Thursday, March 09, 2017

Protons: The destruction of complex I

It is quite difficult to express how exciting this paper is to a Protons thread True Believer:

The CoQH2/CoQ Ratio Serves as a Sensor of Respiratory Chain Efficiency.

The paper covers essential everything I've worked at over the past few years about the ratio of NADH to FADH2 as inputs to the electron transport chain. They even give Dr Speijer an honourable mention. I like these people.

Look at the Graphical Abstract, it's pure Protons:

And these are their highlights:

But, as hinted in the highlights, they take it to another level, beyond where I've gotten to in Protons:

The destruction of complex I by eating saturated fat. Or by generating a few ketones.

Fantastic stuff.



Samuel William said...

Yumm coconut oil. Weirdly coQ10 liquid bottle on the counter.

Tucker Goodrich said...

The link to ischemia / reperfusion injury at the end is interesting, as it links our two interests:

"The Mitochondrial-Targeted Compound SS-31 Re-Energizes Ischemic Mitochondria by Interacting with Cardiolipin"

Peter said...

Tucker, you have to wonder if SS-31 makes PUFA derived cardiolipin behave like saturated fat based cardiolipins. An interesting peptide.


Kenneth Strain said...

@Peter and @Tucker. Since Protons 44 I've been dipping into the topic of cardiolipin and linoleic acid therein, but the more I read the greater my state of confusion about this topic. I believe I've read all the relevant papers linked here, and many others. Most appear to be written from a "more LA in CL is better" point of view, except perhaps for some of the papers on brain mitochondria (e.g. the beautiful mass and charge spec results in the Kiebish et al paper which all seems very reasonable), and one or two other exceptions.

The comments to Protons 44 seem to suggest that there is a paper (or papers) that I'm missing. As my searches go round in circles are there any pointers that could help me out?

Any thoughts on whether the "18:2 is good" theme has any merit (e.g. as part of a complicated feedback process to modulate mitochondrial ETC efficiency) or is it just the "usual" bias caused by what's in the food of the experimental animals?

Any clues would be most welcome!

Peter said...

Hi Kenneth,

Tucker is really the cardiolipin go-to person. It's a bit far down the ETC form my area of current interest! Bit like reactive nitrogen species and oxygen/hypoxia sensing......


Kenneth Strain said...

Thanks Peter,

add Ca2+ to the list (with CL, 02-, H202, NO and ON00-) and that's what I'm trying to get my head around. Many of the papers deal with the strong effects that eventually lead to apoptosis, centred around cytochrome c. That subject is well studied but this catestropic condition is not our main interest.

Instead, I'm wondering what feedback loops operate closer to the normal physiolgical regime. I would expect there to be "something going on", based on the idea that evolution tries every trick given time, and there has been plenty of time for mitochondria to try every trick.

I am therefore interested in what goes on when there is only the normal physiological ROS from complex I (and perhaps a bit from the Q cycle). Does this, over time, affect the cardiolipin binding cytochrome c, or is there a threshold effect protecting from damage at low ROS levels? If so why is there a change with age? The cardiolipin spectrum depends on the tissue and changes with age even though there is renewal and replacement on a much shorter timescale. So the "usual" competitive regulation is working to some extent, but what processes regulate the balance. Probably ROS? If this is all physiological, the complex I ROS could play a role. My understanding does not go much further, but to evaluate the significance of 18:2 in cardiolipin it is surely necessary to figure out what balance evolution has come up with in the normal state. I can see the argument could go either way (or even both ways) - LA in cardiolipin good and/or bad.

These are all rhetorical questions, of course, I don't expect answers to be easily found.


karl said...

I'm having trouble with the phrase "Destruction of Complex I"

Is CI really "destroyed" or just deactivated and later reactivated?

'Destruction' makes it sound like the mitochondria are permanently damaged - which would be a bad-thing - but I think it is really just the method of how the control loop works.

From the paper:
"... we demonstrate that CI is degraded when reduced ubiquinone (or CoQH2) accumulates in the presence of oxygen. The resulting reverse electron transport (RET) generates superoxide, which oxidizes critical CI proteins, inducing their degradation. Finally, we show in vivo that modulation of FADH2 electron flux to CoQ acts as a metabolic sensor, allowing mitochondria to rebalance the relative proportions of respiratory complexes to accommodate variable electron flux ratios from NADH and FADH2.


"Our results allow us to propose a molecular mechanism through which mitochondria reconfigure the mETC to adjust to the ratio of electron flux from NADH and FADH2 (Figure 7). ..."

I think a better word would be 'deactivate'.

Peter said...


I have a lot more detailed reading to do but to me, "degraded" means destroyed. Once you have oxidised a series of cysteines I don't see them being re-reduced...... It has some bearing on how long it would take to synthesise new complex Is if a long term sat fat eater would like to re convert to glucose levels of complex I. Longer than the three days needed to re instate glucokinase in the pancreas, I suspect.


Tucker Goodrich said...

@Kenneth Strain:

Cardiolipin (CL) composition is diet-dependent. On an industrial diet, that means a lot of LA, which is preferentially incorporated. LA is uniquely susceptible to oxidation in cardiolipin, replacing with other fats reduces that susceptibility. LA-rich CL is in constant contact with cytochrome c, and is oxidized by cyt c, producing ROS. It's also susceptible to ROS from complexes I and III. LA metabolites from CL oxidation like 4-HNE also oxidize LA in CL, which creates a self-perpetuating cascade.

If you have CL that is oxidized, then adding fresh LA allows the body to repair, hence notion that LA is beneficial, as it's better than oxidized CL.

The body contains antioxidants like glutathione and other processes to manage CL ox, problems arise when these are overwhelmed. Primary symptom of this is depletion of glutathione in affected tissues.

This increases with age (and is intimately related to worsened aging process) because the LA metabolites produce damage DNA, protein and lipids throughout the cells, impairing cell function.

I did two posts on cardiolipin and what happens, that answer a lot of your questions:


Tucker Goodrich said...


"Is CI really "destroyed" or just deactivated and later reactivated?

"'Destruction' makes it sound like the mitochondria are permanently damaged - which would be a bad-thing - but I think it is really just the method of how the control loop works."

It's destroyed, along with the mitochondria (mitophagy) and the cell (autophagy).

Cardiolipin is the backbone of the ETC, if CL is damaged, the ETC collapses, and all the topics that Peter discusses in the Proton thread cannot occur. This is bad.

"Indeed, CL is required for optimal activity of complex I (NADH–ubiquinone oxidoreductase) [7], [8] and [9], complex III (ubiquinone–cytochrome c oxidoreductase) [7], [10] and [11], complex IV (cytochrome c oxidase) [12], and complex V (ATP synthase) [13]."

"Functional role of cardiolipin in mitochondrial bioenergetics"

@Peter Thanks for the vote of confidence!

Tucker Goodrich said...


Sorry, wasn't really answering your question... Cardiolipin on the brain.

From what I understood about that paper, they're running mitochondria to desctruction in non-physiological conditions. In the body there are regulatory systems that would prevent that outcome.

Tucker Goodrich said...


"Tucker, you have to wonder if SS-31 makes PUFA derived cardiolipin behave like saturated fat based cardiolipins. An interesting peptide."

SFA or MUFA-cardiolipin, yes. We don't have any data on what low-LA cardiolipin behaves like over the long term, but I have certainly come to the conclusion that you state. SS-31 or SkQ1 block oxidation of LA-rich CL, and prevents many of the common bad outcomes we associate with MetSyn. In animal models, so far, of course. But across species.

Peter said...

karl and Tucker,

This is in one of their more severe models of extreme CoQH2 excess:

"The specific labeling of mtDNA-encoded subunits was resolved by 2D BN-SDS-PAGE. CytbKO, Cox10KO, and CytcKO cells are able to assemble CI, but the instability of CI in these cells is revealed by the presence of labeled assembly intermediates or degradation products (Figure S2). Expression of AOX prevented accumulation of these abnormal assemblies, stabilising CI either as a free complex or in SC CI+CIII in cell lines in which CIII is assembled (Figure S2)".

Note AOX is an alternative oxidase for CoQH2, just keeps the amount low so stops RET.

I feel the individual complex I assemblies are probably destroyed. In Fig S2 A we are looking at a very severe model of extreme CoQH2 accumulation and complex I is in pieces as individual proteins. Whether they are failed assemblies or broken pieces of a full complex I is impossible to say but we know that switching from glucose to fatty acids will require a reduction in no-longer-needed but fully intact complex I, to the correct physiological level for dealing with fat. If, and it’s a big if, the process is similar to their complex III deletion model, then the complex I from glucose oxidation will end up in oxidised pieces. I sort of doubt this is a mothballing, more like a recycling of the amino acids. I would stress we are talking physiology here, the removal of surplus complex I to free up more isolated complex III to interact with mtETFdh’s “free” CoQH2 pool.


Tucker Goodrich said...


OK, that makes a lot of sense (he says, doubting he fully understands this).

This would certainly explain the low-carb flu, and the adaptation period that changing your diet requires.

The data I've seen on Respiratory Quotient led me to think that fat-burning capability can atrophy, no reason why carb-burning capability can also atrophy. And it certainly makes sense that that would happen at the point where those fuels are actually used...

Kenneth Strain said...

thank you - the links are much appreciated. I had them bookmarked, but had not realized they were yours. I'll follow them up now. I'm on the search for the original research that backs up your points, which are in my view all quite reasonable.

Kenneth Strain said...

(with apologies for the typo in your name in my previous reply).
Fantastic blog! (I run barefoot too). I've got about 10 posts of yours and some assoicated papers that I need to digest more fully, but I understand LA a little better now, thank you! As you note in a post, with the appropriate terms the search engines start to hit the right seam.

I'd forgotten there was sense in early posts by S.G. (pre-PhD.)


tomer aviad said...

I think as Peter that the complex I is destroyed and not just block as feedback mechanism. I have seen more evidence for this claim here:

"Studies of mitochondrial function also have been carried out in rodent models of type 2 diabetes. Boudina et al. (34) examined heart mitochondrial function in saponin-permeabilized heart muscle fibers isolated from insulin-resistant, diabetic, leptin receptor–deficient db/db mice compared with lean controls. These investigators reported decreased respiration on complex I substrates and palmitoyl-carnitine, associated with proportionately reduced ATP production and therefore no change in ADP/O ratios."

(34 source) -
The original article -

Another main evidence is that people with insulin resistance cant "go back" for being insulin sensitive as they used to be. This means that even when this people eat LCHF and keep there insulin low they still can eat carbs and keep their sugar low like they used to when they were young/

Peter said...


I suspect the inability to return from type 2 diabetes might be more to do with the selection of mitochondria and their mtDNA over time following exposure to high glucose and FFAs for an extended period. The removal of excess complex I when substrate availability shifts from NADH to FADH2 related substrates looks to be physiology rather than pathology, so should be reversible given a switch back in substrate availability. Some degradation of complex I can be picked up within hours, even by comparing fed to fasted rodents, i.e. it must be equally rapidly reversible. But what happens when the emphasis is on FADH2 substrates for months and then metabolism is suddenly asked to switch to NADH based substrates is another matter. Full fat adaptation (NADH to FADH2 shift), for me, took several months. Adaptation to carbohydrate might take take as long going the other way but show as poor glucose control through this period.


tomer aviad said...

Hi, thanks for the comment. Does the exposure to high glucose and FFAs for an extended period makes the mitochondria dysfunction consistently? can damaged mitochondria be replaced?

Peter said...

My suspicion is that there is a continuous pathological ROS generation when FFAs persist with elevated glucose. Apart from effects on complex I this will also affect the mtDNA and there will be a pressure to select for those mitochondria best able to survive under these conditions. In all probability it will be those which have the best antioxidants rather than the best ETC match. The ROS will give chronically damaged complex I and the selection pressure will give upregulated antioxidants. This is the classical finding in PD, alzheimers and ALS neurons. Of course these neurons will not be oxidising FFAs or (except rarely) ketones. Their CoQH2 excess will come from mtG3Pdh and glycolysis. Hence neurons should only use lactate, not glycolysis. But there's no arguing with hyperglycaemia, if it's high enough it will force glycolysis....


tomer aviad said...

What do you mean by saying "able to survive"? does it mean they dont function or destroyed?
I saw a viedo on cancer from Thomas Seyfried where he shows that in tumor cells the mitochondria inner membrane is destroyed:
The image:
The video:

Maybe the same effect happens too under high FFA and glucose?

DLS said...

Tucker Goodrich said...

@Kenneth Strain:

Thank you for your kind words! I got into the barefoot running thing when Born to Run came out. Unlike most, I'd bought a pair of Vibrams a couple of years prior, but had stopped using them about six months before the book arrived (for a silly reason, in hindsight).

I got into the diet thing via S.G.'s blog, as he was also a Vibram-wearer, and the proprietor of the Barefoot Shoes blog Justin Owings sent me a link to WHS. After six month of reading, I was sold.

Let me know if you have any questions.

Tucker Goodrich said...


"This is the classical finding in PD, alzheimers and ALS neurons."

Yes indeed, and the mechanism seems to be ROS oxidation of cardiolipin, which breaks down the ETC and causes mitochondrial dysfunction. Or, as these folks put it:

"Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage"

The problem here is that antioxidants cannot protect cardiolipin, as the molecules of the natural antioxidants are too large and of the wrong charge to reach the membrane where cardiolipin resides. Which suggests that the current high levels of n-6 consumption are far outside our evolutionary milieu.

Tucker Goodrich said...

@tomer aviad:

The study Seyfried's talking about is this one:

"Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer"

If cardiolipin is too damaged, the electron transport chain cannot function, and the cell either dies (apoptosis, triggered by cytochrome c release), or reverts to more primitive methods of fueling itself. The latter option is what cancer cells do, and is the root of the Warburg effect.

"Accumulating data suggest that a decrease in cardiolipin content in the IMM correlates with a similar decrease in the amount of membrane-bound cytochrome c in the mitochondria. Hence, ROS-mediated cardiolipin peroxidation has been experimentally shown to cause detachment of bound cytochrome c from the IMM in both in vitro and in vivo models (for review see Orrenius et al. 13). Furthermore, selective peroxidation of cardiolipin was recently demonstrated by Kagan et al. 5 to precede mitochondrial cytochrome c release during apoptosis. Searching for the mechanism of cardiolipin oxidation, the authors found that cytochrome c, in complex with cardiolipin, catalyzes H2O2-dependent cardiolipin peroxidation which, in turn, triggers the detachment of cytochrome c from its binding to the outer surface of the IMM and its subsequent release into the cytosol through pores in the OMM... Conversely, a host of recent studies have shown a correlation between preserved cardiolipin content and resistance to apoptosis upon manipulation of various mitochondrial antioxidant enzymes..."

"Role of cardiolipin in cytochrome c release from mitochondria" (PDF)

karl said...

OK - so this raises the question if this is reversed by the replacement with new ETC or new Mitochondria? And how fast do we think that happens? (what is the half life of a MT?

How might this effect the ability to reduce the number of senescent cells?

And thus, how might this effect cancer rates?

What happens on a potato diet?

So if getting fat (via consumption of a bunch of LA and high carb diet) then causes FFA. Which might well cause pathological ROS generation and selection pressure on MT.

If there is enough loss of mtDNA for normal conditions - this really could be a one-way street. (really bad news - never get fat!).

... Now how can I respond when someone parrots the old 'healthy fats' narrative? I have trouble holding all this in my aging brain - there is no easy way to explain this. Not easy to simplify - and probably more complex than we are aware of.

So I wonder if there is a similar bit on both ends of the spectrum - super low-carb and super high-carb - changing MT via selection.

The bit about this that is so important - is the selection of MT. What happens when eggs are produced with an abnormal mtDNA selection?

I am intrigued by the idea that the haploid generation - eggs and sperm - tend to have gauntlet selection - only a few survive to reproduce - and on the egg side - that carries the mtDNA - with pressures to match nDNA and then later this mtDNA is further selected over the life of the soma... Life is so strange!

Peter said...

Hi Tucker, thanks for the links. I've been thinking I ought to go to some of the original papers Seyfried cites, they sound interesting. Seyfried badly blots his copybook with "or reverts to more primitive methods of fueling itself". True primitive metabolism is based on reduced ferredoxin, either directly or using its energy to maintain a sodium or proton gradient. This is primitive energy generation. Glycolysis is a Johnny Come-lately new kid on the block for ATP generation, look at all those enzymes needed. Probably fat is even newer but chemiosmosis came first and is by far the most "primitive", for something which has been developing for 4 billion years.

Soooo, oxidised CL is Badness, what is the trigger for ROS generation in the region of CytC? I’m guessing there are clues in the papers, I’ve not had time to read them yet.

The results in distal ETC component knockouts in cell culture show a severely reduced CoQ couple which will perform RET at the drop of a hat. But the damage from RET through complex I seems largely limited to regions around the FMN moiety and CoQ docking site....... What initiates the damage at CytC?


tomer aviad said...

Thank for the links Tucker I need to read and to think about this :)

Peter said...

I can’t access the full text, dunno on that one.
The peanut oil was hydrogenated, i.e. converted to palmitic acid, my favourite. Nice find.
This simply reflects the ability to generate RET. RET at high levels is extremely pathological. This is mentioned in as pathology, i.e. physiology taken to unhealthy extremes. It is completely compatible with the blockade of the ETC at complex IV (i.e. no oxygen) which will cause reducing equivalents to back up to CoQH2. Any FADH2 input will then drive RET and destroy complex I. This level of RET has nothing physiological about it. Oddly enough severe starvation to ketosis improves survival after MI due to hypoxia adaptation as per D’Agostino’s diver paper. FFAs are very high, ketones are very high, infarct is smaller…… Timing the injury to early fasting, so that FFAs are high but ketones not yet developed, gives a bigger infarct too.


George Henderson said...

The previous model of complex 1 inactivation;

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin.

Remodelling the ETC - I like that. The idea of the ETC as a modular assembly that will be reconfigured as the substrate balance shifts. Directly by the effects of the substrates on its outputs. Neat.

Tucker Goodrich said...


"What initiates the damage at CytC?"


"All of these complicating factors make it quite difficult to sort out the exact signaling mechanisms of ROS. It would appear that Nature has developed a highly sophisticated system to regulate specifically the generation and scavenging of ROS and to modulate the downstream effects of physiologically induced ROS emission on mitochondrial and cell activity."

"Mitochondrial Reactive Oxygen Species Production in Excitable Cells: Modulators of Mitochondrial and Cell Function"!po=13.8889

But they note: "In addition to induction of ROS by hypoxia, ischemia, hypothermia, and mitochondrial toxins, ROS per se may lead to even greater ROS generation in a self-amplifying manner..."

CytC and cardiolipin (CL) can autooxidize, because CytC contains an iron atom, and LA is oxidized by iron. CytC deforms to expose this atom to tetra-linoleoyl CL (TLCL), and only TLCL, and this reaction can continue until no TLCL is left. In the process they generate singlet oxygen and lipid peroxides such as 4-HNE, which can also induce ROS generation.

"It is important to note that Kagan’s group reported an enhancement of up to 1000-fold in the peroxidase activity of cyt c–CL complexes in the presence of fatty acid hydroperoxides"

And "Cytochrome c/cardiolipin relations in mitochondria: a kiss of death"

Rotenone inhibits complex I, and does so by oxidizing cardiolipin, but only the LA components. From my site above:

"Moreover, singlet oxygen formation was specifically observed for tetralinoleoyl CL species and was not observed with monounsaturated and saturated CL species."


"Notably, linoleic acid in sn-1 position was the major oxidation substrate yielding its mono-hydroxy- and epoxy-derivatives whereas more readily “oxidizable” fatty acid residues (arachidonic and docosahexaenoic acids) remained non-oxidized."

"LC/MS analysis of cardiolipins in substantia nigra and plasma of rotenone-treated rats..."

Supra-natural levels of LA in the diet, alone, can cause ROS generation that overwhelms the inherent capability to contain ROS generation and lipid peroxide damage in the mitochondria.

The symptom of this process is glutathione depletion in tissues affected by oxidative stress, as glutathione detoxifies HNE.

Tucker Goodrich said...

OK, case closed.

"Brief episode of STZ-induced hyperglycemia produces cardiac abnormalities in rats fed a diet rich in n-6 PUFA"

Abstract (note, they started as PUFA-boosters):

"Diabetic patients are particularly susceptible to cardiomyopathy independent of vascular disease, and recent evidence implicates cell death as a contributing factor. Given its protective role against apoptosis, we hypothesized that dietary n-6 polyunsaturated fatty acid (PUFA) may well decrease the incidence of this mode of cardiac cell death after diabetes. Male Wistar rats were first fed a diet rich in n-6 PUFA [20% (wt/wt) sunflower oil] for 4 wk followed by streptozotocin (STZ, 55 mg/kg) to induce diabetes. After a brief period of hyperglycemia (4 days), hearts were excised for functional, morphological, and biochemical analysis. In diabetic rats, n-6 PUFA decreased caspase-3 activity, crucial for myocardial apoptosis. However, cardiac necrosis, an alternative mode of cell death, increased. In these hearts, a rise in linoleic acid and depleted cardiac glutathione could explain this “switch” to necrotic cell death. Additionally, mitochondrial abnormalities, impaired substrate utilization, and enhanced triglyceride accumulation could have also contributed to a decline in cardiac function in these animals. Our study provides evidence that, in contrast to other models of diabetic cardiomyopathy that exhibit cardiac dysfunction only after chronic hyperglycemia, n-6 PUFA feeding coupled with only 4 days of diabetes precipitated metabolic and contractile abnormalities in the heart. Thus, although promoted as being beneficial, excess n-6 PUFA, with its predisposition to induce obesity, insulin resistance, and ultimately diabetes, could accelerate myocardial abnormalities in diabetic patients."

Money quote:

"n-6 PUFA feeding induced a profound loss of cardiac cardiolipin levels (Fig. 5, top), together with a drop in total ATP: 8.9 ± 0.6, 8.1 ± 0.8, and 6.9 ± 0.6 nmol ATP/mg protein in the NC, ND, and PC groups, respectively, vs. 5.1 ± 0.2 nmol ATP/mg protein in the [PUFA diabetic (PD) group] group (P < 0.05). Inasmuch as changes in cardiolipin have also been associated with mitochondrial structural alterations, we scrutinized mitochondrial morphology in all the above groups. Figure 5A depicts a mitochondrion with a double membrane and lamellar cristae, which are typical in NC, ND, and PC hearts. A novel observation in this study was abnormal condensed mitochondria, but only in the PD group (Fig. 5B)."

So n-6 + hyperglycemia caused their mitochondria to collapse, and their hearts to necrotize.

They must have had Peter in mind when they wrote this:

"Interestingly, in the PD group, although glucose oxidation dropped further compared with the ND and PC groups, palmitate oxidation remained unchanged"

tomer aviad said...

Thanks for the data Tucker.
If I understand correctly PUFA + sugar causes excess ROS which destroy the mitochondria inner membrane?

Tucker Goodrich said...

@tomer aviad:

That's what I would infer, yes. I recall seeing a paper somewhere that said CI is a big source of ROS under hyperglycemia, but haven't been able to find it again.

But the fact that CI has apparently stopped functioning implies that's what has happened.

I tweeted this study to a couple of scientists in this area, and they seemed to think it was pretty impressive. "A thing of beauty."

Tucker Goodrich said...

@tomer aviad:

I think this is about as good as we're going to get:

"Although mitochondria in vivo are rarely, if ever, in this unmitigated state, it is theorized that mitochondria in diabetes, when exposed to high glucose and fatty acid concentrations, may be driven toward greater oxygen use and higher potential, thereby forming more ROS (87, 237, 335). Under state 4 conditions in liver or heart mitochondria, ROS production has been estimated to account for as much as 2% of oxygen consumed (57)."!po=4.88722

tomer aviad said...

Its a good article, I have read him.

What do you think about the ability to recycle disfucntioned mitochondria? Can they be replaced?

Peter said...

That depends on what mtDNA is left. My feeling is that grossly dysfunctional mitochondria disappear very rapidly, along with their damaged mtDNA. What's left is what copes best with elevated glucose and FFAs together. "Normal" mitochondria, which deal best with either glucose or FFAs, but not both, may have been selected out. If all "normal" mitochs have been deleted, what is there left to generate a normal population from? If the most serious defect is the selection of rubbish complex I then we can, to a large extent, side step it with FADH2 from the first step of beta oxidation of saturated fats, down regulate the defective complex I and get on with life. PUFA won't do this.........


tomer aviad said...

Do you think that switching to LCHF diet will restore the defective mitochondria such that the insulin sensitivity will return to be normal?

Peter said...



BigWhiskey said...

It sounds so extremely foreboding...

Peter said...

No, not really. Living on fat is not so bad. It's just that I don't think a few years of eating LCHF(saturated) will get you back to being able to eat pizza and chips with garlic bread on the side followed by Ben and Jerry's with impunity. People have to get used to this. How close you can get, should you wish to, probably depends on how injured you are, we won't all be the same. That applies to hepatocytes, adipocytes, pancreas, neurons and mitochondria and maybe things I've not thought of. There was a cry in the Paleosphere once "We are not broken" or something similar. Bollocks. Live long enough and you will wear out. Where is any one of us in that process?????


Tucker Goodrich said...


"...How close you can get, should you wish to, probably depends on how injured you are, we won't all be the same. That applies to hepatocytes, adipocytes, pancreas, neurons and mitochondria and maybe things I've not thought of...."

In reading about damage caused by n-6 metabolites, they state that some of the protein modifications cannot be repaired. This is likely the mechanism behind SDLDL and high particle count: body can't clear the damaged particles quickly enough, if at all. If your DNA is damaged (which happens) that may be the end of you...

"Role of lipid peroxidation derived 4-hydroxynonenal (4-HNE) in cancer: Focusing on mitochondria"

My experience (Mark Sisson said the same) was that it took five years for me to completely start feeling 'right' after starting no-gluten, LC, L6.

But I don't doubt for a minute that if I went back to junk food I'd start accelerating mortality again.

Tucker Goodrich said...

@Tomer Aviad:

"What do you think about the ability to recycle disfucntioned mitochondria? Can they be replaced?"

Yes, dysfunctional mitrochondria are destroyed via mitophagy (eating of mitochondria), and new mitochondria are constantly produced through biogenesis.

Exercise is a great way to induce biogenesis.

"Do you think that switching to LCHF diet will restore the defective mitochondria such that the insulin sensitivity will return to be normal?"

It may (we don't know) return to your 'normal', but it won't go to 'ideal'. Meaning a bad diet will have the same effect if you try it again. :)

"Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet"

Tucker Goodrich said...


"The review highlights a potential mechanism of the KD involving the production of low levels of redox signaling molecules such as H2O2 and electrophiles e.g. 4-hydroxynonenal (4-HNE), which in turn activate adaptive pathways such as the protective transcription factor, NF E2-related factor 2 (Nrf2). This can ultimately result in increased production of antioxidants (e.g. GSH) and detoxification enzymes which may be critical in mediating the protective effects of the KD."

"Modulation of oxidative stress and mitochondrial function by the ketogenic diet"

Kenneth Strain said...

I'm still working my way through the papers linked from your blog, and I'm still very impressed at the work you've done on this topic.

One loose-end I'm struggling with is the role of Ca2+, and I suspect it is important particularly in the understanding of cellular homeostasis.

Ca2+ is most often mentioned in the context of the catastrophe of apoptosis, but there is enough other material to indicate a process of regulation at lower concentrations than lead to apoptosis. I'm thinking in part of the review by Glancy and Balaban (Biochemistry . 2012 April 10; 51(14): 2959–2973. )

I was not surprised to learn that Ca2+ binds to the head group of cardiolipin (among many other sources: Hwang et al Cell Death and Differentiation (2014) 21, 1733–1745; though again thats about CplxII destruction, not normal homeostatic conditions). This is intriguing and I wonder if the change in CL structure caused by Ca2+ binding is a modulator of -amongst other things- the interaction between tetralinoleoyl cardiolipin and cytochrome-c. As such it would form a connection between mitochondrion and host cell.

ROS feeds out to insulin sensitivity, but Ca2+ seems to be one of the controlling factors in the inward direction (to the mitochondrion). It binds to much more than CL as pointed out in the first reference above, but that binding is especially intriguing.

I think the point I find most puzzling is the tendency of Ca2+ to lead to ROS, so H202 and that increases the CL peroxidation by cytochrome-c. There seems to be too many positive feedback paths and I'm missing what ensures enough stability of tetralinoleoyl cardiolipin in the first place.

Kenneth Strain said...

Ah - I've just found the link Yelling Stop to the Paradies 2009 paper "Role of cardiolipin peroxidation and Ca2+ in mitochondrial dysfunction and disease". That's now high on my list to read.

Tucker Goodrich said...

@Kenneth Strain:

"...I'm still very impressed at the work you've done on this topic."

Thanks. It all basically came down to figuring out the right search term to use, and then putting the pieces together was easy. I owe Nina Teicholz's "Big Fat Surprise" for that, and highly recommend it. She's the one who finally made me realize it was the lipid metabolites that were the problem, not LA per se. After she commented on how harmful they were, I started to look for evidence of them endogenously.

I'm glad you found the Paradies paper (he has a number of good ones) as I've not really focused on that aspect, other than to speculate that the calcium in the arteries that is a hallmark of atherosclerosis has to come from somewhere, and maybe it's coming from the mitochondria dying from lipid peroxidation?

"...I'm missing what ensures enough stability of tetralinoleoyl cardiolipin in the first place."

I don't think anything ensures its stability, it seems to be inherently unstable in the mitochondria. There are mechanisms to deal with the fall out, glutathione's ability to trap toxic products of lipid peroxidation like 4-HNE, for instance. The "Q" paper Peter linked to here:

Had this bit:

"Strong indications that UCPs evolved in association with pressure to reduce ROS formation during β-oxidation can be found in regulation of expression and activity. The first important experiments were performed by the Brand group using yeast mitochondria expressing mammalian UCP-1 [137]. Here, palmitate (saturated C-16) and an oxidized (!) FA (4-hydroxy-2-nonenal) enhanced UCP proton transport. Synergistic effects were observed using both [137]. Oxidized FAs like 4-hydroxy-2-nonenal signal encountering ROS. The uncoupling predicted by the kinetic model indeed should be enhanced during β-oxidation, and even more so upon ROS production, as monitored by oxidized FAs."

The "(!)" is his, so we know how surprising he found this pathway!

4-hydroxy-2-nonenal is 4-HNE, and so the breakdown of TLCL is a fundamental part of mitochrondial function and regulation.

The problem occurs, based on my reading, when TLCL overwhelms the regulatory/reactive systems. One sees this is occurring when glutathione (GSH) is diminished, and HNE is escaping the mitochondria unbound to GSH to wreak havoc on surrounding structures, like DNA. The presence of HNE bound to various other things is a marker for every part of the MetS, broadly defined, which includes cardiovascular disease and Alzheimer's. It's everywhere, along with the other N-6 peroxides.

Since TLCL load is determined by diet, and a high-LA diet is a novelty, I think the logical conclusion is just that we eat too much LA. Skulachev's work with SkQ1, and the success of SS31, demonstrate quite clearly that endogenous antioxidants are not able to protect TLCL, and all they do is clean up after the fact.

It's important to note that "excess" LA is harmful, LA is in everything, so a zero-LA diet's an impossibility, even if it's not actively harmful. The trick is keeping intake in a band that is withing the body's ability to handle, and to note that LA intake was probably also seasonal, so giving the body a chance to repair (see the !Kung and their high-LA mongongo nut diet).

So the short answer to your implied question above, is "Rarity".

Please let us know if you learn enough about Ca2+ to explain it succinctly!

Bob said...
This comment has been removed by the author.
Bob said...


"...maybe it's coming from the mitochondria dying from lipid peroxidation?"

From Hyperlipd :

"Which hormone converts a vascular smooth muscle cell to an osteogenic cell (calcium phosphate secreting) in the vascular media?

Answer: Insulin"

I never forgot this reference, and it seems appropos here. Might this be an indirect effect of LA?

tomer aviad said...


There is something I dont understand and maybe you can explain to me.
From the Q paper ( FA increase ROS in the mitochondria and so there is UPC and antioxidant to deal with the ROS. From this face we can conclude the a mitochondria fueled by glucose is the main target to lower our ROS but as we can see high glucose level makes the ROS high enough to cause disease and other problems in the cell. How can we explain this fact from the mitochondria point of view? Why the mitochondria uses fat and antioxidant when we can use glucose and stay with low levels of ROS.

Tucker Goodrich said...

@Bob: "I never forgot this reference, and it seems appropos here. Might this be an indirect effect of LA?"

"The Natural PPAR Agonist Linoleic Acid Stimulated Insulin Release in the Rat Pancreas"

The scientists on twitter seemed to think it was an impressive bit of work:

Tucker Goodrich said...

@tomer aviad:

That question is what those on the fringes of science are working hard to answer. You'll notice that paper has lots of "may"s and "might"s in it.

Glucose, based on the longevity studies, does seem to have some ill effect that fatty acids don't, but I've never seen a paper that describes a plausible mechanism.

The paper I posted on hyperglycemia and n-6 effects on mitochondria in the heart is the best test I've seen, but I don't know of any similar work absent excess n-6.

If you find some, let us know!

tomer aviad said...

My guess is that UPC decrease the membrane potential and thus the RES and the ROS. UPC are activated from FA and not from glucose as far as I know, so maybe this is the reason for the low ROS.

tomer aviad said...

Kenneth and Bob look at this one:

Oxidative stress promotes mitochondrial Ca2+ overload and the opening of the mitochondrial permeability transition pore, leading to the release of mitochondrial pro-apoptotic proteins into the cytosol that initiate the intrinsic cell death. Ca2+ and ROS therefore engage in a self-amplifying cascade that culminates in cell death, but the molecular mechanisms of this vicious cycle are unknown

Kenneth Strain said...

Thanks tomer aviad - that paper adds another interesting link.

I'll try to convey my somewhat wooly thinking about this (my day job involves much simpler control systems this complicated stuff is new to me).

One of the major themes I'm learning about biological systems is the tendency for populations to exist with slight variations among their members. On the macro scale this variation lets evolution operate, on the smallest biological scale it is how epigenetic modifiers appear work to let (thermal) randomness "smooth out" the digital nature of genetics to produce (effectively-) continuous responses to the environment. (Sorry, that's probably a whole book condensed to one sentence, I hope the flavour comes across.)

Somewhere in between those scales we have mitochondria. We know something about how they are regulated by the nucleus to satisfy the energy demands of the host cell to achieve the proper physiological balance (or balances, across different tissues).

Now we start to learn that ETCs in mitochondria come in different configurations. I begin to wonder how the driving processes and the resulting structures are kept in balance, and what the normal (physiological) mix of the different configurations should be (probably not all one thing or all another, but a regulated mixture of different ETC configurations).

The structural changes resulting from ROS must feed back to the structures at low levels as well as at high levels. One of those ways at least at high levels is known from the catastrophic case of Ca2+ feedback leading to the PTP and apoptosis. Peter has discussed how ROS feedback works at physiological levels, and I'm wondering whether and how Ca2+ feedback regulation also operates in the healthy cell. It can't all be positive feedback of course, so at low levels at least there must be stabilising feedback.

If LA in CL degrades all the time that reminds me of the constant low-level electron leak at CplxI. I assume it is likewise "allowed" (by evolution) to be non-zero exactly because it is regulating something and part of a negative feedback process.

My next question is does Ca2+ have a role? In particular, how does attaching Ca2+ rather than protons to the head groups of CLs affect the CL configuration? We know what it means when there is a lot of Ca2+ (cytochrome-c release among other things). Could CL, in all its varieties, be "holding" several of the ETC components in place in a way that is regulating the structure (Ca2+ also feeds back to configure some proteins - I would expect multiple processes to be involved, there is no need for the answer to be simple given how long the mitochondrion has been around)? Does Ca2+ help regulate ETC structure and hence mitochondrial performance by feeding back to control the structure of the ETC in these multiple subtle ways?

Thanks to having read Tucker's blog, I've got backlog of ideas and concepts to digest, to say the least, and still several (more like several dozen) papers to read.

I'll definitely respond with anything that I can express compactily enough and which might be of interest.

Bob said...


Great find ("The Natural PPAR Agonist Linoleic Acid..."). If I'm understanding this right (in the context of the Protons thread), LA may not only enhance insulin sensitivity in various cells, it may enhance insulin production as well. Talk about a double-whammy!

And I suppose you could add, once the pancreas burns out and can't produce much insulin anymore, insulin injections (to the peripheral circulation yet) come in and finish the patient off.

Tucker Goodrich said...


Yeah, it's a hat-trick, as LA-modified LDL has been shown to be toxic to pancreatic beta cells (where insulin is produced), killing them off...

Tucker Goodrich said...

@tomer aviad, Bob:

"...and ROS therefore engage in a self-amplifying cascade that culminates in cell death, but the molecular mechanisms of this vicious cycle are unknown..."

They're well-described, in fact, and the molecular mechanisms are in the papers in those two blog posts.

BTW, when you read "oxidative stress", always translate it to "n-6 toxicity". All the markers for oxidative stress are n-6 metabolites... The same holds true when you read "lipid peroxidation".

It makes it much simpler to understand what's going on in these papers. ;)

Kenneth Strain said...

A short note about about Ca2+ and CL, while there may still be some interest among those who likely know more, in case there is something of interest.

The key to finding useful information was to search back a bit, as Ca2+ and CL interactions are supposedly old hat. Also finding references looking at the basic chemical properties.

There have been several chemical-physical studies, with results often reported in terms of phase transitions of CL structure in membranes, artificial or real. There are descriptions in several books including the nicely-named "Water: A Comprehensive Treatise" by Felix Franks. Vol 4 Ch 4 section 3 has a little on lipids, including CL.

Pardon me, but as I've read this on Google books, I paraphrase very roughly: Divalent cations cause tighter head group packing which is transferred to the tails and raises the critical temperature for the gel-liquid phase transition*. In the presence of Ca2+ the motion of the protons in the tails becomes more anisotropic. These observations show that the stability and permeability of the membrane depend on what is bound to the head group.

*and in mitochondrial inner membranes Tc for some CL arrangements could be in the physiological temperature range, though that's speculation on my part.

That's got the kind of behaviour I was wondering about.

The next step would be to look for clues about how the presence of norml (low) physiological levels of Ca2+ affect peroxidation of the tails (I mean directly, because of the above conformational changes, not because of control of phosopholipases or any other enzymes, which also happens, of course).

I started looking at this because there are hints that Ca2+ is protective at low levels, and as its concentration increases (due to feedback following stress in the way widely-discussed in the context of apoptosis) the protection disappears, and at much higher levels starts to trigger cytochrome-c release, the PTP etc.

I continue to wonder if low-level protection and stabilisation of the membrane operates by restricting peroxidation. If so, it seems quite reminiscent of the F:N switch from Protons 8 (etc.), which is why I was looking for an effect like this in the first place. Therefore it may provide a second such bang-bang feedback loop for us to get our teeth into. (Like F:N switch it won't really be bang-bang control as the ensemble of mitochondria will work to smooth things out and we end up with cellular IR or whatever after the "vote" is taken.)