Insulin, are you hungry?

An apology. This is a dry post, I had to edit the zombies out as it was getting way too long, maybe another day. It's a bit difficult to know where to start on quite how bad this paper is. Obviously, having read the abstract, we can flick down pretty well immediately to Fig 1 in the full text.



There are a few oddities. First is the flat line in weight gain on days 1, 2 and 3. This is the suppression of hunger by insulin, maybe. There was a full seven days on insulin. This I will return to in the next post.

Next is the sudden increase in weight gain through days 4, 5, 6 and 7 in the insulin infused groups, giving a final set of weight gains on day 7 which are not statistically distinguishable from controls. Except in the group on 2iu/24h of course. The group receiving 2iu/24h is special.

Then there are the data from days 11, 12, 13 and 14. By this time the insulin infusion had stopped (which occurred around day 7ish). Look at the 2iu/24h group. Waaaay after the insulin infusion had stopped their weight gain was still much slower per day than the other three groups. Oddly this didn't reach p < 0.05, despite standard errors which were far from overlapping those of the other three groups. But trying to see what the final weights gains were is difficult because these "post pump" weight gains have been, err, umm, sort of, err. I'm not sure what the word I need is...

You see the data from these last four time points are slightly moved. Each plot has been pulled down, and by a different amount each. No one is going to say by how much. It's pretty obvious that the control line can simply be moved back up to show a linear increase in weight from the insulin infusion period as these rats never got any insulin. But all lines have been shifted down so their day 11 values are set to their day 7 values, whatever the intermediate weight gain on days 8, 9 and 10 was. It is quite likely that the 6iu/24h and the 1iu/24h rats gained weight fairly linearly and so possibly ended up on day 14 at exactly the same weight as the control group. Or heavier.

It's also very likely that the 2iu/24h group also gained weight fairly linearly but slowly, ie their "pulling down" of day 11 values to those of day 7 didn't involve much of a drop compared to the other three groups. Who knows outside the lab?

Here are the data from Fig 1 in tabular form:




Anyhoo, the 2iu/24h rats, however much they did or didn't eat/gain on days 8, 9 and 10, only gained 1.39g/d on days 11, 12, 13,and 14. Food intake per day was down significantly through this later period, 27.7g/d vs at least 30g/d in all other groups. This is very important. The implication is that if you get yourself set up with just the right insulin infusion for a week, then you still won't be hungry a week later! Wow. Insulin is a satiety hormone blah blah blah.

But if you under-dose at 1iu/24h then it's, oh-oh, back up to pre-infusion weight gain rate, or possibly slightly more. Ditto if you over-dose at 6iu/24h, just the same thing happens. Fascinating. Do you think there might be something odd about this 2iu/24h group? Perhaps someone should repeat the experiment at this infusion rate? Then we might see if the result for these rats, on which the whole concept of suppression of weight gain over 7 days rests, was a quirk. No stats were done on the zero weight gain days, ie days 1-3 on insulin. The only p< 0.05, on which the title of the paper rests, was the 2iu/24h group at day seven.

If we lose the 2iu/24h group all we can say is that an insulin infusion reduces weight gain for three days, with complete restoration of any lost weight gain by the seventh day of a continuing infusion.


So, has the experiment been repeated? Luckily it has. By this very group. And the results are in this very same paper! But well buried. You have to be a dissonant pedant to find it. It's all in Figure 4.





This not quite the same experiment as Fig 1, the timings are slightly changed, but the basic design with insulin at 2iu/24h for seven days is identical.

In the main experiment time "on pump" was 7 days and they looked at all of these days, averaging everything over this time.

In Fig 4 they did the same 2iu/24h pump for seven days but only analysed days 3, 4 and 5 as time "on pump". Go figure. They also chose days 8, 9 and 10 as their "post pump" days vs days 11-14 in the first part of the study. Again, go figure. But eyeballing the graphical weight changes in Fig 1, I doubt this matters.

The data in Fig 4 look at meal size and meal frequency because that's how you bury data. But we can reverse engineer Fig 4 to get total food intake per day. Take a ruler to the graph. Multiply meal size by meal frequency and you get food intake per day, neat huh?

The rats on 2iu/24h ate 25.5g/d during "on pump" days 3, 4 and 5. This is pretty much the same as the total 7 day value from Fig 1 and Table 1. Happy researchers? Well done for correct choice of days. But...

Does the depressed food intake continue even after insulin has finished? Do you get sustained appetite control if you get the insulin infusion "just right" for a week? Eyeballing Fig 4's "post pump" values, these are about 3.4g/meal, 9.8 meals/day giving over 33g/d food intake...........

My, those are bloody hungry rats! This is the highest food intake per day in any group in the whole paper. It's the direct opposite of the findings presented in Fig 1 "off pump" section. The sustained depression of food intake shown in both Fig 1 and Table 1 could not be repeated in the Fig 4 experiment.

It doesn't happen.

The 2iu/24h group are no different to any other infusion rate when you look at Fig 4 "post pump" section. Quite why the rats on 2iu/24h used to generate Fig 1 data showed depressed weight gain long term is a complete mystery. Personally I'd want to have had a pathologist check out the pumps in the lowest food intake rats in this group, looking for low grade peritonitis. The pumps are in the abdominal cavity. Maybe some surgeon dribbled in to the wound during implantation. I've worked with surgeons. Ultimately we'll never know.

But ANYONE quoting the data presented of Fig 1 to you WITHOUT even mentioning the results of Fig 4 to you is, well, hmmmmm..... probably in obesity research.

I was going to go on to discuss the flat line of weight gain on days 1, 2 and 3 (at all insulin infusion rates) next but I'll leave that to another post as it has nothing to do with the "insulin at 2iu/24h causes sustained decreased food intake" claim.

Which is complete bollocks.

Peter

The Zombie paper

Just a brief thank you to Julianne and Beth for the full coffin nail paper. I was going to leave zombie rats alone after the last post and didn't think the paper itself was needed to see what was going on. But the quick scan I've had of the coffin nail shows it to be very interesting. Want to read some execrable science? I'll deconstruct it soon but there is an on call night tonight and a family weekend coming up, so I might not be as quick as it deserves. But don't worry, there are still plenty of zombies around...

Perhaps best not comment on this post, comments can stay on the last one all grouped together. I'll take this one down as the next one comes through.

Oh, Hi Melchior. Nice to see you about. It's good when facts plus logical consistency ultimately win through. As they must.

Peter

Insulin, the Un-dead and coffin nails

Things keep getting in the way of the next post, which is roughed out but needs tidying. I've also been meaning to post on the Somogyi overswing effect in diabetes treatment for some time, so here is a minor diversion down that route, mostly because it's very illuminating.

The Somogyi effect is quite common in those unstable diabetic dogs which tend to get shunted in my direction at work. Any clinician will recognise the effect. A dog is given 8iu of lente insulin at 7am with a meal of utter crap (ultra low fat Chappie usually). Blood glucose spikes to 22mmol/l by 9am from the carb load then falls progressively until about mid day, as the slow onset insulin struggles with the hyperglycaemia. It looks like there is a nadir at about 6mmol/l around mid day. The 1pm reading is unexpectedly high at 30mmol/l. No food, no behavioural signs. Just sudden hyperglycaemia. The Somogyi overswing. This fades slowly to around 15mmol/l by the next meal time at 7pm. The cycle repeats.

Management (if you can't change anything else) is to reduce the dose rate of insulin, which stops that sudden surge in blood glucose at 1pm. Somogyi attributed the effect to a reflex release of glucose from the liver to prevent catastrophic hypoglycaemia in response to insulin overdose. Most clinicians seem to still think in these terms.

Logical but incorrect. The advent of continuous glucose meters has pretty well disposed of the "hidden hypo" explanation and people are now looking at the effects of hyperinsulinaemia per se. The sudden rise in blood glucose appears to be associated with progressively rising or even peak levels of insulin in the blood.

Let's have a think about what is happening. Under insulin deficiency conditions glucose can still be used as a fuel, in a somewhat unregulated manner, using concentration driven supply through GLUT1, independent of insulin. Hyperglycaemia is essential for this. It's not good. The poorly regulated glucose supply generates free radicals in the electron transport chain. Superoxide is the main one and this appears to be the key to causing insulin resistance. Hyperglycaemia causes insulin resistance. This is not controversial, as far as I am aware.

As the insulin kicks in we have a period where glucose levels are falling so GLUT1 transport is decreasing and insulin regulated GLUT4 transport is increasing. Initially excess glucose above cellular needs diverts to glycogen and the respiratory chain is kept happy by insulin. As insulin levels continue to rise above physiological needs we end up with a situation where insulin is putting a ton of GLUT4s out, far more than are needed. This happens because we have inadvertently injected a supraphysiological dose of insulin.

All those excess GLUT4s allow glucose molecules to pour in to the cells. You might as well have hyperglycaemia and GLUT1 mediated oversupply, as far as the respiratory chain is concerned. Glucose in excess of the cell needs generates superoxide. Superoxide triggers, as an antioxidant defence mechanism, insulin resistance. With thanks to Dr Guyenet. Again. It is difficult to emphasise how good this paper is.

Somogyi overswing is likely to be caused by acute onset insulin resistance occurring as a direct result of excess glucose uptake in to cells due to supraphysiological insulin concentrations.

The temporal association with hypoglycaemia, which misled Somogyi, comes from the time course of switching sources of glucose oversupply. The hypoglycaemia is not causative, it is just common for it to occur at around the same time that insulin/GLUT4s oversupply substrate to the mitochondria and they say no to it, using insulin resistance.

Let's summarise. This is very, very important:

Excess insulin causes insulin resistance

End summary.

This is just day to day internal medicine. You have to pay the mortgage somehow.




If anyone is interested there is a rather nice discussion paper here, it's pay per view and doesn't say much more than is in the abstract but it has a nice set of references. I have access to a great Athens account. All the comments on insulinomas ring so true to clinical life too.

It's also interesting to go back to the controversies around the Somogyi effect, you can read Somogyi's ideas here and the continuous glucose monitoring evidence here. All very fascinating stuff (well it is to me!) but what does it have to do with shooting fish in a barrel?



Question: Who are the Un-dead?

Which can be rephrased as: Can we control the Somogyi effect?

If we take the average bodybuilder from a few years ago and watch him self-inject with insulin for its anabolic effects and then forget to eat the carb load needed to balance it, we can see the acute effects of insulin overdose. Insulin rises very rapidly from the regular insulin used and every GLUT4 receptor in his body pops on to every cell surface which uses them. There is a free fall of glucose from plasma in to the cells, blood glucose plummets and the chap ends up in A&E or, quite possibly, in a mortuary. There is no time for the massive cellular caloric overload from over-translocation of GLUT4s to generate enough insulin resistance to stop the hypoglycaemia. Glucose pours out of the bloodstream until it drops to levels low enough to kill the brain. Sad but true. Somogyi effect is too late, too little. Insulin overdosed bodybuilders are not the Un-dead.

So who really are the Un-dead?

What if you give insulin as a constant rate infusion, initially at a low rate and gradually crank it up?

Think it through. Progressively increasing insulin levels allow progressively greater amounts of glucose in to cells. If the cellular glucose supply is greater than cellular needs there is increased generation of superoxide by the respiratory chain which signals the cell to become resistant to insulin. A balance is achieved. Increase the insulin CRI, overcome the insulin resistance, generate more superoxide, generate greater insulin resistance, achieve a balance. Do it again. And again. More. Again. How high can you get plasma insulin by playing this sort of game? Here's the table we need:



Okay, they stopped at a total of 6iu/24h/per rat. They could possibly have gone higher but hell, we have here a set of rats with a mean insulin level of 588.9microIU/ml. No, that is not a typo. The SEM was 89.7. Anyone like to guess how high the highest insulin level measured was? Quite high perhaps?

These are the Un-dead. They walk around, without any genetic modification, with an insulin level which, if achieved acutely, would have put them rapidly in to a clinical waste bag. They are very, very, very, (repeat ad nauseam) insulin resistant, otherwise they would look like the bodybuilder in the mortuary.

*****************************************************************
WARNING: There is a black box paradox warning about the paper providing Table 1. I'll stick an addendum on the end of the post.
*****************************************************************

Soooooooo. They are, undoubtedly, hyperinsulinaemic. Are they fat? Of course not. Why should they be fat? They are the Un-dead. If they were remotely sensitive to insulin they would be not be the Un-dead, quite the contrary. But insulin induced insulin resistance does not spare adipocytes. These have mitochondria and generate superoxide. They too will ignore insulin, to a level determined by their mitochondrial superoxide production.

Here's a bit of an aside: The process is physiological. It involves a careful titration of cellular insulin resistance to the cellular energy needs. This is no blanket insulin blocking drug. The responsiveness to insulin is carefully adjusted to just allow enough glucose in to cells to meet their needs. This applies to adipocytes as well as well as to muscle cells. With the number of GLUT4s being translocated by the residual insulin sensitivity, in an environment of 588microIU/ml of insulin, you don't need much of a blood glucose level to supply glucose needs. Table 1 suggests the body settles to a plasma glucose of about 71mg/dl, as opposed to 148mg/dl in the control rats. Metabolism is still largely glucose based, with some responsiveness to insulin preserved despite the need for resistance to survive at 588microIU/ml. Transplanting tissues to a petri-dish allows you to pick up this responsiveness. Free fatty acid release from adipocytes is not significantly inhibited because the adipocytes are insulin resistant to a level where they maintain normal function. Weight gain is similar to that of control rats.

And another BTW. The process is cellular. Bugger the hypothalamus.



A nail in someone's coffin?

Apparently these rats are a nail in the coffin of the insulin hypothesis of obesity.

The actual coffin nail [nb if the link comes up with a failed log-in just refresh the page] is a pay per view article in a journal not covered by Pubmed and I'm unwilling to shell out $40 for it. Perhaps I could ask The Good Doctor for a copy. Fortunately the information on CRI rodent models is freely available in the paper which provided Table 1 above. What is crashingly obvious is the utter lack of understanding of insulin induced insulin resistance by people who are fixated on insulin as a satiety hormone.

This might have been acceptable in 1980 when the physiology of insulin resistance was completely unknown. But to see this explanation promoted by the same obesity researcher who provides us with the concept of insulin resistance as a cellular antioxidant defence mechanism, mediated through superoxide, is utterly depressing. We are, after all, talking about a complete failure to understand the basic physiology of insulin resistance, with the key paper sitting as a free download from Pubmed.

Does the Good Doctor not understand his own citations or is he stuck with terminal cognitive dissonance?

Or perhaps he's just utterly confused.

I feel the coffin nail is misplaced.

Peter



OK the paradox: In the paper providing Table 1 the rats have a blood insulin level of 588microIU/ml with physiological blood glucose levels. BUT isolated muscle and fat cells taken from these rats are highly insulin sensitive, more so than those from the control rats. How is this possible? I can imagine the Good Doctor or some other idiot shouting that the rats aren't insulin resistant at all, because the paper clearly shows their tissues are extra insulin sensitive, ergo the insulin hypothesis of obesity is wrong. Peter is misquoting a paper, you know what I'm like!!!! Gotta read all those papers cited, the Good Doctor knows how few people follow the links.

But the rats are definitely Un-dead.

If you culture adipocytes at consistently supra maximal insulin levels they behave exactly as the whole rats do. So if you pull out a muscle or fat cell from an Un-dead rat, having made it an un-Un-dead rat by decapitation, how long will the insulin resistance last? This is probably determined by the elimination half life of superoxide. Which is, err, not very long... Actually, it's probably determined by the cellular redox state providing the superoxide, which should last at least a few seconds after decapitation.

Confused

Oh dear,

Back in this post I discussed the study by Knudsen et al on forced overfeeding. It found, very clearly, that acute overfeeding produces acute fasting hyperinsulinaemia, provided you feed utter crap.

The hyperinsulinaemia moderates progressively over the next two weeks, at which time the study ended.

In comments after the post this one came up from Dr Guyenet:

"You are utterly confused Peter. These people only gained 0.8 kg of fat mass. Over the course of the study, they went from lean to slightly less lean. 

If you look at studies where overfeeding produced greater fat gains, you see a consistent increase in fasting insulin that corresponds with fat gain, just as those silly obesity researchers would predict:



www.ncbi.nlm.nih.gov/pubmed/18171910

www.ncbi.nlm.nih.gov/pubmed/20814413

www.ncbi.nlm.nih.gov/pubmed/21127472



In animal models of diet-induced obesity (rodents and dogs), blocking the hyperinsulinemia has no effect on the rate of fat gain.

The carbohydrate-insulin-obesity hypothesis is dead and buried, and all that remains are rearguard attempts to salvage it using increasingly complex theories. Just let go of the cognitive dissonance man."


with those three studies to back up the comment.

So, being a bit of a dissonant pedant, I checked the studies for information on changes in fasting insulin with time. Here they are, with Knudsen's data at the top:



nm stands for not measured.

As you can see 18171910 covers none of the acute changes in insulin levels discussed in the post. I've spent a great deal of time discussing adipocyte distension induced insulin resistance and this will be the end effect of sustained adipocyte distension. It will kick in eventually and certainly affects fasting insulin levels.

The second study clearly shows on day 14 and day 28 EXACTLY the same changes seen by Knudsen et al in their 14 day study. Anyone want to how high fasting insulin peaked on day 3 in 20814413? Not measured, but answers on a postcard to... Hint, probably very high.

The third study measured, but doesn't report, fasting insulin only at > day 56 of overfeeding. Oh, and day 0 of course.

These are the classic half truths so typical of modern obesity research, technically correct but comprehending nothing.

But here's the real giggle, again quoting the Good Doctor:

"These people only gained 0.8 kg of fat mass"

These people gained 1.5kg of fat mass. From 10.5kg to 12.0kg. The drop to 11.29kg of fat mass occurred AFTER the overfeeding had finished.

I don't suppose reading the studies matters that much in obesity research.

Personally, I'd be embarrassed to be Dr Guyenet.

Peter, as confused as always.

A glimmer of light

For those who do not have the supplies of anti emetic necessary to read main stream nutrition opinion it's worth noting that not all obesity researchers are idiots. I rather like this article. Woo might too, after her last foray on the WHS for the Noddy guide to obesity.

Peter

Insulin and the Rewards of overfeeding

I've been tempted away from the electron transport chain, origins of life and the suspected paleo prompt nuclear criticality on Mars by a neat paper from Liz.

This is what they did in the study: Over-fed 9 slim, young, fit, healthy blokes for two weeks while limiting their exercise. They establish an energy surplus of about 2000kcal/d. Here we go:




Needless to say, they gained weight. Here we go again, Table 3:



A total of about 1.5kg in two weeks.

Now, all you have to do is to go and ask any cutting edge, state of the art obesity researcher and you can be told that hyperinsulinaemia is a consequence of obesity, not a cause, and that carbohydrates are the worlds greatest slimming aid because insulin is a satiety hormone and, oh, did I fall asleep there?????? Sorry.

Back to the paper. The overfeeding was with utter crap



and produced a rise in fasting insulin from 35pmol/l to 74pmol/l in 3 days.

Personally, I found this quite amusing.

Aside: What would have happened if they had overfed with lard? That's another post, it has been done, rather badly, in Schwartz's lab using dogs. They gained weight. End aside.

By 3 days the fat mass had increase by an average of 100g. My, that is potent fat! Here's the table, day nought vs day three is the place to look:



We could leave it at that and just go away scratching our heads about what goes on in the minds of obesity researchers, but it does get quite interesting. Obviously, as obesity progressed the level of insulin should increase if insulin resistance is caused by fat mass. It doesn't. Fasting insulin falls progressively after the initial spike, and in a fairly linear manner, through days 3, 7 and 14, while weight (and especially visceral fat) actually increases over this time period.

So the idea that fasting insulin rises as a consequence of rising fat mass is, well, you know what it is.

Ah, but if insulin stores fat, why should the level of insulin fall progressively during a sustained hypercaloric eating episode? Surely you must need insulin to store those extra calories? In fact, as insulin levels fall, so does the rate of fat storage. The chaps gained, from Table 3, 1kg of fat mass in the first week and only 0.5kg of fat in the second week... Oh, I guess this must be because the subjects either (a) sneaked off to the gym in the second week or (b) flushed their Snicker Bars down the loo in the second week, without passing them through their gastro intestinal tract first (good idea!) or (c) got bored with Snickers and stopped finding them rewarding. And of course they disconnected their Actiheart monitors at the gym.

Otherwise how you can eat 2000kcal over your energy expenditure, equivalent to nearly 200g of fat gain per day, and gain a kilo of fat in the first week, then continue to eat an excess 2000kcal/d for a second week and only gain half a kilo of fat? Calories in, calories out, you know the rules. Hmmm, in the second week there are 14,000 excess calories-in, 5,000 stored, very interesting.

We all know the obese lie about calories. It seems probable that so too must experimental subjects, in direct proportion to the duration of their over eating! Now we know. Bit of a milestone paper this one.

So what is really going on? What appears to be happening is the insulin system working exactly as it should do. Insulin resistance protects cells from caloric excess, when forced in to the body by a study protocol. Think of it in these terms, with thanks to Dr Guyenet from back when I used to read him. The vast majority of free radicals will be generated at complex I.

The mitochondria say they have too many calories. It's easy for mitochondria to refuse calories from glucose by using insulin resistance, working at the whole cell level. In the presence of massive oral doses of glucose this must elevate insulin to maintain normoglycaemia. The elevated insulin diverts calories from dietary fat in to adipocytes, away from muscle cells. And inhibits lipolysis at the same time, look at the FFA levels in Table 3 on days 7 and 14, waaaay down from pre and post study values. I wonder why they didn't measure FFAs on day 3? So insulin goes up to maintain normal blood sugar levels, overcomes insulin resistance to run cells on a reasonable amount of glucose and shuts down FFA release to counterbalance its action in facilitating the entry of glucose in to cells.

Core to this is (a) there is no hyperglycaemia, insulin still successfully controls glucose flux and (b) insulin inhibits lipolysis. So you store fat. These subjects are both young and healthy. They do not have insulin resistant adipocytes, mitochondrial damage or a fatty liver. The system works as it should.

As time goes by fasting insulin levels fall and weight gain slows. Calorie intake doesn't drop. The only plausible explanation is that the subjects generate more heat and radiate that heat during the second week of the study. Total energy expenditure was estimated using the Actiheart device. You have to wonder how well its computer algorithms coped with the massive overfeeding. It looks like the weakest link in the protocol, assuming the the subjects really ate their muffins and Snicker bars. The device is supposed to be very good but where else did those calories-in go? So let's consider uncoupling proteins. These decrease the inner mitochondrial membrane potential and so decrease free radical production, which decreases insulin resistance. You can afford to allow more glucose in to cells when the UCPs are in place and working. More posts on this to come when we get back to the electron transport chain and free radicals. Free radical generation at complex I is VERY dependent on the inner mitochondrial membrane voltage.

Unfortunately the clamp studies were only performed on days 0 and 14, so all we can say about the insulin sensitivity by clamp (gold standard) is that it was worse on day 14 than day 0. Who knows what the results would have been on days 3 and 7? This protocol is understandable as clamps are a pain to do, but there is no way of ascertaining what glucose disposal per unit insulin was through the body of the study.

So forced weight gain fits quite nicely with the role of insulin in fat storage. It's a nice study because it looked at insulin before fat gain had occurred, and kept on looking too. But how much does forcing people to overeat tell us about "accidental" weight gain in people who have spent good money on some slimming plan to lose weight temporarily with enormous difficulty? Under these circumstances calories are offered to cells at very reasonable levels, mitochondria are already dysfunctional and signal (using free radicals) excess calorie warnings (free radical leakage) and so induce insulin resistance inappropriately. So calories, especially those from dietary fat, get diverted to adipocytes through the subsequent hyperinsulinaemia. And are kept there due to fasting hyperinsulinaemia.

The situations are quite different, I'm not sure that overfeeding healthy subjects tells us too much about accidental obesity, except that they both seem to work through insulin and mitochondria. But this study does tell us a great deal about the idea of reward.

This study is the ultimate affirmation of the Reward Hypothesis of obesity:

If you reward people with enough Danish Krone for over eating, they gain weight.

Peter

Cholesterol: More epidemiology

Too hilarious not to comment on!

Lipitor because your TC "number" is "bad"?.

Ah you can say, but what about psLDL? They never measured psLDL! Mmmm, sucrose...

Thanks to Karl for the heads up

Peter

On GLUT5

Presentation-on-video is one of the least accessible sources of information for me. For a stack of practical reasons I don't get a lot of listening time. I made the effort the other day and got depressed. Why?

We all know hereditary fructose intolerance tends to produce death when some joker sets up an intravenous fructose infusion in the intensive therapy setting.

But why should anyone want to use IV fructose in the ITU setting? Calories. Critically ill people need calories. Lots of them. Meeting this need has been managed in many different ways over the years. Nowadays we do it through an enteral feeding tube or, if you can't get anything in through the gut, using intravenous emulsified soyabean oil (pardon the aside vomit) or emulsified MCTs (ah, that's better, let's have a few ketones in the ITU). You can't use glucose. Unless you add exogenous insulin to a glucose infusion (plus some potassium please) it just makes you hyperglycaemic and you then pee glucose out through your kidneys, especially if you are already as insulin resistant as a lot of critically ill patients are.

Not so with fructose. Infuse fructose in to a peripheral vein and it immediately disappears in to liver tissue to replenish glycogen stores before any excess becomes intra hepatic fat. But it also disappears in to muscle cells, big time. And adipocytes too. In fact, in to any cell expressing the GLUT5 transporter* on its surface. Even bits of the brain do this. No insulin required. Fructose gets calories in to tissues without wasting too much through exceeding its renal excretion threshold. That's why it got chosen for ITU work back in the 1970s. It works. A pity it kills people with hereditary fructose intolerance.

* Did you note "testis" in there? Human sperm appear to run on fructose. There is a post on sperm, fructose and nitrous oxide injected Morris Minors brewing for some day.

I've been particularly interested in whether oral fructose might cause whole body insulin resistance directly, especially adipocyte insulin resistance, rather than just being limited to hepatic de novo lipogenesis and the systemic sequelae associated hepatic insulin resistance. You have to ask whether fructose really stops at the liver, especially in human beings. After all, those GLUT5 transporters are not sitting there on myocytes and adipocytes awaiting an intravenous fructose infusion... So what is the plasma fructose concentration after something a bit like a large McDonald's Coca-Cola Classic®, the 32oz serving? With or without breakfast, and compared to assorted ratios of glucose and fructose? Here are the (very old) data:



Fructose penetrates well past the liver in humans after a 32oz cola. There are GLUT5 receptors on multiple non hepatic cell types. Fructose effects do not appear to be limited to the liver. I was really interested in this some time ago but never got to post about it. It's quite speculative.

The question is: Why are some of the nurses I work with skinny despite subsisting on sucrose in various guises?

Apart from the possibility that skinny folks who eat a sucrose based diet are chronically hungry (some certainly are), why else might they not be fat?

Perhaps: If you can dump a decent dose of fructose on to adipocytes you might well limit their response to insulin. These folks would still get the classic fatty liver and hyperinsulinaemia but fail to store large amounts of fat in adipocytes because fructose exposed adipocytes would become insulin resistant and so release inappropriately large amounts of FFAs for a given level of insulin. They are, after all, resistant to insulin's fat storage effect. No one would suggest that this is a healthy situation and terms such as "skinny-fat", "metabolically obese but slim" and "slim type 2" all come to mind. Weight per se is unimportant, chronic hyperinsulinaemia comes with its own costs.

Where did all of this come from? Tom Naughton and Makro both provided the link to this video.

I agree with a great deal of what Lustig has to say. But I find it hard to swallow an argument when certain easily disprovable biochemical statements are made. Saying that fructose uptake and its transporter are limited to the liver is disappointing. We have enough problems with misinformation on carbohydrate restriction from folks with carbophilia. It's depressing when we simplify our message to the point where even a politician can understand it, ie to the point where it is no longer true.

Sigh.

Peter

Addendum on skinny fructose fed rats:

Here's a typical paper, there are probably loads more out there.

Fructose fed rats are lighter than CIAB fed rats. Here's the table we want:



Despite the lack of gross obesity, the epididymal fat pads are bigger, the actual adipocytes in these pads are bigger than those from controls and the adipocytes are, of course, insulin resistant. Just look at the FFA levels in the table.

Now, let's look at the OGTT results:



The fructose fed rats clearly produce a ton of insulin in response to an oral glucose load, far more than the CIAB fed controls. If they can produce this much insulin, surely they must be able to overcome adipocyte insulin resistance and make themselves FAT? You can certainly store a whole load of extra fat in the adipocytes of an insulin resistant human by adding exogenous insulin in to their diabetes meds...

The group doesn't specify the diet exactly (they must be in obesity research) but it's made by Teklad Laboratories and is probably TD.89247. About 67% of calories from fructose, no other source of carbohydrate.

If so, the bottom line is that these fructose fed rats are on a mix of casein, lard and fructose. Only fructose. They NEVER eat starch. They NEVER eat glucose. They NEVER eat sucrose. Fructose per se is only mildly insulinotropic. An OGTT tells you NOTHING about the insulin response to a meal of TD.89247.

Which these rats live on.

Now what happens if you add fructose AND glucose to a rat's lard/casein diet? It's called sucrose. It is, err, obesogenic. Especially in the presence of easily storable fat.

Because here you spike insulin while simultaneously causing insulin resistance, which will cause a MUCH bigger insulin spike than even the rats on CIAB produce after each meal. Adipocytes can be forced to enlarge under these circumstances. It's called obesity.

Of course it might just be that sucrose is more rewarding than that significantly sweeter molecule, fructose. Perhaps fructose is sooooooo sweet it becomes unrewarding? Or boring. Or perhaps the idea of reward from sweetness is complete bollocks and we are dealing with a simple biochemistry process. I rather like biochemistry.




If we go back to my skinny workmates you can now start to ask questions about a person's ability to absorb fructose from the gut, the ability of their liver to mimimise passage of fructose to the systemic circulation and the level of expression of GLUT5 on adipocytes etc. You can even think about whether these people may, by going to Weight Watchers, limit their intake of starch which is a significant drive to insulin production when added on top of the insulin resistance from their sucrose fix.

But ultimately we're still thinking about biochemistry.

GSD type I vs GSD type III: Cornstarch vs ketones

Very briefly: I hit this by accident. Some time ago there was an exchange of comments about whether VLC eating might function as a management for Glycogen Storage Disease type I, von Gierke's disease. I think the question was firmly, and possibly incorrectly, settled by mnature pointing out that raw cornstarch was THE answer. No choice. I realise GSD type III is not GSD type I, but many of the clinical signs are the same, especially hypoglycaemia. There is choice in type III.

The group have tried a high protein diet to provide hepatic glucose from gluconeogenesis, a mildly ketogeneic diet to AVOID providing exogenous glucose because it just drops in to a bottomless pit of stored glycogen and some synthetic ketones à la Veech to provide some glucose-independent ATP.

Just a case report, it worked. So far so good. Interesting.

Peter

PS The new blogger format. I hate it. Can you make the links live in preview????

FIRKO-isation without all the hassle?

OK, I've treated myself to a few hours at the blog.

If we look at Veech's 2011 paper we can see that he is driving towards a drug which induces ketosis, side stepping all of that starvation or very low carbohydrate eating normally required, outstripping octanoate for carbohydrate defying ketosis. You can even FIRKO-ise your mice without all that standing around on a hill top in mid winter. It would, essentially, allow all of the benefits of ketosis on metabolic efficiency while still consuming a diet of utter crap. That's not something which interests me terribly much, though I can completely see where he is coming from. It will do some good but probably do very little to influence the underlying progress of the disease.

We had friends of friends round to supper the other day. They were interested (as Veech is) in ketosis as a tool to try and modify the progress of Parkinson's disease. So we had a baked mushroom each, filled with bacon and melted soft cheese with a smidge of fried onions and a micro smidge of spinach then topped with half a round of goat cheese for starters, belly pork goulash with soured cream, broccoli and asparagus for main course and Optimal ice cream (minimal dose of honey, no sugar) with a few raspberries for desert. Coffee and double cream. Modest red wine portions. The funniest part was trying to explain to them that serious researchers/nutritionists genuinely explained away the reduced appetite on LC diets as being due to either boredom or lack of palatability.

As Veech commented:

"Further, to achieve effective ketosis with KG diets, almost complete avoidance of carbohydrates is required to keep blood insulin levels low to maintain adipose tissue lipolysis. Such high-fat, no-carbohydrate diets are unpalatable, leading to poor patient compliance."

Eeeh, the stuff people come up with. Personally, I think Veech should sack his chef. Or stop eating F3666 and hire a chef.

The other gem from the paper was this line here, talking about his ketone ester fed mice:

"The ketone levels are similar to those found in humans during prolonged fasting (33, 34) and are 3- to 5-fold higher than the levels reported for mice fed KG diets (13, 15)."

It's the last section of that quote that really made me sit up. Both ref 13 and ref 15 are sitting on my hard drive. They use F3666, 8.6% protein, 3.2% carbs and lots of fat. They found ketone levels of 1.3mmol/l and 1.6mmol/l.

That is amazing. Amazingly pathetic ketosis. Nine percent protein, minimal carbs, the rest fat. If you or I ate this diet for more than a few days we would be peeing brilliant purple on our Ketostix.

What is really special about this Veech study and the other two mouse ketosis papers is not what they tell us about how to get in to ketosis (or not), it's much more what they tell us about C57BL/6 mice. That's right, C57BL/6 mice.

These mice are very special.

I've long thought that these poor rodents behave, when fed a high fat diet, rather like MSG lesioned, ventromedial hypothalamic lesioned or gold thioglucose lesioned animals. Their VMH breaks. They develop neuronally mediated acute insulin hypersensitivity in their adipocytes, they then abnormally store fat at low levels of insulin, increase eating to compensate for this calorie loss in to adipocytes and eventually develop adipocyte distention induced insulin resistance, which shows as metabolic syndrome.

It is impossible to over emphasise how important these ketosis studies are to C57BL/6 mice. Especially if you happen to be a C57BL/6 mouse.

BUT let's pretend none of us is a C57BL/6 mouse, just imagine you are a Wistar rat on 11% protein added to your traditional diet of neat Crisco (Mmmmm, Crisco, yumeeee). You will be in to ketosis with a beta hydroxybutyrate at 4.8mmol/l, and probably develop another mmol/l of AcA, within days and stay there. Are humans more like C57BL/6 mice or Wistar rats? I have no doubt that a human can damage their VMH by the same process by which they become obese. I doubt very much that this has anything to do with eating fat. Sucrose is much more likely. But even if you are obese and have damaged your VMH while becoming obese, you can still get your BHB over 7mmol/l. It may take some time, or even a little water fasting, but you can do it.

BTW Crisco induced ketosis is neuroprotective although I'd personally rather do the same with butter!

If you are a human looking to manage Parkinsons you can quite easily get to 6mmol/l of ketones in your bloodstream. You are not a C57BL/6 mouse. You don't even need Crisco, selected Food will do it.

The massive benefit of a ketogenic diet over the "SAD spiked with ketone esters" approach is that ketogenic diets avoid hyperglycaemic episodes. If you think hyperglycaemia is good for neurons you are probably well in to some nasty neurodegenerative disease!

Peter

BTW Apologies for the total lack of contribution to conversation in the comments. I have the choice between the occasional post or trying to get comments answered and a lot of the time neither gets done. Here's the occasional post. Obviously the next step, given time, is back to Veech 1995 where he talks electron transport chain, mitochondrial inner membrane voltages, proton leakage and a whole load more about very basic concepts, some of which are quite fascinating. Including the benefits of insulin. He then is talking in Nick Lane territory. And I hope everyone noticed that Stan has been to Nick Lane's website and has linked to this publication. I just loved this quote about the acetyl CoA pathway:

It's "a free lunch that you're paid to eat," in the words of Everett Shock.

My own light reading at the moment is this one, as a kid I thought tunnel diodes were cool.

Still not on line

Hi all,

People may realise that I don't really have any great interest in, or knowledge about, the blogging platform I use, so it's a real pain when Google changes odds and ends and I end up unable to even view comments using Firefox and with Safari I get access to comments but no ability to even delete spam from the blog itself. This is a bit of a pain and is markedly limiting.

Meantime the cutting and stitching business is taking rather a lot of our time and even deleting the spam is not looking like happening in the immediate future.

I'll get back on line properly when I get more sorted. At the moment our priority in the evenings is to try and get ahead of running the house so we can have a smidge of free time as a family at the weekends. Serious stuff for the blog is reverse transport of electrons through complex 1 as a major route to mitochondrial free radical generation in the ETC and why it might occur. Preliminary reading would be Lucas Tafur from a year ago here. A great post. I spent hours on Veech's 1995 paper before tripping over Lucas' rather tidy discussion!

All the best

Peter

Fruit and vegetables

Still no net-time to speak of but life goes on. Look at these brightly coloured fruit and vegetables:



They look so good I just can't help myself. I just have to shred them



and feed them to the chickens



who can convert them to Food



Eggcellent (sorry)

Peter

OK, I do have to admit to eating the occasional vegetable as flavouring for Food.

NASH on a ketogenic diet

Just a brief post on the development of NASH in long term ketogenic fed mice because I've been side tracked by some other papers: Starvation (Ethan), metformin (tripped over this one myself, things are moving forward from PSS and Oxygen), UCPs and fat oxidation (kindke)...

NASH, it all comes down to this paper (thanks Liz).

Does sustained ketosis produce NASH? Well yes, or at least something like it. Just look at the picture:



The dark staining patch is made up of neutrophils. You don't want clumps of them aggregating in your liver.

That's what you get if you feed F3666 to C57BL/6 mice. It is, undoubtedly, intensely ketogenic. But is it anything else?



"These are typical amounts of nutrients calculated from
available information. Actual assay results may vary.
For more information contact Jaime Lecker, Ph.D."

The paper (and the F3666 pdf) specifies 16% of PUFA in the fat, which seems rather low considering that USA produced lard (the main ingredient) appears to have 32% PUFA (almost all omega 6) but, even at only 16%, these PUFA are 16% of 95% of your total calories. That's an awful lot of PUFA...

Do PUFA matter for the development of NASH? Probably. From this N and M paper:


"The lipid composition of the different diets which induce steatohepatitis (see Table 4) [19,20,51,52,54], were lard and corn oil, both oils rich in unsaturated fatty acids. We can observe that fat of all the diets inducing steatosis and inflammation (Table 4) were richer in MUFA and PUFA (>30% and >20% of total fat respectively) as compared to our diet (5% and 2%). The injurious effect of unsaturated fatty acids, and particularly n-6 polyunsaturated fatty acids, was associated with enhanced lipid peroxidation and decreased concentrations of antioxidant enzymes, implicating oxidative stress as a causal factor. Indeed, different studies showed the pro-inflammatory effect of polyunsaturated n-6 fatty acids which exacerbate liver oxidative stress [60,61] and promote the development of NASH."


So there is a message: If you are going to eat a very high fat diet in the long term, you should pay serious attention to PUFA creep. Having cirrhosis is not likely to help you make old bones in good shape. Perhaps go easy on the commercial mayonnaise and just spread some butter on your cheese... Actually I tend to add butter to my beef if it's too lean (only happens when eating out, no way we would choose lean beef to cook at home!)...

It makes me wonder about saturophobes who might be swilling PUFA to lower their LDL and triglycerides, with or without a LC diet... More potential victims of the cholesterol hypothesis I suppose.

Peter

FIRKO-ise

Question: How do you convert a C57BL/6 mouse in to a FIRKO mouse?

Answer: Easily.


First, break your mouse. Both the grey squares and white triangles represent C57BL/6 mice on good old "high fat" D12451, so beloved of obesity researchers. They get a broken brain and gain weight. You can also feed chow CIAB to get the black diamonds:



Now let's do a little magic and render all of the adipocytes of the white triangle D12451 injured mice insulin resistant, in both brown and white adipose tissue. This is slightly tricky. Are you all standing in a circle holding hands? On a hill top in mid winter? OK, say the magic words while turning withershins. You have to chant:

Calories-in, calories out, turn this stupid mouse about! FIRKO-ISE...




OK, well done, you can all put your clothes back on now and stop dreaming of UCP-1.


I guess everyone realises by now we've been talking about the graph from this paper



with the classic use of ketosis to normalise the bodyweight of D12451 injured mice. Ketosis renders adipocytes insulin resistant, just like those of FIRKO mice. Of course, because ketogenic dieted C57BL/6 mice are functionally FIRKO, they don't cut calories. They eat as many calories as the D12451 injured mice. But they produce more heat:

"Thus, total heat output was 15% higher, averaged over 24 h, in KD animals (KD 0.538 ± 0.01 kcal/h vs. HF 480 ± 0.01 kcal/h, P < 0.05, n = 4). Oxygen consumption was increased by 34% averaged over 24 h (KD 4.370 ± 0.062 ml·kg–1·h–1 vs. HF 3.248 ± 0.052 ml·kg–1·h–1, P < 0.01, n = 4; Fig. 6C). Weight and CLAMS results were replicated in two additional independent cohorts using the same paradigm. CLAMS analysis also revealed that spontaneous dark-phase locomotor activity in KD animals was ∼30% lower than in HF animals."

[*Brownie point for spotting the typo in the quote. Hot mice indeed!]

They also avoid the gym. Heard that before?

We know that simply not eating for a while induces whole body physiological insulin resistance. We also know we can do exactly the same thing, without all the pesky death involved in sustained not-eating, by simply going in to deep ketosis without cutting calories.

Ketogenic dieting is slightly controversial. I've heard it said that the weight loss in these ketogenic fed mice is not real weight loss. I've even heard the change described as organ shrinkage. Interesting. The ketogenic mice become relatively hypoinsulinaemic and glycogen depleted. Do these affect lean bodyweight directly?

I had the full text of this paper sent to me by a friend and it has a nice summary of the effects of carbohydrate restriction on fluid balance (excuse the rather condescending tone, it's written by a leading obesity researcher):

"...energy is stored in the body as protein, fat, and glycogen, which is a form of carbohydrate. Any imbalance between the intake and use of these macronutrients will lead to an alteration of body composition since the stored protein, fat, or glycogen must change to compensate the imbalance. The energy stored per unit mass of carbohydrate, fat, and protein varies considerably, especially when accounting for the intracellular water associated with stored glycogen and protein.7 Furthermore, dietary carbohydrates have an effect on renal sodium excretion via insulin,60 which results in concomitant changes of extracellular fluid."


As far as I can make out losing liver glycogen, muscle glycogen, excess sodium and, in particular, the water associated with these body components shows up as lean body mass loss on a DEXA scan. Reducing your insulin-induced sodium retention may be good or bad depending on many factors (such as your starting blood pressure!), but it will show as a non adipose tissue body deficit in Fig 2, graph B, second column. There might even be a little muscle reduction, I can't say...

We know from the DEXA scans in Table 5 that ketogenic dieted, post-obese mice had a significant deficit (by mouse standards) of fat compared to the D12451 injured mice and most of it showed up in the whole body scans rather than the hind limb scans. This would suggest to me that they lost central, probably visceral, fat. I think we all pretty well agree that visceral fat is Bad Fat (maybe). It's usually the first to go on ketogenic dieting in humans. I don't see fluid loss or visceral fat loss as big worries.

There seem to be a whole stack of benefits to sticking D12451 injured mice on to an extreme ketogenic diet.

Real FIRKO mice live about 18% longer than CIAB fed mice.

What about these ketogenic fed, pseudo-FIRKO mice? Alas the sad story of their premature demise will have to be left for the next post...

Peter

The books

As we all know, there is a spate of new books out at the moment. I see that Dr Briffa is on Jimmy Moore's LLVDLC.

John sent me a copy of Escape the Diet Trap and it almost immediately disappeared to my wife's mother, to be returned disappointingly rapidly a week later. She has, happily, bought her own copy. No one where she works believes a word of it. I like it.

Not had chance to read Richard's primer or Chris's Hillfit but I know neither need any help from me and I'm looking forward getting a chance to read both as I get a little more time...

Anyone who has moved from Scotland to East Anglia will know how envious you feel browsing http://cairn-in-the-mist.blogspot.com/.

Thanks guys.

Peter

FIRKO mice

Okay. I have an apology to make. I'm not sure there will be an MCQ test on the FIRKO mouse to parallel that on the LIRKO mouse. At this stage of the proceedings I'm not sure that I can muster the motivation which is needed to do justice to such an Herculean task of applied sarcasm. The difficulty is compounded by the loss of my trowel somewhere between Berkshire and Norfolk via Glasgow. You really do need a trowel. I know, excuses, excuses. Mea culpa.

With that apology, I think it's time to discuss this paper.

So now we have the FIRKO mouse. This mutant mouse has been cleverly engineered to fail to express insulin receptors on its adipocytes. Everything else is normal. Functionally the adipocytes are severely insulin resistant. It does not matter how much insulin the pancreas secretes, adipocytes will not, cannot, listen to it. You know the rules. The function of insulin is to store dietary fat in adipocytes. In the almost complete absence of any insulin receptors on any adipocytes, this just ain't gonna happen. So FIRKO mice stay slim, slightly slimmer than a control mouse, and live a bit longer. All on CIAB and without cutting calories of course.

They also fail to develop age related insulin resistance. Please note as a complete aside; those mice on F9, boring old low fat CIAB, do develop age related insulin resistance and glucose intolerance. Wanna stay as healthy as a mouse on F9 with age acquired insulin resistance? Go ahead and eat low fat, about 10% of your calories will do. Try not to get too bored.

I could stop here with this comment from the authors:

"Our data further show that insulin signalling in adipocytes is crucial for triglyceride storage and the development of obesity and its associated metabolic abnormalities"

It would be fun to just thumb your data at those fixated on the central effects of insulin but that would be leaving a whole can of worms unopened. You know how it ticks you off to get partial information on a given study. The selective information rationing typical an obesity researcher. The data are actually quite complex.

Let's get a tin opener.

Sooooooo, what if you take a FIRKO mouse and inject it with gold thioglucose? Obviously you will bust its VMH. You could equally use a electrical ice-pick or a big meal at a Chinese restaurant (jk).

To summarise the last post: This injury increases the ability of adipocytes to divert calories away from metabolism and in to storage, by an increase in their sensitivity to insulin. Fat should simply pour in to the adipocytes of a VMH injured rodent and they should start eating big time. You could be forgiven for thinking you had removed their brain satiety centre or upped their fat set-point.

But the FIRKO mouse has very few insulin receptors on its adipocytes. The brain can scream, shout and have a temper tantrum to demand fat storage. Adipocytes stay cool as a cucumber and don't even give the finger to the brain. Pure ignore-ance. The brain has lost its tool for fat storage. You know the one, the tool which stops you being hungry (snigger) and helps you lose weight (sigh). Insulin.

Now let's look at some of the graphs. We'll start with the supportive one:



We can ignore the middle two columns, they're from different knockout mice. FIRKO mice with a gold thioglucose brain injury (right hand column) weigh the same as, or even a non significant smidge less than, WT mice (or FIRKO mice) without a gold thioglucose injury. Now that's no surprise. Brain:Adipocyte:Insulin.

But there is a shock in store. Here's the next graph, the columns are the same:



FIRKO mice eat MORE if they are injured by gold thioglucose than if they aren't. They eat almost exactly the same extra food as a wild type gold thioglucose injured mouse. While staying slim, of course. But they do eat more.

Does this mean that the VMH really controls appetite rather than the ability to divert calories to fat storage?

FIRKO mice have markedly reduced insulin receptors on both white and brown adipose tissue. The consequence of this on white adipose tissue is simple, insulin causes fat storage, lack of receptors limits fat storage. BAT is more complex. We do have a BATIRKO mouse which has had the insulin receptors knocked out on its brown adipose tissue only. This leads to combined atrophy of BAT (the normal lipid droplets in BAT never form) with marked up regulation of UCP1 production. They stay slim compared to controls while being fed CIAB (aside: although slim they do eventually become diabetic, the reasons for which are utterly unclear to anyone, see the discussion). As the authors comment on "normal" BATIRKO mice:

"Interestingly, the lack of IR leads to the over expression of the UCP-1 and also UCP-2 in the remnant BAT from BATIRKO as compared with controls. These data could be interpreted as a form of compensatory mechanism for the brown fat lipid content and mass loss observed in BATIRKO and may result in a potential increase in the thermogenic capacity of the remnant BAT that may account for the lean phenotype of BATIRKO mice compared with controls"

A lack of insulin receptors on your BAT up regulates thermogenesis. This has nothing to do with the brain and everything to do with the periphery. Why should thermogenesis be increased by VMH injury? I don't know. The control of BAT is complex and I don't think the work has been done yet. There are hints that insulin reduces UCP1 production in mice, bringing us back to changes in insulin signalling and thermogenesis. You might expect a system which activates fat storage might turn off fat burning and vice versa.

At the moment, for FIRKO mice, it looks like an open question as to whether gold thioglucose VMH lesions really increase appetite directly or increases thermogenesis in BAT causing a calorie loss, with compensatory hyperphagia. You can imagine which option I think may be the case, but I do have certain biases.

It's frustrating that there is no information to follow through on this. The group's last publication on the FIRKO mouse was in 2007 and was interesting in its own right.

The FIRKO mouse has white adipose tissue which, with age, gets to have better and better mitochondria. Probably more of them too. The authors talk about increased whole body oxidative metabolism but don't seem to consider BAT seperately from WAT... But having your adipocytes live in [what to them is] an hypoinsulinaemic environment seems to be rather good for them. And the mouse


Anyway, summary:

Remember what is special about FIRKO mouse is that its adipocytes never see insulin, whatever the blood insulin level. Lacking IRs on all of your adipocytes keeps you slim, keeps your insulin levels low and extends your life expectancy by about 18%. It gives you shiny new mitochondria in your adipocytes as you age. If you are a mouse.

It it possible to mimic this state in non-FIRKO mice?

Perhaps it's time to revisit ketogenic diets in mice. Oh, and cirrhosis too.

Peter

Used brain for sale: One careful owner, only slightly broken

Let's start with the old Stranglers track, "No More Heros", take an ice pick to a rat's brain and make its ears burn. OK, chew up its ventromedial hypothalamus with an electrolysis needle. This French paper is a pdf.

Here's the interesting table from the results:



At week one, when weight gain has started but not gone very far, fasting insulin was unchanged but blood glucose was LOWER than that of control rats. These rats, with their brain injury, have increased whole body insulin sensitivity. Mostly prominently in their adipocytes. The paper mentions in the discussion that these rats also hyper secrete insulin in response to secretagogues. Now, as we all know, insulin is both anorexic and unimportant to weight control. But if you just imagined, as I do, for a second that insulin does have something to do with weight gain, what would you expect to happen if you dropped hyper-secreted insulin on to exquisitely insulin sensitive adipocytes? Their job is to store fat under the influence of insulin so...

They would store fat. They would hang on to it. As Taubes might comment, the rats then over-eat because they are losing calories in to their adipocytes. They over eat because they are becoming fat. How do you check this? Well, let's pair feed ice-picked rats with control rats. Limit their calories. Make them go to uncheatable Weight Watchers in a prison cage. From the discussion:

"However pair-feeding rats with controls does not prevent excessive lipogenesis, fat accumulation and hyperinsulinemia [48, 49], suggesting that the disturbances of metabolism and not hyperphagia are the primary factors leading to obesity."

You injure the brain, alter the adipocytes and they store fat. Hyperphagia is an epiphenomenon of calories lost to adipocytes. They store fat even WITHOUT hyperphagia. This was quite obvious in 1992.

Enough frivolity. Let's get slightly more up to date with some Spanish MSG rats.

You have to be a bit careful with MSG injured rats. MSG is a potent neurotoxin and kills or injures almost any cell sporting glutamate receptors. This includes large numbers of nerve cells in the VMH, the target of the electrical ice-pick. However it also blunts growth hormone production, shuts down thermogenesis from brown adipose tissue and, very interestingly, adipocytes themselves probably use glutamic acid for cross talk purposes, so it's hard to know exactly what we do to MSG treated rats in addition to busting their VMH. You have to wonder whether the adipocytes themselves are injured by MSG.

Anyway, at a month of age, MSG injured rats have highly insulin sensitive adipocytes. Before the rats have become visibly obese their fat cells are already somewhat swollen and ready for the off in to full blown blobby-ness, come puberty. So again, you bust the VMH, increase adipocyte insulin sensitivity, adipocytes suck in fat and your rat simply has to eat to maintain access to enough energy to stay alive and cart the inaccessible fat around its cage. It probably doesn't dream of going to the gym.

If adipocytes are hypersensitive to insulin, what would you expect fasting FFAs, glucose and insulin look like before obesity developed?



Eyeball the HOMA score! These rats are a picture of glucoregulatory health! Unfortunately you need an energy supply from somewhere and some extra FFAs might just sort that out. You really need to develop some adipocyte distension induced insulin resistance by becoming obese to get the FFAs up to an appropriate level for a fasting rat. That's just what they do...

Look at the numbers from some Slovakian MSG injured rats as adults.



Cool, huh? Unfortunately we don't have the FFA level in the paper but, looking at the adipocyte size, they will be leaking FFAs in defiance of their double-the-control-rat level of insulin.

Now, are these adult, distended adipocytes insulin sensitive or resistant?



Well, they do bugger all to increase glucose uptake with increasing insulin exposure. So yes, they are insulin resistant. But look at this:



What glucose they do take up is diverted to fat. Might we say they are behaving like muscle cells which lack metabolic flexibility? Mitochondrial injury?

So, as obesity becomes established we end up in the age old situation of insulin resistant distended adipocytes leading to more FFA leakage than appropriate for a given level of insulin and so hyperinsulinaemia develops to try to keep blood glucose normal in the face of chronically elevated FFAs. This is absolutely not the case in the very early days, but rapidly becomes so with time.

This is all quite straight forwards and nothing you wouldn't expect if you accept the importance of insulin in obesity, adipocyte hypertrophy induced insulin resistance and the fact that adipocytes have a nerve supply which regulates their insulin sensitivity. In fact there are interesting papers on the role of adrenal hormones and the vagus as well as the sympathetic nervous system in MSG injury induced obesity. The end result is always increased insulin sensitivity of adipocytes until they become over-distended.

You can, of course, do exactly the same with gold thioglucose. Getting bored with all this? I'd basically come to the conclusion that VMH injuries give the impression of causing hyperphagia when what they actually do is increase lipid loss in to adipocytes, under the influence of insulin.

Okaaaay.

Let's look at a Long-Evans rat. If you feed it on D12492, which has been described as a high fat diet, for just three days, its brain breaks.

What if it is the fat that breaks the brain?

Well, my brain is then going to be completely f*cked.

I really do think that it might just be the fat that does it. How do I know? God told me. Okay, okay, only kidding. About god.

No, James emailed me a link to the latest Schwartz offering. I suspect that Dr Schwartz does not like Gary Taubes. We can also skip to the blog of the 4th author, who certainly does not like Gary Taubes, load up on ondansetron and have a browse. The blog says:

"Based on previous studies, the dietary fat itself is probably an important component that makes D12492 fattening in rodents"

The man is correct.

If you have quite recovered from that, let's look at the simpler aspects of the study. We can come back to the superb electron micrographs of dying mitochondria some other time. BTW, they are very, very cool pictures. I've been looking for similar photomicrographs all over the place. Who would have thought I would have found them here? Anyhoo:

First off, let's look at Figure 1, skip to graph H.



Start some ratties on D12492 and they will immediately double their calorie intake, on day 1. After living your whole short life eating CIAB I can understand this. D12492 tastes so good you just can't help yourself and it must be quite easy to eat enough of it to break your brain. It must be very rewarding. Luckily your brain recovers a bit and soon, by day seven, you're not eating any more calories of D12492 than a rat on CIAB and that's how it stays for the full 28 day period. We can tell this from Graph G. Here the average 28d food consumption on D12492 is only just above the 14d average consumed as CIAB. This excess is mostly accounted for by the first seven days of hyperphagia.

The bit of the brain which breaks "in association" with the massive 60% of calories from fat is the good old VMH. If we go back to the ice-pick rats, the MSG rats and the gold thioglucose rats we might just develop the suspicion that breaking the brain of a Long-Evans rat might affect the insulin sensitivity of its adipocytes.

If it does, fat from the diet will simply pour in to the adipose tissue and the unfortunate rattie will then have to eat extra to supply some energy to run its metabolism on in addition to that used for filling its adipocytes. Initially twice the amount it ate on CIAB. As the adipocytes fill they will become intrinsically less sensitive to insulin and fat accumulation, with its necessary compensatory hyperphagia, will slow. But not stop, if they behave anything like adipocytes in other VMH injured rat models.

On a high fat diet there is plenty of fat to pour in to adipocytes, no lipogenesis is needed. Adipocytes can distend quickly and it would be interesting to see if the fasting hypoinsulinaemia seen in the MSG rats (fed on high carbohydrate CIAB) occurs in D12492 injured rats. Probably it would still occur but be very transient, but obviously no one in the Schwartz lab would be interested in insulin.

Of course, one has to wonder which component of the D12492 might injure a rat's VMH. We are all familiar with the conversation (scroll up to get to the text) between Chris Masterjohn and the good doctor, where the omega 6 PUFA content of D12492 was noted to be 32% of fat and the omega 6:3 ratio was 14 or 16:1. All fascinating background. But my favourite obesity researcher correctly thinks it is the fat, not the type of fat, which breaks the VMH.

You can do exactly the same with butter oil (plus a smidge of soybean oil), which I'm guessing is a bit like ghee. Which I rather like. This is what butter oil at 20g/100g of food does to a Long-Evans rat in this study:



In particular look at what happened to the group HF. They switched from non purified (NP) diet on day 1 on the graph, spiked their intake to about 50% extra calories by day 5ish and were almost back down to the NP group's caloric intake by day 10. Exactly the same pattern as the D12492 also produces in Long-Evans rats.

It is impossible to emphasise how important both studies are to you if you are a Long-Evans rat.

Does three days of high fat eating break your brain if you are a human being? I have to admit that I appear to have singularly failed to become obese on 80% of my calories as fat over nine years. Possibly because 80% of your calories from fat becomes protective? I dunno. I can't help but recall those chaps in Aberdeen eating 66% of their calories as fat and refusing to finish off their allotted 2000kcal/d...




I have to be open to the idea that humans may not respond to high fat feeding in quite the same way as Long-Evans rats do. OK, they just don't. Their VMH doesn't acutely break. The rat is in trouble on 60% of calories as any sort of fat. Humans just say "no thank you" to the extra slice of bacon, in Aberdeen anyway. Oh, and in Lowestoft too.

In summary: Injuring your VMH in any way (even by eating butter oil if you are a Long-Evans rat) does nasty things to your adipocytes. They will store fat even if you cut calories. You will then be very hungry and, unless you do eat more, you will chew up your muscles for energy, get cold and move as little as possible. Oh, and still get fat. People will say you lie about your calorie intake.

Now, is it possible to become obese without breaking your VMH? Of course it is. Does it matter? That depends.

I think the chronic changes in both the Long-Evans rats and the C57BL/6 mice are very important and are quite likely different from the initial fat induced injury to the VMH. They appear to be more related to the chronic hyperinsulinaemia and hyperglycaemia which follow on from adipocyte insulin resistance and elevated FFAs especially in the presence of a high dietary carbohydrate intake. That will lead us to back to mitochondrial injury (which is probably where all of this comes from, did I even mention that obesity is a mitochondrial problem?), free radicals and I might even throw in gliosis. Which is interesting.

Peter

More of the 17% solution

There are various little one-liner papers which I've tripped over in the last few weeks which are probably worth a post although are not related to the main things I'm interested in at the moment.

The first is an isolated oddity. We all remember Dr Axen and the 17% trans fat diet for rats? Followed by the Complete Idiots with their 14.4% solution.

Of course, no one would ever suggest feeding this much trans fat to humans in a weight loss study. Would they? No, surely not. Except I guess it depends on what you have to prove...

How about this study:



Let's do the math. The ketogenic diet provided bulk calories as fat, 100g/d. A scrummy 35 grams were saturated fat, nice. Next comes 34 grams of OK-ish monounsaturated fat. The obligatory 14g/d of disgusting PUFA is included. Now, maths is complex subject.

One plus one is, err, about, I mean, err, somewhere about one and three quarters. About. I think

OK, let's simplify. 35+34+14=100

Wrong. Yea Gods, I always was bad at math. My worst A level grade. Let's try again

100-(35+34+14)= n, where n is the trans fat content of the fat in the ketogenic diet.

Congratulations Dr Sears. You get the Axen Prize for the maximum undeclared trans fat content of an experimental diet used on humans.

Peter