Raphi tweeted this paper recently
Nutritional Ketosis Increases NAD+/NADH Ratio in Healthy Human Brain: An in Vivo Study by 31P-MRS
which is nice provided, as he comments, it can be replicated. There is absolutely no possible conflict of interest anywhere so long as you accept it looks like an in-house Nestlé study. I haven't knowingly bought a Nestlé product in over 30 years.
Anyway. The study looks at healthy brain biochemistry under MCT induced ketosis. The ketone oxidation (or possibly the CNS oxidation of MCTs) increases the NAD+:NADH ratio, ie moves it in the Good direction.
There is a lot of talk about the NADH generation and NAD+ depletion during glycolysis to pyruvate, shifting the ratio in the Bad direction. The assumption (with which I disagree) is that the glycerophosphate shuttle is a rescue mechanism to regenerate essential NAD+ to allow glycolysis to continue, to which I will return in a moment.
The beauty of ketones is that they do not deplete cytoplasmic NAD+ at all and only consume one mitochondrial NAD+ during the conversion of BHB to AcAc. Because this happens within the mitochondria this, plus any NADH generated at the pyruvate dehydrogenase complex, is sitting next to complex I, the most prolific re-generator of NAD+ in the cell...
All well and good and bully for ketones and the manufacturers of Peptamen®1.5 Vanilla (Nestlé Health Science SA).
This got me thinking.
Of course no one in their right mind would expect glycolysis to be arranged in such a manner as to require the glycerophosphate shuttle for simple NAD+ regeneration. This is a wasteful loss of four pumped protons and this energy will appear as heat. Think of brown adipose tissue, full of mtG3Pdh, assuming insulin is plentiful. The correct pathway for the metabolism of glucose without insulin is to lactate without any overall depletion of cytoplasmic NAD+. Lactate can then be taken up by mitochondria exactly as ketones are. Lactate will, in the mitochondria, be reconverted to pyruvate, depleting mitochondrial NAD+ in exactly the same way as the conversion of BHB to AcAc does. Equally this happen right next door to complex I, just waiting to regenerate NAD+ and keep that NAD+:NADH ratio nice and high.
The whole point of the glycerophosphate shuttle (in Protons terms) is to facilitate insulin signalling. Insulin is the hormone of plenty, used to encourage caloric ingress in to cells. Loss of the four pumped protons due to bypassing complex I and using mtG3Pdh instead as part of insulin signalling appears perfectly reasonable under conditions of active caloric ingress. Sustained insulin signalling causes sustained loss of cytoplasmic NADH, which generates NAD+. Once this has happened there is no longer the surfeit of cytoplasmic NADH over NAD+ from glycolysis, which is essential to drive lactate formation. Glycolysis must therefor stop at pyruvate under insulin.
Summary: For insulin signalling the glycerophosphate shuttle is active and loss of NADH requires glycolysis to abort at pyruvate.
Without insulin signalling glycolysis runs to lactate which enters mitochondria without any depletion of cytoplasmic NAD+. The lactate should enter the mitochondria, under normal physiology.
Sooooooo. This had me thinking about what would happen if, in the presence of copious glucose and copious oxygen, there was to be a sudden profound fall in absolute insulin levels. I was particularly interested in systemic lactate levels.
A sudden, profound fall in insulin levels in the presence of glucose is pathology. It generates ketoacidosis, classically from acute beta cell destruction during the onset of DMT1. There is always a profound metabolic acidosis from the failure to suppress glucagon-induced lipolysis and subsequent massive acidic ketone generation. Under the canonical view the absence of insulin should not stop NAD+ regeneration by the glycerophosphate shuttle.
What I wanted to know was whether the Protons predicted shutting down of the glycerophosphate shuttle due to hypoinsulinaemia would result in diversion past pyruvate to lactate as the end result of glycolysis. In the presence of massive levels of ketones I would also expect this lactate to appear in the systemic situation.
Does it?
Yep. Ten seconds on Google says so.
Lactic acidosis in diabetic ketoacidosis
Very nice. I had no idea this was the case because it has no direct influence on treating DKA clinically...
Peter
Of course you have to think about the chicken and egg situation with insulin and mtG3Pdh activation (I have been for years!). Which comes first? I think insulin appears to be essential, as above. I do wonder if the insulin receptor will turn out to dock with the glycerophosphate shuttle in some way...
Sunday, November 25, 2018
Saturday, November 24, 2018
Metallic iron and the origin of metabolism
Over the years I've been convinced that carbon monoxide derived formaldehyde/formate are probably the initial molecular precursors of acetate at the origin of life. All that is needed is a supply of electrons at a sufficiently negative potential to reduce CO2 to CO and so to CH2O then to HCOOH, formate. Clearly a 1.5 volt battery applied across an anoxic CO2 rich reactor might do this. In the Life series of posts the best candidate in reality is the alkaline hydrothermal vent environment such as the Lost City complex, working under anoxic, CO2 rich Hadean ocean conditions.
This paper:
Native iron reduces CO2 to intermediates and endproducts of the acetyl-CoA pathway
from a french institute, suggests that metallic iron alone might provide electrons of sufficiently negative potential to perform the process, this is the basic premise:
Fe0 → Fe2++ 2e-
These electrons have a sufficiently negative potential to allow:
CO2 + 2e- + H2O → HCOOH + O2-
Obviously the Fe2+ would combine with the O2- to give FeO, leaving a formate moiety as the start of the process essential for the origin of pre-biotic metabolism.
In the event the two most common experimental products were acetate and pyruvate, a highly plausible step or two onward from formate, which they also found under certain conditions.
The circumstances of temperature and pressure were, in some experiments, plausible for pre-biotic chemistry.
The problems, compared to the Lane and Martin hydrothermal vents concept, seem to be:
The products are bound to the surface of the iron deposit, potassium hydroxide was needed to hydrolyse them off for measurement.
The process is reactive rather than catalytic, ie the metallic iron is consumed in the process of providing electrons. This contrasts starkly with the continuous supply of electrons supplied by hydrothermal vent conditions over geological time scales.
Then there is the concentration problem. If the organic products were to be freed from the iron surface they need to be somewhere other than the open deep ocean or they will simply be lost by dilution.
Finally the group did not cite any of the work from Nick Lane and his lab excepting one rather general review link. Naughty.
So. Some interesting chemistry and it's good to have multiple groups thinking about a given problem but I don't see the hydrothermal vent hypothesis being abandoned any time soon. Certainly not by believers like myself.
Peter
This paper:
Native iron reduces CO2 to intermediates and endproducts of the acetyl-CoA pathway
from a french institute, suggests that metallic iron alone might provide electrons of sufficiently negative potential to perform the process, this is the basic premise:
Fe0 → Fe2++ 2e-
These electrons have a sufficiently negative potential to allow:
CO2 + 2e- + H2O → HCOOH + O2-
Obviously the Fe2+ would combine with the O2- to give FeO, leaving a formate moiety as the start of the process essential for the origin of pre-biotic metabolism.
In the event the two most common experimental products were acetate and pyruvate, a highly plausible step or two onward from formate, which they also found under certain conditions.
The circumstances of temperature and pressure were, in some experiments, plausible for pre-biotic chemistry.
The problems, compared to the Lane and Martin hydrothermal vents concept, seem to be:
The products are bound to the surface of the iron deposit, potassium hydroxide was needed to hydrolyse them off for measurement.
The process is reactive rather than catalytic, ie the metallic iron is consumed in the process of providing electrons. This contrasts starkly with the continuous supply of electrons supplied by hydrothermal vent conditions over geological time scales.
Then there is the concentration problem. If the organic products were to be freed from the iron surface they need to be somewhere other than the open deep ocean or they will simply be lost by dilution.
Finally the group did not cite any of the work from Nick Lane and his lab excepting one rather general review link. Naughty.
So. Some interesting chemistry and it's good to have multiple groups thinking about a given problem but I don't see the hydrothermal vent hypothesis being abandoned any time soon. Certainly not by believers like myself.
Peter
Wednesday, November 14, 2018
A brief aside in to statins and FH and all cause mortality
I must admit that I have not read this paper, just the abstract. My excuse is, once again, that I have no access to any ondansetron.
Statins in Familial Hypercholesterolemia: Consequences for Coronary Artery Disease and All-Cause Mortality
As always the results of statin therapy are, to say the least, dramatic.
"In patients with heterozygous FH, moderate- to high-intensity statin therapy lowered the risk for CAD and mortality by 44%".
Wow. But why the need for a composite end point?
If we leave aside soft end points which include coronary re-vascularisation (never influenced by serum lipid levels. No laughing at the back there!) and concentrate on the hard end point of all cause mortality we end up with, for non statinated people:
9 deaths per 4,892 person-years, which I make 1.8 deaths per 1000 person-years.
On a statin we have 17 deaths per 11,674 person-years, 1.5 per 1000 person-years.
That looks like a reduction in mortality of 0.3 people per 1000 person-years.
Or, being more whole numberish, 1 person saved by treating for 3,300 person-years on a statin.
Does that convert to treating 100 people for 33 years to avoid one premature fatality? We're all going to die one day so no one avoids death permanently, even by taking a statin. Unbelievable as that sounds.
If you have heterozygous FH your chances of dying tomorrow are rather low but not quite zero. If you take a statin it will reduce this chance by a vanishingly small amount.
Taking the difference between "rather-low-but-not-quite-zero" and "a-vanishingly-small-amount less than rather-low-but-not-quite-zero", dividing this difference by "rather-low-but-not-quite-zero" and multiplying by 100 we get a massive 17% reduction in all cause mortality. Which means diddly squat, but sounds good if you are a statinator. Admittedly not as good as 44% for the composite end point but hey... Neither means anything.
The main benefit of a statin appears to be that the number it gives you on a lab report might just influence a cardiologist to leave your coronary arteries alone.
Peter
Statins in Familial Hypercholesterolemia: Consequences for Coronary Artery Disease and All-Cause Mortality
As always the results of statin therapy are, to say the least, dramatic.
"In patients with heterozygous FH, moderate- to high-intensity statin therapy lowered the risk for CAD and mortality by 44%".
Wow. But why the need for a composite end point?
If we leave aside soft end points which include coronary re-vascularisation (never influenced by serum lipid levels. No laughing at the back there!) and concentrate on the hard end point of all cause mortality we end up with, for non statinated people:
9 deaths per 4,892 person-years, which I make 1.8 deaths per 1000 person-years.
On a statin we have 17 deaths per 11,674 person-years, 1.5 per 1000 person-years.
That looks like a reduction in mortality of 0.3 people per 1000 person-years.
Or, being more whole numberish, 1 person saved by treating for 3,300 person-years on a statin.
Does that convert to treating 100 people for 33 years to avoid one premature fatality? We're all going to die one day so no one avoids death permanently, even by taking a statin. Unbelievable as that sounds.
If you have heterozygous FH your chances of dying tomorrow are rather low but not quite zero. If you take a statin it will reduce this chance by a vanishingly small amount.
Taking the difference between "rather-low-but-not-quite-zero" and "a-vanishingly-small-amount less than rather-low-but-not-quite-zero", dividing this difference by "rather-low-but-not-quite-zero" and multiplying by 100 we get a massive 17% reduction in all cause mortality. Which means diddly squat, but sounds good if you are a statinator. Admittedly not as good as 44% for the composite end point but hey... Neither means anything.
The main benefit of a statin appears to be that the number it gives you on a lab report might just influence a cardiologist to leave your coronary arteries alone.
Peter
Listeriosis is no fun
Just doing my bit
Vegetables, nine dead of listeriosis
Quick edit for when the link dies:
"9 people dead following Listeria outbreak – Tesco, Aldi, Waitrose, Iceland, Lidl, Aldi – Issue Product recall. Please please check on old people and loved ones who may not be in the loop, listeria can be more serious for people who have weakened immune systems.
Full 43 product list for recall is shown as follows issued by the FSA".
All vegetables.
Peter
Vegetables, nine dead of listeriosis
Quick edit for when the link dies:
"9 people dead following Listeria outbreak – Tesco, Aldi, Waitrose, Iceland, Lidl, Aldi – Issue Product recall. Please please check on old people and loved ones who may not be in the loop, listeria can be more serious for people who have weakened immune systems.
Full 43 product list for recall is shown as follows issued by the FSA".
All vegetables.
Peter
Wednesday, November 07, 2018
Green Tea Extract; superb antioxidant?
Here is a little more from this paper:
High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS
This is insulin signalling under massively supra-physiological insulin exposure in cell culture:
This is, obviously, their best gel, that's the one you publish. The insulin resistance (fainter P-Akt band) when insulin and Se are both used compared to insulin w/o Se exposure does appear to be there. At physiological levels of insulin this differential seems likely to be maintained.
This implies blunting of insulin signalling, which allows more FFA oxidation, which generates greater levels of ROS than would occur under continued insulin action. These ROS would be physiological on a ketogenic diet or under extended fasting but are not so in cells under culture using 11mmol glucose (which is what I think is in the medium they used, they don't actually say) plus whatever insulin is present in 10% FBS. So we have this:
Control is from cells under RPMI 1640 alone, traditionally 11mmol/l glucose. Excess selenium blunts insulin signalling so allows FFA release from intracellular triglyceride stores, so increases ROS (in the same way as metformin does but w/o the suppression of gluconeogenesis intrinsic to metformin's action). Adding rotenone, as you would expect, blocks RET so blocks ROS generation. CCCP uncouples respiration, drops delta psi so blocks RET/ROS. Etomoxir blocks access of FFAs to mitochondria so blocks input at mtETFdh, so blocks RET/ROS. MitoQ powerfully targets all mitochondrial ROS so over-rides the FFA oxidation ROS generation effect. Chromium picolinate restores insulin signalling by repleting the Cr depletion induced by Se. MSA is an inhibitor of glutathione peroxidase, so it eliminates the effects of excess GPX. And SS, sodium salicylate, appears to block intracellular lipolysis in hepatocytes, so suppresses fatty acid supply to mitochondria, much as insulin or etomoxir would.
All a very plausible narrative.
Except for oligomycin. What does anyone expect the blockade of ATP synthase to do to ROS generation, throughout the electron transport chain? It is going to increase delta psi, reduce all of the redox complexes and generate a ton of RET and ROS through complex I and probably at ton at complex III too. It is specifically used to generate ROS in many other studies, example here:
The specificity of neuroprotection by antioxidants
I'm not very comfortable with oligomycin as a suppressor of FFA oxidation induced ROS. It is another, rather serious, blight on the paper. It certainly should have been discussed.
I would usually ignore the whole paper except Tom Naughton gave us all the heads up on a recent report of a chap taking what might have been a hefty dose of green tea extract who went in to liver failure. Obviously most folks just excrete antioxidants like GTE with little harm done. I just wonder if he got unlucky or took a huge dose while walking round with the sort of liver full of lipid so beloved of Public Health England. Losing the protection of insulin's inhibition of lipolysis simply dumped a ton of unregulated intra-hepatocyte FFAs from lipid droplets on to his mitochondria, which then popped their clogs.
Who knows? It's another nice narrative. I just wish I wasn't so suspicious of the selenium paper...
Both reports also play rather too well to my biases against antioxidants, but that's how it is...
Peter
High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS
This is insulin signalling under massively supra-physiological insulin exposure in cell culture:
This is, obviously, their best gel, that's the one you publish. The insulin resistance (fainter P-Akt band) when insulin and Se are both used compared to insulin w/o Se exposure does appear to be there. At physiological levels of insulin this differential seems likely to be maintained.
This implies blunting of insulin signalling, which allows more FFA oxidation, which generates greater levels of ROS than would occur under continued insulin action. These ROS would be physiological on a ketogenic diet or under extended fasting but are not so in cells under culture using 11mmol glucose (which is what I think is in the medium they used, they don't actually say) plus whatever insulin is present in 10% FBS. So we have this:
Control is from cells under RPMI 1640 alone, traditionally 11mmol/l glucose. Excess selenium blunts insulin signalling so allows FFA release from intracellular triglyceride stores, so increases ROS (in the same way as metformin does but w/o the suppression of gluconeogenesis intrinsic to metformin's action). Adding rotenone, as you would expect, blocks RET so blocks ROS generation. CCCP uncouples respiration, drops delta psi so blocks RET/ROS. Etomoxir blocks access of FFAs to mitochondria so blocks input at mtETFdh, so blocks RET/ROS. MitoQ powerfully targets all mitochondrial ROS so over-rides the FFA oxidation ROS generation effect. Chromium picolinate restores insulin signalling by repleting the Cr depletion induced by Se. MSA is an inhibitor of glutathione peroxidase, so it eliminates the effects of excess GPX. And SS, sodium salicylate, appears to block intracellular lipolysis in hepatocytes, so suppresses fatty acid supply to mitochondria, much as insulin or etomoxir would.
All a very plausible narrative.
Except for oligomycin. What does anyone expect the blockade of ATP synthase to do to ROS generation, throughout the electron transport chain? It is going to increase delta psi, reduce all of the redox complexes and generate a ton of RET and ROS through complex I and probably at ton at complex III too. It is specifically used to generate ROS in many other studies, example here:
The specificity of neuroprotection by antioxidants
I'm not very comfortable with oligomycin as a suppressor of FFA oxidation induced ROS. It is another, rather serious, blight on the paper. It certainly should have been discussed.
I would usually ignore the whole paper except Tom Naughton gave us all the heads up on a recent report of a chap taking what might have been a hefty dose of green tea extract who went in to liver failure. Obviously most folks just excrete antioxidants like GTE with little harm done. I just wonder if he got unlucky or took a huge dose while walking round with the sort of liver full of lipid so beloved of Public Health England. Losing the protection of insulin's inhibition of lipolysis simply dumped a ton of unregulated intra-hepatocyte FFAs from lipid droplets on to his mitochondria, which then popped their clogs.
Who knows? It's another nice narrative. I just wish I wasn't so suspicious of the selenium paper...
Both reports also play rather too well to my biases against antioxidants, but that's how it is...
Peter
Friday, November 02, 2018
Stone Agers in the Fast Lane?
A destruction of Paleo Diet™ as a management tool for metabolic syndrome in modern humans surfaced recently in a tweet from Miki Ben-Dor, along with his comment that he views meat as the default paleo food.
Plants used as "food" come and go and are nowadays developed in to reduced toxicity versions which are what we call vegetables. Meat is meat and even the invention of factory farming does not seem to be able to convert it in to anything as toxic as a courgette. Remember this?
Courgette stew kills pensioner in Heidelberg
Anyway, back to the Pacific Islanders. This is the book chapter we're interested in and it's entertaining.
Stone Agers in the Fast Lane? How Bioarchaeologists Can Address the Paleo Diet Myth
These people appear to have read (and cite) essentially every paper on gout in archeological record of the paleo Pacific Islander population. They are using gout as a skeletally preserved marker for metabolic syndrome, a fairly reasonable approach to my mind. The assumption that meat causes gout (lots of purines don'tchano) which is threaded throughout the chapter is less acceptable.
So we end up with this as a core summing up close to the end, for anyone who doesn't want to slog through the various straw men they set up to knock down:
"We have also used a case study of Pacific Islanders’ experiences with MetS and paleopathology evidence of gout to reexamine the very basis for the “necessity” of a return to a Paleo Diet. As discussed, the ancestral diet (based on tuberous root crops, not cereals) and population history of Pacific islanders are completely different to the Old and New Worlds where the Paleo Diet debate is entrenched. Yet the burden of MetS is extremely high in the Pacific. While the adoption of westernized diets has exacerbated the expression of MetS conditions in modern Polynesians, the paleopathological evidence (especially gout) suggests the origins of these conditions stems from their Lapita ancestors, who in turn trace their roots back to Island Southeast Asia".
Gout was widespread in the pre-Westernisation Pacific Islanders, despite their paleo diet. The core quote re this paleo diet is that it is "based on tuberous root crops, not cereals".
Translation: A Paleo Diet diet based on paleo tuberous root crops gives you paleo gout.
Eat some meat and get your calories from fat. Vegetables can be viewed as a recreational indulgence if you so wish. But maybe don't over do them unless you want Paleo Diet™ gout.
Peter
Plants used as "food" come and go and are nowadays developed in to reduced toxicity versions which are what we call vegetables. Meat is meat and even the invention of factory farming does not seem to be able to convert it in to anything as toxic as a courgette. Remember this?
Courgette stew kills pensioner in Heidelberg
Anyway, back to the Pacific Islanders. This is the book chapter we're interested in and it's entertaining.
Stone Agers in the Fast Lane? How Bioarchaeologists Can Address the Paleo Diet Myth
These people appear to have read (and cite) essentially every paper on gout in archeological record of the paleo Pacific Islander population. They are using gout as a skeletally preserved marker for metabolic syndrome, a fairly reasonable approach to my mind. The assumption that meat causes gout (lots of purines don'tchano) which is threaded throughout the chapter is less acceptable.
So we end up with this as a core summing up close to the end, for anyone who doesn't want to slog through the various straw men they set up to knock down:
"We have also used a case study of Pacific Islanders’ experiences with MetS and paleopathology evidence of gout to reexamine the very basis for the “necessity” of a return to a Paleo Diet. As discussed, the ancestral diet (based on tuberous root crops, not cereals) and population history of Pacific islanders are completely different to the Old and New Worlds where the Paleo Diet debate is entrenched. Yet the burden of MetS is extremely high in the Pacific. While the adoption of westernized diets has exacerbated the expression of MetS conditions in modern Polynesians, the paleopathological evidence (especially gout) suggests the origins of these conditions stems from their Lapita ancestors, who in turn trace their roots back to Island Southeast Asia".
Gout was widespread in the pre-Westernisation Pacific Islanders, despite their paleo diet. The core quote re this paleo diet is that it is "based on tuberous root crops, not cereals".
Translation: A Paleo Diet diet based on paleo tuberous root crops gives you paleo gout.
Eat some meat and get your calories from fat. Vegetables can be viewed as a recreational indulgence if you so wish. But maybe don't over do them unless you want Paleo Diet™ gout.
Peter
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