Wednesday, August 02, 2017

Metformin (02) The dose makes the poison

Before the days of interest in metformin as an anti-neoplastic agent, a performance enhancing drug or a longevity promoter, it was just given to T2DM patients to help lower blood glucose levels. These folks, as a group, quite often have significant renal disease. Which can render metformin and lactate cumulative in the blood stream and lead to a life threatening lactic acidosis.

This paper looked at a series of 10 hapless folk to whom this happened:

Metformin overdose causes platelet mitochondrial dysfunction in humans

The mean blood concentration which gets you an ITU bed was 32mg/l. Now this is a clinical paper, written by clinicians. Nothing wrong with that, except they use Noddy units which makes the metformin concentrations extremely difficult to relate to the vast body of metformin research, which uses units of millimolar or micromolar.

So we really need to take this image

and think of it in these terms when we're looking at research papers using mmol or micromol concentrations:

Bear in mind that these are very chronic exposure values and metformin is thought to be progressively cumulative within the mitochondria on chronic exposure. Of course, complex I is intra mitochondrial and there will be some dependency on cumulation in getting significant effects at this site. What we can say is that, in the above diagram, there is not enough inhibition of complex I to raise lactate production in platelets, an extra-hepatic tissue (hepatocytes may be slightly different), unless we are using near-death concentrations.

What is not hidden away inside the mitochondrial matrix is mtG3Pdh. It's on the outer surface of the inner mitochondrial membrane and will be exposed to whatever metformin concentration that manages to get inside the cell.

From the classic paper

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

we have this graph from Figure 3, using a slurry of mashed up mitochondria and some glycerol phosphate:

Here we have a significant effect on the oxidation of glycerol-3-phosphate at micromolar concentrations. Admittedly by 50μmol we are looking at very much the upper end of therapeutic concentrations but an effect is clearly visible at this level. We can say from the platelet paper that exposure to 250μmol (black circles at the bottom of the graph), if sustained, will put you in the ITU with potentially fatal lactic acidosis.

Because mtG3Pdh is exposed to cytoplasmic (non cumulative vs mitochondrial) metformin levels it will see the drug at plasma concentrations (or slightly less) and it will see these concentrations as soon as metformin enters the blood stream.

If you want a performance enhancing drug for endurance exercise, say a cycle race taking about three hours, you can pop a single metformin 500mg tablet one hour before the start of the race and extend your time to exhaustion from 167 minutes to 191 minutes. That might make some difference to winning vs not finishing.

Metformin improves performance in high-intensity exercise, but not anaerobic capacity in healthy male subjects

Equally, there is no acute effect on lactate levels in the same study. This is no surprise as I find it difficult to envisage acute complex I blockade, to lactate generating levels, as a performance enhancing ploy.

TLDR: metformin probably works in the cytolasm on mtG3Pdh. Rising lactate may well indicate mitochondrial cumulation and some degree of complex I inhibition. Extrapolating benefits from studies based around millimolar concentrations in-vitro may well put you in to the ITU if you try them in-vivo.


Tuesday, July 25, 2017

Metformin (01) Insulin

This image is taken from the paper Insulin requirement for the antihyperglycaemic effect of metformin and it deserves a little consideration.

They are using BB/S rats which spontaneously develop T1DM if fed standard rodent chow. In the absence of exogenous insulin they die but giving them a little Ultratard twice daily keeps them alive for quite some time. Stopping the Ultratard allows exogenous insulin withdrawal to produce an acute, alive, an-insulinaemic rodent model. This is the model used and at the start of this experiment the rats had no detectable insulin in their blood.

At time point -60 these an-insulinaemic rats were given metformin intrajejunally. Over the next 60 minutes the metformin did nothing to lower plasma glucose. At time point zero they were given a small intravenous bolus of glucose. Metformin had no effect on the additional hyperglycaemia induced.

At time point +90 they were given neutral insulin intravenously. In the control group plasma glucose concentration dropped to a nadir of 20mmol/l at time point +150 but in the metformin treated rats the same dose of insulin continued to reduce the plasma glucose to 10mmol/l at time point +180, when p dropped below 0.05.


Insulin is essential to demonstrate any effect of metformin on blood glucose.

Any idea about how metformin works, be that via the inhibition of mtG3Pdh or via inhibition of complex I, has to accommodate the essentiality of insulin.

That's an interesting constraint.


Monday, July 24, 2017

An update

Hi All.

We've moved house. It has not been the simplest of moves. OK, it was awful. However it was also worth it as we live here now.

While the house is in pretty good order the acre and a half of ground needs some TLC before we can get the chickens strip grazing and maybe some stock in, so I sort of doubt there will be a huge amount of free time to blog. Maybe a little musing on metformin might be possible...

Anyway, we're alive and busy and now live some distance from the nearest main road (in Norfolk terms).


Saturday, June 03, 2017

Why stop at formaldehyde?

If we consider the dissociation of hydrogen:

the right hand side of the equation can supply electrons to another reaction. The tendency for this to occur is in part dependent on the pH of the solution. If we consider alkaline hydrothermal vents we have a pH of around 11, this drives the reaction to the right because the protons avidly combine with hydroxyl ions to give water:

Which means that there is a marked tendency to supply electrons for any electron-accepting reaction. The electrons can hop on to an FeS barrier (each changing the charge on an Fe from 3+ to 2+) which separates the vent fluid from CO2 rich, acidic oceanic water:

Deriving from fluid with a pH of 11 these electrons have a redox potential of -650mV, ie they are highly reducing.

If we now look at the situation on the oceanic side of the barrier we have:

and by adding on the factor of an acidic pH, with lots of protons driving the reaction to the right we have this:

Under these conditions electrons supplied at -650mV are very able to allow the reaction to proceed to the right yielding CO. Repeating the process yields CH2O and metabolism is on its way.

OK. Nick Lane makes these points in his paper:

1. There is no contact between the H2 in the vent fluid and the CO2 in the ocean fluid. The two Hs in the formaldehyde come from oceanic protons combining with vent H2 derived electrons.

2. I've shown the reaction occurring once to CO and again to CH2O. Why stop at twice? Given a supply of -650mV electrons why not keep generating CO and inserting it, along with e- and H+, in to whatever hydrocarbon you have already got in the vent fluid? Nick Lane has reaction sketches for generating almost all of the Krebs cycle components on this basis.

Theoretically, if you wanted to make an origin of life reactor to test whether you can generate a multitude of the hydrocarbons at the core of metabolism you don't actually need a supply of alkaline hydrogen rich fluid. This only supplies electrons at -650mV. An alternative supply would be a 1.5 volt battery with some sort of voltage reduction to get from -1500mV to -650mv and you're away.

A microporous FeS electrode in Perrier water, energised by an AA battery via a couple of resistors and you might just be set up. Getting the apparatus anoxic and detecting the products might be more of a challenge!

Edit Finally followed Nick Lane's final reference. These folks have reached pyruvate via an energised FeS electrode. It's a lot more complex than Perrier water but it works. End edit


Thursday, June 01, 2017

Nick Lane on Proto-Ech

Nick Lane has a few more downloadable papers available on his website, two of which focus on ideas I've thought a lot about. Here are a few quotes:

Iron Catalysis at the Origin of Life

"Why does the reduction of ferredoxin via Ech depend on the proton-motive force? The answer is as yet unknown, but cannot relate to reverse electron flow [as originally proposed (49)] as these methanogens do not possess an electron-transport chain (37,38). A more pleasing possibility is that pH modulates reduction potential at the active site of the enzyme. The flux of protons through Ech from the relatively acidic exterior could lower the pH at the active site of the enzyme, which should facilitate reductions that depend on protons, including CO2 as well as some ferredoxins (50)".

My italics. Next:

Proton gradients at the origin of life

Aside: If you read the full text of Lane's paper you will take note of Jackson JB (2016) Natural pH gradients in hydrothermal alkali vents were unlikely to have played a role in the origin of life. And this passed scrutineering. Nick Lane does not seem impressed. End aside.

"One possibility is that prebiotic carbon and energy metabolism entailed the synthesis of reactive thioesters analogous to acetyl CoA, such as methyl thioacetate, coupled to substrate-level phosphorylation, generating acetyl phosphate and ultimately ATP [1, 17, 27, 60–63] as still happens in bacteria [14, 31]".

"Across the barrier, in acidic conditions, CO2 is more easily reduced, and so is more likely to be reduced by Fe2+ in the barrier. The semiconducting barrier should transfer electrons from Fe2+ on the alkaline side to Fe3+ on the acidic side. The thickness of the barrier does not matter, so long as it is semiconducting. The two phases do not come into direct contact - H2 and CO2 do not react directly (Fig. 3)".

This is really neat, it puts in to a published paper many of the logical concepts that went in to the Life series. I really like the pre biotic ideas of electron transfer across any-thickness FeS barriers. No need for membranes, indeed insulating "crud" membranes would hinder electron transfer from the FeS wall to the enzyme, necessitating the generation of a pore like structure (ancestor to NuoH) to get the voltage generating acidic pH to the active enzyme's site.

This ferredoxin reduction plus subsequent substrate-level phosphorylation is where it should all start. NuoH starts as a pH channel, not part of a nano machine. That comes later with reversal of proton flow and the development of complex I, a true advanced nano machine.

I still don't buy ATP synthase (another very complex nano machine) as running on the primordial vent proton gradient as Nick Lane holds to. Later developing Na+ energetics look much more likely, these following on from Proto-Ech's pore duplication to form a Na+/H+ antiporter, giving a usable Na+ gradient. That clearly post-dates some sort of membrane, which ferredoxin based metabolism must precede when using a geochemical proton gradient. NuoH becomes essential only after a crude membrane forms to impede this process of ferredoxin reduction.

Nice papers.


Tuesday, May 30, 2017

Adrian Ballinger on Everest

Back at the end of 2015 Mike Brampton and I had a conversation about climbing Everest.

Based on Graph A from Fig 3 in D'Agostino's rat paper

Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats

our conclusion was that summiting Everest might be best achieved using a ketogenic diet. I know nothing about extreme climbing or the culture which goes with it but it came as no surprise, via Mike, that they carb loaded and carb loaded and carb loaded. You know, sugar has its own partial oxygen supply built in to the molecule. No point trying to burn fat if there's no oxygen*. Understandable but, obviously, completely incorrect. I think Mike had been trying (frustratedly) to convert altitude folks to fat centred thinking for some years before this.

*It's true that there is no point trying to burn fat under anoxia. But given some oxygen ketosis pays dividends.

So it was interesting to pick up this link on Facebook:

How Adrian Ballinger Summited Everest Without Oxygen

This fits in with Veech's concept of increased metabolic efficiency per unit O2 consumed when burning ketones and D'Agostino's discovery of an "unexpected" rise in arterial PO2 in rats gavaged with a betahydroxybutyrate/acetoacetate combination precursor, while they were breathing room air (PaO2 from 100mmHg to 130mmHg, pardon the archaic units).

Very gratifying, even if completely different from the approach taken by Naked Mole Rats and their fructolysis.


Fructose and lactic acid in Naked Mole Rats

Naked Mole Rats appear to use fructose as their preferred metabolic substrate when exposed to both physiological hypoxia (which is common in their lifestyle) or complete anoxia under experimental conditions. It's irresistible to go and find out a little about why they might do this.

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

I suppose the first thing to say is that the fact that fructose is protective against hypoxic cellular injury has been known for a long time, this paper coms from 1992:

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi

There was a lot of work done in the 1980s and 90s looking at ways of preserving liver cells under anoxia. I'd guess this was looking to improve the survival of harvested livers within the transplant program.

If we look at ATP levels compared to an externally supplied control (MDPA) we have this graph, with hypoxia imposed at one hour and relieved at three hours:

ATP falls faster within the first 30 minutes of anoxia with fructose. Although the trends are interesting, all else is ns after 45 minutes. So fructose causes a more severe ATP depletion than glucose. However a better marker is the ratio of ATP to Pi (phosphorylation potential), here plotted as the inverse for some reason, ie the lower the better in the graph:

So under fructose there is less ATP in the cytoplasm than under glucose but the phosphate level is even lower, giving a similar or more favourable ratio of ATP to Pi except at the 30 minute mark. So the next question is: Where has the phosphate gone?

This might be related to the protective effect of cytoplasmic acidosis. It doesn't seem to matter how you acidify the cytoplasm (fructose is as good a way as any), it's the acidosis which appears to protect against mitochondrial failure. There's a nice paper here

Protection by acidotic pH and fructose against lethal injury to rat hepatocytes from mitochondrial inhibitors, ionophores and oxidant chemicals

and here

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

So if we go back to Gasbarrini's paper we can look at a surrogate for intracellular pH and how it differs between fructose and glucose:

Fructose produces a much more profound acidosis. If we look at that basic ETC doodle I used in the rho zero cell post, but eliminate complexes I, II, III and IV we have this:

We have here two process which can be driven by an excess of protons in the cytoplasm over those in the mitochondrial matrix. Transport of Pi in to the mitochondria and synthesis of ATP. Which of these is most important to ensure cell survival is hard to say. It is even quite possible that it's neither and that maintaining an excess of protons outside the mitochondria maintains delta psi so defers the commitment to apoptosis or the occurrence of necrosis.

Later changes which confirm the commitment to cell death are an influx of extracellular calcium in to the cytoiplasm. This is marked under glucose and stays within tolerable limits with fructose. I strongly suspect the metabolic decision making is being controlled by the pH drop and the Ca2+ influx is consequent to a mitochondrial decision as to how badly damaged the cell might be. But it's hard to be sure with the data we have in these rather elderly papers.

About that acidosis:

Here are the reactions relevant to the pH change in lactic acidosis, all taken from the wiki entry on lactic acid. They are interesting. This is the situation down to pyruvate:

There are two protons generated to acidify the cytoplasm. Now look at this step where pyruvate is converted to lactate. The molecules in the red oval are needed to form the lactate.

So where did the two acidifying protons go to? They are consumed in converting pyruvate to lactate. Does lactic acid generation actually acidify the cytoplasm? It appears not to do so here but it must do because the overall reaction is:

So where are these two protons? They are in the two ATP molecules:

The conversion of ATP to ADP releases them. So lactate causes acidosis only when the ATP generated during glycolysis/fructolysis is consumed... Obviously ATP depletion is common in anaerobic exercise or hypoxia/anoxia. Hence lactic acidosis shows under these two conditions.

The Naked Mole Rat paper is very descriptive, with lots of experimental results but is light on insight as to hows and whys. I think the above scenario might well have explanatory power and might have been extended from the liver to the rest of the body in NMRs.


Thursday, May 18, 2017

Fructose and metabolic syndrome: Uric acid

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

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

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

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

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

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

This is all basic biochemistry.

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

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

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

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

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

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

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

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

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

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


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

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

Phylogenomic Analysis of Type 1 NADH:Quinone Oxidoreductase

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

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

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

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

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

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

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

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

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