Insulin resistance (16) Yes. Vitamin E can cause weight loss

Part 3

It's time to set out a logical explanation for this graph:

taken from 

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

The first thing we have to do is to ask the correct question:

To me the correct question which has to be answered is why an high fat diet (35% of calories) with only 1% of calories from LA is obesogenic, at all. If you come from the point of view that the mechanism of obesity is down to LA alone, irrespective of macros, you need to be teasing this paper apart in great detail.

We know from

Differential effects of saturated versus unsaturated dietary fatty acids on weight gain and myocellular lipid profiles in mice

that a mouse eating an high fat diet based on D14521, if you choose your fat correctly, will not become obese at all provided LA is limited to 1.4% of energy intake. We can simplify Figure 1 from the paper:

to this:

This makes it abundantly clear that if you hold one variable constant, that is the percentage of energy supplied by linoleic acid, the nature of the rest of the fat in the diet is not insignificant.

Okay.

Here is the fat used in Graham's study, which is obesogenic with fat at 35% of calories:

and here is the non obesogenic diet with 45% of calories from fat in the D12451 study. I've combined the saturated fats together and expressed all as percent of total energy to allow easy comparison with the obesogenic diet from Graham's study:

They are identical in composition, the only difference is between 35% and 45% of energy from fat causing an absolute difference in overall calories from fat. A complete coincidence, but still very neat. Both studies look like they could have used the same jar of fat to make up the two diets. Except...

Those 27% of calories from saturated fat in Graham's study were largely from hydrogenated coconut oil. In total, hydrogenated coconut oil supplied 89.1g/kg of saturated fat to the diet.

We can ignore all fatty acids of C16 or greater as they are, as always, completely absorbed as chylomicrons through the thoracic duct and in to the systemic circulation, bypassing the liver completely.

For the shorter chain fatty acids I think we can ignore the myristic acid (~17% of the coconut oil) as it too is mostly carried in chylomicrons and treated much like palmitic acid. Only around 5% of it goes directly to the liver as FFAs.

The lauric acid which makes around 45% of coconut oil is partly packaged in to chylomicrons but something around 30% is delivered directly to the liver as FFAs.

Capric acid (C10) and caprylic (C8) (~15% combined in coconut oil) are completely transported directly to the liver as FFAs and do not enter the systemic circulation.

This medium chain triglyceride inclusion is the major difference between the 35% fat obesogenic diet used by Graham et al and the non obesogenic diet based on the stearate/palmitate/oleate mix in the cocoa butter D12451 study.

This matters.

To get some idea of what is happening we have to go somewhat in to reductio as absurdum and use pure MCT oil with a splash of soybean oil giving 50% of calories as fat in this study

A rich medium-chain triacylglycerol diet benefits adiposity but has adverse effects on the markers of hepatic lipogenesis and beta-oxidation

and look at this, Figure 1:

from which we can extract the relevant lines like this:

Here we can see that, on 4% of linoleic acid, MCT oil at 45% (no lard in this group) of energy intake is obesogenic when compared to 5% LA on a low fat background ie without the MCTs.

MCTs, in any significant amount, are obesogenic in their own right.

In this last study the obesity is made much worse by adding lard containing significant LA, that's what the faded-out lines on the unaltered graph show. I'll just add in the all-lard fed group as we need it when we look at pAKT levels:

So now we can look at the degree of insulin signalling present in hepatocytes at the time of euthanasia after a six hour fast. The full panel C from Figure two looks like this:

which we can simplify down to the interesting bits like this:

Clearly there is a marked reduction of insulin signalling in the lard fed mice. They are the most obese and are providing long chain fatty acids to the fasting liver, some LA but also palmitate and oleate. This is the normal physiological insulin resistance of fasting augmented by elevated basal lipolysis.

The lard-free highest MCT oil fed mice also demonstrate hepatic insulin resistance but far less than the lard fed mice. Which looks like a paradox.

The main problem with interpreting the pAKT signal is timing. The levels were measured in liver tissue after a six hour fast, which is quite a long time for a mouse. If you feed an MCT rich meal you would expect to flood hepatocytes with FFAs via the portal vein during the peak absorptive phase, probably around 1-2h post intake.

It is this flood of MCTs, which enter hepatocyte mitochondria with minimal restriction, which necessitates the resistance to insulin. Never forget that insulin resistance, at it's core, protects against the damaging levels of ROS generated by unrestrained elevation of delta psi. Insulin resistance automatically reduces insulin catabolism.

MCTs are not stored, so the excess lipid within the liver or exogenous lipid from the systemic circulation by six hours post intake will reflect the highly controlled oxidation of longer chain FFAs, not the un-restrained catabolism of MCTs at peak nutrient absorption. As the mice consuming MCTs are less obese than those on lard, it is reasonable for the level of insulin resistance to be lower because basal lipolysis is also lower.

In fact there are various influences on the high MCT fed hepatocytes pushing insulin sensitivity in conflicting directions, especially under fasting. But we can tease out what matters, and when, from a few extra studies. 

The Protons prediction from hepatic insulin resistance with associated reduced hepatic insulin extraction is the facilitation of elevated systemic plasma insulin, primarily at the time of peak delivery of MCTs to the liver via the portal vein circulation.

We can get an idea of whether this genuinely happens from this human intervention study here:

Thermic effect of medium-chain and long-chain triglycerides in man

which gives us this graph:

There is a small but significant rise in systemic insulin after MCT ingestion. It was triggered by 400kcal of MCT oil. You can enhance the effect in rats, who can't object to the GI distress caused, by giving them close to half a full day's calorie intake as a gavage. That's in this paper

Relation of ketosis to metabolic changes induced by acute medium-chain triglyceride feeding in rats

including this effect on systemic insulin

which we can tidy up like this:

It's also quite simple to ask how much insulin is extracted by the liver from the portal vein before being passed out through the hepatic vein and in to the general circulation. All you have to do is compare systemic insulin levels after an MCT bolus in a normal person with those generated by people with severe cirrhosis and multiple porto-systemic shunts, which by-pass whatever dysfunctional liver tissue they have remaining in-situ. That will be this study:

Stimulation of Insulin Secretion in Manby Medium Chain Triglycerides

After just 30ml (270kcal) of MCT oil we get this doubling in systemic insulin in cirrhotic patients during the time of peak MCT absorption, but the effect is gone by three hours post ingestion:

This effect is only present under MCT ingestion. Neither corn oil nor other LCFA containing fat sources do the same. My expectation is that, at peak MCT absorption times, flooding hepatocytes with MCTs will generate an high ROS signal, confined to those hepatocytes. This ROS generation can be dealt with by storing the acetyl-CoA as intra-hepatocyte LCFAs derived from acetyl-CoA (to be later exported as VLDLs), by off-loading acetyl-CoA indirectly as ketone bodies and, finally, by simply resisting insulin.

Which allows systemic hyperinsulinaemia.

The action of insulin is the inhibition of lipolysis.

Which is obesogenic.

The Surwit diet does this.

Here we have a fundamentally different form of obesity compared to linoleic acid induced obesity. In LA obesity the fundamental problem is at adipocyte level and here *inadequate* FADH2 driven ROS generation allows excess insulin signalling to distend those adipocytes.

In Surwit's hydrogenated coconut oil diet derived obesity, the fundamental problem is an *excess* of ROS from the MCTs in coconut oil, delivered in high levels to the liver only, allowing passage of insulin through the liver to give systemic hyperinsulinaemia which acts directly to cause simple obesity.

I suppose that the addition to this is that the lauric acid which reaches the systemic circulation might be a factor in the obesity, the shorter chain length gives poorer ROS generation compared to palmitate or stearate. 

MCT obesity is the only form of obesity which should be *reduced* by limiting ROS signalling.

Drop the ROS, restore hepatic insulin sensitivity, allow the liver to extract insulin, so shrink peripheral adipocytes via normalisation of peripheral insulin levels. *Hepatic* ROS scavenging does this:

and the converse is ROS scavenging does this:

Or, more relevantly

Or, with a minor change of one arrow's colour:

I'll leave it at that for the time being. There are, as far as I can find, no studies looking at the effect of vitamin E on Surwit-like diets. That's understandable. Why should vitamin E, considered to stabilise PUFA, have any effect on saturated fat induced obesity? So why bother looking at this?

But, given the ROS hypothesis of obesity, a plausible mechanism for the action of Surwit like diets is clear.

Peter

Insulin resistance (15) Vitamin E for weight loss? No

Part 2

Here is the next study.

α‐Tocopherol suppresses hepatic steatosis by increasing CPT‐1 expression in a mouse model of diet‐induced nonalcoholic fatty liver disease

From the methods:

"Experiment 1: Mice were divided into seven groups (n = 10 in each) and given the following diets for 8 weeks: standard diet (control group; 30% protein, 68% carbohydrate, and 12% fat including vitamin E acetate [500 IU/g]; Research Diet); HF diet (HF group; 20% protein, 20% carbohydrate, and 60% fat including vitamin E acetate [500 IU/g]; Research Diet) and HF diet with α‐tocopherol (α‐Toc) which is one of the natural vitamin E forms supplementation (20, 50, 100, 150, and 200 mg/kg)."

Okay, a ghastly typo.

We have no idea which of the Research Diets these mice were fed on. I am going to assume that the chow resembles PicoLab Rodent Diet 20/LabDiet 5053 and contains, as per my last post, ~100iu/kg of synthetic vitamin E acetate yielding, also as per last post, 44.5mg/kg of active vitamin E in the food.

The typo is to state that this chow contains 500iu/g. That's quite a lot of vitamin E. The correct amount is (almost certainly) 100iu/kg, not 500,000iu/kg.

I feel it is reasonable to assume the high fat diet was something similar to, or in fact was, D12942 which also probably contains around 100iu/kg vitamin E. Obviously the mice would eat less weight of D12942 than chow because they eat to caloric need. This will be met by a lower weight of D12942 so their intake of d-α-tocopherol would also be a little lower than if they ate the chow. More like 40mg/kg if you taken in to account the reduced weight of food eaten.

I am also going to assume that they added their supplementary vitamin E to this standard high fat diet so we're looking at intakes based on diets containing totals of 60mg/kg to 240mg/kg of d-α

-tocopherol per kilogram.

Anyhoo. For the time being I'm going to ignore the changes in everything other than total body weight.

There are no data presented for the effects of most of the supplement levels used, though these were recorded as per the methods. I think it's safe to assume that the effect on weight was consistent across all vitamin E intakes used, otherwise they would have mentioned it. This is what they actually presented:

There are, undoubtedly, effects from vitamin E supplementation on parameters other than total body weight. People may find the liver damage induced by high dose vitamin E fascinating. I do. But that's another story.

So I think we can say that, in a poorly described study, vitamin E supplementation has absolutely no effect on the body weight of mice over eight weeks of feeding an high fat diet. Over a wide range of dose rates.

To continue the catalogue of appalling vitamin E focused studies, it's now time to look at this one:

Effects of d-α-tocopherol supplements on lipid metabolism in a high-fat diet-fed animal model

How bad is it? It's this bad:

"After the adaptation period, the mice were randomly divided into three groups. Nine mice were placed in the control group [CON, regular diet (10% of calories derived from corn oil) and distilled water as a vehicle (0.1 ml, p.o.)]. Another set of nine mice were placed in the high-fat group [HF, high-fat diet (45% of calories derived from lard) with distilled water as a vehicle (0.1 ml, p.o.)], while the rest of the mice were placed in the high-fat diet with daily oral administration of 100 IU/kg B.W. of d-α-tocopherol group [HF-E, high-fat diet (45% of calories derived from lard)]."

So we know nothing about anything. We have no idea of the vitamin E levels of the control chow or of the high fat diet. We don't even know if the high fat diet was manufactured specifically or whether they just added lard to chow to make 45% of calories from the lard which diluted the chow's vitamin E. We don't know what the lard was composed of in terms of LA either. Or even if it was Japanese or from the USA. We *do* know it was high enough in LA to make the mice fat.

None of this matters too much because the vitamin E supplementation was given by oral gavage of 100iu/kg once daily. This was pure d-α-tocopherol so the arithmetic is easy. The 100iu gavage provided 67mg of active d-α-tocopherol, not the racaemic mix and not the acetate ester. The mice weighed 32g so each got ~2mg/d by the end of the study.

If we reverse engineer to translate this in to how much vitamin E would need to be added to food to deliver that same dose we can do this. We can say that a mouse eats ~2.8g/d of high fat diet. So there would need to have been 2mg in 2g of food or 1000mg/kg of food. Though if you used the synthetic acetate ester then around twice that. This is a massive dose of vitamin E and guess what effect it had on body weight? You're waaay ahead of me:

We might also take note of the caloric intake per day which comes out as exactly what anyone would expect for mice on an high fat diet, in contrast to the last post. The current study calorie intake is picked out in blue:

I hope you're not getting too bored with this. I suffered for weeks with these studies. Now it's your turn. 

I guess I'm not selling you vitamin E as a weight loss hack. That's good.

So how do we square these studies (and many others, it's been a rough three weeks of reading) with the results from

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

where there is a marked decrease in weight gain on an high fat diet with modest vitamin E supplementation?

I absolutely accept that these data are correct as reported.

These are the sorts of findings which test your hypothesis of obesity. It's what makes slogging through the typos and brain farts and shifting definitions of high vs low vs unspecified levels of vitamin E in diet trials worthwhile.

Ultimately we are looking for circumstances where reducing ROS with vitamin E allows weight loss. Some weight loss anyway.

I think there might be an explanation.

Peter

Insulin resistance (14) The beginnings of vitamin E for weight loss

Part 1

I've spent an inordinate amount of time on this, and related, studies over the last few weeks

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

There are serious problems with panel A of Figure 1:

Ordinary mice on chow typically eat in the region of 14kcal/d, ie 98kcal/wk. High growth rate mice can eat 18kcal/d, ie 140kcal/wk. So the chow column is plausible as energy intake per mouse per week. Some mice are reported to eat as little as 9kcal/d on chow. There's a range.

What is not plausible is that mice on high fat diets ate 20% less total calories than the chow fed mice while gaining 20-40% more bodyweight, all as adipose tissue. This doesn't make sense.

In general rodents on obesogenic high fat diets consume very slightly *more* calories per day over the weeks than chow fed mice. The weight of food needed to get those calories will obviously be reduced because there are extra calories per gram. So calorie intake usually ends up slightly increased but on a lower food ingestion weight.

Just for illustration here are the energy intakes from mice fed a 45% lard based diet discussed in the next post. They are typical. Outlined in blue are the numbers we need:

Call me a Cicotard if you like, but the panel A graph from Figure 1 suggests either massive, and I mean massive, uncoupling in the chow fed mice. Which I doubt. Or a catastrophically reduced daily energy expenditure in the high fat diet fed mice, *irrespective* of linoleic acid intake. Which I also doubt. Greatly.

It almost looks like there has been a mix up of weight of food consumed and the total calories consumed per week.

But that doesn't reverse engineer easily. If we say the 110kcal/week specified for the chow mice is actually 110 grams/week then that's 16g of chow each day. No mouse can eat this much chow per day. If we assume that it's for the whole of the group of four in the cage we get a food intake of 4g/d/mouse. Which is plausible.

But who knows? That's a lot of guesswork.

It's a bugger because we need to know how much food each mouse ate in order to determine how much actual vitamin E each consumed.

We are given the vitamin E per kg diet, and the number looks plausible. Looking at Table S2 we have the data for the chow (PicoLab Rodent Diet 20/LabDiet 5053) and can calculate the value for this well recognised and standardised diet.

Diet 5053 contains 99iu/kg of vitamin E acetate on the product sheet. This gives, assuming synthetic vitamin E, 89.1mg/kg vit E. From this calculator we have:

Also assuming the synthetic vit E is a 50:50 mix of the d and l isomers, we have, of active form, half that, giving 45.55mg/kg, as here in Table S2

On this basis I think it is very likely that the absolute amount of vitamin E in all of the diets in this paper are correct in Table S2.

If we deconstruct the (excellent) Table S2 to remove the arithmetic lines we can summarise the amounts of vitamin E in different diets like this

If we take Lab Diet 5053 to represent some semblance of a normal to generous level of vitamin E for normal growth and reproduction of rodents eating one of the most widely available chows in the world we have the low LA diets with less absolute vitamin E than 5053 and the high LA diet containing rather more vitamin E than the 5053 chow diet.

Working through the rest of the (largely appalling) vitamin E-HFD literature most groups would consider vitamin E at 50mg/kg of diet to be low dose and 200mg/kg to be high dose. Those would be dose rates of d-α-tocopherol. So we are looking at low versus moderate in this study.

Now we are in a position to look at the weight loss effects of various levels of vitamin E in various diets.

Peter

Insulin resistance (13) NAC and UCP3

I think we just need to tidy up the effect of NAC on bodyweight before we go on to look at vitamin E in a related role.

The effect of NAC on total body weight of chow fed mice is zero. We have this paper from 2022

Complexity of NAC Action as an Antidiabetic Agent: Opposing Effects of Oxidative and Reductive Stress on Insulin Secretion and Insulin Signaling

which includes this highly convincing graph:

This is a reiteration of the findings in this (not cited!) 2016 paper

N-acetylcysteine Protects Mice from High Fat Diet-induced Metabolic Disorders

which is where I started this thread because I was interested in whether the drop in ROS generation mediated by uncoupling could be recapitulated using an antioxidant to reduce ROS rather than UCPs themselves. The answer is clearly both yes and no.

If we look at the weight graph here:

and remove all data points except the mice on chow and the mice on chow plus NAC we get this:

I think it is reasonable to suggest thet NAC does nothing to the body weight of healthy mice on chow. Except this is not the case for body composition. Here we have, from the same figure, lean and adipose masses of all groups

As always, we have to look slightly deeper than total body weight. In this latter paper we do have an assessment of both lean mass and total adipose mass, shown here for the chow groups only: 

where, on a group size of five, we do have a statistically significant reduction in total adipose mass. It's probably real. If we reverse engineer the figure we get, in crude approximation, 15% body fat in the chow only mice:

and for the chow plus NAC group we have 7% body fat:

Ignoring the p < 0.05, I think most people could identify which group of people at any gym had 15% body fat compared to a similar weight group but with 7% body fat.

You might, or might not, be surprised that the lower body fat group were scarfing more (ns) calories per day than the chunkier group. As a dietician looking at humans you would just assume the fatter folks were lying about food intake, as dieticians do (sic), but in mice you can weigh the food.

Of course the answer is in mild uncoupling. Here are the data for UCP1 in the chow-only fed mice, a trivial increase, also ns. This is not about bulk thermogenesis:

However this is not the case for UCP3. Here the p value is small and the effect large:

This suggests that the increase in gene expression for UCP3 is doing something different to the routine function of UCP1. Undoubtedly UCP3 is not UCP1. A superficial glance at even the abstract of

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate

shows that activation of UCP3 has far more to do with metabolism control than heat generation. A flavour comes from another superficial glance at

Muscle-UCP3 in the regulation of energy metabolism

This latter paper even suggests that UCP3 in muscle (in this case) might be related to control of ROS production. Well, whodathunkit? The authors seem unable to comprehend that all of the numerous "effects" of UCP3 activation can be most simply attributed to a decrease in insulin signalling.

So why does NAC increase UCP3 gene expression in the BAT of mice on NAC, as it does? Probably because it increases UCP3 gene expression everywhere in the mouse, although this wasn't checked.

Let's go back to what is happening on the ROS basis in mice eating chow with NAC.

Cells should uptake energy substrate until "full" and then refuse excess. NAC removes the ROS that signal this "fullness". Substrate continues to enter the cell, a process exactly analogous to the failure to resistance to insulin's signal resulting from the inadequate ROS generation due to LA's low FADH2 supply.

Results should be increased lipid storage and a pathological increased supply of reducing equivalents in to the ETC.

This provides the potential for very high delta psi and activating UCP3 appears to be an adaptation to avoid this. This is a very basic hypercaloric situation. Uncoupling is protective.

Under fasting conditions, ie under low (but not zero) insulin levels, NAC removes the small component of insulin signalling still present in adipocytes so allowing more FFA release than is appropriate. Again, excess FFA delivery supplies excess reducing equivalents to the ETC with potentially damaging high delta psi generation. Again, uncoupling is protective.

So NAC over distends adipocytes in the fed state due to suppressing physiological insulin resistance, comparable to linoleic acid's effect. Under fasting conditions NAC allows excess lipolysis, giving weight loss from shrinking those distended adipocytes. In many ways this is analogous to the blunting of insulin signalling by metformin.

The end result will depend on the dose rate of NAC and the nature of the chow (or high "fat" diet) being fed.

The fundamental point is that scavenging ROS and avoiding weight gain does NOT imply that weight gain is caused by excess ROS. Weight gain is caused by *inadequate* ROS post prandially which can be offset by increased lipolysis during fasting. Same drug, same action, different conditions.

A nice paradox unless viewed from the ROS perspective.

Peter

Insulin resistance (12) Lipopolysaccharide

I've mentioned this paper by Nowotni from Shulman's lab on several occasions

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

It is a great resource on many levels, provided you completely ignore any of the paper's conclusions.

The most entertaining quote is this one from the control intervention period which involved the iv injection of a very small dose of lipopolysaccharide to induce insulin resistance. As the authors say:

"Only endotoxin administration led to mild flulike symptoms with a maximum after 3 h, while other interventions had no side effects."

What they didn't report was that the volunteers, after the endotoxin administration, came up with any spontaneous comment along the lines of:

"I could murder a steak now"

Almost certainly because it didn't happen.

I conclude that either a few hours of insulin resistance doesn't make you hungry or Nowotni is keeping shtum about the hunger generating effects of insulin resistance.

I struggle to identify inflammatory insulin resistance as a trigger for obesity. Even if it is chronically present. The last serious viral infection I had was probably in the late 1980s. If I recall correctly I lost 4kg in a weekend and only part of that was fluid loss. I wasn't hungry. I also went through a period of chronic inflammatory problems while I lived through mycoplasmal arthritis in the months soon after qualifying. I got through an awful lot of paracetamol before the medics got the diagnosis and subsequently I got through an awful lot of oxytetracycline at high dose to get it sorted out. No weight gain from inflammation though.

The endotoxin in the study caused the same degree of insulin resistance as ~900kcal of oral or iv soybean oil. But it provided no caloric input, so this insulin resistance is apparently not an obvious satiety signal.

And it's not pathology either.

Let's actually do some thinking about the advantages of insulin resistance during acute infection.

It's probably most illustrative to initially consider the insulin resistance of fasting. While this is often the situation during infection it is also a normal feature of simply not eating overnight. It is essential for survival, especially if fasting has to be extended.

The primary advantage of this aspect of insulin resistance is to spare glucose for the brain. While our brain can run perfectly well on ketones, these take time to develop and the default setting in modern agricultural society is to run your brain on glucose and leave daily ketosis to oddballs like myself and a few others.

So the ROS generation from FFA oxidation under fasting spares glucose for the brain, useful because the brain preferentially utilises glucose in modern times. Muscles and related tissues are very happy to run on fat and so resist insulin.

There is another part of physiology which is highly dependent on adequate calories from glucose for its optimal function and that is the immune system.

The case is laid out here

Metabolic syndrome and robustness tradeoffs

and here

Insulin resistance and inflammation in an evolutionary perspective: the contribution of cytokine genotype/phenotype to thriftiness

Obviously both sets of authors have zero understanding of the causes of metabolic syndrome but have successfully linked the insulin resistance of starvation to maintaining brain function and the insulin resistance of infection to maintaining optimal immune function.

The second paper also throws in the insulin resistance of exercise, which I hadn't thought of, but makes sense.

Please, please, please bear in mind that NONE of these folks understand the cause of metabolic syndrome from the Protons perspective and so have no idea of why the previously adaptive trait of insulin resistance should now appear to be maladaptive. Modern life is suggested to allow "pathological" insulin resistance through a deficiency of mammoth-killing exercise and/or via a deficiency of starvation on bad hunting days. So they say.

Stop sniggering at the back!

So what *should* happen in infection is that adipocytes and hepatocytes release both FFAs and glucose to optimise immune function. Flooding the immune system with caloric substrate is pro-survival in infection or trauma.

At the same time all non immune essential cells should limit their caloric uptake, especially that of glucose, to their needs and no more. Predominantly from FFAs.

During the early evolution of organisms which have a recognisable immune system and separate organ systems for caloric storage all that would be needed is for evidence of tissue damage to trigger caloric substrate release to facilitate effective pathogen attack and/or tissue repair.

The simplest signal for this would be fragments of damaged tissue, ie 4-HNE, or pieces of damaged pathogen, ie LPS (fragments of the cell wall from gram negative bacteria). Or similar substances. A basic system would have the energy storage tissues "seeing" evidence of  distant damage with a resultant generation of ROS in response. Those organisms which failed to do this failed to survive.

Because metabolic substrate is being flooded in to the circulation, use of the glucose component by tissues which can preferentially use fatty acids with their ROS generation should be limited. The ROS from FFA oxidation will automatically control caloric ingress to optimal for each insulin sensitive cell. The excess substrate, especially glucose to drive NADPH derived ROS generation through NOX enzymes, is then available to attack the pathogen and improve survival.

I suppose the next step to improve would be for immune cells involved in active duty at the site of damage to send a more reliable surrogate messenger and clearly tell the adipocytes/hepatocytes to respond decisively and release substrate without waiting to see how the untidy message of fragments of damage debris might work. So cytokines were developed as long distance messengers. I would hazard a guess that they might be derived from stabilised sections of proteins used in defence of integrity. Of course the same signalling molecules could be used to tell non involved insulin sensitive cells that a flood of emergency calories is on its way and better get resisting insulin now, in anticipation.

The next move is to have a sub population of immune cells actually sitting among adipocytes and hepatocytes, listening for cytokines, ready to act as local amplifiers of incoming signals. Their function would be to signal, via local cytokines and ROS, to adipocytes/hepatocytes to resist insulin, spill caloric substrate and gear up for battle.

There is nothing wrong with doing any of this. We can, with this perspective, regard the insulin resistance induced by inflammatory cytokines as the warning that a caloric flood is on its way, that that caloric flood is destined for the immune system, and it is now essential to enter excess calorie mode, ie resist insulin, in anticipation. I think of these higher level signals as a form of looking in to the future.

Although it might not be obvious initially, the sudden release of metabolic substrate in infection provides a surplus of energy availability at the cellular level despite anorexia. Exactly as fasting does. Running your metabolism on FFAs fundamentally generates ROS without needing insulin facilitated glucose uptake. Fasting, once metabolism is running of FFAs and ketones, is an energy replete situation at both the individual cell level and the central nervous system level. For modern glucose oxidisers the first three days might be a bit rough.

What *is* wrong is for adipocytes (and, presumably, hepatocytes) to accumulate so much lipid that they die by catastrophic failure secondary to over distension. At which point all hell breaks lose within the local tissue to form highly inflammatory crown-like structures and to signal over long distances via cytokines that a disaster is happening. The pathologically augmented inflammatory signalling in adipose tissues is not causative of obesity. It is a response to individual adipocyte obesity. This is most obvious in the most insulin sensitive adipocytes, ie visceral/omental adipocytes. Which become "inflammed" first because they distend soonest, ie because they are the *most* insulin sensitive.

The modern maladaptive inflammatory problems of obesity all stem from the failure to limit insulin mediated distention of adipocytes (and probably hepatocytes).

It's not gluttony and sloth.

It's not failure to find food for a week or failure to attack a mammoth.

Linoleic acid is, of course, the primary problem which breaks the system.

Peter

PS There is a follow on in this paper

Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers

which suggests that in modern organisms that the release of FFAs and glucose under endotoxin exposure is now mediated by active lipolysis via sympathetic drive and lipolytic hormones. Not really surprising. Eukaryote evolution has had a couple of billion years to produce functional heifers.

Insulin resistance (11) NAC might cure your diabetes

The next scenario is to look at the effect of NAC on metabolic syndrome with or without marked hyperinsulinaemia. We're looking at this section of the curve roughed out in this previous post, mostly for completeness before getting on to other aspects.

So this is our region of interest:

which we can re label as showing combined base load FFA oxidation ROS plus insulin mediated ROS generation via elevated delta psi, which limits further caloric ingress but without damage by virtue of the ROS signal being limited to an hypothetical equivalent to 0.3mM H2O2:

Now let's add in the situation where, despite ROS being high and insulin signalling being blunted, calories continue to enter the cell. Where might this caloric substrate be coming from?

Life is simplest if we think about an hyperinsulinaemic normoglycaemic clamp. If we just infuse insulin to give a high post prandial level, classically ~1000pmol/l and then infuse glucose to maintain 5mmol/l we can supply almost all of an individual cell's needs using glucose once steady state has been achieved.

Ideally the 1000pmol/l of insulin will suppress plasma FFAs to around 100μmol/l. These 100μmol/l will supply a base load of ROS from which glucose will top up energy supply until the ROS from FFA oxidation plus the ROS from glucose oxidation reaches the equivalent of H2O2 at 0.3mM on the graph, which resist further ingress of glucose via fully physiological insulin resistance.

In metabolic syndrome the same thing happens but the fixed 1000pmol/l of insulin cannot suppress the FFA levels to the preferred 100μmol/l because distended adipocytes are releasing FFAs independent of insulin while, under clamp conditions, insulin is fixed at ~1000pmol/l. Now the base load ROS generation from FFA oxidation will be higher. The amount of glucose required under fixed insulin levels will be lower and our subject will be labelled as insulin resistant, ie "unwell" or pathologically insulin resistant.

But individual cells are merely responding to having a higher base load oxidation of FFAs. There is nothing wrong with them. Drop the FFAs with acipimox and, given a little time, insulin sensitivity would return to normal. Except the subject's adipocytes would get even bigger and release extra "pathological" FFAs as soon as the drug wears off.

The only pathology is with adipocyte diameter. This is caused by inadequate FADH2 supplied by linoleic acid in the Protons hypothesis.

The situation in DMT2 is even worse in vivo, rather than under exogenous infused insulin, because there is a failure to produce adequate insulin. This leads a failure of suppression of FFA release from adipocytes, so an higher base load of ROS is invariably generated. There is a concurrent inability to suppress glucose release from hepatocytes, so the small amount of glucose needed to "top up" ROS generation to that 0.3mM H2O2 equivalent is minimal. The higher the plasma glucose level rises the less insulin is needed to allow a cell's energetic needs to be met.

If a cell is having all of its metabolic needs met by a combination of FFAs and "insulin facilitation free" glucose ingress then the 0.3mM ROS signal to resist insulin could easily be exceeded. Insulin resistance will become profound.

Let's imagine the level gets up to the equivalent of 1.0mM H2O2 in cell culture. This will fully resist insulin's action but fail to limit the caloric ingress, because this is happening independent of insulin facilitation.

ROS generation will be marked and profoundly damaging.

At this stage the nature of the fatty acids generating elevated base load ROS is immaterial. In fact saturated fats, with their optimal reverse electron transfer abilities, will be *worse* than PUFA in augmenting ROS generation. The downstream problems of ROS damage (4-HNE and its related compounds) will obviously be worse in an high PUFA environment which will be present because the primary pathology (adipocyte distention) is only possible in an high PUFA environment (pax fully hydrogenated coconut oil or butter induced obesity, which are real and different).

In to this soup of problems let's drop enough NAC to drop ROS levels by ~1.0mM of H2O2 equivalents, indiscriminately:

There may still be 1.0mM of H2O2 equivalent of ROS being produced, but they disappear in to the NAC sump.

Suddenly we are working to the left of the peak of insulin's action and, lo and behold, we have restored insulin sensitivity and everything looks much better. Bear in mind that it's not!

So. Can NAC "cure" your diabetes?

Of course. And not.

Can it "cause" diabetes, if you are currently metabolically healthy?

Of course. And not.

It's all quite straight forward, given the Protons perspective.

Peter

Insulin resistance (10) NAC might make you diabetic

Here is the next paper

Complexity of NAC Action as an Antidiabetic Agent: Opposing Effects of Oxidative and Reductive Stress on Insulin Secretion and Insulin Signaling

Amongst the many, many things that this group did there is this section from Fig 9. They injected a set of mice with a dose of insulin typical for an insulin tolerance test. Fifteen minutes later they euthanised them and extracted muscle tissue for assessment of insulin signalling as represented by the phosphorylation of AKT. Half the mice had been on NAC in their drinking water, half hadn't. All were eating chow.

Here are the Western Blots

and here is the darkness of the blots converted to numerical form:

Both groups of mice received exactly the same dose of insulin, 0.75iu/kg, high enough to seriously kick the insulin signalling cascade but still with a fairly low incidence of serious hypoglycaemia. The intention is to get a near maximal insulin response *without* lethal hypoglycaemia.

If we go to the doodles from the last post this is what that dose is trying to achieve:

The 0.75iu/kg will produce *exactly* the same ROS signal in all of the mice, on or off of NAC. But in those mice on NAC a large proportion of the signalling ROS are simply mopped up by the NAC. So instead of getting the phosphorylation of AKT we expect we get much less:

The dose of insulin is identical,  the ROS signal is identical but the NAC non specifically reduces those ROS which carry the onward signal.

The insulin effect is mediated through ROS, NAC limits it.

At low levels on insulin the small ROS signal would be completely wiped out.

At higher levels of exposure insulin will actually signal and should increase its downstream target activation, but not as effectively as it should. We're looking at the three points circled in red here if Fig 9B :

which are responsible for a significant increase in the AUC calculated for glucose.

The initial rise in glucose is unaffected by NAC, which might be surprising if you don't view it from the Protons perspective.

We are sightly stuck because we don't know the insulin exposure at times 15 and 30 minutes, which may not be comparable between the two groups.

We can guesstimate the insulin levels in the control group using the data from this paper for after a six hour fast. It looks like this, added on to the above. No units included for the insulin, it's the pattern I'm interested in:

Now, an OGTT is designed to elicit a maximal insulin response within the physiological range. A maximal physiological response will allow peak utilisation of glucose and, at some time point when combined with elevated plasma glucose, will produce insulin-induced insulin resistance as soon as the the mitochondrial delta psi rises high enough.

The signal to resist is, obviously, ROS.

A significant proportion of which disappears in to the soup of NAC so insulin-induced insulin resistance is blunted, so glucose continues to be disposed of at peak insulin. We're thinking about events somewhere within the time points circled in red:

If you choose your dose of NAC very carefully you can generate the above curves. This is NOT normality, this is balancing pharmacology against differing aspects of physiology.

How well this happens depends on your dose of NAC and probably on the metabolic state of your mice. Not all control groups are as free of metabolic disease as you might like.

Hence the results in JustPeachy's nice paper:

The Antioxidant N-Acetylcysteine Does Not Improve Glucose Tolerance or β-Cell Function in Type 2 Diabetes

I can't find a paper where NAC actually precipitated DMT2 but I had a vague recall that vitamin B3, a favourite of the orthomolecular practitioners, could occasionally precipitate glucose intolerance.

It turns out you need a massive study to pick up the small effect demonstrated in the second hour of the mouse OGTT above using the effective antioxidant B3 rather than NAC but I did find this bias-confirming meta-analysis (you know, one, two, skip a few, 99, 100):

Niacin therapy and the risk of new-onset diabetes: a meta-analysis of randomised controlled trials

Personally I avoid antioxidants. A few decent ROS is my preferred approach.

Peter