Tuesday, November 20, 2012

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.

34 comments:

Chip Spitter said...

And just when I think my head is going to explode from concentrating too hard, through comes slivers of enlightenment.

I am in a tunnel. I can see the train. I already know (roughly) what is going to happen, I just need to go back and understand how burning coal makes it move.

Thanks for making me think.

Kindke said...

Peter I may of missed some bits, but do we definitely know it is the saturated fats that damage the hypothalamus in C57BL/6 mice?

Or maybe its the sucrose that is usually fed with the high-fat lab diets?

I was reading this study on rats, and in this strain of rodent it seems to be the sucrose causing the problems...

"It has been established that rats prone to develop diet-induced obesity have decreased basal brain alpha 2-adrenoceptor levels compared with rats that are resistant to obesity."

The part I find interesting is that apparently only 50% of the rats will develop obesity on the high-fat + sucrose diet. I.E, gene's count, not just calories.

IcedCoffee said...

This always makes me want to go back and reanalyze convention teachings with a more discerning eye. Saturated fat is bad because it doesn't help you get fatter. Fish oil is good because it does help you get fatter. So where does something like a trans-fat fit in? Are they bad simply because they don't help you get fatter? (I think CLA might actually kill fat cells.) So for someone on the borderline of T2D, would a simple switch from their soy oil to a more saturated fat (and no other change) actually be a detrimental change, as it would put a halt to their bodies ability to get fatter, and start the cascade into full blown diabetes?

Rod said...

Peter,
Sometime when you have nothing much to do and feel a need to soften the blow to those of us who are intellectually challenged, perhaps you could summarize the last 3-4 months of your posts.I'm pretty sure you are on to something here. I just wish the mists would part for me and I could see what it is. I love your stuff but it makes me feel inadequate.

Pierre said...

Peter or anyone else with a theory...

What other factors besides food intake would prevent me from going into ketosis? Currently I eat one meal a day...have to say achieving ketosis is a frustrating adventure. I have managed a high of 1.5 mmol blood ketones after a 3 day fast.

karl said...

@Pierre

Most likely if you are really not eating the carbs ( a glucose meter can help identify food with hidden carbs), it is too much protein.

Excess protein is broken down and increases BG.

Pierre said...

Anything physical that would make it difficult to achieve ketosis?

Thanks

karl said...

This needs clarification: There are a lot of folks encouraging O-3 consumption to "balance" O-6 - reducing inflammation.

The important question is what happens if we instead focus on reducing O-6?

,.,.

I'm not sold that we can ignore the form of O-3 - long chain (fish-oil ) has different effects than 18:3 ω-3 alpha linolenic acid or ALA. I'm not saying that these effects are via insulin sensitivity, but I find the blockage of AA formation explaination feels like a bit of hand waving?

Grass fed beef has significant differences to beef of the mass produced, metabolically sick, corn fattened beef :
http://www.nutritionj.com/content/9/1/10/table/T2

Corn fed beef is cheaper to produce by the pound as corn fattens them up much faster. They will hype that corn-fed tastes better - well-marbled etc - that is all BS IMO.

Even the wild deer here in Kansas eat too much corn(they steel it)!

There is also some effect of CLA (more in grass fed beef) - CLA totally confuses me - it is a family of FA - not sure we should even think of it as a PUFA... need Petro to write a
column exploring CLA (I did a google seach of petro's columns for CLA - metions, but not a full treatment.)

I'm wondering what becomes of the old O-3 egg laying chickens? I want to eat them!

George Henderson said...

@ Karl, studies show replacing o-3 redresses 6:3 balance promptly, restricting o-6 does not. In the short term - but longer term, both would be wise.

@ Peter, mayhap we can explain all these effects of the F:N ratio through a single mechanism; upregulation of FOXO1 due to inhibition of phosphorylation by mitochondrial ROS.

Fasting, burning palmitate at physiological levels, increased F:N = increased ROS = upregulated FOXO1 = upregulated gluconeogenesis = no hypoglycaemia when fasting.

PUFA doesn't do this.

Ditto: upregulated FOXO1 = increased sensitivity of TLR4 to LPS = good antibacterial immunity.

Immunosuppressant effect of omega-3s can be deadly in sepsis.

Ditto: upregulated FOXO1 = stregthening of antioxidant defenses.

Feeding PUFA increases susceptibility to oxidative insults.

http://www.hindawi.com/journals/edr/2012/939751/

http://www.ncbi.nlm.nih.gov/pubmed/16865227

However, supraphysiological levels of glucose will also elevate ROS and gluconeogenesis; this is not adaptive.

These mechanisms might explain high fasting BG on diets high in SFA, low in PUFA and antioxidants (which?).
Delivering a little omega 3 in an antioxidant matrix - e.g. krill oil - inhibits gluconeogenesis in CBA/J mice fed a low-fat AIN93M diet.

http://www.frontiersin.org/Nutrigenomics/10.3389/fgene.2011.00045/full

karl said...

If PUFA induces obesity via increased insulin sensitivity (short term) which produces insulin resistance(long term) once they can't gain anymore, then is this also the effect of transfats?

I'm totally bollixed about CLA - I'm thinking it might be because I'm looking at CLA as a group?

George Henderson said...

I think CLA is probably more likely to turn out to be hormetic than just about anything else.
Which is a lazy way of saying a mysterious, but dose-sensitive, action. More than you could get in the diet naturally will not be better for you.
A fat so hard to beak down that dealing with it it stimulates the metabolism of all fat, perhaps.

IcedCoffee said...

Re: CLA,
I was really interested in CLA around a year ago. Studies seem to suggest that it results in the arbitrary destruction of some fat cells. They proposed that our mitochondria are unable to appropriate break down the fat because of the placement of the trans-bond, and that the fatty acid remnants accumulate leading to cell death. Although technically there are two isomers of CLA, while only one is naturally occuring (in dairy and grass fed animals.)

When marketed as a weight loss drug, it did show some promise as promoting favorable body composition (more muscle and less fat). But they always stuck in the caveat that it seemed to exacerbate diabetes. Although just about every study on CLA seemed to use a combo of the two isomers, despite acknowledging that one seems to be mostly harmful, while the other seems to be mostly beneficial.

I tend to think of all trans-fats in a similar way. Our mitochondria can break down trans bonds, but only if they are appropriately situated. If we come in contact with a trans-fat with a poorly situated bond, the end result is fatty acid derivative accumulation and cell death.

I'll try to dig up some old references for these.

George Henderson said...

@ Icedcoffee
Like this, you mean?
http://www.ncbi.nlm.nih.gov/pubmed/17869086

http://www.ncbi.nlm.nih.gov/pubmed/16563722

Apoptopic of rogue cells; and alters arachidonic acid distribution:

http://www.ncbi.nlm.nih.gov/pubmed/11768161

IcedCoffee said...

@George,
Yea its definitely some interesting stuff. Ultimately I tend to think the effect is too small to make a really appreciable difference, but it provides another reason to opt for grass fed meat. Interestingly enough, one of the best sources is Kangaroo. Makes me wish i lived closer to Australia, its super expensive here.

George Henderson said...

This is probably another F:N ratio mediated fatty acid differential:

http://www.ncbi.nlm.nih.gov/pubmed/7591016

Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism.
Davda RK, Stepniakowski KT, Lu G, Ullian ME, Goodfriend TL, Egan BM.
Source
Division of Nephrology, Medical University of South Carolina, Charleston 29425-2251, USA.
Abstract
Many obese hypertensive individuals have a cluster of cardiovascular risk factors. This cluster includes plasma nonesterified fatty acid concentrations and turnover rates that are higher and more resistant to suppression by insulin than in lean and obese normotensive individuals. The higher fatty acids may contribute to cardiovascular risk in these patients by inhibiting endothelial cell nitric oxide synthase activity. To test this hypothesis, we quantified the effects of oleic (18:1[cis]) and other 18-carbon fatty acids on nitric oxide synthase activity in cultured bovine pulmonary artery endothelial cells by measuring the conversion of [3H]L-arginine to [3H]L-citrulline. Oleic acid (from 10 to 100 mumol/L) caused a concentration-dependent decrease in nitric oxide synthase activity at baseline and during ATP and ionomycin (Ca2+ ionophore) stimulation. At 100 mumol/L, linoleic (18:2[cis]) and oleic acids caused similar reductions of nitric oxide synthase activity, whereas elaidic (18:1[trans]) and stearic (18:0) acids had no effect. Oleic acid also inhibited the endothelium-dependent vasodilator response to acetylcholine in rabbit femoral artery rings preconstricted with phenylephrine (P < .05) but had no effect on the response to nitroprusside. The pattern of 18-carbon fatty acid effects on nitric oxide synthase activity in endothelial cells is consistent with activation of protein kinase C. Although oleic acid increased protein kinase C activity in endothelial cells, neither depletion of protein kinase C by 24-hour pretreatment with phorbol 12-myristate 13-acetate nor its inhibition with staurosporine eliminated the inhibitory effect of oleic acid on nitric oxide synthase.

FOXO1 drives iNOS in hyperglycaemia
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2750207/

Christopher said...

CarbSane debunks nutritional ketosis AND exposes Jimmy Moore: http://carbsanity.blogspot.com/2012/11/amazing-and-not-so-amazing-low-carb.html.

Peter said...

George, yes, NO (which seems to be mostly generated further down the ETC than superoxide) is interesting too. It also has functions macroscopically which clearly affect mitochondrial oxygen delivery. Nitrogen derivatives certainly are another large ball game.

Peter

karl said...

@George Henderson

The paper about oxLDL is interesting (oxLDL is something I've spent some time obsessing over). High BG ups oxLDL.

oxLDL is a much better predictor of CAD than LDL - and I've not seen any paper that shows that LDL is a risk-factor at all if you hold oxLDL constant.

It is oxLDL that goes into the macrophages in artery walls.. not LDL and via Lox-1 - the deal that statins block.

Why mainstream medicine isn't testing for oxLDL (it is an inexpensive test) instead of the standard cholesterol panel is just wrong. (Some are now obsessed with LDL size - which the only intervention that corrects the pattern is a low-carb diet.) For most people, it appears the best prevention of CAD is keeping postprandial BG below 110.

Purposelessness said...

wow, carbsane really does have an unhealthy obsession with jimmy moore.

IcedCoffee said...

@karl,
The real question is: do statins lower oxLDL? Because if they don't, then we have your answer about why we don't test for it. How else will we justify prescribing half of the country lipitor?

http://high-fat-nutrition.blogspot.com/2009/08/cholesterol-statins-and-oxldl.html

Gadfly said...

(Bites tongue and does best not to encourage Christopher...)

George Henderson said...

NO is generated from arginine directly, not from mitochonrial ROS, but it looks from the fatty acid differentials as if mito ROS may play a role in activating the synthase.

Extracellular superoxide will quench NO to peroxynitrite, so we don't want much of that. Uric acid will quench it too.

biopterin - very interesting little non-vitamin co-enzyme, mainly required for synthesis of neurotransmitters - dopamine, serotonin, NO.



karl said...

@IcedCoffee

Statins Don't lower oxLDL - They appear to only reduce the non oxidized LDL - (lowering LDL is NOT how I think statins do the good they do - not going to mention the bad they do)

IcedCoffee said...

@karl,
I totally agree. Statins are pretty garbage, and probably extremely misunderstood. I wonder how much they have to do with the relatively recent surge in Alzheimer's and other neuro-degenerative diseases.

Jane said...

Peter,
There is another way this could work besides the F:N ratio. The fish oil PUFA could be oxidised and activate PPAR gamma

'Peroxisome proliferator activated receptor gamma and oxidized docosahexaenoic acids as new class of ligand'
http://www.ncbi.nlm.nih.gov/pubmed/18193404

PPAR gamma makes you fat

'Deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance'
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC556131/

It also corrects metabolic syndrome, probably by raising adiponectin

'Effect of PPAR-gamma agonist on adiponectin levels in the metabolic syndrome: lessons from the high fructose fed rat model'
http://www.ncbi.nlm.nih.gov/pubmed/17261469

PPAR gamma in the hypothalamus proliferates peroxisomes which keeps ROS levels too low for POMC neurons to signal properly

'Peroxisome proliferation-associated control of reactive oxygen species sets melanocortin tone and feeding in diet-induced obesity'
http://www.nature.com/nm/journal/v17/n9/full/nm.2421.html

Peter said...

Still no time to comment properly but @jane:

These are all excellent refs. To me you are looking at the issues a layer up from the mitochondria. These are control systems. They are controlling a basic process which takes place in the ETC. It's fascinating but not unexpected that they use derivatives of the core process for signalling. It's looking like PUFA oxidative derivatives also control iron release from storage in the mitochondria (for yeasts at least). Now there's a link to think about...

Peter

George Henderson said...

The oxidised DHA can also activate PPAR-alpha, which will tend to make you thin (elevated in ketosis, promotes use of FFAs).
This is more consistent with normal effect of fish in diet.


I wonder what the difference is...

Jane said...

George, yes!
Peter, if this all links in with iron I can't wait to hear about it.

I've been trying for ages to find out how mitochondrial superoxide prevents GLUT4 translocation, which the authors of that paper say isn't due to AMPK or FOXO1 or anything else in the insulin pathway. I'm homing in on the cytoskeleton, because preventing actin dynamics can prevent GLUT4 translocation. My present idea (could be rubbish) is that superoxide leaves the mitochondria as H2O2 (this does happen of course) and activates the pentose phosphate pathway and NADPH oxidase (this happens in hypoxic pulmonary vasoconstriction) which can drive actin dynamics by means of glutathionylation.

Jane said...

Forgot to say, actin glutathionylation happens in neutrophils.
'Reactive Oxygen Species-Induced Actin Glutathionylation Controls Actin Dynamics in Neutrophils'
http://www.ncbi.nlm.nih.gov/pubmed/23159440

'Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation.'

DePaw said...

So how much PUFA is too much on a ketogenic diet?

The rats eating that soybean oil based diet with the 'cotton wool' were eating 16% of calories as PUFA.

Jaminet recommends less than 4% calories as PUFa (but he also recommends lower in fat in general).

Would something like 6-8% be fine, such as when a ketogenic diet includes a lot of pork but also plenty of other lower PUFA fats?

Thanks.

Peter said...

DePaw,

I just don't know. Under deep ketosis we would be looking at marked whole body insulin resistance and I think adipocytes would release sat fats preferentially. I feel the main problems with PUFA are undoubtedly related to their inability to generate appropriate insulin resistance, but trying to put a number to it is sticking your neck out.

Personally I live in the real world and I eat commercial UK pork and even some UK chicken. Admittedly both get diluted with copious butter. Worring about the minutiae may do more damage than correcting the minutiae!

It reminds me a bit of the balance between any hormetic benefit of living down wind of either Chernobyl or Fukushima vs the adverse effects of the pauperisation induced by evacuation from the contamination zone. We all know that money is phenomenally protective against ill health and social displacement is disastrous.

Peter

DePaw said...

Thanks for the reply Peter.

I saw elsewhere in your blog you said "My estimate varies between 10 and 20g/d," for how much PUFA you eat.

I eat ~10-15g PUFA, this is on a 80% calories from fat diet (and ~19% from protein, <1% from carbs ~5g/d). I looked it up and calculated ~6% of my fat is from PUFA, or ~5% of calories. This is lower than I though it was, and is very close to Jaminet's suggested 4%.

So I think I'm safe, the real issue is probably with using vegetable oils instead of animal fats or coconut oil. My fats come from a mix of pork, dairy (cream, ghee, cheese), eggs, and beef drippings (tallow to the yankies).

Tim Lundeen said...

This is a fascinating article. It explains why the Kitavans eating a traditional diet have an average fasting blood glucose of 67 -- because they don't eat much if any long-chain sat fat. So they are running on glucose and ketones from coconut oil all the time, and they just can't stabilize their blood glucose except by eating. I suspect this is also why they appear to be relatively short-lived, despite not having CHD, strokes, cancer, diabetes, etc. Their bodies just wear out because they can't switch to fat-burning mode.

For me, it suggested that I can tune my fasting blood glucose based on the ratio of long-chain to short-chain fats in our diet. So I tried it, and it appears to be working :-)

My wife and I were eating a fairly LC paleo diet, with about 100c/day of starchy carbs. We've been sensitive to butter, so were eating mostly beef tallow, goat tallow, and lamb tallow, all of which have a high percentage of c16+ SFA. It was very difficult to get a fasting BG of 83, we had to really tune meal content/timing, and not eat extra o6 fat so no olive oil, pork, duck, or chicken, etc.

So, when I saw this article, we started eating coconut oil instead of adding extra animal fat, so just eating the fat that comes in our beef/goat/lamb. Within the last couple of weeks we've been able to increase starchy "safe" carbs to 400+c/day, and our fasting bg is very stable at 83.

Another interesting effect is that we are eating fewer calories than before. Increasing carb intake seems to have been something we needed, and of course it also increases potassium from the additional root veggies.

Peter said...

Interesting!

Peter