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.

Peter

17 comments:

George Henderson said...

This is great Peter.
you might even be able to make sense of this:
http://www.ncbi.nlm.nih.gov/pubmed/18093684
Why does replacing some corn oil with MCTs make NASH worse (but replacing them all makes it much better)?

Does the unusual energy arrangement in prostate cells have anything to do with the fructose requirement of sperm?
I think of certain high-CICO bee workers using honey for hive-warming purposes, or hummingbirds buzzing on pollen.

M said...

Peter, in case you don't have a PDF of the book "Pure, White & Deadly", and would like one, here is a download. I've downloaded it and ran it through the scan and it contains no virus.

https://docs.google.com/open?id=0B2FsXfJXQyd2QnNWYWpHQVJfLXM

George Henderson said...

M, that's a great book. I also have The Penguin Encyclopedia of Nutrition, 1985, which is witty and wise and where Yudkin nods to Paleo with sections on Neolithic Revolution and Dietary Instinct.
I'd like to see everything Yudkin wrote republished with introductions by various modern experts.
Especially This Slimming Business, if anyone knows where that is online.
I can only find this:
http://www.ncbi.nlm.nih.gov/pubmed/13787548
:(

@ Peter - humming birds drink nectar of course, not pollen.

With regard to citrate as fuel, remember that malate is touted as helpful in fibromyalgia because NADH is immediate TCA product.
And NADH supplements are useful in some brain disorders - is it MS? And would NAD+ be as useful, or more so?

George Henderson said...

NADH as a medicine:
http://www.nadh-apotheke.eu/NADH-Studien/THE%20ENERGIZING%20EFFECT%20OF%20NADH-COENZYME.pdf

randian said...

Completely unreasonable superoxide generation appears to trash the mtDNA

I probably misunderstand this, so here goes: does this imply that metformin, which generates high superoxide levels (as I understand another article here to mean), causes DNA damage?

George Henderson said...

"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."

I always wondered how and why fruit becomes full of citric acid and other TCA intermediates. It doesn't seem like the sort of thing that should be full of mitochondria. This kind of thing helps to resolve that old mystery.
oh, and fruit > seed > sperm - there's even a theme of sorts there.

Peter said...

Thanks M, have downloaded. I did start scanning my copy but it was going to take waaaay more time than I had available.

George, about a year ago I wanted to get on to PUFA, iron overload, copper overload and what a poor hepatocyte can do with more calories than it wants yet it fails to generate insulin resistance. Might try and link the two ideas together...

I can see NAD+ precursors being helpful. I wonder how much good NADH per se would be without being pre-reduced to NAD+. Possible, but I'd like to see some sort of esoteric model of the sort I love!

radian, I think it may be a matter of degree and duration. I'd have to go back to the original protons threads and check out succinate generated superoxide, palmiate superoxide and metformin superoxide. The trouble is you end up looking at different numbers from different papers, different experimental set ups and different methods of measurement of ROS. Metfomin certainly generated free radicals in the short term, what happens after a week or two when intracellular lipids are depleted and insulin sensitivity improves is an interesting set of questions which may not be direct follow ons from the acute effects. AMP kinase does, without a doubt, upregulate and this will have all sorts of non-acute follow ons. There are a whole lot of similar questions as regards degree about hyperglycaemia per se which activates a whole stack of antioxidant processes in the cytoplasm which effectively wipe out the insulin activation signal. You have a ton of H2O2 in the cytoplasm which both causes insulin resistance at high doses combined with the response of antioxidant systems which, while stabilising the redox state under hyeprglycaemia, concurrently obliterate insulin activation once we really want it to work.

Long term it seems to be the overactivity of the antioxidant systems and associated failure of insulin activation which are the bug bear in neurodegeneration.

Probably elsewhere too.

Peter

Rocket said...

Thanks, Peter.

I am into health and endurance sports. I greatly appreciate articles and enjoy your writing style. I find your posts adds to my understanding of metabolism.

-Robert Martin.

George Henderson said...

Re: the hummingbird's metabolism:

Metabolic sources of energy for hummingbird flight 1986

It has been known for some two decades that hovering flight in hummingbirds is the most energetically expensive muscle work known among vertebrates, but the metabolic support for such work has never been clarified. Measurement of the maximum activities of key enzymes of carbohydrate, fat, and amino acid catabolism in flight muscle and heart of rufous hummingbirds (Selasphorus rufus) reveals that the high ATP requirements of short-term hovering flight can only be supported by the oxidation of carbohydrate. Fat oxidation can support a substantially lower maximum rate of ATP turnover, indicating that this process can power only the lower +.++energetic requirements of long-term forward or migratory flight. Mitochondria isolated from flight muscle oxidize pyruvate and palmitoyl-CoA equally well. The inhibition of pyruvate oxidation by palmitoyl-CoA oxidation provides a mechanism by which fat oxidation inhibits carbohydrate oxidation in the transition from short- to long-term flight.

http://ajpregu.physiology.org/content/251/3/R537

Zorica Vuletic said...

"Metfomin certainly generated free radicals in the short term, what happens after a week or two when intracellular lipids are depleted and insulin sensitivity improves is an interesting set of questions which may not be direct follow ons from the acute effects. AMP kinase does, without a doubt, upregulate and this will have all sorts of non-acute follow ons. There are a whole lot of similar questions as regards degree about hyperglycaemia per se which activates a whole stack of antioxidant processes in the cytoplasm which effectively wipe out the insulin activation signal. You have a ton of H2O2 in the cytoplasm which both causes insulin resistance at high doses combined with the response of antioxidant systems which, while stabilising the redox state under hyeprglycaemia, concurrently obliterate insulin activation once we really want it to work.

Long term it seems to be the overactivity of the antioxidant systems and associated failure of insulin activation which are the bug bear in neurodegeneration."

My understanding is that this is saying that long term metformin use obliterates insulin production (or is it insulin sensitivity)? How long term is long term? Is it only at excessive doses?

I ask b/c my mom uses metformin for the past ~8 years now. Thanks.


". Measurement of the maximum activities of key enzymes of carbohydrate, fat, and amino acid catabolism in flight muscle and heart of rufous hummingbirds (Selasphorus rufus) reveals that the high ATP requirements of short-term hovering flight can only be supported by the oxidation of carbohydrate. Fat oxidation can support a substantially lower maximum rate of ATP turnover, indicating that this process can power only the lower +.++energetic requirements of long-term forward or migratory flight. Mitochondria isolated from flight muscle oxidize pyruvate and palmitoyl-CoA equally well. The inhibition of pyruvate oxidation by palmitoyl-CoA oxidation provides a mechanism by which fat oxidation inhibits carbohydrate oxidation in the transition from short- to long-term flight."

I know we are not humming birds, but the idea of being in ketosis during long distance (ultra-marathon as opposed to regular marathons) is quite beneficial and for HIT type activities, more glucose is required. Fascinating.

I also believe that for cows, although they eat grass/vegetation, and this is carbohydrate, that it is converted to fatty acid (ends up being processed in a fat fuel) is also quite interesting.

I don't believe it's wise to copy the diets of other species, but the mechanisms under which work in other species seems quite appropriate in human contexts.

Peter said...

Hi Zorica,

Metformin, however it pans out long term, produces enough free radicals/depletes enough ATP to improve the health of diabetics. It is one of the few drugs ever to have profound anticancer effects long term. The correct amount of free radical production maximises mitochondrial biogenesis. Long term studies don't seem to be available in similar vein to the short term ones for metformin. Benefits of metformin seem to kick in after a few weeks, in people...

Respiration, and by this I think we mean oxygen consumption, is key to life extension in many models. Fat requires more oxygen per unit ATP generation than carbohydrate. That looks like a Good Thing to me. I have an interesting paper by Cynthia Kenyon to discuss. Other models of life extension have genetically DAMAGED respiratory chains which respire very poorly. Life is complex.

Pete

randian said...

Is free radical production part of the mechanism of action of metformin? Does that mean that taking, say, large doses of vitamins C or E (both potent antioxidants) interferes with the action of metformin?

Peter said...

C and E certainly blunt the beneficial effects of exercise. Why not those of metformin?

Peter

Zorica Vuletic said...

"C and E certainly blunt the beneficial effects of exercise..."


Is this only in high doses and not coming from diet?

Peter said...

http://www.pnas.org/content/106/21/8665.full

1000mg C 400iu E, as supplements. I can live with food content of these antioxidants but not as supplements...

Peter

Sam said...

Hey Peter

Can you explain a little what you mean by "take off with a large supply of NADH from the TCA" ? I dont have access to the fulltext of the paper. Do you mean the NADH (and NAD) ends up lost in semen or is metabolised to niacinamide and not recycled fast enough in the salvage pathway or ?

Thx

Peter said...

What I was driving at is that prostate cells would normally generate far more citrate than they need because it is usually exported in to the prostatic fluid. This appears to come from the first step of the TCA, using Zn to inhibit aconite. So if the cell is set up to meet it’s energy needs largely through glycolysis, without using it’s Zn-inhibited TCA, suddenly turning the TCA by loss of Zn inhibition will generate large amounts of NADH, for which the cell has no use. The ratio of NADH to NAD ratio appears to determine the malignancy, i.e. the cell has far more energy to grow with than it needs. Converting the NADH to NAD+ needs ox phos and will generate enlarge amounts of ATP in the process. The phenotype is a glycolysis dependent cell which cannot stop the glycolysis when presented with mitochondrial biogenesis.

Peter