Monday, October 22, 2012

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.


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

Saturday, October 20, 2012

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.


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:

More info here

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

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.


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

Wednesday, October 03, 2012

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.