Life (32) On the correct side of the fence

Preamble. On page 143 of Transformer (I'm assuming everyone has a copy of Transformer) Nick Lane writes:

"The deepest requirement for the proton-motive force might therefore be CO2 fixation. The prime example is the "energy converting hydrogenase" or Ech. This membrane protein has four iron-nickle-sulphur clusters, which transfer electrons from H2 to ferredoxin. Two of the clusters sit right next to a proton channel in the membrane, and their properties depend on proton binding, which is to say, the local pH. So when Ech binds protons, it can accept electrons from H2 (in the jargon, it is more easily reduced). And when the protons detach, Ech becomes more reactive, and can now force its electrons on to ferredoxin, which in turn pushes them on to CO2. Then the incoming protons bind Ech again, and the cycle repeats itself."

Which takes me back to my thoughts on pH differential driving prebiotic chemistry here:

Life (22) FeNi hydrogenase

which contains a serious logical flaw regarding the location of the site for pre-biotic carbon dioxide reduction:

In this diagram organic synthesis is happening on the acidic (pH 6), oceanic side of the FeS barrier. From where it would simply wash away in to the rest of the ocean. Metabolism actually happens on the alkaline, vent side in a constrained cell-like structure with walls made up of iron sulphide mineral.

So I've had to rethink the whole basic process with some ideas triggered by various lines in Transformer. Here we go.

The reaction which summarises the origin of life is

CO2 + H2 -> HCOOH

After formic acid formation the generation of the core building blocks of metabolism is, energetically, all down hill. I think it was Nick Lane who described this as a "free lunch you're paid to eat".

Fascinatingly, you could mix hydrogen with carbon dioxide in a jar and, even if you watched it for 4 billion years, nothing would happen. The problem is that the initial step of the conversion, which is this:

CO2 + H2 -> CO + H2O

is not a free lunch. It requires energy, it is a lack of activation energy which stops the reaction occurring spontaneously. Note it is not remotely as simple as written, the water on the right hand end just there to balance the equation, see below.

It *will* occur spontaneously if the CO2 is held at pH 6 and the H2 is held at pH10, in the presence of an iron-sulphur catalyst. An iron/nickel sulphur catalyst works even better but simple FeS seems to do the job.

So this is a crystal of iron sulphide, part of the wall of a tubule structure in a "white non-smoker" alkaline hydrothermal vent. Imagine it's a few billion atoms thick and many billions of atoms long.

In some areas of the tubule wall vent fluid is trying to pass outwards and ocean fluid is trying to pass inwards. I've depicted an interface between two flow areas as a wavy line:

To make life simpler I've now ignored the billions of other FeS layers to just show the layer adjacent to the inner side of the vent tubule. I've put in the pHs of the fluids too.

The two FeS clusters at the interface are at markedly differing pH conditions.

The red part of the intrinsically catalytic FeS surface can split a molecule of vent derived H2 in to two protons and two electrons. The protons react with hydroxyl ions in the alkaline vent fluid while the electrons hop "down hill" to the acidic FeS part of the surface in blue.

This, ephemerally, provides a negatively charged FeS surface. The electrons are clearly destined to eventually react with protons from the acidic fluid but Nick Lane explains in great detail how that process can be indirect, via CO2 interacting with the charged surface, to allow the proton and electron to recombine as an hydrogen, but this time as part of a hydrocarbon rather than as hydrogen gas. He's not particularly forthcoming on the origin of the charged surface, hence my above doodles to suggest how it might plausibly develop.

Lane goes through the conversion of CO2 to acetate at a charged surface in a step by step guide. The title of the section is "Magic surfaces" and it runs from p133 to 140 of Transformer. I've yet to find a better description. This is a wholely abiotic process. My limited summary of the "difficult" initial step is like this:

The process is laid out in Transformer, organics can be formed on the alkaline side of an FeS permeable barrier.

Nick Lane addresses the formation of this set up on page 146. He has shown that protons cross FeS barriers very easily and hydroxyl ions do so only slowly, facilitating the situation I've outline above. Nice. Electrons move over the surface of the FeS barrier briefly before combining with CO2, protons travel through the FeS barrier to provide the driving pH differential.

I've taken these concepts and overlain them on a cartoon of the power module from the membrane bound hydrogenase (MBH) of pyrococcus furiosus (I have a better cartoon for MBH than I do for Ech, they share a functionally homologous power unit so this part is interchangeable). This is Fig4 A from

Structure of an Ancient Respiratory System

which I doodled on 2019 like this:

and which I can now overlay the origin of organic synthesis scenario from my above doodles (rearranged slightly and new colour scheme) to give this

If we blank out the enzyme we can compare the two electron/proton pathways one above the other. Oh, and I've put in the Ni atom which pyrococcus uses in its hydrogenase like this:

The enzyme just needs to recapitulate the pH differential at the active site to allow the reaction to proceed. Quite how you get from this pH driven abiotic process to the same process embedded in a massive and complex enzyme system is not important here, what matters is that the basic core is clearly preserved. Getting the system to run in reverse and coupling it to an antiporter as a pump is another story.

Just in case I haven't mentioned it, the reason that the power units of Ech and MBH are so important is that they are directly homologous to the power unit of complex I and the multiple other related membrane pumps. Which means that the system, clearly derived from the inorganic process at the origin of metabolism, is pretty well ubiquitous.

The next step is to examine how a abiotic system using hydrogen as fuel can function as a proton pump which generates hydrogen as waste and what it might do with any spare electrons. That can wait for another day.

Peter

Dalton Graham

I'm in a bit of a quandary about blogging. I've had to rethink some of my thoughts on the origin of life and the function of the membrane bound hydrogenases which kicked it all off and how they might have lead to superoxide being used as the core growth signal on Earth.

I've also got a number of ideas I want to get written down about the differences between hydrogenated coconut oil driven obesity and linoleic acid driven obesity. Plus I'm about to run out of free time in the very near future so argh...

Anyhoo, Tucker sent me the link to this discussion he and Brian Kerley had with Dalton Graham.

As in

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

discussed in a slightly tangential way here 

Insulin mimesis without NOX enzymes (5) MCT plus linoleic acid

Which gives me about four more  things I'd like to blog about but might not get time for. Especially listen to what chow does to liver fat content in the long term and about unpublishable but highly enlightening observations. Oh, and a shared view of peer review. Oh and what it's like to be junior linoleio-phobe in a conventional lab. Oh, and... Never mind, just give it a listen:

Ep. 10: Dalton Graham: How to Induce Fatty Liver

Peter

Credo

There was a line by karl in a  comment to my last post which triggered a summary of my current thinking about life and signals. A lot of this covers ideas I've had for a long time but not blogged about and, as I can never be sure a post will write itself, here is a thought dump to lay it out without any detail or references. As karl said:

"My hunch is the ROS signal is more primitive than insulin itself?"

In response:

Hi karl,

Superoxide is crucial to bacterial (and archaeal) growth, division and death.

Mitochondria retain this signalling system based on a bacterial ~200mV membrane potential.

The cell surface membrane (archaeal derived) has relinquished its 200mV potential to mitochondria.

Cell surface signalling still uses superoxide but this is now NOX controlled.

It provides the same signal as mitochondrial superoxide but has differing cues and locations.

Larger multicellular organisms use a pancreas to condense/combine these ROS signals in to a redox signal carried by a pair of -S-S- double bonds (ox stress marker, think of as a glutathione G-S-S-G mimetic) between two short peptides to encode and transmit an assessment of whole body redox status from the circulation, via the circulation.

Individual cells respond/resist this signal by making superoxide in response to it then modulate it using their own locally derived NOX/RET superoxide signals.

There is no need for it to be the insulin/insulin receptor combination that carries this signal, any -S-S- di-peptide and an appropriate receptor will do. Most metazoans use insulin/insulin receptor but plants, protozoa and yeasts made different choices but they all do the same job.

These assorted signalling systems cross react across all eukaryotes because the underlying ROS signal is fundamental.

Mitochondrial (bacterial style) ROS from RET in ETC -> mitochondrial division (biogenesis) = Good™.

Cell surface (eukaryote invented) NOX ROS -> cellular division -> tissue growth (+ cancer) = potentially Bad™.

Aside: Bad™ stolen from @KetoCarnivore on X/Twitter. End aside.

That LUCA, even before her division in to bacteria/archaea and while living in an anoxic hydrothermal vent, had a globin ancestor to bind O2, an SOD ancestor (based on Fe) to convert superoxide to H2O2 and a catalase to detoxify H2O2 suggests to me that O2 was available, rare/precious and used as a signal (by accepting an electron -> superoxide) of "membrane" potential (ie opportunity to grow, ie superoxide signal) which had to be processed and terminated at a time even before cell membranes were genetically specified.

Obviously catalase derived O2 would be re-stored by the globin for re-use. LUCA (in a deep anoxic ocean) would have derived a meagre supply of precious O2 from the radiolysis of water, a process still used by deep earth-crust bacteria living at a mile below the nearest O2 supply at the earth's surface, even today.

This is my most simplistic view. Without it I can't understand mice becoming obese eating an "high fat" diet. With it I’m spared the deeper intricacies of intermediary metabolism, a complex system if ever there was one.

Peter

Insulin mimesis without NOX enzymes (6) MCT versus linoleic acid

I have a preference for median lifespan when thinking about the potential effect of an intervention on a population because it is looking at the population as a whole, rather than a few (quirky?) isolated individuals who make up the maximum longevity crowd. Or should I say the maximum longevity chosen few? I guess it confirms my biases too. Which I like.

Let's recap the Jim Johnson's lab findings about the Surwit diet effects described here.

Reduced Circulating Insulin Enhances InsulinSensitivity in Old Mice and Extends Lifespan

I have removed the lowest insulin gene group data and just included those with a normal insulin phenotype. A number metabolic snapshots were taken at around the 80 weeks of age mark. Surwit fed animals were heavier on the scales and fatter by DEXA compared to chow fed:

No one should be surprised that they were also hyperglycaemic and hyperinsulinaemic, ie insulin resistant:

What should be extremely surprising is that these Surwit fed mice while being obese, hyperinsulinaemic, hyperglycaemic and insulin resistant, lived over 100 days longer than their slim chow fed relatives. The data are in here:

which I can simplify down with some very crude curve fitting by eye in Powerpoint to give this:

Interesting.

Now let's look at some rats fed an obesogenic diet based on lard. There are some good data in here although it does not give us the linoleic acid content of the lard:

An isocaloric moderately high-fat diet extends lifespan in male rats and Drosophila

Oh, and their data presentation is not great.

Here we have the weights. Colour scheme is different to the first study

You can't tell if the weight loss toward the end was from the surviving rats eating less or that the fattest rats died earliest. Probably a bit of both. But the lard fed rats were fat.

They were hyperinsulinamic

and they were hyperglycamic.

and obviously they too were insulin resistant.

But this time the longevity curves are reversed and the fat rats die younger than the slim control fed rats, by about 100 days:

So we have the mice in Jim Johnson's lab and the rats in the lab in Harbin, China. Both have comparable levels of obesity and insulin resistance. Both are oxidising FFAs when insulin should be suppressing FFA availability in peripheral tissues. In both cases excess energy is being supplied from fatty acids so there is an absolutely normal physiological reduction/rejection of some of the calories which are being taken up by cells using insulin facilitation.

This normal physiological response is mediated by reverse electron transfer through complex I acting to inhibit insulin signalling at the insulin receptor/substrate level.

In animals made obese using fully hydrogenated coconut oil in a Surwit diet the mitochondria are normal and mitochondrial/cytoplasmic membranes lipids are low in linoleic acid as you would expect from 1-2% LA in the diet. So high-physiological ROS from oxidising fatty acids will inhibit the insulin cascade, as they must, but in the process will only encounter "physiological" levels of linoleic acid and only generate "physiological" levels of 4-HNE, 13-HODE, 9-HODE  etc. These lipoxides, like superoxide, are normal signalling molecules. A low level of generation is normal and generally beneficial. Probably essential.

Generating ROS via RET in the ETC is pro-survival and pro-longevity. See here, discussion on another day.

The obesity induced by increased dietary linoleic acid is different. Here the adipocytes are large because they fail to limit their insulin cascade adequately. Under these conditions adipose tissues will store an excess of lipid from all sources without insulin being elevated, indeed in the earliest stages insulin signalling will have been enhanced and IR subnormal. The core initiating problem being that linoleic acid is present in levels which generate too small an ROS signal, so fail to limit caloric ingress and storage. Also there is good evidence that linoleic acid is preferentially oxidiseg compared to saturated fats.

However excess basal lipolysis secondary to adipocyte size will release all species of FFAs, arguably with some favourites, the problem now is that adipocytes *ignore* insulin. It doesn't matter how insulin sensitised you might have been via LA in order to become obese. If basal lipolysis is up, it's up. And insulin no longer matters. The exact mix of FFAs delivered to the periphery (especially muscle) becomes less important. All cells oxidising any type of fat generate more ROS than cells oxidising glucose.

One of the secondary problems with high linoleic acid diets is that, separate from their obesogenic effects in mitochondria, the LA molecules are also present at increased levels in tissue lipids.

The problems really present when ROS are being generated on a background of an intake of (in this human observational study) of 17% of calories as linoleic acid. Apart from making you obese this provides an environment where ROS which *should* meet a "normal" level of LA (ie derived from 2% in the diet) are actually encountering an environment derived from 17% LA in the diet. Generating *physiological* levels of 4-HNE etc is out of the question when all the ROS can "see" is freely available double bonds to interact with. Lipids are converted to lipoxides in supraphysiological quantities, cellular damage ensues and this chain of redox damage manifests as what we describe as pathology.

That's what I think is happening.

I think I've said enough for one post. I'll have to write a separate post about RET and Superfly,

the fly featured in the paper nominated for the best graphical abstract of all time, ever. By me anyway.

And about ROS and mitochondrial biogenesis.

Peter

Insulin mimesis without NOX enzymes (5) MCT plus linoleic acid

You can become obese by having your adipocytes fail to limit insulin signalling. That is what linoleic acid plus an insulogenic diet does, pure Protons.

You can also become obese by making your liver alone insulin resistant in combination with an insulogenic diet. The Surwit diet approach.

These concepts put us in to a position where we can examine this study, HT to Tucker:

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

In particular we can look at these curves:

Obviously blue circles are the chow and red triangles show the 8% linoleic acid, high overall fat diet. The latter is as obesogenic as you might expect. The surprising curve is the beige 1% LA line. This diet is clearly less obesogenic than the high LA diet but is not exactly slimming when compared to the chow line. And it supplies only 1% of calories from LA.

So do *all* high fat diets make you (and rodents) obese?

The macro of the diets are described in Table 1. I've cut off the vitamin E parts as they are a different subject altogether and might be worth a post some day.

If you come at this from the point of view that saturated fats are slimming and LA is obesogenic then the low LA diet causing a significant level of obesity is a paradox.

In the legend to Table 1 there is a link to supplementary information which I followed, trying to find whether fructose was included in the diets (the paper doesn't actually say) but I did find out that the bulk of the fat in both the 35% fat diets is, as you may have guessed, fully hydrogenated coconut oil.

The 1% low LA diet is a classical Surwit derivative (possibly without the sucrose, I don't think it matters) and is inducing hepatic insulin resistance via ETC derived high levels of ROS to allow penetration of glucose/insulin to peripheral sites. The adipocytes see too much insulin for too much time and get fat.

The 8% high LA diet has exactly the same problem from its MCT components as the 1% LA diet but this time it's compounded by peripheral adipocyte exposure to high levels of LA causing pathological insulin sensitivity, delivered directly via chylomicrons from the gut, bypassing the liver. They get high insulin exposure secondary to MCTs acting in the liver to allow increased glucose/insulin to the systemic circulation PLUS a pathological inability to resist insulin's fattening signal in the periphery via LA, ie adipocyte Protons effect on top of localised hepatic insulin resistance. Double wammy, double weight gain.

Peter

Insulin mimesis without NOX enzymes (4) MCT

Before I get to talk about this (highly recommended) paper:

Ability of high fat diet to induce liver pathology correlates with the level of linoleic acid and Vitamin E in the diet

which came up in a discussion between Tucker and Brad a few months ago, I have to clarify as well as I can how fully hydrogenated coconut oil, one of the most saturated fat sources on the planet, is obesogenic, even under low linoleic acid conditions. The post is already part written but I want to get the MCT mechanism down here as clearly as possible using one of my long term doodles.

Apologies if it's getting repetitive, this is really an executive summary. Here we go.

One basic concept of the Protons hypothesis is that saturated fats shut down insulin signalling when fatty acids are freely available and glucose is scarce. Classically this is under fasting conditions where allowing glucose in to muscles etc would rapidly deplete the body of glucose stores which are helpful to maintain brain function under fasting conditions. It applies whole-body. Like this:

Under high fatty acid availability the ROS derived from reverse electron transfer through complex I of the mitochondrial electron transport chain inhibit insulin signalling at the insulin receptor/receptor substrate level, ie at the cell surface. Simple and obviously adaptive. This is also the system which fails when linoleic acid in bulk is introduced to the diet.

The next concept is that metabolic substrates which do not cause insulin secretion from beta cells will generate their own insulin-mimicking ROS signal using NADPH oxidases, also at the cell surface of nutrient recipient cells. At low levels these ROS inhibit the "insulin signalling suppression system" and allow the insulin signalling to take off, in the absence of insulin per se:

Mostly this happens in the liver because that's where most of the fructose/ethanol/acetate ends up. High doses are a separate story.

Aside: An high concentration of these substrates, ie two cans of cola giving >17mmol/l fructose in the systemic circulation or doing ethanol shots, will generate severe inhibition of the insulin cascade at the insulin receptor/insulin receptor substrate level whole-body, via NOX hyperactivation. And cellular damage from ROS spilling everywhere will result, ie Badness. Back to low level discussions. End aside.

The situation with MCTs is different from both of the above. They too are delivered primarily to the liver, not to the systemic circulation, and taken up rapidly by the mitochondria. All undergo beta oxidation to acetyl-CoA but only some of this acetyl-CoA will enter the Krebs cycle to provide FADH2 and NADH to the electron transport chain. The rest will be diverted to ketone bodies without providing any input to the ETC, and so without generating the inhibitory ROS you would expect from a saturated fat. I consider this is what happens, almost only in hepatocytes:

Low levels of MCTs (in this doodle I've suggested 50micromol/l, this is just for a thought experiment, not data based) arrive at the liver and have absolutely nothing to do with NADPH oxidase enzymes while generating low levels of ROS via mitochondria. These "low" levels are high enough to disable the "insulin signalling inhibitory system" and allow the insulin cascade to be activated but not high enough to directly inhibit the insulin receptor/substrate complex. This activation is insulin "mimesis" without substrate going anywhere near NOXs 1,2, 4 etc. But only in hepatocytes because that's where MCTs are diverted to.

The result is to sequester glucose in to hepatocytes so reduce penetration of glucose past the liver and in to the systemic circulation. The pancreas sees reduced glucose, so secretes less insulin. Lipolysis increases, you might lose weight.

Under Surwit-like diet conditions with coconut oil at 35-60% of calories things are different. Here large amounts of MCTs are delivered to the liver, I've suggested 600micromol/l but who knows what the portal vein concentrations really are? These MCTs still "waste" acetyl-CoA to produce ketones but the increased bulk supply means more acetyl-CoA can enter the Krebs cycle/ETC. Enough to generate ROS at levels which will inhibit activation of the insulin cascade at the insulin receptor/substrate level:

Under conditions of suppressed hepatocyte insulin signalling dietary derived glucose will penetrate past the liver and in to the systemic circulation. This will require the pancreas to crank out more insulin. And so inhibit lipolysis. As in the obesogenic Surwit diet.

That is all for now.

Peter

Insulin mimesis and NOX enzymes (3) MCT

So now it's time to see how it is possible to understand the obesogenic effects of fully hydrogenated coconut oil in Surwit diets and also the recognised potential for MCT induced weight loss effects. Oooh, another paradox!

I've been skirting around this subject for years and I still do not have nice solid answers but this is getting closer.

It's worth thinking, initially, about fructose as a weight loss agent.

Inclusion of low amounts of fructose with an intraduodenal glucose load markedly reduces postprandial hyperglycemia and hyperinsulinemia in the conscious dog

Going back to this canine model of intraduodenal glucose infusion +/- 5% fructose we have these curves:

The fructose supplemented line (open circles) shows a markedly reduced insulin excursion, a direct result of the reduced penetration of glucose past the liver. Fructose, acting solely at the liver, is imitating insulin via ROS from an NADPH oxidase. Glucose gets stored:

The pancreas sees a markedly reduced glucose level, so responds with a markedly reduced insulin secretion (200pmol/l down to 100pmol/l).

If you were an adipocyte in the periphery you are not going to see the fructose acting as insulin via ROS to sequester glucose as hepatic glycogen. This all happens in the liver. All an adipocyte sees is 100pmol/l of insulin instead of 200pmol/l.

What is that going to do to your rate of lipolysis?

I would expect it to be higher in the fructose supplemented state.

Aside. There is s small rise in lactate with the fructose infusion but I doubt it would offset an halving of the insulin level. End aside.

Does this cause weight loss? I don't know that anyone has asked that question in this form for humans using fructose. 

Acetate, which I consider to induce a similar signalling response to fructose, certainly does. Drinking vinegar for weight loss appears to work. The effect is not huge but appears real and is mechanistically logical:

Vinegar intake reduces body weight, body fat mass, and serum triglyceride levels in obese Japanese subjects

So maybe fructose is a weight loss drug, in modest amounts, on a background of a starch/linoleic acid diet.

So if we get back to medium chain triglycerides for weight loss, here they used either coconut oil or MCT oil (both worked):

Coconut oil and medium-chain fatty acids attenuate high-fat diet-induced obesity in mice through increased thermogenesis by activating brown adipose tissue

You can get a marked decrease of weight gain in a (mouse) model of lard-driven obesity by small amounts of MCTs.

Like fructose and to some extent acetate, low doses of MCTs are predominantly diverted directly to the liver via the portal vein from the gut. If they generate modest doses of ROS, ie at insulin mimetic levels, in the liver alone, they will allow the diversion of glucose in to hepatic storage and so reduce penetration of glucose to the systemic circulation and that will reduce the need for insulin secretion. Peripheral adipocytes will see less insulin and so store less, or release more, fatty acids.

To me that makes sense. Increasing hepatic insulin-like signalling derived from very modest MCT ROS generation protects the peripheral adipocytes from glucose/insulin exposure.

Exposure to higher levels of MCTs, ie Surwit-like diets, is undoubtedly obesogenic.

As we increase the proportion of MCTs in the diet this gut-to-liver channel increases delivery giving increased storage of glucose as hepatic glycogen (no problem) and an attempt at hepatic storage of MCT fatty acids. But MCTs aren't stored, they are rapidly oxidised to give ketones plus mitochondrially derived ROS. At high enough exposure there will be enough ROS to finally resist insulin signalling within the hepatocytes.

Hepatic insulin resistance allows more insulin to penetrate past the liver to the systemic circulation and so to reach peripheral adipocytes. It's not essential for MCTs per se to reach those adipocytes in any quantity, though if they do so at insulin mimetic levels they will compound the problem.

Back in March this year I pointed out how mixed coconut MCTs at "physiological" concentration are experimentally confirmed generators of ROS at a level which will phosphorylate Akt in isolated neurons, insulin mimesis. I was also clear that "supra-physiological" exposure to octanoate inhibited correct development of adipocytes, ie caused insulin resistance. These effects applied to hepatocytes, which are the primary target of dietary MCTs, would be quite enough to explain the Surwit effects and weight loss effects, depending on dose.

Summary: Low dose MCTs are insulin-mimetic and primarily delivered to the liver only. They protect peripheral adipocytes from insulin exposure and allow weight loss/limit weight gain.

High dose MCTs provide insulin resistance levels of ROS in hepatocytes and facilitate insulin's penetration to peripheral adipocytes. Any low levels of MCTs reaching peripheral adipocytes will provide low levels of ROS to augment fat storage by insulin per se.

It is perfectly possible to generate obesity with highly saturated MCT based diets. Even at 2% linoleic acid.

Peter

Insulin mimesis and NOX enzymes (2) Lactate

I wasn't going to torture lactate in the usual manner but Alan Couzens on Twitter suggested the two essentials for weight loss were to oxidise lipids and to minimise lactate. I was vaguely aware that lactate is a potent inhibitor of lipolysis and that rapidly rising lactate with exercise is going to tank your fatty acid availability. So I assumed lactate is an insulin mimetic and this is a quick hit searching Pubmed for lactate and lipolysis. GPR81 and HCAR1 are different names for the same receptor:

Lactate inhibits lipolysis in fat cells through activation of an orphan G-protein-coupled receptor, GPR81

which obviously suggests that this might be happening:

However there was nothing here to relate this to NADPH oxidases, so I added some more search terms and got this paper:

Lactate-upregulated NADPH-dependent NOX4 expression via HCAR1/PI3K pathway contributes to ROS-induced osteoarthritis chondrocyte damage

which is more interesting.

Recall that acetate primes NOX4 in neutrophils to be ready to protect you from severe sepsis? By phosphorylating it.

Lactate does something similar and allows me to add this part to the doodle:

This, in chondrocytes, is a positive feedback loop. When they want to to reject insulin signalling, they really will generate toxic levels of ROS, which will ruin your knees. I wonder if this positive feedback is a generic trait too. Okay, I'll leave lactate alone soon.

Interestingly, in people undergoing total knee replacement surgery serum lactate can be as high as 13mmol/l at rest. Even in "normal" controls one individual had a lactate of 9mmol/l at rest. One feature of metabolic syndrome seems to be that you walk around with the lactate levels of a young athlete under exercise while sitting still, even without a cup of sugary tea by your hand...

Fascinating.

Peter

Insulin mimesis and NOX enzymes (1) A principle

This one of my most scribbled upon diagrams. It's how insulin works:

This is the signal, without insulin, generated by mild hyperglycaemia:

and this is the signal generated by "physiological" fructose exposure

You can do exactly the same doodle with ethanol. This paper summarises the low and high dose exposure to ethanol:

Biphasic effects of chronic ethanol exposure on insulin-stimulated glucose uptake in primary cultured rat skeletal muscle cells: role of the Akt pathway and GLUT4

and I hope no one is surprised that it is NOX mediated:

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: a role for angiotensin type 1 receptor

"Ethanol induced systemic and vascular oxidative stress, evidenced by increased ... NAD(P)H oxidase-mediated vascular generation of superoxide anion... "

I also hope I can be forgiven for suggesting that this is the situation. There may be more than one receptor involved so just look at the general picture

The general principle which drops out of all of this is that low concentrations of substrates which enter cells without requiring insulin signalling are set up to provide their own insulin signal, largely through the NADPH oxidases. Low levels of glucose, fructose and ethanol are all insulin mimetics by virtue of their activation of assorted NADPH oxidases to generate an insulin-like ROS signal which eventually activates Akt signalling downstream. They give the impression of being insulin "sensitisers". Really they are mild insulin mimetics.

If pushed to extremes any of these substrates will generate enough ROS to resist insulin signalling. If more ethanol is entering a cell than it needs for metabolic requirements, and that ethanol ingress cannot be "turned off", then the body simply shuts down insulin signalling by the correct amount to compensate and so maintains metabolic stability.

If the system is so overloaded that simply resisting insulin is not enough to normalise caloric ingress then it will generate markedly elevated ROS both from NOX sources and from excess mitochondrial delta psi sources and so ROS mediated damage ensues. This is Badness. 

Addding 5% fructose to an intra duodenal glucose infusion decreases systemic glycaemia and systemic insulinaemia, feeding 70% of calories as fructose doesn't, neither does putting it in the drinking water when fructose makes rodents thirsty yet all they have access to is fructose-water.

Low dose ("physiological") exposure is no problem. High dose is.

All of this leads to acetate.

Acetate enters cells without the assistance of insulin. Sooooo....

Would you expect low doses of acetate to inhibit lipolysis by mimicking insulin? If it is likely to generate its own insulino-mimetic ROS signal? And if it does, would you then expect supra "physiological" exposure to reliably inhibit insulin signalling?

I would.

Short-Chain Fatty Acids Differentially Affect Intracellular Lipolysis in a Human White Adipocyte Model

The paper suggest that this happens

and the the fact that high concentration of acetate enhance lipolysis is a paradox. Of course if you alter the pathway to this one:

then the paradox disappears and you have another logical piece of understanding where excess acetate gives excess ROS and blocks the insulin like effect, leading to increased lipolysis.

But is it true? As far as I have read low levels of acetate are well recognised as insulin sensitising but no one is looking at NOX and ROS.

However this paper suggests I might be on the correct track:

Acetate sensing by GPR43 alarms neutrophils and protects from severe sepsis

How might acetate save your life in sepsis?

"Indeed, GPR43 activation by acetate triggered p47phox S345 phosphorylation [of NADPH oxidase] (Fig. 1d, Supplementary Fig. 1d), thereby confirming that GPR43 activation primes neutrophils."

In my mind I couple the two papers together and I like the inference.

Ketone bodies?

High dose plus hyperglycaemia (aka diabetic ketoacidosis) and NOX4:

Hyperketonemia (acetoacetate) upregulates NADPH oxidase 4 and elevates oxidative stress, ICAM-1, and monocyte adhesivity in endothelial cells

Under more physiological conditions

Activation of G protein-coupled receptors by ketone bodies: Clinical implication of the ketogenic diet in metabolic disorders

Ketones inhibit lipolysis, GRP43 activates NOX, probably NOX4:

"...lipid profile amelioration by KDs could be ascribed to the actions of acetoacetate via GPR43 and of β-OHB via GPR109A on lipolysis..." ie they shut it down.

My world view says that ketone bodies at low concentrations are ROS generators using a signal through a G protein from GRP43 to activate an NADPH oxidase to imitate insulin's ROS activating signal. High levels of ROS do the opposite.

It's a general principle. You know the doodle by now.

Life makes sense.

Peter