High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates
is today's obligatory reading. What is it all about? They are trying to tease out what is happening directly within the mitochondria when different metabolic substrates are offered to the citric acid cycle. This is not easy. Their mitochondrial model is far from reality but is the best we can do at the moment, certainly in 2008. The authors are well aware of this and discuss the issue in some detail. I feel, personally, that what they have found makes perfect sense and can be extended, as is my tendency, to make a few more links in to basic physiology and, eventually, insulin resistance.
It's also worth pointing out that they are dealing with isolated mitochondria. No cells, no insulin, no adipocytes, not even any cytoplasm to support glycolysis. You can't run mitochondria on glucose! A model.
Here is their starting point:
"While it is generally accepted that mitochondria are the main site of cellular ROS production, studies in isolated mitochondria have shown that the amount of H2O2 released by mitochondria (H2O2 originates from the dismutation of O2•− , and is much easier to measure than O2•−) undermost conditions is rather modest (<0 .1="" 1="" 2000="" a="" and="" are="" being="" br="" by="" cellular="" chain="" complex="" condition="" derives="" diverse="" diverted="" driven="" electron="" electrons="" exception="" far="" formation="" frequently="" from="" h2o2="" highest="" holds="" i="" in="" indeed="" is="" isolated="" largely="" leak="" measurements.="" mg="" min="" mitochondria="" mitochondrial="" notion="" of="" only="" passing="" per="" pmol="" protein="" quoted="" rates="" release="" reverse-electron="" ros="" source="" strongest="" succinate="" superoxide="" that="" the="" these="" this="" through="" tissues="" to="" transfer="" true="" under="" up="" value="" yielding="">
And what is "needed" for this spewing of electrons on to molecular oxygen?
"In isolated mitochondria, reverse-electron transfer through complex I occurs when the ubiquinol pool is in a highly reduced state and a strong membrane potential is present, i.e. the energy of the membrane potential drives the ubiquinol (with electrons provided by succinate)-dependent reduction of NAD+ to NADH with electrons passing in the reverse direction through complex I ."
We talked about the CoQ couple in the last post. Here it is being referred to as ubiquinol, the reduced form of CoQ. A reduced CoQ couple is essential for reverse electron transport.
Also note the necessity of "a strong membrane potential".
Back to the study:
They isolated mitochondria and studied them asap, in the short time-window during which they remain remotely functional. They fed them with various components of the citric acid cycle and looked at H2O2 production, a reasonable surrogate for superoxide.
Adding in glutamate mixed with malate is a classic combination for driving NADH utilisation through complex I. The mitochondria generate about 30pmol/min of H2O2 under these conditions. This is not a lot and some labs report the amount as being close to zero.
Driving complex II (succinate dehyrdogenase) directly, using succinate but not supplying any NADH generators, produces rather more H2O2, around 400pmol/min. A ten fold increase.
Driving both complex I and complex II with a combination of all three of the above substrates can produce over 2000pmol/min H2O2. Sometimes but not always, as we shall see.
All of these findings where achieved at physiologically plausible concentrations of substrate. However oxaloacetate, formed in-situ from the malate supplied, turned out to be a confounder for that last result. Oxaloacetate is an inhibitor of complex II, so reduces reverse electron transport through complex I. With succinate dehydrogenase partially blocked the CoQ pool is less reduced, so more ready to accept electrons from Complex I, rather than driving them the wrong way through it to generate superoxide.
The end conclusion the paper came to is that anything which depletes oxaloacetate will disinhibit succinate dehydrogenase, reduce the CoQ pool and at the same time increase the likelihood of reverse electron transport through complex I, leading to superoxide generation.
The follow on from this is that anything supplying large amounts of acetyl-CoA will automatically deplete oxaloacetate because citrate synthetase consumes oxaloacetate as it combines it with acetyl-CoA to start the citric acid cycle with, err, citric acid. The group did it with pyruvate. They did it with palmitoyl carnitine. Pyruvate = carbohydrate. Palmitate = fat.
In the real world the cycle turns and the acetyl-CoA source which initially depleted oxaloacetate eventually restocks the oxaloacetate supply (except in the ketogenic liver of course). But the initial oxaloacetate depletion sends a signal. The mechanism is the activation of succinate dehydrogenase, which both allows the citric acid cycle to cycle and promotes a significant reduction of the CoQ couple which can generate superoxide.
This is a basic physiology paper. There is no suggestion or mention of gluttony, however coded. But excess acetyl-CoA might be suggestive of freely available metabolic substrate. But no comment in this paper, except my me. You could simply say that overeating supplies too much acetyl-CoA. Hmmm, maybe I should go in to obesity research? But there are additional considerations, like this one:
I would just like to point out that beta oxidation, through electron-transferring flavoprotein dehydrogenase (previously described in Peter terms as complex II-like), reduces the CoQ pool independent of succinate dehydrogenase (genuine complex II). Would you expect a reduced CoQ pool from beta oxidation to predispose to superoxide generation? Might this be a Good Thing or a Bad Thing? Think carefully about the semantics here. What do we mean by "good" and "bad"?
Superoxide is important. It speaks to tissues far away from the mitochondria which generated it in addition to the cell containing them. We need to translate this in to more familiar terms, which has been hard work avoiding slipping in to in this post!
The next post is about insulin resistance. You would be dead without it.