Wednesday, November 27, 2013

Protons (30) Uncoupling and metabolic rate in insulin resistance

I wanted to look at insulin resistance, uncoupling and metabolic rate. If we just review the effect of an intravenous bolus of palmitic acid on an anaesthetised rat we can see that the bolus produces a period of increased oxygen consumption, ie an increased metabolic rate, in a then-uncoupled healthy rat. From Curi again:

This next graphic is looking at glucose oxidation, rather than oxygen consumption, from the same paper.

The left hand graph shows what happens under basal metabolism, ie glucose is being taken up by isolated muscle cells without help from insulin facilitated GLUT4 translocation. Fatty acids uncouple, ATP levels fall, there is an increase in glucose oxidation to compensate, ATP levels are corrected. Presumably some of the fatty acids get metabolised too.

On the right is the effect under supra maximal insulin. We have no idea of dose response curve to insulin from this study, it describes either using none or using 10mU/ml. That's mU, not microU! At a concentration of 10mU/ml insulin will overcome any suggestion of insulin resistance, short of a knockout model. Insulin supports glucose oxidation, uncoupling increases this. The set up is not designed to look at insulin resistance as might be related to that uncoupling. But the effects on overall metabolic rate, at 5.6mmol of glucose, are quite clear cut.

What if that elevated FFA level was maintained long term?

As adipocytes become progressively more resistant to the the anti-lipolytic effect of insulin (why is another whole ball game), plasma free fatty acids rise even under levels of insulin which should be suppressing them. Unless these free fatty acids are converted to CoA derivatives they are will uncouple respiration. This should reduce delta psi and increase metabolic rate.

A reduced delta psi will not support reverse electron flow through complex I. The essential insulin induced pulse of superoxide, converted to H2O2, will not occur. There will be fasting insulin resistance. This paper spelled it out. In brief:

The insulin signalling cascade is tonically restrained by a phosphatase which deactivates the insulin/receptor complex (which activates itself by auto-phosphorylation) as soon as the process tries to get started. For insulin to signal you need to have an elevated delta psi which allows a nanomolar pulse of H2O2 to cripple this phosphatase and so allow the insulin/receptor complex to get signalling.

With reduced delta psi this isn't going to happen. We have enhanced insulin resistance of starvation until the time when food arrives.

You can increase delta psi, of course, to get insulin working. Increasing delta psi requires increased electrons in to the respiratory chain through complex I. In the blunted insulin signalling situation of low delta psi we can't use GLUT4 transporters but we can, given high enough plasma glucose levels, get sufficient glucose in to the cell to generate enough of a delta psi to allow the pulse of H2O2 and its downstream effects to occur. So overcome the inability of insulin to signal in the presence of FFAs.

The cost is that an elevated blood glucose level is needed. This is what we look at with the ratio of fasting glucose to fasting insulin, the HOMA score. An elevated HOMA score is a marker of UNCOUPLING at the delta psi level in mitochondria. It should be associated with an increased metabolic rate in proportion to the degree of fatty acid induced uncoupling.

Does this happen in real life? These data are from the Pima but the pattern is generic in insulin resistant states:

Note first that the changes in FFA concentrations are statistically non significant, but that the trend is nicely upwards with insulin levels

The authors comment:

"Alternatively, FFA may contribute to increased RMR via stimulation of mitochondrial uncoupling proteins (UCPs) (53,54). As early as 1976, Himms-Hagen (53) suggested that FFAs stimulate UCP-1. More recently, elevated FFA concentrations were reported to stimulate UCP-3 expression in rats (54)"

Bear in mind that as people progress from NGT through IGT to diabetes there are not only progressive changes in blood lipid and glucose levels but also progressive damage to mitochondria per se, which makes comparing a healthy rat to a metabolically challenged human being slightly dubious.

But the changes make sense.

If we look at the post prandial situation we have, after a carbohydrate containing meal, the combination of chronically elevated FFAs with acutely elevated glucose. There will be a high delta psi as soon as blood glucose rises high enough (supra physiological) to allow non-GLUT4 uptake to elevate delta psi high enough for insulin signalling. At this time point the mitochondria allow insulin to function. We can now translocate GLUT4s to the cell surface and start to lower postprandial hyperglycaemia by pouring metabolites through glycolysis or in to glycogen stores.

At the same time, among insulin's many diverse functions, the activation of free fatty acids to their CoA form increases, with a view to anabolic or storage functions. I would presume a different CoA ligase directs the activated fatty acids to oxidation. There is quite a group of LCFA CoA ligases.

At this point we lose the freedom of free fatty acids, they are ligated to CoA and suddenly become effective inhibitors of uncoupling rather than facilitators. We are now set up for the post prandial state. As soon as we have access to glycolysis combined with beta oxidation we have the possibility to generate marked reverse electron flow through complex I and re-inhibit the action of insulin using much greater generation of H2O2 than is needed for insulin's initial activation.

Metabolically, the mitochondria do this at this time to stop caloric overload in any individual cell, by diverting excess calories to storage in adipocytes. From the NADH:FADH2 ratio, long chain saturated fats do this best. Monounsaturates appear to be designed to allow a normal combination of glucose and fatty acid oxidation and PUFA fail to generate adequate insulin resistance to protect an individual cell (including adipocytes) from an overload of metabolic substrate. In the immediate post prandial state saturated fats can best protect cells from an absorptive excess of metabolites. These fatty acids are already present in supraphysiological levels, whatever has been eaten.

The elevated insulin needed to maintain post prandial normoglycaemia will, if the adipocytes are able to respond at all, divert fat from the circulation and into those adipocytes. This is the simple ability of insulin, and cellular resistance to insulin, to limit metabolic injury when calories are present in excess of immediate needs.


Under inappropriately elevated FFAs there is an initial failure of insulin's action due to depressed delta psi during fasting and a subsequent post-meal failure of insulin's action due to inappropriately elevated fatty acid metabolism through electron transport flavoprotein dehydrogenase's FADH2 when combined with high delta psi.

To me, this looks very much like impaired metabolic flexibility, reduced to the level of delta psi and superoxide. I like it.



Anonymous said...

Brilliant work, Peter. This ties together the entire Protons series for me.

The more metabolic issues I investigate, the more I bump into failures of the basic mitochondrial machinery of cellular energy production, e.g. my 2013 AHS presentation on metabolic flexibility (sadly unavailable as yet on video). And despite the protestations of one obesity researcher, the scientific literature in this field appears to be accumulating at a rapidly accelerating rate.

If you (or your readers) are interested in the consequences of your work at the functional level, you might appreciate my 2013 bibliography...particularly van de Weijer 2013, Chomentowski 2011, Jenkins 2013, and Heilbronn 2007.

Meanwhile, the aside in the final paragraphs ("...PUFA fail to generate adequate insulin resistance to protect an individual cell (including adipocytes) from an overload of metabolic substrate") explains a great deal about why excess PUFA "improves" test numbers yet is injurious in the long term.


Kindke said...

Hopefully im reading this right, so in theory the higher we can get plasma FFA, the higher the metabolic rate? PROVIDED plasma glucose levels are kept in check.

Slightly off topic but I just want to mention a few months ago I tried the uncoupler DNP and noticed it failed to produce any weight loss while I was eating high carb, despite my body temperate going up dramatically and insane sweating. Once I went ketogenic the weight started to fall off very quickly. Also the increase inbody temperature was more bearable and sweating declined while ketogenic.

I stopped the DNP after 4 weeks because it does have some serious side effects. I think its commonly believed DNP increases oxidative stress but actually its been reported to decrease it.

karl said...

This has me thinking about a MD I know that is fiddling with cold exposure to treat CFS/FM. Daily exposure of 2min to -20C in nothing but underwear and slippers ( takes access to a meat locker ) does not sound like a lot of fun, but I'm thinking it would stimulate production of brown-fat. ( Seems a very cold shower could have the same effect).

Apparently, this treatment seems to work, which makes me wonder if the cause of CFS/FM may have to do with insulin and FFA? It is going to hard to market this uncomfortable treatment - (There was a similar treatment once used to treat anxiety (cold-wet-sheet-packs) back in the days before benzodiazepine.)

All of which makes me wonder about the inflammatory effects of FFA - and inflammation may well be behind CFS/FM and many affective disorders.

Wow - taking DNP is pretty wild in my mind. The risk of cataracts is non trivial.. The idea that producing a fever

Unknown said...

Amazingly done!

Bill said...

iirc, ACS1 targets fatty acids to oxidation, according to Rosalind Coleman. And ACS5 to triacylglycerol synthesis. She also believes this is a property intrinsic to the enzymes as opposed to their cytosolic locations... but had no interest in a mitochondrially-targeted ACS5 (?).

Anonymous said...

Fantastic post, Peter, on so many levels.

Ken said...

Peter, I do not know if you have read anything of Brownlee's theories and work on intracellular excess ROS damage in diabetes. But this last post seems to dovetail well with it. Here are some links: (2004 Banting Lecture) (2010 paper with more detail on macrovascular complications)

In the Banting Lecture I suggest that the half-page section entitled "How does the unifying mechanism explain diabetic macrovascular disease?" is of the most interest with respect to the post. The later paper goes into more detail on the same subject.

Hopefully Brownlee's cited work might be interesting enough to merit a new blog post.

Peter said...

Thanks all,

J, I very much appreciate that you think this all makes sense, as you have been thinking about this for some time. I have a few things on insulin induced thermogenesis which seem necessary and then I might take a break from Protons. Funny how it all started with the simple question of what is the difference between fatty acid metabolism and glucose metabolism, when viewed by the ETC...

Bill, I'm not sure it matters too much where the acyl-CoAs are headed, acutely, so long as they are bound they won't uncouple and delat psi will increase. Of course packing your muscles with trigs will have significant long term effects on the ability to control FFA flux.

Ken, Brownlee is interesting and I see him as correct on the pathways of complications. But if you inhibit the generation of, or effect the removal of, mitochondrial derived free radicals you may well limit hyperglycaemic complications, but the cost is of failing to develop insulin resistance and electrons will pour down the ETC as glycolysis will not back up. You are now in to making glucose behave like PUFA and will supply unstoppable pyruvate the the ETC. PUFA in bulk do this and an excess of energy supply might well provide the correct signal for a cell to divide. Not so good if you really wish to avoid neoplasia. There really is no substitute for chronic normoglycaemia.

karl, might ketogenic uncoupling do a similar job without the freezer?

Kindke, yes, that is how it looks to me. Rising glucose should, if all is well, automatically shut down FFA supply. The problems come when this fails of course.


John said...

"...PUFA in bulk do this and an excess of energy supply might well provide the correct signal for a cell to divide. Not so good if you really wish to avoid neoplasia."

So it seems this would happen with lauric acid as well? I guess that is the only medium/short chain fat capable being consumed in bulk?

Kindke said...

So Peter do you think youll ever get around to that post on adipocyte insulin resistance and obesity as an addition to the proton series?

I was thinking about this link last night, Its common knowledge that oxidative stress is increased in obesity, and in particular in adipose tissue.In this study They found massively increased h2o2 in adipose tissue of obese rodents but not in muscle.

If increased h2o2 is what allows insulin to start working then this seems to suggest that a forced increase in insulin sensitivity in adipose tissue in obesity.

I also wonder how it ties together with this paper where the authors claim Hyperinsulinemia increases the amount of GLUT4 selectively in adipose tissue compared to muscle. The increased GLUT4 facilitating increased glucose metabolism in adipocytes and therefore increased h2o2 and insulin sensitivity in adipocytes.

BTW is it only insulin-mediated h2o2 that allows the insulin receptor to work or is it just general inreases in h2o2. The former wouldnt make sense.

Peter said...

john, no. Lauric acid, along with other MCTs, never enters chylomicrons. They are transfered directly to the portal vein as free fatty acids for immediate metabolism by the liver in to harmless ketones. The liver is designed to meet metabolite throughput like this at reasonable levels, ditto fructose. But the F:N ratio, combined with the superoxide/delta psi view of insulin function, tells us exactly why MCTs are dealt with differently to LCFAs. The Protons ideas have quite good explanatory power...

Kindke, those are useful links, thanks. I've not even started on adipocytes really. But at the start of the protons thread I had no idea that it would all pan out as it has. I keep coming back to the idea that life is logical and self consistent. It hasn't let me down yet, nor gotten me chowing down plain boiled spuds. Thought I have to come back to those spuds re insulin induced thermogenesis quite soon.

Obviously adipocytes should be the most insulin sensitive cell type under caloric overload. They are there as a buffer. If they behave as a hoover, rather than a buffer, we are in to hunger secondary to fat loss in to adipocytes, ie obesity.


Brad Reid said...


Off topic, a bit, but so very well written and it has aspects of it that relate to topics and discussions often noted here with regard to gene expression (versus gene mutation) and diet. Hope everyone reads this: Brad Reid

Peter said...

Brad, Ta, I enjoyed that


Kindke said...


Just speculation, but my guess is that drinking a 1/2 pint of cream in isolation ( no protein/carbs with it ), should cause additional adipocyte insulin resistance?

What im trying to get at is, the more fat ( saturated ) you eat the more adipocyte insulin resistance you create, provided glucose and protein intake are again kept in check.

I have to say I really like the idea that losing weight ( fat ) is all about making your adipocytes insulin resistant. Keeps things simple.

Unknown said...

Hi Peter

Thanks for all your great bloggings over time.

Off-topic, but hopefully you may find this interesting:

"New research shows obesity is an inflammatory disease

Diet-induced obesity, adipose inflammation, and metabolic dysfunction
correlating with PAR2 expression are attenuated by PAR2 antagonism.
FASEB J December 2013 "

Nothing absolutely new, I guess, but focusing on a particular mechanism.


Kindke said...

Heres a quote from the below paper....

"suppression of insulin signalling in adipose tissue prevents obesity"

Butyrate "might" be a treatment for obesity but its almost impossible to get hold of significant therapeutic quantities, 10g per day.

Peter said...

Hi Tom,

I just love these ideas. I love the way that steroids, the most potent anti inflammatory drugs ever found, do not seem to be front runners in obesity management. The word which comes to mind is, of course, varespladib, and Malcolm Kendrick's post below. Cardiovascular disease is supposed to be an inflammatory process too, now cholesterol has been pretty well pardoned (by the Nissen no less).

You have to keep asking, what is going on at the level of the mitochondria that produces a shitload of changes which morons can try to modify without sorting out the underlying problem. The body heap grows. You can get more about inflammation and CVD here

The other hysterical snippet is that I see from MIMMS that lomitapide is now license for hFH treatment. It lowers cholesterol but has, as yet, no discernible effect on survival. I'm looking forward to it's withdrawal. Medicinal memes are so strange...

Adipocyte inflammation (which is real of course) will go the same route as CV inflammation (equally real) under varespladib.

Ta Kindke!


karl said...

Peter wrote:
It lowers cholesterol but has, as yet, no discernible effect on survival

I've wondered if PUFA's lower cholesterol by inducing inappropriate insulin sensitivity - so LDL and trygly go down as the fats are promptly stored - as people get ever fatter...

All seems pretty good until FFA starts to go up (leaking out or distended Adipocytes). clothes no longer fit, blood pressure increases, and systemic inflammation stimulate macrophages that end up in the intima wall - gobbling up oxLDL until they become distended foam-cells that damage arteries.

It could very well be that the government promotion to have us eat plant antifreeze (AKA PUFAs ) is doing a lot of damage.

Scott Russell said...

All of this seems to be premised on the idea that there is a disparity between the insulin sensitivity caused by PUFAs on the cellular level, and the insulin secretion generated by the pancreas. After all, PUFA generated the least pancreatic insulin secretion, while SFA generated the greatest. Why would there be this mismatch in secretion/sensitivity? If we're going on the premise that life typically makes sense,(which I agree with), I tend to think that vitamin E might play a role. (Might look into this if I ever get free time.) After all, the more PUFA we see in nature, the more vitamin E we seem to see packaged with it.

@Kindke, I doubt its quite that simple. Although the pint of cream will certainly make cells more insulin resistant, it will also give the body a lot more energy to deal with. The increase in uncoupling is not unlimited.(Although the DNP would help here.) And even if we could burn through a pint of cream and just feel toasty warm, burning through the pint would only get you back to baseline.

Unknown said...

The last two posts Peter are bring your ideas and my ideas closer. When you see physics and biology collide things are good. How electrons and protons work from food, it offends common sense. In my opinion this is perfectly good, because so does Relativity, Quantum Mechanics, that's the way nature works. People think the brain needs way more glucose than it really does because they do not realize via the weak force and the electromagnetic force we can alter the information in food to suit our needs physiologically. The field of action of your mitochondria determine how it works to handle electrons and protons and their interaction with photons which are the force carrier for electrons......and interact with all forms of matter via the electromagnetic force.

karl said...


Re the the premise that life typically makes sense.

Yes BUT! Do not assume that life makes sense as if it was 'engineered'.

Our bodies are evolved - not designed and that has consequences in understanding our biology. Think of a car, where the steering wheel, car wheels, flywheel, differential gear were all the same part - with optimization pressures to do a good job at all of these things. The compromise makes it confusing to understand the function from the form - and if we are not aware of the other selection pressures - life does not make sense.

Unknown said...

Life is not parsimonious, it is a biologic belief that persists that it is.......just look at the 30 steps in photosynthesis. Everyone is quantized. Look at how actin and myosin work, look at how rhodopsin and melanopsin work with melatonin. Occam's razor and parsimony are bullocks in biology but treated as dogma. Riddle me this? How do Wendall seal’s live in extreme cold at the poles and can dive as deep in icy water as seals dive in warmer water, and that their trip to the surface for air is from l0 to 30 minutes. The male will stay in the icy water for 3 to 4 days, while the female has to feed her young on the ice. The antarctic seal lives a much easier life than the penguin that walks and toboggans great distances to raise its young (100 miles), but all in all they collectively defy laws that look to food and oxygen for power and heat. How do they do that? Was relativity or the photoelectric effect parsimonious?

karl said...

Yes, but I don't want to disagree with Petro, but only to point out that our evolutionary history is murky and affects many details of why things work they way they do.

What does bother me is that many in biology ( I'm talking about those that accept evolution) keep drifting back to looking at these systems as if they were 'engineered' - when they are not - they are 'evolved'. These systems only "make sense" if you keep in mind that they are evolved.

If we were engineered our DNA would have a cyclic-redundancy-check-sum, our eyes would not have features that obscure parts of the retina, we would breath 'through' instead of in-and-out etc,etc,etc,etc....

There was a paper about Ghrelin ..

Which led me on a track of reading - and there is this bit about ghrelin inducing apoptosis.. (could be that suffering regular fasting - which would expose us to higher levels of ghrelin, might rid us of apoptosis resistant cells that leak inflammatory interleukins?) Seems it has lots of stress related effects - again all these control loops seem to overlap each other - very hard to understand the whole. I think we will have to develop computer models that takes in all these control loops - and they we can fiddle with one at a time to see how the whole reacts.

ItsTheWooo said...

Peter thank you for addressing the unique state of post obesity; it's almost never spoken about by teh shoddy 2 bit paleo gurus out there who would sell fantasy fairy tales that they have the magic cure for obesity.

I am extremely insulin sensitive meaning to say, I respond with exaggerated response to any challenge that is insulinogenic. I seem to have exaggerated SNS response to insulin (e.g. eating a large, moderate carb meal will cause insomnia, activation, and tremor that lasts many hours). Even paleo darling food like coconut, I have observable hypoglycemic symptoms because the MCTs -> insulin that promotes a drop in blood nutrients. NORMAL PEOPLE are supposed to feel full from coconut; I get hypoglycemia because I am so sensitive to the insulin of MCT.

Once I did challenge self with 50 gram dextrose and blood glucose dropped to 40. I was thinner then, so perhaps would not be so egregious now.

Post obese people MUST follow a VLC diet if they hope to feel even slightly normal, sadly most prefer to eat 2 lindt truffles crash diet, binge on carbs, regain all weight, and then write an entire authoritative blog stating that carbs have nothing to do with weight control, it should seem.

The only people I've known who have a sort of relaxed/not eating disordered exercise obsessive weight loss strategy are people on low carb diets like me. We don't set ourselves up to fail by viewing VLC diet as some kind of punishment or deprivation either. Low carb is really the only way we can be sorta normal.

I have whole twitter feed/ instagram account filming the yummy stuff I eat. Better than cutting up my guts and being knife edge from binging from hypoglycemia... or being big fatass anonymous internet warrior diet expert pretending I am deprived / setting myself up for failure by romanticising eating bagels or whatever.

I eat this, its delicious, and I'm skinny, I'm happy about that. THe cake is made of cream cheese, sour cream, walnuts, eggs, splenda, sugar free jam. It tastes really good. Even this will make me gain weight however; most people would have ZERO appetite and chornic ketotic nausea eating stuff like this . (more !)

JohnN said...

Brad et all,
I can't resist another plug for Aeon, my favorite magazine. There's a companion piece to that excellent article on the selfish gene. John.