Protons (30) Uncoupling and metabolic rate in insulin resistance

I wanted to look at insulin resistance, uncoupling and metabolic rate. If we just review the effect of an intravenous bolus of palmitic acid on an anaesthetised rat we can see that the bolus produces a period of increased oxygen consumption, ie an increased metabolic rate, in a then-uncoupled healthy rat. From Curi again:

This next graphic is looking at glucose oxidation, rather than oxygen consumption, from the same paper.

The left hand graph shows what happens under basal metabolism, ie glucose is being taken up by isolated muscle cells without help from insulin facilitated GLUT4 translocation. Fatty acids uncouple, ATP levels fall, there is an increase in glucose oxidation to compensate, ATP levels are corrected. Presumably some of the fatty acids get metabolised too.

On the right is the effect under supra maximal insulin. We have no idea of dose response curve to insulin from this study, it describes either using none or using 10mU/ml. That's mU, not microU! At a concentration of 10mU/ml insulin will overcome any suggestion of insulin resistance, short of a knockout model. Insulin supports glucose oxidation, uncoupling increases this. The set up is not designed to look at insulin resistance as might be related to that uncoupling. But the effects on overall metabolic rate, at 5.6mmol of glucose, are quite clear cut.



What if that elevated FFA level was maintained long term?

As adipocytes become progressively more resistant to the the anti-lipolytic effect of insulin (why is another whole ball game), plasma free fatty acids rise even under levels of insulin which should be suppressing them. Unless these free fatty acids are converted to CoA derivatives they are will uncouple respiration. This should reduce delta psi and increase metabolic rate.

A reduced delta psi will not support reverse electron flow through complex I. The essential insulin induced pulse of superoxide, converted to H2O2, will not occur. There will be fasting insulin resistance. This paper spelled it out. In brief:

The insulin signalling cascade is tonically restrained by a phosphatase which deactivates the insulin/receptor complex (which activates itself by auto-phosphorylation) as soon as the process tries to get started. For insulin to signal you need to have an elevated delta psi which allows a nanomolar pulse of H2O2 to cripple this phosphatase and so allow the insulin/receptor complex to get signalling.

With reduced delta psi this isn't going to happen. We have enhanced insulin resistance of starvation until the time when food arrives.

You can increase delta psi, of course, to get insulin working. Increasing delta psi requires increased electrons in to the respiratory chain through complex I. In the blunted insulin signalling situation of low delta psi we can't use GLUT4 transporters but we can, given high enough plasma glucose levels, get sufficient glucose in to the cell to generate enough of a delta psi to allow the pulse of H2O2 and its downstream effects to occur. So overcome the inability of insulin to signal in the presence of FFAs.

The cost is that an elevated blood glucose level is needed. This is what we look at with the ratio of fasting glucose to fasting insulin, the HOMA score. An elevated HOMA score is a marker of UNCOUPLING at the delta psi level in mitochondria. It should be associated with an increased metabolic rate in proportion to the degree of fatty acid induced uncoupling.

Does this happen in real life? These data are from the Pima but the pattern is generic in insulin resistant states:

Note first that the changes in FFA concentrations are statistically non significant, but that the trend is nicely upwards with insulin levels

The authors comment:

"Alternatively, FFA may contribute to increased RMR via stimulation of mitochondrial uncoupling proteins (UCPs) (53,54). As early as 1976, Himms-Hagen (53) suggested that FFAs stimulate UCP-1. More recently, elevated FFA concentrations were reported to stimulate UCP-3 expression in rats (54)"

Bear in mind that as people progress from NGT through IGT to diabetes there are not only progressive changes in blood lipid and glucose levels but also progressive damage to mitochondria per se, which makes comparing a healthy rat to a metabolically challenged human being slightly dubious.

But the changes make sense.

If we look at the post prandial situation we have, after a carbohydrate containing meal, the combination of chronically elevated FFAs with acutely elevated glucose. There will be a high delta psi as soon as blood glucose rises high enough (supra physiological) to allow non-GLUT4 uptake to elevate delta psi high enough for insulin signalling. At this time point the mitochondria allow insulin to function. We can now translocate GLUT4s to the cell surface and start to lower postprandial hyperglycaemia by pouring metabolites through glycolysis or in to glycogen stores.


At the same time, among insulin's many diverse functions, the activation of free fatty acids to their CoA form increases, with a view to anabolic or storage functions. I would presume a different CoA ligase directs the activated fatty acids to oxidation. There is quite a group of LCFA CoA ligases.


At this point we lose the freedom of free fatty acids, they are ligated to CoA and suddenly become effective inhibitors of uncoupling rather than facilitators. We are now set up for the post prandial state. As soon as we have access to glycolysis combined with beta oxidation we have the possibility to generate marked reverse electron flow through complex I and re-inhibit the action of insulin using much greater generation of H2O2 than is needed for insulin's initial activation.

Metabolically, the mitochondria do this at this time to stop caloric overload in any individual cell, by diverting excess calories to storage in adipocytes. From the NADH:FADH2 ratio, long chain saturated fats do this best. Monounsaturates appear to be designed to allow a normal combination of glucose and fatty acid oxidation and PUFA fail to generate adequate insulin resistance to protect an individual cell (including adipocytes) from an overload of metabolic substrate. In the immediate post prandial state saturated fats can best protect cells from an absorptive excess of metabolites. These fatty acids are already present in supraphysiological levels, whatever has been eaten.

The elevated insulin needed to maintain post prandial normoglycaemia will, if the adipocytes are able to respond at all, divert fat from the circulation and into those adipocytes. This is the simple ability of insulin, and cellular resistance to insulin, to limit metabolic injury when calories are present in excess of immediate needs.

                                                    *****

Under inappropriately elevated FFAs there is an initial failure of insulin's action due to depressed delta psi during fasting and a subsequent post-meal failure of insulin's action due to inappropriately elevated fatty acid metabolism through electron transport flavoprotein dehydrogenase's FADH2 when combined with high delta psi.

To me, this looks very much like impaired metabolic flexibility, reduced to the level of delta psi and superoxide. I like it.

Peter

Death by dogma

It's amazing what you can find on the internet when you click on a link. I stumbled over this recently:

Premature aging in mice activates a systemic metabolic response involving autophagy induction

I picked up this gem of a paper from, of all places, a blog with somewhat limited enthusiasm for ketogenic eating. I'll just go through the results section of the paper, giving a staccato summary of each paragraph, because you have to be sure of exactly what a group have found before you consider whether you agree with their conclusions. These mice lack the ability to form prelamin A correctly, instead they form progerin, and they age very rapidly. Here we go with the results section:

A mutation which damages nuclear architecture and causes premature aging also increases autophagy.

The abnormal protein formed (that prelamin A precursor known as progerin) appears to be the cause of premature aging and to be associated with increased autophagy (in this model).

Other models, XPF and CSB/XPA, of rapidly aging mice (both with defective DNA repair processes) do the same thing but without accumulating progerin, especially they increase basal autophagy. So this upregulated autophagy is common to several models of premature ageing, not just the prelamin A model.

mTOR signalling is switched off. Really switched off.

The PI3K-Akt pathway, which usually activates mTOR, is not the explanation.

AMPK is switched on. Really switched on.

Stopping the response to DNA damage (p53 knockout) does not stop enhanced AMKP activity. So we are not looking at extra autophagy to recycle damaged DNA.

Next we get on to glucose. The five hour starved level of glucose is low, around 38% of control value. Insulin is low too.

In the liver things are strange.

Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are up-regulated, both are important for gluconeogenesis.

Puryvate kinase (a glycolysis regulator) is not up-regulated. So where is the glucose going if it's not going to glycolysis?

Glucose from gluconeogenesis appears to end up in the liver as glycogen granules, without needing glycogen synthase to be up regulated. This glycogen can be accessed if needed.

At the same time fatty acid producing genes are up regulated. Glucose is being converted to fatty acids. Genes associated with fatty acid oxidation are up regulated too. And a fatty liver develops. Very interesting.

Pyruvate dehydrogenase kinase-4, key for switching from glucose to fat burning, is strikingly upregulated. These mice burn fat. They reject glucose. And they die of precocious aging!

All of the "good" markers indicating longevity in many models are fantastic in these mice. The end product is early death.

Metabolically, everything appears to come down to PGC 1-alpha. It's production is very upregulated. This cofactor appears to responsible for the switch from glucose to fat based metabolism.

End of results summary.

This is where the paper stops.

On the basis of these findings a concern expressed in the discussion is that elevated PGC 1-alpha drives autophagy, which is initially adaptive but might become maladaptive when chronically activated. This is a potentially valid concern (there is an autophagy triggered form of cell death distinct from apoptosis and necrosis) but we have to bear in mind that there is zero data to support this specific concern provided in the paper. That's all of the results section summarised up above.

The authors are well aware that the reason for rapid aging is the genetic defect in nuclear architecture formation. This leads, indirectly, to genomic instability which immediately puts this model in to the same category as other premature aging models such as XPF and CSB/XPA, both of which have defects in DNA repair, also as mentioned above.

So why do the cells of these animals go in to a state of AMPK driven, mTOR inhibition dependent, persistent autophagy?

They do this because they have a severe ATP deficit. High levels of AMP per unit ATP drive AMPK.

Why is there insufficient ATP?

Let's have a read at Nick Lane's essay Mitonuclear match: optimizing fitness and fertility over generations drives aging within generations. Here's the quote:

"Oxidative phosphorylation involves the transfer of electrons through a succession of redox centres in respiratory chains, from an electron donor such as NADH, to a terminal acceptor such as oxygen. A slowing of electron transfer means that respiratory complexes become more highly reduced, which increases their reactivity with oxygen, corresponding to a rise in free-radical leak [25]. A slowing of electron transfer is the most likely outcome of any mismatch between mitochondrial and nuclear genomes. The reason relates to the mechanism of electron transfer. If the gap between adjacent redox centres in respiratory chains is increased by just 1 A, electron transfer by quantum tunnelling slows down by an order of magnitude [26] and free-radical leak should rise accordingly. Given that hydrogen bonds and Van der Waal’s forces act over distances in the range of 1–2 A, it is likely that any changes to optimal subunit interactions would disrupt the distance between redox centres by more than 1 A, slowing electron flow and increasing free-radical leak. Thus, a rise in free-radical leak is the predicted outcome of virtually any subunit mismatch, and indeed has been reported [27, 28]"


Question: How well might the electron transport chain function in the mitochondria of a cell which has a permanent defect in its ability to repair nuclear DNA damage?

Answer: Three separate mouse models, engineered to have defective DNA repair, have chronically activated AMPK.

I think these mice are "starving" with full stomachs because they cannot generate ATP. They have progressive un-repaired changes to the nuclear DNA coding for (amongst many things) electron transport chain components which means these proteins simply don't fit the proteins from mtDNA.


Soooooooo. Essentially all of the changes in these mice could be traced back to inadequate ATP generation, with the added bonus of excess ROS generation to further damage the "unrepairable" DNA.

The extended autophagy is a total red herring, sort of.

I picked this paper up as a FB link "liked" by Chris Highcock. The linked blogger is so impressed by the lethal effect of autophagy that he appears to consider that diet-induced autophagy is a "worthless dogma" and avoids it, not liking dogma. That's fine by me, we all have our quirks.

Unfortunately for this autophago-phobic concept, and possibly for the poor chap himself, it turns out that if you take a prelamin A mutant mouse and treat it with rapamycin you will actually promote even MORE autophagy. So, if autophagy is the cause of premature aging, things should get worse. Yes? The actual result is that:

"Here, we report the discovery of rapamycin as a novel inhibitor of progerin [defective prelamin A], which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts..."

or just have the title:

Autophagic degradation of farnesylated prelamin A as a therapeutic approach to lamin-linked progeria.

As an approach, it works. Further increasing autophagy rescues prelamin A mutant mouse cells. RIP autophagy as a "cause" of accelerated aging.




I like Chris a lot, he's a really nice chap. Luckily the sort of hill walking he does (still love your Pentland pictures on FB, if you read this Chris) he will be regularly and repeatedly activating AMPK with subsequently increased autophagy. Exercise does this. So does the "misery" of ketogenic eating.

Actively changing your diet to avoid autophagy, based on a progerin model which can actually be rescued by increasing autophagy, just might be a booboo. Imitate with caution.

Peter

BTW It's sort of nice looking at technical papers which (superficially) challenge my low carbohydrate biased perspective. Knowing you are in a correct paradigm makes dissecting them a rather relaxed process when you start form a position where the world makes sense. Perhaps this is dogma.

Ketoacidotic death in lactation!

Laura forwarded me the link to this epic study:

A gestational ketogenic diet alters maternal metabolic status as well as offspring physiological growth and brain structure in the neonatal mouse.

It has a very very clear message. This is the first paragraph of the results section:

"Lactation in KD [ketogenic diet fed] Dams Leads to Fatal Ketoacidosis. In the initial trial phase, 4 KD dams remained with their biological litters post parturition, and during lactation. However, lactation appeared to cause severe physiological strain to all KD dams. Soon after parturition they started exhibiting signs of high stress, as was also apparent by them cannibalizing their litter. Measurements of their blood glucose and ketone revealed elevated values (Glucose: ∼20 mmol/L and Ketone: ∼6 mmol/L), indicating development of ketoacidosis. This ketoacidosis rapidly progressed to a fatal metabolic state, leading to the death of the KD dams within a few days post parturition"

I hope that is clear. No beating about the bush.

These folks are cutting edge. They know how to get impressive results.

Take home message: If you wish to survive lactation do not, UNDER ANY CIRCUMSTANCES, base your diet on CRISCO. Do not do it.

My wife seems to have survived two ketogenic lactations in pretty good shape. Daniel's school is well in to spelling test this term. He is the only kid in his class to have made no mistakes so far. It doesn't look as if we have broken his brain. No Crisco.

Of course Sussman et al blame the ketosis. They keep very quiet about the Crisco:

TD.96355 Ketogenic Diet: "Vegetable Shortening, hydrogenated (Crisco) 586.4g/kg"

Want to die? Always ask for Crisco by name. Crooks.

Peter

Protons (29) Uncoupling with fatty acids

Trying to work out exactly what is happening in a given functional component of the inner mitochondrial membrane is not the most simple of undertakings. For anyone who wishes to bend their brain a little you could do worse than working through Federenko's paper on the likely structure/function of UCP1 in brown adipose tissue. I think the original pdf was sent to me by Alex, some time ago. Although it is looking at UCP1 I think we can reasonably assume other UCPs work in much the same manner. This set of line doodles summarises the paper:

Here's the explanatory text:

(A) The simplest mechanism of steady H+ IUCP1 induced by LCFAs. UCP1 operates as a symporter that transports one LCFA and one H+ per the transport cycle. First, the LCFA anion binds to UCP1 on the cytosolic side at the bottom of a hypothetical cavity (1). H+ binding to UCP1 occurs only after the LCFA anion binds to UCP1 (1). The H+ and the LCFA are translocated by UCP1 upon conformational change, and H+ is released on the opposite side of the IMM, whereas the LCFA anion stays associated with UCP1 due to the hydrophobic interactions established by its carbon tail (2). The LCFA anion then returns to initiate another H+ translocation cycle (3). Charge is translocated only in step 3 when the LCFA anion returns without the H+.


All nice and simple, compared to how complex it was to work all of this out. In plain English: The (removable) fatty acid flip-flops back and forth, passing a proton each time. UCPs absolutely MUST have access to long chain fatty acids to function. The LCFAs must be free, ie NOT be activated. Sticking a large CoA moiety on to the red dot (which is the carboxylic end of the LCFA) will completely inhibit any activity in UCP1 as there is then no negatively charged binding site on the fatty acid to allow it to ferry protons, repeatedly, down their concentration gradient back in to the mitochondrial matrix.

So fatty acids may just be slightly important to uncoupling. Uncoupling may be a Good Thing or the converse, depending on your point of view or your carbohydrate intake.



This leads us on to Curi's group and their 2006 attempt to look for the effect of FFAs on uncoupling in tissue culture, in isolated mitochondria and even in whole live rats. When you see a line in the methods which specifically states "in Krebs–Ringer bicarbonate buffer containing 5.6 mM glucose" you know you are in for a treat. You have to have read as much cell culture literature as I have to realise how precious a line like this is. All you normally get is the type of medium, probably the brand name and the supplier, but oops, forgot to mention it has, or might have, or might not have, 25mmol of glucose in it. Or some other random amount. Duh. The easy way to tell (when not specified) is that if saturated fats cause apoptosis in cell culture, be certain that glucose is >20mmol.

Anyhoo. Here we go:

This is from first generation harvested and cultured skeletal muscle cells. The fluorescence rises with inner mitochondrial membrane potential, delta psi. Antimycin A blocks ATPase so hyperpolarises the membrane, CCCP is a chemical uncoupler and so reduces it. These are just tests to show the prep works. The caprylic acid is useless (it works well on isolate mitochondria but the cytoplasm probably stops MCTs reaching the mitochondria without adding CoA while they are on their way there). Palmitic acid and linoleic acid uncouple, like a mild version of CCCP.

You can show exactly the same effect in chronically cultured myotubules, just another cellular prep.

This is our first look at isolated mitochondria. This is a graph of the same delta psi sensitive fluorescent dye plotted against time. The graph is upside down because, in real life, delta psi is negative. You add SMM (skeletal muscle mitochondria) to the observation medium. They fire up and delta psi increases. This is the curve dropping downwards from the SMM arrow. In the middle of the 2 minute bar, palmitic acid is added and delta psi decreases (upward flick in the upside down graph) marked by the PA arrow. The bottom line is what happens if you have added guanidine di phosphate, GDP, an inhibitor of UCPs. At the end they also added CCCP to fully uncouple the mitochondrial membrane and show that delta psi collapses.

Note, you can only show the inhibitory effect of GDP when the CoQ couple is oxidised. A reduced CoQ couple stops GDP's inhibitory effect on uncoupling. This gives a mass of contradictory studies in the literature, needless to say. The control of uncoupling as a fascinating area, perhaps another post.

And this graph is looking at the rise in oxygen consumption induced by uncoupling using assorted fatty acids. Again, mitochondria, so caprylic acid works, though never as well as the longer fatty acids. Note the lack of GDP effect, the CoQ couple must be reduced in this prep. BSA (bovine serum albumin) eliminates the uncoupling as it "hoovers-up" all available FFAs.



Does this uncoupling happen in intact animals? Yes. A stack of caveats, but yes. Ratties were bolused with intravenous palmitate or alcohol (As the control??!!?? Alcohol is not inert, even if it doesn't uncouple) and then oxygen consumption was measured:

Palmitic acid uncouples.

Right, that will do for now. The plan is to look at FFAs and insulin resistance next, within this framework.

Peter, chronically uncoupled.

Polycystic Kidney Disease and mTOR

I think I'm pretty well out of blogging time for the next month or so, so the post looking at fatty acids, uncoupling and insulin resistance will have to sit on the back burner while I work on an anaesthesia project. A friend emailed me a paper with this rather nice diagram of the probable aetiopathology of polycystic kidney disease. Whenever I see mTOR driving pathology I always think of ketogenic eating to obtund the process. If you carry PKD genetics I don't see any other remotely sensible option...

No Reverse Warburg Effect here. I love the 2-deoxyglucose too. If there was ever a way of faking a ketogenic diet, this is it. More so than ketone esters! A bit of physiological insulin resistance or a drug to block glycolysis? Or how about a modern mTOR inhibitor? I'll leave it open to guesswork (or Pubmed if you must) what a ketogenic diet does to mTOR signalling.

Peter

Electron Transport Chain image

Just off to bed. Wow, do I have wild Saturday nights! Had to share this lovely pic. One of the better representations of the ETC I've ever seen, lifted from here.

Like.

Peter

Wooo and the snps

There seems to be quite a bit of interest going on in the LC Hardcore at the moment and I'm sat here, under my stone, looking at UCPs as prime mediators of the insulin resistance of fasting and membrane pumps in the origin of life as relates to lactate and extracellular pH in cancer. And I should really be working on a dead-lined anaesthesia project. Wooo and Toxic ("I read your potatoes, and the news will never be good" LMAO) have both brought up 23andme and, surprise surprise, Wooo has enough snps on assorted ion channel genes to have her in a loony bin several times over. She concludes that a ketogenic diet gets her a normal life.

Obviously I have nothing to disagree with here.

What I would comment on is that a very, very large chunk of the "normal" population may not be quite as normal as she suspects. Having frank bipolar disease with severe enough presentation to give you a clear cut "label" is quite rare. Having severe depression to the point of complete loss of functionality or schizophrenia to the point of obeying the voices completely, whatever they command, are all equally rare. But shades of grey appear to be very common and I don't see that many normal people are all that normal. Undoubtedly we all have snps on all sorts of genes. That's genetic variability and is essential to provide a pool for the species to adapt through.

That many mental illnesses are essentially metabolic, and that ion channels have a great deal to do with neural energy demands, is not exactly unexpected. Sid Dishes emailed me this rather interesting review (BTW finding this in Nature is rather like reading a massive endorsement of the Atkins diet in the Sun or the Daily Mail, Sid feels paradigm shift) looking at exactly the metabolic aspect. Yet another email needing a thankyou not sent yet. Thanks Sid. Look at this quote from the abstract talking about central neurons:

"it is now clear that they [psychiatric illnesses] are associated with impairments of synaptic plasticity"

and tie that back to peripheral neuropathy, here's Chowdhury on peripheral nerves:

"The consequences of suboptimal ATP supply for the distal nerve fiber are numerous: (1) collateral sprouting and plasticity will be retarded, (2) this will lead to gradual pruning of the axonal network and shrinkage of sensory innervation fields, and (3) end organs of myelinated fibers within the dermis will lose innervation and function (see Fig. 3)"

My italics.

The parallels, to me, make it sound like we are talking about the same process. I would suggest that hyperglycaemia breaks the mitochondrial population and ketosis is an excellent sticking plaster. Which snps you have determine which neurons break first.

If you also have metabolic snps which limit your ability to avoid hyperglycaemia on the SAD in addition to neural snps which make for "upper limit of normality" energy demands within hyperglycaemia compromised neurons, you are on your way to the Funny Farm. Or a ketogenic diet.

I, for one, am very glad Wooo hit the ketogenic diet arm.

I have said that I think it is unlikely that humans are in any way adapted to a diet which regularly and severely induces hyperglycaemia. Had Wooo been born in to a normoglycaemic environment, what would the effect have been of her ion channel snps on perception?

If you think Wooo is "normal", you are crazy.

Personally, I think we need people who are three standard deviations from the population norm when it comes to insight and perception. This may well be down to ion channels and snps of the Wooo flavour... I don't see that we would get too far, species wise, if we were all Taterheads with ion channels which allowed tolerance of hyperglycaemia until Alzheimers (type 3 diabetes) kicked in, and yet only allowed as much insight in to anything as a turnip has. Of the latter, there is a lot of it about, we have more than enough.

Blog on Wooo.

Peter

Omega 3s and G-protein coupled receptors

Let's just summarise the role of omega 6 fats in Sauer's rat model of cancer:

In the lab situation rapid hepatoma tumour growth needs either arachidonic or linoleic acids. The acids must be taken up in to the hepatoma cells, they must be acted on by lipoxygenase to produce 13-hydroxyoctadecadienoic acid, better known as 13-HODE. 13-HODE appears to be the mitogen which promotes rapid cancer growth. 13-HODE looks like a repair signal gone wrong in cancer cells. Omega 3 fatty acids block omega 6 fatty acid uptake in to hepatoma cells. That's all well and good but the reason I got in to this paper was omega 3 PUFA signalling, rather than those omega 6 issues...



OK, Sauer starts to give some pointers on the function of omega 3 fatty acids in health. That's interesting, as I'm no great lover of any sort of PUFA when I view them from the Protons perspective, yet omega 3s seem to come out pretty well, certainly at low doses. You know my fall back, omega 3 PUFA don't always behave like omega 6 PUFA because they get used as signalling molecules blah blah blah. My own inability to tie the molecular structure of omega 3s to their clinical effects is very frustrating! That they probably act are sites "above" the ETC suggest that they act as what I view as 'high level signals".


Well they do.


The signalling appears to be through a G-protein linked receptor with all of the usual cAMP cascade that follows binding of a ligand to such a receptor. What I found particularly interesting was the effect produced on fat pads of normal rats when EPA (other papers from Sauer suggest all omega 3s act on whichever receptor is involved) was added to the perfusate.

OK, here is a neat little graph taken from here:

This is from fed rats. In the fed state the FFA uptake by the inguinal fat pad of a rat is about 6 mcg/min/gram, white open squares.

Adding EPA at 0.84mmol/l (a bit supraphysiological for EPA but let's let that ride) and FFA uptake by the fat pad drops to zero, or close to zero. Or, in fact, you could argue a suggestion of fatty acid relase, shown as a negative uptake value. Black circles. Fatty acids are not taken up, they end up in the venous effluent in the experiment, plus a little extra.

Whooooah, so do FFAs go through the roof when you take fish oil IRL??

Well no. That's because of this graph from here in the same paper:

Here we have the free fatty acid release from the inguinal fat pad of a healthy rat who has been starved for 48 hours. Fatty acid release is trundling along at about 3mcg/min/gram until EPA is added, again at around 0.8mmol/l. The release of FFAs, in the fasted state, is eliminated. Table 1 in the same paper shows you can get this effect of halting lipolysis in starved rats with under 0.3mmol/l EPA.

Both effects are mediated through a G-protein coupled receptor, ie high level signalling compared to electrons and superoxide in the electron transport chain.

Obviously there are a number of serious problems with this paper but, as a proof of concept, I buy it. I doubt DHA or alpha linolenic acid would work as well (or the group would have used them for this proof of point exercise!) and I think the levels of EPA used produce a very artefactual "switch-like" effect which is probably a graded response. I doubt 0.8mmol/l or even 0.3mmol/l of EPA is exactly physiological but...

Let's suggest that there is a progressive removal of the influence of adipocytes from the FFA flux in/out of plasma as the level of omega 3s in arterial blood increases. Omega 3 fatty acids render adipocytes irrelevant to free fatty acid levels in the plasma.

That is one hell of an idea.

Next we need a brief look at hepatoma cells, again the graph is provided by Sauer and it shows that omega 3 fatty acids, in a G-protein coupled receptor manner, completely turn off the uptake of ALL fatty acids in to hepatoma cells.

If, and it's quite a big "if", the same effects apply to hepatocytes as well as hepatoma cells, we then have a very straightforward mechanism for the protective effects of omega 3 fish oils on hepatic lipidosis. From my point of view this is quite real as there are pretty convincing papers showing that cats, in real life, can be largely protected against the potentially fatal hepatic lipidosis of rapid weight loss by modest doses of omega 3 fatty acids.

Soooo while omega 3s stop the release of all FFAs from adipocytes, they simultaneously stop the uptake of all fatty acids in to the two primary storage organs for fatty acids, adipocytes and liver.

Do plasma FFAs go up or down?

They do, of course, go down. A paradox? Next paper.

Health warning: This paper is so steeped in VLDL and ApoB lipophobia that it makes difficult reading. But there is so little published on FFAs and omega 3 supplementation that it's worth the ondansetron to read it. It's looking at how omega 3 supplements might lower fasting triglycerides, which are the devil incarnate for CVD risk. A huge chunk of VLDL comes from FFAs released from adipocytes and their subsequent repackaging by the liver. Apparently, and I quote from the abstract:

"FO [fish oil] counteracts intracellular lipolysis in adipocytes by suppressing adipose tissue inflammation"

A bit like insulin resistance is caused by "inflammation". Well, maybe it's that simple. They have taken the concept of high level signalling to its 2013 pedestal without looking for basic mechanisms. They have placed the G-protein coupled receptor on to macrophages in the fat pads, which subsequently control the adipocyte lipolysis using cytokines. I haven't checked how good this concept is. Sauer never looked that deeply. Looks a bit modern to me.

Personally I would guess that there are similar receptors on both adipocytes and hepatocytes, but the review does not seem to cover the ability of omega 3s to inhibit general fatty acid uptake by these two tissues. Ah well.

What they do argue is that omega 3 fatty acids upregulate lipoprotein lipase, pretty well whole body. Of course liver and adipocytes ignore this fatty acid bonanza, as above. LPL upregulation is what I needed to know from this paper.

So where do "spare" fatty acids go to? They go to muscles. Upregulated lipoprotein lipase (heralded as the saviour from elevated fasting triglycerides) allows increased lipid release from VLDL to lower those fasting triglycerides. But it's worth bearing in mind that cells do not "see" VLDL, the LPL is on the vascular endothelium and the cells behind the vessel wall only ever receive "free" fatty acids. These are not labelled as from albumin, VLDL or chylomicrons.

Slight aside for later: It seems likely that chylomicrons are going spill their lipids via that same LPL, worth remembering.

The story in the review can be sumarised as omega 3 fatty acids block the release of FFAs from adipocytes and increase the activity of lipoprotein lipase pretty well whole body. VLDL drops, FFAs drop. All is happy in the cardiovascular system. If you believe.


Various "bits" of omega 3s, especially the lipid peroxides of DHA, are signals for mitochondrial biogenesis. I had a paper which specified which lipoxide was most effective but must have missed the "save" button. Mea culpa yet again. There are hints here.


That's a very neat story, which has more than a grain of truth to it.

Why is it like this? What does it mean, physiologcally? Speculation time:


Omega 3 fats come from plants. Mostly from chloroplasts. Where do humans get their omega 3s from? Certainly not from plants. If we did then the rabid Dr Furhman would not be (correctly) recommending DHA supplementation (along with B12) to avoid brain collapse on veg*n diets. Actually, this link is quite funny when cited by Mc-Starch-Dougall:

"There is no evidence of adverse effects on health or cognitive function with lower DHA intake in vegetarians"

Well, I found it amusing. It's almost the converse of the neurological truism which states that being concerned about having a neurodegenerative disease probably means you don't actually have one.

Anyhoo. Away from the coast we have to get our DHA from animals (or buy algae derived supplements). They get it from grass. There is DHA present in adipose tissue of herbivores just as much as it is present in lipid membranes of their cells. My suspicion is that DHA is a signal to your metabolism that you have just eaten animal fat, from an animal who's food chain starts with grass [or algae]. The more fat you eat, the stronger the signal. We do not need much DHA overall for our brains as it is well protected in this site, but we might well be using it at low levels as a [G-protein coupled receptor sensed] signal to target metabolic adaptation to process fat. So is McDougall correct that veg*n "brain" tissue is OK, despite their periphery being depleted? Shrug.

Fish oil supplements? Well, using our "dietary fat is here" marker to pharmacologically modify some perceived CVD risk factor, without the appropriate change in source of metabolic fuel supply, looks to me to be of very limited value. Large intervention trials do show some benefit from omega 3s provided you do your stats well enough, you have a large enough population to pick up a very small effect and you give a high enough dose. But they do not seem to be any sort of panacea. Especially of you are avoiding dietary fat while "faking" the signal that you have eaten dietary fat...

This is not exactly surprising when you try to pick the likely physiology apart. I like the concept of DHA as an animal fat signal.

Peter

Final thought: Do we need omega 3 PUFA at anything above the most minimal levels if we are in saturated fat based ketosis? Of course I don't know. But the signal to cope with starvation is palmitic acid (physiological insulin resistance), not DHA. I live in starvation mode, not on a mixed diet with only intermittent access to healthy ruminant fat. I have long wanted to look at the selective release of FFAs from adipocytes in extended starvation. My suspicion is that in the early days after glycogen depletion palmitic acid is preferentially released over other lipids, PUFA are not needed/wanted. By a few weeks all the palmitate is gone and whatever is left then gets released. People like David Blaine suddenly start to feel weak, wobbly and are probably hypoglycaemic once they run out of palmitate and have to release less saturated fats. Two to four weeks if you carry some spare weight. Sauer's rats had only ever been fed a low fat omega 6 based diet and had no serious palmitate reserves, PUFA release came early for these.

Axen and Axen: The tradition continues

Another superb abstract, hot off the press, on LCHF diets. The data are suggested to be utterly clear cut, solid, and supportive of the physiology of LC eating which I have been espousing for many years now.



The conclusions are criminal. There are 13 authors. None seems to have read Kinzig et al's 2010 paper showing not only this very effect but also showing its complete reversal by a few carbohydrate based meals.


That omission, by 13 people on the author list, is what is criminal. The lack of understanding of the basic physiology of carbohydrate restriction even without Kinzig, by people in what claims to be nutrition research, is criminal. The blood on their hands, from dialysis patients, is criminal.



If even one of the authors has read Kinzig it leaves you wondering about the ethics in the group. If they haven't read the literature...

Disclosure: I haven't seen the full text.

Peter

BTW I just Pubmeded "ketogenic" + "insulin" + "resistance" and Kinzig was hit 24, primarily because 2010 is ancient history and the hits come in date order.

Hat tip to Liv for the link.

EDIT

Laura gave us this in the comments: "They actually cite the Kinzig 2010 paper in their discussion saying that their results match and that the results "could" be reversible... and then they leave it at that".

You have to ask what the lead in time to a study like this is. It strikes me as possible that Kinzig pipped them at the post by 3 years but they went on with the study as they had the funding but couldn't add a re feeding arm because they had no study ethics approval for this. Just trying to be kind, probably a mistake. Or perhaps they are just LC bashers as per usual. The cardinal sign, of using the name "Atkins" in the abstract, is not a good marker for an ethical group. But Kinzig is correct. Reversal is absolutely nothing a newbie LCer wouldn't pick up in 10 minutes on the internet.

END EDIT

Ketones, without the side order of Danish Pastry please

Here is a discussion paper from Denmark. It is a deeply satisfying read, well worth overcoming the slight oddities of grammar which seem to have come from it being written in Danish as a first language. What they are doing is taking the concept discussed by Nick Lane (which they cite) about the intracellular selection of mitochondria under bioenergetic stress and putting a testable molecular framework in place. Obviously, from the Hyperlipid perspective, just look what they place at the top of the list as one of their factors for mitochondrial health. Here's the whole conclusion section:

Conclusions

This perspective deals with the notion that adaptive stress responses to respiratory challenges and stimulation drive natural selection of genetically and epigenetically inherited properties of mitochondria:

• When brain energy turnover increasingly depends on ketone body or fatty acid metabolism rather than on glucose, sparing of complex I and proliferation of mitochondria is beneficial to overall mitochondrial health.
• High glucose availability for oxidative phosphorylation, on the other hand, establishes a state of low selection pressure with increased accumulation of lesions.
• Intermittent non-chronic insults with increased ROS production benefit mitochondrial health and promote healthy aging and increased longevity.
• In healthy tissue, transient non-lethal insults such as chemotherapy, hypoglycemia, or hypoxic challenges, select mitochondria that are more resilient to subsequent challenges. These mitochondria are better adapted and more numerous.
• Stressful challenges with increased ROS levels, followed by subsequent recovery and treatment with biogenesis-promoting agents, yield mitochondria with greater respiratory capacity than mitochondria treated with biogenesis-promoting agents alone.

These claims have specific and testable implications, the resolution of which can revise the general understanding of the role of mitochondrial challenges in healthy aging.




OK, 6.06am here, time to fry some egg yolks in butter. No taters for me.

Peter

Starvation and cancer growth: Sauer vs Lisanti

Preamble: There has been some puzzlement recently among the LC hard core about the main stream antagonism against LC as an approach to disease management. I share their puzzlement. Stan currently has a post here and Wooo has one here. Why do people have to fake data to support an incorrect idea or to abandon techniques which work? Weird. But that's their problem. What frightens me more is when people have an idea in serious science but are disturbing in the conclusions which they come to based on it (I don't usually doubt the data, I'm innocent that way). It particularly scares me when supportive citations are cherry picked while closely related contradictory citations are omitted. Especially when the omitted citations have enormous explanatory power. I came across this unpleasant clash of research thinking accidentally through ron's comment on a Protons post. I was unaware of Sauer's work but have been meaning to blog about cancer and metabolic coupling for some time. Finding that Sauer was in the citation list of Lisanti's coupling paper has pushed me enough to stick this post up. The refs (all free full text) you need to make up your own mind are in the text below. I'm not sure that "enjoy" is quite the correct word. Here we go.




Over in the comments to the Protons summary post ron linked to this paper showing, rather nicely, that sustained fasting markedly promotes cancer xenograft growth. Sauer comments in the paper that the group had noticed this previously and this study appears to be a formalisation of that observation. They had an "Oh, that's interesting" moment and, being scientists, investigated rather than burying it. Here is one of a choice of several graphs:

The bottom lines are the tumour weights, the top lines the animal weights.

Sooooooooo, living with normal-for-starvation levels of ketones and and free fatty acids promotes cancer growth in these particular models, like wildfire.

I have been heard to comment, on more than one occasion, that I have personally been "fasting" for the last 10 years. I just keep replenishing my fat loss using dietary butter. I have had elevated ketones and free fatty acids, 24/7, for much of the last 10 years, probably to starvation levels.

I sort of like Sauer. He wanted to know what happened in starvation to promote cancer growth. As a 1980s physiologist he then did lots of operations on lots of rats which we would rather not go in to in great detail. But he got results.

Amongst the things they did was to perfuse cancer xenografts in live rats with blood from non cancer bearing rats who were in the fed or fasted state. Joined the "donor" rats directly to the arterial supply to the tumour, using various bits of tubing, all very cunning. The cancers only grew rapidly when perfused with blood from starved rats.

They then took blood from fed rats and engineered it in to various reconstituted blood-like fluids resembling the blood from starved rats, by adding assorted fatty acids, and perfused the tumours to see what it was that made them grow.

Palmitic, stearic and oleic FFA supplementation was inactive in promoting tumour growth.

Linoleic and arachidonic promoted growth, really well. That is very scary.

Aside: When people come to look in earnest at ketogenic diets for cancer management the omega 6 content of adipocytes is going to be one hell of a confounder. You will almost need to eliminate weight loss in order to eliminate or at best reduce the release of omega 6 PUFA if the patient has been living on soy oil or Flora for a lifetime... Not easy. End aside.

Got high cholesterol? Want to lower it? Use polyunsaturated acid based margarine! Want to grow a cancer? Hmmmmmm.

Personally I'll settle for butter or 90% cocoa chocolate with palmitic or stearic acids. I suppose I ought to 'fess up about ketones. Well, no. There has to be a pause here.

If you had a concept which ought to show that ketones were a super-fuel for cancer (there are folks with this viewpoint) you might want to cite Sauer and the papers which show that something about fasting or ketosis promotes cancer growth. Which is exactly what this group did in this paper:

"Ketones and lactate “fuel” tumor growth and metastasis. Evidence that epithelial cancer cells use oxidative mitochondrial metabolism".

Nice title. These are the refs they used:

21. Sauer LA, Dauchy RT. Stimulation of tumor growth in
adult rats in vivo during acute streptozotocin-induced
diabetes. Cancer Res 1987; 47:1756-61.
22. Goodstein ML, Richtsmeier WJ, Sauer LA. The effect
of an acute fast on human head and neck carcinoma
xenograft. Growth effects on an ‘isolated tumor vascular
pedicle’ in the nude rat. Arch Otolaryngol Head
Neck Surg 1993; 119:897-902.


Now, this group is very, very good. They have this concept that fibroblasts are enslaved by cancer cells and forced to perform glycolysis but then abort their own TCA and ox phos, supplying lactate and ketones, both derived from pyruvate, to the cancer cells which then use their own mitochondria to fuel cancer cell growth. It's probably correct.

In support of this concept they injected, intraperitoneally, half a gram per kg of lactate or half a gram per kg of beta hydroxybutyrate daily and got increased metastasis with lactate and increase cancer growth with beta hydroxybutyrate. Probably this really happens.

But there are some holes in this study. The ketones supplied to the mice carrying the cancer xeonografts were given by intraperitoneal injection and no one knows what blood levels were reached. Possibly quite high for a while. They never measured them, that I can see. Even well funded dieters measure their ketones.... Let's assume they go so high, whole body, as to actually mimic the sort of levels produced in the minute extracellular gap between a slave fibroblast converting glucose to ketones and pumping them directly on to the surface of an adjacent master cancer cell. We don't know what that level is either, but both get the desired effect on cancer growth to support the paradigm.

BTW, another complete aside, the locally-supplied, fibroblast-generated ketones and lactate are UTTERLY glycolysis dependent. If the Warburg effect is not happening in cancer cells, the reverse Warburg effect looks to be VERY susceptible to sudden onset normoglycaemia affecting the fibroblasts in metabolically coupled systems. The ketones/lactate come from glucose in this set up, not from lipolysis or anaerobic exercise! End aside.

EDIT: Not the ketones, those almost certainly still come from FFAs. END EDIT.

So the question is, when comparing Sauer and Lisanti, what happens when you feed an in-vivo cancer xenograft with PHYSIOLOGICAL doses of ketones by continuous perfusion, using starvation levels?

Sauer of course, did check this. He took blood from fed rats, added ketones to it without omega 6 FFAs and used the blood to directly perfuse a series of cancer xenografts. He doesn't actually give us a concentration for the ketones he used (Edit; without looking up ref 10: OK, I checked ref 10, 4-ish mmol/l in the control rats, just about where I live) but he does appear to be a very interested in teasing out the cause of the effect, so I'll buy that he used the concentration he had measured in the blood of starved rats, which supported cancer growth so well.

When he had finished with the neutral  effects of palmitic, stearic and oleic acids and the growth promoting effects of linoleic and arachidonic acids, this is what he has to say about ketones:

"Finally, perfusion of normolipemic blood enriched in the ketone bodies (10) had no effect on [3H]thymidine incorporation in tumors growing in fed adult rats (data not shown)."

Doesn't bode too well for therapies based on the Reverse Warburg effect from Lisanti's group targeting mitochondria. Did they not read all of Sauer's papers? Or did they really read them all and cherry picked the ones they wanted? Which idea scares you most? The cancers grow under the influence of omega 6 PUFA derivatives, NOT ketones. Sauer says so. Believe which ever group you like. I'm biased and I rather like Sauer.

Peter



Addenda.

It is very simple to fit omega 6 PUFA FFAs in to the Protons concept of cancer fuelling. I'm still working at why omega 3 fatty acids are protective in these models, they shouldn't be. In cirrhosis models they behave exactly as they should do, promoting cirrhosis as the omega 6s do, but more so. There is a link missing here somewhere. Sigh! I hate "higher level signalling" as an explanation, always seems like a cop out to me. What happens at basic energy metabolism level should give the answer...


Also Sauer specifically looked at cancer utilisation of ketones, lactate and assorted other fuels in some detail here. Some cancers can and do use ketones, but I don't see plasma ketones or lactate as superfuels for cancers in the real world. They get used, but I still see local glycolysis in fibroblasts as the major pathway supplying them. I'm fine with the Reverse Warburg effect. Targeting mitochondria will be a booboo.

Protons so far, some sort of summary!

Edit: I no longer think this first paragraph is correct, there is an update here. End edit.

We appear to have two basic states of the electron transport chain. There is the situation under fasting or ketogenic dieting conditions. Here delta psi is low, complex I throughput is low and there is plenty of FADH2 input through electron transporting flavoprotein dehydrogenase coming from the first step of beta oxidation of real fats, like palmitic acid. With a low delta psi it is near impossible to generate reverse electron flow through complex I so activation of insulin signalling is rapidly aborted by the continuing action of tyrosine phosphatase.

This is the insulin resistance of starvation. Without it death from hypoglycaemia would be routine after a day or so without food.

Next is the state of the electron transport chain proteins under the influence of insulin signalling. How this is achieved is currently outside my reading but I think it is perfectly reasonable to assume that specific electron transport chain proteins will be phosphorylated as a direct result of insulin signalling being active. With a large supply of NADH to complex I and a restricted supply of fatty acids due to insulin acting on adipocytes there is a high membrane voltage, high throughput of electrons down the ETC via complex I but no reverse flow because there is a minimal input via electron transporting flavoprotein dehydrogenase's FADH2.

These are the two simple extremes of organisation under "isocaloric" conditions and neither generates significant reverse electron flow, ie there is minimal superoxide production at complex I.

Under hypercaloric conditions, usually an elevated supply of both glucose and fatty acids, we have the high delta psi, high FADH2 input through electron transporting flavoprotein dehydrogenase from beta oxidation and so significant reverse electron flow through complex I to signal that more than enough calories are available to the cell.

Under simple glucose based caloric overload mtG3P dehydrogenase steps in in the place of electron transporting flavoprotein dehydrogenase and supplies an FADH2 input to signal the need for hypercaloric insulin resistance. This seems a perfectly reasonable approach to hyperinsulinaemic hyperglycaemia.

Under normal physiology I would expect blood glucose to remain under 7mmol/l at all times, probably under 6mmol/l, provided the food eaten is food and the physiology processes used are undamaged. Even under caloric overload with a baked spud.

What do we really mean by caloric overload?

Overload is the utterly normal response to eating any meal. ANY meal. As soon as the rate of calorie absorption exceeds the post prandial metabolic requirement, we need to store the excess calories. The development of individual cell insulin resistance is utterly normal under these conditions. Blood glucose, blood lipids and blood insulin rise. Fat is diverted to adipocytes. Glucose is diverted to glycogen stores.

All of this is achieved by reverse electron flow through complex I generating a physiological response. The acute storing of calories is essential. This is how we do it.

The diversion of glucose to the brain in starvation is induced by failure to sustain insulin activation due to lack of sufficient mitochondrial membrane potential needed to signal that it's OK to respond to insulin. Low insulin is helpful and low glucose is essential for this process.

I think this summarises the Protons thread to date.

Perhaps we can go on to look at some pathology sometime. Mix 'n' match of the two situations is not a good idea.

Peter

Prostate cancer and citrate and maybe omega 3s

A while ago, when I was looking through various publications from Chowdhury, I found this one: Prostate cancer cells over express mtG3P dehydrogenase. That's interesting. Why?

Normal prostate cells are special. They don't do the TCA. Glycolysis is fine. Pyruvate conversion to citric acid is also fine. Aconitase is not. Aconitase is deliberately inhibited by Zn retention and the citric acid of the citric acid cycle, which cannot be further metabolised in the said cycle, is then exported in to the prostatic fluid. In large amounts. Mitochondria are not used (much). This is hardly a recipe for over expression of mtG3P dehydrogenase.

Aside: I'm assuming the citrate is used to fuel the mitochondria of sperm. Simply dropping citrate on to the TCA of sperm looks like adding N2O/petrol injection to a standard saloon car engine. Maximum power output at the cost of maximum stress. Only the fastest get to the egg and only best survive the journey, which seems like a good idea when looking for the sperm with the best nuclear-mitochondrial match for fertilisation... End aside.

If we look at the paper on Zn, the TCA and mitochondria in prostate cancer (PCa) we can see that PCa cells lose Zn induced inhibition of aconitase and take off with a large supply of NADH from the TCA, a smidge of FADH2 through complex II and go towards that metastatic ratio of NAD+/NADH. Of course citrate concentration in semen plummets.

So PCa cells use the TCA and oxidative phosphorylation, ie they use mitochondria, to burn citrate derivatives. Normal prostate cells don't. Prostate cancer cells routinely perform beta oxidation. Not so normal prostate cells.

Equally interesting, as Loda's group point out, Fatty Acid Synthase (FAS) appears to be an oncogene in PCa cells. That, to me, suggests that while some of the citrate may well enter the TCA there is also a net synthesis of fatty acids outside the mitochondria. Fatty acid synthesis is a cytoplasmic process. Exported citrate provides acetyl CoA as the raw material for fatty acid synthesis.

BTW I don't doubt that prostate cells do use fatty acids in combination with "normal" levels of glycolysis, but Liu's fascinating paper here, supporting near exclusive fatty acid oxidation in PCa cells, is a classic example of stacking the deck to prove a point, with subtle transitions in graph labelling between tritiated 2-deoxy-glucose (an inhibitor of glycolysis!) and "glucose". There was no glucose, except the deoxy molecule. Oddly enough, glucose and 2-deoxy-glucose are not the same! While I'm completely accepting of the up-regulation of beta oxidation in this cancer, the near complete shutting down of glycolysis looks like pure artefact. They compare metabolic preference by looking at palmitate depletion from the palmitate-only culture medium, which is normal. Then they looked at 2-deoxy-glucose depletion from the 2-deoxy-glucose medium. The whole point of 2-deoxy-glucose is that, while it can be phosphorylated by hexokinase, further metabolism is completely blocked by the lack of hydroxyl group on the second carbon of the molecule. It may get taken up by cells, but it is never bulk metabolised. So it never gets depleted from the growth medium. Duh. I wonder if they expected this result...



I've also looked at Load's ideas about "futile cycling". This is the concept that acetyl CoA, from beta oxidation of fatty acids within the mitochondria, is exported as citrate to form cytosolic acetyl CoA to be converted to palmitate, which is re-imported in to the mitochondria to provide acetyl CoA to re-export as citrate.... Doesn't make sense to me. If you have functional mitochondria and a functional ETC, why bother if it's futile?

But we have seen something very similar in the past. FAS activation seems to be an important feature of TFAM knock out adipocytes. There is no functional complex I in TFAM knockout cell mitochondria and acetyl CoA provides limited FADH2. Without complex I you need FADH2 to drive the ETC, NADH won't hack it. Converting acetyl CoA from any source repeatedly to palmitate generates significant FADH2 during its re-oxidation. It's cycling, but it's not futile. You get something from it which you cannot normally get from pure acetyl CoA, so long as complex I is dysfunctional. Of course you get horrible levels of NADH too, but...


So you have to ask yourself: Do prostate cancer cells lack complex I? Logic says they must do.

Well, what do you know, Parr et al point out:

"For example, a 3.4∆ associated with PCa, removes the terminal region of ND4L, all of ND4, and nearly all of ND5 (Maki et al., 2008; Robinson et al., 2010)"

ie there is commonly a 3.4kb deletion of mtDNA which codes for a very large chunk of complex I in prostate cancer cells. This deletion, the paper suggests, appears to occur BEFORE the cells convert to aggressively cancerous forms.

So what cripples complex I? Well you could make all sorts of guesses about this, especially if you are a lipophobe. There is no doubt elevated free saturated fatty acids, in the presence of hyperglycaemia, will drive completely unreasonable numbers of electrons the wrong way through complex I and a great deal of collateral damage might well result from this process. If you have elevated FFAs you would be insane to raise your blood glucose level. "That's Mr Potato Head to you" (Toy Story 1).

How about simple hyperglycaemia? If you can generate enough free radicals from hyperglycaemia to induce some mitochondria functional you are then in a position to start using those mitochondria. Feeding through mtG3P dehydrogenase's FADH2 to the CoQ couple, while the NAD+/NADH ratio is horribly low from glycolysis, allows plenty of reverse electron flow when you really don't want it. For neurons, which don't do a great deal of beta oxidation, this is my guess for the extensive oxidative damage to complex I seen in PD and AD. Loss of complex I in a neuron, which doesn't do beta oxidation, is going to be disatrous. But in prostate cancer cells? Completely unreasonable superoxide generation appears to trash the mtDNA, as Parr pointed out. Conversion of citrate to fats allows survival under these conditions.

Now let me see, what did Chowdhury say about PCa cells and mtG3P dehydrogenase?????????? Up-regulated is the word. No cell is going to produce mtG3P dehydrogenase without functional mitochondria (and glycolysis) and mtG3P dehydrogenase bypasses a broken complex I, in a similar manner to electron transferring flavoprotein dehydrogenase does. Hyperglycaemia is an interesting concept for generating this cancer.






So....... Do PUFA, particularly omega 3 PUFA, give you prostate cancer? As per the suggestion from the observational association here. Probably not. No more than butter or FAS-produced palmitate give you prostate cancer. But PUFA are really quite special, certainly once the damage is done. They supply significantly less FADH2 input to the electron transport chain per molecule than saturated fats do under beta oxidation conditions, omega 3 PUFA being significantly worst than omega 6 PUFA. So here we have specific fats behaving as suppliers of NADH in rather higher amounts than saturated fats do and FADH2 in rather lower amounts. We have a lack of complex I in PCa cells, so supplying NADH is a recipe for metastasis and a poor fuel for the electron transport chain... In PCa cells acetyl CoA from PUFA is a sitting duck for export as citrate with conversion to palmitate and re-beta oxidation, to maximise FADH2 production. Oxidation of omega 3s via acetyl CoA and its subsequent synthesis and re oxidation as palmitate is not futile.

I have no issue with omega 3 fatty acids as signalling molecules, we clearly need some. I would be very cautious about bulk omega 3s, as I would about bulk omega 6s, as a source of calories.

We are looking here at a potential survival/growth mechanism in the behaviour of cells with severely damaged mitochondria, using any pathway they can to generate ATP. But thinking that it was the the omega 3 PUFA which broke the mtDNA in the first place might be a big mistake. Hyperglycaemia appears to be a far better recipe for mtDNA damage through hypercaloric insulin resistance, N-1a, reverse electron flow, etc gone to excess. PUFA are poor generators of FADH2 during beta oxidation so probably don't drive a lot of reverse electron transport through complex I. And never forget that even the bête noire of fatty acids, palmitate, is harmless in the face of normoglycaemia despite being an excellent generator of FADH2 and reverse flow.



Finally, Parr's group consider the damaged mitochondrial genome to be en-route to a situation where apoptosis becomes very difficult:

"As deletion-driven mtgenome depletion advances, cells become more resistant to cell death stimuli, in comparison to their parental cell lines (Cook and Higuchi, 2012), allowing proliferating cells to escape apoptotic control."

One step towards immortality for PCa cells, excepting the unfortunate destruction of their host organism.

Peter

Physiological insulin resistance again

I started the Protons thread with the simple question: What is the difference, from the metabolic point of view, between the energy supplied by fat vs that supplied by glucose derivatives.

This gives a simple picture of insulin resistance as a metabolic technique to limit caloric entry in to an individual cell under conditions of excess availability. NADH, tending to come from glucose, drives complex I to generate a decent inner mitochondrial membrane potential (delta psi). Feeding substrate in at other access points to the electron transport chain's CoQ couple, be that electron transporting flavoprotein dehydrogenase, mtG3Pdehydrogease, NADPH dehydrogenase or others, reduces that CoQ couple and promotes reverse electron flow through complex I, superoxide generation and insulin resistance. This is the insulin resistance seen so clearly when you pay folks to over-eat, assuming you feed them crapinabag. The exact mechanism of this failure of insulin to act is not clear, but large amounts of H2O2 act at several points to inhibit the activation pathway. Of course an intramitochondrial mechanism would be really neat, or some sort of complexing of the insulin/receptor with ETC proteins. Hard to say what we will find here in future, but an interesting area.

What about the insulin resistance of starvation? Do we have the same phenomenon of reverse electron flow through complex I as the mechanism?

So now we have to think about ketones with normolglycaemia. Back in my early days of looking at mitochondria I spent many hours with Veech's seminal paper on mechanical work generated by isolated rat hearts, pumping fat-free fluids spiked with glucose, ketones, glucose/insulin or glucose/insulin/ketones.

Ketones alone do exactly what maximal glucose/insulin do in terms of mechanical work, but by a completely different mechanism. Ketones produce a DROP in delta psi. This reduces uncoupling because there is a much lower voltage pushing protons back in to the mitochondrial matrix. This means that even with a lower delta psi ATP production is maximised (plus a few other changes) and so is the ability of the muscle to pump.

Insulin/glucose together maintain a high delta psi but modify the ETC proteins to improve efficiency, probably involving covalent bonding. I would assume phosphorylation is key.

Mechanical work was perfectly well maintained on ketones vs glucose/insulin, no need for a high delta psi with the ketones. Of course, no one is going to generate superoxide from complex I when the mitochondrial matrix is at a mere -120mV. To generate reverse electron flow it is the high value of delta psi which, when there is enough NADH per unit NAD+, puts an electron on to oxygen via FeS N-1a in complex I.

But under conditions of ketosis, be that ketogenic isocaloric eating or simple starvation, it is axiomatic that somatic insulin resistance is essential to spare adequate molecules of glucose for that little bit of brain metabolism which cannot be met by ketones alone. You need insulin resistance exactly when ketones remove the key driving potential needed for insulin resistance...

The trick lies in insulin activation. Insulin's action both generates and requires a small burst of superoxide. The superoxide is generated intramitochndrially by reverse electron flow through complex I. The superoxide is converted to H2O2 which diffuses to the cytoplasm where it inhibits the enzyme which normally deactivates the insulin/insulin receptor complex. With the reduced delta psi induced by pure ketones this is not going to happen, the insulin receptor rapidly deactivates and we have a simple mechanism for the physiological insulin resistance of ketosis/starvation.

To summarise: Superoxide in large amounts from complex I signals excess calories in the cell and inhibits insulin's action for cellular protection.

Superoxide in nano molar concentrations is essential for insulin's activation and is not made when ketosis lowers the potential across the inner mitochondrial membrane.

The two phenomena are both utterly essential and quite separate.

That makes me happy

There is a mass of detail of this process laid out in this paper H2O2 Signalling Pathway: A Possible Bridge between Insulin Receptor and Mitochondria. It makes interesting reading. I love the stuff on antioxidants causing insulin resistance and Ian recently resurfaced the old paper about supplementing with Vitamins C and E blunting exercise induced improvement in insulin sensitivity.

Bear in mimd that an awful lot of this work comes from tissue culture, transgenic mice, isolated mitochondria, all the usual suspects, so accept with caution.

But it makes sense to me

Peter

Crabtree and cardiovascular surgery

Just a brief update as it appears to relate to the last post on the problems of acute normoglycaemia, via Heartwire.

Sudden onset attempted normoglycaemia is not a good idea 48 hours before cardiovascular surgery.

Hard to say whether the strokes might be associated with exposing the Crabtree effect (mothballed mitochondria) or the risks of letting a surgeon loose with a bottle of insulin and a low fat diet, ie blood glucose <40mmol!

Peter

Chowdhury and Crabtree play with mitochondria

I still have a stack of comments needing a reply and a number of emails outstanding. Some awaaaaaay outstanding. Mea culpa, that's life. But this post has being lying around for so long that if I wait until all is tidy before I hit post it may never get posted at all, so here it is...



I have been reading a great number of Chowdhury's publications on diabetic neuropathy recently. I rather like his ideas and, although he limits his thinking to the NAD+/NADH ratio, his line of thought fits very well with the Protons thread and the FADH2/NADH ratio concept, which obviously influences NAD+/NADH. There is quite a lot of overlap and repetition within the recent papers as primary publications tend to blend with reviews. But a picture emerges about chronic hyperglycaemia and, very interestingly, of the problems from pathological relative or absolute hypoinsulinaemia.

In this paper [Aside: Note the association of DECREASED superoxide production with diabetes in the abstract, I rather like superoxide!] he mentions the Crabtree Effect. This is the fully reversible switch from a mixture of glycolysis and oxidative phosphorylation to almost pure glycolysis, classically seen in yeasts for alcohol production, when glucose supply is copious. Of course yeasts are fully able to dump their mitochondria completely, given enough sugar. A bit like certain types of cancer cells. Any cancer or yeast cell without mitochondria is utterly dependent on glycolysis for ATP production. These are the cancers which might well respond reliably to a ketogenic diet.

This is rather important as it explains why some cancer cells, which retain mitochondria but which show the Warburg effect in DMEM at 25mmol of glucose, might simply revert to oxidative phosphorylation on glucose restriction assuming FFAs or ketones are available. A nice Warburg/Crabtree paper is here and, while I've only had time to skim read it, it looks good. But I digress, back to Chowdhury:

He is primarily looking at neural failure in diabetes, from the bioenergetic and mitochondrial point of view. SIRT1, AMP kinase and good old PGC-1alpha are his areas of interest. This is from that first link:

"However, in the longer term, the high intracellular glucose concentration provides an ample supply of ATP via several nonmitochondrial-dependent pathways. Consequently, the metabolic phenotype of the cell adapts and functions in the absence of a dependence on the tricarboxylic acid cycle and oxidative phosphorylation for ATP production, possibly by initiating a process homologous to the “Crabtree effect” (35). Thus, rates of electron donation to the respiratory chain are suboptimal in neurons in long-term diabetic rats and may predispose to lower rates of mitochondrial respiratory chain activity and oxidative phosphorylation. Key metabolic activity sensors and/or regulators such as AMPK and NRF-1 are putative candidates for this modulation..."

and

"...our preliminary data demonstrated a significant reduction in activity of AMP kinase, a regulator of PGC1-α, in DRG [Dorsal Root Ganglia, cell bodies of sensory neurons] in type 1 diabetic rodents (manuscript in preparation)."


There is a nice summary of the metabolic sensors likely to be involved in his paper here. SIRT1 senses the NAD+/NADH ratio. Hyperglycaemia, in excess of insulin supply, depletes NAD+, increases NADH and, as a result, SIRT1 says to PGC-1alpha "Hey, no need for mitochonrdia, shut down mitochondrial biogenesis". Let's not forget nicotinic acid, NAD+, Hoffer and cancer. You could argue that nicotinic acid, by increasing NAD+, is an indirect activator of SIRT1... Of course SIRT1 controls a whole barrel of genes of great importance to health. I think FOXO has had many honourable mentions in comments by George. Also, no one should read SIRT1 without thinking of Cynthia Kenyon and her nematodes.

Chowdhury continues with AMP kinase, which can be viewed as a sensor looking at the ATP status of a cell, much as SIRT1 looks at the NADH status. If glycolysis is in overdrive there will be depleted AMP (also ADP and inorganic phosphate) as ADP gets converted by substrate level phosphorylation to ATP and needs replacing. The complexities of AMP, inorganic phosphate, ADP and ATP seem to be addresses in the Crabtree effect paper. If anyone wants to go there before me, feel free. But the simplistic picture is that excess ATP will act in a similar manner to excess NADH and both sensors interact with PGC-1alpha to say goodbye to mitochondrial biogenesis.

Dumping your mitochondria seems to be fine so long as you have continuous access to hyperglycaemic levels of glucose and can run on glycolysis. OK, I'll rephrase that: Dumping your mitochondria is an utter total complete disaster which is survivable, at a cost, so long as glucose is available in excess. Hyperglycaemia makes you hyperglycaemia dependent. If you really are running your nerve cells (and the rest of your body) on hyperglycaemia facilitated glycolysis then there appear to be a few follow on speculations available:

You get hungry if you lose your hyperglycaemia. I can remember being desperately hungry, in pre LC days, and being disappointed to see a blood glucose of 4.7mmol/l on the hand held glucometer at work. Hardly a low enough level to explain the driving hunger which I used to feel so commonly in those days...

Acute normalisation of glucose levels is going to make you feel utterly CRAP. This takes me back to the concept of "Atkins Flu" and J Stanton's musing as to its origin. The Crabtree effect is whole body, not limited to neurons. A dependence on hyperglycaemic glycolysis makes sudden onset normoglycaemia quite a shock. It's correctable, more rapidly in some people than others.

Then there is the phenomenon of initial worsening of retinopathy with acute onset normoglycaemia for diabetics. This has been noted in a couple of trials (link stolen from Jenny Ruhl). It's particularly worrying because it shows up in conventional "intensive therapy" for diabetes control, which is actually pretty poor in terms normalising blood glucose levels. So the risks are quite low compared to something like Bernstein's target of 4 or 5 mmol/l for blood glucose, 24-7, using an adequate protein, high fat, mildly ketogenic LC diet. For diabetes treatment you need something like this level of control for long term health. In the short term you seem to have to pay some sort of price to get yourself out of the hyperglycaemic corner you are stuck in. It looks like the Crabtree-like effect to me. Of course these short term problems pale compared to the long term benefits of normoglycaemia. But they are there.

Interestingly insulin, even without normoglycaemia, has some ameliorating effect on mitochondrial dysfunction in Chowdhury's lab models of diabetes. We might need to go back to Veech's early work on ketones vs glucose/insulin to take this further. This brings home nicely that diabetes, with relative or absolute hypoinsulinaemia is significantly worse than the earlier stage of impaired glucose tolerance, where hyperinsulinaemia still predominates.

So Chowdhury's ideas give some nice pointers as to why chronic hyperglycaemia is so bad. In his lab animals he is mostly looking at 22 weeks of streptozotocin diabetes to get the mitochondrial pathology, you can't pick up the changes to p<0.05 at 16 weeks. So we really are looking at long term neural damage in his papers.

Whatever the effects of acute hyperglycaemia, chronic hyperglycaemia does your mitochondria no good at all.

I rather like my mitochondria...

Peter

Food: Burgers

OK, buy some cheap beef mice [OMG should say mince!], the fattier the better. There's 600g in six completed burgers here. Add about 100g of grated cheddar cheese, extra mature. One whole egg binds it nicely. Include one very small onion, very finely chopped. Now here's the dangerous bit. You can add salt AND pepper. Oh oh, that old "one spice two spice" obesogenic effect, could be in trouble here.

Now here's the next trick: Instead of making the usual six burgers, make 12 very thin burgers. Put a decent square of feta cheese in the middle of one burger, cover with a second one, squish the edges to make one double thickness burger, enclosing the feta. Make six double thickness feta-enclosing burgers.

Now. Burn them. AGE the surface... Glycate those amino acids over glowing charcoal.



Two is more than enough, for me anyway. I don't care how good they taste, I manage two and have pigged out. I guess polishing off the left over feta during preparation may have something to do with this!

We have a plan to add shredded tarragon or fennel leaves to the next batch. Salt, pepper AND a herb. Livin' dangerously here.

Sorry for no chance to reply to emails or comments. Too busy barbecuing after a heavy weekend on call.

Peter

Diagnosing and Treating Vitamin B12 Deficiency video

Just briefly: People know I don't have the sort of life which leads to watching YouTube videos. This is a long one. I watched it all when I should have been at comments on the blog or answering emails (oops).

Diagnosing and Treating Vitamin B12 Deficiency

I have a (severely coeliac) friend who has recently, and rather belatedly, been diagnosed with catastrophic neurological B12 deficiency. The biggest problem with getting a diagnosis in most cases is folic acid supplementation. Folate eliminates the anaemia associated with the term "pernicious anaemia". Your haematology is normal. Your myelin falls to pieces. Your doctor diagnoses all sorts of things except the one which might avoid neurological melt down.

As a vet I use serum low B12/folate as surrogate markers for GI malabsorption problems and, in these days of crapinabag, we do a lot of testing. In my patients B12 deficiency is very, very common and we probably miss a fair number of "atypical" B12 deficiencies. We treat the ones we see. It's a good idea.

Peter

BTW I've never seen a low folate in a cat or dog. They do happen, but B12 is the very common one.

EDIT WTF, Paula had a low folate, normal B12 result from a dog today. Upper GI signs and vomiting. Well, there you go! Bed time now. END EDIT

BTW two, myelin failure as a loss of insulation is self explanatory. But the schwann cells also supply the lactate for normal neurological energy generation. Loss of lactate is a metabolic catastrophe for nerve cells.