Another brief housekeeping post

OK. I published the "awaiting moderation" comments. Lots of them are insightful and need a response. Time for this is not looking good at the moment! The main upset is that I mis-clicked delete instead of publish on a comment by Nicolás Flamel which I can't find any way of recovering. Sorry for that. Please feel free to re comment if you wish.

Peter

Insulin makes you hungry (5) except when you resist

TLDR: The function of insulin is the inhibition of lipolysis. Does resisting insulin facilitate lipolysis?

This the next paper:

Improving Influence of Insulin on Cognitive Functions in Humans

Aside: Oooooh, look. No control group! How can I already tell they are going to find that insulin suppresses appetite? End aside, sniggering excepted.

The only parts I am interested in are the clamps and the appetite scores (no food intakes in this one).

So we have a low insulin clamp and a high insulin clamp looking a lot like this:

These are the hunger ratings during the clamps:

I think we can say that hunger kicked in at around 150 minutes under the low dose insulin clamp and at around 300 minutes in the high dose insulin clamp. When would hunger kick in w/o insulin? We'll never know because...

So anyway, there we have it. Insulin, entering the brain by the physiological route, suppresses appetite  in real live humans with a dose response. Time to shut up shop and go back to kayaking in my free time.

But just one moment. I think it might be worth looking at this study in the units of insulin concentration that we are most used to working with. I used this website to do the conversions.

We have here a fasting insulin somewhere around 40-50pmol/l. In old money that is 6.0microU/ml, quite reasonable.

The low clamp is around 900pmol/l, just over 130microU/ml. This is pretty well "normal" for physiological post prandial insulin after eating a meal of junk.

The "high" insulin clamp is around 30,000pmol/l (it's a log scale) by the six hour mark. I'll try to be careful with decimal points here, but I think this equates to just over 4000microU/ml. I'm not sure that even an insulinoma patient would run an insulin of 4000microU/ml. If anyone thinks this sort of insulin level has anything to do with appetite control in the physiology of humans, then they may be mistaken. But this level of insulin exposure does delay the onset of hunger, by over two hours in this study...



Now, here is a sideways way of looking at CNS insulin.

If the physiological role of insulin in the VMH is to augment fat storage, what might be the effect of CNS insulin resistance? Go on. I dare you to say that the effect might be partial failure to suppress lipolysis, less suppression of FFAs and so reduced appetite augmentation. Resisting insulin allows you to resist its hunger generating effects. How's that for a bizarre idea? Feel free to point out faults in the logic.

I've long been interested in the concept that exposure to insulin itself induces insulin resistance. There are a whole slew of papers to suggest this, some better than others. Overall I find the concept quite convincing.

If we want to actually see insulin resistance kick in rapidly, and measure it, we have to go to cell culture. Here we can overdose by decent amounts, using nanomoles rather than picomoles, and watch the reduction in signalling triggered by these high insulin concentrations. Insulin receptor (IR) phosphorylation drops within 45 minutes from the initial peak at 10 minutes after exposure. It's in this paper if anyone is interested in the details

Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

but this is a typical graph:

If we convert the nanomolar concnetration from cell culture to the picomoles of in-vivo clamps then the two higher concentrations used in this graph would be 17,000pmol and 170,000pmol, similar to those from the high dose clamp used in the human study. Especially when you consider that 17,000pmol and 170,000pmol have indistinguishable effects on IR phosphorylation (and acute generation of resistance to that phosphorylation effect) in cell culture.

So it seems quite feasible to me that a physiological clamp at 900pmol/l would produce normal "insulin hunger" due to lipolysis suppression and that a clamp at 30,000pmol/l would induce significant insulin resistance. That limitation on insulin signalling would obtund the normal inhibition of lipolysis (but not eliminate it entirely) and so defer the normal onset of hunger routinely induced by the physiological action of insulin. So very large doses of insulin (170,000pmol in cell culture imitating 30,000pmol/l as a clamp) should postpone the onset of hunger compared to mildly supra-physiological doses (5000pmol in culture imitating 900pmol/l as a clamp) because the higher doses should cause less inhibition of lipolysis, with the proviso that you choose your target concentration very carefully. Obviously no one does a study without a pilot trial to make sure they will get the results they want. A saline infusion produces no significant increase in hunger over 150 minutes, though the trend in hunger is upwards because supper was a long time ago by the end of that particular study!

I'll take a pause here because I think this current concept is quite controversial enough to be stated as simply as possible. Obviously there are more studies which we can look at to explore the usefulness of this idea.

Peter

Holiday reading

I'm off on vacation for a while so the next posts are likely to be delayed. If anyone would like a light summary of the ideas I'm thinking about, this is a nice review:

Yin and Yang of hypothalamic insulin and leptin signaling in regulating white adipose tissue metabolism

For a level deeper of understanding you just need to add in that saturated fats have an FADH2:NADH ratio around 0.49 which is the physiological signal for insulin resistance and MUFA generate one of around 0.47, giving the signal for insulin sensitivity. This is physiology.

Adding in PUFA as a bulk calorie source, with an insulin hyper-sensitivity generating FADH2:NADH ratio of well below 0.47, leads to expanded adipocytes. This is pathology.

Because a core function of insulin is the inhibition of lipolysis.

Peter

Insulin makes you hungry (4) unless you keep it out of your brain

TLDR: The function of insulin is the inhibition of lipolysis. Especially via the brain. Where insulin detemir doesn't go.

People will be aware that insulin detemir is really strange stuff. There are perfectly respectable papers showing that it cannot enter the brain (and blocks the entry of normal insulin in to the brain too) or that it is fantastic at entering the brain, much better than more normal insulins.

There are probably more studies in the latter camp but my biases push me towards the former camp. The nature of the researchers also tends to push me towards the former camp. I posted on insulin detemir here and here to explain my point of view.

Now there is this paper:

Euglycemic Infusion of Insulin Detemir Compared With Human Insulin Appears to Increase Direct Current Brain Potential Response and Reduces Food Intake While Inducing Similar Systemic Effects

OK. After an overnight fast, a 90 minute euglycaemic hyperinsulinaemic clamp and a 20 minute wait, subjects consistently eat less food (303kcal, 17% reduction) after an insulin detemir clamp (1475kcal) than they do after a neutral insulin clamp (1782kcal). Just eyeballing the insulin doses used we can assume that the plasma insulin levels were a reasonable approximation for humans in the normal post prandial period, ie physiological fed-state rather than pharmacological.

The research group is completely wedded to the idea that central insulin is an appetite suppressant and that weight gain from any insulin therapy is only a reaction to recurrent hypoglycamia. As there is no hypoglycaemia during the clamps their presumption is that this neutral insulin infusion results in a reduced food intake. As insulin detemir gives less food intake after a normoglycaemic clamp than neutral insulin does, then their conclusion is that insulin detemir is having a more potent central appetite suppressing effect than the neutral insulin.

They are so confident about this that the inclusion of a control situation, where saline was infused without any insulin and appetite was checked after this, was considered un-necessary. This really is the level of research in the "satiety" insulin camp.

Fortunately for us we do still have results from 1985 where food intake after a physiologoical post-prandial level clamp at 150microU/ml for 150 minutes using neutral insulin was compared to saline control and these give us this table cited in a previous post ("liquid drink" is a calorie containing soup-like food):

which allows us to calculate that saline reduces food intake by 40% compared to neutral insulin. Or to rephrase that more plausibly: a clamp using neutral insulin increases food intake by 60%. You can see why a control group was omitted by the "satiety from insulin" paper. I rather like insulin determir compared to any other insulin but you can see it has its work cut out to beat saline as a satiety hormone!

Simplest explanation: Insulin in the brain decreases peripheral lipolysis. This makes you hungry after an hyperinsulinaemic clamp. Insulin detemir doesn't enter the brain so has no CNS augmentation of its peripheral suppressive effect on lipolysis at a given level of glucose control, so it generates less hunger.

I'm a simple sort of a person. That's how I see it. Could be wrong of course.

Peter

Insulin makes you hungry (3) a matter of semantics and free fatty acids

OK. There is absolutely nothing technically incorrect with the description of the results contained within the title of this paper:

Effects of insulin-induced hypoglycaemia on energy intake and food choice at a subsequent test meal

They gave a small dose of insulin (low enough to not need rescue glucose within the study period) as a single bolus, waited for 20 minutes then offered the subjects an eat-as-much-as you-like buffet. This is what the glucose levels did

and this is the subsequent food intake in kcal:

That will be 1700kcal after an hypoglycaemic glucose of 2.0mmol/l or 1400kcal after a blood glucose of 4.5mmol/l. Glucose drop and kcal increase are both p less than 0.05.

Hypoglycaemia makes you hungry.

Next is this one, not quite so good because the title omits the word "insulin", but never mind.

Short-term nocturnal hypoglycaemia increases morning food intake in healthy humans

Here they infused insulin to a glucose nadir of 2.2mmol/l, stopped the insulin infusion and then infused glucose to normalise blood glucose concentration within 30 minutes. On one occasion the nadir was induced at the start of REM sleep (as early as possible in the night) and on another occasion it was induced about 3.5 hours later. Total sleep time was about six and a half hours on each occasion. At feeding time all of the subjects were normoglycaemic. The white column is the control, the hatched is from when the hypo was about six hours before feeding and black is from when the hypo was about three hours before feeding.

The conclusion is that an hypo soon before you eat makes you hungry (p less than 0.05). An hypo just after you have fallen asleep the evening before might do as well but the p value is ns for this test. In general we can say any hypo leaves you hungry.

But both studies did two separate things. They generated hypoglycaemia and they gave insulin. The assumption is that it was the hypoglycaemia which drove the hunger. Even if the hypoglycaemia was long gone at feeding time.....

Hallschmid (second paper) is a dynamo of publications showing central insulin (via the intranasal route) is an appetite suppressor. You've noticed that this second paper did not have a group given insulin combined with intravenous glucose to protect against hypoglycaemia, isolating the effect of the insulin alone. But then we all know what that would have shown.

Let's look at this from the real world point of view, which is: The function of insulin is the inhibition of lipolysis.

What happened to FFA levels in either study? We'll never know.

Many years ago I posted on a Spanish study cited by Dr Davis. It has all of the pictures you might need embedded in the post. From this we can say that spiking your insulin by eating a small (40 gram) high carbohydrate snack will produce a rise in insulin from 50picomol/l (normal fasting) to around 70picomol/l (a little bit higher but not much, full post-prandial insulin would be at least several hundred picomol/l) at one hour, with return to 50picomol/l by two hours. Despite this tiny rise in insulin the FFAs drop from over 400micromol/l to 100micromol/l at two hours (by which time insulin has actually normalised) with persistence of some degree of hypolipidaemia for up to another three more hours. This is a tiny increment of insulin compared to the above studies.

We know that hypoglycaemia without hyperinsulinaemia does not drive appetite. My hypothesis is that it's the insulin itself which drives a fall in FFAs, which in turn drives the hunger to rise, all secondary to insulin's anti-lipolytic effect. Note, there is no need for any direct appetite modulation from insulin to explain these results.

So: you eat a Mars Bar at 15.30, just before evening consults ('cos you had a long theatre list, you had no lunch and there was nothing else to eat. Looong time ago, but I can still remember those days). By 17.30 you are ravenous and a bit shaky. Ha! Reactive hypoglycaemia! Oh, but your BG is 4.5mmol/l. Huh?

Best measure FFAs.............

Peter

Insulin makes you hungry (2) even in the presence of hyperglycaemia

TLDR: The function of insulin is the inhibition of lipolysis. Really.

This is the paper cited by Woo:

Effect of Insulin and Glucose on Feeding Behaviour

The research group used real live humans. They looked several protocols but the ones we are interested in are those where they clamped insulin between 100 and 150microU/ml (high but plausible for post prandial on something like the SAD) and clamped glucose at either 150mg/dl (highish normal) or 300mg/dl (frank hyperglycaemia). They also had a control group (saline infusion) and an eu-insulinaemic with mild hypoglycaemia (50mg/dl) group. This is what the insulin/glucose values looked like, excluding the controls:

They asked the folks how hungry they were and got this sort of result:

I think the asterisks give the correct impression. However just for fun, at the end of the clamps, the subjects were allowed to suck a liquid meal via a straw through a barrier (so they couldn't see how much they were drinking). They ate/drank as much as they wished to. This is what happened:

The hypoglycaemic group was excluded from the eating trial because they had had all sorts of things done to get stable hypoglycaemia with eu-insulinaemia which involved somatostatin/replacement insulin and so precluded feeding in the immediate post-clamp period. We just have to accept the subjective appetite ratings for this group.

You would have thought that it would have been settled, back in 1985, that insulin in humans is an appetite stimulant. And that mild hypoglycaemia of 50mg/dl is not, provided there is no hyperinsulinaemia.

Of course this is a simplistic interpretation and quite possibly incorrect. Let's have a look at it from a more metabolic perspective.

What does insulin do?

The function of insulin is the inhibition of lipolysis.

So if we look at this current paper in the light of the 2011 paper we can make a more insightful interpretation of what is happening.

The people under hyperinsulinaemic clamps are not eating, because that is the study protocol. They are being infused with insulin at a time when they have no access either to any food or to their adipocyte lipid stores (and their hepatic glucose output will be near zero under the insulin clamp). Even the people under an hyperglycaemic hyperinsulinaemic clamp, who received around 700kcal of glucose over 150 minutes, are losing much of this glucose in to glycogen stores while simultaneously being deprived of adipocyte sourced FFAs.

The hunger may well come from metabolic energy deprivation rather than the insulin itself being an "hunger signal" of any sort. Insulin is the signal to store ingested calories. If it stores calories as if you have just had a meal when you haven't just had said meal, you are going to be hungry for those lost calories!

Peter

Insulin makes you hungry (1)

TLDR: The function of insulin is the inhibition of lipolysis. In the brain. In the periphery.

If you want to think about the central effects of insulin you could do a great deal worse than working through this paper:

Brain insulin controls adipose tissue lipolysis and lipogenesis

It is jammed full of exquisite quotes:

"To reduce the likelihood of pharmacological effects of the insulin doses administered, we choose a dose of insulin that is more than 15,000–fold lower than those commonly used for ICV [intra cerebro ventricular] insulin infusions (Air et al., 2002; Brief and Davis, 1984; Rahmouni et al., 2004)"

and

"An ICV (5 μl/h) or MBH [medial basal hypothalamus] (0.18 μl/h per side) infusion with either vehicle (artificial cerebrospinal fluid (aCSF) (Harvard Apparatus, Holliston, MA) or insulin (ICV 30μU; MBH 2 μU; Humulin R, Lilly) was started and maintained for 360 min"

and

"Both ICV and MBH insulin administration markedly suppressed the rate of appearance (Ra) of glycerol under basal and clamped conditions indicating that brain insulin, and more specifically MBH insulin signaling, suppresses lipolysis (Figs. 1B and C)"

and

"Hyperinsulinemia [systemic] induced by a 3 mU · kg−1 · min−1 clamp decreased the Ra glycerol by about 65% compared to a 1 mU · kg−1 · min−1 clamp in vehicle infused animals (Fig. 1C). Thus, at the doses administered, brain insulin infusion inhibited lipolysis to a similar extent as that achieved with peripheral hyperinsulinemia"


Here it is in pictures.

What does a CNS infusion of insulin do to lipolysis, at what are purported to be physiological dose rates? Obviously, it does exactly what peripheral insulin does, but using minuscule amounts; it suppresses lipolysis:

Of course, you have to ask how physiological is this "physiological" infusion rate? Insulin in the brain is thought to be derived from insulin secreted by the pancreas, so we are at liberty to ask what level of plasma insulin would produce a comparable level of suppression of lipolysis. The answer is an hyperinsulinaemic clamp of 3mU/kg/min, as in the above quote. That will be a clamp of somewhere around 70microU/ml clinically (3.5ng/ml in the paper), the sort of level of systemic insulin a healthy human might produce following a meal of real food, it's the column on the right of the graph:

Obviously the fall in lipolysis results in depressed FFA levels during the central insulin infusion, as you might expect:

The researchers didn't check the fall in FFAs from the peripheral 3mU/ml/kg clamp but I would expect it to be comparable to the central infusion level fall, glycerol appearance was equally suppressed by the central and peripheral administrations.

So one question (among many) is: Does this fall in plasma FFA levels result in hunger?

Oddly enough there are relatively few studies where humans have had experimental cannulae inserted in to either their cerebral ventricles or directly in to their ventomedial hypothalamus for insulin infusion. Ok, there are none.

But there are plenty of studies where humans have had systemic hyperinsulinaemic euglycaemic clamps performed. What is rare is to ask what the effect of such a clamp might be on hunger. Which brings us to another gem of a paper, this one from Woo. And it's good.

Peter

Butter gives you fatty liver! Again.

This paper is an absolute gem:

Saturated Fat Is More Metabolically Harmful for the Human Liver Than Unsaturated Fat or Simple Sugars

Obviously you have to be very careful in reading it. It contains no trace of understanding in its entirety, but the numbers in the results are fascinating.

How do we sum it up?

If you pay people to over eat 1000kcal per day for three weeks they gain weight and they gain liver fat. The group eating extra butter/coconut oil gain the most liver fat (IHTG is intra hepatic triglyceride). From Figure 1:

Unsaturated fat (22% omega six PUFA) causes less IHTG accumulation than saturated fat, similar to an excess 1000kcal of (mostly) sucrose. So saturated fat is bad for your liver and PUFA or sucrose are less problematic. Shrug.

Aside: Almost no-one gets fat because they deliberately over eat. People get fat accidentally, bit by bit plus the occasional splurge, which they cannot then lose. In this study people did NOT accidentally get fat against their will. They over-ate because they were paid to, whether they were hungry or not. Any resemblance to real life is purely accidental. End of ranty aside.

So anyway, let's get to the interesting bit. Lipolysis. The group measured the rate of glycerol appearance, a perfectly reasonable surrogate for the release of FFAs from adipocytes. Under fasting conditions I think you would agree that saturated fat group increased their rate of lipolysis, just a trend, ns, over the three weeks.

Here are the changes in the rates of glycerol release under an hyperinsulinaemic clamp:

OK. Under an insulin infusion of 0.4mIU/kg/min, plus a bit of glucose, the adipocytes which have been exposed to saturated fats are STILL releasing glycerol (and so FFAs). Eating a mix of olive oil, pesto and pecans for three weeks allows lipolysis to drop like a stone when you infuse insulin, p less than 0.01 between these two groups.

This is pure Protons in action. Saturated fat provides an high input of FADH2 at electron transporting flavoprotein dehydrogenase, so reduces the CoQ couple, so promotes reverse electron transport through complex I which will generate superoxide when the NADH:NAD+ ratio is high, ie under caloric surplus. Superoxide is the signal used for setting up insulin resistance, to stop caloric ingress. Beta oxidation of PUFA skips one FADH2 generation for each double bond present so they are crap at signalling insulin resistance by this mechanism. Even under caloric excess, insulin continues to act and packs more calories in to adipocyes. And it refuses to allow lipolysis. It even very slightly (and ns) reduces fasting lipolysis (in graph B above), when insulin is as low as it's going to get on the SAD (in Finland).

Now for some context.

What do we know about the adipocytes of the subjects at the start of the study?

The BMIs were 30, 31 and 33 in the three groups and of the 38 people involved in the study, 22 had impaired fasting glucose at admission.

So these people already have PUFA induced obesity plus complications. That means that their adipocytes have gravitated to a certain (large) size related to their absolute exposure to insulin combined with a PUFA enhanced insulin sensitivity. This adipocyte size is larger than it would have been had the adipocytes mounted the normal resistance to insulin's action which is provided by saturated fats. As caloric ingress made each adipocyte "full", this "fullness" should have be communicated from the mitochondria (as superoxide) to the adipocyte (as insulin resistance) and eventually the brain as satiety (that signal is VERY interesting, another day perhaps). Under PUFA any distended adipocyte does not feel "full", it behaves as if it is still hungry. Whole body hunger follows on from this. Thanks to the cardiological community and their love of PUFA.

Along come three weeks of palmitic acid (plus a few other nice saturated lipids). Now there is plenty of FADH2, a reduced CoQ couple etc etc and the adipocytes are suddenly able to resist insulin... They suddenly realise that they are grossly distended and there is now no way they are going to accept any more calories (even with insulin infused at 0.4mIU/kg/min). In fact, given this new-found "awareness" of their bloated size, they are going to off load as much lipid as possible, in resistance to insulin's bloating signal. This is why they release glycerol (and associated FFAs) in the face of an hyperinsulinaemic clamp...

Of course lipolysis is fine if you accept the fat from your adipocytes and stop feeling hungry, so stop eating. Developing adipocyte insulin resistance gets fat out of distended adipocytes, saturated fat delivers this.

Of course, if you are pouring FFAs out of your adipocytes but some clown is paying you to eat 1000kcal above your preferred daily intake, you are going to have to do something with those FFAs. Failing to take the chance of a hike to the top of some 1000kcal high hill, the fat will end up in your liver. The more lipolysis, the more fat in your liver. In real life you would simply eat less, we know that supplying small amounts of fat to the liver via the portal vein is a potent suppressor of appetite, at least in rats.

This why I love rodent studies of obesity. You cannot pay a mouse to overeat. Any obese mouse gets to be that way because it is hungry. If you make it hungry using linoleic acid to sequester dietary fat in to its adipocytes then its liver will be fine until adipocyte distension releases enough FFAs to then allow fatty liver to develop.

Sorry if all this sounds like a scratched vinyl record about Protons but people will take this current study as proof that saturated fat is bad for you, which is bollocks of course. Or simply as incomprehensible. One of my biggest problems is that the Protons concept provides a logical explanation for many of the "paradoxes" of different fat types. However it is something of a language of its own and I feel I have no shared vocabulary to explain what is going on with people who do not have the concept... Ah well.

Peter

BTW The sucrose arm is interesting too but that's another story for another day. You noticed the ns-reduced weight gain in the sucrose arm? I digress...

Oops

OMG, just seen how many comments are awaiting moderation now I'm back to occasional posting. Groan. I'll see what I can do, if desperate I'll just delete the spam and hit post for them all. Apologies for the inattention over the past few weeks.....

Peter

Acipimox and insulin

Woo had a bit of a rant about acipimox. Here's my simplified idea.

I've been interested in acipimox, in a round about sort of a way, for a very long time. To me, the core fascination is that it is not only an effective suppressor of lipolysis, but it is pretty well weight-neutral and it most certainly does not result in weight gain.

Which, you have to admit, is interesting.

How can this be? I feel something of a clue can be found in the studies using a similar drug, nicotinic acid. Both drugs effectively suppress plasma free fatty acids via the same receptor but the neatest study happens use nicotinic acid.

People may recall that I posted about the role of FFAs in the secretion of insulin as demonstrated by an isolated rodent pancreatic preparation, some time ago. The core concept here is that insulin secretion is dependent on the chain length and saturation of the FFAs used for perfusing the pancreas along with the glucose. This phenomenon appears to be well appreciated by the authors of this next paper (same research group):

Circulating fatty acids are essential for efficient glucose-stimulated insulin secretion after prolonged fasting in humans

So what happens to in-tact humans when you fast them for 24 hours (to raise FFAs) and then bolus them with intravenous glucose? Or fast them, artificially drop their FFAs with nicotinic acid, and then bolus them with glucose? This is what happens:

I think it is reasonable to state that dropping FFAs acutely, using nicotinic acid, results in a 50% drop in the area under the curve for insulin secretion over 60 minutes for a given bolus of glucose. The more speculative idea is that dropping insulin might reduce lipid uptake in to adipocytes. I don't know. It's an interesting idea.

If we simply consider acipimox to be a long acting analogue of nicotinic acid we have here a potential explanation for why it fails to induce weight gain. It might just simultaneously lower insulin levels. Understanding acipimox appears to require some insight in to the insulin hypothesis of obesity, not a notable feature in certain areas of obesity research.

Failure to appreciate the roll of insulin in obesity will limit any sort of understanding of the condition or the drugs which might or might not influence it. Seems that way to me.

Peter

Speculation on the effect of subcutaneous adipocytes implanted in to the mesentery of mice

Taking a piece of subcutaneous fat from a sacrificed mouse and implanting in to the mesentery of a recipient mouse causes weight loss in the recipient, starting once the surgery has healed. We know that healing takes something just over six weeks:

Is the effect replicable? Yes, fairly well, here it is in a second cohort by the same group:

While this is pretty certain, the explanation is very speculative. Note the effect kicked in slightly sooner in cohort 2, I would assume that healing was a little quicker for this cohort. Maybe the researchers got better at performing the surgery. The 12 week weights are essentially identical between cohorts.

So......... Adding extra adipocytes causes overall fat loss, as in this image from Cohort 1 featured in the last post:

I find this rather interesting, to say the least. The black squares represent mice which have had subcutaneous adipose tissue implanted around their mesentery so that a significant proportion of its venous drainage enters the portal vein and goes directly to the liver.

I've looked through the data from the paper for any suggestion as to an explanation of why the weight loss occurred. There is nothing anywhere to suggest uncoupling while the mice where in the CLAMS apparatus (though calorie restriction is a good way to suppress uncoupling acutely). Calorie malabsorption through surgical damage to the mesentery seems unlikely as similar surgery to implant visceral fat gave normal weight gain compared to control mice.

So I think the mice in the SC-VIS group simply cut their ad-lib calories. Food intake was only measured over the one day in the CLAMS apparatus so we'll never know what the overall food intake was relative to controls, but I can't see any other explanation.

Which leads to the question as to why they might have cut calories, other than the obvious: They we not as hungry as the control mice. This effect occurred with mesenteric implanted subcutaneous adipocytes, but not with implanted visceral adipocytes. So we have to ask what the difference might be between the adipocyte types.

Visceral adipocytes are more insulin sensitive than subcutaneous adipocytes. They will store fat more easily and refuse to release it until insulin drops down to absolutely basal levels. On an ad-lib high carbohydrate diet this will not happen very often. As far as the liver is concerned, the visceral fat in the mesentery and omentum is non existent for most of the time. Adding extra visceral adipocytes at this site will not change this.

Subcutaneous adipocytes are less sensitive to insulin, they will store fat at high insulin levels but release FFAs easily as insulin levels fall. Obviously, they are always smaller than visceral adipocytes and they stay that way when implanted in to the recipient mice. If they are implanted in a location from which their easily released FFAs go directly to the liver they are in a position to have a metabolic effect.

Summary of the speculation so far: Visceral fat adds nothing to the FFA level of portal vein blood unless insulin level is well below that of a mouse on ad lib standard mouse chow. Placing subcutaneous adipocytes where their venous drainage goes directly to the liver supplies supplementary FFAs directly to the liver when insulin levels are merely low rather than rock bottom.



It has been known for a very, very long time that infusing FFAs in to the portal vein suppresses appetite and that this effect requires a functional vagal nerve supply to the liver. This group of rats is receiving an oleic acid infusion (if you are going to secure a long term cannula in to the portal vein then something larger than a mouse might make the surgery slightly more practical and less challenging) in to their portal vein:

Hepatic-portal oleic acid inhibits feeding more potently than hepatic-portal caprylic acid in rats

Oleic acid infused in to the portal vein at 14mcg/min for six hours reduced food intake during the 12 hour dark period from 23g in the controls to 17g in the oleic acid group. At 14mcg/min the infusion supplies 840mcg/h and over six hours this supplies just over 5mg, ie 0.005g in total dose, roughly 0.05kcal, 0.21kJ of oleic acid. This is enough to drop food intake by six grams of food at 12.4kJ/g, ie 74kJ.

This group looked at 14mcg/min because they knew it would work. We don't know how low an infusion rate could go while still having an effect on appetite (from this paper anyway). The two unanswerable questions we are left with are: What was the augmentation of FFA supply to the liver from SC adipocytes in the mesentery of the operated rats in Kahn's study? Was this enough to limit food intake? We don't know the answers but you can imagine what my guess is.

Getting access to the portal vein to either measure or infuse anything is a complete surgical nightmare. Kahn's group looked at all sorts of systemic messengers in terms of cytokines and adipokines and found absolutely nothing to explain their phenomenon.

Perhaps they were looking in the wrong place.

Peter

I guess the third question is why the SC adipocyte recipient mice actually ate a little more than the controls in the CLAMS apparatus (as judged by RQ). I'd still bet on differences in total fat mass available over-riding the stress induced refusal to eat, irrespective of degree of hunger in any mouse through the rest of the study... You could throw in cortisol, adrenalin and the lack of neural innervation of transplanted adipoctes but let's leave it simple (and possibly incorrect).

More supplementary speculation: Is the normal profound fall in appetite on induction of ketogenic eating in humans directly related to the sudden access to visceral abdominal fat, secondary to the major reduction in circulating insulin? This would suggest that the satiating effect of ketogenic diets might be more marked in people with significant visceral obesity. It would become less obvious as weight loss progresses until the majority of remaining fat is where it should be, in the non-visceral adipocytes of a normal shaped human being... The effect might even be virtually non existent in young, fit, healthy folks who would simply eat under ketosis to maintain their current rather normal bodyweight.

Guddling in the dark for a respiratory quotient (2)

OK. I've been bugged by the RQ values which come out of CLAMS equipment. For months. Multiple papers have values that don't make sense, which is an issue making me doubt my sanity and is damaging to my personal extensive set of confirmation biases. They also produce basic contradictions of the physiology of the oxidation of glucose vs the oxidation of fatty acids. But now I think I can go back and explain much of the peculiarity in this image from Kahn's group, the one which triggered this previous post. Two sets of mice, on the same chow, having sustained differences in RQ, in a way I couldn't understand:

Well now I think I can. The insight needed comes from a non related paper on the effect of knocking out the sweet taste receptor in mice.

So, let's have a look at the RQ data from the CLAMS apparatus in this paper:

Disruption of the sugar-sensing receptor T1R2 attenuates metabolic derangements associated with diet-induced obesity

And in particular from this next set of graphs. They are interesting because they cover two complete days in the CLAMS equipment.

Look at the grey and the black lines first, these are control and sweet receptor KO mice, being fed pretty standard low fat crapinabag. We can see that between day one dark period and day two dark period in the CLAMS equipment that a) the peak height of the RQ rises, b) carbohydrate oxidation rises and c) lipid oxidation falls. No one did stats to compare these two days with each other so I don't know what the p value might be, but the trend certainly looks very real to me. No one altered the food macros between day one and day two.

I think mice, like humans, cut their calories when you put them in to a respiratory chamber or in to a CLAMS apparatus. Certainly for the first two or three days of mice in CLAMS.

In this T1R2 KO study you can see evidence for the the improving intake of (carbohydrate based) food between day one and day two which is inseparably linked to the rising RQ. There had been a day of acclimatisation before the two study days which will have minimised this trend but it's very much still visible. The change is less obvious (but still present) for the fat fed mice (red coloured traces) but it's not so clear cut in these mice because they are largely either oxidising fat from their diet or fat from their adipocytes, both of which look pretty much the same from the RQ point of view.

Core insight. You can alter RQ away from the macros of a (high carbohydrate) diet by simply eating less. Adipocyte fat makes up the difference in calories and this automatically drops the RQ. Let's take this idea and look at the RQ figure from Kahn's paper.

In a normal mouse, on normal low fat chow, the RQ during the early feeding period rises to above 1.0 due to de novo lipogenesis combined with fat repletion/storage. Kahn's control mice (SHAM) started with a RQ at around 0.88 and they slept through the light period so dropped their RQ to around 0.75. When they started to feed with the arrival of the dark period (on their high carbohydrate chow) in the CLAMS apparatus the RQ only rises to 0.88. In real mice eating this sort of chow the RQ should rise to 1.2 for a few hours.

The RQ will not rise to 1.0 if the mice fail to eat enough carbohydrate to meet or exceed their energy needs. If they don't eat enough they will continue to utilise body fat and this will lower their RQ below that of a mouse eating to weight stability. These mice did not eat enough carbohydrate to suppress fat oxidation. The RQ says so. The absolute food intakes are below but these are fairly irrelevant compared to the RQ data.

They were put in to the CLAMS apparatus with no suggestion (in the methods) of an acclimatisation period. I think the mice were frightened and dropped their food intake. We can see from the RQ of both groups that at no point does this even approach, let alone exceed, 1.0 (in the acclimatised mice in the T1R2 KO paper figure the RQ reaches 1.1 at the peak of feeding on day 2, ie CLAMS day 3).

Summary: Both of Kahn's groups are oxidising fat to give a low RQ on an high carbohydrate diet and the only way they can be doing this is if the fat is coming from their adipocytes. They are not eating enough of their carbohydrate based diet to maintain fat storage or to raise the RQ.

That just leaves us to ask why the control group are oxidising more fat than the intervention group. Is this a magical preference of the intervention group to oxidise glucose? Is that why they are so slim? I know that sounds strange but that's the conclusion in the paper. Logically, oxidising FAT makes you slim. Storing fat makes you fat. But that's under ad-lib conditions. At the time of the hypocaloric 24 hours for Kahn's mice in the CLAMS apparatus we have these data available from which we can estimate fat stores at a given time, week ten in particular:

The white diamonds are still the controls/sham operated. They carry about 10g of fat at week ten. The black squares are the intervention group, they carry about 5g of body fat at this time. If both groups cut calories acutely during their day of anorexia in the CLAMS apparatus, I would expect the animals with the most adipose tissue to release the most FFAs. And oxidise the most FFAs. That has to be the control group, because they're a lot fatter to start with. The intervention group has less stored fat available, so oxidises less fat. Interestingly the fatter control group ate 3.9g of carbohydrate based food in the CLAMS vs 4.2g for the slimmer intervention group (ns), which just might reflect less hunger in the fatter control mice because they are accessing their bigger fat stores, ie the control mice appear to run on fat (because they are doing so on this one single day). A lack of stored fat in the intervention group is why they ate more (carbohydrate based) food and utilised less adipose derived fat. So this group appears to run on carbs, judging by their RQ. But the RQ over the rest of their lives, in either group, while eating normally, to satiety, outside the CLAMS apparatus, is a mystery.

But yay! The CLAMS equipment is almost certainly working correctly. The RQ graph is very explicable. It probably tells us NOTHING about the substrate oxidation under non-CLAMS conditions, but we can speculate about that in another post.

Trying to make inferences about overall post-intervention metabolism, based on a single day in a very novel (and possibly frightening) environment under acute hypocaloric conditions will set you up for a hiding to nothing as far as comprehension and understanding of your intervention are concerned. But given enough thought the results at least become comprehensible.

Now I'm happy.

Peter

Now that I'm happy I might have a think about why the slim, subcutaneous to visceral fat-transplanted mice in Kahn's study might have come to remain slim. Clearly, from the fat mass graph, the transplantation of subcutaneous fat in to the location of visceral fat does do something...

Nighttime Eaters have an elevated RQ on a given macro ratio diet. They're getting fat

I picked up this paper via Face-ache so cannot recall to whom I should credit for the find. Sorry. The post is also highly speculative.

Higher 24-h Respiratory Quotient and Higher Spontaneous Physical Activity in Nighttime Eaters

It's worth noting that the difference is small but probably biologically significant. Statistically p is less than 0.05. Before we think about it we need some background. That comes from the same group in an earlier paper:

Nighttime eating: commonly observed and related to weight gain in an inpatient food intake study

They looked at accurately measured food intake for Nighttime Eaters (NEs) under in-patient conditions at near identical macros to non-NEs:

and at weight gain over the subsequent 3.4 years, while the subjects were free living. Weight gain is, not surprisingly, higher in the NEs:

To me, NEs wake up in the night and go to the fridge and eat some food because they are hungry. Control subjects do not get up at night and do not go to the fridge and do not eat food, because they are not hungry. Note that hunger is a slippery term and these researchers have a psychiatry based view of obesity*. In Table 2 there is no significant difference in "cognitive hunger" between NEs and non-NEs. However in the methods the term used is "perceived hunger" and this is described thus:

"... perceived hunger (ie, the susceptibility of eating in response to subjective feelings of hunger)"

So NEs may not eat any more than non-NEs when they are "subjectively" hungry. My argument is that they are simply hungry more frequently, including right through the night in fact.


*Aside: But they are learning! Quote of the century from the paper: "These differences in substrate oxidation and SPA indicate that the night eating behavior phenotype may have physiologic underpinnings"

OMG gluttony may just be physiology!!!!!!!!!!!!!! End aside.


Anyhoo.

Why might NE people be hungry more often than non-NEs? From the adipocentric view of obesity, when dietary fat falls in to their adipocytes and stays there, NE subjects "lose" this fat. In the absence of a decent supply of metabolisable fat there is nothing left to oxidise except carbohydrate, with its high associated RQ (pax protein). Once the bulk of the ingested carbohydrate is metabolised and the fat is in the adipocytes for the duration, there is nothing for it but to get some more carbohydrate to eat and metabolise. The signal for this need to eat is called hunger. The fat loss phenomenon is not huge, the RQ for NEs is 0.85 and for non-NEs is 0.83. But I think that is enough.

Eating another mixed meal or mixed macro snack supplies necessary glucose for oxidation but the fat is again "lost" in to adipocytes. This keeps happening.

So. Are these folks going to get heavier? Of course they are. That is intrinsic to the elevated RQ compared to non-NEs while eating a similar macro ratio diet. It can only occur during fat accumulation. People with high RQs gain weight over the years. The high RQ is a direct result of the loss of dietary fat in to adipocytes. From the respiratory chamber study it would be about ten grams of fat per day "lost" in to adipocytes. In the background paper the NEs weight gain over 3.4 years was actually roughly five grams per day rather than ten grams per day, but people are more active when outside a respiratory chamber! Obviously fat "lost" in to adipocytes is fat gained on the scales.

Are NEs insulin sensitive or insulin resistant?

That's easy. Their RQ is high, they are losing fat in to storage. The fat is staying in storage within the adipocytes. They must be insulin sensitive. Think of the Laron dwarf humans, genetically GH-receptor deficient with subsequent exquisite insulin sensitivity. Short of stature and seriously obese at the same time as maintaining that exquisite insulin sensitivity...

To look for data to confirm my biases we have to move along to

Circadian rhythm profiles in women with night eating syndrome

This is by a different group. They are less psychiatric in outlook but fail to perceive that obesity might be a significantly adipocyte related problem. They mention stomach and liver and circadian rhythms, but not adipocytes. Their data are a little shaky but certain features come through as plausible. They measured a ton of (mostly) hormones but the only two parameters which grab my attention are insulin and glucose.

Now, the x axis is as clear as mud (like much of the rest of the paper). It really is "time of day", sort of. The first sample was taken at 8am, this is the start of each of the graphs, eight is interpolated between six and ten. Twenty four hours later, 8am next day is at 32 on the x axis and we get an extra data point taken at 9am on the second morning, ie 25 hours in to the study, it's at 33 on the x axis. Simple huh? Sorry if I've insulted the clarity of mud.

Three meals were served during daytime and snacks were available and consumed ad-lib, including through the night if so needed. Solid lines are controls, dashed lines are NEs. Macros of intakes were not controlled.

Control group (non-NEs) eat through the day. Glucose and insulin peak at around 5pm, probably around evening meal time, and both trough at around 4am because these folks would like to sleep through the night (they were blood sampled once an hour so...) and don't eat while asleep or wanting to be asleep.

The insulin peak is lower for the NEs as (I am assuming) they are insulin sensitive so they easily distend their adipocytes. Insulin sensitive adipocytes need less insulin to squirrel away diet derived fat. The insulin peak is delayed because these people are NEs, nighttime eaters, by definition. Eating later gives a later insulin peak. The NE insulin curve also never shows that drop in the early hours because NEs continue to eat at night. Because they're hungry at night. That's why they are called... You get the gist.

The glucose curve is equally explicable in terms of pathological insulin sensitivity. Just a little insulin lowers the blood glucose and facilitates its oxidation (daytime dip in glucose) and facilitates uptake in to adipocytes to generate glycerol for triglyceride sequestration. Gradually falling insulin due to lower (but not zero) food intake in the early hours of the morning allows some recovery of blood glucose levels, probably assisted by the surge of growth hormone in the early hours of the morning with its mild glucose raising, insulin resistance effect.

Night Eaters are pathologically insulin sensitive. Like the Laron dwarf humans and Laron mice, extreme insulin sensitivity causes obesity, given ad lib food. Unlike the Laron individuals with their genetic oddity of long term preserved insulin sensitivity, NE people will eventually distend their adipocytes to the level of leaking free fatty acids.

But until their adipocytes become dysfunctional NE people are insulin sensitive, hungry, and lose the fat component of their diet in to their adipocytes. Of course, once their adipocytes become distended and start to leak FFAs they will stop getting fatter and start to access the spilled fat. Oxidising this will drop their RQ but they will at this time become IGT/diabetic due to unregulated and inappropriate FFA release.

If I had to suggest an explanation for NE patho-physiology it would be PUFA, mostly linoleic acid... I'd expect NEs to be people who avoid saturated fat and prefer corn oil, over the long haul. More victims of the cardiologists.

Peter

AHA approved egg!

One of the full size chickens miss-fired yesterday and produced this minute egg:

I was pretty sure this would be an AHA approved egg. Zero fat. Zero cholesterol. Tiny amount of protein. You don't have to include the eggshell (unless you feel a calcium supplement is a good idea. Could treat your acid reflux at the same time!).

Of course it should be fried in corn oil. Still hungry?

Fill up on sugar!

Ah, the decades of stupidity that have now been mostly overturned. Just the corn oil to get rid of.

Peter

Pasta for weight loss

This paper hit T'internet recently and has been cited all over the place:

Effect of pasta in the context of low-glycaemic index dietary patterns on body weight and markers of adiposity: a systematic review and meta-analysis of randomised controlled trials in adults

Obviously the sole claim to fame for the paper is the conflict of interest statement. I've greyed it out so no-one is tempted to read it in full, the flavour is all you need:

"Competing interests: All authors have completed the Unified Competing Interest form (available on request from the corresponding author) and declare: LC has worked as a clinical research coordinator at Glycaemic Index Laboratories, Toronto, Ontario, Canada. CWCK has received research support from the Advanced Food Materials Network, Agriculture and Agri-Foods Canada (AAFC), Almond Board of California, American Pistachio Growers, Barilla, California Strawberry Commission, Calorie Control Council, Canadian Institutes of Health Research (CIHR), Canola Council of Canada, International Nut and Dried Fruit Council, International Tree Nut Council Research and Education Foundation, Loblaw Brands Ltd, Pulse Canada, Saskatchewan Pulse Growers and Unilever. He has received in-kind research support from the Almond Board of California, California Walnut Council, American Peanut Council, Barilla, Unilever, Unico, Primo, Loblaw Companies, Quaker (Pepsico), Pristine Gourmet, Kellogg Canada, WhiteWave Foods. He has received travel support and/or honoraria from the American Peanut Council, American Pistachio Growers, Barilla, Bayer, California Walnut Commission, Canola Council of Canada, General Mills, International Tree Nut Council, Loblaw Brands Ltd, Nutrition Foundation of Italy, Oldways Preservation Trust, Orafti, Paramount Farms, Peanut Institute, Pulse Canada, Sabra Dipping Co., Saskatchewan Pulse Growers, Sun-Maid, Tate & Lyle, Unilever and White Wave Foods. He has served on the scientific advisory board for the International Tree Nut Council, McCormick Science Institute, Oldways Preservation Trust, Paramount Farms and Pulse Canada. He is a member of the International Carbohydrate Quality Consortium (ICQC), Executive Board Member of the Diabetes and Nutrition Study Group (DNSG) of the European Association for the Study of Diabetes (EASD), is on the Clinical Practice Guidelines Expert Committee for Nutrition Therapy of the EASD and is a Director of the Toronto 3D Knowledge Synthesis and Clinical Trials foundation. DJAJ has received research grants from Saskatchewan Pulse Growers, the Agricultural Bioproducts Innovation Program through the Pulse Research Network, the Advanced Foods and Material Network, Loblaw Companies Ltd., Unilever, Barilla, the Almond Board of California, Agriculture and Agri-food Canada, Pulse Canada, Kellogg’s Company, Canada, Quaker Oats, Canada, Procter & Gamble Technical Centre Ltd., Bayer Consumer Care, Springfield, NJ, Pepsi/Quaker, International Nut & Dried Fruit (INC), Soy Foods Association of North America, the Coca-Cola Company (investigator initiated, unrestricted grant), Solae, Haine Celestial, the Sanitarium Company, Orafti, the International Tree Nut Council Nutrition Research and Education Foundation, the Peanut Institute, the Canola and Flax Councils of Canada, the Calorie Control Council (CCC), the CIHR, the Canada Foundation for Innovation and the Ontario Research Fund. He has received in-kind supplies for trial as a research support from the Almond Board of California, Walnut Council of California, American Peanut Council, Barilla, Unilever, Unico, Primo, Loblaw Companies, Quaker (Pepsico), Kellogg Canada, and WhiteWave Foods. He has been on the speaker’s panel, served on the scientific advisory board and/or received travel support and/or honoraria from the Almond Board of California, Canadian Agriculture Policy Institute, Loblaw Companies Ltd, the Griffin Hospital (for the development of the NuVal scoring system, the Coca-Cola Company, EPICURE, Danone, Diet Quality Photo Navigation (DQPN), Better Therapeutics (FareWell), Verywell, True Health Initiative, Institute of Food Technologists (IFT), Saskatchewan Pulse Growers, Sanitarium Company, Orafti, the Almond Board of California, the American Peanut Council, the International Tree Nut Council Nutrition Research and Education Foundation, the Peanut Institute, Herbalife International, Pacific Health Laboratories, Nutritional Fundamental for Health, Barilla, Metagenics, Bayer Consumer Care, Unilever Canada and Netherlands, Solae, Kellogg, Quaker Oats, Procter & Gamble, the Coca-Cola Company, the Griffin Hospital, Abbott Laboratories, the Canola Council of Canada, Dean Foods, the California Strawberry Commission, Haine Celestial, PepsiCo, the Alpro Foundation, Pioneer Hi-Bred International, DuPont Nutrition and Health, Spherix Consulting and WhiteWave Foods, the Advanced Foods and Material Network, the Canola and Flax Councils of Canada, the Nutritional Fundamentals for Health, Agri-Culture and Agri-Food Canada, the Canadian Agri-Food Policy Institute, Pulse Canada, the Saskatchewan Pulse Growers, the Soy Foods Association of North America, the Nutrition Foundation of Italy (NFI), Nutra-Source Diagnostics, the McDougall Program, the Toronto Knowledge Translation Group (St. Michael’s Hospital), the Canadian College of Naturopathic Medicine, The Hospital for Sick Children, the Canadian Nutrition Society (CNS), the American Society of Nutrition (ASN), Arizona State University, Paolo Sorbini Foundation and the Institute of Nutrition, Metabolism and Diabetes. He received an honorarium from the United States Department of Agriculture to present the 2013 W.O. Atwater Memorial Lecture. He received the 2013 Award for Excellence in Research from the International Nut and Dried Fruit Council. He received funding and travel support from the Canadian Society of Endocrinology and Metabolism to produce mini cases for the Canadian Diabetes Association (CDA). He is a member of the International Carbohydrate Quality Consortium (ICQC). His wife, ALJ, is a director and partner of Glycemic Index Laboratories, Inc., and his sister received funding through a grant from the St. Michael’s Hospital Foundation to develop a cookbook for one of his studies. JLS has received research support from the Canadian Institutes of health Research (CIHR), Diabetes Canada, PSI Foundation, Banting and Best Diabetes Centre (BBDC), Canadian Nutrition Society (CNS), American Society for Nutrition (ASN), Calorie Control Council, INC International Nut and Dried Fruit Council Foundation, National Dried Fruit Trade Association, The Tate and Lyle Nutritional Research Fund at the University of Toronto, and The Glycemic Control and Cardiovascular Disease in Type 2 Diabetes Fund at the University of Toronto (a fund established by the Alberta Pulse Growers). He has received in-kind research support from the Almond Board of California, California Walnut Commission, American Peanut Council, Barilla, Unilever, Unico, Primo, Loblaw Companies, Quaker (Pepsico), Kellogg Canada, WhiteWave Foods. He has received travel support, speaker fees and/or honoraria from Diabetes Canada, Canadian Nutrition Society (CNS), Mott’s LLP, Dairy Farmers of Canada, Sprim Brasil, WhiteWave Foods, Rippe Lifestyle, mdBriefcase, Alberta Milk, FoodMinds LLC, Memac Ogilvy & Mather LLC, PepsiCo, The Ginger Network LLC, International Sweeteners Association, Nestlé Nutrition Institute, Pulse Canada, Canadian Society for Endocrinology and Metabolism (CSEM), Barilla Centre for Food and Nutrition (BCFN) Foundation, and GI Foundation. He has ad hoc consulting arrangements with Winston & Strawn LLP, Perkins Coie LLP, and Tate & Lyle. He is a member of the European Fruit Juice Association Scientific Expert Panel. He is on the Clinical Practice Guidelines Expert Committees of Diabetes Canada, European Association for the study of Diabetes (EASD), Canadian Cardiovascular Society (CCS), and Canadian Obesity Network. He serves as an unpaid scientific advisor for the Food, Nutrition, and Safety Program (FNSP) and the Technical Committee on Carbohydrates of the International Life Science Institute (ILSI) North America. He is a member of the International Carbohydrate Quality Consortium (ICQC), Executive Board Member of the Diabetes and Nutrition Study Group (DNSG) of the EASD, and Director of the Toronto 3D Knowledge Synthesis and Clinical Trials foundation. His wife is an employee of Unilever Canada. No competing interests were declared by CRB, SBM and LAL".

Quite what is wrong with CRB, SBM and LAL that they have no competing interest to declare is not specified.

So. This a BMJ publication. The critical aspect to me is the publication date.


Was it April the first?


Well. It should have been, except April the first this year was Easter Sunday. Not even at BMJ do they hit the "publish" button on a Sunday morning. Easter Monday appears fair game and someone at BMJ appears to have been at work to hit said publish button on April the 2nd.

Ah, the twists of fate produced by the lunacy of the movement of Easter through the calendar.

I think someone at the BMJ may have a sense of humour. Reading the conflict of interest statement, I wonder if the authors do too and whether there was some collusion in the choice of publication date.

Otherwise it's not funny.

Peter

Guddling in the dark for a respiratory quotient

Here's a paradox: How can two groups of mice, on exactly the same chow, have different 24h averaged RQs, p less than 0.05?

It's from here if anyone wants to peek at the methods. Two sets of animals on the same chow. It's 9F 5020, 21% of calories from fat (7% of calories from PUFA) and 55% from carbohydrate.

At all time points the SC-VIS mice have an higher RQ, ie are oxidising more glucose, than the SHAM mice. But they are all fed the same chow, which should average out at the same overall RQ.

Clearly you can increase the RQ, even above 1.0, during de novo lipogenesis, especially when hungry mice suddenly eat carbohydrate. But there is either a payback during the sleep phase where RQ falls below the food derived RQ while that carbohydrate-derived fat is oxidised or there can be no fall in that fasting RQ if the DNL generated fat is "lost" in to adipocytes and stays there, ie under weight gain. Of course simply sequestering dietary fat in to adipocytes will generate an RQ more typical of glucose oxidation because less fat is being oxidised, full stop, during weight gain.

During on-going fat loss the extra low RQ from adipose derived fat oxidation does not have to be payed back either. "Food" of very low RQ, has been supplied from adipocytes. It's gone out of the body as CO2 and water.

But the black square mice are weight stable or actually losing adipose weight (ie should have an extra low RQ) at the time these RQs were measured, while the open diamond mice are actively gaining weight (including adipose tissue), so should have that higher RQ.

Food intakes are describes as "no significant difference" between the groups, despite the differential weight shifts.

To me this is inexplicable and should have been discussed in the paper. My feeling is the CLAMS equipment is generating a totally illogical result.

Unless I've totally missed something. I would really like to know whether I have totally missed something.

Just on general principles of substrate oxidation, never mind what they have done to the mice.......

Peter

Eating lots of meat and nothing much else

Wooo has posted a couple of times about Dr Shawn Baker who eats an all meat, very high protein diet, maybe over 400g/d protein intake. His HbA1c is reported as 6.3%. Personally I have absolutely no interest in this style of eating but the underlying mechanism is obviously interesting.

How about this for a hypothetical marked protein ingestion scenario:

A person eats a lot of meat. In response to the insulinogenic amino acids present they secrete insulin. This will be amino acid specific, I’ve not looked in to how amino acids trigger insulin secretion in detail but it will NOT be through pancreatic glucokinase and subsequent glucose metabolism, as is the case for glucose triggered insulin secretion. So they secrete post prandial insulin but not using glucokinase. The insulin will be exactly in balance with the glucagon for that specific protein meal.

The expression of the gene for generating pancreatic glucokinase is controlled by the carbohydrate content of the diet. Glucose means glucokinase is required. All amino acid diet, no glucose, down-regulate glucokinase.

So, as glucose is subsequently and gradually produced from gluconeogenic amino acids and then released from the liver over several hours (in the presence of only basal insulin), there is only a mild glucose derived stimulus to trigger insulin secretion, and this slow release of glucose by the liver also provides only a minimal drive to express the gene for pancreatic glucokinase. Also hepatic glucose output shouldn’t trigger any of the gut derived insulin secretion potentiating hormones (GLP-1 and the like).

So pancreatic glucokinase is mothballed. Modest glucose release from protein metabolism won’t trigger insulin secretion without the glucose sensor. End result is low insulin with moderately elevated glucose, especially during the time protein is being processed. Which I'd guess is pretty well all of the time on greater than 300g/d. How high should glucose go? High enough to allow a slow trickle to be taken up by constitutive transporters and so deal with hepatic glucose output in this way, without insulin facilitated augmentation. Facilitated by exercise if you like that sort of thing.

How toxic is glucose in the absence of hyperinsulinaemia, given that HbA1c over 6%? Dr Baker will let us know over the next 15 years!

Of course exactly the same happens on LCHF eating, just fat does not provoke chronic glucose release from its metabolism outside of a little glycerol derived gluconeogenesis… It probably happens too in some of the weird sucrose based weight loss diets where the mice (it's mostly mice but we all know that you can do "carbosis" in humans too) are hypoinsulinaemic (otherwise they would be fat!) but glucose intolerant. A diet based on a non-insulinogenic sugar (fructose) and its palmitate derivative will mothball pancreatic glucokinase too.

Peter

On phosphorylating AKT in GHrKO Laron mice

OK. I started this whole adipocyte thread because I was interested in the longevity effect in the GHrKO mouse, the Laron mouse. These posts get written because I am compelled to, I have no choice in it. I never know where they are going to end up as they start. This one has involved a lot of looking at the various types of adipocytes and how they function in normal physiology and what happened when transplanted to more unusual places. Much of it makes sense, and it does put a very different perspective on the roles of visceral and subcutaneous adipose tissue. What I was looking for was what might be special about Laron dwarf mouse derived adipocytes. You can't quite find all of the answers you want to because not all of the questions have really been asked directly, but I think you can get close. I think this is going to be the last post in the series, a relief to me, and possibly to readers too.

Laron mice (GHrKO) are the longest lifespan mice ever engineered by humans. They are dwarf and obese and the obesity tends to be central. They have exquisitely low blood insulin levels and it is thought that the reduced signalling through the GH/IGF-1/insulin system is responsible for their longevity. Adding GHrKO adipocytes to the abdomen of normal mice improves their glucose tolerance significantly.

The role of transplanted visceral fat from the long-lived growth hormone receptor knockout mice on insulin signaling

N-S mice are normal mice with a sham implantation which adds no extra adipose tissue, N-N are normal mice receiving extra intra-abdominal adipose tissue from normal mice (these should really have had some enhanced glucose tolerance but all of these transplant models differ slightly in technique) and in this case the GTT was done at about eight days post op, ie there may well have been a lot of healing derived IL-6 visiting the liver. The N-GHrKO mice are Bl/6 mice which have received adiopcytes from GHrKO dwarves, shown as black squares:

The GHrKO adipocytes are clearly a bit more effective than the normal eWAT adipocytes from the last post. Personally, I was surprised at how relatively small the enhancement of the glucose tolerance was, but then there is always that IL-6 to overcome, so perhaps they really are Super Adipocytes.

GHrKO mice develop extremely elevated GH levels, probably through a total lack of IGF-1 negative feedback, but this GH does nothing. Without a receptor the GH, functionally, isn't there. The lack of GH induced lipolysis pushes the balance of adipocyte size towards the obese phenotype. It seems to affect pretty well all adipose depots fairly equally. If we then go on to look at adipose specific FaGHrKO mice, these are obese too but lack any of the insulin sensitising effects of the whole body GHrKO mice:

The Role of GH in Adipose Tissue: Lessons from Adipose-Specific GH Receptor Gene-Disrupted Mice

"Surprisingly, FaGHRKOs shared only a few characteristics with global GHR−/− mice. Like the GHR−/− mice, FaGHRKO mice are obese with increased total body fat and increased adipocyte size. However, FaGHRKO mice have increases in all adipose depots with no improvements in measures of glucose homeostasis".

My assumption that lack it is the of growth hormone signalling in adipocytes which promotes obesity may not be the whole explanation. It is also true that these FaGHrKO adipocytes, which are possibly very insulin sensitive, are working in a mouse with normal insulin signalling outside of those KO adipocytes. This means that the mice will have normal levels of systemic insulin sensitivity/resistance. Putting calories anywhere other than their special adipocytes will have the potential to induce insulin resistance and any increase in insulin to deal with this will undoubtedly put more triglyceride in to the insulin hyper-sensitive FaGHrKO adipocytes.

So much for GH.

The second effect in whole body GHrKO mice is that there is essentially no IGF-1 produced either by the liver or as a local tissue hormone in response the "invisible" GH. This is not the case in FaGHrKO mice, their adipocytes may never see GH but they see plenty of IGF-1 which is coming from the perfectly normal GH sensitive liver of the recipient mouse. So is it a lack of IGF-1 signalling which underlies the insulin sensitising effect of GHrKO mice?

"Maybe" is the definitive answer and "probably" the more borderline answer... I guess "dunno" still has to rate pretty well too.

If you disrupt the IGF-1 receptor of cell lines in tissue culture post-developmentally (using siRNAs) or if you study genetically IGF-1 knockout foetal derived fibroblasts, they all show marked increases in insulin signalling, which is inducible by the siRNAs when these are used. I've stuck the studies down at the end of the post. Note that none of the studies used adipocytes, but the effect appears generic to pretty well all cell lines tested.

Now, this is either receptor suppression or a receptor absence being used to generate this effect in all of the studies I've found. It has nothing to do with IGF-1 signalling, ie it's not a metabolic effect, it mostly seems to be that IGF-1 receptors associate with insulin receptors and stop them working as well as they can do. So it doesn't appear a loss of ligand induced signalling effect (though obviously there is no signalling if there is no receptor), it's the physical lack of IGF-1 receptors which causes the effect. Clearly GHrKO mice do have IGF-1 receptors, they just never manufacture any IGF-1 to stimulate them. Do these unused receptors have the effect of suppressing long term insulin signalling? I suppose it is possible that permanent, lifelong, severe elimination of all IGF-1 exposure might actually down-regulate IGF-1 receptor gene expression, so allow insulin receptors to work more effectively. We'd need an IGF-1 receptor count to be done on some true GHrKO Laron mice to find out if this is the case. The study hasn't been done that I can find but, if IGF-1 receptor genes are mothballed in GHrKO mice, this would provide a complete explanation of the Laron insulin sensitivity effect and the rest of this post is irrelevant. Just in-case it's not so, here is the rest of the post. It is very, very speculative. And might be wrong:

There is just one paper which suggests that setting up a near-complete cessation of IGF-1 exposure, with normal IGF-1 receptor genes still present, at around 10 days of age (mice again) has a long term effect to enhance insulin receptor gene expression. To emphasise: these mice have the IGF-1 receptor gene (so they should be making IGF-1 receptors), just minimal IGF-1 exposure, rather like the Laron mice. Inducing this state very early in life appears to be key for sensitising to insulin signalling.

IGF-1 Regulates Vertebral Bone Aging Through Sex-Specific and Time-Dependent Mechanisms

"Within 3 months of a loss of IGF-1, there was a 2.2-fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1".

The ad hoc hypothesis in this last paper is that insulin signalling increases to meet metabolic needs, despite there still being IFG-1 receptors present to potentially interfere with insulin receptor function. This is the suggestion that makes me think that total loss of IGF-1 signalling, with genetically preserved IGF-1 receptor genes (but which no longer get expressed), might underlie the Laron GHrKO mouse insulin sensitivity effect. It's not just in adipose tissue, it's whole body. Everything becomes insulin sensitive and the level of insulin needed to maintain normoglycaemia plummets. Low insulin signalling = long life.

You then have to ask why this might happen if we are looking for a metabolic effect in excess of insulin receptor function modification.

IGF-1, in addition to it's anabolic role, also facilitates glucose ingress, much as insulin does. We know that this is the case from a number of studies including those involving humans with defective insulin receptors (Donohue Syndrome or Leprechaunism) or in severe lipodystrophy (such as Berardeinelli-Seip Syndrome) where IGF-1 facilitates glucose uptake clinically. This would allow tonic insulin-independent uptake of glucose to generate NADH for activation of the glycerophosphate shuttle (mtG3Pdh) and set bias for reducing the CoQ couple.

Overlaid above this we have genuine insulin signalling, used to facilitate closely controlled caloric ingress and glycolytic NADH generation for CoQ couple reduction. Only small amounts of insulin would be needed in excess of IGF-1 delivered glucose to allow enough extra ingress to instigate insulin signalling. Under caloric excess smaller than anticipated amounts of insulin would be need to induce insulin resistance promptly because there is the background IGF-1 facilitated glucose ingress.

Without the tonic IGF-1 facilitated glucose supply all glucose would have to come via insulin signalling. Insulin would find each cell calorically "emptier" of glucose than it would be had IGF-1 been signalling. With an enhanced extracellular to intracellular glucose gradient more glucose should enter the cell per GLUT4 translocated. In the post prandial state all glucose entry would be via insulin alone. There would be much less need to activate insulin-induced insulin resistance, or at least it would be significantly delayed, in the process of controlling caloric ingress. Much of the time insulin could be allowed to signal and that signalling would still merely supply cellular needs without needing to induce any insulin resistance for negative feedback.

You could describe the whole body lack of an IGF-1 background glucose supply as making all cells chronically "hungry", so improving both insulin signalling and glucose ingress per unit insulin signalling enacted.

This apparent  chronic hunger due to lack of IGF-1 signalling might be where the longevity effect come from and might be why genuine caloric restriction of GHrKO mice does not add to their already considerable lifespan.

That's how it looks to me.

I'd sort of hoped that would be it for this thread but certain adipocyte transplant studies keep niggling at the back of my mind. I'm trying to ignore them.

Peter





Here are those IGF-1 receptor disruption/deletion studies:

Down-regulation of Type I Insulin-like Growth Factor Receptor Increases Sensitivity of Breast Cancer Cells to Insulin

"We used small interfering RNA (siRNA) to specifically target down- regulation of IGF1R and found that IGF1R was efficiently suppressed without affecting IR expression. However, IGF1R down-regulation by siRNA sensitized cells to insulin. Our results suggest that specific targeting of IGF1R alone enhances insulin signaling, which may be an undesirable effect in breast cancer cells".

Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

"A striking observation from our studies was the increase in insulin responsivity in osteoblasts following deletion of the IGF-1R".

Insulin Receptor (IR) Pathway Hyperactivity in IGF-IR Null Cells and Suppression of Downstream Growth Signaling Using the Dual IGF-IR/IR Inhibitor, BMS-754807

"The insulin receptor (IR) pathway in IGF-IR null MEFs was hypersensitive to insulin ligand stimulation resulting in greater AKT phosphorylation than in wt or het MEFs stimulated with the same ligand".

Differential Roles of the Insulin and Insulin-like Growth Factor-I (IGF-I) Receptors in Response to Insulin and IGF-I

"In IGFRKO cells, insulin-induced phosphorylation of IRS-1 was enhanced, suggesting that IGFR may actually inhibit IR signaling to some extent".