Friday, September 06, 2013

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.


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.


SS Biker said...

Fascinating post Peter.

I have been investigating the role of Intra-muscular TG in endurance exercise. In particular I'm trying to find out if in the absence of dietary fat whether IMTG is replenished by adipose FA to any degree (as a fat loss mechanism). Most studies I see suggest that dietary fat is required however the studies only looked at HF and LF (ie HC) diets in the "athletic" context and these showed dietary fat is essential to replenish IMTG, of course they didn't look at a LCLF diet or LC Athletes and the interventions are necessarily short as usual.

In the course of my reading it seems that Hormone sensitive lipase activity is reduced in the case of increasing unsaturation of adipocyte FA composition. I assume your comment re: Hypoglycaemia refers to "inappropriate insulin sensitivity" due unsaturated fat? looks like a double whammy?

Presumably for someone on a long term high sat fat/low carb diet w3 supplementation isn't going to enhance FA availability to muscle and thus w3 supplementation might not be a useful strategy for replenishment of IMTG - (at a stretch I appreciate you weren't talking about this).

So long term, if a former fatty (like me) can drop serious fat weight and replace adipose unsat fats with saturated fats does maintenance of adiposity with diet/exercise ultimately become much easier?

Adam Kosloff said...

Hi Peter,

a) not to butter your non-bread, but you're insanely brilliant. I wish there were a Hyperlipid 101, so that some of your theories and insights could be translated for the lay folks.

b) on a totally unrelated note, many moons ago, you posted about how you used a ketogenic diet to treat AS. I have a friend with the same condition, who's doing a low carb/starch diet (a la Life without Bread) to try to conquer AS. Other than your own posts, are there materials you might recommend that he check out? Ideally, he'd like to find a dietician here in L.A. to help him.

Would that we could ALL have our own personal dieticians (trained correctly!).

thanks and cheers :)

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Sabine said...

In cold climates, this omega 3 effect could also allow for burning of fat in brown adipose tissue, in addition to burning fat in muscles?

Rob Cosimo said...

To omega 3 or not to omega 3; that is the question

marie said...

Peter, you gave me a laugh and a trip down memory lane with "13-hydroxyoctadecadienoic acid". In college we used that one instead of "supercalafragalistic expialidocious" to test our sobriety ;)

Your ideas in this post are fascinating, I just wish you could arrange to do the experiments to test your suspicion about the temporal pattern of FFA release in starvation! Are you ever tempted to expand your earlier research experience into the metabolic field? You spend a lot of thought on it already.

Purposelessness said...

Here is some more pesky higher level signalling to think about, the obesogenic effects of O6 PUFAs seem to depend on the protein content of the diet, or better, on the glucagon/insulin ratio (a cAMP-PKA-COX-prostaglandin axis) (in vitro and mouse study) (open access) (I enjoyed it):

Unknown said...

Great study, thanks for sharing.

Puddleg said...

This is wonderful stuff.
A girl I knew went to Sakhalin (Eastern Russian island off Siberia) in the dying days of the Soviet empire. She said that a popular snack there was fish preserved in a jar of hard fat. At the time it didn't sound appetising but, these days, I'd queue to buy some.

Puddleg said...

@ Purposelessness,

LOVE your reference. "We show that n-6 PUFAs were pro-adipogenic when combined with a high carbohydrate diet, but non-adipogenic when combined with a high protein diet in mice."

js290 said...

Anything (glucose, ethanol, PUFAs, etc) that stops the adipocytes from releasing FFA could be interpreted to mean the body is trying to oxidize it as energy ASAP. "Preferentially" may mean the body prefers not to have it in the system.

Unknown said...

Peter PUFA omega 3's are difficult to understand from PUFA 6 chemistry because how they work is based upon quantum principles in the CNS using their pi electron clouds with iodine and the water in CSF. In the human neuro-immune system omega 3 PUFA's are converted by macrophage's to an intermediate compound called 13S and 14 S epoxy-maresin. Maresins are produced by macrophages from DHA and they exert potent pro-resolvin and tissue homeostatic actions on the MHC1 proteins. These alter their action. Resolvins are compounds that are made by the human body from the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). They are produced by the COX-2 pathway especially in the presence of aspirin. Experimental evidence indicates that resolvins reduce cellular inflammation by inhibiting the production and transportation of inflammatory cells and chemicals to the sites of inflammation. Maresin's promote the conversion of macrophages from M1 phenotype to M2 phenotype that specifically does not stimulate inflammation.

karl said...

There is something to Dr. Davis's high dose fish oil - it really does appear to reduce Lp(a) which if elevated and some bad genes appears to cause CAD ( to be precises oxLP(a)).

The problem is we don't know if reducing Lp(a) via high dose fish oil improves long term outcomes or just treats a lab number. (Way to much of medical practice consists of treating lab numbers instead of the disease - the medical community seems to be real suckers for erroneously thinking correlation=causation (You would think the MDs would need to take some basic science/statistics etc along the way?))

I'm looking for a study where they fed mice equal calorie diets - one with high PUFA fats - the other with low PUFA and looking at the effect on weight - any one have the reference? (email )

Puddleg said...

Resolvins are formed by addition of acetyl group from aspirin, the one which inhibits COX2, to DHA.
Which makes the old folk remedy of cod liver oil plus cider vinegar for arthritis look pretty reasonable.

Karl, I don't see weights in the abstract for this, but surely they will be in the full-text

Puddleg said...

This looks like the book we need

Metabolic Syndrome Pathophysiology: The Role of Essential Fatty Acids
By Undurti N. Das

Anonymous said...

you can download it from library genesis:

Puddleg said...

@gen, thanks

@ Karl, this seems close; high fat diets, mice, different amounts of SFA, linoleate, n-3 (table 1)
Interestingly uses hydrogenated coconut oil as main SFA source.

Figure 2 - high-fat diets (60%) with 1% linoleate are not fattening, with 8% linoleate they are, 1% omega 3 attenuates this somewhat. Also true for 35% fat diet (control).

Nice touch - feed efficiency is measured as well as intake. Not sure exactly what that means but looks good.

karl said...

@George Henderson

"uses hydrogenated coconut oil as main SFA source." (This would be a pretty big confounding variable - but the rodent studies are full of them - it is as if they didn't learn about the scientific method. I suppose if they did decisive studies it would make writing new grant studies harder... )

I can't find a clean study on this. Seems pretty straight forward:

Change out the PUFA for real SFA. Equal calorie intake.

My guess on what would happen - the PUFA rats would store more fat - thus have less calories to burn - so they would move slower - perhaps block T4=>T3 conversion? Autonomic system slows down? In the end you have calories-in = calories-out, but in this case both groups would have equal calories-in. Seems like someone must have done this?

Dr. B G said...

Too thought provoking for words....!

I concur, DHA is an animal fat signal and the receptors are 'food sensors' (GP120, FFA1/GPR40).

DHA is also very effective at lowering cortisol, improving HRV (heart rate variability), and decreasing SNS outflow for stressed people. I don't know if this is elucidated yet but I suspect it's mediated by either GP120 and/or GPR40 and/or PPAR because these are all neuroendocrine repectors/fat-sensors.

Chart: Metabolite-sensing G protein-coupled receptors

Diagram: G-protein Receptors, Insulin

What amazes me is that SCFAs/propionate, ketones, and LCFA/omega-3 all have varied mechanisms of action on NFkB, PSNS/SNS systems and immunity (and Sauer cancer rats) that appear tightly coordinated by diet, movement and the microbiota/pathogens.

We co-evolved escaping pathogens, predators and starvation. GPR120 has interesting immunity functions. "Whereas GPR120 is highly expressed on adipocytes and inflammatory macrophages, GPR40 is not expressed on the latter (65). GW9508, which is an agonist of GPR120, was found to inhibit the response of cultured macrophages to endotoxin, and this involved maintenance of cytosolic IkB and a decrease in production of TNFa and IL-6 production, effects that are similar to those of EPA and
DHA. Thus, it appears that GPR120 is involved in antiinflammatory signaling. GPR120-mediated gene activation was enhanced by EPA and DHA. Furthermore, the ability of DHA to inhibit responsiveness to endotoxin was abolished in GPR120 knockdown cells. These findings indicate that the inhibitory effect of DHA (and probably also EPA) on NFkB occurs via GPR120."
Mechanism of action of n-3 fatty acids

Ketones and gut SCFAs are also biological proxies too (weak agonists of PPAR) but I think are more markers and agonists for metabolic flux/demands and energy states (fed v. fasting v. extended exercise). I didn't know this but ketones regulate heart rate and reduce SNS outflow too (more 'calmness') via GPR41 (and probably PUMA-G). I esp like Figure 4E...

D said...

Some interesting studies here. Including fat mice and thin mice, both on a high fat diet but with a different 0-6 intake.

Puddleg said...

@ Karl,

google "cross ralk between PUFA and thyroid"


male lean and obese Zucker rats (5 weeks initial age) were fed either a low-fat (15% calories) or one of two high-fat diets (65% calories; predominant fat source of either soybean oil or palm olein) for 8 weeks.
Body weight gain and carcass lipid content were increased (16%-17%; P < 0.005) in obese rats fed the high-fat palm olein diet as compared to those fed the low-fat diet. These parameters were not increased in obese rats fed the high-fat soybean oil diet. In contrast, indirect calorimetry measurements indicated a moderate increase in heat production (Kcal/effective body mass/day; 14.5%) and decrease in energy balance (44.8%) in the obese rats fed the high-fat soybean oil diet as compared to those fed the low-fat diet. The differential response of the lean and obese Zucker rats to this short-term dietary manipulation demonstrate that genetic background can influence an individual's response to dietary fat type and level.

This is a bit confusing and I'd like to see the tables. But maybe 65% fat is past the low-carb tipping point where LA ceases to be obesigenic, has the opposite effect (see my blog )

this one's a lot clearer:

Rats carrying one copy of the fa allele are predisposed to diet-induced metabolic disturbances which contribute to hyperinsulinemia, obesity and dyslipidemia. To investigate the role of dietary carbohydrate and fat in the development of these conditions, we fed 6-week old male heterozygous (fa/+) lean rats carbohydrate-free diets containing primarily saturated fat either ad libitum or pair-fed. These diets were compared to standard chow and to a high saturated fat mixed diet containing 10% energy from sucrose for 4 weeks. The carbohydrate-free diet resulted in significantly lower circulating glucose levels compared to all other groups (p = 0.006). Weight gain was negligible in the carbohydrate free groups compared to standard diet and 10% sucrose diet (p = 0.03). This was reflected in energy efficiency which was markedly reduced (90%) in the carbohydrate-free groups compared to the other groups (p = 0.04). Corresponding changes were noted in fat pad mass. The subscapular and epididymal fat pads were increased 42% and 44%, respectively, in animals consuming the 10% sucrose diet compared to all other groups (p < 0.01). Comparable changes in fatty acid synthase (FAS) mRNA were observed in response to the carbohydrate-free diet, which resulted in a 53% decrease in adipocyte FAS mRNA (p < 0.001). Addition of 10% sucrose to the diet completely reversed this effect resulting in a 69% increase in adipocyte FAS mRNA compared to the carbohydrate-free groups (p = 0.01). Similarly, hepatic FAS mRNA was elevated by 51% and 66% in the 10% sucrose and standard diet groups respectively, compared to the carbohydrate-free groups. Therefore, diets that contain minimal carbohydrate may minimize net lipid storage and adiposity.

Puddleg said...

@ D,

I actually linked to that mouse paper by Hibbeln et al. in my blog a few days ago (comments section).
It is mentioned here too with a link.
These are some cool papers, if you take all the different reference links there as a whole body of knowledge. They show macronutrients interacting to alter one another's metabolic effects.

Unknown said...

Hibbeln et al., Crawford and Cunnane's work is nothing short of a miracle. This is 90% of their studies make up my brain gut series. we need people making the cross to other disciplines, like physics, to understand how it all works in the brain.

Kevin FST said...

Possible reason for n-3 PUFA being OK (or better than):

Dr Ling Gao has found that when the omega-3 fats EPA and DHA are oxiduzed, they significantly activate the Nrf2 pathway. For years researchers have noted decreased levels of free radical damage in individuals who consume fish oil... with this new research, the relationship between fish oil and antioxidant protection is now clear. As Dr Gao reported, "Our data support the hypothesis that the formation of... compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based anrioxidant and... detoxification defense systems."38
Perlmutter (2013), Grain Brain, pp144-145.

38. L. Gao et al., "Novel n-3 Fatty Acid Oxidation Products Activate Nrf2 by Destabilizing the Association Between Keap1 and Cullin3," Journal of Biological Chemistry 282, no.4 (January 26, 2007): 2529-37.

Peter said...

Hi Kevin,

Nice, yes, DHA fragments signal mitochondrial biogenesis. I like this. On an evolutionary basis I can see some logic. If the nucleus senses damaged bits of mitochondrial structural lipids they might be used as signals indicating the need for more mitochondria to limit on going damage. I dare say enough antioxidants would f*ck this response, well and truly. Vitamins C and E come to mind.

I also still feel that using omega 3s as bulk fuel will be as disastrous as as using omega 6s. But as signalling molecules...


Unknown said...

Peter Schmitt said...

Hello Peter,

I recently discovered your blog and am now up to binge-read your proton-series from the start. I do understand some biochemestry but I'm probably not as well equipped as your usual commenters. So please excuse, if my question is trivial.

The main problem, which seems to arise with the consumption of pufa seems to be their oxidation in mitochondria and the effects which arise from there via reduced ROS-production. But does this not only affect linoleic and linolenic acid ( meaning up to 18 carbon-molecules ) ? Shouldn't "overly" consumed arachidonic, docosahexaenoic acid or docosapentaenoic acid be oxidised by the peroxisomes down to C8 including, as far as I have read, higher ROS-production as the oxidation of similar unsaturated fatty acids in mitochondria ?

I hope, you get my question despite it being asked under an older article. Keep your work up,
Peter Schmitt