Tuesday, December 06, 2016

Dunnigan-type Familial Partial Lipodystrophy

Ivor posted a link to this paper on FB:

Premature atherosclerosis associated with monogenic insulin resistance

Obviously the role of hyperinsulinaemia as a driver for CVD was his main point, reiterated from the paper. Which I think it would be rather hard to disagree with.

But what got me really interested was that here we have a monogenic form of insulin resistance. One (rare) gene defect and all the rest of insulin resistance follows, with early inset CVD etc. So what does this single gene do, which results in the failure of insulin signalling? Well, it seems to have nothing to do with insulin signalling per se, oddly enough.

The affected gene codes for a laminin, one of a family of important structural proteins essential for normal nuclear function, mitosis and important in the control of apoptosis. The particular mis-sense mutation found in the folks detailed in the paper appears to target adipocytes. The gene causes Dunnigan-type familial partial lipodystrophy. Children are born normal and stay pretty well normal until puberty. At that time they lose peripheral fat, maintain central fat and become IGT/diabetic. They lose adipocytes, ie they lose the ability to effectively store fatty acids.

As you lose your sump for fatty acid storage the ability of insulin to inhibit lipolysis in the remaining, overly distended adipocytes, fails so serum free fatty acids rise.

Now, I would be the last person to suggest free fatty acids per se inhibit insulin's action (any more than intracellular accumulated triglycerides do), but a metabolite of fatty acids almost certainly does. Be that acyl-carnitine or acyl-CoA, be that at the redCoQ-complex III docking site or elsewhere, be that via free radicals or not, elevated free fatty acids are a precursor for a molecule which generates insulin resistance. This is quite separate from my ideas on the Protons thread where it is the oxidation of fatty acids which acts as the switch for insulin signalling.

So, does Dunnigan-type partial lipodystrophy cause elevated fatty acids to levels which might be potentially facilitative for insulin resistance? Well, the Hegele paper doesn't report FFA levels. He is to be commended for his perception that insulin per se might have something to do with CVD but he, and most of the rest of the researchers on lipodystrophies, focuses on the elevated triglyceride and related lipoproteins. As they would.

But anyway, I found one paper which delivered the goods on free fatty acids:

Elevated Serum C-Reactive Protein and Free Fatty Acids Among Nondiabetic Carriers of Missense Mutations in the Gene Encoding Lamin A/C (LMNA) With Partial Lipodystrophy

Free fatty acids in affected people: 0.66±0.05mmol/l.

In unaffected people: 0.43±0.03mmol/l, p less than 0.0001.

Makes sense to me, like a mild, late onset version of Berardinelli-Seip lipodystrophy and compatible with the concept that getting fat is fine until you can't gain more weight, so leak FFAs from adipocytes when you really shouldn't. Berardinelli-Seip folks are born emaciated, with no fatty acid storage capability... And yes, they are very diabetic.

Now, there are other many other issues based around elevated free fatty acids and many of them give some interesting insights. I'm not sure which I want to go to next. I'll have a think about it.

Peter

Thursday, December 01, 2016

Inhibiting lipolysis using acipimox

Okay, time to start doing a little blogging again. I've been thinking about various aspects of free fatty acids largely derived from studies using acipimox, like this one:

Inhibition of Lipolysis Ameliorates Diabetic Phenotype in a Mouse Model of Obstructive Sleep Apnea

I consider that one primary action of insulin is to inhibit lipolysis. Which makes it a driver of weight gain. Or, rather, it makes it a mediator of calorie trapping within adipocytes, which drives hunger (you needed those trapped calories), said hunger then gets the blame for the swollen adipocytes. You know, humans only get fat by eating too much. Ask any obesity researcher.

So what is the effect of other inhibitors of lipolysis on adipocyte size? The classic, freely available inhibitor of lipolysis is acipimox. Does acipimox make you fat? There is nothing on the patient information leaflet or data sheet about weight gain. Being hungry while you take it is only mentioned as a side effect in anecdotal reports from the poor folks taking the stuff. Of course the link between being hungry and gaining weight is easily eliminated by a simple matter of willpower. Again, ask any obesity researcher.

The published clinical research with acipimox (which is interesting) is usually of too short a duration to show weight changes, most studies usually last a few days or a couple of weeks.

So eventually I found an animal model using acipimox. It was looking at intermittent hypoxia (termed IH below) and weight loss (also very interesting, another day) but it came up with this little gem:

"Acipimox treatment [prevented IH-induced lipolysis and] increased epididymal fat mass and adipocyte size by 19% and 10%, respectively".

Acipimox, given to mice eating standard mouse crapinabag, causes weight gain, more especially fat gain. It does not cause hypoglycaemia and any appetite stimulation is likely to be because adipocytes have accepted dietary fat and are not letting it go.

Just as insulin denies lipolysis and so distends adipocytes, so too does acipimox. Acipimox, unlike insulin, does not drive potentially fatal hypoglycaemia with subsequent life saving food ingestion to explain away the weight gain.

This is where I started with acipimox: does it cause weight gain? Yes, inhibiting lipolysis, without using insulin, causes weight gain.

Of course, no one uses acipimox to cause weight gain. It is usually used to decrease plasma free fatty acids with a view to improving some aspect of metabolic function.

Which of course leads on to the monogenetic insulin resistance paper cited by Ivor on Facebook...

Peter

Thursday, November 03, 2016

An Error

Back in January of this year I made a significant artimetical error which, combined with the massive confirmation bias from which I suffer, led to the incorrect conclusion that fats require less O2 per ATP than glucose. This is incorrect. They require about 5% more. There are a lot of implications from this in my head and for subsequent posts on the blog. I’ll be working at tidying up follow on-posts but the initial incorrect post is still there to stand as a warning.

Sorry folks, shouldn’t have made the mistake and certainly should have spotted it without help.

With thanks to Mateusz re reworking the arithmetic.

Peter

Thursday, September 29, 2016

Flow Mediated Dilation: What does it mean?

Flow mediated dilation (FMD) is one of the last bastions of low fat dogma. FMD is particularly interesting as you appear to be able to prove almost anything with it. I suspect that results in general are very dependent on exactly how you set up a given experiment and how you report the numbers but most of the papers I've read take the background physiology as given and just supply you with the percentage change. Frequently FMD is enhanced under low fat weight loss conditions and obtunded under low carbohydrate weight loss or even after a single high fat meal.

Let's take a fairly typical study such as this one:

Benefit of Low-Fat Over Low-Carbohydrate Diet on Endothelial Health in Obesity

It uses the term "Atkins" and was published in 2008, a time when ketogenic dieting was less acceptable than nowadays. It's nice because the food intake was tightly controlled and the low carbohydrate group was in ketosis throughout. And it also gives me lots of numbers to play with from the results section.











Table 2 of the results is the most interesting. From the bottom line we can see that there is nothing wrong with the ability of the brachial artery to dilate, if you supply nitroglycerin as a nitric oxide source then there is no difference between groups at any time point for the ability of the artery to respond. What changes is the willingness (or ability/need) of the endothelium to generate the nitric oxide in situ to dilate the artery as flow increases in the low carbohydrate group compared to the low fat group. This shows in line three.

Nothing throughout Table 2 is statistically significant except for line three, that ability of the brachial artery to dilate after occlusion. It's a ratio of before and after occlusion, expressed as a percentage. This went down a little after six weeks ketogenic eating and up a little under low fat eating, giving p as less than 0.05, which means ketogenic eating is Bad For You. That's where the paper stopped. I carried on because the numbers making up the ratio are provided and they are interesting.

So next place to look is line two. This is the maximum diameter of the artery after occlusion is released. There is basically no difference in this parameter with time in either group. A little up and down but basically static.

Then there is line one. Line one shows the basal diameter of the artery in the LC group increasing linearly from 3.42mm to 3.58mm over time. The LF group does an up and a down, ending up dropping from 3.94mm to 3.85mm over the study.

[Addendum, see final conclusions: Does the upping and downing of the LF group reflect changes in FFA release with weight loss, high at 2 weeks and low at 6 weeks as the weight loss bottoms out for the LF group? We don't have enough numbers for weight change or FFA levels to check this, but it fits my ideas]

It is the behaviour of the resting diameter which differs between the groups on a background of a fairly static peak diameter. ie the peak diameter doesn't change much but LC/ketogenic eating progressively increases the basal diameter of the brachial artery. The artery doesn't need to dilate as much after 6 weeks of ketogenic eating to get to the maximal diameter needed to supply the post-occlusion hyperaemia of the forearm.

Conclusion: LC arteries are dilated at rest, they don't need to dilate much more to reach the "normal" size of a post occlusion hyperaemia distended artery.



You could leave it at that.

But I can't.

We have a set of diameters and a set of measured blood velocities. So I can crudely estimate the blood flow, aka the oxygen delivery. That's πr2 times the mean speed of the blood, not forgetting to convert the cm/s velocity to mm/s.

Under basal conditions the blood flow increases for the LC group over the six weeks, from 3326mm3/s to 3876mm3/s, up by 17%.

Basal blood flow for the low fat group decreased from 3939mm3/s to 3668mm3/s over the same period, that's down by 7%.

I have no idea whether these changes are statistically significant but they might well be clinically significant. If I had occlusive peripheral vascular disease I know what I would eat. But you knew I'd say that anyway!


Post occlusion hyperaemic blood flow for the LC group started at 6542mm3/s and increased to 7760mm3/s by six weeks, up by 19%.

For the low fat group the start was 8587mm3/s and this increased to 8752mm3/s, up by, err, 2%.

The gain for ketogenic eating, in peak hyperaemic blood flow, is ten times that of the low fat group. Both ended up about the same, but the LC group had a lower initial value. What would have happend on a level playing field at entry point? That we'll never know because it's probably set by the physical size of the forearms of the subjects...

Conclusion: As a percentage of entry point, the maximal post occlusion flow for LC shows a much greater increase over the six weeks of the study.



I could leave it at that.

But I can't.

Ketones work some magic on the redox spans of the ETC and on the energy yield of ATP hydrolysis. The ketogenic dieters were genuinely positive for urinary acetoacetate every day. So they actually need less oxygen, yet they had a greater oxygen supply to their forearm... Of course, we don't know the mixed venous oxygen tension but we've all read D'Agostino's rats and know it's probably high under ketones, if the arterial oxygen tension is anything to go by.

It's pretty obvious that these ketogenic folks were uncoupling. They ate 100kcal more per day for 6 weeks than the LF group and lost more weight, more fat and less lean mass. Hard to believe I know, but I tend to believe the results in this paper because the numbers trash low fat eating again and again, while the authors are clearly lipophobes.

Uncoupling may be wasteful of oxygen but is advantageous for protecting our mitochondria, plus the excessive use of oxygen is offset by the oxygen sparing effect of ketones. With the onset of limited oxygen availability the ATP levels will drop and so uncoupling will stop, while ketones will continue to be oxygen sparing. Ketosis is a pretty good state to survive an occlusive episode.

No wonder ketogenic eating limits the need for vasodilation after brachial artery occlusion...

Conclusion: Ketogenic eating is the Bee's Knees for surviving ischaemic episodes.




That's pretty well it for this post. I'm about to stop, except to speculate as to why limited FMD is a good predictor of poor cardiovascular health.

Fat. Low FMD is a marker of using fat as a major metabolic fuel. Under fat oxidation your brachial artery will already be dilated so it won't dilate much more after an occlusion episode. If you are eating a diet of total crap based on carbohydrate it is quite possible to have elevated FFAs combined with elevated blood glucose, once your adipocytes have been stuffed so full that they won't take any more and leak FFAs despite elevated glucose and insulin. Innapropriately elevated FFAs is the disaster recipe, a hallmark of metabolic syndrome.

Elevated free fatty acids (and their oxidation) is Bad For You under metabolic syndrome but it is completely normal under ketogenic eating.


My overall conclusion: Reduced FMD is an epiphenomenon cause by fat oxidation. Fine when it's appropriate. Bad if you are overweight, insulin resistant and eating sugar.

Peter

Monday, September 19, 2016

On the bike

Just back from a pushbike/teaching tour from (my bit, Mike went much further) Orkney to Inverness. I'll try to get up to speed on emails and comments as soon as I can!

Peter

Thursday, August 11, 2016

Protein catabolism should generate an RQ of around 0.8

Anyone who has read Martha's story and put her narrative together with the folks in Phinney's 1980 study will have immediately wondered: How many of Phinney's subjects were lactating? Even just a little bit?

I think we can say, pretty categorically, that none of them were lactating. Gluconeogenesis from lipid is very likely to have been occurring but obviously (now) this can only drop the RQ when the glucose produced is not being oxidised. Clearly my initial idea expressed in the Phinney post is wrong.

Martha is easy, her child took the sugars hence the spectacularly low RQ. Trying to explain why a protein supplemented fast should drop the RQ below 0.69 needs a little more thought.

This is what Phinney thought might be happening under moderate exercise:

"The low RQ value of 0.66 observed during the final exercise test was surprising, as long chain fatty acid oxidation occurs at an RQ of 0.69. (The only common fuel oxidized at a lower ratio is ethanol at 0.67). The answer to this disparity may lie, at least in part, in the rise in serum ketone concentration observed during exercise. As the hepatic production of ketones from long-chain fatty acids occurs at an RQ of zero, a net retention of ketones in body fluids will result in a reduction in observed RQ due to non steady-state conditions. By calculating the increase in the whole body ketone pool associated with exercise, one can account for approximately half of the decrement of CO2 production that would be necessary to explain the decrease in RQ below 0.69. Other factors that could contribute to this low RQ include losses of ketones in the urine and loss of acetone in the breath after decarboxylation of acetoacetate in the blood, as well as CO2 utilized in urea genesis".

However, the non steady state accumulation of ketones does not apply to the at-rest readings from the Eskimo in Heinbecker's study.

I'd like to have a guess at the more "steady state" condition.

Full oxidation of a "typical" protein such as albumin produces a value of around 0.8 for the RQ. So I've invented a single mythical amino acid which gets close to the average RQ of protein. It looks like this:

NH2 - CH - COOH
            CH2
            CH3

Two of these amino acids oxidise using nine molecules of oxygen to give one molecule of urea and seven molecules of CO2, giving an RQ of 0.78. If this was replaced with a dietary equivalent the RQ would stay around 0.8 and the RQ of 0.69 from saturated fat would be increased somewhat. If the oxidised amino acid was not replaced the RQ change would be exactly the same but muscle wastage would occur.

What if, as a ketosis induced protein sparing effect, certain non-essential amino acids, were synthesised from urea plus carbon from fats plus a little oxygen. I'm not suggesting for a moment that this is exactly what happens, but the equation must balance whatever pathways might be used.

I've spent quite some time with scraps of paper working out how much oxygen has to be added to a couple of -CH2-CH2- moieties from saturated fat, along with a urea molecule, to reassemble the above pair of amino acids. "Mythical" protein turnover...

It takes 3O2 and liberates one CO2.

Combining this with the 9 O2 and 7 CO2 from oxidation, the whole repalcement of this amino acid would use:

12 O2 and generate 8 CO2 giving and an RQ of 0.67.

So the replacement of one "typical" amino acid using part of the acyl-chain of a saturated fatty acid generates an RQ of 0.67.

That's getting us somewhere below 0.69, what then matters is how general this effect might be which obviously depends on protein turnover, protein intake, protein quality and anything else anyone can think of. The value is pushed further down by the loss of oxygen rich ketone molecules through the breath and urine.

I'm very aware that minor errors in logic or arithmetic might alter the above calculations.

What an RQ well below 0.69 speaks very clearly against is gross muscle catabolism (which pushes the RQ upwards towards 0.80). Clearly, muscle loss does occur but I can see no reason why muscle loss should be an essential pre requisite for fat oxidation during fasting. The ability to minimise muscle loss under fasting (or ketogenic eating) might just provide some advantage on an evolutionary basis.

Marking out amino acid oxidation (ie loss of protein) as an essential pre requisite to fatty acid oxidation (in the absence of carbohydrate) suggests a rather odd view of reality. If it were correct it should show as elevated RQ's above 0.69 in proportion to the amount of amino acid oxidation which might be going on.

Which is not the case.

Peter

Edit for raphi: the arithmetic:










Tuesday, August 09, 2016

Glucose from fatty acids: RQ of 0.454


This is a section from Table V of Heinbecker's 1928 paper:

Studies on the Metabolism of Eskimos








This tells us certain very, very interesting things.  The subject is a young Eskimo woman. Column 6 gives her RQ and, by day 3.5 of fasting, it is 0.454. Which is clearly impossible. Maybe. It took me a few minutes to realise that the result is probably correct, certainly within the limits of measuring RQ in 1928 in a tent in the Arctic. Let's assume it's ballpark correct.

I've been through this too many times. An RQ below 0.69 suggests the generation of oxygen rich molecules from fatty acids. An RQ of 0.454 suggests a huge amount of (probable) gluconeogenesis from fat is going on.

The other thing which becomes obvious from simple logic is that any oxygen rich molecule generated from fat must NOT be oxidised for it to drop the RQ. If you oxidise stearic acid to CO2 and water you will get the same amount of CO2 per unit O2 consumed whether that process goes via acetyl-CoA (as it usually does) or via ketones, oxaloacetate or glucose.

The girl, Martha, was breast feeding a baby throughout the study:

"Subject II. Nursing female".

She has eaten nothing for 3.5 days, she is excreting both glucose and galactose in her milk. She has used up her glycogen stores. Where is the glucose/galactose for the milk coming from?

The RQ is 0.454, the milk sugars are coming from fat.

Sooooooo. Question:

How much gluconeogenesis is possible from fatty acids?

Answer:

A lot.

How much is a lot? It's not really practical to put a number to this, but enough to drop the RQ to 0.454 or, equally, enough to make a continuous supply of human breast milk. Both seem to be reasonable answers.

Unless you have an agenda.

Peter

Sunday, August 07, 2016

Just a heads-up

Protons (44) has been markedly updated. Just a heads up in case the update doesn't come through as a "New Post" to anyone who follows. Perhaps best not leave comments here on this little notification post.

Peter

Thursday, August 04, 2016

Protons (44) Does fatty acid oxidation really drive reverse electron transport and superoxide generation at complex I?

This post has been extended and adjusted quite considerably in the light of further information. The first five comments in the comments section are from pre update.

I suppose I should say now that I am particularly interested in data which trash the Protons hypothesis. I am so deeply biased in its favour that contradictory evidence has to be taken very seriously. Hence the initial post (preserved and embedded below) and the current extension of it based on another paper, also via Mike. It just goes to show how deeply selective people can be with the information which they pass on and how limited they are in coming forward with what they really think is happening. Personally, I'm interested in how stuff works. That's what I write about. Any agenda comes from the biases I have about how well the Protons hypothesis, largely self generated, fits most of the data.

Needless to say, other papers (Back in Protons 3) using mitochondrial preparations show they generate significant amounts of superoxide using palmitoyl carnitine. Anyway, here we go with the edited post:


The original post:

Well, should I develop any leisure time not taken up with the beach, crabbing, canoeing or any one of the hundreds of school holiday activities which are on-going, I have some serious reading to do!

From Mike Eades:

Mitochondrial fatty acid oxidation and oxidative stress: lack of reverse electron transfer-associated production of reactive oxygen species

The group seem pretty good and are supportive of succinate and mtG3Pdh driven RET, but not of ETFdh driven RET. You can imaging how much that gives me to think about! Needless to say, in view of the age of the paper, the group has interesting stuff published more recently which may have something to say about FFA oxidation and ROS generation.

Life is never as simple as you might like it to be!!!!

More to come, will take time.

Peter

End of original post.


It's worth adding this quote from the results to make things absolutely clear:


"The rate of ROS release from heart mitochondria oxidizing carnitine esters of long- and medium-chain fatty acids was much lower than that in the presence of succinate (Fig. 1A, B, C and D) and comparable to that with NAD-linked substrates, pyruvate or glutamate (not shown). An increase of acylcarnitine concentration from 0.5 mM up to 5 mM (examined with butyryl- and octanoylcarnitine) did not enhance ROS production (not shown)".

You can get ROS to be produced in this preparation, but only by using an ETC inhibitor. That's not physiological. Okay.



Now here is some more current (2013) thinking from Schönfeld and Reiser. This is the Schönfeld, as in the first author of the above (2010) paper. Here is what he says in this review:


Why does brain metabolism not favor burning of fatty acids to provide energy? - Reflections on disadvantages of the use of free fatty acids as fuel for brain


"This should be substantiated by the following quantitative analysis: during complete degradation of one glucose molecule, two molecules FADH2 and 10 molecules of NADH are formed, which corresponds to a FADH2/NADH ratio of 0.2. In contrast, b-oxidation of palmitic acid generates 15 molecules of FADH2 and 31 molecules of NADH, with an FADH2/NADH ratio of approx 0.5. Consequently, during b-oxidation there is competition of NADH and FADH2 electrons for oxidized ubiquinone as electron acceptor. This situation would most likely enhance oxidative stress in neurons for two reasons. Thus, slow NADH oxidation maintained the redox state of the electron carriers upstream of complex III in a highly reduced state, a situation similar to rotenone inhibition of complex I. Such situation enhances the superoxide generations by ETC. Moreover, at a high FADH2/NADH ratio, more FADH2 becomes oxidized by the electron transfer flavoprotein-ubiquinone oxidoreductase, a reaction known to be a potent source for superoxide generation".

Let's zoom in:

"Consequently, during b-oxidation there is competition of NADH and FADH2 electrons for oxidized ubiquinone as electron acceptor".

The whole quote and most especially the crucial snippet could have been lifted almost directly from the Protons thread. This is exactly the argument I made for the use of lactate rather than palmitate in neurons. This is simply one facet of the overall Protons concept, which is largely based on the FADH2/NADH ratio.

NB In the 2010 paper there was no difference in total ROS generated between feeding the mitochondria on pyruvate or palmitoyl canitine. Go figure!

Bear in mind that in his 2010 paper Schönfeld found very low generation of superoxide from any fatty acid source (using heart and liver mitochondria) and, although the group have some info since then from brown adipose tissue mitochondrial ROS, they don't appear to have generic data to support Schönfeld's (roughly correct) Protons-like hypothesis above. You can read their quote as well as I can. FADH2 via ETFdh is accepted as driving ROS generation via CoQ reduction. i.e. ROS are generated in proportion to FADH2 which is generated in proportion to the length and saturation of beta oxidised FFAs. They don't specify RET, the ROS may come from ETFdh directly, but I can live with that (should it turn out to be correct). It's the CoQ reduction and FADH2 input that speak to me.

They didn't find anything like this in their 2010 paper comparing ROS from butyric acid to octanoic acid to palmitic acid! All three substrates generated ROS comparable to pyruvate despite the FADH2/NADH ratio being very different.

My presumption is that Schönfeld considers his version of the Protons FADH2/NADH concept to be correct and I'd be willing to bet he even knows exactly why the 2010 model doesn't show this.

But he ain't sayin' nuffing. There's a lot of it about.

Summary: I don't think I'm about to discard my pet hypothesis quite yet!

Peter

Tuesday, August 02, 2016

Acetone to oxaloacetate

Some time back in May this year I posted on what I considered to be gluconeogenesis from acetoacetate via acetone (You don't have to read that post, the next link is far more important. OK, read the post then, I enjoyed writing it). It's a simple four or five step conversion from ketone to oxaloacetate, which can enter the Krebs Cycle allowing the regeneration of citric acid. This is my illustration:






















Notice the oxaloacetate (and the citrate off to the bottom right). Now, back in 2012 Chris Masterjohn posted on gluconeogenesis from acetoacetate via acetone and oxaloacetate (Follow the link and this time READ the post). This is his illustration:


















Perhaps not as pretty or as comprehensive as my pic but pretty well saying the same thing.

I think it is reasonable to say that both Dr Masterjohn and myself are both fully aware, on record, that acetone from acetoacetate is a substrate for the generation of oxaloacetate.

As Dr M says, fat burns in the flame of oxaloacetate. This does not have to come from glucose. It does not have to come from amino acids. He seems to have forgotten his own post from 2012. Fat provides oxaloacetate. Can anyone imagine that a period of food deprivation would not supply the necessary metabolites to utilise ketone bodies? Well duh.

I know this. He knows this. Now, try this video from this post, starting at about 3 minutes in. I gave up at around 8 minutes, so apologies if he returns to acetone as a source of oxaloacetate later on. It doesn't seem likely from the bits I listened to.

So, the big question is: What happened to Dr Masterjohn's knowledge about ketones and oxaloacetate between July 2012 and August 2016?

This is beyond me. I find it incomprehensible. In 2012 he knew... In 2016??????????

Peter

Thanks to Karl for the heads-up.

Sunday, July 31, 2016

A load of crap in Gartnavel

This came to me via Facebook from Wally Courie:

Poop Pill’s Surprise Failure Shows That the Microbiome is Still a Mystery

The original faecal transplant work was done in Glasgow and got written up here:

Faecal transplant for recurrent Clostridium difficile-associated diarrhoea: a UK case series.


Many years ago, probably around 2006 when the above paper was being written/published, I attended a presentation at a probiotics meeting in central London by a medic from Gartnavel General Hospital in Glasgow. Might have been by MacConnachie himself. He was quite deadpan, described their very simple procedure for obtaining, filtering and administering the faecal transplant by NG tube. You can read the abstract of the paper to get some idea of the success rate of the procedure. Hint; very high.

What really stuck in my memory was his comment was that, as they removed the NG tube, the patient cured. Many of these these folks hadn't left the house, often never left the loo, for years before referral. Their next bowel movement was going to be normal.

With a success rate as high as documented there has never been a double blind placebo controlled trial. I rather like that.

So I can imagine him watching the development (as I did) of the multiple probiotic capsule which was (or wasn't!) going to cure C. difficile gut-rot without all of the "ick-factor" of the trip to Gartnavel. Then quietly going in to work to cure some more people, assisted by a pooh sample.

Peter

Wednesday, July 06, 2016

Arteriosclerosis (6) and Subbotin

It seems a very long time ago that I wrote a series of posts on the mechanism of the development of the arterial tunica intima from a single layer of cells through to a thickened structure of similar or greater thickness than the muscularis layer. I've gone back and re-labelled them under Arteriosclerosis (1) through to (5).

Very briefly: Arteriosclerosis (1) covered the development of diffuse intima thickening (DIT) from birth through to about three years of age. DIT occurs over areas of disrupted elastin, is on the lumenal side of the elastin and is largely composted of glycosaminoglycans (GAG) and a few cells.

Arteriosclerosis (2) covered the role of platelets in supplying ILGF-1 which generates GAG at the site of platelet adhesion. That is where the platelets stick to damaged endothelium and strengthen an area at which the elastic tissue has been ruptured. It is a physiological process of tissue reinforcement.

Arteriosclerosis (3) covered the condition of von Willebrand's Disease, where platelets fail to adhere to damaged tissue. In this condition the intima stays thin and there is no DIT with age, assuming the vWD is not fatal at a young age. The elastin layer gets completely trashed as there no ability to generate reinforcement.

Arteriosclerosis (4) illustrated this process by showing the formation of surface microthombi to deliver platelets and generate GAG, it covered mucoplysaccaride disorders with massive intimal thickening when GAG cannot be degraded/remodelled and introduced the idea of intimal thickening in the arterial supply to the atrioventricular node as a precursor to sudden cardiac death.

Arteriosclerosis (5) is just the pictures to complete part (4).

To summarise: Arteriosclerosis begins as non pathological DIT which is a reinforcement system to maximise the correct development of the arterial tree to best withstand the loads to which it is subjected. It gives a tailor-made cardiovascular system for a given individual.

That's normal.

Subbotin has recently published a review article documenting his view that pathological arteriosclerosis, with lipid infiltration, commences when the tunica intima becomes too thickened to receive adequate oxygenation without developing a vascular supply of its own. It is normally avascular, getting oxygen and nutrients by diffusion from either the arterial lumen or the arterioles embedded in the muscularis layer. Once the thickened tunica intima develops a vascular supply all hell breaks loose with apoB labelled lipids attaching themselves to the GAG on the mistaken basis that this the extra cellular matrix of damaged tissue.

This is a highly plausible scenario and fits with the microscopy much better than any fairytale about apoB lipoproteins sneaking through the arterial endothelium and burrowing deep, deep in to the intima before depositing themselves on the border with the muscularis layer, leaving the surface layer completely unaffected. Which is what the histology shows. Shrug. Never forget: Only purple spotted cholesterol causes CVD, anything else is bollocks.

There are several things which spring to mind about DIT, insulin and oxygenation. If, as I think likely, a general thickening occurs under the effect of chronic hyperinsulinaemia acting on the ILGF-1 receptors which are on the lookout for platelets, we have a reason why insulin, not glucose, drives CVD. Anything which hastens thickening of the tunica intima hastens CVD. Insulin.

Second is that not only do VLC diets drop insulin, but they also reduce tissue oxygen extraction while maintaining normal ATP production. If Subbotin is correct that localised tissue hypoxia is what converts a benign reinforcement area to a fragile pool of lipid, then acetoacetate and beta-hydroxybutyrate (and stearic acid) are your first obvious port of call. AcAc/BHB looks like an excellent tool to blunt the impact of hypoxia on the need for blood vessel invasion of a given tissue.

Third: What happens if you keep insulin high with a diet of complete crap while supplementing ketone esters to reduce tissue oxygen needs? My guess, and it's only a guess, is that intimal thickening will progress apace but will only develop vascularisation at a later stage of the process, when the slack provided by the exogenous ketones has been taken up. Equally, the worry might be that you take ketone esters for a year, living on crap, and then run out of funds while you have thickened avascular DIT which was quite happy under ketones but is then going to need more oxygen per unit ATP under glucose oxidation. At least by going the ketogenic diet route you should have had basal insulin to minimise DIT progression in combination with your AcAc/BHB, up until the time you feel that you'd rather die than continue to live without eating pizza.......

Peter

Monday, May 23, 2016

The degradation of mitochondrial research


Does everyone remember this?



















Most especially this bit:























Control is using pyruvate at 5 mmol/l and the ATP synthesis shut down is generated using palmitoyl carnitine at 10 micromol/l. Got that?

Try this one too, plots of pyruvate against palmitoyl carnitine:


















And how about this one, increasing doses of palmitoyl carnitine alone, showing self destruction of ATP synthesis with rising doses of palmitoyl carnitine:
















Is everyone convinced that allowing anything over, say 5 micromolar palmitoyl carnitine anywhere near a mitochondrion is going to crash ATP synthesis? Lots of experiments, lots of evidence.

Now, this is pretty basic science. What happens when a lab takes their basic science and goes clinical? This is the same lab:

Chronic Reduction of Plasma Free Fatty Acid Improves Mitochondrial Function and Whole-Body Insulin Sensitivity in Obese and Type 2 Diabetic Individuals

Here is the key statement, from the methods:

"Mitochondrial ATP synthesis rate was measured ex vivo with a chemiluminescence technique as previously described (16)".

Reference 16 is the one from which all of the above graphs have been taken. The isolation, washing and feeding of the mitochondria have not been changed. Yet now, in a clinical study showing the wonders of free fatty acid reduction, we get this:












We can ignore the acipimox groups and use the pre treatment open columns. Look at ATP yield from Pyr, this is pyruvate 2.5 mmol/l. Now look at PMC 0.5 and PMC 1. Here we have palmitoyl carnitine being added at either 0.5 mmol/l, ie 500 micromol/l or even 1000 micromol/l, giving comparable rates of ATP synthesis to pyruvate 2.5 mmol/l.  That 1000 micromol/l is one hundred times the concentration used in their first paper to shut down electron flow and collapse delta psi.

Where did the inhibition of electron transfer from reduced CoQ to complex III by palmitoyl carnitine go to? What changed?

They went from basic science to a clinical application. Was the basic science correct? Is the clinical paper correct? An interesting set of changes. Makes me thing of the degradation we see so commonly in research, from something which looks sound to something which looks incomprehensible.

Peter

Will fasting destroy your mitochondria? (No).

I started with this paper:

Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

"Prolonged" here means 60 hours. I had to look up "dysfunction" in a dictionary as I feel it carries negative connotations. It does.

"abnormality or impairment in the operation of a specified bodily organ or system"

Not eating for a couple of days renders your mitochondrial either abnormal or impaired. That's a big assertion to put in to a title.

So I don't like this group. They are fully aware that fasting requires insulin resistance and that this insulin resistance is physiological. Under such conditions there are, undoubtedly, changes in mitochondrial function. What sort of a label you apply to the changes says rather more about the mindset of the authors than it does about the mitochondria. These folks are deep lipophobes.

There are two respiration states under which the mitochondria from fed people outperform those from the same people after a 60 hour fast:

State 3 respiration is when you supply so much ADP that the ATP synthase complex can run at its absolute maximum rate. It's a measure of the ability of the ETC to pass a very high electron flow while using the membrane voltage. Oxygen consumption (a surrogate for electron flow) is reduced in preparations from fasted people.

The second condition where mitochondrial oxygen consumption is reduced by fasting is under pharmacological uncoupling.  FCCP, just such an uncoupler, allows the absolute maximum flow of electrons down the ETC, completely unfettered by any need to drive ATP synthase at all and probably with no delta psi to work against when pumping protons. Under fasting conditions there is measurably less oxygen consumed under FCCP than when tissue is isolated from fed subjects.

Out of interest they also treated a set of mitochondria with oligomycin (which blocks ATP synthase) and checked for physiological uncoupling (state 4o respiration). There is no difference in oxygen consumption under either nutritional state.

How badly are the humans crippled by this level of mitochondrial "dysfunction" under fasting?

"Twenty-four hour energy expenditure during the last 24 h of the 60 h intervention was slightly but significantly reduced upon prolonged fasting (10.88+/- 0.33 vs. 10.30+/- 0.30 MJ/day, in fed versus fasted, respectively, P less than 0.02). The difference was mainly caused by a reduction in diet-induced thermogenesis, and not caused by a decrease in resting metabolic rate (data not shown)".

They are normal.

These subjects went from a free fatty acid concentration of just over 200 micromol/l after an overnight fast to just under 2000 micromol/l (no typo) after a 60 hour fast. Fatty acid oxidation is supposed to be a supply controlled system. With a ten fold rise in a metabolic substrate perhaps we might expect a ten fold rise in ATP production driven by an attempted ten fold rise in membrane potential? Reductio ad absurdum. Do you think we might need some sort of control system to be applied? Like a brake?

The brake looks interesting to me. The next layer to dig in to is this group:

Deleterious action of FA metabolites on ATP synthesis: possible link between lipotoxicity, mitochondrial dysfunction, and insulin resistance.

OK, you've read the title. Are they lipophobes? No Brownie points there I'm afraid!

Now, you have to be careful. The 60h starvation folks used permeablised muscle tissue. What every had happened to the mitochondria under starvation is (pretty much) still in place and didn't budge. This next group of lipophobes washed and isolated their mitochondria. We are now dealing with squeaky clean mitochondria with all of the cytoplasm washed off. And all of the fatty acids and their derivatives, along with any cytoplasmic enzymes to interconvert them, also washed off.

In the following diagram they added palmitoyl carnitine to mitochondria being fed on pyruvate and ATP production went through the floor. They could do something similar using palmitoyl-CoA and oleoyl-CoA, even without added carnitine. They could also simply wash those mitochondria to restore pre-lipotoxicity ATP synthesis rates:


















Treating mitochondria with palmitoyl carnitine reduces ATP synthesis by 90%. Washing those mitochondria restores normal function. It looks very much like the site of action of the fatty acid derivatives is a) on the outside of the mitochondria and b) a non-covalently bound effect.

This is what the authors say:

"We therefore postulate that a rise in intramyocellar fatty acyl-CoA interferes with mitochondrial ATP synthesis by inhibiting the electron transport chain and decreasing the inner mitochondrial membrane potential. As a result, fatty acyl-CoA oxidation is reduced, leading to a further rise in intracellular FACoA concentration and exacerbation of the mitochondrial dysfunction. This sequence of events leads to a self-perpetuating negative feedback cycle whereby a small rise in intramyocellar FACoA impairs mitochondrial function and further increases the intramyocellar fatty acyl-CoA concentration".

NB I think they mean a positive feedback loop, to be producing runaway mitochondrial dysfunction and an ATP supply crisis. You know, skip a meal and "phut" your mitochondria snuff out.

My impression is slightly different. I look at the lethal effects of my favourite fatty acid's carnitine derivative and ask, scratching my head: How do people function with FFAs of 2000 micromol/l if 5 micromol/l of palmitoyl carnitine is going to kill them?

For a reality check we can just look at those 60 hour fasted people in the respiratory chamber. Are they wanting to die or wanting to go out for a steak?

Mmmmmmmm.... Steak......


The mitochondrial-washers are suspicious (but have no evidence) that fatty acid derivatives are dropping in to the binding pocket on complex III where reduced CoQ should be docking and so blocking the ability for electrons to pass from reduced CoQ to complex III. If this is true, and yes the docking site really is very close to the outer surface of the inner mitochondrial membrane, we have a system where fatty acid derivatives can limit the flow of electrons down the ETC to the levels needed for perfect ATP production while maintaining a physiological insulin resistant state to keep the brain supplied with glucose.

We know this system works because people can go without eating for 60 hours without looking anything other than slightly slimmer than they did when they walked in to the respiratory chamber.

We also know that people can go for six weeks on a protein supplemented fast and actually improve their exercise ability, based purely on fatty acid catabolism.

Of course, we must ask what happens to the redox state of the CoQ couple if you point blank refuse to allow reduced CoQ to hand on its electrons on to complex III. All of the CoQ will end up fully reduced. Reverse electron transport through Complex I is inevitable, given a decent membrane voltage. That means targeted superoxide production and the Good Things that flow from this. That means physiological insulin resistance. I view this as one layer up in control systems from the NADH:FADH2 ratio within the mitochondria. Note that the this particular effect of fatty acid derivatives does NOT require active oxidation of those derivatives. There are a number of papers out there where fatty acids induce insulin resistance even when beta oxidation is pharmacologically blocked. The data in this paper are the explanation, they make complete sense (more than you can say for the authors).

Peter

Sunday, May 15, 2016

Fruit Flies and NDI1

The Protons thread originated when I asked myself: What is the difference between fat oxidation and glucose oxidation? This rapidly led to the redox state of the CoQ couple as a driver for reverse electron transport (RET) through complex I, superoxide generation and the benefits of ROS signalling. It was a period of deep insight, especially about electron transporting flavoprotein and its dehydrogenase. Core to the Protons thread is the redox state of the CoQ couple and the generation of reverse electron transport.

In amongst the recent flurry of blogging about calorimetry and associated physiology Mike Eades forwarded this text to me:

Mitochondrial ROS Produced via Reverse Electron Transport Extend Animal Lifespan

Mostly on the basis of its title, I think. It's a very complex paper, drosophila based, couched in terminology which is probably completely routine if you work with flies but as clear as mud if you don't. I think we can reduce the paper to its title, discussion headings and three diagrams, one of which I'm going to butcher, the way you do. It's free full text if anyone wants to bend their brain.

Those titles:

ROS Production Increases with Age and Correlates with a Decrease in Complex I-Linked Respiration

Over-Reduction of the CoQ Pool Increases ROS Production and Extends Lifespan

CoQ-Mediated ROS Signalling Can Rescue Pathology Induced by Oxidative Stress

Loss of CoQ-Mediated ROS Signalling Accelerates Ageing

I mean, to a superoxidophile, what more could you ask? Just so long as the superoxide is RET derived from complex I...

OK, now the images. This one is core. It's taken from the Graphical Abstract and looks to be hand drawn in pencil. Note the NDI1 super-fly logo on the chest. This research group is crazy. I like that:























We've met NDI1 before. Its a small NADH oxidase from yeasts which reacts the reducing equivalents from NADH with oxygen to give water, reducing the CoQ couple in the process. It drives, given an adequate delta psi, RET through complex I. Inserting the gene for this protein in to a fly does this to life span:













Obviously, the line off to the right represents the NDI1 positive flies. NDI1>daGAL4, as "fly people" might say. They then went on to use an almost infinite supply of other tricks, in other flies, to show that it really is RET through complex I via CoQ reduction that extends life via site specific ROS generation. I won't slog through the arguments, you now know where the paper is!

And here is the lamb to the slaughter picture, presented as part of Figure 2:















There's NDI1 in blue, using NADH to reduce the CoQ couple and generate ROS. The main thing I dislike about this image is that the ROS seem to be popping out of the CoQ couple. They actually come from RET through complex I, so let's change it so it really looks that way:












That's better. Now I, personally, don't have and don't really want to have an NDI1 sitting on the matrix side of my inner mitochondrial membrane. Perhaps there is some similar enzyme available? Ah yes, let's mentally substitute ETFdh:












And of course, if we want to drive this process, we need FADH2, transported to ETFdh via electron transporting flavoprotein, generated by the first step of beta oxidation of saturated fats. Any double bonds mean this step gets skipped and all we supply is NADH. If we want FADH2 it's palmitate and stearate all the way:
















I have absolutely no idea whether using FADH2 from beta oxidation will do, in mammals, what NDI1 does in fruit flies. But I like the paper, and I like that idea.

And the fly doodle of course!

Peter

The addendum; because this post is not totally irrelevant to my recent blogging ideas:

Veech doesn't like fatty acid oxidation. He has little time for acetoacetate but loves beta hydroxybutyrate because it, specifically, reduces the NAD+/NADH couple while oxidising the CoQ couple, increasing the redox span. ie BHB oxidises the CoQ couple.

Kwasniewski is very pro saturated fat and rather anti ketosis. He wants FADH2 driven metabolism which enters the ETC by reducing the CoQ couple. He has a disturbing habit of being correct without providing any science.

The flies suggest going with Kwasniewski rather than buying bulk BHB by the tanker-load when it eventually hits the market as an affordable ketone ester. But they're only flies...

Friday, May 13, 2016

Uncoupling in a can?

In response the suggestion from E-S, at top of comments to the last post, that caloric output should be measured using a calorimeter:

Direct calorimetry identifies deficiencies in respirometry for the determination of resting metabolic rate in C57Bl/6 and FVB mice

"In a carefully controlled study, Walsberg and Hoffman (10) examined the accuracy of respirometry in multiple species, including the kangaroo rat (Dipodomys merriama Mearns), dove (Columbina inca Lesson), and quail (Coturnix communis Linnaeus), by comparing simultaneous outputs from animals studied with both direct and respirometry methods. Those authors concluded that when disparate species were studied under various conditions that estimations of heat production by RER-based respirometry calculations led to errors averaging 21% for kangaroo rats, 15% for doves, and 17% for quail".

"Here, we demonstrate that the rate of inaccuracy of respirometry [for the CL57Bl/6 mouse] is roughly 10–12% and posit that this magnitude of inaccuracy, given the target range, is unacceptably large. We conclude that the challenges faced by the obesity therapeutics research community in identifying or validating novel therapeutic targets in mice (and likely other species as well) may be compounded by the inappropriate yet almost universal and sole reliance upon respirometry".


RER underestimates calorie output, compared to a calorimeter. But you can't buy a calorimeter off the shelf.

So folks use RER and that table. "Everyone does it". Yeah. Well.

Peter

Wednesday, May 11, 2016

Uncoupling and weight loss

I've spent the last three posts making the point that fatty acid oxidation (supplemented by ketosis) increases the amount of ATP (and energy yield of ATP hydrolysis) available per unit oxygen consumed. This is particularly clear under the conditions of extended, intensely hypocaloric eating described by Phinney, where exercise can be sustained for longer, at a lower VO2, than on a mixed diet.

Now, oxygen consumption is a surrogate for caloric output. How many calories you "spend" per unit oxygen consumption is a complex calculation and depends on your fat to carb ratio.

But we don't run on calories. We run on ATP (mostly), or rather we run on the energy yielded from ATP hydrolysis.

To make that absolutely clear: We know, from Phinney, that under pure fat oxidation, we can generate enough ATP energy (physical treadmill load) to sustain moderate exercise by using less calories (ie lower VO2) on fat oxidation than on mixed diet oxidation. The increase in ability shows as a 25% drop in VO2, ie a 25% drop in calories needed to get enough ATP energy to move at 70% VO2 max.

That, to me, is pure survival adaptation. It's elegant, neat, cool etc.

It's not providing a metabolic advantage for weight loss.



Now, once again, I must wander off in to rodent studies.

If you take a rat or a mouse and feed it a genuine ketogenic diet you get some interesting effects. Let's look at this small study in rats. Here's heat output. Red is chow fed, grey is ketogenic:








Day or night, energy output is lower for the ketogenic rats compared to the chow fed rats. Phinney got a 25% drop in VO2 on his treadmill, the rats have calorie output down by an average of 11%, at a similar RQ. Running on fat (+/-ketones) requires less calories to generate adequate ATP levels.

Note, these are not real heat outputs in the rats. No one measured heat flux in any way. They're calculated from the VO2. They're done using the software built in to a CLAMS device around well accepted values of calories used per litre of oxygen consumed. This drop in calculated heat output, in itself, is not a surprise in view of Phinney's work.

What is surprising is that VO2 actually increased to generate this reduced heat output:









The rats should be using less oxygen per minute to produce their whole-body ATP energy requirement running on fat, according to Phinney. And me. And the chart. They're not, they're using more, in absolute terms.

The conclusion here is that the VO2 has gone the wrong way. So we have to ask: What is the difference between a fasting, exercising human on an RQ of 0.66 and a ketogenic rat slumming around its cage with a very similar RQ of 0.7?


The rats are uncoupled. They pump protons through complexes I, III and IV but a significant number of those protons drop straight back in to the mitochondria through open uncoupling proteins. Calories and oxygen are used (at the same RQ as any other more productive oxidation) but no ATP is produced from any protons which do not use ATP synthase. VO2 moves in two directions. It goes down (and so do calories used) due to switch from glucose oxidation to fat oxidation. It goes up due to uncoupling. The overall effect, up or down, on VO2 depends on the relative effects of RQ change, uncoupling, gluconeogenesis, NEAT and actual exercise.

Phinney's treadmill walkers had high FFAs and high ketones but absolutely no suggestion of any sort of uncoupling. Why?

To get any further we have to go to the Protons thread back in 2014.

Uncoupling proteins are kept closed by cytoplasmic ATP. And there is always enough cytoplasmic ATP in a functional cell to keep UCPs closed. There is one particular way (of several) to open them. The inhibition from cytoplasmic ATP can be overcome by an excess of mitochondrial ATP. Mitochondrial ATP, obviously, enters the UCP from the opposite end to cytoplasmic ATP and gets in the way of the latter's binding. Mitochondrial ATP cannot reach far enough into the UCP to induce the closed conformation itself, so the pore opens. The blog post has nice images and a more thorough description. Here's my fave picture:























How do we keep mitochondrial ATP levels low?

Phinney had six week starved humans on a treadmill showing every probability of low mitochondrial ATP and UCPs closed tighter than the proverbial monkey's @rsehole.

On the opposite front we have rats in a cage whose biggest effort is to move over to the hopper of ketogenic pellets and have a munch. These animals uncouple like mad while eating to satiety. They also either maintain low fat reserves or lose fat reserves if previously made obese from fat/sucrose feeding. We've all read this mouse study even if today's rat epic is very inaccessible (thanks Mike).


It seems to me that it is possible to maximise the efficiency of energy usage to ensure survival under near starvation conditions. However fat based your metabolism, you are not going to uncouple your oxidative metabolism unless you have adequate ATP within the mitochondrial matrix.

It's very clear that an ad libitum ketogenic diet allows uncoupling and metabolic inefficiency down to a lean bodyweight, certainly in rodents. This is not arguable. Here's the graph. No mouse was forcibly calorie restricted:



Days 1-4 after switch to ketosis they ate less, by day eight after the switch to ketogenic eating they were eating more calories (ns) than other groups but staying weight stable.

The question to me is: By how much do you have to deliberately restrict the calories of a ketogenic diet fed human to eliminate the uncoupling effect? Or, more simply, turn the question round: How do you get a human to lose weight most effectively on a ketogenic diet? This is easier to answer.

As Amber O'Hearn suggests:

Eat meat.
Not too little.
Mostly fat.

Perhaps someone should tell Dr Hall this. Better still, make it his epitaph as science progresses.

Peter

Monday, May 02, 2016

On Stephen Phinney and an RQ of 0.62

****Edit****
There is an update on this post here
****End edit****

Now, oxidising long chain saturated fat gives you an RQ of 0.69. Lower than this needs a supplementary process of some sort. In the last post I had Table II from Stephen Phinney's 1980 paper. There are RQs below 0.69 all over the place and even the mean RQ of the 6 week fasting exercise test was 0.66,  with some individuals down at 0.62.
















So how can we manipulate RQ values?

This is a graph taken from that nice paper on ketogenic diets for rats. The black line is the RQ of the chow fed rats. They are on 17% or so calories from fat, 64% of calories from starch and the rest is protein. Grey zones are night, white zones are daytime. Ratties are nocturnal, they eat their high carbohydrate diet at night. While they are eating they run their metabolism on glucose. This should give an RQ of 1.0 but we can see the RQ is greater than 1.0 during the times at which the rats are feeding:











We've seen this before during an OGTT in massively weight reduced people. Show them some glucose and they will immediately convert it to lipid and store it. After a mere 75g of glucose during an OGTT, these post obese ladies will develop an RQ over 1.0, see the top dashed line:











This is de novo lipogenesis, either routine in the rats on a 64% carbohydrate or pathological in the post obese ladies. Glucose arrives as an oxygen rich molecule. During the reorganisation to a very oxygen poor molecule oxygen is provided without it needing to be taken up through the lungs. Smaller oxygen flux per unit CO2 produced gives an RQ greater than 1.0.

So it's pretty easy to get a RQ above 1.0. How easy is it to get an RQ below 0.69?

As we all know, acetoacetate is unstable, spontaneously decarboxylating to acetone and CO2. On its own this isn't fast enough to be useful so we have acetoacetate decarboxylase to speed the process up. You find it in the liver and in the brain, mostly. The sorts of places where glucose might be useful.

Apart from being exhaled, what is the fate of acetone in the body? I can't imagine that we are deliberately forming the stuff enzymatically just to breathe it out... Well, here's a pathway I cribbed earlier, can't remember from which paper but one on basic acetone metabolism:























Soooo theoretically ketone bodies, via acetone and oxaloacetate , are glucose precursors. If you radio label acetone with (14)C, where does it end up?

"Radioactivity from (14)C acetone was not detected in plasma free fatty acids, acetoacetate, beta-hydroxybutyrate, or other anionic compounds, but was present in plasma glucose, lipids, and proteins".

Ketones to glucose. How much?

“On the basis of our specific activity data, we have calculated that 4-11% of plasma glucose production could theoretically be derived from acetone”.

The 11% was calculated for 21 day starved humans.

The most logical explanation for an RQ of 0.62 is that the person is performing a significant conversion of fat to glucose. This is completely plausible via acetoacetate, acetone and oxaloacetate. The exact steps are unimportant. What matters is that there will be an increased consumption of oxygen per unit CO2 produced. The RQ is just a ratio so increasing oxygen use will make it drop, possibly below that 0.69 of saturated fat oxidation.

Summary: We already know that total O2 consumption must and did drop on fat adaptation. We know from simple arithmetic that CO2 production drops even more that O2 usage when fat (vs glucose) is oxidised, to give us that normal RQ of 0.69.

If there is a further usage of O2 in the process of converting ketones derived from fat in to glucose, this would explain an RQ of 0.62.

Despite this "waste" of oxygen you still use less O2 per ATP from fat oxidation, even if doing some gluconeogenesis. We know this from the absolute VO2 measurements combined with the RQ values in Phinney's Table II and my back of envelope calculations.

I sit in awe of fat oxidation. We carry fat as long term energy storage for use in times of need. Under those conditions of privation this long term energy store allows very efficient ATP generation per unit oxygen, at the same time as reducing CO2 production, at the same time as generating a significant amount of glucose. Fatty acids and beta oxidation, with ketones thrown in, are just awesome.

I'm also hugely impressed by how far ahead of its time Stephen Phinney's paper was and how well it still stacks up against modern papers.

Peter

Friday, April 29, 2016

Endurance and oxygen flux during fatty acid oxidation

I was browsing Pubmed looking for other things when I came across this ancient publication by Stephen Phinney, from back in 1980. Those were the days. That was the Summer I contracted mycoplasmal arthritis as a final year vet student seeing practice in North Devon. Lots of NSAIDs then six weeks on tetracyclines once the serology came back positive. Yum. Anyway:

Capacity for Moderate Exercise in Obese Subjects after Adaptation to a Hypocaloric, Ketogenic Diet

Free full text, nice read. Replicable, if you have the equipment.

This is table II. For the first endurance test the treadmill was set at 3 mph with a personalised incline. The same test was then repeated after one and six weeks of a protein supplemented fast. For the six week treadmill run the settings were identical to the first sessions but the subjects wore backpacks containing weights equivalent to the bodyweight that they had lost during the 6 weeks of their fast. There was no exercise training in the interim.

















The column to look at is the one in the middle labelled VO2 and expressed as ml/min. On a mixed diet the treadmill settings required a flux of oxygen of 1875ml/min to maintain this significant effort. After 6 weeks on a protein supplemented fast, wearing weights in a backpack to offset the bodyweight loss, with the same treadmill settings, subjects required 1497 ml/min of oxygen. That's a big drop.

Some of this is from the magic of ketones, BHB increasing the free energy of hydrolysis of ATP. A large chunk of this component should have been in place by the one week treadmill test as everyone was well in to ketosis at this time. There was some drop in VO2 needed by this time point, but nowhere like the drop by six weeks.

We can tell by the RQ values that at baseline (mean RQ 0.76) the subjects were oxidising a mixture of fat and carbohydrate (and some protein I guess). At one week there was more fat oxidation occurring (RQ 0.72). By six weeks we have the rather strange RQ of 0.66, lower than is produced even by the oxidation of pure saturated fat.

Oxidation of a fully saturated fat produces an RQ of 0.69. Hmmmmm...

I worked out that oxidising fat, on a simple mathematical basis, should give you a 5% improvement in ATP production per unit oxygen consumed compared with glucose. With Veech's increased Gibbs free energy of ATP hydrolysis while oxidising ketones we gain a combined 5% improvement in ATP supply alongside a decreased total ATP requirement. These are components of the decreased VO2 which didn't make it in to the discussion as they were either unknown (free energy of ATP hydrolysis) or hadn't been worked out (ATP per O2). Not a bad gain for six weeks of fasting. I can see, very clearly, that anyone with a chronic lung disease would be crazy to eat any way other than for a metabolism based on lipid oxidation with ketosis. It just makes sense.

I'll leave it now for this post and put up some ideas about the subnormal RQ figure as the next post.

Peter

Tuesday, April 26, 2016

Dave Asprey and Dr Veech

Dave Asprey has a very interesting extended discussion with Dr Veech on his Bulletproof website, mostly about ketones but also about the history of biochemistry and a number of other subjects. Dr Veech is very pro ketones while being surprisingly anti high fat diets, an interesting combination and clearly far from my own perspective. Much of the interview is simply fascinating in its own right but I'd just like to talk about the aspects with which I disagree. As one must.

Exclusive: Interview with Ketone Expert Dr. Richard Veech

The section of interest is around one hour five minutes in to the discussion. For those who would like some flavour of Dr Veech's uncomfortable stances on a fat based diet, cardiologists and cholesterol you can grind your teeth through the ten minutes leading up to that point. It's pretty obvious that Dr Veech has a lot of reading to do on cholesterol and cardiovascular health, should he so wish.

I had to correct a few relatively minor typos, nothing to do with what was said, just how it got written down, so I've put up the original text followed by my edited version. I've listened to this section three times now and I think my modified transcript is correct:



Original:

Dr. Veech: However, when you’re burning fat, you’re going through beta-oxidation. One reducing equivalent goes to NAD and one goes to pflavo protein. You’ve already lost 1/3 of your ATP in that step. Go back to your lennature Lehninger, beta-oxidation, you do one NADH, you do one NADH, you do one pflavo protein. You do that to keep from blowing the mitochondria up.

Tidied up:

Dr. Veech: However, when you’re burning fat, you’re going through beta-oxidation. One reducing equivalent goes to NAD and one goes to flavo protein. You’ve already lost 1/3 of your ATP in that step. Go back to your Lehninger, beta-oxidation, you do one NADH, you do one flavo protein. You do that to keep from blowing the mitochondria up.




I sold my copy of the biblical Lehninger at the end of my second year at vet school when we finished biochemistry as a subject in its own right. I have to say I really enjoyed the book. I also midnight question-spotted the urea cycle for my biochemistry written exam and it came up. Yeaha!

Sooooooo. Yes, beta oxidation (of saturated fats) does indeed yield one FADH2 which is embedded in a flavoprotein as well as generating an NADH. Electron transporting flavoprotein (ETF), which docks with ETF dehydrogenase, reduces the CoQ couple and omits pumping the four protons which could have been pumped had a second NADH been generated instead. My back-of-an-envelope calculations suggest an NADH could have been generated instead of the FADH2. This does indeed waste a four protons worth of the ATP which might have been generated.

And yes, the FADH2 is generated to stop damage to our mitochondria. So fats are bad?



With some slight discomfort I have to re-cite (yet again) the Protons thread. The whole point of generating inputs which reduce the CoQ couple is to drive reverse electron flow through complex I. Low levels to trigger insulin signalling, high levels to resist insulin signalling. H2O2 is the second messenger.

The waste of proton pumping by supplying FADH2 at the CoQ couple is offset by it being used to regulate the system. This applies to mitochondrial glycerol-3-phosphate dehydrogenase, driven by glycolysis, or ETFdh driven by beta oxidation of saturated fats. Both initially assist insulin signalling and, as substrate throughput increases, they facilitate resistance to the signal from insulin to accept more calories.

Another sooooooo. FADH2 is, I agree, wasteful but more importantly it also drives reverse electron flow through complex I. And undoubtedly you can have too much of a good thing. Waaaay back in the early Protons posts I spent a lot of time looking at FADH2:NADH ratios and decided that somewhere around palmitic or stearic acids there was a maximum healthy FADH2:NADH ratio, somewhere around 0.48. At that time Dr Speijer was kind enough to supply his paper looking at the development of the peroxisome in LECA, the Last Eukaryote Common Ancestor (not to be confused with LUCA, of the hydrothermal vents).

One major function of peroxisomes is to deal with very long chain fatty acids using a beta oxidation version which does not generate FADH2. Problem solved, no need to blow a gasket in our mitochondria. In fact the peroxisomes shorten VLC fatty acids to octanoate, much beloved of Dave Asprey and Dr Veech.

Minor aside: How might you explode your mitochondria if Dr Veech's concerns were correct re FFAs and his idea about FADH2 being used to reduce the supply of pumped protons from complex I? There is a technique to completely flood your mitochondria with NADH. It's called beta-hydroxybutyrate, particularly if supplied in large amounts from ketone esters. This enters the mitochondria using the monocarboxylate transporters, generates NADH as it converts to acetoacetate then goes on to generate three more NADHs from each of the two acetyl-CoA entering the TCA. These NADHs are potentially capable of pumping membrane popping numbers of protons through complex I, without concerning Dr Veech. And without actually doing any damage, that I can tell. End aside.

Next minor aside: Free fatty acids are not going to explode your mitochondria: Under ketogenic high fat eating there is a combination of elevated free fatty acids (happy happy), low insulin (so no loss of FFAs through insulin induced triglyceride formation), plenty of ATP in the mitochondria to allow uncoupling proteins to function (UCPs must have generous ATP on their mitochondrial matrix end to allow them to function) and those ad lib FFAs are necessary and available to actually carry the protons through the uncoupling proteins. FFAs are essential for uncoupling, no FFAs = no uncoupling, pax mtG3Pdh. FFAs = uncoupling = no exploding mitochondria. End second aside.

I'm left here with my view of a healthy metabolism as one based around beta oxidation of saturated fatty acids. Ketones as they happen, no stress. Nothing originating from the discussion has budged my entrenched position in the least, interesting though it has been to listen to.

Peter

Sunday, April 24, 2016

When is a ketogenic diet not a ketogenic diet?

I've listened to a few youtube/podcasts videos recently. Seyfried, Veech and D'Agostino appear to besettled in to what looks like a "Ketone Ester" corner. There are many, many things which make me splutter a little in some of the things they say but I think it's hard to decry ketone bodies too badly. They clearly do things. It's quite possible that the main effect of flooding the TCA with acetyl-CoA is the inhibition of glycolysis. If it does nothing else, that seems worth doing.

The flip side is Ron Rosedale's view, shared by a few others. Towards the end of his presentation, largely about mTOR, he takes a position on ketones. I don't think he has anything against them per se, but what he really wants is a metabolic state based around beta oxidation of fatty acids. Just enough protein, minimal carbs, oxidise fats. If that throws off some ketones, so be it.

I clearly recall Wooo posting some time ago about the ketogenic diet for epilepsy and pointing out that what matters is compliance with the diet, not levels of a given ketone in blood or urine. If fatty acid oxidation dominates I'd be willing to bet the glial cells generate a ton of ketones which enter neurons without a dipstick in sight.

I think I might be in that camp. So I'm a little uncomfortable with medium chain triglycerides, octanoate alone, ketone salts and ketone esters. They clearly have benefits but they are not a route I would take currently.

I like ketones as a surrogate for fatty acid oxidation. Or should I say that I like beta oxidation, and ketones are a reasonable surrogate.

For the beta oxidation "camp" we have this paper:

Induction of ketosis in rats fed low-carbohydrate, high-fat diets depends on the relative abundance of dietary fat and protein.

It's a no nonsense sort of a paper. For the LC rats the diet was beef dripping. With added casein at 5.5%, 11.8% or 19.1% of calories and a few vitamins and minerals. The only carbs came from the vitamin/mineral mix. Anyone could get any rodent diet manufacturer to formulate it:










Rule one of a scientific paper: The methods must supply enough information to replicate the study.

Replication = validation. Without it your paper is worthless.

So rats on a LC diet are only in ketosis when over 90% of calories are supplied as beef dripping:
















A BHB blood level of 28mg/dl is reasonable ketosis. That would be around 3.0mmol/l in new money. That's for the rats on 5.5% casein in their beef fat. All else was ns compared to the chow fed rats. There we have it. Decent ketosis in a rat is reliably achievable by feeding beef tallow. No MCTs, no ketone esters, no octanoate. Anyone could do this with their rodents. One niggle:

For the CICOtards: The LC animals were pair fed to the calories eaten by the chow fed rats, a feature of the paper I dislike a little. It's equal calories all round but the low carb rats probably ate less than their appetite would have dictated. This might have accentuated ketosis (but we'll never know...) and ad lib feeding might have blunted ketosis.

The weight gains themselves are fun:

















Obviously the red squares are the ketogenic animals. There is a table of blood insulin, glucose, FGF-21 and FFAs but, well, we all know what the number have to be so there's not much need to go in to it in any detail. KDs work.

So if you want to know what a ketogenic diet does in a given medical condition, this is the one diet you have to use on a rat model. Maybe use butter instead of beef dripping (I'd prefer this for myself) but it looks like a gold standard to me... Ketones as a spin off of a whole body FFA based metabolism. The metabolic state is what interests me. Replicate at will.


Let's compare this with a ketone ester camp paper. This is the diet:

"...mice received KD-USF, a custom ketogenic diet designed by the authors and produced by Harlan Laboratories, fed ad libitum".

What's it made of? Dunno. Here are the macros:





What sort of fat? Dunno. Just fat. Maybe: Crisco? Fish oil? Canola? Coconut? Mmmm, butter? Replication anyone? If you can't replicate the study how can you tell whether the results were made up or real? Personally, I think the results are absolutely true. I have no doubt. Why? Because this is the level of ketones generated by the diet, where it says KD:

















The ketogenic diet generated, estimated by zooming in on the above chart, something around 0.1-0.2mmol/l of BHB. No one would make up a figure that low, these are honest results. With added ketone esters we get up to almost exactly the level of ketones found in Bielohuby's truly ketogenic tallow fed rats, without the crippling expense of the ketone esters.

People shouldn't get me wrong. I have nothing against trying to use a ketogenic diet for management of cancer. I even think using ketone esters might be reasonable for folks who can't cope with cream, butter, eggs and a wide variety of meats and non starch veggies.

What I would prefer is for a diet described as ketogenic to actually generate some ketones. You cannot describe a diet generating 0.1mmol/l of BHB as ketogenic! Especially when close on 3.0mmol/l is easily achieved on a beef tallow based true ketogenic diet. It's not exactly surprising that adding 3.0mmol/l BHB derived from ketone esters should out performed a non-ketogenic "ketogenic" diet for cancer management! And the non ketogenic high fat diet did help a little, presumably by eliminating starch triggered insulin signalling...

I was a bit shocked that non of this was discussed in the discussion. Being driven by a love of ketone esters is no excuse for sloppy science. When you are on the winning side there is no need for this.

Peter