Protons (77) Shulman PUFA and insulin sensitisation. Or not. Or so.

Over in the comments to the last post on metformin and Shulman's lab, Tucker pointed out that Shulman was an author (penultimate, so a senior author) on Nowotny et al's 2013 paper

Mechanisms Underlying the Onset of Oral Lipid–Induced Skeletal Muscle Insulin Resistance in Humans

which starts its discussion with the controversy about whether PUFA, particularly from soybean oil,  induce insulin resistance or insulin sensitivity. The most contradictory paper they cite is Xiao et al from 2006. In my head I think of this as the Hot Chocolate or the Cocoa study, which I discussed here as pure Protons in a cup of hot chocolate:

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

The two studies have diametrically opposite conclusions and this is an obvious opportunity for insight.

To me, it contradicts Protons, so let's go! Sadly, as Tucker points out, the chance for insight is completely missed by the Shulman group. They settled for PUFA -> insulin resistance. They lack the Protons perspective.

Equally sadly I have no handy mental label for the Nowotny/Shulman paper, probably because it produces no confirmation bias induced dopamine release in my head. My bad.

So let's compared the two.

The Cocoa study fasted obese subjects for 12 hours then fed them almost nothing but fat (with just a little carbohydrate) in small aliquots over 28-30 hours depending on how you read the methods. These subjects, by the time they started their hyperglycaemic clamp, had been either fully fasting or running their metabolism on the study fat, for approaching forty hours. In the control section of the study there were minimal calories supplied throughout, just a little carbohydrate in each drink. We can consider our control section to be, of necessity, in the functional insulin resistance of (very mild) starvation.

They are in a functional, very physiological, insulin resistance which is essential secondary to fasting or a fat based metabolism.

In Nowotny/Shulman their subjects were fasted for 10 hours in the aftermath of three days of a "high carbohydrate" eating period. By the start of the study period the subjects might have been ready for breakfast but they will have had absolutely no need (with a liver still full of glycogen) for the physiological insulin resistance adaptation to an absence of carbohydrate food intake such as would be necessitated by 40 hours of near fasting.

Subjects either drank 900kcal of soybean oil, started a 6h infusion of ~900kcal of intravenous soybean oil (Intralipid) or, for the control group, a 6h infusion containing approximately 54kcal of glycerol, ie mild fasting of 16h in total for the controls. Glycerol is primarily used by the liver for gluconeogensis so it will be a bit like the carbohydrate from the drink in the Cocoa study.

So Nowotny/Shulman  only looked at soybean oil vs nothing on a deliberately carbohydrate based metabolism of their mildly fasted control group. After carb loading to ensure a glucose based metabolism.

And soybean oil triggered insulin resistance. Glycerol didn't. QED, PUFA cause insulin resistance, directly. Diacylglycerols blah blah and all that crap as a mechanism, which is what the study seems to have been all about.

But the soybean oil was only being compared to a fully primed glucose based metabolism. Using an hyperinsulinaemic clamp and glucose supply to normoglycaemia.

In the Cocoa study the various fats were being compared to what was approaching a 40 hour fast. These subjects were already physiologically insulin resistant.

Adding more fat will (at approximately the correct 24h metabolic requirement for calories) either increase or decrease the degree of fasting insulin resistance based on Protons, the F:N ratio and reverse electron transport derived ROS.

Here PUFA cause a decrease in the physiological insulin resistance of 40 hours of fasting. Saturated fat augments it. MUFA is neutral:

You have to be very, very careful about what you are comparing to what.

Without thinking through the methods sections you could easily be forgiven for believing that the Nowotny/Shulman paper shows that PUFA cause insulin resistance. Possibly uniquely.

They clearly do, compared to to glucose. But they are *less* effective than saturated fats in performing the essential metabolic function of resisting insulin when glucose is in short supply. Shulman missed this, despite having Xiao point it out (*sarcasm warning*) in words of one syllable. PUFA are insulin sensitising when compared to whatever FFAs an obese person has available after nearly 40 hours of fasting.

Peter

Afterthought: The Cocoa SFA group required just under 40μmol/kg/min of glucose to maintain 20mmol/l in their plasma. The PUFA group needed about 55μmol/kg/min of glucose.

Given an infinite supply of donuts, which state would result in you eating the most?

Mmmmmm PUFA. The fat that makes you fat. By limiting insulin resistance.

Metformin (16) The LaMoia Shulman review

I first came across Gerard Shulman and his research group at Yale here:

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

and, although they are now looking at other targets for metformin's action, mitochondrial glycerol-3-phosphate dehydrogenase inhibition appears to be adequate to explain most of its clinical features.

I finally looked up who he is because, while looking for papers about certain aspects of metformin, I found this comprehensive review paper:

Cellular and Molecular Mechanisms of Metformin Action

which contains the bias confirming lines:

"Taken together, these studies indicate that metformin’s effect to increase insulin-stimulated peripheral glucose uptake is secondary to improved glycemic control and reversal of glucose toxicity, which can mostly be attributed to metformin’s ability to directly inhibit hepatic gluconeogenesis and HGP."

My own turn of phrase was:

"It [metformin] *appears* to improve insulin sensitivity, lowering the plasma level of insulin and glucose, but this is because it inhibits hepatic gluconeogenesis via inhibiting mtG3Pdh. That drops hepatic glucose output and that is what lowers the insulin level." I'm slightly cautions about the glucotoxicity aspect.

If you want more of an idea about how Shulman works there is a relatively short interview here which gives the flavour.

https://www.youtube.com/watch?v=qXxZ-I9N7Kc

Obviously he needs to take about four more steps backwards up the course of insulin resistance before he reaches perilipins and basal lipolysis. Whether he will ever go a step further beyond that and realise how linoleic acid controls the adipocyte size which controls the perilipins is possibly another order of magnitude further away. He also has zero concept that insulin resistance, which he notes is utterly preserved across all of those metazoan species which use insulin (which is most of us), is a functionally protective mechanism. As in here:

Insulin resistance is a cellular antioxidant defense mechanism

Until you realise insulin resistance is an antioxidant defence mechanism you will keep trying to "cure" it.

Never the less, he's a bright guy.

Tracy LaMoia, who is first author on the above two author review, seems to be a recent addition to the Shulman lab and is deeply steeped in metformin function. To the point where she is first author of this paper in addition to the review:

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis

I've yet to examine how convincing the complex IV part of the study might be, there's a lot to read, but it does pretty convincingly destroy any residual notion that metformin acts clinically by inhibiting complex I.

Or that a one millimolar or higher concentration of metformin is in any way related to clinical usage/efficacy. In fact actually measuring plasma metformin and reporting it in your research appears to be unusual.

This has consequences.

If you read any paper where they are using metformin at 1mM, 5mM or even 20mM to blockade complex I, crash ATP supply and thus activate AMPK, you can absolutely bin all of the cell culture sections of the paper. On diabetes, cancer, ageing etc. All of them. It is always the first thing I check.

Any section of such studies describing in-vivo work, be that mouse, rat or human, will give results that are likely to be believable. Though interpretation of the findings will be unreliable when swathes of the research population still mistakenly believe that metformin is an insulin sensitiser which works by blockade of complex I after being concentrated within mitochondria to 1000 times plasma level.

Which is preposterous. See piericidin A in LaMoia's paper above.

Peter

Protons (76) Those D12492 fed mice (Speakman and Tucker again)

For people who are thinking about re-listening to Tucker's discussion with Prof Speakman, at

Ep. 22: John Speakman—What Causes Obesity?

you could do worse than to check the section from time point of 24 minutes through to 26m 20. Speakman is describing exactly the phenomenon in the graph below, beautifully illustrated from the Schwartz laboratory. I may just have mentioned this many times in multiple blog posts:

He also describes, in brief, the concept of Reward as applied to these data.

To me, the Reward hypothesis has approximately zero explanatory power for the phenomenon in the graph and Speakman eloquently describes this deficit. He and Tucker discuss how an addictive drug drives progressively increasing consumption, but an high fat diet clearly has a decreasing drive to eat until near normal consumption resumes by about a week.

But always with residual obesity and slow, on-going weight gain.

Let's consider a better explanation for the behaviour of the mice in the Schwartz lab.

Linoleic acid in the D12492 is around 18% of total calories, according to a table I downloaded from Research Diets in 2011. This is well above the insulin sensitising dose noted for humans in the last post.

The whole argument from the Protons hypothesis is that linoleic acid has the ability to facilitate insulin signalling to a) increase post-prandial fat storage b) inhibit fasting fatty acid oxidation. That is a recipe for an acute loss of calories in to adipocytes and an hypocaloric crisis.

Which is easily corrected by eating some more. As in the above mice.

Now, before we look at the next paper, some ground rules need to be set out.

Metformin.

This is the most mis-represented drug ever investigated and almost all of the conclusions published about it are incorrect.

Metformin is an inhibitor of insulin signalling which therefore results in a decreased phosphorylation of AKT. Every time. See here here

here and many more places. It *appears* to improve insulin sensitivity, lowering the plasma level of insulin and glucose, but this is because it inhibits hepatic gluconeogenesis via inhibiting mtG3Pdh. That drops hepatic glucose output and that is what lowers the insulin level.

And don't forget SHORT syndrome, discussed here.

Having established that, let's put some ideas in to perspective. Linoleic acid is a pathological insulin sensitiser. Metformin is an insulin desensitiser.

The converse drugs to metformin are the glitazones. In vivo these *increase* the phosphorylation of AKT. What else would you expect? They really are insulin sensitisers. Their standard side effect is a worsening of obesity. Of course.

We are now in a position to explain the "hyperphagia" of mice fed high fat, high linoleic acid diets such as the D12492 used in the Schwartz lab.

We need to look at this paper:

Metformin Reduces Body Weight Gain and Improves Glucose Intolerance in High-Fat Diet-Fed C57BL/6J Mice

The mice were offered something very similar to D12451 (45% fat rather than the 60% fat of D12492)  but we don't know from which company it was purchased or even if the lard included was from Japan or America. No gas chromatography was used this time so a best guess might be around 10-15% of total calories as LA.

In the first hour of access each mouse eats roughly 5.5g of it, ie 28kcal, that's roughly a third of the 70kcal/d that the Schwartz mice would eat in a full 24h period while on a chow diet:

But the really interesting finding is what happens when you either reduce insulin signalling with metformin or increase it with pioglitazone.

Blunting insulin signalling (metformin 300mg/kg p/o) before access to the food decreases the one-hour food consumption by 80%.

That's 80 per cent.

5.5kcal in an hour to 1.0kcal in an hour.

The food is still yummy, it will still light up the endogenous opioid, endocannabinoid and serotonin systems (dopamine too I guess) of the hypothalamus but the hyperphagia essentially disappears. The hyperphagia is made worse by pioglitazone, of course.

It's simply about pathological insulin sensitivity being corrected by an insulin signalling inhibitor.

It is an energy supply problem.

So metformin is a partial rescue drug for LA toxicity. It's not perfect but it illustrates basic physiological principles. Obviously the correct solution to obesity is the reduction of linoleic acid in the diet to around or just below 2% of calories. Ruminant fat. Not metformin. Not a GLP-1 agonist.

Oh, almost forgot. Near normalisation of calorie intake: I've said it before, hyperphagia ameliorates over a week because distended adipocytes increase their basal lipolysis and will raise FFAs high enough to a) induce enough insulin *resistance* to reduce LA's lipid storage effect and b) overcome the blockade of CPT1 from malonyl-CoA. Adequate calories then become available *provided* adipocytes stay distended. Under-eating simply shrinks the adipocytes, reduces basal lipolysis mediated FFA release and re-establishes pathological insulin sensitivity. Because there is now a need to maintain adipocyte size, food intake must trickle along at levels just high enough to maintain adequate obesity for adequate caloric availability from increased basal lipolysis to resist insulin.

Peter

Protons (75) Tucker; Speakman; Astrup and linoleic acid. And insulin sensitivity

Tucker has a podcast episode in which he chats to John Speakman about obesity. It's one of the more interesting podcasts I've listened to in many years.

Ep. 22: John Speakman—What Causes Obesity?

A very large part of the core discussion is contained within this paper, a massive collaboration, with Speakman as first author:

Total daily energy expenditure has declined over the last 3 decades due to declining basal expenditure not reduced activity expenditure

Basically total daily energy expenditure in the studied populations is down slightly over the last 30 years, despite daily activity energy expenditure going up. This means that basal metabolic rate must have dropped.

Which, of course, begs the question of what might cause basal metabolic rate to fall.

The answer is not obesity.

There are certain groups of people who *do* have a decreased BMR, the most obvious of whom are the post-obese.

The post-obese, like the pre-obese, come with a cluster of abnormalities the two most prominent of which are an enhanced insulin sensitivity and a defect in fat oxidation. And sometimes a depressed metabolic rate, especially BMR. 

To me, the enhance insulin sensitivity is causal, the impaired fat oxidation is secondary. The decreased metabolic rate is simply a longer term downstream effect of chronic under supply of calories to metabolism.

Aside: I haven't discussed it yet but, obviously, pathological insulin sensitivity should also show as an exaggerated ability to over-store fat under peak insulin effect. This shows rather nicely under an hyperinsulinaemic euglycaemic clamp in Astup's lab. See top panel of Fig 2. But currently I'm mostly thinking about fasting conditions. End aside.

So. The core feature of pre or post obesity following on from the pathological insulin sensitivity is a decreased ability to oxidise lipid and a facilitated ability to oxidise carbohydrate. The RQ should rise.

What would happen if you took eight apparently healthy men and fed them, for a week, a complete diet providing 2% PUFA then switched them to a 10% PUFA diet for another week, as a crossover study?

This is the paper, from 1988:

Polyunsaturated:Saturated Ratio of Diet Fat Influences Energy Substrate Utilization in the Human

You can clearly alter the RQ under fasting conditions, on a fixed food quotient diet, simply by altering the dietary fat from 2% of calories as PUFA to 10% PUFA, switching palmitate in or out to balance the PUFA, which was mostly linoleic acid. MUFA were kept constant, as were all other macros.

Within seven days this happened to the fasting RQ values.

Obviously there are three interesting subjects. One showed a decrease in RQ, suggesting enhanced lipid oxidation under linoleic acid. That's unusual. It is normal for linoleic acid to augment the thermic effect of food because it is preferentially oxidised but that is finished well before an overnight fast is finished. Hard to say what was going on with that subject. It wasn't a hospitalised study but all food was provided by the investigators. File it under odd.

The rise in RQ, signifying a change away from lipid and towards carbohydrate oxidation while fasting, was (pax the exception) ubiquitous across all other subjects, but in two subjects there was such a rise in RQ that the investigators seriously considered that there might be a problem with their measurement system. There wasn't. Their comment:

"Although a fasting RQ of 0.9 is unusual, reanalysis of the calibration parameters of the respiratory gas exchange system obtained prior to tests on these subjects revealed no abnormality in analyzer response. No reason for rejection of these RQ values could be determined."

Clearly 10% of LA in the diet moves almost all subjects towards a "pre-obese" phenotype. In two of the eight this move was dramatic. It seems very, very likely to me that these two individuals are at serious risk of obesity in an omega-6 rich environment. Follow up weights over the years would have been lovely but was not remotely the purpose of the study.

You can, within seven days, convert normal people in to pre-obese people, as viewed from metabolic substrate oxidation perspective.

All you have to do is make sure they are eating 10% of their calories from linoleic acid.

Some people will get bitten by this feature of linoleic acid more rapidly than others.

Eventually the whole population will.

Thank your cardiologist.

Peter

Addendum. The world is full of U shaped curves. Adding linoleic acid to the diet causes an initial excess insulin sensitivity. This distends adipocytes. As adipocytes distend they increase their basal lipolysis and release FFAs which cannot be suppressed by insulin. This, at some point, appears to normalise fasting insulin sensitivity at the cost of distended adipocytes, ie obesity, and chronically elevated FFAs. On a starch based diet the high level of post prandial insulin needed to overcome the still (unsupressable) FFA induced insulin resistance at peak absortption will sequester more lipid in to adipocytes, from where they will again leak, via basal lipolysis, leading to frank insulin resistance, hyperinsulinaemia and metabolic syndrome.

Under fasting conditions the pathological insulin sensitivity activates malonyl-CoA formation and the subsequent inhibition of CPT1 mediated entry of fatty acids in to mitochondria. This would, if it occurred in isolation, simply lead to hypometabolism unless enough glucose alone was available to run metabolism. However, it doesn't happen in isolation. It happens combined with obesity, which increases the supply of FFAs irrespective of insulin sensitivity. All that is needed is to elevate FFAs high enough to get adequate substrate in to mitochondria (there is not 100% inhibition of CPT1) and enough lipid derived ROS can then inhibit insulin, reactivate CPT1 and restore metabolism. Hence obese people have high metabolic rates.

The crux comes with conventional dieting. As adipocytes shrink the supply of FFAs from basal lipolysis drops, insulin sensitivity is restored and people get right back to where linoleic acid takes them: obtunded fat oxidation, carbohydrate dependency and hypometabolism. The classical post-diet hungry person.

Why is BMR falling in the developed world despite obesity being rampant? Because everyone is being drugged with linoleic acid to become obese and no one wants to be fat. The more you resist obesity, the more your caloric restriction shows as decreased BMR. The BMR is falling in response to Weight Watchers, Slimming World etc. People are not as fat as linoleic acid "wants" them to be.

Ultimately obesity "fixes" the pathological insulin sensitivity from linoleic acid on both fronts, at the cost of weight gain. But it's not a real fix, it's a sticking plaster and we call it metabolic syndrome.

End.

Protons (74) Arne Astrup and the formerly obese

I have a soft spot for Arne Astrup. Back in the days of the depths of the Danish fat taxation stupidity, he was one of the voices of reason speaking out against the tax. It was a near miss for sanity. Academics have since argued that the tax was repealed too soon (and the sugar tax never got started) and that it was actually "working", at least among those who couldn't hop over the border to buy their (Danish?) butter in Germany. Had it been allowed to continue to "work" we might have successfully forced a whole nation to avoid fat, especially saturated fat. Where might that have led? If you wish to compose the answer on a postage stamp it is just three letters long, which will fit neatly on to even the smallest stamp.

Anyhoo. People may have noticed that I like this paper from the Astrup led lab

Fat metabolism in formerly obese women

mostly because Table 3 confirms all of my biases by showing formerly obese women are exquisitely insulin sensitive, which is pure Protons:

The rest of the paper is more difficult.

The formerly obese are, as expected, only deriving around 35% of their energy from fat oxidation at time zero on the graph below while the never-obese controls are deriving just under 80% of their energy needs from lipid oxidation, time zero again. These values are while sitting still on a bicycle ergonometer, after an over night fast. Not quite basal metabolic rate or resting energy expenditure but pretty close:

It is also worth noting that performing exercise at 50% of VO2 max (previously individually measured) completely normalises energy production derived from fat oxidation. Those are time points 15-60min. All we need here is for AMPK to instigate oxidation of the fatty acids available while suppressing their formation. Then the pathological insulin sensitivity is bypassed. There are several posts possible on AMPK but again, here is not the place to explore the control of insulin signalling by AMPK and vice versa. Both happen.

Finally, the really strange thing is that these formerly obese women have modestly *elevated* FFAs, both at rest and throughout exercise, consistently around 300µmol/l greater than controls.  If the formerly obese have all this extra lipid available, why don't they oxidise it?

We can say, quite conclusively, that these FO women have normal electron transport chains. Under exercise they oxidise lipids exactly as well as control women do. My assumption is that there has to be a signalling problem which is inhibiting fatty acid oxidation but can be over-ridden by AMPK activation.

We know, from a mass of rodent and human studies, that when you allow a subject access to carbohydrate food (or an OGTT) after an extended fast, they perform de novo lipogenesis, giving an RER > 1.0, for about an hour. The duration is interesting. Insulin-induced insulin resistance (which is complex and probably involves the glycerophosphate shuttle) usually comes in to effect at around about an hour in many models. This will reduce insulin signalling from its peak action under "hungry" conditions to a more moderate "fed" signal.

So peak insulin signalling after a fast, but before establishment of some physiological limitation, is a potential major driver of de novo lipogenesis with storage as triglyceride and, as we shall see, an effective inhibitor of fat oxidation.

Mechanistically we have to look briefly at the Randle Cycle.

Two of the many actions of insulin are to activate the pyruvate dehydrogenase complex and the acetyl-CoA carboxylase complex. This generates malonyl-CoA which inhibits CPT1 mediated transport of fatty acids in to mitochondria. Hence FFAs are available but not oxidised. But there is no problem with the mitochondrial ETC itself, all that is needed is for the insulin signal to be reduced.

During that initial refeeding period both the RQ of >1.0 and the inability to oxidise lipid can be viewed as manifestations of marked insulin sensitivity. Carbohydrate uptake is enhanced by insulin and the products of this carbohydrate catabolism are diverted, by insulin, to metabolites which inhibit fat oxidation and away from the Krebs Cycle and the electron transport chain.

Aside: My interest is in ROS based control systems so I have tended to ignore such details. But here the downstream effects of excessive insulin signalling on the Randle Cycle do matter. My bad. End aside.

My premise is that obesity is cause by a pathological sensitivity to the hormone insulin, mediated by linoleic acid. If this is correct then we would expect pathological lipid synthesis/storage to be combined with an inhibition of fatty acid oxidation. The normal "one hour" of peak insulin sensitivity is extended or even becomes continuous by using linoleic acid as a significant energy source (pax uncoupling intakes).

Here we have the formerly obese who are, without a doubt, destined to become obese again in the future. We also have people with obese parents who are not yet obese themselves. Both show the accentuated insulin sensitivity in combination with depressed fatty acid oxidation, both at rest and post prandially. All that is required to do this is to allow insulin to continue to act at peak efficacy under conditions where a functional limitation should have been imposed. Linoleic acid replacing palmitate/stearate under the Protons hypothesis provides exactly this.

So, in Astrup's particular group of formerly obese subjects described in the current study, it has proved possible to have inhibited fatty acid oxidation with sufficient severity that it leads to elevated plasma FFAs, because they cannot be used for energy generation. All as a consequence of augmented insulin signalling.

This particular group of FO subjects do appear to be a rather extreme example. Other FO people assessed by Astrup's group in previous studies do not feature the elevated FFA or profoundly depressed fasting insulin aspects, though the inability to oxidise lipid to produce adequate energy is a consistent feature over many studies. I think this current group of women are probably outliers who give an insight in to mechanisms. That's good.

It's also clear that these formerly obese women have a metabolic rate under fasting conditions significantly lower than that of the never obese controls. This is not surprising. Fat oxidation is being largely inhibited by elevated malonyl-CoA and a significant portion of glucose is being diverted from energy production to form that malonyl-CoA and its derived and stored lipid.

The FO women's resting energy expenditure is 3.77kJ/min, ie 0.9kcal/min, 1296kcal/24h. Never obese women expend 4.88kJ/min at rest, 1.2kcal/min, 1728kcal/24h. Except of course the FO women are not oxidising fat, because they are unable to effectively oxidise FFAs. They are using glucose.

So they are 461kcal/24h "hungrier" than never obese controls and are running on limited supplies of glucose from glycogen. The obvious solution is to access more glucose, which insulin has actively locked in to the liver/muscle stores of glycogen.

Traditionally this is solved in the real world by raiding the fridge at 3am. For something sweet. Much of which will be diverted to storage.

Weight gain.

It happens.

Peter

Protons (73) Spanish vs Canadian studies or 3% LA vs 10.3% LA

I hope no one has forgotten the "Spanish Study"

Distinctive postprandial modulation of beta cell function and insulin sensitivity by dietary fats: monounsaturated compared with saturated fatty acids

which was very, very carefully set up to demonstrate post prandial insulin resistance following the ingestion of the saturated fats from butter while also demonstrating a progressively improving insulin sensitivity using oils with increasing content of (mostly) linoleic acid.

A brief look at reference 15 methods section confirms they measured the test meal lipids by gas chromatography.

Obviously, to anyone lacking the Protons perspective, the clear cut message is that saturated fat causes insulin resistance. Insulin resistance is BAD. Saturated fats are BAD.

What Protons actually predicts is that resisting insulin by ingesting saturated fats limits insulin-facilitated obesity, so eliminates the subsequent adipocyte distension derived release of FFAs (which cannot be suppressed by insulin), which would lead to metabolic syndrome.

Resisting insulin prandially resists obesity and so resists obesity-derived insulin resistance. Read that very carefully.

Aside: Resisting insulin in the immediate post ingestion period is a short term effect for a few hours. It's physiological. Constant presence of FFAs secondary to increased basal lipolysis from distended adipocytes is present 24/7 irrespective of what you eat. These fatty acids supply calories and if you are eating carbohydrate then you must resist cellular glucose ingress to take in to account that FFA supply of calories. This insulin resistance is different (still the correct physiological response to FFA availability) because it follows on from pathology related to adipocyte lipid droplet size. The third type of insulin resistance is much more complicated. So is the fourth. Here is not the place to discuss them. End aside.

Sooooo. I really, really like this:

This is *not* demonstrating saturated fat induced pathological insulin resistance. Here the insulin "resistance" will simply stop you getting fat. I would define this as the "normal" response to an high fat meal, assuming a low linoleic acid based fat.

What it *is* demonstrating is pathological insulin sensitivity following the ingestion of 10.3% (actually slightly under this but close enough) of calories as linoleic acid. Here as little as 140pmol/l of insulin will rapidly clear your plasma of calories and leave you hungry. The meal is largely lost in to your adipocytes. You WILL want to eat again, and soon.

This is fundamental and simply falls out of the Protons hypothesis.


Now, there are problems with the study and the authors are to be congratulated on the result generated (though not on their conclusions of course), especially the composition and size of the meals they had to design to get there. But here we are looking at a dynamic response to a single meal. Is it possible to examine their important findings under more steady state conditions? Would the relationship of PUFA ingestion to pathological insulin sensitivity still hold?

What would we find if we kept plasma FFAs forcibly elevated for 24h using repeated small oral fat loads supplying 2430kcal over a 24h period (but no other food) instead of a single oral ingestion of 800kcal as one mostly fat meal?

Then, instead of tracking the insulin response to a small amount of starch/protein along side the fat of the 800kcal meal, we could assess insulin sensitivity during an hyperglycaemic clamp at 20mmol/l of glucose in plasma. The more glucose needed to achieve this level, the greater the insulin sensitivity.

We are now well away from normal physiology but we are asking essentially the same question under more constrained conditions.

I'm assuming people have realised that I'm now describing Xiao's study from the last post

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

which produced this chart from the above protocol:

To me, the results of Xiao's study and the Spanish study concur beautifully.

Not everyone will agree with me that these reflect a core reality, I wouldn't expect that. But in my NSVHO this is how physiology works. Linoleic acid is insulin sensitising.

However, when you have confirmation bias as badly as I do, and you find two non related studies which neatly corroborate each other while confirming your biases, you know you are trapped. That's me.

You have been warned.

Peter

Addendum: Obviously palm oil at around 8% of calories as LA is already insulin sensitising, it's in the same ball park as the 10.3% LA arm of the Spanish Study. A true SFA arm to the study would need to have LA at around 2% of calories and I would predict a GIR well under 40μmol/kg/min.

Protons (72) Humans: 8% LA vs 74% LA by sustained oral ingestion

This study in humans is very different to the previous rat study. People were fed repeated oral fat loads (you could call this Bulletproof Cocoa rather than Bulletproof Coffee), once an hour for 12 hours then every two hours overnight until the start of an hyerglycaemic clamp at 24h, through which the oral fat loading continued. Glucose was infused to a stable 20mmol/l and insulin allowed to respond as best it might. Insulin sensitivity was determined by the glucose infusion rate in the last 30 minutes of the clamp. In some ways this is more physiological than the hyperinsulinaemic euglycaemic clamp, which is considered the gold standard. The oral fat ingestion was slightly above calculated 24h caloric requirements for these subjects.

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

Over a 24h period the ingestion of safflower oil, with linoleic acid providing in the region of 70% of total calories, ought to demonstrate the initial Protons predicted insulin sensitising effect, which would only be later replaced by the uncoupling effect if the study had been continued for a week or two. 

Again we can assess insulin sensitivity by how much glucose was needed to be infused during the last 30 minutes of the clamp to maintain an hyperglycaemia of 20mmol/l. This is what happens:

I don't think I have to make any qualifications here. SFA oral ingestion for 24h causes a very similar degree of insulin resistance to oral ingestion of a minimal calorie supplying control chocolate drink. Tallow rather than palm oil would have accentuated the effect.

Ingesting 70% of your calories as linoleic acid over a 24h period is insulin sensitising compared to ingesting SFA, p less than 0.001. Or ingesting virtually nothing at all, p < 0.05.

Linoelic acid is insulin sensitising.

This is BAD. When fasting you *must* resist even basal insulin or that insulin will lower fasting glucose, lower fasting FFAs and you will be hungry. And raid the fridge at 2am. And get fat.

Protons.

Peter

Protons (71) Rats: 13% LA vs 61% (mostly) LA by infusion

I have a certain, very specific, idea of how linoleic acid produces obesity. It seems as though relatively few people share this point of view. That is absolutely fine. Bright people have their own ideas and, eventually, if the core process is consistent, all views of the development of obesity and its associated insulin resistance will eventually converge. I spend a great deal of time thinking about whether linoleic acid enhances insulin sensitivity -> directly causing obesity or whether linoleic acid causes insulin resistance directly -> reactive hyperinsulinaemia -> obesity. The data make me favour the former.

This is the first paper I have come across where various fatty acid mixtures were assessed, in rats, for their acute effects on insulin sensitivity in vivo. In particular I was interested in the effect on glucose utilisation under hyperinsulinaemic euglycaemic clamp conditions. The higher the infusion rate, the more insulin sensitive the rat is.

In vivo effects of polyunsaturated, monounsaturated, and saturated fatty acids on hepatic and peripheral insulin sensitivity

They infused intravenous oil emulsions continuously for five hours and then continued throughout the exogenous hyperinsulinaemia over the following two hours, while clamping glucose at around 6.5mmol/l. So this is looking at normoglycaemia combined with fasting levels of FFAs until the clamp period. The rate of deliver per hour was roughly comparable to a 24h intake of calories for a rat of this size, averaged to an hourly rate.

Everything is fairly physiological until you add in the insulin/glucose infusions for the clamp while maintaining the lipid supply. Then you are looking at the situation where FFA supply cannot be suppressed by insulin, so you have a model for metabolic syndrome.

The results are quite clear. Whole body insulin responsiveness is suppressed by any fatty acid availability.

Clearly the glucose infusion rate, representing whole body insulin sensitivity, is lower in the SATU group (lard oil) compared to the PUFA group (soybean oil) but this is not remotely statistically significant (p = 0.2849). However there is no suggestion that linoleic acid is uniquely triggering insulin resistance compared to saturated fats, bearing in mind that modern (2015) Canadian lard is higher in insulin sensitising LA at 15% than my preferred fats such as beef tallow or suet which are around 2% LA (correctly ignoring any CLA content).

So the Protons concept could be suggested to have earned some marks here, there is more insulin resistance in the saturated fat group (GIR 43micromol/kg/min) when compared to the less insulin resistant linoleic acid infused group (GIR 73 micromol/kg/min). But not statistically significant.

However, there is no suggestion that linoleic acid per se causes enhanced insulin resistance, so causing obesity via secondary hyperinsulinaemia. In fact the trend is in the reverse direction.

In these rats.

Humans next.

Peter

Foie Gras (11) Hepatocyte mitochondria

Another tidy up, this time related to 

Fat Quality Influences the Obesogenic Effect of High Fat Diets

and the paradox of mitochondrial uncoupling in section B of Fig 4:

It is patently obvious from this plot that mitochondria extracted from the liver tissue of lard fed rats (consuming an obesogenic level of linoleic acid) do have an higher uncoupled oxygen consumption at all values of membrane potential when compared to the level of oxygen consumption in those rats fed the high safflower oil diet.

That is exciting and paradoxical.

We know from Figure 1 that the safflower oil fed rats were more uncoupled overall than the lard fed rats. They were synthesising much more UCP-1/cell in their brown adipose tissue and they had a greater absolute mass of brown adipose tissue by the end of the study.

They were also actively expending more energy per day at the end of the study compared to the lard fed rats. This is stated in the legend to Figure 2:

"Percent contribution of lipids, proteins and carbohydrates to total daily energy expenditure (lard = 380 ± 15, safflower-linseed = 410 ± 25 kJ/day x kg^0.75) in rats fed lard or safflower-linseed high fat diet."

I would expect all rats/mice fed high safflower oil diets to use this technique and so eventually normalise their weight to that of chow fed rats/mice on a long term basis, as was found (in mice) here:

Prevention of diet-induced obesity by safflower oil: insights at the levels of PPARalpha, orexin, and ghrelin gene expression of adipocytes in mice

Okay, let's summarise:

Safflower oil induced an initial obesity by increasing insulin sensitivity which was, by day 14, in the process of being reversed by UCP-1 reducing that excessive insulin sensitivity in WAT, assisted by activating BAT.

No one would expect hepatocytes to express UCP-1, they just don't do this. The liver deals with excess calories by sequestering them as triglycerides under the influence of insulin, sequestering them as triglycerides under the influence of succinate derived from peroxisomal omega oxidation or by signalling to BAT using FGF21 as a mediator to increase UCP-1 expression so as to bulk off-load calories as heat. But not in the liver.

Soooooo.

Safflower oil (~70% linoleic acid) produces whole-body uncoupling in the rats in the current study, apparently with the exception of within liver tissue.

Hepatocytes *do* use UCPs, they definitely synthesise UCP-2 and UCP3, but not for bulk lipid oxidation. Current thinking is they are used to fine tune their inner mitochondrial membrane potential while other signals deal with bulk caloric overload.

So the paradox is that lard fed rats have more uncoupled mitochondria than safflower fed rats. That's what the graph at the top of the page shows, ie hepatocytes are doing the opposite of what the whole rat is doing...

They measured delta psi of isolated mitochondria with a dye (safranin O) calibrated back (through 4 layers of references) to the standard technique which gives us our best estimation (don't ask) of membrane potential. They then fed isolated mitochondria in the presence of oligomycin (to block ATP synthesis) and rotenone (to prevent RET through complex I). At this point all oxygen consumption is from uncoupling. If you add increments of malonate to progressively inhibit complex II you can progressively lower the delta psi and look at the degree of uncoupling at a given titrated delta psi.

On the face of it it looks very much as if the liver really is doing the opposite to the rest of the body, which seems counter intuitive:

The degree of uncoupling is being assessed at a fixed potential, here the group chose to use 150mV (the blue line) for their example, giving an uncoupled oxygen consumption of 41.9 in lard fed vs 22.2ngatoms/(min x mg protein) of oxygen if safflower oil fed.

But is this the case in vivo? The lard fed rats are chronically underfed and have lipid locked in to adipocytes by excessive insulin sensitivity so what little lipid is being released is via augmented basal lipolysis. It is completely plausible (but also completely made-up) that they might be running a membrane potential, in vivo, as low as 120mV. Like this, blue line:

At 120mV you are not going to making a lot of ATP so uncoupling would be actively disadvantageous. In this example the uncoupled oxygen consumption would be low, in the region of 19ngatoms/(min x mg protein) of oxygen, red line.

The safflower oil fed rats went through an initial hypocaloric episode during their initial weight gain phase, but now they are uncoupling in WAT which will blunt insulin signalling and release a surfeit of FFAs, enough to supply large amounts of FFAs the liver mitochondria and (in parallel) accumulate as lipid droplets to the point of cellular damage occurring.

Under this level of direct hepatic caloric excess the mitochondrial membrane potential is likely to be high. If we run another thought experiment (ie make up) a potential of 160mV, just under that 170mV threshold for marked ROS generation, this would give us an uncoupled oxygen consumption of 29ngatoms/(min x mg protein) like this:

So, if the membrane potential differs between groups in vivo, so would the level of uncoupling. It is completely plausible that (safflower) lipid overloaded mitochondria are running an high delta psi, so need more uncoupling. Mitochondrial will never have a fixed delta psi of 150mV. It is absolutely possible that, in vivo, the safflower oil fed rats had more uncoupled hepatic mitochondria compared to the lard fed rats.

I feel much more comfortable with having hepatocytes uncouple *more* with safflower oil than with lard. The whole study is bias confirming of multiple aspects of the Protons hypothesis. Things have to make sense.

I have no problem with the mitochondrial preparation the group developed here and how they have used it. It's no better/worse than any other mitochondrial preparation. What is crucial is how you interpret the data it provides you with in the light of what must be happening physiologically. Then extrapolate backwards to the most plausible in-vivo situations, with caveats.

I have my biases.

Peter

Late addendum.

The mitochondrial uncoupling curve I have been discussing was generated from mitochondria treated with FFAs to facilitate uncoupling. Of course, if you argue that the lard fed, hypocaloric rats had lower levels of FFA in the fed state in vivo (in the fasted state there is no difference in FFA level) then there would be much less uncoupling than that discussed above, emphasising the point. Without FFA supplementation, at 160mV in the lard fed rats uncoupled oxygen consumption was as low as ~5ngatoms/(min x mg protein) in section A of Figure 4. In reality it would be somewhere between this value and the FFA supplemented value. As much as any mitochondrial prep reflects reality.

Foie Gras (10) Liver

Just to tidy up my thoughts on

A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue

TLDR: I suppose all I really have to say is that the title is incorrect and the scrutineers are completely incompetent.

We have these data for mRNA production from "pro-inflammatory" genes in liver tissue:

which we know, from their section of adipose tissue, have absolutely zero correlation with active inflammation, which they assessed in adipose tissue using the activity of the myeloperoxidase system.

The liver is full of resident macrophages, known as Kuffper cells, which are very good at activating their myeloperoxidase system. The group has an assay for this activity. They didn't use it on liver tissue. Why not?

I have no idea whether the liver macrophages actually used any of the reported mRNA products to generate inflammation. 

Go figure.

The group did, for some reason, measure plasma CRP levels, CRP being an acute phase protein produced by the liver in response to any inflammation, *anywhere* in the body. It might have been raised in response to the activity of the myeloperoxidase in the adipose tissue of the HF fed mice, that might be logical. We'll never know because they omitted to measure CRP in the plasma of the HF fed mice.

Go figure. I have not edited this chart in any way:

So we know essentially nothing about inflammatory changes in the liver and we know nothing about the levels of CRP produced (or not) in the plasma of HF diet fed mice. Which did have inflamed adipose tissue and *might* have had inflamed liver tissue.

Oh, their one interesting finding was that saturated fat is suppressive of "pro-inflammatory" gene expression in liver tissue. But not in adipose tissue.

Would this be protective against inflammatory liver damage? There is no way you can assess this from this study, but the idea is nice.

But ultimately the liver section of the paper is complete dross.

I said it before, these people are rank amateurs.

Peter

Foie gras (9) Adipocyte ROS

Time to look at the Vaughan mouse weight gains and actual diet compositions. Here is a recap of the weight gains:

and here is Table 1 with the percentages of energy from linoleic acid added in red by myself:

It's clear that the saturated fat group were only fed 6% of their calories as insulin sensitising linoleic acid. In addition to this relatively low amount of LA, the stearic acid in the saturates has an high F:N ratio and will in part offset the low F:N ratio of the LA component.

If adipocytes resist insulin they stay small.

The oleic acid group were fed 7% of calories as LA but with only 12% of calories as saturated fat there is nothing to oppose the insulin sensitisation effect of LA so they gained a significant amount of weight.

The high (35% of calories) LA diet is uncoupling, produced low levels of ROS as a consequence, and so limited weight gain. Given long enough it would normalise bodyweight.

The high omega 3 diet contained an obesogenic level of LA coupled with an uncoupling level of alpha linolenic acid, though putting a number to the level of ALA needed to uncouple is difficult, but it is lower than the level of LA needed. This is another combination which I expect, given long enough, would normalise body weights. 

I feel that some explanation is needed as to why the LA/ALA 18:3n3 diet mice, and to some extent the high LA diet mice, weigh more than the saturated fat diet mice, to the point that the 18:3n3 diet looks obesogenic.

There are, initially, two effects of fatty acids with multiple double bonds. The first is the reduction in RET through complex I which itself has two effects. Under peak insulin action there is reduced negative feedback so adipocytes distend more than they should. Second is that during fasting, when fat is the primary fuel, RET should occur to limit glucose utilisation in order to spare glucose for the brain/hypothalamus and so limit hunger. With the blunted RET from LA/ALA more glucose is used by muscle etc so more food must be eaten, so breaking the fast, in order to keep glucose levels adequate to limit hunger. Much of this extra food then gets stored.

These two together put fat in to adipocytes and demand more food intake. This is the classic situation under D12492.

This is also likely to be the initial situation when using 35% LA or a mix of 11% LA with 23% ALA in the period before uncoupling becomes established.

The second effect is via uncoupling.

There will be weight loss in these latter two diets, but only once UCP-1 is activated in white adipocytes to lower delta psi and so reduce insulin signalling. At that point adipocyte FFAs can either be oxidised to release heat in situ (beiging of WAT) or transferred to BAT where high levels of UCP-1 can oxidise them to release heat in bulk.

This concept suggests that, by 14 days, the mice on the "18:2n6" diet would be in weight loss and should have low ROS generation due to uncoupling, after an initial weight gain.

The effect should be more marked in the "18:3n3" group, ie an higher initial weight gain, then incipient weight loss by 14 days.

This is why I like the Schwartz data, daily resolution of food intakes and fat mass changes allow you look at things more mechanistically. It would have been nice to have these data from the current pro-linoleic acid study but thats not what the study was all about.

Here are the mRNA data for inflammatory gene expression in adipocytes in vivo (or immediately post euthanasia!), which I am taking to be a surrogate for ROS generation:

 

From the ROS perspective the SFA adipocytes are generating ROS by RET, so are limiting insulin signalling-induced lipid droplet distension. The mice are slim. And healthy.

The 18:2n6 mice are uncoupled, have low ROS due to this and are actively losing weight after an initial gain. Same for the 18:3n3 mice.

The HF fed mice (9.6% LA, obesogenic, failing to limit insulin signalling) have low ROS because they are failing to generate enough of them via RET to limit caloric ingress, ie have "pathological" insulin sensitivity. "Healthy" insulin sensitivity, through healthy ROS, is shown by the SFA group. The HF group are simply sequestering calories in to lipid droplets without oxidising them. Here weight gain is on-going but there is no issue with high ROS because they are effective at sequestering calories. Except...

Now the HF fed mice are really interesting. They have levels of inflammatory gene mRNA expression comparable to all of the other groups, including the SFA group (p>0.05 for the comparison) but look at their MPO activity, an indicator of active inflammation. I've rearranged chart G so all columns are on the same scale:

All groups of mice have comparable levels of inflammatory gene mRNA expression (pax the SFA fed group) but only the HF group have actively inflamed adipose tissue.

Why?

We can say that generating mRNA from pro-inflammatory genes alone is not sufficient to activate the inflammatory cascade to the extent of activating the myeloperoxidase system.

I have to ask myself what, exactly, is the function of these genes we are looking at, within physiology, at the most basic level.

I would suggest that they might be to deal with normal ROS generated from normal metabolism. The SFA diet induces high levels of healthy ROS via RET. It generates a large, effective response in ROS mitigating genes. All other groups, at the 14 day mark of the study, have low levels of RET derived ROS, so low levels of mRNA from inflammatory (or rather mitigating) genes. Including the HF diet group.

What is different about the HF diet group is that they are, through Protons, unable to limit caloric ingress. As much of the excess calories as possible will be rendered in to harmless stored triglycerides but all that is needed to generate frank inflammation is the generation of a delta psi in excess of 170mV. This leads to ROS which are only in a small part derived from RET, ie are mostly pathologically derived.

I think the HF diet fed mouse adipocytes are doing this. There is tissue damage occurring and lipid peroxides are produced at levels which signal danger of serious injury and so macrophages move in to sort out the damage. Probably making incorrect assumptions about the source of the damage, leading to pathology. This appears, in this study, to be independent of the expression of what are considered, in this study, to be pro-inflammatory genes.

Activation of the myeloperoxidase system, as observed in the current study, is not a simple consequence of activating mRNA generation of inflammatory genes.

It just strikes me that expressing a gene and using its product may be greatly influenced by factors this study doesn't address.

So there are at least three descriptions possible for the state of ROS generation in the adipocytes of these mice. There are no simple linear relationships. You need some sort of framework to understand what is going on.

Protons.

Which makes me happy.

Peter

Foie Gras (8) Vaughan's macrophages

Okay, back to the Vaughan paper

A High Linoleic Acid Diet does not Induce Inflammation in Mouse Liver or Adipose Tissue

and a look at their macrophages in cell culture. The pattern is consitent so I'll just run through MCP-1 gene mRNA expression. As in the last post I am going to work on the basis that these genes are, in the absence of trauma or infection, going to respond to mitochondrial/NOX ROS production. It's a graph of relative expression, the control represents the response to the ROS being produced by supplying glucose at around 30mmol/l. That will be in response to NOX-2 being activated through calmodulin kinase II as we would expect. Even without insulin, these ROS will activate the phosphorylation of AKT and translocate GLUT4s to the cell membrane. It's the black column:

Adding 50 μmol/l of stearic acid (white column) will generate ROS by reverse electron transport though complex I, via an increase FADH2:NADH ratio, physiologically to limit insulin facilitated glucose ingress. Even if there is no insulin present, it still generates the ROS, which will limit the facilitation provided by NOX-2 derived ROS. Saturated fatty acids do this at low delta psi levels, there is no multi-enzyme/complex ROS generation, they just generate neat and tidy, fully physiological RET. The cell responds appropriately with superoxide dismutase and catalase and a coincidentally extended lifespan. Okay, I can't resist it, here he is again:

Oleic acid is another whole series of posts, so I'll mostly gloss over it. Oleic acid is designed to facilitate insulin signalling as a physiological balance to palmitic acid. It generates some ROS but not enough to significantly resist insulin signalling. There are many concepts stemming from this and here is not the place to discuss them.

Linoleic acid (hatched column) utterly fails to generate enough RET to limit glucose ingress, so glucose continues to enter, while at the same time the LA provides calories in excess of what the cell needs. The problem is that LA cannot shut down glucose ingress because it fails, through its double bonds, to generate the RET facilitated ROS to limit that glucose ingress.

The cell is full of ATP and depleted of ADP so ATP synthase cannot turn. Delta psi rises and above 170mV large numbers of ROS are generated. These come from a combination of complex II, complex III, electron transporting flavoprotein dehydrogenase and mitochondrial glycerol-3-phosphate dehydrogenase. Oh, and I guess some from RET. All secondary to high delta psi.

All stemming from inadequate RET derived ROS from LA's surfeit of double bonds. A few prophylactic ROS, produced without an high delta psi, stops an awful lot of problems. I think the phrase is that a stitch in time saves nine...

I think here is a good place to point out that ROS generate a response in inflammatory gene mRNA expression, probably at any level of exposure. Low levels will result in normal physiology, high levels will result in frank inflammation,  but also will induce inflammation limiting gene expression, in which I would include multiple uncoupling protein genes. Failure to limit high levels of ROS generation clearly results in apoptosis or, given a catastrophic failure of the ETC secondary to 4-HNE and associates, necrosis.

This paper is quite unique in view of the low dose of fatty acids used. If they had chosen the standard route of 1000μmol/l FFAs in addition to glucose at 25mmol/l (utterly non physiological) then RET would have dominated, giving apoptosis/necrosis levels of ROS, meaning stearic acid would be bad (usually palmitic is used), oleic less so and LA would be almost harmless. There is an infinite supply of such papers to reinforce the stupidity of saturophobia.

Okay, the in vivo mice next.

Peter

Foie Gras (7) What do you mean by inflammation?

This paper contains a very superficial and bullet point overview of the inflammatory cascade. It omits swathes of anti inflammatory pathways and many of the boxes on the chart could be massively expanded. The paper is interested in using phytotoxins (mis-termed phytonutrients) as anti inflammatory agents, so all I take from the paper is their doodle

Animal Models of Inflammation for Screening of Anti-inflammatory Drugs: Implications for the Discovery and Development of Phytopharmaceuticals

A legible version of the diagram is in the paper if anyone would like to actually read it.

We can simplify the process slightly with the red arrow. In addition I've circled in blue the component that is being used as a marker of inflammation in the Vaughan paper:

We can now consider the basic process a little more carefully. This massive review (no, I've not read it all) brings to prominence the central role of ROS in inflammation

Reactive Oxygen Species in Inflammation and Tissue Injury

and has this nice illustration featuring mitochondria as sources of ROS. Superoxide generation is never accidental. It is a tightly controlled and highly specific process to achieve certain ends. Mitochondria and NOXs are not making mistakes or having accidents:

BTW MPO is the myeloperoxidase we've seen recently in Vaughan's paper. HOCl is bleach, it's an inflammatory tool. We use it to kill germs.

So this lets us redraw this doodle of inflammation with a small modification:

Right up at the top of the inflammation doodle is a small red circle labelled PLA2, phospholipase A2. Its job, in the event of tissue injury, signalled by ROS and derivatives, is to release arachidonic acid from lipid membranes which then allows the generation of a raft of inflammatory mediators using cyclooxygenase and lipoxygenase enzymes.

Corticosetroids are potent suppressors of PLA2. They also releases both glucose and FFAs from the liver and adipocytes in to the blood stream. Physiological cortisol does this to deal with the sort of situation where you and a bunch of your mates are about to go and drive an angry mammoth into a bog, to kill it and butcher it. But high FFAs and high glucose together generate metabolic ROS, which are pro inflammatory.

You want raw energy, not to be crippled by an acute inflammatory reaction. So cortisol also down regulates the inflammatory cascade which *should* have been triggered, via ROS from the ETC, while flooding mitochondria with large amounts of substrate in anticipation of incipient need.

So cortisol floods your metabolism with calories, simultaneously controlling inflammation, and your mitochondria convert those calories in to the ability to successfully kill a mammoth, ie obtain food for a month for you and your tribe. Those calories get used successfully. Nothing is overloaded, nothing is damaged. Glucose and FFAs get used up during the hard work involved, facilitated by AMP-kinase rather than insulin. There is no generation of bulk inflammation, and what minor ROS mediated lipid peroxidation does occur has the routine inflammatory response suppressed. 

Evolution sets the levels correctly. It may be blind but it recognises functionality when it sees it.

Now imagine you're an Homo modernus visiting your bank manager in the 1960s to get a mortgage with a borderline adequate income and a just passable deposit, for the house of your dreams. The manager is a local petty Hitler and, even if he intends to grant the mortgage, he's going to really make you suffer, mentally, for it. Because he can.

You're stressed pre interview. So cortisol floods your system. Glucose and FFAs are made plentiful to allow you to fight at your hardest during a necessary mammoth kill. But there are consequences to killing the bank manager and most people, understandably, decide not to do so. Much as they might wish to. Especially if he refuses to grant you the debt you can barely afford. For your safety. There is no energy usage, no AMPK activation, just a serious availability of calories.

If the levels are high enough they will flood your mitochondria and provide substrate well in excess of what is needed for current ATP demand. If ATP synthase refuses to turn because ATP is high and ADP is low, while NADH and FADH2 keep supplying electrons to the ETC at the CoQ couple, delta psi will rise above that safe figure in the region of 170mV and ROS will be produced in large amounts from multiple sites, giving both insulin resistance and tissue damage.

You can use pharmaceuticals to inhibit phospholipase A2 as much as you like to avoid any response to this damage, classically using prednisolone or dexamethasone, but there is still the flood of calories generating ROS at tissue damaging levels. You might feel okay because the whole inflammatory cascade is suppressed, but cells are still dying from high ROS levels. If these happen to be endothelial cells lining your coronary arteries you are in trouble. Some will be.

So I would posit that the increased risk of CVD under corticosteroids could be (in part, there are many other issues) an effect of acute ROS injury. Under corticosteroids the inflammatory response is suppressed but the excess calories are still generating excess ROS. The damage is still done.

Of course the same applies to metabolic syndrome.

I define metabolic syndrome as the inability to shut down FFA availability in the presence of insulin and elevated glucose. FFAs are high from basal lipolysis at the same time as glucose from the diet causes hyperglycaemia. If substrate supply is high enough ROS will be generated and damage will be done. This time there will be an actual inflammatory response, no corticosteroid involved, and people can make statements like "obese people are chronically inflamed". They are producing mitochondria mediated ROS to physiologically resist insulin. If they are forced to continue to accept calories despite resisting insulin, the ROS become simply damaging. Inflammation follows. 

If you do not link this "inflammation" back to ROS then you would, logically, treat it with dexamethasone. Which would be a booboo, as we say in the UK. You would suppress inflammation while supplying even more ETC input, so worsening ROS mediated damage. Those poor coronary artery endothelial cells. In the absence of slaughtering a mammoth of course.

It's also worth thinking about the role of 4-HNE. Just a skim of the abstract of this book chapter is worth a moment:

Oxidative Stress-Induced Lipid Peroxidation: Role in Inflammation

4-HNE is, in itself, a product of ROS damage. It is also a generator of ROS per se. Much of the inflammatory cascade is self amplifying (which is why we have systems to turn inflammation off as well as on). Taking exogenous 4-HNE has profound effects on ROS mediated signalling in the insulin cascade, at low dose by injection to enhance, at high dose (with other lipid peroxides) orally to inhibit. It matters in the inflammatory cascade as much as in the insulin cascade.

My interest, getting back to Vaughan's obese mice without hepatic inflammation, is rooted in the factors which generate 4-HNE in situ around the ETC and which factors stop this. I'm talking about ROS generation.

Back to those mice and their non-inflamed hepatocytes and adipocytes.

Peter