Metformin (04) Pre and Post Prandial

The next metformin paper to look at is this one:

Beneficial effects of metformin on energy metabolism and visceral fat volume through a possible mechanism of fatty acid oxidation in human subjects and rats

Here are the RQ data from 16 healthy humans after an overnight fast and for the three hours following a mixed carbohydrate/fat meal tolerance test (type of carbohydrate and fat not specified).

Aside: Here is the test "food" description: "meal tolerance tests (592 kcal, 75g of carbohydrate, 28.5g of fat; Saraya Co., Osaka, Japan)". It's great to know that there is a company called Saraya and that they have headquarters in Osaka. But I can't even find out what sort of "meals" Saraya make. Quite how anyone might replicate this study using the methods section is beyond me. In addition to these omissions the test "meal" is repeatedly described as "cookies". Go figure. Still, let's assume the measurements of RQ is numerically accurate, fingers crossed. End aside.

These healthy people, who haven't eaten overnight, have an RQ of 0.8 and the test meal produced a downward trend in RQ indicating that the "cookies", providing roughly 50% of calories as fat, tended to increase fatty acid oxidation or decrease carbohydrate oxidation. I can't be arsed to criticise their stats methods. Let's stick with the gross changes.

After two weeks on metformin at an eventual dose rate of 500mg three times daily there is a significant fall in fasting RQ indicating an increase in non-fed fat oxidation compared to the control state.

Under metformin the "cookies" produce a rising RQ, suggesting preferential metabolism of glucose in the immediate post prandial period.

So metformin promotes fat oxidation during fasting but promotes glucose oxidation during the first three hours after a plate of "cookies".

Interesting.

We should see if we can explain these effects on RQ in terms of mitochondrial glycerol-3-phosphate dehydrogenase (mtG3Pdh), electron transporting flavoprotein dehydrogenase (ETFdh) and the redox state of the CoQ couple driving reverse electron transport (RET) through complex I.

Peter

Succinate doesn't drive reverse electron transport. Maybe.

Mike Eades sent me this paper:

Reactive oxygen species are generated by the respiratory complex II – evidence for lack of contribution of the reverse electron flow in 
complex I

suggesting that RET through complex I, when driven by succinate oxidation at complex II, is a pure artefact of the pathologically high level of succinate used in the mitochondrial preparations involved. Bearing in mind that trying to work out exactly what the physiological concentration of succinate might be, in the region of the active site of a complex II in a working, oscillating, in-situ mitochondrion, involves an awful lot of guesswork.

However, the paper might well to be correct, within the limitations of the mitochondrial preparations they are using.

If you feed mitochondria with 5.0mmol/l succinate there is profuse ROS generation, 85% of which can be blocked by rotenone, ie this 85% is RET generated. The other 15% comes from other places, including complexes II and III, at least. But if you feed mitochondria with 0.5mmol/l succinate, or even 1.0mmol/l, there is no ROS generation at all. The case is made that ROS from RET are not a feature of "normal" levels of succinate driving the reduction of the CoQ couple.

Fine.

But this is a mitochondrial preparation. It has no cytoplasm, no glycolytic enzymes, no source of glycerol-3-phosphate, no FFAs, no carnitine. You can't buy a vial of FADH2 bound to electron transferring flavoprotein to feed in at ETFdh. This makes manipulating the CoQ couple in a way which is physiologically significant very difficult. In the current study we have no input to the CoQ couple other than complex II using succinate.

Those folks like myself, who feel that the redox state of the CoQ couple is the main sensor of the energy status of the cell, would never expect a single input in to the CoQ couple to be the sole representative of energy status. Even during glycolysis there is some fatty acid oxidation providing electron transferring flavoprotein to ETFdh. And succinate from FFA derived acetyl-CoA will also supply to complex II during lipid oxidation. And conversely some glycolysis will occur, even when FFA oxidation predominates, supplying glycerol-3-phosphate to mtG3Pdh.

Until we can set preparations up in which these inputs can be adjusted we are not able to say much about what might be happening in-vivo to RET. And once you start smashing the mitochondria to pieces and reassembling them as inside-out vesicles (so you can supply metabolites to the intra-mitochondria binding sites that would normally be hidden away from your extra-mitochondrial culture fluid) you are a very, very long way from in-vivo indeed.

Just saying...

Peter

Metformin (03) In-vivo experiments require non-lethal dose rates!

Just before I move on to metformin-induced substrate oxidation changes in healthy volunteers, I think it's worth looking at this neoplasia paper in a little detail. It's fairly typical of the work done on metformin as an anti-cancer agent and focuses on the highly reproducible inhibitory effect of metformin on complex I.

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.

Most of this work is very clever and very carefully done, but lives with the problem that the experiments usually use concentrations of metformin in-vitro which would be lethal in-vivo because, well, everybody does it and there is no effect if you don't... However the mouse xenograft studies have to use clinically relevant therapeutic doses of metformin otherwise the mice would be, well, a bit dead. There are other problems which will become apparent as we work through the data.

The figure I'd like to focus on is supplementary data section three of figure seven.

Graphs B and C look like this:

This is what they did to generate them. They took A549 tumour cells and injected them in to immuno-incompetent mice then measured the growth of the resulting tumour.  A549 cells are highly sensitive to metformin, so graph B comes as no surprise. Graph C is much, much cleverer. They wanted to prove that metformin was actually working on complex I. So they destroyed complex I with a shRNA targeting NDUSF3, an essential subunit of this complex. To keep the cell line functional they replaced complex I with our old friend the yeast derived NADH dehydrogenase NDI1. This enzyme does not bind metformin nor pump protons but does reduce NADH to NAD+ and does feed electrons to the CoQ couple and the downstream complexes. You can see from graph C that replacing complex I with NDI1 protects the A549 cell derived tumours from the growth slowing effects of metformin.

Look at B. Look at C. Protection from metformin in C. Yes?

Now, you have to ask: What is the effect of knocking down complex I in cancer cells? If you cannot reduce NADH to NAD+ then the TCA cannot turn. Citrate cannot be metabolised to alpha ketoglutarate so is exported from the mitochondria and can be used for tumour anabolism. The tumour becomes highly aggressive. Like this:

Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism

or this, blogged about many years ago:

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression

This illustrates my marked discomfort with accepting complex I blockade as the mechanism of anti-cancer action of metformin. Blockading complex I will admittedly decrease ATP supply from oxidative phosphorylation but at the cost of supplying a large amount of citrate to the cytoplasm ready for anabolic processes, while glycolysis continues unabated, supplying cytoplasmic NADH and ATP.

So in the current paper, by knocking down NDUSF3, they should have generated an aggressive phenotype. They didn't, because they also engineered-in NDI1, which will reduce cytoplasmic NADH to NAD+ very effectively. Dropping the NADH to NAD+ ratio suppresses tumour aggressiveness in the above papers.

Does the engineered A549 NDUSF3 + NDI1 tumour in nude mice show reduced or increased aggressiveness compared to the A549 unmodified tumour? We are looking to compare the top line in graph B above (dark squares) with the pale squares in graph C. By eyeball they actually look pretty much the same.

Except for the x axes. Graph B is 40 weeks, graph C is 50 weeks. Hard to compare the two... But if we stretch graph C so that weeks 10-40 align with weeks 10-40 of graph B, then superimpose the two graphs we can generate the following, rather more informative, image:

It looks to me as if inserting NDI1 in to the mitochondria of a cell line, (probably) made aggressive by knockdown of NDUSF3, renders the in-vivo tumour growth rate much lower than the natural tumour cell line and remarkably similar to that of metformin treated natural tumour cell line. Probably by reducing the NADH:NAD+ ratio.

This doesn't automatically suggest that metformin might be acting by reducing the NADH:NAD+ ratio, though it might be, but it does illustrate how nicely you can still pull interesting snippets out of papers full of experiments with metformin at lethal concentrations.

The difference between isolated mitochondrial preparations and mouse models is that the mouse models have a supply of insulin, glycerol-3-phosphate and the enzyme to use cytoplasmic NADH to reduce the CoQ couple, facilitating insulin signalling and so cancer growth. This is much more likely to be the process which we can block with metformin at therapeutic concentrations.

Peter