Tuesday, July 31, 2012

Protons: 25mmol/l

This post is a slight aside based on minor details in the paper "Insulin resistance is a cellular antioxidant defense mechanism". Which shows, quite clearly, that palmitate at levels as low as 0.05mmol/l causes some degree of insulin resistance. By 0.15mmol/l it's significant and by 0.5mmol/l it's worse. The graphs are from myocytes in cell culture.



This graph show GLUT4 count on the surface of myocytes. Left is control, next is the count after an acute exposure to insulin, taken as the 100% response. Adding more and more palmitate decreases the percentage response.

No one, not even a fatphobic vegan, has palmiatate levels in the FFAs of their blood as low as 0.05mmol/l.



This graph shows the effect of exposure time to 0.15mmol/l on GLUT4 translocation. Things get worse by the hour. Is this real?

Let's have a look in the methods:

"Palmitate (PALM) treatment was performed essentially as described in ref. 5"

So let's go to ref 5:

5. Hoehn KL, et al. (2008) IRS1-independent defects define major nodes of insulin resistance. Cell Metab 7:421–433.

In results we get this superb snippet:

"In our preliminary investigations, we observed that high (>300 μM) palmitate doses were toxic to cells, resulting in morphological changes and even detachment from the substratum."

Palmitate is clearly pretty nasty stuff. And I feed it to my daughter!

In the methods section under "Oxidative stress" we get a description of "stepdown medium", as used in both of the studies discussed. The composition of cell culture medium may be common knowledge to people using cell culture for a living but it was news to me. This is virtually a throw away comment:

"...while total glucose levels (measured with an Accu-Chek II glucometer [Roche]) decreased slightly from 24.7 ± 1.6 mM to 23.3 ± 1.9 mM".

The DMEM cell culture medium used here contains 25mmol/l of glucose!

So let's rephrase that toxicity of palmitate:

"Palmitate at 0.3mmol/l is severely toxic to cultured cells in the presence of 23mmol/l of glucose".

That I can certainly believe.

Aside: It is very interesting to note that palmiate at 0.15mmol/l is low-physiological for a human and yet is described as toxic, without mention of the grossly pathologic 25mmol/l of glucose in the culture medium. I suspect that whenever you look at a cell culture based study demonstrating palmitate toxicity this will apply.

So, does palmitic acid cause insulin resistance (aka superoxide production) under low glucose conditions? Where there is no caloric overload?

Studies looking at palmitic acid in the presence of low glucose are as common as hen's teeth...

Peter

18 comments:

O Numnos said...

I have despaired at many a paper (analytical chemistry - mostly in the "nutritional" field) but the stuff you highlight leaves me stunned.

Dave Bridges said...

Looking at anything from tissue culture in 5 mM glucose is exceedingly rare.

Kindke said...

Good work Peter. Im too lazy to read methods in most papers but it seems you pretty much have to :(

Actually Im skeptical of using cell culture studies to "prove" anything for the simple fact as soon as you remove cells from the organism you immediately disconnect it from all the various feedback loops that are present in vivo, for example the organisms nervous system.

How safe is extrapolating from in vitro to in vivo ???

George Henderson said...

The elephant in the metabolic room has stomped all over this experiment - and how many others like it?

This might be more nuanced:
"Bergman and others, for example, documented the role of nocturnal flux of FFA to the liver in promoting insulin resistance, arguably by impairing insulin suppression of HGP (Bergman and Ader, 2000). But in light of the Kadowaki paper showing that insulin sensitivity is maintained by Irs2 during fast, FFAs could also provide grist for the lipogenic mill, driving VLDL and LDL secretion, and thus setting off the ill-starred combination of excessive glucose and lipid production. "

I discuss this paper in my latest post, The Metabolic Push-Me-Pull-You

http://hopefulgeranium.blogspot.co.nz/2012/07/the-metabolic-push-me-pull-you-hcv-core.html

But feel I've hardly scratched the surface, I haven't even worked those pesky FFAs in yet.

CharlesVegas said...

Stunning indeed. You should send a comment to the journal that published this trash.

js290 said...

Yet another example of so-called "scientists" trying to decouple a coupled system. How did any of these people pass a differential equations class much less put in a lab to conduct science? I mean, would you trust a mechanic who didn't know the difference between a hammer and a screwdriver?

Jane said...

DMEM contains iron.

'..[iron] is frequently added to cell culture media as a nitrate or sulfate salt. .. Free or ineffectively sequestered iron can be very toxic to cells. .. In view of the above facts, one should strongly consider whether adding iron salts directly to a medium is appropriate. ..'
http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/iron.html

Saturated fatty acids like palmitate can bind iron and take it into cells. I don't see how else this tiny amount of palmitate could be a problem. Of course, the very high glucose won't help.

Jane said...

It seems saturated fat is only bad for you if your diet is very high in iron.

'.. we analyzed total cholesterol and the number of LDL particles and blood sugar measurements and inflammation measurements, and we didn’t see anything particularly dangerous occurring when people ate lots of red meat but kept the saturated fat very low...

'.. we expected that because these diets have low carbohydrate, when we fed the high saturated fat level along with the red meat, we would see a pretty benign metabolic risk profile. ..

'.. the surprise was that the combination of the high beef diet and the high saturated fat diet caused very serious increases in all of the cholesterol related risk factors we had been measuring, including total particle numbers, small LDL, total LDL cholesterol, inflammation, whatever we looked at, we saw an adverse effect. ..

'.. certain kinds of saturated fat, beef tallow being one of them, and the saturated fat called stearic acid being another, both promote the absorption of heme iron. ..'
http://www.meandmydiabetes.com/2012/04/17/ron-krauss-saturated-fat-red-meat-it-depends/

Matthius said...

What happens if you replicate ketophysiologic conditions in vitro and run a similar analysis? Does anyone do this?

fortune said...

Hi Jane:

My impression from reading the full back-n-forth commentary over 2010/2011 in the journal was that the Siri-Torino/Hu/Krauss et al study was actually pretty thoroughly destroyed by the end. It seems certainly as if Hu at least just gave up defending it. As a result I've been hesitant to continue to cite Krauss' works, alas. Do you believe otherwise? (Hope springs eternal!)

Jane said...

Hi Fortune
Do you mean 'Saturated fatty acids and risk of coronary heart disease: modulation by replacement nutrients'?

I've just read it, it seems to be a review rather than a study. Can you give me more details please?

Krauss seems to be in the process of finding out what people who work on iron such as Eugene Weinberg have been saying for years. But most interestingly, Weinberg does not seem to know about the effect of saturated fatty acids on iron absorption. At least it isn't mentioned in his recent review 'Iron loading in humans'.

So Krauss knows something Weinberg might not, that could solve the mystery surrounding saturated fat and heart disease.

Saturated fatty acids have been found to increase iron absorption and decrease absorption of copper and manganese. Since copper and manganese are needed for dealing the the adverse effects of excess iron, this is potentially HUGE.

It was found by Leslie Klevay years ago that a high-fat diet which caused heart disease in mice did not do so if they were given extra copper. Now we need to know whether the brain damage and obesity caused in rodents by a high-fat diet can be prevented by extra copper and/or manganese.

George Henderson said...

@Jane,
tests of mineral uptake differentials are usually done in near-deficiency states, because this is what is considered clinically important.
Most research into iron uptake is based around improving it in anaemic females.
Vit C improves iron uptake when this has been inhibited by dietary polyphenol but there is (SFAIAA) no clinical evidence it promotes iron excess.

Pierre C. said...

A different point of view.
Palmitate here is acting as it is supposed to do. It is a natural and necessary component that enables the cell to trigger its defensive program in this abnormal environment full of glucose : to limit this glucose continually streaming into the cell and damaging the mitochondria and else, let's become insulin resistant.
(Maybe palmitate in the cell is simply a signal that mithocondria can burn something else that glucose, so "you can get that damaging xxxx out of there" -> "OK to start insulin resistance" )

The lack of palmitate in this full-of-glucose-environment is just an unfortunate state where the cell cannot work properly, and cannot trigger its insulin resistance. Then mitochondria will be slowly but definately damaged by running only on glucose, etc.
("you have nothing but glucose to burn then... burn it as long as you can before you die")

Maybe a sort of "stop looking at the finger and look at the moon that it is pointing".

Insulin resistance is not a problem, it is a solution (not very efficient in the long term, though) to an abnormal state : chronic glucose overload.


Thanks for your exciting blog, btw.

blogblog said...

@Jane,

wild mice NEVER eat fatty diets. They are seed eaters that eat >80% carohydrate and <5% fat. NO mouse dietary study is relevant to humans.

Jane said...

Glucose is actually a very necessary antioxidant. It gets processed through the pentose phosphate pathway to produce NADPH, which works together with antioxidants such as glutathione to prevent oxidative damage.

Cells in culture are exposed to unnaturally high levels of oxygen.

Peter said...

Hi Pierre C, That is the main point of the paper, the next post here looks at other survival benefits of insulin resistance. There's been no time to tidy it up in the last week or so.

Jane, OMG, I never realised saturated fat caused heart disease!!!! Must have been living under a stone for too long.

Peter

Jane said...

Hahaha!

Jack Kruse said...

Jane you hit upon something huge that I have been saying but of course is not heard due to the current meme in the states that is Paleo when you said," It seems saturated fat is only bad for you if your diet is very high in iron. " Well, this is what Cordain found too but he could never make sense of it in his original book. He got pounded for it by the current meme. Now we have AHS in the books and some of the more prominent biochemists saying saturated fat is always OK.....that has been settled. I nearly died when I saw it because If you look at the work of Cunnane and Crawford in neural lipid chemistry of DHA and brain evolution, you can not assimilate DHA into a brain in the presence of a lot of saturated fat or a ton of iron. This means that from an evolutionary standpoint you cant form a human brain eating pastured meat alone. A recent tweet of AHS said exactly the opposite. To make a human brain you need ridiculous amounts of DHA with iodine to protect it from oxidation. Fe destroys that. I am so glad to see Peter hitting on this because this science needs to get out from under the current meme. Remko Kuipers was a presenter at AHS and he parting tweet was a warning to paleo (paraphrased)........"I dont understand why there were not more seafood papers at an ancestral meeting?" The current meme is protecting a franchise in my view. The science will lead the way with the real data but we need to read it and think about it. It is clear you all do that here and I applaud you all.