Tuesday, August 07, 2012

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).


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.


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?


ItsTheWooo said...

"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."

One of many excerpts on this blog which illustrate your brilliance.
It explains everything; why metformin has so many mechanisms of action to help weight loss and type 2 diabetes. It causes weight loss because adipocytes cannot store fat, and are increasingly using fat for energy at rest. FFA are reduced and insulin sensitivity improves. Like alcohol metformin prevents the liver from generating glucose at rest, and explains the hypoglycemia risk in mildly diabetic or non-diabetic patients.

The TZD drugs are the polar opposite and help diabetes at the expense of increased fatness (and cancer). The generation of new, small adipocytes with these drugs also increases adiponectin, which can further help type II. TZD drugs dial things back to the early diabetic state, when your fat tissue isn't yet overdistended and you still have a lot of obesity left before inflammation and genetics stops the fattening process, rendering you with high A1c.

Kindke said...

Peter I was looking over this paper which seems to be a followup from the other paper about insulin and the brain.

They mention that in common obesity, adipocytes are still insulin sensitive but its the brain that develops the insulin resistance. Your comment in the last post was interesting, about how brain IR stops generation of palmitoleate thus contributes to whole body IR.

PS. I was trying to find the composition of the diet they fed the rats in that paper, they mention it as (Modified Lab w/10% Lard 57IR, LabDiet, St.
Louis; MO ) However when I looked at LabDiet's website I couldnt find 57IR diet in any of thier products. Hmmm!

Kindke said...

Effect of thiazolidinediones on body weight in patients with diabetes mellitus.

Treatment of diabetes mellitus with medications, including insulin, sulfonylureas, and thiazolidinediones (TZDs), often leads to weight gain through a variety of mechanisms.

Data indicate that with TZD treatment, there is a favorable shift in fat distribution from visceral to subcutaneous adipose depots that is associated with improvements in hepatic and peripheral tissue sensitivity to insulin

A weight-management program combining a low-calorie, low-sodium diet with education and behavior modification has been shown to be effective in patients with type 2 diabetes being treated with TZDs.

I.E. we need to inject more "willpower" into our T2DM patients to overcome the affect of the drug attempting to make them fat!!!!

Also moving visceral fat to subQ fat is a double edged sword. Losing Visceral fat on a ketogenic diet is super easy, however losing subQ fat on any kind of diet is substantially harder.

majkinetor said...

Thx for the Metformin post. It looks like Metformin is one of those rare "big pharma" good inventions.

Do I view this thing correctly when I think about Metformin correlation with low carb diet: since glucose generates 5 times more NADH+ then FADH2 it increases complex 1 activity. Beta oxidation makes more balanced use of C1 and C2 thus, compared to high carb diet it will decrease activity of C1 just like Metformin.

majkinetor said...

... and from Nick Lane book it looks like balanced utilization of C1-C4 leads to healthy and happy mitochondria.

Jane said...

If metformin is only a weak inhibitor of complex 1, how can it make so much superoxide? The paper says nothing about the possibility that mitochondrial superoxide is going to the cytoplasm and generating more superoxide via NADPH oxidase.

This does happen. Mitochondrial ROS can activate the pentose phosphate pathway to produce NADPH for NADPH oxidase.

Palmitate can activate NADPH oxidase, BTW.

George Henderson said...

Voltage-dependent Anion Channels Control the Release of the Superoxide Anion from Mitochondria to Cytosol

Cytosolic NADH excess is relieved by the plasma membrane oxidoreductase complex, transferring electrons to plasma O2.

I though pyruvate was the complex 2 substrate and minimized complex 1 involvement? Must reread that bit.

George Henderson said...

Oxybarbiturates (secobarbital, amobarbital, amytal) also inhibit complex 1.
"Mitochondrial depolarization with reduced ATP synthesis can
result from impaired electron transport (or substrate supply) or
enhanced inner membrane proton permeability. In cells with
active glycolysis, the former will result in only a partial depolarization, because ATP synthase reversal [is this what is meant by running it backwards?] can maintain a suboptimal membrane potential even in the face of total respiratory
inhibition by consuming ATP generated by glycolysis"


Respiratory inhibition in more ways than one;
Goodnight Marilyn.

George Henderson said...

If loss of membrane potential can cause ATP synthase to run backwards, this is another way drugs can stimulate mitochondrial ROS?

Polar metabolites of retinol-plus-alcohol, opioids, NSAIDs can all destabilize MMP.

What if all drugs that suppress respiration (breathing) in the CNS also inhibit respiration in mitochondria?
Wouldn't that be a case of metabolism driving higher regulatory functions?

liv said...

I thought that Metformin increases insulin sensitivity (turned out it is not!). How can it have so many benefits (after reading about it on Wikipedia I have the strong impression that it is a wonder drug, activating AMPK and all), if it makes SO much superoxide? I mean, it is a poison for mitochondria, how can it have systemic metabolic beneficial effects at all?

Peter said...

Iva, metformin appears to correct the changes in adipocytes which lead to systemic insulin resistance. There will come a point where, as adipocytes shrink, they will become functional again and generate appropriate levels of fatty acids for the metabolic conditions prevailing and release an appropriate mix of saturated and monounsaturated fats for normal body energy homeostasis. This is the direction we're going in next.


Peter said...


I think it's 5001 standard lab chow with 10% lard added. Obesity prone mice (and probably rats) do awful damage to their VMH on ANY high fat diet, ghee supplemented included. Non obesity prone rodents won't. But no one in obesity research would use an obesity resistant mouse, would they? I think humans gain their obesity by completely different mechanisms, but this does not alter the importance of what these models tell us. Especially as chronic hyperglycaemia is particularly toxic to those patches of the brain which do accept fatty acids. There are some. This is a long way ahead in the series but I suspect the VMH is one such area.


George Henderson said...

"It has been known for more than a century that animals accumulate fat on fat-free, high-carbohydrate diets. Even in the steady state, as shown by tracer techniques, there is a major metabolic flux that can be summarized as carbohydrates => fatty acids => CO2 + H2O. However the rate of lipogenesis is not equally rapid under all circumstances. Synthesis of fatty acids is disturbed by a number of conditions. If the total calories of the diet are restricted and become insufficient to maintain body weight, the rate of lipogenesis may fall to 5% of its normal value.
Similar decreases in the rate of lipogenesis have been observed in the thiamine-deficient animal, compatible with the role of thiamine pyrophosphate in the transfomation of pyruvate into acetyl-CoA."
(Principles of Biochemistry, White, Handler and Smith, 1954)

The beri-beri weight loss diet, anyone?

Peter said...

I am trying very, very hard not to suggest who should be the lead promoter, first victim and centre fold boy for this amazing weight loss concept. I'm succeeding. Just.


Peter said...

Its, never got to comment. Yes. We look at things the same way. BTW My nurses tell me I'm probably the craziest person they work with. Actually they seem to think our Italian new grad is fairly crazy too but I think I beat her hands down. The voices say so.


George Henderson said...

Well, now we know why those societies that eat most of their calories as white rice don't get obese.

ItsTheWooo said...

That's okay, because if people think you're crazy there's a good chance you have some interesting new ideas no one believes in.

George Henderson said...

Damn it, I actually think I might be onto something here.
Consider the triage theory of micronutrient disposal, and the fact that deficiency of TCA co-enzymes like thiamine suppresses appetite.
Consider low obesity in the past on unfortified white bread and sugar, and low obesity in white rice cultures.

On a high-carb diet that is optimal for TCA co-enzyme vitamins, lipogenesis is favoured and appetite increased so that fat can be stored.

On a high-carb diet that is marginal for TCA co-enzymes, direct conversion of glucose to ATP is favoured, appetite is decreased, and lipogenesis is suppressed.

For example, alcohol depletes thiamine and absence of normal body fat often results from alcoholism.
Yet beer drinkers can gain weight; beer is a source of thiamine and other TCA co-enzymes.

Pathological reactions to toxins (such as alcoholic fatty liver) may be somewhat less influenced by triage than processes which are part of the evolutionary round.

Paul Jaminet considered the epidemiological evidence against flour supplementation a while ago.
I'll go back and have a look at that.
The effect ought to differ on a case by case basis; genetics and other factors influence micronutrient uptake and usage.

Jack Kruse said...

Peter this is why you are at the top of my must do list. I use metformin in my practice like mad. It is the pharmocologic equivalent of what the Okiniwans major benefit is to longevity. they have a genetic defect that limits ROS at cytochrome one and it has nothing to do with their diet. Tanaka paper in 1998 said it and it appears the rest of the kitavin loving paleo crowd dont read past Cordains work. Nick Lane has the reference in his books too. You my friend are a rock star and a patient advocate. I have been using this drug as a longevity booster and mTor regualtor for some time. It has amazing affects on changing the gut flora of the obesity phenotype and also decreases intestinal permeability to limit BBB assault. My latest blog is from two of my forum members who came to visit me on my 3 week vacation and guess what we talked about. Metformin. Thanks Pete. Love your work. Dr. Kruse

Jack Kruse said...

Another comment for you to consider too. metformin also has deep synergy clinically with curcumin, black pepper, resveratrol, and NAC to alter the obesity flora. Moreover we can upgrade the effect when we make sure the resveratrol is partially ingested via the buccal or SL mucosa. Another reason swishing before one swallows is a very smart healthy decision.

Tim Lundeen said...

If metformin reduces convertion of glucose to lactate, and the brain runs on lactate, what is the effect on brain health?

Also, if metformin increases superoxide production and the brain doesn't like superoxide, is this a problem for brain cells?

Peter said...


Metformin increases lactate production, it doesn't decrease it. On very rare occasions the increase is to fatal levels. It also generates superoxide when cells are metabolising fatty acids, not something neurons do a great deal of...