Back in 1995 Veech was looking at a ketone mixture as physiologically equivalent to insulin/glucose. In order to limit his variables the isolated rat myocardia used in the study were perfused with Krebs-Henseleit buffer containing the metabolic milieu of interest. The buffer has no free fatty acids so takes the provision of acetyl CoA from beta oxidation right out of the equation. It also eliminates any uncoupling from free fatty acids in the perfusate. It took me a while to twig that this was potentially a very long way from the situation under fasting or ketogenic diet conditions where free fatty acids might well be at the maximal physiological levels whenever ketones hit 5.0mmol/l.
The idea was certainly in mind when the group published this, in 2004:
“Current ketogenic diets are all characterized by elevations of free fatty acids, which may lead to metabolic inefficiency by activation of the PPAR system and its associated uncoupling mitochondrial uncoupling proteins. New diets comprised of ketone bodies themselves or their esters may obviate this present difficulty.”
By 2012 the problem with ketogenic diets had been reduced to one of impossible compliance, rather than metabolic inefficiency of free fatty acid metabolism:
"Further, to achieve effective ketosis with KG diets, almost complete avoidance of carbohydrates is required to keep blood insulin levels low to maintain adipose tissue lipolysis. Such high-fat, no-carbohydrate diets are unpalatable, leading to poor patient compliance."
You notice the uncoupling, previously a potential problem, is now in the title of the paper. Ketones in real life, even from ketone esters, work in a milieu of free fatty acids. If you flood the mitochondria with ATP-generating ketones, which generate no ATP in the cytoplasm, you just might expect to open that uncoupling pore and allow a few FFAs to translocate some protons, to limit over production of ATP within the mitochondria.
Currently, in 2014, the delectable savour-the-flavour of ketone esters allows this:
“…the ester can be taken as an oral supplement without changing the habitual diet.”
I watch this stuff with some degree of amazement. There is a suspicion that AD incidence is increasing rather faster than an ageing population would explain. The suggestion is that it has an environmental component. Now, many potential explanations are possible but I would like to think it is the saturophobic, cholesterophobic, fructophilic low fat based dietary advice from the American Heart Association which is the prime driver. Seems likely.
If AD (also known as type 3 diabetes) is a dietary disease, much as type 2 diabetes is largely a dietary disease, providing a crutch which will allow you to cling to the diet which got you in to AD in the first place strikes me as the biggest risk from ketone esters.
Excepting the stale urine/sweaty socks yummy aroma of course. Bring on the egg yolks fried in butter as an alternative, please.
A ketogenic diet features several things in addition to ketones. There is the chronic normoglycaemia which is anathema to the Crabtree effect. There is the physiological rock bottom basement insulin levels in a system where insulin signalling is f*cked. There are the elevated free fatty acids. These are the best.
Those free fatty acids are taken up by astroglial cells and used to generate in-situ ketone bodies. What sort of levels do they supply in vivo? That's an unknown (as far as I can tell), but I'm willing to bet that FFA supply under true ketogenic eating is both high and consistent, irrespective of fed/fasted state.
This is not quite the case if you are on the old MCT kick or mainlining sweaty socks while munching crapinabag.
Peter
A little background about Dr and Mr Newport and ketones which triggered this post off:
I have been unable to tease out, from Dr Newport's original article, that of Emily Deans or from the abstract of the case report above, quite what level of carbohydrate Mr Newport consumed in the original MCT phase, during the drug trial or while on ketone esters. I suspect it might have been more than a banana a day.
Oh, and another addendum. I, personally, clearly have issues with faking a ketogenic diet. This is true. But let me not decry ketones or their esters per se. If MCT oil or ketone esters get you out of bed and let you get dressed without needing assistance, that's great. They sure as hell knock spots off of anything which Big Pharma has to offer for AD management. The fact that I have yet to die as a direct result of eating less than one banana a day means that I hope never to need ketone esters. I feel a ketogenic diet should be high on the agenda for those with neurodegenerative diseases, with ketone esters or MCTs as a fall back. But then I would, wouldn't I...
Tuesday, October 28, 2014
Sunday, October 12, 2014
Where has the superoxide gone?
This is the first section of Fig 1 section C from the paper using dihydroethidium (DHE) to view in vivo superoxide production in control and diabetic kidneys, though not in the figure below.
It's a very important figure as it shows, very convincingly, that sudden onset hyperglycaemia has zero effect, none whatsoever, on superoxide production in their model of normal, non diabetic kidney tissue, that's the second column, identical to the first.
I have a lot of time for the failure to generate superoxide in diabetic kidneys, especially with pyruvate dehydrogenase complex down regulation limiting input to mitochondria from the end stage of glycolysis. But I have a concept that acute hyperglycaemia in normal, non Crabtree affected, tissues SHOULD generate superoxide, it should come from the respiratory chain and it should more particularly come from complex I in the region of the FAD moiety, preferably via the FeS cluster N1-a.
Now, if I had an in vivo tool for viewing superoxide generation, how would I do this? Well, I would use it in vivo. I would set up an iv glucose infusion, or perhaps a large intragastric glucose bolus, inject the DHE, wait a while, then look for superoxide/DHE derivative with my lovely optical scanner.
To keep the scrutineers happy I might have repeated the findings ex vivo, using the technique of paramagnetic detection of a superoxide/spin trap derivative, but the core finding, that superoxide generation on acute hyperglycaemia does NOT occur has to be shown in vivo. We already know it DOES occur ex vivo in multiple models, and the authors cite the studies to show this.
So, if hyperglycaemia triggers superoxide generation ex vivo in assorted non Crabtree adapted cells, why doesn't it do so in this study?
I don't know. There is a piece of core information which the scrutineers failed (miserably) to demand to be included in the study methods.
Figure 1C was not obtained in vivo. Column Ctrl was from a tissue homogenate of health kidney from non diabetic mice fed with pyruvate, malonate and ADP, subsequently flooded with 25mmol of glucose to produce the +HG column. That is not so bad. It's a model and it's clearly able to get GrantAid quality results.
But is it real?
Let's look at the equipment used. This is what they say:
"These studies were carried out in a MiniScope MS200 Benchtop EPR Spectrometer (Magnettech), which is designed to allow tight control of pO2 and temperature".
Why do they need tight control of pO2? You can obtain utterly rigid control control of pO2 by exposing your preparation to room air. Correct pressure to 760mmHG and pO2 is fixed at 21% of this.
To me the question is: What was the pO2 which failed to generate any superoxide when a mush of cytosol and mitochondria was exposed to 25mmol of glucose?
Was it 159.6mmHg, i.e. room air? Was it 40-50mmHg as other groups suspect mitochondria run at? Or was it 22mmHg?
This might matter. I got the 22mmHg value from the previous paper by the same authors which gave 3% oxygen as the likely conditions for normal mitochondrial function. This was a non referenced, throw away comment:
"Because the physiologic concentration of oxygen in mammals in vivo is less than 3% in most organs, we carried out a series of studies to determine whether ethidium or 2-hydroxyethidium was the specific oxidation product of DHE in vivo (i.e., in the intact animal, not cell culture/tissue slice) using several different validated animal models of increased or decreased superoxide".
Why it matters to me so much is that if an electron is thrown out of complex I due to hyperglycaemia triggered reverse electron flow through complex I, would it generate superoxide if the pO2 had been set to below physiological limits? Or if the guesstimate of 3% oxygen is correct and there is no superoxide generated, is there no reverse flow occurring? Or does the reverse flow occur, the electron is ejected, but it drops on to the surrounding protein structure rather than oxygen to be used as a distant signal via superoxide/H2O2/insulin receptor?
Using the in vivo technique would have told us exactly what was happening, at a true but non measured tissue pO2. I'm worried that the in vivo technique showed the anticipated (by me) hyperglycaemic superoxide and an ex vivo technique had to be developed and adjusted to maintain the fund generating core finding of no extra superoxide.
There was no reply to a simple polite email query as to the pO2 used.
Peter
It's a very important figure as it shows, very convincingly, that sudden onset hyperglycaemia has zero effect, none whatsoever, on superoxide production in their model of normal, non diabetic kidney tissue, that's the second column, identical to the first.
I have a lot of time for the failure to generate superoxide in diabetic kidneys, especially with pyruvate dehydrogenase complex down regulation limiting input to mitochondria from the end stage of glycolysis. But I have a concept that acute hyperglycaemia in normal, non Crabtree affected, tissues SHOULD generate superoxide, it should come from the respiratory chain and it should more particularly come from complex I in the region of the FAD moiety, preferably via the FeS cluster N1-a.
Now, if I had an in vivo tool for viewing superoxide generation, how would I do this? Well, I would use it in vivo. I would set up an iv glucose infusion, or perhaps a large intragastric glucose bolus, inject the DHE, wait a while, then look for superoxide/DHE derivative with my lovely optical scanner.
To keep the scrutineers happy I might have repeated the findings ex vivo, using the technique of paramagnetic detection of a superoxide/spin trap derivative, but the core finding, that superoxide generation on acute hyperglycaemia does NOT occur has to be shown in vivo. We already know it DOES occur ex vivo in multiple models, and the authors cite the studies to show this.
So, if hyperglycaemia triggers superoxide generation ex vivo in assorted non Crabtree adapted cells, why doesn't it do so in this study?
I don't know. There is a piece of core information which the scrutineers failed (miserably) to demand to be included in the study methods.
Figure 1C was not obtained in vivo. Column Ctrl was from a tissue homogenate of health kidney from non diabetic mice fed with pyruvate, malonate and ADP, subsequently flooded with 25mmol of glucose to produce the +HG column. That is not so bad. It's a model and it's clearly able to get GrantAid quality results.
But is it real?
Let's look at the equipment used. This is what they say:
"These studies were carried out in a MiniScope MS200 Benchtop EPR Spectrometer (Magnettech), which is designed to allow tight control of pO2 and temperature".
Why do they need tight control of pO2? You can obtain utterly rigid control control of pO2 by exposing your preparation to room air. Correct pressure to 760mmHG and pO2 is fixed at 21% of this.
To me the question is: What was the pO2 which failed to generate any superoxide when a mush of cytosol and mitochondria was exposed to 25mmol of glucose?
Was it 159.6mmHg, i.e. room air? Was it 40-50mmHg as other groups suspect mitochondria run at? Or was it 22mmHg?
This might matter. I got the 22mmHg value from the previous paper by the same authors which gave 3% oxygen as the likely conditions for normal mitochondrial function. This was a non referenced, throw away comment:
"Because the physiologic concentration of oxygen in mammals in vivo is less than 3% in most organs, we carried out a series of studies to determine whether ethidium or 2-hydroxyethidium was the specific oxidation product of DHE in vivo (i.e., in the intact animal, not cell culture/tissue slice) using several different validated animal models of increased or decreased superoxide".
Why it matters to me so much is that if an electron is thrown out of complex I due to hyperglycaemia triggered reverse electron flow through complex I, would it generate superoxide if the pO2 had been set to below physiological limits? Or if the guesstimate of 3% oxygen is correct and there is no superoxide generated, is there no reverse flow occurring? Or does the reverse flow occur, the electron is ejected, but it drops on to the surrounding protein structure rather than oxygen to be used as a distant signal via superoxide/H2O2/insulin receptor?
Using the in vivo technique would have told us exactly what was happening, at a true but non measured tissue pO2. I'm worried that the in vivo technique showed the anticipated (by me) hyperglycaemic superoxide and an ex vivo technique had to be developed and adjusted to maintain the fund generating core finding of no extra superoxide.
There was no reply to a simple polite email query as to the pO2 used.
Peter
Friday, October 10, 2014
The Crabtree Effect and superoxide in diabetes
I started with this paper about in vivo superoxide detection in the brain but, apart from the technique, there was no examination of the response to hyperglycaemia so I moved on. The next paper by the same group is looking at superoxide and mitochondrial function/health in the kidney under various models of diabetes. The general principles appear similar in neurons and kidney cells.
An in vivo technique to view superoxide is really useful. I have alluded to a certain discomfort in examining electron/oxygen interaction in mitochondria within cells/mitochondrial preparations under room air, with a partial pressure for oxygen of around 150mmHg (sorry for the non SI units, showing my age there!). There is no way that normal mitochondria are exposed to this much oxygen, a little browse around pubmed suggests that the best in vivo estimate is around 40-50mmHg, subject to some debate. That's without even thinking about what CO2 partial pressure you should use for cell culture... So observation in vivo takes care of a lot of this. If an electron is thrown out of the respiratory chain (I feel nothing in the ETC is accidental) the chances of it dropping on to an oxygen molecule seem somewhat higher if we have three times the oxygen partial pressure than the system was designed to work under. If the electron wasn't destined for an oxygen molecule, where else might it have been going?
The first point has to be that in two models of type one diabetes there is less superoxide production in the kidneys of diabetic mice in vivo than control mice, that's Figure 1 section C. Going ex vivo (I probably have a full post on the problems with this ex vivo section) we have the same effect demonstrated using a paramagnetic technique, that's Fig 2C. The reduced superoxide in diabetic kidneys was confirmed in the tissue homogenates under relatively normal metabolic substrate supply. Exposing the preparations to glucose at 25mmol/l has no effect on superoxide generation from the control kidney homogenate but actually reduces it, rather a lot, in the diabetic derived homogenate.
Interesting.
NOTE If you follow the text through about Fig 1C and their SOD2+/- mice you will find that the data is not very accurately described. So caution here. The SOD2+/- had a non significant increase in mitochondrial superoxide in Fig 1C, so it is hardly surprising this did not rescue the diabetic mice from renal disease in Fig 3A and F. I don't like their writing about this whole SOD2+/- section. Definite caution. END NOTE.
The paper has, amongst its problems, a lot of very perceptive points which make a great deal of sense. It's quite hard to know where to start. Let's begin with the failure of superoxide production.
So this paper flies in the face of the Protons concept of hyperglycaemia driving reverse electron flow from mtG3Pdh through complex I to generate insulin resistance. That too is probably another post, comparing the diabetic state with the non diabetic hyperglycaemic state. Anyhoo. The group rather like Crabtree. So do I. The Crabtree effect, the shutting down/mothballing of mitochondrial function, is an adaptation to oversupply of glycolysis derived substrates. It allows a limit to be set on the throughput of pyruvate to mitochondria and jettisons any excess as lactate. This situation, once it is established, is probably quite different to the situation which leads to its adoption.
Chronic hyperglycaemia induces the Crabtree effect and down regulates mitochondrial biogenesis, mitochondrial repair and electron transport chain function. It not only does this but it also phosphorylates the pyruvate dehydrogenase complex, very specifically, and this directly shuts down input to the TCA from glycolysis (or input from lactate itself, if we want to apply this concept to neurons, as we might). This is all in the paper. Of course I would add that it doesn't affect ketone derived acetyl-CoA input to the TCA, although the ketone derived acetyl-CoA will be processed by a degenerate electron transport chain...
Under sustained hyperglycaemia there is an excess of calories which leads to a failure to activate AMP kinase, a core sensor of energy abundance which is phosphorylated under hypo caloric conditions. AMPK regulates PGC 1 alpha, a messenger to trigger mitochondrial biogenesis. But the central link, the activation of AMKP, is mitochondrial derived superoxide. And, oddly enough, one of the functions of AMPK activation is the generation of mitochondrial superoxide. A self sustaining loop.
The group administered rotenone to control mice. Now, the effect of rotenone on superoxide generation appears (in general) to be rather dose rate related. In the present study the dose rate was chosen so that there was a near complete suppression of superoxide production from the ETC of the mice. Acute suppression of superoxide results in the reduced phosphorylation (reduced activation) of AMPK and increased phosphorylation of PDH, which shuts it down. This loss of superoxide is a short term mimic of the long term established Crabtree effect. No superoxide, no mitochondrial maintenance. Consider that chronic high dose rotenone poisoning is a standard model for Parkinsons Disease and you begin to see the importance of superoxide in the brain. Long term hyperglycaemic failure to generate superoxide is probably a more normal route to neurodegeneration than rotenone in most (but not all) neurodegenerate humans...
The fall in superoxide production in diabetic tissue homogenates again pulls me back to brain function. Crabtree suppresses hyperglycaemic superoxide production, i.e. the effect is antioxidant. Let's look at what glucose does to neurons from this paper which we've chatted about before. Here's the only bit I'm interested in today:
"Indeed, it has been shown that glucose is used by neurons to maintain their antioxidant status via the pentose phosphate pathway (PPP), which cannot be fueled by lactate (Magistretti, 2008; Herrero-Mendez et al., 2009)"
It's impossible over emphasise the importance of that sentence. It says it all about why neurons should run on lactate! To avoid upregulating antioxidant status.
What does increasing antioxidant status do to superoxide signalling? The term f*cked comes (unavoidably South Park-ishly) to mind. There are a swathe of papers showing that the antioxidant status in neurons of AD and PD patients is upregulated.
Once you go with Crabtree you can see that glucose and PPP driven antioxidant upregulation might be all that is needed to lose superoxide signalling and destroy mitochondrial function. Lactate does not do this. Lactate does not induce the Crabtree effect.
Let's be very specific: Glucose, under the Crabtree effect, triggers a cascade which ends up with failure to generate superoxide and this maintains mitochondrial shutdown. Up regulating antioxidant status may theoretically be helpful in dealing with non mitochondrial superoxide generation, but it's not going to help signal for mitochondrial biogenesis.
High glucose exposure generates glucose dependence. This is a recurring theme and is core to neurodegeneration. I look at safe starches and can see that, if you are living with the Crabtree effect in key neurons, some starch/glycolysis might make you feel better if you are ketogenically hypoglycaemic, but it's not going to help un-Crabtree your mitochondria. On the other hand I can't see that pushing starch to a level which produces hyperglycaemia is anything other than damaging, as opposed to merely neutral as it might be when your pancreas does its job effectively.
I'll take a break before going on to the sections on mitochondrial deletions and respiratory chain oxidative damage elsewhere in the paper. Or maybe I should talk about the bits I deeply dislike related to oxygen pressure and superoxide.
Peter
An in vivo technique to view superoxide is really useful. I have alluded to a certain discomfort in examining electron/oxygen interaction in mitochondria within cells/mitochondrial preparations under room air, with a partial pressure for oxygen of around 150mmHg (sorry for the non SI units, showing my age there!). There is no way that normal mitochondria are exposed to this much oxygen, a little browse around pubmed suggests that the best in vivo estimate is around 40-50mmHg, subject to some debate. That's without even thinking about what CO2 partial pressure you should use for cell culture... So observation in vivo takes care of a lot of this. If an electron is thrown out of the respiratory chain (I feel nothing in the ETC is accidental) the chances of it dropping on to an oxygen molecule seem somewhat higher if we have three times the oxygen partial pressure than the system was designed to work under. If the electron wasn't destined for an oxygen molecule, where else might it have been going?
The first point has to be that in two models of type one diabetes there is less superoxide production in the kidneys of diabetic mice in vivo than control mice, that's Figure 1 section C. Going ex vivo (I probably have a full post on the problems with this ex vivo section) we have the same effect demonstrated using a paramagnetic technique, that's Fig 2C. The reduced superoxide in diabetic kidneys was confirmed in the tissue homogenates under relatively normal metabolic substrate supply. Exposing the preparations to glucose at 25mmol/l has no effect on superoxide generation from the control kidney homogenate but actually reduces it, rather a lot, in the diabetic derived homogenate.
Interesting.
NOTE If you follow the text through about Fig 1C and their SOD2+/- mice you will find that the data is not very accurately described. So caution here. The SOD2+/- had a non significant increase in mitochondrial superoxide in Fig 1C, so it is hardly surprising this did not rescue the diabetic mice from renal disease in Fig 3A and F. I don't like their writing about this whole SOD2+/- section. Definite caution. END NOTE.
The paper has, amongst its problems, a lot of very perceptive points which make a great deal of sense. It's quite hard to know where to start. Let's begin with the failure of superoxide production.
So this paper flies in the face of the Protons concept of hyperglycaemia driving reverse electron flow from mtG3Pdh through complex I to generate insulin resistance. That too is probably another post, comparing the diabetic state with the non diabetic hyperglycaemic state. Anyhoo. The group rather like Crabtree. So do I. The Crabtree effect, the shutting down/mothballing of mitochondrial function, is an adaptation to oversupply of glycolysis derived substrates. It allows a limit to be set on the throughput of pyruvate to mitochondria and jettisons any excess as lactate. This situation, once it is established, is probably quite different to the situation which leads to its adoption.
Chronic hyperglycaemia induces the Crabtree effect and down regulates mitochondrial biogenesis, mitochondrial repair and electron transport chain function. It not only does this but it also phosphorylates the pyruvate dehydrogenase complex, very specifically, and this directly shuts down input to the TCA from glycolysis (or input from lactate itself, if we want to apply this concept to neurons, as we might). This is all in the paper. Of course I would add that it doesn't affect ketone derived acetyl-CoA input to the TCA, although the ketone derived acetyl-CoA will be processed by a degenerate electron transport chain...
Under sustained hyperglycaemia there is an excess of calories which leads to a failure to activate AMP kinase, a core sensor of energy abundance which is phosphorylated under hypo caloric conditions. AMPK regulates PGC 1 alpha, a messenger to trigger mitochondrial biogenesis. But the central link, the activation of AMKP, is mitochondrial derived superoxide. And, oddly enough, one of the functions of AMPK activation is the generation of mitochondrial superoxide. A self sustaining loop.
The group administered rotenone to control mice. Now, the effect of rotenone on superoxide generation appears (in general) to be rather dose rate related. In the present study the dose rate was chosen so that there was a near complete suppression of superoxide production from the ETC of the mice. Acute suppression of superoxide results in the reduced phosphorylation (reduced activation) of AMPK and increased phosphorylation of PDH, which shuts it down. This loss of superoxide is a short term mimic of the long term established Crabtree effect. No superoxide, no mitochondrial maintenance. Consider that chronic high dose rotenone poisoning is a standard model for Parkinsons Disease and you begin to see the importance of superoxide in the brain. Long term hyperglycaemic failure to generate superoxide is probably a more normal route to neurodegeneration than rotenone in most (but not all) neurodegenerate humans...
The fall in superoxide production in diabetic tissue homogenates again pulls me back to brain function. Crabtree suppresses hyperglycaemic superoxide production, i.e. the effect is antioxidant. Let's look at what glucose does to neurons from this paper which we've chatted about before. Here's the only bit I'm interested in today:
"Indeed, it has been shown that glucose is used by neurons to maintain their antioxidant status via the pentose phosphate pathway (PPP), which cannot be fueled by lactate (Magistretti, 2008; Herrero-Mendez et al., 2009)"
It's impossible over emphasise the importance of that sentence. It says it all about why neurons should run on lactate! To avoid upregulating antioxidant status.
What does increasing antioxidant status do to superoxide signalling? The term f*cked comes (unavoidably South Park-ishly) to mind. There are a swathe of papers showing that the antioxidant status in neurons of AD and PD patients is upregulated.
Once you go with Crabtree you can see that glucose and PPP driven antioxidant upregulation might be all that is needed to lose superoxide signalling and destroy mitochondrial function. Lactate does not do this. Lactate does not induce the Crabtree effect.
Let's be very specific: Glucose, under the Crabtree effect, triggers a cascade which ends up with failure to generate superoxide and this maintains mitochondrial shutdown. Up regulating antioxidant status may theoretically be helpful in dealing with non mitochondrial superoxide generation, but it's not going to help signal for mitochondrial biogenesis.
High glucose exposure generates glucose dependence. This is a recurring theme and is core to neurodegeneration. I look at safe starches and can see that, if you are living with the Crabtree effect in key neurons, some starch/glycolysis might make you feel better if you are ketogenically hypoglycaemic, but it's not going to help un-Crabtree your mitochondria. On the other hand I can't see that pushing starch to a level which produces hyperglycaemia is anything other than damaging, as opposed to merely neutral as it might be when your pancreas does its job effectively.
I'll take a break before going on to the sections on mitochondrial deletions and respiratory chain oxidative damage elsewhere in the paper. Or maybe I should talk about the bits I deeply dislike related to oxygen pressure and superoxide.
Peter
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