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

[alex3619]

perhaps it could be a virus instead of a genetic or epigenetic issue.

It was unlikely to be an active virus. I think they would have seen that. Now a non lytic virus is possible.

 

[alex3619]

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

(Satire) Well, if this research continues then expect to hear about mental waves from remote patients influencing cells in the petri dish - psychogenic magic or psychic energy might be their next big thing.

 

[Bob]

No insulin problem at rest.

See figure 3 in the paper. I think it may suggest that insulin is also not a problem when the cells are stimulated.

 

[Bob]

Would that matter? Metformin also targets insulin resistance.

You might have a point. I suppose it depends on the mechanism by which the insulin resistance is mediated. Metformin seems to have an indirect mechanism with regards to its effect on insulin, that involves AMPK, and so that's why I thought it looks like it might have potential. But it's all a bit of a guessing game at the moment.

 

[Simon]

Just a few question marks having read the paper:

1. Small sample size, and full sample not used in any individual test
10 CFS, 7 controls (age- but not sex-matched), but not all are used for each test, making the samples smaller still:

• 9 CFS & only 5 controls for glucose tests
• 8 CFS & all 7 controls for AMPK test
  
• all 10 CFS & only 5 controls for IL-6 test

2. AMPK differences only seen after 16 hours, not 4 hours or 24 hours and they don't explain why.
From a position of relative ignorance I would have expected to see a difference at all exercise timepoints, and the authors don't comment on this time difference. (nb glucose only done at baseline and 16 hours, IL-6 testing only done at baseline, 4 & 24 hours, not 16 hours)

3. Flat 2-D muscle culture (single layer on base of dish) doesn't allow proper muscle myotubules to form:

While monolayer cultures provide a good model for studying the response to exercise in vitro, a potential limitation of the model is that in 2D cultures the myotubes are not completely aligned. One way to address this is by developing a 3D cell culture system. A number of studies have now been reported where either C2C12 myoblasts [32, 33] or primary rat myoblasts [34–36] have been grown and differentiated in a 3D culture environment which mimics more closely the structural, functional and myogenic properties of native muscle. A recent study has also described the 3D culture properties of primary human skeletal muscle cells [37]. Exercise stimulation in 3D culture is not yet well described but is an important consideration for future experiments.

More reasons to hope for a replication, though it's still an exciting study.

 

[Bob]

2. AMPK differences only seen after 16 hours, not 4 hours or 24 hours and they don't explain why.
From a position of relative ignor nce I would have expected to see a difference at all exercise timepoints, and the authors don't comment on this time difference. (nb glucose only done at baseline and 16 hours, IL-6 testing only done at baseline, 4 & 24 hours, not 16 hours)

Yes, I wondered about that too. Perhaps it indicates that the observed variables are at the end of a chain of events? (And perhaps might reflect differences seen only in severely affected patients?) Perhaps they need to look for other variables that might be initiating a chain of events? Like you say, its a fascinating start, and more research needed. (Big hint to the UK's MRC - let's have some research funding for follow-up studies, please.)

 

[charles shepherd]

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!

 

[adreno]

You might have a point. I suppose it depends on the mechanism by which the insulin resistance is mediated. Metformin seems to have an indirect mechanism with regards to its effect on insulin, that involves AMPK, and so that's why I thought it looks like it might have potential. But it's all a bit of a guessing game at the moment.

You're walking into dangerous territory here, unless you are very good at biochemistry

I'm certainly not able to elucidate the difference.

They both seem to activate AMPK, both through different mechanisms:

Notes: As discussed in the text, there are many known activators of AMPK. This noncomprehensive list highlights many of the well-established and newly discovered AMPK activators that have positive effects on T2D. Many activators activate AMPK via an increased AMP:ATP ratio (shown in red), causing AMP to bind to the γ-subunit. However, a subset of compounds stimulate AMPK activation via other mechanisms, such as binding directly to the α-subunit (shown in green), stimulating phosphorylation of αThr172 through CaMKKβ (orange), or binding directly to the β-subunit (blue). The mechanism by which rooibos activates AMPK is not known. AiCAR is phosphorylated to ZMP, an analog of AMP that can activate AMPK via the γ-subunit.

On metformin:

Zhou et al reported that metformin activates AMPK, and its insulin-sensitizing actions have been attributed to AMPK. Metformin does not activate AMPK directly; instead, it has been shown to inhibit complex 1 of the mitochondrial respiratory chain, which promotes a switch from aerobic to anaerobic glycolysis, thus increasing the AMP:ATP ratio and promoting AMPK activation.

On lipoic acid:

Shen et al showed that the mechanism by which α-lipoic acid activates AMPK is through the CaMKKβ-mediated phosphorylation of Thr172. The authors reported that the selective inhibitor of CaMKKβ STO-609 prevented α-lipoic acid-stimulated AMPK activation and subsequent ACC phosphorylation.

http://www.dovepress.com/ampk-activ...or-type-2-diabetes-peer-reviewed-article-DMSO

 

[charles shepherd]

Bob

Re MRC getting involved with future funding: This is obviously up to Julia et al in Newcastle.

But she is on the Board of the CM Research Collaborative (who will know about this research), has received MRC grants, is currently receiving an MRC grant, and clearly knows what to do if she feels that this research is worth pursing with an MRC grant.

The ring fenced money from the MRC for ME/CFS research that followed on from the high priority research recommendations that the Expert Group produced has now been used and there are no plans to repeat this initiative.

So it's up to researchers to apply to the MRC if they want an MRC research grant.

 

[Sidereal]

I just want to comment on iNOS. It is usually not a big driver except in septic shock.

Montoya said his cytokine data looks like SIRS.

 

[Valentijn]

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 does seem more likely ... though when looking through OMIM regarding mutations on certain genes, there are quite a few where a genetic disease is triggered by something, rather than being present since birth. Basically the gene is a bit dysfunctional from the start, but manages well enough until a stressor (illness, hypoxia) hits it and then all hell breaks loose. I think these problems are usually acute and episodic, rather than becoming chronic - but perhaps it's possible that something gets stuck on the fully dysfunctional setting at that point.

 

[Sidereal]

On metformin:

Zhou et al reported that metformin activates AMPK, and its insulin-sensitizing actions have been attributed to AMPK. Metformin does not activate AMPK directly; instead, it has been shown to inhibit complex 1 of the mitochondrial respiratory chain, which promotes a switch from aerobic to anaerobic glycolysis, thus increasing the AMP:ATP ratio and promoting AMPK activation.

In an non-ME/CFS population, say people with diabetes, inhibiting complex I of the ETC to jack up AMPK trick works but I've read a number of anecdotes of ME/CFS patients being prescribed metformin and finding that it depletes what little energy they had to begin with. You can use other stuff like stimulants or T3 to raise AMPK but as we all know, the sicker you are with ME/CFS, the more likely to crash from such approaches. Some would argue energy production is downregulated on purpose because there's something wrong with the enzymes that deal with ROS which are created when energy is created.

 

[Marco]

@Bob

An expert opinion would be useful but I'm not sure we can rule autoimmunity out altogether as autoantibodies can be detected in cultures of mucous membrane biopsies (and may even be more diagnostically accurate than serum testing).

http://www.ncbi.nlm.nih.gov/pubmed/21885984

 

[adreno]

In an non-ME/CFS population, say people with diabetes, inhibiting complex I of the ETC to jack up AMPK trick works but I've read a number of anecdotes of ME/CFS patients being prescribed metformin and finding that it depletes what little energy they had to begin with. You can use other stuff like stimulants or T3 to raise AMPK but as we all know, the sicker you are with ME/CFS, the more likely to crash from such approaches. Some would argue energy production is downregulated on purpose because there's something wrong with the enzymes that deal with ROS which are created when energy is created.

Yes, it certainly sounds as though metformin would be an unpleasant experience for most of us.

I would actually prefer lipoic acid in this case, as it is also a mitochondrial antioxidant.

 

[Bob]

@Bob

An expert opinion would be useful but I'm not sure we can rule autoimmunity out altogether as autoantibodies can be detected in cultures of mucous membrane biopsies (and may even be more diagnostically accurate than serum testing).

http://www.ncbi.nlm.nih.gov/pubmed/21885984

We probably shouldn't rule anything out. This sort of study might throw out completely novel and unexpected findings. But an intestinal biopsy (as per the study you've cited) would perhaps include a range of immune cells that aren't seen in muscle tissue. The gut lining has an immune role and secretes various substances including antibodies, so perhaps it wouldn't be surprising to find antibodies in the tissue culture in that particular study?

 

[Marco]

We probably shouldn't rule anything out. This sort of study might throw out completely novel and unexpected findings. But an intestinal biopsy (as per the study you've cited) would perhaps include a range of immune cells that aren't seen in muscle tissue. The gut lining has an immune rule and secretes various substances including antibodies, so perhaps it wouldn't be surprising to find antibodies in the tissue culture in that particular study?

Bottom line is Bob that I don't know so as you say let's not rule anything out.

 

[alex3619]

They both seem to activate AMPK, both through different mechanisms:

The way this diagram is organized is misleading. There are loads of different mechanisms combined there, just grouped into convenient categories. Resveratrol for example is a phosphodiesterase-4 inhibitor and initially raises cAMP. Many of these mechanisms are still being researched, and there may be surprises.

Let me add another layer to this discussion. I was interested in the idea of raising cAMP to modify the physiology. Things that mess about with AMP levels might do that. When cAMP goes up, Ca++ goes down (but can spike) and it changes the entire intracellular messaging balance.

 

[Bob]

Re MRC getting involved with future funding: This is obviously up to Julia et al in Newcastle.

But she is on the Board of the CM Research Collaborative (who will know about this research), has received MRC grants, is currently receiving an MRC grant, and clearly knows what to do if she feels that this research is worth pursing with an MRC grant.

The ring fenced money from the MRC for ME/CFS research that followed on from the high priority research recommendations that the Expert Group produced has now been used and there are no plans to repeat this initiative.

So it's up to researchers to apply to the MRC if they want an MRC research grant.

Thanks Charles. I suppose my point was: I hope the MRC are going to support follow-up studies and similar studies, if applications are made by any researchers with a robust research plan. Yes, an application must be made, but the MRC must also be willing to fund large and expensive studies into ME/CFS. An application is only going to be successful if the MRC is willing to support ME/CFS.

 

[alex3619]

Bottom line is Bob that I don't know so as you say let's not rule anything out.

I would describe it this way ... this study does not rule anything out, but it does challenge a few hypotheses.

 

[alex3619]

See figure 3 in the paper. I think it may suggest that insulin is also not a problem when the cells are stimulated.

That is not what I see.

I find this statement confusing:

. Both basal and insulin stimulated glucose uptake were significantly increased in CFS compared to control (p = 0.001 and p<0.05, respectively). However, this does not alter the finding that CFS was unable to increase glucose uptake in response to EPS since an increase in glucose uptake was observed only in the presence of insulin indicating that the cells are not maximally stimulated in the basal state.

And this:

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

We also need further research on what IL6 is doing.

In figure 3 there was a big increase in EPS/insulin over just insulin in controls, but not in CFS. However this might be due to the issue that the insulin response was quite large without EPS.

However CFS patient muscle also had a larger basal glucose uptake. What does this mean? The basal rate should reflect normal metabolic processes. Do we need more energy just to survive? Is the impact of low energy made worse by this? Is it just low energy or is it high demand and low supply in combination? Too many questions, not enough answers.

Let me put it another way. One interpretation is we need more energy at rest, and don't increase energy enough during activity.