Thursday, May 18, 2017

Fructose and metabolic syndrome: Uric acid

Some weeks ago a friend sent me a full text copy of the Naked Mole Rats (NMR) paper

Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat

which demonstrated that they (NMRs) appear to generate and use fructose as a coping stratagem for dealing with hypoxia or even anoxia. This is fascinating and leads back to research in the late 1980s, mostly looking at anoxia in liver or liver cells. I'm guessing that this liver work was funded to look at ways of improving the condition of transplant grafts. Fructose is significantly better than glucose for supporting anoxic liver cells, possibly something you might expect, possibly not. Perhaps in another post.

Anyway. So I've been looking at why fructose is different to glucose and to do this you end up asking rather difficult questions about the upper sections of both glycolysis and fructolysis.

Fructose enters the fructolytic pathway by being phosphorylated very rapidly to fructose-1-phosphate. Given a large enough supply of fructose this phosphorylation can deplete the ATP supply in a cell, most obviously in hepatocytes which bear the brunt of metabolising fructose. This takes place before aldolase generates the trioses which probably (or don't, in the case of fructose) control insulin signalling through mtG3Pdh and the glycerophosphate shuttle.

If this initial ATP depletion by fructokinase is profound it is perfectly possible to take two "waste" ADP molecules and transfer a phosphate from one to the other. This generates one ATP and one AMP. The ATP is useful to the cell and the excess AMP is degraded to uric acid.

This is all basic biochemistry.

In the Protons series I have worked on the (incorrect) basis that fructose should drive the glycerophosphate shuttle hard enough to generate RET (reverse electron transport) and so signal insulin resistance. The degree of insulin resistance should neatly reduce insulin mediated glucose supply by an appropriate amount to offset the fructose and so maintain a stable flux of ATP generation from the combined fructose and glucose. That's not quite how it appears to work. Even before the aldolase step in fructolysis, the body is starting to prepare the process of insulin resistance. This paper is not unique but shows general principles:

Uric acid induces hepatic steatosis by generation of mitochondrial oxidative stress: potential role in fructose-dependent and -independent fatty liver

The title of the paper is sneaky, it doesn't give away the answer! Nor does the abstract. If you don't want to read the paper, the missing link is NOX4.

NADPH oxidase 4 (NOX4), if exposed to uric acid (present here from fructolysis induced AMP degradation), translocates to the mitochondria and starts to generate enough hydrogen peroxide* to down regulate aconitase, abort the TCA and divert citrate out of the mitochondria through the citrate/malate shuttle for DNL. This will not just affect fructose metabolism, acetyl-CoA from glucose, entering the TCA as citrate, will also be diverted to DNL.

*The NOX family appear to be the only enzymes with no function other than to produce ROS, mostly superoxide. NOX4 is unique in that it always produces hydrogen peroxide. There is uncertainty if the "E loop" of the enzyme converts superoxide to hydrogen peroxide directly or if this is a docking site for superoxide dismutase, which does the conversion as an accessory module to NOX4.

I think it is a reasonable assumption that the hydrogen peroxide generated by NOX4 will be what signals the insulin resistance induced by fructose, rather than RET via mtG3Pdh. Quite why fructose doesn't drive the glycerophosphate shuttle is a difficult question to answer. Obviously the aldolase products of fructose-1-P (fructolysis) differ from those of fructose-1-6-bisphosphate (glycolysis) but these pathways are very difficult to get at experimentally and I've not found any papers looking at what controls why dihydroxyacetone phosphate from fructolysis doesn't drive mtG3Pdh, but that appears to be the case. There are hints that some activation of the glycerophosphate shuttle does occur but NOX4 seems to be the main player. It might relate to the consumption of NADH in the conversion of glyceradehyde to glycerol and so reducing the need to decrease it using the glycerophosphate shuttle. Hard to be sure.

So. Uric acid is the evil molecular link between fructose and metabolic syndrome via NOX4. And yes, yes, you can block metabolic syndrome using allopurinol to reduce uric acid production in rats but you have to give them a sh*tload of it. After that NOX4 might be considered evil or hydrogen peroxide is evil or aconitase is evil when it's on strike. Lots of drug targets available for molecular cleansing.


My own concept is that there is the necessity to developing insulin resistance when fructose is available so as to limit glucose ingress to offset the ATP from that fructose ingress. If that is done by NOX4, so be it. The facility to deal with fructose by the generation of hydrogen peroxide is not random, it's not some accidental mistake perpetrated by evolution on hapless humans who munched on a few Crab apples or found a little honey. It is an appropriate evolutionarily response to a relatively common occurrence. The fact that uric acid mediated insulin resistance is common to alcohol metabolism as well as to fructose metabolism suggests that this mechanism is a general approach to dealing with a calorie input which takes priority over metabolising glucose.

Developing a drug along the lines of allopurinol to block uric acid production, or an inhibitor of NOX4, or a hydrogen peroxide scavenger to avoid insulin resistance is simply trying to block a perfectly adaptive response to a reasonable dose of fructose.

All that's needed to avoid a pathological response to fructose is to avoid ingesting a pathological dose of the stuff. There is actually quite a lot of evidence to suggest that physiological levels of uric acid production might be beneficial...

Peter

Mulkidjanian: Na+ pump or Na+/H+ antiporter?

Mulkidjanian is a co-worker with Skulachev and extremely wedded to the primacy of Na+ bioenergetics, which is good. He has been looking at NuoH and NuoN subunits of complex I and their phylogenetics. In contrast to this, in the past I've discussed similarities between NuoH and NuoL. You just have to accept we're never going to be certain which component of complex I is most closely related to another... Anyway, I like this paper:

Phylogenomic Analysis of Type 1 NADH:Quinone Oxidoreductase

"Two recently published works independently noted the structural similarity between the NuoH and NuoN subunits and suggested their origin by some ancestral membrane protein duplication [13, 14]. Our analysis does not exclude the possibility that this duplication may have occurred even before the LUCA stage. In this case the initial NDH-1 form [proto-Ech in my terminology] had only one type of membrane subunit (the ancestor of NuoN and NuoH), which could function as a sodium transporter. The duplication of the gene would result in a different subunit, which improved the kinetic effectiveness of the redox-dependent sodium export pump (that participated in maintenance of [K+]/[Na+] greater than 1 in a primal cell) by facilitating proton translocation in the reverse direction".

Bear in mind that none of us can be certain exactly what a given protein might have been doing based on these family trees of genes.

I think there is general agreement that ancestor of NuoH and NuoN is a membrane pore and that it is primordial. In Mulkidjanian's scenario that pore is associated with a redox driven hydrogenase. His idea is that the hydrogenase is using preformed ferredoxin, or something similar, to extrude Na+ ions from the cell. This requires an external source of energy and his concept is for ZnS catalysed photosynthesis giving a localised organic "soup", ie heterotrophy. The refs are here and here. The source of K+ for the primordial cell cytoplasm is suggested here. I have to say, I'm not a convert to these aspects of his ideas, I'm staying more aligned with autotrophic thinking...

My own view is that the pore was a duct to localise oceanic acidic pH tightly to an NiFeS hydrogenase within alkaline vent "cytoplasm" to allow the hydrogenase to reduce ferredoxin, the primary energy currency of the proto-cell. The power source is the pH differential across an internal FeNiS moiety within the hydrogenase, combined with molecular hydrogen as the electron donor to reduce ferredoxin and so, eventually, CO2.


Given the almost certain ancestral gene duplication it is not difficult to make an antiporter out of NuoH/NuoN, whether you consider the ancestor to have been a proton pore or part of a Na+ pump. Even today, the membrane component of Complex I functions as an antiporter for Na+/H+ provided you separate it off from the hydrophilic matrix section:

The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter

Given an antiporter sitting in a Na+ opaque membrane we can antiport a ton of Na+ out of the cell using a geological proton gradient to give us the result of a low intracellular Na+ concentration. Excess Na+ extrusion can be converted, by electrophoresis, to an elevated K+ inside giving the modern intracellular composition. In the early days the electrophoresis might not have been K+ specific, theoretically any positive ion other than Na+ would do. K+ is the long term preferred option.

As soon as we leave the vent there is no free antiporting so we need to have a system which provides energy to generate a Na+ potential (buffered by K+ electrophoresis). The power available to do this becomes very limited in the absence of a geothermal proton gradient, when all that is available is the reduction of CO2 using H2, the Wood–Ljungdahl pathway. The Na+ chemiosmotic circuit then comes in to it's own as a system for combining small amounts of free energy in to units large enough to generate one ATP molecule. Recall how the modern pyrophosphatase Na+ pump requires the hydrolysis of four PPi to give one ATP via chemiosmotic addition. Until the advent of photosynthesis and the possibility of heterotrophy, all free living prokaryotes would have been autotrophic and living on a meagre energy budget.

The switch from luxurious hydrothermal vent conditions to lean autotrophic conditions goes a long way to explaining the universality of chemiosmosis. Alkaline hydrothermal vents may be stable on geological time scales but not for 4 billion years of un-interrupted flow and if the Wood–Ljungdahl pathway is all there is to replace the vent power supply it's going to be chemiosmosis all the way...

Peter

Thursday, April 20, 2017

Skulachev addendum

This is the final paragraph in the discussion section of the paper by Skulachev, regarding the use of a Na+/K+ concentration gradient across a membrane to store potential energy, convertible to a Na+ or H+ gradient as needed, and why elevated K+ does not have to be a primordial feature of proto-cells:

"One might think that Na+ ions are incompatible with life and this is the reason why K+ is substituted for Na+ in the cell interior. Apparently, it is not the case as, e.g., in halophilic bacteria [Na+]int can reach 2 M [41]. The very fact that some enzyme systems work better in the presence of K+ than of Na+, may be considered as a secondary adaptation of enzymes to the K+-rich and Na+-poor conditions in the cytosol [40]. Besides, it would have been dangerous to couple any work performance with Na+ influx to the cytoplasm if Na+ were a cell poison".

That makes perfect sense to me.

Peter

Wednesday, April 19, 2017

From Skulachev to LUCA

TLDR: Cells become islands of raised K+ ion concentration when energy is supplied.


Okay, here come the doodles based on Skulachev's paper

Membrane-linked energy buffering as the biological function of Na+/K+ gradient

This is the scenario in ultra modern bacteria, the pinnacle of about 4 billion years of evolution. The membrane is tight to all significant ions at reasonable temperatures and concentration gradients. In this set of pictures the proton population represented within the red circle is holding a membrane voltage of 180mV, as per usual:






The trans-membrane potential from the pumped protons is stable while ever the pumping and the consumption of protons is balanced. The problem is that it doesn't need many protons to generate that 180mV. Pumping any more than basic needs generates too great a membrane voltage. The converse is that it doesn't take much excess proton consumption to collapse the potential. So you need a buffer which does not waste the energy used to pump.

If a bacterium suddenly increases proton pumping by eating some glucose we have this problem of a spike in membrane voltage:









We can get around this by allowing a positive ion to travel in the opposite direction. This will stop the rising membrane potential as the ion uses the membrane potential to enter the cell against a concentration gradient. It uses an ion-specific channel, in this case for potassium. This process is electrophoresis down the electrical gradient, against a concentration gradient, powered by the electrical component rather than the pH component of the rising proton gradient:










The number of K+ ions matches the excess protons pumped. The electrical potential is thus maintained at 180mV at the "cost" or "benefit" (semantics here!) of K+ entering the cell. But there is a problem in that the more protons pumped and the more K+ entering the cell, the higher the pH of the intracellular medium becomes. That K+ pool is actually tied to the OH- left behind by pumping out H+. Caustic potash...










This is not good for metabolic processes. But it is easily surmounted using a 1:1 ratio Na+/H+ (electro-neutral) antiporter to get some protons back in to the cell to offset the excess OH-












while still maintaining an electrical gradient of 180mV using H+, keeping an electro-neutral Na+/K+ gradient as an energy store:










Obviously the Na+/H+ antiporter is being driven by the pH component of the proton gradient. It's neat how evolution has separated out the pH and electrical components of a proton gradient!

The whole system is fully reversible so if there is a sudden drop in proton pumping the transmembrane Na+/K+ gradient can be reconverted to a proton gradient to "buffer" changes in proton translocation. This seems to be how modern, proton pumping bacteria with superbly proton tight membranes work. In E coli the ion channel and antiporter are ATP gated.

That's how Skulachev looked at modern bacteria in 1978.


I'm now going to wander off on my own and speculate about LUCA with a proton leaky but Na+/K+ tight membrane. This is just me from here onwards:

Let's have a think about LUCA, with a cell membrane which is tight to Na+, and probably K+ too, but highly leaky to both protons and hydroxyl ions. Metabolism is based on Na+ pumping and a Na+ specific ATP synthase. The initial Na+/H+ antiporter (from the Life series) is gone as a source of Na+ gradient as soon as LUCA leaves the alkaline hydrothermal vents.

I like the idea that LUCA used a pyrophosphatase to pump Na+ but with any Na+ pump we have the same problem as in modern bacteria: You can only store a small amount of energy as a 180mV Na+ gradient, as per H+ above:










But excess Na+ pumping can be easily be accommodated by K+ electrophoresis:










There is no need for the Na+/H+ antiporter in this scenario because there is no pH change associated with pumping Na+ ions, so all we need is the ion specific channel for K+.

This sets up a non-electrical energy store which is "accessible" to form an electrical gradient when primary Na+ pumping is low.

The buffer automatically implies the generation of a raised intracellular K+. We have here, based on a tiny step beyond Skulachev's ideas, a place within LUCA which is potassium rich. It's simply produced to buffer changes in ion pumping by the primary Na+ pump (or usage by ATP synthase) across relatively primitive membranes. And driving intracellular K+ higher is an indicator to the cell that there is excess of energy available, which should select for increased enzyme activity based on rising intracellular K+ concentration. Many of the "core" LUCA enzymes do indeed use K+ as a cofactor to function optimally.

Summary: Cells become islands of raised K+ ion concentration when more than basal a level of energy is supplied. Remember that for our later discussion about Mulkidjanian's ideas on the origin of life on Earth.

Peter

Monday, April 17, 2017

Skulachev in 1978

We know from papers like

Effect of Very Small Concentrations of Insulin on Forearm Metabolism. Persistence of Its Action on Potassium and Free Fatty Acids without Its Effect on Glucose

that, as we raise the concentration of insulin perfusing a tissue bed, the first effect is the suppression of lipolysis. Then it promotes potassium translocation in to cells. If you keep the concentration low enough there is zero effect on glucose translocation.

More practically: Anyone in first line general practice will be well familiar with the moribund cat with an obstructed bladder (thank you Go Cat) and a plasma K+ of 11.0mmol/l. You know the intravenous dose of Ca2+ you've given will stave off a-systole for a while and you've started to correct the acidosis with bicarbonate but the ECG still looks awful, as does the rest of the cat. Neutral insulin, covered by glucose, will usually drive potassium back in the cells where it belongs and keep the patient alive for long enough to allow you to get to work on the underlying problem. Pure potassium pragmatism.

So I have always wondered: Why does insulin facilitate active K+ translocation in to cells?

This strikes me as a very deep question. Always has.


There are hints as to why in Skulachev's paper from 1978.

Membrane-linked energy buffering as the biological function of Na+/K+ gradient.

I've only just found this paper and skimmed through it so far. It's a really interesting piece of theoretical bioenergetics from a close friend of the late Peter Mitchell. It was published in the year that Mitchell received his Nobel Prize for elucidating the principles of chemiosmosis. The paper is one of those which needs a note pad, a pencil and a pencil sharpener to work through. On the to-do list but I think it is saying that K+/Na+ translocation is an energy buffer to smooth out rapid changes in proton translocation energetics. That is a deep process.

I hope that's what Skulachev is saying!

And the follow on: Insulin signals a flood of calories. You're going to either spike delta psi or need to buffer it. That needs K+ to enter the cytoplasm to limit the voltage spike induced by the subsequent increase in H+ exit via pumping... Is insulin pre-empting this need? I'll try and get some doodles together but off-blog is getting busy at the moment.


Skulachev is still publishing important stuff today and his department is deeply involved in the evolutionary primacy of Na+ bioenergetics and, as a recent foray in to clinical pragmatism, the development of mitochondrial targeted antioxidants which appear to extend healthspan as well as lifespan.

Interesting chap and the 1978 paper strikes me as very perceptive and very prescient. You don't get many that good.

Peter

Wednesday, April 05, 2017

Rho zero cells

Well, this post is about rho zero °) cells. TLDR: It's even more obscure than usual.

This is the basic ETC plus the ATP:ADP antiporter (ANT) and the Pi:H+ symporter (Slc25a3) added:









Most of this is very obvious but it's worth pointing out that ANT exchanges one ATP outwards with 4 negative charges for an ADP inwards which has 3 negative charges. The ADP needs an inorganic phosphate to reform ATP and this Pi carries one negative charge and enters the mitochondria via Slc25a3, facilitated by consuming one proton of the proton gradient. All is hunky dory with electrical balance, accepting some delta psi consumption.

ρ° cells are man made constructs which have no mitochondrial DNA, usually deleted by exposure to ethidium bromide. They live by glycolysis and need supplementary pyruvate and uridine to survive. They have no electron transport chain proteins because they lack core components needed to form complexes I, III, IV and the F0 (membrane) component of their F0F1 ATP synthase.

They do still form "petit" mitochondria. The F1 component of ATP synthase is present and it works. ANT and Slc25a3 are present and functional. There is CoQ, which is permanently reduced because there is nowhere for it to hand its electrons on to... A number of other cellular processes are also blocked, those which need to reduce CoQ to CoQH2 to occur. From

Restoration of electron transport without proton pumping in mammalian mitochondria

we have:

















The really strange thing is that ρ° cells have a mitochondrial membrane potential and a proton gradient. This is what happens:









ATP which has been made in the cytoplasm enters the mitochondria via ANT running in reverse. The F1 component of the ATP synthase breaks down the ATP to ADP and Pi. ADP is exchanged outwards via the ANT antiporter and Pi is carried outwards in combination with a proton via the Slc25a3 symporter. This proton flux maintains the proton gradient across the inner mitochondria membrane, all of this process is being powered by glycolytic ATP synthesis.

I became interested in ρ° cells because the are so strange. But there are some practical things they tell us too. There's a venerable mini review here:

Cells depleted of mitochondrial DNA (ρ°) yield insight into physiological mechanisms

They cannot perform reverse electron transport through complex I, because there is no complex I. So no superoxide. Equally, there is none from complex III either. Clearly this has implications for what type of apoptosis they can perform and how they sense oxygen tension but more interestingly you can make ρ° versions of pancreatic beta cells.

These can't secrete insulin.

Back in the 1990s no one was thinking about RET as being essential to insulin secretion but they were pretty sure the process was based around mitochondria as well as needing glycolysis. In pancreatic beta cells glycolysis specifically inputs to the ETC at mtG3Pdh in large amounts, which will generate RET and the superoxide needed for insulin secretion. This occurs in other cells as part of insulin responsiveness, but not to the same degree as in the beta cells.

Placing some functional mitochondria in to ρ° beta cells restores insulin secretion ability.

The review suggests mtG3Pdh in beta cells acts as a sensor for cytoplasmic NADH levels. That's a nice idea. Just struck me as interesting.

Peter

Saturday, April 01, 2017

Loki and its membrane potential

Nick Lane makes some interesting comments about Loki, currently accepted as being the closest living descendent of the archaeon which merged with an alpha proteobacterium to generate LECA, the Last Eukaryote Common Ancestor:

Lokiarchaeon is hydrogen dependent

Loki is fascinating. We don't quite have all of its genome, roughly 92% of it. There are bits missing for parts of ATP synthase and for the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex but we can be pretty sure these are in that missing 8% of the genome which we have yet to find and sequence. After all, prokaryotes don't carry junk DNA. Having most of the genes for a functional complex suggests that the rest of those needed to make it work are present.

What is completely absent is anything suggesting any sort of respiratory chain. That's not so unusual, especially in anaerobes.

Or any sort of membrane pump.

No membrane pump? I suspect that there must be small ion pump of some sort either tucked away in the missing 8% of the genome or within some of the currently uninterpretable DNA. Certainly none of the large modern complex pumps are present in any part, so Ech, Rnf and MtrA-H are all out, although the MtrH gene alone is present. I'd assume MtrH is transferring methyl groups to somewhere other than to the absent MtrA-G Na+ pump, over to CODH/ACS seems most likely.

The core energy process appears to be based on using electron bifurcating hydrogenases to generate very low potential ferredoxins. This allows the CODH/ACS complex to generate acetyl phosphate or acetyl-CoA. Substrate level phosphorylation can then give ATP and it's all down hill, energetically speaking, from there onwards. This gives a strict anaerobic metabolism based on an external source of hydrogen.

Obviously a membrane gradient has many uses in addition to ATP synthesis so I wouldn't doubt for a moment that one is present. Keeping it energised is the trick.

It left me thinking about how you might generate a membrane potential in the absence of any obvious relative to modern day ion pumps. I recalled that Koonin had mentioned some very ancient sodium pumps based around either decarboxylation reactions or around pyrophosphate cleavage.

In Evolutionary primacy of sodium bioenergetics he comments:

"These ancestral ATPases [ATP synthase in reverse] would pump Na+ along with the Na+-transporting pyrophosphatase [62] and chemically-driven Na+-pumps, such as Na+-transporting decarboxylase [29,63], which, being found in both bacteria and archaea, appear to antedate the divergence of the three domains of life".

From which ref 62 is a good read

Na+-Pyrophosphatase: A Novel Primary Sodium Pump

"The role of Na+-PPase can be most easily conjectured in the thermophilic marine bacterium, T. maritima, which utilizes Na+ as the primary bioenergetic coupling ion and employs a Na+-ATP-synthase (35, 36). In this organism, Na+-PPase may work in concert with Na+-ATP-synthase to scavenge energy from biosynthetic waste (PPi) in order to maintain the Na+ gradient, especially under energy-limiting conditions".

And for Na+ pumping via conversion of succinate to proprionate:

Bacterial Na+- or H+-coupled ATP Synthases Operating at Low Electrochemical Potential

"A prominent example is Propionigenium modestum, which grows from the fermentation of succinate to propionate and CO2 (Schink and Pfennig, 1982; Dimroth and Schink, 1998). The free energy of this reaction is about -20 kJ/mol whereas approximately -70 kJ/mol is required to support ATP synthesis in growing bacteria (Thauer et al., 1977). To solve this apparent paradox, 3–4 succinate molecules must be converted into propionate before one ATP molecule can be synthesized".


This last process is somewhat more complex than pyrophosphate hydrolysis and looks less of a candidate for "hidden" membrane potential generation than the Na+PPase. After all, CODH/ACS is providing ATP and many reactions which need to be "one-way" cleave ATP to AMP and PPi. The PPi "waste" would then be available to pump Na+.

My guess would be that Loki will turn out to use Na+ membrane energetics...

Time will tell.

Peter

Thursday, March 30, 2017

Amgen share price and PCSK9 inhibition with Repatha (2)

I had an email from a PR company representing Amgen, re Repatha and all cause mortality. Here's the bit of interest:

******************************************************************

I respect your opinion, but did want to share some additional information with you regarding the 2-year length of the study.

FOURIER was an event-driven study and was to conclude when least 1,630 hard major adverse cardiovascular event (MACE) events were accumulated. Amgen expected the study to run for 43 months with a 2 percent annual event rate in the placebo arm. However, the annual event rate in the placebo arm exceeded 3 percent and led to a faster accumulation of hard MACE events. Since the relative risk reduction in the hard MACE composite endpoint grew from 16 percent in the first year to 25 percent beyond 12 months, Amgen anticipates that a longer duration trial would have led to further relative risk reduction.

Would you please consider correcting this sentence of your post?

“The study was stopped early, presumably to stop the hard end points of dead patients from becoming too obvious.”


******************************************************************



You can see how Amgen made their decision. Am I incorrect in my presumption about why the study was terminated early?

Well, technically yes. The protocol is laid out. That's unarguable. So they have a point and have designed the study well, from their point of view.

The fact that 444 people died in the treatment arm vs 426 in the placebo arm was not statistically significant, despite representing a 4.2% increased relative risk of death over the study duration.

What seems to concern Amgen is the implication that all cause mortality had any influence on the decision to terminate the study early. Obviously I cannot know whether this is the case and Amgen are certain that my presumption is incorrect. So maybe some compromise:

If we go with this I can reword the sentence to:

“The study was stopped early due to an unexpected excess of combined cardiac adverse end points in the placebo arm. At this time point the 4.2% increase in relative risk of all cause mortality in the treatment arm was not statistically significant”.

I don't think these facts are arguable with.

Well, that's been interesting. I feel somewhat honoured to have been contacted by a company representing Amgen to correct my presumptions!

Peter

Sunday, March 26, 2017

The pathology of evolution

Aaron posted the link to this paper via Facebook:

Selection in Europeans on Fatty Acid Desaturases Associated with Dietary Changes

As the authors comment in the discussion:

"Agricultural diets would have led to a higher consumption of grains and other plant-derived foods, relative to huntergatherer populations. Alleles that increase the rate of conversion of SC-PUFAs to LC-PUFAs would therefore have been favored".

Or to rephrase it slightly, from the legend of Fig 6:

"The adoption of an agricultural diet would have increased LA and decreased ARA and EPA consumption, potentially causing a deficiency in LC-PUFAs".

This is something I have thought about, in more general terms, for some time.

At the time of the switch from hunting animals for their fat to growing grains for their starch the paper suggests that there was a population-wide potential deficiency of the longer chain PUFA, arachidonic acid, EPA and DHA.

This applied a selection pressure to the population. Within the population there was a random distribution of the ability to elongate and desaturate linoleic and alpha linolenic acids to their longer chain derivatives.

People who had this ability in generous amounts did well. Those without, didn't.

What happened to those people who were "without" the lucky gene snps to survive well without animal derived lipids? They didn't "develop" the genes, no individual suddenly develops a better gene. Their intrinsic inability means they didn't reproduce as successfully.

Their genes are currently under represented in the gene pool today.

It has always struck me that the process of getting poorly adapted genes out of the gene pool is what we describe as pathology, illness. Trying to patch it up is what we call medicine. Individuals don't adapt. They either do well or badly. The population "adapts" through the illnesses of those whose genes are not appropriate to the new environment.

The adaptation of our species to the novel situation of agriculture is far from complete. On-going adaptation of a species to a new environment is via the suffering of the individuals with genes more appropriate to the previous long term stable environment. The default for a person with on-going pathology might be to step back 10,000 years rather than continuing to assist evolution of the species via personal pathology. A lot of pathology will be needed.

Miki Ben-Dor has a nice post along these lines this on his blog.

Peter

Of course the adaptation to sucrose and bulk seed oils has only just begun. LOTS of pathology needed to adapt the species to those two! Juvenile onset type 2 diabetes is what we call the process.

Saturday, March 25, 2017

Amgen share price and PCSK9 inhibition with Repatha

Just a one-liner to bookmark the death of Repatha.

PCSK9 inhibitors have bombed and the cardiology community is in complete denial. Now this is nothing new, it happens on a regular basis. Repatha produced a massive drop in LDL cholesterol and a small drop in cardiac end points. It also produced a small (ns) rise in both total mortality and cardiovascular mortality.


EDIT
I have altered the sentence which used to be here in response to a request from a PR company representing Amgen!!!!!! There's a post about it here.
END EDIT


No-one should ever listen to the cardiovascular community on a cholesterol lowering drug. Instead, just look at the share price of Amgen:


















The red arrow indicates the release date of the FOURIER study data on March 17th 2017. It's a acute adverse event signalling the failure of a blockbuster drug. The trend in share price also indicates the conversion of an upward trend in price to a downward trend at the time of this adverse event.

As always, the cholesterol hypothesis is dead. It keeps on being killed but, obviously, it never lies down!

Amgen have invested an unimaginable amount of money in their PCSK9 inhibitor. This is a gross failure of basic research. A nerd with smart phone could have told them they were gambling on a very long shot.

They lost.

Peter

Friday, March 24, 2017

Will palmitic acid give you cancer or fuel metastasis?

Again thanks to Mike Eades for the full text of this paper and to Marco for poking me about it.

Targeting metastasis-initiating cells through the fatty acid receptor CD36

The executive summary: Both feeding a high fat diet to mice then implanting a certain type of cancer cells or feeding palmitic acid to that certain type of cancer cells pre-implantation makes the cancer much more aggressive once implanted. Up-regulating CD36 (described as a fatty acid transporter) has the same effect.

So. The question is: Should we all abandon high fat diets because fat, particularly palmitic acid, appears to be a promoter of aggressive metastasis?

I have thee things I'd just like to discuss.

I suppose the first is CD36. This is long accepted as a fatty acid transporter which facilitates the entry of FFAs in to those cells which express it on their surface. As far as I was aware this was all it did. My bad. The authors do mention that it promotes the uptake of other substances, including oxLDL, as an aside (they didn't look at this) and they do cite Hale's study using glioblastoms, which is rather more explicit about what CD36 really is:

Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression.

"We confirmed oxidized phospholipids, ligands of CD36, were present in GBM [glioblastomas] and found that the proliferation of CSCs [cancer stem cells], but not non-CSCs, increased with exposure to oxidized low-density lipoprotein".

CD36 is a scaveneger receptor which promotes the uptake of all sorts of lipids and oxidised phospholipids. Of course you can't help but think of 13-HODE and all of the other oxidised omega 6 PUFA derivatives which might or might not have been available to be taken up using extra CD36 receptors. This was not the point of the study, the study was aimed at nailing palmitic acid, to which I will return.

The second point relates to the mice fed the high fat diet.

The mice were fed TD.06414, essentially the same as D12492. Scroll to the bottom of the page to see the metabolic effects!

Lard and sucrose/maltodextrin, designed to produce obesity, hyperglycaemia, hyperinsulinaemia and hyperleptinaemia. No one measured the linoleic acid content of the diet so we can assume, very safely, that the approximate 16% of PUFA in the fat suggested by the manufacturer, is a gross under estimate. No one would expect a diet like this to be anything other than cancer promoting. Throwing in a few extra CD36s will make it worse. Is palmitate the problem in these "high fat" fed mice or is it 13-HODE, other PUFA oxidation products, insulin or leptin?

Point three is the one I'm currently interested in.

Pre incubation of the CD36+ cancer cells with 400micromolar unadulterated palmitic acid, for just 48 hours pre-implantation, promotes markedly increased metastasis when they are injected in to the mouse model. No PUFA, no 13-HODE, no hyperinsulinaemia. Just palmitic acid.

This is undoubtedly the money shot for the research group.

Now, what is going on here? From the focus of my blogging at the moment it's clear that palmitic acid is the highest driver of FADH2 input in to the ETC short of stearic acid. What will 48 hours of high level, uncontrolled FADH2 drive do to reverse electron transport (RET) and the structural integrity of complex I?

This is a model. A concentration of 400micromol palmitate, with no other FFAs, just never happens in real life. This model of extreme palmitate induced RET will force mitochondria to disassemble a pathological amount of their complex I. That's pretty obvious from the work of Guarás. The function of complex I is to reduce the NADH:NAD+ ratio and so disassembling complex I will do the inverse and raise NADH per unit NAD+. I went through the relevance of changes in this ratio, specifically for the generation of aggressive metastatic cancer phenotypes, in 2013 when I posted about Hoffer and B3 therapy for cancer prophylaxis and the modern versions using all of the clever stuff we do nowadays.

Of course you have to wonder about point two above; how much of the cancer promoting effect of obesity might be from the pathology of concurrently elevated fatty acids (reducing complex I availability so NAD+ generation) combined with elevated glucose (supplying the maximum amount of NADH) acting via the NADH:NAD+ ratio, never mind 13-HODE etc. A double whammy.

Personally I'm not about to give up eating butter on the basis of this paper. But that's just me I guess.

Peter

BTW, will blocking CD36 be an anti-cancer adjunct? Quite possibly, especially if it blocks 13-HODE entry in to the cell. Or even if it blocks FFA entry when people can't be ars*d to avoid hyperglycaemia while ever they have chronically elevated FFAs.

Thursday, March 23, 2017

Just a little on complex I and models

OK, just a brief summary of the parts I like most from the excellent

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

We should all have a picture of the ETC looking a bit like this, omitting mtG3Pdh and ignoring supercomplex formation:











Guarás et al set up a model, a proof of principle extreme. They blocked the ETC completely at either complex III, or at cytochrome C or at complex IV. They even discussed short term oxygen deprivation as a generator of ROS, equivalent of blocking the extreme end point of the ETC. If you completely block the ETC in this way then any input through FADH2 containing enzymes to CoQ will always generate a massively reduced CoQ pool, one prerequisite for reverse electron transport (RET),











leading to the near complete disassembly of complex I. It's a model, an extreme version of reality. Fascinating in its own right, as was the ability of the fungal CoQH2 oxidase (AOX) to protect complex I completely from any of the engineered ETC defects:
















AOX may not pump any protons but it does preserve complex I by reducing the extreme levels of CoQH2 which drive RET.

They subsequently went on to look at more physiological ways to generate RET and came to the conclusion that, as the balance of inputs from NADH vs FADH2 shifted, then the amount of complex I relative to complex III would need to be altered. RET is the physiological signal to balance complexes I and III against NADH and FADH2 supply.

Elegant is not the word.

They even went on to ascertain which cysteine residues were preferentially oxidised to disassemble the complex. They group around the FMN and the CoQ docking area, surprise surprise... OK. If you insist, here is the ribbon diagram from fig 5:






















There's a lot of explanation in the text of what the colours and the asterisks mean.

All I really wanted to lay down with this post is that there is a physiological process where FADH2 inputs control complex I abundance. That is how it should be.

When you want a pathological model of complex I destruction, pathological levels of FADH2 input will deliver.

Peter

Thursday, March 16, 2017

Protons: More from Dr Speijer

Mike Eades forwarded this paper to me, by Dr Speijer:

Being right on Q: shaping eukaryotic evolution

How good is it?

He covers a vast field including loads on

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

and

Supercomplex assembly determines electron flux in the mitochondrial electron transport chain

and even

Mitochondrial fatty acid oxidation and oxidative stress: lack of reverse electron transfer-associated production of reactive oxygen species (another gift from Mike Eades).

I've far from finished reading the paper, these are just some of the gems. At some point I really will get round to a bit more on complex I and RET to regulate susbstrate processing but there is rather a lot happening at home and there might be something of a delay. To say the least.

Peter

Stearic acid, FADH2, complex I and cancer

Just a quick aside:

George cited this study in the comments of the last but one post:

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis

While I think it is possible that metformin might inhibit mtG3Pdh at levels below those which inhibit complex I, the complex I effect may well still be equally real.

He goes on to say:

"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".

Remodelling the ETC using RET from FADH2 inputs might be antineoplastic too ie, less complex I would then be available for whatever ox phos the cancer is capable of…

Now: What normal food will generate the highest FADH2 input to the ETC per unit NADH? Correct, stearic acid will.

So what does stearic acid do to breast cancer cells in culture?

Stearate preferentially induces apoptosis in human breast cancer cells


Does it work in a rodent model?

Dietary stearate reduces human breast cancer metastasis burden in athymic nude mice


Do people with breast cancer have low stearate levels in their cell membranes?

Erythrocyte membrane fatty acid composition in cancer patients


Does stearate-driven RET down regulate complex I availability in cancer cells which are partially dependent of glucose derived NADH oxidation via said complex I? And so kill them?

Maybe…

Peter

That poor old C57Bl/6 mouse

There is a is a link within the Guarás et al CoQ paper I put up in the last post (which I'll probably go back to in some detail in future posts). It's to a paper by Lapuente-Brun et al:

Supercomplex Assembly Determines Electron Flux in the Mitochondrial Electron Transport Chain

Here is my doodle, butchered from elsewhere, of the  Supercomplex (SC), also known as the Respirasome:












The things to note are the assembly of complexes I, III and IV in to one unit and that there are enclosed molecules of both CoQ and Cytochrome C within the SC. These electron transporters are isolated from their respective general membrane pools so as to ensure maximal efficiency of electron transfer within the integrated SC.

The SC doesn't just happen. It's glued together by specific assembly proteins. In particular C III and C IV are joined by a protein called Cox7A2l (to be renamed supercomplex assembly factor I, SCAFI).

Unless you are a C57Bl/6 mouse.

If you are a C57/Bl/6 mouse your SCAFI  is 4 amino acids too short. It doesn't work.

The whole, excellent, paper by Lapuente-Brun et al is really about supercomplex formation and preferential assembly of the basic complexes. But because it uses the broken C57Bl/6 as an example of defective respiration it does bring home how irrelevant this particular mouse might be to more humans with a more normal metabolism.

Quite how this defect of SC assembly might make that the C57Bl/6 mouse in to the strange metabolic item which it is is not clear.

But, at the core of normal respiratory supercomplex formation, the Bl/6 mouse broken. That's an awful lot of mouse research which is broken. You could almost feel sorry for obesity researchers. But not quite.

Just sayin'.

Peter

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.

Peter



Saturday, March 04, 2017

Trans fats vs linoleic acid

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TLDR: Trans fats may not be as bad as they are made out to be.
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This paper is comparing a high linoleate diet (using lard) to a soya oil derived (Primex) diet where much of the linoleate has been industrially hydrogenated in to trans fats and fully saturated fats.

A comparison of effects of lard and hydrogenated vegetable shortening on the development of high-fat diet-induced obesity in rats

The thing I like best about it is that, wait for it, they measured the fatty acid composition for their diets! HPLC and all that. Then, they put the results in the paper! On the down side their concepts about energy balance are pure CICO and rats have lingual receptors for fat which link to "hedonistic" centres in the brain. So they understand nothing, but we can forgive them for that.

Here are their results:

















I think this might suggest that linoleic acid is obesogenic. Not that I've ever mentioned that before. Now obesity is obesity. What about health? Here are the numbers which matter, obviously insulin is the one we want to look at:















So, clearly, eating 11% of your calories as linoleic acid makes you fat and ill. The fat in the HVF (and the NF control) diet is made of:

"Diet compositions are presented in Supplementary Table 1. Primex pure vegetable shortening, a mixture of partially hydrogenated soybean and palm oil, was used by Dyets Inc. (Bethlehem, PA, USA) to formulate NF and HVF experimental diets".

Compositions panned out as :























Now these are measured, nothing is accepted from USDA food tables etc. So..... If we look at health outcomes (Table 3) in conjunction with diet composition (Table 1) it become pretty evident that, in these rats, trans fatty acid (TFA) feeding at 15% of total calories is positively health generating compared to linoleic acid feeding at 11% of calories. I did not expect this.



Aside: If any diet trial does not have its fatty acid composition measured you have no idea how much linoleic acid it contains. Usually more than you think. Also, joke of the century so far:

Question: If you are in a position of power over innocent folks who are trying to eat healthy food, which fat would you ban?

Answer: The wrong one!

End aside.



I was on a PubMed search looking for trans fat toxicity. In this current study trans fats not only fail to cause insulin resistance, they render insulin-glucose parameters identical to the NF fed rats, despite the TFA fed rats carrying an extra 100g of adipose tissue.

That is very interesting. You can't answer the whys and wherefores from this paper. The missing piece of information is probably FFAs.

Trans fats are very odd. On acute exposure to TFAs isolated adipocytes release stored FFAs. This is what Cromer et al have to say in their study:

Replacing Cis Octadecenoic [Oleic] Acid with Trans Isomers in Media Containing Rat Adipocytes Stimulates Lipolysis and Inhibits Glucose utilization

"Overall, results of this study clearly show that conversion of octadecenoic acid from the cis isomer [oleic acid] to the trans isomer [elaidic acid] in adipocyte media will substantially increase lipolysis and inhibit glucose oxidation and conversion to cell lipid".

Just to clarify: Acute exposure of freshly isolated adipocytes to trans-oleic acid causes fat release, decreased glucose uptake and decreased glucose incorporation in to lipids. Sounds like a weight loss drug to me.

I was expecting this lipolysis to show up as elevated fasting FFAs and elevated fasting insulin. But there isn't any insulin resistance under fasting conditions visible in Table 3 so I think it is reasonable to assume there is no elevation of FFAs at this time either......

The only explanation I can come up with is that the adipocytes of the TFA fed rats are not as "full" as they should be, due to trans fatty acid induced lipolysis. Giving some "space" within an adipocyte allows the insulin sensitising effect of PUFA oxidation to show, certainly while fasting and lipolysis is the correct state to be in. Hence the fasting insulin levels are normal despite the 100g of extra bodyweight.

Or you could theorise that excess lipolysis from the trans fats is being almost exactly matched by decreased lipolysis from the insulin sensitising effects of linoleic acid. The combination just happens to pan out close to normal, provided the rats carry 100g of excess adipose tissue.

Some degree of post prandial hyperinsulinaemia/insulin signalling seems essential just to maintain those extra 100g of accumulated fat, but it's clearly not visible in the post absorptive phase.

If anyone can come up with a better explanation, I'm all ears.



BTW, this obviously relates to Axen and Axen's work. Their hyper-obesogenic diet was based on something Crisco-ish from the 1990's:

"The hydrogenated vegetable fat contained ∼25% long-chain saturated, ∼44% monounsaturated and ∼28% PUFA, with ∼17% of total fat as trans fatty acids (manufacturer’s communication)".

With fat making up 60% of the calories in the diet, and that fat being 28% PUFA, this is somewhere around 17% of total calories as linoleic acid. That is a LOT of linoleic acid. At the time I thought that the trans fatty acids would be to blame. Nowadays I'm not so sure.


How much of the bad rap that trans fats have received is from the PUFA which travelled with them at the time? Without mentioning the amount of fructose in the biscuits. Modern Primex is much more hydrogenated, so lower in PUFA, than whatever Axen and Axen used. It's far less obesogenic too.

Peter

Other odd final thought: People who are obese and insulin sensitive: Are they the folks who eat most trans fats along with their hearthealthypolyunsaturated linoleic acid???????

Friday, March 03, 2017

Mitochondria in cancer cells

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TLDR: This is just a speculative post about a paper which is interesting from the Protons point of view but is not really related to anything else.
******************************************************************

This is the paper by Catalina-Rodriguez et al:

The mitochondrial citrate transporter, CIC, is essential for mitochondrial homeostasis

It is interesting on many levels. At the most basic, it gives you the actual concentration of glucose used to grow the cancer cells. This is pretty unusual.

I picked the paper up while looking at what Lisanti has published recently on the fuelling of cancer growth using ketones derived from glucose via cancer associated fibroblasts. He appears to have been asked to write a commentary on the Catalina-Rodriguez paper. Here it is:

Genetic Induction of the Warburg Effect Inhibits Tumor Growth

The basic idea of the commentary, partly agreed with by Catalina-Rodriguez, is that genetically inducing the Warburg Effect in cancer cells kills them. This is obviously very compatible with Lisanti's ideas, that cancer cells generate ATP via ox phos, and is the diametric opposite of the Warburg Effect.

But the paper by Catalina-Rodriguez, while it does discuss the Warburg Effect, also provides enough information that other explanations are tenable beyond those supported by Lisanti. Anyone looking at Lisanti's work without considering other explanations needs to be more circumspect. The paper is all about the mitochondrial citrate transporter, CIC.

We had better put CIC in to context first. It exports citrate from the mitochondria to liberate mitochondrial derived acetyl-CoA to the cytoplasm for anabolic processes.

It's an antiporter, it exchanges citric acid for malate. Citrate out, acetyl-CoA released, residual oxaloacetate reduced to malate via NADH, malate re enters the TCA.


















We can add this to the TCA like this:

















So CIC exports citrate and imports malate. Because citrate carries 3 negative charges and malate only two, a proton is carried out with the citrate which maintains electrical neutrality but generates a pH gradient. It's also clear that a cytoplasmic NADH is consumed converting oxaloacetate to malate and this is regenerated within the mitochondria by the TCA as malate reconverts back to oxaloacetate.

So we can look at this as a cycle:

















It bypasses all of the energetic processes in the rest of the TCA so delivers very little to the ETC. The normal function of this pathway appears to be a safety valve for the cell when the mitochondria are presented with far too much acetyl-CoA.

In some cancer cells things go very awry.

Two hallmarks of cancer cells appear to be abnormal electron transport function and free radical leakage from the ETC. The free radical leakage is at levels which cause proliferation rather than apoptosis.

Complex I primarily reduces NADH to NAD+. If we have a dysfunctional complex I (either directly or by poor function downstream in the ETC) there is going to be a great deal of NADH per unit NAD+ within the matrix. High levels of NADH inhibit isocitrate dehydrogenase and so drive citrate export in to the aberrant CIC cycle. The cost is limited to one imported cytoplasmic NADH, the benefits are reduced generation of TCA derived NADH and FADH2... Plus cytosolic citrate is an inhibitor of glycolysis, so works as a brake on the excessive supply of pyruvate derived acetyl-CoA.

So some degree of poor ETC function can be accommodated by exporting acetyl-CoA rather than turning the TCA. The red cycle in the above doodle predominates and the TCA is almost inactive.

Under these conditions the mitochondria are largely protected from their dysfunctional electron transport chains. There are ROS (mostly superoxide) being generated but at levels which are not fatal to the cell although they can drive mutagenesis, in both mitochondrial and nuclear genomes.

What happens if you inhibit CIC in a cancer cell which is using CIC to both limit ROS production and to deliver cytoplasmic acetyl-CoA for anabolism? The whole extra-mitochondrial cycle should shut down, meaning acetyl-CoA has nowhere to go other than around the TCA. Less citrate can be exported, so cytosolic citrate levels fall. Falling cytoplasmic citrate allows increased glycolysis and this can feed even more acetyl-CoA in to the TCA. Complex I is still dysfunctional so we are then in to a massively elevated NADH:NAD+ ratio. We will also increase FADH2 driven CoQ reduction via complex II as the TCA turns. A high NADH:NAD+ is the prerequisite to getting an electron on to an oxygen atom to generate superoxide in complex I, back in Protons (22) I speculated this might be at FeS N-1a. A reduced CoQ couple also drives this via reverse electron transport.

Putting an electron on to oxygen aborts all of its downstream proton pumping. What might happen if a very high percentage of electrons jumped ship at complex I due to a grossly elevated NADH:NAD ratio and a reduced CoQ couple?

Increased superoxide, more oxygen consumption (though not necessarily at complex IV), much more ROS. Might the ROS release cytochrome C by oxidation of its cardiolipins, so giving collapse of the mitochondrial membrane potential and triggering apoptosis?

This is not the "genetic induction of the Warburg Effect", however Lisanti describes it. This is over driving a dysfunctional electron transport chain to the point of mitochondrial destruction. The most telling results section graph is this one:
























These are the death rates for cancer cell cultures. On the left are three control cultures with functional CIC, on the right are those treated with BTA, a CIC inhibitor. NAC is n-acetyl cysteine. Which is the most lethal treatment? I think we must all agree with the devastating effect of pyruvate alone as being the most lethal treatment. Which gets zero mention in the text anywhere that I can find...

My idea would be that pyruvate is toxic because it bypasses glycolysis so cannot be shut down by the glycolytic inhibitory effect of raised cytoplasmic citrate. There is a spike of acetyl-CoA which cannot be avoided and which CIC levels are not set up to deal with. More acetyl-CoA enters the TCA, more NADH, more superoxide from complex I... The authors focus instead on the fifth column where pyruvate rescues cells from BTA toxicity. I can see no logic to this and have no explanation for that particular result. At least I do actually mention the results which don't fit my hypothesis!

The next very interesting result is the effect of uncoupling. CCCP is an uncoupler which reduces the MMP. This leads to mitophagy +/- apoptosis. A functional CIC cycle saves cells from CCCP damage. You could argue that CIC is just a Saviour of Distressed Cells. But on a more interesting level: Uncoupling normally markedly increases electron flow down the ETC. In normal mitochondria this reduces ROS generation. But if the ETC is leaking electrons to generate ROS under even normal flow it's easy to see why uncoupling can allow massive ROS generation. CIC is protective because it keeps electrons out of the ETC.

They also found that CCCP induces CIC production. My assumption would be that CIC is induced by ROS and CCCP generates ROS. The same thing happens with rotenone by the opposite process. Rotenone does not uncouple, it blocks the ETC at the exit of complex I. This gives reduced conversion of NADH to NAD+, markedly increase the NADH:NAD+ ratio, electrons jump to O2 to N-1a to form superoxide... CIC induction is the solution to limit this.

There are some very interesting results here.

I have absolutely nothing against looking at CIC inhibition as a management for certain subgroups of cancers. It may well help, but thinking about what is actually happening might give more insight.

Pete

Wednesday, February 15, 2017

Linoleic acid and Tuberous Sclerosis

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TLDR: I don't like linoleic acid much.
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Tuberous Sclerosis (TS) is a genetic disease which affects mTOR signalling and predisposes to many problems, one of which is early onset kidney tumours. People with TS also have a tendency to suffer from intractable epilepsy so, almost by accident, a number of them have had their tumours closely monitored while eating a deeply ketogenic diet for the epilepsy. Does the KD help slow tumour growth?

No.

To look at this in more detail a group in Poland has used a TS rat model and tried to manage the disease with a KD. This is the study:

Long-term High Fat Ketogenic Diet Promotes Renal Tumor Growth in a Rat Model of Tuberous Sclerosis

It is a very interesting paper. All rats were euthanased at 14 months of age, having spent differing periods of time eating a close derivative of the F3666 ketogenic diet. The longer the rats had spent on this diet, the more aggressive the renal tumour progression was found to be, especially in the rats which ate it for eight months (the longest duration).

Other than this there were some additional interesting findings. Insulin and glucose did (almost) exactly what you would expect:






   


















What is more interesting is that growth hormone increased progressively with duration of time spent on the KD:




















This was noted by the authors and was considered as one of the potential explanations of the increase in tumour burden after 8 months on KD:

"Likewise, it has been shown that the growth hormone activates the MAPK pathway, thus its overproduction in the ketogenic groups may also boost ERK1/2 phosphorylation. We believe that HFKD induced the ERK1/2 activation results as a cumulative effect of the renal oleic acid accumulation and the systemic growth hormone overproduction".

I'm not convinced by the oleic acid idea but no one would argue against identifying elevated growth hormone as a stimulant for tumour growth.



Overall we have progressively falling levels of both glucose and insulin, with a progressively rising growth hormone concentration, over the eight months of a ketogenic diet. I asked myself if there might be an explanation for the nature of these reciprocal changes, before thinking about the tumour growth.

Looking at the insulin-glucose levels we can say that insulin sensitivity increased with time on the ketogenic diet, using the surrogate HOMA score based on the product of insulin and glucose. That's despite a concurrent tripling of GH levels, which should induce insulin resistance.

The obvious concept is that GH was being used to maintain normoglycaemia in a set of rats which were developing progressively increasing (pathological) insulin sensitivity and might, theoretically, have become hypoglycaemic on a very low carbohydrate, very low protein diet. That is, despite having been on a high fat diet, they failed to maintain adequate (ie physiological) insulin resistance to spare glucose for the brain.

Does GH sound like a metabolic solution for the problem of pathologically increasing insulin sensitivity? Pathological insulin sensitivity: Is anyone thinking linoleic acid? Well, I am (now there's a surprise).

The rats were raised on a standard chow of un-stated composition before switching to their KD. It seems a reasonable assumption that the chow was relatively low in fat. Exactly how much linoleic acid was supplied is unknown but other rodent chows I've seen described or analysed tend to provide about 2% of calories as linoleic acid.

The F3666 derived diet looks, depending on the lard composition, to be in the region of 18% omega 6 PUFA. That's high (yes, another rodent study has shown that mice develop NASH on this diet, no surprise there).

The rats were raised initially on a starch based diet so their juvenile adipose tissue would probably be composed of DNL derived saturated and monounsaturated fats, supplemented by a little PUFA from the diet. Transition to F3666, which provides approximately 18% of calories in the form of linoleic acid, generates a metabolism much more dependent on linoleic acid. The younger the rats were when they switched to F3666, the less "normal" adipose tissue they would have had available and the more rapidly they would end up with adipose tissue (and plasma) high in linoleic acid.

Rats on true ketogenic diets do not become obese, even on F3666. So we have slim, omega 6 fed rats. Small adipocytes, no excess FFA release, no insulin sensitivity differential between adipocytes and the rest of the body. There is nothing to over-ride the insulin sensitising effect of linoleic acid. Both adipocytes and the rest of the body become progressively more insulin sensitive mediated through linoleic acid. They don't become obese because insulin stays so low due to the lack of carbohydrate and protein. The excess insulin sensitivity only kicks in gradually because their pre-stored, chow derived adipose tissue provides a supply of physiological FFAs which can act as a buffer to the sensitising effect of 18% linoleic acid for a while.

Glucose falls progressively due to the development of progressively increasing pathological insulin sensitivity, linoleic acid induced. GH may well be a stress response to maintain normoglycaemia under these conditions. The GH may or may not be acting as a tumour promoter, but we cannot ignore the role of linoleic acid in its elevation.


Now the tumours.

We all remember Sauer's rats with their xenografts which grew like wildfire as soon as he starved them? Yes. The tumours grew because they were exposed to linoleic acid released from their adipocytes under starvation. Linoleic acid is a precursor for 13-hydroxyoctadecadienoic acid, better known as 13-HODE. Sauer demonstrated that this was the problem very neatly, at the cost of extensive vivisection. I doubt anyone would be allowed to replicate his work today.

We have no idea of either the linoleic acid or the 13-HODE concentration in the plasma of the F3666 fed TS rats. It would be interesting to know. It might matter...

I particularly think it might matter because F3666 is going to be the "off the shelf" KD that a lot of researchers are going to use.....



At the end of the last post I mentioned that fact that any person who is currently obese through following conventional advice to replace healthy saturated fats with 13-HODE generating linoleic acid is probably carting around kilos of a tumour growth-promoting precursor. In Sauer's study all that was needed to release the linoleic acid was starvation. I would suggest that ketogenic eating might do the same, especially if it is based around saturophobic stupidity (think of kids in the USA with tuberous sclerosis on a "medical" ketogenic diet, or the rats in the above study). There is also anecdote on tinternet that patients of Dr Atkins did fine if they had CVD but those with cancer did badly. I find this plausible. They were obese because they were loaded with linoleic acid and they may well have followed an Atkins diet high in hearthealthypolyunsaturates. That's a good way to grow a cancer.

Sauer found a solution in the form of fish oil to limit tumour growth in his rats, most especially EPA. The very long chain omega 3 PUFAs activate g-protein coupled receptors to reduce lipolysis from adipocytes and activate fatty acid oxidation from the diet. VLC omega-3 fatty acids do not promote excessive insulin sensitivity via the Protons based FADH2:NADH ratio concept because they are specifically oxidised in peroxisomes, not mitochonria. The peroxisomes shorten them to C8 length and then pass this to mitochondria as caprylic acid which has a "palmitate-like" FADH2:NADH ratio of 0.47 which is fine for maintaining physiological insulin resistance.

You do have to wonder whether the benefits of fish/oil in a population loaded with linoleic acid might stem largely from this effect of limiting adipocyte release of that linoleic acid. An interesting idea.

I still find it breathtaking how much the lipid hypothesis of heart disease might have done to injure individual people exposed to its recommendations. Which includes much of the world.

Peter

Thursday, February 09, 2017

Musing about linoleic acid

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TLDR: I have long wanted to know how you could have differential insulin resistance between adipocytes and the rest of the body. Linoleic acid appears to be the answer
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This is a set of thoughts, jotted down without references, that have been of interest to me over the last six months while I have neglected poor old Hyperlipid.


Linoleic acid produces excessive whole body insulin sensitivity.

Adipocytes distend under this effect.

Distended adipocytes release unregulated FFAs.

FFAs cause insulin resistance which eventually overcomes the excess systemic insulin sensitivity.

Hyperinsulinaemia results.



Here we go.

I have nothing against the role of both sucrose and refined starch in the pathogenesis of both obesity and insulin resistance. Anyone who has read Weston Price or Vilhjalmur Stefansson will be only too aware that the substances most easily transported over long distances to affect unacculturated peoples were sugar and flour. No one was carting margarine or corn oil to the Arctic in the early part of the last century. Both fructose and alcohol, both of which deliver largely uncontrolled calories in to cells, can clearly generate aspects of metabolic syndrome. However I am interested in linoleic acid and free fatty acid release from adipocytes at the moment, so I'll leave the case against sugar for the time being. So, linoleic acid:



One of the core ideas which came out of the Protons thread was that palmitic acid is a generator of physiological insulin resistance. The complementary fatty acid is palmitoleate and this generates less insulin resistance for when insulin action is desirable.

The function of physiological insulin resistance is to limit ingress of calories in to a cell when there is a surfeit of calories available. Or to limit the ingress of glucose when glucose is in short supply and it's best not to waste it on non-glucose dependent tissues.

In to this well balanced system comes linoleic acid as a bulk nutrient. The oxidation of linoleic acid, by the Protons hypothesis, produces even less insulin resistance than palmitoleate and so undermines the ability of a given cell to refuse caloric ingress in excess of its needs. Failure to develop insulin resistance means that insulin continues to act.

Continued action of insulin in a calorie replete cell results in diversion of excess calories to intracellular triglycerides (+/- glycogen). This is very reasonable in adipocytes, at the cost of obesity, but less acceptable in tissues such as muscle, liver and pancreas.

The underlying pathology is continued inappropriate insulin sensitivity.



But obesity is a condition more normally associated with insulin resistance.

From the Protons point of view the question is: How can linoleic acid acid, which results in pathological insulin sensitivity whole body, eventually result in insulin resistance, also whole body?

Insulin activates lipoprotein lipase and inhibits hormone sensitive lipase. Combined, these effects facilitate fat storage in adipocytes. But there is another lipase which controls both basal and stimulated lipolysis known as Adipocyte Triglyceride Lipase (ATGL). One, amongst the several, factors which control ATGL is perilipin A, a protein which surrounds the lipid droplet in adipocytes. It is probably an interaction between ATGL and perilipin A which determines the increase in basal lipolysis as adipocyte lipid droplet size increases.


So there is a balance. Linoleic acid is allowing increased insulin action and so causing fat accumulation with a suppression of both FFA release and adipocyte lipid turnover. ATGL is looking to limit adipocyte distension by allowing lipolysis, so raising FFAs, outside of the control of insulin. But will only act on basal lipolysis in response to progressive lipid droplet expansion.

For as long as the pathological sensitivity to insulin exceeds the FFA release driven by ATGL we can have worsening obesity but metabolic syndrome is delayed.

Once ATGL mediated lipolysis raises systemic FFA levels enough, despite insulin continuing to act on adipocytes, we can then have systemic insulin resistance with insulin sensitive adipocytes. Insulin resistance when combined with a carbohydrate based diet requires elevated insulin levels which will continue to act on the insulin sensitive adipocytes. Which will increase ATGL driven lipolysis...

This is metabolic syndrome.

Once the elevated glucose from insulin resistance kills off enough beta cells then insulin levels drop, glucose levels rise, HSL is disinhibited so FFAs rise. You might even get ketoacidosis. This is type 2 diabetes. ATGL might even take a break.

The first approach to correcting it is carbohydrate restriction, so dropping hyperinsulinaemia and minimising the vicious cycle. Doing something about the kilos of linoleic acid stored in an obese person's adipocytes is an altogether longer term project.

Peter

Can linoleic acid keep you slim?

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TLDR This is a weird study which might show the insulin sensitising effect of linoleic acid in obesity resistant rats.
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Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites


I deeply dislike this paper on so many levels. I'm not going to go through everything which is wrong with it, I'll just highlight a few aspects which strike me as interesting.

The first (unexpected) finding is that none of the groups of rats on high fat diets were significantly heavier than the chow fed rats. The PUFA fed rats were slightly lighter (you read that correctly) and the SAT fed rats were slightly heavier, but all ns at eight weeks of feeding. Who can buy rats which can eat coconut/lard and not gain weight? Korean rats? Korean lard? Who knows. But neither the safflower oil nor the lard fed rats become obese. I find this very, very odd in a set of "we bought them off the shelf" Wistar rats. But well, maybe. Just seems odd.

Second point (expected) is that not only did the SAT fed rats develop insulin resistance, but the PUFA fed rats did not, in fact they showed enhanced insulin sensitivity when compared to either chow or SAT fed rats. This is compatible with the Protons view of PUFA oxidation in non-adipocyte distended rats.

Third (expected) is that as insulin acted on muscles under PUFA those muscles developed the highest levels of stored triglycerides. This is exactly what you would expect, insulin diverts excess calories to be safely sequestered as triglycerides.

The fourth point (very unexpected) is that while triglycerides were being happily sequestered in to muscle, they weren't being sequestered in to adipocytes. Adipocyte sequestration of triglycerides (obesity) happens routinely in a huge swathe of linoleic acid feeding trials in many different species.



Sooo, can you extract anything from this mass of contradictions? I'm just wondering whether if you happened upon a generally obesity resistant rat strain you might be left viewing the insulin sensitising effect of PUFA oxidation without the insulin resistance generating effect of adipocyte distension.

Maybe it separates out the the opposing drives on the adipocyte under PUFA exposure...

Maybe.

Peter