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
Monday, July 22, 2013
Tuesday, July 02, 2013
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
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
Sunday, June 23, 2013
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
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
Monday, June 03, 2013
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
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
Tuesday, May 28, 2013
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.
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.
Tuesday, May 21, 2013
What do I eat? 2013 update
OK, what do I eat? It's been a long time since I posted anything about myself. Another hastily written post during Hazel's nap.
Breakfast is always the same. I melt about 100g of butter in a frying pan. I crack 11 eggs yolks in to this (I fry the whites later for the chickens!) and fry them until they hold their shape. I then pour 8 yolks for myself and Hazel, with all of the free butter, in to a bowl and mash them with a fork before stirring the mix in to an "egg soup". Hazel has a dollop, I have the rest. We like it quite solid. I finish any Hazel doesn't want, hopefully I get six yolks worth. Daniel likes his yolks just fried, he has the other three.
If I feel like it, I have a creamy cocoa, ie about 4g cocoa powder, 2ml honey, maybe somewhere between 30 and 60ml double cream. Usually a decaff coffee too. Sometimes with caffeine, though I tend to prefer my caffeine in the evenings.
Lunch on work days is portable stearic acid as 100g of Lindt 90% cocoa solids chocolate. Sometimes with 100g macadamias, sometimes not. Occasionally 300ml of soured cream along side some chocolate.
Supper is a meal. It varies a lot. If you scroll down the index to the set of posts starting with "Food" you get the idea of the sort of things I eat for a main meal. I have been known to eat green leaves with supper. I have been known to eat parsnip chips. I have been known to have gluten free home made cake for desert under a centimetre of butter or drowned in soured cream. If fat has been a little low with supper I might make ice cream with just a little added sugar. Or rhubarb baked in cream and flavoured with cinnamon. Or another creamy cocoa.
Alcohol, some. I like dry wine and gluten free beer. Never a huge amount.
I don't weigh any food nowadays (I went through the Fitday kick years ago), especially since the digital scales passed away. I weigh myself about once a year. My jeans are 28" waist, depending where I buy them.
There are oddities that need to be borne in mind, ie that I have never been overweight. I eat this way as a result of an accident at an anaesthesia meeting, chatting with a friend who had dropped from 18 stone to 12 stone on Atkins induction. I had just discovered Pubmed and devoured studies voraciously.
It didn't take long to realise that Kwasniewski's Optimal Diet was where I wanted to go. I don't eat enough offal and I don't make enough bone broths, but otherwise I keep fairly close. My biggest transgression is excess protein.
Quite why excess protein is bad is interesting. If you read the DNA chapter in Nick Lane's "Life Ascending" it brings home quite how closely integrated amino acid metabolism is to the TCA. It's way too complex to see how any given protein would interact, so aiming for high grade low quantity is how I work, when practical. If you eat more than you need it goes in to the TCA.
In my Fitday days I used to run at around 2000kcal/day for weight stability but I probably run a fair bit higher than than nowadays, most days. But no two days are the same and there is far too much to enjoy in life without agonising over +/- 5g of butter.
Is it worth it? Well, I sort of forget that eating this way is odd. You have to giggle at the nurses with their little tubs of salad and half an apple but they're just kiddies... But I went from being early middle aged to pretty much how I felt as a teenager, once I had adapted (full adaptation took me about 6 months). I don't feel I have changed much, beyond the needs of a second family, in the last 10 years healthwise. Beard is a little greyer.
I feel well. You sort of forget what it used to be like to need to eat NOW. And to fall asleep for three hours after a huge bowl of rice before being awake half the night. Anyone with a bad back will be very familiar with using one foot to pull your underwear up the other leg to where you can actually reach it without bending down. All gone, along with the little pot belly.
I have no expectation of living for ever. But I have no intention to going back to where I was at 40 years of age. Aging un-noticed would be nice. When you find something which works this well you don't throw it away.
Peter
Breakfast is always the same. I melt about 100g of butter in a frying pan. I crack 11 eggs yolks in to this (I fry the whites later for the chickens!) and fry them until they hold their shape. I then pour 8 yolks for myself and Hazel, with all of the free butter, in to a bowl and mash them with a fork before stirring the mix in to an "egg soup". Hazel has a dollop, I have the rest. We like it quite solid. I finish any Hazel doesn't want, hopefully I get six yolks worth. Daniel likes his yolks just fried, he has the other three.
If I feel like it, I have a creamy cocoa, ie about 4g cocoa powder, 2ml honey, maybe somewhere between 30 and 60ml double cream. Usually a decaff coffee too. Sometimes with caffeine, though I tend to prefer my caffeine in the evenings.
Lunch on work days is portable stearic acid as 100g of Lindt 90% cocoa solids chocolate. Sometimes with 100g macadamias, sometimes not. Occasionally 300ml of soured cream along side some chocolate.
Supper is a meal. It varies a lot. If you scroll down the index to the set of posts starting with "Food" you get the idea of the sort of things I eat for a main meal. I have been known to eat green leaves with supper. I have been known to eat parsnip chips. I have been known to have gluten free home made cake for desert under a centimetre of butter or drowned in soured cream. If fat has been a little low with supper I might make ice cream with just a little added sugar. Or rhubarb baked in cream and flavoured with cinnamon. Or another creamy cocoa.
Alcohol, some. I like dry wine and gluten free beer. Never a huge amount.
I don't weigh any food nowadays (I went through the Fitday kick years ago), especially since the digital scales passed away. I weigh myself about once a year. My jeans are 28" waist, depending where I buy them.
There are oddities that need to be borne in mind, ie that I have never been overweight. I eat this way as a result of an accident at an anaesthesia meeting, chatting with a friend who had dropped from 18 stone to 12 stone on Atkins induction. I had just discovered Pubmed and devoured studies voraciously.
It didn't take long to realise that Kwasniewski's Optimal Diet was where I wanted to go. I don't eat enough offal and I don't make enough bone broths, but otherwise I keep fairly close. My biggest transgression is excess protein.
Quite why excess protein is bad is interesting. If you read the DNA chapter in Nick Lane's "Life Ascending" it brings home quite how closely integrated amino acid metabolism is to the TCA. It's way too complex to see how any given protein would interact, so aiming for high grade low quantity is how I work, when practical. If you eat more than you need it goes in to the TCA.
In my Fitday days I used to run at around 2000kcal/day for weight stability but I probably run a fair bit higher than than nowadays, most days. But no two days are the same and there is far too much to enjoy in life without agonising over +/- 5g of butter.
Is it worth it? Well, I sort of forget that eating this way is odd. You have to giggle at the nurses with their little tubs of salad and half an apple but they're just kiddies... But I went from being early middle aged to pretty much how I felt as a teenager, once I had adapted (full adaptation took me about 6 months). I don't feel I have changed much, beyond the needs of a second family, in the last 10 years healthwise. Beard is a little greyer.
I feel well. You sort of forget what it used to be like to need to eat NOW. And to fall asleep for three hours after a huge bowl of rice before being awake half the night. Anyone with a bad back will be very familiar with using one foot to pull your underwear up the other leg to where you can actually reach it without bending down. All gone, along with the little pot belly.
I have no expectation of living for ever. But I have no intention to going back to where I was at 40 years of age. Aging un-noticed would be nice. When you find something which works this well you don't throw it away.
Peter
Monday, May 20, 2013
A bit more on ketones and diabetic nephropathy
Just in haste, sorry for the missed typos! Liz sent me the full pdf of this paper:
Treatment of Diabetes and Diabetic Complications With a Ketogenic Diet
It's primarily a review, both of the work by Phinney and Westman and of the lab animal studies by the group producing the review.
Deranged glucose metabolism appears to be what causes the problems in diabetes, type 1 or 2. More on this when I get back to the chronic hyperglycaemia post. It's not forgotten.
Ketones, the metabolism of which remains normal, appear to be key in replacing abnormal glucose metabolism, more so than simply achieving normoglycaemia without ketosis.
Protein limits ketosis and the use of low carbohydrate diets with unrestricted protein intake may explain some of the failures to maintain the benefits of carbohydrate restriction. I have to say, Jimmy Moore comes to mind here. I like his success with ketosis.
It looks to be as easy to substantially reverse diabetic nephropathy in mice as it is to limit nephropathy progression in humans, but ketones are essential.
Mouse, human, diabetic nephropathy, reversal with ketosis. Shrug.
The take home message is very simple. If you have severe metabolic problems the answer is not simply carbohydrate restriction. It also involves protein limitation, to adequate but not gluconeogenic levels. Low carb, adequate protein, high fat. With the emphasis on the fat.
Taterism is fine for any Tato Head without metabolic problems. As everyone will eventually develop metabolic problems, so Taterism will eventually injure everyone. Some folks probably have dialysis blood on their hands already.
The high fat brigade are correct.
Peter
Treatment of Diabetes and Diabetic Complications With a Ketogenic Diet
It's primarily a review, both of the work by Phinney and Westman and of the lab animal studies by the group producing the review.
Deranged glucose metabolism appears to be what causes the problems in diabetes, type 1 or 2. More on this when I get back to the chronic hyperglycaemia post. It's not forgotten.
Ketones, the metabolism of which remains normal, appear to be key in replacing abnormal glucose metabolism, more so than simply achieving normoglycaemia without ketosis.
Protein limits ketosis and the use of low carbohydrate diets with unrestricted protein intake may explain some of the failures to maintain the benefits of carbohydrate restriction. I have to say, Jimmy Moore comes to mind here. I like his success with ketosis.
It looks to be as easy to substantially reverse diabetic nephropathy in mice as it is to limit nephropathy progression in humans, but ketones are essential.
Mouse, human, diabetic nephropathy, reversal with ketosis. Shrug.
The take home message is very simple. If you have severe metabolic problems the answer is not simply carbohydrate restriction. It also involves protein limitation, to adequate but not gluconeogenic levels. Low carb, adequate protein, high fat. With the emphasis on the fat.
Taterism is fine for any Tato Head without metabolic problems. As everyone will eventually develop metabolic problems, so Taterism will eventually injure everyone. Some folks probably have dialysis blood on their hands already.
The high fat brigade are correct.
Peter
Wednesday, May 15, 2013
A Peek at Paleo
I don't eat a paleo diet, I'm just a saturophile. If it's saturated fat and it happens to come from a neolithic block of butter, that's fine by me. But I hadn't realised going paleo could be actively worse for your health than eating some version of the SAD. You can download Eric Trexler's thesis from here. Catchy title is:
"Paleolithic Diet is Associated With Unfavorable Changes to Blood Lipids in Healthy Subjects"
Some people may have noticed that I have minimal interest in blood lipid levels. I know people angst about them, but I've yet to be convinced that they have anything to do with heart disease other than as a surrogate for how much sugar you eat.
The thesis reports pre and post diet lipids but only gives end-of-paleo-diet food breakdown, and only the fat/saturated fat at that. Go figure. Guess they forgot to ask what folks were eating to have better lipids than their study diet was going to produce! Perhaps they had a defective crystal ball.
I've always viewed HDL as a surrogate for saturated fat intake. The sub population with the best HDL on pre diet lifestyle (around 82mmol/l) dropped it to around 69mmol/l on paleo diet plus exercise and weight loss. My assumption is that these folks actually dropped their saturated fat intake or increased their PUFA intake by so much that even the exercise induced rise in HDL and weight loss induced rise in HDL couldn't offset the fall in HDL induced by the study diet. Impressive.
Triglyceridess rose non significantly. I view trigs as a surrogate for sugar intake. You have to guess how much SAD high fructose corn syrup was replaced by paleo fruit. Or whether fruit juice [Peter vomits quietly in the corner] was allowed. Well, the trigs went up (slightly), not down... Gathering was good that day, every day, for 10 weeks!
OK, so what sort of a paleo diet was this? Quote:
"Subjects were advised to increase their consumption of lean meat [Peter vomits quietly in the corner again], fish, eggs, nuts, fruit, and vegetables and were instructed to strictly avoid all grains, dairy products, and legumes."
Obviously spuds appear to have been on the menu in paleoland and animal fat is the devil incarnate!
What was their source material for these well thought out recommendations? They were based on Eaton and Konnor's 1985 paper:
"Paleolithic nutrition. A consideration of its nature and current implications"
You can't get at the full text or even an abstract on line. Luckily Anna, over at Lifextension, fills in the details for us. She pasted a copy of her information over on ItsTheWoo's blog here, explaining both where Eaton got the data and pointing out the 2000 correction he published, amending his paleo fat intake estimates (upwards of course). I get a faint impression that Anna may not be best impressed by Eaton's ideas. Or by Taterism in general. BTW, did anyone run through the list of references? Given a year or two I might try one day, but perhaps just sticking with simple saturophilia might be easier.
I rather like Anna's commentary. I like her suggestion that Eaton's ideas seem uncomfortably influenced by politically correct beliefs aligned with the AHA's diet advice. Reading Trexler's thesis I was also struck that it could easily have been written by an AHA cardiologist. The naked fear of LDL cholesterol shines through the whole text.
My take home message is that if you are going to align your paleolithic diet advice with the AHA, people are going to get hurt.
Peter
"Paleolithic Diet is Associated With Unfavorable Changes to Blood Lipids in Healthy Subjects"
Some people may have noticed that I have minimal interest in blood lipid levels. I know people angst about them, but I've yet to be convinced that they have anything to do with heart disease other than as a surrogate for how much sugar you eat.
The thesis reports pre and post diet lipids but only gives end-of-paleo-diet food breakdown, and only the fat/saturated fat at that. Go figure. Guess they forgot to ask what folks were eating to have better lipids than their study diet was going to produce! Perhaps they had a defective crystal ball.
I've always viewed HDL as a surrogate for saturated fat intake. The sub population with the best HDL on pre diet lifestyle (around 82mmol/l) dropped it to around 69mmol/l on paleo diet plus exercise and weight loss. My assumption is that these folks actually dropped their saturated fat intake or increased their PUFA intake by so much that even the exercise induced rise in HDL and weight loss induced rise in HDL couldn't offset the fall in HDL induced by the study diet. Impressive.
Triglyceridess rose non significantly. I view trigs as a surrogate for sugar intake. You have to guess how much SAD high fructose corn syrup was replaced by paleo fruit. Or whether fruit juice [Peter vomits quietly in the corner] was allowed. Well, the trigs went up (slightly), not down... Gathering was good that day, every day, for 10 weeks!
OK, so what sort of a paleo diet was this? Quote:
"Subjects were advised to increase their consumption of lean meat [Peter vomits quietly in the corner again], fish, eggs, nuts, fruit, and vegetables and were instructed to strictly avoid all grains, dairy products, and legumes."
Obviously spuds appear to have been on the menu in paleoland and animal fat is the devil incarnate!
What was their source material for these well thought out recommendations? They were based on Eaton and Konnor's 1985 paper:
"Paleolithic nutrition. A consideration of its nature and current implications"
You can't get at the full text or even an abstract on line. Luckily Anna, over at Lifextension, fills in the details for us. She pasted a copy of her information over on ItsTheWoo's blog here, explaining both where Eaton got the data and pointing out the 2000 correction he published, amending his paleo fat intake estimates (upwards of course). I get a faint impression that Anna may not be best impressed by Eaton's ideas. Or by Taterism in general. BTW, did anyone run through the list of references? Given a year or two I might try one day, but perhaps just sticking with simple saturophilia might be easier.
I rather like Anna's commentary. I like her suggestion that Eaton's ideas seem uncomfortably influenced by politically correct beliefs aligned with the AHA's diet advice. Reading Trexler's thesis I was also struck that it could easily have been written by an AHA cardiologist. The naked fear of LDL cholesterol shines through the whole text.
My take home message is that if you are going to align your paleolithic diet advice with the AHA, people are going to get hurt.
Peter
Sunday, April 28, 2013
Hyperglycaemia is bad
Hyperglycaemia does whatever you want it to. Want to show it increases glycolysis and/or oxidative phosphorylation? No problem. Want to show it decreases both? Equally no problem. Choose your tissue, choose your duration, choose your insulin level, choose your glucose level, choose your tissue culture medium before test, choose... With the correct combination you can show anything.
But certain patterns emerge from lots of papers. In the short term hyperglycaemia increases both glycolysis and oxidative phosphorylation. Acute hyperglycaemia in neurons induces an equally acute hyperpolarisation of the inner mitochondrial membrane (a pre requisite for reverse electron flow through complex I), followed by a burst of free radicals (from reverse electron transport in the face of a low NAD+/NADH ratio?), followed by a collapse of the inner mitochondrial membrane potential (from free radical induced loss of cytochrome c?), soon to be followed by apoptosis, as you might expect
These guys set out the events nicely but suggest the mechanism is unclear. I would be willing to bet on G-3-P dehydrogenase as driving reverse electron flow using the high membrane potential from glycolysis. It seems that, under "mitochondrial preparation" conditions, ignoring reverse electron flow, G-3-P dehydrogenase also spills a reasonable dose of free radicals not only inwards towards the matrix but also outwards to the inter membrane space, in roughly equal amounts. As does complex III of course, but complex III is not specifically driven by a short side branch of hyperglycaemia-induced hyperactive glycolysis. Goodness only knows if this happens in-vivo, but let's accept that it does. Cytochrome c is on the outer surface of the inner mitochondrial membrane and spilling free radicals outwards seems a good way to oxidise the cardiolipin anchors and release one of the most important pro apoptotic proteins we have, cytochrome c.
So acute hyperglycaemic injury, in a cell type where glucose entry is essentially concentration driven, is potentially apoptotic if the injury is severe enough. Lesser but sill significant injury may come from spills of superoxide from complex I on to the mitochondrial DNA, another potentially interesting effect. Research on G-3-P dehydrogenase is still in its infancy and there are no clear cut answer as to how important this scenario might be, but I rather like it. Is it true? Who knows. It's hard to tell.
Exactly how difficult it is to transfer information from "preparations" to any semblance of "in vivo" is reviewed by Martin Brand. I like this chap, he really looks at the limitations of how much we currently know (not much, it appears) plus he came up through Naked Mole Rat research, another positive. Here's his summary of where free radicals might be produced:
Outwards spillage, directly on to cytochrome c, from G-3-P dehydrogenase and complex III...
It's quite clear that hyperglycaemia is not invariably acutely fatal to all neurons on first exposure. It takes years of following the advice of the ADA and AHA to develop diabetic neuropathy or to kill off enough central neurons (around 70%) to get the clinical label of Alzheimers and, while recurrent hyperglycaemia might get us there directly, the indirect effects are much more interesting to a mitochondriac like myself.
Chronic hyperglycaemia is where we have a depressed inner mitochondrial membrane potential, reduced glycolysis and electron transport with subsequent failure to generate superoxide.
Badness too.
Peter
But certain patterns emerge from lots of papers. In the short term hyperglycaemia increases both glycolysis and oxidative phosphorylation. Acute hyperglycaemia in neurons induces an equally acute hyperpolarisation of the inner mitochondrial membrane (a pre requisite for reverse electron flow through complex I), followed by a burst of free radicals (from reverse electron transport in the face of a low NAD+/NADH ratio?), followed by a collapse of the inner mitochondrial membrane potential (from free radical induced loss of cytochrome c?), soon to be followed by apoptosis, as you might expect
These guys set out the events nicely but suggest the mechanism is unclear. I would be willing to bet on G-3-P dehydrogenase as driving reverse electron flow using the high membrane potential from glycolysis. It seems that, under "mitochondrial preparation" conditions, ignoring reverse electron flow, G-3-P dehydrogenase also spills a reasonable dose of free radicals not only inwards towards the matrix but also outwards to the inter membrane space, in roughly equal amounts. As does complex III of course, but complex III is not specifically driven by a short side branch of hyperglycaemia-induced hyperactive glycolysis. Goodness only knows if this happens in-vivo, but let's accept that it does. Cytochrome c is on the outer surface of the inner mitochondrial membrane and spilling free radicals outwards seems a good way to oxidise the cardiolipin anchors and release one of the most important pro apoptotic proteins we have, cytochrome c.
So acute hyperglycaemic injury, in a cell type where glucose entry is essentially concentration driven, is potentially apoptotic if the injury is severe enough. Lesser but sill significant injury may come from spills of superoxide from complex I on to the mitochondrial DNA, another potentially interesting effect. Research on G-3-P dehydrogenase is still in its infancy and there are no clear cut answer as to how important this scenario might be, but I rather like it. Is it true? Who knows. It's hard to tell.
Exactly how difficult it is to transfer information from "preparations" to any semblance of "in vivo" is reviewed by Martin Brand. I like this chap, he really looks at the limitations of how much we currently know (not much, it appears) plus he came up through Naked Mole Rat research, another positive. Here's his summary of where free radicals might be produced:
Outwards spillage, directly on to cytochrome c, from G-3-P dehydrogenase and complex III...
It's quite clear that hyperglycaemia is not invariably acutely fatal to all neurons on first exposure. It takes years of following the advice of the ADA and AHA to develop diabetic neuropathy or to kill off enough central neurons (around 70%) to get the clinical label of Alzheimers and, while recurrent hyperglycaemia might get us there directly, the indirect effects are much more interesting to a mitochondriac like myself.
Chronic hyperglycaemia is where we have a depressed inner mitochondrial membrane potential, reduced glycolysis and electron transport with subsequent failure to generate superoxide.
Badness too.
Peter
Wednesday, April 24, 2013
Axen and Axen (4) Ketogenic insulin resistance. It's all over now...
I have so many posts I want to get finished, all of which are inter-related and all of which need waaaaaay too much work, that I thought I would just throw this one out in the interim. I began with this paper which came as a pdf from Liz. While I was getting the pubmed link to it I noticed the same group had another rather similar paper out which was equally interesting and then the third link down the page was an accidental find which is this one, subject of this post.
I don't know if it's worth going through the figures individually, they are very similar to those from Axen and Axen which produced a series of posts a few years ago, except that the feature of COMPLETE reversal of insulin resistance is, here, presented right up front in Figure 6 and in the abstract too:
That figure for insulin looks a little dubious at 120 minutes but I'll let that go, I guess p was still > than 0.05... Pretty close to full reversal.
It's quite hard to know exactly how much this group understand about their results. They give roughly equal weight to the adverse (sic) effects of a ketogenic diet as they do to the fact it is reversible within a week (or less, they only checked at a week) of re-introducing carbohydrate.
What they seem to lack is the concept that rats fed a very restricted carbohydrate diet MUST be insulin resistant to survive, as happens in starvation. But maybe they are creeping towards some sort of understanding. It's about time. Good.
When people cite Axen and Axen to prove ketogenic diets are going to make you diabetic (there are folks who believe this, or at least wish you to believe it!) you have an answer in Kinzig et al 2010.
BTW, the links which led me here relate to using ketogenic diets to control both pain and inflammation. This is a potentially very useful tool but the beneficial effect does appear to be as rapidly reversible as the physiological insulin resistance... Ketogenic diets are a fix, not a cure (in the short term anyway). But inflammation appears to be a feature of ageing, long term, and if KDs work in "ageing inflammation" all we have to decide is the age at which we should all start on a KD. Unless someone has a method of stopping the ageing process of course....
Peter
I don't know if it's worth going through the figures individually, they are very similar to those from Axen and Axen which produced a series of posts a few years ago, except that the feature of COMPLETE reversal of insulin resistance is, here, presented right up front in Figure 6 and in the abstract too:
That figure for insulin looks a little dubious at 120 minutes but I'll let that go, I guess p was still > than 0.05... Pretty close to full reversal.
It's quite hard to know exactly how much this group understand about their results. They give roughly equal weight to the adverse (sic) effects of a ketogenic diet as they do to the fact it is reversible within a week (or less, they only checked at a week) of re-introducing carbohydrate.
What they seem to lack is the concept that rats fed a very restricted carbohydrate diet MUST be insulin resistant to survive, as happens in starvation. But maybe they are creeping towards some sort of understanding. It's about time. Good.
When people cite Axen and Axen to prove ketogenic diets are going to make you diabetic (there are folks who believe this, or at least wish you to believe it!) you have an answer in Kinzig et al 2010.
BTW, the links which led me here relate to using ketogenic diets to control both pain and inflammation. This is a potentially very useful tool but the beneficial effect does appear to be as rapidly reversible as the physiological insulin resistance... Ketogenic diets are a fix, not a cure (in the short term anyway). But inflammation appears to be a feature of ageing, long term, and if KDs work in "ageing inflammation" all we have to decide is the age at which we should all start on a KD. Unless someone has a method of stopping the ageing process of course....
Peter
Sunday, March 31, 2013
TCA rap
From Bert. Not advocating carb consumption but I can see that electron transferring flavoprotein dehydrogenase would need some serious effort to rap... So enjoy glycolysis and the TCA:
http://www.youtube.com/watch?v=aMBIs_Iw0kE&feature=player_embedded
Click-able, sorted!
Ta Bert. I enjoyed.
Peter
Now if I can get Ryan started on beta oxidation. Hmmmm....
http://www.youtube.com/watch?v=aMBIs_Iw0kE&feature=player_embedded
Click-able, sorted!
Ta Bert. I enjoyed.
Peter
Now if I can get Ryan started on beta oxidation. Hmmmm....
Wednesday, March 20, 2013
Sta'ins, CoQ, diabetes and Dr Andreas Eenfeldt's link
I'm not very conscientious about reading many blogs as I don't really have time to look after my own blog properly, but I will occasionally flick through the links from Stan's site and I felt that Dr Andreas Eenfeldt's link to the official Swedish data sheet for simvastatin was rather excellent. Via Google translation:
"Diabetes is a possible side effect. This is more likely if you have high blood sugar and high blood fat levels, are overweight and have high blood pressure. Your doctor will monitor you while you are taking this medicine."
Statins deplete CoQ. This means that for every electron carried from any input, NADH or FADH2, down the ETC there will be less CoQ available in the redox couple and the CoQH2 levels will be relatively high. An highly reduced CoQ couple (ie low CoQ per unit CoQH2) will drive reverse electron flow through complex I and generate superoxide. Insulin resistance. Diabetes.
I suspect that EVERYONE on a statin will step their insulin resistance up by an amount proportional to the CoQ depletion. Everyone. Just a few will cross the arbitrary boundaries between "normality", "impaired glucose tolerance" and "diabetes".
Hyperglycaemia doesn't care about labels or boundaries. You get it, you suffer.
Obviously this rather nasty side effect can be COMPLETELY avoided by putting the statin script in the bin.
If you are going to take simvastatin anyway then some coenzyme Q10 might ameliorate some of the damage you have chosen to do to yourself.
Peter
"Diabetes is a possible side effect. This is more likely if you have high blood sugar and high blood fat levels, are overweight and have high blood pressure. Your doctor will monitor you while you are taking this medicine."
Statins deplete CoQ. This means that for every electron carried from any input, NADH or FADH2, down the ETC there will be less CoQ available in the redox couple and the CoQH2 levels will be relatively high. An highly reduced CoQ couple (ie low CoQ per unit CoQH2) will drive reverse electron flow through complex I and generate superoxide. Insulin resistance. Diabetes.
I suspect that EVERYONE on a statin will step their insulin resistance up by an amount proportional to the CoQ depletion. Everyone. Just a few will cross the arbitrary boundaries between "normality", "impaired glucose tolerance" and "diabetes".
Hyperglycaemia doesn't care about labels or boundaries. You get it, you suffer.
Obviously this rather nasty side effect can be COMPLETELY avoided by putting the statin script in the bin.
If you are going to take simvastatin anyway then some coenzyme Q10 might ameliorate some of the damage you have chosen to do to yourself.
Peter
Let them eat fat: Ron Rosenbaum
Tuesday, March 19, 2013
Protons: Aside to T cells
Just because I like it. This is obscure. We can ignore the upper section as this is quite specific to T cells (which the paper is all about). What I love is the consideration that mitochondrial glycerol-3-phosphate dehydrogenase (marked as GPD2), sitting on the outer surface of the mitochondria, is very likely to be driving reverse electron flow through complex I to generate free radicals. Something I would avoid, personally (except perhaps in my T cells).
The bit I love is the big red outline arrow from QH2 going to the left towards complex I.
The whole mechanism and specific purpose here is linked to activation of the drive for T cells to divide, a free radical mediated phenomenon through NF-kappaB. What interests me at the moment is what might happen to a cell which cannot divide when mG3Pdh is driven, say by hyperglycaemia acting on a neuron... Lots of papers to wade through on this.
Peter
The bit I love is the big red outline arrow from QH2 going to the left towards complex I.
The whole mechanism and specific purpose here is linked to activation of the drive for T cells to divide, a free radical mediated phenomenon through NF-kappaB. What interests me at the moment is what might happen to a cell which cannot divide when mG3Pdh is driven, say by hyperglycaemia acting on a neuron... Lots of papers to wade through on this.
Peter
Saturday, March 16, 2013
Protons: Meet the glycerol 3 phosphate shuttle
The next thing we have to think about is the glycerol 3 phosphate shuttle. This is a route in to the electron transport chain for cytoplasmic NADH, directly from the cytoplasm, no complex I involved.
There are two glycerol 3 phosphate dehydrogenases which make up the shuttle, just to confuse matters. Free in the cytosol there is cytosolic G3P dehydrogenase, which actually uses NADH to add a pair of hydrogens to a glycolysis intermediate (dihydroxyacetone phosphate) to form G3P.
The other G3P dehydrogenase really does dehydrogenate G3P, back to dihydroxyacetone phosphate. But this second G3P dehydrogenase is embedded in the outer surface of the inner mitochondrial membrane. And it contains an FAD/FADH2 moiety which takes these two hydrogens and uses them to reduce the CoQ couple, feeding electrons in to the electron transport chain.
So we are putting electrons from cytosolic NADH directly in to the ETC through FADH2. From the outside. And pumping no protons.
The G3P shuttle is very, very important.
In healthy cells the signal to reject excess calories picks on glucose, in the form of the development of insulin resistance, mediated by superoxide generated at complex I of the mitochondria. At iron sulphur cluster N-1a. The most simple way of doing this is to oxidise fully saturated fats (mmmmm, butter), generate a lot of FADH2, post a few electrons the wrong way through complex I and shut down glucose acceptance by the cell.
You can make glucose act as if it were butter through the G3P shuttle.
Think what happens if you are a Taterhead, just finishing your 4th plate of plain boiled, unsalted, unseasoned, unpeeled spuds.
Your FFAs, especially palmitate, are through the floor. Your glucose, given its own way, would be through the roof. Insulin is demanding that all cells accept glucose because no one wants a blood glucose of 30mmol/l. There is a shedload of NADH in both the cytoplasm and in the mitochondrial matrix. Electrons are pouring down the ETC but, in your post spud-prandial insulin induced stupor, you are not exactly sprinting to the gym.
You have to stop the supply of NADH pouring through complex I but, unless the NADH level is over three times the NAD+ level in mitochondria, you are not exactly going to get an electron on to N-1a excepting when there is a markedly reduced CoQ couple and a strong membrane potential. In the absence of palmitic acid (you're pigging out on fat-free spuds, don't forget) you need mitochondrial G3P dehydrogenase to pour electrons on to the CoQ couple, which allow the insulin/glucose induced membrane potential to push electrons back up the ETC to N-1a. And then SHUT DOWN THE BLOODY GLUCOSE SUPPLY.
You might just be able to do the same to deal with excess insulin. If insulin (from exogenous injection or an insulinoma) is allowing a free fall of glucose in to the cell and the cell really doesn't want all of this metabolic substrate, it has to say no. It was quite a while ago now but we have discussed insulin induced insulin resistance. Here's a possible metabolic mechanism. And the mechanism would kick in when the G3P shuttle goes in to overdrive, not when glucose becomes too low. Back to when we had the discussions about the Somogyi overswing... It's just a mimic of pigging out on spuds but without the spuds.
But the queen of insulin resistance generators is, of course, fructose. Fructose free falls through glycolysis to levels that the cells really cannot expect to use immediately. Of course a lot of it gets off loaded as lactate but there is still way more pyruvate than a cell can reasonably be expected to oxidise immediately. I consider this effect to be dose related. Eating the occasional apple might not kill you (gasp, there, I said it) but three Big Gulps per day probably has you well on your way.
Answer to fructose exposure is to shut down glucose supply to a level which compensates for the calories coming through from fructose. There can be no easier way than to reduce the CoQ couple using an FADH2 input. G3P dehydrogenase does this directly from the cytoplasm. It, like electron transferring flavoprotein dehydrogenase, is what I would describe as "complex II -like" in its action.
This smacks of physiological regulation to me.
Things get slightly more pathological where hyperglycaemia is overcoming insulin resistance. Or overcoming absolute insulin deficiency, as Sonksen and Sonksen pointed out. I'll come back to this in future posts.
There is quite a lot of support for this concept in Pubmed but here is an abstract I particularly enjoyed. Anyone thinking of indulging in a bit of Taterism should have a read first. A real giggle while you boil your spuds.
The wild type mice were funniest. For your delectation:
"The high carbohydrate diet induced hyperglycaemia, hyperinsulinaemia, and islet hyperplasia in the wild-type [mice]"
Oh, for the love of Taterism, does anyone remember the discussion of Barnard's victims and their progression of diabetes under a low sugar, high complex carbohydrate diet? Well, mice are not so different from people! In people we call this Taterism. Well, some of us do.
And look at the tweaked physiology in the same abstract. If you knock out mG3P dehydrogenase (ie you eliminate this "complex II-like" FADH2 reduction of the CoQ couple) you get increased insulin sensitivity (no reverse electron flow as there is no FADH2 route in to the ETC except complex II and we're not feeding fat).
Is this a good or a bad state to be in? That depends on degree and whether you are happy to push far more electrons down your ETC than you could possibly have a use for. An interesting question. I'm not in the queue to trial a mG3P dehydrogenase inhibitor (actually, it's called diazoxide and it does seem to help). Not eating carbs seems a rather safer bet.
[BTW if anyone has this high carb paper it would be nice to know which mice they used and what the diet was actually made of... Ta.]
Edit: Got it, many thanks Paul and Purposelessness. End edit
Which leads straight on to neurons.
Peter
There are two glycerol 3 phosphate dehydrogenases which make up the shuttle, just to confuse matters. Free in the cytosol there is cytosolic G3P dehydrogenase, which actually uses NADH to add a pair of hydrogens to a glycolysis intermediate (dihydroxyacetone phosphate) to form G3P.
The other G3P dehydrogenase really does dehydrogenate G3P, back to dihydroxyacetone phosphate. But this second G3P dehydrogenase is embedded in the outer surface of the inner mitochondrial membrane. And it contains an FAD/FADH2 moiety which takes these two hydrogens and uses them to reduce the CoQ couple, feeding electrons in to the electron transport chain.
So we are putting electrons from cytosolic NADH directly in to the ETC through FADH2. From the outside. And pumping no protons.
The G3P shuttle is very, very important.
In healthy cells the signal to reject excess calories picks on glucose, in the form of the development of insulin resistance, mediated by superoxide generated at complex I of the mitochondria. At iron sulphur cluster N-1a. The most simple way of doing this is to oxidise fully saturated fats (mmmmm, butter), generate a lot of FADH2, post a few electrons the wrong way through complex I and shut down glucose acceptance by the cell.
You can make glucose act as if it were butter through the G3P shuttle.
Think what happens if you are a Taterhead, just finishing your 4th plate of plain boiled, unsalted, unseasoned, unpeeled spuds.
Your FFAs, especially palmitate, are through the floor. Your glucose, given its own way, would be through the roof. Insulin is demanding that all cells accept glucose because no one wants a blood glucose of 30mmol/l. There is a shedload of NADH in both the cytoplasm and in the mitochondrial matrix. Electrons are pouring down the ETC but, in your post spud-prandial insulin induced stupor, you are not exactly sprinting to the gym.
You have to stop the supply of NADH pouring through complex I but, unless the NADH level is over three times the NAD+ level in mitochondria, you are not exactly going to get an electron on to N-1a excepting when there is a markedly reduced CoQ couple and a strong membrane potential. In the absence of palmitic acid (you're pigging out on fat-free spuds, don't forget) you need mitochondrial G3P dehydrogenase to pour electrons on to the CoQ couple, which allow the insulin/glucose induced membrane potential to push electrons back up the ETC to N-1a. And then SHUT DOWN THE BLOODY GLUCOSE SUPPLY.
You might just be able to do the same to deal with excess insulin. If insulin (from exogenous injection or an insulinoma) is allowing a free fall of glucose in to the cell and the cell really doesn't want all of this metabolic substrate, it has to say no. It was quite a while ago now but we have discussed insulin induced insulin resistance. Here's a possible metabolic mechanism. And the mechanism would kick in when the G3P shuttle goes in to overdrive, not when glucose becomes too low. Back to when we had the discussions about the Somogyi overswing... It's just a mimic of pigging out on spuds but without the spuds.
But the queen of insulin resistance generators is, of course, fructose. Fructose free falls through glycolysis to levels that the cells really cannot expect to use immediately. Of course a lot of it gets off loaded as lactate but there is still way more pyruvate than a cell can reasonably be expected to oxidise immediately. I consider this effect to be dose related. Eating the occasional apple might not kill you (gasp, there, I said it) but three Big Gulps per day probably has you well on your way.
Answer to fructose exposure is to shut down glucose supply to a level which compensates for the calories coming through from fructose. There can be no easier way than to reduce the CoQ couple using an FADH2 input. G3P dehydrogenase does this directly from the cytoplasm. It, like electron transferring flavoprotein dehydrogenase, is what I would describe as "complex II -like" in its action.
This smacks of physiological regulation to me.
Things get slightly more pathological where hyperglycaemia is overcoming insulin resistance. Or overcoming absolute insulin deficiency, as Sonksen and Sonksen pointed out. I'll come back to this in future posts.
There is quite a lot of support for this concept in Pubmed but here is an abstract I particularly enjoyed. Anyone thinking of indulging in a bit of Taterism should have a read first. A real giggle while you boil your spuds.
The wild type mice were funniest. For your delectation:
"The high carbohydrate diet induced hyperglycaemia, hyperinsulinaemia, and islet hyperplasia in the wild-type [mice]"
Oh, for the love of Taterism, does anyone remember the discussion of Barnard's victims and their progression of diabetes under a low sugar, high complex carbohydrate diet? Well, mice are not so different from people! In people we call this Taterism. Well, some of us do.
And look at the tweaked physiology in the same abstract. If you knock out mG3P dehydrogenase (ie you eliminate this "complex II-like" FADH2 reduction of the CoQ couple) you get increased insulin sensitivity (no reverse electron flow as there is no FADH2 route in to the ETC except complex II and we're not feeding fat).
Is this a good or a bad state to be in? That depends on degree and whether you are happy to push far more electrons down your ETC than you could possibly have a use for. An interesting question. I'm not in the queue to trial a mG3P dehydrogenase inhibitor (actually, it's called diazoxide and it does seem to help). Not eating carbs seems a rather safer bet.
[BTW if anyone has this high carb paper it would be nice to know which mice they used and what the diet was actually made of... Ta.]
Edit: Got it, many thanks Paul and Purposelessness. End edit
Which leads straight on to neurons.
Peter
Thursday, March 14, 2013
Protons: NAD+/NADH some more
This is an interesting paper (from George I think). There are a stack of caveats about it, but the core findings appear to hold water.
They are dealing with cancer cell lines (ie cancer cells which are immortal and live in tissue culture) which have lost complex I of the electron transport chain. These cells have elevated levels of NADH per unit NAD+, ie their NAD+/NADH ratio is low. Obviously NADH is high because there is no complex I to oxidise it back down to NAD+. Any NAD+ which gets converted to NADH simply stays there as NADH.
Glycolysis continues to generate NADH and the TCA generates more NADH because that's all you can do with acetyl CoA (pax complex II derived FADH2), barring the scenario we looked at in a previous post.
You can manipulate the NAD+/NADH ratio.
It doesn't seem to matter whether you manipulate the absolute NADH levels down or the absolute NAD+ levels upwards and it doesn't seem to matter how you manipulate either level. Having excess NADH combined with relatively depleted NAD+ makes these cancers very much more aggressive in terms of metastasis. It's fascinating to see terms like Ki67 bandied around as aggression markers, we are just starting to use Ki67 clinically to try to assess to seriousness of that almost invariable "Grade 2" score assigned to the vast majority of mast cell tumours which get as far as histopathology. Are they a good Grade 2 or a bad Grade 2? Scoring cancer aggression is not the easiest thing to do and Ki67 looks interesting for those of us who have to manage mast cell tumours with a scalpel and/or a tyrosine kinase inhibitor. Anyway, back to the paper:
The paper brings up Ndi1, a fascinating little enzyme stolen from yeasts and engineered in to cancer cells. Ndi1 is a rather small, relatively simple enzyme which inserts itself in to the inner surface of the inner mitochondrial membrane and happily converts mitochondrial matrix NADH to NAD+ in a process linked to reducing the CoQ couple, but it pumps no protons in the process. It bypasses the broken complex I completely, skipping electrons straight from NADH to CoQ while effectively lowering NADH and raising NAD+. And it tames the cancer's metastatic behaviour.
Compare this Ndi1 route in to the ETC to the FADH2 route in from fat metabolism, which uses electron-transferring-flavoprotein dehydrogenase, and also reduces the CoQ without pumping any protons.
The two produce rather similar effects. Ndi1 is essentially making NADH behave like an FADH2 based input to the CoQ couple. Ndi1 may be more effective because it is purely focused on relieving the excess NADH and could, theoretically, completely normalise the NAD+/NADH ratio, whereas running metabolism on fat will only bias electron supply to FADH2 without stopping some NADH generation.
Both mice and cell cultures are being run on glucose in this paper. If all that matters is the NADH to NAD+ ratio, what might happen on a diet which intrinsically generates less NADH? An interesting but un-asked question.
Perhaps you might not need that Ndi1 enzyme inserted in to your inner mitochondrial membrane?
The very simple converse approach, similar to giving B3 as used by Hoffer, was to add an NAD+ precursor to the drinking water. I can see that this might well be effective in raising NAD+ but the thought of long term megadosing on any nutrient to achieve this effect is beyond what I might personally want to do. I might change my mind if I had cancer.
I do think that is very interesting in its own right, but it leads on to a host of other questions. In fact there are too many questions for a simple thread. It's particularly interesting to think about what an excess of NADH to NAD+ signifies to a cell. This strikes me as a core decision making signal about a cell's future. How this ties in with superoxide production from complex I, or the lack of it when complex I is dysfunctional, is also related. Couple that with the fact that high levels of NADH providing substrates (pyruvate, malate or glutamate) massively increase superoxide generation when FADH2 generating substrates (succinate) are simultaneously provided to isolated mitochondria from normal tissues. The whole area looks to have lots of potential for working out what is going on at the primary switching point of the electron transport chain.
That looks OK but life is never quite that simple....
It's very interesting to note that there are now several publications from this group which are pretty convincing that many breast cancers, in vivo, have hyperactive mitochondria and perform oxphos to a very high level to support their aggressive growth pattern. Including both increased expression of the genes for complex I components and the enhanced ability (in mitochondria from fresh frozen human surgical breast cancer biopsies) to process large amounts of NADH to NAD+. Certainly compared to both non neoplastic surrounding epithelial cells and most especially compared to the surrounding stromal cells (mostly fibroblasts).
The results from the two groups are both very convincing and utterly incompatible.
Nice.
To square the circle you get some help from this paper which suggest that at least one of the cell lines used in Santidrian's study are, in fact, melanoma derived cells, not breast cancer cells at all. In terms of aggressive phenotype most clinicians might be significantly more concerned about melanoma vs breast adenocarcinoma. I would anyway.
You may have to be married to a pathologist to realise quite how difficult it is to differentiate highly malignant cell types as both their genes and their appearance seem to eventually degenerate or evolve in to some sort of identical "cancer cell".
That seems to be the point being made by this chap.
So whether MDA-MB-435 cells are melanoma cells or breast cancer cells, they are degenerate/evolved enough to be indistinguishable from either family of cancer of origin and may well be behaviourally indistinguishable too.
The logical explanation is that MDA-MB-435 cells represent a more advanced cancer development than the still rather "normal" cells in routine surgical patients, most of the cells from which will be running on oxphos fueled by manipulating their surround fibroblasts (which will be running on glycolysis, with shut down mitochondria) to donate large amounts of TCA substrates to their controlling cancer cells
Aside: You still have a massive fuel source for cancer cells based on glycolysis here, it's just moved from the cancer cells themselves to the surrounding fibroblasts. But the down side is that while you might starve the fibroblasts by running on low glucose and high FFAs (they have lost their mitochondria) you still have active cancer cells willing to use ketones, FFAs or lactate through their very active mitochondrial electron transport chains... End depressing aside.
I really like this idea of degrees of degeneration as a possible explanation for the irreconcilable results. Again, you might need to be married to a pathologist to appreciate the phenomenal importance of fibroblasts in coming to a histopathological diagnosis about many cancer families. Fibroblasts are VERY important. Metabolic coupling may well be why.
Not sure whether to go on to mitochondria in Parkinsons or have a break and look at this whole concept of metabolic coupling between cancer cells and fibroblasts. The two subjects are vaguely convergent, eventually.
Peter
They are dealing with cancer cell lines (ie cancer cells which are immortal and live in tissue culture) which have lost complex I of the electron transport chain. These cells have elevated levels of NADH per unit NAD+, ie their NAD+/NADH ratio is low. Obviously NADH is high because there is no complex I to oxidise it back down to NAD+. Any NAD+ which gets converted to NADH simply stays there as NADH.
Glycolysis continues to generate NADH and the TCA generates more NADH because that's all you can do with acetyl CoA (pax complex II derived FADH2), barring the scenario we looked at in a previous post.
You can manipulate the NAD+/NADH ratio.
It doesn't seem to matter whether you manipulate the absolute NADH levels down or the absolute NAD+ levels upwards and it doesn't seem to matter how you manipulate either level. Having excess NADH combined with relatively depleted NAD+ makes these cancers very much more aggressive in terms of metastasis. It's fascinating to see terms like Ki67 bandied around as aggression markers, we are just starting to use Ki67 clinically to try to assess to seriousness of that almost invariable "Grade 2" score assigned to the vast majority of mast cell tumours which get as far as histopathology. Are they a good Grade 2 or a bad Grade 2? Scoring cancer aggression is not the easiest thing to do and Ki67 looks interesting for those of us who have to manage mast cell tumours with a scalpel and/or a tyrosine kinase inhibitor. Anyway, back to the paper:
The paper brings up Ndi1, a fascinating little enzyme stolen from yeasts and engineered in to cancer cells. Ndi1 is a rather small, relatively simple enzyme which inserts itself in to the inner surface of the inner mitochondrial membrane and happily converts mitochondrial matrix NADH to NAD+ in a process linked to reducing the CoQ couple, but it pumps no protons in the process. It bypasses the broken complex I completely, skipping electrons straight from NADH to CoQ while effectively lowering NADH and raising NAD+. And it tames the cancer's metastatic behaviour.
Compare this Ndi1 route in to the ETC to the FADH2 route in from fat metabolism, which uses electron-transferring-flavoprotein dehydrogenase, and also reduces the CoQ without pumping any protons.
The two produce rather similar effects. Ndi1 is essentially making NADH behave like an FADH2 based input to the CoQ couple. Ndi1 may be more effective because it is purely focused on relieving the excess NADH and could, theoretically, completely normalise the NAD+/NADH ratio, whereas running metabolism on fat will only bias electron supply to FADH2 without stopping some NADH generation.
Both mice and cell cultures are being run on glucose in this paper. If all that matters is the NADH to NAD+ ratio, what might happen on a diet which intrinsically generates less NADH? An interesting but un-asked question.
Perhaps you might not need that Ndi1 enzyme inserted in to your inner mitochondrial membrane?
The very simple converse approach, similar to giving B3 as used by Hoffer, was to add an NAD+ precursor to the drinking water. I can see that this might well be effective in raising NAD+ but the thought of long term megadosing on any nutrient to achieve this effect is beyond what I might personally want to do. I might change my mind if I had cancer.
I do think that is very interesting in its own right, but it leads on to a host of other questions. In fact there are too many questions for a simple thread. It's particularly interesting to think about what an excess of NADH to NAD+ signifies to a cell. This strikes me as a core decision making signal about a cell's future. How this ties in with superoxide production from complex I, or the lack of it when complex I is dysfunctional, is also related. Couple that with the fact that high levels of NADH providing substrates (pyruvate, malate or glutamate) massively increase superoxide generation when FADH2 generating substrates (succinate) are simultaneously provided to isolated mitochondria from normal tissues. The whole area looks to have lots of potential for working out what is going on at the primary switching point of the electron transport chain.
That looks OK but life is never quite that simple....
It's very interesting to note that there are now several publications from this group which are pretty convincing that many breast cancers, in vivo, have hyperactive mitochondria and perform oxphos to a very high level to support their aggressive growth pattern. Including both increased expression of the genes for complex I components and the enhanced ability (in mitochondria from fresh frozen human surgical breast cancer biopsies) to process large amounts of NADH to NAD+. Certainly compared to both non neoplastic surrounding epithelial cells and most especially compared to the surrounding stromal cells (mostly fibroblasts).
The results from the two groups are both very convincing and utterly incompatible.
Nice.
To square the circle you get some help from this paper which suggest that at least one of the cell lines used in Santidrian's study are, in fact, melanoma derived cells, not breast cancer cells at all. In terms of aggressive phenotype most clinicians might be significantly more concerned about melanoma vs breast adenocarcinoma. I would anyway.
You may have to be married to a pathologist to realise quite how difficult it is to differentiate highly malignant cell types as both their genes and their appearance seem to eventually degenerate or evolve in to some sort of identical "cancer cell".
That seems to be the point being made by this chap.
So whether MDA-MB-435 cells are melanoma cells or breast cancer cells, they are degenerate/evolved enough to be indistinguishable from either family of cancer of origin and may well be behaviourally indistinguishable too.
The logical explanation is that MDA-MB-435 cells represent a more advanced cancer development than the still rather "normal" cells in routine surgical patients, most of the cells from which will be running on oxphos fueled by manipulating their surround fibroblasts (which will be running on glycolysis, with shut down mitochondria) to donate large amounts of TCA substrates to their controlling cancer cells
Aside: You still have a massive fuel source for cancer cells based on glycolysis here, it's just moved from the cancer cells themselves to the surrounding fibroblasts. But the down side is that while you might starve the fibroblasts by running on low glucose and high FFAs (they have lost their mitochondria) you still have active cancer cells willing to use ketones, FFAs or lactate through their very active mitochondrial electron transport chains... End depressing aside.
I really like this idea of degrees of degeneration as a possible explanation for the irreconcilable results. Again, you might need to be married to a pathologist to appreciate the phenomenal importance of fibroblasts in coming to a histopathological diagnosis about many cancer families. Fibroblasts are VERY important. Metabolic coupling may well be why.
Not sure whether to go on to mitochondria in Parkinsons or have a break and look at this whole concept of metabolic coupling between cancer cells and fibroblasts. The two subjects are vaguely convergent, eventually.
Peter
Protons: Back to N-1a and a nice quote
Eureka moment when I tripped over this gem by Vinogradov
"The redox potential of one binuclear [FeS] center (N-1a) is so negative that it can not be reduced by NADH"
Couple this with this group's conclusion:
"These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a".
You can't reduce N-1a to generate superoxide using NADH at "normal" concnetrations. The easiest way you can generate superoxide at N-1a is by reverse electron flow through complex 1 under conditions of a strong membrane potential and a high FADH2 input, in this case using succinate. Very satisfying. They also point out that, if you can get the NAD+/NADH ratio high enough you can get it far enough from its electrical mid point to pass electrons "down gradient" to N-1a. At ratios of less than about 3 parts NADH to one part NAD+ the transfer is uphill and isn't going to happen. As they say:
"...the reduction of the *ROS site [they consider that it is probably N-1a] is regulated by the NADH/NAD+ ratio rather than the NADH level (eqns 7 and 8)..."
which sort of takes us back to B3 and some cancer cells which have probably lost N-1a so fail to develop insulin resistance, ie they don't limit their energy generation to their needs. They also develop metastatic aggressiveness in proportion to their elevated NADH:NAD+ ratio (even if they expressed it as a reduced NAD+/NADH ratio!). I wrote that post a while ago, time to check it and hit publish...
Peter
BTW Vinogradov pointed out in his review paper that very few labs have the massively expensive and complex gear to look at this sort of redox research and both of the papers discussed here are from groups who know each other, Vinogradov being thanked for reading through the manuscript of the second paper. But I think they are correct.
"The redox potential of one binuclear [FeS] center (N-1a) is so negative that it can not be reduced by NADH"
Couple this with this group's conclusion:
"These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a".
You can't reduce N-1a to generate superoxide using NADH at "normal" concnetrations. The easiest way you can generate superoxide at N-1a is by reverse electron flow through complex 1 under conditions of a strong membrane potential and a high FADH2 input, in this case using succinate. Very satisfying. They also point out that, if you can get the NAD+/NADH ratio high enough you can get it far enough from its electrical mid point to pass electrons "down gradient" to N-1a. At ratios of less than about 3 parts NADH to one part NAD+ the transfer is uphill and isn't going to happen. As they say:
"...the reduction of the *ROS site [they consider that it is probably N-1a] is regulated by the NADH/NAD+ ratio rather than the NADH level (eqns 7 and 8)..."
which sort of takes us back to B3 and some cancer cells which have probably lost N-1a so fail to develop insulin resistance, ie they don't limit their energy generation to their needs. They also develop metastatic aggressiveness in proportion to their elevated NADH:NAD+ ratio (even if they expressed it as a reduced NAD+/NADH ratio!). I wrote that post a while ago, time to check it and hit publish...
Peter
BTW Vinogradov pointed out in his review paper that very few labs have the massively expensive and complex gear to look at this sort of redox research and both of the papers discussed here are from groups who know each other, Vinogradov being thanked for reading through the manuscript of the second paper. But I think they are correct.
Thursday, February 21, 2013
Complex I, Hoffer and B3
Over the years I have read some pretty far out stuff. I think I've commented previously on the late Dr Abram Hoffer's use of B3 for controlling schizophrenia. Interesting and a source of thought trains which I never got the time to follow through. What I personally enjoyed were the accounts of recovered schizophrenics as to what it was like to have been inside the syndrome and then recover. Re-reading the site, it all looks very anecdotal now.
I also went so far as to purchase his book, written in conjunction with Linus Pauling, on treating cancer with vitamins B3 and C. You can just google the ISBN number 18970251141897025114 to find out a bit more about it. I see it's quite financially valuable nowadays!
Before we settle down to the nuts and bolts of the deeply involved models which the latest complex I paper (thanks George) used, the bottom line is that adding 1% nicotinamide (similar effects could be obtained with vitamin B3) to the mouse breast cancer models was very impressive at extending lifespan of those mouse models, especially through suppression of metastasis.
I find this slightly eerie. I can hear Hoffer in my head, sounding rational and being regarded as a quack for treating cancer (and many other illnesses including schizophrenia) with B3. Yet here are these guys in 2013 with their multiple mouse models, multiple tumour types, multiple interventions all showing that B3 or its analogue is very, very effective in suppressing breast cancer metastasis. In mouse models of course.
Slightly déjà vu...
A lot more to say about this paper but the B3 aspect is just so weird I had to put this aside up.
Peter
I also went so far as to purchase his book, written in conjunction with Linus Pauling, on treating cancer with vitamins B3 and C. You can just google the ISBN number 18970251141897025114 to find out a bit more about it. I see it's quite financially valuable nowadays!
Before we settle down to the nuts and bolts of the deeply involved models which the latest complex I paper (thanks George) used, the bottom line is that adding 1% nicotinamide (similar effects could be obtained with vitamin B3) to the mouse breast cancer models was very impressive at extending lifespan of those mouse models, especially through suppression of metastasis.
I find this slightly eerie. I can hear Hoffer in my head, sounding rational and being regarded as a quack for treating cancer (and many other illnesses including schizophrenia) with B3. Yet here are these guys in 2013 with their multiple mouse models, multiple tumour types, multiple interventions all showing that B3 or its analogue is very, very effective in suppressing breast cancer metastasis. In mouse models of course.
Slightly déjà vu...
A lot more to say about this paper but the B3 aspect is just so weird I had to put this aside up.
Peter
Thursday, February 14, 2013
Protons: TFAM and behenic acid
Well, Peter on Hyperlipid is still messing about with KO mice! Ah, but they really do provide some level of insight and are a slightly more productive occupation compared to the Times crossword. Hee hee, so...
Aside: If you click on the TFAM link immediately below this paragraph and then go to "see all" under "related citations" you will find that there are a sh!tload of genes which can be knocked out to protect hapless mice from the dire consequences of D12492. Let's re appraise that as: Evolution has provided us with a stack of genes giving protection (read "normal processing") against the damage (lack of "normal processing") intrinsic to foods which are caricatured by D12492. They do this by inducing a decrease in glucose metabolism when serum FFAs are elevated. Knocking out these genes will reverse an unimaginably large number of years worth of evolution and I would anticipate that we might just end up dead slim when effective blocking drugs are developed to "cure" the obesity epidemic. Really dead as well as really slim. Anyhoo, back to the mice...
This is the paper where the gene for TFAM was knocked out in the adipocytes of some mice. Edit: Thanks to Purposelessness for the link through comments, end edit. Two consequences of this appear to be that complex I doesn't seem to be there and that complex IV, while present, doesn't seem to be up to much either. With a large chunk of their adipocyte ETC disabled these mice seem to be a little stunted overall but they stay slim and simply refuse to develop insulin resistance with either age or with the feeding of a ton of sugar, described throughout as a "high fat" diet, good old D12492. Don't snigger, this isn't funny!
If you have just come from Kushnareva et al's paper on complex I you could just ask whether the FeS complex N-1a is present or absent in these mice, but then you hardly need to ask this because the mice don't become insulin resistant, ie they don't generate superoxide at N-1a, even though the adipocytes have increased lipid and DNA damage, probably through them spewing electrons at complex IV. So there's really no need to trawl back through the layers of refs to the development of this mouse model to know that there is no FeS cluster N-1a. Or certainly nothing feeding electrons to it. If there were, there would be insulin resistance... I find this difficult to get excited about, useful though it is.
But the paper is utterly fascinating in several other ways. I went through the results to Fig 6 where TBARS and 8-OHdG are shown for D12492 fed mice, being surprised that no damage was reported when the mice were fed crapinabag (this is not quite as true as it sounds but, again, it's not important here). Not so good for D12492 feeding of course. Moral: Don't eat toffee fudge as your sole diet. There is a great deal else which could be said about free radical damage in these mice but that's not for today.
What I noticed, immediately below the TBARS figure and still within Fig 6 of the main paper, was the relative abundances of assorted activated fatty acids from both brown and white adipose tissue:
Of particular interest to me was the rather high relative abundance of activated (carnitine linked) behenic acid, C22:0, over on the far right of each chart. Now bear in mind that there is not a lot of C22:0 in the fat of normal mice anyway, so the massive relative abundance may be, in part, an artifact of a rather low level of C22:0 in control mice. But there is probably a lot more in mice with a crippled ETC. Behenic acid is fully saturated, it's long, it's F:N ratio is as close to 0.5 as you can get and it should be rapidly degraded in peroxisomes. But these mice have markedly down regulated peroxisome promoter genes and the organelles shown in the photomicrograph look, apparently, pretty crappy.
So let's have a think about how this might fit together. These adipocytes have no complex 1. What can you do with NADH in mitochondria if there is no complex I? Diddly squat is, I think, the correct term. Glycolysis works fine and generates a smidge of ATP by substrate level phosphorylation. And some NADH too, which is of no use what so ever. Complex II, succinate dehydrogenase, is fully conserved and can feed in to the CoQ pool to run the ETC, the abnormal complex IV doing the best it can. No matter how much the CoQ pool is reduced there will be no reverse electron flow through complex I because complex I ain't there.
But running from pyruvate dehydrogenase through the TCA to succinate dehyrogenase generates 3 utterly useless NADHs and completing the TCA gets you yet another NADH. What's a cell to do?
Well, it can start by generating a ton of pyruvate by glycolysis. These cells do have a lot of pyruvate. You can feed this in to mitochondria and through pyruvate dehydrogenase (or even pyruvate carboxylase, see below) then generate citric acid from oxaloacetate. If you really don't want to generate more useless NADH than you have to, then why not export the citrate to the cytoplasm through the malate shuttle and convert it to long chain fatty acids? Fatty acids only half-care about putting NADH in to complex I. They will, of course, generate some NADH but what they also do, which is really useful, is to generate FADH2 within electron-transferring flavoprotein which can be used via electron-transferring-flavoprotein dehydrogenase to reduce the CoQ pool, without all of that cyclical messing around with the TCA. And without any need for complex I of course.
As we know, the longer the saturated fat the more FADH2 it generates per unit NADH. These cells desperately need FADH2 input to reduce the CoQ pool. A 22 carbon fully saturated fat is a good option. Behenic acid. Peroxisomes are NOT wanted, it's FADH2 to CoQ all the way.
Of course as acetyl CoA is taken for fatty acid synthesis the citrate yields malate again which, if it re enters the TCA, will give another useless NADH as it converts to oxaloacetate. So why not convert the malate back to pyruvate (replacing the NADPH used to generate that malate) and then use pyruvate carboxylase to generate oxaloacetate without the obligatory NADH resulting from when malate is converted to oxaloacetate? You can then allow citrate regeneration which can be re-exported to cycle around through more FFA generation, yielding usable FADH2 again.
Looking at Fig S6 from the supplementary data we can see that, of the TCA metabolites, only citrate is significantly increased. Pity they didn't measure oxaloacetate.
Pyruvate is increased as you might expect. Glycolysis really needs to be generating NADPH (pentose phosphate pathway) for fatty acid synthesis but I can't see from the paper if this might be occurring. I think it's a reasonable assumption that it is, because the behenic acid is not coming from the diet and NADPH is needed for the fatty acid synthesis which generates it in-situ. There is plenty of lactate derived from the excess of pyruvate over what is needed by the crippled mitochondria.
I also can't tell from the paper how much the glycerol phosphate shuttle is up regulated to feed in through it's own FADH2 route to reduce the CoQ couple either. I suspect it's rather active.
Anything which inputs to the CoQ couple other than complex I seems to be essential for these cells to survive. The impression I get is of adipocytes which are simply converting glucose to fat and running on a (genuine) high fat diet at the mitochondrial level, despite the oral diet being crapinabag.
The photomicrographs of the mitochondria doing this show that they look pretty sick, but what would you expect with a big chunk of the ETC missing?
Just look at the internal structure loss in the mitochondria of the KO mice. Not so good...
Interestingly there are a number of quite severe complex I deficiency mitochondrial diseases in human clinical medicine. Current management is with a high fat diet, hats off to the clinicians for this. Somehow I doubt that sucrose is classified as a fat for these poor folks!
Overall, nice adipocytes. It still looks to me as if the F:N concept is essential for getting any sort of an idea of what might be going on in this type of model.
Peter
Aside: If you click on the TFAM link immediately below this paragraph and then go to "see all" under "related citations" you will find that there are a sh!tload of genes which can be knocked out to protect hapless mice from the dire consequences of D12492. Let's re appraise that as: Evolution has provided us with a stack of genes giving protection (read "normal processing") against the damage (lack of "normal processing") intrinsic to foods which are caricatured by D12492. They do this by inducing a decrease in glucose metabolism when serum FFAs are elevated. Knocking out these genes will reverse an unimaginably large number of years worth of evolution and I would anticipate that we might just end up dead slim when effective blocking drugs are developed to "cure" the obesity epidemic. Really dead as well as really slim. Anyhoo, back to the mice...
This is the paper where the gene for TFAM was knocked out in the adipocytes of some mice. Edit: Thanks to Purposelessness for the link through comments, end edit. Two consequences of this appear to be that complex I doesn't seem to be there and that complex IV, while present, doesn't seem to be up to much either. With a large chunk of their adipocyte ETC disabled these mice seem to be a little stunted overall but they stay slim and simply refuse to develop insulin resistance with either age or with the feeding of a ton of sugar, described throughout as a "high fat" diet, good old D12492. Don't snigger, this isn't funny!
If you have just come from Kushnareva et al's paper on complex I you could just ask whether the FeS complex N-1a is present or absent in these mice, but then you hardly need to ask this because the mice don't become insulin resistant, ie they don't generate superoxide at N-1a, even though the adipocytes have increased lipid and DNA damage, probably through them spewing electrons at complex IV. So there's really no need to trawl back through the layers of refs to the development of this mouse model to know that there is no FeS cluster N-1a. Or certainly nothing feeding electrons to it. If there were, there would be insulin resistance... I find this difficult to get excited about, useful though it is.
But the paper is utterly fascinating in several other ways. I went through the results to Fig 6 where TBARS and 8-OHdG are shown for D12492 fed mice, being surprised that no damage was reported when the mice were fed crapinabag (this is not quite as true as it sounds but, again, it's not important here). Not so good for D12492 feeding of course. Moral: Don't eat toffee fudge as your sole diet. There is a great deal else which could be said about free radical damage in these mice but that's not for today.
What I noticed, immediately below the TBARS figure and still within Fig 6 of the main paper, was the relative abundances of assorted activated fatty acids from both brown and white adipose tissue:
Of particular interest to me was the rather high relative abundance of activated (carnitine linked) behenic acid, C22:0, over on the far right of each chart. Now bear in mind that there is not a lot of C22:0 in the fat of normal mice anyway, so the massive relative abundance may be, in part, an artifact of a rather low level of C22:0 in control mice. But there is probably a lot more in mice with a crippled ETC. Behenic acid is fully saturated, it's long, it's F:N ratio is as close to 0.5 as you can get and it should be rapidly degraded in peroxisomes. But these mice have markedly down regulated peroxisome promoter genes and the organelles shown in the photomicrograph look, apparently, pretty crappy.
So let's have a think about how this might fit together. These adipocytes have no complex 1. What can you do with NADH in mitochondria if there is no complex I? Diddly squat is, I think, the correct term. Glycolysis works fine and generates a smidge of ATP by substrate level phosphorylation. And some NADH too, which is of no use what so ever. Complex II, succinate dehydrogenase, is fully conserved and can feed in to the CoQ pool to run the ETC, the abnormal complex IV doing the best it can. No matter how much the CoQ pool is reduced there will be no reverse electron flow through complex I because complex I ain't there.
But running from pyruvate dehydrogenase through the TCA to succinate dehyrogenase generates 3 utterly useless NADHs and completing the TCA gets you yet another NADH. What's a cell to do?
Well, it can start by generating a ton of pyruvate by glycolysis. These cells do have a lot of pyruvate. You can feed this in to mitochondria and through pyruvate dehydrogenase (or even pyruvate carboxylase, see below) then generate citric acid from oxaloacetate. If you really don't want to generate more useless NADH than you have to, then why not export the citrate to the cytoplasm through the malate shuttle and convert it to long chain fatty acids? Fatty acids only half-care about putting NADH in to complex I. They will, of course, generate some NADH but what they also do, which is really useful, is to generate FADH2 within electron-transferring flavoprotein which can be used via electron-transferring-flavoprotein dehydrogenase to reduce the CoQ pool, without all of that cyclical messing around with the TCA. And without any need for complex I of course.
As we know, the longer the saturated fat the more FADH2 it generates per unit NADH. These cells desperately need FADH2 input to reduce the CoQ pool. A 22 carbon fully saturated fat is a good option. Behenic acid. Peroxisomes are NOT wanted, it's FADH2 to CoQ all the way.
Of course as acetyl CoA is taken for fatty acid synthesis the citrate yields malate again which, if it re enters the TCA, will give another useless NADH as it converts to oxaloacetate. So why not convert the malate back to pyruvate (replacing the NADPH used to generate that malate) and then use pyruvate carboxylase to generate oxaloacetate without the obligatory NADH resulting from when malate is converted to oxaloacetate? You can then allow citrate regeneration which can be re-exported to cycle around through more FFA generation, yielding usable FADH2 again.
Looking at Fig S6 from the supplementary data we can see that, of the TCA metabolites, only citrate is significantly increased. Pity they didn't measure oxaloacetate.
Pyruvate is increased as you might expect. Glycolysis really needs to be generating NADPH (pentose phosphate pathway) for fatty acid synthesis but I can't see from the paper if this might be occurring. I think it's a reasonable assumption that it is, because the behenic acid is not coming from the diet and NADPH is needed for the fatty acid synthesis which generates it in-situ. There is plenty of lactate derived from the excess of pyruvate over what is needed by the crippled mitochondria.
I also can't tell from the paper how much the glycerol phosphate shuttle is up regulated to feed in through it's own FADH2 route to reduce the CoQ couple either. I suspect it's rather active.
Anything which inputs to the CoQ couple other than complex I seems to be essential for these cells to survive. The impression I get is of adipocytes which are simply converting glucose to fat and running on a (genuine) high fat diet at the mitochondrial level, despite the oral diet being crapinabag.
The photomicrographs of the mitochondria doing this show that they look pretty sick, but what would you expect with a big chunk of the ETC missing?
Just look at the internal structure loss in the mitochondria of the KO mice. Not so good...
Interestingly there are a number of quite severe complex I deficiency mitochondrial diseases in human clinical medicine. Current management is with a high fat diet, hats off to the clinicians for this. Somehow I doubt that sucrose is classified as a fat for these poor folks!
Overall, nice adipocytes. It still looks to me as if the F:N concept is essential for getting any sort of an idea of what might be going on in this type of model.
Peter
Wednesday, February 06, 2013
Protons: Where is FeS cluster N-1a?
I picked up this paper from one of Nick Lane's books, can't remember which one but probably PSS. The paper itself is very, very interesting and delves deeply in to redox potentials, especially within the iron-sulphur centres of complex I, to the sort of level which is way beyond my ability to critique. So I'm accepting their findings pretty much as presented, with the usual caveats about mitochondial studies. In particular it worries me that we can study superoxide generation in mitochondria at partial pressures of oxygen around 100mmHg (room air). No mitochondria see this concentration of oxygen in vivo.
The group make a pretty good case that the FeS centre N-1a is the primary source of superoxide generation from complex I when mitochondria are being fed on NADH generating substrates, in particular when there is a large excess of NADH per unit NAD+.
It also appears to be the source of superoxide under succinate driven activation of complex II, probably secondary to reverse electron flow up the FeS clusters secondary to markedly reducing the CoQ couple.
The group didn't look at activated fatty acids but the F:N ratios would suggest to me that palmitic acid and upwards (chain length of fully saturated fats) would behave much as succinate does, by reducing the CoQ couple in a very similar manner. I've argued for some time that superoxide is the physiological switch to turn off glucose metabolism (ie trigger insulin resistance at the individual cell level) when generous levels of saturated fats predominate for oxidation.
This leaves open the simple question of what, exactly, is the N-1a FeS cluster of complex I? What does it do? What does complex I look like anyway? How much iron-sulphur does it contain? I did a quick Google image search for the structure of iron-sulphur chain of complex I and it turned up some beautiful pictures. I rather like this one from the Netherlands:
Look at the string of FeS clusters, shown in red, running through the protein bed. Cluster N-1a is the one right adjacent to FMN, probably set behind it rather than as close as it looks. The one off to the right is N7. Modern complex I almost certainly never originated as a proton pump but its deepest ancestor rather looks to have been a layer of iron pyrites, FeS. Up at the top of the picture, in yellow, is the flavin mono nucleotide which transfers electrons from NADH to the FeS chain. At the bottom, in pink, is ubiquinone of the CoQ pool, ready to transfer electron equivalents to complex III. The CoQ pool is fascinating as having it reduced (in this paper by supplying succinate to complex II) appears to cause reverse flow up the FeS chain as far as (and perhaps beyond) the side branch to N-1a, with superoxide generation as the result. Palmitic acid probably does the same but obviously reduces the CoQ pool using electron-transferring-flavoprotein dehydrogenase (which lacks a catchy name) rather than succinate dehydrogenase (complex II), as we've discussed previously.
Both NADH and CoQ move electron equivalents around on a macroscopic scale by moving physically. The FeS clusters shuttle electrons by quantum tunnelling. This is a very short distance phenomenon. Below are the sorts of distances between the various FeS clusters in complex I. If anyone thinks the arrangement of FeS clusters is in any way a random set up I suspect they have no concept of what 4 billion years means.
We can ignore the N7 cluster as it's not really in the FeS chain at all, being over 20Å out to one side and it is not highly conserved. Cluster N-1a is highly conserved but it too is not really part to the chain of FeS clusters. It also sits out to one side, about 11Å out from FMN. This is a perfectly reasonable tunnelling distance to transfer electrons to and from FMN, but to hand an electron on to the N3 cluster (and so to the rest of the FeS chain) is over 19Å, a relatively low probability electron transfer. So N-1a looks as if it might be functioning as a reflection of the redox state of FMN, which reflects the reduction of the NADH/NAD+ couple. With a highly reduced NADH/NAD+ couple there is a spare electron on N-1a just waiting for an oxygen molecule to convert to superoxide and signal "no more" at the macroscopic (cellular) level... It will be interesting to see how this message is carried from N-1a to wherever it affects cell function but for the time being it's the superoxide generation which I find interesting per se.
I think it is worth pointing out that N-1a is directly sat at the very first step of the first complex of the electron transport chain. Where else would you put the control point? Very neat...
This gives us a nice view of complex I and N-1a linked superoxide generation which sets us up to look at what happens when you destroy complex I by growing mice with the TFAM gene knocked out in their adipocytes... Let's go there soon, if not next.
Peter
The group make a pretty good case that the FeS centre N-1a is the primary source of superoxide generation from complex I when mitochondria are being fed on NADH generating substrates, in particular when there is a large excess of NADH per unit NAD+.
It also appears to be the source of superoxide under succinate driven activation of complex II, probably secondary to reverse electron flow up the FeS clusters secondary to markedly reducing the CoQ couple.
The group didn't look at activated fatty acids but the F:N ratios would suggest to me that palmitic acid and upwards (chain length of fully saturated fats) would behave much as succinate does, by reducing the CoQ couple in a very similar manner. I've argued for some time that superoxide is the physiological switch to turn off glucose metabolism (ie trigger insulin resistance at the individual cell level) when generous levels of saturated fats predominate for oxidation.
This leaves open the simple question of what, exactly, is the N-1a FeS cluster of complex I? What does it do? What does complex I look like anyway? How much iron-sulphur does it contain? I did a quick Google image search for the structure of iron-sulphur chain of complex I and it turned up some beautiful pictures. I rather like this one from the Netherlands:
Look at the string of FeS clusters, shown in red, running through the protein bed. Cluster N-1a is the one right adjacent to FMN, probably set behind it rather than as close as it looks. The one off to the right is N7. Modern complex I almost certainly never originated as a proton pump but its deepest ancestor rather looks to have been a layer of iron pyrites, FeS. Up at the top of the picture, in yellow, is the flavin mono nucleotide which transfers electrons from NADH to the FeS chain. At the bottom, in pink, is ubiquinone of the CoQ pool, ready to transfer electron equivalents to complex III. The CoQ pool is fascinating as having it reduced (in this paper by supplying succinate to complex II) appears to cause reverse flow up the FeS chain as far as (and perhaps beyond) the side branch to N-1a, with superoxide generation as the result. Palmitic acid probably does the same but obviously reduces the CoQ pool using electron-transferring-flavoprotein dehydrogenase (which lacks a catchy name) rather than succinate dehydrogenase (complex II), as we've discussed previously.
Both NADH and CoQ move electron equivalents around on a macroscopic scale by moving physically. The FeS clusters shuttle electrons by quantum tunnelling. This is a very short distance phenomenon. Below are the sorts of distances between the various FeS clusters in complex I. If anyone thinks the arrangement of FeS clusters is in any way a random set up I suspect they have no concept of what 4 billion years means.
We can ignore the N7 cluster as it's not really in the FeS chain at all, being over 20Å out to one side and it is not highly conserved. Cluster N-1a is highly conserved but it too is not really part to the chain of FeS clusters. It also sits out to one side, about 11Å out from FMN. This is a perfectly reasonable tunnelling distance to transfer electrons to and from FMN, but to hand an electron on to the N3 cluster (and so to the rest of the FeS chain) is over 19Å, a relatively low probability electron transfer. So N-1a looks as if it might be functioning as a reflection of the redox state of FMN, which reflects the reduction of the NADH/NAD+ couple. With a highly reduced NADH/NAD+ couple there is a spare electron on N-1a just waiting for an oxygen molecule to convert to superoxide and signal "no more" at the macroscopic (cellular) level... It will be interesting to see how this message is carried from N-1a to wherever it affects cell function but for the time being it's the superoxide generation which I find interesting per se.
I think it is worth pointing out that N-1a is directly sat at the very first step of the first complex of the electron transport chain. Where else would you put the control point? Very neat...
This gives us a nice view of complex I and N-1a linked superoxide generation which sets us up to look at what happens when you destroy complex I by growing mice with the TFAM gene knocked out in their adipocytes... Let's go there soon, if not next.
Peter
Subscribe to:
Posts (Atom)
