Friday, March 15, 2019

Tucker on omega six PUFA

While I've been day dreaming about antiporters at the origin of life Tucker has been busy. I guess most people reading here would read Tucker anyway but just in case not, here's the link

Response to Gary Taubes on Omega-6 Fats (Seed Oils) and Obesity

Personally I have no issue with the two concepts being complementary. Carbohydrate in quantities which get glucose past the liver will drive up systemic insulin. Omega six (and 18C omega three) fatty acids will make adipocytes hyper-respond to the obesogenic signal of elevated systemic insulin. Fat (largely dietary sourced) is then lost in to adipocytes. Loss of this fat is the equivalent of not having eaten it, so you are left hungry. It's the old weight gain causes hunger paradigm. I like it.


Wednesday, March 06, 2019

Life (24) Porting over CCCP

Just to summarise: The membrane bound hydrogenase (MBH) of Pyrococcus furiosus pushes a proton outwards through a proton permeable membrane which then returns through a subunit of MBH which looks very much like three quarters of the Mrp antiporter. The Mrp antiporter is derived from a very, very ancient antiporter which appears to have been one of the core systems of the last universal common ancestor (LUCA). Which ion travels in which direction in the modern families of Mrp is currently not particularly clear and may depend on all sorts of factors. OK.

No one has worked out the detailed structure/function of the Mrp antiporter, but there is a very interesting paper from back in 2001 which might give us some insight about function at least.

Mrp‐dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters

The study used modern E coli whose plasma membrane is tight to both protons and Na+. A strain with all of its antiporters deleted was used and then plasmids were engineered to supply a single gene antiporter of the nahA type (also from E coli) or an Mrp from Bacillus pseudofirmus OF4 (yup, that's its name) or one from Bacillus subtilis.

Respectively we have strains ending in -118 endowed with the blank plasmid, -nhaA for the monogenic antiporter, -BSmrp (B subtilis Mrp) or -OFmrp (B pseudofirmus Mrp). They stuck the engineered E coli in to 25mmol of NaCl, fed it and looked at the intracellular Na+ concentration. Very simple.

The first two columns are exactly what you would expect. Having no antiporter gives an intracellular Na+ in equilibrium with culture medium Na+ at around 25mmol, near enough. Any antiporter rescues this, the nhaA monogenic antiporter somewhat better than either of the Mrp antiporters.

Now here comes the clever bit. They added CCCP, a classical protonophore which drops the membrane potential from 150-ish mV to 15-ish mV (obviously the membrane voltage should be zero but the E coli complex I will be working flat out to stop this fatal occurrence plus I suspect the dose of CCCP used was less than supramaximal, though I've not checked this), and then they looked at the intracellular Na+ concentration. So they have suddenly converted a modern E coli to an E coli with a proton permeable, Na+ impermeable cell membrane. Like Pyrococcus but without the boiling water. Or like LUCA. Both organisms to which Mrp antiporting is/was very important.

The simple nhaA is utterly dependent on a proton tight membrane with a transmembrane proton gradient and it fails to antiport anything in the presence of CCCP, as you would expect (line two, both right hand columns). The nhaA is not primordial.

Both of the Mrp type antiporters maintain an intracellular Na+ between 13.4mmol and 16.9mmol, which is "high-but-physiological", using a CCCP proton "leaky" membrane voltage of 15 mV to effectively pump out Na+.

Quite how the Mrp antiporters do this is unclear. Most of the work has been done on the big subunits, A and D. One is probably a proton channel and one a Na+ channel but even this is not completely clear and in Yu et al's Structure of an Ancient Respiratory System they consider both MrpA and D to have proton channels. So it's messy. The combined small subunits, MrpE, MrpF and MrpG, plus the tail end of MrpA appear to be a proton/Na+ antiporter in their own right. I'll refer to this section as a "simple" antiporter.

Clearly, trying to get a Na+ gradient from a proton leaky membrane by making use of a monogenic nhaA type antiporter doesn't work. Using an Mrp antiporter does.

OK, wild speculation time.

I consider the arrangement in Pyrococcus furiosus is necessary because the cell membrane is permeable to protons. Pump a proton outwards and generally it will boomerang back. Pump it directly in to the mouth of an antiporter and it will return while antiporting a Na+ outwards. The Na+ stays outside. It does this using much of the Mrp machinery.

The end game is to drop a precious proton down the throat of the simple antiporter, without losing it through a leaky membrane. I think the Pyrococcus MBH keeps this proton "in-complex" to avoid losing it. Power is probably supplied to the left hand proton channel from the FeNi hydrogenase by the loop cluster and helix HL.

Like this, yellow circles are antiporters:

Looking at Mrp, I think it is the precursor of the MBH and is doing exactly the same thing but geochemically, ie it is an adaptation to a low geochemical proton gradient across a proton leaky membrane. It still takes the proton from a 15mV proton motive force but it initially uses this to antiport another proton outwards, protects this one from loss through the leaky membrane by keeping it "in-complex" and uses this to antiport Na+ outwards, which stays outside:

In both diagrams everything is identical to the right of the FeNi hydrogenase or MrpA N-terminal domain. All that differs is the method for "elevating" the guarded proton to the entrance of the simple antiporter on the right.

TLDR: Mrp is an adaptation to a failing geochemical proton gradient. Membrane bound hydrogenase is the adaptation to a failed geochemical gradient.

It is amazing to me that Mrp keeps its core function today despite the radically different tasks (saline and alkaline tolerance) which it is needed for nowadays. All we need is a proton leaky membrane to return it to do what it did initially. No change.

Okay, it's not an envelope, its the back of a chocolate wrapper. Speculation is such fun, given enough chocolate.


Life (23) Antiporting: Choose your ion well

Sorry to go on about Yu et al's paper

Structure of an Ancient Respiratory System

but there's an awful lot in it. Here again is their working model of the MBH from Pyrococcus furiosus:

The Na+ channel on the right hand end shown as MbhC was looked at in great detail in the paper and is very convincingly a Na+ channel. The group never looked at the actual structure of the unpowered antiporter Mrp so they are working with other people's assumptions, some of which are quite likely better than others as regards the nature of the ion favoured by a particular channel. So here is the membrane arm of MBH they ended up with, labelled to represent the unpowered original Mrp antiporter. Obviously the hydrogenase has been replaced by the MrpA N-terminal:

Now the problem with this, apart from the highly electrogenic charge exchange of three protons in for one Na+ out, is that the left hand MrpA module is actually a Na+ channel. There's lots of pretty convincing evidence for this but I won't waffle on any more about it. So lets set up the Mrp antiporter as a pure H+/Na+ exchanger like this:

Two protons inwards, two Na+ antiported outwards, what could be nicer? This is the structure favoured by group in Sweden comparing MrpA and MrpD with the antiporter-like subunits of complex I.

Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN

 I think it might be completely wrong.

I like this paper but I'm still left with a niggling thought that having two antiporters in one complex, apparently both doing the same thing, is just a little wasteful. The normal bacterial approach is one where the jettisoning of genes is developed to a fine art.

So Yu et al and Sperling et al have rather different ideas about the function of various channels in the Mrp complex. No one is really talking about why there should be four channels...

The modern Mrp antiporter is very interesting in it's own right. It needs a little post of its own.


Thursday, February 28, 2019

Thinking about things

I'm just following trails from the Mrp antiporter derived subunit of the membrane bound hydrogenase of Pyrococcus furiosus. The original Mrp antiporter is, as you would hope, among those 60-odd gene families going right back to LUCA. Excellent stuff in here:

One step beyond a ribosome: The ancient anaerobic core

The final comment in the conclusions is this

"With regard to the most primitive forms of microbial physiology, microbiologists reached the same conclusion 45 years ago [26], namely that methanogens and acetogens probably represent the most ancient lineages [36]. We required 2000 genomes and powerful computers for our conclusions, while Decker et al. just thought about it. Evidently, just thinking about things can be a source of scientific progress".

That is so cool.


Tuesday, February 26, 2019

The internet is a strange place (2)

Tom Naughton has a post up which pretty well sums up why I don't use twitter. It also encapsulates why I do respect those people who continue to do so and are willing to endure Twitter Dumb as a result of actively promoting the consumption of Food to crowds with wisdom. As opposed to the Hyperlipid approach of: "Here's the info, do what you like with it". Mea culpa.

However, I do feel Tom is a little harsh in places.

In particularly he is grossly insulting to the mental abilities of some of the more common root vegetables. If a person in category three of Twitter Dumb has insight comparable to that of a turnip, how does that make the turnip feel? More fascinatingly, how do Twitter Dumb folks survive in the real world?

Happily the explanation for the continued existence of the average category three Twitter Dumb is summarised in this paper. Most of which is composed of category three Twitter Dumb concepts

Processed foods and food reward

but it includes this gem sentence:

"All organisms must procure energy to survive, and most lack higher-order brain functions that support consciousness".

There you go.


Also relevant:

Sunday, February 24, 2019

Life (22) FeNi hydrogenase

OK, more doodles. More on Yu's paper.

Back in 2015 I produced this diagram based around Nick Lane's ideas and labwork:

Please note that the inclusion of three FeS clusters in the diagram is a complete fluke. No prescience involved! I went on to concentrate on the left hand side of the diagram to give this:

I apologised at the start of the following post because the diagram is upside down by modern convention. So let me turn it the correct way up here and alter the shapes a little, not changing anything basic. Like this:

This is the basic plan for a membrane bound FeNi hydrogenase. Obviously the exact shape is a cheat.  Lets look at the basic structure of a real life type 4 FeNi hydrogenase, say the one from the MBH of Pyrococcus furiosus. Which looks like this, ignoring the pumping/antiporting subunits (not shown):

Which is clear as mud. Until you overlay the doodle:

Look at those three embedded FeS clusters from the nickel catalytic core to the ferredoxin docking site, perfectly set up for electron tunneling! The H+ exit track is really as shown (though the blue arrow is my guess) and the hollow core of the gold section (MbhM) does connect to the outside. I omitted, by accident, that the track to ferredoxin is that of electrons freed from hydrogen. I'm showing the hydrogenase splitting hydrogen as per vent conditions. Nowadays it runs the other way (usually fed on fructose of all things) with hydrogen as waste.

Stuff makes sense.


Thursday, February 21, 2019

Life (21) Pyrococcus furiosus

Pyrococcus furiosus is an interesting organism. It has a penchant for living in environments at around 100degC. It looks like it has occupied this niche for a very, very long time. It has a proton permeable, Na+ impermeable cell membrane. At 100degC constructing a proton tight membrane appears to be bloody difficult.

At some point Pyrococcus hopped from an alkaline hydrothermal vent to a volcanic black smoker type hydrothermal vent. It went, as I've argued, with a proton leaky membrane using Na+ energetics to generate ATP. Given the tools available, how did this work and what do the metabolic fossils look like?

An alkaline vent driven proto-Ech is core. It uses the proton gradient to reduce ferredoxin using molecular hydrogen. Proton (and hydroxyl) permeability is essential to neutralise the entering protons and allow the process to be continuous. At the time I wrote the Life series I felt this was unlikely to be a reversible process. I was wrong, it is.

Here is the initial proto-Ech generating reduced ferredoxin using molecular hydrogen, taken from here and here.

And here it is slightly tweaked, running in reverse, pumping protons pointlessly out through a proton permeable membrane and generating hydrogen as waste:

The second core evolutionary development is the vent proton gradient driven antiporter. This uses vent conditions to reduce the intracellular Na+ concentration:

Whatever the initial advantage of extruding Na+ from the cell might have been the major subsequent development was the formation (twice) of the Na+ ATP synthase. This Na+ ATP synthase is, ultimately, powered by the ocean to vent proton flow and permeability to protons (and hydroxyl ions) is still essential to maintain the influx of protons.

At this time life has available a proton pump, a proton leaky membrane, a proton/Na+ antiporter and a Na+ ATP synthase.

There is no point in pumping protons across a proton permeable membrane, especially if you leave the vent and every ferredoxin molecule becomes precious and life must become frugal.

What if you physically joined the proton pump and the antiporter together? So that the pumped proton stayed within the pump-antiporter complex and was never actually freed in to the environment, but was simply delivered to the proton entrance of the antiporter? So as to return to the cell, exchanging a Na+ ion outwards? Like this:

The cell membrane is still proton leaky, Na+ opaque and an Na+ ATP synthase is driven by the Na+ gradient. Like Pyrococcus furiosus today.

Does Pyrococcus have this reverse proto-Ech physically coupled to an antiporter?

Here is the image taken from this superb paper Structure of an Ancient Respiratory System:

The blue protein is what I've called proto-Ech running in reverse, generating waste H2 from reduced ferredoxin. It uses this redox reaction to pump a proton out (downwards) through the left side of the "H+ translocation module" which then (in my head) returns directly (upwards) through the right hand side of the "H+ translocation module" which is the proton half of the antiporter. This gives an associated antiported Na+ outward (downwards) via the "Na+ translocation module", second half of the antiporter. This goes off to drive Na+ energetics.

That's how it is. I think what Pyrococcus does is a derivation of exactly what LUCA would have done to leave alkaline hydrothermal vents. It's preserved due to the chance environment which makes proton tightening of the cell membrane impracticable...

Genuine Ech and Complex I are different, they use similar subunits but the redox component is on the opposite end of the intramembrane/antiporter section and this looks to be a later derivative to me, secondary to a proton tight membrane and the change to using proton pumping with blocked Na+ ingression, a far more complex process (accidental pun). And there are other Na+ pumps too. But this one in Pyrococcus, it's the one which should be there. And it is.

Made my day.


Saturday, February 16, 2019

The internet is a strange place

I don't do twitter or facebook (If you are a metabolic person and have friend-requested me and I've ignored it please don't take it personally, it's just not what I do with faceache) to any extent and almost never use them for metabolic subjects. By an accident of twittard I picked up this tweet by Chris Masterjohn (via raphi and Mike Eades). It made me laugh out loud and still has me giggling occasionally:

"No! Carbohydrate restriction is the stupidest approach to fatty liver ever devised. If it “works” in any case it is almost certainly by supplying more methionine and choline, not by lowering carbs. It is impossible to make more fat from carb than you get by eating fat"

I can't help but think "chylomicron", "thoracic duct" and "physiology".

Then I giggle some more.


I guess it ranks along side of "Masterjohn", "Martha" and "RQ 0.454".

Edit on 27th Feb: Published 2 days ago, very post hoc. This is what happens to surrogates for NAFLD when people apply the “stupidest approach to fatty liver ever devised”.

Post hoc analyses of surrogate markers of non-alcoholic fatty liver disease (NAFLD) and liver fibrosis in patients with type 2 diabetes in a digitally supported continuous care intervention: an open-label, non-randomised controlled study

End edit.

Sunday, February 10, 2019

Cell surface oxygen consumption (3) Alternative options

Have a look at this:

Limits of aerobic metabolism in cancer cells

"To gain a better understanding of cell metabolism as a function of the growth metabolic demand we performed a back-of-the-envelope calculation focusing on the major biomass components of mammalian cells".

In these days of "shotgun metabolomics" two people appear to have sat down with one or more sheets of paper (possibly larger than an envelope, though you can get some quite large envelopes I guess) and have gone back to basic first principles. They then published in a Nature journal. I love this. I feel it rates alongside getting this image published in Cell Metabolism.

TLDR for the paper:

Glucose drops through glycolysis to lactate at a rate where ATP generation per minute massively outstrips that available from mitochondrial oxphos. In redox balance. It's fast.

Anabolism from glucose consumes pyruvate (and phosphoenolpyruvate) which then forbids the pyruvate -> lactate NAD+ regeneration step. This imposes a need to avoid or deal with a cellular NADH excess.

In the Cell surface oxygen consumption (2) post I hypothesised that the regeneration of NAD+ at the cell surface would be in direct proportion to anabolism derived from pyruvate (ie glucose/glycolysis anabolism), to maintain redox balance (ie get rid of excess NADH, cycling it back to NAD+). It is particularly a feature of highly glycolytic cancer cells.

These folks appear to be saying the same thing but looking at differing cellular techniques to avoid NADH cumulation.

There's lots of other good stuff in there too. Like the rate of mitochondrial ATP generation from HeLa cell mitochondria compared to that of normal cardiac myocyte mitochondria. The ATP production via oxphos is an order of magnitude greater in mitochondria from the cardiac myocytes.

Oh, and glutaminolysis as another NADH avoiding ploy. This is the quote:

"Glutamate can be converted to citrate via reductive carboxylation. In this pathway the NAD(P)H production by glutamate dehydrogenase is compensated by the reverse activity of the NAD(P) isocitrate dehydrogenase (Fig. 1). Glutamate can be taken from the medium or generated from glutamine by glutaminase. Interestingly, arginine and proline can be produced from glutamate with concomitant consumption of NADH (Fig. 4a). This could provide an additional mechanism for NADH turnover".

Note that the glutamate is not being oxidised, it is running a small section of the TCA backwards to generate citrate for lipid synthesis, ie anabolism. This is not glutamate turning the TCA in the normal direction toward oxaloacetate to generate ATP via NADH and oxphos, because the mitochondria of cancer cells don't seem to do oxphos very well. Somewhat Seyfried supportive.


Sunday, February 03, 2019

Lactate as bulk energy transport

Using RQ to track whole body substrate oxidation is pretty straight forward. An RQ of 1.0 means glucose oxidation and of 0.69 indicates fat oxidation. Mixtures come out in between. It is very simple to show that glucose is routinely converted to fatty acids because in the immediate post prandial period for any rodent fed standard low fat crapinabag the RQ becomes greater than 1.0. We would expect that during the later period when the rodent is asleep/not eating there would be a lower than expected RQ (lower than the calculated food quotient, FQ) while predominantly stored fat is oxidised. But on a high carb, very low fat diet we would expect the overall averaged RQ over 24h to be a little under 1.0, ie pretty much the same as the FQ. For an hypothetical "all glucose" diet part of the glucose diverts thus via fatty acids:

Eating: Glucose minus a little O2 -> fat      RQ > 1.0

Sleeping: Fat plus lots of O2 -> CO2 + H2O    RQ < 1.0

CO2/O2 = 1.0 on average over 24h.

If that 24h averaged RQ was all we had to work with we would not suspect that de-novo lipogenesis ever occurred. Nice and simple.

Much more difficult to pick up is the bulk conversion of fatty acids to glucose. This produces an unusually low RQ in the short term. But if the glucose is being produced to fuel the brain during starvation then its prompt oxidation would "correct" the unusually low RQ back upwards to a fatty acid RQ. The obvious exception was noted in a metabolically fat adapted and lactating young lady during extended fasting. She made glucose and galactose from fatty acids and gave them to her baby, rather than oxidising the sugars herself. End result was an RQ of 0.454 after just over three days of fasting with continued breast feeding.

She was making sugar out of fatty acids in bulk. She might or might not have been doing the same without lactation but in the absence of donating the sugars to her infant this would never show.

So the RQ and the FQ always average out to be the same unless something very specific is happening, ie as with Martha.

Much more difficult is to ask how do you tell whether glucose is being converted to pyruvate which then enters the mitochondria to join the TCA or whether glucose converts to lactate which is then shipped in to the mitochondria. And what if you have the absolutely crazy idea that glycolysis almost always leads to lactate and that this lactate is a transferable currency between cells? Glucose is then viewed as a one way gift from liver to tissues, to be shared out between cells/tissues as lactate.

That latter view has to use tracers to look at lactate or glucose flux. Label some lactate with carbon-13 and infuse it to steady state in the plasma of a mouse. Kill the mouse promptly and humanely and look where the C-13 atoms have ended up in glycolysis and/or TCA intermediates. Repeat the process with C-13 labelled glucose. Then glutamate. And then any other intermediary metabolite which might remotely shift bulk energy around the body.

It turns out that in starch fed mice glucose and lactate are the bulk plasma energy carriers, lactate slightly more so than glucose in the fed state and much more so in the fasted state. Certainly on a molar basis, bearing in mind that a mole of glucose has twice the carbon of a mole of lactate, which makes the situation slightly more complex. But lactate labels the TCA more strongly than glucose. Not surprisingly glucose labels glycolytic intermediates better than lactate.

Free fatty acids and ketones are a separate subject in high carbohydrate/low fat fed mice but they flux remarkably little energy, at least when fasting is limited to eight hours. Brain metabolism is also another separate subject.


Glucose feeds glycolysis to lactate. Most of this glycolytic lactate enters the plasma pool. Plasma lactate feeds the TCA in other cells.

Now the insightful bit from near the end of the letter:

"Among the many metabolic intermediates, why does lactate carry high flux? Lactate is redox-balanced with glucose. The rapid exchange of both tissue lactate and pyruvate with the circulation may help to equate cytosolic NAD+/NADH ratios across tissues, allowing the whole body to buffer NAD(H) disturbances in any given location. Nearly complete lactate sharing between tissues effectively decouples glycolysis and the TCA cycle in individual tissues, allowing independent tissue-specific regulation of both processes. Because almost all ATP is made in the TCA cycle, each tissue can acquire energy from the largest dietary calorie constituent (carbohydrate) without needing to carry out glycolysis. In turn, glycolytic activity can be modulated to support cell proliferation, NADPH production by the pentose phosphate pathway, brain activity, and systemic glucose homeostasis. In essence, by having glucose feed the TCA cycle via circulating lactate, the housekeeping function of ATP production is decoupled from glucose catabolism. In turn, glucose metabolism is regulated to serve more advanced objectives of the organism".

What I think this is saying is that lactate supplied to the TCA/OxPhos is for "housekeeping" ie ATP production. Glycolysis is for anabolism. Neither is absolute, but I find it an interesting point of view.

So the ultimate TLDR is:

Ox-phos = housekeeping
Glycolysis = anabolism

There is probably significant fudge-room.


Saturday, February 02, 2019

Lactate: Is the astrocyte-neuron lactate shuttle scuppered?

You don't usually learn much from statements which you, personally, consider likely to be correct. Annoying statements are far more productive.

Working through Seyfried's paper

Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis

I came across this snippet which galled me a little:

"It is glucose and not lactate that primarily drives brain energy metabolism (Allen et al., 2005; Dienel, 2012; Nortley and Attwell, 2017), making it unlikely that lactate could serve as a major energy metabolite for neoplastic GBM cells with diminished OxPhos capacity".

Now, people will realise that the astrocyte-neuron lactate shuttle is more than a little inflammatory as a subject, to say the least. Currently it is not doing too well in the face of experimental data, which are not at all straightforward to obtain. I went to Nortley and Attwell as the most recent reference. As a rather pro-lactate shuttle sort of a person I found their straw-man setting up of the shuttle rather annoying but their data potentially convincing, though I am far from certain about this. Here is the link:

Control of brain energy supply by astrocytes

This left me wondering what more pro lactate-shuttle people might be thinking nowadays, so I went via the "see all" button to locate this commentary by Tang:

Brain activity-induced neuronal glucose uptake/glycolysis: Is the lactate shuttle not required?

which is a rather more circumspect but still accepts a decreasing probability that the lactate shuttle is in any way crucial to astrocyte-neuron energetic coupling. The silver lining was this link, used to point out that in Bl6 mice whole-brain lactate extraction from plasma is essentially zero under the reasonably normal physiological conditions studied:

Glucose feeds the TCA cycle via circulating lactate

The basic concept in the paper, that lactate is the predominant metabolic substrate for the TCA is fine to me but that the source of lactate is predominantly extracellular is very counterintuitive. But the data presented are quite convincing. So I'm interested. I think it needs a little aside before talking about the paper itself and the situation in the brain in particular, so I'll post some random thoughts before looking at the paper in more detail. The biggest down side to the paper is the authors' failure to mention Schurr, the main proponent of lactate as a redox-balanced product of glucose, a very deeply insightful and much neglected observation. But then Schurr is a serious proponent of the astrocyte-neuron lactate shuttle...


Sunday, January 20, 2019

Metformin (10) Macavity


Macavity, Macavity, there's no one like Macavity,
He's broken every human law, he breaks the law of gravity.
His powers of levitation would make a fakir stare,
And when you reach the scene of crime - 
Macavity's not there!
You may seek him in the basement, you may look up in the air
But I tell you once and once again, - Macavity's not there!

I think people might have noticed over the years that I'm not a great fan of metformin acting clinically by blockade of complex I. Particularly over the last few months I have waded deep through layers of references looking at the morass of "paradoxes" about metformin. In particular that 250micromolar in your plasma will kill you but 4000 micromolar can be justified in cell culture because meformin "accumulates in the mitochondria" at up to 1000 times the level in the blood/cytoplasm, which is a deeply held belief structure on which an unimaginable amount of grant funding hangs. Does metformin cumulate in mitochondria?

It doesn't. I really enjoyed this review from last year. Talk about reinforcement of confirmation bias:

Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences

But when you have spent years slogging through papers thinking: That's crap! it comes as a huge relief to find that it's not just you who thinks this, no matter how politely the reaction is phrased.

So. Is metformin research all garbage? No, of course not. Anything involving a live animal on oral doses which do not cause death by lactic acidosis is worth thinking about. Any parallel cell culture research in the same paper using a 4millimolar concentration can be junked. In vivo effects are real, at real dose rates. Cell culture at 1000 times overdose is fiction.

Just to summarise my own speculations:

Under fasting the component of insulin signalling facilitated by the glycerophosphate shuttle can be replaced by saturated fatty acid oxidation via electron transporting flavoprotein dehydrogenase. This maintains insulin signalling at the "cost" of increased fat oxidation. Hence the weight loss.

In the peak absorptive period after a carbohydrate based meal the normal development of insulin-induced insulin resistance is blunted and glucose oxidation continues for longer than without the metformin. If you are eating the absolute crap suggested by any cardiologist or diabetologist this might be of some benefit. If you are already LC the increased fasting fatty acid oxidation is probably where the benefits accrue from.

Cancer benefits are likely to be real, off "target" and secondary to reduced insulin exposure.


Edit: These people seem to be looking at the real world too. Worth spending some time on this

Low metformin causes a more oxidized mitochondrial NADH/NAD redox state in hepatocytes and inhibits gluconeogenesis by a redox-independent mechanism

Saturday, January 19, 2019

Cell surface oxygen consumption (2)

How does cell surface oxygen consumption happen? Here is the main reaction overall (it's probably not that simple!):

4e- + 4H+ + O2 -> 2H2O

A certain amount of hydrogen peroxide is also generated and just a little superoxide (probably for cell-cell signalling). Other electron acceptors can stand in for, or compete with, oxygen as the terminal electron acceptor. The reaction occurs on the outer surface of the plasma membrane.

The source of the electrons is cytoplasmic NADH, which is oxidised to NAD+ and H+ on the inner surface of the plasma membrane. Each of the electrons is transported via a plasma membrane ubiquinone:semi ubiquinone cycle which then donates them to the redox enzyme on the external cell surface. Here is a simplified version of Herst and Berridge's Figure 1:

Although I doubt that there is very much NADH or NAD+ in normal extra cellular fluid there has to be an NADH docking site on the outer redox enzyme as one of the hallmarks of cell surface oxygen consumption is that it can be halted completely by flooding the cell culture medium with NADH. Electrons from this then follow the dashed line to fully reduce the ubiquinone to ubiquinol and the system grinds to a halt.

So you can measure cell surface oxygen usage by the fall in consumption which occurs when you add exogenous NADH just as you can measure mitochondrial oxygen usage by the fall in consumption which occurs when you add myxothiazol.

Why is the system there?

Let's go back to the two routes of glycolysis. Without the glycerophosphate shuttle (insulin signalling driven/driving) we have

Glucose -> lactate -> mitochondria -> pyruvate -> TCA

and there is no depletion of cytoplasmic NAD+ as one is consumed and one produced in the glucose -> lactate process. With insulin signalling we have two parallel processes:

Glucose -> glycerophosphate shuttle -> CoQ -> ETC

which consumes cytoplasmic NADH, leaving none to convert pyruvate to lactate. So in parallel we have to abort glycolysis at pyruvate:

Glucose -> pyruvate -> mitochondria -> TCA

which balances the cytoplasmic NADH:NAD+ ratio nicely.

Now, let's consider a cell undergoing rapid growth with a view to divide. For today I will ignore mitochondrial biosynthesis and consider what happens if cytoplasmic pyruvate is consumed for amino acid biosynthesis. For each molecule of pyruvate which has been diverted to an amino acid there will be one less available to provide cytoplasmic NAD+ by conversion to lactate, which will limit glycolysis because cytoplasmic NAD+ is essential for the oxidation of glyceraldehyde-3-phosphate.

Under these conditions cell surface oxygen consumption appears to be able to step in to oxidise cytoplasmic NADH to cytoplasmic NAD+, which then allows glycolysis and its associated ATP production. This looks to be particularly important if there is any sort of a problem with the ETC and the glycerophosphate shuttle.

In rho zero cells, where the ETC is deleted (and there is no glycerophosphate shuttle) and glycolysis is the sole source of ATP production, cell surface oxygen consumption has to supply NAD+ in direct proportion to how much pyruvate is lost to anabolism rather than being used to supply NAD+ via lactate generation. In rho zero anabolic cancer cells cell surface oxygen consumption can be as much as 90% of the total oxygen consumption of the parent cell line.

TLDR: Anabolism requires cell surface oxygen consumption to regenerate NAD+. Its importance rises if there is defective ox-phos.


Friday, January 18, 2019

Cell surface oxygen consumption (1)

Back in the comments thread to the More on insulin and the glycerophosphate shuttle post there has been some discussion as to whether Warburg/Seyfried was/is correct about cancers being glycolysis driven or whether ox-phos is the core processes driving cancer metabolism. Raphi and Altavista have fairly opposite views.

Altavista rather liked this paper, using supra pharmacological concentrations of metformin to block complex I in tissue cultured cancer cells. It is true that this intervention produced a dose dependent decrease in oxygen consumption, but we have no idea of what the absolute oxygen consumption of the cells was, only the relative fall from control cell levels. I have huge problems with this paper. Relative change smells like relative risk, as in cardiology...

So I went looking to find out whether cancer cells do consume oxygen in decent amounts and whether this oxygen is simply metabolised by their mitochondria.

It seems that oxygen metabolism is not the sole prerogative of mitochondria.

This paper is fascinating reading and Table 1 gives us the actual oxygen consumption rates of assorted cancer cell lines in culture.

Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines

Total oxygen consumption varies from as high as almost 28pmol/sec/10^6 cells in the HeLa line to as low as 5pmol/sec/10^6 cells in the P815 cell line.

So there is no doubt that cancer cells in culture do consume oxygen.

Does that imply they are using it for ox-phos? Fascinatingly, the answer is no. Not completely.

A significant proportion of the oxygen consumption is occurring at the cell surface plasma membrane. If you acutely block the ETC of the mitochondria using myxothiazol this plasma membrane oxygen consumption continues and appears to account for around half of the total oxygen consumption, the exact percentage varies.

What is really interesting is what happens if you generate ρ° derivatives of your cell line. These have no functional ETC at all but still consume significant amounts of oxygen, usually in the order of around 90% of the total amount consumed by the parent cell line. They have obviously adapted to their lack of mitochondrial ETC by hugely up regulating cell surface oxygen consumption.

Why? How?

I'll probably put posts up about these questions as we do have some ideas. But the whole reason I went looking was to decide whether cancer cells perform ox-phos. It seems that at least some of them do. Those which don't (major mutations of complex I genes) tend to be very, very unpleasant in patients. Amongst other cancers it's possible that the degree of dysfunction in ox-phos and its replacement by plasma membrane oxygen consumption may correlate with the degree of malignancy of the cancer.

Nothing is black and white. Many cancers respire to various degrees. Not always very well.

You can't tell from simple oxygen consumption if a cancer cell line is respiring using the mitochondrial ETC or performing plasma membrane oxygen consumption. Your Clark electrode can't tell you. That explains a chunk of why people can't agree on whether cancers use ox-phos or not. All consume oxygen, but some use more ox-phos than others.

I just thought it was interesting.


Friday, January 11, 2019

How primordial is oxidative phosphorylation?

It is perfectly possible to run a sophisticated metabolism using the energy available from an hydrogen rich geochemical proton gradient of the type still found in locations such as Lost City in the Atlantic. This provides an (almost) endless supply of electrons via FeS catalysts of sufficiently negative potential to reduce carbon dioxide to carbon monoxide. From here it is all down hill, energetically speaking, to acetate and metabolism. The process is completely dependent on geochemical conditions for the free-ride. These basic steps are still embedded in the carbon monoxide dehydrogenase/acetyl-CoA synthase complex discussed rather nicely in this 2018 paper (how did I miss it?):

Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes

Back in the Life series of posts I argued in favour of Koonin's concept that Na+ provided the primary electrochemical gradient which was used to drive a Na+ transporting rotary ATP synthase. It's all in here:

Evolutionary primacy of sodium bioenergetics

My own idea is that primordial Na+ energetics derived from a geochemical proton gradient which was converted to a Na+ gradient by a H+/Na+ antiporter. In order to detach from the geochemical proton gradient what is needed is a Na+ pump to replace this geochemically driven antiporter. Acetobacterium woodii has the most highly conserved version of such a Na+ coupled system and back in 2009 the Rnf complex was the primary suspect for being the site of Na+ pumping.

The ins and outs of Na+ bioenergetics in Acetobacterium woodii

By 2010 this was pretty well confirmed:

Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase

In its most autotrophic guise A woodii can still run its metabolism on molecular hydrogen. The process of electron bifurcation allows the generation of reduced ferredoxin from H2 and so can provide electrons of a potential to use NAD+ as their electron acceptor, with sufficient energy left over to pump a single Na+ ion, in imitation of the H+/Na+ geochemical driven antiporter used soon after the origin of life. This Na+ pumping allowed prokaryotes to leave hydrothermal vents, provided there was access to molecular hydrogen as food.

This core process which freed early life from the ties to geochemical alkaline hydrothermal vents is clearly the oxidation of reduced-ferredoxin, ie this is an oxidative reaction driving an electrochemical Na+ gradient to phosphorylate ADP to ATP using a Na+ driven rotary ATP synthase.

It is oxidative phosphorylation.

Oxidative-phosphorylation is as primordial as the exodus of prokaryotes from hydrothermal vents.