Chewing the FAT

CD36 is another of those cell surface proteins with an interesting use. It actually does quite a few things, but the one I'm thinking about is its role as Fatty Acid Translocase, hence its other name, FAT. The late 1990s seems to have been a fashionable time for CD36 research and this era has provided a number of interesting papers.

CD36 takes a molecule of free fatty acid, frequently palmitic acid, and transports it through the cell membrane to the cytoplasm, en route for beta oxidation to provide a bucket load of ATP. Of course, the palmitic acid will also signal the induction of insulin resistance. No point burning glucose if you have palmitic acid. Nowadays any competent lab can knock out a specific gene from a mouse and see if the gene loss does anything much. So in a CD36 knockout mouse we have the ability to make the cell membrane largely opaque to palmitic acid. What does this lack of intracellular fatty acids do to insulin sensitivity?

CD36 knockout mice have lower blood glucose than wild type mice.



Now this may be hunky dory for a mouse with ad lib access to mouse chow. You can have a nice low blood glucose and probably a nice low insulin level. But what if you were to glycogen deplete the mouse and then make it run to escape from a cat? Its glucose is already low. It can mobilise fatty acids perfectly well, but they can't enter the cells. So no insulin resistance forms and the mouse muscles continue to run on a progressively falling glucose concentration, in a sea of unusable fatty acids, until its brain stops working and the cat gets a mouse sized meal. This hypothesis is untested so far!

These mice also have lower blood insulin too (can't check this fully as it's a Nature pay-per-peep publication and insulin is not important enough to make it in to the abstract, so I'm taking Hajri's word on this). All of this is pretty much as you would expect. These mice are born and bred on glucose and virtually never use any palmitic acid unless they make it de-novo, intracellularly, from glucose. They are probably exquisitely insulin sensitive, for what good that might do them in the wild.




Over expressing CD36 gives the facility to get lots of fatty acids in to cells and this increases both blood glucose and blood insulin due to insulin resistance.


Again, a simple balance, put lots of fatty acids (probably as acyl-CoAs) in the cytoplasm and cells say no to glucose. Hence you need increased levels of insulin to keep blood glucose normal. So is there pathological insulin resistance here?



Apparently not.

It's worth noting that response to an IV glucose load was NOT damaged in these mice, though there is a "trend towards" higher glucose levels from 30-120 minutes after the bolus in mice over expressing CD36 (open circles).

I think it is a reasonable assumption in these CD36 over expressing mice that the insulin surge following the glucose bolus from the IVGTT can still reduce FFAs, and so increase insulin sensitivity, in exactly the way it should to normalise glucose.

It seems quite likely that the blood insulin will peak at a higher level in the CD36 over-expressing mice. In general, over secreting insulin is probably a Bad Thing. So should a low carbohydrate, high fat eating person be afraid of eating a portion of chips with their roast bellypork? Will it spike glucose and/or insulin to unreasonable levels?

Certainly not. You may have a slightly higher insulin level than a carb eater for an hour or two after 30 grams of potato derived glucose, of course. But a carb eater will not eat a small potato, they will eat ten times that weight of carbohydrate in 24 hours. At least. So a low carbohydrate eater's 24 hour exposure to insulin will be vanishingly small compared to someone nurturing chronic illness using the USA Food Pyramid and eating 300 grams of carbohydrate a day.

So OK, is it acceptable to have a portion of chips with your roast (after being marinaded in lemon juice and then rubbed with a Mexican spice mix) belly pork ? Well, that's a personal decision. Me, I'm fine with it.

Actually, last night the pork was chip-less but followed by two gluten free muffins for dessert (mostly almonds and millet flour, living dangerously with the millet perhaps).

Peter

Overfeeding humans: Jebb

"Obesity implies a failure of autoregulatory homeostatic responses to caloric excess"

The quote comes from this paper first authored by Mario Siervo but with Susan Jebb as the group leader. I'll discuss the paper in a moment.

Who is Susan Jebb?

From the Medical Research Council website:

Cross-government 2007- Chair, Expert Advisory Group on Obesity - Susan Jebb

Department of Health 2006 - Chair, Expert Group developing the Healthy Living Social Marketing Programme - Susan Jebb

Department of Health/Food Standards Agency 2004-2005 Expert Working Group on Nutrient Profiling - Susan Jebb

Government Office for Science 2006-2007 Science Advisor, Foresight Project 'Tackling Obesities: Future Choices' - Susan Jebb

You get the idea. An obesity politico. This sort of politico. Also check the date on that link. 2003. I was just starting on low carbohydrate eating at the time. Jebb was all over the papers. One very obvious thing, to someone who had just read Atkins' "New Diet Revolution" from cover to cover, was that none of the experts being quoted had read the book!

So that puts Jebb in context. Here's the interesting study on overfeeding humans rather than dogs. This is the feeding protocol:

3 weeks run in feeding
3 weeks 20% extra calories
1 week rest, eat as much of a Jebb diet as you feel like
3 weeks 40% extra calories
1 week rest
3 weeks 60% extra calories
3 weeks rest

This is the table detailing exactly what was eaten.

These are the weight changes, also subdivided in to tissue composition.


This is the executive summary: People got fat on the excess calories and couldn't loose the weight within 3 weeks. Some people couldn't loose any of the weight at all.

Jebb's conclusion. People pig out at Christmas and fail in their New Year diets. Greed and sloth, greed and sloth. Once you've pigged out, if you're greedy, you'll keep troughing.


Now, let's ignore Jebb and look what happened.

Protein was increased from 85g/d through 101g/d, 112g/d to 126g/d from baseline through over feeding protocol. Some increase, but not unreasonable.

Fat was used to increase the "energy density" of the diet and so was increased from 120g/d through 158g/d, 196g/d to 231g/d. The later being Kwasniewski levels for optimal health (but without the carbs!).

Carbs started at 322g/d and ramped up through 375g/d, 409g/d to 446g/d. Okayyyyy. Interestingly, just reducing these carbs to a tenth of this overfeeding level would have given quite easy weight loss for most people, with the fat left alone!

There were snacks too but they don't affect the basic argument.

Jebb is a calories in calories out sort of a person, so fructose is the same as glucose to her. We'll never know how much fructose was fed.

Now let's look at substrate oxidation. With all that increased fat intake what happened to fat oxidation? (All of the values are approx and from the figure)


With a fat intake of 4.5MJ/d fat oxidation was 4.5MJ/d. Neat that!

On 20% overfeeding fat intake went up to 6MJ/d and fat oxidation DROPPED to 3.8MJ/d.

On 40% overfeeding fat intake increased to about 7.7MJ/d and oxidation DROPPED FURTHER to 3.5MJ/d.

On 60% overfeeding fat intake was 8.5MJ/d and fat oxidation seems to have bottomed out at 3.5MJ/d, no further drop.

Three points,

Carbohydrate oxidation went up as carbohydrate intake increased. This cannot happen without insulin. Increased carbohydrate oxidation means increased insulin, certainly at this level of increase of glucose oxidation. Jebb either doesn't know this, and is an idiot, or does know this and didn't measure insulin for a personal agenda. I favour the idiot theory with Jebb. I guess you could argue insulin sensitivity increased but this is a study of gross overfeeding, so that's unlikely.

Fat oxidation decreased with increased calories. What controls lipolysis? Insulin. More insulin, less lipolysis. Less lipolysis means less fat oxidation. Fat is stored more effectively and is locked in to storage. You can't oxidise stored fat.

Body water went up. Water retention means sodium retention (water retention without sodium retention = hyponatraemia = death). Sodium retention is a hallmark of elevated insulin acting on the kidneys.

In a short communication the same group measured leptin and ghrelin levels, which indictaed everything should be hunky dory for return to normal body weight, but clearly things weren't. That's assuming leptin satiates and ghrelin makes you hungry. In a simple balance of energy in vs energy out, weight is controlled by appetite. This being a Jebbish paper, they didn't measure insulin. They didn't measure the primary fat storage hormone. Oh, Susan, how could you not do this?

So what really happened in this study?

Weight gain, to anyone with half a brain, is a phenomenon of the diversion of ingested calories to storage as adipose tissue. Metabolic fuel requirement must be met at the cellular level, above that calories can go to storage as fat. Weight loss means the body gaining access to stored fat calories. Hunger controls eating behaviour when there is no artificial requirement to over eat by 60%. Hunger will adjust food intake until there is an adequate supply of metabolic fuel for the whole body.

If a large chunk of those calories consumed go in to storage, even without overeating, you will maintain hunger until you achieve enough AVAILABLE calories which are needed to run your metabolism. Whether these come from food or bodyfat depend on blood insulin level. High insulin levels lock energy in to fat, so you must eat more food to obtain metabolic fuel. Hence you don't lose weight because energy locked in to bodyfat isn't being used.

You don't need to measure insulin to know it goes through the roof when you eat nearly half a kilo of carbohydrate in a day. You don't need to measure insulin to know it is elevated when you see fatty acid oxidation plummet. You don't need to measure insulin to know it is elevated when you see glucose oxidation rise.

You MUST measure insulin if you want the readers of your scientific publications to think you remotely know anything about weight control and are in a position to advise the nation.

Ultimately, the verdict on Susan A Jebb will be that she she did not measure insulin.

Peter

Oh, and weight loss was impossible for some people, they were the ones who got most fatty liver infiltration per unit fructose ingestion. As a guess.

Thanks to Robert for the link to the papers in this post.

Physiological insulin restisance: Guess what?

There are a series if papers from back in the 1950s by Drury and Wick plus occasional others. I had the misfortune to read the methods of a couple in some detail and, unless you have a strong stomach or are intrinsically sadistic, I suggest you don't. Physiologists in the 1950s had a different view of animal welfare to that now prevalent. The studies would not be allowed in any civilised country today or published in any reputable journal if carried out. I'm not going to link to them.

The main finding is that the oxidation of glucose can blocked, even in the presence of large amounts of injected insulin, by a modest quantity of a particular small molecule. This form of insulin resistance, if you want to call it that, does not seem to occur at the cell surface, so it's probably not mediated through the failure of insulin to mobilise GLUT4s. And, as glucose seems to enter the cells and disappear, the presumption has to be that it is "non oxidatively disposed" as the modern parlance has it. Probably to glycogen, there's not really anywhere else for it to go.

So what is this evil chemical which blocks glucose oxidation even in the face of hyperinsulinaemia?

Beta hydroxy butyrate. That's it. Ketone bodies (acetoacetate seems to work as well) are triggers for insulin resistance. Hence the appalling problems of type two diabetics on the Atkins induction diet. What problems? Oh, normoglycaemia and weight loss! Well, maybe there are problems long term or or or...

Again ketone bodies, one of the hall marks of carbohydrate or total calorie restriction, channel glucose away from muscles, toward brain and add a modest supplementary energy supply to brain tissue too.

It's exactly what you expect on an adaptive basis, exactly the same function as palmitic acid performs and clearly the two metabolic pathways are closely linked, though ketones seem to work downstream of the action of palmitic acid.

The fact that ketones do still allow insulin to move plasma glucose in to cells, and probably store it as glycogen, might be of interest to those who's blood glucose seems to do strange things after they eat medium chain triglycerides. MCTs (in rats anyway) undoubtedly spike both insulin and ketones, but usually result in normoglycaemia (insulin resistance?). But this is in a carbohydrate fed, glycogen replete rat. If you are initially glycogen depleted the shift to replete glycogen under ketones from MCTs might just leave you hypoing. No one has looked at this as far as I'm aware but ItsTheWoos' experience is interesting on this front...



Actually, looking carefully at the graph from Yeh and Zee, glucose does dip through an amount which might be clinically noticeable...

Anyway, there you have it. Metabolic poison number three, beta hydroxy butyrate. Evolution sure made a lot of b@lls-ups on the way to where we are today. I'm doomed, as always.

Peter

Mortality and cholesterol

People might enjoy O Primitivo's latest excel plot here. I did. I also suspect the effort involved in this is stupendous. Thanks!

Peter

Physiological insulin resistance and palmitic acid again

I like palmitic acid. It causes insulin resistance. Thank goodness.

Ted sent me this link. It's depressing.


I'm going to discuss a thought drug. I'm going to call it Palmitofake, and it can be developed by Pfizer, no, Fort Dodge. I particularly dislike FD for anaesthesia related reasons.

So what does Palmitofake do? BTW, if you didn't need any other hint you can tell this drug is going to bomb as there is neither an x, y or z in its name. Trust FD to screw up (in my mind).

Palmitofake is a fluoride substituted analogue of palmitic acid which irreversibly binds to the acyl-CoA interaction site of JNK1 and so inhibits the pathway by which palmitic acid keeps GLUT4 transporters off of the cell surface membrane, whole body-wide.

The logic to this is that the lipotoxin, palmitic acid (nature's second biggest mistake, the biggest was obviously cholesterol) can no longer keep glucose out of cells and metabolism can run, unimpaired by fat, for ever on glucose. Woo hoo bring on the glucose.

This concept is so obviously safe and utterly in keeping with modern thoughts on type 2 diabetes that no safety testing is deemed necessary and it can be sold direct to the public via placement in the drinking water. OK, maybe as an over the counter pill. Let's look at a case study:

Jim has just done a heavy workout at the gym. Like really heavy and, catastrophe of all time, he forgot his Sportzaide. Sportzaide is a glucose drink used to maintain blood glucose levels during workouts, it promotes sufficient insulin secretion that no fat is ever burned and no glycogen ever depleted. We wouldn't want him to lose weight from exercise would we?

So Jim is modestly glycogen depleted for the first time in his life. It's an odd situation but, in the last few million years, it has been known to happen occasionally to the hominids who eventually became us. It's called not having anything to eat for a week before having to chase your diner.

If Jim is in government you might argue that brain function is unimportant, but you would be wrong. Jim needs a functional brain, just to stay alive. Whatever else happens, he needs some glucose for his brain. There is no active transport of glucose, it runs down a concentration gradient in to brain cells using GLUT1 and GLUT3. However many transporters are present, if blood glucose drops below 2.0mmol/l Jim is going to be unwell and if it goes below 1.0mmol/l he's going to be very dead.

Jim's blood glucose drops. His liver would happily pump out lots more, but it's got none left. His pancreas has stopped producing insulin above basal rates some time ago and is now powerless to mobilise glucose in any way that doesn't need protein catabolism, and this is not exactly a supply on demand source.

In the natural order of things Jim will, by now, be mobilising enormous amounts of free fatty acids from his 40kg of beer gut. These free fatty acids rush to his muscles and provide an almost inexhaustible supply of energy. They don't rush to his brain. His brain wants glucose. His brain needs glucose. His brain will have a temper tantrum for glucose. Ultimately it will kill Jim if it doesn't get it.

Jim's body, metabolically, is in starvation mode. It needs to stop wasting glucose on his biceps and give it to his brain. The biceps do fine on free fatty acids, the brain dies in a sea of energy without glucose. The trick to staying alive when glycogen depleted is to keep glucose out of any tissue that can cope without it and save almost all of it for brain use.

So the rule is, when the body is flooded with free fatty acids, all fat using tissues should stop using glucose. They should see those free fatty acids and internalise their GLUT4 transporters so they don't waste brain glucose on dumb muscle.

The message to put this change in place is palmitic acid.

Jim has a very specific and very serious problem. He just started on Palmitofake yesterday as part of the initial clinical trials. As soon as he floods his muscles with palmitic acid he should have internalised his GLUT4 glucose transporters. Palmitofake stops this. He got in to the lift as an irritable exec with a blood glucose of 2.0mmol/l, got out of the lift on a stretcher with a blood glucose of 1.0mmol/l and died before the paramedics could get a glucose infusion up on him, with a blood glucose of 0.1mmol/l

PALMITIC ACID CAUSES INSULIN RESISTANCE. YOU WOULD BE DEAD WITHOUT IT. IT'S ADAPTIVE.

We should be looking at what gets broken in metabolic syndrome at the cellular energy processing level, not shooting the messenger. And we all know that low fat diets reduce mitochondrial number and high fat diets, especially if ketogenic, increase mitochondrial numbers. I really must get back to those high fat fed mice from 10 posts ago!

It's Saturday night. I need a glass of wine and bed!

Peter

Hepatic insulin resistance through caloric overload

It seems from Dr Lustig's commentary that one specific method of developing insulin resistance is by increasing fatty acid acyl-CoA moieties within a cell. Acyl-CoA is a single fatty acid molecule joined to a CoA group and represents an "activated" fatty acid, ready to do things metabolically. In both muscle and liver it appears to be these activated lipids, rather than stored triglycerides, which are the metabolic signal, via JNK1 and serine phosphorylation of IRS1, which is used to down regulates the activity of insulin on glucose control.

This paper suggests high levels of free fatty acids are taken up by the liver and inhibit it's response to insulin. My feeling is that the FFAs can come from hepatic lipase (as above), from overstuffed adipocytes leaking FFAs or even from dietary intake, as lipoprotein lipase spills diet derived FFAs from chylomicrons in to plasma as well as in to adipocytes.

Reducing FFA delivery to the liver by inhibiting hepatic lipoprotein lipase does nothing to get rid of hepatic lipid droplets (they have to go out as VLDLs) but the decrease in FFA delivery lowers Acyl-CoA and allows normal liver response to insulin.

Is it possible to overload the delivery of FFAs to the liver without in-situ generation of acyl-CoA from either fructose or alcohol?

Apparently yes. You can do it by diet. It's not easy, but if anyone wants to try it here's the technique. It works in dogs anyway.

This is a post I've had around for a few days while the little palmitate storm blew over:

There is a key concept in veterinary medicine which states that cats are not small dogs. No one would argue with this, especially those who associate with the superior species.

However, dogs might well be reasonably viewed as small humans, it makes a great deal more sense than considering mice to be very, very small humans.

So, if someone tells you that they fed a high fat diet to a group of dogs, restricting their caloric intake to weight stability, and that they all developed virtually complete hepatic insulin resistance within a few weeks, you might just have to sit up and take notice. Especially as the fat was cooked bacon grease, provided by the university canteen. Don't ask. The dogs didn't get the bacon (as far as I can tell). This is that study, free full text.

Anyway. They initially fed these poor dogs a "can a day" of an Hills "prescription" diet (that's the sum total of the methods info, except the macronutrient ratio in the can) and enough dry diet (some random food made by Wayne Dog Food) to maintain weight stability. They did this for two weeks then took away some of the dry cr*p in a bag and replaced it with bacon grease. About 2g/kg bodyweight of bacon grease. They kept it almost isocaloric with those first two weeks of eating traditional dog food. The idea was weight stability. The agenda was to prove that, under isocaloric and weight stable conditions, fat was bad, bad, bad. Replacing as little as 8% of mixed calories with bacon grease will cause total hepatic insulin resistance.

Like wow!

So what does this group consider a caloric intake to maintain weight stability in a 27kg mongrel while it is eating cr@p in a bag? Are you sitting down?


Cr@p in a bag: Total calories 3,885kcal/d

For "less cr@p in a bag but plus 2g/kg bacon grease": more like 3,945kcal/d


This is for a 27kg dog sitting in a cage.

Go on, read that again; 3,945kcal/d. I'm not joking.

OK, so the first question is whether these dogs were weight stable. Ha ha ha ha. The amazing thing is that they only gained about 2kg during the study period. Make that 3kg if you include the weight gained in the pre study "weight stability" period.



Aside: look at how they wangled the weight stability. Pre study admission time until study week zero, about 1kg weight gain. Not statistically significant. From study week zero to week 12 there was a 1.9kg gain which fluctuated in and out of statistical significance WHEN COMPARED TO STUDY WEEK ZERO. Had they compared the on-going weights to the weight at the time they first got their hands on these pooches (minus 2 weeks), virtually all weights from about week 4 would have been significantly up on the enrollment weight. Funny that. Back to the dogs:

3.0 kg in 14 weeks is >10kg per year. In three years, at this feeding rate, the dogs would weigh >60kg. Some weight stability!

Also, there is no control group. I would love to see what 3,885kcal/d of cr@p in a bag would do to a dog's weight in 12 weeks. Waltham's daily energy requirement for a 27kg "typical" adult dog is 1300 kcal/d and for an active dog 1480 kcal/d. Personally I doubt that chronically catheterised laboratory mongrels are getting a huge amount of exercise.

So this is another study where the introduction and discussion are utterly divorced from the methods and the results (and from reality). It's worth just flicking through the methods and, in your mind's eye, look at how much money was used on these dogs. A clinical MRI was around about £1000 a shot in the UK Home Counties in 2009.

For all this money spent, is there anything of interest in the study?

Fortunately yes, lots.

The first thing is that if, like me, you eat somewhere in excess of 2g per kilogram bodyweight of dietary fat every day DO NOT, under any circumstances, add 3000 kcal of carbohydrate to it. If you do this you will develop virtually complete hepatic insulin resistance within a few months. You will also get very very very very fat. Not in a week, but certainly in a couple of years. Thank goodness for this study, saved my liver.

Second is that you will not immediately develop peripheral insulin resistance. This will take significantly longer to develop. That's interesting. The liver is the initial site of injury in caloric overload, just as it is from fructose poisoning, or alcohol too for that matter. I might have guessed at muscle/fat for caloric overload.

Third is how would Garry Taubes view the achievement of getting a group of medium sized dogs to consume 4000 kcal/d? The equivalent of how do you get a 64kg human to consume 10.000 kcal/d? Challenging.

I would guess a mass of uncoupling proteins and elevated insulin to cover hepatic glucose leakage...

Anyway, if anyone has personally managed to consume 10,000kcal per day for a few years I'd love to know how you are getting on. Foie gras?

Peter

BTW the really scary features of this paper are that it got through it's grant proposal, it got through scrutineering and it spawned another, even more expensive, project using the same model which also got approved, completed and published. As my wife says, the peer review process is awful, but no one can think of anything less bad so far. Fortunately the group are wasting USA tax payer's dollars rather than my pounds sterling. Phew.

Addendum: What's the physiology behind the pathology? Well a dog never eats carbs in the wild, beyond the gut contents of herbivores. It usually takes in a massive caloric load of fat. It needs insulin to store that fat, so fat intake ought to make the liver a little insulin resistant, leak a little glucose and then it's up to the pancreas to sort out the glucose, taking the lipids along with it in to fat cells. This is normality. Adding massive carbs to massive fat will simply break a perfectly adaptive system... That's my take. Don't do it!

Palmitic acid based food vs olive oil or corn oil supplements

Just before I get back to hepatic insulin resistance I thought I'd just put this topical paper up, in view of the discussions emanating from the "palmitic acid is going to kill you by hyperphagia" post.

It's a classic, coming to me via Barry Groves' book Eat Fat, Get Thin. It took ages to find but is happily available in full text nowadays. Thank you once again to the USA for PubMed.

Back in the early 1960s there were still a number of clinicians alive who thought that that Ancel Keys was an arrogant idiot, a crook, or (more likely) both. The concepts that cholesterol caused heart disease and that drinking corn oil might prevent heart disease via cholesterol lowering were both ideas suitable for contempt.

Hard to say if Rose, Thompson and Williams were part of that perceptive group but anyway, this is the study they carried out.

They had three comparable groups of heart attack victims. One third were left alone to eat eggs, cream, sausages etc, you get the message. I was there in the 1960s in England and we ate that sort of stuff all the time, it was just food. Olive oil was a novelty and I'd never heard of corn oil. The other two groups got oil supplements.

So here's the protocol (all the jpgs just click to enlarge):



What did the macronutrient intake end up like?



Interestingly, here are the changes in TC. Of course back in the 1960s the goal posts were still centred on TC. Look at that cracking drop in the TC of the corn oil victims:



And here are the body counts, the top two lines are the dead people. Bottom right hand corner is the "event free survival" percentages at 2 years.



Fascinatingly they had two cases of diabetes, one in the olive oil group and one in the corn oil group. Both occurred on adding the oil and ameliorated on withdrawal. BUT BUT BUT you gasp, saturated fat, PALMITIC ACID for crying out loud, causes insulin resistance. Lovely oleic acid, darling of Dr Clegg's massive project, does not cause insulin resistance. Surely diabetes is insulin resistance caused by saturated fat? Well, it's your life. Clegg says oleic acid is the health nectar of the gods. Rose has noticed a reversible diabetes trigger and has a body count. Your choice!



I'll leave the summary to Rose et al:



Whenever the lipid hypothesis receives yet another fatal blow, as it does repetitively, there has to be an editorial rushed out with a death reversing ad hoc hypothesis which makes the dead people in the corn oil group pale in to insignificance.

My summary of the editorial:

Heart attack victims still need corn oil but shouldn't be so greedy and should loose a little weight too. Ad hoc hypothesis number two thousand five hundred and twenty five. Still the body count grows.

Finally my view about olive oil:

Not as bad as corn oil but butter is better!

Oh, and for anyone who is thinking of having an indwelling catheter placed in to their third ventricle for palmitic acid infusion: Don't.

Peter

Want some acid? Bad trip on palmitic...

Well, it's Thursday night, only another 20 hours to go until my next doner kebab. Our Friday night habit has become something of a ritual and Glasgow is graced with the most enormous choice of doner shops, certainly compared to Newbury. But the quality is a little suspect on occasions. We have had two kebabs where the grease left in the bottom of the container HAS NOT SOLIDIFIED.

This is worrying and I certainly do not revisit those particluar shops. The Anniesland shop by the railway station has turned in to this category. A real doner kebab should leave solid white fat in its container and a coating of thick grease on your lips. This is mutton fat, predominantly stearic and palmitic acids. Real saturated fat is hard when cooled. Runny stuff makes me think it's adulterated with soy oil or sunflower oil... No thank you! Gimme the hard stuff.

Anyone with the sort of doner habit I have is well aware of the catastrophic effects of palmitic acid on appetite control. You know what it's like. You go in to a kebab pusher's den, I mean shop, for just "two small doners, no bread, no salad, no sauce", eliciting the ritual response: "What, just the meat?" in a heavy Glaswiegan accent. "Aye, that's right" you confirm, usually with a double thumbs up (I'm learning the lingo, does it show? I haven't dared add "laddie" to this intonation, yet. I value my teeth). Use the same shop twice and you become well known (infamous?). You've promised yourself that you're only going to eat one portion and your wife intends to share the other with your toddler son.

Anyhoo. Half a pound of doner meat down and you are now just ravenous. You fight the hunger off for another 10 minutes, but you know you are on to a looser. You blow another £3.20 on a second portion. Sitting in Mothercare's car park, finishing your second kebab, you promise yourself that now you will just drive home and stop eating, and you actually turn on the ignition before the palmitic acid driven hunger breaks your will like a matchstick and you go back for a third portion. This time you don't leave the shop and wolf down your fourth portion, an extra large one, which gets you up to well over the two pounds of meat mark, and you need more. After that it's a race within the family to spent the week's food budget on Friday night doner kebabs. With five or six pounds of meat eaten you hopefully run out of money and the palmitic acid pusher mercilessly and mercifully kicks you out on the street, half a sheep in your stomach and ravenous from the palmitic acid flooding your brain. That hunger is going to go on for days and you already are aware that there is no money until the next giro comes through...


WHAT? You don't recognise the scenario? Well that must just be your ignorance of this study and this newspaper article summarising it.

I have to say I quite like what I have seen of the study. It's really very weird, in that it actually gives you the exact diets used, in full. That's a bl**dy first in recent "fat bashing" studies. It is also published in a free access journal. This too is very good. It has pretty good control groups etc. I will actually read it in full some time but, at the moment, I just have to comment that it is utterly, totally and completely divorced from my experience of reality. Does anyone else develop driving hunger from a single exposure to lamb fat (or butter, as in the study)? That goes on for days?

Which planet do these rats and mice live on? Possibly the same one as the researchers, ie not the Earth!

Peter

EDIT: Thanks for the heads up Mark

NAFLD model based on fish oil

Just briefly, another post on NAFLD, in frivolity:

"We fed a highly unsaturated fat diet (30% fish oil) to female Sprague-Dawley rats (180-200g), consumed ad libitum for 8 weeks"

"We propose that female rats fed with a diet containing highly unsaturated fatty acids are an extremely useful model for the study of NAFLD"

I propose this is bullsh*t.

If you are on 3000kcal/d and are going to drink 100ml/day of fish oil as 30% of those calories, PLEASE do not make up the rest of your non protein calories from dextrose. I warned you.

Also, if you think this in any way represent the human NAFLD which is rampant in the developed world, please desist from this idiotic idea and send your funding money to me, preferably in used fivers. And go clean that toilet.

Peter

Hepatic insulin resistance

Jenny Ruhl pointed out this study not so long ago:

"These data demonstrate that IHTG [intra hepatic triglycerides], not VAT [visceral adipose tissue], is a better marker of the metabolic derangements associated with obesity"

which leads neatly to this article:

"These results show that progressive increases in IHTG content are associated with progressive impairment of insulin action in liver, skeletal muscle, and adipose tissue in nondiabetic obese subjects"

Both of these are interesting as they make the liver the centre of the changes commonly found in type 2 diabetes and its precursor, IGT. There are now quite a few observational studies suggesting that VAT is not particularly tightly associated with insulin resistance.

I don't think anyone would consider the central role of the liver surprising when you think about the involvement of fructose in this syndrome. Because fructose barely penetrates beyond the liver you would really expect the liver to be the site of its effect on the body.

The development of insulin resistance is usually associated with the accumulation of lipid in a given tissue (or I guess you should say the processes leading to that accumulation, it may not be the lipid per se). The metabolism of fructose guarantees that this occurs in the liver. There's a nice paper describing an intervention study here:

"A 7-d high-fructose diet increased ectopic lipid deposition in liver and muscle and fasting VLDL-triacylglycerols and decreased hepatic insulin sensitivity"

This is a study in PEOPLE, not rats!

Note that it only induced hepatic insulin resistance, systemic insulin resistance should take longer.

If the liver stops listening to insulin it will not take up glucose or store it as glycogen in response to "physiological" concentrations of insulin. It will then leak glucose in to the blood stream in totally unacceptable amounts. It is up to a combination of muscle and adipose tissue to keep the blood glucose level remotely acceptable. This will require lots of insulin, because there is lots of glucose available to leak from the liver. The palmitic acid from fructose will be shipped out as VLDLs and stored as fat by the enormous quantitiy of insulin needed to keep blood glucose remotely normal...

Ultimately, in the longer term, adipocytes will become so full that they too will become insulin resistant, which can then lead to inappropriate NEFA release, muscle insulin resistance and type two diabetes. Obviously there will be genes which determine at what size your adipocytes give up their unfair struggle. So we can look at the genetics of resistance to a functional poison or we can just avoid the poison. I know that latter is a silly idea, but I like it!

Makes it a bit hard to see how abdominal "fat-ectomy" might cure diabetes in rats and mice, which it does seem to do... Mind you, in dogs, visceral fat-ectomy improves peripheral insulin sensitivity modestly, so this would fit in with VAT accumulation being a down stream effect spreading hepatic insulin resistance to the periphery by NEFA, and only when VAT has given up listening to insulin itself...

So the accumulation of visceral fat becomes a consequence of fatty liver rather than a cause... In the short term fructose is perhaps the best trigger for hepatic insulin resistance. It can be achieved in other ways, I'll discuss giving a dog some bacon fat alongside its cr*p in a bag on another day...

Peter

Tom Naughton's FSA post

Just in case anyone doesn't follow Tom Naughton's blog, now would be a good time to start!

Peter

Sweden and diabetes again: Salty beer in Kiwiland

If you go to the homepage of the Lancet and search on Mann and Nye you will get this preview of their comment on the woeful state of diabetes management in Sweden. If you want to spend $31.50 (which I don't) you can obviously read the full text. Here's the preview text via the Lancet homepage, there's no abstract on PubMed:

"There will be much greater interest than is usually the case in the outcome of a review of the scientific evidence for dietary recommendations for patients with diabetes commissioned by the National Board of Health and Welfare in Sweden. This review is not yet finalised and thus not yet published. The appearance of fad diets offering near miraculous health outcomes invariably attracts hordes of adherents hoping for a magic bullet, the books describing them often becoming bestsellers. The ultra-low-carbohydrate Atkins diet was no exception, spawning specialist products, supermarkets, and restaurants worldwide"

If you want the basic flavour of the full text, while keeping your small change, you can go to Medical News Today's website. I've kept a copy of the text for when the link goes down but for now here is the biggest giggle in the article:

"A group of experts which is a branch of the Board was scheduled to publish a report on nutritional recommendations for people with diabetes. However two of the experts on the panel were withdrawn by the newly appointed Director-General. He considered that their links to the food industry via the Swedish Nutrition Foundation might represent a conflict of interest. The Foundation receives food industry funding. It also provides independent advice to the industry through expert scientists. The two scientists involved (Bengt Vessby and Nils-Georg Asp) are internationally respected. Their dismissal caused outrage in the rest of the Board and the scientific community."

I think the journalist is using the term "scientist" very loosely here.

Personally, if I were either Mann or Nye I'd be thinking what I might do to earn my living if I got the boot in NewZealand in the same way as did Vessby and Asp in Sweden. As always, lavatory attendant would be my preferred re training option for them.

Less crying in their beer would be a nice too. But then, if I was as wrong as they are, I'd be crying in my beer too!

Peter

Saatchi on the OD?

I happened on Front Row (BBC radio 4) by accident an hour ago. They were discussing Charles Saatchi's new book. There was recounted an anecdote: Saatchi had been asked if it was true he had gone on a diet which required the eating of at least 6 egg yolks a day. The answer was yes, and that its effect was that he had gone from fat and ugly to being thin and ugly.

Too true, I can relate to this. The OD will not alter your face beyond weight change.... Can't think that any other diet involves that many eggs and works...

Peter

When is a high fat diet really a high fat diet?

The paper I mentioned some time ago about the preservation of both fat oxidation and cardiac function on a high fat (60% of calories from fat) diet compared to Western diet (45% from fat) is now available in full text for free. The paper is long (10 pages) and detailed and makes anyone touting an apoE-/- mouse, or a fat stupid one, look pretty dumb. It will take some analysis and it's the World Congress of Veterinary Anaesthesia at the moment so I'm not doing detailed reading this week.

A minor snippet in abstract form which further backs up the idea that you have to manipulate your experimental set up to show the adverse effects of high fat diets is this one. Having lots of mitochondria appears to be a good thing. This fits with Power, Sex and Suicide. One approach to minimising free radical damage is to have lots of mitochondria running at tickover (with uncoupling proteins in abundance). It also suggest that if you are going to have a coronary occlusion, better be in ketosis if you wish to recover! Of course the occlusion is a bit unlikely if you are in ketosis anyway....

Are either of the papers dubious? Well, they fit with my personal experience of what it feels like to eat a high fat diet. But then I'm not eating 16% of calories as refined sucrose!

Peter

Cholesterol, never mind LCAT, try ACAT

Just a quickie. More from the killing fields.

Those poor folks with heterozygous FH are such a playground for cardiologists. My latest accidental find was ACAT inhibition using pactimibe. Medscape describes it this way:

"Pactimibe was in development as an ACAT1 inhibitor, intended to make available more free cholesterol for reverse cholesterol transport, which theoretically could reduce lipid accumulation within atherosclerotic lesions."

Oh, this link works in Safari but not Firefox... Not worth reading, the quote is all you need anyway!

OK, stop foam cell generation and ship out the evil cholesterol (maybe using enhanced LCAT? That'll be next) to HDL for reverse transport. CAPTIVATE-ing idea. I think they were a bit unlucky with this one.

Combined endpoint was deaths, heart attacks and strokes. One out of 438 on placebo, 10 out of 443 on treatment. That's a relative risk of errr, umm... arithmetic fails me.

My biggest worry about drugs being developed based on the lipid hypothesis is that at some time they're going to repeat, again by fluke, the small success of the statins. We'll get a drug which does a little good, a lot of harm and conveniently forget about those who died on pactimibe, torcetrapib or which ever LCAT enhancing drug gets developed. Keeping up the failure rate must be quite difficult, but this continued failure is our best hope for getting some research on heart disease started.

Peter

BTW this drug works really well in genetically hypercholesterolaemic rabbits. No one is suggesting the the WHHL rabbit is a crap model for any sort of arteriosclerosis based on the people injured in this study. OK, I am. It looks like crap model to me.

Low carbohydrate high protein and ApoE-/- mice (2)

I'm waiting to hear about the mineral content of the high protein low carbohydrate diet used to generate arteriosclerosis in mice. Personally I feel the whole study is totally irrelevant but I'm interested because (a) it is a major achievement in its own right and (b) it is being used politically to specifically warn against low carbohydrate diets. The press release is very cleverly done, anecdote and personal testimony is, I am very well aware, powerful stuff. Just like blogging really!

OK, it's time to talk about renal disease in rats and mice. I'm afraid quite a lot of it involves "models" but, well, that's just how it goes. Much of the information here comes from this paper. I think it took Malcolm Kendrick, to whom I am greatly indebted, about 5 minutes to find it on PubMed.

We're going to talk about CKD-MBD, that is Chronic Kidney Disease-Mineral Bone Disorder. CKD-MBD has three components: 1) abnormal serum biochemistries, 2) abnormal bone remodeling, and 3) vascular calcification.

Especially interesting is the last of these three, vascular calcification.

Using acute models of renal failure, particularly the 5/6ths nephrectomy, it is very very hard to demonstrate arterial calcification within the the lifetime of the animal unless you include a genetic modification, typically apoE-/- or LDLR-/- knockouts. So, if you wish to get calcification of arteries, choose an apoE-/- mouse as it speeds up the process. Adding a high content of phosphorus to the diet is also normal as this accelerates the kidney failure and is a known trigger for the conversion of a smooth muscle cell in to an osteoblast.

The phosphorus in the diet has to be bioavailable. That provided by grains is mostly there as phytic acid and, although rodents do have a phytase (humans don't), the amount of phosphate absorbed from grain based diets is much lower than from casein based diets.

In the face of early renal damage it seems like 0.7% phosphate in a casein diet (0.7g/100g) progresses the renal failure where as 0.2% keeps renal damaged animals similar to controls for many weeks. The control groups being given non bioavailable phosphate from grains.

So the next question you have to ask is whether rats or mice on lab chow have renal damage. Well, I blogged about that here.

Enough to say that nephrocalcinosis was absolutely routine until the NTP-2000 diet was introduced. This improved diet still does this:

"The NTP-2000 diet prevented nephrocalcinosis and decreased the severity of nephropathy and cardiomyopathy, the common lesions of F344 rats in 13-week studies"

Note the time scale for nephropathy. Less than 13 weeks. And this is a grain based diet with its phosphate mostly inaccessible to the rat/mouse. Whether you could get changes in the week between weaning and starting on a specialist diet is an interesting point.

But a casein based diet will allow phosphate uptake far better than a grain based diet. Two question then come to mind. Normal rat/mouse chow usually runs at just under 20% of calories from casein. Does increasing this to 45% increase the phosphate uptake? I don't know.

And perhaps you would still need to add supplementary phosphorus? Again I don't know.

So let us summarise: Casein based diets markedly facilitate the toxic action of phosphate on mildly damaged kidneys. Renal failure causes arterial calcification. Arterial calcification will occur in apoE knockout mice with renal failure under circumstances where it is impossible to get it with normal lipid metabolism. With enough tweaks it is possible to get arteriosclerosis in mice by careful manipulation of the model.

Epithelial progenitor cells are produced in the bone marrow under the influence of erythropoietin. Erythropoietin is produced by normal kidneys but only in reduced amounts by nephrotic kidneys. Vascular damage and regeneration appear to be heavily influenced by these cells.

If you wanted to make a diet for a mouse which triggered both arterial calcification and depressed EPC numbers a reasonable stratagem might be to feed a casein based high phosphate diet. To really make things happen choose an apoE knockout mouse. A normal mouse might not oblige.

You really need to know exactly how to manipulate renal function, genetics and EPC numbers in lab mice. This might not happen by accident and would require a great deal of knowledge about renal function and arterial damage.

I think that, for the time being, we will have to await the composition of both the mineral supplement used in the diets and the phosphate content of the calcium salts used as a partial replacement for that supplement in the high protein arm of the study.

But the thought train is interesting.

I was wishing for histopath on the kidneys but, if this hypothesis is correct, all that would have come out would have been that high protein diets damage kidneys.

Peter

Low carbohydrate, high protein and ApoE-/- mice

OK, here's the hot abstract from back in 2007.

And here's the press release from 2009, I've put the text up on my odds and sods blog as press releases don't last for ever on the net. The paragraphs are a bit chewed up but you can get the gist OK.

Cardiologists are impatient people. If they want to study aortic aneurisms they tend to do things like placing a balloon in the aorta via the femoral artery, inflating it and then pulling. Down the aorta, with the balloon inflated. Or they might go in there surgically, cross clamp the aorta in two places, perfuse the isolated section of aorta with some unpleasant chemical, then set all back to normal and try out the latest drug for aneurism treatment on the preparation. The prime requirement is the suspension of disbelief that the "model" has anything to do with human senile dissecting aortic aneurisms. It doesn't.

Obviously the cholesterol fed Syrian hamster is a great model for arteriosclerosis, but it's boring. There's nothing sexy about feeding a herbivore cholesterol. Sexy needs genetically modified mammals to make it happen.

So you want a mouse to get atheroma? Well, they don't. Feed them mouse chow and they get arterial damage and fibrosis all right, but not nice big juicy cholesterol filled plaque. What to do? Delete a gene.

One offspring from the impatience of cardiologists is the apoE-/- mouse. This mouse is a genetic cripple who's ability to process fat has been severely damaged. There are a very, very, very small number of people in the world who are homozygous for defective apoE. They are functionally apoE-/-. Nature does not allow this commonly. Contrast it with FH where there are hundreds of different types of FH, ie breaking your LDL receptor gene is easily done and evolution has not attempted to conserve it particularly highly.

Feeding a high fat diet to apoE-/- mice is bad news for the mice. Until anyone gives us the full text of the paper we'll have no idea of exactly what they fed to the mice but, ultimately, they broke the mice first. Actually, if Dr Murray is anything to go by, even the full text won't tell us much about what they fed the mice!

If you are apoE -/- I wish you luck. Statistically, you're not. Neither is the cardiologist, Dr Rosenzweig, who gave up his LC diet on the basis of this study. But then, he thinks the transgenic apoE-/- mouse is a model for human arteriosclerosis.

Peter

EDIT: OK, I now have the full text (thanks H) and here is the total information supplied in the methods section about the diets:

"Male pups were placed on one of the three study diets 1 week after weaning: standard chow diet (Harlan Teklad #2018 rodent chow), high-fat ‘Western’ diet (Harlan Teklad # 88137) and a custom-ordered low-carbohydrate diet manufactured to our specifications (Harlan Teklad)."

That's it. It is traditional to give enough information in the methods section to allow another group to repeat your protocol. If the problems in these mice are NOT from being apoE-/- then Foo et all are to be congratulated on developing a diet to produce more problems than the Western or Cafeteria diet, but they ain't telling anyone how to do it! No answer from Murray on the same query.

Prompt reply from Dr Rosenzweig with the table of diet compostion, just asking now about the Ca modification and if it involves PO4 changes

High fat diets make you fat and stupid (2)

While trying to understand what was going on in the "Deterioration of physical performance and cognitive function in rats with short-term high-fat feeding" paper by Andrew Murray I came across a similar paper by PubMed-ing "fructose insulin memory" or the like.

This paper provides more information and is very up front about added fructose! Here is what they did:


"In the present study we fed rats a diet high in saturated fats and simple sugars, and supplemented their water with high-fructose corn syrup. This diet increased fasting blood glucose levels and serum cholesterol and triglycerides. Additionally, we found that the diet impairs hippocampus-dependent learning, synaptic plasticity, and dendritic spine density. These adverse effects on brain function were associated with reduced levels of BDNF in the hippocampus and suggest that “Western” diets impair synaptic function and cognition by a mechanism involving reductions in BDNF and atrophy of dendritic spines"

The control diet was good old "standard NIH chow".

The high calorie diet was high in both saturated fat and glucose but was also supplemented with high fructose corn syrup as 20% of the drinking water.

This means that it was impossible for a rat to take a mouthful, any mouthful of food without a glucose load. It was also impossible to drink any water without a fructose load. Rats have to drink.

The most notable thing to me was the fructose in the water. If it was possible to do this with a fat/glucose combo, why add fructose to the drinking water? Answer: It's probably not possible to do this without the fructose. Is there any other possible answer? I guess you could suggest it's the equivalent of the Fanta with the big mac and extra fries... So would the big mac and fries be OK without the Fanta? That we'll not find out from this study because including a fat/glucose combo without fructose was not on the agenda.

I would guess that the fat stupid rats in Andrew Murray's study will have had a fair dose of fructose (as sucrose) in the 16% of calories they derived from carbohydrate. The control diet contained zero fructose.

There are many other points in the paper which are interesting when you take the Taubes view of obesity but they're not core to what is going on. Ultimately there is a lot of insulin sloshing around in the rats on their high fat diet.

Peter

High fat diets make you fat and stupid (1)

Dear Dr Murray,

I have read with great interest your recent publication

Deterioration of physical performance and cognitive function in rats with short-term high-fat feeding

in FASEB.

I have been unable to determine the carbohydrate composition of the diets used from the materials and methods section.

I note that Rat and Mouse No. 1 Maintenance (Special Diet Services, Witham, UK) is composed of wheat, barley, wheat middlings, soyabean meal, dried whey, soya oil and vits/minerals and, as such, is a standard lab rodent maintenance diet.

The composition of the custom diet (diet code 829197; Special Diet Services) is not given in the methods section of your paper. Is it a simple replacement of a proportion of the cereals with a lipid mixture to produce the fat composition detailed, or has the nature of the carbohydrate source used been altered in addition to the reduction in quantity? This seems to be quite an important aspects to the study.

I would very much appreciate a breakdown of the diets.

Thanks in advance.

Beyond that, fascinating paper.

Best wishes

Petro

Cholesterol: LCAT and rabbits

Just a brief word about LCAT and rabbits before getting to the "high fat food makes you lazy and stupid" paper. The full text of this is quite interesting but it will take some time to go through and it's the weekend with houses to view and stuff to do...

Back to LCAT. I was checking the spelling of the non abbreviated text of LCAT and this grant proposal from 1995 was the first hit. I just had to read in in the aftermath of Franceschini's findings.

This is the logic to what they want to do, it's straightforward, hard core lipid hypothesis:

"SUMMARY: In humans, the development of atherosclerosis is positively and inversely correlated with the plasma levels of low density lipoproteins (LDL) and high density lipoproteins (HDL) respectively. LCAT, the major enzyme involved in the esterification of free cholesterol present in circulating plasma lipoproteins, is a major determinant of plasma HDL concentrations. Recent studies have established that transgenic rabbits overexpressing human LCAT have 6-7 fold higher plasma HDL levels than control, non-transgenic siblings. In addition, LCAT transgenic rabbits have reduced plasma concentrations of the atherogenic LDL and apoB-containing lipoproteins. This lipoprotein phenotype characterized by elevated plasma HDL and reduced LDL levels leads to marked protection against the development of diet-induced atherosclerosis in LCAT transgenic rabbits compared to control animals."

This is what they actually want to do:

"1) Evaluate the feasibility of gene therapy utilizing the LCAT gene and suitable vectors as a treatment approach for the prevention of atherosclerosis in animal models as well as patients with premature cardiovascular disease; and,

2) Evaluate the use of gene therapy to correct LCAT deficiency in LCAT knockout mice models systems and patients with LCAT deficiency; and,

3) Develop and evaluate the anti-atherogenic properties of pharmacological agents that raise plasma concentrations of LCAT."

Franceschini thinks they will kill people doing this. OK, he's a bit more polite than that:

"This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk."

He has probably read about torcetrapib!



Anyway the bit which really absolutely grabbed me and had me rolling around giggling is this bit, selected from the above:

"This lipoprotein phenotype characterized by elevated plasma HDL and reduced LDL levels leads to marked protection against the development of diet-induced atherosclerosis in LCAT transgenic rabbits compared to control animals."

Okaaaaaay. Genetically engineered rabbits which make too much LCAT are protected against DIET INDUCED atherosclerosis. These people are talking about the cholesterol fed rabbit. Excess LCAT protects against cholesterol poisoning in rabbits.

Franceschini found DEFICIENCY of LCAT, in real humans, eating real human food (such as is eaten in Italy in 2009), is apparently protective against arteriosclerosis.

I find it very difficult to make a better case for the irrelevance of the cholesterol fed rabbit to anything other than the study of cholesterol poisoning in rabbits. This is rare in clinical practice. Ok, it's non existent outside of cardiology institutes!

Personally I'm not a rabbit.

Peter