Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individua

[msf]

Wow, I like the way this research seems to link up with that of other researchers in the UK, now if only they could link up with other researchers around the world. The only thing that worries me is that the patient cohort is a CFS one, even though it was funded by the ME association, The quote about about two different muscle types suggests that they are aware that ME is different from CFS, however, so perhaps they are just using a CFS cohort for political reasons.

 

[Gijs]

(...)''Recent studies suggest that graded exercise therapy (GET) has benefits for patients with CFS [38] although these benefits have been small. Frequently patients with CFS feel that exercise in fact makes their symptoms worse. Our recent MRS studies suggested that there were at least two muscle phenotypes grouped together through the entirely symptom based diagnostic classification of CFS. This might explain, in part, the limited benefits seen in the PACE trial for GET. We believe that the results of the current study further emphasise the potential for a peripheral component to CFS and the need to fully characterise the muscle phenotypes in CFS before generically prescribing exercise as an effective intervention. Further work is needed to understand the muscle biochemical abnormalities in CFS and the impact that exercise might have upon these.''

I think this is an important warning for GET!!! First do no harm!!!

 

[Simon]

Wow, this is potentially an incredibly important study: though the standard proviso about replication applies, especially for such a small study.

As far as I know it's the first study to develop an 'in vitro' (lab bench) study that isn't just looking at individual cells taken from the body but grows functioning muscle cells outside the body. Critically, these are new cells grow away from any factors like autoantibodies, autonomic problems such as blood flow, cytokines or even faulty illness beliefs, and still they find a difference. As others have said that throws up challenges to other models of mecfs if these results are replicated.

Certainly it's the strongest evidence to date of a primary problem in muscles (or in cells at any rate since we don't know the situation tissues other than muscle).

Even more fascinating, the researchers highlight a problem the AMP-Kinase, a key sensor and regulator of the eneryg level of the cell, which is a plausible molecule to have a key role in this illness. As @nandixon says, AMP-K is active in other tissues, including the brain (they study doens't look at other tissues for comparison) and problems with AMP-K in the brain could explain problems with mental fatigue too. Interestingly, the finding of reduced IL-6 production by the muscle cells from CFS patients ties in with the low AMPK activation as IL-6 is an AMPK activator.

The big question is how an effect in the body transfers to an effect in cell culture. The study suggests genetics though presumably all the patients were healthy before they got mecfs so the genes they were born with were presumably ok. Epigenetics (long-term, inheritable changes in gene activation) is one mechansim, though presumably infection is another.

This looks to me like it could be an incredibly fruitful area of research and I hope others will try to replicate pronto.

I wonder why they didn't also consider stochastic (autoimmunity) in addition to genetic/epigenetic.

Presumably because autoantibodies couldn't explain defects in the cultured muscles, where the muscles were grown away from the influence of autoantibodies?

 

[lansbergen]

Or is it that if there were an infectious cause, the epigenetic changes in the muscle would persist ex vivo?

I guess that is posseble. The infection I suspect does not disapear in culture.

 

[Marco]

Presumably because autoantibodies couldn't explain defects in the cultured muscles, where the muscles were grown away from the influence of autoantibodies?

Fair point although autoimmune mediated demyelination in MS wouldn't disappear if you biopsied nerve cells to test their electrical conductivity. Whether the same could apply to a physiological process such as this I don't know but don't see why not in principle.

 

[Bob]

Of course there is a possibility that some of the changes could be deconditioning ....

I hope that the leading proponents of the deconditioning theory have now abandoned the theory since the recent PACE trial mediation analysis debunked it:
http://www.thelancet.com/journals/lanpsy/article/PIIS2215-0366(15)00054-1/fulltext

 

[Bob]

I wonder why they didn't also consider stochastic (autoimmunity) in addition to genetic/epigenetic.

If they are culturing muscle cells only, and no blood cells are present, then that rules out many factors, including B-cell mediated issues and other immune related issues. There's got to be some dysfunction within the muscle cell itself. Also, the cell culturing process suggests that the dysfunction is replicated when cells divide. So (based on my basic understanding of biology) that does seem to suggest some genetic or mitochondrial component.

Of course, B-cells may also be affected by the same issue so this research doesn't rule out the possibility of dysfunctioning immune cells. This research seems to suggest that all our cells might be dysfunctioning, which would make a lot of sense to me. That would fit with a multi-system, systemic illness.

Edit: crossed posts.

 

[Keela Too]

@Bob ... you missed my point.

Deconditioning as a theory of why we are ill is obviously nonsense.

However we do over time loose condition... ie our muscles do become weaker.

Therefore when comparing muscle tissues, ME patients' muscle really should be compared to muscle from healthy people who are also deconditioned. That is not easy, because I cannot imagine any healthy couch potato physically doing as little as I do.

Hence the reason I was suggesting the NASA bedrest folk - the healthy volunteers asked to spend a year in bed... if I remember right.

Those folk would make excellent controls for Severe ME patients.

 

[Gijs]

It doen't rule out infection. Viral DNA/RNA replicated in cells as well (it also depends were they are hiding). They could mess up the Gene function. Retroviral can do this. I think this point to an aquired state of mitrodysfunction. Mitrodysfunction is Multi systemic, brain, cardio/vasculair, muscle contracion stomach/gut etc...

 

[Bob]

Loads of great comments in this thread. I'm tempted to quote my fave comments, but there are too many. It's a great discussion.

Thanks for the research papers that have been posted - some of those seem very pertinent to this research. It looks like it would be worth testing Metformin if Julia Newton is testing drugs in vitro. And it would be worth testing for ZAG down-regulation if it might lead to any deeper understanding of epigenetic processes.

After reading this thread, and the research paper, I'm even more excited about this research than I thought I would be - and I was already excited about it. I've always thought that my illness involved a cellular dysfunction that affected muscles, brain and immune system. And this research would support all of that. It supports a multi-system and systemic disease process. It doesn't rule out immune abnormalities or brain abnormalities (including immune abnormalities in the brain), but it does suggest that such abnormalities are secondary to another underlying disease process possibly involving genes, HERVS, mitochondria, or perhaps viral infection. My pet theory has often been mitochondrial abnormalities, but I'm open to many other possibilities.

I've just attempted to read through the full paper, and the complexity is extraordinary! No wonder it took so long to get published. But I think it's definitely worth waiting for. I think the significance of this paper may grow over time, as the potential implications are digested and follow-up research is carried out. I don't understand the methodology, and I'm in utter awe of the investigators for being able to carry out such extraordinarily complex research. This seems to be an amazing feat of science.

I think the main findings probably only touch the surface in terms of understanding the underlying cellular dysfunction, but it seems like a great start. Now we need the MRC to put its money where its mouth is, and to fund a large scale follow-up studies looking deeper for a wide range of potential abnormalities, and testing as many drugs in vitro as possible. The idea of testing drugs in vitro, on Julia Newton's cell cultures, seems very promising. Let's get going, MRC!

 

[Bob]

ALA and ALCAR might help:

Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle

Acetyl-l-carnitine inhibits TNF-alpha-induced insulin resistance via AMPK pathway in rat skeletal muscle cells

Actually, the study suggests that there's no problem with insulin...

Interestingly, glucose uptake in response to insulin was normal in the chronic fatigue syndrome cultures, pointing to a specific exercise related defect.

 

[Marco]

If they are culturing muscle cells only, and no blood cells are present, then that rules out many factors, including B-cell mediated issues and other immune related issues. There's got to be some dysfunction within the muscle cell itself. Also, the cell culturing process suggests that the dysfunction is replicated when cells divide. So (based on my basic understanding of biology) that does seem to suggest some genetic or mitochondrial component.

Of course, B-cells may also be affected by the same issue so this research doesn't rule out the possibility of dysfunctioning immune cells. This research seems to suggest that all our cells might be dysfunctioning, which would make a lot of sense to me. That would fit with a multi-system, systemic illness.

Edit: crossed posts.

Thanks Bob.

I hadn't picked up on the cell culturing issue..

 

[Bob]

@Bob ... you missed my point.

Deconditioning as a theory of why we are ill is obviously nonsense.

However we do over time loose condition... ie our muscles do become weaker.

Therefore when comparing muscle tissues, ME patients' muscle really should be compared to muscle from healthy people who are also deconditioned. That is not easy, because I cannot imagine any healthy couch potato physically doing as little as I do.

I'm always missing the point, but I thought that we'd established that deconditioning was not a significant factor in this illness, in any sense. I can't remember exactly what I'm basing this conclusion on, but I thought there had been various bits of research and anecdotes that have trashed the idea that deconditioning has any significant bearing on our function. Sorry I can't be more specific - I'm going from vague memories here. But one such anecdote is how folk who respond to rituximab don't complain of deconditioning after they respond to treatment - they just get up and go.

But, also, importantly, the muscle cultures were standardised in this study, and I imagine that the in vitro cells would likely be conditioned or deconditioned to the same extent by the time the experiment took place.

But, just to be certain that muscle conditioning was standardised, perhaps they should have subjected the cells to a course of CBT before the experiment took place... ... oh wait... ... Erm...

(Edit: BTW, the CBT comment is not directed at you, Keela. Just mocking our 'friends'.)

 

[adreno]

Actually, the study suggests that there's no problem with insulin...

Would that matter? Metformin also targets insulin resistance.

 

[alex3619]

The lactic acid cycle runs off pyruvate, not glucose specifically.

The primary driver is glycolysis. It is hard to see how a lot of lactate could be produced by any other method. If lactate is increased then where is it coming from?

I am concerned they might be making a conceptual error. I first saw it in Wong et. al. 1986?. 1996? I can get a reference, but its not important. That paper interpreted some findings as increased glycolysis. I pointed out, which was unpopular, that a mitochondrial defect such as a block at aconitase would do the same thing.

In this current paper there might be some decrease in mitochondrial function. It might have similar effects. I have not looked into how AMPK would fit with that hypothesis though.

In other words I think this paper is part of the story, but we have more mysteries to unravel.

One possibility ties right back to NO. This hypothesis is that our eNOS is defective, and pumping out peroxynitrite. As eNOS function is increased during physical activity this might mean that increased activity leads to more peroxynitrite, damaging iron center mitochondrial enzymes, including aconitase. Peroxynitrite destroys aconitase. Glutathione oxidation might also decrease the rate of new aconitase synthesis (its needed to fold the protein in the mitochondria, and aconitase is imported into mitochondria unfolded). One issue I have raised before is that normal aconitase protein levels might not mean much if its not folded or improperly folded. Its about function, not how much there is. Aconitase is also not the only target, there are other enzymes including in the electron transport chain. If a bunch of them are targeted then the total degradation could be high even if the isolated degradation on any one is small.

I just want to comment on iNOS. It is usually not a big driver except in septic shock. Sure individual cells can pump out high concentrations, but I think the volume is usually small. Our blood vessel endothelial lining on the other hand, while producing much less per cell, is pervasive through most tissue, and this includes the brain.

 

[lansbergen]

But, just to be sure of no deconditioning, perhaps they should have subjected the cells to a course of CBT before the experiment took place...

 

[alex3619]

Actually, the study suggests that there's no problem with insulin...

No insulin problem at rest. We have yet to fully figure out what the problem during exercise is, and I am not sure the paper is clear on this, it will need more research.

 

[lansbergen]

The primary driver is glycolysis. It is hard to see how much lactate could be produced by any other method. If lactate is increased then where is it coming from?

But was the acid lactate? I never saw a definitive answer.

I still bet on superoxide caused by SOD2 dislocation.

 

[Bob]

It doen't rule out infection. Viral DNA/RNA replicated in cells as well (it also depends were they are hiding). They could mess up the Gene function. Retroviral can do this.

I agree, but I don't think it's limited to retroviruses as a potential candidate for the observed abnormalities - I think it could potentially be any virus that can thrive in infected muscle cells. And if it's a virus causing the problem then I don't think it has to be a genetic or epigenetic issue. In other words, perhaps it could be a virus instead of a genetic or epigenetic issue. Something that wasn't explored in this paper. Unless there's something that I haven't considered that would make this unlikely.

 

[alex3619]

But was the acid lactate? I never saw a definitive answer.

I still bet on superoxide caused by SOD2 dislocation.

Carbonic acid is even more likely. However some of the other research, for decades, points right at lactic acid.

There is something anomalous here, but what it is looks difficult to determine.