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

[Bob]

I agree with you Alex. This study raises many interesting questions, and gives no answers.

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.

I interpreted this to mean that there was no further increase when EPS was added to insulin, which suggests a problem with EPS only, but I may have misinterpreted.

 

[Sidereal]

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.

A channelopathy or a chronic infection would explain increased resting energy expenditure. You see the same problem in HIV or hepatitis C, for instance. The cell has to work harder to maintain membrane potential in order to merely exist/survive, let alone exercise.

 

[alex3619]

A channelopathy or a chronic infection would explain increased resting energy expenditure. You see the same problem in HIV or hepatitis C, for instance. The cell has to work harder to maintain membrane potential in order to merely exist/survive, let alone exercise.

We have had reasons to suspect one or more channelopathies for decades. Yet so far nobody has proven one.

 

[jimells]

It also raises the question as to how a putative psychogenic disorder causes long term muscle changes that persist in vitro.

I asked Sir Simon about this and he determined that some of the petri dishes were abused at the glass factory. In addition, some of the factories are located in bad neighborhoods

 

[Snow Leopard]

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.

Not necessarily.

The chronic fatigue syndrome and control cultures were studied under identical conditions at passage 7, making it highly probable that the defects in the chronic fatigue cultures are due to retained defects within the cultures of an epigenetic and/or genetic basis.

That is exactly as it sounds, but we haven't really seen any fruitful results from SNP studies, a genetic mitochondrial disease is unlikely, making epigenetic factors most likely. Those epigenetic factors are due to conditioning* of the cells over time, as adaptations to various biological conditions.

*(not necessarily behavioural muscular deconditioning!)

I had forgotten about the comments I had made several years ago, mentioned by @nandixon

I can't remember all the details, but the idea is that the symptoms are indeed mediated through increased oxidase stress, issues with fatty acid metabolism, there were a few suggestions at the time: http://www.ncbi.nlm.nih.gov/pubmed/21205027 (was part of ongoing research, should be a followup at some time)
This relates to some of the things mentioned:

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

But the mediation of the symptoms, is not the same as the primary/central cause, which remains unknown and may result from B-cell issues for example.

Getting back to AMPK, working out the role that it plays is subtle, as like many proteins in the body, it plays a role in a variety of pathways, being a fairly central factor. So it is hard to tell which issues in the cell are leading to the differences.

Some more speculation:

http://www.ncbi.nlm.nih.gov/pubmed/14707762
"Protecting muscle ATP: positive roles for peripheral defense mechanisms-introduction."

AMPK has been described as a 'sensor for cellular stress':

Abstract
Skeletal muscle has evolved an impressive array of mechanisms for peripherally mediated control of ATP homeostasis. Some of these mechanisms are intracellular, and others are extracellular and include influences on the cross-bridge cycle itself and substrate supply. This paper introduces three distinctly different topics that nevertheless all have ATP defense in common. The role of ADP in fatigue is controversial but has recently been more clearly delineated so that an effect on alleviating force declines during extreme fatigue is plausible. AMP plays its role by activating the protein-kinase, AMPK, which is a key sensor of cellular energy stress. AMPK has different isoforms, is not uniformly distributed in the cell, and its activation is carefully controlled. It has multiple effects including improvements in substrate supply for the metabolic pathways producing ATP and inhibition of anabolic processes to further spare ATP. Red blood cells have the capacity to sense hypoxia and to release vasodilators where there is a locally increased demand for blood supply. The papers in this series emphasize the important positive roles of metabolites and sensors of fatigue in the balance between ATP supply and demand.

From Wikipedia:

The function of ACC is to regulate the metabolism of fatty acids. When the enzyme is active, the product, malonyl-CoA, is produced which is a building block for new fatty acids and can inhibit the transfer of the fatty acyl group from acyl CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria.

So the AMPK-ACC (Acetyl-CoA carboxylase) pathway is a key switch regulating fatty acid metabolism as a result of cellular stress.

Eg:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165558/
"Hypoxia Triggers AMPK Activation through Reactive Oxygen Species-Mediated Activation of Calcium Release-Activated Calcium Channels"

Also,

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274756/
"Muscle Fatigue and Cognition: What is the Link?"

We hypothesize that mechanisms contributing to muscle damage after strenuous exercise may be the same as those that could be caused by high doses of AICAR. Free oxygen radicals are generated during exercise as a side product of oxidative metabolism. In particular, increased production of nitric oxide (NO) derivates is a desired consequence of exercise for proper muscle function but higher levels of NO can cause contractile dysfunction, resulting in muscle fatigue. Strenuous exercise can accelerate the generation of NO to levels that result in oxidative stress (Nikolaidis et al., 2008), sustained for days after exercise (Appell et al., 1992). NO induces mitochondria biogenesis in skeletal muscle via upregulation of PGC1α, and interacts with AMPK. Pharmacological activation of AMPK with AICAR and the subsequent induction of GLU4 are blunted by inhibition of NO production. AMPK phosphorylates and activates eNOS and nNOS, and is necessary for NO-dependent increase in the expression of PGC1α, mitochondrial gene expression, and respiration in skeletal muscle cells. It was proposed that NO and AMPK interact through a positive feedback loop in skeletal muscle (Lira et al., 2010). Moreover, in neurons NO production is elicited by AMPK, and in turn, increases AMPK activity (Murphy et al., 2009). Altogether, high doses of AICAR may be harmful for body and brain.

Interestingly, there are links with the exercise studies by Light et al, with eNOS etc being regulated by beta adreneric receptors.

Lastly, in terms of epigenetic factors, they can be induced over time by different levels of stimulating factors (vs controls).
Such as:
"Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independent of no synthase"
http://www.jbc.org/content/early/2010/02/03/jbc.M110.108688

"AMP-Activated Protein Kinase Signaling Stimulates VEGF Expression and Angiogenesis in Skeletal Muscle"
http://circres.ahajournals.org/content/96/8/838

"The role of AMP-activated protein kinase in endothelial VEGF signalling"
http://theses.gla.ac.uk/1129/

The endothelium acts to maintain vascular homeostasis, including the regulation of vascular tone, blood fluidity and coagulation. Endothelial dysfunction, a condition largely characterised by reduced NO bioavailability, is an important feature associated with the aetiology of several pathophysiological disorders including type 2 diabetes and cardiovascular disease. AMPK is the downstream component of a protein kinase cascade important in the regulation of cellular and whole body metabolism. AMPK has been demonstrated to mediate a number of physiological responses in the endothelium, including the stimulation of eNOS phosphorylation and NO synthesis; and as such AMPK represents a therapeutic target in the dysfunctional endothelium. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. VEGF stimulates NO production, proposed to be a result of phosphorylation of Ser-1177 on eNOS, a residue also phosphorylated upon AMPK activation in cultured endothelial cells. The present study, utilising HAEC as a model, provides the first demonstration that AMPK is activated by physiological concentrations of VEGF; and furthermore, partially mediates VEGF-stimulated phosphorylation of eNOS on Ser-1177 and subsequent NO production. In addition, the present investigation demonstrates that the upstream AMPK kinase CaMKK is responsible for these VEGF-mediated effects. VEGF is known to increase intracellular calcium levels in endothelial cells via the generation of DAG and IP3. DAG increases Ca2+ influx through a family of non-selective cation channels, whereas IP3 promotes the release of Ca2+ from intracellular stores. High potassium-induced depolarisation, which reduces the driving force for Ca2+ entry through non-selective cation channels in endothelial cells, abolished VEGF-mediated AMPK activation, whereas the IP3 receptor blocker 2-APB was without effect. Exposure of HAEC to a DAG mimetic (OAG) also stimulated AMPK, an effect which was sensitive to the CaMKK inhibitor STO-609 and high potassium induced depolarization. The functional effects of VEGF-stimulated AMPK were also assessed in HAEC. Ablation of AMPK abrogated VEGF-stimulated HAEC migration and proliferation, two key features of the angiogenic process. While AMPK was necessary for VEGF-stimulated endothelial cell proliferation direct activation of the kinase was insufficient to induce this process. AICAR-stimulated AMPK activation has been demonstrated to stimulate fatty acid oxidation in endothelial cells. However, exposure of HAEC to VEGF did not alter fatty acid oxidation in the present study. Together, the current investigation suggests that a VEGF-Ca2+-CaMKK-AMPK-eNOS- NO pathway is present in HAEC, and furthermore, that AMPK is required, albeit insufficient, for the VEGF-stimulated angiogenic response.

As from the above, it is all quite subtle and there are multiple pathways at work, but the key is that the cells in the ME patients had been conditioned over time due to physiological differences, (relying on a different state of regulation of the pathways of the type mentioned above) to have a different AMPK response.

Note, even though I mentioned VEGF above, I'm not suggesting that there is an issue with VEGF (which has not been observed in cytokine studies), I suspect different growth factors and issues with the receptor rather than the growth factor itself, but all speculative at this point...

 

[lansbergen]

I asked Sir Simon about this and he determined that some of the petri dishes were abused at the glass factory. In addition, some of the factories are located in bad neighborhoods

 

[nandixon]

That is exactly as it sounds, but we haven't really seen any fruitful results from SNP studies, a genetic mitochondrial disease is unlikely, making epigenetic factors most likely. Those epigenetic factors are due to conditioning* of the cells over time, as adaptations to various biological conditions.

*(not necessarily behavioural muscular deconditioning!)
............
As from the above, it is all quite subtle and there are multiple pathways at work, but the key is that the cells in the ME patients had been conditioned over time due to physiological differences, (relying on a different state of regulation of the pathways of the type mentioned above) to have a different AMPK response.

In order to rule out the results of the study being due to possible epigenetic changes due to behavioral muscular deconditioning, it seems they may have needed to look at three groups: ME/CFS patients, healthy controls (who are not avid runners or weightlifters, etc.), and, e.g., long-distance runners.

I hate to think about it, but is it possible that the bar charts might look (relatively) the same when comparing ME/CFS patients to healthy (non-vigorous exercising) controls, versus those same healthy controls compared to say long-distance runners?

 

[WillowJ]

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.

That's my understanding as well.

I think these problems are usually acute and episodic, rather than becoming chronic

I'm told that some chronic genetic diseases can present this way.

 

[WillowJ]

I hate to think about it, but is it possible that the bar charts might look (relatively) the same when comparing ME/CFS patients to healthy (non-vigorous exercising) controls, versus those same healthy controls compared to say long-distance runners?

I sometimes wonder if the physiology of ME patients might not look more like athletes during a triathalon, than healthy people at rest.

 

[Sean]

I sometimes wonder if the physiology of ME patients might not look more like athletes during a triathalon, than healthy people at rest.

I recall somebody commenting a while back that it is interesting that patients are not more classically deconditioned, almost as if their bodies are actually working hard, at least at the cellular level, even when apparently doing nothing at the macro level.

 

[adreno]

I doubt it. Athletes have upregulated antioxidant systems able to deal with the ROS created during exercise. We don't.

 

[xks201]

I have heard a lot about b cell dysfunction from bad vaccine reactions. The above info of course encompasses dysfunctional mitochondria and or NO metabolism. if indeed a NO deficiency was present I would think arginine or something would easily fix it....bit something tells me it isn't that simple in all cases.

 

[Bob]

@Jonathan Edwards, if you haven't seen this paper yet, i'm sure you'll be interested. It's Julia Newton's latest.

 

[wastwater]

Isnt (S)CMV a known genetic mutator

 

[Jonathan Edwards]

@Jonathan Edwards, if you haven't seen this paper yet, i'm sure you'll be interested. It's Julia Newton's latest.

Thanks Bob,
I had not got around to looking at it properly until today.

I have to admit to being puzzled. A genetic difference giving rise to the illness pattern of ME that would show up in culture seems unlikely. When people talk about epigenetic in this long term proliferative culture situation I guess it would be methylation and I find it hard also to see how that would work. There is of course the other possibility that ME muscle biopsies tend to contain satellite cells more of a subtype less frequent in controls and that this is a differential growing out effect. Again, I find that a bit unlikely.

I think any effects of antibodies would disappear in culture but there is just one other mechanism that could be relevant. In scleroderma it seems likely that autoantibody attack over a period of years leaves endothelial and muscle cells with exhausted telomeres. Maybe ME muscle cells have something like a telomere problem. It would not seem to be telomere as such because the cells divided OK, but maybe there is some other mechanism that works like this. Maybe it takes us back to epigenetic methylation and repeated autoantibody based damage to muscle leads to gradual methylation of certain genes. Trouble is that there are so many clues missing that one might expect to find - no long term rise in CK, no ANA, no fibrosis...

Then there are the data. They look OK as they stand but I don't get a feel for how they slot into a clear story. I do not understand the absence of the 16 hour peak for AMPK. I do wish people would not use these histograms with error bars - I find it pretty impossible to get a feel for the data. I think it should be banned!

It is going to be very difficult to replicate this study because getting muscle biopsies is not easy - it is a fairly unpleasant procedure in my experience. The previous MRI spectroscopy approach seems to me much more practical - and also a much more direct indication of what is going on in real life. Maybe now that there are these data from the cultures they can go back and look at related changes with MRIS.

If this is the first sign that a primary muscle abnormality can be pinned down that has to be important news. However puzzling it might be, if it is real it would shift the framework for thinking into a new gear. I think we need to see what follow up there is.

 

[Bob]

As Alex said, lots of questions, not many answers!

 

[PDXhausted]

I'd be really tempted to offer up my muscle for biopsy. Skeletal muscle problems (weakness, cellular metabolism problems) are my biggest complaint.

The thing that made them go from moderate to severe is amoxicillin... But by way of gut damage. I initially felt better the first few weeks, and after my gut/microbiome became damaged by week 6, I started to not be able to move my muscles and rapidly declined to nearly paralyzed.

The thing that has improved them is Equilibrant. Hard to say why, but my instinct says inducing interferon gamma for various reasons. I'm still completely bedridden, my leg muscles have actually wasted since becoming bedridden and even with improvements I can't seem to build the muscle back, though I'm better using the muscle I still have while on Equilibrant.

These were some quick off-the-cuff comments from my husband, a hematopathologist, that I thought were interesting:

"Yes, the mechanics of the cell metabolism are dysfunctional. As to how, needs to be further determined. Is there viral disruption, epigenetic (methylation aberrancies) or gene down regulation? Is there a cell structural problem? Is there an electrochemical problem? (Keep in mind the electrochemistry is incredibly important for routine cell function.)

Some viruses will integrate into host DNA and hijack their cellular machinery. Their genetics can be passed on through cell division or possibly by co-infection of progeny cells.

I would be taking a very close look at the glycolysis pathway... Looking too at ultra structural cell changes by light and electron microscopy.

It would be very interesting to test for infectious elements in these cells, by multiple methods, like pcr, immuno stains, culture, co-infection.

There are so many mysteries out there... Just look at prion disease. I mean, where did that come from? Infectious protein? So much to discover. I don't let conventional wisdom preclude possibilities."

And some follow up comments:

"Could there be a paracrine (or lack thereof) phenomenon in the CFS muscle cells responsible for the findings? (Paracrine as in cell-to-adjacent-cell signaling). What if CFS cells were cultured along with normal muscle cells? Would the normal cell induce changes in CFS cells that allow them to function properly? Or conversely, would the CFS cells produce signals that cause normal muscle cells to become dysfunctional? If it is infectious, perhaps the CFS cells could inoculate normal cells?

In addition, comparisons should be made to cells with known mitochondrial disease like mitochondrial myopathy, as well as cells from patients with autoimmune conditions (MS and Lupus) and cells from non-diseased patients with atrophy of skeletal muscle.

Ultra structural studies using electron microscopy would be helpful to assess for structural anomalies that may hint at underlying pathogenesis.

Clever biochemical studies could be performed to assess what step(s) of glucose metabolism are being disrupted, if that is the case.
Nested PCR studies could be performed to look for viral nucleic acids in CFS cells. Immunohistochemical stains, like VP1 for enterovirus that Dr Chia uses, could be performed. "

 

[RustyJ]

The disregulated glucose uptake in the muscle might be, dare I say it, systemic - could it explain the SPECT scan abnormalities (referred to often as hypoperfusion) in the brain. What about the gut...?

 

[lansbergen]

The disregulated glucose uptake in the muscle might be, dare I say it, systemic - could it explain the SPECT scan abnormalities (referred to often as hypoperfusion) in the brain. What about the gut...?

There are muscle cells in the gut, so why not?

 

[lansbergen]

There are so many mysteries out there... Just look at prion disease. I mean, where did that come from? Infectious protein?

The protein that was blamed, nowadays usely is called a surogate marker.

Some TSE strains probably are very old.

Anyway the cellulair prion protein is very very very very old.