Thursday, August 29, 2013

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:


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.


Sunday, August 25, 2013

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.

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.



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.

Thursday, August 08, 2013

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.


Monday, August 05, 2013

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.