Tuesday, October 28, 2014

Are ketone esters dangerous?

Back in 1995 Veech was looking at a ketone mixture as physiologically equivalent to insulin/glucose. In order to limit his variables the isolated rat myocardia used in the study were perfused with Krebs-Henseleit buffer containing the metabolic milieu of interest. The buffer has no free fatty acids so takes the provision of acetyl CoA from beta oxidation right out of the equation. It also eliminates any uncoupling from free fatty acids in the perfusate. It took me a while to twig that this was potentially a very long way from the situation under fasting or ketogenic diet conditions where free fatty acids might well be at the maximal physiological levels whenever ketones hit 5.0mmol/l.

The idea was certainly in mind when the group published this, in 2004:

“Current ketogenic diets are all characterized by elevations of free fatty acids, which may lead to metabolic inefficiency by activation of the PPAR system and its associated uncoupling mitochondrial uncoupling proteins. New diets comprised of ketone bodies themselves or their esters may obviate this present difficulty.”

By 2012 the problem with ketogenic diets had been reduced to one of impossible compliance, rather than metabolic inefficiency of free fatty acid metabolism:

"Further, to achieve effective ketosis with KG diets, almost complete avoidance of carbohydrates is required to keep blood insulin levels low to maintain adipose tissue lipolysis. Such high-fat, no-carbohydrate diets are unpalatable, leading to poor patient compliance."

You notice the uncoupling, previously a potential problem, is now in the title of the paper. Ketones in real life, even from ketone esters, work in a milieu of free fatty acids. If you flood the mitochondria with ATP-generating ketones, which generate no ATP in the cytoplasm, you just might expect to open that uncoupling pore and allow a few FFAs to translocate some protons, to limit over production of ATP within the mitochondria.

Currently, in 2014, the delectable savour-the-flavour of ketone esters allows this:

“…the ester can be taken as an oral supplement without changing the habitual diet.”

I watch this stuff with some degree of amazement. There is a suspicion that AD incidence is increasing rather faster than an ageing population would explain. The suggestion is that it has  an environmental component. Now, many potential explanations are possible but I would like to think it is the saturophobic, cholesterophobic, fructophilic low fat based dietary advice from the American Heart Association which is the prime driver. Seems likely.

If AD (also known as type 3 diabetes) is a dietary disease, much as type 2 diabetes is largely a dietary disease, providing a crutch which will allow you to cling to the diet which got you in to AD in the first place strikes me as the biggest risk from ketone esters.

Excepting the stale urine/sweaty socks yummy aroma of course. Bring on the egg yolks fried in butter as an alternative, please.

A ketogenic diet features several things in addition to ketones. There is the chronic normoglycaemia which is anathema to the Crabtree effect. There is the physiological rock bottom basement insulin levels in a system where insulin signalling is f*cked. There are the elevated free fatty acids. These are the best.

Those free fatty acids are taken up by astroglial cells and used to generate in-situ ketone bodies. What sort of levels do they supply in vivo? That's an unknown (as far as I can tell), but I'm willing to bet that FFA supply under true ketogenic eating is both high and consistent, irrespective of fed/fasted state.

This is not quite the case if you are on the old MCT kick or mainlining sweaty socks while munching crapinabag.


A little background about Dr and Mr Newport and ketones which triggered this post off:

I have been unable to tease out, from Dr Newport's original article, that of Emily Deans or from the abstract of the case report above, quite what level of carbohydrate Mr Newport consumed in the original MCT phase, during the drug trial or while on ketone esters. I suspect it might have been more than a banana a day.

Oh, and another addendum. I, personally, clearly have issues with faking a ketogenic diet. This is true. But let me not decry ketones or their esters per se. If MCT oil or ketone esters get you out of bed and let you get dressed without needing assistance, that's great. They sure as hell knock spots off of anything which Big Pharma has to offer for AD management. The fact that I have yet to die as a direct result of eating less than one banana a day means that I hope never to need ketone esters. I feel a ketogenic diet should be high on the agenda for those with neurodegenerative diseases, with ketone esters or MCTs as a fall back. But then I would, wouldn't I...

Sunday, October 12, 2014

Where has the superoxide gone?

This is the first section of Fig 1 section C from the paper using dihydroethidium (DHE) to view in vivo superoxide production in control and diabetic kidneys, though not in the figure below.

It's a very important figure as it shows, very convincingly, that sudden onset hyperglycaemia has zero effect, none whatsoever, on superoxide production in their model of normal, non diabetic kidney tissue, that's the second column, identical to the first.

I have a lot of time for the failure to generate superoxide in diabetic kidneys, especially with pyruvate dehydrogenase complex down regulation limiting input to mitochondria from the end stage of glycolysis. But I have a concept that acute hyperglycaemia in normal, non Crabtree affected, tissues SHOULD generate superoxide, it should come from the respiratory chain and it should more particularly come from complex I in the region of the FAD moiety, preferably via the FeS cluster N1-a.

Now, if I had an in vivo tool for viewing superoxide generation, how would I do this? Well, I would use it in vivo. I would set up an iv glucose infusion, or perhaps a large intragastric glucose bolus, inject the DHE, wait a while, then look for superoxide/DHE derivative with my lovely optical scanner.

To keep the scrutineers happy I might have repeated the findings ex vivo, using the technique of paramagnetic detection of a superoxide/spin trap derivative, but the core finding, that superoxide generation on acute hyperglycaemia does NOT occur has to be shown in vivo. We already know it DOES occur ex vivo in multiple models, and the authors cite the studies to show this.

So, if hyperglycaemia triggers superoxide generation ex vivo in assorted non Crabtree adapted cells, why doesn't it do so in this study?

I don't know. There is a piece of core information which the scrutineers failed (miserably) to demand to be included in the study methods.

Figure 1C was not obtained in vivo. Column Ctrl was from a tissue homogenate of health kidney from non diabetic mice fed with pyruvate, malonate and ADP, subsequently flooded with 25mmol of glucose to produce the +HG column. That is not so bad. It's a model and it's clearly able to get GrantAid quality results.

But is it real?

Let's look at the equipment used. This is what they say:

"These studies were carried out in a MiniScope MS200 Benchtop EPR Spectrometer (Magnettech), which is designed to allow tight control of pO2 and temperature".

Why do they need tight control of pO2? You can obtain utterly rigid control control of pO2 by exposing your preparation to room air. Correct pressure to 760mmHG and pO2 is fixed at 21% of this.

To me the question is: What was the pO2 which failed to generate any superoxide when a mush of cytosol and mitochondria was exposed to 25mmol of glucose?

Was it 159.6mmHg, i.e. room air? Was it 40-50mmHg as other groups suspect mitochondria run at? Or was it 22mmHg?

This might matter. I got the 22mmHg value from the previous paper by the same authors which gave 3% oxygen as the likely conditions for normal mitochondrial function. This was a non referenced, throw away comment:

"Because the physiologic concentration of oxygen in mammals in vivo is less than 3% in most organs, we carried out a series of studies to determine whether ethidium or 2-hydroxyethidium was the specific oxidation product of DHE in vivo (i.e., in the intact animal, not cell culture/tissue slice) using several different validated animal models of increased or decreased superoxide".

Why it matters to me so much is that if an electron is thrown out of complex I due to hyperglycaemia triggered reverse electron flow through complex I, would it generate superoxide if the pO2 had been set to below physiological limits? Or if the guesstimate of 3% oxygen is correct and there is no superoxide generated, is there no reverse flow occurring? Or does the reverse flow occur, the electron is ejected, but it drops on to the surrounding protein structure rather than oxygen to be used as a distant signal via superoxide/H2O2/insulin receptor?

Using the in vivo technique would have told us exactly what was happening, at a true but non measured tissue pO2. I'm worried that the in vivo technique showed the anticipated (by me) hyperglycaemic superoxide and an ex vivo technique had to be developed and adjusted to maintain the fund generating core finding of no extra superoxide.

There was no reply to a simple polite email query as to the pO2 used.


Friday, October 10, 2014

The Crabtree Effect and superoxide in diabetes

I started with this paper about in vivo superoxide detection in the brain but, apart from the technique, there was no examination of the response to hyperglycaemia so I moved on. The next paper by the same group is looking at superoxide and mitochondrial function/health in the kidney under various models of diabetes. The general principles appear similar in neurons and kidney cells.

An in vivo technique to view superoxide is really useful. I have alluded to a certain discomfort in examining electron/oxygen interaction in mitochondria within cells/mitochondrial preparations under room air, with a partial pressure for oxygen of around 150mmHg (sorry for the non SI units, showing my age there!). There is no way that normal mitochondria are exposed to this much oxygen, a little browse around pubmed suggests that the best in vivo estimate is around 40-50mmHg, subject to some debate. That's without even thinking about what CO2 partial pressure you should use for cell culture... So observation in vivo takes care of a lot of this. If an electron is thrown out of the respiratory chain (I feel nothing in the ETC is accidental) the chances of it dropping on to an oxygen molecule seem somewhat higher if we have three times the oxygen partial pressure than the system was designed to work under. If the electron wasn't destined for an oxygen molecule, where else might it have been going?

The first point has to be that in two models of type one diabetes there is less superoxide production in the kidneys of diabetic mice in vivo than control mice, that's Figure 1 section C. Going ex vivo (I probably have a full post on the problems with this ex vivo section) we have the same effect demonstrated using a paramagnetic technique, that's Fig 2C. The reduced superoxide in diabetic kidneys was confirmed in the tissue homogenates under relatively normal metabolic substrate supply. Exposing the preparations to glucose at 25mmol/l has no effect on superoxide generation from the control kidney homogenate but actually reduces it, rather a lot, in the diabetic derived homogenate.


NOTE If you follow the text through about Fig 1C and their SOD2+/- mice you will find that the data is not very accurately described. So caution here. The SOD2+/- had a non significant increase in mitochondrial superoxide in Fig 1C, so it is hardly surprising this did not rescue the diabetic mice from renal disease in Fig 3A and F. I don't like their writing about this whole SOD2+/- section. Definite caution. END NOTE.

The paper has, amongst its problems, a lot of very perceptive points which make a great deal of sense. It's quite hard to know where to start. Let's begin with the failure of superoxide production.

So this paper flies in the face of the Protons concept of hyperglycaemia driving reverse electron flow from mtG3Pdh through complex I to generate insulin resistance. That too is probably another post, comparing the diabetic state with the non diabetic hyperglycaemic state. Anyhoo. The group rather like Crabtree. So do I. The Crabtree effect, the shutting down/mothballing of mitochondrial function, is an adaptation to oversupply of glycolysis derived substrates. It allows a limit to be set on the throughput of pyruvate to mitochondria and jettisons any excess as lactate. This situation, once it is established, is probably quite different to the situation which leads to its adoption.

Chronic hyperglycaemia induces the Crabtree effect and down regulates mitochondrial biogenesis, mitochondrial repair and electron transport chain function. It not only does this but it also phosphorylates the pyruvate dehydrogenase complex, very specifically, and this directly shuts down input to the TCA from glycolysis (or input from lactate itself, if we want to apply this concept to neurons, as we might). This is all in the paper. Of course I would add that it doesn't affect ketone derived acetyl-CoA input to the TCA, although the ketone derived acetyl-CoA will be processed by a degenerate electron transport chain...

Under sustained hyperglycaemia there is an excess of calories which leads to a failure to activate AMP kinase, a core sensor of energy abundance which is phosphorylated under hypo caloric conditions. AMPK regulates PGC 1 alpha, a messenger to trigger mitochondrial biogenesis. But the central link, the activation of AMKP, is mitochondrial derived superoxide. And, oddly enough, one of the functions of AMPK activation is the generation of mitochondrial superoxide. A self sustaining loop.

The group administered rotenone to control mice. Now, the effect of rotenone on superoxide generation appears (in general) to be rather dose rate related. In the present study the dose rate was chosen so that there was a near complete suppression of superoxide production from the ETC of the mice. Acute suppression of superoxide results in the reduced phosphorylation (reduced activation) of AMPK and increased phosphorylation of PDH, which shuts it down. This loss of superoxide is a short term mimic of the long term established Crabtree effect. No superoxide, no mitochondrial maintenance. Consider that chronic high dose rotenone poisoning is a standard model for Parkinsons Disease and you begin to see the importance of superoxide in the brain. Long term hyperglycaemic failure to generate superoxide is probably a more normal route to neurodegeneration than rotenone in most (but not all) neurodegenerate humans...

The fall in superoxide production in diabetic tissue homogenates again pulls me back to brain function. Crabtree suppresses hyperglycaemic superoxide production, i.e. the effect is antioxidant. Let's look at what glucose does to neurons from this paper which we've chatted about before. Here's the only bit I'm interested in today:

"Indeed, it has been shown that glucose is used by neurons to maintain their antioxidant status via the pentose phosphate pathway (PPP), which cannot be fueled by lactate (Magistretti, 2008; Herrero-Mendez et al., 2009)"

It's impossible over emphasise the importance of that sentence. It says it all about why neurons should run on lactate! To avoid upregulating antioxidant status.

What does increasing antioxidant status do to superoxide signalling? The term f*cked comes (unavoidably South Park-ishly) to mind. There are a swathe of papers showing that the antioxidant status in neurons of AD and PD patients is upregulated.

Once you go with Crabtree you can see that glucose and PPP driven antioxidant upregulation might be all that is needed to lose superoxide signalling and destroy mitochondrial function. Lactate does not do this. Lactate does not induce the Crabtree effect.

Let's be very specific: Glucose, under the Crabtree effect, triggers a cascade which ends up with failure to generate superoxide and this maintains mitochondrial shutdown. Up regulating antioxidant status may theoretically be helpful in dealing with non mitochondrial superoxide generation, but it's not going to help signal for mitochondrial biogenesis.

High glucose exposure generates glucose dependence. This is a recurring theme and is core to neurodegeneration. I look at safe starches and can see that, if you are living with the Crabtree effect in key neurons, some starch/glycolysis might make you feel better if you are ketogenically hypoglycaemic, but it's not going to help un-Crabtree your mitochondria. On the other hand I can't see that pushing starch to a level which produces hyperglycaemia is anything other than damaging, as opposed to merely neutral as it might be when your pancreas does its job effectively.

I'll take a break before going on to the sections on mitochondrial deletions and respiratory chain oxidative damage elsewhere in the paper. Or maybe I should talk about the bits I deeply dislike related to oxygen pressure and superoxide.


Thursday, September 25, 2014

Uncoupling control in defence of FFAs

I've been reading this review on beta hydroxybutyrate and am struck by the concerns expressed throughout about the potential damage caused by free fatty acids, due to uncoupling, a sentiment I have picked up in several of Veech's publications which are heavily cited in the review.

I was particularly struck by how two papers I've recently discussed were described, so it's topical for me. One was the puzzling toxicity of a LCKD diet as published by Wang et al. This is the one using vegetable shortening of indeterminate trans fat concentration, a point sadly un-noted (or considered unimportant?) by the review. And second is the Kuwait study, described as LCKD in the review, which was not exactly glycogen depleting for a rodent.

Aside: This cited study starved rats for three days before ischaemia/reperfusion. That should have depleted glycogen AND raised raised FFAs (neither of which was checked, but any lipophobe should expect uncoupling combined with backup anaerobic glycogen reserve loss to be disastrous in ischaemia/reperfusion) as well as predictably increasing B-OHB. Combined starvation changes in fact reduce the damage produced and improve recovery. End aside.

So I'm a little ambivalent about the review and how much of the rest of their ideas I might take at face value.

Ultimately, thinking about free fatty acids, we have to talk about the control of uncoupling.

Recall this image from this study in part 29 of the Protons thread:

Free fatty acids are essential for proton transport across the inner mitochondrial membrane to uncouple oxygen consumption from ATP synthesis and to maximise electron flow down the electron transport chain with minimal resistance and minimal non essential superoxide generation.

No free fatty acids, no uncoupling. Free fatty acids are core to uncoupling.

But they are far from the only factor. For protons to be transported through the channel of the UCP by free fatty acids the channel must undergo a conformational change, which is highly dependent on the ATP status of the cytoplasm and the mitochondrial matrix.

So we have this picture from this very impressive study:

ATP in the cytoplasm fits in to a specific binding site, with each phosphate moiety of ATP fitting up against a specific arginine, all three aligning results in closure of the channel and inhibition of uncoupling, whatever the FFA concentration. Here is what the authors say:

"Moreover, residues R79 and R279 correspond to the arginines involved in nucleotide binding and protein inhibition in UCP1. According to the three-step binding model proposed for UCP1, β-phosphate of PN [phospho-nucleotide] binds first to R182 (helix IV, loose binding). The second step is the binding of γ-phosphate to R83 after protonation of E190 (tight binding). After the subsequent binding of α-phosphate to R276 (helix VI) the protein switches to the inhibited conformation"

Cytoplasmic ATP (and GTP) inhibit uncoupling. But not all of the time, despite the fact that there is normally always enough cytoplasmic ATP to inhibit uncoupling. So yet another factor comes in to play.

It is quite possible to inhibit the inhibition of uncoupling produced by cytoplasmic ATP.

You do this with mitochondrial ATP. ATP binding from the mitochondrial side of the channel interferes with the binding of cytoplasmic ATP but cannot reach the R83 arginine itself to close the channel. So elevated mitochondrial ATP keeps the uncoupling channel open, even in the face of rather high cytoplasmic ATP levels.

The logic to this is that if there is plenty of ATP within the mitochondria there is no need to preserve delta psi and it's fine to uncouple. If there is ATP in the cytoplasm but very little in the mitochondria the implication appears to be that ATP synthase is not generating enough mitochondrial ATP, i.e. we are either hypoxic or over-uncoupled. Continued glycolysis generates ATP on the cytoplasmic side so allows the uncoupling channel to close using this cytoplasmic ATP.

It's pretty logical.

So. Under hypoxia, whatever the level of FFAs, what happens to uncoupling?

It stops due to a lack of mitochondrial ATP. Should you fear FFAs? Only if you think you will continue to uncouple respiration under hypoxia. The balance of mitochondrial to cytoplasmic ATP should shut down uncoupling very rapidly when needed.

Just say no to Crisco (if that's how Wang et al got their result).

It has long worried me that in Veech's seminal paper on glucose, insulin and ketone metabolism in an isolated heart preparation the group was very, very careful to run the study without any involvement of free fatty acids. For those of us living in a temperate latitudes, lounging on the beach under a coconut palm while waiting for lunch to drop on our heads is not an option. Have you ever been to Lowestoft beach? No ketones without elevated FFAs at latitude 52 deg N on the North Sea coast. Fasting, or living on meat for a while, seems more likely than eating MCTs outside the tropics. I fail to see how the body would manufacture the miracle of ketones at exactly the same time as it releases the devil incarnate of free fatty acids.

Some folks like free fatty acids. Me, for one.

Some of us like uncoupling too, in the right place, at the right time.


Tuesday, September 23, 2014

Ketones for ALS?

OK. I've been thinking a lot about ketogenic diets and motor neuron disease, which appears to me to be just one facet of Alzheimers, Parkinsons and a number of other neurodegenerative diseases.

The first thing to say is that they (ketones) don't seem to work terribly well. I picked up this paper via the Deanna Protocol website. I wrote an unpublished post at the time setting down what an abysmally written paper it is but I thought I would stick to the basics today. Feeding a ketogenic diet to the mice engineered to have an ALS-like disease delays their time to falling off a log but does not extend their lifespan:

"There was no statistically significant difference in the age at death between KD fed animals compared to SOD1-G93 transgenic mice fed a standard laboratory diet (133 ± 4 vs. 131 ± 4 days, p = 0.914)"

Some improved motor function, for a while, may be worth having if you suffer from ALS but I don't think it's exactly a cure or remission.

Although the methods section is very reticent about the diet, it is high in carbohydrate (20% of calories) and protein (20% of calories), so must be MCT based to achieve ketosis.

The findings are confirmed by a nicely written, very clear paper using caprylic acid as a supplement to standard CIAB mouse chow:

"SOD1-G93A animals on caprylic triglyceride diet had a median survival of 135 days. Although it was longer than the median survival of SOD1-G93A animals on control diet (129 days), it did not reach statistical significance (Mantel-Cox test, p=0.165)"

Things weren't much better here in the Deanna Protocol paper. Their ketogenic diet supplied 77% of calories from fat, type unspecified, and essentially zero from carbohydrate. Protein was high at 22% of calories. This is what you get as a result:

"Although the mean survival of SOD1-G93A animals was longer in all three treatment groups than the control group, this difference reached statistical significance only in the KD+DP (4.2%, p = 0.006) and SD+DP groups (7.5%, p = 0.001, Fig. 5, Table 5, Data S2)."

i.e. the ketogenic diet, without the alpha ketoglutarate of the Deanna Protocol, was no better than control mice on CIAB.

All of which is quite interesting and should be quite depressing for groups working with MCTs, ketone esters or ketone salts as managements for neurodegenerative diseases.

We can say certain things about the first two studies. Using MCTs on a moderate to high carbohydrate diet is unlikely to lead to the metabolic changes of a true ketogenic diet. Normoglycaemia is probably not on the menu. It will not lead to the sort of effects of minimal carbohydrate, just adequate protein, very high fat diet. The effect of such a diet has been described as unique.

Of course, a few grams of MCTs on a diet of standard lab chow will generate ketones. That is hardly equivalent to a true ketogenic diet with its reduced glycaemia and basement value insulin levels.

As the paper on the Deanna protocol reports:

"Blood glucose was not significantly different between the diet groups", not exactly what was reported for mice eating D12336.

Ultimately, no one yet appears to have looked at a true ketogenic diet in ALS.

The focus is on the ketones. Ketones are good, but they are not magic. There are people who believe that the ketones themselves are simply a surrogate for very low insulin levels, which is magic (You know who you are Wooo!) and that the benefits of ketogenic diets may stem from the low insulin levels rather than the ketones per se, certainly for obesity management. For neurodegenration I find this idea very appealing. I think that the low glucose/insulin might be particularly important within the brain. I can't see that the work has been done yet, too much of a focus on ketones.

As something of an aside, the Deanna Protocol is interesting in its own right. The core supplement is (arginine-linked) alpha ketoglutarate. From the Protons point of view, if the alpha ketoglutarate enters the TCA at alpha ketoglutarate dehydrogenase and leaves it at malate, it would appear to be a very FADH2 selective input at complex II, generating an NADH:FADH2 ratio of 1:1, i.e. it is functioning as a rather specific FADH2 input. We're all aware that complex I dysfunction is a hallmark of neurodegenerative diseases and, in the absence of beta oxidation (we're in neurons here), complex II is the primary route in to the CoQ couple for electrons via FADH2. Along with mtG3Pdh of course, if that happens to be active. I can see the logic to using this AKG to push complex II without the excess rise in non-usable NADH, which large amounts of acetyl-CoA provide. I'm not surprised AKG is the core component of the Deanna Protocol and hats off to her father for picking this up.

A further aside, Deanna tried coconut oil, caprylic acid and MCTs early on in her disease. Not a lot of help. Adding extra acetyl-CoA from ketones will be of limited help in a condition with complex I dysfunction. My interest still lies in ketones combined with low blood glucose, not as an add on to healthy starches.

It is quite clear from the last post featuring cardiac ischaemia and ketones that any old ketones will do when hypoxia is the problem: Bring on the MCTs. Logically ketones are fats, part pre-oxidised in the liver, so require less oxygen to complete their metabolism in the cardiac muscle. They do not uncouple protons from oxidative phosphorylation either, which we will probably come back to. And while normal fatty acids do uncouple ox phos, this effect is (under normal circumstances) completely lost when mitochondrial ATP levels fall. This probably happens rather quickly under hypoxia.

The energetic failure of neurodegenerative diseases is only partially amenable to ketones. We are looking at a rather different phenomenon to ischaemia and it might be worth looking at the problems of burning glucose in neurons next. And the problems from failing to generate adequate superoxide for maximal health. There's a lot to think about.


Sunday, September 21, 2014

Should idiots be allowed to write the methods section of any "scientific" paper?

Time to get back to the blog. We had a great summer and life has just kept on being more interesting than blogging. There are about 20 comments I need to read and approve which I'll do my best to get around to, but I thought it was time to hit the keyboard after the summer holidays.

I thought I would just post briefly on the struggles of trying to work out exactly what a given paper is describing in dietary research. I did set out a post a few weeks ago, being rather derogatory, about this paper on ALS. Here is the preamble:

I feel I should like the paper. Really. What with all this iced water being poured over people's heads in the name of ALS research etc. But it's hard.

OK. We're looking at a ketogenic diet for mice endowed with an engineered model of ALS which is quite similar to one of the familial forms of human ALS.

Being me, I go to the methods section first, to see what they fed the poor mice on. From the philosophical point of view I expect the methods section of a paper to allow me to duplicate a given research protocol. All I am told in this case is that the ketogenic diet is 60% fat, 20% carbs and 20% protein and that it was made by Research Diet, Inc. New Brunswick, NJ. That's it. Now, until RD are bought out by some other multinational company, they have a website and this tells me that they supply only one ketogenic diet, D12336, which is 11% protein 89% fat and zero carbohydrate, pretty much what you need to get a rodent in to mild ketosis. So this research group are using a custom diet, what goes in to it is anyone's guess.

My guess is medium chain triglycerides. I don't think you can get a mouse in to ketosis with protein at 20% of calories and carbohydrate at 20%. You'd have trouble getting a human in to ketosis with this, unless you used MCTs.

This is important because I'm interested in teasing out whether there is any point in the enormous effort and endless tedium of eating a low carbohydrate driven ketogenic diet with thyroid deficiency, lethargy, brain fog, glucose deficiency and auto immune disease predisposition as routine sequelae, not to mention the constipation and halitosis (is this me?), when merely popping down to my local Caribbean corner store for a bottle of coconut oil might do the job equally well.

What goes in to the diet matters. Coconut oil is not safflower oil, is not butter. What goes in to the methods matters. Research must be replicable.

End of preamble. I wasn't best impressed.

Before I go on to think about ALS and what help medium chain triglycerides may or may not provide in another post, I thought I would just like to revisit the lethal effects of a VLC keogenic diet paper on the outcome of induced ischaemia and reperfusion of the myocardium in some hapless rats.

It is fairly clear that using "vegetable shortening" as your primary ketogenic source of calories is likely to destroy your myocardium if you have an ischaemic episode. Your first heart attack might be your last. It took an email to Research Diets to get the information about the probable trans fat content of their diet and confirmed to me that the research group had written a methods section which put their paper, and probably the researchers, in to the garbage category.

The same appears to apply to the flip side. I had wondered what a non vegetable shortening based ketogenic diet might do under the same circumstances. Well I'd missed the study, which fully reinforces my pro-LC confirmation bias. Ketogenic diets are the bees-knees for surviving a period of cardiac ischaemia, Crisco excepted.

So, what does the miracle diet for surviving your next coronary look like? I don't know. You don't know. You can read the full text. You still won't know.

The diet is 60% protein by calories! And 10% carbohydrate. The remaining 30% is "oil". Now you know as much as in the paper. Can you replicate the study, based on the methods? No.

BTW: They didn't even check ketone levels! I think we have to assume MCTs again and assume some degree of ketosis.


The scrutineers also need to be up against the wall come the revolution.

For all three papers. Crap. Even though I like the results.

Hiya all!


Friday, July 11, 2014

Neuron fuel and function

In the comments following a previous post Dustin linked to this rather lovely paper from the early 1970s, back when I was still at school and marathon racing my kayak.

This is one of the nicest figures:

The lower section is the interesting part. CJ had not eaten for 50 days and had a (very) fasting blood glucose of 4.0mmol/l when he received around 0.1 IU/kg of insulin by intravenous injection. Please don't try this at home. You can see that a) he is still insulin sensitive and b) his blood glucose bottomed out at around 0.5mmol/l by 60 minutes. Throughout this period he was asymptomatic. No hypo.

CJ was not running his brain on glucose. The upper section shows a rapid and sustain increase in B-OHB extraction (the dark hatching between the arterial line and jugular bulb line for B-OHB)  by his brain through this period of time. Ketones, at levels in excess of 11.0mmol/l, can sustain apparently normal brain function. Given an alternative fuel source this would seem to put the level of blood glucose needed for normal neural function at some (non determined) value of less than 0.5mmol/l.

Drenick et al did not look at fatty acid extraction by the brain. That's a pity, but understandable. No one expects the brain to metabolise palmitic acid. Well, perhaps we should say that neurons should not metabolise palmitic acid. Astrocytes do. Astrocytes are ketogenic and are in a perfect position to supply ketone bodies to neurons using the monocarboxylate transporters ubiquitous on them. I hope to come back to this by the end of the post.

Of course, a 50 day starvation period is not exactly the normal human predicament and we could argue that the normal human brain is glucose dependent. This too, I think may be a very debatable point.

Work has been done with humans under hypoglycaemia with brain function supported by either lactate or pyruvate by intravenous infusion. They're fairly effective, not perfect, but there are limits in how far you can push the metabolism of human volunteers.

Rats are not so fortunate.

If we go to Figure 1 from the rather nice paper emailed to me by Edward

we can see that, under the influence of a massive 20 IU/kg of insulin, there is an almost complete loss of plasma glucose and a slightly more complete loss of brain response to limb stimulus in the insulin-only, profoundly hypoglycaemic group, top row of  section A. This has occurred by two and a half hours. The next row has had the hypoglycaemia corrected with glucose (i.e. it's essentially a control group) and has a normal response to stimulus at 4 hours. The lower row shows the effect of lactate in supporting brain function during four hours of persistent, profound, uncorrected hypoglycaemia. You have to note that progress from left to right is time in milliseconds after stimulus and that there is a clear cut delay of about 10 ms in the response time under pure lactate compared to under glucose. This is reiterated in section D, where the response can be seen to be delayed and blunted when compared to the glucose supported data of curve C.

This has led the authors to speculate that, heresy of heresies, there may actually be an absolute need for some glucose by the brain! Strange I know, but... They're not sure of this, just speculating. There are other potential explanations.

Now, most people do not walk around with a blood lactate of 9.0mmol/l. Perhaps most people really do run their brain on glucose?

This seems very unlikely. Or, rather, it seems very unlikely that the neurons in the brain run on glucose. Astrocytes certainly do. But one of the main functions of astrocytes appears to be to manufacture lactate from glucose (directly or from stored glycogen) and deliver it to neurons as a one step conversion fuel giving pyruvate, which can enter the TCA as acetyl-CoA without any messy glycolysis. There is an awful lot of information in this paper.

I put up this nice illustration previously, in the Protons thread:

Neurons are spared glucose. Why?

Mitochondrial glycerol-3-phosphate dehydrogenase. Glucose, during glycolysis, is quite able to input to the electron transport chain through an FADH2 based input at mtG3Pdh, which can reduce the CoQ couple and set the ETC up for reverse electron flow through complex I, with the generation of superoxide as this occurs. Modest superoxide is a Good Thing, especially if you want to signal for mitochondrial biogenesis or cell division. Excess superoxide is a potent signal for apoptosis. Apoptosis is verboten for CNS neurons because any information stored in their synaptic connections will be lost along with the cell. Replacing the cells will hardly replace the memories and appears equally forbidden. There is a need for immortality without reproduction in neurons, for their survival in excess of the lifespan of an organism needing a functional memory in a learning brain.

Feeding lactate through pyruvate to acetyl-CoA does not drive CoQ pool reduction "ahead" of the throughput of electrons coming in from complex I. Neurons do not want to generate insulin resistance. Avoiding glycolysis looks (to me) to be the way they do this. Generating hyperglycaemia looks like a way to overcome the normal lactate shuttle and of forcing glucose directly in to neurons. Enough apoptosis and eventually neural loss just might show as memory loss.

Hyperglycaemia and Alzheimer's...

Generating large amounts of superoxide in astrocytes during glycolysis is not damaging to the neurons supported by the derived lactate. Astrocytes are certainly replaceable, although there seems to be some debate about cell division vs stem cell recruitment. Astrocytes are also able to divide unreasonably rapidly and form various grades of brain tumour. They are common and frequently aggressive. Neuron derived tumours are much rarer and are usually derived from embryonic cells giving medullablastomas rather than being derived from mature neurons. That seems to fit the metabolic arrangements in the brain rather neatly.

This takes us back, eventually, to palmitate as a ketogenic energy supply to the brain via astrocytes. Again, an FADH2 input through electron transporting flavoprotein dehydrogenase can couple with hyperglycaemia to generate reverse electron flow through complex I giving excess superoxide generation. I consider this to be why free fatty acids are excluded from neurons. It's not that FFAs generate excess superoxide per se, they don't. But combined with hyperglycaemia they certainly do, especially palmitate and the longer of the saturated fatty acid series. You really don't want this happening in a cell whose remit is immortality.

Ketones and lactate do not drive reverse electron flow through complex I. Glucose can. Palmitate certainly can. What you want from a metabolic fuel depends on the remit of your cell types. Neurons within the brain preserve information by their continued existence. This is best done by burning lactate or ketones. NOT glucose and, of course, not FFAs. Anyone who claims that glucose is the preferred metabolic fuel of the brain has not though about what a neuron has to do and what an astrocyte actually does do. Or much about the electron transport chain.