Thursday, February 14, 2013

Protons: TFAM and behenic acid

Well, Peter on Hyperlipid is still messing about with KO mice! Ah, but they really do provide some level of insight and are a slightly more productive occupation compared to the Times crossword. Hee hee, so...

Aside: If you click on the TFAM link immediately below this paragraph and then go to "see all" under "related citations" you will find that there are a sh!tload of genes which can be knocked out to protect hapless mice from the dire consequences of D12492. Let's re appraise that as: Evolution has provided us with a stack of genes giving protection (read "normal processing") against the damage (lack of "normal processing") intrinsic to foods which are caricatured by D12492. They do this by inducing a decrease in glucose metabolism when serum FFAs are elevated. Knocking out these genes will reverse an unimaginably large number of years worth of evolution and I would anticipate that we might just end up dead slim when effective blocking drugs are developed to "cure" the obesity epidemic. Really dead as well as really slim. Anyhoo, back to the mice...

This is the paper where the gene for TFAM was knocked out in the adipocytes of some mice. Edit: Thanks to Purposelessness for the link through comments, end edit. Two consequences of this appear to be that complex I doesn't seem to be there and that complex IV, while present, doesn't seem to be up to much either. With a large chunk of their adipocyte ETC disabled these mice seem to be a little stunted overall but they stay slim and simply refuse to develop insulin resistance with either age or with the feeding of a ton of sugar, described throughout as a "high fat" diet, good old D12492. Don't snigger, this isn't funny!

If you have just come from Kushnareva et al's paper on complex I you could just ask whether the FeS complex N-1a is present or absent in these mice, but then you hardly need to ask this because the mice don't become insulin resistant, ie they don't generate superoxide at N-1a, even though the adipocytes have increased lipid and DNA damage, probably through them spewing electrons at complex IV. So there's really no need to trawl back through the layers of refs to the development of this mouse model to know that there is no FeS cluster N-1a. Or certainly nothing feeding electrons to it. If there were, there would be insulin resistance... I find this difficult to get excited about, useful though it is.

But the paper is utterly fascinating in several other ways. I went through the results to Fig 6 where TBARS and 8-OHdG are shown for D12492 fed mice, being surprised that no damage was reported when the mice were fed crapinabag (this is not quite as true as it sounds but, again, it's not important here). Not so good for D12492 feeding of course. Moral: Don't eat toffee fudge as your sole diet. There is a great deal else which could be said about free radical damage in these mice but that's not for today.

What I noticed, immediately below the TBARS figure and still within Fig 6 of the main paper, was the relative abundances of assorted activated fatty acids from both brown and white adipose tissue:

Of particular interest to me was the rather high relative abundance of activated (carnitine linked) behenic acid, C22:0, over on the far right of each chart. Now bear in mind that there is not a lot of C22:0 in the fat of normal mice anyway, so the massive relative abundance may be, in part, an artifact of a rather low level of C22:0 in control mice. But there is probably a lot more in mice with a crippled ETC. Behenic acid is fully saturated, it's long, it's F:N ratio is as close to 0.5 as you can get and it should be rapidly degraded in peroxisomes. But these mice have markedly down regulated peroxisome promoter genes and the organelles shown in the photomicrograph look, apparently, pretty crappy.

So let's have a think about how this might fit together. These adipocytes have no complex 1. What can you do with NADH in mitochondria if there is no complex I? Diddly squat is, I think, the correct term. Glycolysis works fine and generates a smidge of ATP by substrate level phosphorylation. And some NADH too, which is of no use what so ever. Complex II, succinate dehydrogenase, is fully conserved and can feed in to the CoQ pool to run the ETC, the abnormal complex IV doing the best it can. No matter how much the CoQ pool is reduced there will be no reverse electron flow through complex I because complex I ain't there.

But running from pyruvate dehydrogenase through the TCA to succinate dehyrogenase generates 3 utterly useless NADHs and completing the TCA gets you yet another NADH. What's a cell to do?

Well, it can start by generating a ton of pyruvate by glycolysis. These cells do have a lot of pyruvate. You can feed this in to mitochondria and through pyruvate dehydrogenase (or even pyruvate carboxylase, see below) then generate citric acid from oxaloacetate. If you really don't want to generate more useless NADH than you have to, then why not export the citrate to the cytoplasm through the malate shuttle and convert it to long chain fatty acids? Fatty acids only half-care about putting NADH in to complex I. They will, of course, generate some NADH but what they also do, which is really useful, is to generate FADH2 within electron-transferring flavoprotein which can be used via electron-transferring-flavoprotein dehydrogenase to reduce the CoQ pool, without all of that cyclical messing around with the TCA. And without any need for complex I of course.

As we know, the longer the saturated fat the more FADH2 it generates per unit NADH. These cells desperately need FADH2 input to reduce the CoQ pool. A 22 carbon fully saturated fat is a good option. Behenic acid. Peroxisomes are NOT wanted, it's FADH2 to CoQ all the way.

Of course as acetyl CoA is taken for fatty acid synthesis the citrate yields malate again which, if it re enters the TCA, will give another useless NADH as it converts to oxaloacetate. So why not convert the malate back to pyruvate (replacing the NADPH used to generate that malate) and then use pyruvate carboxylase to generate oxaloacetate without the obligatory NADH resulting from when malate is converted to oxaloacetate? You can then allow citrate regeneration which can be re-exported to cycle around through more FFA generation, yielding usable FADH2 again.

Looking at Fig S6 from the supplementary data we can see that, of the TCA metabolites, only citrate is significantly increased. Pity they didn't measure oxaloacetate.

Pyruvate is increased as you might expect. Glycolysis really needs to be generating NADPH (pentose phosphate pathway) for fatty acid synthesis but I can't see from the paper if this might be occurring. I think it's a reasonable assumption that it is, because the behenic acid is not coming from the diet and NADPH is needed for the fatty acid synthesis which generates it in-situ. There is plenty of lactate derived from the excess of pyruvate over what is needed by the crippled mitochondria.

I also can't tell from the paper how much the glycerol phosphate shuttle is up regulated to feed in through it's own FADH2 route to reduce the CoQ couple either. I suspect it's rather active.

Anything which inputs to the CoQ couple other than complex I seems to be essential for these cells to survive. The impression I get is of adipocytes which are simply converting glucose to fat and running on a (genuine) high fat diet at the mitochondrial level, despite the oral diet being crapinabag.

The photomicrographs of the mitochondria doing this show that they look pretty sick, but what would you expect with a big chunk of the ETC missing?

Just look at the internal structure loss in the mitochondria of the KO mice. Not so good...

Interestingly there are a number of quite severe complex I deficiency mitochondrial diseases in human clinical medicine. Current management is with a high fat diet, hats off to the clinicians for this. Somehow I doubt that sucrose is classified as a fat for these poor folks!

Overall, nice adipocytes. It still looks to me as if the F:N concept is essential for getting any sort of an idea of what might be going on in this type of model.



Puddleg said...

Looks like you might be right. Alternative explanations, anyone?

just got this from Tess; retinol (as retinol) is essential for pyruvate utilization by mitochondria
low retinol = low energy.

It is puzzling why metabolic regulation by the pathway described in this report depends on retinol that vertebrates cannot synthesize de novo. In limiting vitamin A to nutritional sources, there must be an evolutionary advantage of such import as to override the physiological needs for vitamin A in vision and retinoic acid-dependent transcription. The answer may lie in the scenario that finite amounts of vitamin A are subject to depletion during periods of severe starvation when an organism is forced to conserve energy. Our observation that in the absence of vitamin A energy generation by respiration adapts downwards appears relevant in this context. Accumulation of triglycerides in the livers of vitamin A-deficient mice (47) may also signify a metabolic switch to fat for energy generation to offset limited utilization of pyruvate from glycolytic sources. It is also predictable that chronic deviations of vitamin A transport will lead to metabolic disease. Recent observations that the circulating levels of retinol binding protein 4, the major transporter of vitamin A in plasma, are elevated in obesity illuminate this point

Nigel Kinbrum said...

Peter, D12492 doesn't contain "a ton of sugar". D12492 is 8.9% sugar by weight. Fudge is 64-80% sugar by weight.

Sabine said...
This comment has been removed by the author.
Sabine said...

Re. Vitamin A: High carbohydrate diets cause deficiencies in Vitamins and minerals, vitamin A, D, K,......

Natural high-fat diets don't.

Puddleg said...

"The answer may lie in the scenario that finite amounts of vitamin A are subject to depletion during periods of severe starvation when an organism is forced to conserve energy."

not only that, but ketone bodies are a substrate that, like alcohol, promote degradation of retinol. So retinol stores are also depleted more directly by starvation to conserve energy. I always wondered what was the point of an essential nutrient like retinol being destroyed by a normal product of fasting, now we know.

Puddleg said...

And maybe this action of beta-hydroxybutyrate, depleting retinol, is the ultimate cause of metabolic slowdown on ketogenic diets, the so-called thyroid syndrome. In which case it ought to be possible to protect retinol against degradation in some way.

Sabine said...

It seems to be important to distinguish between ketosis from starvation, and a normocaloric or hypercaloric ketogenic diet (=sufficient or more than sufficient nutrients).

Puddleg said...

@ Sabine,
the only fully adaptive ketogenic diet we know of, the Inuit, is very high in retinol.

Paul N said...

So what you are saying, George, is that if you are going to eat high fat paleo/primal/WAPF style, to bring on the cod liver oil - where have I heard that before?!

Stipetic said...

D12492 also contains maltodextrose, which is quickly converted to glucose, so that's another source of an immediately available sugar. The lard in this "diet" is high in omega-6 polyunsaturates (not to mention anything about the yummy soybean oil too). Sounds like a pretty decent approximation of the type of herbivorous food, and sugar rush, a mice would encounter in the wild. ;-)

Scott Russell said...

Great stuff as always. I'll probably have to read that a few more times for it all to sink in!

re: retinol, I would be a bit more hesitant to lay the blame for metabolic slowdown solely on retinol deficiency. Mostly because I'm trying to imagine a keto diet that isn't high in retinol, and it would be a challenge. Aren't eggs the poster-child of the low-carb family of diets?

Sabine said...

Beef liver, pan fried, 3 ounces 6,582 micrograms of preformed Vitamin A.

HMMMMMM, yummy liver! One of the staples of a high-fat ketogenic diet.

Sabine said...

100 grams of butter = 950 micrograms of preformed Vitamin A.

Manythings said...

Perhaps less vitamin A if we've cooked the butter

Manythings said...

Iced Coffee, if you are on a keto diet and consuming your own fat stores (to lose weight) then you might well end up retinol deficient.

tess said...

and never forget -- ingestion doesn't ensure absorption. i "shouldn't" have to supplement iron, but because of my peculiar physiology, i must.

Sabine said...

Vitamin A is very heat resistant (fat soluble), and easily absorbed (from animal sources = complete retinols with fat), we also store a lot of it in our own fat, (unless, of course, we made ourselves deficient by consuming a high grain/high carbohydrate diet, which frequently also leads to malabsorption syndromes).

John said...


Do you have the link where the ketone bodies lead to retinol degradation?

Concerning shutdown, I've had that in my head for a long time regarding vitamin A. There are lot of results regarding its influence on TSH, t3 uptake, UCPs, body fat, etc. You can find even more perhaps with retinoic acid, but I don't know how that goes along with retinol intake.

I think there are a few papers showing that low vitamin A actually increases metabolic rate, but it's usually the opposite.

I always assumed Inuit, polar bears, etc consumed so much retinol to induce uncoupling in the cold weather.


There is an Inuit paper that reports 30,000IU per day. Very few people I would guess eat that much; probably a decent amount of liver/liver oil is needed.

Sabine said...

I am just wondering about this theorizing regarding thyroid function, vitamin status and the lot...
Why did the Inuit load themselves with ketogenic foods before going out on the ice in order to stay warm?
Why do people report increased resilience, increased vigor, and increased energy on ketogenic diets?
A properly done ketogenic diet is not known for causing any deficiencies, why would people be expected to have deficiencies?
Why can I go outside at -10 degrees Celsius without a coat or a decent shirt on and feel comfortable on a long-term ketogenic diet?

No signs of low thyroid function!

No signs of deficiencies!

Any explanations?

Manythings said...

I remember reading an interview (years ago) with Inuit women. They talked about about raw seal liver. After a hunt, this was a real treat - eating it gave them a warm, pleasant feeling within a few minute. I wonder if this was the vitamin A (supplementing retinol is giving me loads of extra energy).

Bill said...

Peter, great post. Can you can make the connection here for me: is this the very same behenic acid that is suppressed in plasma of liver disease patients but increased in those on a ketogenic diet?

P.S. @ Tess, what about the oysters?

Purposelessness said...

Hi Peter!

You made more sense of that paper than I ever would..

I have something totally offtopic to share, it's about protein, insulin and glucagon. I recently ran across that study:
The whey meal is the most insulinogenic one, but it has the least suppressive effect on actual fat oxidation (while all the meals lower plasma FFA). Whey also released the most glucagon.. Yet here I was thinking glucagon only impacted fat metabolism in pharmacological doses? Or am I simply wrong (ie having read too much Good Doctor lately) and this isn't controversial at all?
I would appreciate a quick word from you on this topic..

love, Purp

Puddleg said...

@ John, I came across ketones (beta-HB) inducing conversion of retinol to polar metabolites (which disrupt mito membrane potential) when I was studying alcohol-plus-retinol hepatotoxicity. Not sure I can find it easily, it was listed in a table of chemicals that had that effect.
Probably the dehydrogenase enzymes overlap, and high levels of substrate for one will induce the others. Hydroxybutyrate dehydrogenase, alcohol dehydrogenase, retinol dehydrogenase.

@ Peter, behenic acid elevates LDL.
Yet it should also produce insulin resistance(?): are these 2 effects compatible?

tess said...

@Bill, if only they weren't such a pain in the "roundsteak" to open! snout-to-tail eating in one bite!

karl said...

In this paper Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance. What was the control diet?

Petro's blog where we once again have inappropriate insulin sensitivity (tm) has me back to a question I've been pondering...

We know that insulin regulates the FA flux of adipose tissue - ( at least when the food is tasty ) - we also know that insulin sensitivity regulates this flux.

Insulin sensitivity is controlled in part by the type and quantity of FA.

So the question is, which is the dominating control? It appears to me that looking at insulin levels without knowing the state of sensitivity can be seriously misleading.

And - when insulin sensitivity of adipocytes is low - what happens to other insulin receptors? Could muscle and brain receptors be effected in a similar way?

As sexual creatures we have overlapping control loops - (we need them to overlap to avoid the consequences of genetic variation ) - rather amazing that these mice in spite of missing one of these control loops manage to survive.

The control of FA flux I would think would be normally be subservience to glucose control. Elevated glucose is harmful in so many ways ... ( sadly reminds me of when I recently saw the hospital serve sugared soda pop and a high carb meal to a friend of mine who was in the hospital for diabetic neuropathy - he could no longer feed himself. )

@George Henderson
What I remember was that a lack of thyroid could reduce vit A ( not the other way round). .. ...

see :


There was some talk about a ratio of vitamin A to vitamin D to vitamin k(which one?) that I've heard, but could not find the papers that the ideas were based on.

I have never liked liver - but think I should eat it anyway..

Purposelessness said...


"Animals and Diets
aP2-Cre transgenic (Abel et al., 2001) and TFAM-floxed (TFAMf/f) mice (Larsson et al., 1998) have previously been described. All mice were housed in a mouse facility on a 12 h-light/dark cycle in a 22oC temperature-controlled room. Mice were maintained on a standard chow diet containing 22% of the calories from fat, 23% from protein and 55% from carbohydrates (Mouse Diet 9F 5020; PharmaServ) or subjected to a high fat diet (HFD) containing 60% calories from fat, 20% from protein and 20% from carbohydrates (OpenSource Diet D12492, Research Diet) beginning at approximately 6 weeks of age as indicated. Animal care and study protocols were approved by the Animal Care Committee of Joslin Diabetes Center and were in accordance with the National Institutes of Health guidelines."

karl said...


Thanks - again it appears that these are supplied by two different companies - thus the control diet may vary in non obvious ways besides the difference in sugar and fat.

I suppose these folks missed Richard Feynman's Cargo-cult lecture - or just found it old fashioned.. What a waste of research resources.

Bill said...

Agreed 100%. Differences in the phenotypes of rodents fed chow and purified diets could be due to almost anything… and every once in a while, one of these little gems pops up to remind us of all the physiologically relevant nutrient AND non-nutrient components present in chow but not purified diets:

On another note, can you [or anyone] explain why you say these mice have ‘inappropriate insulin sensitivity?’

Bill said...

Agreed 100%. Differences in the phenotypes of rodents fed chow and purified diets could be due to almost anything… and every once in a while, one of these little gems pops up to remind us of all the physiologically relevant nutrient AND non-nutrient components present in chow but not purified diets:

On another note, can you [or anyone] explain why you say these mice have ‘inappropriate insulin sensitivity?’

Purposelessness said...

You make a good point, karl. Though I tend to read these studies not really in terms of different diets, when I see high fat diet I just think "diet that makes mice fat and sick" amd if I read chow I think "diet that doesn't make mice as fat or sick".

Scott Russell said...

I think the vitamin overlap you're thinking of has to do with absorption. AKA high vitamin a intake can be damaging, but this effect is protected against with adequate vitamin d and k intake. I think it was based on fat soluble vitamin transporters.

@George, I like the idea of dehydrogenase overlap. I definitely know that hangovers while fully keto are miserable. Did a little digging and found that BHB and acetaldehyde overlap in their dehydrogenase activity. Sigh.

I'd still be amazed if low-carbers are actually retinol deficient on a wide scale. I know personally, my retinol intake is massive.

Puddleg said...

@ karl, the hyperthyroid state is upregulating beta-carotene conversion to retinol to increase mito energy production. But if there WAS a deficiency of retinol or carotene, what would happen to the thryroid?
I think the term would be "ketosis-induced retinol sink".

It is even possible that carotenes may be important in their own right for delivering retinol to some tissues when status is low. Retinol formed in situ may be advantageous at times, just as NAD+ from tryptophan might be occasionally preferred over that from dietary niacin.

Bill said...

@GH, that’s an interesting point. I had always only considered carotenoids as to be either digested into retinols or sloughed off in skin cells (depending on need). Never really thought of them as potentially active storage forms of retinol. Any data?

Puddleg said...

None whatsoever Bill! But restricting carotene might be worthwhile in hyperthyroidism. Or not. It's unusual that there are no conditions where restriction of dietary carotenoid is mooted as a therapy.

My idea was, that biosynthetic pathways for things usually found in diet but able to be synthesised, like niacinamide or retinol, may involve enzymes adjacent to the sites where they are needed. And in some cells or under some conditions this might be an advantage, for example if there was some stressor promoting breakdown of pre-formed retinol on its travel from its normal storage site, or NAD sinks elsewhere in the cell.
A question of location, location, location.

Puddleg said...

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression

The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

Bill said...

Location, indeed! Presence of the enzyme responsible for cleaving beta-carotene (BCMO1) in liver, kidney, brain, testis, and adipose, certainly bodes well for your theory

Peter said...

Hi all,

@George, I was wanting to get on to all sorts of ideas related to failure to develop insulin resistance but your ref

is just too closely related to the current posts and to where I'm thinking of going that it needs a post in its own right. It's pulling me away from membrane bioenergetics and all sorts of evolutionary stuff! Half term has very little free time.

Q How do you limit NADH production (with associated NAD+ depletion)?

A Produce FADH2 rather than NADH

And the follow on:

Q If you are running on FADH2 and are protected against NADH excess by shutting down glucose supply (read insulin resistance), what happens when you side step insulin resistance?

A NADH excess (read cancer).

And finally:

Q How do you side step the protective effect of insulin resistance on the excess production of NADH per unit NAD+?

A Hyperglycaemic glucose uptake, fructose or alcohol. Oh, and PUFA of course.

The effect of the last answer (PUFA excepted) has to be big enough to over ride any effect they have on limiting glucose usage, ie there is a threshold for each.

This is really cool!


Gadfly said...

@ Nigel

Tomorrow, I want you to take 10% of all your meals (by weight) as sugar. Get back to us about whether or not that seems like a lot.

Puddleg said...

Ketone bodies help to correct the NAD+/NADH imbalance too:

In the study by Maalouf et al. (2007b), ketone bodies decreased NADH levels in intact neurons and in isolated mitochondria but did not affect glutathione levels. Furthermore, ketone bodies prevented the inhibition of mitochondrial respiration by calcium in the presence of pyruvate and malate but not succinate. Given that NADH oxidation correlates with decreased mitochondrial formation of reactive oxygen species (Duchen, 1992; Kudin et al, 2004; Sullivan et al, 2004a) and that pyruvate and malate drive mitochondrial respiration through complex I, the source of reactive oxygen species in neurons (Turrens 2003), these findings strongly suggested that ketone bodies decreased the production of reactive oxygen species by enhancing complex I-driven mitochondrial respiration rather than increase antioxidant factors such as glutathione.

Ketone bodies improve mitochondrial respiration and, as a result, increase NAD levels relative to NADH, decrease reactive oxygen species (ROS) formation and enhance ATP production. (fig 2)