Wednesday, December 12, 2012

More yawns on insulin and knockout mice

Ok, I've just got 10 minutes to spare. What shall we play?

Last Saturday was the pathology lab's Christmas Party. In my cracker I was graced by a small and extremely nasty quality plastic magnifying glass which, sadly, I discarded. It might have come in useful in searching for any intellectual understanding in the Good Doctor's latest demolition of the role of insulin in obesity. Actually, the loss of the hand lens is no real problem as I doubt whether even an electron microscope would allow us to find something which is not there.

Let's begin with TNFalpha knock out mice. First thing is that these are based on C57BL/6 mice and, as I have discussed on many occasions, these mice develop an hypothalamic injury which decreases sympathetic outflow to adipocytes and so increases their ability to store fat. You need some technique to make fat mice fat and C57BL/6 are your candidate critters. They are really useful in that they lose fat in to adipocytes, just don't imagine they have anything to do with human obesity. But as a tool, they're great.

Here are the diagrams we need, taken straight from the Good Dr's blog:



All very clear cut.

But what does it mean? Fasting insulin goes up when adipocytes become insulin resistant and so leak FFAs, especially palmitic and stearic acids. Adipocytes become insulin resistant when they become over stuffed with lipid. They don't want any more fat, so they refuse to respond to insulin. Remember that the role of insulin is the storage of dietary fat, plus a little DNL if dietary fat is very low. If adipocytes don't want to respond to insulin they will signal to the rest of the body to do the same. That's working on the basis that adipocytes control whole body insulin resistance based on the ratio of palmitic acid to palmitoleic acid they release, which is in turn dependent on their own insulin sensitivity and SCD1 activity.

Now I have yet to delve in to the mechanics of adipocyte distention induced insulin resistance but I can tell you something here and now for free. It involves the TNFalpha. If you knock out TNFalpha your adipocytes (and pretty much the rest of your body) cannot become insulin resistant. No one gets fat due to insulin failing to act. You become fat due to the action of insulin on adipocytes. When adipocytes refuse to listen to insulin you stop getting fatter, but become hyperglycaemic (unless you eat LC of course!).

So the difference between wild type high fat fed mice (plus sucrose of course, the "cookie dough" they "can't get enough of") and TNFa-/- HFD fed mice is that the wild type mice are sending the signal to the rest of the body that they are fat enough and would like to stop accepting any more calories, fat or glucose. A combination of hypothalamic injury, a sucrose rich diet and a pancreas of steel makes these wild type C57BL/6 mice continue to become obese because they need massive levels of insulin to maintain normoglycaemia. All because insulin resistant adipocytes are signalling that insulin resistance should be produced in insulin controlled cells throughout the body.

Summary: Insulin sensitive adipocytes distend in response to insulin. Insulin resistant adipocytes don't. Hyperglycaemia needs to be corrected. The large dose of insulin needed for this continues to drive obesity by force-enlarging insulin resistant adipocytes. With TNFalpha knocked out the adipocytes become "effortlessly" obese. They, and the rest of the body, will stay insulin sensitive and there will be "easily achieved" normoglycaemia. Healthy obese, but still very obese. Obese due to modest insulin acting on very insulin sensitive adipocytes.


Now, on to iNOS knock out mice. These are really interesting. Again, they are based on C57BL/6 freak mice. A useful model for basic physiology, so long as you have some concept of what is wrong with them. These mice have also had inducible nitric oxide synthetase knocked out ONLY from their muscles. Adipocytes are normal, or as normal as any C57BL/6 mouse can be. Here are the weights etc from the results:



Just look at those gluttonous food intakes and fat gains! Wow.

We can get a basic handle of what is going on from this set of graphs:



Graph a is worthy of the Good Doctor.  HFD fed Nos2-/- mice are not only enormously obese, but they manage it on a fasting insulin which is LOWER (admittedly not significantly so) than that of the WT mice fed crapinabag! Blooooodie hell!

I could spend days discussing graph b, but I'm already half way through my 10  minutes so let's leave it, fascinating though it is...

Graph c. This is the pay dirt. Here they injected insulin in to the various WT and KO mice and tracked the fall in blood glucose levels. The ruler-drawn straight line of black squares is the HFD Nos2-/- mice. These massively obese mice are the MOST insulin sensitive, whole body, of all groups, certainly at the 60 minute mark.

Their adipocytes aren't. These are insulin resistant. All that remains sensitive to insulin are the targeted knock out muscles. On insulin injection these mice simply pour glucose in to their Nos2-/- muscles because the muscles ignore the signals from the insulin resistant adipocytes. Their muscle cells are like a black hole in to which glucose pours. Now, at the risk of quoting the Good Dr yet again: Hypoglycaemia is a very, very potent driver of hunger. Eat, or die.

What's on the menu? Ah, a bowl of lard sweetened with sugar. Death is not an option, let's eat the lard to get the sugar. Good idea, stayin' alive. Now, we've used the sugar, what shall we do with the lard? Ah, as a brain injured C57BL/6 mouse we have adipocytes which are rather more willing to accept fat than a genuine wild type mouse might have. Bye bye fat, in to the adipocytes you go! But at least death due to hypoglycaemia is avoided, all be it at the cost of greater obesity!

If you have been following the protons thread you can see that linoleic acid, ie corn oil, is a mild mimic of TNFa-/- mice and of Nos2-/- mice. It's obesogenic while preserving insulin sensitivity. Your cardiologist made you fat.

Now, in my last 30 seconds: Why are adipocyte insulin receptor knock out (FIRKO) mice healthy and slim? Well you could ask the Good Dr for some sort of platitude, but, hey, that would be stupid.

No. Adipocytes control whole body insulin sensitivity. They see no insulin if they have had their insulin receptors knocked out. They sport minimal (zero?) GLUT4s on their surface. What fat they contain has been accumulated without the assistance of insulin. I think it is reasonable to assume they have some GLUT1s on their surface. Any glucose taken up will be available for lipid synthesis but, without insulin's action, there will be no insulin induced SCD1 desaturase activity. So palmitate it is and, in the absence of insulin's action, this will be freely released and should signal whole body insulin resistance. But it doesn't. It does exactly the same as the palmitic acid does in SCD1 knockout mice. Peroxisiomes. FIRKO mice eat more, weigh less and (probably) generate more heat than WT mice do. They are insulin sensitive everywhere except for their adipocytes. They behave exactly as SCD1-/- mice do but get there by a rather indirect route. Excess palmitate is burned in peroxisomes and the C8 end product in mitochondria.

Life is, in the end, logical. Having the correct tools helps. It must be awful to be wallowing in the mire of the Reward hypothesis.

Peter

25 comments:

karl said...

Excellent post. The insulin-resistance boogie-man is a non-sequitur unless it is inappropriate insulin resistance.

People throw insulin resistance around as if it is a disease in itself - failing to realize that someone that is obese needs a bit of insulin-resistance to help direct the flux of fat out of adipose tissue if they ever want to lose weight.

There is another way to think of this: in electronics we talk about control loops with both negative and positive feedback. Looking at the feedback signal is often confusing as the feedback signal can be tiny if the loop gain is high. (It only takes enough feedback to change the output).

In the case of adipose tissue the signals are summed to determine the direction and magnitude of the FA flux. Looking at the absolute level of a single feedback signal is not going to illuminate understanding of what is going on.

If we look at the signals controlling adipose FA flux, the level of insulin has to be seen in relation to the level of insulin sensitivity, leptin, sympathetic stimulation etc.

Insulin obviously is central to this control loop and it takes a lot of effort to produce the cognitive dissidence required to try to stigmatize low-car ( Or should we say normal-carb?) diets. I would hate to be married to the dear doctor's thesis.



Peter said...

Karl,

Sorry for not acknowledging the heads up, post really was written in haste. Thanks. I never read that blog myself and I was amazed at how universally awful it has become. The food looks disgusting. But not as bad as the "science" on insulin. Finding a number which supports your point of view and keeping quitet about what it actually means is pretty shocking.

The Good Dr will go far.

Peter

BTW I have a great scene from Cars 1 which I might edit in to the top of the post. Just need the time.

john said...

A similar argument was used for JNK knockouts. It seems that he chooses words carefully, constructing strawmen that look real to his followers.

How do we know that the nos2-/- have insulin resistant adipocytes before becoming obese? It is only after significant muscular glucose uptake that they become fatter?

Peter said...

John,

Adipocyte insulin stimulated PI 3-kinase and Akt phosphorylation was equally suppressed in "normal" obese C57BL/6 mice and muscle Nos2-/- C57BL/6 obese mice at the end of the experiment. Slightly more so in the fatter Nos2-/- mice, exactly as you would expect from their greater obesity producing more distention-induced adipocyte insulin resistance.

Very interestingly graph b (in the fig I put up on the blog) shows that there is actually pretty severe insulin resistance (whole body, ie must include muscles) in a glucose tolerance test. I suspect these mice allow so much glucose in to their muscle cells that we get a burst of superoxide from supra physiological entry of glucose in to the myocytes. The core self protection system of superoxide mediated insulin resistance over rides the crippled control system of engineered Nos2-/- for the first 30 minutes, it's fading by 60 minutes and is gone by 90 minutes. I said it was interesting.

Also I doubt any adipocytes in C57BL/6 mice are insulin resistant until they are very distended. They start off over-sensitive to insulin, that's why the mice become fat...

The group are not looking so much at obesity as at insulin resistance and the role of iNOS in this, which leaves the data wide open for the GDr to misappropriate. It's also fascinating to see how little overall grasp of what is going on both this and the TNFa group have. Absolutely fascinating.

I have the full pdf if you would like it.

Peter

ItsTheWooo said...

Observing peter dismantle these models of "low insulin induced obesity" and the deceptive conclusion that insulin "is not required for obesity" on behalf of neophytes like Guyenet produces of feeling of all encompassing shame for the state of obesity research.

One is forced to come to the following conclusion:

1) They don't understand the physiology and implications of their own models (i.e. they're stupid)

2) They are moralistic religious zealots on a quest, at all costs, to prove that reckless hedonism and novel pleasures drive modern obesity in the same way plant opiates trigger junkiehood. Insulin, endocrine, metabolic dynamics simply do not figure in except perhaps reactive to chronic caloric overconsumption from food addiction.


There really is no other option. They either fail to understand their own models, or they are so biased in their pathetic little moral crusade they are willing to consciously or unconsciously totally distort data and fact to justify their weird eating rituals involving gruel and self flagellation.

I tend to think the latter. I believe they are intelligent and capable of sufficiently understanding these "low insulin models of obesity" if they want to. On the other hand, evidence of moral/hygiene neurosis is ubiquitous among all individuals preoccupied with framing obesity as a hedonism disorder.

It would be like asking an active pot head his opinion of drug laws. Don't expect a rational unbiased answer.

FrankG said...

I think you missed the most obvious agenda Woo... the almighty dollar!

I find it disingenuous and hypocritical when some try to discredit the likes of Gary Taubes for "selling a book" but somehow Guyenet etc.. are immune from such accusations because they are "true scientists".

He still has bills to pay (maybe a massive student loan?), he still has to ensure his future as a researcher: with grants and possibly tenure at a prestigious university, honoraria and speaking fees for taking part in industry-funded galas... maybe even a book of his own someday... and he's less likely to see any of that if he rocks the establishment boat or upsets his mentor :-)

FrankG said...

I hasten to add...

NOT that I am disagreeing with your assessment of the moralising and judgemental attitude of this "sect"

AND I heartily agree and find it enlightening that Peter: by applying straightforward common-sense along with his own technical expertise, is able to so quickly demolish fabrications that obviously seem plausible to some. Please keep it up Peter!

I've long since stopped reading Guyenet's blog myself -- even to my untrained but scientific thinking, what he asserts does not hold up to scrutiny -- and I hope that the IQ of the readership over there continues to fall.

karl said...

There is a bit the feedback loops have me thinking about: ( I might well be wrong ), but if dietary PUFA fats increase insulin sensitivity, that is a similar effect to increasing insulin - and would tend to lock FA in adipose tissue.

The huge increase in PUFA consumption is sold as being healthy based on short term studies, but life is a long term endeavor. Could this be part of the cause of the T2D pandemic?

,.,.,
As far as the grain based diet fanatics: It appears there is a pattern of quoting short term studies besides using hi-fat diets with lots of sugar.

A low-carb (or what I would call a normal carb diet) produces metabolic changes towards keto-adaption that takes a few weeks to happen. It is quite easy to show transient effects that just are not there long term.

One of the effects of going on a low carb diet is a temporary increase in LDL. Now this may come as a surprise to the low-fatters, but we did not evolve LDL to cause heart disease. One of it's functions is to transport fats (it also has immunological functions). If there is an increase flux of fat out of adipose tissue (AKA weight loss), there needs to be an increase in LDL. Once the weight is lost, the LDL goes back down.

OT-meander:
What puzzles me, is that it appears well established that only oxLDL is taken into the macrophages in artery walls - yet the medical community tests for LDL. (The oxLDL is not expensive - they just don't do it!) It is quite possible to lower oxLDL without independent of LDL. One of the things that drives the oxidization of LDL is BG - which tends to increase if you follow a low-fat diet.

Andrew S said...

Woo, I have to disagree. Or pick nits, you decide.

1. Their IQs are too low
2. They're on a moral crusade

and I offer:

3. They are poor thinkers

The vast majority of bad science I see comes from people that aren't rational or objective. They're biased, sure, but they never internalized enough about objective thought to realize that they're biased. Many of them even know what "argument from authority" means -- but totally fail to see that's what they're doing. Among the engineers I work with, there's very little acknowledgement of the difference between intelligence and rationality.

The standard model presented by scientists is that their discipline is "so complex" that no mere mortal could understand it. This probably comes about because *they* don't understand it -- to them, it's just a mishmash of rules that they memorized.

And there's also a lot of IQ worship: "how could the GDr possibly be wrong, he has a high IQ!" and "how could any medical doctor possibly be wrong, given how much they had to memorize in med school!"

tess said...

Peter, i seldom comment any more, because ... if i DO understand your posts, they're so clear and self-evident that i don't even have questions to ask! but i want you to know how much i appreciate your blogging, and what a valuable service i feel you give to anyone interested in the subject of nutrition and health. THANK YOU! :-)

Jack Kruse said...

Pete you continue to hit home run after home run while the " in crowd" continues to back slide into obscurity.....with the leaders of a template that is not our solution. Tip of the hat to you. Fat rules all mitochondrial bio energetics and this is where rubber meets road. Your blog continues to show every mistake we make in our own health/science creates an opportunity for a healthy recovery. We cant just live our life, we must create it and live that result.

blogblog said...

@andrew S,

"how could any medical doctor possibly be wrong, given how much they had to memorize in med school!"

The vast majority of medical doctors have an extremely rudimentary knowledge of basic science. They simply memorise enough to pass their exams and soon forget most of it it once they graduate.

DLS PABLO said...


so we are back 2 teh evil insulinz?

http://ajcn.nutrition.org/content/early/2012/12/10/ajcn.112.043976.abstract

btw, slow and fast fat? mind is full of fuck .. should i ditch teh butter peter?

DLS PABLO said...

edit, teh milk. this one is on butter
http://jn.nutrition.org/content/134/5/1110.long

Gadfly said...

"btw, slow and fast fat? mind is full of fuck .. should i ditch teh butter peter?"

I'm thinking that ditching the cryptography would be the best place to start.

DLS PABLO said...

sorry but if u have problemz deciphering my code then ur not really thinking much me thinks. kby

karl said...

Looks like there is a part II of the dear doctors post - and JJ one of the authors made some comments.

I find it amazing that he apparently believes that insulin is not part of the control loop that controls FA flux into and out of adipose tissue.

ItsTheWooo said...

I imagine DLS pablo posting surrounded by empty cheap beer cans, mumbling in spanish and laughing to himself as he writes down 1 liners such as this:

"sorry but if u have problemz deciphering my code then ur not really thinking much me thinks. kby"

Score another masterpiece of comedy for pablo!

Dave said...

"I find it amazing that he apparently believes that insulin is not part of the control loop that controls FA flux into and out of adipose tissue."

As Stephan points out in his own blog to the exact same comment you made, you have reading comprehension problems to make such a statement.

Principal Quattrano said...

@Andrew S:

Besides
1. Their IQs are too low
2. They're on a moral crusade
3. They are poor thinkers

there are other possibilities, like brain fog and blood sugar fluctuations from meals of gluten and sugar.

Adam Kosloff said...

Peter,

Your blog and woo's (and the smart comments of folks like FrankG and Tess) give me hope for humanity.

Keep it up!

Adam

nancan said...

Psychology may be more important here than science. The truth may set us free, but then some people really like the chains and whips.

Naxossa said...

What I love especially is that your posts are not just clearcut, easy to ready but also hilarious. On top of that you draw in excellent commentators. Thanks Peter, and yes you have wide readership. This post was referred to among others on the Dutch blog by Melchior Meijers : Paleo Perspectief. Merry Christmas,James

Robert Andrew Brown said...

Hi Peter

Thought provoking and fascinating material as ever.

This may be of interest.

http://dvr.sagepub.com/content/3/1/26.long

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