Wednesday, July 25, 2012

Protons: Superoxide

Here 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•− [6], and is much easier to measure than O2•−) undermost conditions is rather modest (<0.1%electron leak). The exception to this is succinate driven reverse-electron transfer through complex I, a condition yielding by far the highest rates of H2O2 release in isolated mitochondria [7–10]. This holds true in mitochondria isolated from diverse tissues (up to 2000 pmol of H2O2/min per mg of mitochondrial protein [7–15]). Indeed, the frequently quoted value of 1–5% of electrons passing through the chain being diverted to the formation of superoxide is only true under this condition [16,17], and the notion that mitochondria are the strongest source of cellular ROS largely derives from these measurements."

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 [18]."

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.

Peter

20 comments:

George Henderson said...

"good" is adaptive, supportive of homeostatis, "bad" is maladaptive, disruptive of homeostatis?
When they said "fat burns in a carbohydrate flame" back in the 1940s, they meant oxaloacetate

bopes said...

Heh. I'll have to wait for the translation. Based on the paucity of comments I guess everyone else is too . . .

While we wait, superoxide looks like a Good Thing and a Bad Thing, at least according to wikipedia: "Superoxide is biologically quite toxic [sounds bad!] and is deployed by the immune system to kill invading microorganisms [sounds good!]. In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for use in oxygen-dependent killing mechanisms of invading pathogens [sounds good!]. . . . Superoxide is also deleteriously produced [sounds bad!] as a byproduct of mitochondrial respiration (most notably by Complex I and Complex III), . . . Superoxide may contribute to the pathogenesis of many diseases . . . , and perhaps also to aging via the oxidative damage that it inflicts on cells [sounds bad!]."

Eva said...

It's so amazing that tiny little mitochondria even by themselves can be so complex and adaptable!
-Eva

john said...

Hi Peter,

Mostly this is over my head, and I can only sort of see where this is going, but anyway... Complex 1 activity is generally associated with markers of aging. But inhibiting complex 1 with a variety of things *usually* ends up badly. Ketones protect against this "toxicity," and arctigenin, by seeming to inhibit complex 1, actually shows positive effects in ob/ob mice. I believe I have seen life extension in nematodes from reducing complex 1, but I forget the inhibitor.

The above is pretty clear...So you are making a "small-scale" argument analogous to how FIRKO, etc explains low carb diet trials?

Glutamine supplementation and methionine restriction both increase fat oxidation, and both have plausibility for life extension.

Donny, who comments here sometimes, has written on Krebs/citric acid, while Art Ayers has written on superoxide. One must peruse many sources to understand this properly.

Sidereal said...

I'm hoping there'll be a For Dummies version eventually. :)

George Henderson said...

Just posted a nicely confusing blog on calories vs carbs as drivers of de novo lipogenesis.

http://hopefulgeranium.blogspot.co.nz/2012/07/what-does-hypercaloric-actually-mean.html

Or click on my link by the Blogger B above

Peter said...

George, absolutely agree. I'm looking at water fasting as hypercaloric nutrition on a cellular basis. Palmitic acid >0.5mmol/l +/- total FFA deliver at 1.5mmol/l is the result of starvation macroscopically but that may not be how it looks to an individual cell... Unless you don't make a whole lot of used of FFAs!

Sorry for the delay in the next post, but at least the BBQ is assembled during Hazel's second nap and the w/e shop is done before the w/e! Woo hoo, getting on!

Peter

Jane said...

Very interesting paper. I've been wondering for years whether mitochondrial superoxide might oscillate, because it would explain such a lot. In pancreatic beta cells, if you add oxaloacetate to inhibit succinate dehydrogenase, things oscillate. Have a look at this paper.


Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentrations of effectors.
http://www.ncbi.nlm.nih.gov/pubmed/9392486

Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic beta-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 micromol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 micromol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. ..

Peter said...

Jane, the other related insulin secretagogue is succinic acid ester. This appears to drive complex II directly and probably generates reverse electron transport superoxide. There are old papers and a few rat models but no free radical examination. Also nothing on cells other than pancreatic tissue culture... Obviously no consideration of oscillatory activity. I also noted during reading about insulin induced insulin resistance that this does not occur if the insulin administration is pulsatile.

Peter

George Henderson said...

An interesting aspect of mitochondrial H2O2 is that protection against it is almost wholly dependent on an antioxidant use of (heme) iron. Catalase is the most efficient of the antioxidant enzymes. Unlike the selenium-based peroxidase enzymes (which also reduce H2O2 to H20) it doesn't require reduced glutathione.
Iron, pro-oxidant?
You'd die of oxidative stress without it.

Sarah said...

Peter, I have been following your posts on mitochondrial function with great interest although the details are way over my head. I have, for many years, been suffering from CFS caused from mitochondrial dysfunction - as proven by Dr Myhill's blood test. My ATP cycle was not working properly and my mitochondrial membrane was much too permeable. My overall energy output was estimated (from the results) at about 50%.

I struggled for a long time to stick to a high fat diet as it is so alien to what we are 'told' is nutritionally correct. However, for the past couple of weeks I have been eating only natural meat, fish, eggs, butter, cream and some unhomogenised milk in coffee (to keep my carbs up a little bit) and I feel so much better. I don't think I will bother going back to eating vegetables because I feel I should! All my stomach and digestive issues have cleared up and I already feel like I have more energy. Plus, I don't really feel hungry much and I have lost weight.

I think your blog is great and I just wish I had found it years ago. I, almost daily, refer to it for your thoughts and clarification on certain matters.

Please keep it up!!

Sarah

Jane said...

Peter, do you have any ideas about how mitochondrial superoxide prevents GLUT4 translocation?

I thought it must be AMPK inhibition, but superoxide activates it. I suspect calcium is involved, and the PPP. Apparently the PPP can be activated by mitochondrial ROS, and it can regulate both calcium and AMPK.

George Henderson said...

@ jane, a likely mechanism is ROS inhibition of Fox01 phosphorylation.
this increases Fox01 nuclear transcriptional activity, promoting gluconeogenesis and IR. Fructose can do this too.

@peter, this is interesting:

Activation of SIRT1 has been reported in the hearts of
fructose-fed mice through increases in NADC
/NADH ratio
(Pillai et al. 2008). Similarly, increased NADC
/NADH ratio is
associated with increased SIRT1 protein levels in the livers of
fasted rats (Rodgers et al. 2005). Fructose-induced gluconeogenesis can be suppressed by inhibition of the electron
transport chain. This may be due to a decrease in NAD+/NADH ratio that occurs in parallel (Pryor et al. 1987), but the role of SIRT1 in the hepatic response to fructose has yet
to be examined.
In this study, we demonstrate that fructose induces hepatic gluconeogenesis and lipogenic gene expression through a SIRT1-dependent mechanism. This occurs through a
complex I-mediated increase in NAD+/NADH ratio. This study indicates that SIRT1 activators may cause adverse metabolic consequences if used as a therapeutic strategy
for T2DM.
(so much for resveratrol!)

Fructose-Sirt1 PDF

George Henderson said...

By NADC is meant NAD+, lost in the change of code from pdf to HTML and not caught by me.

This is really interesting; reduction of mitochondrial intermembrane electron carriers by complex 2 beats fructose DM2 effects?
So carb restriction does defuse the fructose bomb, and here is the mechanism?

George Henderson said...

A quick question:
does increase in reduction of ubiquinone automatically mean increased NADH/NAD+ ratio?
I am assuming this is so, but don't want to take it for granted.

Peter said...

George, sorry I'm so slow on responses to the biochemistry. It's quite hard to tease this out. I think the whole idea that CoQ reduction generates reverse electron transfer (aka superoxide) is based on mitochondrial studies where they feed substrates directly to complex II, and then either do or don't add complex I feeding NADH generators. Using FADH2 from beta oxidation in isolation from the rest of beta oxidation (ie some NADH and a ton of acetyl-CoA) would be basically impossible. You would need to supply, to the inner mitochondrial membrane, FADH2 reduced electron transfer flavoprotein, that's not quite like getting some succinate or glutamate in there. You might be able to use mitochondrial fragments but you are getting far from reality by then.

The point is very interesting as I suspect that an increased NADH level relative to NAD+ would favour superoxide production as there is no where for the reverse transported electrons to go to. No NAD+ available, NADH isn't going to accept anything, where's the oxygen??? Seems likely...

And of course statins deplete the size of the CoQ pool, so the degree of reduction per electron transported should increase. Increased incidence of T2DM is pretty ubiquitous in the statin studies. These are the tip of the iceberg. Everyone probably has depletion of the CoQ pool on a statin, just a few cross the arbitrary boundry in to diabetes during the duration of a statin trial.

Peter

George Henderson said...

Anything that inhibited CoQ10 synthesis would also inhibit conversion of vitamin K1 to K2 MK4, as far as I can tell.
This could have negative consequences on brain sulfatide levels that may help account for memory loss syndrome.

In real life it is probably impossible to run mitochondria on a single substrate, there is always other fuel in the cell, but the ratios - the fuel mix - changes. Dietary composition, physiological state, cell type, and so on influence the mix.

George Henderson said...

Vit K2 MK4 conversion from phylloquinine requires HMG-CoA reductase:
http://jhs.pharm.or.jp/data/56(6)/56_623.pdf
(p. 629)

Vit K1 conversion and brain sulfatides:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2891353/

If you really must take statins, it might pay to supplement vit K2 (as MK4) as well as ubiqinone.

(are some of the nugatory benefits of statins related to a wafarin-like effect?)

George Henderson said...

Just posted a blog on statins and Vitamin K2

majkinetor said...

Vitamin K2 is actually next to CoQ10 and it looks like some mitos prefer Q10 and some K2 in ETC.

Thats recent eureka discovery.


Vos, Melissa, Giovanni Esposito, Janaka N Edirisinghe, et al. 2012Vitamin K2 Is a Mitochondrial Electron Carrier That Rescues Pink1 Deficiency. Science (New York, N.Y.) 336(6086): 1306–1310. http://www.ncbi.nlm.nih.gov/pubmed/22582012, accessed August 5, 2012.