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