Wednesday, May 6, 2009

Atherogenesis in Mice

Another day, another plug for the diet-heart hypothesis in BBC Health, even when the study being reported on Scientists pinpoint fats danger is really about molecular genetics(Thorp et al.,Reduced Apoptosis and Plaque Necrosis in Advanced Atherosclerotic Lesions ofApoe and Ldlr Mice Lacking CHOP, Cell Metabolism ,Volume 9, Issue 5, 474-481, 6 May 2009, subscription required). The study showed that mice lacking a gene (CHOP) which helps to trigger cell death (apoptosis) had a 35% smaller area of plaques and 35% less apoptosis and 50% less necrosis (dead tissue) in plaques. To quote the researhers directly (as reported by the BBC):

Lead researcher Dr Ira Tabas said that previous research had suggested that this mechanism might be involvedin plaque rupture, but the magnitude of the effect uncovered in the latest study was a surprise.
He said: "The fact that we were able to isolate one gene encoding one protein with such a profound effect on plaque necrosis (death) was a big surprise."
Dr Tabas said the finding raised hopes of new drugs which could act on the key gene, or the associated mechanism, to cut the risk of dangerous plaques.
"Just about everybody in our society has atherosclerosis (thickening of the arteries) by the time we reach 20," he said.
"So the wave of the future in treating atherosclerosis will be in preventing harmless lesions in young people from becoming dangerous ones, or soothing dangerous plaques so they don't rupture as we age."

Never mind what effect such a treatment might have on necessary cell death (e.g. to deal with emerging cancers) in other parts of the body.

Anyway, what does this have to do with diet and the heart? Well, again from the BBC article:
Scientists have identified a genetic mechanism which appears to determine which fatty deposits in the arteries have the potential to kill us. Most of these plaques pose no risk to health, but a minority burst, forming blood clots, which can cause heart attacks or strokes. .....
Fatty deposits begin to form in the arteries of most people in their teens, but the vast majority are harmless.

Here we see the perpetuation of the myth that fat just floats around in the bloodstream clogging up our arteries like it would a drainage pipe. Plaque formation is a much more complex process than that and its genesis is still not fully understood (see for example, extensive discussion here or here).

But, ah you say, just read on ...
The researchers bred mice prone to develop plaques, and fed them a high-fat diet for 10 weeks.

So what was this high-fat diet? It was the TD.88137 Western Diet (Teklad Lab Animal Diets, Harlan Laboratories, Madison, WI) which consists of:

Corn Starch150.0
Anhydrous Milkfat210.0
Mineral Mix, AIN-76 (170915)35.0
Calcium Carbonate4.0
Vitamin Mix, Teklad (40060)10.0
Ethoxyquin, antioxidant0.04

(Data from this pdf.)
This diet is 17.3% protein, 48.5% carbohydrate and 21.2% fat by weight, but 15.2% protein, 42.7% carbohydrate and 42.0% fat by energy, thus approximating a typical Western-style diet which is high in fat and simultaneously high in carbohydrate. Note that of the carbohydrates 70.4% by weight is sucrose! The mice are eating 30% of calories as sucrose. Now mice are not little people, but what does that kind of intake do to people?

How does this compare to a mouse's real diet?
From The Mouse in biomedical science (James G. Fox, Stephen W. Barthold, Muriel T. Davisson, Christian E. Newcomer, 2nd ed., Academic Press, 2007) p. 28 we learn that it is still debated whether mice are granivores, eating a wide range of cereals, oilseeds, and a variety of grass and plant seeds, or whether they live on a mix of plant and animal sources. However from the evidence presented in this book it appears that in many environments, mice eat small invertebrates for at least part of the year (i.e. when seeds are in short supply) or to supplement plant seed diets.

In short, the typical composition of a diet of invertebrates is high in fat and protein e.g. from p.41 in
Marsupial nutrition, (Ian D. Hume, Cambridge University Press, 1999) it can range from 20-60% fat and 10-75% protein (by weight of dry matter) for typical things that a mouse might eat (insects and insect larvae). Cereals are typically 68-79% carbohydrate, around 10-15% protein and 2-7% fat, legumes are as much as 25% protein, typically 50-60% carbohydrate and only 1-2% fat whereas nuts (e.g. hazelnut) and oilseeds (e.g. sunflower) are typically about 15-25% protein, 50-60% fat and 15-20% carbohydrate (from various tables in On Food and Cooking, H. McGee, 1st ed. Unwin Hyman, 1984).

From this we can conclude that a typical wild mouse would for part of the year eat a diet that was mainly protein and fat and for another part of the year eat a diet that was high in carbohydrate - at least if it ate cereals, but not so much if it ate other types of seeds - but low in fat. It would not however be eating a lot of sucrose. The carbohydrate in grains and seeds is starch which is a polymer of glucose and does not contain fructose. As further support of this analysis here Peter of Hyperlipid considered data on what wild-type mice eat when given free choice: about 12% protein, 6% carbohydrate and 82% fat (all as proportions of energy).

A final note about the mice. The mice used in the experiment were either apoe or ldlr mice. Apoe mice lack a particular lipoprotein (apolipoprotein E) which is important in both the HDL and vLDL cholesterol transporters, in particular:
ApoE mediates high affinity binding of chylomicrons and vLDL particles to the LDL receptor, allowing for specific uptake of these particles by the liver, preventing the accumulation of cholesterol rich particles in the plasma
Mice develop normally, but exhibit five times normal serum plasma cholesterol and spontaneous atherosclerotic lesions
Ldlr mice lack a proper LDL receptor and essentially mimic (familial) hypercholesterolaemia with a very high circulating LDL level and an increased propensity to develop atherosclerotic lesions amongst other things.

Does this not indicate that fat is the root cause? Well not necessarily.

vLDL is made in the liver to transport triglycerides (made from excess carbohydrates intake) to the tissues for use and storage. At this stage, it does not contain apoE: it has to pick that up from HDL on the way. ApoE contributes to its recognition and re-uptake by the liver after it has performed its delivery task or it loses its apoE and becomes an LDL particle and is taken up by body cells with an LDL receptor. So, this process will become disrupted in an apoe mouse which does not have a proper apoE protein. No wonder it ends up with excess blood cholesterol (which really means excess circulating lipoproteins). Similarly as ldlr mice lack the LDL receptor, they cannot remove the LDLs left at the end of the described process. On the other hand, after digestion, fat is absorbed either directly into the bloodstream - if the molecule is small - which gets it to the liver (where it may contribute to triglyceride production) or, for larger molecules, as chylomicrons which go via the lymphatic system into the bloodstream and from there directly to fat tissue for storage or to the liver to be used to provide fuel.
So which is more likely to contribute to the problem - the 21.2% of food (by weight) that comes as fat (most of which doesn't go straight to the liver anyway) or the 48.5% of food (by weight) that comes as carbohydrate - three-quarters of which is sucrose and half of that is fructose (i.e. 17% by weight of the total food intake) which goes straight to the liver and comes out as triglycerides.

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