Insulin detemir (3)

I think it is quite clear how I view insulin detemir. Kindke was unable to resist finding the link to the abstract with the diametrically opposing view. I'll just stick both links in here to keep them together, so people can look at both research findings and draw their own conclusions.

Insulin detemir is not transported across the blood-brain barrier.

versus

Insulin detemir is transported from blood to cerebrospinal fluid and has prolonged central anorectic action relative to NPH insulin.

I think it is reasonable to assume that at least one of these two papers is factually incorrect.

If you search on Begg and Woods as co-authors you will find papers redolent with words like "reward", "hedonic" and "dopamine".  That's Begg and Woods, if anyone can stomach it.

I was, in my normal confirmation biased way, much more interested in the sort of work produced by Banks, Morley and/or Mooradian. These folks appear to be scientists rather than psychiatrists and they have some great publications. They include major work on the blood brain barrier, leptin transport, insulin transport, leptin resistance, gerontology, diabetes, antioxidants, the list goes on and on.

Here are a few little gems I particularly enjoyed in abstract form which might be worth a mention.

I dislike antioxidants. This is quite interesting from Banks and Morley:

Effect of alpha-lipoic acid on memory, oxidation, and lifespan in SAMP8 mice.

Alpha lipoic acid is a mitochondrial component present in normal cells and is available in mega doses as a supplement. It's a serious and deeply mitochondrial penetrative antioxidant. It helps a lot with diabetic neuropathic pain. SAMP8 mice are oddities which have been bred for early onset senility and memory loss. They are used (probably totally inappropriately) for Alzheimers Disease research. Treating them with antioxidants improves their memory performance. You might think this is a good idea. The cost is measured by a shortening of their life as elderly SAMP8 mice from 34 weeks to 20 weeks after start of treatment (started at 11 months of age). This may or may not be a good thing if you are an SAMP8 mouse (death might be a release). How it applies to a person managing their diabetic neuropathy or trying to delay the progression of their Alzheimers Disease is fascinating and slightly worrisome. I'll stick to a life based around beta oxidation, normglycaemia and a little superoxide signalling, stuff the antioxidants. Last sentence of the abstract "These findings are similar to studies using other types of antioxidants". Sweet, provided you avoid sugar. And antioxidants.

The next snippet includes Morley and Mooradian as authors and relates to making your blood sweet,  literally this time, using intravenous glucose:

Mechanism of pain in diabetic peripheral neuropathy. Effect of glucose on pain perception in humans.

Simple hyperglycaemia in a normal person reduces the threshold for feeling pain. It reduces the severity of pain you can tolerate. This applies to a normal human being on a glucose infusion or a diabetic person on a diet designed by a diabetologist, no glucose infusion needed. If hyperglycaemia makes a tolerable stimulus in to a painful experience and makes just bearable pain become unbearable, how many chronic pain syndromes would go in to remission with sustained normoglycaemia? Fat phobia makes this question currently un-answerable. The paper was published in 1984. Does anyone fancy having a gangrenous foot amputated for diabetic complications and waking up on a dextrose saline infusion in recovery? And then being offered the "diabetes diet" on the post op ward?

Banks and Morley were also instrumental in the generation of data for the concept that trigycerides in plasma induce leptin resistance at the blood brain barrier, a few years old now but still quite a useful concept:

Triglycerides induce leptin resistance at the blood-brain barrier.

I find the cream bashing in this last paper a little distasteful and I have to admit that Banks appears to be unaware that high saturated fat low carbohydrate diets are THE way to reduce fasting trigycerides in real people. Can't have everything I suppose. But even if the cream effect applies to people, who cares if I am leptin resistant with a full stomach provided leptin will work perfectly well in the post absorptive (low triglyceride) period? I am a human, not a mouse. I ate a high fat meal without sugar last night, ergo I'm not hungry today. Low trigs equal leptin sensitivity...

I'll call a halt there. Life is full of interesting snippets which make sense. They usually come from the sort of people who say insulin detemir does not cross the blood brain barrier.

Peter

Insulin detemir (2)

Morphine is a rather odd opioid analgesic. It has a complex multi-ring structure with two rather prominent hydroxyl groups which render it rather more hydrophilic and significantly less lipid soluble than many of its relatives. If you bolus a patient with IV morphine there is a delay in its passage across the blood-brain barrier due to this relatively poor lipid solubility. Time to peak effect is significantly delayed to somewhere around 15 minutes because the brain concentration lags way behind the rapidly changing plasma concentration. The brain never "sees" the peak plasma concentration due to this delay.

Now, if you boil some morphine up with acetic acid you can form ester linkages joining acetate on to those two hydroxyl radicals to give you di-acetyl morphine, better known as diamorphine or heroin. Masking the hydroxyl radicals markedly increases the lipid solubility of the drug and so the brain concentration rapidly follows the plasma concentration. In general lipid soluble agents cross the blood brain barrier rather faster than more water soluble agents. Peak plasma concentration will give a rapid onset peak brain concentration, which appears to be associated with effects rarely seen with morphine itself. Giving the enhanced recreational potential. This is all basic anaesthesia pharmacology with excerpts from Trainspotting thrown in.

Insulin detemir was developed to give an insulin with a very flat glycaemia controlling effect for use as a basal or background insulin. The clever people at Novo Nordisk deleted the terminal threonine from the B chain and attached a medium chain fatty acid to the now terminal lysine at position B29. The rather nice 14 carbon saturated fat, myristic acid, sticks out from the insulin molecule and neatly binds to the fatty acid binding site of albumin. It does this very rapidly and keeps the insulin bound and ineffective. Over the hours which follow there is a slow dissociation of the insulin from albumin which allows a very shallow dose response rate for glucose control. Ideal for a basal insulin.

There is a suggestion that this tagging of insulin might facilitate its transport in to the brain, a sort of heroin-insulin tweak. The idea is that myristic acid might facilitate the transport of insulin in to the brain and lead to a massive suppression of eating and subsequent weight loss. Assuming you are a true believer in the central anorectic effect of insulin. Which, sadly, I'm not.

Years ago, when insulin determir was first paraded as the living proof of the central anorectic effect of insulin, I looked up its structure and thought, as you do, that FFAs in general have very limited access to the brain. Insulin is not morphine and the myristic acid is not acetic acid. That big, long side chain of detemir is directly related to the sorts of free fatty acids which are specifically excluded from the brain. My own prediction would be that insulin detemir would have a significantly REDUCED effect within the brain.

It turns out that, at least in some labs, that my idea was slightly correct. But my idea was limited compared to the actual effect. Insulin detemir not only fails to cross the blood brain barrier itself but it also blocks the ability of ordinary human insulin to pass from plasma in to the brain. There is probably a specific insulin transporter which is nicely blockaded by an insulin molecule with the fatty acid tail of detemir sticking out. This paper says it all:

Insulin Detemir is Not Transported Across the Blood-Brain Barrier

Not a lot of mincing of words there.

If we go to labs with an outlook on life which I find comprehensible we can clearly see that physiological doses of insulin, within the brain, augment lipid uptake in to adipocytes, enhance adipocyte sensitivity to insulin, increase lipogenesis and augment fat gain. Largely through the sympathetic nervous system. I can't see how anyone would be surprised by this. Quite why anyone would expect central insulin to do the opposite of what peripheral insulin does at a comparable concentration is beyond me. I enjoyed this paper:

Central insulin action regulates peripheral glucose and fat metabolism in mice

"Moreover, chronic intracerebroventricular insulin treatment of control mice increased fat mass, fat cell size, and adipose tissue lipoprotein lipase expression, indicating that CNS insulin action promotes lipogenesis. These studies demonstrate that central insulin action plays an important role in regulating WAT mass and glucose metabolism via hepatic Stat3 activation".

How clearly does it need to be spelled out? This one is fun too:

Brain insulin controls adipose tissue lipolysis and lipogenesis.

"Here, we show that insulin infused into the mediobasal hypothalamus (MBH) of Sprague-Dawley rats increases WAT lipogenic protein expression, inactivates hormone-sensitive lipase (Hsl), and suppresses lipolysis. Conversely, mice that lack the neuronal insulin receptor exhibit unrestrained lipolysis and decreased de novo lipogenesis in WAT".


If you go looking you can find papers from Oz and Cincinatti which show that insulin detemir DOES cross the blood brain barrier and DOES suppress food intake, far better than neutral insulin does. In their own labs of course.

But I cannot forget that if you transport a researcher out of a Cincinatti psychiatry department and put her in to an industrial insulin lab she cannot get any effect of centrally infused insulin detemir or neutral insulin for that matter. Novo Nordisk cannot demonstrate this marvellous effect of insulin, even their own special insulin, in their own lab. We all know that much of the mindset of obesity research is not particularly effective at producing results which work. How they get the results derived from their ideas in their labs is what fascinates me! You couldn't make stuff up this counter intuitive. Maybe in another post.

Back in the real world we have this:

Insulin detemir results in less weight gain than NPH insulin when used in basal-bolus therapy for type 2 diabetes mellitus, and this advantage increases with baseline body mass index

Insulin detemir causes a small weight loss in morbidly obese patients, those with BMI >35kg/m2. Why? Because it blocks the brain entry of the chronically (and markedly) elevated levels of insulin so common in the morbidly obese. It has limited or zero effect within the brain in its own right. The brain simply loses awareness of the systemic pathologically elevated insulin. If plasma insulin is high enough this sudden loss of insulin's access to the brain can result in a decrease in brain driven, neurologically mediated, forced lipid storage in adipocytes, i.e. a little weight loss.

In the absence of marked hyperinsulinamia, i.e. in less obese type 2 diabetics, insulin detemir causes weight gain because there is less tonically elevated plasma insulin for the central uptake blockade to neutralise. There is no weight loss effect, although gain is undoubtedly blunted.

Insulin detemir is the best indicator I have seen that the central role of physiological concentrations of insulin within the brain is to augment fat storage. This makes sense to me.

I wouldn't ask a psychiatrist to develop an anaesthetic protocol. Or a weight loss protocol!

Peter

Insulin detemir (1)

There is a certain belief structure within obesity research which maintains that the central action of insulin is to limit appetite. Obviously, not everyone agrees with this. I would like to do some wild speculating (any resemblance to real life events is purely accidental) about this paper:

Evaluation of the lack of anorectic effect of intracerebroventricular insulin in rats

The paper is very interesting, partly for what they failed to reproduce but mostly for the affiliations of the authors.

The first thing to say is that, if you work in the pharmaceutical industry, you want drugs which work. Hardcore. It’s no good fudging your results when working in pharmaceutical R&D because you’re going to get caught out as soon as anyone tries to actually use your drug. Which is guaranteed to happen. The drug has GOT to work. Industry has no fudge factor. You might have to lie, evade, obfuscate, misplace computer files and massage data to hide the serious adverse effects of your functional patented drug, but you wouldn’t want to have to do this for a molecule which is ineffective in the first place. Statins are very, very effective. At lowering cholesterol. The fudge factor comes from whether this does any good for any person and what multiple adverse effects the drugs might generate.

So I have respect for the integrity, within certain defined limits, of a drug company R&D team. The managers and PR crowd are another matter altogether. Think Dilbert.

Let’s look at the authors of this paper:

There are three. Jessen is the first author, so probably did the bulk of the work and wrote much of the paper. She works in the department of insulin pharmacology at Novo Nordisk, the company which makes insulin detemir. Bouman is last author so is possibly Jessen's line manager and also works for Novo Nordisk. Insulin detemir is interesting because it is the only insulin ever to have been shown to cause weight loss in any patient group. OK, this is limited to morbidly obese (BMI>35) type two diabetics and the weight loss is very small. But it does happen. Quite amazing really and quite different to any other insulin formulation on the market, all of which reliably cause weight gain. Hence I suspect the project at Novo Nordisk was to find out the hows and whys of this strange effect.

Jessen and Bouman will have started with generous (by academic standards) funding, because the drug industry will work at a potentially rewarding idea in a rather more motivated manner than an academic department. Neither author has any track record of publishing on the central anoretic effect of exogenous insulin. Their job is to get reliable and repeatable results about how insulin detemir is special. In this project they failed to achieve any sort of anorectic effect of insulin detemir, or of any other sort of insulin, within the brain. Mucho problemo.

Clegg is middle author and works in the Department of Psychiatry, University of Cincinnati. She has a vast number of publications, several of which feature the successful anorectic effect of insulin when administered directly in to the brain. In at least one such study she is the lead author.

Jessen has co published with Clegg back in 2001 on a non insulin related subject, presumably before Jessen moved to work for Novo Nordisk. They know each other and have worked together before.


I have this image of two industrial pharmacologists setting out to investigate the CNS effects of their rather promising systemic drug, insulin detemir, comparing it to routine and more obesogenic neutral insulin. They fully expect central insulin to be anorectic because they've read all of the papers. That's their job. They expect insulin detemir to be extra effective. In the first run of experiments using intra cerebral administration they failed to get any effect, of any type of insulin, on food intake. None.

This is big. And bad. EVERYONE in obesity research KNOWS that insulin, within the brain, suppresses appetite (excepting the few people who think this idea is bollocks of course, there are always a few people who think logically).

Jessen and Bouman probably think they have made a mistake somewhere along the line. They know that Clegg can, in academia at least, deliver results that show a suppression of appetite in rats following centrally administered insulin. They call her over from of Cincinatti to trouble shoot their problems.

In a hard nosed, financially driven situation, she can't do it. From the abstract of the study:

“Although we varied rat strain, stereotactic coordinates, formulations of insulin and vehicle, dose, volume, and time of injection, the anorectic effect of intracerebroventricular insulin could not be replicated”.

It seems to me that there are differences between academia and industry. It’s the difference between holding a religious belief in the central anorectic effect of insulin and looking for an effect which might suggest a marketable drug which will actually work to assist weight loss. I would call the latter "The Real World".

Time to discard the idea that centrally acting insulin is an anorectic agent? Kudos to the researchers for publishing.

Peter

Random musings on carbon monoxide

There are occasional days at work when I get a lunch break. Sometimes it is long enough to get home and back, often it’s not. Given an hour of free time in the basement, what might you do?

Well, usually I go and have a look whether Nick Lane's group has any new publications up. Pondering the origins of life is a great relief from the problem solving with limited information that makes up clinical work.

In recent months I’ve usually had three browser windows open to try and get an accurate understanding of what he and his coworkers are specifically saying.

As a preamble to discussing those methanogens and acetogens which do not use cytochromes I would just like to lay it on with a trowel that these strict anaerobic organisms are not “living fossils” or in anyway primitive. They appear to be using a method of carbon fixation which is developed from that of the first prebiotic synthesis. That does not make them simple. If this is the first technique for carbon fixation it has been developing for well over 3 billion years. It’s still used nowadays because it works.

So lets go to Figure 1 sections a and b of An Origin-of-Life Reactor to Simulate Alkaline Hydrothermal Vents to have a look at the energetics of fixing organic carbon:

This shows that hydrogen is unable to reduce CO2 to formaldehyde at pH 7. However, if you introduce a pH difference across a thin FeS barrier, it appears to be possible to put an electron or two on to FeS clusters which can then drive the reaction. At the risk of spoiling the paper, it works. Yield is low, but it's there.

Of much greater interest (to me anyway) is the ability to reduce CO2 to CO. I've added these potentials to Nick Lane's diagram like this:

There is a lower barrier to be overcome and, given the suggested pH gradient, greater likelihood of the reaction proceeding. I think the group are interested in formate and formaldehyde because they have the potential to do many things other than generate acetate/methane plus the reactions from there onwards are exergonic.

I'm more interested in CO formation because the carbon monoxide dehydrogenase (CODH) enzyme appears to be core to carbon fixation and is conserved between archaea and eubacteria, certainly in the methanogens and acetogens which lack cytochromes and are strictly anaerobic in their metabolism. So too is acetyl CoA synthase, which is directly linked to CODH. This suggests that this enzyme complex, or the abiotic reaction it preserves, is very ancient. We need to look at Early bioenergetic evolution, Fig 2 section b for a suggestion of how prebiotic acetate synthesis might have occurred. This is the top section:

If we work down from the CO2 at the top of the dashed line rectangle we can see that CO2 is being reduced by electrons from a very low potential FeS cluster (just where it says +2[H]), generated as in my modification of the blue bar chart above, using the pH gradient across a thin FeS barrier. The FeS cluster on the right is the actual catalytic cluster and the green sphere is a Ni atom crucial to its function.

The CO diffuses to a second FeS catalytic cluster which is doped with two more Ni atoms, shown as two green spheres. This reaction does not need a low potential FeS group, so I'm not sure why the black/yellow cluster is shown to the left of the "Ni" in the diagram, excepting that modern ACS does have an FeS cluster covalently attached to the FeNiS catalytic cluster. Doesn't make the diagram particularly easy to interpret. Anyhow, the CO binds to one of the two Ni atoms and waits for a methyl donor.

The methyl source is on the far left of the diagram, shown as CH3-X. This has to be of abiotic origin in an origin-of-life scenario and the simplest is undoubtedly CH3-SH. This methyl group attaches to the CO on the Ni atom and we then have CH3-CO- attached to the catalytic site. Thiolysis using a source of sulphydryl (shown as HS-R in the diagram, below the CH3CO-Ni, still within the dotted rectangle) generates a thioester of acetate, shown (outside the rectangle) as CH3CO-SR.

We've already speculated the availability of CH3-SH in vent fluids to supply methyl groups, so we could simply replace HS-R with HS-CH3 i.e. CH3-SH can provide both the methyl group and the sulphydryl group for the above reactions. The only step requiring any energy input is the initial reduction of CO2 to CO using a low potential FeS cluster.

So when you supply CO and CH3-SH to a slurry of FeS and NiS you get CH3CO-S-CH3 of abiotic origin. This was done back in 1997 by Huber and Wächtershäuser. The trick of reducing CO2 to CO is the hard knot to pick and which needs FeS and a proton gradient, not available in the slurry.

The thioester can hydrolyse to heat and acetate but more interestingly it carries enough energy to form a phosphate derivative which retains slightly more available energy than ATP.

After translating this in to a system which has developed genes and proteins, we still the have remnants visible.

Let's now look at the lower section of Figure 2 section b. The complex series of reactions down the left is how modern acetogens generate their methyl donor source, down the right is the system from methanogens. These are both replacing the speculated abiotic CH3-SH. These pathways are not particularly relevant to any origin of life speculation but clearly are interesting in their own right as they also support CO2 reduction by H2. There are some interesting speculations as to why tungsten or molybdenum are required cofactors... Today it's still the central rectangle we are interested in:

This is the modern version of what we have been looking at previously, with the two Ni doped FeS clusters on the right hand side. The reduction of CO2 to CO is no longer accomplished using a proton gradient, in fact these archea and eubacteria use a sodium gradient rather than a proton gradient. Instead, a low potential ferrodoxin protein with two FeS clusters is generated in the cytoplasm using electron bifurcation from molecular hydrogen (not shown, discussed here, that's the third window in my browser!). The systems used differ between methanogens and acetogens but the core energy currency for both is still reduced ferredoxin. This ferredoxin looks, to me, like a fossil of the FeS on a proton gradient which provided reducing power in the initial scenario.

The transport of CO to the second FeSNi cluster is down a molecular tunnel in the modern enzyme but the binding to a Ni atom at the end of that tunnel persists. Source of the methyl group is quite different between archaea (methanogens) and eubacteria (acetogens). This differing arrangement suggests development after the divergence of the two lineages.

The use of CH3-SH to form a thioester has been replaced by the use of coenzyme A, giving us the familiar acetyl-CoA (shown as CH3CO-SCoA) below the rectangular box. This can be used for substrate level phosphorylation of ADP to ATP or can be used as a source of cell carbon for metabolism. There is a lot of interest along these lines in this paper: The stepwise evolution of early life driven by energy conservation.

There are some interesting ideas which stem from this believable scenario.

Substrate level phosphorylation is clearly very ancient. There are suggestions from the speculated origins of ATP synthase that ATP usage may have been common before this enzyme complex was developed.

The possibility of generating an ATP-like moiety was present when all that was available was a proton gradient derived reduced FeS cluster and some CH3-SH.

Carbon fixation does not appear, initially, to have involved proton translocation, though the gradient was essential.

Carbon monoxide is utterly intrinsic to the formation of life. Strange, that.

Peter

Unclean

People may have seen this quote on Facebook. Lovely to see Steven Rentaquote Nissen publicly acknowledging that the death toll from cardiovascular medicine's lethal low fat diet has finally been halted by a couple of investigative journalists. Thank you Mr Taubes and Ms Teicholz. Oh, he missed that bit...... Here's the quote:



Steven Nissen, chairman of cardiovascular medicine at the famed Cleveland Clinic: For years, "we got the dietary guidelines wrong. They've been wrong for decades."

Advice to avoid foods high in fat and cholesterol led many Americans to switch to foods high in sugar and carbohydrates, which often had more calories. "We got fatter and fatter," Nissen says. "We got more and more diabetes."

Recent studies even suggest that longtime advice on saturated fat and salt may be wrong, Nissen says.




Personally I feel a little contaminated, unclean, by Nissen's falling in to line with what any sensible person with a laptop and net access realised fourteen years ago. Yeugh. Anyway: I thought I would help out by sketching out his next press release:



Steven Nissen, chairman of cardiovascular medicine at the famed Cleveland Clinic: For years, "we got the cholesterol guidelines wrong. They've been wrong for decades."

Advice to take drugs to lower cholesterol led many Americans to pay for statins which made them diabetic and increased their cancer risk. "We got sicker and sicker,” Nissen says. "We got more and more dementia.”

Recent studies even suggest that longtime advice in favour of statins was a bad as that against saturated fat and salt, Nissen says.


Y'all know it's coming! You saw it here first.

Peter

Ketosis and Protein

I just wanted to throw out a few comments about the inhibition of ketogenesis by protein. The obvious effect, that of stimulating gluconeogenesis, appears to be at best a partial explanation of what happens.

I've long been interested in how amino acids feed into (and are derived from) the citric acid cycle and related pathways. Clearly any amino acid which metabolises to oxaloacetate within the liver is simply going to remove that void in the citric acid cycle (oxaloacetate deficiency) which results in acetyl-CoA being diverted to ketogenesis. Aspartate is one such. Those metabolising to pyruvate are also going to do essentially the same thing. There is no need for increased gluconeogenesis in this scenario. Gluconeogenesis may happen at a increased rate. It may not. Providing a source of oxaloacetate in the liver mitochondria will stop ketogenesis, whatever gluconeogenesis does, whatever insulin does.

Personally I am very ketoadapted. I've drifted in and out of ketosis since just after that start of the current century, probably around the summer of 2001 if I recall correctly. I find carbohydrate restriction effortless. Limiting to 30-40 grams per day is easy. Protein limitation is much more difficult. With about 20grams of protein in each breakfast and a few grams derived from cream, chocolate or macadamias at lunch time this does not leave a huge allowance for meat intake at suppertime. At around 65kg bodyweight nowadays keeping to 1g/kg is not the easiest target. A decent steak and I miss it. Life is too short to stress about this, but I certainly don't eat steak every day. Urinary ketones are always there at the + or ++ level. Exercise (distance walking) usually gives +++ as does the evening meal post prandial period, unless there have been excess chips with supper or I've gone significantly over my protein limit.

So I've limited protein, mildly, for years. My degree of keto adaptation still allows free generation of ketone bodies, certainly to a level where I can detect acetoacetate in my urine.

For some reason the concept of amino acids being derivatives of (and inputs towards) the TCA pulls me back to Nick Lane's ideas, the origin of life and the throwing together of metabolism. I'm willing to buy the reduction of CO2 by H2 to give formate as the starting point of metabolism. There is an energetic cost to this initial step but once going it's all down hill, energetically, to pyruvate. Many amino acids are formed from pyruvate and close derivatives. This makes sense. Evolution doesn't plan but does progress within the framework of what is available.

In modern biology DNA doesn't do very much other than replicate (I simplify). RNA is much more active, it carries the message out, assembles itself in to ribosomes and does all of the picking and choosing of amino acids etc to make a protein. I like to think of DNA as a rather stable "hard copy" of the information which was originally carried by the less stable RNA. As such DNA is the fossilisation of the amino acid preferences of primordial RNA. Written in to DNA are the remnants of what was probably a chemical associations of RNA with specific amino acids. If DNA specifies a cytosine at the start of a triplet then the amino acid chosen via transport RNA will be derived from alpha-ketoglutarate, if an adenine the coded amino acid will be oxaloacetate derived, if thymine it comes from pyruvate and if guanine the amino acid will be derived from any one of several possible small molecules. The second base specifies how hydrophobic/hydrophilic the chosen amino acid might be and these two cover a high proportion of the biological amino acids. The third base is degenerate, i.e. it doesn’t carry any specific information but does allow a wider pool of amino acids to be selected.

I'm afraid this is all rather cool to me.

I love these glimpses in to the early mish-mash of chemicals and how they might have interacted before life became seriously organised. What you can and cannot say about LUCA, the last universal common ancestor, and the first steps away from prebiotic chemistry, is largely determined by such biochemical fossils.

All of this random musing came from wondering whether amino acids might supply oxaloacetate and so suppress ketosis. Some do.

Well. Eating a steak is not very ketogenic. It’s hard to separate this from the origins of metabolism and of life, for me anyway,

Peter

Shewanella and electrons

Have we found alien life? This interview did the rounds via Facebook recently. There is only one answer.

No.

We have found bacteria that can run their electron transport chain using an electrical connection to an external electron acceptor. In more ordinary bacteria electrons travel down the ETC to complex IV where, under oxidative metabolism conditions, they are passed to oxygen as the terminal acceptor, still within the cell.

Bacteria are the most sophisticated metabolists on Earth. To stick a series of cytochromes together to form a wire, from the end of an electron transport chain to an extracellular acceptor of electrons, is no big deal. Once a bacterium is using such a wire (which will, undoubtedly, be using quantum tunnelling effects much as the FeS clusters of complex I do) to access extra cellular terminal acceptors it is absolutely no problem to make that terminal acceptor anything with an appropriate redox potential.

Under these conditions a cathode electrode will allow the metabolism of NADH from the cytosol, via complex I and the CoQ couple down the ETC and out along the cytochrome wire. A cathode electrode, whatever it is made of, is an electron acceptor. That's the definition.

So growing bugs on a cathode is utterly unremarkable. NADH comes from "food". Electrons from NADH drive the ETC to make ATP and are dumped out-of-cell on to the cathode.

Where things get slightly interesting is that you can also apply a negative voltage to the end of the wire, making it an anode, a net supplier of electrons. Certain types of bacteria can function under these conditions without any carbon input. From the article:

“A lot of organisms that can put electrons onto an electrode can also do the opposite and take electrons from one”—though not at the same time—Rowe says. That ability to reverse course surprises me, and Rowe, too. “I’d think it would be really hard on the organisms. You’re basically stealing energy from them. But they do okay.”

This is incorrect, it lacks perception.

It is perfectly possible to run the ETC in reverse. Bacteria do this on occasions, for reasons best known to themselves, especially if their normal metabolic substrates cannot generate NADH directly. Usually ATP is used to drive ATP synthase in reverse to maintain the membrane potential. Electrons flow in reverse to reduce NAD+ to NADH which can then be used for anabolism. All that Shewanella spp need to do is to use the negative voltage of an external electrode to facilitate reverse electron flow and they can be generating NADH for anabolism without any carbon source. About a third of a volt does the trick, from the interview.

Whether this process would allow the fixation of atmospheric carbon dioxide to actually allow growth is irrelevant. What matters is that there is absolutely nothing about these bacteria living on "pure electricity" which suggests anything other than a clever piece of wiring added on to the end of a  fairly normal ETC.

This is what Shewanella oneidensis looks like with its complex I and CoQ couple:

A clever bacterium? Yes, with very busy periplasm. But anyone who thinks that any bacteria are simple is stupid. What if you think that these particular ones are alien life forms?

"Kenneth Nealson is looking awfully sane for a man who’s basically just told me that he has a colony of aliens incubating in his laboratory".

It is only an alien life form if aliens developed the same electron transport chain as any Earthly organism which uses oxidative phosphorylation to chemical acceptors.

How much does Nealson know?

Peter

GSD IIIa, ketones, MAD and Veech again

I mentioned the high protein/exogenous ketone approach to Glycogen Storage Disease IIIa in a recent post. It's very nice that an effective treatment can actually be achieved through a Modified Atkins Diet (MAD at <10g of carbs per day) involving food, w/o faking ketosis though those exogenous synthetic ketones.

Robert gave me the heads up on the latest paper on GSD IIIa using MAD, available as a free text through Pubmed. It's rather good as, again, it shows that there are medics out there who think matters through and occasionally come to correct conclusions. I love the clinical details of compliance/non compliance too. And that the early hypoglycaemia, treated with corn starch (bleugh), was asymptomatic under ketosis.

That's nice.

He also sent me the full text of Veech's

The Determination of the Redox States and Phosphorylation Potential in Living Tissues and Their Relationship to Metabolic Control of Disease Phenotypes

which is a fascinating personal insight in to what it was like to work in Hans Krebs' lab, combined with the sort of hard core math which implies rather more understanding of biochemistry than simply adding MitoSOX red to some cells and looking for colour changes to show oxidative stress. That's some complex reading to work through when clinical and home life combine to give me a chance!

Peter

Palmitic acid and hyperglycaemia in diabetic heart failure (1)

I've been gifted a paper of great interest. The authors of the paper are very good in their explicit discussions of the limitations of their models, the reasons for the choices they made and the limitations of all current models and probes. This is good and, needless to say, there are an awful lot of "ifs, buts and maybes" to the data! On the down side the paper is an epub of the crudest type, retaining numbered lines, the figures at the very end of the whole paper and all of the captions in a lump between the references and the figures. So not easy reading for a very complex paper, with very complex figures and very complex captions. I spent a large amount of time on this paper over the Christmas vacation but never hit post on any of it. Here is an introduction.

Over the years we have seen, largely via Veech, the ability of ketone bodies alone to rescue myocardial function to give mechanical work performance comparable to the combination of insulin with a relatively normal level of glucose in the perfusion of isolated rat hearts. Equally there is the concept that ketones essentially bypass insulin resistance to rescue metabolism.

The hearts in Veech's study were perfused with oxygenated buffer which was devoid of the free fatty acids which are a normal metabolic substrate for heart muscle.

As a lipophile I have long wondered whether, outside of the neurons, palmitic acid might be a reasonable substitute for ketones in rescuing muscle metabolism damaged by hyperglycaemia.

From the start of the discussion section of Bhatt's paper:

"We investigated the role of substrate-driven redox status on the contractile/energetic performance of heart trabeculae from the T2DM ZDF rat. The main findings are: i) HG exerts a detrimental action on contractility of T2DM heart trabeculae that Palm is able to rescue; ii) Palm prevents oxidative stress exacerbated by HG, an effect independent from insulin action; iii) insulin appears to worsen the negative effect of HG through higher oxidative stress and lower GSH; iv) ZDF heart mitochondria emit less ROS and display higher ROS scavenging capacity of the GSH/Trx antioxidant systems; v) cardiac redox balance in HG appears to play a causal rather than correlative role in the preservation of contractile performance in ZDF trabeculae"

Palmitic acid will rescue hyperglycaemia induced myocardial contractility failure. OK, I'm happy.

This was the core finding in the paper. There are a whole slew of spin off concepts which grow from it but if I wait until I have them all sorted out it might be a very long time before I get another post out!

But here we have it. Hyperglycaemia is bad. Palmitic acid rescues hyperglycaemia induced dysfunction. A paradigm shift in glucose vs FFAs!

Peter

ACCORD and musings on insulin

There are a couple of things I would just like to mention in passing. Jenny Ruhl has just posted a nice entry about ACCORD. This is very important. Lowering your blood glucose is significantly protective against CVD events. This is the exact opposite of the initial analysis of the results where a flawed interpretation of the data led to vociferous suggestions that lowering the HbA1c of diabetics might be actually dangerous. Hopefully this reanalysis will put an end to such stupid ideas which are still dangerously prevalent today.

Before Jenny posted the above I had been thinking about both metformin and insulin for the management of diabetes. I have posted on insulin, which is probably the ideal drug for diabetes management provided it is combined with low carbohydrate eating, in the past but it bears reiterating.

This is my opinion. If you can control your diabetes with metformin and wish to eat lots of carbohydrate, by all means get on with it, that's your choice. Not checking or not worrying about your blood glucose excursions might be a mistake. What you mean by good control might not involve HbA1c values in the 5% region or below.

If you need insulin to control your blood glucose, you have no choice. It's low carb. Live with it.

Why?

Some researchers (who write third rate papers using a rather inappropriate pancreatectomised dog model) are now starting to wake up to the fact that the pancreas secretes insulin in to the portal vein, not the subcutaneous tissues. In normal individuals insulin/glucose arrives at the liver via the portal vein and insulin facilitates transport of the glucose in to the liver, being metabolised in the process. Relatively little post pranial insulin or glucose penetrates to the peripheral circulation in a normal individual.

EDIT: Gretchen has kindly pointed out that the liver uptakes glucose through GLUT2, not GLUT4. The function of insulin, acting on its receptor (facilitating its degradation), is to suppress gluconeogenesis and hepatic glucose output. This is the correct function of the elevated portal insulin level. It makes no difference to the issues with peripheral vs portal insulin, but the correction is welcome. END EDIT

Once a person needs insulin to control their blood sugar levels they inject it subcutaneously. This will invariably elevate the systemic concentration of insulin. It will only modestly elevate the portal vein level. This is very important.

In the cited paper the dogs get a meal with 50% of calories from carbohydrate, 30% from fat and 20% from protein. In control dogs, instrumented but not pancreatectomised, the portal vein insulin after the meal is, at certain time points, ten times the peripheral systemic concentration. This is what is needed to allow the liver deal with the glucose load from the meal while simultaneously protecting the body from both hyperglycaemia and hyperinsulinaemia.

Subcutaneous insulin will make the rest of the body do the liver's job of clearing post prandial glucose, the liver can't manage because it never sees the requisite ten times the normal peripheral insulin concentration needed to deal effectively with the portal glucose load.

What happens when you use the rest of the body as a glucose sump? From the paper:

"Peripheral hyperinsulinemia is associated not only with increased risk of hypoglycemia, but also an increase in catechol and cortisol secretion and lipolysis [14], deleterious effects on vessel walls [6], ischemic heart disease, hypertension and hyperlipidemia [8], and abnormalities in hemostasis [10]"

As always, I would add that it's the obese, blind, legless person in the queue for dialysis who pays the bill for eating the carbohydrate.

This is the situation for all type I diabetics and the more advanced type 2 diabetics. The only route round it is to use intra peritoneal insulin which is, in part, absorbed through the mesenteric veins so is partially portal vein selective. There are, needless to say, a stack of complications to intra peritoneal insulin infusion. Tight control of glucose using subcutaneous insulin from a blood glucose controlled pump is no solution. Though glycaemia is better controlled it is still at the cost of too little insulin in the portal vein and too much in the periphery, using the body as a glucose sump. Over the years I have never been quite able to decide whether hyperinsulinaemia or hyperglycaemia is the primary factor which kills nerves and kidneys. It's a difficult call. And a fascinating discussion in its own right.

What happens if you eat a diet very low in starch?

Very little insulin is ever secreted by the pancreas, especially as glucokinase down regulates. Very little glucose ever needs to be taken up by the liver. Very little insulin will be metabolised by the liver. The insulin gradient between the portal vein and the systemic circulation will be as low as you can practically get it. If someone still needs to inject insulin alongside a very low carbohydrate diet, and many might not, injecting a very small amount subcutaneously will deliver an arterial concentration to the gut, pancreas and eventually to the portal vein and liver which is still quite close to what the portal vein might have supplied. If the body is not using insulin the tissues will not extract it, so portal and systemic concentrations will converge. Everything pans out at some near basal level.

A very low carbohydrate diet is not perfect for insulin dependent diabetics but it is streets ahead of anything else. What people do or do not consider a "normal" human diet will not get around this. Need exogenous insulin? You are not in a position to eat ancestral starch. It's a simple matter of anatomy, physiology and biochemistry.

Peter

The P479L gene for CPT-1a and fatty acid oxidation

In order to work out what is happening with a given child having an episode of hypoglycaemia as a result of having the P479L version of CPT-1a, we need some information.

My thanks to Mike Eades for the full text of the paper on the Canadian Inuit, which does include a certain amount of useful clinical data.

Here is the snippet about a young girl having a hypoglycaemic episode while hospitalised:

“Plasma free fatty acid was 3.8 mmol/L and plasma 3-hydroxybutyrate was 0.5 mmol/L”

Blood glucose was 1.9 mmol/l at the time. An FFA level of 3,800 micromol/l is impressively high. She was generating a small amount of ketones.

No one would argue with intravenous glucose at this point, the question is about how she got here.

So. The problem here does not (as I'd initially thought) appear to insulin induced suppression of FFAs to a level at which beta oxidation fails to support metabolism. FFAs are very high, even for an P479L person after a short fast. With ketones starting to be produced (and low blood glucose) I feel it is reasonable to assume that her liver glycogen is depleted and, while some fatty acids are entering the hepatocytes, not enough of them are being oxidised to support ketogenesis. Glycogen is being depleted to keep liver cells functional. Gluconeogenesis from protein is unable to meet the hepatic (and whole body) demand for glucose calories in the situation of limited access to FFA calories.

However much glycogen derived glucose you consider that the ancestral diet contained I feel it is very, very unlikely to be greater than the glucose and fructose of a modern diet. I feel that getting enough glycogen in to the liver to fully fuel its metabolism in the absence of adequate fatty acid oxidation is a non starter. The P479L mutation was not "permitted" by high oral carb loading, it was permitted by conditions which facilitated fatty acid oxidation. You don't have to agree.


What starts to look much more interesting is what controls CPT-1a activity and how this might vary from the ancestral diet to the modern diet.

The paper makes the point that omega 3 fatty acids appear to up regulate fatty acid oxidation (in rats at least) by the liver. If this is true in humans then a high level of omega 3 fatty acids from marine fats might up regulate fatty acid oxidation to a level which no longer necessitates the depletion of hepatic glycogen derived form oral glucose intake or protein catabolism.

In support of this is that the distribution of P479L within Alaska is not uniform, it's significantly commoner in the coastal regions compared to the inland areas.

"The allele frequency and rate of homozygosity for the CPT-1a P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the Inuit or Inuvialuit in the two western territories"

I'm open to other explanations, there are papers suggesting that the mutation helps to preferentially dispose of omega 6 PUFA, with omega 3 fatty acids as the facilitator.

In summary: Maintaining adequate FFA oxidation to avoid glycogen depletion looks to be the core need in P479L. A high fat diet with a large proportion of omega 3 fats might be a plausible way of maintaining adequate hepatic fatty acid oxidation. Hyperglycaemia (via Crabtree effect) looks to be anathema. Glycogen loading with a normal starch/sugar based modern diet is clearly ineffective to prevent hypoglycaemia for some individuals. Resistant starch as a reliable nightly adjunct to infant feeding seems very unlikely in the ancestral diet. Repeated periods of fasting were probably routine when hunting was poor and does not appear to have selected against P479L in weaned children. Unweaned children are unlikely to be exposed to fasting, provided milk was available from lactation.

Well, there are some more thoughts on the biochemistry.

People clearly have very differing ideas of what the Inuit did or did not eat as an ancestral diet. The P479L gene eliminates the need for source of dietary glucose to explain very limited levels of ketosis recorded in the Inuit. While it is perfectly possible to invoke a high protein diet to explain a lack of ketosis in the fed state this goes nowhere towards explaining the limited ketosis of fasting. P479L fits perfectly well as an explanation.

I have some level of discomfort with using the Inuit as poster people for a ketogenic diet. That's fine. They may well have eaten what would be a ketogenic diet for many of us, but they certainly did not develop high levels of ketones when they carried the P479L gene.

However. Over the months Wooo and I seem to have come to some sort of conclusion that, while systemic ketones are a useful adjunct, a ketogenic diet is essentially a fatty acid based diet with minimal glucose excursions and maximal beta oxidation. Exactly how important the ketones themselves are is not quite so clear cut. From the Hyperlipid and Protons perspective I would be looking to maximise input to the electron transport chain as FADH2 at electron-transferring-flavoprotein dehydrogenase and minimise NADH input at complex I. Ketones do not do this. Ketones input at complex II, much as beta oxidation inputs at ETFdh, but ketones also generate large amounts of NADH in the process of turning the TCA from acetyl-CoA to get to complex II, which ETFdh does not. I'm not a great lover of increasing the ratio of NADH to NAD+. These are my biases.

Confirming that the Inuit are not poster boys for ketosis is a "so what?" moment for me. Using their P479L mutation to argue against ketogenic diets is more of a problem. It's a massive dis-service to any one of the many, many people out there who are eating their way in to metabolic syndrome to suggest that a ketogenic diet is a Bad Thing because no one has lived in ketosis before. Even the Inuit didn't! My own feeling is that everyone comes from stock who occasionally practiced and survived intermittent fasting so we are should be adapted to this. I'd guess that if you are of Siberian, Inuit or First Nations extraction you might benefit from Jay Wortman's oolichan oil as part of a ketogenic diet.

I'm always amazed by the concept that a ketogenic diet might be temporarily therapeutic but must be discontinued because it eventually becomes Bad For You. It reminds me so much of the converse concept that low fat diets, which might worsen every marker of health which people may care to look at, will deliver major benefits at some mythical future date.

Ultimately, point scoring on the internet about what the Inuit did or didn't eat shouldn't destroy people's chances of health. Destroying a circular argument about Inuit diets may may the destructor feel good. Destroying the feet, eyes and kidneys of a person with type 2 diabetes, who need a ketogenic diet, as a spin off from that victory must be difficult to live with. I don't know how anyone can do this.

I think that's probably all I have to say for now.

Peter

Coconuts and Cornstarch in the Arctic?

EDIT There is a follow on post to this one including some clinical data on the hypoglycaemia episodes. I'll put a link in here now it's up. END EDIT

Remi and Ken both pointed me toward this paper:

A Selective Sweep on a Deleterious Mutation in CPT1A in Arctic Populations

The paper itself is largely an account of the detective work involved in pinning down a specific mutation which has been positively selected for in a Siberian population living in the Arctic. The same mutation is also present in non related groups inhabiting the Arctic areas of northern America. The mutated gene is very common and frequently homozygous. It puts a leucine in the place of a proline in CPT-1a, the core enzyme for getting long chain fatty acids in to mitochondria. Putting a leucine where there should be a proline means the protein is basically f*cked. The mutation is linked, not surprisingly, to failure to generate ketones in infancy and can be associated with profound hypoglycaemia, potentially causing sudden death.

From the evolutionary point of view we have here a mutation which is significantly lethal at well below reproductive age, so it should have been weeded out because affected individuals are less likely to live long enough to pass on the gene. But it has been highly positively selected for in several populations, the common factors being cold climate and minimal access to dietary carbohydrate. It's a paradox.

Following a link in the paper gives us this abstract, with this snippet:

"Investigation of seven patients from three families suspected of a fatty acid oxidation defect showed mean CPT-I enzyme activity of 5.9 ± 4.9 percent of normal controls"

A value 6% with an SD of 5% suggests to me that some of these people may well have a CPT-1a function very close to zero. How common is the mutation?

"We screened 422 consecutive newborns from the region of one of the Inuit families for this variant; 294 were homozygous, 103 heterozygous, and only 25 homozygous normal; thus the frequency of this variant allele is 0.81"

I think "very common" is a reasonable description.

How dangerous is it?

"Three of the seven patients and two cousins had hypoketotic hypoglycemia attributable to CPT-Ia deficiency"

Quite dangerous.

The next thing we can do is google CPT-1a deficiency and have a look what needs to be done to stay alive if you carry this gene.


Clearly, if you can't transport LCFAs in to your mitochondria, you should run your metabolism on glucose/pyruvate and avoid the dysfunctional fatty acid transporter. This means raw corn starch, as we have seen used (probably wrongly) for glycogen storage diseases. Properly cooked starches are too short acting to reliably keep a child alive all through the night. They aren't safe enough.

Of course MCT oils have a role too. A CPT-1a defect has no effect on MCT metabolism so these can be used either directly by tissues or indirectly via liver/glial produced ketones.

LCFAs, unable to be metabolised, accumulate in the tissues as a storage disease. The advice is to avoid them as far as possible.

So the archetypical CPT-1a defect tolerant environment would seem to be a person sitting on a South Seas Island beach by a pile of coconuts chewing on a raw yam, with copious flatus night and day.

But it's not.

The CPT-1a defect evolved in multiple non related populations where both starch and MCT were very notable by their near-complete absence. It's an Arctic selected gene. No starches. No coconuts.



Let's take a speculative look at what is going on.

Living on a very low carbohydrate diet is associated with chronically elevated free fatty acids, chronically low levels of insulin and an ignorance of glucose. i.e. the body ignores glucose. Synthesise what glucose is needed but, beyond that, who cares?

Living in a sea of free fatty acids, which are taken up in to cells in a largely concentration dependent manner, allows an increased gradient to push FFA-CoA at any residual function in CPT-1a. It would appear, from the evolutionary perspective of Arctic inhabitants, that near ketogenic levels of FFAs are adequate even if you have the proline to leucine substitution at amino acid 479 in CPT-1a. You can do enough beta oxidation to cope.

Of course, the minute you lower free fatty acids, perhaps to the level of a post prandial starchivore, beta oxidation is going to grind to a halt without the concentration gradient effect. This is pathological. The temporary fix of substrate level ATP synthesis and related pyruvate supply to the mitochondria is fine for a while, but any reactive hypoglycaemia is going to be potentially fatal, especially if you are asleep or food deprived at the time. We know that insulin suppresses lipolysis at levels which don't budge GLUT4s. When insulin has suppressed lipolysis and blood glucose is low, FFAs might be fatally limited.

If you have the mutation but you never do the starchivore thing your FFAs are high 24/7, whether you have just chewed on a lump of seal blubber or not. No paper in the reference list appears to have looked at the FFA levels of children with this mutation on a mixed diet, let alone on the ancestral fat based diet of the polar regions. Given sustained very high levels of FFAs, you might even make some ketones.

If free fatty acids are high and there is no insulin to divert them in to storage, all of the nasty storage diseases associated with CPT-1a dysfunction might well disappear. This is the situation where the mutation allows carriers to thrive.

I think elevated free fatty acids, without elevated insulin, is a recipe for the tolerance of this mutation.




But the mutation is not just tolerated. This is no neutral mutation, it is positively advantageous. The prevalence of the mutated gene is far from random. Why is it beneficial?

This is not quite so simple.

Uncoupling is one component. Uncoupling respiration generates heat. There might just be a positive advantage to running your metabolism fairly uncoupled in a very low temperature environment. Elevated FFAs are completely essential to uncoupling and heat generation. Limiting fatty acid removal from the cytoplasm to the mitochondria might be a facilitator of uncoupling. It's FFAs on the cytosolic side of UCPs which facilitate proton translocation. Having a higher level of cytoplasmic FFAs at a given level of plasma FFAs might give an advantage over the normal level of uncoupling seen under near ketogenic diet conditions.

The second possibility is that, once you have established high enough levels of FFAs to push through the CPT-1a bottle neck, you simply run at this level flat out, all the time. One of the features of the CPT-1a from the modified gene is that it fails to be inhibited by malonyl-CoA.  Even with limited CPT-1a activity there must be times at which ATP synthesis exceeds metabolic requirements and fatty acid transport ought to slow. There is no longer any brake to be applied to FFA transport if excess acetyl-CoA, exported to form malonyl-CoA in the cytoplasm, fails to inhibit CPT-1a . Oversupply of ATP within the matrix is likely to provide optimal uncoupling conditions, in excess of those from a ketogenic diet with regulated fatty acid uptake. That would be my guess. If it's cold enough, this might make the difference between survival or not. It keeps you warm, especially when you are asleep and the TCA should be quiescent.

Flicking through other references in the paper it does appear that indigenous Siberian people do have an elevated resting metabolic rate. In fat free mass it is 17% above calculated values i.e. they are uncoupled.

Finally, adults are not affected by the hypoglycaemia syndrome. My presumption is that, after puberty, they are sufficiently insulin resistant to have adequate FFAs present to maintain relatively normal mitochondrial function. It's the children who need their ancestral diet.



People with glycogen storage diseases die of hypoglycaemia (amongst other problems). We know that a deeply ketogenic diet both protects from hypoglycaemia and sets the body up to run perfectly well without any dietary glucose, which might be lost to glycogen stored permanently in the liver/muscles. There is every justification for giving the finger to cornstarch here and the folks suggesting a modification of ketogenic eating appear to be on fairly safe biochemical ground.

For the P497L mutation everything from the evolutionary perspective suggest that a very high FFA inducing diet may be equally efficacious. But the risks associated with failure, from the occasional safe starch meal or unsafe birthday cake at a party, carries the potential for catastrophe once insulin puts free fatty acids in to free fall.

Peter

BTW: You just have to wonder if any other CPT-1 mutations might behave in a similar manner to the P497L change in the Arctic... Could it be bye-bye time for cornstarch?

Are ketone esters dangerous?

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

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

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

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

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

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

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

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


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

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

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

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

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

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

Peter

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

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

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

Where has the superoxide gone?

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

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

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

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

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

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

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

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

But is it real?

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

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

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

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

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

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

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

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

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

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

Peter

The Crabtree Effect and superoxide in diabetes

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

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

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

Interesting.

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

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


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

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

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

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

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

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

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

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

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

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

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

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

Peter

Uncoupling control in defence of FFAs

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

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

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

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

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

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

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

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

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

So we have this picture from this very impressive study:

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

"Moreover, residues R79 and R279 correspond to the arginines involved in nucleotide binding and protein inhibition in UCP1. According to the three-step binding model proposed for UCP1,17 β-phosphate of PN [phospho-nucleotide] binds first to R182 (helix IV, loose binding). The second step is the binding of γ-phosphate to R83 after protonation of E190 (tight binding). After the subsequent binding of α-phosphate to R276 (helix VI) the protein switches to the inhibited conformation"

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

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

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

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

It's pretty logical.

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

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

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

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

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

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

Peter

Ketones for ALS?

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

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

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

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

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

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

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

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

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

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

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

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

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

As the paper on the Deanna protocol reports:

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

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

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




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

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

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

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

Peter