who continued to service the passengers at some level throughout the infection period, were exposed to SARS-CoV-2 containing aerosols much of the time. 

In this case 145 contracted COVID-19. None died.

Considering that the HMS Queen Elizabeth's COVID-19 count is likely to be incomplete you have to ask yourself what, exactly, has the vaccine achieved?

Then if you look at the UK, which had a decent COVID-19 wave in spring of 2020, a completion of that wave in autumn 2020 and a marked atypical spike in Jan/Feb 2021 coincident with vaccine roll-out, the two summer nadirs are indistinguishable, you could even argue that summer 2020 had a slightly lower 7 day average death rate than we have currently.

Matt Hancock oversaw massive care-home fatalities in the first wave and failed to set up any way of separating COVID-19 patients from the elderly needing hospital treatment during last winter. So many of the people who might die of COVID-19 today are already dead. Another thing which disgusts me. But if this winter turns out to be a standard influenza year, with real influenza, no doubt the vaccines will get the credit.

Finally, I'd missed Peter Doshi's BMJ letter making some pertinent points about the Pfizer initial trial (or should I call it an advertising campaign?)

Peter Doshi: Pfizer and Moderna’s “95% effective” vaccines—we need more details and the raw data

I guess the real question is: Can you develop and market a massively profitable product to the whole world which doesn't actually work?

The pharmaceutical industry did this in slow motion with the biggest blockbuster drugs of all time.

The statins.

Are the vaccines any better? I hope so. Not looking good at the moment.

Peter

Lockdowns Summit

You can register for free on-line access. Donations are optional.

Lockdowns Summit

Someone has to map out a route out of the current political cesspit. I wonder if the press will turn up or report it? Last anti-lockdown demo in London was probably genuinely over half a million people, nothing on the BBC.

Peter

Mongongo nuts

Just recently Raphi had a very interesting and very thought provoking chat with Herman Pontzer.

They touched upon honey and the Hadza but didn't mention mongongo nuts and the !Kung San people.

So I will. I might get back to honey in another post.

Mongongo nuts are a major problem for the ROS hypothesis of obesity.

The !Kung San people live on the edge of the Kalahari Desert, as do mongongo trees. The nuts are freely available, storable and edible cooked or raw. They sound quite nice. They go by several names, Manketti nut is the one used in this paper:

Characterization of Schinziophyton rautanenii (Manketti) nut oil from Namibia rich in conjugated fatty acids and tocopherol

With a linoleic acid content just over 30%, and frequently providing a large proportion of the !Kung San people's calories, they should cause obesity, by the ROS hypothesis. If you read the abstract and look at the commas very carefully it almost suggests that the LA is actually conjugated linoleic acid but absolutely doesn't confirm this in the fine print of the full text. With the locations specified for double bonds at 9 and 12 this really is your normal, common-or-garden LA.

So the !Kung should be obese and/or hungry. And they're not.

How come? Another 30-ish% of the fatty acids in the nuts are from alpha eleostearic acid, a triple double bond isomer of alpha linolenic acid. This really is a conjugated fatty acid with double bonds at 9, 11 and 13. Conjugated means the double bonds alternate with single bonds. For ordinary PUFA there are two single bonds between each double bond.

Alpha eleostearic acid is something of a wonder drug, curing everything from cancer to whatever you fancy. It also is very easily converted (by rats at least) in to conjugated linoleic acid (CLA), presumably by hydrogenating the 13 double bond to give cis-9, trans-11 CLA:

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

CLA is, undoubtedly, a weight/fat loss drug. I glossed over it when it was reported in this paper

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

but it seems to be real as in

Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism

The CLA/safflower paper was using 6.4g of mixed CLA isomers per day, on a high linoleic acid background (by definition, the subjects were type 2 diabetics with BMI >30, ie LA intoxicated), and got steady weight loss over 18 weeks from this small supplement.

Eating a 1000kcal portion of mongongo nuts would give around 30g of alpha eleostearic acid to convert to CLA. Subsisting on primarily mongongo nuts might supply twice that. Sixty grams of eleostearic acid being converted to just under 60g of cis-9, trans-11 CLA might be enough to offset the LA content.

The situation for the !Kung San seems quite unique and I can't quite imagine any other nut providing an almost year round supply of high fat calories. Any examples gratefully received. In temperate climates nuts are very seasonal and largely supply linoleic acid.

Peter

Addendum from Tucker via twitter; it's not completely clear how important mongongo nuts really are to the !Kung:

Mongongo: The ethnography of a major wild food resource

however there will always be a roughly 1:1 ratio of LA to CLA precursor when they are consumed, in whatever quantities.

Obesity and diabetes (3) Acipimox

I first went looking for papers on Acipimox in 2014. I had read that it was an inhibitor of lipolysis and I was interested in how much weight gain it caused. Back in those days I was still fairly attached to the most basic of carbohydrate-insulin-models of obesity. If you consider that insulin causes weight gain by the inhibition of lipolysis, giving a non-insulin inhibitor of lipolysis should do the same... Shouldn't it?

Well, no, it doesn't. Acipimox produces a profound fall in free fatty acids and a marked improvement in glucose tolerance. Very, very occasionally I found snippets in discussion fora that it could increase hunger but this was not by any means routine. These give the flavour:

Effect of the Antilipolytic Nicotinic Acid Analogue Acipimox on Whole-Body and Skeletal Muscle Glucose Metabolism in Patients with Non-insulin-dependent Diabetes Mellitus

Effect of a Sustained Reduction in Plasma Free Fatty Acid Concentration on Intramuscular Long-Chain Fatty Acyl-CoAs and Insulin Action in Type 2 Diabetic Patients

Effects on insulin secretion and insulin action of a 48-h reduction of plasma free fatty acids with acipimox in nondiabetic subjects genetically predisposed to type 2 diabetes

All of which sounds very good (unless you are into the CIM of obesity!) and you have to wonder quite why Acipimox has not become standard of care and have largely reversed the current global diabetes pandemic. In fact, a recent 2020 meta-analysis of niacin (the parent compound from which Acipimox is derived) trials suggests we might be remiss in failing to do so:

Effectiveness of niacin supplementation for patients with type 2 diabetes: A meta-analysis of randomized controlled trials

But then you could go on to ask why giving niacin itself  might actually make people with impaired glucose tolerance flip in to frank type two diabetes (amongst other medical catastrophes) with worrying regularity

Effects of extended-release niacin with laropiprant in high-risk patients

Of course you could blame the laropiprant, given to suppress the niacin flushing. Or you could more usefully think about the metabolic consequences of dropping plasma FFAs by using a potent inhibitor of lipolysis.

If we work on the basis that DMT2 is essentially the down stream consequence of the inability of distended adipocytes to limit basal lipolysis, it comes as no surprise that artificially shutting down release of FFAs might improve markers of metabolic health.

The cost would be larger adipocytes.

But this doesn't happen, at least not much. The explanation is contained in this paper from 1992, largely looking at the reasons for the long term failure of Acipimox to control FFA levels:

Effects of prolonged Acipimox treatment on glucose and lipid metabolism and on in vivo insulin sensitivity in patients with non-insulin dependent diabetes mellitus 

It's simple. Making adipocytes retain their lipids increases their size. There is no suggestion that tolerance develops to this. All that happens is that there is a rebound increase in basal lipolysis as the Acipimox wears off. The drug-induced transient fall in FFAs produces a transient decrease in the oversupply of calories from FFAs, so cells should and must adapt to by reducing insulin resistance. Numbers improve at the cost of bigger adipocytes. As soon as the drug wears off the adipocytes, now bigger, reinstate basal lipolysis at their previous high rate plus some extra due to the extra distending effect of Acipimox. As they off-load their extra size by releasing FFAs, the physiological need of other cells in the body to resist insulin is both restored and augmented.

There is no net benefit and all the drug might do, if it does produce any increase in adipocyte size, is to convert IGT people, with some reserve function remaining in their adipocytes, in to very sightly heavier diabetics who have less ability to suppress adipocyte size-induced increased basal lipolysis.

If you are pre diabetic but not glycosuric and you become glycosuric in the periods between Acipimox/niacin doses you will convert from pre-diabetic to diabetic, assuming you use glycosuria as your marker for diabetes.

Peter

Obesity and diabetes (2) Basal lipolysis and weight gain

This is a paper at the "dislike" end of my bias spectrum:

In Vitro Lipolysis is Associated with Whole Body Lipid Oxidation and Weight Gain in Humans

which can be summed up by the first line of the introduction

"Positive energy balance results in greater triglyceride storage in adipose tissue and resultant accumulation of body fat."

which explicitly states that they have the arrow of causation at 180 degrees to the correct direction. So don't expect too much from the paper. I also hate that they omitted to mention in the title that the association with weight gain is negative.

Beyond that the methods are sketchy and the results are limited to a number of model derived correlations subjected, eventually, to multiple unspecified adjustments. So not a lot of hope for the group or for the largely Pima Indian population under their misguided care. But I digress.

Once again adipocyte size is quite tightly correlated with basal lipolysis, like this

which looks quite linear until we convert the log numbers to normal numbers like this

which shows us that basal lipolysis rises progressively sharply with adipocyte size. There is an upper limit to adipocyte size, and this will be set by rising basal lipolysis equaling the obesogenic effect of linoleic acid facilitating the over action of insulin.

They fed the subjects a fixed macro, calorically calculated diet for three days before a day in a metabolic chamber, where they ate three similarly fixed macro/calorie meals.

People turned out to have differing RQs (they use RQ, respiratory quotient, as their term rather than RER, respiratory exchange ratio. My brain works this way too, it's about the only bit of the paper I like, even though RER is probably the correct term) on a fixed macro diet. So clearly something is happening on a fuel partitioning basis.

People who oxidised the most fat in the metabolic chamber were the least likely to gain weight over the following eight or so years. Those oxidising the most carbohydrate were likely to gain the most weight.

Funny that.

I can't see any explanation in the discussion of why that might be.

From the ROS/Protons perspective it is quite clear that people with smaller adipocytes have not finished gaining weight. They are part way to becoming obese because they are consuming LA in combination with an insulogenic diet so are over-storing fat. When they eat a fixed macro/calorie diet they sequester lipid in to adipocytes, fail to retrieve it and run their metabolism on the more accessible carbohydrate. They're probably the most hungry, in my book.

Those with maximum sized adipocytes eat the same fixed macro diet, sequester the same lipids in to their large adipocytes via LA augmentation of insulin's fat storage signal but then go on to release much of that extra stored fat by the increase in basal lipolysis which is associated with trying to further stretch large adipocytes. This supplies continuously elevated FFAs, with subsequent fat oxidation, despite the presence of glucose and insulin at the same time. They should be the least hungry.

What can a cell do when presented with a ton of FFAs and a ton of glucose, both having their uptake facilitated by insulin?

That's right. Resisting insulin is the correct option. That's what the systemic cells do.

The cost shows as elevated glucose in combination with the elevated fat oxidation (eventually FFAs rise measurably but early on the fat oxidation increase precedes the rise in plasma FFAs).

It is metabolic inflexibility encapsulated.

Of course the obvious question is whether that increased fat oxidation, associated with reduced rate of weight gain, is positively associated with insulin resistance. Alongside the reduced weight gain.

From the same institution, back in 1991 and forgotten about during the last 30 years, we have this paper:

Insulin Resistance Associated with Lower Rates of Weight Gain in Pima Indians

Insulin resistance is, indeed, associated with limited weight gain. As you would expect.

Summary overall:

Fat oxidation a major mechanism of insulin resistance. Increased basal lipolysis is a mechanism of both increased fat oxidation and decreased weight gain. Linoleic acid is the mechanism of increased adipocyte size to drive increased basal lipolysis. Some degree of insulin signalling is essential for linoleic acid to drive adipocyte size increase.

Life is logical.

Peter

Extra thoughts: During weight gain, while calories are being lost in to adipocytes, the rest of the body is in caloric deficit. Calories lost to adipocytes must be replaced by extra food. This is the correct arrow of causation. There is insulin sensitivity.

Once adipocyte basal lipolysis equals or occasionally outstrips fat sequestration in to adipocytes, the rest of the body is being provided with supplementary FFAs. It is in caloric surplus. Insulin resistance is then physiologically appropriate.

There is a gradual transition between the two states.

Obesity and diabetes (1)

There is absolutely no doubt in my mind that adipocytes can become insulin resistant. The most convincing paper I've come across is this one

Basal lipolysis, not the degree of insulin resistance, differentiates large from small isolated adipocytes in high-fat fed mice

They took adipose tissue from a group of mice which had been made insulin resistant, and obese, by feeding a high coconut fat/sucrose (ie Surwit-like) diet and extracted a supply of adipocytes. Whether these cells were large or small, provided they had been exposed to Surwit-derived conditions for eight weeks, they were all equally insulin resistant. Big ones and little ones. Size made no difference. Insulin resistance is real but not associated with adipocyte distention.

What they also found was that size of adipocytes was very closely and positively associated with basal lipolysis, that is with the rate of lipolysis in the absence of insulin or sympathomimetic agents.

This is not a new finding. From 1972:

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

I like this paper. I have certain biases, one of the strongest of which is that I like papers in which you are given the concentration of glucose which is used in their cell culture medium. Here they happen to have used 1mmol/l, which might rightly be considered a little low, but at least they tell you. Unlike many papers.

Basal lipolysis correlated well with cell diameter. If basal lipolysis is related to the function of lipid droplet surface proteins this is completely plausible and almost predictable. Here's their graph:

The ability to suppress basal lipolysis using insulin appears to be completely determined by the conditions you use to incubate your cells, higher glucose appears to make insulin more effective on suppressing basal lipolysis, but that's an aside. This current study used a glucose concentration of 1mmol/l and showed absolutely no effect of insulin on basal lipolysis. In fact, as you increase the concentration of insulin from zero through high physiological to gross pharmacological the rate of basal lipolysis actually increases. Like this:

If anyone thinks this might represent insulin induced insulin resistance I might be tempted to agree, though the significance to anything physiological of exposure to 100,000microU/ml of insulin seems somewhat dubious.

They did find that physiological insulin exposure suppressed sympathomimetic induced lipolysis, an effect blunted by a grossly pharmacological overdose of insulin. Again, something confirmatory to my biases:

The take home message is that insulin is not particularly effective at suppressing basal lipolysis and that basal lipolysis increases with adipocyte cell size.

We have certain inter related processes here. Insulin facilitates lipid storage in adipocytes and we know that this function is both normally self limiting via ROS generation and is augmented pathologically by the oxidation of polyunsaturated fatty acids, leading to increased adipocyte size.

As adipocyte size increases the separate process facilitating basal lipolysis progressively increases, which cannot be suppressed by hyperinsulinaemia, certainly not completely.

Above a certain size it becomes almost impossible to suppress free fatty acid release from adipocytes using insulin. Under these circumstances there is a continuous supply of free fatty acids, despite the presence of insulin and a copious supply of post prandial glucose.

At a certain point the processes of augmented lipid storage and increasing basal lipolysis will come in to something approaching equilibrium.

The size of adipocytes at this point will be determined by the level of insulin being generated by the diet, the degree of augmentation of that insulin signalling by linoleic acid and whatever factors influence the rate of rise of basal lipolysis with increasing adipocyte size (currently unclear).

That's assuming a pancreas of steel which can crank out insulin at adequate levels to control glycaemia to non diabetic levels despite an increasingly un-suppressible fatty acid supply.

This will be relatively easy, provided adipocytes remain insulin sensitive. We know from 

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

that it is perfectly possible to have insulin sensitive adipocytes, adipocyte distention and systemic insulin resistance without those adipocytes becoming insulin resistant themselves. Under these circumstances the is ample scope for further weight gain. Provided the pancreas can hypersecrete insulin, glycaemia can stay fairly well controlled as weight increases.

Whatever causes adipocytes to change from insulin sensitive, as in this last paper, to insulin resistant, as in the first paper, will markedly increase the demand for insulin to maintain normoglycaemia. If the pancreas cannot meet this demand or beta cells start to undergo apoptosis secondary to exposure to elevated glucose and free fatty acids in combination, progression to DMT2 occurs where insulin secretion can no longer control glycaemia, let alone FFAs.

With the onset of DMT2 weight gain will stall. It is perfectly possible to facilitate the process of further weight gain (and associated increased basal lipolysis) by continuing to squeeze more insulin out of the pancreas using a sulphonyl urea drug or by the injection of exogenous insulin. Both approaches appear to be favourites with diabetologists once they've started with metformin.

To summarise: Excess insulin sensitivity from linoleic acid, combined with elevated insulin secretion, expands adipocytes. Expanded adipocytes increase basal lipolysis, which is difficult to suppress. With increased basal lipolysis fat oxidation, whole body, increases and induces the normal physiological insulin resistance which occurs via ROS/Protons mechanism. Eventually reduced pancreatic function leads to diabetes primarily via the inability to secrete enough insulin to normalise glucose levels while FFAs are elevated.

There's more to this.

Peter

Metformin (12) You don't need to be SHORT

Metformin is a drug which blunts the action of insulin.

Metformin is the most widely prescribed insulin sensitising agent in the world.

Discuss.

It is quite core to any logical self consistent view of the world that metformin is an agent which blunts insulin signalling, as per the ROS/Protons hypothesis via inhibition of mitochondrial glycerophosphate dehydrogenase.

The first clinical hint that this might be correct was from this study

Metformin paradoxically worsens insulin resistance in SHORT syndrome

discussed in this post.

If you are unlucky enough to be born with with SHORT Syndrome, a genetic defect in the insulin signalling pathway, you need to be hyperinsulinaemic to maintain normoglycaemia, especially during an OGTT. If some joker puts you on to metformin for 4 days then repeats the OGTT the level of insulin needed to maintain normoglycaemia goes from extremely high (690microU/ml, pax the typo on the graph) up to way too high to measure (well over 1000microU/ml for over an hour), like this:

Note, apart from the typo for insulin units, that the colours were switched between the graphs. Oops.

SHORT Syndrome is rare. Finding studies of the effect of acute metformin administration on the results of an OGTT in normal people is quite difficult. The closest I have is looking at the effect of metformin on an OGTT in obese people who still have a normal OGTT result. This is the paper

Effects of short-term metformin treatment on insulin sensitivity of blood glucose and free fatty acids

and here are the results for the obese normal OGTT people. Dashed line is under metformin after a ten day course, solid line before metformin:

The increased level of insulin secreted is not quite enough to effectively control blood glucose. As I might have mentioned, metformin blunts insulin signalling. No surprises there then. 

However, if you give the metformin to someone who is already insulin resistant or has type 2 diabetes, the opposite happens, same study:

This has a lot of bearing on what we mean by diabetes but might be better left for a few posts while I run though the transition form health through obesity to impaired glucose tolerance to frank DMT2.

Peter

Ossabaw pigs

I notice that Brad Marshall has a great post out on PUFA/insulin sensitivity and especially ALA. Brad is seriously thinking along ROS/Protons lines. He looks at ideas I've toyed with over the years but never found the papers to follow through on, finds the papers and follows through. Enjoy.

Obesity prone pigs go from Normal to Pathological Insulin Sensitivity to Torpor when given enough PUFA

Peter

Random musings on uncoupling (7) DNP and metformin

NAFLD, NASH, ALD and alcoholic steatohepatitis (ASH?) are all associated with the accumulation of lipid within liver cells. The two primary culprits are fructose and alcohol. Both undergo rapid metabolism to acetyl CoA (+/- lactate) with the potential to generate lipid within hepatocytes as a result.

Sadly life is never quite that simple. Certainly some of the liver lipid does indeed come from the metabolism of fructose or ethanol, but back in this post there are the papers which suggest fructose acts systemically to induce acute insulin resistance in adipocytes and so releases fatty acids which transfer to the liver (and visceral fat) stores:

Fructose and lipolysis

and this post points out the same about ethanol:

Alcohol and weight loss

Hepatic lipid delivery should trigger hepatic insulin resistance and the resultant persistence of metabolic substrate in the blood should signal to the hypothalamus that there are plenty of calories available, ie it's not time to eat yet. You have only to look at the hepatic response of FGF21 production, which increases thermogenesis, in response to both alcohol or fructose to see this in action. FGF21, when not produced in response to starvation (which it is), signifies that the liver sees enough calories to stimulate thermogenesis in excess of obligate needs.

So what goes wrong in fatty liver disease?

The action of insulin on hepatocytes is to suppress glucose release, facilitate lipogenesis and facilitate triglyceride formation. You just have to ask yourself, is there any dietary component which facilitates the excessive action of insulin? Which might make a perfectly reasonable process into a lipid-storage overload pathology?

Could that be linoleic acid? Which induces a failure to resist caloric ingress at times when that would be appropriate.

Fatty liver disease, from fructose or ethanol, looks to me very much like the result of excess insulin action on hepatocytes. The same linoleic acid which produces this accumulation of lipid in adipocytes  will also facilitate accumulation in hepatocytes and facilitate the conversion of benign fatty liver into inflamed hepatitis though its lipoxide derivatives.

We've known for years that a high saturated fat diet protects against NASH: 

Long term highly saturated fat diet does not induce NASH in Wistar rats

provided it is very low in PUFA. In fact the low PUFA is probably more important than the high saturated fat content.

If we accept this chain of thought, fatty liver represents an accumulation of lipid in response to linoleic acid facilitated excessive action of insulin. It happens because while the oxidation of linoleic acid generates enough ROS to allow insulin signalling to occur, it does not allow the generation of enough ROS to limit insulin's actions when the hepatocytes are full. Exactly as for adipocytes.

So hepatic lipid accumulation is a consequence of excess insulin signalling, and only once the ability to accumulate any more intrahepatic lipid has been exceeded does the generation of ROS become adequate to resist insulin's caloric ingress/retain signal. After that, hepatic insulin resistance will occur, glucose will no longer be retained and the liver will no longer be a sump for absorbing FFAs.

Systemic levels of FFAs and glucose will rise and the rest of the body will have to go in to anti-oxidant defence mode, AKA whole body insulin resistance. Hunger will plateau and weight will stabilise.

So. The primary problem is the excess storage of (largely adipocyte derived) FFAs as intra hepatocellular triglyceride, beyond the point where this is adaptive.

It cannot happen without the LA facilitated augmentation of insulin signalling. This does not happen if the lipids being oxidised within the liver are predominantly saturated, as in the NASH prevention paper above.

Looking at hepatic lipid accumulation in these terms suggests that blunting insulin signalling might he a simple solution. Hence the efficacy of 2,4-dinitrophenol. You could view DNP as acting as a caloric sump for hepatocytes, burning off the fat and introducing a caloric deficit. Or you could speculate that all that is needed is a small drop in mitochondrial membrane potential, to produce a reduction of insulin signalling to approximately offset the augmentation induced by LA, and the problem would self correct.

I tend to favour the latter option. But then I would.

My personal view is that this is what low dose DNP does. It blunts insulin signalling in hepatocytes. Blunted insulin signalling blunts lipid accumulation and the liver never accumulates enough lipid intermediates to generate insulin resistance. Without the enhanced insulin signalling sequestering calories into lipid stores the liver will allow more glucose and FFAs in the systemic circulation which will reduce hunger. This might not be enough to generate detectable weight loss in a few weeks of a rodent study but it just might over a few years.

The parallel with metformin is that I consider metformin's core action at therapeutic dose rates is the inhibition of the mitochondrial component of the glycerophosphate shuttle, limiting FADH2 input to the CoQ couple and so limiting the ROS generation which is needed to maintain insulin signalling (and to markedly reduce insulin-induced insulin resistance, but that's another story). It does this at micromolar concentrations in the cytoplasm, where it can easily access mtG3Pdh.

Metformin and DNP both reduce the generation of ROS needed to maintain insulin signalling, all be it by different mechanism. Insulin signalling is blunted. Excess lipid (and glucose) storage is inhibited. There might be a trivial loss of weight due to reduced hunger.

ASIDE Obviously as metformin/DNP reduce ROS and insulin signalling they allow increased fat oxidation, largely via AMPK, and some "new" ROS will be generated to replace those suppressed by metformin/DNP. But the "cost" of these "new" ROS is fat loss. Which is a win overall for metformin/DNP/obesity END ASIDE.

Interestingly both metformin and vintage DNP increase lactate formation systemically, presumably because glycolysis is still on going, especially when glucose levels are raised post prandially, and the activation of the pyruvate dehydrogenase complex is blunted in proportion to the blunting of insulin signalling. Hence pyruvate to lactate becomes the preferred route to continue glycolysis.

Also both are longevity drugs, even using old fashioned plain DNP in rodent drinking water

Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity

Blunting insulin signalling certainly does interesting things.

I have tried to resist insulin for decades. So far, so good...

Peter

Random musings on uncoupling (6) Nouveaux DNP

 I started here with with DNP

2,4 Dinitrophenol as Medicine

Several links came out of the paper. First was this one from Shulman's group

Controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats

The paper contains a great deal of information about the development of the sustained release DNP formulation, which sounds good. All we know about the rats and diets are that they were Sprague Dawley rats or Zucker Diabetic Fatty rats and the diets are minimally described as safflower oil 60% fat for NAFLD or methionine/choline deficient for NASH.

Bottom line is that a sustained release hepatic targeted DNP preparation is enormously safe and produces marked amelioration of liver disease in all of the models tested.

Using Bl/6 mice they also show that the degree of hepatocyte mitochondrial uncoupling was so minor as to be undetectable in a CLAMS apparatus.

Next is this one, again from Shulman's lab, where the hydrogen of the DNP hydroxyl group was replaced with a methyl moiety, rendering this DNP derivative inactive. This was then converted to active DNP primarily in the liver by cytochrome P450, with no detectable toxicity and no detectable increase in oxygen consumption on a whole body basis:

Reversal of Hypertriglyceridemia, Fatty Liver Disease and Insulin Resistance by a Liver-Targeted Mitochondrial Uncoupler

There is pretty convincing evidence that both of the above modified DNP delivery systems were fairly tightly targeted to the liver. Relatively little appeared to act on other organs and there is no information about the action on adipose tissue, but then these experiments were not looking for weight loss, merely controlling the liver damage/dysfunction of metabolic syndrome.

And the drugs do control metabolic syndrome. Here are the intraperitoneal glucose tolerance test results for the high fat fed Sprague Dawley rats, red being the treatment groups throughout:

and the insulin levels at the same times:

and the results for the Zucker Diabetic Fatty rats are even more impressive:

and insulin levels:

All of this is merely by limiting lipid accumulation within hepatocytes.

And the rats stayed fat.

You have to look at this and wonder: Here we have an intervention which primarily blunts insulin signalling originating from the mitochondria of hepatocytes. A drug which reduces insulin signalling and yet leads to a dramatic improvement of of whole body insulin sensitivity.

The parallels with metformin are striking

Peter

Random musings on uncoupling (5) Vintage 2,4-dinitrophenol

Thinking about uncoupling leads to the idea that it is wasteful if it occurs in excess of that needed for useful thermogenesis. Being energetically wasteful on a fixed calorie input means you do not have adequate calories left for your metabolism (though you might cut a few metabolic corners) so you should be hungry in proportion to the extra calories-out as heat. On a non restricted diet you should just eat more.

When I started thinking about the activation of uncoupling proteins by linoleic acid and its derivatives it seemed logical to have a look at the metabolic effects of other mechanisms of uncoupling, the best known of which is 2,4-dinitrophenol (DNT). It is, at first glance, a much simpler situation than that of LA because there is no feature of its metabolic effects which might promote excess caloric storage.
All it does is uncouple respiration and turn food, mostly fat, in to heat.

A more nuanced reflection would be that such a huge calories-out might be the equivalent to climbing Ben Nevis several times a day. Which ought to make you hungry.

While high dose DNP undoubtedly does make you hungry, the increase in hunger can be relatively easily offset by mild stimulators of lipolysis such as caffeine and/or sympathomimetics.

It would take more than a couple of cups of coffee to get you up and down Ben Nevis four times in a day.

So DNP not only increases calories-out, it must also be increasing access to calories-stored, allowing them to become calories-in so as to convert them to calories-out without excessive hunger.

Reverse electron transport, needed to generate the ROS which are essential to maintain insulin signalling, is highly dependent on the mitochondrial membrane potential. The core function of DNP is to lower that membrane potential and it should lower ROS generation and so blunt insulin signalling.

The effect is non specific, it doesn't matter where the FADH2 and NADH inputs are coming from, if membrane potential is artificially lowered, all of insulin's signalling will be reduced.

Not eliminated, but enough to access adipose tissue's stored fat in proportion to the blunting of insulin's action. Clearly there is no obvious need for the decrease in insulin signalling to exactly offset the increased heat generation. It happens to be close and a bit of caffeine appears to match things up nicely.

This is how I view the high dose rate fat loss facilitating effect of DNP. It still seems to be used as such in cultures where rapid loss of residual fat is required to get the perfect physique for a competitive edge in physical culture circles. Risk of death is of little concern, after all, exogenous insulin is used to bulk up muscle before cutting fat with DNP, if you are dedicated enough.

There are currently attempts to rehabilitate low dose/sustained release DNP as a useful drug. This review was written by a researcher heavily committed to such a drug development project. He's biased, of course, but that doesn't stop him being correct:

2,4 Dinitrophenol as Medicine

In some ways I approve, getting rid of people's fatty liver for them, or even correcting full blown NASH, is a Good Thing. Except I'm uncomfortable with the concept of applying a sticking plaster to metabolic syndrome and then waiting to see what other catastrophes turn up further down the road. After all, you only have metabolic syndrome because you have chronic linoleic acid intoxication.

However this might be quite a good sticking plaster, as sticking plasters go.

Peter

Random musings on uncoupling (4) coconut

Feeding mice on a high sucrose, low linoleic acid diet activates FGF21 production by the liver which stimulates heat generation in brown adipose tissue, leading to a lean phenotype, marked insulin sensitivity and poor glucose tolerance secondary to down regulated glucokinase in the liver. This latter is not surprising as fructokinase has a much higher rate constant for fructose phosphorylation than glucokinase does. Use it or lose it applies, even if only temporarily, so glucokinase down regulates. A bit like eating a low carb diet also down regulates glucokinase.

This is the post and this is the paper:

Chronic high-sucrose diet increases fibroblast growth factor 21 production and energy expenditure in mice

Edit, this one too

Ingestion of a moderate high-sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon-like peptide-1 secretion

End edit

My basic feeling was that fructose generated a caloric overload in the liver. Rather than dealing with this issue using hepatocyte mitochondrial uncoupling the task of dealing with the excess was delegated to brown adipose tissue and FGF21 was the messenger. "Higher  level" signalling. BAT uncouples on behalf of the liver. 

Of course that immediately suggests that other caloric overloads, especially if uncontrolled, might do the same thing. George Henderson tweeted this paper, which I've known about for years but have never gone in to in great detail:

Long term highly saturated fat diet does not induce NASH in Wistar rats

I hadn't realised how much uncoupling these rats were doing. They all weighed pretty much the same but caloric intake was way higher in the butter fed rats and even higher still in the coconut fed rats. That's interesting compared to coconut oil used in the Surwit type diets but these current diets are low in PUFA and sucrose free. Here are the caloric intakes:

The coconut based diet was particularly interesting as the rats were consuming twice the calories of the chow fed rats and weighed exactly the same. You could argue that coconut just tastes better than chow and the rats over ate then uncoupled. Or, more interestingly, you could suggest that medium chain fatty acids enter liver mitochondria in an unregulated manner and generate large amounts of input to the electron transport chain. If hepatocytes are experiencing a caloric overload what else should they do other than generate FGF21 and sub contract a calorie disposal solution to the BAT?

This arrangement would benefit, as with fructose, from the hepatocytes being the primary cells targeted to receive the unregulated caloric supply and is a good reason for keeping MCTs out of chylomicrons and passing them directly to the liver via the portal vein. Which is what happens.

So we can look at this study (just ignore everything about stress response and how a few ketones will do horrendous things to you):

Dietary Manipulations That Induce Ketosis Activate the HPA Axis in Male Rats and Mice: A Potential Role for Fibroblast Growth Factor-21

Here is what gavaging a chow fed rat with MCT oil does to FGF21 an hour later

LCT stands for corn oil. The acute effect of a low dose is almost nothing. Corn oil enters the systemic circulation in chylomicrons via the thoracic duct. It will be obesogenic as per ROS/Protons and only very mildly stimulating of FGF21 generation. Long term at high dose rates it will, as we've noted, uncouple enough to offset the metabolic syndrome induced as per ROS/Protons and result in a slim rodent which needs to over eat mildly to compensate for the side effect of uncoupling.

After coconut oil the uncoupling effect via FGF21 is marked so the compensatory eating has also got to be marked because the primary source of calories floods liver mitochondria with medium chain fatty acids.

So......... Localised hepatic caloric overload is a stimulus for FGF21 production leading to BAT thermal caloric disposal. As far as the rest of the body is concerned there is just the BAT caloric loss induced deficit to be perceived. There is a hypercaloric state in hepatocytes and a hypocaloric state in other systems, hypothalmus included. Food intake rises to maintain a normal energy supply to avoid weight loss.

Note the arrow of causality. The rats/mice are not over eating and burning off the excess. They are eating extra using an appropriate appetite to cope with BAT calorie expenditure/loss. They might not want to be hot but they have no choice. They eat to make up for it.

Peter

BTW there is this:

FGF21 treatment ameliorates alcoholic fatty liver through activation of AMPK-SIRT1 pathway

with alcohol being another hepatocyte caloric overload source which also generates FGF21 to "dispose" of the excess hepatic calories via BAT.

Using AMPK.

Which is where things get complicated.

Random musings on uncoupling (3) oxygen consumption

This is an nice little study from Japan looking at the effect of fat composition of diet on oxygen consumption before and after a meal:

Diet-Induced Thermogenesis Is Lower in Rats Fed a Lard Diet Than in Those Fed a High Oleic Acid Safflower Oil Diet, a Safflower Oil Diet or a Linseed Oil Diet

We can accept changes in oxygen consumption as a pretty good surrogate for the degree of uncoupling.

These are the diets used

The sucrose content is around 5% of calories so no confounder there. Fat is consistently 40% of calories and they measured the composition of the fats used. Like this

This lard is Japanese lard, produced in the early years of the 1990s. It's 7% linoleic acid. With lipids at 40 % of the calories in the diet this means that overall the LA provides 2.8% of the calories. So this is not an obesogenic diet*. However we get very little information about that because the rats were grown under a time restricted, calorie restricted protocol. An energy intake value was chosen as about the amount of food that a hungry rat would eat in an hour. This amount was fed twice a day. So there is no browsing allowed which suggests that some modest degree of caloric restriction is in place and the absolute supply of calories will be the same for all groups, despite each group have differing overall metabolic needs.

*[If the old anecdote about pork consumption in Okinawa is true this lipid profile might explain any possible longevity effect.]

The high oleic safflower oil diet provides 6% of calories as LA, the safflower oil diet provides 30% of calories as LA and the linseed oil diet provides 6% LA but this is combined with 21% as ALA. Alpha linolenic acid, from the ROS/Protons perspective, is an extreme version of LA although hard evidence for this is very thin on the ground. There are many studies using ALA which show that it is marvellous stuff at any dose rate but these studies almost always use "pair fed" or fixed calorie, mildly restricted protocols.

The protocol here also describes confirming that none of the diets generated lipid peroxides before being fed to the rats. The group is quite meticulous, so refreshing.

Here are the oxygen consumptions, indicating the degree of uncoupling present in the immediate post-meal period:

We can see that lard at 2.8 % of calories as LA shows very little uncoupling. Diets with LA between 6% and 30% LA uncouple more, although there is no evidence of a graduated response, and that a mix of 6%LA with 21% ALA uncouples the most, although this latter is only statistically ns greater than for the other PUFA groups, with a group size of six rats.

So. Given a fixed, mildly restricted calorie intake, we can take the lard fed rats as being very close to metabolically "normal", and eating closest to what they might want if fed ad-lib. Then we have three groups of rats, fed exactly the same number of calories, whose diet has been modified to induced a significant amount of uncoupling. All animals are at the same room temperature so obligatory thermogenic needs should be equal. So higher PUFA groups of animals will waste some of their consumed energy and not be allowed to replace it. What happens to body weights?
 

The low PUFA lard fed rats are the heaviest and carry about 5% more carcass fat than the PUFA fed rats. I consider them to be at normal body weight. Visceral fat, a surrogate for metabolic damage, is identical across the groups, all are free from clinical metabolic syndrome.

I would argue that the heavier rats are the least hungry, the high oleic acid safflower fed rats are a little more hungry and the two high PUFA diet rats are the most hungry. Quite what would have happened if the study had included an ad-lib fed arm we will never know.

So here's some speculation about "what if" there had been an ad-lib arm to the study:

The lard fed rats (this time) are our normal group and would weigh only a little more due to relief from calorie restriction. It would be nice if the ad-lib fed 6% LA group might have ended up with modest excess energy storage as fat gain and shown extra food consumption to match the extra weight gain. The high ALA fed group should have consumed even more food with less or similar weight gain because uncoupling is having a significant effect. Finally the group with 30% calories as LA should have come out somewhere between. The group having 30% of LA calories seems to be just on the border between where the ROS calorie storage effect transitions to the uncoupling effect in terms of dominance. From other studies 45% of calories as LA might have had the uncoupling effect absolutely dominating, so a slim phenotype would show, but that would be impossible in a diet where only 40% of calories are from fat in total.

How does this fit in to cellular "hunger" concept? At levels of linoleic acid where facilitated diversion of calories to storage predominates, excessive insulin signalling is dominant. The cell is dealing with an hypercaloric state.

Under marked uncoupling conditions the cellular state is the opposite. A separate defence mechanism against caloric excess is being activated, whether there is a caloric excess or not, by dropping the mitochondrial membrane potential because the closer a diet gets to 45% LA diet the more it provides a supra-physiological level of LA for the uncoupling protein function to maximise, combined with copious supplies of oxidative products such as 4-HNE to activate those uncoupling proteins.

At this point ROS generation is suppressed because, whatever the FADH2:NADH ratio, high mitochondrial membrane potential is still essential for reverse electron transport. There will be a transition from failure to limit ROS generation (forcing a cellular energy surfeit) to a reduction in insulin signalling as the result of a process which also involves the direct loss of calories by uncoupling (an hypocaloric state). The fact that suppressed insulin signalling releases fatty acids is over ridden by their loss through overactive uncoupling.

So PUFA are able to produce both cellular repletion and cellular hunger depending on the concentration.

It feels counterintuitive that a metabolite should, by one action, generate an hypercaloric state with excess energy storage and yet, by a separate process, go on to produce the opposite effect via uncoupling at higher levels of exposure.

But this appears to be the case.

It becomes much clearer using pharmacological uncoupling, which takes us back to 2,4-dinotrophenol.

Peter

Random musings on uncoupling (2) revised

Okay, here is how I ended the last post:

"It's also worth pointing out that this appears to be an ancient system and that high PUFA exposure might uncouple in anticipation of the cellular caloric influx which PUFA signify. It has become pre emptive and has, certainly in rodents, largely been shifted from "all" cells primarily to the brown adipose tissue. The PUFA signal might also be very central to the browning of white adipose tissue to beige. That's a process you would never want to have to use, being in a situation where generating beige adipose tissue might be helpful is not somewhere you want to go."

which is wooly thinking, to say the least.

Uncoupling is triggered by ROS generation using a locally available PUFA derived lipoxide signal combined with whatever fatty acids are available in the immediate vicinity of the mitochonrial inner membrane uncoupling proteins. A supply of PUFA is absolutely needed for the signalling molecule generation (4-HNE and related) and intact PUFA have been selected to uncouple better than saturated fats do. These features might be related.

PUFA are always present in the inner mitochondrial and have many functions. this function of acting as a safety valve appears to be one of them. It will not need to be specifically linked to bulk PUFA induced cellular caloric excess. I envisage it as a response to any excess caloric ingress, hyperglycaemia or markedly elevated FFAs post prandially (or even elevated levels of systemic fructose) when the law of mass action (ie a large concentration gradient) overwhelms the normal response of insulin resistance when cells are replete.

I view this aspect as the ancient system. It applies to any caloric overload and happens to use a PUFA/ROS signal to limit excessive mitochondrial membrane potential using uncoupling.

The fact that this system is functional at levels of PUFA intake far in excess of those that a particular species (humans) might be adapted to is perhaps unexpected but does seem to be the case, but this is more understandable if it is viewed as a generic safety mechanism.

Whether those slim rodent models consuming 45% of their calories as linoleic acid are dealing with excess caloric ingress by uncoupling or whether they are actually under caloric deficit because the emergency uncoupling system is being activated inappropriately due to oversupply of signalling precursors/uncoupling facilitating fatty acids is not clear.

Peter