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".

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

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.

Peter

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.

Peter

How much linoleic acid to get fat and insulin resistant?

Tucker and Mike both gave me the heads-up on this paper recently.

Linoleic acid causes greater weight gain than saturated fat without hypothalamic inflammation in the male mouse

It's nice and simple: feeding 22.5% of calories as linoleic acid (LA) to a mouse makes it obese and insulin resistant. Feeding 15% of calories as LA makes it identically obese but without the insulin resistance. The argument can be made, very convincingly, that LA is converts to 4-hydroxynonenyl (4-HNE) in proportion to the LA content of the diet. 4-HNE is a powerful driver of insulin resistance so the hypoglycaemic response to exogenous insulin is markedly blunted in the 22.5% LA group of mice. As in the top line here:

Here are the weight gains, which need a little consideration:

With the eye of faith you can see that the top line (22.5% LA) starts off gaining weight faster than the next line down (15% LA) but converges from around 7-8 weeks onward. My guess is that this is when insulin resistance from 4-HNE started to over-ride the insulin sensitising effect of LA which caused the weight gain in the first place.

Now the third line down is the interesting one. This diet only contained 1% linoleic acid. OK, these mice are statistically significantly slimmer than the 15% and 22.5% LA fed mice (p less than 0.05) but they are hardly exactly svelte when compared to the crapinabag (CIAB)-fed control mice (black line down at the bottom). And the CIAB food contained 4.22% of calories derived from LA.

That needs some thinking about.

It makes me ask: Why do the vast majority of high fat fed mice/rats become obese? Apart from the fact that they have been selected for this response.

There's probably another post or two needed on that one.

Peter

As an aside I would just comment that while I agree with Tucker that 4-HNE and related products of free radical modified PUFA are the best explanation for this study, my feeling is that the 15% LA group with sustained insulin sensitivity allowing sustained weight gain probably explains the situation in the current human population rather better. As sustained adipocyte distension progresses then we eventually get FFAs released in the presence of glucose and insulin, a Bad Thing. The ROS from this combination will eventually generate 4-HNE too but rather further down the obesity road compared with the 22.5% LA situation.

Urinary c-peptide

It's the Winter Solstice tomorrow, greetings to all! My favourite astronomical event of the year, even though we do the major feasting on Christmas day in our house. Anyway, here's a fairytale for the depths of Winter (in the northern hemisphere anyway). Happy Solstice!

                                            *********

Here we go: Just occasionally you come across a statement like this:

"(B) Insulin secretion throughout the day was assessed by 24-hr urinary c-peptide excretion and was significantly reduced only following the RC diet".

Okay, what does it suggest to us when the reduced 24-hr urinary c-peptide group lost less fat  than the higher c-peptide group? Less insulin but less fat loss??? Perhaps it suggests that the insulin hypothesis of obesity is incorrect?

C-peptide is part of pro-insulin. Each molecule of insulin produced provides one molecule of c-peptide within the pancreas. Assuming (not completely accurately) that c-peptide is not consumed within the body we can use its 24-hr average urinary excretion as a surrogate for overall insulin production. With an awful lot of caveats, this seems fair to me. I think statement B is correct.

So 24-hr urinary c-peptide gives us an idea of how many molecules of insulin are being manufactured per day by the islets within the pancreas. Insulin is broken down by insulin degrading enzyme as part of its signalling process, not exactly proportionally, but as a general principle this is correct. I went through it in some detail when thinking about the Potato Diet, a sub-category of carbosis. The more signalling, the more degradation.

On average around 50% of secreted insulin (in dogs on a mixed diet) is removed by the liver on first passage from the portal vein through to the hepatic vein (termed first past extraction, FPE). Humans are very similar. None of this hepatic first pass extracted insulin ever arrives in the general circulation. The rate of extraction varied from as low as just over 20% up to almost 80% in the dog study. If you have 100 molecules of c-peptide produced, somewhere between 20 and 80 of the associated molecules of insulin will never arrive in the systemic circulation.

Does anything specific alter the FPE? Well, yes, of course. Does anyone think it might be free fatty acid delivery to the liver? Much as this paper tells us

Free Fatty Acids Impair Hepatic Insulin Extraction in Vivo

So under a weight stable modest LC diet (or more accurately; under whole body adipose tissue mass stability) the reduction of insulin secretion from the pancreas under that modestly reduced carbohydrate intake will undoubtedly occur, but would be offset by reduced hepatic FPE and enough insulin will penetrate to adipocytes to keep them full. In the real world this is extremely difficult to make happen, people just want to eat less on a LC diet because as insulin falls more fat exits adipocytes and hunger diminishes. As in Aberdeen. But you can approximate it by artificially controlling (increasing) food intake, ie you pay people to eat more dietary fat than they would like to (which keeps insulin secretion unchanged but increases fatty acid delivery to the liver), hepatic FPE falls and more insulin reaches adipocytes to keep them full.

Under ketosis (let's say with carbs less than 20g/d) there is so little insulin secretion that having an FPE which is probably approaching zero doesn't matter much. Near basal physiological insulin is secreted, almost none is FPE-ed by the liver but there is still minimal exposure of adipocytes to insulin because almost none is being secreted in the first place. So appetite plummets as FFAs rise as they pour out from the adipocytes, despite a minimal hepatic FPE. This should make it even harder to overeat. However, if you do manage it, minimal hepatic FPE by the liver is one of your methods to maintain fat stores under ketosis!

Conversely you can achieve low FFA delivery to the liver by using acipimox. People do not necessarily gain weight with this lipolysis inhibiting drug because it decreases hepatic FFA delivery, so increases hepatic insulin signalling and so increases hepatic insulin metabolism. I assume this increased insulin metabolism will increase FPE and this will decrease insulin delivery to the systemic circulation. I also think this is also how carbosis works, hence the need for very low fat provision in any carbosis inducing diet.

Anyway, here's a thought experiment using made-up numbers. Any resemblance to real life is totally accidental. If a moderate carb diet (say 140g/d) allows a 22% fall in 24-hr urinary c-peptide, does this mean there is a 22% fall in 24h exposure of adipocytes to systemic insulin? Well no...

Say 100 molecules of insulin are secreted and FPE pre-study is 50% then 50 molecules of insulin survive passage through the liver to suppress systemic lipolysis. If only 78 molecules of insulin are secreted under mild carbohydrate restriction but FPE falls to 20% (exaggeration to make the point!) due to increased lipolysis delivering extra FFAs to the liver, then 62 molecules of insulin will make it through FPE and as far as the adipocytes. This insulin could conceivably allow less lipolysis when compared to a carbosis inducing diet, despite reducing insulin secretion.

On a diet in which fat is so restricted as to allow almost none to be spared for oxidation so that FFA delivery to the liver falls precipitously, we can suggest an 80% FPE might be the result. This would be the situation under carbosis, say with a human eating 7.7% fat as part of a severely calorie restricted diet. So of the 100 molecules of insulin still being produced under these circumstances (typified by no fall in 24-hr urinary c-peptide) with an 80% FPE only 20 of those molecules of insulin will eventually hit the adipocytes and so lipolysis would then be greater than under the modest LC diet.

Please bear in mind that these numbers are a reductio ad absurdum example, but they do make a point about what is possible. There are other effects which would kick in but that's not my point here.

My grossly biased opinion is that any study which shows an intervention with superiority in fat loss will be associated with either lower insulin exposure of adipocytes or with an induced failure of insulin to act on those adipocytes (ie by metformin, alcohol, fructose or of course palmitic acid, plus a few others) than is achieved by any comparison diet.

But then I would say that..........

Peter

A post not about Walter Kempner

I've had this paper on my hard drive for a while. It's been sitting somewhere near the front of the back of my mind but was doing nothing to really grab my interest.

Chronic high-sucrose diet increases fibroblast growth factor 21 production and energy expenditure in mice

I've got a draft of a post from mid summer this year which I wrote simply because I like the attitude of the authors. They say things like:

"Excess carbohydrate intake causes obesity in humans".

That's the first line of the abstract. You know, it's that "nailing your colours to the mast" sort of a statement. Even though I do think life is a little more complex than that.

Anyway, I like these folks who are looking at the slimming effect of sucrose in BL6 mice. That's correct, sucrose is a slimming drug/food in mice, under the correct circumstances. People too. The data in the 2017 paper is an extension of the work they did in 2012, written up in this paper:

Ingestion of a moderate high‐sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon‐like peptide‐1 secretion

I don't intend to go through either paper in detail, it's just that the 2012 paper has some rather special macro ratios that caught my eye.

This is what they did to the mice in that original paper:

"After adaptation for 2 weeks, they [the mice] were divided into three groups and fed a normal chow diet (NC), a high‐starch diet (ST) supplemented with 38.5% corn starch or a SUC containing 38.5% sucrose; the latter two diets were prepared by the addition of corn starch or sucrose, respectively, to CE‐2 (Table 1)"

Essentially they are diluting chow with starch or sucrose. Here is Table 1 for the diet compositions, note my red rectangle:

With group sizes of n=4 and five weeks on the diet very little of anything reached statistical or biological significance. The 2017 study used a slightly modified version of the diet to keep a low fat percentage identical across the diets but still had 38.5% of calories from sucrose, was run for 15 weeks and had group sizes of n=8-10. Results were statistically significant all over the place and suggest that the sucrose diet is decidedly good for metabolic health and gives a slim phenotype on ad lib consumption. Just so long as fat calories are very, very low. This looks very much like what Denise Minger described as carbosis, based in part around Walter Kempner's very effective, very unpleasant, ultra low fat, high sucrose medical diet. The Rice Diet is very real.




This post is not about any of the above.




Now, watch carefully. I'm going to sneak in some more macros

If you wanted a "reduced" fat diet which induces carbosis in human beings I recon the red text is pretty well it. I particularly enjoyed that exactly 7.7% of calories came from fat in each diet, this could almost be deliberate. If you combine what is almost certainly a very effective spontaneous weight loss diet with a 30% calorie restriction I suspect you might be on to a winner when comparing it against a reduced carbohydrate diet. Of course to really nail it you would have to compare it to an absolutely non ketogenic diet, say one supplying a total of 140g/d of carbohydrate. Does carbosis beat a middling carbohydrate mixed diet? You bet.

Oh, the scribbled-in red numbers came from Table 2 of this paper.

Most people in respectable CICO based mainstream nutrition have never heard of carbosis, Walter Kempner, the Rice Diet and have probably never heard of Denise Minger.

But Kevin Hall has. My respect for his knowledge-base and ingenuity is vast. Such a pity it's wasted on constructing props for his bizarre pet theories of weight control.

While the 7.7% of calories as fat in both studies is something which amuses me greatly, I do have to admit it may just be an hysterical accident.

At least I'm up front about my rather pronounced personal biases and rather peculiar sense of humour.

Peter

An exchange of half bricks

I would guess that everyone is aware of the study by Ebbeling et al, Ludwig's group, looking at the metabolic effect of low carbohydrate diets on total energy expenditure (TEE, all graphs show kcal/d) in the aftermath of weight loss on a conventional diet.

Effects of a low carbohydrate diet on energy expenditure during weight loss maintenance: randomized trial

I'd like to summarise their data using numbers taken from Tables 2 and 3 which, with a little arithmetic, allows me to produce this graph of TEE at various time points. These are as follows: when the subjects walk off the street (Pre on the graph), after a period of semi-starvation on a conventional diet (Start) and then during weight stability on a high, medium or low carbohydrate diet (End). The plot looks like this:

In the study they compared the change from the Start TEE to the End TEE, ie they used these data points:

They took the absolute changes from Start to End thus and got a resultant p of less than 0.05

This, obviously, is completely unacceptable. Well, it is if you are Kevin Hall. So now we have this

No Significant Effect of Dietary Carbohydrate versus Fat on the Reduction in Total Energy Expenditure During Maintenance of Lost Weight

What Ludwig's group did wrong (amongst the many other things pointed out by Hall and Guo) is that they used the wrong data points.

Recall the original graph:

According to Hall: If you want to ask about the effect of low carbohydrate diets on the depression in TEE produced by conventional semi-starvation you should NOT compare the semi-starved TEE (as in Start) to the TEE on a high, medium or low carbohydrate diet (End). You should instead use the TEE expenditure at randomisation (Pre on the graph). Like this:

Using Pre as your anchor point you can draw the same data thus:

Which obviously gives us p greater than 0.05 and all of the benefits of low carbohydrate diets are lost. Phew. Happy Hall. But why should anyone use the Pre values as an anchor point?

Now, no one is an unbiased researcher. Hall is, surprisingly, no exception. Hence the current exchange of half bricks in the BMJ.

As I see it the Ebbeling paper looks at the effect of LC eating on the damage done to TEE by conventional dieting.

What Hall wants the analysis to do instead is to look at the overall effect of damage done to TEE by conventional semi-starvation combined with partial rescue during weight-stable LC eating vs the combined damage done by conventional semi-starvation followed by maintained damage done by HC weight-stable eating. As he writes:

"However, the final analysis plan was modified to make the diet comparisons with the TEE measurements collected in the immediate post-weight loss period rather than at the pre-weight loss baseline"

To me Hall is stating that Ebbeling et al almost did make the "Hall" mistake of using the "Pre" TTE as anchor point but corrected this at the 11th hour, still before blinding was unmasked. What puzzles me is how Ebbeling could have ever even considered using the "pre weight loss baseline" as the anchor point in the original study design.

The massive benefit to Hall of including the conventional semi-starvation active weight loss period along with the intervention weight stability period is to dilute the remedial biological effect of LC eating out of statistical significance.

The core information which the study provides is about the remedial effect of LC eating on correcting the damage done by a conventional semi-starvation period. That effect only happens between "Start" and "End", which is when carbohydrate restriction is applied.

That's one of the MASSIVE problems with carbohydrate restricted eating. It only provides benefit when you don't eat carbohydrate!

Including data from "Pre" right through to "End" dilutes the clearly demonstrable biological effect of carbohydrate restriction on reduced TEE post conventional dieting.

So what doe the title and text of Hall's rebuttal tell us? Either about Hall or about TEE? Don't over think it!

I would also declare that my own biases are a conflict of interest but if you need me to say that then you have probably arrived here by accident, you know where the back button is.

However I would say that I am ambivalent about the importance of the TEE changes, though I suspect they do happen. What really matters to me is what happened in Aberdeen over a decade ago.

Peter

More on insulin and the glycerophosphate shuttle

Raphi tweeted this paper recently

Nutritional Ketosis Increases NAD+/NADH Ratio in Healthy Human Brain: An in Vivo Study by 31P-MRS

which is nice provided, as he comments, it can be replicated. There is absolutely no possible conflict of interest anywhere so long as you accept it looks like an in-house Nestlé study. I haven't knowingly bought a Nestlé product in over 30 years.

Anyway. The study looks at healthy brain biochemistry under MCT induced ketosis. The ketone oxidation (or possibly the CNS oxidation of MCTs) increases the NAD+:NADH ratio, ie moves it in the Good direction.

There is a lot of talk about the NADH generation and NAD+ depletion during glycolysis to pyruvate, shifting the ratio in the Bad direction. The assumption (with which I disagree) is that the glycerophosphate shuttle is a rescue mechanism to regenerate essential NAD+ to allow glycolysis to continue, to which I will return in a moment.

The beauty of ketones is that they do not deplete cytoplasmic NAD+ at all and only consume one mitochondrial NAD+ during the conversion of BHB to AcAc. Because this happens within the mitochondria this, plus any NADH generated at the pyruvate dehydrogenase complex, is sitting next to complex I, the most prolific re-generator of NAD+ in the cell...

All well and good and bully for ketones and the manufacturers of Peptamen®1.5 Vanilla (Nestlé Health Science SA).

This got me thinking.

Of course no one in their right mind would expect glycolysis to be arranged in such a manner as to require the glycerophosphate shuttle for simple NAD+ regeneration. This is a wasteful loss of four pumped protons and this energy will appear as heat. Think of brown adipose tissue, full of mtG3Pdh, assuming insulin is plentiful.  The correct pathway for the metabolism of glucose without insulin is to lactate without any overall depletion of cytoplasmic NAD+. Lactate can then be taken up by mitochondria exactly as ketones are. Lactate will, in the mitochondria, be reconverted to pyruvate, depleting mitochondrial NAD+ in exactly the same way as the conversion of BHB to AcAc does. Equally this happen right next door to complex I, just waiting to regenerate NAD+ and keep that NAD+:NADH ratio nice and high.

The whole point of the glycerophosphate shuttle (in Protons terms) is to facilitate insulin signalling.  Insulin is the hormone of plenty, used to encourage caloric ingress in to cells. Loss of the four pumped protons due to bypassing complex I and using mtG3Pdh instead as part of insulin signalling appears perfectly reasonable under conditions of active caloric ingress. Sustained insulin signalling causes sustained loss of cytoplasmic NADH, which generates NAD+. Once this has happened there is no longer the surfeit of cytoplasmic NADH over NAD+ from glycolysis, which is essential to drive lactate formation. Glycolysis must therefor stop at pyruvate under insulin.

Summary: For insulin signalling the glycerophosphate shuttle is active and loss of NADH requires glycolysis to abort at pyruvate.

Without insulin signalling glycolysis runs to lactate which enters mitochondria without any depletion of cytoplasmic NAD+. The lactate should enter the mitochondria, under normal physiology.


Sooooooo. This had me thinking about what would happen if, in the presence of copious glucose and copious oxygen, there was to be a sudden profound fall in absolute insulin levels. I was particularly interested in systemic lactate levels.

A sudden, profound fall in insulin levels in the presence of glucose is pathology. It generates ketoacidosis, classically from acute beta cell destruction during the onset of DMT1. There is always a profound metabolic acidosis from the failure to suppress glucagon-induced lipolysis and subsequent massive acidic ketone generation. Under the canonical view the absence of insulin should not stop NAD+ regeneration by the glycerophosphate shuttle.

What I wanted to know was whether the Protons predicted shutting down of the glycerophosphate shuttle due to hypoinsulinaemia would result in diversion past pyruvate to lactate as the end result of glycolysis. In the presence of massive levels of ketones I would also expect this lactate to appear in the systemic situation.

Does it?

Yep. Ten seconds on Google says so.

Lactic acidosis in diabetic ketoacidosis

Very nice. I had no idea this was the case because it has no direct influence on treating DKA clinically...

Peter

Of course you have to think about the chicken and egg situation with insulin and mtG3Pdh activation (I have been for years!). Which comes first? I think insulin appears to be essential, as above. I do wonder if the insulin receptor will turn out to dock with the glycerophosphate shuttle in some way...