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...


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...


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.


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.


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.


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.


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.


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!