I don't do twitter or facebook (If you are a metabolic person and have friend-requested me and I've ignored it please don't take it personally, it's just not what I do with faceache) to any extent and almost never use them for metabolic subjects. By an accident of twittard I picked up this tweet by Chris Masterjohn (via raphi and Mike Eades). It made me laugh out loud and still has me giggling occasionally:
"No! Carbohydrate restriction is the stupidest approach to fatty liver ever devised. If it “works” in any case it is almost certainly by supplying more methionine and choline, not by lowering carbs. It is impossible to make more fat from carb than you get by eating fat"
I can't help but think "chylomicron", "thoracic duct" and "physiology".
Then I giggle some more.
Peter
I guess it ranks along side of "Masterjohn", "Martha" and "RQ 0.454".
Saturday, February 16, 2019
Sunday, February 10, 2019
Cell surface oxygen consumption (3) Alternative options
Have a look at this:
Limits of aerobic metabolism in cancer cells
"To gain a better understanding of cell metabolism as a function of the growth metabolic demand we performed a back-of-the-envelope calculation focusing on the major biomass components of mammalian cells".
In these days of "shotgun metabolomics" two people appear to have sat down with one or more sheets of paper (possibly larger than an envelope, though you can get some quite large envelopes I guess) and have gone back to basic first principles. They then published in a Nature journal. I love this. I feel it rates alongside getting this image published in Cell Metabolism.
TLDR for the paper:
Glucose drops through glycolysis to lactate at a rate where ATP generation per minute massively outstrips that available from mitochondrial oxphos. In redox balance. It's fast.
Anabolism from glucose consumes pyruvate (and phosphoenolpyruvate) which then forbids the pyruvate -> lactate NAD+ regeneration step. This imposes a need to avoid or deal with a cellular NADH excess.
In the Cell surface oxygen consumption (2) post I hypothesised that the regeneration of NAD+ at the cell surface would be in direct proportion to anabolism derived from pyruvate (ie glucose/glycolysis anabolism), to maintain redox balance (ie get rid of excess NADH, cycling it back to NAD+). It is particularly a feature of highly glycolytic cancer cells.
These folks appear to be saying the same thing but looking at differing cellular techniques to avoid NADH cumulation.
There's lots of other good stuff in there too. Like the rate of mitochondrial ATP generation from HeLa cell mitochondria compared to that of normal cardiac myocyte mitochondria. The ATP production via oxphos is an order of magnitude greater in mitochondria from the cardiac myocytes.
Oh, and glutaminolysis as another NADH avoiding ploy. This is the quote:
"Glutamate can be converted to citrate via reductive carboxylation. In this pathway the NAD(P)H production by glutamate dehydrogenase is compensated by the reverse activity of the NAD(P) isocitrate dehydrogenase (Fig. 1). Glutamate can be taken from the medium or generated from glutamine by glutaminase. Interestingly, arginine and proline can be produced from glutamate with concomitant consumption of NADH (Fig. 4a). This could provide an additional mechanism for NADH turnover".
Note that the glutamate is not being oxidised, it is running a small section of the TCA backwards to generate citrate for lipid synthesis, ie anabolism. This is not glutamate turning the TCA in the normal direction toward oxaloacetate to generate ATP via NADH and oxphos, because the mitochondria of cancer cells don't seem to do oxphos very well. Somewhat Seyfried supportive.
Peter
Limits of aerobic metabolism in cancer cells
"To gain a better understanding of cell metabolism as a function of the growth metabolic demand we performed a back-of-the-envelope calculation focusing on the major biomass components of mammalian cells".
In these days of "shotgun metabolomics" two people appear to have sat down with one or more sheets of paper (possibly larger than an envelope, though you can get some quite large envelopes I guess) and have gone back to basic first principles. They then published in a Nature journal. I love this. I feel it rates alongside getting this image published in Cell Metabolism.
TLDR for the paper:
Glucose drops through glycolysis to lactate at a rate where ATP generation per minute massively outstrips that available from mitochondrial oxphos. In redox balance. It's fast.
Anabolism from glucose consumes pyruvate (and phosphoenolpyruvate) which then forbids the pyruvate -> lactate NAD+ regeneration step. This imposes a need to avoid or deal with a cellular NADH excess.
In the Cell surface oxygen consumption (2) post I hypothesised that the regeneration of NAD+ at the cell surface would be in direct proportion to anabolism derived from pyruvate (ie glucose/glycolysis anabolism), to maintain redox balance (ie get rid of excess NADH, cycling it back to NAD+). It is particularly a feature of highly glycolytic cancer cells.
These folks appear to be saying the same thing but looking at differing cellular techniques to avoid NADH cumulation.
There's lots of other good stuff in there too. Like the rate of mitochondrial ATP generation from HeLa cell mitochondria compared to that of normal cardiac myocyte mitochondria. The ATP production via oxphos is an order of magnitude greater in mitochondria from the cardiac myocytes.
Oh, and glutaminolysis as another NADH avoiding ploy. This is the quote:
"Glutamate can be converted to citrate via reductive carboxylation. In this pathway the NAD(P)H production by glutamate dehydrogenase is compensated by the reverse activity of the NAD(P) isocitrate dehydrogenase (Fig. 1). Glutamate can be taken from the medium or generated from glutamine by glutaminase. Interestingly, arginine and proline can be produced from glutamate with concomitant consumption of NADH (Fig. 4a). This could provide an additional mechanism for NADH turnover".
Note that the glutamate is not being oxidised, it is running a small section of the TCA backwards to generate citrate for lipid synthesis, ie anabolism. This is not glutamate turning the TCA in the normal direction toward oxaloacetate to generate ATP via NADH and oxphos, because the mitochondria of cancer cells don't seem to do oxphos very well. Somewhat Seyfried supportive.
Peter
Sunday, February 03, 2019
Lactate as bulk energy transport
Using RQ to track whole body substrate oxidation is pretty straight forward. An RQ of 1.0 means glucose oxidation and of 0.69 indicates fat oxidation. Mixtures come out in between. It is very simple to show that glucose is routinely converted to fatty acids because in the immediate post prandial period for any rodent fed standard low fat crapinabag the RQ becomes greater than 1.0. We would expect that during the later period when the rodent is asleep/not eating there would be a lower than expected RQ (lower than the calculated food quotient, FQ) while predominantly stored fat is oxidised. But on a high carb, very low fat diet we would expect the overall averaged RQ over 24h to be a little under 1.0, ie pretty much the same as the FQ. For an hypothetical "all glucose" diet part of the glucose diverts thus via fatty acids:
Eating: Glucose minus a little O2 -> fat RQ > 1.0
Sleeping: Fat plus lots of O2 -> CO2 + H2O RQ < 1.0
CO2/O2 = 1.0 on average over 24h.
If that 24h averaged RQ was all we had to work with we would not suspect that de-novo lipogenesis ever occurred. Nice and simple.
Much more difficult to pick up is the bulk conversion of fatty acids to glucose. This produces an unusually low RQ in the short term. But if the glucose is being produced to fuel the brain during starvation then its prompt oxidation would "correct" the unusually low RQ back upwards to a fatty acid RQ. The obvious exception was noted in a metabolically fat adapted and lactating young lady during extended fasting. She made glucose and galactose from fatty acids and gave them to her baby, rather than oxidising the sugars herself. End result was an RQ of 0.454 after just over three days of fasting with continued breast feeding.
She was making sugar out of fatty acids in bulk. She might or might not have been doing the same without lactation but in the absence of donating the sugars to her infant this would never show.
So the RQ and the FQ always average out to be the same unless something very specific is happening, ie as with Martha.
Much more difficult is to ask how do you tell whether glucose is being converted to pyruvate which then enters the mitochondria to join the TCA or whether glucose converts to lactate which is then shipped in to the mitochondria. And what if you have the absolutely crazy idea that glycolysis almost always leads to lactate and that this lactate is a transferable currency between cells? Glucose is then viewed as a one way gift from liver to tissues, to be shared out between cells/tissues as lactate.
That latter view has to use tracers to look at lactate or glucose flux. Label some lactate with carbon-13 and infuse it to steady state in the plasma of a mouse. Kill the mouse promptly and humanely and look where the C-13 atoms have ended up in glycolysis and/or TCA intermediates. Repeat the process with C-13 labelled glucose. Then glutamate. And then any other intermediary metabolite which might remotely shift bulk energy around the body.
It turns out that in starch fed mice glucose and lactate are the bulk plasma energy carriers, lactate slightly more so than glucose in the fed state and much more so in the fasted state. Certainly on a molar basis, bearing in mind that a mole of glucose has twice the carbon of a mole of lactate, which makes the situation slightly more complex. But lactate labels the TCA more strongly than glucose. Not surprisingly glucose labels glycolytic intermediates better than lactate.
Free fatty acids and ketones are a separate subject in high carbohydrate/low fat fed mice but they flux remarkably little energy, at least when fasting is limited to eight hours. Brain metabolism is also another separate subject.
TLDR:
Glucose feeds glycolysis to lactate. Most of this glycolytic lactate enters the plasma pool. Plasma lactate feeds the TCA in other cells.
Now the insightful bit from near the end of the letter:
"Among the many metabolic intermediates, why does lactate carry high flux? Lactate is redox-balanced with glucose. The rapid exchange of both tissue lactate and pyruvate with the circulation may help to equate cytosolic NAD+/NADH ratios across tissues, allowing the whole body to buffer NAD(H) disturbances in any given location. Nearly complete lactate sharing between tissues effectively decouples glycolysis and the TCA cycle in individual tissues, allowing independent tissue-specific regulation of both processes. Because almost all ATP is made in the TCA cycle, each tissue can acquire energy from the largest dietary calorie constituent (carbohydrate) without needing to carry out glycolysis. In turn, glycolytic activity can be modulated to support cell proliferation, NADPH production by the pentose phosphate pathway, brain activity, and systemic glucose homeostasis. In essence, by having glucose feed the TCA cycle via circulating lactate, the housekeeping function of ATP production is decoupled from glucose catabolism. In turn, glucose metabolism is regulated to serve more advanced objectives of the organism".
What I think this is saying is that lactate supplied to the TCA/OxPhos is for "housekeeping" ie ATP production. Glycolysis is for anabolism. Neither is absolute, but I find it an interesting point of view.
So the ultimate TLDR is:
Ox-phos = housekeeping
Glycolysis = anabolism
There is probably significant fudge-room.
Peter
Eating: Glucose minus a little O2 -> fat RQ > 1.0
Sleeping: Fat plus lots of O2 -> CO2 + H2O RQ < 1.0
CO2/O2 = 1.0 on average over 24h.
If that 24h averaged RQ was all we had to work with we would not suspect that de-novo lipogenesis ever occurred. Nice and simple.
Much more difficult to pick up is the bulk conversion of fatty acids to glucose. This produces an unusually low RQ in the short term. But if the glucose is being produced to fuel the brain during starvation then its prompt oxidation would "correct" the unusually low RQ back upwards to a fatty acid RQ. The obvious exception was noted in a metabolically fat adapted and lactating young lady during extended fasting. She made glucose and galactose from fatty acids and gave them to her baby, rather than oxidising the sugars herself. End result was an RQ of 0.454 after just over three days of fasting with continued breast feeding.
She was making sugar out of fatty acids in bulk. She might or might not have been doing the same without lactation but in the absence of donating the sugars to her infant this would never show.
So the RQ and the FQ always average out to be the same unless something very specific is happening, ie as with Martha.
Much more difficult is to ask how do you tell whether glucose is being converted to pyruvate which then enters the mitochondria to join the TCA or whether glucose converts to lactate which is then shipped in to the mitochondria. And what if you have the absolutely crazy idea that glycolysis almost always leads to lactate and that this lactate is a transferable currency between cells? Glucose is then viewed as a one way gift from liver to tissues, to be shared out between cells/tissues as lactate.
That latter view has to use tracers to look at lactate or glucose flux. Label some lactate with carbon-13 and infuse it to steady state in the plasma of a mouse. Kill the mouse promptly and humanely and look where the C-13 atoms have ended up in glycolysis and/or TCA intermediates. Repeat the process with C-13 labelled glucose. Then glutamate. And then any other intermediary metabolite which might remotely shift bulk energy around the body.
It turns out that in starch fed mice glucose and lactate are the bulk plasma energy carriers, lactate slightly more so than glucose in the fed state and much more so in the fasted state. Certainly on a molar basis, bearing in mind that a mole of glucose has twice the carbon of a mole of lactate, which makes the situation slightly more complex. But lactate labels the TCA more strongly than glucose. Not surprisingly glucose labels glycolytic intermediates better than lactate.
Free fatty acids and ketones are a separate subject in high carbohydrate/low fat fed mice but they flux remarkably little energy, at least when fasting is limited to eight hours. Brain metabolism is also another separate subject.
TLDR:
Glucose feeds glycolysis to lactate. Most of this glycolytic lactate enters the plasma pool. Plasma lactate feeds the TCA in other cells.
Now the insightful bit from near the end of the letter:
"Among the many metabolic intermediates, why does lactate carry high flux? Lactate is redox-balanced with glucose. The rapid exchange of both tissue lactate and pyruvate with the circulation may help to equate cytosolic NAD+/NADH ratios across tissues, allowing the whole body to buffer NAD(H) disturbances in any given location. Nearly complete lactate sharing between tissues effectively decouples glycolysis and the TCA cycle in individual tissues, allowing independent tissue-specific regulation of both processes. Because almost all ATP is made in the TCA cycle, each tissue can acquire energy from the largest dietary calorie constituent (carbohydrate) without needing to carry out glycolysis. In turn, glycolytic activity can be modulated to support cell proliferation, NADPH production by the pentose phosphate pathway, brain activity, and systemic glucose homeostasis. In essence, by having glucose feed the TCA cycle via circulating lactate, the housekeeping function of ATP production is decoupled from glucose catabolism. In turn, glucose metabolism is regulated to serve more advanced objectives of the organism".
What I think this is saying is that lactate supplied to the TCA/OxPhos is for "housekeeping" ie ATP production. Glycolysis is for anabolism. Neither is absolute, but I find it an interesting point of view.
So the ultimate TLDR is:
Ox-phos = housekeeping
Glycolysis = anabolism
There is probably significant fudge-room.
Peter
Saturday, February 02, 2019
Lactate: Is the astrocyte-neuron lactate shuttle scuppered?
You don't usually learn much from statements which you, personally, consider likely to be correct. Annoying statements are far more productive.
Working through Seyfried's paper
Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis
I came across this snippet which galled me a little:
"It is glucose and not lactate that primarily drives brain energy metabolism (Allen et al., 2005; Dienel, 2012; Nortley and Attwell, 2017), making it unlikely that lactate could serve as a major energy metabolite for neoplastic GBM cells with diminished OxPhos capacity".
Now, people will realise that the astrocyte-neuron lactate shuttle is more than a little inflammatory as a subject, to say the least. Currently it is not doing too well in the face of experimental data, which are not at all straightforward to obtain. I went to Nortley and Attwell as the most recent reference. As a rather pro-lactate shuttle sort of a person I found their straw-man setting up of the shuttle rather annoying but their data potentially convincing, though I am far from certain about this. Here is the link:
Control of brain energy supply by astrocytes
This left me wondering what more pro lactate-shuttle people might be thinking nowadays, so I went via the "see all" button to locate this commentary by Tang:
Brain activity-induced neuronal glucose uptake/glycolysis: Is the lactate shuttle not required?
which is a rather more circumspect but still accepts a decreasing probability that the lactate shuttle is in any way crucial to astrocyte-neuron energetic coupling. The silver lining was this link, used to point out that in Bl6 mice whole-brain lactate extraction from plasma is essentially zero under the reasonably normal physiological conditions studied:
Glucose feeds the TCA cycle via circulating lactate
The basic concept in the paper, that lactate is the predominant metabolic substrate for the TCA is fine to me but that the source of lactate is predominantly extracellular is very counterintuitive. But the data presented are quite convincing. So I'm interested. I think it needs a little aside before talking about the paper itself and the situation in the brain in particular, so I'll post some random thoughts before looking at the paper in more detail. The biggest down side to the paper is the authors' failure to mention Schurr, the main proponent of lactate as a redox-balanced product of glucose, a very deeply insightful and much neglected observation. But then Schurr is a serious proponent of the astrocyte-neuron lactate shuttle...
Peter
Working through Seyfried's paper
Mitochondrial Substrate-Level Phosphorylation as Energy Source for Glioblastoma: Review and Hypothesis
I came across this snippet which galled me a little:
"It is glucose and not lactate that primarily drives brain energy metabolism (Allen et al., 2005; Dienel, 2012; Nortley and Attwell, 2017), making it unlikely that lactate could serve as a major energy metabolite for neoplastic GBM cells with diminished OxPhos capacity".
Now, people will realise that the astrocyte-neuron lactate shuttle is more than a little inflammatory as a subject, to say the least. Currently it is not doing too well in the face of experimental data, which are not at all straightforward to obtain. I went to Nortley and Attwell as the most recent reference. As a rather pro-lactate shuttle sort of a person I found their straw-man setting up of the shuttle rather annoying but their data potentially convincing, though I am far from certain about this. Here is the link:
Control of brain energy supply by astrocytes
This left me wondering what more pro lactate-shuttle people might be thinking nowadays, so I went via the "see all" button to locate this commentary by Tang:
Brain activity-induced neuronal glucose uptake/glycolysis: Is the lactate shuttle not required?
which is a rather more circumspect but still accepts a decreasing probability that the lactate shuttle is in any way crucial to astrocyte-neuron energetic coupling. The silver lining was this link, used to point out that in Bl6 mice whole-brain lactate extraction from plasma is essentially zero under the reasonably normal physiological conditions studied:
Glucose feeds the TCA cycle via circulating lactate
The basic concept in the paper, that lactate is the predominant metabolic substrate for the TCA is fine to me but that the source of lactate is predominantly extracellular is very counterintuitive. But the data presented are quite convincing. So I'm interested. I think it needs a little aside before talking about the paper itself and the situation in the brain in particular, so I'll post some random thoughts before looking at the paper in more detail. The biggest down side to the paper is the authors' failure to mention Schurr, the main proponent of lactate as a redox-balanced product of glucose, a very deeply insightful and much neglected observation. But then Schurr is a serious proponent of the astrocyte-neuron lactate shuttle...
Peter
Sunday, January 20, 2019
Metformin (10) Macavity
TLDR:
Macavity, Macavity, there's no one like Macavity,
He's broken every human law, he breaks the law of gravity.
His powers of levitation would make a fakir stare,
And when you reach the scene of crime - Macavity's not there!
You may seek him in the basement, you may look up in the air
But I tell you once and once again, - Macavity's not there!
Macavity, Macavity, there's no one like Macavity,
He's broken every human law, he breaks the law of gravity.
His powers of levitation would make a fakir stare,
And when you reach the scene of crime - Macavity's not there!
You may seek him in the basement, you may look up in the air
But I tell you once and once again, - Macavity's not there!
I think people might have noticed over the years that I'm not a great fan of metformin acting clinically by blockade of complex I. Particularly over the last few months I have waded deep through layers of references looking at the morass of "paradoxes" about metformin. In particular that 250micromolar in your plasma will kill you but 4000 micromolar can be justified in cell culture because meformin "accumulates in the mitochondria" at up to 1000 times the level in the blood/cytoplasm, which is a deeply held belief structure on which an unimaginable amount of grant funding hangs. Does metformin cumulate in mitochondria?
It doesn't. I really enjoyed this review from last year. Talk about reinforcement of confirmation bias:
Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences
But when you have spent years slogging through papers thinking: That's crap! it comes as a huge relief to find that it's not just you who thinks this, no matter how politely the reaction is phrased.
So. Is metformin research all garbage? No, of course not. Anything involving a live animal on oral doses which do not cause death by lactic acidosis is worth thinking about. Any parallel cell culture research in the same paper using a 4millimolar concentration can be junked. In vivo effects are real, at real dose rates. Cell culture at 1000 times overdose is fiction.
Just to summarise my own speculations:
Under fasting the component of insulin signalling facilitated by the glycerophosphate shuttle can be replaced by saturated fatty acid oxidation via electron transporting flavoprotein dehydrogenase. This maintains insulin signalling at the "cost" of increased fat oxidation. Hence the weight loss.
In the peak absorptive period after a carbohydrate based meal the normal development of insulin-induced insulin resistance is blunted and glucose oxidation continues for longer than without the metformin. If you are eating the absolute crap suggested by any cardiologist or diabetologist this might be of some benefit. If you are already LC the increased fasting fatty acid oxidation is probably where the benefits accrue from.
Cancer benefits are likely to be real, off "target" and secondary to reduced insulin exposure.
Peter
Edit: These people seem to be looking at the real world too. Worth spending some time on this
Low metformin causes a more oxidized mitochondrial NADH/NAD redox state in hepatocytes and inhibits gluconeogenesis by a redox-independent mechanism
Saturday, January 19, 2019
Cell surface oxygen consumption (2)
How does cell surface oxygen consumption happen? Here is the main reaction overall (it's probably not that simple!):
4e- + 4H+ + O2 -> 2H2O
A certain amount of hydrogen peroxide is also generated and just a little superoxide (probably for cell-cell signalling). Other electron acceptors can stand in for, or compete with, oxygen as the terminal electron acceptor. The reaction occurs on the outer surface of the plasma membrane.
The source of the electrons is cytoplasmic NADH, which is oxidised to NAD+ and H+ on the inner surface of the plasma membrane. Each of the electrons is transported via a plasma membrane ubiquinone:semi ubiquinone cycle which then donates them to the redox enzyme on the external cell surface. Here is a simplified version of Herst and Berridge's Figure 1:
Although I doubt that there is very much NADH or NAD+ in normal extra cellular fluid there has to be an NADH docking site on the outer redox enzyme as one of the hallmarks of cell surface oxygen consumption is that it can be halted completely by flooding the cell culture medium with NADH. Electrons from this then follow the dashed line to fully reduce the ubiquinone to ubiquinol and the system grinds to a halt.
So you can measure cell surface oxygen usage by the fall in consumption which occurs when you add exogenous NADH just as you can measure mitochondrial oxygen usage by the fall in consumption which occurs when you add myxothiazol.
Why is the system there?
Let's go back to the two routes of glycolysis. Without the glycerophosphate shuttle (insulin signalling driven/driving) we have
Glucose -> lactate -> mitochondria -> pyruvate -> TCA
and there is no depletion of cytoplasmic NAD+ as one is consumed and one produced in the glucose -> lactate process. With insulin signalling we have two parallel processes:
Glucose -> glycerophosphate shuttle -> CoQ -> ETC
which consumes cytoplasmic NADH, leaving none to convert pyruvate to lactate. So in parallel we have to abort glycolysis at pyruvate:
Glucose -> pyruvate -> mitochondria -> TCA
which balances the cytoplasmic NADH:NAD+ ratio nicely.
Now, let's consider a cell undergoing rapid growth with a view to divide. For today I will ignore mitochondrial biosynthesis and consider what happens if cytoplasmic pyruvate is consumed for amino acid biosynthesis. For each molecule of pyruvate which has been diverted to an amino acid there will be one less available to provide cytoplasmic NAD+ by conversion to lactate, which will limit glycolysis because cytoplasmic NAD+ is essential for the oxidation of glyceraldehyde-3-phosphate.
Under these conditions cell surface oxygen consumption appears to be able to step in to oxidise cytoplasmic NADH to cytoplasmic NAD+, which then allows glycolysis and its associated ATP production. This looks to be particularly important if there is any sort of a problem with the ETC and the glycerophosphate shuttle.
In rho zero cells, where the ETC is deleted (and there is no glycerophosphate shuttle) and glycolysis is the sole source of ATP production, cell surface oxygen consumption has to supply NAD+ in direct proportion to how much pyruvate is lost to anabolism rather than being used to supply NAD+ via lactate generation. In rho zero anabolic cancer cells cell surface oxygen consumption can be as much as 90% of the total oxygen consumption of the parent cell line.
TLDR: Anabolism requires cell surface oxygen consumption to regenerate NAD+. Its importance rises if there is defective ox-phos.
Peter
4e- + 4H+ + O2 -> 2H2O
A certain amount of hydrogen peroxide is also generated and just a little superoxide (probably for cell-cell signalling). Other electron acceptors can stand in for, or compete with, oxygen as the terminal electron acceptor. The reaction occurs on the outer surface of the plasma membrane.
The source of the electrons is cytoplasmic NADH, which is oxidised to NAD+ and H+ on the inner surface of the plasma membrane. Each of the electrons is transported via a plasma membrane ubiquinone:semi ubiquinone cycle which then donates them to the redox enzyme on the external cell surface. Here is a simplified version of Herst and Berridge's Figure 1:
Although I doubt that there is very much NADH or NAD+ in normal extra cellular fluid there has to be an NADH docking site on the outer redox enzyme as one of the hallmarks of cell surface oxygen consumption is that it can be halted completely by flooding the cell culture medium with NADH. Electrons from this then follow the dashed line to fully reduce the ubiquinone to ubiquinol and the system grinds to a halt.
So you can measure cell surface oxygen usage by the fall in consumption which occurs when you add exogenous NADH just as you can measure mitochondrial oxygen usage by the fall in consumption which occurs when you add myxothiazol.
Why is the system there?
Let's go back to the two routes of glycolysis. Without the glycerophosphate shuttle (insulin signalling driven/driving) we have
Glucose -> lactate -> mitochondria -> pyruvate -> TCA
and there is no depletion of cytoplasmic NAD+ as one is consumed and one produced in the glucose -> lactate process. With insulin signalling we have two parallel processes:
Glucose -> glycerophosphate shuttle -> CoQ -> ETC
which consumes cytoplasmic NADH, leaving none to convert pyruvate to lactate. So in parallel we have to abort glycolysis at pyruvate:
Glucose -> pyruvate -> mitochondria -> TCA
which balances the cytoplasmic NADH:NAD+ ratio nicely.
Now, let's consider a cell undergoing rapid growth with a view to divide. For today I will ignore mitochondrial biosynthesis and consider what happens if cytoplasmic pyruvate is consumed for amino acid biosynthesis. For each molecule of pyruvate which has been diverted to an amino acid there will be one less available to provide cytoplasmic NAD+ by conversion to lactate, which will limit glycolysis because cytoplasmic NAD+ is essential for the oxidation of glyceraldehyde-3-phosphate.
Under these conditions cell surface oxygen consumption appears to be able to step in to oxidise cytoplasmic NADH to cytoplasmic NAD+, which then allows glycolysis and its associated ATP production. This looks to be particularly important if there is any sort of a problem with the ETC and the glycerophosphate shuttle.
In rho zero cells, where the ETC is deleted (and there is no glycerophosphate shuttle) and glycolysis is the sole source of ATP production, cell surface oxygen consumption has to supply NAD+ in direct proportion to how much pyruvate is lost to anabolism rather than being used to supply NAD+ via lactate generation. In rho zero anabolic cancer cells cell surface oxygen consumption can be as much as 90% of the total oxygen consumption of the parent cell line.
TLDR: Anabolism requires cell surface oxygen consumption to regenerate NAD+. Its importance rises if there is defective ox-phos.
Peter
Friday, January 18, 2019
Cell surface oxygen consumption (1)
Back in the comments thread to the More on insulin and the glycerophosphate shuttle post there has been some discussion as to whether Warburg/Seyfried was/is correct about cancers being glycolysis driven or whether ox-phos is the core processes driving cancer metabolism. Raphi and Altavista have fairly opposite views.
Altavista rather liked this paper, using supra pharmacological concentrations of metformin to block complex I in tissue cultured cancer cells. It is true that this intervention produced a dose dependent decrease in oxygen consumption, but we have no idea of what the absolute oxygen consumption of the cells was, only the relative fall from control cell levels. I have huge problems with this paper. Relative change smells like relative risk, as in cardiology...
So I went looking to find out whether cancer cells do consume oxygen in decent amounts and whether this oxygen is simply metabolised by their mitochondria.
It seems that oxygen metabolism is not the sole prerogative of mitochondria.
This paper is fascinating reading and Table 1 gives us the actual oxygen consumption rates of assorted cancer cell lines in culture.
Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines
Total oxygen consumption varies from as high as almost 28pmol/sec/10^6 cells in the HeLa line to as low as 5pmol/sec/10^6 cells in the P815 cell line.
So there is no doubt that cancer cells in culture do consume oxygen.
Does that imply they are using it for ox-phos? Fascinatingly, the answer is no. Not completely.
A significant proportion of the oxygen consumption is occurring at the cell surface plasma membrane. If you acutely block the ETC of the mitochondria using myxothiazol this plasma membrane oxygen consumption continues and appears to account for around half of the total oxygen consumption, the exact percentage varies.
What is really interesting is what happens if you generate ρ° derivatives of your cell line. These have no functional ETC at all but still consume significant amounts of oxygen, usually in the order of around 90% of the total amount consumed by the parent cell line. They have obviously adapted to their lack of mitochondrial ETC by hugely up regulating cell surface oxygen consumption.
Why? How?
I'll probably put posts up about these questions as we do have some ideas. But the whole reason I went looking was to decide whether cancer cells perform ox-phos. It seems that at least some of them do. Those which don't (major mutations of complex I genes) tend to be very, very unpleasant in patients. Amongst other cancers it's possible that the degree of dysfunction in ox-phos and its replacement by plasma membrane oxygen consumption may correlate with the degree of malignancy of the cancer.
Nothing is black and white. Many cancers respire to various degrees. Not always very well.
You can't tell from simple oxygen consumption if a cancer cell line is respiring using the mitochondrial ETC or performing plasma membrane oxygen consumption. Your Clark electrode can't tell you. That explains a chunk of why people can't agree on whether cancers use ox-phos or not. All consume oxygen, but some use more ox-phos than others.
I just thought it was interesting.
Peter
Altavista rather liked this paper, using supra pharmacological concentrations of metformin to block complex I in tissue cultured cancer cells. It is true that this intervention produced a dose dependent decrease in oxygen consumption, but we have no idea of what the absolute oxygen consumption of the cells was, only the relative fall from control cell levels. I have huge problems with this paper. Relative change smells like relative risk, as in cardiology...
So I went looking to find out whether cancer cells do consume oxygen in decent amounts and whether this oxygen is simply metabolised by their mitochondria.
It seems that oxygen metabolism is not the sole prerogative of mitochondria.
This paper is fascinating reading and Table 1 gives us the actual oxygen consumption rates of assorted cancer cell lines in culture.
Cell surface oxygen consumption: A major contributor to cellular oxygen consumption in glycolytic cancer cell lines
Total oxygen consumption varies from as high as almost 28pmol/sec/10^6 cells in the HeLa line to as low as 5pmol/sec/10^6 cells in the P815 cell line.
So there is no doubt that cancer cells in culture do consume oxygen.
Does that imply they are using it for ox-phos? Fascinatingly, the answer is no. Not completely.
A significant proportion of the oxygen consumption is occurring at the cell surface plasma membrane. If you acutely block the ETC of the mitochondria using myxothiazol this plasma membrane oxygen consumption continues and appears to account for around half of the total oxygen consumption, the exact percentage varies.
What is really interesting is what happens if you generate ρ° derivatives of your cell line. These have no functional ETC at all but still consume significant amounts of oxygen, usually in the order of around 90% of the total amount consumed by the parent cell line. They have obviously adapted to their lack of mitochondrial ETC by hugely up regulating cell surface oxygen consumption.
Why? How?
I'll probably put posts up about these questions as we do have some ideas. But the whole reason I went looking was to decide whether cancer cells perform ox-phos. It seems that at least some of them do. Those which don't (major mutations of complex I genes) tend to be very, very unpleasant in patients. Amongst other cancers it's possible that the degree of dysfunction in ox-phos and its replacement by plasma membrane oxygen consumption may correlate with the degree of malignancy of the cancer.
Nothing is black and white. Many cancers respire to various degrees. Not always very well.
You can't tell from simple oxygen consumption if a cancer cell line is respiring using the mitochondrial ETC or performing plasma membrane oxygen consumption. Your Clark electrode can't tell you. That explains a chunk of why people can't agree on whether cancers use ox-phos or not. All consume oxygen, but some use more ox-phos than others.
I just thought it was interesting.
Peter
Friday, January 11, 2019
How primordial is oxidative phosphorylation?
It is perfectly possible to run a sophisticated metabolism using the energy available from an hydrogen rich geochemical proton gradient of the type still found in locations such as Lost City in the Atlantic. This provides an (almost) endless supply of electrons via FeS catalysts of sufficiently negative potential to reduce carbon dioxide to carbon monoxide. From here it is all down hill, energetically speaking, to acetate and metabolism. The process is completely dependent on geochemical conditions for the free-ride. These basic steps are still embedded in the carbon monoxide dehydrogenase/acetyl-CoA synthase complex discussed rather nicely in this 2018 paper (how did I miss it?):
Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes
Back in the Life series of posts I argued in favour of Koonin's concept that Na+ provided the primary electrochemical gradient which was used to drive a Na+ transporting rotary ATP synthase. It's all in here:
Evolutionary primacy of sodium bioenergetics
My own idea is that primordial Na+ energetics derived from a geochemical proton gradient which was converted to a Na+ gradient by a H+/Na+ antiporter. In order to detach from the geochemical proton gradient what is needed is a Na+ pump to replace this geochemically driven antiporter. Acetobacterium woodii has the most highly conserved version of such a Na+ coupled system and back in 2009 the Rnf complex was the primary suspect for being the site of Na+ pumping.
The ins and outs of Na+ bioenergetics in Acetobacterium woodii
By 2010 this was pretty well confirmed:
Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase
In its most autotrophic guise A woodii can still run its metabolism on molecular hydrogen. The process of electron bifurcation allows the generation of reduced ferredoxin from H2 and so can provide electrons of a potential to use NAD+ as their electron acceptor, with sufficient energy left over to pump a single Na+ ion, in imitation of the H+/Na+ geochemical driven antiporter used soon after the origin of life. This Na+ pumping allowed prokaryotes to leave hydrothermal vents, provided there was access to molecular hydrogen as food.
This core process which freed early life from the ties to geochemical alkaline hydrothermal vents is clearly the oxidation of reduced-ferredoxin, ie this is an oxidative reaction driving an electrochemical Na+ gradient to phosphorylate ADP to ATP using a Na+ driven rotary ATP synthase.
It is oxidative phosphorylation.
Oxidative-phosphorylation is as primordial as the exodus of prokaryotes from hydrothermal vents.
Peter
Evolutionary history of carbon monoxide dehydrogenase/acetyl-CoA synthase, one of the oldest enzymatic complexes
Back in the Life series of posts I argued in favour of Koonin's concept that Na+ provided the primary electrochemical gradient which was used to drive a Na+ transporting rotary ATP synthase. It's all in here:
Evolutionary primacy of sodium bioenergetics
My own idea is that primordial Na+ energetics derived from a geochemical proton gradient which was converted to a Na+ gradient by a H+/Na+ antiporter. In order to detach from the geochemical proton gradient what is needed is a Na+ pump to replace this geochemically driven antiporter. Acetobacterium woodii has the most highly conserved version of such a Na+ coupled system and back in 2009 the Rnf complex was the primary suspect for being the site of Na+ pumping.
The ins and outs of Na+ bioenergetics in Acetobacterium woodii
By 2010 this was pretty well confirmed:
Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase
In its most autotrophic guise A woodii can still run its metabolism on molecular hydrogen. The process of electron bifurcation allows the generation of reduced ferredoxin from H2 and so can provide electrons of a potential to use NAD+ as their electron acceptor, with sufficient energy left over to pump a single Na+ ion, in imitation of the H+/Na+ geochemical driven antiporter used soon after the origin of life. This Na+ pumping allowed prokaryotes to leave hydrothermal vents, provided there was access to molecular hydrogen as food.
This core process which freed early life from the ties to geochemical alkaline hydrothermal vents is clearly the oxidation of reduced-ferredoxin, ie this is an oxidative reaction driving an electrochemical Na+ gradient to phosphorylate ADP to ATP using a Na+ driven rotary ATP synthase.
It is oxidative phosphorylation.
Oxidative-phosphorylation is as primordial as the exodus of prokaryotes from hydrothermal vents.
Peter
Monday, December 31, 2018
How much linoleic acid to get fat and insulin resistant?
Tucker and Mike both gave me the heads-up on this paper recently.
Linoleic acid causes greater weight gain than saturated fat without hypothalamic inflammation in the male mouse
It's nice and simple: feeding 22.5% of calories as linoleic acid (LA) to a mouse makes it obese and insulin resistant. Feeding 15% of calories as LA makes it identically obese but without the insulin resistance. The argument can be made, very convincingly, that LA is converts to 4-hydroxynonenyl (4-HNE) in proportion to the LA content of the diet. 4-HNE is a powerful driver of insulin resistance so the hypoglycaemic response to exogenous insulin is markedly blunted in the 22.5% LA group of mice. As in the top line here:
Here are the weight gains, which need a little consideration:
With the eye of faith you can see that the top line (22.5% LA) starts off gaining weight faster than the next line down (15% LA) but converges from around 7-8 weeks onward. My guess is that this is when insulin resistance from 4-HNE started to over-ride the insulin sensitising effect of LA which caused the weight gain in the first place.
Now the third line down is the interesting one. This diet only contained 1% linoleic acid. OK, these mice are statistically significantly slimmer than the 15% and 22.5% LA fed mice (p less than 0.05) but they are hardly exactly svelte when compared to the crapinabag (CIAB)-fed control mice (black line down at the bottom). And the CIAB food contained 4.22% of calories derived from LA.
That needs some thinking about.
It makes me ask: Why do the vast majority of high fat fed mice/rats become obese? Apart from the fact that they have been selected for this response.
There's probably another post or two needed on that one.
Peter
As an aside I would just comment that while I agree with Tucker that 4-HNE and related products of free radical modified PUFA are the best explanation for this study, my feeling is that the 15% LA group with sustained insulin sensitivity allowing sustained weight gain probably explains the situation in the current human population rather better. As sustained adipocyte distension progresses then we eventually get FFAs released in the presence of glucose and insulin, a Bad Thing. The ROS from this combination will eventually generate 4-HNE too but rather further down the obesity road compared with the 22.5% LA situation.
Linoleic acid causes greater weight gain than saturated fat without hypothalamic inflammation in the male mouse
It's nice and simple: feeding 22.5% of calories as linoleic acid (LA) to a mouse makes it obese and insulin resistant. Feeding 15% of calories as LA makes it identically obese but without the insulin resistance. The argument can be made, very convincingly, that LA is converts to 4-hydroxynonenyl (4-HNE) in proportion to the LA content of the diet. 4-HNE is a powerful driver of insulin resistance so the hypoglycaemic response to exogenous insulin is markedly blunted in the 22.5% LA group of mice. As in the top line here:
Here are the weight gains, which need a little consideration:
With the eye of faith you can see that the top line (22.5% LA) starts off gaining weight faster than the next line down (15% LA) but converges from around 7-8 weeks onward. My guess is that this is when insulin resistance from 4-HNE started to over-ride the insulin sensitising effect of LA which caused the weight gain in the first place.
Now the third line down is the interesting one. This diet only contained 1% linoleic acid. OK, these mice are statistically significantly slimmer than the 15% and 22.5% LA fed mice (p less than 0.05) but they are hardly exactly svelte when compared to the crapinabag (CIAB)-fed control mice (black line down at the bottom). And the CIAB food contained 4.22% of calories derived from LA.
That needs some thinking about.
It makes me ask: Why do the vast majority of high fat fed mice/rats become obese? Apart from the fact that they have been selected for this response.
There's probably another post or two needed on that one.
Peter
As an aside I would just comment that while I agree with Tucker that 4-HNE and related products of free radical modified PUFA are the best explanation for this study, my feeling is that the 15% LA group with sustained insulin sensitivity allowing sustained weight gain probably explains the situation in the current human population rather better. As sustained adipocyte distension progresses then we eventually get FFAs released in the presence of glucose and insulin, a Bad Thing. The ROS from this combination will eventually generate 4-HNE too but rather further down the obesity road compared with the 22.5% LA situation.
Thursday, December 20, 2018
Urinary c-peptide
It's the Winter Solstice tomorrow, greetings to all! My favourite astronomical event of the year, even though we do the major feasting on Christmas day in our house. Anyway, here's a fairytale for the depths of Winter (in the northern hemisphere anyway). Happy Solstice!
*********
Here we go: Just occasionally you come across a statement like this:
"(B) Insulin secretion throughout the day was assessed by 24-hr urinary c-peptide excretion and was significantly reduced only following the RC diet".
Okay, what does it suggest to us when the reduced 24-hr urinary c-peptide group lost less fat than the higher c-peptide group? Less insulin but less fat loss??? Perhaps it suggests that the insulin hypothesis of obesity is incorrect?
C-peptide is part of pro-insulin. Each molecule of insulin produced provides one molecule of c-peptide within the pancreas. Assuming (not completely accurately) that c-peptide is not consumed within the body we can use its 24-hr average urinary excretion as a surrogate for overall insulin production. With an awful lot of caveats, this seems fair to me. I think statement B is correct.
So 24-hr urinary c-peptide gives us an idea of how many molecules of insulin are being manufactured per day by the islets within the pancreas. Insulin is broken down by insulin degrading enzyme as part of its signalling process, not exactly proportionally, but as a general principle this is correct. I went through it in some detail when thinking about the Potato Diet, a sub-category of carbosis. The more signalling, the more degradation.
On average around 50% of secreted insulin (in dogs on a mixed diet) is removed by the liver on first passage from the portal vein through to the hepatic vein (termed first past extraction, FPE). Humans are very similar. None of this hepatic first pass extracted insulin ever arrives in the general circulation. The rate of extraction varied from as low as just over 20% up to almost 80% in the dog study. If you have 100 molecules of c-peptide produced, somewhere between 20 and 80 of the associated molecules of insulin will never arrive in the systemic circulation.
Does anything specific alter the FPE? Well, yes, of course. Does anyone think it might be free fatty acid delivery to the liver? Much as this paper tells us
Free Fatty Acids Impair Hepatic Insulin Extraction in Vivo
So under a weight stable modest LC diet (or more accurately; under whole body adipose tissue mass stability) the reduction of insulin secretion from the pancreas under that modestly reduced carbohydrate intake will undoubtedly occur, but would be offset by reduced hepatic FPE and enough insulin will penetrate to adipocytes to keep them full. In the real world this is extremely difficult to make happen, people just want to eat less on a LC diet because as insulin falls more fat exits adipocytes and hunger diminishes. As in Aberdeen. But you can approximate it by artificially controlling (increasing) food intake, ie you pay people to eat more dietary fat than they would like to (which keeps insulin secretion unchanged but increases fatty acid delivery to the liver), hepatic FPE falls and more insulin reaches adipocytes to keep them full.
Under ketosis (let's say with carbs less than 20g/d) there is so little insulin secretion that having an FPE which is probably approaching zero doesn't matter much. Near basal physiological insulin is secreted, almost none is FPE-ed by the liver but there is still minimal exposure of adipocytes to insulin because almost none is being secreted in the first place. So appetite plummets as FFAs rise as they pour out from the adipocytes, despite a minimal hepatic FPE. This should make it even harder to overeat. However, if you do manage it, minimal hepatic FPE by the liver is one of your methods to maintain fat stores under ketosis!
Conversely you can achieve low FFA delivery to the liver by using acipimox. People do not necessarily gain weight with this lipolysis inhibiting drug because it decreases hepatic FFA delivery, so increases hepatic insulin signalling and so increases hepatic insulin metabolism. I assume this increased insulin metabolism will increase FPE and this will decrease insulin delivery to the systemic circulation. I also think this is also how carbosis works, hence the need for very low fat provision in any carbosis inducing diet.
Anyway, here's a thought experiment using made-up numbers. Any resemblance to real life is totally accidental. If a moderate carb diet (say 140g/d) allows a 22% fall in 24-hr urinary c-peptide, does this mean there is a 22% fall in 24h exposure of adipocytes to systemic insulin? Well no...
Say 100 molecules of insulin are secreted and FPE pre-study is 50% then 50 molecules of insulin survive passage through the liver to suppress systemic lipolysis. If only 78 molecules of insulin are secreted under mild carbohydrate restriction but FPE falls to 20% (exaggeration to make the point!) due to increased lipolysis delivering extra FFAs to the liver, then 62 molecules of insulin will make it through FPE and as far as the adipocytes. This insulin could conceivably allow less lipolysis when compared to a carbosis inducing diet, despite reducing insulin secretion.
On a diet in which fat is so restricted as to allow almost none to be spared for oxidation so that FFA delivery to the liver falls precipitously, we can suggest an 80% FPE might be the result. This would be the situation under carbosis, say with a human eating 7.7% fat as part of a severely calorie restricted diet. So of the 100 molecules of insulin still being produced under these circumstances (typified by no fall in 24-hr urinary c-peptide) with an 80% FPE only 20 of those molecules of insulin will eventually hit the adipocytes and so lipolysis would then be greater than under the modest LC diet.
Please bear in mind that these numbers are a reductio ad absurdum example, but they do make a point about what is possible. There are other effects which would kick in but that's not my point here.
My grossly biased opinion is that any study which shows an intervention with superiority in fat loss will be associated with either lower insulin exposure of adipocytes or with an induced failure of insulin to act on those adipocytes (ie by metformin, alcohol, fructose or of course palmitic acid, plus a few others) than is achieved by any comparison diet.
But then I would say that..........
Peter
*********
Here we go: Just occasionally you come across a statement like this:
"(B) Insulin secretion throughout the day was assessed by 24-hr urinary c-peptide excretion and was significantly reduced only following the RC diet".
Okay, what does it suggest to us when the reduced 24-hr urinary c-peptide group lost less fat than the higher c-peptide group? Less insulin but less fat loss??? Perhaps it suggests that the insulin hypothesis of obesity is incorrect?
C-peptide is part of pro-insulin. Each molecule of insulin produced provides one molecule of c-peptide within the pancreas. Assuming (not completely accurately) that c-peptide is not consumed within the body we can use its 24-hr average urinary excretion as a surrogate for overall insulin production. With an awful lot of caveats, this seems fair to me. I think statement B is correct.
So 24-hr urinary c-peptide gives us an idea of how many molecules of insulin are being manufactured per day by the islets within the pancreas. Insulin is broken down by insulin degrading enzyme as part of its signalling process, not exactly proportionally, but as a general principle this is correct. I went through it in some detail when thinking about the Potato Diet, a sub-category of carbosis. The more signalling, the more degradation.
On average around 50% of secreted insulin (in dogs on a mixed diet) is removed by the liver on first passage from the portal vein through to the hepatic vein (termed first past extraction, FPE). Humans are very similar. None of this hepatic first pass extracted insulin ever arrives in the general circulation. The rate of extraction varied from as low as just over 20% up to almost 80% in the dog study. If you have 100 molecules of c-peptide produced, somewhere between 20 and 80 of the associated molecules of insulin will never arrive in the systemic circulation.
Does anything specific alter the FPE? Well, yes, of course. Does anyone think it might be free fatty acid delivery to the liver? Much as this paper tells us
Free Fatty Acids Impair Hepatic Insulin Extraction in Vivo
So under a weight stable modest LC diet (or more accurately; under whole body adipose tissue mass stability) the reduction of insulin secretion from the pancreas under that modestly reduced carbohydrate intake will undoubtedly occur, but would be offset by reduced hepatic FPE and enough insulin will penetrate to adipocytes to keep them full. In the real world this is extremely difficult to make happen, people just want to eat less on a LC diet because as insulin falls more fat exits adipocytes and hunger diminishes. As in Aberdeen. But you can approximate it by artificially controlling (increasing) food intake, ie you pay people to eat more dietary fat than they would like to (which keeps insulin secretion unchanged but increases fatty acid delivery to the liver), hepatic FPE falls and more insulin reaches adipocytes to keep them full.
Under ketosis (let's say with carbs less than 20g/d) there is so little insulin secretion that having an FPE which is probably approaching zero doesn't matter much. Near basal physiological insulin is secreted, almost none is FPE-ed by the liver but there is still minimal exposure of adipocytes to insulin because almost none is being secreted in the first place. So appetite plummets as FFAs rise as they pour out from the adipocytes, despite a minimal hepatic FPE. This should make it even harder to overeat. However, if you do manage it, minimal hepatic FPE by the liver is one of your methods to maintain fat stores under ketosis!
Conversely you can achieve low FFA delivery to the liver by using acipimox. People do not necessarily gain weight with this lipolysis inhibiting drug because it decreases hepatic FFA delivery, so increases hepatic insulin signalling and so increases hepatic insulin metabolism. I assume this increased insulin metabolism will increase FPE and this will decrease insulin delivery to the systemic circulation. I also think this is also how carbosis works, hence the need for very low fat provision in any carbosis inducing diet.
Anyway, here's a thought experiment using made-up numbers. Any resemblance to real life is totally accidental. If a moderate carb diet (say 140g/d) allows a 22% fall in 24-hr urinary c-peptide, does this mean there is a 22% fall in 24h exposure of adipocytes to systemic insulin? Well no...
Say 100 molecules of insulin are secreted and FPE pre-study is 50% then 50 molecules of insulin survive passage through the liver to suppress systemic lipolysis. If only 78 molecules of insulin are secreted under mild carbohydrate restriction but FPE falls to 20% (exaggeration to make the point!) due to increased lipolysis delivering extra FFAs to the liver, then 62 molecules of insulin will make it through FPE and as far as the adipocytes. This insulin could conceivably allow less lipolysis when compared to a carbosis inducing diet, despite reducing insulin secretion.
On a diet in which fat is so restricted as to allow almost none to be spared for oxidation so that FFA delivery to the liver falls precipitously, we can suggest an 80% FPE might be the result. This would be the situation under carbosis, say with a human eating 7.7% fat as part of a severely calorie restricted diet. So of the 100 molecules of insulin still being produced under these circumstances (typified by no fall in 24-hr urinary c-peptide) with an 80% FPE only 20 of those molecules of insulin will eventually hit the adipocytes and so lipolysis would then be greater than under the modest LC diet.
Please bear in mind that these numbers are a reductio ad absurdum example, but they do make a point about what is possible. There are other effects which would kick in but that's not my point here.
My grossly biased opinion is that any study which shows an intervention with superiority in fat loss will be associated with either lower insulin exposure of adipocytes or with an induced failure of insulin to act on those adipocytes (ie by metformin, alcohol, fructose or of course palmitic acid, plus a few others) than is achieved by any comparison diet.
But then I would say that..........
Peter
Wednesday, December 12, 2018
A post not about Walter Kempner
I've had this paper on my hard drive for a while. It's been sitting somewhere near the front of the back of my mind but was doing nothing to really grab my interest.
Chronic high-sucrose diet increases fibroblast growth factor 21 production and energy expenditure in mice
I've got a draft of a post from mid summer this year which I wrote simply because I like the attitude of the authors. They say things like:
"Excess carbohydrate intake causes obesity in humans".
That's the first line of the abstract. You know, it's that "nailing your colours to the mast" sort of a statement. Even though I do think life is a little more complex than that.
Anyway, I like these folks who are looking at the slimming effect of sucrose in BL6 mice. That's correct, sucrose is a slimming drug/food in mice, under the correct circumstances. People too. The data in the 2017 paper is an extension of the work they did in 2012, written up in this paper:
Ingestion of a moderate high‐sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon‐like peptide‐1 secretion
I don't intend to go through either paper in detail, it's just that the 2012 paper has some rather special macro ratios that caught my eye.
This is what they did to the mice in that original paper:
"After adaptation for 2 weeks, they [the mice] were divided into three groups and fed a normal chow diet (NC), a high‐starch diet (ST) supplemented with 38.5% corn starch or a SUC containing 38.5% sucrose; the latter two diets were prepared by the addition of corn starch or sucrose, respectively, to CE‐2 (Table 1)"
Essentially they are diluting chow with starch or sucrose. Here is Table 1 for the diet compositions, note my red rectangle:
With group sizes of n=4 and five weeks on the diet very little of anything reached statistical or biological significance. The 2017 study used a slightly modified version of the diet to keep a low fat percentage identical across the diets but still had 38.5% of calories from sucrose, was run for 15 weeks and had group sizes of n=8-10. Results were statistically significant all over the place and suggest that the sucrose diet is decidedly good for metabolic health and gives a slim phenotype on ad lib consumption. Just so long as fat calories are very, very low. This looks very much like what Denise Minger described as carbosis, based in part around Walter Kempner's very effective, very unpleasant, ultra low fat, high sucrose medical diet. The Rice Diet is very real.
This post is not about any of the above.
Now, watch carefully. I'm going to sneak in some more macros
If you wanted a "reduced" fat diet which induces carbosis in human beings I recon the red text is pretty well it. I particularly enjoyed that exactly 7.7% of calories came from fat in each diet, this could almost be deliberate. If you combine what is almost certainly a very effective spontaneous weight loss diet with a 30% calorie restriction I suspect you might be on to a winner when comparing it against a reduced carbohydrate diet. Of course to really nail it you would have to compare it to an absolutely non ketogenic diet, say one supplying a total of 140g/d of carbohydrate. Does carbosis beat a middling carbohydrate mixed diet? You bet.
Oh, the scribbled-in red numbers came from Table 2 of this paper.
Most people in respectable CICO based mainstream nutrition have never heard of carbosis, Walter Kempner, the Rice Diet and have probably never heard of Denise Minger.
But Kevin Hall has. My respect for his knowledge-base and ingenuity is vast. Such a pity it's wasted on constructing props for his bizarre pet theories of weight control.
While the 7.7% of calories as fat in both studies is something which amuses me greatly, I do have to admit it may just be an hysterical accident.
At least I'm up front about my rather pronounced personal biases and rather peculiar sense of humour.
Peter
Chronic high-sucrose diet increases fibroblast growth factor 21 production and energy expenditure in mice
I've got a draft of a post from mid summer this year which I wrote simply because I like the attitude of the authors. They say things like:
"Excess carbohydrate intake causes obesity in humans".
That's the first line of the abstract. You know, it's that "nailing your colours to the mast" sort of a statement. Even though I do think life is a little more complex than that.
Anyway, I like these folks who are looking at the slimming effect of sucrose in BL6 mice. That's correct, sucrose is a slimming drug/food in mice, under the correct circumstances. People too. The data in the 2017 paper is an extension of the work they did in 2012, written up in this paper:
Ingestion of a moderate high‐sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon‐like peptide‐1 secretion
I don't intend to go through either paper in detail, it's just that the 2012 paper has some rather special macro ratios that caught my eye.
This is what they did to the mice in that original paper:
"After adaptation for 2 weeks, they [the mice] were divided into three groups and fed a normal chow diet (NC), a high‐starch diet (ST) supplemented with 38.5% corn starch or a SUC containing 38.5% sucrose; the latter two diets were prepared by the addition of corn starch or sucrose, respectively, to CE‐2 (Table 1)"
Essentially they are diluting chow with starch or sucrose. Here is Table 1 for the diet compositions, note my red rectangle:
With group sizes of n=4 and five weeks on the diet very little of anything reached statistical or biological significance. The 2017 study used a slightly modified version of the diet to keep a low fat percentage identical across the diets but still had 38.5% of calories from sucrose, was run for 15 weeks and had group sizes of n=8-10. Results were statistically significant all over the place and suggest that the sucrose diet is decidedly good for metabolic health and gives a slim phenotype on ad lib consumption. Just so long as fat calories are very, very low. This looks very much like what Denise Minger described as carbosis, based in part around Walter Kempner's very effective, very unpleasant, ultra low fat, high sucrose medical diet. The Rice Diet is very real.
This post is not about any of the above.
Now, watch carefully. I'm going to sneak in some more macros
If you wanted a "reduced" fat diet which induces carbosis in human beings I recon the red text is pretty well it. I particularly enjoyed that exactly 7.7% of calories came from fat in each diet, this could almost be deliberate. If you combine what is almost certainly a very effective spontaneous weight loss diet with a 30% calorie restriction I suspect you might be on to a winner when comparing it against a reduced carbohydrate diet. Of course to really nail it you would have to compare it to an absolutely non ketogenic diet, say one supplying a total of 140g/d of carbohydrate. Does carbosis beat a middling carbohydrate mixed diet? You bet.
Oh, the scribbled-in red numbers came from Table 2 of this paper.
Most people in respectable CICO based mainstream nutrition have never heard of carbosis, Walter Kempner, the Rice Diet and have probably never heard of Denise Minger.
But Kevin Hall has. My respect for his knowledge-base and ingenuity is vast. Such a pity it's wasted on constructing props for his bizarre pet theories of weight control.
While the 7.7% of calories as fat in both studies is something which amuses me greatly, I do have to admit it may just be an hysterical accident.
At least I'm up front about my rather pronounced personal biases and rather peculiar sense of humour.
Peter
Tuesday, December 04, 2018
An exchange of half bricks
I would guess that everyone is aware of the study by Ebbeling et al, Ludwig's group, looking at the metabolic effect of low carbohydrate diets on total energy expenditure (TEE, all graphs show kcal/d) in the aftermath of weight loss on a conventional diet.
Effects of a low carbohydrate diet on energy expenditure during weight loss maintenance: randomized trial
I'd like to summarise their data using numbers taken from Tables 2 and 3 which, with a little arithmetic, allows me to produce this graph of TEE at various time points. These are as follows: when the subjects walk off the street (Pre on the graph), after a period of semi-starvation on a conventional diet (Start) and then during weight stability on a high, medium or low carbohydrate diet (End). The plot looks like this:
In the study they compared the change from the Start TEE to the End TEE, ie they used these data points:
They took the absolute changes from Start to End thus and got a resultant p of less than 0.05
This, obviously, is completely unacceptable. Well, it is if you are Kevin Hall. So now we have this
No Significant Effect of Dietary Carbohydrate versus Fat on the Reduction in Total Energy Expenditure During Maintenance of Lost Weight
What Ludwig's group did wrong (amongst the many other things pointed out by Hall and Guo) is that they used the wrong data points.
Recall the original graph:
According to Hall: If you want to ask about the effect of low carbohydrate diets on the depression in TEE produced by conventional semi-starvation you should NOT compare the semi-starved TEE (as in Start) to the TEE on a high, medium or low carbohydrate diet (End). You should instead use the TEE expenditure at randomisation (Pre on the graph). Like this:
Using Pre as your anchor point you can draw the same data thus:
Which obviously gives us p greater than 0.05 and all of the benefits of low carbohydrate diets are lost. Phew. Happy Hall. But why should anyone use the Pre values as an anchor point?
Now, no one is an unbiased researcher. Hall is, surprisingly, no exception. Hence the current exchange of half bricks in the BMJ.
As I see it the Ebbeling paper looks at the effect of LC eating on the damage done to TEE by conventional dieting.
What Hall wants the analysis to do instead is to look at the overall effect of damage done to TEE by conventional semi-starvation combined with partial rescue during weight-stable LC eating vs the combined damage done by conventional semi-starvation followed by maintained damage done by HC weight-stable eating. As he writes:
"However, the final analysis plan was modified to make the diet comparisons with the TEE measurements collected in the immediate post-weight loss period rather than at the pre-weight loss baseline"
To me Hall is stating that Ebbeling et al almost did make the "Hall" mistake of using the "Pre" TTE as anchor point but corrected this at the 11th hour, still before blinding was unmasked. What puzzles me is how Ebbeling could have ever even considered using the "pre weight loss baseline" as the anchor point in the original study design.
The massive benefit to Hall of including the conventional semi-starvation active weight loss period along with the intervention weight stability period is to dilute the remedial biological effect of LC eating out of statistical significance.
The core information which the study provides is about the remedial effect of LC eating on correcting the damage done by a conventional semi-starvation period. That effect only happens between "Start" and "End", which is when carbohydrate restriction is applied.
That's one of the MASSIVE problems with carbohydrate restricted eating. It only provides benefit when you don't eat carbohydrate!
Including data from "Pre" right through to "End" dilutes the clearly demonstrable biological effect of carbohydrate restriction on reduced TEE post conventional dieting.
So what doe the title and text of Hall's rebuttal tell us? Either about Hall or about TEE? Don't over think it!
I would also declare that my own biases are a conflict of interest but if you need me to say that then you have probably arrived here by accident, you know where the back button is.
However I would say that I am ambivalent about the importance of the TEE changes, though I suspect they do happen. What really matters to me is what happened in Aberdeen over a decade ago.
Peter
Effects of a low carbohydrate diet on energy expenditure during weight loss maintenance: randomized trial
I'd like to summarise their data using numbers taken from Tables 2 and 3 which, with a little arithmetic, allows me to produce this graph of TEE at various time points. These are as follows: when the subjects walk off the street (Pre on the graph), after a period of semi-starvation on a conventional diet (Start) and then during weight stability on a high, medium or low carbohydrate diet (End). The plot looks like this:
In the study they compared the change from the Start TEE to the End TEE, ie they used these data points:
They took the absolute changes from Start to End thus and got a resultant p of less than 0.05
This, obviously, is completely unacceptable. Well, it is if you are Kevin Hall. So now we have this
No Significant Effect of Dietary Carbohydrate versus Fat on the Reduction in Total Energy Expenditure During Maintenance of Lost Weight
What Ludwig's group did wrong (amongst the many other things pointed out by Hall and Guo) is that they used the wrong data points.
Recall the original graph:
According to Hall: If you want to ask about the effect of low carbohydrate diets on the depression in TEE produced by conventional semi-starvation you should NOT compare the semi-starved TEE (as in Start) to the TEE on a high, medium or low carbohydrate diet (End). You should instead use the TEE expenditure at randomisation (Pre on the graph). Like this:
Using Pre as your anchor point you can draw the same data thus:
Which obviously gives us p greater than 0.05 and all of the benefits of low carbohydrate diets are lost. Phew. Happy Hall. But why should anyone use the Pre values as an anchor point?
Now, no one is an unbiased researcher. Hall is, surprisingly, no exception. Hence the current exchange of half bricks in the BMJ.
As I see it the Ebbeling paper looks at the effect of LC eating on the damage done to TEE by conventional dieting.
What Hall wants the analysis to do instead is to look at the overall effect of damage done to TEE by conventional semi-starvation combined with partial rescue during weight-stable LC eating vs the combined damage done by conventional semi-starvation followed by maintained damage done by HC weight-stable eating. As he writes:
"However, the final analysis plan was modified to make the diet comparisons with the TEE measurements collected in the immediate post-weight loss period rather than at the pre-weight loss baseline"
To me Hall is stating that Ebbeling et al almost did make the "Hall" mistake of using the "Pre" TTE as anchor point but corrected this at the 11th hour, still before blinding was unmasked. What puzzles me is how Ebbeling could have ever even considered using the "pre weight loss baseline" as the anchor point in the original study design.
The massive benefit to Hall of including the conventional semi-starvation active weight loss period along with the intervention weight stability period is to dilute the remedial biological effect of LC eating out of statistical significance.
The core information which the study provides is about the remedial effect of LC eating on correcting the damage done by a conventional semi-starvation period. That effect only happens between "Start" and "End", which is when carbohydrate restriction is applied.
That's one of the MASSIVE problems with carbohydrate restricted eating. It only provides benefit when you don't eat carbohydrate!
Including data from "Pre" right through to "End" dilutes the clearly demonstrable biological effect of carbohydrate restriction on reduced TEE post conventional dieting.
So what doe the title and text of Hall's rebuttal tell us? Either about Hall or about TEE? Don't over think it!
I would also declare that my own biases are a conflict of interest but if you need me to say that then you have probably arrived here by accident, you know where the back button is.
However I would say that I am ambivalent about the importance of the TEE changes, though I suspect they do happen. What really matters to me is what happened in Aberdeen over a decade ago.
Peter
Sunday, November 25, 2018
More on insulin and the glycerophosphate shuttle
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...
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...
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
Tuesday, October 30, 2018
Selenium induced glutathione peroxidase generation
TLDR: excess selenium induces excess glutathione peroxidase which blunts physiological superoxide/H2O2 signalling within a cell. This, as you might expect, is a Bad Thing.
I stumbled across this paper quite by accident. It has a number of problems, not least of which is that none of the authors appears to be a native english speaker and this tends to show through. It also carries a rather catastrophic typo/brain fart in the abstract, where TCA, as in tricarboxylic acid cycle, was written out in full as trichloroacetic acid cycle. Ouch. Never the less, they don't seem to like antioxidants.
High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS
Over all they don't seem to like suppressing the physiological levels of ROS needed for insulin signalling, certainly while feeding their rats a high carbohydrate diet. Not that they tell us what they fed the rats on!
This is the intraperitoneal glucose tolerance test result, look at "control" and "Se" for the effect of the high selenium diet:
Among many of the problems with the paper another is the difficulty distinguishing which line is which on the graphs. I think we can say the top line is clearly the selenium supplemented group and the bottom line is probably the control group. The main effect is at the 30 minute mark. This suggests to me that there is an inadequate first phase insulin response (needing ROS from RET induced by mtG3Pdh) to overcome the systemic failure of insulin action (also needing ROS from RET via mtG3Pdh to diffuse as far as the insulin receptor). By 60 minutes the difference is negligible. Had they measured insulin I'd bet the second phase response was exaggerated.
Here is the intraperitoneal insulin tolerance test result:
This time the bottom line is clearly the control group, top line the selenium treated group. From the control group I see no suggestion that this particular dose of insulin is inducing insulin-induced insulin resistance, so all we see is the failure of the insulin signalling activating action of ROS at the two hour mark. Note the lines are all parallel until the one hour mark. This is what suggests that there is nothing wrong with insulin action per se at the 30 minute mark and is another factor which makes me suspect that there is a failure to secrete insulin during the early stages of the IPGTT. The normal response to exogenous insulin, up until one hour mark, occurs while ever the exogenous insulin can overcome the loss of ROS caused by the glutathione peroxidase excess induced by the high selenium diet.
As humans we deal with huge numbers of xenobiotic antioxidants, mostly from plants. For the vast majority we can simply use our liver and/or kidneys to dump them to where they can do us no harm. Just occasionally we fail, as with selenium. The end result is not pretty.
Peter
There are some more interesting finings in the paper which might be worth chatting about. Maybe another day.
Random throw-away thought: Is this how uric acid induces insulin resistance? By being too good an antioxidant???? Hmmmmmm.
I stumbled across this paper quite by accident. It has a number of problems, not least of which is that none of the authors appears to be a native english speaker and this tends to show through. It also carries a rather catastrophic typo/brain fart in the abstract, where TCA, as in tricarboxylic acid cycle, was written out in full as trichloroacetic acid cycle. Ouch. Never the less, they don't seem to like antioxidants.
High selenium impairs hepatic insulin sensitivity through opposite regulation of ROS
Over all they don't seem to like suppressing the physiological levels of ROS needed for insulin signalling, certainly while feeding their rats a high carbohydrate diet. Not that they tell us what they fed the rats on!
This is the intraperitoneal glucose tolerance test result, look at "control" and "Se" for the effect of the high selenium diet:
Among many of the problems with the paper another is the difficulty distinguishing which line is which on the graphs. I think we can say the top line is clearly the selenium supplemented group and the bottom line is probably the control group. The main effect is at the 30 minute mark. This suggests to me that there is an inadequate first phase insulin response (needing ROS from RET induced by mtG3Pdh) to overcome the systemic failure of insulin action (also needing ROS from RET via mtG3Pdh to diffuse as far as the insulin receptor). By 60 minutes the difference is negligible. Had they measured insulin I'd bet the second phase response was exaggerated.
Here is the intraperitoneal insulin tolerance test result:
This time the bottom line is clearly the control group, top line the selenium treated group. From the control group I see no suggestion that this particular dose of insulin is inducing insulin-induced insulin resistance, so all we see is the failure of the insulin signalling activating action of ROS at the two hour mark. Note the lines are all parallel until the one hour mark. This is what suggests that there is nothing wrong with insulin action per se at the 30 minute mark and is another factor which makes me suspect that there is a failure to secrete insulin during the early stages of the IPGTT. The normal response to exogenous insulin, up until one hour mark, occurs while ever the exogenous insulin can overcome the loss of ROS caused by the glutathione peroxidase excess induced by the high selenium diet.
As humans we deal with huge numbers of xenobiotic antioxidants, mostly from plants. For the vast majority we can simply use our liver and/or kidneys to dump them to where they can do us no harm. Just occasionally we fail, as with selenium. The end result is not pretty.
Peter
There are some more interesting finings in the paper which might be worth chatting about. Maybe another day.
Random throw-away thought: Is this how uric acid induces insulin resistance? By being too good an antioxidant???? Hmmmmmm.
Sunday, October 21, 2018
Metformin (09) Islets
This is another very abstracted study using isolated mouse islets in cell culture to assess the effect of metformin on insulin secretion.
Metformin Inhibits Mouse Islet Insulin Secretion and Alters Intracellular Calcium in a Concentration-Dependent and Duration-Dependent Manner near the Circulating Range
From the Protons perspective the factors which drive insulin signalling are the same ones which drive insulin secretion, certainly at low physiological concentrations. The situation is different under post prandial conditions where, eventually, reverse electron transport increases from low, physiological activating levels of ROS to the high physiological levels which drive insulin resistance rather than activation. Recall this is what I consider to be the cellular repletion signal, the one so easily mistaken for insulin as an anorexic agent. Anyway, here we have metformin acting under 11mmol/l of glucose to suppress insulin secretion.
Just to recap; 20micromolar metformin is therapeutic, 200micromolar is life threatening lactic acidosis and 1mmolar (1000micromolar) is death:
Any findings in the paper using concentrations of 200micromolar or higher can safely be ignored for therapeutic relevance. Except for the confirmation that cells die rather well at 1.0mmol of metformin and are doing rather more apoptosis than you might like at 200micromolar (see Figure 3 in the paper). No surprises there.
Also consider that picking up subtleties of insulin secretion by measuring the concentration in a culture well is a very blunt instrument. But at least they are looking.
So why doesn't metformin cause diabetes on the sort of criminal (up until very recently) diet advised by any diabetologist?
This is partly because the redox changes in the liver suppress gluconeogenesis, though the exact mechanism by which blockade of mtG3Pdh suppresses hepatic glucose production is debatable.
It's also because, certainly in peripheral cells suffering from chronic hyperinsulinaemia-induced lipotoxicity, cessation or reduction of insulin signalling will allow release of fatty acids able to generate their own RET via the oxidation of beta oxidation derived electron transporting flavoprotein at mtETFdh of the electron transport chain and so restore insulin signalling. Or, if there is enough superoxide, insulin resistance. So cells suddenly realise they have a great supply of FFAs, adequate ATP generation and no need for any more caloric ingress. Which generalises to the whole organism as a "no need to eat" state, which might just give weight loss. As metformin does.
Kind of like LC eating in a pill.
Peter
Actually metformin might do the same to lipid in the pancreas as it does in peripheral tissues. Loss of accumulated pancreatic lipid is what people like Dr Roy Taylor consider the mechanism by which the hypoinsulinaemia of semi-starvation induces some degree of remission of diabetes, in a few patients, while they can stick to it.
Metformin Inhibits Mouse Islet Insulin Secretion and Alters Intracellular Calcium in a Concentration-Dependent and Duration-Dependent Manner near the Circulating Range
From the Protons perspective the factors which drive insulin signalling are the same ones which drive insulin secretion, certainly at low physiological concentrations. The situation is different under post prandial conditions where, eventually, reverse electron transport increases from low, physiological activating levels of ROS to the high physiological levels which drive insulin resistance rather than activation. Recall this is what I consider to be the cellular repletion signal, the one so easily mistaken for insulin as an anorexic agent. Anyway, here we have metformin acting under 11mmol/l of glucose to suppress insulin secretion.
Just to recap; 20micromolar metformin is therapeutic, 200micromolar is life threatening lactic acidosis and 1mmolar (1000micromolar) is death:
Any findings in the paper using concentrations of 200micromolar or higher can safely be ignored for therapeutic relevance. Except for the confirmation that cells die rather well at 1.0mmol of metformin and are doing rather more apoptosis than you might like at 200micromolar (see Figure 3 in the paper). No surprises there.
Also consider that picking up subtleties of insulin secretion by measuring the concentration in a culture well is a very blunt instrument. But at least they are looking.
So why doesn't metformin cause diabetes on the sort of criminal (up until very recently) diet advised by any diabetologist?
This is partly because the redox changes in the liver suppress gluconeogenesis, though the exact mechanism by which blockade of mtG3Pdh suppresses hepatic glucose production is debatable.
It's also because, certainly in peripheral cells suffering from chronic hyperinsulinaemia-induced lipotoxicity, cessation or reduction of insulin signalling will allow release of fatty acids able to generate their own RET via the oxidation of beta oxidation derived electron transporting flavoprotein at mtETFdh of the electron transport chain and so restore insulin signalling. Or, if there is enough superoxide, insulin resistance. So cells suddenly realise they have a great supply of FFAs, adequate ATP generation and no need for any more caloric ingress. Which generalises to the whole organism as a "no need to eat" state, which might just give weight loss. As metformin does.
Kind of like LC eating in a pill.
Peter
Actually metformin might do the same to lipid in the pancreas as it does in peripheral tissues. Loss of accumulated pancreatic lipid is what people like Dr Roy Taylor consider the mechanism by which the hypoinsulinaemia of semi-starvation induces some degree of remission of diabetes, in a few patients, while they can stick to it.
Monday, October 15, 2018
Metformin (08) Insulin and AMPK
This is a simplified diagram of most of the pathways around AMP-kinase (AMKP) which I found at the website of a commercial company on tinternet
Obviously most of the diagram shows the Good Things which activating AMPK does. There appear to be three core activating signals; changes in Ca2+ via calmodulin, increasing cyclic AMP and that change in AMP+ADP:ATP triggered by exercise, low glucose, hypoxia and severely toxic doses of metformin. Here they are emphasised in blue:
There is only one inhibitory input shown, I've marked it in red:
When you follow this input back you end up with the one factor which suppresses AMPK signalling.
We know that metformin inhibits mtG3Pdh at normal pharmacological concentrations. From the Protons perspective this blocks the generation of the ROS essential for insulin signaling. No insulin signalling, no inhibition of AMPK. Which nicely fits in with this paper:
Insulin inhibits AMPK activity and phosphorylates AMPK Ser485/491 through Akt in hepatocytes, myotubes and incubated rat skeletal muscle
Of course metformin inhibits complex I (which drops ATP and so activates AMPK) at concentrations which put you in to the ITU with lactic acidosis, around 200micromolar in plasma. Recall this?
It also activates AMPK via inhibition of AMP-deaminase using tissue culture exposure of 10mmolar. That's 10,000micromolar, which would probably put you in to the morgue rather than the ITU.
A quick reality check suggests that taking one 500mg metformin tablet an hour before a bike race might just help you win... Somehow I don't think blockade of complex I would do that! Freeing up fatty acids by dumping insulin signalling might just do the job.
Peter
Obviously most of the diagram shows the Good Things which activating AMPK does. There appear to be three core activating signals; changes in Ca2+ via calmodulin, increasing cyclic AMP and that change in AMP+ADP:ATP triggered by exercise, low glucose, hypoxia and severely toxic doses of metformin. Here they are emphasised in blue:
There is only one inhibitory input shown, I've marked it in red:
When you follow this input back you end up with the one factor which suppresses AMPK signalling.
Insulin.
We know that metformin inhibits mtG3Pdh at normal pharmacological concentrations. From the Protons perspective this blocks the generation of the ROS essential for insulin signaling. No insulin signalling, no inhibition of AMPK. Which nicely fits in with this paper:
Insulin inhibits AMPK activity and phosphorylates AMPK Ser485/491 through Akt in hepatocytes, myotubes and incubated rat skeletal muscle
Of course metformin inhibits complex I (which drops ATP and so activates AMPK) at concentrations which put you in to the ITU with lactic acidosis, around 200micromolar in plasma. Recall this?
It also activates AMPK via inhibition of AMP-deaminase using tissue culture exposure of 10mmolar. That's 10,000micromolar, which would probably put you in to the morgue rather than the ITU.
A quick reality check suggests that taking one 500mg metformin tablet an hour before a bike race might just help you win... Somehow I don't think blockade of complex I would do that! Freeing up fatty acids by dumping insulin signalling might just do the job.
Peter
Saturday, September 15, 2018
Insulin makes you hungry (9) Women
Intranasal insulin does not, under any circumstances, reduce food intake or bodyweight in women.
That's half the population.
Hallschmid's lab gives us all of these:
Intranasal insulin reduces body fat in men but not in women
Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin
Comparable sensitivity of postmenopausal and young women to the effects of intranasal insulin on food intake and working memory
So. What about this via Wooo:
Postprandial administration of intranasal insulin intensifies satiety and reduces intake of palatable snacks in women
It's free full text if anyone want's to read the details of the convoluted protocol involved. Take my word, you will regret it. Here's the executive summary
You can summarise the results by saying intranasal insulin puts women off of Chocolate Chip Cookies. At least those made by Coppenrath of Geest in Germany. Maybe this will happen with chocolate Hobnobs in the UK or some sort of Oreo in the USA. Who knows? The subjects just filled up on the Premium Spritz Cookies and the Crunchy Coconut Cookies, all made by the same firm. There are lots of soft end points with low p values in the study. Hard end point to me is calories.
Does intranasal insulin affect total calorie intake of cookies, even in the convoluted protocol used?
No.
I have no idea why intranasal insulin is ineffective to speed cellular repletion in 50% of the world population but it makes me suspicious that women deal with calories slightly differently to men and that we're not going to find out how or why from squirting insulin up anyone's nose. And that insulin is not a satiety hormone.
I think I need to get outside more.
Peter
That's half the population.
Hallschmid's lab gives us all of these:
Intranasal insulin reduces body fat in men but not in women
Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin
Comparable sensitivity of postmenopausal and young women to the effects of intranasal insulin on food intake and working memory
So. What about this via Wooo:
Postprandial administration of intranasal insulin intensifies satiety and reduces intake of palatable snacks in women
It's free full text if anyone want's to read the details of the convoluted protocol involved. Take my word, you will regret it. Here's the executive summary
You can summarise the results by saying intranasal insulin puts women off of Chocolate Chip Cookies. At least those made by Coppenrath of Geest in Germany. Maybe this will happen with chocolate Hobnobs in the UK or some sort of Oreo in the USA. Who knows? The subjects just filled up on the Premium Spritz Cookies and the Crunchy Coconut Cookies, all made by the same firm. There are lots of soft end points with low p values in the study. Hard end point to me is calories.
Does intranasal insulin affect total calorie intake of cookies, even in the convoluted protocol used?
No.
I have no idea why intranasal insulin is ineffective to speed cellular repletion in 50% of the world population but it makes me suspicious that women deal with calories slightly differently to men and that we're not going to find out how or why from squirting insulin up anyone's nose. And that insulin is not a satiety hormone.
I think I need to get outside more.
Peter
Wednesday, September 12, 2018
Insulin makes you hungry (8) Blokes
Now it's time to look at the simple situation of men given intranasal insulin. We have a chronic study using 40iu and an acute study using 160iu.
Edit. Oops, forgot the links!
Manipulating central nervous mechanisms of food intake and body weight regulation by intranasal administration of neuropeptides in man
Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin
End edit.
I've not found an acute study using 40iu but we can be pretty sure it works in the same manner as the acute 160iu dose. The only real difference is that 160iu spills over in to the systemic circulation whereas the 40iu doesn't. This is unimportant for acute studies as the effects of peripheral and central insulin are identical before the development of CNS insulin resistance occurs.
Both allow facilitated ingress of calories in to peripheral cells, and help keep those calories there, until the cell signals that it wants no more.
With supplementary CNS insulin peripheral cells become "full" quicker, generate ROS sooner and resist insulin sooner. The brain senses that calories are no longer being taken up. So men stop eating sooner. This is not a central effect on appetite. It's a peripheral effect of allowing calories to fill peripheral cells sooner. So eating stops about (in the 160iu study) 200kcal prematurely. Adipocytes (and other cells) are full. The main difference compared with placebo is that there is less food present within the gut.
We know, at 40iu of intranasal insulin four times daily, that there is no weight loss over three weeks. After one dose of 160iu caloric intake after the first meal is reduced compared to placebo. If that were to translate to 600kcal/d (assuming three meals) there should be some weight loss. Ignoring changes to uncoupling, ie metabolic rate, this suggests that at subsequent meals there is a greater calorie intake, or more snacking between meals.
You could simply say that insulin makes you hungry (have I mentioned this before?). But that would be wrong. Extra insulin allows cell fullness to signal to the brain to stop eating when there is less food in the gut than there should be. Less food in the gut means you get hungry sooner. That means you eat more subsequently. By just enough to make up for the deficit. Because you skimped on a meal. So there is no weight loss for three weeks...
My hypothesis is that after three weeks on 40iu of intranasal insulin four times a day the VMH develops CNS insulin resistance. At this point the effect of intranasal insulin is lost. Also lost is the physiological CNS effect of augmented calorie storage derived from endogenous pancreatic insulin entering the brain.
People will then eat more at each meal because it takes longer for their peripheral cells to get full, so there will be more food in the gut at the time of satiation kicking in. But, with less CNS augmentation of insulin's peripheral action, lipolysis is free to proceed at a higher rate per unit insulin in the blood stream. So hunger is deferred by the augmented availability of stored FFAs. Oxidising these stored FFAs is, by definition, fat loss.
Men lose fat mass once they lose the CNS mediated fat storage effect of insulin.
That makes sense to me and explains why men who are already insulin resistant derive no benefit from intranasal insulin.
Peter
BTW, I'd expect the 40iu dose to work better long term than the 160iu dose provided the systemic leakage of insulin after 160iu outweighs the localised effect of developing insulin resistance within the CNS only. At four times the dose the CNS insulin resistance should occur sooner, to facilitate fat loss, but the systemic spill-over will always augment fat storage. Which would win?
The Hallschmid lab does not seem to have done a long term study of insulin at 160iu four times daily. It would be interesting to know what might have happened. Or maybe they did a pilot study and decided not to go there.
Edit. Oops, forgot the links!
Manipulating central nervous mechanisms of food intake and body weight regulation by intranasal administration of neuropeptides in man
Differential sensitivity of men and women to anorexigenic and memory-improving effects of intranasal insulin
End edit.
I've not found an acute study using 40iu but we can be pretty sure it works in the same manner as the acute 160iu dose. The only real difference is that 160iu spills over in to the systemic circulation whereas the 40iu doesn't. This is unimportant for acute studies as the effects of peripheral and central insulin are identical before the development of CNS insulin resistance occurs.
Both allow facilitated ingress of calories in to peripheral cells, and help keep those calories there, until the cell signals that it wants no more.
With supplementary CNS insulin peripheral cells become "full" quicker, generate ROS sooner and resist insulin sooner. The brain senses that calories are no longer being taken up. So men stop eating sooner. This is not a central effect on appetite. It's a peripheral effect of allowing calories to fill peripheral cells sooner. So eating stops about (in the 160iu study) 200kcal prematurely. Adipocytes (and other cells) are full. The main difference compared with placebo is that there is less food present within the gut.
We know, at 40iu of intranasal insulin four times daily, that there is no weight loss over three weeks. After one dose of 160iu caloric intake after the first meal is reduced compared to placebo. If that were to translate to 600kcal/d (assuming three meals) there should be some weight loss. Ignoring changes to uncoupling, ie metabolic rate, this suggests that at subsequent meals there is a greater calorie intake, or more snacking between meals.
You could simply say that insulin makes you hungry (have I mentioned this before?). But that would be wrong. Extra insulin allows cell fullness to signal to the brain to stop eating when there is less food in the gut than there should be. Less food in the gut means you get hungry sooner. That means you eat more subsequently. By just enough to make up for the deficit. Because you skimped on a meal. So there is no weight loss for three weeks...
My hypothesis is that after three weeks on 40iu of intranasal insulin four times a day the VMH develops CNS insulin resistance. At this point the effect of intranasal insulin is lost. Also lost is the physiological CNS effect of augmented calorie storage derived from endogenous pancreatic insulin entering the brain.
People will then eat more at each meal because it takes longer for their peripheral cells to get full, so there will be more food in the gut at the time of satiation kicking in. But, with less CNS augmentation of insulin's peripheral action, lipolysis is free to proceed at a higher rate per unit insulin in the blood stream. So hunger is deferred by the augmented availability of stored FFAs. Oxidising these stored FFAs is, by definition, fat loss.
Men lose fat mass once they lose the CNS mediated fat storage effect of insulin.
That makes sense to me and explains why men who are already insulin resistant derive no benefit from intranasal insulin.
Peter
BTW, I'd expect the 40iu dose to work better long term than the 160iu dose provided the systemic leakage of insulin after 160iu outweighs the localised effect of developing insulin resistance within the CNS only. At four times the dose the CNS insulin resistance should occur sooner, to facilitate fat loss, but the systemic spill-over will always augment fat storage. Which would win?
The Hallschmid lab does not seem to have done a long term study of insulin at 160iu four times daily. It would be interesting to know what might have happened. Or maybe they did a pilot study and decided not to go there.
Sunday, September 09, 2018
Insulin makes you hungry (7) superoxide is satiety
What causes satiety, if it's not insulin? This has to be understood if there is to be any chance of understanding the effects of intranasal insulin from the metabolic point of view.
Let's begin with individual cells, these are the entities which need to control metabolic substrate availability.
You eat some food. Plasma glucose and chylomicrons/FFAs rise, delivering energy to peripheral tissues. In the early stages of food absorption both glucose and FFAs enter cells under the facilitation of insulin. They do this easily, the cells are "hungry".
As an individual cell becomes replete it has to signal that it doesn't want any more metabolic substrate. This is achieved via the CoQ couple acting as the master sensor for metabolic energy status. It is being reduced using NADH from the cytoplasm (the glycerol-3-phosphate shuttle), by FADH2 input via complex II (acetyl-CoA in the TCA) and via FADH2 input from ETFdh (from beta oxidation of saturated fats). And of course from mitochondrial NADH via complex I. Given a high delta psi (ie minimal consumption of the proton motive force because ATP is already plentiful) this CoQ reduction eventually facilitates RET through complex I to give superoxide generation in order to stop insulin signalling. Which then limits cellular caloric ingress. This can be thought of as the "cellular satiety" signal. It is ROS generated. Let's say that again:
Satiety in peripheral cells is an ROS signal. It is generated in the mitochondria. This is pure Protons.
Now let's scale that up.
As more and more peripheral cells decide that they no longer need to respond to insulin then there is less and less of a "sump" available for absorbed calories to drop in to. The availability of calories which no longer have anywhere to go is the whole-body driver of the need to signal satiety. This surfeit of calories will be sensed in the VMH and the cessation of eating will be ROS mediated.
These people have the correct sort of idea:
Fuel utilization by hypothalamic neurons: roles for ROS
and so do these
Role for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake
There is no need, to my mind, for CNS insulin to be involved. While insulin clearly has many effects in the brain, neurons do not appear to use insulin signalling to control caloric ingress, so insulin signalling should not influence ROS generation. Astrocytes might use insulin as peripheral cells do, but not neurons. After a meal, during food absorption, the brain can just get on with its normal insulin-induced physiological function, which is the facilitation of calorie storage in adipose (and other) tissues. Until they are full.
I'll make that clear: Satiety occurs when the brain senses that calories are no longer being accepted by the peripheral tissues using an ROS signal. Superoxide will be that signal.
That's what I consider to be the normal physiology.
We can now apply this to the various studies using intranasal insulin.
Peter
Let's begin with individual cells, these are the entities which need to control metabolic substrate availability.
You eat some food. Plasma glucose and chylomicrons/FFAs rise, delivering energy to peripheral tissues. In the early stages of food absorption both glucose and FFAs enter cells under the facilitation of insulin. They do this easily, the cells are "hungry".
As an individual cell becomes replete it has to signal that it doesn't want any more metabolic substrate. This is achieved via the CoQ couple acting as the master sensor for metabolic energy status. It is being reduced using NADH from the cytoplasm (the glycerol-3-phosphate shuttle), by FADH2 input via complex II (acetyl-CoA in the TCA) and via FADH2 input from ETFdh (from beta oxidation of saturated fats). And of course from mitochondrial NADH via complex I. Given a high delta psi (ie minimal consumption of the proton motive force because ATP is already plentiful) this CoQ reduction eventually facilitates RET through complex I to give superoxide generation in order to stop insulin signalling. Which then limits cellular caloric ingress. This can be thought of as the "cellular satiety" signal. It is ROS generated. Let's say that again:
Satiety in peripheral cells is an ROS signal. It is generated in the mitochondria. This is pure Protons.
Now let's scale that up.
As more and more peripheral cells decide that they no longer need to respond to insulin then there is less and less of a "sump" available for absorbed calories to drop in to. The availability of calories which no longer have anywhere to go is the whole-body driver of the need to signal satiety. This surfeit of calories will be sensed in the VMH and the cessation of eating will be ROS mediated.
These people have the correct sort of idea:
Fuel utilization by hypothalamic neurons: roles for ROS
and so do these
Role for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake
I'll make that clear: Satiety occurs when the brain senses that calories are no longer being accepted by the peripheral tissues using an ROS signal. Superoxide will be that signal.
That's what I consider to be the normal physiology.
We can now apply this to the various studies using intranasal insulin.
Peter
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