Protons: SCD1 and the bomb

Our daughter has taken to watching DVDs. When we are not being tortured by Postman Pat (Special Delivery Serrrrrrviccccce, you know the tune) or the Mickey Mouse Clubhousssse we do at least get some amusement when she requests that blistering LC comedy "A Matter of Loaf and Death", by Nick Park.

Just how funny you can make a story about an obese cereal killer (no typo, the subtitles specify cereal killer, I said Park is funny!), murdering bakers as revenge for her obesity ("Are you ballooning?") has to be seen to be appreciated. It's a lot more amusing than Postman Pat.

One of the funniest scenes is where Gromit cannot get rid of Paella's bomb. It's a direct tribute to the 1960s Batman scene where "Some days you just can't get rid of a bomb".

You know, with the ducks

and the nuns. Park has kitten-enhanced the nuns



and included Yorkshire as the preferred site for bomb disposal. The Wars of the Roses are, apparently, over but not forgotten.

This post is about how physiology uses SCD1 to dispose of the metabolic bomb of hyperglycaemia in the presence elevated levels of palmitic acid.

It was pancreatic beta cells in culture which produced this picture:



I love this group because not only do they tell you in the methods section EXACTLY what glucose concentration they used in culture (5mmol/l vs 11-25mmol/l) without making you go back through three layers of references (to bury the 25mmol/l most groups use, but never discuss), but they also describe 11mmol/l as hyperglycaemia. That is, pathology.

This is Figure 4 from the same paper showing markers of apoptosis, superb:




Note the increase from palmitate to stearate. Note the complete protective effect of oleic acid and very modest toxic effect of linoleic acid. Aside: Note also the complete and total protection provided by limiting glucose to 5mmol/l, with any fatty acid, at any concentration. You have adipocytes leaking FFAs? Your best hope of keeping a functional pancreas is to limit your glucose to 5mmol/l. How? Answers on a postage stamp to...

It's also worth noting that stearic acid had to be reduced from the original 0.4mmol/l to 0.25mmol/l because at the higher concentration with high glucose they found exactly the same thing as Dave Lister did in Red Dwarf when Holly brought him out of stasis. Everyone is dead Dave. Everyone. Is. Dead. Dave. You have to U-tube the clip. Stearic acid at 0.4mmol/l with glucose at 25mmol/l, in cell culture, is utterly lethal to beta cells. If you pharmacologically block apoptosis the cells simply undergo the rather messy collapse of necrosis. This is a non survival insult.

Okay, okay, here's the clip:



The group went on to do quite intersting things with blockade of acyl-CoA synthetase and also with inhibition of fatty acid oxidation, which leads to all sorts of other threads which are, in part, where I have been wandering for the last few weeks. Far too much for this post.

So let's look at SCD1 knockout mice which have been rendered obese by also knocking out their leptin gene. Here we have rapid onset obesity due to adipocyte fat storage, free fatty acid leakage due to adipocyte insulin resistance and a complete inability to place a double bond in to palmitic acid or stearic acid. They have elevated FFAs and these are almost all saturated. This paper describes the study. It has to be noted that to obtain the FFA levels you have to reverse engineer Fig4 part A:



A ruler and calculator gives FFAs for the normal ob/ob mice as 0.32mmol/l and for the SCD1 k/o ob/ob mice as 0.56mmol/l. Any group which makes you reverse engineer in this way to get something as simple as FFA levels is, in my book, highly suspect. Does anyone think that 0.32mmol/l is quite low? Despite the greater obesity. Partly due to maintained insulin sensitivity in adipocytes (that's why they distend) while ever de novo lipogenesis produces palmitoleate using SCD1 and partly due to the higher levels of insulin production (normal beta cell mass) working on those insulin sensitive adipocytes... These mice are still in a slightly privileged position, metabolically, as they have yet to become obese enough for their SD1 equipped adipocytes to become seriously insulin resistant, they are still only six weeks old.

And here is the % of types of FFAs.



The column on the left is the one which represents about 0.32mmol/l of total FFAs and the column on the right is around 0.56mmol/l, as above. Glucose varies but fasting levels can be as high a 700mg/dl. So what happens to beta cells?

They divide up in to two types. The health ones and the dying ones.

The basic finding is that young ob/ob mice need either oleic or palmitoleic acid to maintain a functional beta cell mass. Exposure to high levels of glucose combined with palmitic and/or stearic acids induces apoptosis plus some necrosis in beta cells. Most non pancreatic tissues in the SCD1 knock out mice appear to be able to upregulate beta oxidation, especially in peroxisomes, of fatty acids which minimises both obesity and insulin resistance.

The beta cells of the pancreas do not appear to have this luxury.

They need to lower that F:N ratio with palmitoleate or oleate, otherwise they are left holding the bomb.

Peter

Dalcetrapib fails as it should

Still no time to post but this one liner is another gem. Dalcetrapib, another (yawn) HDL raising drug has bombed. Who is surprised?

Funnily enough Dr Nissen (Rentaquote-you-have-a-statin-deficiency) feels dacletrapib failed because it was too weedy to do any good, only a 30% increase in HDL.

Evacetrapib (Nissen's gravy train) and anacetrapib will REALLY work because they double HDL and slash LDL.

No they won't. They will do exactly what torcetrapib did as they are as potent as torcetrapib was. Body count will rise. Dalceptrapib killed no one as it is not potent enough to do so!

Peter

Skirting around leptin

I've been wanting to post about Wallace and Gromit, Batman and ob/ob SCD1 k/o mice for weeks now and it keeps not happening. Before we go there, just a word or two about leptin and weight gain. You can't work through anything relating to ob/ob mice (+/- SCD1 k/o) without having to, finally, sit down and read something about leptin. Or at least ob/ob mice...

To me the core question to ask is whether ob/ob mice are gaining weight because they have a brain disorder giving overeating or an adipocyte disorder storing calories. As always, there is an infinite supply of data suggesting a brain disorder, there's no denying leptin does things in the brain. The question is: Are ob/ob mice in caloric excess as they gain weight? ie Do they eat too much so gain weight or do they primarily lose calories in to their adipocytes and so have to eat more to just meet metabolic needs?

We have various rodent models of obesity which have the common feature of reducing the sympathetic nervous system drive to adipocytes, so failing to oppose insulin's lipogenic action. These animals gain fat even if you calorie restrict them. I'm thinking about hypothalamic ice-picks, various VMH neurotoxins or unprotected free radical generation. But the common thread is the loss of sympathetic nervous system driven lipolysis, facilitating insulin driven fat storage.

When confronted with the overwhelming literature on leptin it's hard to know where to start, especially when people are not asking the sorts of question which interest me, looking at the data from my very particular perspective.

I accept that ob/ob mice get fat. So too do brain injured rats and mice. Is there a common mechanism here? The smoking gun would be a period of enhanced insulin sensitivity in adipocytes, due to decreased sympathetic tone, which allows both fat gain and the preservation of insulin sensitivity in the early weeks, until adipocyte distension induced insulin resistance kicks in for the swelling adipocytes and systemic insulin resistance develops.

Of course the easy part, with absolute leptin deficiency, is asking whether leptin increases hypothalamic sympathetic nervous system outflow. That took about 30 seconds on pubmed and this was the 6th or 7th hit.

Leptin increases sympathetic drive. I think it's reasonable to conclude leptin deficiency does the converse and reduces sympathetic drive from the hypothalamus. So I'll take that as a yes. Don't forget I'm biased.

Sooooooo. Does leptin deficiency defend insulin sensitivity during rapid weight gain? As it should if the mechanism is enhanced lipid storage. And weight gain in young ob/ob mice is, well, rapid. To say the least. There will only be a very narrow window to pick up preserved insulin sensitivity before adipocyte insulin resistance and hyperinsulinaemia set in. By which time researchers have a usable model of established obesity.

I'm interested in what goes on before the model becomes "usable". We know that by rewarding volunteers to overeat we can spike their insulin levels massively within three days, probably faster. Does this happen with ob/ob mice as they over eat?

I've been working through a whole stack of papers on ob/ob mice, FFA levels, insulin levels, ketogenic diets... All the usual stuff. I ended up in a review by Lindström, giving this little gem of a quote:

"The muscle insulin resistance is not observed in very young [ob/ob] mice, but develops after 3–4 weeks [131]"

The abstract of ref 131 supports the concept of preserved insulin sensitivity, looking at muscle rather than adipocyte insulin resistance. The papers on palmitoleate as a lipokine suggest that muscle insulin resistance is controlled by adipocyte insulin resistance, via SCD1 and palmitoleate. The paper is rather nice because it is looking at ob/ob mice as ob/ob mice, not as some completely inappropriate model for hyperleptinaemic obese humans. It was, after all, 1980 when it was published.

So to summarise:

Danish volunteers who are paid to overeat spike insulin from 35pmol/l to 74pmol/l in just three days. Mice with zero leptin overeat massively, but do not show the same insulin spike. The insulin spike signifies insulin resistance, that characteristic antioxidant defence response to an excess of calories in the metabolic milieu. This does not happen with the early overeating phase of ob/ob mice. They are in metabolic caloric deficit, which they make up by eating enough to remain vaguely functional.

Absolute leptin deficiency appears to be a very harsh driver of fat storage. Losing this many calories makes you hungry. I guess some bit of the brain is involved in converting this state of actual calorie deficit in to a feeling of hunger, but that's not what interests me nearly as much as what is happening at the adipocyte level of calorie storage.

Peter

Now we can get on to Wallace and Gromit and knocking out SCD1 in ob/ob mice.

Protons: Love your superoxide (outside your brain)

Two off topic posts in a day! How come? I had the weirdest morning today. A three hour consulting session with only six appointments, all straight forward. Bloody hell, was I lucky for a Saturday! Can't blog at work so I had a quick browse to see what Nick Lane has been up to recently. He has a cracking article up (as a pdf) on heteroplasmy which rewards careful reading in its own right, but look at these two "throw away" quotes.

First on ROS, good old superoxide from reverse electron transport:

"ROS leak seems to optimize ATP synthesis by stimulating mitochondrial biogenesis (mtDNA copy number), an interpretation supported by the fact that antioxidants lower not only ROS leak but also mtDNA copy number and ATP synthesis. ROS leak, in effect, signals low capacity relative to demand, stimulating compensatory mitochondrial biogenesis".

How do we minimise mitochondrial biogenesis? By running metabolism on glucose of course, but don't forget the lack of superoxide generation when oxidising PUFA. But who needs mitochondria when you can lower LDL levels by swilling corn oil? Ah cardiology, you have a lot to answer for. Executive summary: Want mitochondria? Burn PALIMTATE.

And on cerebral metabolism:

"In the brain, where further mtDNA biogenesis is limited, neurons would then become compromised whenever energy demands were high, possibly causing acute cognitive and behavioral abnormalities".

The brain neurons are running on lactate under crapinabag conditions. We considered this before. No fatty acids. No glycerol 3 phosphate. No FADH2 input to the CoQ couple. No free radicals. No signal for mitochondrial biogenesis. No mitochondrial biogenesis. You could substitute ketones and maybe get a few mitochondria back if you were canny, but most medics aren't canny. What happens to the lactate supply for neurons when hyperglycaemia drops on to chronically elevated FFAs and triggers apoptosis in glial cells? I think we can attach various labels, depending on which neuronal cell types die first. Alzheimers seems a nice name for the commonest scenario.

Lovely pair of quotes. Glad I got the browse time. But don't ignore the heteroplasmy discussion at the core of the article, it's good stuff.

Peter

Look AHEAD trial stopped

Eat less, move more, have your heart attack on time!

With apologies for lack of any attention to the blog recently (which may be set to continue for some time) but this snippet just had to get passed on. This link from Karl:

http://www.theheart.org/article/1458351.do

More info here

http://www.nih.gov/news/health/oct2012/niddk-19.htm

And the glowing anticipation of success here from the planning stage:

https://www.lookaheadtrial.org/public/home.cfm

Pubmed gives a series of genuine success stories from the early days on all sorts of parameters. But the cardiovascular end points show how utterly useless these interventions are long term.

However the massive omission, from the quick look I've managed, is of any intention to report the all cause mortality. It seems very likely to me that more people died in the intervention group than in the usual care group, but p was > 0.05.

Call me a cynic, but I think they stopped the trial because they could see where that p number was heading. Has anyone seen a body count from anywhere in the trial?

Also, what might the outcome have been if the intervention group had been repeatedly bullied, harassed and indoctrinated to maintain a normoglycaemic, low grade ketogenic diet for 13.5 years? Say to an HbA1c of around 5%?

Ha ha ha bloody ha.

Peter

EDIT: Have started on the SCD1 k/o ob/ob mice. The thread WILL continue.

Protons: Zero fat

A bit speculative here, read with caution!

How do we lower free fatty acids? Obviously, with nicotinic acid. What does this do to insulin secretion in response to a glucose challenge? I'll just work through this figure from the same paper which gave us the insulinotropic effects of various FFAs a couple of posts ago.



Section A is very simple, it just shows that they succeeded in clamping glucose at just over 200mg/dl, about 12mmol/l, ie just in to supraphysiological levels.

Section B shows FFA levels, which they manipulated very carefully. All rats started at about 0.6mmol/l. Nicotinic acid lowered FFA levels to 0.1mmol/l. These are the black squares. Two other intervention groups were included. The white triangles had their lipolysis shut down using nicotinic acid but then had FFAs clamped back up again using a soyabean oil infusion (mostly omega 6 PUFA) and the black triangle group had an infusion of lard based lipids (a mix of lipids but with a significant palmitic acid content) to restore and hold FFAs at about 0.8mmol/l.

The nicotinic acid group, with FFAs of 0.1mmol/l, cannot secrete insulin in response to glucose. Flat line at the bottom of graph C.

The open squares are the control group. These rats show the normal response to an hyperglycaemic clamp. They reduce FFAs in response to the inhibition of lipolysis from secreted insulin, down to 0.2mmol/l. Insulin inhibits lipolysis. But the reduced FFAs also reduce insulin secretion. There is a balance struck with only a modest rise in insulin, sustained throughout the clamp. You can see this in section C, open squares.

The two lipid infused groups have clamped glucose and clamped FFAs. They secrete insulin in proportion to the amount of palmitate in the lipid infusion. A bit extra over control if you use low F:N ratio omega 6 PUFA, a ton extra when you include some palmitate. Section D is simply a summary of this.

Step by step at the mitochondrial level: The lower fatty acid supply results in decrease reduction of the CoQ couple in beta cells. This reduces the reverse electron transport and associated superoxide triggered by glucose as it feeds NADH in to complex I, so limits insulin secretion. You can virtually ablate the insulin response to glucose by eliminating beta cell fatty acid supply.

Now, nicotinic acid is one way of reducing FFAs. There have to be other, perhaps more physiological, methods. Maybe we could use insulin per se? From food perhaps? Let's try eating around 40g of carbohydrate and look at the Spanish study graph again. Insulin rises from 50pmol/l to 75pmol/l. This is enough to reduce FFAs from 0.5mmol/l to just over 0.1mmol/l. Look at the FFAs, especially the circles between 120 and 300 minutes:



Now (again, sorry!) look carefully at the insulin levels after the small carb load, bottom circles.



By 180 minutes insulin is actually lower than fasting, and FFAs are still well below fasting levels too. The rat model appears to hold in humans, not what the study was looking at, and a small effect. But I think the effect is real.

How about scaling this up to a massive dose of potato induced insulin and limiting dietary fat? Severely limiting dietary fat. And never mind pussy footing around at 40g of mixed carbs and protein. There is a limit to how low FFAs can be driven, and it seems safe to assume that a baked potato or three might just inhibit lipolysis maximally and keep it that low for rather a long time. But if you deprive beta cells of free fatty acids you blunt their ability to secrete insulin. Very, very high carbohydrate diets really ought to be able to inhibit lipolysis to the point where the knock on effect is the inhibition of insulin secretion, provided you don't supply exogenous fat. Look at the nicotinic acid treated rats...

Once you get FFA levels low enough to inhibit insulin secretion you will start to move in to the sort of territory where insulin secretion might be blunted enough to allow hyperglycaemia. But the feedback effect of reduced insulin levels is also the re commencement of lipolysis. This will restore enough FFAs to maintain functional insulin secretion and so avoid potential hyperglycaemia, which the body tries to avoid. Of course you have to throw in the increased insulin sensitivity of muscles deprived of exogenously supplied FFAs too.

So is it possible to eat an ad lib, calorie unrestricted diet based on near pure carbohydrate and lose weight? Working from the premise that lowered insulin is a pre requisite for hunger free weight loss, as I always do, the answer is possibly yes. We all remember Chris Voight on his all potato diet (plus 20ml of olive oil, low in palmitate, per day) who lost a great deal of weight over a few weeks, the rate of weight loss accelerating as the weeks progressed? I had a think about it here, well before I had any inkling as to what might be happening in the electron transport chain.

We need to know what the interaction of insulin and FFAs was during this particular n=1 self experiment, and we don't. The rats suggest to me that insulin levels were initially raised post prandially and FFAs were not then available from peripheral adipocytes. Assuming the fall in lipolysis persisted in to the post-absorptive period (the primary function of insulin, especially at low levels, is the inhibition of lipolysis rather than facilitation of glucose diffusion, we've all read Zierler and Rabinowitz) we have a method for limiting insulin secretion late post prandially using reduced free fatty acid levels.

As an aside I personally wonder it might be the ectopic lipid supplies typically found in muscle, liver and visceral adipocytes which might still be available for metabolism by the tissues when exogenous supplies are shut down.  It reminds me of how metformin most likely depletes ectopic lipid to improve insulin sensitivity, despite having complex I inhibition as its primary action. You need lipid from somewhere. So reducing FFA supply by inhibiting systemic lipolysis may well be a route to lower fasting insulin levels. Especially if you are not far in to metabolic syndrome.

Once ectopic lipid becomes depleted then lipolysis would accelerate in peripheral adipocytes as systemic insulin resistance falls and fasting insulin levels too, which might be what was reported as progressively increasing weight loss by Chris Voight. Insulin levels would be low, especially during fasting, and appetite low at the same time due to hypoinsulinaemia facilitated lipolysis, much as appetite is low under LC induced hypoinsulinaemic eating. There is more than one way to skin a.... Oops let's not complete that phrase!

What would happen to a healthy person under these conditions, long term, is anyone's guess. Chis Voight gave up after a few weeks when weight loss became alarmingly rapid. But we know from the crucial study by the vegan apologist Barnard that, for diabetic people at least, that a long term, whole food, low sucrose and low fat diet is a complete disaster, once the initial weight loss ceases.

This is playing with fire (possibly near literally, at the mitochondrial level) if you are a diabetic. Please don't go there.

But the physiology of weight loss on ultra low fat diets is basically comprehensible, especially once you look at lipids and superoxide at the ETC level, and what the body needs to function effectively. Running your metabolism on pure glucose would induce, theoretically, an infinite glucose sensitivity and low fasting insulin. If we do reductio ad absurdum you would end up with no fat stores and experience death from hypoglycaemia if you ever depleted your glycogen stores. Mitochondria like (saturated) fatty acids. Fatty acids keep them in control.

I think someone in obesity research used Chris Voight's experience to support some cock and bull story about food reward and a set point of body fat. We can wait for the recant on that one, if you could care less about it. The biochemistry is, as always, the fascinating stuff.

Peter

Never forget, never forgive

For recreational purposes only, from here:



How toxic is palmitate at any concentration from zero to 0.4mmol/l if glucose is held at 5mmol/l? It's not.

How toxic is glucose at 25mmol/l in the presence of increasing palmitate? Glucose at this concentration becomes progressively more toxic within increasing but still physiological levels of palmitate.

Glucose at 25mmol/l is UTTERLY non physiological.

There is potentially a huge amount to discuss from this paper, one day, maybe. Even though they never delve in to the ETC, which they should do. But anyway, someone commented that papers using 5mmol/l glucose in the culture medium were rare. This one is a hen's tooth.

Back to thread next.

Peter

Protons: The pancreas

We've seen the concept of superoxide being used to produce insulin resistance as a means of limiting (glucose derived) energy input in to cells which really don't want it. Superoxide appears to be the primary marker of energy excess at the cellular level.

We know from isolated mitochondrial preparations that superoxide is physiologically produced by reverse electron transport through complex I and is driven, gently, by succinic acid alone working through complex II. Far more is produced when the NADH level is high as well as having a reduced CoQ couple through FADH2 input, be that from complex II or from fatty acid oxidation products. Macroscopically fat and glucose together should produce enough superoxide to show as cellular insulin resistance, rejecting glucose from the cell, while allowing continued fatty acid oxidation. That's simple and logical.

But if you are building an energy sensor, it would be a bit dumb to restrict access to the very energy molecules which you are trying to look at to judge overall energy status, especially when energy status is high: You need to decide when to store calories...

The beta cells appear to use both fatty acids and glucose to generate superoxide, but instead of signaling beta cell insulin resistance, they signal insulin secretion. Several lines of evidence fit in with this.

You can get succinic acid itself directly in to beta cells by providing it as a methyl or ethyl ester. As a metabolic fuel source this acts as a near pure complex II substrate, pushing electrons in to the ETC through the FADH2 of succinate dehydrogenase to reduce the CoQ couple and set the scene for reverse electron transport and superoxide production, especially when NADH from glucose metabolism rises. In a commonly used model of functional beta cells, succinic acid methyl ester is a marked insulin secretion potentiator, especially at higher glucose concentrations. Glucose supplies NADH, succinate supplies FADH2, they clash at the CoQ couple and the generation of superoxide signals that there is a ton of energy available. Better store it. Better secrete insulin.

Succinic acid methyl ester drives complex II. This drives insulin secretion in response to glucose. But it's a drug. There is nothing physiological about this drug. So shall we go a little more physiological?

To recap from previous posts: Superoxide generation is directly proportional to the ratio of FADH2 generated to the amount of NADH generated for a given substrate, the F:N ratio.

Here's a nice graph of insulin secretion stimulated in response to 12.5mmol glucose on a background of assorted free fatty acids from an isolated pancreas preparation:



If you can't be bothered to work out the F:N ratios (shame on you), here they are added to the graph:



Please excuse the C8 value; as we all know, MCTs are shunted directly to the liver via the portal vein. They do not seem to feature too prominently in pancreatic superoxide generation and insulin secretion. It would take a ton of reading to see why and how they are handled differently to longer chain fatty acids. For the time being let's stay looking at C16 and longer as these make a much tidier story...

So, for the four longer fatty acids tested, the amount of insulin secreted is remarkably closely associated with the F:N ratio of the fatty acid available.

Does this work in people?

Of course it does. Remember the Spanish study? I lo0ked at it in some detail here.

In particular look at this graph:



From the top downwards we have butter, high palmitic acid seed oil, refined olive oil and a mix of fish and vegetable oils as the white triangles. It is very clear that the insulin secretion here is in direct proportion to the saturation and length of the fatty acids in the meal, in an intact group of volunteers..

Aside: Obviously, there is a glaring error in the graph. All of the curves except the control use 800kcal of total food, of which 40g is carbohydrate/protein and the bulk is fat. The graph is missing a group where 800kcal was supplied as pure carbohydrate. We can all imagine where this much bulk glucose would have put the insulin curve, needless to say there is absolutely no way it would fit on to the presented graph. We would need a much taller vertical axis, which would show the mixed meals in their true context!

But the principle, that insulin secretion at a given level of glucose is elevated in direct proportion to the F:N ratio of the background fat, holds perfectly well in this carefully contrived human study.

BTW, lucky for me they didn't include a coconut oil group!

The obvious conclusion from this finding is that to lower insulin maximally we should, taking as given that replacing carbohydrate with fat is the biggest step by far, all go for vegetable oil with some fish oil. Not butter. But in the original post on the Spanish paper I went on to discuss what appeared to be happening to the lipid from the meal. It could stay in the bloodstream and be used for metabolism, as the butter did, or it could be cleared rapidly in to adipocytes allowing metabolism to return to being glucose based. At the cost of expanding the adipocyte stores of fat.

The high PUFA meal really was rapidly stored as fat in adipocytes. The F:N based explanation is because we are supplying a low F:N ratio fat and so not generating insulin resistance phyiologically; we are allowing lipid easily in to adipocytes because the lipid does not generate adipocyte insulin resistance. We are going back to glucose metabolism as rapidly as possible. PUFA facillitates fat storage and glucose based metabolism. All is fine until you can't get any fatter. Butter limits fat storage and runs metabolism of palmitic and stearic acids. Those high PUFA-fed mice generate obesity when fed their high PUFA diets from pre-conception onwards:



In the butter group there is some excess insulin. Does this matter if no one (cellularly) is listening to it?

I next want to look at the flip side, the reduction of supply of free fatty acids to the pancreas. This was done in the same paper. You can certainly do this in intact rats (and humans if you so wish). Then we might get back to the fat mice.

I think that had better be another post as this one is getting overly long and it's light enough to let the chickens out.

Peter

Protons: Linoleic acid in the hypothalamus

Hi all, just getting my head above water now that we have two or three locums at work to cover some of the (rather difficult) gaps in the rota!



Before we look at the fat mouse study which wins the prize for most miserly hoarding of data, I just wanted to put up a brief post, based on that paper, about breaking your hypothalamus with a high fat diet. Just to re emphasis: This is NOT what happens to a human after 7 days on a high fat diet.

Remember Schwartz's rats? Put them on a high fat diet and this happens to food intake:



Note the very sudden and dramatic spike in the intake of food, shown by the red line which I've added to emphasise the abrupt change from baseline chow consumption. We can ignore the red oval for this post. What happens in the VMH neurons of these rats?

This is what happens:



The dark brown staining cells on the right are dying, the rats have been eating "cookie dough", which they "can't get enough of", for seven days. The nice healthy cells in the left hand photomicrograph are from rats on crapinabag. The basic idea appears to be that feeding rats a bit of fat and sugar makes them eat so much, starting in just one day, that by seven days their VMH is killed by over indulgence. You eat too much, you kill your brain. Simple. This is, of course, absolute bollocks.

At the risk of repetition, we can produce exactly the same lesions in the VMH with MSG or gold thioglucose (or an ice pick if you must be crude and don't want nice pictures). This injury results in fat gain which must be compensated for by overeating. Rats will gain weight more slowly if they are on low fat diets than on high fat diets because of the effects of increased de novo lipogenesis which I've discussed in previous posts.

Want pretty pictures from GTG injured rats? Here's some random immuno from a random paper, there's a lot of it around, only black and white though:



Gold thioglucose on the right, arrow marks the injury area. And I just noticed the same pics, also in black and white, from fat injured rats from elsewhere in the Schwartz paper (mirror imaged compared to the GTG pics, random choice of which side of brain got sectioned!) after just a week on their high fat diet:



So we can produce the pretty black stains of dying cells with gold thioglucose (or MSG if we looked at neonatal immuno) but this injury preceeds the loss of calories in to adipocytes and subsequent "hyperphagia". THE INJURY COMES FIRST.

Let's really look at the bizarre idea that non-forced "overeating" causes subsequent damages your VMH. This is how it works for over eating by a gold thioglucose injected rat, no yummie high fat diet needed: It simply decides to over eat crapinabag because this has suddenly and randomly become delicious and so it becomes obese. We all know overeating CAUSES the VMH injury in fat fed rodents. So how do GTG injured rats get the injury first and over eat secondarily? Gold thioglucose obese rodents might SEEM to have a chemical lesion causing obesity but clearly they get fat first, travel back in time (squeezing in to a time machine as obese chrononaughts) and retrospectively force the researchers to give them the injection of GTG to obtain the lesion in their VMH which they are going to produce in the future by eating too much crapinabag. Got that? You've all watched Back to the Future. I watched parts I and II but never managed part III. It's simple time travel. Ditto MSG and ice-pick (ouch!) obese rodents. Self inflicted injuries using time travel.

Or we could abandon such stupidity and say that high fat diets injure the VMH first and this injury increases fat storage by decreasing sympathetic tone to adipocytes, as it does.

And I suspect it's superoxide, generated by a high F:N ratio (classically derived from palmitic acid at an F:N ratio of 0.47) in POMC neurons, which probably does the damage. You all know POMC neurons, the ones in the VMH with both gluokinase to sense (via metablism) glucose and CD36 to monitor FFA status (via metabolism again). No lactate for the energy status sensing neurons of the VMH...

So the question is, as always, what happens to the VMH of a C57BL/6 mouse (bred to get fat on a high fat diet) when put on a high fat diet which does NOT generate superoxide in POMC neurons? You can do this.

No one has done the necessary immuno staining under these conditions to get the pretty pictures of dying (or non dying) cells, as far as I know. But it's easy to look at the weight gains, which are a reasonable surrogate for POMC injury. Schwartz again using rats:



Not the most lucid graph, but it gives the basic idea. The control weight gains on the left are comparable to the weight gains shown for day 14.

Now, here is what happens if you take a C57BL/6 mouse and put it on to 35% of calories from fat if you keep the F:N ratio of that fat well below 0.47, using omega 6 PUFA with an F:N ratio of 0.42, as the primary source of fat:



Ignore the top two lines (for now) and look at the weight gain of the mice in the bottom two lines. One group weaned on to crapinabag, the other weaned on to 35% of calories from fat, but a fat with a low F:N ratio. There is zero, zilch, nil difference in weight gain over three weeks. There is no excess weight because there is no VMH injury. No one generates significant superoxide from a low F:N ratio fat like linoleic acid. That appears to include the POMC neurons of C57BL/6 mice.

C57BL/6 mice (and Long Evans rats) are specifically bred to get fat on palmitic acid (sometimes plus fructose) based diets. They fail to deal with the absolutely normal levels of superoxide produced in POMC neurons in the VMH which are crucial to energy status sensing. They do not have the luxury of developing insulin resistance as their job is to monitor both glucose and fatty acid levels. They are not allowed to run on lactate with an F:N ratio of 0.2 the way much of the brain does. They take whatever plasma gives them and do their best to cope with it. Or, in the case of rodents bred to become fat on high fat diets, not cope with it.

Before we go looking at the linoleic acid paper a bit more carefully I think it's worth trying to look at energy sensing rather more peripherally than the POMC neurons of the VMH. Then we can come back to the fat mice and try to think about what's going on using the meagre data available. Because it's quite interesting.

Peter

Guess the weight of the mouse competition

Here's the Brownie points quiz.

What is the weight of either mouse?

If you find the answer, in this study, please let we know!

Peter

BTW you are allowed/obliged to scour results, discussion and all supplementary data. Please.

Protons: SCD1 knockout mice

Let's peep inside an adipocyte belonging to a mouse which has had its stearoyl-CoA desaturase gene deleted.

It's busy making lipid, being an adipocyte. Two carbons, four carbons, six, eight, ten, twelve, fourteen and hey, there's the sixteen for palmitic acid. Now, how much glucose and insulin is there around? Ah, lots. Need to signal this with palmitoleate. In goes the double bond to prove it... Oops. No SCD1. Hmmmm. We now have a ton of palmitic acid and no chance to convert any of it to palmitoleic acid. Tricky.

Is the adipocyte going to become insulin resistant? Unless it runs on glucose and never uses any lipid this seems likely. Will the adipocyte stay small? It should do, it's insulin resistant, so won't store fat. Should it export saturated fat as FFAs? Yes. Should we have a slim but insulin resistant mouse? On chow it should become hyperinsulinaemic. Well, you might expect so.

But that's not what happens. The mice stay slim alright, but have excellent insulin sensitivity. Like really, really good insulin sensitivity. You can even feed them on toffee fudge cheesecake and they stay fairly slim and very insulin sensitive.

The SCD1 deleted mice also eat more despite being slimmer than WT mice when on chow, ie they are in CICO-denial:

"On average, the SCD1−/− mice consumed 25% more food than wild-type mice (4.1 g/day vs. 5.6 g/day; n = 9, P < 0.05). Nonetheless, they were leaner and accumulated less fat in their adipose tissue"

Huh. Bloody gym sneaks again. Even while they are asleep:

"The SCD1−/− mice exhibited consistently higher rates of oxygen consumption (had higher metabolic rates) than their wild-type littermates throughout the day and night (Fig. 3A). After adjusting for allometric scaling and gender, the effect of the knockout allele was highly significant (P = 0.00019, multiple ANOVA, Fig. 3B)."

These animals have a hugely increased metabolic rate. The brown adipose tissue "looks normal". That's not the answer.

They are also ketogenic during fasting (daytime is sleep time but they don't really go to the gym while they are asleep). Fasting BHB was 4.4mg/dl. For those watching their ketone meter at home, eat your heart out. They do this even when living on toffee fudge cheesecake.

OK. Utter basics:

What is the F:N ratio within the mitochondria of these mice? Is it:

a) <0.45
b) <0.45
c) <0.45
d) <0.45
e) <0.45
f) Huh????
g) >0.48 (trick answer, don't choose this one!)

The mice are insulin sensitive. They do not have undiluted palmitic acid oxidation going on in their mitochondria. This would produce a ton of superoxide and severe insulin resistance. We know that their mitochondrial F:N ratio must be low. Their metabolism is ketogenic. What fats produce ketones on a high carbohydrate diet? Those MCTs from coconuts and breast milk do. Where do you get C8 caprylic acid from if you are an SCD1 knockout mouse on a low fat diet?

From your peroxisiomes.

Mice with palmitic acid on tap and no ability to lower the F:N ratio by desaturation simply oxidise it in peroxisiomes, FADH2 free, to C8 which is ketogenic, has a low F:N ratio and they produce a lot of heat in the process.

In the words of the paper:

"Northern blot analysis also supports changes in fatty acid oxidation and lipid biosynthesis. Probes for acyl–CoA oxidase (ACO), very long chain acyl–CoA dehydrogenase (VLCAD), and carnitine palmitoyltransferase-1 (CPT-1) indicate increases in β-oxidation"

My emphasis. VLCAD is the main one in peroxisomes, as well as being present in mitochondria. The authors do not come up with any comprehensive explanation of what is going on. The F:N ratio delivers.

I think I mentioned some time ago the explanatory ability of the F:N ratio is awesome. It just goes on.



I was going to leave it there, back to work next week so blogging will diminish, but here is some idle rambling which followed on from this post.

Now here's the question. If some guy like me set out to maintain the lowest practical insulin level (which will minimise SCD1 activation) and bases his diet on the very longest chain, most fully saturated fat practical, would you expect me to activate my peroxisomes? Might the result be that I might stay slim and be cold tolerant?

When we moved in to our current house I unpacked the scales after they had spent nearly a year in a box provided by Pickfords. I was 63.8kg after a year of not checking anything, down by about a kilo from Glasgow. But I was getting a great deal of hill walking in Scotland and probably had more muscle. I forgot about the scales for another year but dug them out recently. Down to 62.8kgs. I eat a huge amount of palmitic acid. I generate enough superoxide to maintain the needed physiological insulin resistance to eat LCHF and I suspect I might have quite active peroxisomes.

I still run a dawn phenomemon FBG of around 5.5mmol/l, if I get up early enough to check it beforehand it's about 4.3mmol/l, once 3.9mmol/l. Random BG through the day vary from 3.3mmol/l after a half day of walking to and from the beach while the car was being MOTed to 6ishmmol/l post prandial if I had parsnip chips (yum) with my high fat beef burgers. Yes, I pour the cooking fat over the chips. A big carb load will get me above 7.0mmol/l easily but only for a couple of hours. I try not to do this too often.

Posting-wise I have no idea what time will allow next week but beta cell failure in SCD1 knockout ob/ob-ve mice tells us interesting things about cells which have minimal antioxidant defences and are deprived of palmitoleic and oleic acids.

Peter

Protons: de novo lipogenesis

Okay, time to think about whole body insulin sensitivity, adipocytes and insulin.

First the core process; adipocytes which are listening to insulin will post GLUT4s on their surface, accept glucose and do enough de novo lipogenesis to both store and release palmitoleate. The palmitoleate is a signal that there is plenty of glucose around, let's use it.

Adipocytes are accepting glucose for signalling purposes and DNL lipid formation, plus they are sequestering away whatever lipid is available from the diet. The core function of insulin is the storage of DIETARY fat under the influence of carbohydrate. Boy, that is an old post! But the fact that there is plenty of glucose around means that the body should maintain insulin sensitivity, to make use of that glucose. But DNL in adipocytes which are insulin sensitive makes you, err, fat. As Cao et al point out in their lipokine paper:


"Additionally, genetic or pharmacological manipulations that boost de novo lipogenesis in adipose tissue (even though this sometimes leads to expansion of the fat depot) are associated with improved metabolic homeostasis (Kuriyama et al., 2005; Waki et al., 2007)."


I think this is a long winded paraphrase of the Hyperlipid concept "Getting fat is bad when you stop".


Increased insulin sensitivity in adipocytes makes you fat. That's as you would expect.

Back to the ice pick rats with their acute onset insulin hypersensitivity in adipocytes. During rapidly increasing bodyweight (on a low fat diet) there is a marked increase in obesity with excellent insulin sensitivity. Ended by six weeks.

Ditto MSG rats, but for in 4 weeks rather than 6 weeks. Can't tell from the gold thioglucose abstract, but at least a few weeks. Probably depends on all sorts of minutiae.

While ever these rodent models are gaining weight they maintain insulin sensitivity because they are doing DNL to get fat. On a low fat diet increasing obesity means DNL, palmitoleate and the ability to run metabolism on glucose. Logical.

Only once a brain-damaged rat becomes obese enough does hyperinsulinaemia set in, with attendant glucose intolerance. By this stage adipocytes are insulin resistant so have reduced ability to respond to insulin, reduced GLUT4 expression and, presumably, reduced palmitoleate synthesis. From the adipocyte's point of view there is not a lot of insulin around, whatever the blood concentration might be.

Lets look at the converse to obesity:

What does a lack of insulin signify? No food (or no carbohydrate, pax protein). Starvation requires insulin resistance as an obligate state for survival. How much good is palmitoleate going to do you under starvation or ketogenic dieting? Not a lot, unless you enjoy dropping precious glucose in to muscles until you brain falls to pieces.

An adipocyte which sees no insulin will not generate palmitoleate. If it generates anything at all (doubtful) it will be palmitate. Releasing some residual palmitoleate from adipocytes is fine for a few days, as long as there is glycogen hanging around. By three days this will be gone and so too should the palmitoleate. You are now in to hard core survival driven insulin resistance.

That's where I live.

Adipocyte distension induced insulin resistance is completely different. Here the adipocytes see low insulin when there is a ton of it around. There is a ton of glucose around too. But an adipocyte acts as per starvation and does what would be absolutely the correct thing under starvation circumstances. It releases palmitic acid and stops generating palmitoleate. Doing this while the macroscopic organism is eating bagels and french fries is bad. It's bound to generate massive hyperinsulinaemia to normalise glucose in the face of a ton of palmitic acid.



I'm just wondering whether there is time to look at the C57BL/6 mice. Just briefly as there is a lot of Mickey to be extracted on this subject when we get to idiots in detail. Briefly:

By an utter quirk of metabolism the VMH of C57BL/6 mice breaks under high dietary fat levels. So they have access to ample dietary fat when their VMH is injured, by definition. They store this fat because sympathetic tone to adipocytes is acutely lost and adipocytes become exquisitely insulin sensitive. Fat falls in to adipocytes as soon as the injury occurs, probably within hours of eating some butter, almost any amount of butter, however small.

But the fat they store is dietary fat. No DNL. They do not need to gain fat by DNL as it is there in the hopper. Dietary fat falls in to adipocytes. Palmitoleate synthesis? When fat is distending adipocytes so fast they are leaking FFAs despite losing lipolytic sympathetic tone? There is a ton of dietary fat dropping in to adipocytes, this is what gets released as they distend. By day three C57BL/6 mice are systemically insulin resistant. Their palmitoleate levels will be low and palmitic acid levels high. They are just a modification of the ice-pick/MSG/gold thioglucose family, but the process happens at warp speed due to the availability of DNL-free bulk fat.

Even on high fat plus high sucrose diets humans do not injure their VMH, at least not immediately. But C57BL/6 mice do and they have taught me a great deal over the years, made me think a great deal too. But they are still just a model, as explicable as the rest of the models, from the insulocentric point of view.

Once you have enough data.

To summarise: Palmitoleate is released by adipocytes when glucose and insulin are plentiful. Palmitate is released when glucose is sparse and insulin is low.

The sh!t hits the fan when glucose and insulin are plentiful but adipocytes are so distended that they THINK glucose and insulin are low. When both insulin and glucose are high you want palmitoleate. If your adipocytes give you palmitate under these circumstances you had better have a pancreas of steel or diabetes here you come.


I think we might go to PUFA and SCD1 in adipocytes before hepatic DNL in this series.


BTW It's nice to see people in comments being a post or two ahead! At least this isn't complete gobbledegook to everyone!

Peter

Protons: Palmitoleate

I think we have to start with the results section of Cao et al's very interesting (and free to study if you want all the detail) paper:

Identification of a Lipokine, a Lipid Hormone Linking Adipose Tissue to Systemic Metabolism

As always, the paper is a superb piece of detective work featuring a superabundance of genetically engineered mice from the C57BL/6J background fed an high fat diet, the nature of which doesn't make it in to the methods, but we can just assume that it's all the usual fare. They started from the protective effect of knocking out certain fatty acid receptors in the mice, which prevented the development of metabolic syndrome, and ran with this concept for the massive project detailed in the paper. It's big. It ended up with them doing the following to confirm that they got it correct. From the very end of the results:

"To define the effects of individual fatty acids on metabolic regulation, we prepared Intralipid with triglycerides composed of a single fatty acid, either TG-palmitoleate or TG-palmitate. Infusion of either lipid resulted in a two-fold increase in total plasma FFA levels with similar dynamics (Figure S13). While TG-palmitate suppressed the entire proximal insulin-signaling pathway including activation of insulin receptor and phosphorylation of insulin receptor substrate 1, 2 and AKT in liver, TG-palmitoleate strongly potentiated these insulin actions (Figure 7A). We observed similar effects of both lipids on muscle tissue where palmitoleate enhanced and palmitate impaired insulin signaling (Figure 7B)."

It's a switch, at the crude level of Intralipid infusions. Viewed macroscopically:

Palmitoleate = insulin sensitive
Palmitate = insulin resistant

I may have mentioned this before!

If you take a light switch apart, under the plastic there are some metal parts. The metal provides a sea of probability through which electrons can flow, provided the metal is continuous from light bulb to the powerstation (pax transformers). Or not flow, if we replace a few mm of metal with a few mm of room air.

If we accept that superoxide from complex I reverse electron transport is insulin resistance, then fatty acid binding proteins are a macroscopic overlay over this process, they are part of the plastic of the switch.

Superoxide never leaves the mitochondria, it probably converts to H2O2 to talk to the nucleus or acts locally to activate transcription factors which then talk to the nucleus. Adipocytes don't talk to muscles using superoxide either. The intermediary they use appears to be palmitoleate, probably the ratio of palmitoleate to palmitic acids, once you get away from bulk Intralipid infusions.

Why is it arranged this way? The body has to know what substrates are available. Ignoring protein, carbohydrate talks to the body through insulin, and through insulin transporting glucose in to adipocytes. That's the next post.

There: Not a mention of FADH2 or NADH. Even if I'm thinking about them, as per the last post...

Peter

BTW, Charles commented on the depressing amount of superoxide associated with a high fat, low carb diet. True, but about as scary as going for a walk at the brisk-but-not-excessive pace which is reputed to burn fat best. Burning fat is what LCHF eating is all about. Useful if you don't have the hours a day to walk for health purposes. Walking seems to be quite good for you!

Protons: FADH2:NADH ratios and MUFA

A few more thoughts building on F:N ratios of differing metabolic substrates:

Each cycle of beta oxidation (assuming an even numbered carbon chain fully saturated fatty acid) produces one FADH2, one NADH and one acetyl-CoA. This gives a total of 2FADH2 inputs and 4 NADHs per cycle of beta oxidation. But the very last pair of carbon atoms in a saturated fat do not need to go through beta oxidation as they already comprise acetate attached to CoA, so they can simply enter the TCA as acetyl-CoA. This last step only produces 1 FADH2 and 3 NADHs, with no extras.

So the shorter the fatty acid, the less FADH2 per unit NADH it produces. Short chain fatty acids like C4 butyric acid have an F:N ratio of 0.43 while very long chain fatty acids, up at 26 carbons, have an F:N ratio of about 0.49.

As Dr Speijer points out, differing length fatty acids are dealt with differently. Very short chain fatty acids head straight for the liver and get metabolised by hepatic mitochondria immediately. Any excess acetyl-CoA gets off-loaded as ketones.

Very long chain fatty acids end up in peroxisomes for shortening, usually to C8, which is then shunted to mitochondria for routine beta oxidation. Of course peroxisomal beta oxidation generates zero FADH2, except that from acetyl-CoA, because peroxisomal FADH2 is reacted directly with oxygen to give H2O2. And heat, of course.

Bear in mind that the ratio of F:N generated by a metabolic fuel sets the ability to generate reverse electron flow through complex I and subsequent superoxide production, macroscopically described as insulin resistance.

So fatty acids up to C8 are cool, dump them to the liver and make a few ketones. Very long chain fatty acids over C18, shorten to C8 in peroxisomes, shift them to mitochondria and make some ketones if needs must. The F:N ratio of C8 is about 0.47, a value chosen by metabolism as the end product of peroxisomal shortening. The number is important. Actually the number is even lower as peroxisomal beta oxidation generates the NADHs of beta oxidation, just not the FADH2s, but why allow facts like this to spoil a great argument. C8 from breast milk and/or coconuts seems fine and has that F:N ratio of 0.47.

Now the area of interest is, of course, C16, palmitic acid. This has an F:N ratio of about 0.48, almost as superoxide generating as a C26 fatty acid up at 0.49. And palmitic acid does, without any shadow of a doubt, produce macroscopic insulin resistance. That's 15 FADH2s and 31 NADHs.

So an F:N of 0.47 is not a serious generator of superoxide and an F:N of 0.48 is.

What happens when we drop a double bond in to palmitic acid? Mitochondrial beta oxidation generates FADH2 as it drops a double bond in to the saturated fat chain. If the double bond is already there, hey, no FADH2!

Palmitoleate has one double bond. This of course gives 14 FADH2s and 31 NADHs, an F:N ratio of 0.45.

Palmitate 0.48
C8 caprylic 0.47, chosen by peroxisomes to hand to mitochondria
Palmitoleic 0.45

Adding a single double bond to palmitic acid drops its F:N ratio from significantly superoxide generating to minimally superoxide generating. It looks like a switch to me.

I just love the way the numbers pan out. Of course we can now go on to what these number signify and what determines unsaturation. And uncoupling too, I guess. We are then back to insulin and stearoyl-CoA desaturase and also de novo lipogenesis. It might be worth an aside to PUFA and how these behave too, especially in adipocytes.

Peter

Protons: Lactate

I've been aware for some time that there is a reasonable idea that the brain runs on lactate. Dr Speijer emailed me a link to a very recent paper which supports this concept at the cutting edge of modern research, without having to go back to that old stuff from over five years ago which no one ever reads because it has no lovely photomicrographs and no ultracool transgenic mice.

The editorial has this nice diagram which sums up what might be going on:




Let's get back to electron donors. The brain hates superoxide. It hates fatty acids. It's a bit ambivalent about glucose (gasp). I don't think I would say it rejects glucose, just there are better fuels.

Is there anything the brain does like? Well, ketone bodies seem to be okay, but what the brain really seems to like is lactate. Perhaps I should rephrase all of this and say that the neurons of the brain love lactate. The rest of the brain seems fine on glucose and will even dabble with fatty acids at a pinch. But glucose is fed to neurons, pre digested by the glial cells, as lactate. The FFAs are fed as ketones, yes the glial cells in the brain are ketogenic, it's not just the liver that does this. I suppose the neurons might use glucose directly, but they become quite sick if you knock out lactate supply by eliminating MCT1 (mono carboxylic acid transporter 1).

Neurons are irreplaceable, more or less. They aim for zero superoxide production. This means behaving like a mitochondrial preparation which is being fed on glutamate, a provider of NADH only. Near zero free radical production is the closest you can come to having no mitochondria at all, yet still have the powerhouse of the electron transport chain at your command. When thinking about apoptosis that is. Apoptosis is not a good idea in non-replaceable cells...

This means minimising FADH2 utilisation. Fatty acids, with their beta oxidation derived FADH2, are out. No way in neurons.

Glucose is not ideal either. Why not? Well glucose supplies the best possible neuronal FADH2:NADH (F/N) ratio of 0.2, ie it gives one FADH2 for 5 NADHs. Usually. This is superb for minimising superoxide production (and maintaining insulin sensitivity). But not always. What about glycerol-phosphate dehydrogenase or glycerol-phosphate oxidase? Both of these, in much the same manner as the FADH2 moiety within electron-transporting flavoprotein dehydrogenase, can reduce the CoQ couple and promote superoxide production. That's without thinking about simply over driving the TCA with pathological hyperglycaemia. There is absolutely no doubt that hyperglycaemia generates superoxide production. Unfortunately most of the people discussing this on pubmed have no real concept of F:N ratios or what exactly goes on in the respiratory chain to generate superoxide. There is no nice neat diagram to copy paste. My own assumption is that massive enough inputs of glucose drive huge amounts of NADH production which cannot be accommodated once FADH2 from succinate dehydrogenase reaches a critical level or is supplemented by glycerol-phosphate dehydrogenase based FADH2. At this point a cell says no to glucose calories, ie it makes superoxide and becomes insulin resistant. As has been observed, insulin resistance is an antioxidant defence mechanism, you need it. If pushed hard enough to overcome insulin resistance a cell will take one step closer to apoptosis.

Not so with lactate. Lactate supplies acetyl-CoA (which itself has an F:N ratio of 0.25) along side a couple of extra NADH molecules (one each from lactate dehydrogenase and pyruvate dehydrogenase) which reduce the overall F:N ratio to 0.2, the same as glucose). Yet pre-prepared lactate does not need any glycolysis to take place in the neurons themselves. It has no possibility of supplying ANY FADH2-like input to the CoQ couple, outside of succinate dehydrogenase (complex II) activation in the turning of the TCA. It's the purest of complex I inputs available to any intact organism. No wonder the brain loves lactate. Lactate usage appears to be the best way of postponing apoptosis, short of abandoning mitochondria altogether. Glucose comes second.

I had always though of lactate in the brain as a sort of direct mitochondrial fuel injection system. Lactate dehydrogenase then mitochondrial uptake of pyruvate. Just a fast response time. But looking at FADH2 to NADH ratios gives a much deep insight in to what is going on.

What about fat????? Not for the brain.

But for the rest of the body? What makes mitochondria happy? Hint: It's not glucose.

Peter

Mmmmmm eggs!

Eggs will kill you!!!!!

As a UK resident: Thank god it's not London, London but London, Ontario. Phew. Thought the goons in epidemiology at Imperial College had been at it again. Happily the shame for this has to go to Canada. Oh dear, sorry Canada.

Peter

We are not alone

Obviously anyone with even a basic interest in origin of life questions will be watching the progress of Curiosity on Mars. Those of us who buy in to the serpentine and alkaline hydothermal vents concept will be interested in whether the crustal chemistry of Mars is olivine based and whether major water bodies were even present. Or equally, whether a semblance of white non-smokers might be present when acidic ground-water interacts with olivine, without needing an ocean and vents... An interesting time for testing hypotheses about whether there is life "out there", in our own back yard...

EDIT: A quick google shows olivine, serpentine and methane plumes are all present on Mars. The methane could easily be abiotic in origin, the question is whether it actually is or not...

On the more down to Earth front, if anyone thinks my basic ideas about the ratio of FADH2 based input vs NADH input to the ETC determining superoxide production are not totally incomprehensible, we are definitely not alone. I had a very nice email from Dr Speijer in Amsterdam, a fellow thinker along these lines. He has come to exactly the same conclusions and published an hypothesis paper in Bioessays back in 2011. The first section is just excellent. We may diverge in interpretation (but not FADH2:NADH ratios) very slightly late in the essay on PUFA, but it really is full of very good thinking and an excellent paper.

His ideas about peroxisomes (a very early eukaryotic invention) of course addresses that age old question of "Who's (macroscopic) fat is it anyway?", the answer being that the gut bacteria own it. On the sub cellular front, fat is primarily made in cytoplasm but at the behest of the mitochondria, only secondarily in peroxisomes and, as peroxisomes are probably a response to deal with overly long (ie excessively high FADH2 generating) fatty acids, the answer would seem to be mitochondria order fatty acid production, they own them and they have their own agenda for them. It's a sort of intracellular parallel the the fiaf series on gut bacteria and adipocytes. Very interesting concept.

If mitochondria own fatty acids I would expect them to enjoy burning fatty acids. Whatever the generation of controlled superoxide is, it's what keeps mitochondria happy. Then there is the brain to think about, its avoidance of fatty acids, it's love of ketones for an occasional fling and its very probable long term love affair with lactic acid. All based on FADH2 to NADH ratios of course.

There's a lot to post about. Back to the Protons series next (I think).

Peter

Insulin in the brain: Hyperphagia?

Let's start with this quote from Brain insulin controls adipose tissue lipolysis and lipogenesis:

"Insulin is considered the major anti-lipolytic hormone. Its anti–lipolytic effects are thought to be exclusively mediated through insulin receptors expressed on adipocytes (Degerman et al., 2003). Cyclic–AMP (cAMP) signaling represents the major pro–lipolytic pathway in WAT, which is chiefly regulated by the sympathetic nervous system (SNS)."

and then go on to this one from the discussion:

"We draw this conclusion from the finding that denervation of WAT leads to no change in lipogenic protein expression, but completely abrogates Hsl activation leading to increased adipose depot mass (Buettner et al., 2008)".

OK, got that? Brain insulin makes you fat by damping down lipolytic neurotransmission to adipocytes. Turning off your sympathetic nervous system supply to your fat cells allows insulin to go on an obesity spree.

Then there is this quote (MBH is medial basal hypothalamus, better known as VMH, ventro medial hypothalamus):

"Our studies raise several questions. One is which neuronal subtype within the CNS and the MBH mediates the effects of insulin on the regulation of WAT metabolism".

That first one really is an interesting question, one which we can go some way towards answering. We know that the cell type is, as already noted, part of the sympathetic nervous system. In the paper they found either surgical or chemical sympathectomy of adipose tissue increases both lipogenesis and inhibits hormone sensitive lipase in that tissue. I think this is straight forward. The sympathetic nervous system is tonically opposing insulin's lipogenesis effect and insulin's inhibition of hormone sensitive lipase.

The next thing we can say is that these cells sport glutamate receptors. We can safely assume this because, if neonatal rats are injected with the excitotoxin MSG, these are some (among many) of the cells which actually die. That is, the sympathetic nervous system supply to adipose tissue dies. Lipogenesis is unrestrained. Hormone sensitive lipase shuts down. Carbohydrate easily pours in to adipocytes and stays there. Blood glucose levels are low, free fatty acid levels are low, insulin sensitivity is excellent. While ever adipocyte expansion is on going that is. As the adipocytes stretch they eventually become insulin resistant. Here's the table of metabolic parameters from pre-obese MSG injured and control rats, from a previous post:



We know you can do exactly the same by killing these cells with gold thioglucose. This neurotoxin kills those nerve cells which inhibit lipogenesis.

As the authors say: After gold thioglucose injection "systemic insulin sensitivity is preserved [actually it's increased, but these are obesity researchers, so don't quibble] during the early phase of the obesity syndrome, resulting in extensive fat production".

These hypothalamic cells don't seem to take too kindly to the application of an ice pick either:

"In this study, we have measured the expression of the insulin-sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty-acid synthase and acetyl-CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH-lesioned rats".

All of these interventions allow calories to pour in to adipocytes and stay there. So what does the poor rat do? It's losing a sh*t load of calories in to its adipocytes but, luckily, it has access to a massive hopper of crapinabag in its cage. It simply has to eat enough calories to supply the loss in to adipocytes, plus enough to run its metabolism on. This can be described, by non comprehending people, as hyperphagia. Metabolically it is normophagia.

These rats are calorically neutral or even in mild energy deficit. They have to be running "hyperphagic" just to stand still, metabolically. They are NOT showing "voluntary" overeating. They DO NOT have an injury to any sort of "satiety" centre. They have low insulin, low FFAs and low glucose. They are NOT being paid to over eat! They will NOT be producing a ton of superoxide, despite having a hugely increased caloric intake. Until...


When does this stop? It stops when FFA leakage due to the resistance to insulin induced by adipocyte distention exactly matches the FFA releasing effect which the (now non-existent) sympathetic nervous system would have been having on non distended adipocytes. Sorry for the convoluted sentence, can't simplify it! The distension process was complete by six weeks in the ice pick rat study cited above. Obese rodents then end up with a crudely normal metabolic rate. But this injured system is a complete bodge. We are looking at the replacement of a finely tuned fuel switching system which exactly matches fuel availability to fuel needs with a system where broken adipocytes are simply leaking FFAs at a level which constantly supplements glucose use, without any semblance of fine tuning to metabolic needs. The chronic elevation of fatty acids drives, through the NADH/FADH2 ratio, superoxide production and insulin resistance. Eating glucose then becomes unacceptable because there is inappropriate whole body insulin resistance from excess and inappropriate FFAs. A large amount of insulin is need to control hyperglycaemia under these conditions. Failure to supply adequate insulin to do this, for any reason, is labelled diabetes.

What has this to do with the current obesity epidemic? If you are overweight I would suggest you should take the ice pick out of your brain. No ice pick? Hmmmmm, damn! Back to the drawing board on that one then.

Ah, but maybe you are a C57BL/6J mouse?

Before we can tackle such a stupid question I think we need to go back to superoxide and fatty acids, to about where we were before this digression began.

Peter

Protons: Metformin

I'll just stick this post up to get it out of the way. I was going to go on to acute uncoupling next but the link from O Numnos in the last post comments is too good not to post about. It goes some way to tying weight gain in to LACK of superoxide, so brings the thread of insulin as a "satiety" hormone and this thread on weight gain as a failure to generate superoxide in adipocytes (good and bad) together. Might take more than one post... The summary of what's coming: Is insulin a satiety hormone? Only in so far as becoming stable-obese limits your hunger. Anyway, here are a few more thoughts on superoxide first.

Metformin is generally considered to be a Good Drug.

Interestingly it is an inhibitor of complex I of the respiratory chain, which is almost certainly its primary site of action. It aborts glycolysis to lactate because pyruvate is not much use to mitochondria with blocked complex I. Acute exposure to metformin in tissue culture generates a ton of superoxide. Just what you would expect to benefit someone with T2DM!

Let's have a look at this rather nice paper.

They are using differentiated 3T3-L1 adipocytes, a strange beast if ever there was one, but "everyone does it".

They are working under room air with 25mmol/l glucose, supplemental pyruvate, glutamine and 1000pmol/l insulin. These cells are being driven, hard, generating NADH which works through complex I. Complex II will be supplying some FADH2 but there is zero beta oxidation, unless the fatty acids in the adipocyte stores are being accessed. With insulin at 1000pmol/l this is not going to be happening.

Here is the effect of metformin on oxygen consumption:



A dose dependent fall, exactly what you would expect when blocking complex I. Here is the effect of 1.0 mmol/l metformin on oxygen consumption with time:



Nice curves! And here is the effect on ECAR, a surrogate for lactate generation, over 24 hours:



Metformin is only a relatively weak inhibitor of complex I, the incidence of life threatening lactic acidosis is very low. Not so for the more effective biguanides, phenformin and buformin. Obviously the latter two are no longer used clinically, there were too many hiccups.

Now, here is the level of DHE fluoresence, a specific marker of superoxide production. It's being compared to rotenone (remember Coopers Demodectic Mange Dressing? Thought not!), a serious complex I inhibitor.



Metformin is pretty good at generating superoxide. A bit counter intuitive for a drug which is the best treatment, short of insulin, for managing T2DM, a condition essentially defined by failure to overcome insulin resistance (aka superoxide production).

Hmmmmmmmmmmm.

Now, do 3T3-L1 adipocytes like being in forced to live on ATP from glycolysis plus whatever oxphos can be squeezed through metformin inhibited complex I? Annexin V is a marker of very, very unhappy cells. This is what metformin does to the % of cells which are moribund in culture:



So what is going on? Is metformin going to kill our fat cells in vivo?

It's all back to tissue culture conditions. Glucose at 25mmol/l makes the cells utterly dependent on a combination of glycolysis and NADH oxidation at complex I, plus a little FADH2 from succinate metabolism. Our adipocytes are not in this situation.

Now look at this graph:



This is in starvation medium. Only 2.5mmol/l glucose, no pyruvate, no glutamine. I think insulin is still supramaximal, but who cares about insulin when glucose is down at 2.5mmol/l in tissue culture? But here is the really interesting bit: They had also added 0.3mmol/l of palmitic acid to both the control cells and to the metformin cells. Compare it to the graph below, which is the same situation but with glucose and NADH drivers replacing palmitate:



So: Starvation medium plus palmitate completely reverses the fall on oxygen consumption produced by metformin. Palmitate plus starvation medium, even with metformin, actually allows more oxygen consumption that cells running flat out on glucose in the absence of metformin. It's what you would expect, the respiratory quotient is lower for fatty acids than for carbohydrate.

Metformin does not stop fatty acid oxidation. You do need some complex I activity to provide the NAD+ for beta oxidation, but no one is suggesting there is a complete block of complex I by metformin, it's not mange dressing.

So where do the free radicals come from with metformin? I would guess that the citric acid cycle still cycles, there is a build of of NADH due to complex I inhibition and complex II still reduces the CoQ couple. This could allow reverse electron transfer through whatever complex I functionality is left. There are absolutely no data on this, but I like the idea.

The group didn't look at superoxide production under starvation conditions or under starvation plus palmitate. I had a nice email reply to my query from the corresponding author along these lines, there are no data about this, as yet. I would expect the levels of superoxide to be comparable, with metformin being able to mimic palmitate based metabolism in the face of massive fat-free glucose supply, certainly for superoxide generation.

So, superoxide is insulin resistance. Adipocytes under metformin make a ton of superoxide. Are they insulin sensitive or resistant? Resistant of course.

Does an adipocyte which is insulin resistant listen to insulin's orders to store fat? Of course not. "Normal" insulin resistant adipocytes spew free fatty acids to the limit of albumin's transport provisions, with a few other moderating factors.

A metformin poisoned adipocyte is desperate for proton pumping substrate and complex I is doing bugger all to help. But electron-transfering flavoprotein dehydrogenase works perfectly well to allow an alternative electron supply...

Adipocytes under metformin have no choice but to burn fat. In vivo they have a barrel load of the stuff available as soon as they stop listening to insulin. They appear to use fatty acids for metabolism rather than dumping them as FFAs to plasma. Sounds like a recipe for treating metabolic syndrome to me.

Oh, that's what metformin is used for! Well I never...

So, do I think metformin causes adipocytes to become insulin resistant? Of course I do. Is this a Good Thing? You decide.

Peter

BTW Want an opposite to metformin? You can make adipocytes more sensitive to insulin with the thiazolidinediones. They allow insulin to become more effective on already over-distended adipocytes and generate lots of extra, nice, new, ready-to-stuff-with-fat adipoctes. They make you fatter. What would you expect?

Insulin in the brain: off topic giggle

I had my septic tank emptied a fortnight ago. The contents were a load of crap, but less crappy that the paper purporting to show that insulin is a satiety hormone as quoted by some obesity researcher.

What REALLY happens when you infuse insulin in to the cerebro spinal fluid of a mouse? You know, the satiety hormone... Just in to the brain, nothing systemic, no hypoglycaemia.



Insulin = big fat adipocytes. Big fat mice. Lovely micrographs.

http://www.jci.org/articles/view/31073/figure/5 will give you the legend.

http://www.jci.org/articles/view/31073 will give you the full text. Might discuss the paper better in a few months time!

But main conclusion:

The brain fine tunes the storage of lipid under the influence of insulin (by increasing fat storage via lipoprotein lipase and also by DNL from glucose). It uses the sympathetic nervous system outflow from the ventromedial hypothalamus to do this. Interpret with caution as these are C57BL/6, mice who may well have some very specific weakness in their ventromedial hypothalamus.

OMG did I laugh when I found this one.

Wanna loose some weight, go eat some potatoes. LMFAO!

Sorry for the crudity. Been on call too long this weekend!

Peter