Essential fatty acids are essential

If you take a cow and feed it on grass it gets quite a lot of omega 3 fatty acids. If you feed it on a barley based concentrate feed it doesn't get nearly so many, just loaded up on omega 6s. Because cows have a rumen they actually live on a combination of volatile fatty acids produced by bacteria, which breaking down that otherwise useless fiber in grass, plus bacterial protein. Not much of the grass itself actually gets through to the cow. Most bovine fat is self assembled from things like butyric acid or acetate, so it's fully saturated or monounsaturated, ie typical mammalian produced fat. But some essential fatty acids do get through, after all they're essential to the cow just as much as they are to you and me.

How much PUFA get through the intensely reducing environment of the rumen? This paper gives some idea of the input and transformations which occur.

Table 1 shows the amounts of linoleic and linolenic acids in grass, concentrates and sliage. Grass and silage are pretty much the same, with one part omega 6 (linoleic, 18:2) to three parts omega 3 (linolenic, 18:3). That is, grass has a rather huge excess of omega three over omega six. Before it hits the rumen.

Concentrates don't. They're not quite as bad as the "prudent" diet of the Lyon Heart study (only a cardiologist could design a diet that bad) but, at roughly eight parts omega 6 to each part omega 3, this is still cardiological profit making nirvana for the AHA.

What comes out of the rumen? The paper next looks at the fatty acid composition of intramuscular fat, the results are in Table 3. Grass only fed cattle have about 2.33 times as much omega 6 as omega 3 fatty acids in their muscles.

I firmly believe that humans evolved with an excellent ability to hunt herbivores, grass fed herbivores. On the basis that hunting provided the bulk of the lipids to a hunter-gatherer, this looks like a pretty good fatty acid ratio to aim at. Eating wild herbivores seems to be what we were good at and what should provide us with a healthy diet. Plus a bit of fishing too I guess.

The concentrates-only fed cattle were actually given some hay too, because cows tend to die if you feed them on concentrates alone, and they came out with a 4.15 parts omega 6 to each part omega 3 fats in their muscles. It's worth noting that the worst quality of grain fed Irish beef still provides an omega 6 to omega 3 ratio as good as the intake in the best ever dietary intervention trial! Still, a ratio of 2:1 looks to be even better. In both groups the PUFA made up about 5% of the fat.

The other place worth looking is Kitava , full text here, keep scrolling down to find it and try to ignore the more weird papers written by Cordain. These subsistence farmers got their lipids from fish and coconuts. There are some omega 6 fats in both fish and coconuts, but the omega 3 from the fish predominate, ie they eat less than one part omega 6 to each part omega 3. No heart disease, despite smoking. PUFA made up 10% of the lipids eaten, which were low in total at 20% of calories.

Back to cattle. What comes out in the milk? Important if you are as dairy dependent as I am. I only have data for grass fed cattle. You can see from table 3 in this paper* that PUFA run at around 5% of lipids and that there is almost a 1:1 ratio. Omega 6 come out at or just above 1% of total lipids, omega 3 at just below 1%. Hang on, that's only 2%... What are the other 3% to make up the 5% PUFA? It's mostly conjugated linoleic acid, CLA. The good stuff, the anti-cancer, anti-this pro-that CLA. Non synthetic, straight from the cow. You can see why I like dairy fats. Cows intend calves to be healthy.

That's the grass fed stuff. In general grass is cheap and concentrates are expensive, certainly here in the UK. In areas where grass will grow and wheat won't, we grow cows. Via grass. It makes quite good silage for winter use too. If you are running a dairy unit you will feed the maximum possible of grass/silage and a minimum of cattle cake. Economics dictate this. It's a hard market for dairy farmers. But even the worst case lipid scenario, using a maximum of cattle cake, would be a 1:4 ratio in cream. This is as good as the Lyon investigators got with their gloop.

Obviously neither chickens nor pigs have a rumen, so their fatty acid balance will be far more affected by the high omega six content of their diet. This is the primary reason I add 5g/day of fish oil to my diet. It goes some way to getting an essential fatty acid ratio of about one part omega 3 to, at worst, 2 parts omega 6 overall. PUFA make up about 5% of my total lipid intake, which obviously is quite high in absolute terms, due to the total amount of fat I eat.

This seems to be a very reasonable approach to PUFA for me.

Obviously all vegetable oils except olive oil are banned from the house. Banning these oils is the biggest step needed to make balancing lipids straightforward. It's possibly more important than the gloop to the Lyon heart study success. Once you crack a bottle of corn oil, sunflower oil or a pot of margarine you will never get your omega 6 intake low enough to balance things out with a few grams of fish oil. I guess that's why it's impossible to show overall benefit form one or two cod liver oil capsules a day in a "normal" diet...

Olive oil gets used in our house as a flavouring, never for bulk calories. Actually, so does a small amount of sesame oil too...

The food has to taste good as well as being nutritious!

I don't regard fish oil as a supplement. I look on it as a tool for correcting the fatty acid defect ubiquitous in UK non ruminant fat. It even makes the excellent dairy lipids better.

Peter


*Oh, I just found that the milk-lipids paper is on my hard drive as a pdf and it's not on pubmed. No idea where I got it from! It's:

Elgersma, A., S. Tamminga, and G. Ellen. 2003. Effect of grazing versus stall-feeding of cut grass on milk fatty acid composition of dairy cows. Proceedings of the Int. Occ. Symp. of the European Grassland Federation, Pleven, Bulgaria, May 2003. Grassland Science in Europe 8: 271-274.

if anyone want's to chase it!

Omega 3s and G-protein coupled receptors

Let's just summarise the role of omega 6 fats in Sauer's rat model of cancer:

In the lab situation rapid hepatoma tumour growth needs either arachidonic or linoleic acids. The acids must be taken up in to the hepatoma cells, they must be acted on by lipoxygenase to produce 13-hydroxyoctadecadienoic acid, better known as 13-HODE. 13-HODE appears to be the mitogen which promotes rapid cancer growth. 13-HODE looks like a repair signal gone wrong in cancer cells. Omega 3 fatty acids block omega 6 fatty acid uptake in to hepatoma cells. That's all well and good but the reason I got in to this paper was omega 3 PUFA signalling, rather than those omega 6 issues...



OK, Sauer starts to give some pointers on the function of omega 3 fatty acids in health. That's interesting, as I'm no great lover of any sort of PUFA when I view them from the Protons perspective, yet omega 3s seem to come out pretty well, certainly at low doses. You know my fall back, omega 3 PUFA don't always behave like omega 6 PUFA because they get used as signalling molecules blah blah blah. My own inability to tie the molecular structure of omega 3s to their clinical effects is very frustrating! That they probably act are sites "above" the ETC suggest that they act as what I view as 'high level signals".


Well they do.


The signalling appears to be through a G-protein linked receptor with all of the usual cAMP cascade that follows binding of a ligand to such a receptor. What I found particularly interesting was the effect produced on fat pads of normal rats when EPA (other papers from Sauer suggest all omega 3s act on whichever receptor is involved) was added to the perfusate.

OK, here is a neat little graph taken from here:

This is from fed rats. In the fed state the FFA uptake by the inguinal fat pad of a rat is about 6 mcg/min/gram, white open squares.

Adding EPA at 0.84mmol/l (a bit supraphysiological for EPA but let's let that ride) and FFA uptake by the fat pad drops to zero, or close to zero. Or, in fact, you could argue a suggestion of fatty acid relase, shown as a negative uptake value. Black circles. Fatty acids are not taken up, they end up in the venous effluent in the experiment, plus a little extra.

Whooooah, so do FFAs go through the roof when you take fish oil IRL??

Well no. That's because of this graph from here in the same paper:

Here we have the free fatty acid release from the inguinal fat pad of a healthy rat who has been starved for 48 hours. Fatty acid release is trundling along at about 3mcg/min/gram until EPA is added, again at around 0.8mmol/l. The release of FFAs, in the fasted state, is eliminated. Table 1 in the same paper shows you can get this effect of halting lipolysis in starved rats with under 0.3mmol/l EPA.

Both effects are mediated through a G-protein coupled receptor, ie high level signalling compared to electrons and superoxide in the electron transport chain.

Obviously there are a number of serious problems with this paper but, as a proof of concept, I buy it. I doubt DHA or alpha linolenic acid would work as well (or the group would have used them for this proof of point exercise!) and I think the levels of EPA used produce a very artefactual "switch-like" effect which is probably a graded response. I doubt 0.8mmol/l or even 0.3mmol/l of EPA is exactly physiological but...

Let's suggest that there is a progressive removal of the influence of adipocytes from the FFA flux in/out of plasma as the level of omega 3s in arterial blood increases. Omega 3 fatty acids render adipocytes irrelevant to free fatty acid levels in the plasma.

That is one hell of an idea.

Next we need a brief look at hepatoma cells, again the graph is provided by Sauer and it shows that omega 3 fatty acids, in a G-protein coupled receptor manner, completely turn off the uptake of ALL fatty acids in to hepatoma cells.

If, and it's quite a big "if", the same effects apply to hepatocytes as well as hepatoma cells, we then have a very straightforward mechanism for the protective effects of omega 3 fish oils on hepatic lipidosis. From my point of view this is quite real as there are pretty convincing papers showing that cats, in real life, can be largely protected against the potentially fatal hepatic lipidosis of rapid weight loss by modest doses of omega 3 fatty acids.

Soooo while omega 3s stop the release of all FFAs from adipocytes, they simultaneously stop the uptake of all fatty acids in to the two primary storage organs for fatty acids, adipocytes and liver.

Do plasma FFAs go up or down?

They do, of course, go down. A paradox? Next paper.

Health warning: This paper is so steeped in VLDL and ApoB lipophobia that it makes difficult reading. But there is so little published on FFAs and omega 3 supplementation that it's worth the ondansetron to read it. It's looking at how omega 3 supplements might lower fasting triglycerides, which are the devil incarnate for CVD risk. A huge chunk of VLDL comes from FFAs released from adipocytes and their subsequent repackaging by the liver. Apparently, and I quote from the abstract:

"FO [fish oil] counteracts intracellular lipolysis in adipocytes by suppressing adipose tissue inflammation"

A bit like insulin resistance is caused by "inflammation". Well, maybe it's that simple. They have taken the concept of high level signalling to its 2013 pedestal without looking for basic mechanisms. They have placed the G-protein coupled receptor on to macrophages in the fat pads, which subsequently control the adipocyte lipolysis using cytokines. I haven't checked how good this concept is. Sauer never looked that deeply. Looks a bit modern to me.

Personally I would guess that there are similar receptors on both adipocytes and hepatocytes, but the review does not seem to cover the ability of omega 3s to inhibit general fatty acid uptake by these two tissues. Ah well.

What they do argue is that omega 3 fatty acids upregulate lipoprotein lipase, pretty well whole body. Of course liver and adipocytes ignore this fatty acid bonanza, as above. LPL upregulation is what I needed to know from this paper.

So where do "spare" fatty acids go to? They go to muscles. Upregulated lipoprotein lipase (heralded as the saviour from elevated fasting triglycerides) allows increased lipid release from VLDL to lower those fasting triglycerides. But it's worth bearing in mind that cells do not "see" VLDL, the LPL is on the vascular endothelium and the cells behind the vessel wall only ever receive "free" fatty acids. These are not labelled as from albumin, VLDL or chylomicrons.

Slight aside for later: It seems likely that chylomicrons are going spill their lipids via that same LPL, worth remembering.

The story in the review can be sumarised as omega 3 fatty acids block the release of FFAs from adipocytes and increase the activity of lipoprotein lipase pretty well whole body. VLDL drops, FFAs drop. All is happy in the cardiovascular system. If you believe.


Various "bits" of omega 3s, especially the lipid peroxides of DHA, are signals for mitochondrial biogenesis. I had a paper which specified which lipoxide was most effective but must have missed the "save" button. Mea culpa yet again. There are hints here.


That's a very neat story, which has more than a grain of truth to it.

Why is it like this? What does it mean, physiologcally? Speculation time:


Omega 3 fats come from plants. Mostly from chloroplasts. Where do humans get their omega 3s from? Certainly not from plants. If we did then the rabid Dr Furhman would not be (correctly) recommending DHA supplementation (along with B12) to avoid brain collapse on veg*n diets. Actually, this link is quite funny when cited by Mc-Starch-Dougall:

"There is no evidence of adverse effects on health or cognitive function with lower DHA intake in vegetarians"

Well, I found it amusing. It's almost the converse of the neurological truism which states that being concerned about having a neurodegenerative disease probably means you don't actually have one.

Anyhoo. Away from the coast we have to get our DHA from animals (or buy algae derived supplements). They get it from grass. There is DHA present in adipose tissue of herbivores just as much as it is present in lipid membranes of their cells. My suspicion is that DHA is a signal to your metabolism that you have just eaten animal fat, from an animal who's food chain starts with grass [or algae]. The more fat you eat, the stronger the signal. We do not need much DHA overall for our brains as it is well protected in this site, but we might well be using it at low levels as a [G-protein coupled receptor sensed] signal to target metabolic adaptation to process fat. So is McDougall correct that veg*n "brain" tissue is OK, despite their periphery being depleted? Shrug.

Fish oil supplements? Well, using our "dietary fat is here" marker to pharmacologically modify some perceived CVD risk factor, without the appropriate change in source of metabolic fuel supply, looks to me to be of very limited value. Large intervention trials do show some benefit from omega 3s provided you do your stats well enough, you have a large enough population to pick up a very small effect and you give a high enough dose. But they do not seem to be any sort of panacea. Especially of you are avoiding dietary fat while "faking" the signal that you have eaten dietary fat...

This is not exactly surprising when you try to pick the likely physiology apart. I like the concept of DHA as an animal fat signal.

Peter

Final thought: Do we need omega 3 PUFA at anything above the most minimal levels if we are in saturated fat based ketosis? Of course I don't know. But the signal to cope with starvation is palmitic acid (physiological insulin resistance), not DHA. I live in starvation mode, not on a mixed diet with only intermittent access to healthy ruminant fat. I have long wanted to look at the selective release of FFAs from adipocytes in extended starvation. My suspicion is that in the early days after glycogen depletion palmitic acid is preferentially released over other lipids, PUFA are not needed/wanted. By a few weeks all the palmitate is gone and whatever is left then gets released. People like David Blaine suddenly start to feel weak, wobbly and are probably hypoglycaemic once they run out of palmitate and have to release less saturated fats. Two to four weeks if you carry some spare weight. Sauer's rats had only ever been fed a low fat omega 6 based diet and had no serious palmitate reserves, PUFA release came early for these.

AGE RAGE and ALE: VLDL degradation

Malonyldialdehyde (MDA) is a small molecule formed by the random oxidation of a polyunsaturated fatty acid. The exact chemistry seems quite complex but needs, as an absolute minimum, two double bonds in the fat molecule, three omega numbers apart. But a feature of organic chemistry makes the free radical attack much more successful if there is a third double bond, located three omega numbers away from that bare minimum pair. So linoleic acid, that good old omega 6 fatty acid, can form some MDA because it has a double bond at the omega 9 position and at the omega 6 position, but it struggles to do it. Alpha linolenic acid, with its third double bond down at the omega 3 position, really allows MDA production to get going. That's chemistry. There's the briefest of summaries here.

So omega three fatty acid supplementation will increase MDA production. Adding vitamin E will largely eliminate this effect in the short term and the theory is that the vitamin E protects the omega 3 fatty acids in the chylomicrons en route to the liver. So more undamaged PUFA reach the liver, which has a greater impact on fasting triglycerides (VLDLs). This much comes from this paper.

But what I found really interesting is what happens within the liver itself. This paper comes up with some answers. The VLDL particles are manufactured as per normal, but if there is enough lipid peroxidation, the particle is degraded and never released. The liver appears to use iron to generate MDA from PUFA as a decider as to whether to release the VLDL particle or degrade it. The more MDA generated within the liver cell, the lower the plasma VLDL levels drop.

Why should that be? The liver makes a VLDL particle, tests how stable it is in terms of lipid peroxidation, and refuses to release any VLDLs deemed too unstable. This peroxidation is what omega 3 fats do, far better than omega 6 fats do it. The message I get from this is that our liver does not want lipid peroxidation prone VLDLs being released in to the circulation. So, if we accept that VLDLs from carbohydrate are stable (palmitic acid based) lots can be safely released. Render then unstable with fish oil and they, and their components, stay in the liver. Is this good or bad?

Well, taking fish oil makes your fasting triglyceride value look like it belongs to a LC eating person, even though you may not be a LC eating person. Does your cardiac risk belong to the the LC person or the mixed diet person? Draw the comparison with torcetrapib. Fantastic lipids, increased death risk. Now look at atrovasatain, quite "good" lipids and permission to trade in your heart attack death certificate for a cancer one, with the same date. Then look at LC eating and wonder about the blank cause of death and date.

Where do fish oils fit in to this spectrum? Dropping your triglycerides is treating a number. Fine for cardiologists. Eating like a Greenland eskimo requires strict LC in addition to 15g/d of EPA+DHA. This is a double triglyceride lowering approach but one which, in addition, dramatically minimises the glycation of apoB containing lipoproteins too. Is it the LC, the low trigs or the changes in tissue lipids which helps reduce CV risk? The mixed diet eating person with fish oil induced LC style triglycerides may well be munching lots of fruit as healthy low fat snacks to stave off hunger pangs between mixed meals. VLDL gycation?

If you have already lived your way to a heart attack, just "doing" EPA+DHA at 3.5g/d is as effective as 4g/d of corn oil for prevention of that second heart attack within the next 12 months! It's okay, unless you are one of the 25% of heart attack victims in each group re infarcting. Of course the fasting triglycerides were MUCH lower in the omega 3 group...

So do I think fish oils are useless? Not at all, but I think that using them as a tool to manipulate a number is, well, not a good idea. I do know that low dose EPA+DHA seems to benefit me, at around 1g/day. Whether this is "treating" the amount of omega 6 fatty acids I get from chicken and pork, I wouldn't like to say. But there's a lot more to omega 3 supplements than meets the eye.

What does seem lacking to me is convincing evidence of toxicity. The Greenlanders (back in the 1950s) were at low CV risk on high omega three intakes and I think it's reasonable to assume they were at the same low cancer risk as the Inuit described by Stefansson, see Stephan's post here. So I don't rate omega 3 as coming with the same toxicity as omega 6s.

There's another post on hepatic VLDL stability testing, but I'll call it a day on this rambling entry...

Peter

On drinking varnish

Dietary linoleic acid elevates the endocannabinoids 2-AG and anandamide and promotes weight gain in mice fed a low fat diet.

Raphi sent me this link early in the New Year. It’s nice. It demonstrates, at some level of complexity, that omega 6 PUFA at 8% of calories are obesogenic in mice, even if they are fed otherwise fat free CIAB. It’s all about endocannabinoid ligands and receptor activation. Potentially useful when folks get round to starting class actions against the cardiological community and any other health advisors warning against saturated fat. If you limit fat to 30% of calories and saturated fat to 10% you still have 20% PUFA/MUFA in your diet. That’s easily obesogenic. Your cardiologist made you fat. Sue now.

But all of this endocannabinoid stuff is what I call high level signalling. At the core mitochondrial level we know that omega 6 PUFA fail to limit insulin activity under situations where a saturated fat would shut down insulin mediated calorie ingress. In an adipocyte this means that, during oxidation of omega 6 PUFA, insulin continues to signal and fatty acids (and glucose) fall in to the adipocytes, stay there, and you get really hungry. Modified chemicals derived from this system of omega six fatty acids are overlaid on top of the core mitochondrial signalling. A modified derivative of arachidonic acid becomes an endocannabinoid ligand and makes you hungry and fat. The system takes something basic and develops an overlay of enormous complexity, this is what I call higher level signalling.

I hate higher level signalling. Give me the core process anyday.

On this front people may realise I have issues with omega 3 PUFA fats. From the ETC perspective they are worse than omega 6 PUFA and should be more obesogenic. But, in general they’re not. In fact there is a massive industry showing us how good they are for us. But there are suggestions that the core process which makes omega 6 PUFA obesogenic really do apply to the omega 3s. Bear in mind that we are only talking about linoleic and alpha linolenic acids here. Longer fatty acids go to peroxisomes for oxidation and have little influence on core mitochondrial processes, though they do perform a great deal of high level signalling. Here we go:

Sucrose counteracts the anti-inflammatory effect of fish oil in adipose tissue and increases obesity development in mice.

Notice the obesogenic effect of fish oil only shows when sucrose is present in the diet. Replacing sucrose with protein eliminates the effect. Fructose is an unstoppable source of cellular energy intake which needs insulin resistance to limit insulin signalling facilitated ingress of glucose. As insulin continues to act, fat cells sequester calories. Fish oil combined with sucrose is the worst, corn oil is intermediate and, without sucrose, none of the fats are obesogenic.

This makes me happy. I can see the core process at work, never mind what EPA and DHA say to g-protein coupled receptors.

There is another paper which shows a similar effect and I like it rather a lot because the cognitive dissonance, which shines through every word of the text, is rather entertaining. How can you get a life-sustaining source of funding if your data show that omega 3 PUFA are grossly obesogenic? They improve insulin signalling exactly as the ETC effects would predict. The cost of improved insulin responsiveness in adipocytes is obesity. Here we go again:

Adipose tissue inflammation induced by high-fat diet in obese diabetic mice is prevented by n-3 polyunsaturated fatty acids.

The values to look at begin with the weight gain. All we have to do is to subtract weight at the start of the study period from weight at the end (perhaps the authors don't do arithmetic?). Low fat group gained a gram, added saturated fat group gained 0.6 g, added omega 6 group lost* 2.4g and omega 3 group gained 10.4g.

Ten point four grams.

These are db/db mice which lack a functional leptin receptor. They are diabetic and I feel their chronic hyperglycaemia represents a similar drive to obesity as the fructose loading in the last study, ie an unregulated source of calories which drop in to adipocytes and which require insulin resistance to shut down whatever further caloric ingress it can practically do. Free fatty acids, a reasonable surrogate for the action of unmeasured insulin, are low so this suggests adipocyte sensitivity to insulin is high, hence the weight gain.

Weight gain in the alpha linolenic acid group was over 17 times that of the saturated fat group and 10 times that of the low fat group. Notice saturated fat protected (admittedly ns) against the weight gain seen on the low fat diet. The logic is obvious. What do the authors say? Well, I can find no mention in the discussion of this massive weight gain in the omega 3 group. Zilch. This is the quote from the only mention it gets, in the results section:

"Body weight at the end of the study was somewhat higher in db/db mice fed HF/3 compared with HF/S (Table 1)".

My emphasis.

There is no other mention of the hard fact that omega 3 fats are obesogenic. Also note that in relatively normal, non hyperglycaemic db/+ mice, the omega 3s are not obesogenic. Much the same as for non-fructose fed mice in the previous study.

Now look at the * I put in above. The omega 6 diabetic group LOST 2.4g. Ouch, at the core mitochondrial function level! How can this be? This needs no mention at all in the paper because p is greater than 0.05 (in the twisted stats used by the authors). But brownie points if you have noted the oddity about this particular group of mice.

Well done! Yes, in a group of 5 animals the standard deviation at the end of omega 6 feeding is 8.6. No other group had a standard deviation greater than 3 at any time. How do you get a standard deviation of 8.6? These are diabetic mice. Four gained weight, one became ill and this one lost a lot of weight. That's my guess, just trying to reverse engineer information out of the data supplied by a group of dissonant thinkers...

So, I went to an on-line standard deviation calculator and fed in various options where 4 mice gained some weight and one mouse lost a tonne of weight. Using a 2g gain for 4 possibly healthy mice and a 20g loss for the fifth poorly mouse we get four mice at 44g and one at 22g. This gives a mean weight at the end of the study of 39.5g to with an SD of just over 9. I think something like this is what happened. Would this group notice one skinny mouse in with four fat ones? Hahahahaha!

Summary: When PUFA are being oxidised in the mitochondria of adipocytes, those adipocytes are unable to resist the signal from insulin to distend with fat. The more double bonds in the PUFA has, the greater the effect. Linseed oil should be used for making varnish.

Peter

More on drinking varnish

This paper is a gem.

Reducing the Dietary Omega-6:Omega-3 Utilizing α-Linolenic Acid; Not a Sufficient Therapy for Attenuating High-Fat-Diet-Induced Obesity Development Nor Related Detrimental Metabolic and Adipose Tissue Inflammatory Outcomes

What did they do? They fed rats chow or they fed them on one of four other diets enriched in PUFA. The extra PUFA were based around various mixtures of linoleic acid with alpha-linolenic acid, some  were mostly corn oil, some were slanted towards varnish (flax/linseed oil). Total 18-C PUFA made up 9.4% of calories, ie was obesogenic, and this was identical for all of the high fat diets. Overall macros were identical in all of the high fat diets too. There was no sucrose. The rats were fed ad lib.

Here is the link to Table 1 which lists the compositions, it's too big for putting it up as a jpeg. Just look at how utterly fair the composition of the high fat diets were. Even if the absolute amount of linoleic acid in the lard is not accurate, there will be a consistent error across the diets and the results stay plausible. My only complaint is that there was no group where the omega-3 lipids predominated in the diet PUFA, a 50:50 mix was the maximum. Whereas the maximum omega-6 fed group got essentially all of their PUFA from omega-6 PUFA.

The second excellent feature is that the rats were neither semi-starved nor forcibly overfed. Rats are not people. They cannot be verbally asked to overeat to maintain a stable bodyweight nor to calorie restrict to lose weight. They will simply eat until they are no longer feeling hungry. If that happens while they are svelte or not until they are morbidly obese, the rats don't care.

What happened?

Almost nothing. The chow fed rats, with around 3.5% of calories as PUFA, stayed at a reasonable weight. The obesogenic high fat diets (ie nearly 10% of total calories as PUFA) each caused almost exactly the same progression of obesity:

Why almost?

Can you see that the open squares group gain weight slightly more slowly than the other PUFA diet groups? This shows between week six and week 17. The two hashtags mark out a couple of time points where this achieved statistical significance. This slightly less obese group of rats is the group which ate the least alpha-linolenic acid, the most linoleic acid. This suggests that omega-6 PUFA are less fattening than omega-3 PUFA. I like that. Protons likes that.

The effect was fairly small and only shows as an early facilitation of weight gain. By the end of the study the rats and their adipocytes were all about as fat as they were going to get on 9.4% of calories from any family of PUFA.

You can easily hide this effect by under feeding (pair feeding to the same calories as a chow fed group or arbitrarily reducing overall caloric availability) or overfeeding (paid humans or intragastric cannula over-fed rats). If you are an omega-3 lover this can be necessary. But, given a decent study, it shows.

Consuming the 18-C omega-3 rich linseed oil/flax oil/varnish may not make you terribly much fatter than corn oil will eventually make you, but it should get you there quicker. The situation for EPA and DHA is different. Oxidising these will increase the cytoplasmic NADH:NAD+ ratio via peroxisomal oxidation (bad) and give reasonable mitochondrial function from oxidising the residual saturated caprylic acid C-8 (good), which is the normal fate of very long chain fatty acids of any ilk.

Executive summary: Omega-3 18-C fatty acids are more obesogenic than omega-6 18-C fatty acids. The effect is small but real, it might show better if all of the PUFA were alpha-linolenic acid rather than to 50:50 mixture used. It still makes me happy.

Peter




The Protons view (skip this if you're fed up with hearing it over and over again)...

I consider that the mitochondrial oxidation of PUFA will always show as increased peak insulin sensitivity. The cost of that increased insulin sensitivity is fat gain. The fat gain eventually eliminates any benefit from the initial increase in insulin sensitivity. Forced manipulations of the food intake downwards will preserve the intrinsic insulin sensitivity at the cost of chronic hunger. So when high PUFA-fed lab-rats are "pair fed with the chow group" the PUFA rats will look really good, metabolically. The converse, encouragement to overeat, based on avoiding "accidental" weight loss (weight loss is a huge confounder in studies of hepatic lipid accumulation from almost any intervention, PUFA included) by weekly weighing to maintain weight will mask any benefits from saturated fat induced adipocyte insulin resistance. Stacking the deck is crucial to the result you want to get.

The P479L gene for CPT-1a and fatty acid oxidation

In order to work out what is happening with a given child having an episode of hypoglycaemia as a result of having the P479L version of CPT-1a, we need some information.

My thanks to Mike Eades for the full text of the paper on the Canadian Inuit, which does include a certain amount of useful clinical data.

Here is the snippet about a young girl having a hypoglycaemic episode while hospitalised:

“Plasma free fatty acid was 3.8 mmol/L and plasma 3-hydroxybutyrate was 0.5 mmol/L”

Blood glucose was 1.9 mmol/l at the time. An FFA level of 3,800 micromol/l is impressively high. She was generating a small amount of ketones.

No one would argue with intravenous glucose at this point, the question is about how she got here.

So. The problem here does not (as I'd initially thought) appear to insulin induced suppression of FFAs to a level at which beta oxidation fails to support metabolism. FFAs are very high, even for an P479L person after a short fast. With ketones starting to be produced (and low blood glucose) I feel it is reasonable to assume that her liver glycogen is depleted and, while some fatty acids are entering the hepatocytes, not enough of them are being oxidised to support ketogenesis. Glycogen is being depleted to keep liver cells functional. Gluconeogenesis from protein is unable to meet the hepatic (and whole body) demand for glucose calories in the situation of limited access to FFA calories.

However much glycogen derived glucose you consider that the ancestral diet contained I feel it is very, very unlikely to be greater than the glucose and fructose of a modern diet. I feel that getting enough glycogen in to the liver to fully fuel its metabolism in the absence of adequate fatty acid oxidation is a non starter. The P479L mutation was not "permitted" by high oral carb loading, it was permitted by conditions which facilitated fatty acid oxidation. You don't have to agree.


What starts to look much more interesting is what controls CPT-1a activity and how this might vary from the ancestral diet to the modern diet.

The paper makes the point that omega 3 fatty acids appear to up regulate fatty acid oxidation (in rats at least) by the liver. If this is true in humans then a high level of omega 3 fatty acids from marine fats might up regulate fatty acid oxidation to a level which no longer necessitates the depletion of hepatic glycogen derived form oral glucose intake or protein catabolism.

In support of this is that the distribution of P479L within Alaska is not uniform, it's significantly commoner in the coastal regions compared to the inland areas.

"The allele frequency and rate of homozygosity for the CPT-1a P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the Inuit or Inuvialuit in the two western territories"

I'm open to other explanations, there are papers suggesting that the mutation helps to preferentially dispose of omega 6 PUFA, with omega 3 fatty acids as the facilitator.

In summary: Maintaining adequate FFA oxidation to avoid glycogen depletion looks to be the core need in P479L. A high fat diet with a large proportion of omega 3 fats might be a plausible way of maintaining adequate hepatic fatty acid oxidation. Hyperglycaemia (via Crabtree effect) looks to be anathema. Glycogen loading with a normal starch/sugar based modern diet is clearly ineffective to prevent hypoglycaemia for some individuals. Resistant starch as a reliable nightly adjunct to infant feeding seems very unlikely in the ancestral diet. Repeated periods of fasting were probably routine when hunting was poor and does not appear to have selected against P479L in weaned children. Unweaned children are unlikely to be exposed to fasting, provided milk was available from lactation.

Well, there are some more thoughts on the biochemistry.

People clearly have very differing ideas of what the Inuit did or did not eat as an ancestral diet. The P479L gene eliminates the need for source of dietary glucose to explain very limited levels of ketosis recorded in the Inuit. While it is perfectly possible to invoke a high protein diet to explain a lack of ketosis in the fed state this goes nowhere towards explaining the limited ketosis of fasting. P479L fits perfectly well as an explanation.

I have some level of discomfort with using the Inuit as poster people for a ketogenic diet. That's fine. They may well have eaten what would be a ketogenic diet for many of us, but they certainly did not develop high levels of ketones when they carried the P479L gene.

However. Over the months Wooo and I seem to have come to some sort of conclusion that, while systemic ketones are a useful adjunct, a ketogenic diet is essentially a fatty acid based diet with minimal glucose excursions and maximal beta oxidation. Exactly how important the ketones themselves are is not quite so clear cut. From the Hyperlipid and Protons perspective I would be looking to maximise input to the electron transport chain as FADH2 at electron-transferring-flavoprotein dehydrogenase and minimise NADH input at complex I. Ketones do not do this. Ketones input at complex II, much as beta oxidation inputs at ETFdh, but ketones also generate large amounts of NADH in the process of turning the TCA from acetyl-CoA to get to complex II, which ETFdh does not. I'm not a great lover of increasing the ratio of NADH to NAD+. These are my biases.

Confirming that the Inuit are not poster boys for ketosis is a "so what?" moment for me. Using their P479L mutation to argue against ketogenic diets is more of a problem. It's a massive dis-service to any one of the many, many people out there who are eating their way in to metabolic syndrome to suggest that a ketogenic diet is a Bad Thing because no one has lived in ketosis before. Even the Inuit didn't! My own feeling is that everyone comes from stock who occasionally practiced and survived intermittent fasting so we are should be adapted to this. I'd guess that if you are of Siberian, Inuit or First Nations extraction you might benefit from Jay Wortman's oolichan oil as part of a ketogenic diet.

I'm always amazed by the concept that a ketogenic diet might be temporarily therapeutic but must be discontinued because it eventually becomes Bad For You. It reminds me so much of the converse concept that low fat diets, which might worsen every marker of health which people may care to look at, will deliver major benefits at some mythical future date.

Ultimately, point scoring on the internet about what the Inuit did or didn't eat shouldn't destroy people's chances of health. Destroying a circular argument about Inuit diets may may the destructor feel good. Destroying the feet, eyes and kidneys of a person with type 2 diabetes, who need a ketogenic diet, as a spin off from that victory must be difficult to live with. I don't know how anyone can do this.

I think that's probably all I have to say for now.

Peter

Protons: Physiological insulin resistance, addendum two

George put up the link to this paper, which allows you to tease information out about omega-3 PUFA as bulk calories vs lard as bulk calories. We are not talking about essential supplies of essential lipids here. We are looking at serious bulk calorie supply. This is quite, quite different.

Aside: The basic conclusion that FO is protective against endotoxin shock is fascinating but may be restricted to C57BL/6 mice. Pity everyone uses them. But it's interesting, and on file, never the less. End aside.

Here's the composition of the diets:



Fairly typical research diets, a little more sucrose than I would like but, well, everyone does it.

What about weight gains? Here they are:



Although body weights, at all time point, are not significantly different between groups this is just due to the initial group weights being different. If you look at weight gain rather than absolute weights, the lard group gains more weight than the fish oil group.

The insulin and glucose levels do support the idea that insulin sensitivity is controlled by the degree of unsaturation of the bulk lipid in the diet, ie PUFA diets increase insulin sensitivity. But there is no excess weight gain in the fish oil group. Why not?

C57BL/6 mice suffer an injury to their hypothalamus on exposure to a saturated fat based diet, especially if combined with sucrose. Omega-6 PUFA do not seem do this and I doubt omega-3 PUFA do either. I considered this back here.

So we are comparing obesity in an un-injured group carrying omega-3 enhanced insulin sensitive adipocytes versus a hypothalamic injured group carrying adipocytes which are partially resistant to insulin due to dietary palmitic acid and partially sensitive to insulin due to decreased sympathetic outflow from the hypothalamus to adipocytes. C57BL/6 mice are very special in their response to saturated fats.

This is a knotty problem to try and untangle. This paper helps a lot.

I just want to look at two of the control groups, both of which are C57BL/6 mice, both of which are exposed to a high palmtic acid diet and so both will have an hypothalamic injuy.

So we can have, among many, two groups of C57BL/6 mice, one fed a high fat diet to make it fat and the next fed a high fat diet to make it fat, but then add in a significant dose of omega-3 PUFA. Just to add some insult to injury. The first group gets a whammy. The second group gets a double whammy. Want to see the graph? Ok, ok, here it is:



First, strain your eyes to follow the open triangles. This is the high fat only control group. These are C57BL/6 freak mice with a brain injury triggered by palmitic acid. They have limited weight gain but, as they store palmitate without DNL, hence without desaturase activation, hence without palmitoleate generation, they develop metabolic syndrome. Visceral fat, fatty liver. Of course the group didn't measure either insulin or glucose (they are in obesity drug development), but these mice have metabolic syndrome and have lost the ability to get any fatter. They are in trouble. They don't actually weigh any more than un-injured C57BL/6 mice fed traditional crapinabag.

Now look at the open circles of ever increasing obesity. Fatties or fatties? This is what happens when you add fish oil to the diet of a palmitic acid injured C57BL/6 freak mouse. Impressive waistlines huh? Of course we don't get the insulin or glucose levels here either, but these mice do not have metabolic syndrome. They maintain the insulin sensitivity of their adipocytes, especially peripherally, and continue to become obese with sustained metabolic health. They will stay healthy until their adipocyte distension induced insulin resistance eventually over rides the insulin sensitising effects of the bulk fish oil.

We have a pair of models. Skinny-fat and obese-but-metabolically-slim. Both are explicable by looking at the basic effects of bulk lipid supply from the diet acting on the insulin signalling system within mitochondria.

Summary: These are palmitic acid injured C57BL/6 freak mice which have the added insult of having their adipocytes rendered extra insulin sensitive by the F:N ratio of a significant percentage of the fatty acids in the fish oil of their experimental diet. This postpones metabolic syndrome until they have become fat enough to develop it.

The F:N ratio concept delivers again.

Peter

BTW no one knows the omega-6 content of the fish oil is in this second study! The discussion mentions that there is zero omega-6 in the basic high fat diet, which has no added fish oil. Imagine running a Rimonabant study when you don't know the omega-6 content of the (high fat) diets. But this becomes irrelevant if you look at the basic metabolism at the molecular level. Either family of low F:N ratio PUFA will delay metabolic syndrome, while ever they assist weight gain. And you have to remember that C57BL/6 mice break by eating butter.

Mediterranean France

There is one notable diet intervention trial which succeeded in producing marked improvement in outcome, for both cardiac and cancer mortality. This is the Lyon Diet Heart Study. The final analysis is available in full text here. The sub analysis for cancer protection is available here.

What did the Lyon group do? Well this is a little bit difficult to find out as the early publications are in the Lancet and are not available on line. But, from the subsequent rhetoric, I think we can assume the usual things about increasing fruit and vegetables, skipping fat, especially the dreaded saturated fat, and putting the maximum amount of fiber down the loo were all applied. All well and good, except most of these were done far more effectively by the WHEL study, which failed miserably.

When you look at the macronutrient ratios given in table 3 it's clear that there was a 3% replacement of calories from fat with those from carbohydrate. Not a huge change, but on the basis of the Finland study it was probably significantly deleterious. Vitamin intake? In table 4 of the cancer sub analysis paper you can see that there was minimal difference in daily intake of vitamins E and C between groups.

On the face of it there are remarkably few differences between the two groups, especially when you look at the much larger and completely ineffective changes produced by the WHEL study in nutrient intakes.

So what is so special about the Mediterranean diet in Lyon that is not present in the Mediterranean diet in San Diego?

Surely everyone (in France anyway) knows that there is a centuries long tradition of avoiding all butter and cream in an arc between Perpignan and Nice, and for 50 miles inland. Absolutement mon amis, people there have always, at least since Roman times, eaten an experimental gloop produced by Astra-Calve, a subsidiary of Unilever. Made of partially hydrogenated canola oil. Hopefully that's the low erucic acid version of rapeseed oil. The trans fats listed in table here don't look too traditional but, what the heck, it was free and people ate it, in the study anyway.

As it says in the trial design their Mediterranean/intervention diet included:

"no butter and cream, which were to be replaced with an experimental canola oil–based margarine (Astra-Calve, Paris, France) rich in oleic and alpha-linolenic acids. The oils recommended for salad and food preparation were canola and olive oils exclusively."

The exact composition of this gloop is unclear, except that it had a rather high omega 3 to omega 6 ratio. Skipping back to table 3 from the final analysis paper we can also see that dumping all corn oil and sunflower oil (not allowed for cooking or salad dressing) reduces your omega six intake, while the gloop increases your omega three intake, giving a ratio of 1:4. That's a pretty good ratio. The "prudent diet" diet group trundled along with a ratio, pleasing to any poverty stricken cardiologist who needs more business, of 1:16. Awful.

The message I get from the Lyon study is that an absolute omega three fatty acid deficiency is probably rather bad for you and that correcting the ratio of omega three to omega 6 is probably very good for you.

Want to get hold of Astra-Calve's gloop to correct your fatty acid balance?

Forget it. There are better ways.

Peter

PS The Lyon study final report begins with this sentence:

"Recent dietary trials in secondary prevention of coronary heart disease (CHD) reported impressive reduction of the recurrence rate by a range of 30% to 70%."

It cites three references for this statement.

First is the DART study. This found that reducing total fat while increasing PUFA (probably omega 6 back in the 1980s) was useless. No surprise there. Increased cereal fiber was slightly worse, this produced a small but non significant increase in the risk of being dead at the two year mark. But two or three fish meals a week, without all that fruit 'n' fiber rigmarole, was very useful. A drop of 29% in two year total mortality. Good, though hardly world shattering

Second reference is to Singh. Read more about this particular paper from Singh here in the BMJ and you will see why the WHEL trial did so badly!

Third reference is self citation.

I don't see a huge amount of support for that first statement.

AGE RAGE and ALE: linoleic acid

I'm really sorry about this but I haven't quite finished with figure 1 of the engrossing commentary by Krauss. Let's open it up again here.

Now the first question we have to ask is "What is the most abundant polyunsaturated fatty acid in human LDL particles?"

OK, that's a give away at linoleic acid, our least favourite omega 6 fatty acid. This is pretty obviously the case as we've just discussed how, if you get enough omega 3 fatty acid in to a nascent LDL particle, it becomes a stillborn VLDL particle and leaves it's lipid, along with its apoB protein, in the liver. Linoleic acid based VLDLs get secreted.

So here's the tricky question. Where, in Krauss' diagram, is the linoleic acid? Well it has to be in the liver cell somewhere to get put in to the LDL particles. Clearly some lipid is added to the initial assembly of the nascent LDL, over on the left hand side of the diagram. The rest comes from that lipid droplet in the middle. You would expect that lipid droplet to be mostly saturated fat if it was fructose or alcohol derived, but with the amount of linoleic acid in the modern diet there could easily be plenty of this throughout the liver cell lipid stores.

Why is there linoleic acid throughout the liver? The liver likes linoleic acid! In an utterly artificial model, the cholesterol fed hamster (you'd better believe it!) on moderate fat diets (45% of calories from fat) show an upregulation of the LDL receptor as the proportion of fat from linoleic acid rises. Dietary saturated fat down regulates the receptor. It seems that this holds true across species and it certainly seems to work in humans, diets high in omega 6 PUFA were the classical cholesterol lowering approach pre statins. You can see why the liver should ignore an LDL particle full of saturated fat. This is Krauss' large fluffy non atherogenic lipid, used for delivering calories and cholesterol to wherever they are needed. It came from the liver, why should it go back? But why is the liver so keen to uptake LDL particles when the diet is high in linoleic acid? My guess is that linoleic acid loaded anything is a novel phenomenon and in pre agriculture times linoleic acid was probably very useful and in very short supply, so it got recycled. It is the preferred fatty acid for LDL cholesterol because, in small amounts, it has significant uses and benefits which are not provided by the omega 3 fats. So there is some logic to aborting an LDL particle over-endowed with omega 3 fats. But what were positive benefits when linoleic acid was in short supply have gone awry as the amount in the diet has skyrocketed over the last 10,000 years, especially the last hundred years or so. The knock on effects of a high linoleic acid diet are interesting for atheroma formation.

Stephan also has some interesting thoughts on linoleic acid and violence up on his blog at the moment. The two problems are interesting as while CVD mortality is currently dropping in the USA and UK, the incidence of CV disease is probably static, and might be increasing if it weren't for the decline in smoking. The fact that mortality from gunshots is rising while mortality from heart disease is falling, despite the rise in incidence for both, is a plus mark for cardiologists managing established heart problems. Trauma management has some catching up to do. Or maybe we could just give up eating 10% or so of our calories from those omega 6 fats!

Peter

Prostate cancer and citrate and maybe omega 3s

A while ago, when I was looking through various publications from Chowdhury, I found this one: Prostate cancer cells over express mtG3P dehydrogenase. That's interesting. Why?

Normal prostate cells are special. They don't do the TCA. Glycolysis is fine. Pyruvate conversion to citric acid is also fine. Aconitase is not. Aconitase is deliberately inhibited by Zn retention and the citric acid of the citric acid cycle, which cannot be further metabolised in the said cycle, is then exported in to the prostatic fluid. In large amounts. Mitochondria are not used (much). This is hardly a recipe for over expression of mtG3P dehydrogenase.

Aside: I'm assuming the citrate is used to fuel the mitochondria of sperm. Simply dropping citrate on to the TCA of sperm looks like adding N2O/petrol injection to a standard saloon car engine. Maximum power output at the cost of maximum stress. Only the fastest get to the egg and only best survive the journey, which seems like a good idea when looking for the sperm with the best nuclear-mitochondrial match for fertilisation... End aside.

If we look at the paper on Zn, the TCA and mitochondria in prostate cancer (PCa) we can see that PCa cells lose Zn induced inhibition of aconitase and take off with a large supply of NADH from the TCA, a smidge of FADH2 through complex II and go towards that metastatic ratio of NAD+/NADH. Of course citrate concentration in semen plummets.

So PCa cells use the TCA and oxidative phosphorylation, ie they use mitochondria, to burn citrate derivatives. Normal prostate cells don't. Prostate cancer cells routinely perform beta oxidation. Not so normal prostate cells.

Equally interesting, as Loda's group point out, Fatty Acid Synthase (FAS) appears to be an oncogene in PCa cells. That, to me, suggests that while some of the citrate may well enter the TCA there is also a net synthesis of fatty acids outside the mitochondria. Fatty acid synthesis is a cytoplasmic process. Exported citrate provides acetyl CoA as the raw material for fatty acid synthesis.

BTW I don't doubt that prostate cells do use fatty acids in combination with "normal" levels of glycolysis, but Liu's fascinating paper here, supporting near exclusive fatty acid oxidation in PCa cells, is a classic example of stacking the deck to prove a point, with subtle transitions in graph labelling between tritiated 2-deoxy-glucose (an inhibitor of glycolysis!) and "glucose". There was no glucose, except the deoxy molecule. Oddly enough, glucose and 2-deoxy-glucose are not the same! While I'm completely accepting of the up-regulation of beta oxidation in this cancer, the near complete shutting down of glycolysis looks like pure artefact. They compare metabolic preference by looking at palmitate depletion from the palmitate-only culture medium, which is normal. Then they looked at 2-deoxy-glucose depletion from the 2-deoxy-glucose medium. The whole point of 2-deoxy-glucose is that, while it can be phosphorylated by hexokinase, further metabolism is completely blocked by the lack of hydroxyl group on the second carbon of the molecule. It may get taken up by cells, but it is never bulk metabolised. So it never gets depleted from the growth medium. Duh. I wonder if they expected this result...



I've also looked at Load's ideas about "futile cycling". This is the concept that acetyl CoA, from beta oxidation of fatty acids within the mitochondria, is exported as citrate to form cytosolic acetyl CoA to be converted to palmitate, which is re-imported in to the mitochondria to provide acetyl CoA to re-export as citrate.... Doesn't make sense to me. If you have functional mitochondria and a functional ETC, why bother if it's futile?

But we have seen something very similar in the past. FAS activation seems to be an important feature of TFAM knock out adipocytes. There is no functional complex I in TFAM knockout cell mitochondria and acetyl CoA provides limited FADH2. Without complex I you need FADH2 to drive the ETC, NADH won't hack it. Converting acetyl CoA from any source repeatedly to palmitate generates significant FADH2 during its re-oxidation. It's cycling, but it's not futile. You get something from it which you cannot normally get from pure acetyl CoA, so long as complex I is dysfunctional. Of course you get horrible levels of NADH too, but...


So you have to ask yourself: Do prostate cancer cells lack complex I? Logic says they must do.

Well, what do you know, Parr et al point out:

"For example, a 3.4∆ associated with PCa, removes the terminal region of ND4L, all of ND4, and nearly all of ND5 (Maki et al., 2008; Robinson et al., 2010)"

ie there is commonly a 3.4kb deletion of mtDNA which codes for a very large chunk of complex I in prostate cancer cells. This deletion, the paper suggests, appears to occur BEFORE the cells convert to aggressively cancerous forms.

So what cripples complex I? Well you could make all sorts of guesses about this, especially if you are a lipophobe. There is no doubt elevated free saturated fatty acids, in the presence of hyperglycaemia, will drive completely unreasonable numbers of electrons the wrong way through complex I and a great deal of collateral damage might well result from this process. If you have elevated FFAs you would be insane to raise your blood glucose level. "That's Mr Potato Head to you" (Toy Story 1).

How about simple hyperglycaemia? If you can generate enough free radicals from hyperglycaemia to induce some mitochondria functional you are then in a position to start using those mitochondria. Feeding through mtG3P dehydrogenase's FADH2 to the CoQ couple, while the NAD+/NADH ratio is horribly low from glycolysis, allows plenty of reverse electron flow when you really don't want it. For neurons, which don't do a great deal of beta oxidation, this is my guess for the extensive oxidative damage to complex I seen in PD and AD. Loss of complex I in a neuron, which doesn't do beta oxidation, is going to be disatrous. But in prostate cancer cells? Completely unreasonable superoxide generation appears to trash the mtDNA, as Parr pointed out. Conversion of citrate to fats allows survival under these conditions.

Now let me see, what did Chowdhury say about PCa cells and mtG3P dehydrogenase?????????? Up-regulated is the word. No cell is going to produce mtG3P dehydrogenase without functional mitochondria (and glycolysis) and mtG3P dehydrogenase bypasses a broken complex I, in a similar manner to electron transferring flavoprotein dehydrogenase does. Hyperglycaemia is an interesting concept for generating this cancer.






So....... Do PUFA, particularly omega 3 PUFA, give you prostate cancer? As per the suggestion from the observational association here. Probably not. No more than butter or FAS-produced palmitate give you prostate cancer. But PUFA are really quite special, certainly once the damage is done. They supply significantly less FADH2 input to the electron transport chain per molecule than saturated fats do under beta oxidation conditions, omega 3 PUFA being significantly worst than omega 6 PUFA. So here we have specific fats behaving as suppliers of NADH in rather higher amounts than saturated fats do and FADH2 in rather lower amounts. We have a lack of complex I in PCa cells, so supplying NADH is a recipe for metastasis and a poor fuel for the electron transport chain... In PCa cells acetyl CoA from PUFA is a sitting duck for export as citrate with conversion to palmitate and re-beta oxidation, to maximise FADH2 production. Oxidation of omega 3s via acetyl CoA and its subsequent synthesis and re oxidation as palmitate is not futile.

I have no issue with omega 3 fatty acids as signalling molecules, we clearly need some. I would be very cautious about bulk omega 3s, as I would about bulk omega 6s, as a source of calories.

We are looking here at a potential survival/growth mechanism in the behaviour of cells with severely damaged mitochondria, using any pathway they can to generate ATP. But thinking that it was the the omega 3 PUFA which broke the mtDNA in the first place might be a big mistake. Hyperglycaemia appears to be a far better recipe for mtDNA damage through hypercaloric insulin resistance, N-1a, reverse electron flow, etc gone to excess. PUFA are poor generators of FADH2 during beta oxidation so probably don't drive a lot of reverse electron transport through complex I. And never forget that even the bête noire of fatty acids, palmitate, is harmless in the face of normoglycaemia despite being an excellent generator of FADH2 and reverse flow.



Finally, Parr's group consider the damaged mitochondrial genome to be en-route to a situation where apoptosis becomes very difficult:

"As deletion-driven mtgenome depletion advances, cells become more resistant to cell death stimuli, in comparison to their parental cell lines (Cook and Higuchi, 2012), allowing proliferating cells to escape apoptotic control."

One step towards immortality for PCa cells, excepting the unfortunate destruction of their host organism.

Peter

AGE RAGE and ALE: VLDL degradation and Fish Oil

OK, this is the direct link to figure 1 of Krauss' commentary paper. I just want to run through what seems to be happening and some of the consequences. Best to have the picture open alongside Hyperlipid.

Top left is the nucleus, then there's the stack of endoplasmic reticulum, then a newly synthesised lipid particle stuck on the outer surface of the ER. There is an interesting initial particle labelled I receiving "lipids" (another post there) which goes on to become that unlabelled particle on the outer surface of the ER (call it a nascent lipoprotein). The nascent lipoprotein has two arrows leading away from it representing two metabolic options. The upper arrow goes to a particle labeled III. On this pathway the particle receives saturated fatty acids which, at step V, stop it being destroyed. Destruction is termed PERPP, and the line from PERPP has a flat end meaning its blocked, and the legend "+SFAs" is shown as doing the blocking (by the little curved arrow). Got it? This SFA loaded particle is exported as an IDL (bad) or a large LDL (good). Luckily the IDL appears, on this diagram, to convert to the large form of LDL. But IDL may only be bad if secreted post prandially, I don't know, life is so complex in the ad hoc world of the lipid hypothesis!


Summary so far: Basic particle plus SFA gives the "good" version of "bad" cholesterol. Don't you just love these terms.


Now go back to that nascent lipoprotein and follow the alternative pathway shown by the downwards arrow. This leads to particle labeled IV, having accepted a lipid droplet via the curved arrow. Remember the lipid droplet. It might matter later. Anyway, there is then a dashed arrow showing possible secretion of this particle. If secreted it becomes a large VLDL particle.

While large LDL particles are good guys, large VLDLs are not. In fact they are the precursor to the evil incarnate particle, the small dense LDL. Like Darth Vader, only without the pre death conversion to the good side. Secrete and die.

Fish oils to the rescue! There is an arrow from the PERPP "destructaparticle" system pointing straight to this evil particle, joined by a little "n-3 PUFA-ox" which destroys the evil large VLDL before it ever gets secreted. Less of the large VLDL means less of the small dense LDL. More joy on blood results. Not only do triglycerides drop, it's the evil fraction of triglycerides which drop.


To summarise this pathway: Basic particle plus lipid droplet gives you bad VLDLs and small dense LDLs, unless aborted by omega 3 lipoxidation product (malondialdehyde is claimed but I'll get on to signaling molecules eventually).


All of that is pretty straight forward but it doesn't give us any insight in to anything except how to improve lab numbers.

Here are the nitty gritty questions.

1. Where did the SFAs come from? This is easy. Just follow Krauss' refs and you will find it's diet. Another post there needs writing.

2. Where did the lipid droplet come from? Well, lipid droplets in the liver vary from normal physiological amounts through to hepatic lipidosis. Hepatic lipidosis is a routine feature of the metabolic syndrome. We know that fructose is converted to lipid as rapidly as possible in the liver. We know that insulin inhibits the release of all VLDLs from the liver. Combining fructose with a glucose source (to raise insulin) seems a good way to generate hepatic lipid and block it's release. The bigger the dose and particularly the more continuous the ingestion, the more lipid droplets are likely to form. Sucrose or HFCS would do the job nicely. So might alcohol. Alcohol is interesting. Low doses improve insulin sensitivity and high doses do the opposite. The histpathology of non alcoholic hepatic steatosis is indistinguishable from alcoholic steatosis. They are the same condition. Keeping those lipid droplets in your liver seems a good way to get hepatic lipidosis and subsequent cirrhosis.

3. Where are the lipid droplets going? Well, if you dose up on fish oils the answer is nowhere! Lipid droplets should be off-loaded as small dense LDL precursor particles, the large VLDLs. You're not going to release them if you're on high dose fish oils! So you are trading the drop in "bad" LDLs for a rise in hepatic lipidosis. Are you going to mangle your liver to make Krauss happy? Yes?

Now let's go back and look at the high dose fish oil study from back in 1991.



Here are the triglyceride effects of 30ml fish oil a day for three weeks. Without vitamin E some omega 3s still get to the liver and trigs (probably large VLDLs in this case) drop from 2.6 to 2.0mg/dl. Modest and not statistically significant. Have a wash out period and do it again, but this time preserve the omega 3s with vitamin E. The drop is 48% this time, p<0.01, enough to warm the cockles of a cardiologist's heart.

Remember, these absent triglycerides should have contained lipid droplets which the liver wants rid of. I find it fascinating to see that, in the post washout/pre omega 3+vitamin E session, that the trigs were up at 3.4mg/dl, quite a bit higher than the 2.6 at the start of the study. Is this the liver off loading lipid droplets retained during the first section of the study? With trigs down at 1.8mg/dl by the end of the high dose vitamin E section, how much hepatic steatosis is going on?

Now look at this table, especially insulin and glucose. You'll have to click to enlarge.



All of these FBG values are scarily high, so these volunteers are on the edge of diabetes. What happens when you load up on fish oils? Insulin: No significant change at any time. FBG; both fish oil sessions show increased FBG! For low dose vitamin E section the change had p<0.05, for the vitamin E protected phase it had p<0.001.

That smells of insulin resistance to me and hepatic lipid overload is the easiest explanation.

Aha! So the Greenland eskimo, who refuse to die of cancer on 15g/d of EPA+DHA, must all have been dropping like flies from hepatic cirrhosis. Or type 2 diabetes. Apparently not. This dose of fish oil appears to be fine, just so long as you are not making lipid droplets in the first place. That means no sugar and no excessive alcohol. Remember modest dose alcohol improves insulin sensitivity, so zero alcohol is not needed. The Greenland eskimo were VERY low carb.

So where does that leave fish oil supplements? I think if you have a problem with alcohol they are very bad news and you should be absolutely minimising all forms of PUFA, unless you really want cirrhosis. If you are eating to the ADA or AHA sucrose ladened guidelines and already have "mysterious" raised liver enzymes, you will make an already appalling job worse.

If you are LC and low PUFA in the first place, or even just eating a diet which doesn't generate hepatic lipidosis (minimal sucrose), I think there are advantages to modest dose fish oils for long term changes in insulin sensitivity (another post needed). Dropping your triglycerides in the short term is not one of them, unless you are in to treating numbers, in which case; come back torcetrapib, all is forgiven!

Peter

PS Let's just clarify: There is no "good" or "bad" bad cholesterol. You can fuel your metabolism with saturated fat, and the "good" bad cholesterol goes up as a marker. It's the saturated fat which is really good. Or you fuel your metabolism on sucrose, which raises "bad" bad cholesterol. Stuff the cholesterol. It's a marker you are being evil to your metabolism by eating sucrose, which is what does the damage.

Protons: The linoleic acid fed mice

I've sent a great deal of time trawling through Masseria et al's paper on linoleic acid fed mice. I'm going to try to generate some data which aren't reported and see how much of a transgenerational effect really occurs.

This paper is the one where a group of mice were weaned from chow fed parents on to a moderate fat diet mostly based around linoleic acid. They then were bred for four more generations on this linoleic acid based diet. Replacing starch in your diet with linoleic acid makes you fat. I'm assuming no sucrose was added at the diet switch. Really, they wouldn't have added sucrose without saying? No, no...

The most important variable I'd like to see, which was measured once a week throughout the study, was total body weight. I cannot find ANY body weights for 22 week old mice. All mice were weighed weekly, according to the methods.



We can tell from the photograph of a chow fed mouse along side an HF4 mouse that the HF4 mice weigh more than chow fed mice. Fascinating. This image of these two mice is the total information on weights at 22 weeks of age. Why are no weights reported?

Now, if you had a linear increase in body weights from generations HF1 through HF4, would you report it? Draw a graph perhaps? Bear in mind that you can generate this data with a set of electronic kitchen scales from Argos, no PCR or Western Blot required.

Here are my fabricated data: At 22 weeks of age mice of generations HF1 through to HF4 weighed the same. There is not even an non significant trend to increase. Otherwise it would have beeen reported.

Next, fat pad weights: All mice which made it to 22 weeks of age were euthanased. Now, to weigh their fat pads would need a visit to the local head shop for some slightly dodgy digital scales, but it's still not exactly rocket science. Masseria et al appear to have the scales anyway, because they weighed and reported the weight of plenty of fat pads in selected mice. But, in a research project on adiposity, they appear to have thrown almost all of the fat pads from 22 weeks of age mice in to the clinical waste bin. Of course, as per the body weights, they might have the data. If they do they are not saying. Is there a linear increase in fat mass from HF1 to HF4? I think it is reasonable to assume not.

So this is what is happening:

The "STD" chow fed mice are exposed to a "normal" mouse diet throughout. They stay insulin sensitive and have normal weight.

HF0 mice are unique in the HF generations. In this first omega 6 fed generation there is a total of 19 weeks of exposure to the omega 6 diet, starting from weaning at 3 weeks of age and going through to 22 weeks of age. These mice do become over weight at 22 weeks, as judged by their fat pad weights as given in Fig 1, but not by as much as do the HF2 mice in the same Fig 1. They do not develop insulin resistance.

Then there is a third set of mice, the HF1-HF4 mice, which are all exposed to the same diet for the same duration, ie throughout their gestation (assume 3 weeks for this), breast feeding (assume another three weeks) and weaning until 22 weeks of age, ie for 25 weeks in total. They develop an interesting pattern of insulin resistance.

So to clarify: It looks very much as if we have three groups of mice. Chow mice never get exposed to high omega 6 intake, HF0 mice are exposed for 19 weeks and HF1-4 mice are exposed for 25 weeks. The number of weeks of exposure is what increases the bodyweight and this (exposure time) is what increases from generations Chow through HF0 to HF1-4.

Within generations HF1-4* neither weights nor fat pad weights are reported.

*Except of course the isolated value from Fig 1 part C where we just get HF2 pad weight, but we still can't see the "lack of trend" within these generations.


I might have been tempted to just leave it at that and suggest that eating a high omega 6 fat diet increases you weight in proportion to the percentage of your life you were exposed to it, if it wasn't for Fig 4.

If we pull out Figure 4 (for the key) we see this:



and especially the insulin section:



Which makes it clear that the hormonal milieux is completely different between generations HF1, HF3 and HF4, the right hand three columns. Insulin is up at 4.5 times control in HF1,  drops to 3 times control in HF3 and is down at 2.5 times control in generation HF4. HF2 can be interpolated between HF1 and HF3, I'll fabricate (fabrication warning!) a value of around 3.75 times control.

So the very simple concept that a high PUFA diet, due to a failure to generate superoxide in mitochondria, increases adipocyte insulin senitivity, so facilitates weight gain at a given level of insulin, is not as clear cut as I would like. Damn.


The main hint about what is happening comes from Fig 5. Because of the small group sizes I find it very difficult to accept that these lines mean very much in absolute terms. But there is, certainly, the impression that adipocyte numbers, especially numbers of small adipocytes, really does go up transgenerationally through generations HF1-4. Probably.

So HF0 mice hit weaning with a "chow fed mouse adipocyte count" and get the next 19 weeks on a diet which maintains insulin sensitivity due to the limited production of FADH2 per unit NADH by its dietary fat. These mice get fat but stay insulin sensitive because 19 weeks on an omega 6 diet does not have time to produce enough adipocyte distension to generate enough insulin resistance to over ride the effects of the PUFA diet. Quite fat, but insulin sensitive. Utterly straight forward.

Generations HF1-4 get 25 weeks of omega 6 exposure and all become more obese than HF0. But through the same set of generations they also develop progressively more adipose tissue hyperplasia. ie they have more and more fat cells.

As the total number of fat cells goes up, so does the ease at which they accept fat without becoming so distended as to become insulin resistant. So HF1 have just a little adipose hyperpalsia and each adipocyte manages to hit distension induced insulin resistance by 22 weeks of age. By the time we get to HF4 there are far more small adipocytes available to accept fat, they do so easily and we end up, after 4 generations, with obese but far less insulin resistant mice.

It looks like a simple trade off between the obesogenic effect of omega 6 fats vs the distension induced insulin resistance of the whole population of adipocytes.

Omega 6 fats appear to be great for putting fat in to adipocytes. Adipose hyperplasia appears to be the key to maintaining insulin sensitivity during weight gain. Omega 6 fats are great for generating adipose hyperplasia.

Having adipose hyperplasia means lots of half empty adipocytes are willing to signal that they are not full. Did you notice the leptin levels in Fig 4? Very interesting. Losing weight is not going to be easy if you are a human in this situation.

There are hints from birth weights and pre weaning growth rates that this may be a process which starts in utero. There are also hints from previous publications by this group that the obesogenic effect of omega 6s can be blocked by indomethacin, a cyclo oxygenase inhibitor. That is; there is at least one, possibly several, layers of control in place over the F:N driven process.

At a basic level the F:N ratio concept it is quite clear where the obesogenic effect might come from. That there are additional refinements to this concept is not surprising but, to me, tinkering with the control system when the basic engine of metabolism is broken, is worse than pointless.

Try not to base your diet on corn oil.

Peter

BTW: Most annoying quote from the paper:

"These observations indicate that, despite the fact that glycemia in HF4 mice appeared normal at 22 weeks old (151 ± 30 mg/dl for HF4 versus 170 ± 30 mg/dl for STD mice), continuous exposure to the omega 6HFD led to a sustained increase in plasma insulin levels, which strongly suggests the emergence of insulin resistance of adult animals at later generations."

Technically correct, but here is a repeat of the plot they are describing from Fig5:



Hmmmmmmmmm. Do we heed the voice of Author-ity or read the graph?

Metabolism nuts and bolts PUFA

This post is basic biochemistry that we probably all know. I've just stuck it down as I've slogged through it while I've been working through Chris Masterjohn's treatise on PUFA, it seems a waste not to use it. Ignore if you're happy with elongase and desaturase enzymes.

Omega counted double bonds are very straight forward, they are counted from the methyl end of a long chain fatty acid. Because mammals can't add extra chain length or desaturate at the methyl end, these bonds are "fixed" in their identity. So mammals can pop a double bond in to stearic acid in the omega 9 position to give oleic acid and that's it as far as the methyl end is concerned. Double bonds at the omega 3 and 6 positions are also fixed and come from the diet (mostly, there may be an exception). No chance of elongating at the methyl end, so the 9th/6th/3rd will always be the ninth (or 6th or 3rd) bond down from the omega end of the chain. So oleic acid is an omega 9 fat and all of its derivatives are too. Whatever elongation/desaturation happens, it happens at the carboxyl end. Ditto omega 3s and 6s.

Delta refers to desaturase enzyme's ability to change a single bond to a double bond, extracting two hydrogen atoms in the process. So delta 6 desaturase pops a double bond in to the place of the 6th carbon-carbon bond, counting from the carboxyl group end of a fatty acid. This number is the alpha number, as it's counted from the opposite end to omega number. The desaturases mostly don't care how long the fatty acid is, they just grab the acid end, count six (or seven or nine or five etc) and stick in a double bond.

We definitely have a delta 5 and a delta 6 desaturase. Oddly enough we never put double bonds in to adjacent locations in our fatty acid carbon chains, there is always a gap. The pattern goes double bond, two singles, double, two singles etc, as far as I can see. That is if you want extra double bonds at all.

Fatty acids get elongated. This always happens from the carboxyl end, and always involves adding two extra carbon atoms, using single bonds only. In mammals anyway.

So when the fatty acid with a recently added double bond placed in the alpha 6 position by our delta 6 desaturase gets elongated, that new double bond gets promoted from the alpha 6 position to the alpha 8 position.

To keep the pattern we want two single C-C bonds then the new double. That mean going for the 5th C-C bond using delta 5 desaturase.

This neatly gives us the end product of arachidonic acid from linoleic acid.

We also get to eicosapentaenoic acid (EPA) by the same pathway if we start from the alpha linolenic acid parent. Then a simple elongation and a delta 4 desaturation gives docosahexanoic acid (DHA). If your delta 4 desaturase doesn't work you can try this:

Double elongation w/o desaturation to 24 C chain with first double bond now pushed to the 9 position from the carboxyl end. Delta 6 desaturase places an appropriate double bond at the 6 position, as it always does. This weird fatty acid is then shortened by two c atoms (off of the COOH end of course) to give DHA, the new double bond thus ending up, as it should for DHA, at the 4 position (from the 6 position where it was placed by delta six desaturase).

That last paragraph is from Mary Enig's book "Know Your Fats". The rest is general biochemistry.

There is no arguing with the essentiality of arachidonic acid and probably the same goes for DHA. If we don't get them pre formed in our diet, this is how we make them.

Peter

Clofibrate and PUFA

It's a bit difficult not to post about the cholesterol hypothesis. I'm sheepish to admit that it is in a large part because it is such an easy target. But mostly because it also encapsulates herd stupidity beautifully. And it's wrong, yet keeps giving tricky suggestion that it might be right.

So today I want to look at clofibrate. This drug is a killer on a par with torcetrapib but does, like the statins, reduce cardiac "incidents". So the obvious conclusion is that, deep down, somewhere, anywhere, LDL cholesterol is the cause for heart disease. That's wrong.

Clofibrate is a stimulator of PPAR alpha receptors. It increases the production and activity of peroxisomes, which are cell organelles with many functions, one of which is the burning of lipids. Lowering intracellular lipids lowers insulin resistance. This is generally considered to be a Good Thing. I doubt this, if it is drug induced. Forcing your body to burn fatty acids while overloading it with glucose from your diet seems a bit odd to me. And very much in to the territory of the Law of Unintended Consequences.

Clofibrate also lowers cholesterol. So the cardiologists used to love it (until the body count got too high). But is there causality between the reduced cholesterol and reduced cardiac episodes?

Look what clofibrate does to the PUFA in the myocardium of rats.

It goes some way to correcting the appalling omega 3 to omega 6 ratio produced by the junk described as laboratory rodent "chow". I don't think anyone would object to correcting the omega 3 deficiency which is so ubiquitous in bodies of both lab rats or Food Pyramid munchers. Personally, doing this by eating 5 pence worth of fish oil capsules a day allows me to eat cheap UK beef rather than the seriously nice but expensive grass fed stuff. The latter is a weekend treat only.

No, there is a world of difference between taking a few grams of fish oil per day and putting a large spanner in your metabolic works called clofibrate. Correcting PUFA ratios would be expected to reduce cardiac incidents. Dropping your cholesterol level is probably the cause of the increased all cause mortality.

For statins the effects are about even, for clofibrate the increased mortality effect predominates.

But the message I get from both of these drug classes is that, while they were developed to reduce chloesterol, they do other things. A cholesterol lowering drug only gets out of the lab and in to clinical practice if its unknown, unsought and accidental benefits outweigh the cholesterol lowering problem it causes. It is these accidental benefits which determine whether any cholesterol lowering drug stands the test of time.

Who would have imagined clofibrate had its cardiovascular effects through going some way to correcting the fatty acid balance of the myocardium? That's not why it was developed.

BTW both Actos (pioglitazone) and Avandia (rosiglitazone) also work on PPAR receptors (PPAR gamma this time, rather than alpha) but they do similar things. They are used for their insulin resistance lowering effect. They are much less useful that putting your bagel in the bin and represent a VERY big spanner in your metabolic works. I notice that they don't seem to be too good for you, now they are in general use. Any more than clofibrate is.

Peter