Protons: Metformin

I'll just stick this post up to get it out of the way. I was going to go on to acute uncoupling next but the link from O Numnos in the last post comments is too good not to post about. It goes some way to tying weight gain in to LACK of superoxide, so brings the thread of insulin as a "satiety" hormone and this thread on weight gain as a failure to generate superoxide in adipocytes (good and bad) together. Might take more than one post... The summary of what's coming: Is insulin a satiety hormone? Only in so far as becoming stable-obese limits your hunger. Anyway, here are a few more thoughts on superoxide first.

Metformin is generally considered to be a Good Drug.

Interestingly it is an inhibitor of complex I of the respiratory chain, which is almost certainly its primary site of action. It aborts glycolysis to lactate because pyruvate is not much use to mitochondria with blocked complex I. Acute exposure to metformin in tissue culture generates a ton of superoxide. Just what you would expect to benefit someone with T2DM!

Let's have a look at this rather nice paper.

They are using differentiated 3T3-L1 adipocytes, a strange beast if ever there was one, but "everyone does it".

They are working under room air with 25mmol/l glucose, supplemental pyruvate, glutamine and 1000pmol/l insulin. These cells are being driven, hard, generating NADH which works through complex I. Complex II will be supplying some FADH2 but there is zero beta oxidation, unless the fatty acids in the adipocyte stores are being accessed. With insulin at 1000pmol/l this is not going to be happening.

Here is the effect of metformin on oxygen consumption:



A dose dependent fall, exactly what you would expect when blocking complex I. Here is the effect of 1.0 mmol/l metformin on oxygen consumption with time:



Nice curves! And here is the effect on ECAR, a surrogate for lactate generation, over 24 hours:



Metformin is only a relatively weak inhibitor of complex I, the incidence of life threatening lactic acidosis is very low. Not so for the more effective biguanides, phenformin and buformin. Obviously the latter two are no longer used clinically, there were too many hiccups.

Now, here is the level of DHE fluoresence, a specific marker of superoxide production. It's being compared to rotenone (remember Coopers Demodectic Mange Dressing? Thought not!), a serious complex I inhibitor.



Metformin is pretty good at generating superoxide. A bit counter intuitive for a drug which is the best treatment, short of insulin, for managing T2DM, a condition essentially defined by failure to overcome insulin resistance (aka superoxide production).

Hmmmmmmmmmmm.

Now, do 3T3-L1 adipocytes like being in forced to live on ATP from glycolysis plus whatever oxphos can be squeezed through metformin inhibited complex I? Annexin V is a marker of very, very unhappy cells. This is what metformin does to the % of cells which are moribund in culture:



So what is going on? Is metformin going to kill our fat cells in vivo?

It's all back to tissue culture conditions. Glucose at 25mmol/l makes the cells utterly dependent on a combination of glycolysis and NADH oxidation at complex I, plus a little FADH2 from succinate metabolism. Our adipocytes are not in this situation.

Now look at this graph:



This is in starvation medium. Only 2.5mmol/l glucose, no pyruvate, no glutamine. I think insulin is still supramaximal, but who cares about insulin when glucose is down at 2.5mmol/l in tissue culture? But here is the really interesting bit: They had also added 0.3mmol/l of palmitic acid to both the control cells and to the metformin cells. Compare it to the graph below, which is the same situation but with glucose and NADH drivers replacing palmitate:



So: Starvation medium plus palmitate completely reverses the fall on oxygen consumption produced by metformin. Palmitate plus starvation medium, even with metformin, actually allows more oxygen consumption that cells running flat out on glucose in the absence of metformin. It's what you would expect, the respiratory quotient is lower for fatty acids than for carbohydrate.

Metformin does not stop fatty acid oxidation. You do need some complex I activity to provide the NAD+ for beta oxidation, but no one is suggesting there is a complete block of complex I by metformin, it's not mange dressing.

So where do the free radicals come from with metformin? I would guess that the citric acid cycle still cycles, there is a build of of NADH due to complex I inhibition and complex II still reduces the CoQ couple. This could allow reverse electron transfer through whatever complex I functionality is left. There are absolutely no data on this, but I like the idea.

The group didn't look at superoxide production under starvation conditions or under starvation plus palmitate. I had a nice email reply to my query from the corresponding author along these lines, there are no data about this, as yet. I would expect the levels of superoxide to be comparable, with metformin being able to mimic palmitate based metabolism in the face of massive fat-free glucose supply, certainly for superoxide generation.

So, superoxide is insulin resistance. Adipocytes under metformin make a ton of superoxide. Are they insulin sensitive or resistant? Resistant of course.

Does an adipocyte which is insulin resistant listen to insulin's orders to store fat? Of course not. "Normal" insulin resistant adipocytes spew free fatty acids to the limit of albumin's transport provisions, with a few other moderating factors.

A metformin poisoned adipocyte is desperate for proton pumping substrate and complex I is doing bugger all to help. But electron-transfering flavoprotein dehydrogenase works perfectly well to allow an alternative electron supply...

Adipocytes under metformin have no choice but to burn fat. In vivo they have a barrel load of the stuff available as soon as they stop listening to insulin. They appear to use fatty acids for metabolism rather than dumping them as FFAs to plasma. Sounds like a recipe for treating metabolic syndrome to me.

Oh, that's what metformin is used for! Well I never...

So, do I think metformin causes adipocytes to become insulin resistant? Of course I do. Is this a Good Thing? You decide.

Peter

BTW Want an opposite to metformin? You can make adipocytes more sensitive to insulin with the thiazolidinediones. They allow insulin to become more effective on already over-distended adipocytes and generate lots of extra, nice, new, ready-to-stuff-with-fat adipoctes. They make you fatter. What would you expect?