Thursday, January 16, 2014

Protons (34) Rotenone

So far I've been thinking about insulin resistance as a normal physiological process for regulating the energy input in to each cell, on an individual metabolic needs basis. There is nothing pathological about this process, regulation is essential. To develop insulin resistance in the immediate aftermath of a single 2000kcal meal or during a 5 day fast is exactly what you need to do. Combining the post prandial state with the fasting state is pathological but could be corrected by simply restoring insulin sensitivity to adipocytes, so allowing them to lower free fatty acids when glucose is elevated.

There are times when I would like there to be differing terms for the insulin resistance of the immediate post prandial period, of starvation and of metabolic broken-ness. It would make for a much clearer picture than saying someone is "insulin resistant".

Now I'd like to think about some pathology.

I’m going to start with this paper on rotenone, with thanks to Mike for the full text. I'm not sure how widely rotenone is used nowadays but, in the lab, it is a freely available inhibitor of complex I. I doubt this toxicosis is particularly prevalent in everyday life, I'm more interested in a basic mechanism which we can extend to other injuries of complex I. Adding rotenone to almost any cell line produces this sort of effect on metabolism, here in C2C12 muscle-like cells (which do it best). From Fig4:

Oxygen consumption is depressed, delta psi is depressed and NADH accumulates at the expense of lowered NAD+ levels. BTW, some authors use the NADH/NAD+ ratio, some the inverse. Ah well, it's still badness. These folks talk about "reductive stress".

The interesting point to note from section D is the rise in acetyl-carnitine, a molecule used to export acetyl-CoA from mitochondria to cytoplasm.

If we block the ability of the mitochondria to feed NADH in to the ETC there is no point in turning the TCA because this generates four NADHs for that single FADH2 from succinate dehydrogenase (which still works if you provide succinate exogenously). Instead the acetyl-CoA is exported as acetyl-carninitine or as citrate after combination with oxaloacetate. Once in to the cytoplasm acetyl-CoA is available for de novo lipogenesis. Does it get used for this?

Here are some C2C12 muscle-like cells which are running on a "low" concentration (probably around 5.0mmol/l) of glucose. The ones on the right are also exposed to 5.0 nanomol of rotenone in the complete absence of insulin or any equivalent. Cytoplasm is pale purple, nucleus is dark purple:

The beautiful orange-red staining droplets are lipid, more clearly visible in this enlargement:

How much lipid is deposited? That depends on how much rotenone is used and how long the complex I has been blocked for. From Fig 2:

You can do exactly the same thing by blocking complex I with piericidin A or by knocking down expression of the gene NDUFV1 (codes for a huge chunk of complex I) in C2C12 cells. From Fig 3:

You get the same effect in people with the misfortune to be born with severe defects in complex I; in the affected tissues there is lipid accumulation. By the time you get down to <5% complex one activity there are quite nasty knock on effects on fatty acid oxidation and cell viability, but that's another story.

Very few people would choose to take rotenone nowadays but there are a number of other complex I inhibitors, some of which are quite widely available. Flunarizine, isoniazid and atrazine spring to mind with bisphenyl A as a more general mitochondrial toxin which includes complex I blockade. None are marketed as weight loss supplements.

I see this as a generic mechanism. Complex I dysfunction leads to intracellular lipid accumulation.

What else is interesting?

These cells are being fed with low levels of glucose without insulin. Glucose uptake will be basal and much of the energy from glycolysis will be diverted to lipogenesis. Again, if ox phos is reduced mitochondrial ATP production will be depressed and glycolysis will be the main source of ATP using substrate level phosphorylation. We know lipogenesis is occurring, so we must be deriving NADPH from glycolysis. If we were to add insulin to the culture medium of a rotenone poisoned cell and perhaps increase the glucose level to 25mmol/l, what would happen?

Assuming we can generate enough of a delta psi to allow insulin signalling we would pour glucose in to the cell and down through glycolysis to pyruvate. This gives us a ton of acetyl-CoA in the mitochondria. Complex I is still blocked. What's a cell to do except de novo lipogenesis in its cytoplasm?

As we allow glucose in to a cell, we push de novo lipogenesis. This is a parody of insulin sensitivity. The faster we allow glucose in to a cell, the faster it is lost to lipid. My line of thought is about how well this ability to sequester glucose as lipid might give the impression of being extremely sensitive to insulin, when the actual mitochondria are dysfunctional and should be signalling insulin resistance.

I can't get away from the unarguable fact that post-obese women perform enough DNL to get their RQ > 1.0 on exposure to 75g of glucose in an OGTT.

Perhaps we need to look at the mitochondrial function of people who's "preferred" metabolic state is best achieved by obesity. And how we might damage complex I without mainlining rotenone.

Better hit "post" as things keep getting in the way of extending this particular entry.



Gretchen said...

1. Livestock people still use rotenone to dust animals for insects. I have a bag out in the barn.

2. Metformin also blocks complex I, yet metformin helps with weight loss. How do you explain that?

Gretchen said...


skepticle said...

Hi Peter, I came across this today. Please let us know what you think!

skepticle said... oops

Peter said...

Hi Gretchen. Gulp! Both block complex I but rotenone generates ROS during fwd flow of electrons. Metformin doesn't seem to and actively blocks ROS production under reverse flow, certainly in the longer term. Acute effects may differ.

skepticle, the should be some excellent drugs come out of the research. We will have to see the consequences of blocking food intake when peripheral metabolism is so broken as to only function when pushed by metabolic syndrome. They also do not seem to have the concept that hyperphagia might result from a brain lesion which allows fat loss in to adipocytes.

Likely unintended consequences mean I will not be at the front of the queue for some designer drug. Probably many will be though...


Galina L. said...

"So the dramatic increase in the prevalence of obesity "is caused by "the lack of progress in combating one of the most serious health problems of this century" - "hyperphagia-induced obesity"? I am sure the luck of progress is temporary and a science will help to micro-manage the complex system of a human body as successfully as usual. I just wonder what the most probable bunch of side effects would such drug cause?

Galina L. said...
This comment has been removed by the author.
Tucker Goodrich said...

"Perhaps we need to look at the mitochondrial function of people who's "preferred" metabolic state is best achieved by obesity. And how we might damage complex I without mainlining rotenone."

Here's one way...

"Fatty liver was induced in rats with a choline-deficient (CD) diet for 30 days.... It is concluded that CD diet causes mitochondrial complex I dysfunction which can be attributed to ROS-induced cardiolipin oxidation. These findings provide new insights into the alterations underlying mitochondrial dysfunction in NAFLD."

But NAFLD isn't common in obese people, is it? ;)

"Mitochondrial dysfunction in rat with nonalcoholic fatty liver: Involvement of complex I, reactive oxygen species and cardiolipin"

Anonymous said...

Hi Peter,

This might be off topic, but not entirely, I suspect. People on my blog are fretting about this Dutch experiment, which apparently (paraphrase) 'demonstrates the detrimental long term health effects of a ketogenic diet'.

I smell a very dead rat but I'm too stupid to locate it. Would love to hear your comment, if you have time.

ItsTheWooo said...

very interesting as always Peter, my thoughts regarding obesity is it may not always be mitochondrial damage but often is a nervous system derangement affecting the ans, which controls the metabolism of all cells abd hypothalamic damage or very low Leptin, with exaggerated pns tone and insulin supersensitivity. It ispossible some of these oxidative defects are functional, at least in the larger framework of a hierarchy of superior neuroendocrine derangement.

Michael Frederik said...

The authors are from the LUMC, you should contact them and ask for the diet they used. These anticonvulsant diets are usually very high in PUFA (soya or corn oil). Sometimes something better,, but still terrible is used. Anyway, that would be my guess.

Michael Frederik said...

See these comments, too:

Peter said...

Melchior, Micheal is probably correct. The writing was on the wall some time ago for PUFA based ketogenic diets. The PUFA is worse than people thought.

I’d like to blog on the problems associated with paediatric ketogenic diets. PUFA overload is not the word!

No time for more


Anonymous said...

Hi Michael and Peter,

Thanks! I will follow your links and then mail these guys. I'll let you know if they answer.



NKSL55 said...

Initially, when I looked at the Ellenbroek et al. paper you reference, I found a link from PubMed to a pre-print of what appeared to be the entire paper. There was a table in it that compared the two diets. Alas, now only the abstract of the paper appears to be available. But my memory of the table was that the high fat diet was very high fat, maybe 95%. The fat was one-third of each of the following components: saturated, monounsaturated and polyunsaturated fatty acids.

There was a note below the table mentioning that either 6% or 3% of the PUFAs (again, this is from memory, I don't have this paper in front of me) were of one type. I think it was 6% omega-6.

To me this paper seems to be response to a paper in the same journal from a different group. I think this paper is free to everyone from the journal:

Get it? The US researcher paper is entitled "A very low carbohydrate ketogenic diet improves glucose tolerance in ob/ob mice independently of weight loss" whereas the Dutch group entitled their paper "Long-term ketogenic diet causes glucose intolerance and reduced beta and alpha cell mass but no weight loss in mice". The US experiment spans 50 or 60 days, whereas the Dutch experiment spans 22 weeks.

A few comments:
(1) Seems pretty hard to get mice into ketosis. 95% fat diet? So, all the usual caveats with whether a mouse model is meaningful for comparison to human nutrition.
(2) Peter has written many posts on why insulin resistance is good thing when you are on a high fat diet (or starving).
So the glucose intolerance part would be of little interest. But this paper provides pancreatic histology correlating the loss of glucose tolerance with a decrease in size of the alpha and beta-islets.
Okay, still not earth-shattering. But kind of interesting that long term adaptation to a ketogenic diet might occasion changes visible via histology.
(3) It is fairly well known that a few days back on the carbs and the insulin resistance reverses and your oral glucose tolerance test comes back normal. Peter cites a Kinzig et al. paper in this post demonstrating the effect in rats here:

Is that sufficient to reverse the changes in pancreatic histology? Or would 22 weeks on a KD be enough to permanently commit a mouse to a high fat diet to the extent that subsequent exposure to carbs would result in, well, uncontrolled T2 diabetes?
(4) Probably not, but even if this mouse model is a reasonable facsimile of human physiology, it would only mean that unrelenting ketosis for a large fraction of your life time (decades) might have some untoward effects. Last I checked Peter does OD, not KD. So one could just shrug ones shoulders and say "JK was right again".


Peter said...

Hi Tucker, this is very interesting. Oxidation of cardiolipin inhibits complex I. It is completely reversible by supplying non oxidised cardiolipin. My immediate reaction is that this might be a negative feedback to limit NADH input to the ETC, perhaps when there are enough free radicals being produced to potentially free cytochrome c from its cardiolipin anchors with the possibility of inducing apoptosis. Very interesting concept… Hyperglycaemia has major effects on cardiolipin and its oxidation.

Wooo, yes, but my suspicion is that an awful lot of CNS pathology will be mitochondrial in origin. Even Guyenet’s lab pointed out that VHM lesions develop relatively slowly, needing chronic hyperglycaemia. We all know what VMH lesions do to adipocyte insulin sensitivity. Sid’s scary paper on the psychiatry of mitochondrial diseases has me feeling that the track of neural mitochondrial issues is correct. But folks with PD, with b*ggered strial mitochondria, have grossly abnormal myocardial mitochondria and, I think, in adipocytes too. So I think there are whole-body issues. Adipocyte mitochondrial issues seem likely in obesity with insulin resistance, less mitochondrial involvement if obesity does not induce insulin resistance. Undoubtedly SNS innervation will be important to the size of adipocytes at the time they decide to resist insulin. And size may not be the answer to adipocyte insulin resistance.

Phillip. If the pancreatic “atrophy” is really atrophy (not seen the histo) rather than normal physiological response to low insulin needs, we have two explanations. Keto kills or omega 6 PUFA kill. Simple choice, but this paper won’t help.


Jane said...

Peter, you want to know how to inhibit complex 1 without mainlining rotenone? I found out recently that calcium can do it. Could be interesting because according to Zemel, calcium increases lipogenesis and inhibits lipolysis.

'... we show that ROS generated by Nox activation mobilizes Ca2+ flux from the cytosol to mitochondria, leading to S-glutathionylation of 75- and 50-kDa proteins of the complex I and inhibition of complex I activity, which results in elevated mitochondrial ROS. ...'

NKSL55 said...

Peter wrote:
"Phillip. If the pancreatic “atrophy” is really atrophy (not seen the histo) rather than normal physiological response to low insulin needs, we have two explanations. Keto kills or omega 6 PUFA kill. Simple choice, but this paper won’t help."

Okay Peter, you are throwing me for a loop invoking o6 PUFA in this context. I thought o6 PUFA was a "bad for you liver" thing? Is there a "bad for your pancreas" component to the o6 PUFA story? I have been reading your blog, but have only made it from the beginning to Jan 2009 so far. So I could easily have missed this part of the story...

Anyway, given that previous work in rats by Kinzig et al. showed that the IR, etc. effects of an 8 week KD were quickly reversed upon resumption of a chow diet, doesn't seem likely the story would be any different in mice after 22 weeks.

Biological systems tend to be built on a "use it or lose it" efficiency model. Cranking the macro-nutrient lever so far towards fat that you are deep in ketosis all the time, or cranking it so far into carbs that ketosis never happens -- either would be expected to occasion adaptations that might take a bit to reverse.

But Ellenbroek et al. added some histology to the story. Not that I'm qualified to assess it. That might partially make up for the inability to do a literature search prior to writing a manuscript.


Peter said...

Phillip, I wasn’t really thinking along the lines of PUFA induce apoptosis in pancreatic cells. More a generic feeling that if the main problem with the diet is excess PUFA then maybe it might explain all of the issues. Ultimately I would expect the pancreatic atrophy to be due to lack of usage under ketosis. I’d expect it to be reversible. I’d expect any ketobasher to be damned careful not to check this.

The main problem I see with omega 6 PUFA is their inability to generate insulin resistance when it’s needed. I think a good argument could be made for this being why they promote cancer cell growth as they do. I wasn’t really expecting them to be the cause of reduced beta cell numbers…

I would agree completely that the phraseology of a paper specifies an agenda. I guess we all have agendas, some causing more collateral damage than others.


Jane said...

Peter, the problem with omega 6 PUFA may be to do with iron. Here's a paper showing that fatty acids can carry iron across lipid bilayers. Iron is 'bad for your pancreas'. Linoleic acid carried the most iron, and palmitic acid the least.

'Transport of Fe2+ across lipid bilayers: possible role of free fatty acids'

Peter said...

Jane, I have a paper on my old mac hard drive showing that PUFA release Fe from intracellular stores. In yeasts, I think. As we know, saturated fats are by far the best promoters of iron uptake from the gut in higher organisms and PUFA are by far the best promoters of cirrhosis. I like this. If I had the time to search I would be willing to bet that saturated fats allowing the hoarding of iron in a non reactive form. PUFA release it from storage to fill the cell with hydroxyl radicals, rather than the fairly benign H2O2 of routine signalling purposes.

The question is, as always, why? PUFA limit the ability to generate post prandial insulin resistance as they have limited FADH2 input at ETFdh. If a cell is faced with an onslaught of acetyl-CoA, perhaps iron induced insulin resistance is preferable to utter caloric overload from PUFA generated NADH combined with glycolysis derived acetyl-CoA and the inability to say no to more glucose or fructose.

Failure to generate adequate insulin resistance might be the route to hepatic carcinoma, another gift of corn oil.

Just speculating…


Marco Buquicchio said...

NKSL55 could be useful to see who are the funders of the study!

Tucker Goodrich said...

"Perhaps we need to look at the mitochondrial function of people who's "preferred" metabolic state is best achieved by obesity. And how we might damage complex I without mainlining rotenone."

We know these answers now...