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.
Peter
