Life (31) Chinese whispers

Back in 2008 Noha Mesbha published her excellent PhD thesis

ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS

which introduced the world, via this paper

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

to Natranaerobius thermophilus and its antiporter nt-Nha. Which gives every impression of being a stand alone Na/H+ antiporter very closely related to the invariably operon controlled MrpA protein (named shaA) of Clostridium tetani. As she says

"Gene nt-Nha had 35% identity to the shaA (mrpA) gene of Clostridium tetani. The Mrp proteins belong to the monovalent cation/proton antiporter-3 protein family. ...Sequence analysis of the regions surrounding gene nt-Nha, however, did not show that it was part of an operon. This indicates that gene nt-Nha does not encode a subunit of an Mrp system, but rather a mono-subunit antiporter".

All well and good.


Then in 2010 Morino et al published

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

Although the whole paper was about B subtilis (and how none of its Mrp subunits worked in any incomplete combination to antiport anything) they did make this throw-away comment:

"A MrpA/MrpD homologue encoded by a “stand alone” gene from polyextremophilic Natranaerobius thermophilus was recently reported to exhibit Na+/H+ and K+/H+antiport activity in anaerobically grown E. coli KNabc (24)"

where (24) is Mesbha's PhD paper. Notice that at this stage Mesbha's

nt-Nha ~ shaA, very closely related at 35% conserved gene sequence,

has been changed by Morino in to

nt-Nha ~ An "MrpA/MrpD homologue".

This is a just about acceptable per se because MrpA and MrpD are homologous to each other and nt-Nha is closely related to MrpA (shaA) of C tetani. But Mesbha herself never mentions MrpD in her 2009 paper or in her PhD thesis. And "MrpA/MrpD" is open to mis-interpretation. So we have "definition-creep" here, where nt-Nha could be accidentally seen as some sort of composite of MrpA in combination with MrpD. Ouch.

So next we have the 2017 offering by Jasso-Chávez et al

Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans

where we have this bizarre statement

"On the other hand, a fused MrpA-MrpD homolog in the alkaliphilic Natranaerobius thermophilus displayed Na+/H+ antiport activity when produced in E. coli strain KNabc (5, 28)"

Ref (5) is Morino's paper on B subtilis Mrp, in which one rather misleading citation suggests that nt-Nha is an "MrpA/MrpD homologue". This has developed to the extent that nt-Nha has now "become" a fusion of two genes to give a rather mythical monster.

Ref (28) is just Mesbha's PhD paper in which nothing of the sort was suggested.

So the Jasso-Chávez paper is utterly flawed due to misinterpreting a poorly phrased statement and adding an erroneous modification so as to grossly mis-represent an initial very solid finding by Mesbha. The Jasso-Chávez discussion of nt-Nha can be distilled as:

"Send three and fourpence, we're going to a dance".

The chance of their understanding how nt-Nha or their very own archaeal MrpA subunit work as a stand-alone antiporter appears to be approximately zero.

Very sad.

Peter

BTW The folks who worked from the actual gene to model the nt-Nha protein structure suggest that

"The final model presents 13 transmembrane α-helices organized in a similar arrangement as the NuoL subunit".

You know the picture but here it is again 'cos I think it's lovely

To the gym?

This press release in advance of a poster presentation is doing the rounds on social media at the moment:

Ability to lift weights quickly can mean a longer life

A factual association is probably true between being above the median in ability to do work on a specifically selected gym machine and longevity.

What does this mean? It's observational. Hypothesis generation only.

Perhaps non-athletes with higher power output might live longer because they have excellent mitochondria and our mitochondria are essentially what determine our longevity. Having good mitochondria might well mean that you exercise spontaneously without describing yourself as an athlete. As in I might carry a couple of sacks of chicken food myself rather than wait for Paul the yard-man and his barrow. But this is an effect, not a cause.

Non-athletes with low power output may be the converse. They have mitochondria in which the cardiolipins anchoring their cytochrome-C to the mitochondrial inner membrane are as flimsy as a PUFA in a deep fat fryer, which are willing to trigger apoptosis at the drop of a superoxide molecule. Poor mitochondria, less muscle fibres giving sarcopaenia, shorter longevity. Probably already giving diabetes in-situ. An AHA poster-child.

Taking someone from the second category and making them exercise might convert them to being healthy with a long life span. Maybe.

Or, there again, it might make the gym sessions so unbearable that they quit.

Or, if you don't let them quit, it might simply make no difference.

Or, if you don't let them quit, it might kill them sooner!

As Eeyore said "think of all the possibilities, Piglet, before you settle down to enjoy yourselves".

The study (as per press release) tests nothing. Researchers are giving very specific advice based on an untested observation which might be going to make them look very, very stupid when the results of an intervention to test their hypothesis actually refutes it. But that will take decades and hopefully the researches will be retired by then. More likely it will be ignored or termed a paradox. All the possibilities Piglet.......

EDIT or the intervention might show benefit. That's not impossible! END EDIT

Peter

Declaration: I have nothing against exercise. Nowadays I mostly boulder because it is three dimensional problem solving using muscle groups to failure on a regular basis. Plus I also try to keep my cardiolipins as saturated as practical!

Life (30) Guesses about Na+ channels

Complex I exists in various states, the two main ones being activated and deactivated. The deactivated form is convincingly a Na+/H+ antiporter. There is a pretty good case made in this paper, which has a number of flaws but is generally probably correct:

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

As always, the suspect antiporter is thought to be NuoL, the distal "antiporter-like" subunit. You can see the logic for this which is supported by the stand-alone antiporting homologues of NuoL seen in nt-Nha and at least one archaeal MrpA subunit. Personally I'm not sure this is the case. The phenomenal difficulty in trying to interpret exactly what is happening in a structure as intricate and as minute as complex I allows many views of the available data.

It also appears to be the case that in bacteria which use menaquinone as their electron acceptor (rather than CoQ) some degree of Na+/H+ antiporting occurs under normal active NADH oxidation/proton pumping. That's in here

Respiratory complex I: A dual relation with H(+) and Na(+)?

and here's the energetics doodle from the same Fig 2 as I pinched the NADH:ubiquinone doodle from in the last post:

Because transferring two electrons from NADH to menaquinone only provides 480mV/2e- the complex uses Na+ moving down its concentration gradient to "top up" energy availability and so get the extra energy needed to pump the full four protons.

Aside: The paper has lots of good ideas but they are very wedded to the concept that NuoL, M and N are still antiporters and that loss of "control" by the redox cytoplasmic arm allows this antiporting to re establish. It's a very reasonable idea but I think it can be improved upon, especially now we have more detailed information about the Na+ pumping of the P furiosus MBH, where this is not what has happened. End aside.

Why on earth should it matter whether complex I pumps the full complement of four protons? If there is only enough Gibbs free energy for two or three protons, why not just pump two or three protons?

What occurred to me is that for complex I to pump the four protons it might be necessary to have a full "priming" of the membrane arm with enough Gibbs free energy for a full "push to the left", as in this doodle, discussed in a previous post:

What if you only have 480mV/2e- available, giving a half hearted "nudge" to the left when you need the full shove from 840mV/2e-? Is it possible that, under these circumstances, nothing at all happens? There is no flip of the glutamate/lysine pairs from together to apart, which triggers all of the opening/closing of water channels that allows proton translocation? Zero proton translocation?

If you wanted to restore the full "kick to the left" it might be a reasonable energetic top-up to supply the extra energy from Na+ ingress up near the Q binding site to allow the menaquinone plus Na+ ingress to generate the full Gibbs free energy for activation of the membrane arm. That needs a trans-membrane channel, so we are looking at the really complicated region around NuoH and NuoA/J/K. Not easy.

The best characterised Na+ channel in the whole related set of the complex I, MRP and MBH systems is the one in the MBH of P furiosus, described by Yu et al in

Structure of an Ancient Respiratory System

which gives the Na+ channel looking like

with the red blobs being the modelled Na+ binding sites. This is made up of proteins from four separate genes and is thought to be homologous to the Na+ channel of the MRP antiporter, also a multigene structure. They show them as identical in their discussion doodles, like this for the MRP multigene Na+ channel, simplified to the subunit shown as MrpG:

The equally multigene Na+ channel of the MBH of P furiosus (in this doodle the multiple Mbh genes are simplified to MbhC) is shown in exactly the same location with the same critical broken helix, in the same shade of green:

The final doodle in this figure is complex I. Here is their image:

which keeps things nice and simple between NuoH and the Nuo A/J/K region, where Nqo10 is shown but nothing else. So I wanted to know if there could be a Na+ channel which could be used to either top up the energy of the NADH:menaquinone couple or to allow the antiporting function to occur in deactivated standard CoQ based complex I. Of course no one is looking for Na+ channels in complex I in quite the same way as Yu et al were looking for one in MBH, where they knew it was crucial. Anyway, I went looking. In here

Structure and function of mitochondrial complex I

is where I found find this image (which is unfortunately left to right transposed in its view) that includes a rather nice broken helix shown in pink which I have circled in red:

If we take this broken helix, colour it green and transplant it in to Yu et al's complex I we get this:

Might it be the Na+ channel I need?  No one knows (yet). It is known that there is a conformational change in the region of the CoQ binding pocket when complex I changes from the active to the deactivated state, close to the region of the broken helix head. I would suggest this conformational change might allow the Na+ antiporting function to occur when pumping in complex I is deactivated. Protons would be allowed to enter the cell/mitochondrion in exchange for Na+ expulsion (theoretically reversible but that seems unlikely physiologically unless you are a bacterium using menaquinone where some Na+ ingress is worth it to trigger the membrane arm). Logic says that this trade off would be preserved if antiporting provided a net benefit to the organism under which ever conditions might have favoured deactivation of complex I.

Peter

Addendum. Here are the three complexes lined up. Because PowerPoint lets you do it. No other reason.

Life (29) Applied billiards to MRP and MBH

Here is a nice doodle of complex I taken from this interesting but off topic paper

It shows that taking two electrons from NADH and dropping them to CoQ provides a Gibbs free energy of 840mV for the pair. That's all well and good and is why cells love oxygen based electron transport chains. But I'd like to look at this in reverse. We know that complex I works perfectly well in reverse. So we can say that dropping four protons from extracellular to intracellular down a membrane voltage of 150mV across a proton tight membrane will allow the performance of 840mV of work.

For complex I running in reverse this 840mV of work is always used for NADH generation from NAD+ (or superoxide generation) because that's the unit bolted on to the membrane arm. Other metabolic units can be used in place of NADH dehydrogenase and many of these can be found in various bacteria using various substrates with various numbers of antiporter-like subunits in the membrane arm. All will work in reverse and provide/utilise differing Gibbs free energies.

So I would like to view the membrane arm of all of these complexes as a machine in its own right which, when running in reverse, can harvest the energy available from a proton gradient for whatever function the cell might like. Obviously this is not the current arrangement, but is possible.

So here is the intact membrane arm of the complex I of T thermophilus, once again taken from Luca et al

running in reverse to give 840mV/2e- of usable energy. As in the last post we can imagine each proton passing through each channel "kicks" to the right ('cos we're running in reverse here) until the cumulation of four "kicks" is enough to cause the conformation change in Nqo 8 which drives two electrons back up the FeS chain to an NAD+. Think of the membrane arm as a machine to accumulate multiple "kicks" to give a big Gibbs free energy.

Now let's remove some of the sub units and see what we get. How about this:

Two proton translocations will provide much less Gibbs free energy than four proton translocations. I've faintly allowed a third proton channel but there is no suggestion this is a functional feature of complex I at all. It's Nqo 10, never mentioned in the paper. Note that all of the subunits are kicking in the same direction.

The above is obviously the basis of the MRP antiporter, which looks like this:

where the energy of two protons is cumulated to allow a single Na+ ion to be extruded. How much of a Gibbs free energy will be developed by the two protons will obviously be influenced by the membrane voltage through which the protons fall. It will also be influenced by the permeability to protons of the membrane in which MRP is embedded. If the membrane was 100% permeable to protons there is no point trying to harvest a gradient which isn't there. The more permeable the membrane the less energy can be harvested per proton. But we do know that in E coli under CCCP with a membrane voltage of as little as 15mV that MRP can cumulate enough Gibbs free energy to antiport Na+ when monogenetic antiporters fail completely.

In the ancestral hydrothermal vent scenario a membrane voltage from the pH gradient between vent and oceanic fluids might be, ideally, around 150-200mV. I still consider MRP is most likely an adaptation to allow colonisation of vent environments where apposition of fluids is not perfect or some mixing has already occurred and the available membrane voltage might be very low. Given MRP LUCA might survive on the edges of vents in addition to thriving in the luxurious conditions of perfect apposition near the centre.

A step on from MRP is, hypothetically, to add a power unit which will allow Na+ pumping when membrane voltage is too low even for MRP. You can think of this as an adaptation to the tail end of the vent system. Some membrane voltage is still present as are complex molecules (from dying prokaryotes in the better vent environments) out of which ferredoxin can be synthesised. This is not quite the membrane bound hydrogenase of P furiosus as we will see. It's an augmented "kick-to-the-right" through MRP to assist the generation of enough Gibbs free energy to allow Na+ extrusion:

Notice that all of the "kick" arrows run in the same direction as for all of the illustrations in this post, looking to generate a significant Gibbs free energy at the right hand end.

The final stage of generating the MBH of P furiosus (and freedom from the vent gradient completely) is like this: We have to reverse the direction of the proton travel in MBH subunit H. The "kick" from proton ingress is to the right. We want to impose a kick towards the left to reverse the proton flow to give egress, using ferredoxin power. This requires a barrier between MbhM and MbhH to stop that kick to the right, completely different to complex I and any doodle so far. As Yu at al comment in the legend to Figure 3

"A hydrophilic axis across MbhM membrane interior is also identified but it is separated from that in MbhH due to a gap between the two subunits"

and in the legend to Figure S4:

"Note the large gap between subunits M and H in MBH (A). There are four elongated densities located to the lower region of the gap (A) inset; marked by blue dashed lines), which stack against several hydrophobic regions of subunits M and H. These densities are likely from two phospholipid molecules that may stabilize the structure and prevent ion leakage across the membrane bilayer. The dashed curves in (C) and (D) highlight the fact that the chain of hydrophilic residues found in complex I is continuous (D), but is discontinuous in MBH (C)".

This is the genuine article:

and my version, more crudely:

As well as stopping MbhM "kicking" the central water channel protons of MbhH to the right, the conformation change from the NiFe hydrogenase has to be imposed on MbhH to force a proton outwards.

So the white arrow of proton "kick" in the central water channel of MbhH is reversed by the green cranks using the power from the hydrogenase (green arrow) to enforce this. The bright red proton channel is present as specified by Yu et al and is both essential and functional. Quite what the remnant of this is doing complex I I don't know and quite what the proton channel within MbhM is preserved for I also can't imagine (yet). But the general principles of proton movement as set down here are much more satisfying than my initial thoughts on MRP and MBH.

Peter

Life (28) Proton Billiards

Here we are looking at complex I from from Thermus thermophilus as featured in this paper:

Symmetry-related proton transfer pathways in respiratory complex I

Because it's bacterial it is all labelled up in Nqo terminology. In mammalian complex I we use Nuo terms, where NuoH is Nqo8, NuoL is Nqo 12, NuoM is Nqo 13 and NqoN is Nqo 14. We can ignore all of the other subunits.

Here are the water channels used to allow protons to move through complex I, red beads being water molecules:

The CoQ binding site (and the NADH dehydrogenase unit) are at the right hand end. What is most important is that the water channels are not all open (hydrated) at the same time. In the resting state the N-side channels are open. A conformational change in Nqo8 is induced by CoQ reduction which opens its water channel and allows a proton to enter from the cytoplasm. This triggers a chain of conformational changes horizontally along the central water channel, moving a proton from right to left within each antiporter-like subunit and which also closes the N-side water channels and opens the P-side water channels, to allow protons to move outwards in to the periplasm. This is their doodle from the discussion:

If anyone goes through the diagram in the sort of detail I did they can see that Nqo13 doesn't make sense because glutamic acid E377 is not a lysine (K abbreviation), which it is in the other two subunits. That messes up all of the charge movements and the inter-subunit electrostatic binding. From Fig 1 section B elsewhere in the paper you can see there is an arginine (R163) just "north" of E377 which might be doing this job by binding to the aspartate (D166) of Nqo12 but I can't see that this is addressed anywhere in the paper. So it's just my guess. Still. The basic concept is pretty convincing.


TLDR: The reduction of CoQ to CoQH2 clunks protons horizontally within the central hydrated channel of each antiporter-like subcomplex from their input zone to their output zone.


We have to bear several things in mind. First is that the system is completely reversible today. As in reverse electron transport using a high membrane potential and reduced CoQ couple to reduce NAD+ to NADH, and generate ROS when NAD+ is all used up... Protons will move inwards from periplasm to cytoplasm as this happens.

Also this is complex I, it is a relatively late addition to modern bacterial metabolism dependent on proton tight membranes and the availability of molecular oxygen.

Third is that our best remnant of LUCA is the Na+ pumping membrane bound hydrogenase of P furiosus and this drives from left to right through an Nqo14/NuoN related subunit (and will almost certainly be equally reversible) to its Na+ channel.

I have some Powerpoint doodles to take this a little further.

Peter

Life (27) Alphabet soup

So this is the predicted structure of  the modern MrpA-like functional antiporter from N thermophilus, nt-Nha:

courtesy of

A missing link between complex I and group 4 membrane-bound [NiFe] hydrogenases

As you can see it is a double channel and it is homologous to NouL from mammalian (and others) complex I. The two marked amino acids are a glutamic acid and a lysine which are conserved in NuoL. I would guess that the left hand channel is the proton channel and the right hand one is for the antiported Na+ ion. In the modern bacterium N thermophilus this antiporter is electrogenic, ie it moves more than one H+ inwards for each Na+ outwards. This may or may not have been the case in LUCA.

So nt-Nha looks a lot like NuoL and so quite like NuoM and NuoN (and MrpA, MrpD and MbhH) but it is the only one to antiport. The antiport mechanism really hasn't been worked out yet.

I guess the next thing to look at is from the same paper. This time we're looking at NuoH, the anchor point at the base of all complex I family of proton pumps. Here it is as a model with NuoH in gold superimposed over the "left hand" channel of NuoL in grey:

Half of NuoL is structurally pretty well identical to NuoH.

From the proto-Ech posts I consider NuoH to be derived from the primordial channel designed to allow oceanic pH penetration in towards the NiFe catalytic site (at alkaline hydrothermal vent pH) to generate reduced ferredoxin as the core power molecule of early LUCA, able to reduce CO2 to CO at the CODH/ACS complex.

So a derivative of the ancestral proton channel formed half of NuoL. I think it is very likely that this is the case for all of the "antiporter-like" subcomplexes, though clearly some changes in function have occurred. What made up the other channel of the ancestral antiporter? For a clue as to this one we have to change subunit and change nomenclature. The work has not been done for NuoL but it has been done for the membrane bound hydrogenase of P furiosus (subunit MbhH) which is structurally more closely related to NuoN rather than NuoL.

Let's switch papers and go back to

Structure of an Ancient Respiratory System

Here we are viewing subunit MbhH, remember that we think that the left hand channel is probably derived from the ancestor of NuoH:

Now, here's the clever bit. If we draw a horizontal line across the left hand channel, say just below the "9", we can rotate the channel around this line so all of the labels 9, 10, 11, 12 and 13 end up on the bottom far side of the model. With this done the two channels are superimposable, rather neatly:

So the right hand side of MbhH is simply the inverse of the left hand side.

We only need one protein, the original ancestral proton channel, to make both sides of the original ancestral antiporter. Minor modification would allow one side for H+ and the other for Na+. Then some sort of coupling to convert H+ in to drive Na+ out. But basically we can make all of the modern "antiporter-like" subunits from an ancestral derivative of two conjoined NuoH-like channels.

That is so neat it has to be correct. So it's probably wrong!

That coupling process might be extractable from work being done with modern complex I but I have only skim read the paper so far, so I'm not sure how much further I can progress the current fairy tale.

Peter

Life (26) MrpA MrpD NuoL NuoM and NuoN. Plus nt-Nha.

A few years ago I mentioned this paper

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

In brief they had Bacillus subtilis strains with either an MrpA knockout or an MrpD knockout. The E coli complex I equivalent of NuoL can replace MrpA and the NuoN equivalent can replace MrpD in B subtilis. NouM doesn't seem do either. But all five subunits look very similar to each other and all are clearly related. NuoL, NuoM and NuoN are always described as "antiporter-like". MrpA and MrpD are thought to be antiporters but none ever work alone, the whole complex is needed, so they are probably "antiporter-like" too. They all appear to have been derived from an antiporter but any intrinsic antiporting has been lost.


Which makes me sad because it seems very probable that all of the above subunits are derived from the primordial antiporter at the origin of life which initiated Na+ bioenergetics and all that followed on from that.

Then came Natranaerobius thermophilus. N thermophilus is not really in the league of P furiosus, it's okay growing at up to 57 degC (which will still scald you) but no higher and it has adapted its membrane to remain proton tight at this temperature. BTW it's strictly anaerobic, is an halophile and an alkaliphile. It has (among several) one family of modern antiporters which are clearly genetically related to the MrpA of modern Clostridium tetani (and probably all other MrpAs). Modern nt-Nha is a fully functional antiporter as a stand-alone single gene protein. As these folks say:

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

"Gene nt-Nha had 35% identity to the shaA (mrPA) gene of Clostridium tetani. The Mrp proteins belong to the monovalent cation/proton antiporter-3 protein family. This family is composed of multi-component Na+/H+ and K+/H+ antiporters encoded by operons of six or seven genes, and all genes are required for full function in Na+ and alkali resistance (Ito et al., 2000). Sequence analysis of the
regions surrounding gene nt-Nha, however, did not show that it was part of an operon. This indicates that gene nt-Nha does not encode a subunit of an Mrp system, but rather a mono-subunit antiporter".

EDIT number 2: These people have isolated an MrpA from an archaeal species which will antiport on its own, which makes it very similar to nt-Nha. There is also some evidence that complex I can function as a partial Na+/H+ antiporter as in this paper. NuoL is the main suspect. END EDIT.

Neither the MrpA-like nt-Nha nor the modern MrpA subunit of C tetani is in any way primordial. Both are used to extrude Na+ in exchange for H+ but this is not to drive Na+ energetics, they are much more associated with resistance to high Na+ concentrations and to alkaline pH environments. So it is possible that the N Thermophilus nt-Nha is a relatively modern derivative (it does use a proton tight membrane) of a relatively modern MrpA.

Or, more excitingly, it's possible that an ancestral Na+/H+ antiporter gave rise to both nt-Nha and MrpA. This would be the interesting option as it is possible that the Na+ binding sites, the route across the antiporter for Na+ and the mechanism for activation might just give us the technique used by the original ancestral antiporter. Genetics and structure-function modeling look to be the way to go but I can't see that it's been done yet.

Edit: Found the structure homology studies in here, lying around on my hard drive for years too. End edit.

Peter

Life (25) Left or right hand?

Here is the basic Mrp antiporter structure as suggested in

Structure of an Ancient Respiratory System

Quite how many protons are exchanged for how many Na+ is uncertain but there are papers using modern Mrp set in proton tight membranes that suggest it might be more than one H+ in per one Na+ out. ie Mrp is electrogenic, or rather it consumes pH gradient to extrude Na+. This would be no problem in the hydrothermal vent scenario, protons being freely available there. Personally I'd like electroneutrality but that's just my biases as to how P furiosus works. Anyway, Mrp is much like this, with uncertainty about the numbers of ions:

Here is exactly the same antiporter but broken down in to the main channels:

If we ignore the arrows for the H+ in the diagram all we have to do is remove the bulk of MrpA (the N-terminal) and replace it with a power source "pushing" in from the left and we have the membrane bound hydrogenase of P furiosus, still retaining the MrpG Na+ channel and working as a Na+ pump:

The paper then goes on to talk about Complex I and how that, in the Mrp nomenclature, the combined MrpD plus the fused-on C-terminal of MrpA are flipped around. I spent a long time mentally lining up various channels in my head until I twigged the simplest way to look at it was to keep Mrp channels unchanged but look at the NADH dehydrogenase of complex I as simply pumping in to a completely un-flipped Mrp but being bolted on to the opposite end, in the place of the MrpG Na+ channel. Leaving the N-terminal of MrpA still in place, like this:

I've squeezed in an extra MrpD because that's what complex I appears to have done as a modified duplication of either MrpA or MrpD. In mammalian mitochondrial nomenclature the MrpA N-terminal derivative is NuoL, the narrowed (only in this image, not really) MrpD gives NuoM and the full sized MrpD is NuoN. Yu et al only use the bacterial  complex I terminology based around the Nqo numbers. I've avoided these numbers (just used the Mrp terminology throughout the doodles) as the switches from terminology to terminology did my head in (as we say here in Norfolk) for weeks. MBH, Mrp and Nqo. Alphabet soup for the subunits!

But the core insight for me was that if you supply power from the left you pump Na+. Supply it from the right and you pump protons. This looks very much like motorising Mrp from one end makes it work in the Na+ extrusion antiport mode. Adding the power source to the opposite end, coincidentally removing the Na+ channel at the same time, drives the antiporter in the H+ expulsion direction, reversing the primordial function of Mrp and so forming the origin of the complex I family.

Obviously there is nothing primordial about complex I. It is reliant on a proton tight membrane and the ability to extract large amounts of energy from NADH, which usually means the presence of molecular oxygen. The least altered representative of antiquity is undoubtedly the MBH of P furiosus and even more so is the ancestor of the Mrp antiporter family.

Peter

Looking to the future

I notice the EU is considering approval for a plan to enforce removal of copyright infringing images form pretty well everywhere on T'internet.

Over many years I've felt that this sort of action would clearly be the end of Hyperlipid as any of us knows it. I back up every month or so to keep my ideas safe for myself but if Hyperlipid suddenly disappears I think everyone should have an idea as to why. This will be it.

Anyway, for now something resembling normal service will be resumed when I can tear myself away from Mrp antiporters and derivatives, which is proving rather difficult.

Peter

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.

Peter

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.

Peter

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.

Peter

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.

Peter

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.

Peter

Also relevant:

https://dilbert.com/strip/2019-02-25
https://dilbert.com/strip/2019-02-26

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.

Peter

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.

Peter

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.

Peter

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.

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.

Peter

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.


TLDR:

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.

Peter

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...

Peter

Metformin (10) Macavity

TLDR:

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.

Peter

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

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.

Peter