Thursday, September 27, 2012

Protons: The pancreas

We've seen the concept of superoxide being used to produce insulin resistance as a means of limiting (glucose derived) energy input in to cells which really don't want it. Superoxide appears to be the primary marker of energy excess at the cellular level.

We know from isolated mitochondrial preparations that superoxide is physiologically produced by reverse electron transport through complex I and is driven, gently, by succinic acid alone working through complex II. Far more is produced when the NADH level is high as well as having a reduced CoQ couple through FADH2 input, be that from complex II or from fatty acid oxidation products. Macroscopically fat and glucose together should produce enough superoxide to show as cellular insulin resistance, rejecting glucose from the cell, while allowing continued fatty acid oxidation. That's simple and logical.

But if you are building an energy sensor, it would be a bit dumb to restrict access to the very energy molecules which you are trying to look at to judge overall energy status, especially when energy status is high: You need to decide when to store calories...

The beta cells appear to use both fatty acids and glucose to generate superoxide, but instead of signaling beta cell insulin resistance, they signal insulin secretion. Several lines of evidence fit in with this.

You can get succinic acid itself directly in to beta cells by providing it as a methyl or ethyl ester. As a metabolic fuel source this acts as a near pure complex II substrate, pushing electrons in to the ETC through the FADH2 of succinate dehydrogenase to reduce the CoQ couple and set the scene for reverse electron transport and superoxide production, especially when NADH from glucose metabolism rises. In a commonly used model of functional beta cells, succinic acid methyl ester is a marked insulin secretion potentiator, especially at higher glucose concentrations. Glucose supplies NADH, succinate supplies FADH2, they clash at the CoQ couple and the generation of superoxide signals that there is a ton of energy available. Better store it. Better secrete insulin.

Succinic acid methyl ester drives complex II. This drives insulin secretion in response to glucose. But it's a drug. There is nothing physiological about this drug. So shall we go a little more physiological?

To recap from previous posts: Superoxide generation is directly proportional to the ratio of FADH2 generated to the amount of NADH generated for a given substrate, the F:N ratio.

Here's a nice graph of insulin secretion stimulated in response to 12.5mmol glucose on a background of assorted free fatty acids from an isolated pancreas preparation:

If you can't be bothered to work out the F:N ratios (shame on you), here they are added to the graph:

Please excuse the C8 value; as we all know, MCTs are shunted directly to the liver via the portal vein. They do not seem to feature too prominently in pancreatic superoxide generation and insulin secretion. It would take a ton of reading to see why and how they are handled differently to longer chain fatty acids. For the time being let's stay looking at C16 and longer as these make a much tidier story...

So, for the four longer fatty acids tested, the amount of insulin secreted is remarkably closely associated with the F:N ratio of the fatty acid available.

Does this work in people?

Of course it does. Remember the Spanish study? I lo0ked at it in some detail here.

In particular look at this graph:

From the top downwards we have butter, high palmitic acid seed oil, refined olive oil and a mix of fish and vegetable oils as the white triangles. It is very clear that the insulin secretion here is in direct proportion to the saturation and length of the fatty acids in the meal, in an intact group of volunteers..

Aside: Obviously, there is a glaring error in the graph. All of the curves except the control use 800kcal of total food, of which 40g is carbohydrate/protein and the bulk is fat. The graph is missing a group where 800kcal was supplied as pure carbohydrate. We can all imagine where this much bulk glucose would have put the insulin curve, needless to say there is absolutely no way it would fit on to the presented graph. We would need a much taller vertical axis, which would show the mixed meals in their true context!

But the principle, that insulin secretion at a given level of glucose is elevated in direct proportion to the F:N ratio of the background fat, holds perfectly well in this carefully contrived human study.

BTW, lucky for me they didn't include a coconut oil group!

The obvious conclusion from this finding is that to lower insulin maximally we should, taking as given that replacing carbohydrate with fat is the biggest step by far, all go for vegetable oil with some fish oil. Not butter. But in the original post on the Spanish paper I went on to discuss what appeared to be happening to the lipid from the meal. It could stay in the bloodstream and be used for metabolism, as the butter did, or it could be cleared rapidly in to adipocytes allowing metabolism to return to being glucose based. At the cost of expanding the adipocyte stores of fat.

The high PUFA meal really was rapidly stored as fat in adipocytes. The F:N based explanation is because we are supplying a low F:N ratio fat and so not generating insulin resistance phyiologically; we are allowing lipid easily in to adipocytes because the lipid does not generate adipocyte insulin resistance. We are going back to glucose metabolism as rapidly as possible. PUFA facillitates fat storage and glucose based metabolism. All is fine until you can't get any fatter. Butter limits fat storage and runs metabolism of palmitic and stearic acids. Those high PUFA-fed mice generate obesity when fed their high PUFA diets from pre-conception onwards:

In the butter group there is some excess insulin. Does this matter if no one (cellularly) is listening to it?

I next want to look at the flip side, the reduction of supply of free fatty acids to the pancreas. This was done in the same paper. You can certainly do this in intact rats (and humans if you so wish). Then we might get back to the fat mice.

I think that had better be another post as this one is getting overly long and it's light enough to let the chickens out.



LeonRover said...

Bravo Peter,

A most concise plausible fatty acid hypothesis.


Tucker Goodrich said...
This comment has been removed by the author.
Tucker Goodrich said...

Dr Guyenet posted this yesterday on Twitter:

"Dietary Linoleic Acid Elevates Endogenous 2-AG and Anandamide and Induces Obesity"

From Hibbeln & Co. I think you'll like it.

Scott Russell said...

This makes me wonder about the role of the F:N ratio in the liver. As coconut oil is often converted into ketones, it seems logical that it would be right up there with palmitic and stearic acid, as those would presumably be in abundance while fasting.

Is the implication here that n-6 fatty acids have the same F:N ratio as n-3?

Great stuff as always!

karl said...


Are you thinking that the resistance in via the VMH? Or systematic?

I'm still thinking of what is damaging the VMH in humans - which I'm thinking would be the root of the epidemic of T2D.

I was wondering about a sorted list of suspicious factors in our diets...

Peter said...

Tuck, (and IcedCoffee)

Interesting they never looked at adipocytes, but plenty of grist to the mill there. You even get the bodyweights!!!

As an aside, the biggest problem I have is with the omega 3 PUFA and why they are not obesogenic in the same way as omega 6 PUFA based on F:N ratio. They are equally cirrhosis generating in the liver. But I have a discussion paper, somewhere unlocatable, which suggests omega 6 PUFA increase insulin sensitivity as per this thread, while omega 3s decrease insulin sesnitivity, not as per this thread. You would expect this last effect to be beneficial, and it is, but why does it happen? Lot of thought to go in to that one.


Brian H said...

This post really pulls it together for me... didn't get it when you cited the Spanish Study in your 2010.3.19 entry. I must say stumbling across your blog is one the best things that's happened to me. As a result of your posts I have switched from high protein and moderate fat to high fat and moderate protein, *and" switched from high olive oil to high butter. As a result I am having the most successful diet in 20 years. If I can figure out why even modest amounts of whiskey tend to wreck things for me, I'll be set. -Brian

Peter said...


I'm still thinking that lipids enter the adipocytes easily under the low insulin resistance conditions provided by Omega 6 PUFA until the adipocytes become distended enough to leak FFAs due to distention induced insulin resistance. Once the adipocytes are overfull they should behave as if they are in a low insulin situation, ie preferentially release palmitate. If there is continued carbohydrate intake when the adipocytes are signalling the need for insulin resistance due to perceived hypoinsulinaemia we get hyperglycaemia. In the post above this one there is a graph of the effect of hyperglycaemia on beta cells with a 0.4mmol/l palmitate background. They die.

The VMH is a sophisticated fine tuner of the pancreas and probably behaves in much the same way, even in normal (non C57BL/6) humans. It's hard to reverse cell death in VMH neurons...

That's before we look at autonomic nerve cell death under hyperglycaemia and the knock-on effects of mutliple control system failures.


Peter said...

Brian, good. To me the message from the graph in the post after this one is that palmitic acid is utterly non toxic at almost any plausible physiological concentration until you spike glucose. Insulin resistance is good good good when you need it and don't abuse it!


Oh, Tuck, nice to see the population data on sucrose consumption as well as on seed oils, in the link you put up.

Unknown said...

You said Peter, "As an aside, the biggest problem I have is with the omega 3 PUFA and why they are not obesogenic in the same way as omega 6 PUFA based on F:N ratio." The reason is that we do not use DHA for energy. It is highly conserved. Plant based o-3 is handled differently. In humans, ALA is oxidized and used as a source of energy most frequently, and for carbon backbones to synthesize nonessential compounds like saturated fatty acids and cholesterol. Moreover, ALA in humans is not well converted to long chained DHA in humans because of the enzyme biochemistry in humans is not well developed. We now know because of the work of Kaduce in 2008, that adult neurons can make DHA endogenously, but its ability to do so is sharply limited. In adults humans, the DHA synthesis pathway is very inefficient and essentially stops at DPA omega 3, causing a sick brain to be dependent upon a constant source of new DHA from the marine food chain.
There is an evolutionary reason for this fact buried in performance physiology of DHA itself. DHA is not burned for energy in humans because of this factor. That factor is this: bio-energics of this lipid due to it specific ability to conduct electrical signals. This theory was tested by Turner et al., who demonstrated a positive linear relationship between the high molecular activity of the enzyme Na+K+ATPase (the sodium-potassium pump) and membrane concentration of DHA in the surrounding phospholipids in brain, heart, and kidney tissue of samples from both mammals and birds. Further, the highest concentration of DHA was found in the mammalian brain as was the highest activity rate of the pump. This is significant as the sodium-potassium pump accounts for some 20% of the basal metabolic rate but approximately 60% of the energy utilization in the brain.” This is precisely how evolution got the extra energy for the brain and heart to work well in mammals. In humans this ability is magnified because they collect DHA from the evolutionary cradle. I just wrote a blog post about this called Brain Gut 13. Cite one on that blog post will fascinate you if you bother to read it.

Once it is obtained it is avidly retained and reused. It is protected from oxidation by the brain’s massive antioxidant system. this involves iodine, Vitamin E, glutathione, DHEA, Oxytocin, and melatonin in life. The brain makes up 2-3% of our body weight but consume 20-25% of whole body energy! It is the ultimate energy hog! Using DHA in concert with the Na/K ATPAse increased metabolic efficiency dramatically so DHA is never burned for fuel. It is highly conserved.

Most of the energy in the brain is tied to phospholipid recycling for cell membrane recycling. This was reported by Purdon and Rapoport in 2007. Most people in the research circles think DHA is concentrated in the brain because of conformational fluidity of DHA, but this does not appear to be the case because melting points past the first three double bonds in PUFA’s does not alter melting point abilities of other PUFA’s substantially. Turner’s paper (cited below) has even deeper implications for humans. It appears that DHA lipids allow humans membranes to do some unique electrophysiologic things that few other mammals can do. DHA acts as a metabolic neuro-physiologic pacemaker to amazing biochemical abilities. DHA appears to directly impact and influence the metabolism of the whole organism via an impact on the basal metabolic rate because of the linear relationship in how the Na/K ATPase functions.
Here is the blog:

Here is the Cite

Tucker Goodrich said...

"Oh, Tuck, nice to see the population data on sucrose consumption as well as on seed oils, in the link you put up."

I did say you'd like it. ;)

I guess Stephan's going to have to recant giving up on the PUFA causes obesity hypothesis. And I suspect he'll be happy to do so.

Tucker Goodrich said...

"As an aside, the biggest problem I have is with the omega 3 PUFA and why they are not obesogenic in the same way as omega 6 PUFA based on F:N ratio."

Turns out a critical part of mitochondria is made up of linoleic acid. And the portion composed of linoleic acid increases linearly with dietary intake. And when that happens, as I recall, mitochondria become better at burning glucose.

This may be a fluke that predisposes critters like us to over-consume glucose and linoleic acid, despite other bad effects.

Too late to go digging for that study...

Peter said...


Thanks. Only skim read so far but looks good.

Tuck, I guess there is no limit to how many balls ups the Good Doctor will have to recant on, then re recant on. ItsTheWoo has his number.

Again, no time, but I'm wondering how the linoleic acid induced canabinoid receptor changes compare to food restriction canabinoid changes (I'm assuming these happen). After all, increasing adipocyte insulin sensitivity haemorrhages calories in to adipocytes, the idea of "hypo caloric metabolism during fat gain". I'm wondering if the changes are similar. That would be cool, but a subject I've not even thought about.


Allan Folz said...

Jack, please excuse me asking here instead of your own blog, but based on your comment it seems like you'd have an opinion.

My son showed all the stereotypical boy ADHD & oppositional tendencies both at home and school, though school was far worse. Unfortunately it took my a while to put 2 and 2 together, but I finally realized a mega-EPA supplement solved the issue. The formulation he's taking is 800mg EPA & 400mg DHA. From some reading after I discovered the "fish oil" supplement was what helped his behavior, it seemed early studies that were high in DHA showed no efficacy, and that it wasn't until they tried some high EPA studies that they were able to show a significant efficacy for O3 to treat ADHD. I'm guessing your familiar with this much?

If so, my question is that the aforementioned dosage seems quite high to me relative to what one would get in a typical diet. So, is there likely some other deficiency, a co-factor so to speak, that we're brute-force overwhelming with the mega-EPA? (The boy is 8 and a stout 60 lbs. No wheat belly or fatty liver on him.)

I have reservations about long-term supplementation of so much O3. He's been taking that 5 days/wk since about March. It definitely makes a difference in his mental health, no question on that. I try weaning his dosage now and then and it feels like things fall off fairly quickly as the dosage drops. Thoughts? Anyone?


Allan Folz said...

OK, so Jack, I read your link after writing my question. Seems it addresses almost exactly what I was asking. Would have saved myself a lot of typing at any rate.

Nonetheless, to close the loop, my guess is your answer would be two-fold:

1) 800mg EPA & 400mg DHA isn't so much

2) DHA so readily oxidizes, that dietary intake via a capsule, ie. without the accompanying anti-oxidants found in real food, is not going to be particularly effective

Bonus suggestion: try replacing the supplement with real, whole-seafood intake

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Robert Andrew Brown said...


There is a significant amount of work on ADHD oppositional defiant and supplementation with fish oil DHA EPA GLA.

Digestive disturbances may compromise absorption.

Some groups are genetically poor converters of plant based to long chain fats.

Mineral deficiencies are more common than realised, for a variety of reasons including agricultural practices, food preparation, changing dietary habits, loss of traditional preparation methods etc.

I have seen some suggestion that the risk of ADHD may be increased by mineral / other nutrient deficiencies. This 'essay' raises some thought provoking points.

Alberto Bolognini said...

Hello Peter, you might wish to consider also this seemingly counterintuitive finding
when reasoning on mitochondrial genesis and oxydative stress. It seems there is a class of fats that do not stimulate mitochondria by means of ordinary superoxide mechanisms. I wonder what the deeper rationale behind is.

Peter said...

The old omega 3 paradox... I think Jack has the correct idea re omega 3s. Bulk omega 3s as your primary source of calories may well have direct mitochondrial effects, in the liver at least, which better reflect their F:N ratio, they are more cirrhosis generating than corn oil. The CLA (NB there is a whole family of CLAs) is probably a signalling molecule working above the level of the mitochondria. There again, maybe it's a complex I inhibitor! Interesting.