I started with this paper about in vivo superoxide detection in the brain but, apart from the technique, there was no examination of the response to hyperglycaemia so I moved on. The next paper by the same group is looking at superoxide and mitochondrial function/health in the kidney under various models of diabetes. The general principles appear similar in neurons and kidney cells.
An in vivo technique to view superoxide is really useful. I have alluded to a certain discomfort in examining electron/oxygen interaction in mitochondria within cells/mitochondrial preparations under room air, with a partial pressure for oxygen of around 150mmHg (sorry for the non SI units, showing my age there!). There is no way that normal mitochondria are exposed to this much oxygen, a little browse around pubmed suggests that the best in vivo estimate is around 40-50mmHg, subject to some debate. That's without even thinking about what CO2 partial pressure you should use for cell culture... So observation in vivo takes care of a lot of this. If an electron is thrown out of the respiratory chain (I feel nothing in the ETC is accidental) the chances of it dropping on to an oxygen molecule seem somewhat higher if we have three times the oxygen partial pressure than the system was designed to work under. If the electron wasn't destined for an oxygen molecule, where else might it have been going?
The first point has to be that in two models of type one diabetes there is less superoxide production in the kidneys of diabetic mice in vivo than control mice, that's Figure 1 section C. Going ex vivo (I probably have a full post on the problems with this ex vivo section) we have the same effect demonstrated using a paramagnetic technique, that's Fig 2C. The reduced superoxide in diabetic kidneys was confirmed in the tissue homogenates under relatively normal metabolic substrate supply. Exposing the preparations to glucose at 25mmol/l has no effect on superoxide generation from the control kidney homogenate but actually reduces it, rather a lot, in the diabetic derived homogenate.
NOTE If you follow the text through about Fig 1C and their SOD2+/- mice you will find that the data is not very accurately described. So caution here. The SOD2+/- had a non significant increase in mitochondrial superoxide in Fig 1C, so it is hardly surprising this did not rescue the diabetic mice from renal disease in Fig 3A and F. I don't like their writing about this whole SOD2+/- section. Definite caution. END NOTE.
The paper has, amongst its problems, a lot of very perceptive points which make a great deal of sense. It's quite hard to know where to start. Let's begin with the failure of superoxide production.
So this paper flies in the face of the Protons concept of hyperglycaemia driving reverse electron flow from mtG3Pdh through complex I to generate insulin resistance. That too is probably another post, comparing the diabetic state with the non diabetic hyperglycaemic state. Anyhoo. The group rather like Crabtree. So do I. The Crabtree effect, the shutting down/mothballing of mitochondrial function, is an adaptation to oversupply of glycolysis derived substrates. It allows a limit to be set on the throughput of pyruvate to mitochondria and jettisons any excess as lactate. This situation, once it is established, is probably quite different to the situation which leads to its adoption.
Chronic hyperglycaemia induces the Crabtree effect and down regulates mitochondrial biogenesis, mitochondrial repair and electron transport chain function. It not only does this but it also phosphorylates the pyruvate dehydrogenase complex, very specifically, and this directly shuts down input to the TCA from glycolysis (or input from lactate itself, if we want to apply this concept to neurons, as we might). This is all in the paper. Of course I would add that it doesn't affect ketone derived acetyl-CoA input to the TCA, although the ketone derived acetyl-CoA will be processed by a degenerate electron transport chain...
Under sustained hyperglycaemia there is an excess of calories which leads to a failure to activate AMP kinase, a core sensor of energy abundance which is phosphorylated under hypo caloric conditions. AMPK regulates PGC 1 alpha, a messenger to trigger mitochondrial biogenesis. But the central link, the activation of AMKP, is mitochondrial derived superoxide. And, oddly enough, one of the functions of AMPK activation is the generation of mitochondrial superoxide. A self sustaining loop.
The group administered rotenone to control mice. Now, the effect of rotenone on superoxide generation appears (in general) to be rather dose rate related. In the present study the dose rate was chosen so that there was a near complete suppression of superoxide production from the ETC of the mice. Acute suppression of superoxide results in the reduced phosphorylation (reduced activation) of AMPK and increased phosphorylation of PDH, which shuts it down. This loss of superoxide is a short term mimic of the long term established Crabtree effect. No superoxide, no mitochondrial maintenance. Consider that chronic high dose rotenone poisoning is a standard model for Parkinsons Disease and you begin to see the importance of superoxide in the brain. Long term hyperglycaemic failure to generate superoxide is probably a more normal route to neurodegeneration than rotenone in most (but not all) neurodegenerate humans...
The fall in superoxide production in diabetic tissue homogenates again pulls me back to brain function. Crabtree suppresses hyperglycaemic superoxide production, i.e. the effect is antioxidant. Let's look at what glucose does to neurons from this paper which we've chatted about before. Here's the only bit I'm interested in today:
"Indeed, it has been shown that glucose is used by neurons to maintain their antioxidant status via the pentose phosphate pathway (PPP), which cannot be fueled by lactate (Magistretti, 2008; Herrero-Mendez et al., 2009)"
It's impossible over emphasise the importance of that sentence. It says it all about why neurons should run on lactate! To avoid upregulating antioxidant status.
What does increasing antioxidant status do to superoxide signalling? The term f*cked comes (unavoidably South Park-ishly) to mind. There are a swathe of papers showing that the antioxidant status in neurons of AD and PD patients is upregulated.
Once you go with Crabtree you can see that glucose and PPP driven antioxidant upregulation might be all that is needed to lose superoxide signalling and destroy mitochondrial function. Lactate does not do this. Lactate does not induce the Crabtree effect.
Let's be very specific: Glucose, under the Crabtree effect, triggers a cascade which ends up with failure to generate superoxide and this maintains mitochondrial shutdown. Up regulating antioxidant status may theoretically be helpful in dealing with non mitochondrial superoxide generation, but it's not going to help signal for mitochondrial biogenesis.
High glucose exposure generates glucose dependence. This is a recurring theme and is core to neurodegeneration. I look at safe starches and can see that, if you are living with the Crabtree effect in key neurons, some starch/glycolysis might make you feel better if you are ketogenically hypoglycaemic, but it's not going to help un-Crabtree your mitochondria. On the other hand I can't see that pushing starch to a level which produces hyperglycaemia is anything other than damaging, as opposed to merely neutral as it might be when your pancreas does its job effectively.
I'll take a break before going on to the sections on mitochondrial deletions and respiratory chain oxidative damage elsewhere in the paper. Or maybe I should talk about the bits I deeply dislike related to oxygen pressure and superoxide.