Saturday, October 12, 2019

Metformin (11) a SHORT paradox

I'll just throw this one out there as I found it while looking for something else:

Metformin paradoxically worsens insulin resistance in SHORT syndrome

SHORT syndrome is a (very, very rare) genetic failure of insulin signalling at the PIK3 regulatory subunit 1 level. Insulin binds to its receptor but signalling fails due to a single downstream gene defect giving severe insulin resistance. People with this syndrome are, needless to say, very thin. They maintain normoglycaemia using a very high level of insulin which does, given a high enough concentration, produce normoglycaemia. There doesn't appear to be any problem with secreting insulin from the pancreas. In fact, to overcome the failure of insulin signalling during her OGTT the patient's pancreas produced a plasma insulin of 688mIU/l*. In new money that is just under 5000pmol. As in roughly ten times what you might expect. Severe, but not quite insuperable, insulin resistance.

*The paper specifies insulin in mIU/ml. I'm assuming this is a typo or a font failure because clinical insulin concentrations are usually expressed as microIU/ml or mIU/l. Obviously if it really is 688mIU/ml the concentrations will be 1000 times those quoted. Gulp. People really should use the SI system.

Back to the patient. The obvious thing to do is to give an insulin sensitising agent, number one in popularity nowadays being metformin.

This turned out to be a bit of a boo boo.

During an OGTT under metformin the patient's insulin resistance worsened and mild hyperglycaemia ensured but this was despite a plasma insulin concentration which was simply too high to measure. The lab could measure up to around 7000pmol (pax typos) and it looks like the curve went MUCH higher than that.

That is despite metformin's predictable and recently found ability to suppress insulin release from mouse islets.

From the Protons perspective metformin blunts insulin signalling via blockade of mitochondrial glycerol-3-phosphate dehydrogenase. Its beneficial effects to increase insulin sensitivity come from reduced exposure to insulin signalling in peripheral cells. The peripheral cells of a person with SHORT syndrome barely see insulin signalling at all even without the metformin. You would hardly expect further blocking any residual insulin signalling to help matters. It doesn't.

It's the sort of paradox which only happens when you are in the wrong paradigm of metformin's mechanism of action. Might have been a chance to make progress...

Peter

Addendum: The lady in question did not seem to enjoy her experience with the medics too much:

"As we intended to check the effects of this approach, an extended 75 g OGTT was performed on metformin 4 days later. This showed dramatic and paradoxical worsening of insulin tolerance with insulin concentrations above the upper assay detection limit (Fig. 1b). Metformin treatment was discontinued. She was discharged home on Dydrogesterone and vitamin D supplementation. We planned to perform investigations on other family members, and particularly on her younger brother, but despite several reminders they failed to attend clinic appointments as well as declined admission to the hospital".

Friday, October 11, 2019

Ketogenic diets are unhelpful and dangerous for managing mitochondrial diseases. Maybe.

I think this is probably an abstract from a short communication at a conference. I picked it up from Miki Ben-Dor on twitter



I think we can say that ketogenic diets are pretty rubbish for managing mitochondrial illnesses.

By chance there is also a twitter discussion on-going relating to omega 6 based ketogenic diets.

First, nutritionists LOVE omega 6 PUFA and HATE saturated fats. In case anyone hadn't noticed. This tweet came from laura cooper, this time picked up via Raphi and Tucker.







Supported by this



















It seems very likely that we can combine these two concepts and come up with some sort of an explanation.

I think we can accept that the ketone induced metabolic changes noted by Veech in isolated working rat hearts, resulting in increased energy yield per ATP molecule, still apply even with poorly functional mitochondria, because there is comparable improvement in the control of abnormal mitochondria induced intractable epilepsy vs ordinary intractable epilepsy. The ketogenic diet is clearly doing something...

That's good.

Everything else is bad.

If there is a significant problem with the structure/function of complex I ketones will not be directly helpful. They deliver acetyl-CoA to the TCA and essentially nothing else. There will be a small FADH2 input from succinate dehydrogenase but all other electrons will be presented as NADH, which needs a functional complex I to do anything much.

To bypass a poorly functional complex I we really need input as FADH2 directly to the CoQ couple without having to turn the TCA. That means beta oxidation of fatty acids, in particular it needs those fatty acids to be fully saturated because electron transporting flavoprotein only receives electrons to form FADH2 from the first desaturation step at the start of beta oxidation. Any double bonds skip this step.

Using PUFA immediately reduces energy sourced via this route.

The next thing we need to realise that modern nutritionist derived ketogenic diets cause, amongst other things, pancreatitis. I posted about pancreatitis, Intralipid and propofol here. It should come as no surprise that the side effects (from here) of PUFA based ketogenic diets in children can be severe, they're probably a lot higher in PUFA than even F3666 rodent chow...

"Other early-onset complications, in order of frequency, were hypertriglyceridemia, transient hyperuricemia, hypercholesterolemia, various infectious diseases, symptomatic hypoglycemia, hypoproteinemia, hypomagnesemia, repetitive hyponatremia, low concentrations of high-density lipoprotein, lipoid pneumonia due to aspiration, hepatitis, acute pancreatitis, and persistent metabolic acidosis. Late-onset complications also included osteopenia, renal stones, cardiomyopathy, secondary hypocarnitinemia, and iron-deficiency anemia".

Then there are cardiolipins. Each cytochrome C molecule is anchored to the outer surface of the inner mitochondrial membrane by four lipid anchors. Their nature is largely controlled by the dietary lipid supply. Modern PUFA based ketogenic diets will result in highly unsaturated cardiolipin anchors. Damaged mitochondria produce an excess of ROS. ROS break PUFA based cardiolipins giving apoptosis or, if ATP levels are too low for this, necrosis. Not going to do your ragged red muscle fibres any good. Or you cardiomyopathic cardiomyocytes.

I could go on, but you get the flavour.

Is there any end to the damage done by the lipid hypothesis?

Probably not.

Peter

Tuesday, October 01, 2019

Personal update 2019

Okay, personal update time.

Back in the middle of May this year Paul emailed me to let me know that Dr Kwasniewski had died at the age of 82. The possibility of his having had bowel cancer several years ago is apparently nearly impossible to follow up on but it doesn't appear to have been directly related to his passing away. I'd been meaning to post on this but never quite got round to it until Marco also emailed me with the same news last week. The Optimal Diet (OD) has served me well for about 17 years or so.

May was an interesting time for me. For a set of reasons not at all related to my own health I had been tempted to try the scenario of a paleolithic ketogenic diet, much along the lines of the Paleomedicina PKD protocol. I was basically interested in the level of practicality involved before suggesting it to a friend with a "modern-ketosis" resistant neurological problem. The practicalities eventually proved too problematic so the PKD option was never taken up.

I personally never expected that the PKD would change much for me.

I was wrong.

First, I stopped snoring. As far as I am aware, completely. Within a few days. I have a severely deformed nasal septum, probably traumatic in origin (if playing "toss the caber" as a kid with a larch pole, don't throw it straight up in the air in case it comes vertically back downwards directly on to your nose. Ouch). Both nostrils are severely narrowed. I never expected to ever stop snoring.

Second, the low back pain from which I got enormous relief with the OD, went. I've had three minor positional back injuries in five months but each resolution has been incredibly rapid with minimal analgesic needs.

My minor dry skin problems went within a few days, though this coincided with onset of decent access to sunlight in May, the Spring had been cool here in the UK.

Oh, and I dropped from 66kg to 62kg in a month, 11-12% body fat to 9%, estimated by lower body impedance on a set of Tanita home scales.

I carried on with the PKD.

So now I am stuck.

I really enjoy not being awakened by my wife to get me to roll back to sleeping on my side again, sometimes several times a night. I like having no back pain. I like the continued muscle strength development at the bouldering wall.

On the downside it is very socially uncomfortable. It has really brought home to me how utterly easy standard modern ketogenic eating really is. A bit of cooking and a few sweeteners and there is almost nothing you can't have within the diet.

Over the months on PKD I've added in very occasional cheese and a very, very occasional glass of Proseco on a Friday night, without apparent problems. Adding cauliflower or broccoli triggered low back soreness (I have to wonder if this is a nocebo effect, not exactly double blind!).

So nowadays I'm thinking about protein, GH, IGF-1 and insulin. I've always been cautious about protein levels but there are features about higher protein within a solidly ketogenic background that limits IGF-1 generation per unit GH secretion.

There are a number of posts there.

Currently I am, somewhat reluctantly, almost completely plant free. I'm no guru on this way of eating any more than on anything else, plus I'm very late to the party!

Peter

Monday, September 23, 2019

The paradoxical fat mice (2)

This is the intra-peritoneal insulin tolerance test (ITT) result from the mice in

Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin

as mentioned a post or two ago and which needs some sort of an explanation:


















The two asterisks denote that for both of the calorie restricted groups of mice there is an elevated glucose compared to the ad-lib groups in the late part of the ITT, irrespective of whether the insulin gene dose had been reduced by 50% or 75%. Obviously the effect is biologically trivial but the p value of less than 0.05 makes me think the effect is real.

I think to understand this we have to go back some time and look at the concept that metformin has no effect on blood glucose in the absence of insulin. This is the graph from here, discussed here:



My interpretation was/is that, on a background of no metformin (upper line) that insulin (given at 90 mins) generated insulin induced insulin resistance from about sixty minutes later (time 150 mins) and this become p less than 0.05 by 90 minutes after the insulin (time 180 mins), illustrated by the failure to generate insulin-induced insulin resistance in the metformin treated mice (the lower trace).

This is insulin-induced insulin resistance in type 1 diabetic mice revealed by metformin treatment.

Next we can look at type 1 diabetic mice chronically treated with long acting exogenous insulin for a few weeks before an ITT. These have pre existing insulin-induced insulin resistance before any intra-peritoneal short acting insulin is given, taken from here, previously discussed here:















In this case the mice with established insulin resistance simply developed hyperglycaemia when injected with intra-peritoneal short acting insulin. There are no p values but by eyeball the 120 minute value on the upper curve looks like it might be stastically significantly elevated compared to time zero. The message here is that insulin given to an insulin resistance patient can produce hyperglycaemia.

This is the Somogyi Effect. It is real.

In the real world insulin secretion and insulin sensitivity are carefully balanced. Anything which increases insulin secretion increases tissue exposure to insulin and down regulates insulin action. Insulin is the messenger between insulin secretion at the pancreas and insulin response/resistance at the tissues. This is in addition to the shared use of reactive oxygen species to generate both insulin secretion and insulin responsiveness.

We know that the mice with full Ins2 knockout and with or without Ins1 partial knockout are phenotypically normal and have fairly normal insulin levels in their blood.

Aside: To actually get reduced insulin levels Johnson's lab have more recently used a full Ins1 knockout with or without partial Ins2 knockout. The partial Ins2 knockouts do have lowered insulin, are slim, don't get fatty liver and live much longer than they should do. It's all in here. Nice. End aside.

So reduced insulin genotype mice should be more insulin sensitive than full insulin complement mice, though we didn't have a fully normal group in either of the papers from the Johnson lab.

Without calorie restriction (CR) Ins1 partial knockout have their insulin system in balance. With caloric restriction they are so insulin sensitive that when they have an insulogenic calorie restricted small meal they lose calories in to their adipocytes and enter torpor, ie insulin signalling is verging on pathological. So they get fat too. They are not insulin resistant.

But during an ITT they do not merely have the modest insulin levels they might produce in response to their normal small meals. They get 0.75iu/kg of insulin IP, probably more than they have ever seen before. It works. Insulin signalling drops plasma glucose for about 30 minutes. At this point the tissues realise that they are seeing more insulin than they have ever seen in their lives. Insulin-induced insulin resistance kicks in and with it the Somogyi Effect to give elevated glucose.

I think the graph at the top of this page shows an acute onset insulin-induced insulin resistance.

This insulin resistance effect appears to be releasing glucose from the liver, or failing to oppose glucagon action here. It might also have allowed a release of free fatty acids from those greedy adipocytes which precipitated daily torpor. It is just possible that the transient insulin resistant state during the brief ITT might be the only few hours of the entire life of the CR mice that they were not hungry...

Peter

Saturday, September 21, 2019

Ketones in Tehran

Just a one-liner

This is a quite fascinating paper from Iran. Bear in mind that none of the authors appears to be a native english speaker and that they could really have done with some editorial assistance, but the results seem quite significant to me. Seyfried gets a thank you for input to the study design but clearly was not an author.

Feasibility, Safety, and Beneficial Effects of MCT Based Ketogenic Diet for Breast Cancer Treatment: A Randomized Controlled Trial Study

They recruited patients who were deemed to need chemotherapy before breast cancer surgery. Half got chemo alone and the other half got chemo plus 12 weeks on a calorie restricted, MCT based ketogenic diet. They all went to surgery and were followed up post-op for about 30 months.

As far as I can make out 30 people in each group completed the intervention. Each of the cross ticks ("censored") are patients lost to follow up in some way, around 10 in each group.














It looks very much like none of the available for follow up patients in the intervention group died. Forty percent (about ten people?) died in the control group, p=0.04.

This is from a 12 week ketogenic pre-surgery intervention. If a drug had produced this effect it would be a blockbuster. There was no instruction to stay ketogenic after the 12 week trial period finished, though there are hints that at least some of the women did.

Interesting, to say the least.

Peter

Sunday, September 15, 2019

The paradoxical fat mice (1)

This paper is very interesting. I think I picked it up via Raphi on twitter. It comes from Jim Johnson's lab.

Caloric Restriction Paradoxically Increases Adiposity in Mice With Genetically Reduced Insulin

The background is in these two papers:

Phenotypic alterations in insulin-deficient mutant mice

Compensatory Responses in Mice Carrying a Null Mutation for Ins1 or Ins2

The paradoxical mice all had the Ins2 gene fully knocked out and in addition to this some mice also had one allele for the Ins1 gene knocked out (Ins1+/-). So the mice in the study had either a half or a quarter of the normal mouse insulin gene complement. Some mice were fed ad-lib, some were 40% calorie restricted (CR).

The CR, lowest insulin gene group (Ins1+/-) had significantly elevated total fat mass and a significantly elevated percentage of bodyweight as fat. That's a paradox to the insulin hypothesis of obesity and so really interesting. The Ins1+/+ group also had a (ns) increase in percentage body fat but not in absolute fat mass, so the trend is there too, but only a trend.

Metabolically, the split is between ad-lib vs CR groups.

All mice had the same maximal insulin response to a 2g/kg intraperitoneal glucose tolerance test but the CR groups had a very significantly reduced peak and AUC for glucose, ie they were much more insulin sensitive. The intra-peritoneal insulin tolerance test result might be worth a post in its own right, it's paradoxical too but there won't be space to cover it today.

So let's have a look at energy expenditure (EE) from Fig3 C.















To make things a bit clearer I've copy pasted the light period from the left half of the graph on to the end of the dark period to give more of an idea of the EE curves are really like during dark to light transition:









The red line starts horizontally with no significant difference in EE between ad-lib fed mice or CR mice. There is a modest increase during the dark (active) period when the CR mice get their three very small meals, as indicated. After the last meal a precipitous and highly significant fall in EE occurs. The mice enter torpor, a state of extreme lassitude and hypothermia. At around two hours in to the next light period the mice wake up and EE returns to just below that of the ad-lib mice and the cycle repeats. The ad-lib mice behave like normal mice.

The CR mice have a profound hypometabolic period every day. You could argue, if you are a cico-tard, that this is why they store excess fat. They eat all the food they can get but expend relatively little energy so they become fat: CICO. But I would disagree.

Here's my guess as to what is happening. Speculation warning.

We know that the CR mice are exquisitely insulin sensitive. They are that way because they have a low number of insulin genes and they never get enough food to trigger a major insulin spike anyway. The CR is the dominant factor but it needs the genetic background to get the paradox to occur. Insulin-induced insulin resistance, acute or chronic, does not occur due to lifetime low insulin exposure. The fact that all mice are capable of producing the same maximal insulin response to an IPGTT does not mean that the CR group experience an equivalent insulin exposure to the ad-lib group during their routine lives. They never get enough food to trigger a maximal insulin response.

The CR mice spent the bulk of the light period with a slightly low EE. Dark period arrives and with it food. As the food is eaten there is an upward trend in EE followed by a drop. The second small meal arrives, again an upswing followed by a drop. The third and final meal gives the same upswing but the drop in EE which follows just goes on downward. The mice enter torpor, a state of profound lassitude and hypothermia.

I think torpor happens because the mice simply have no accessible calories.

This is despite the fact that it occurs immediately after the third of their calorie restricted meals. Their problem is that the meals generate an insulin response. The mice are so insulin sensitive that calories are lost in to adipocytes (and probably hepatocytes) under the over-effective action of insulin.

They lose calories in to adipocytes. These are calories out. The adipocytes get bigger with the lost fat.

Torpor occurs BECAUSE the mice have become fatter.

This is the equivalent of the hunger which follows for a human under a euglycaemic (or even hyperglycaemic) hyperinsulinaemic clamp. There is no hypoglycaemia but fatty acids become locked in to adipocytes by the hyperisulinaemia and hunger follows due a lack of available calories. I posted about it here.

At two hours in to the light period insulin drops low enough to allow lipolysis. The mice wake up.

That's all.

Except: Why do the CR mice have paradoxically (although ns) elevated fasting insulin cf the ad lib mice? There are two reasons. Here are the blood sample times added to the EE graph. The arrows are not quite in the correct clock times, as detailed in the methods, but the times related to feeding/metabolism are approximately correct.








The green arrow is the sampling time for the ad-lib fed mice. It is about six hours in to the light period and the mice would normally have been asleep during the hours leading up to it. Light-period snacking, from the respiratory exchange ratio (RER) graph in Fig3 D, would not normally have started by this time so it's a very simple physiological fasting sample.

The blue arrow for the fasting CR mice just hits the end of torpor. I'm not sure these mice ever have a time when they wouldn't eat, given the chance, but here they are in their hypometabolic phase and have minimal access to calories. At this time insulin is actually a little (ns) higher than for the ad-lib groups (Fig1 D). Higher insulin means fat stays in adipocytes. Why is insulin high?

Calorie restriction does many things in addition to dropping metabolic rate. If you fast a hard working group of humans for 5 days they develop a post prandial increase in GIP (glucose-dependent insulinotropic hormone). This was found in the CR mice in both the fasting and fed state (Fig4 C). GIP facilitates insulin release, hence insulin is a little higher the CR mice and loss of calories in to adipocytes more severe, necessitating torpor.

It's interesting as to why GIP might be elevated under hunger conditions. Possibly generating and saving fat becomes a priority when calories are low. The Ins1+/- CR mice certainly have the highest RER (>1.05) after their third meal, suggesting that they prioritise the conversion of glucose to fat. This DNL, should it occur in the liver, might go some way to explaining the elevated triglycerides in both of the CR groups. Maybe. Accentuated DNL in people who have undergone massive weight loss via gastric bypass surgery is routine during an OGTT. Like these people.

Anyway, I'll stop now. This post is about 1/4 the length it started out as, so if corners seem a bit cut then mea culpa.

Peter

Thursday, September 05, 2019

Hyperlipid Protons ambassadors

Many people may have noticed that the blog Hyperlipid is not exactly the most user friendly of blogs. The prose clearly appears to make sense but some of the concepts are not always particularly simple unless you have the Protons idea well understood.

Last year (2018) Mike Eades made a sterling presentation which summarised the concept in a talk at Low Carb Down Under in terms that were much more accessible



and Brad Marshall now has a blog on which, throughout 2019, he has been writing around ideas which derive partly from the Protons thread on Hyperlipid. But in significantly more user friendly language, while still being on the spot.

Fire in a Bottle

is his website, a very neat name. It's good. He farms low PUFA pigs too.

Peter

*Very few things in life are quite so disappointing as finding that insulin interacts directly with the NOX4 (NADH oxidase 4) complex to generate the bulk of the initiating low levels of superoxide/H2O2 which trigger insulin signalling. Some ROS do come from the electron transport chain but NOX4 seems rather important. Sigh. Bulk ROS to terminate/blunt insulin signalling do appear to be ETC derived...

Sunday, September 01, 2019

The sweet taste of DMEM

I mentioned in the last post that few papers ever specify the glucose concentration used for cell culture. This came up in comments from Alex and I think it deserves a mention in a post of its own.

The standard methods description for almost all cell culture usually specifies that DMEM was used, along with assorted ancillary chemicals and a source of growth factors, usually foetal bovine serum, but rarely the glucose concentration.

A brief trip to any commercial supplier's website gives you approximately 50 DMEM formulations. Three or four have zero glucose and are intended to allow you to specify your own glucose concentration. Another five or six use the original 1g/l of glucose giving 5mmol/l, ie a physiological glucose concentration. This is described as "low glucose".

The other forty-odd specify 4.5g/l, ie 25mmol/l.

I've never done cell culture but I gather the normal technique is to use 25mmol/l medium, let the cells use the glucose to grow and when the glucose drops toward 10mmol/l then they are re-fed with new medium at 25mmol/l of glucose. This works. For decades.

It also provides you with enormous information about the behaviour of cells under hyperglycaemic conditions.

Obviously, 25mmol/l of glucose in an intact human is pure pathology but it is well tolerated by the cells in culture because there is no other caloric source which inputs as FADH2. No free fatty acids.

At the most basic level a glucose concentration of 25mmol/l should a) never occur at all and b) if it does occur it should promptly trigger, via insulin acting on adipocytes, a fall in FFAs to 100 micromol/l or less.

This graph is from a paper on pancreatic beta cell death. From the open circles it is clear that, with glucose held at physiological concentration of 5mmol/l (G5), palmitate is harmless at up to at least 400micromol/l.



This should surprise no one because a 60 hour fast in a healthy human will provide 2000micromol/l of mixed free fatty acids with a glucose of just under 5mmol/l. And no multiple organ failure.

Compare that with the closed circles where glucose is pathologically high at 20mmol/l. Physiological fasting levels of FFAs then unmask the gross pathology of glucose at 20mmol/l. Typical of ordinary cell culture concentration.

Given the idiotic stand of the cardiologist community against saturated fat and given the apparent safety of pathologically elevated glucose in fat-free or PUFA supplemented cell culture medium it should come as no surprise that very few labs worry about the consequences of their grossly non-physiological DMEM.

Demonstrating the pathology of hyperglycaemia using (and blaming) saturated fatty acids or the converse lack of acute toxicity from PUFA (low FADH2 input) is a route to funding which would surely discourage any questioning of the techniques of cell culture which have been successfully used for decades. As folks say:

"We've always done it that way".




That's all I really wanted to say but here are a couple of odds and ends:

There are groups for whom glucose matters. Even if stuck in the incorrect complex I inhibition paradigm for metformin's action, the concentration of glucose is recognised as important by some.

This group:

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

have developed a system for glucose homeostasis in cell culture

















Their approach is pretty well unique. It is NOT how cell culture is normally done.


The other brief aside while I'm on cell culture from an outsider viewpoint is FBS, foetal bovine serum. This contains essential growth factors. You can pretty well translate it as "insulin" or "IGF-1".

When cell culture shows that metformin works to reduce cancer cell growth, it is working in the presence of insulin signalling (usually combined with hyperglycaemia). Which metformin blocks at the glycerophosphate shuttle level, without the need for a lethal blockade of complex I.

Peter

Wednesday, August 28, 2019

Who gave you pancreatitis?

Background: Propofol is a mainstay anaesthetic induction agent. Its use is associated with occasional pancreatitis episodes. I won't wander aside about the poor dog which was referred for cataract surgery and which did eventually recovered from its perioperative fulminating pancreatitis.

Propofol is dissolved in what is essentially Intralipid, a soybean emulsion used for parenteral nutrition. The lipid emulsion gives a transient hypertriglyceridaemia. Hypertriglyceridaemia from any cause is associated with pancreatitis.

Me, being me, would automatically blame the PUFA in the Intralipid. But then I would.

I came across this paper by accident this morning:

Distinctive roles of unsaturated and saturated fatty acids in hyperlipidemic pancreatitis

It's good. The group even give you the glucose concentration used in their cell culture, 0.2%, ie 200mg/dl or around 10mmol/l. Not normal but hardly seriously pathological.

Aside: Less innocent groups keep quiet about glucose concentration and can reliably show endoplasmic reticulum stress and any other nasty attributable to palmitic acid. With how much glucose??? End aside.

This is what they found:

"Unsaturated fatty acids at high concentrations but not saturated fatty acids induced intra-acinar cell trypsin activation and cell damage and increased PKC expression"

So. If you have a genetic hypertriglyceridaemia, say lipoprotein lipase deficiency, and you get acute necrotising pancreatitis (not fun) there is every possibility that it was induced by the high content of polyunsaturated fatty acids in those triglycerides and the FFAs derived from them.

Which means that your cardiologist put you in the ITU. Avoid saturated fats, replace them with polyunsaturated oils. Thank you Public Health England and your equivalents world wide.

The converse might well be that loss of the gene for lipoprotein lipase, or similar loss, might not have been a big deal when humans lived by eating elephants. Or even until corn oil took off as a cholesterol lowering scam.

Peter

Protons (50) The video

Dave Speijer has a great video up on YouTube based largely around his recent paper in BioEssays.

Can All Major ROS Forming Sites of the Respiratory Chain Be Activated By High FADH2/NADH Ratios?

It's nice to hear the man himself on a subject about which he has spent a great deal of time thinking. Here it is:



Many thanks to Andrew Moore, editor-in-chief at BioEssays and publisher of many of Dr Speijer's papers, for the heads up that this video had been produced.

Peter

Monday, August 19, 2019

Protons (49) Complex III

Dave Speijer, an extremely insightful person if ever there was one, has a new paper out:

Can All Major ROS Forming Sites of the Respiratory Chain Be Activated By High FADH2/NADH Ratios?

the link to which I am extremely grateful to Bob for forwarding to me. This concept is purely from Dr Speijer. But I like it. A lot.

I'll start with an old doodle I produced about a decade ago depicting the front end of the electron transport chain. Matrix is at the top, cytoplasm at the bottom:

















Electrons travel from NADH to Coenzyme Q, reducing it to QH2 which donates them to complex III being oxidised back to Q in the process.

The unlabelled blobs in the diagram are complex II (succinate dehydrogenase), electron transporting flavoprotein dehydrogenase and mitochondrial glycerophosphate dehydrogenase, all of which compete with complex I for CoQ as an electron acceptor. Given a high membrane voltage, a deeply reduced CoQ couple with most of the CoQ as QH2, then reverse electron transport back through complex I gives superoxide/H2O2 generation (provided the mitochondrial NAD pool is also highly reduced so unable to accept electrons (ie little NAD+, lots of NADH).

This is pretty straight forward and is the gist of the Protons thread. The extension of this is that, under high substrate availability, H2O2 from this process stops insulin facilitated caloric ingress to the cell.

Another major site of ROS generation in the ETC is complex III. Dave Speijer would hold that this is also triggered, like RET, by a deeply reduced CoQ couple. This is why.

So here is a stripped out version of the above doodle:

















The problem here is that it's not that simple. Complex III does rather odd things with its two electrons. Electron bifurcation is a standard enzymic technique perfected very early on by proto-biology and it is exactly what happens here. One electron travels to cytochrome C (which only ever carries one electron at a time) and then on to complex IV and O2. The other does not:

















The second electron is transferred backwards to another  CoQ molecule and so partially reduces it to QH*, the radical semiquinone.

















So (oxidised) Q is a necessary electron acceptor for complex III. Under high substrate availability and with most of the CoQ couple present as QH2 there will be very little Q available.

With one electron securely on cytochrome C, with any delay in the availability of Q, the second electron can be left sitting on one of the two haem groups along its route, easily available for donation to O2 to give superoxide, so adding its ROS signal to that of complex I. Both indicate that enough substrate is present and it is time to limit insulin signalling, to limit caloric ingress.

Nothing, absolutely nothing, about the construction of the ETC is random.

The general principle that a highly reduced CoQ couple is a signal to halt caloric ingress in to the cell applies to complex III just as much as to complex I.

When you want to resist insulin, you really want to resist insulin. Superoxide is then your friend.

If you fail to limit caloric ingress then eventually ROS from complex III are ideally placed to break the cardiolipins which anchor the water soluble cytochrome C to the inner mitochondrial membrane. Destroy these anchors and the ROS signal changes from"resist insulin" (good) to "perform apoptosis" (possibly not quite so good)...

And of course ROS in excess of physiological signalling are going to activate all sorts of inflammatory pathways.

Peter

Aside: The process of ROS generation is probably limited by pairing of complex IIIs (complex III is always a dimer in-vivo) for cooperation to use one Q to accept an electron from each of the pair. This should limit ROS generation as one Q will be available to two complex IIIs, twice that avaiable if they were each working alone. Nothing is random. End aside.


















Tuesday, July 23, 2019

On belief structures in lipidology (2) KODA-CP

Altavista put up an excellent link to an article describing an AI algorithm capable of mining pubmed abstracts and coming up with gold. The exact opposite of modern meta-analysis (repeat after me: the meta-analysis of dross is dross). And the AI algorithm could use old data to predict recent discoveries! Sadly, the university I graduated from is currently teaching final year vet students that any publication over five years old is unimportant/can be ignored. The end of science. Alt was prompted to share his gem following this link put up by Hap:

C‐reactive protein promotes atherosclerosis by increasing LDL transcytosis across endothelial cells

Which is worth thinking about (and ignoring). Unless anyone thinks that LDL transcytosis across human umbilical vein derived endothelial cells (HUVECs) in culture resembles the process of arteriosclerosis, I think we can safely ignore this aspect of the modelling in the paper. Just ask yourself how severe is venous arteriosclerosis, with or without C-reactive protein. And the cells in the model die if you expose them to anything greater than 35mg/ld LDLc! But it brings up the apoE-/- mouse, a truly fascinating subject.

The apoE-/- mouse is also a model. They develop hyperlipidaemia and rapidly progressive "arteriosclerosis". Obviously, the apoE-/- lipid particles, which lack their apoE attachment protein, work their way (using active transcytosis of course) through the endothelium of blood vessels and cause cholesterol to accumulate in the subendothelial space and... Well. It's a pretty neat evolutionary dead end, if you believe it.

The apoE-/- model tells us more than anyone could ever want to know about apoE-/- mice. I've just spent some considerable time on Pubmed and SciHub trying to find out if there is any sort of full blown apoE-/- syndrome in humans. Not apoE2/E2 etc, more like no apoE at all. Zero. Zilch. Like the mice.

No luck finding it so far.

EDIT Yay, Adam found it

Effects of the absence of apolipoprotein e on lipoproteins, neurocognitive function, and retinal function

No suggestion of CVD. Just shows how important adding over 1% by weight of dried (oxidised) egg yolk to the diet to generate the "model" might be!

END EDIT



But apoE-/- mice are interesting in their own right. They really do accumulate lipids on their arterial linings in a manner exuberant enough to make a lipidologist wet their knickers. None of this messing about with the non-lipid intimal thickening so characteristic of real human arteriosclerosis. Lipids, lots of them, just "invade" the arterial walls and stick. Right there, pretty well on the surface.

But.

Every now and then you trip over an interesting paper, in this case about why apoE-/- mice really have vascular problems. Here's one:

TLR2 Plays a Key Role in Platelet Hyperreactivity and Accelerated Thrombosis Associated with Hyperlipidemia

The paper is long and complex and very, very clever. As per usual. And less than five years old.

Here is the scene-setter from the discussion

"Patients with enhanced platelet reactivity are at increased risk for cardiovascular events.4, 37–39 Enhanced platelet reactivity is associated with chronic and acute inflammation, infections, diabetes, and a number of pathophysiological states related to dyslipidemia, including atherosclerosis, diabetes, and metabolic syndrome". My italics.

The mechanism appears to be through CD36, a multifunctional scavenger-type receptor present on most cells but here they are looking at platelets:

"Previously we have linked platelet hyperreactivity in dyslipidemia to accumulation in circulation of specific oxidized phospholipids, oxPC-CD36, which activate platelets via the scavenger receptor CD36"

The "previously" citation is to:

Phosphoproteomic Analysis of Platelets Activated by Pro-Thrombotic Oxidized Phospholipids and Thrombin

which introduces us to KODA-CP, or to put it more elegantly 9-keto-12-oxo-10-dodecenoic-phosphatidyl choline. This was just one of the more effective CD36 activators of the many lipid products present in oxidised lipoproteins.


So. Hyperlipidaemia facilitates the generation of KODA-CP which activates CD36, which activates TLR2, which makes platelets super sticky.

KODA-CP must have linoleic acid or arachidonic acid as part of its parent molecule.

The platelets stick. In apoE-/- mice enough of them stick to form massive aggregates on the arterial surface that look a bit like late stage lipid infiltrated arteriosclerosis plaques. It's a model.

BTW, platelets carry apoB labeled lipoproteins, among the many physiologically appropriate contents of their cytoplasmic granules (link below). Under the more normal generation of arterial intimal hyperplasia which precedes pathology I consider this lipid will simply be used for normal repair/hyperplasia processes. But there is nothing physiological about apoE-/- mice. They look like they should stick a ton of platelets to any damaged vascular wall, with more apoB labels than any-(mouse)-body knows what to do with. Given enough omega 6 PUFA to generate the KODA-CP.

Apart from the cardiologist derived omega 6 PUFA (another link below), did you notice the core involvement of the CD36 receptor? CD36 also facilitates free fatty acid uptake in to many cells. It is stored within cells and translocates to the cell surface, a bit like GLUT4 proteins, when needed. Stored in the cell, translocated when needed.

What controls CD36 translocation to the cell surface?

Insulin, of course (another post there).

Just thought you might like to know.

Peter

Let's just summarise. Lacking the apoE protein limits the utilisation of lipoproteins, much as having a fully non functional LDL receptor does in familial hypercholesterolaemia. This increases the concentration of lipoprotein particles in the circulation.

Applying the Dunning-Kruger effect to lipidology: Lots of LDLc particles = lots of invasion. QED. That's been it for the last 50 years. I don't thing many lipidologists every get past this obvious, unarguable, simple fallacy. Oh, also core to lipid "therapy" has been, and still is, giving corn oil to lower the LDLc count.

In reality the elevated lipoproteins are a marker of reduced utilisation and are associated with an increased residency time in the circulation. Given lipids based on palmitic, stearic or oleic acids I don't think that would matter.

Given lipids filled with linoleic acid, the essential precursor of KODA-CP, you will get a progressive rise in KODA-CP associated with increasing persistence of the lipoproteins. The more KODA-CP the more activation of platelets via CD36/TLR2 (and undoubtedly other pathways) and the stickier the platelets become.

Given the pathological intake of linoleic acid promoted by cardiologists and lipidologists working under their cholesterophobic hypothesis it seems perfectly possible that seed oils (and insulin) may well be drivers of the platelet adhesion which is core to the vascular damage in apoE-/- mice. Platelets even carry apoB100 labeled lipoproteins in their cytoplasmic granules which allows us to immuno-stain lipid accumulations with this LDLc implicating flag.

Given lipoproteins which lack apoE on their surface, accumulation of KODA-CP, hyperreactive platelets and a surfeit of insulin we are in a position to understand how the apoE-/- mouse works. Which is cool for those of us who like to understand things.

How much of this applies to actual human arteriosclerosis? Increasing platelet stickiness will amplify the normal response to arterial injury. I think this may be real. The rest is just a very extreme, rather bad model.

Most models, like this one, are usually useless.

Is it conceivable that cardiological dietary advice represents the exact opposite of the correct approach? That it would actively worsen the problem it is aiming to ameliorate?

Yep. But we knew that anyway.

Increasing linoleic acid in the diet is undoubtedly a facilitator of the generation of KODA-CP and the activation of the subsequent cascade goes a long way to explain the Sydney Diet Heart Study and the Minnesota Coronary Experiment. People died. From corn oil.

I'll stop now.



Some helpful links that didn't integrate neatly in to the text.

Effects of saturated and polyunsaturated fat diets on the chemical composition and metabolism of low density lipoproteins in man (1980, written on papyrus)

Apolipoprotein B release from activated human platelets (1986, probably on parchment, safe to ignore).

Monday, June 24, 2019

On belief structures in lipidology

Dr Thomas "Just-take-the-statin" Dayspring writes on twitter:

"Any apoB-lipoprotein less than 70 nm in diameter can pass be pass thru endothelium - The LDLs are 20.5-25 nm. Remnants and IDLs are less than 70 nm and greater than 30 nm. The term small, dense LDL is way too simplistic - Big LDLs, like small LDs (less than 20.5 nm) if present in excess can invade artery"

This statement makes a prediction. It predicts that the accumulation of lipid in arteriosclerosis will be, initially, sub-endothelial.

As in this review of transcytosis (because passive "leakage" of LDLc down a concentration gradient across an endothelial cell layer is laughably impossible for particles over 6 nm across. No, that 6 nm is not a typo, according to the review). Not that "impossible" means anything to a lipidologist.

"During the initial stages of atherosclerosis, LDL particles are transported [transcytosed] across the EC [endothelial cell] barrier and accumulate in the subendothelial space".

So. All we need to do is a few post mortem examinations, find some poor people who had early arteriosclerosis at the time they died, and look for that lipid which will be sitting neatly under that single layer of endothelial cells lining their arteries. That is the prediction embedded in Dayspring's tweet.

If we go to this paper:

Early Human AtherosclerosisAccumulation of Lipid and Proteoglycans in Intimal Thickenings Followed by Macrophage Infiltration

EDIT: from a link in Subbotin's excellent "Excessive intimal hyperplasia in human coronary arteries before intimal lipid depositions is the initiation of coronary atherosclerosis and constitutes a therapeutic target" END EDIT

we can find real images of real arteries from real people who died of non related causes while carrying different levels of arteriosclerosis:























Left side images are van Giesen stained for histology, central images are with Sudan IV for lipid and right hand are immunostained with anti-CD68 antibodies to show macrophages. The pairs of small black arrowheads indicate the level where the intima stops and the outer media layer begins. The vascular endothelium is a single cell layer at the top of each image.

Let's look at the circled image in detail. This is an example of early atherosclerosis from a real human with real early changes who died of non related causes. It's just the sort of place you might hope to catch an LDLc particle creeping between the cells of a single endothelial layer or freshly spat out after transcytosis by an endothelial cell. Lipid stains bright red:


















Well, there's the lipid, deep, deep down at the junction of the intima and the media, right between the arrow heads...

There is none anywhere near the endothelial cell layer. If you believe that LDLc, as a result of a concentration gradient between artery and sub endothelial layers, "moves" or "invades" across that endothelial cell layer you have to explain how there is none at all in the sub-endothelial area and there is a progressive accumulation at the intima-muscularis junction. How does the lipid get from the top of the image to the deep spaces without any of it showing up in the lipid-free zone between the two?

"Beam me down, Scotty" is undoubtedly the most plausible explanation.

It is very, very hard to explain how utterly disreputable the lipid hypothesis is. All of this angst about increased LDLc and/or apoB counts on LC diets is based on the assumption that somewhere, somehow, cholesterol is the cause of heart disease. How LDLc "invades" (by active and controlled transcytosis!) the sub-endothelial space, disappears from there and then suddenly appears at over 200micrometres deeper, with none showing in the intervening zone requires a belief tenet which bears no resemblance to reality...

This was bollocks in the 1950s. My question is, as always, at what time did it stop being bollocks?

No one would reasonably doubt that the lipid deep down at the intima/media junction level comes from lipoproteins (though there are other plausible explanations). No one would doubt that loading the lipoproteins with with linoleic acid is likely to be a Bad Thing. No one would doubt that generating oxidative derivatives of the lipids in those lipoproteins might be a Bad Thing.

But trans-endothelial "invasion" is beyond belief.

This would suggest that all lipidologists are talking crap.

Nothing new there then.

Peter

Tuesday, May 28, 2019

Metform (11) metformin vs mtG3Pdh knockout

This paper is interesting (and badly written):

A high carbohydrate diet does not induce hyperglycaemia in a mitochondrial glycerol-3-phosphate dehydrogenase-deficient mouse

It uses a mtG3Pdh knockout mouse, which is essentially a mouse which behaves as if it was on an enormous dose of metformin without all of that toxic blockade of complex I which gives a potentially lethal lactic acidosis at high dose rates. If you feed these mice standard crapinabag they are phenotypically normal. If you feed them a diet consisting some casein, a little PUFA to avoid EFA deficiency and the rest of the calories from pure sucrose they become rather interesting.

Eating pure sucrose does not make normal mice fat. It does make them insulin resistant and hyperinsulinaemic and, of course, insulin resistant adipocytes refuse to retain fat unless insulin action is facilitated by the oxidation of PUFA. Hence the normal body weight.

But the knockout mice actually become slim on sucrose. Here are the data, we can ignore the heterozygous (HET) groups:








They are slim because insulin levels are low. From the Protons perspective the function of the glycerophosphate shuttle in the pancreas is to drive enough reverse electron transport through complex I to trigger insulin release. Less RET, less insulin release, less fat storage, less hunger. Here is the isolated response of the perfused pancreas model to hyperglycaemia:























First phase insulin release is about a third of that in the normal mice. The mice are not diabetic because, in the absence of the glycerophosphate shuttle, RET to allow insulin signalling is generated by beta oxidation supplying electron transfering flavoprotein the CoQ couple via mtETFdh. Insulin signalling still happens but at the "cost" of increased lipid oxidation in the peripheral tissues.

What doesn't happen is sucrose induced insulin resistance. Again I consider this is triggered via the glycerophosphate shuttle causing RET at a level to shut down insulin signalling, which simply doesn't happen in the knockout mice. Lack of glycerophosphate shuttle also stops the generation of insulin-induced insulin resistance under conditions of high insulin concentrations coupled with energy replete cells.

Does anyone recall this figure from Metformin (01) post back in 2017?



Insulin was given at 90 minutes. At 150 minutes in the upper (non metformin-ed) rats insulin action starts to fail, at about the correct time for insulin-induced insulin resistance. By 180 minutes that upper trace, the non-metformin group, shows an upward trend in glucose as exogenous insulin levels are no longer high enough to overcome insulin-induced insulin resistance (the rats are DM T1 under insulin withdrawal).

At 180 minutes in the lower line showing metformin treated rats we can see the continued action of insulin being facilitated by the metformin because it blocks insulin-induced insulin resistance. It was mention in the comments to the post that, clinically, this effect of metformin might worsen the possibility of hypos in humans if combined with exogenous insulin usage. Potentially fatal hypos.

So what happens if you inject a sucrose treated mtG3Pdh knockout mouse with exogenous insulin to check their insulin sensitivity? Insulin sensitivity is preserved, to fatal effect:






















All of the mice with the mtG3Pdh knockout died under exogenous insulin. This is exactly how I would expect metformin to behave in humans using insulin. A functional glycerophosphate shuttle allowed a sucrose diet to block this fatal sensitivity to exogenous insulin.

Obviously the mtG3Pdh mice have a normal complex I. Might they still develop lactic acidosis? Sadly the group didn't look at this (they had no idea back in 2003 that they had developed a meformin mimic model mouse). I do think there might be some elevation of systemic lactate despite a normal complex I.

In the absence of the glycerophosphate shuttle glycolysis is going to run directly to lactate to maintain redox balance. If glycolysis proceeds at a rate in excess of oxidation of the lactate within mitochondria (recall oxphos is slow compared to glycolysis) then some glycolytic lactate will spill outwards, though this is never likely to reach ICU-needing levels. No need to have a complex I blockade to generate mild lactic acidosis.

Does this metformin-ed like mouse have the exercise gains seen in human cyclists after popping 500mg of metformin pre-race?

That requires that we look at a different model.

Peter

Wednesday, April 17, 2019

Life (31) Chinese whispers

Back in 2008 Noha Mesbha published her excellent PhD thesis

ANAEROBIC HALOPHILIC ALKALITHERMOPHILES: DIVERSITY AND PHYSIOLOGICAL ADAPTATIONS TO MULTIPLE EXTREME CONDITIONS

which introduced the world, via this paper

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

to Natranaerobius thermophilus and its antiporter nt-Nha. Which gives every impression of being a stand alone Na/H+ antiporter very closely related to the invariably operon controlled MrpA protein (named shaA) of Clostridium tetani. As she says

"Gene nt-Nha had 35% identity to the shaA (mrpA) gene of Clostridium tetani. The Mrp proteins belong to the monovalent cation/proton antiporter-3 protein family. ...Sequence analysis of the regions surrounding gene nt-Nha, however, did not show that it was part of an operon. This indicates that gene nt-Nha does not encode a subunit of an Mrp system, but rather a mono-subunit antiporter".

All well and good.


Then in 2010 Morino et al published

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

Although the whole paper was about B subtilis (and how none of its Mrp subunits worked in any incomplete combination to antiport anything) they did make this throw-away comment:

"A MrpA/MrpD homologue encoded by a “stand alone” gene from polyextremophilic Natranaerobius thermophilus was recently reported to exhibit Na+/H+ and K+/H+antiport activity in anaerobically grown E. coli KNabc (24)"

where (24) is Mesbha's PhD paper. Notice that at this stage Mesbha's

nt-Nha ~ shaA, very closely related at 35% conserved gene sequence,

has been changed by Morino in to

nt-Nha ~ An "MrpA/MrpD homologue".

This is a just about acceptable per se because MrpA and MrpD are homologous to each other and nt-Nha is closely related to MrpA (shaA) of C tetani. But Mesbha herself never mentions MrpD in her 2009 paper or in her PhD thesis. And "MrpA/MrpD" is open to mis-interpretation. So we have "definition-creep" here, where nt-Nha could be accidentally seen as some sort of composite of MrpA in combination with MrpD. Ouch.

So next we have the 2017 offering by Jasso-Chávez et al

Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans

where we have this bizarre statement

"On the other hand, a fused MrpA-MrpD homolog in the alkaliphilic Natranaerobius thermophilus displayed Na+/H+ antiport activity when produced in E. coli strain KNabc (5, 28)"

Ref (5) is Morino's paper on B subtilis Mrp, in which one rather misleading citation suggests that nt-Nha is an "MrpA/MrpD homologue". This has developed to the extent that nt-Nha has now "become" a fusion of two genes to give a rather mythical monster.

Ref (28) is just Mesbha's PhD paper in which nothing of the sort was suggested.

So the Jasso-Chávez paper is utterly flawed due to misinterpreting a poorly phrased statement and adding an erroneous modification so as to grossly mis-represent an initial very solid finding by Mesbha. The Jasso-Chávez discussion of nt-Nha can be distilled as:

"Send three and fourpence, we're going to a dance".

The chance of their understanding how nt-Nha or their very own archaeal MrpA subunit work as a stand-alone antiporter appears to be approximately zero.

Very sad.

Peter

BTW The folks who worked from the actual gene to model the nt-Nha protein structure suggest that

"The final model presents 13 transmembrane α-helices organized in a similar arrangement as the NuoL subunit".

You know the picture but here it is again 'cos I think it's lovely






Sunday, April 14, 2019

To the gym?

This press release in advance of a poster presentation is doing the rounds on social media at the moment:

Ability to lift weights quickly can mean a longer life

A factual association is probably true between being above the median in ability to do work on a specifically selected gym machine and longevity.

What does this mean? It's observational. Hypothesis generation only.

Perhaps non-athletes with higher power output might live longer because they have excellent mitochondria and our mitochondria are essentially what determine our longevity. Having good mitochondria might well mean that you exercise spontaneously without describing yourself as an athlete. As in I might carry a couple of sacks of chicken food myself rather than wait for Paul the yard-man and his barrow. But this is an effect, not a cause.

Non-athletes with low power output may be the converse. They have mitochondria in which the cardiolipins anchoring their cytochrome-C to the mitochondrial inner membrane are as flimsy as a PUFA in a deep fat fryer, which are willing to trigger apoptosis at the drop of a superoxide molecule. Poor mitochondria, less muscle fibres giving sarcopaenia, shorter longevity. Probably already giving diabetes in-situ. An AHA poster-child.

Taking someone from the second category and making them exercise might convert them to being healthy with a long life span. Maybe.

Or, there again, it might make the gym sessions so unbearable that they quit.

Or, if you don't let them quit, it might simply make no difference.

Or, if you don't let them quit, it might kill them sooner!

As Eeyore said "think of all the possibilities, Piglet, before you settle down to enjoy yourselves".

The study (as per press release) tests nothing. Researchers are giving very specific advice based on an untested observation which might be going to make them look very, very stupid when the results of an intervention to test their hypothesis actually refutes it. But that will take decades and hopefully the researches will be retired by then. More likely it will be ignored or termed a paradox. All the possibilities Piglet.......

EDIT or the intervention might show benefit. That's not impossible! END EDIT

Peter

Declaration: I have nothing against exercise. Nowadays I mostly boulder because it is three dimensional problem solving using muscle groups to failure on a regular basis. Plus I also try to keep my cardiolipins as saturated as practical!

Life (30) Guesses about Na+ channels

Complex I exists in various states, the two main ones being activated and deactivated. The deactivated form is convincingly a Na+/H+ antiporter. There is a pretty good case made in this paper, which has a number of flaws but is generally probably correct:

The deactive form of respiratory complex I from mammalian mitochondria is a Na+/H+ antiporter

As always, the suspect antiporter is thought to be NuoL, the distal "antiporter-like" subunit. You can see the logic for this which is supported by the stand-alone antiporting homologues of NuoL seen in nt-Nha and at least one archaeal MrpA subunit. Personally I'm not sure this is the case. The phenomenal difficulty in trying to interpret exactly what is happening in a structure as intricate and as minute as complex I allows many views of the available data.

It also appears to be the case that in bacteria which use menaquinone as their electron acceptor (rather than CoQ) some degree of Na+/H+ antiporting occurs under normal active NADH oxidation/proton pumping. That's in here

Respiratory complex I: A dual relation with H(+) and Na(+)?

and here's the energetics doodle from the same Fig 2 as I pinched the NADH:ubiquinone doodle from in the last post:













Because transferring two electrons from NADH to menaquinone only provides 480mV/2e- the complex uses Na+ moving down its concentration gradient to "top up" energy availability and so get the extra energy needed to pump the full four protons.

Aside: The paper has lots of good ideas but they are very wedded to the concept that NuoL, M and N are still antiporters and that loss of "control" by the redox cytoplasmic arm allows this antiporting to re establish. It's a very reasonable idea but I think it can be improved upon, especially now we have more detailed information about the Na+ pumping of the P furiosus MBH, where this is not what has happened. End aside.

Why on earth should it matter whether complex I pumps the full complement of four protons? If there is only enough Gibbs free energy for two or three protons, why not just pump two or three protons?

What occurred to me is that for complex I to pump the four protons it might be necessary to have a full "priming" of the membrane arm with enough Gibbs free energy for a full "push to the left", as in this doodle, discussed in a previous post:























What if you only have 480mV/2e- available, giving a half hearted "nudge" to the left when you need the full shove from 840mV/2e-? Is it possible that, under these circumstances, nothing at all happens? There is no flip of the glutamate/lysine pairs from together to apart, which triggers all of the opening/closing of water channels that allows proton translocation? Zero proton translocation?

If you wanted to restore the full "kick to the left" it might be a reasonable energetic top-up to supply the extra energy from Na+ ingress up near the Q binding site to allow the menaquinone plus Na+ ingress to generate the full Gibbs free energy for activation of the membrane arm. That needs a trans-membrane channel, so we are looking at the really complicated region around NuoH and NuoA/J/K. Not easy.

The best characterised Na+ channel in the whole related set of the complex I, MRP and MBH systems is the one in the MBH of P furiosus, described by Yu et al in

Structure of an Ancient Respiratory System

which gives the Na+ channel looking like























with the red blobs being the modelled Na+ binding sites. This is made up of proteins from four separate genes and is thought to be homologous to the Na+ channel of the MRP antiporter, also a multigene structure. They show them as identical in their discussion doodles, like this for the MRP multigene Na+ channel, simplified to the subunit shown as MrpG:














The equally multigene Na+ channel of the MBH of P furiosus (in this doodle the multiple Mbh genes are simplified to MbhC) is shown in exactly the same location with the same critical broken helix, in the same shade of green:

















The final doodle in this figure is complex I. Here is their image:













which keeps things nice and simple between NuoH and the Nuo A/J/K region, where Nqo10 is shown but nothing else. So I wanted to know if there could be a Na+ channel which could be used to either top up the energy of the NADH:menaquinone couple or to allow the antiporting function to occur in deactivated standard CoQ based complex I. Of course no one is looking for Na+ channels in complex I in quite the same way as Yu et al were looking for one in MBH, where they knew it was crucial. Anyway, I went looking. In here

Structure and function of mitochondrial complex I

is where I found find this image (which is unfortunately left to right transposed in its view) that includes a rather nice broken helix shown in pink which I have circled in red:

















If we take this broken helix, colour it green and transplant it in to Yu et al's complex I we get this:













Might it be the Na+ channel I need?  No one knows (yet). It is known that there is a conformational change in the region of the CoQ binding pocket when complex I changes from the active to the deactivated state, close to the region of the broken helix head. I would suggest this conformational change might allow the Na+ antiporting function to occur when pumping in complex I is deactivated. Protons would be allowed to enter the cell/mitochondrion in exchange for Na+ expulsion (theoretically reversible but that seems unlikely physiologically unless you are a bacterium using menaquinone where some Na+ ingress is worth it to trigger the membrane arm). Logic says that this trade off would be preserved if antiporting provided a net benefit to the organism under which ever conditions might have favoured deactivation of complex I.

Peter

Addendum. Here are the three complexes lined up. Because PowerPoint lets you do it. No other reason.

















Wednesday, April 03, 2019

Life (29) Applied billiards to MRP and MBH

Here is a nice doodle of complex I taken from this interesting but off topic paper














It shows that taking two electrons from NADH and dropping them to CoQ provides a Gibbs free energy of 840mV for the pair. That's all well and good and is why cells love oxygen based electron transport chains. But I'd like to look at this in reverse. We know that complex I works perfectly well in reverse. So we can say that dropping four protons from extracellular to intracellular down a membrane voltage of 150mV across a proton tight membrane will allow the performance of 840mV of work.

For complex I running in reverse this 840mV of work is always used for NADH generation from NAD+ (or superoxide generation) because that's the unit bolted on to the membrane arm. Other metabolic units can be used in place of NADH dehydrogenase and many of these can be found in various bacteria using various substrates with various numbers of antiporter-like subunits in the membrane arm. All will work in reverse and provide/utilise differing Gibbs free energies.

So I would like to view the membrane arm of all of these complexes as a machine in its own right which, when running in reverse, can harvest the energy available from a proton gradient for whatever function the cell might like. Obviously this is not the current arrangement, but is possible.

So here is the intact membrane arm of the complex I of T thermophilus, once again taken from Luca et al












running in reverse to give 840mV/2e- of usable energy. As in the last post we can imagine each proton passing through each channel "kicks" to the right ('cos we're running in reverse here) until the cumulation of four "kicks" is enough to cause the conformation change in Nqo 8 which drives two electrons back up the FeS chain to an NAD+. Think of the membrane arm as a machine to accumulate multiple "kicks" to give a big Gibbs free energy.

Now let's remove some of the sub units and see what we get. How about this:












Two proton translocations will provide much less Gibbs free energy than four proton translocations. I've faintly allowed a third proton channel but there is no suggestion this is a functional feature of complex I at all. It's Nqo 10, never mentioned in the paper. Note that all of the subunits are kicking in the same direction.

The above is obviously the basis of the MRP antiporter, which looks like this:



















where the energy of two protons is cumulated to allow a single Na+ ion to be extruded. How much of a Gibbs free energy will be developed by the two protons will obviously be influenced by the membrane voltage through which the protons fall. It will also be influenced by the permeability to protons of the membrane in which MRP is embedded. If the membrane was 100% permeable to protons there is no point trying to harvest a gradient which isn't there. The more permeable the membrane the less energy can be harvested per proton. But we do know that in E coli under CCCP with a membrane voltage of as little as 15mV that MRP can cumulate enough Gibbs free energy to antiport Na+ when monogenetic antiporters fail completely.

In the ancestral hydrothermal vent scenario a membrane voltage from the pH gradient between vent and oceanic fluids might be, ideally, around 150-200mV. I still consider MRP is most likely an adaptation to allow colonisation of vent environments where apposition of fluids is not perfect or some mixing has already occurred and the available membrane voltage might be very low. Given MRP LUCA might survive on the edges of vents in addition to thriving in the luxurious conditions of perfect apposition near the centre.

A step on from MRP is, hypothetically, to add a power unit which will allow Na+ pumping when membrane voltage is too low even for MRP. You can think of this as an adaptation to the tail end of the vent system. Some membrane voltage is still present as are complex molecules (from dying prokaryotes in the better vent environments) out of which ferredoxin can be synthesised. This is not quite the membrane bound hydrogenase of P furiosus as we will see. It's an augmented "kick-to-the-right" through MRP to assist the generation of enough Gibbs free energy to allow Na+ extrusion:























Notice that all of the "kick" arrows run in the same direction as for all of the illustrations in this post, looking to generate a significant Gibbs free energy at the right hand end.

The final stage of generating the MBH of P furiosus (and freedom from the vent gradient completely) is like this: We have to reverse the direction of the proton travel in MBH subunit H. The "kick" from proton ingress is to the right. We want to impose a kick towards the left to reverse the proton flow to give egress, using ferredoxin power. This requires a barrier between MbhM and MbhH to stop that kick to the right, completely different to complex I and any doodle so far. As Yu at al comment in the legend to Figure 3

"A hydrophilic axis across MbhM membrane interior is also identified but it is separated from that in MbhH due to a gap between the two subunits"

and in the legend to Figure S4:

"Note the large gap between subunits M and H in MBH (A). There are four elongated densities located to the lower region of the gap (A) inset; marked by blue dashed lines), which stack against several hydrophobic regions of subunits M and H. These densities are likely from two phospholipid molecules that may stabilize the structure and prevent ion leakage across the membrane bilayer. The dashed curves in (C) and (D) highlight the fact that the chain of hydrophilic residues found in complex I is continuous (D), but is discontinuous in MBH (C)".

This is the genuine article:















and my version, more crudely:























As well as stopping MbhM "kicking" the central water channel protons of MbhH to the right, the conformation change from the NiFe hydrogenase has to be imposed on MbhH to force a proton outwards.

So the white arrow of proton "kick" in the central water channel of MbhH is reversed by the green cranks using the power from the hydrogenase (green arrow) to enforce this. The bright red proton channel is present as specified by Yu et al and is both essential and functional. Quite what the remnant of this is doing complex I I don't know and quite what the proton channel within MbhM is preserved for I also can't imagine (yet). But the general principles of proton movement as set down here are much more satisfying than my initial thoughts on MRP and MBH.

Peter