Friday, November 20, 2015

Protons (40) Living without the glycerophosphate shuttle

If we look at a mouse with a deletion of the gene for cytoplasmicG3Pdh we have a reasonable model for elimination of the glycerophosphate shuttle at the level were glycolysis would normally be used to drive reverse electron flow through complex I, to facilitate insulin signalling. These mice are remarkably normal and can swim in deep water, possibly not too happily, for at least 20 minutes with weights on their tails, i.e. they can exercise, at least if necessary to save their lives. They have markedly reduced levels of pyruvate and mildly elevated levels of lactate in muscle tissue compared to control mice.

It's the bottom two lines which I looked at. The lactate to pyruvate ratio reflects the NADH to NAD+ ratio within the cytoplasm, as Krebs puts it:

In a cell [H+] is a constant and K is also a constant (by definition).

So clearly we have a lot more NADH available in the knockout mouse, so more lactate gets formed from pyruvate, which becomes a minor player in the cascade of glycolysis to oxidative phosphorylation allowing lactate to take over. The authors of the mouse paper suggest that the lactate is expelled from the cells and that the Cori cycle, in the liver, is active to deal with the it. I’d prefer to think of lactate as being shunted directly to the mitochondria for use in the TCA.

If you subscribe to the view that the glycerophosphate shuttle is needed to provide NAD+ for glycolysis to proceed you might expect a few problems with glucose processing. There is undoubtedly an accumulation of metabolites upstream of the glycerophosphate shuttle and a depletion of those downstream but glycolysis does proceed. But from my point of view, with no glycerophosphate shuttle, there is nothing to allow the body to facilitate insulin’s drive to self activate using glucose. How do these mice cope?

They cope very well.

If you feel that tying a weight to the tail of a mouse and dropping it a beaker of water is a bit too crude, there are more sophisticated methods of inducing exercise. Worse than making obese people do cardio at any gym where fat shaming rules. It's possible to run a mouse to utter exhaustion and monitor its respiratory quotient while it runs. So you can see whether it burns predominantly glucose or fat and in what balance. You can also measure exactly how long it can run for, before it collapses at the level of exhaustion where it can no longer avoid an electroshock or two or three. Here are the core findings from sending mice to an electro-gym (the Thumb Tack Hypothesis taken to serious levels):

HeA are the knockout mice, By are the control mice. Knockout mice run harder and for longer than control mice. The rest of the graphs use the same coding, solid line is the control mice, dotted line the knockouts.

Taken from the RQ we can ask whether the knockout mice can oxidise glucose. Yes:

Pretty much as well as the control mice.

Can they oxidise lipid? Yes, somewhat better than can the control mice:

I think it is also worth noting that under marked but non-exhausting exercise that glycogen in the muscle of knockout mice does not fall, it does so in control mice:

These modified mice, which cannot use glycolysis to trigger insulin signalling, have a tendency to have MORE glycogen in their muscles (although p is greater than 0.05) at rest and they deplete it less under sustained near-maximal exercise. I'd guess insulin does signal.

It's also worth noting that blood lactate under the same conditions does not rise in the knockout mice whereas it does in control mice (p less than 0.05, yay!). My assumption is that lactate is being metabolised in the muscles of knockout mice and shunted to the liver for the Cori Cycle in control mice:

So what might be going on in these knockout mice? The requirement for insulin signalling is a modest amount of reverse flow of electrons through complex I, ie the CoQ couple must be reduced. The usual, here absent, technique is the glycerophosphate shuttle. But we can reduce the CoQ couple in other ways. My favourite way is via the oxidation of the FADH2 generated by metabolism of saturated fatty acids. How much FADH2 is needed to replace the glycerophosphate shuttle?

From the graph of lipid oxidation above we can see that knockout mice under exercise are oxidising somewhere around 45mg/kg/min of fat. The control mice are oxidising just over 30mg/kg/min. From the Protons perspective the increased fat oxidation is a requirement for normal insulin signalling and this insulin signal cannot limit fatty acid oxidation until the rate is almost 50% higher in the knockout mice than in those where the glycerophosphate shuttle works. These mice oxidise fat because insulin signalling is not being triggered by glycolysis. It also means that lipid oxidation has to be higher before it can trigger insulin resistance and cellular energy influx limitation.

We don't (as far as I know) have a drug to inhibit cytG3Pdh.

We do have one to inhibit mtG3Pdh, the other half of the glycerophosphate shuttle.

It's called metformin. Does metformin do the same thing as having a cytG3Pdh knockout does? Under exercise?  In terms of getting the King Of the Mountains jersey in the Tour de France perhaps?

Possibly so.


Summary: Metformin blocks glycolysis triggered insulin signalling and cells replace this with FADH2 triggered insulin signalling from fatty acid oxidation (at ETFdh). This results increased fatty acid oxidation and in improved high intensity exercise ability. Oh, and I guess weight loss etc...

Aside. I think I might start sticking the refs from a post in at the end. There are times I can't remember in which post a paper was used, searching my own blog/hard drive might be easier if the author or a keyword are actually present rather than there just being a highlighted text field!!! The blog is getting a bit unwieldy.

Glycerol 3-phosphate dehydrogenase 1 deficiency enhances exercise capacity due to increased lipid oxidation during strenuous exercise

Mouse lacking NAD+-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function but abnormal metabolite pattern in skeletal muscle

The Redox State of Free Nicotinamide-Adenine Dinucleotide in the Cytoplasm and Mitochondria of Rat Liver

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


Tucker Goodrich said...

So it seems metformin simulates a low-carb diet, by reducing glucose demand from the cells, and reducing glucose production by the liver.

Not surprising then, that it's useful in the treatment of diabetes. The performance gain is pretty interesting, however.

Have you seen the draft FASTER study from Phinney & Volek that's been floating around of late? Especially the part about glycogen usage/recovery in low-carb athletes? (I can send if not.)

Tucker Goodrich said...

And interestingly, neither "metformin" nor "Glucophage" appears on the World Anti-Doping Code Prohibited list...

Betsy said...
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Peter said...

Tucker, yes. metformin is LC in a pill. The suppression of hepatic glucose output is another subject. Needless to say it is (probably) energetics related.

Also yes re FASTER. It wouldn't have surprised me if the LC athletes had had more glycogen than to HC group. Can't have everything!

The other person of interest was Steve Redgrave. He functioned on insulin w/o metformin as far as goes. Clean, I think.


Tucker Goodrich said...

"It wouldn't have surprised me if the LC athletes had had more glycogen than to HC group. Can't have everything!"

That's the weird part, the glycogen for both groups behaved exactly the same, even though the LC groups was of course using much less glucose for fuel, they started with and used just as much glycogen. And refilled the glycogen stores just as quickly, without ingesting glucose.

They suspect that glycogen might have some use other than as fuel, at least in part.

Betsy said...

Does this mean that a low carb diet along with adequate riboflavin and B5 would be a work around for people with Complex I deficiency diseases such as polymiositis?

Betsy said...

And what happens if there is a CoQ deficiency due to destroyed mevalonate pathway? Some people acquire polymyositis after taking statins. I can't put everything together in my brain. Maybe my questions aren't even valid, I don't know at this point, I'm having a hard time keeping up with all this information.

Unknown said...
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marie said...

Peter, thanks for analyzing this paper. You reminded me that "metformin is LC in a pill" just in time to convince an old college friend I'll see in a few days for thanksgiving (usa). He's just been diagnosed with T2D. He also has gout though. I'm very comfortable with cyclic ketogenic eating for myself and family, but know next to nothing about gout. Would there be any caution or modification needed for my friend do you think? I'm recovering from thromboendoarterectomy right now and don't trust myself to research that alone. Thanks for any pointers, here or email, if you get a chance.

Passthecream said...

@Marie, I used to suffer from gout long before being diagnosed as t2dm. As with diabetes there are different types of gout and everyone responds differently. Initially cutting back starchy ( ie junk) food and eating better generally made a difference to the gout. Metformin didn't eliminate it entirely but since going low carb I have had no recurrences. I don't eat much fruit anymore either, fwiw, you can read what Peter wrote about fructose and gout some time ago. Even so when I get my periodic blood test results back I always have uric acid level near the top of the normal range. It must be innately so.

@Peter, lovely work. Metformin works for its intended purpose very well, but even with 'LC in a pill' I still have mixed feelings about using the stuff in the long term. Genuine LC seems to be errh, sweeter ? Excuse the pun. I mean it feels better, systemically. Sometimes I have to take a holiday from metformin. Perhaps there are some subsystems that don't like having glycolysis supressed to that extent even in the absence of extrinsic glucose supplies? At the back of my mind is the notion that much stronger drugs with similar effects such as rottenone are used to induce a form of Parkinsonism.


Betsy said...

I do also look forward to your answer to Marie's question about gout and diabetes.

In the meantime I will post something about uric acid since I have spent some time thinking about it due to waking up one morning with severe joint pain all over my body. (Yes, I had been drinking fresh pineapple juice.)

See what you think about this view of uric acid. Are the crystals made of copper urate or some other urate? Are copper free radicals floating around in the bloodstream more common in diabetes? Another thing I've been wondering is why does stress bring on a gout attack? Cortisol? I don't know, I just see some dots but can't connect them.

B2 has helped me to be able to eat meat again, but it took a long time. During the time when I couldn't eat it, I couldn't eat anything too acidic, tomato sauces being the worst, pineapple juice and tomato sauce can still cause joint pain to return. So something to do with acidity, and I do think that acidity breaks down ceruloplasmin somehow, causing free copper.

Anyway, here's a couple of paragraphs from the study.
"The ability of urate to scavenge oxygen radicals and protect the erythrocyte membrane from lipid oxidation was originally described by Kellogg and Fridovich,[21] and was characterized further by Ames et al.[4] Although these experiments defined a paradigm, they addressed effects of uric acid under specific conditions in which exogenously added uric acid protected cells from oxidants, which were also added exogenously to aqueous incubation media. This kind of condition is undoubtedly relevant to a variety of physiological situations when circulating uric can scavenge reactive radicals released into the blood by deleterious reactions, such as autoxidation of hemoglobin or peroxide production by macrophages.[4] However, even in the plasma urate can prevent lipid peroxidation only as long as ascorbic acid is present.[22]

One of the major sites where the anti-oxidant effects of uric acid have been proposed is in the central nervous system, particularly in conditions such as multiple sclerosis, Parkinson’s disease, and acute stroke.[23–26] While chronic elevations in uric acid are associated with increased stroke risk,[27,28] acute elevations in uric acid may provide some anti-oxidant protection. For example, uric acid protects cultured rat hippocampal neuronal cells from oxidative stress,[29] and administration of uric acid 24 hours prior to middle artery occlusion also attenuated brain injury induced by acute ischemia in rats.[29]"

Betsy said...
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marie said...

Passthecream and Betsy, thank you both for your thoughts on gout, t2d and diet and for the info pointers, I'll be looking up your references. I'm basically trying to check to make sure that going VLC or even keto for the diabetes won't aggravate the gout (I don't immediately see any reason it woyld). I could just get him to fast in order to improve the insulin resistance and eventually cure the t2d (a la Jason Fung treatment or bariatric surgery) but I dont have great faith that he'll keep up a regular fasting regimen long enough once he's back in his own environment. So, leaning towards VLC.

Betsy said...

In one of Peter's other posts he says that fasting when you are in a bad metabolic state can be very dangerous.

Getting your body out of the diabetic and gout condition can be difficult, but maybe Peter will have some recommendations.

Metformin sounds like it would help ease things up a lot. The workaround that he talks about in this post might be what you aim for with your husband.

Ash Simmonds said...

Gout/joint inflammation is one of the primary reasons I stick to keto/LC/carnivory.

I've put together a shit ton of resources on this stuff here.

Betsy said...

For diabetes I would consider looking into non-alcoholic fatty liver disease. I don't know whose plan to recommend, but Chris Masterjohn has some good ideas which happen to fit in with Kwasniewski's diet.

Bariatric surgery unfortunately has some very serious side effects, which I suppose doctors don't tell you about until the issues pop up, kidney stones being the most obvious.

Link with respect to NAFLD and diabetes.

Passthecream said...

Thanks Ash!

The Taubes letter is fantastic. Your compilation is awesome.
All sorts of loose ends are being tied up for me in there.

Do uric acid levels typically stay a little higher with higher ketone levels?

Did you ever experience the joys of Colchicine?

Ash Simmonds said...

>Do uric acid levels typically stay a little higher with higher ketone levels?

Not really, see this graph:


Basically, in the early stages when not keto-adapted and are pissing them out they compete for excretion with uric acid, but over time when you're no longer wasting tons (months) levels drop to normal or even lower. However yeah there is a higher risk of gout outbreaks during this time, also why it's not a good idea to do silly stuff like "carb backloading" etc. This is also why I'm wary of the idea of exogenous ketones - last thing I need is to deliberately stop my uric acid from being excreted.

Those at higher risk might benefit from an intake of ~50-80g of carbs/day and make the adaptation slower but easier.

>Did you ever experience the joys of Colchicine?

No, after much excrutiating testing I've found simple ibuprofen in 600-1000mg doses and fasting to be the best relief. Of course not a good strategy if you have gut issues, and not one I'd do for more than a few days - but thankfully for me it seems to clear up in 2-3 days doing this.

Betsy said...

@marie, it looks like NAFLD can be tied to gout, too!
Gout and risk of non-alcoholic fatty liver disease

And some info on Kwasniewski's diet and what it works for: diabetes and gout

Chris Masterjohn has plenty of good information showing that choline is the ticket for fatty liver. I just realized that maybe that's one reason that Kwasniewski's diet is helpful for all those conditions, the choline in the egg yolks helps clean out your liver.

karl said...

Petro wrote:
"The blog is getting a bit unwieldy."

Yes - You need to write a book - it is obvious they need it for the med schools!

There is a bit of secret google-foo that might help others - ferinstance - if you want to see what Petro wrote about NADH try this:

Brings up bit in comments as well

karl said...

It should be noted that there are variable responses to metformin - made a friend of mine VERY sick. I'm not surprised as this part of our evolution is still in flux from the recent invention of the plow.

(There are a subset of people that can even get a bad reaction from taking CoQ10 supplements - makes me very absent minded - end up constantly losing my cup of coffee. )

My point is these interventions - pharmaceutical, supplements, potato-diets - have risks that are hard to measure - "Black Swans' and all (see

To me it seems that doing the same thing via diet is a better idea. HFLC diets, IMO, are closer to what our hunter-gather ancestors ate - thus likely to be low in risk.

Peter said...

Thanks Karl. I will get to emails (and comments) soon. Just need to do two days work, finish a little bit of rewiring I'm working at and get stuff sorted for Hazel's birthday and there will be time!!!! All this hiking and 'yaking tends to take a lot of time. Well worth it...


Peter said...

Hi Tucker, glycogen as a signalling molecule is interesting. Must go read the full text.

Betsy, re complex I def. Yes and no… In acquired diseases typified by complex I dysfunction (AD, PD etc) exogenous ketones are only effective so long as complex II is functional. So the TCA is driven hard with NADH accumulation but complex II provides some input to the ETC. I have papers somewhere showing complex II blockers abrogate the benefit of ketosis in neurodegenerative diseases. Clearly neurons do not have the luxury of beta oxidation derived FADH2 inputs, they’re stuck with complex II. CoQ failure = permanently reduced CoQ couple, lots of reverse electron flow, lots of insulin resistance = diabetes. Good old statins.

marie, in gout I would be very, very cautious about any glycolysis substrates other than glucose. Glucose can be rejected by physiological insulin resistance. Fructose cannot. LCHF based on palmitic acid + forced uncontrolled glycolysis = worse gout, would be my bet. Stan (Heretic) has an anecdote about his mother and apples on this. A spud a day for your carbs would be safer. I guess a high PUFA diet might be a problem too as the reverse electron flow to stop glucose ingress will not happen as double bonds fail to generate the FADH2 at ETFdh to stop glucose ingress. I think of gout as calorie overload in the cells of the joints (and other places).

Pass, I have some ambivalence about metformin but for those it doesn’t kill is does, overall, seem to help. But it is messing with a very fundamental control point. The rotenone issue seems to be fully complex I related. Very high dose metformin would do this, the current papers are looking at pharmacological doses/concentrations. Looks (at the mo) like therapy = mtG3Pdh, supra pharmacological = complex I blockade. But even low dose rotenone extends lifespan in simple models like C elegans… Yes, high dose rotenone = PD, of a type. And maybe mtG3Pdh full blockade is equivalent to beneficial low dose complex I blockade. Early days to say yet.

Betsy, uric acid is hugely complex. As a free radical scavenger I expect it to block insulin signalling as it will mop up superoxide or H2O2 that was destined to activate the insulin/receptor complex. So blocking insulin signalling, in general = extending life (as in mice on glucosamine). Under high level H2O2 generation uric acid may mop up the damage done by stupid things like fructose ingestion. But it is also a marker of fructose ingestion per se so is a surrogate for dietary problems. Where does it pan out???? Complicated!

marie, just be careful re helping others. You can explain what works but not everyone who has the desires which resulted in T2DM +/- gout can make the changes needed and not everyone will accept them. The willingness to change has to come from the person, you can’t push them too hard from the outside. Just take care.

Ash, yes, not a lot of fructose in meat.

Betsy, fatty liver = PUFA/fructose and saturated fats are largely effective at ameliorating (unless you are at the level of bridging fibrosis in which case welcome to the colchicine, for what it’s worth, I’ve never prescribed it myself).


Betsy said...

Peter, thank you for your comments, still trying to understand. :)

As for this, "Betsy, re complex I def. Yes and no… In acquired diseases typified by complex I dysfunction (AD, PD etc) exogenous ketones are only effective so long as complex II is functional. So the TCA is driven hard with NADH accumulation but complex II provides some input to the ETC. I have papers somewhere showing complex II blockers abrogate the benefit of ketosis in neurodegenerative diseases. Clearly neurons do not have the luxury of beta oxidation derived FADH2 inputs, they’re stuck with complex II. CoQ failure = permanently reduced CoQ couple, lots of reverse electron flow, lots of insulin resistance = diabetes. Good old statins."

Does that mean that once you have AD, PD, or one of the diseases with complex I dysfunction, if you have Complex II dysfunction also, there is no hope of return to normal cellular function, AND ketosis won't work? It seems as if CoQ failure is difficult to reverse, too. (Does taking CoQ10 supplements actually help?)

One more quick question that refers to the Kwasniewski type diet, does going over a certain amount of grams of carbohydrates mess up the body's use of fats? I just started this about 3 weeks ago, my mind isn't clear enough to make sure I'm doing it properly by measuring and counting grams.


Betsy said...

I admit I don't understand very much of this, but it does seem that with so many pathways and so many possible blocks....nutritional, pathological, chemical, it is amazing that anyone can have normal metabolism with adequate energy levels.

marie said...

Peter, thanks for the caution re.gout, sugar/fructose and physiological insulin resistance in ketosis, it's just the sort of thing I was looking for.
Thanks also for the warning reminder about another type of resistance entirely, lol. Thing is, ever since the ketosis success on chemo for my dad and dramatic weight loss for my sister in law, family and friends think I've got all the answers...which is worse than a natural skepticism/resistance because no matter my urging they don't do their own homework nor start slowly. So I'm very cautious and full of warnings. Of course, this is more trustworthy than any push, leading to yet more fervor on their part. A bit of a vicious cycle there.

Betsy said...

Statins, cholesterol, and Alzheimer's.