Saturday, July 02, 2022

Brand on 4-HNE facilitated uncoupling

This is one of those papers with strong confirmation bias reinforcing properties, so care is needed. I do indeed have some difficulties with the paper, some of which I'll point out as we go along.

Brand again.

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

So. They wanted to look at how mitochondrial membrane potential controls uncoupling. They also wanted to differentiate between "endogenous" uncoupling and 4-HNE facilitated uncoupling. This is not easy. Certain steps need to be taken. Each step moves you away from reality and closer to answering the question you have in mind. Which may or may not reflect reality.

They used adenine nucleotide translocase (ANT), the enzyme which exchanges ADP for ATP across the inner mitochondrial membrane because a) it uncouples and is closely related to standard UCPs and b) there is a ton of the stuff on all mitochondrial membranes which makes quantitative measurements possible. They also have an inhibitor which allows more information to be extracted.

Here's the setup:

They needed to eliminate FFA induced uncoupling (and energy supply) so added BSA to the medium to soak up endogenous FFAs.

They needed to actively control energisation of the mitochondrial membrane so blocked complex I completely with high dose rotenone to stop oxidation of NADH from endogenous malate or glutamate catabolism.

When they wanted to activate energy supply they used succinate alone working through complex II, which is still functional.

Obviously, to look at uncoupling, they had to block ATP synthase with oligomycin.

Next they equilibrated the proton concentration across the inner membrane using the H+/K+ exchanger nigericin to allow a them to use a voltage only sensitive probe to measure membrane potential.

The degree of mitochondrial activation from succinate was controlled by either titrating with malonate (complex II inhibitor) or cyanide (complex IV inhibitor) to give a stable measurable membrane voltage.

Finally, apparently 4-HNE is active at around 1μM but you need higher concentrations to get an easily measurable effect, especially in the presence of de-fatted BSA, so here they used 35μM.

Having done many (but not all) of the above things they end up with this graph in Figure 1:

which makes everything clear.

This is my biggest problem with the paper. From graph A they extract the information in bar chart B and bar chart C which give details of the calculated proton leaks at 2.5 minutes and 5.0 minutes in to activation with succinate. Which demonstrate quite clearly that HNE facilitated uncoupling via ANT has kicked in by 2.5 minutes and is marked by 5 minutes. This is chart B, extracted from the grey lines in graph A at around 137mV, both time points:

And chart C is from the dark lines in the lower right hand end of graph A at 175mV, only at time 2.5 minutes: 

My problem is that, after many hours trying to curve fit and calculate in Powerpoint, I can never quite extract the numbers from graph A to fit charts B and C.

If we assume that the research group have the raw data are getting their calculations correct we can have this as the main summing up:

"In liver, time at high membrane potential in the presence of HNE results in a striking increase in proton conductance, which may be interpreted in two ways. First, HNE may slowly form covalent adducts with ANT, progressively converting ANT into a form with high proton conductance, or secondly, HNE metabolism may progressively form fatty acids [41], which then activate proton conductance [28]."

The authors consider option two to be unlikely.

If this paper really does hold water we have a situation where high membrane voltage, working through 4-HNE, produces long acting uncoupling in proportion to the time spent at high membrane voltage.

Hence you can run a mitochondrial preparation with supra maximal substrate supply and, rather than exploding/imploding, the correct physiological "decision" is to uncouple in proportion to the excess membrane potential and stabilise it at something close to functionality.

High delta psi -> ROS -> 4-HNE -> ANT/UCP -> damage limitation.

That's how things should work.

Of course that's not always how things pan out.