Metabolite-Detecting Gene Expression After Exercise in CFS, MS, and Controls

[CBS]

Differences in Metabolite-Detecting, Adrenergic, and Immune Gene Expression After Moderate Exercise in Patients With Chronic Fatigue Syndrome, Patients With Multiple Sclerosis, and Healthy Controls
Psychosom Med PSY.0b013e31824152ed; published ahead of print December 30, 2011, doi:10.1097/PSY.0b013e31824152ed

Andrea T. White, PhD,
Alan R. Light, PhD,
Ronald W. Hughen, MS,
Timothy A. VanHaitsma, MS and
Kathleen C. Light, PhD

+ Author Affiliations

From the Departments of Exercise and Sport Science (A.T.W., T.A.V.), Anesthesiology (A.R.L., R.W.H., K.C.L.), and Neuroscience (A.R.L.) and The Brain Institute (A.T.W.), University of Utah, Salt Lake City, Utah.

Abstract

Objective: Chronic fatigue syndrome (CFS) and multiple sclerosis (MS) are characterized by debilitating fatigue, yet evaluation of this symptom is subjective.

We examined metabolite-detecting, adrenergic, and immune gene expression (messenger ribonucleic acid [mRNA]) in patients with CFS (n = 22) versus patients with MS (n = 20) versus healthy controls (n = 23) and determined their relationship to fatigue and pain before and after exercise.

Methods: Blood samples and fatigue and pain ratings were obtained at baseline and 0.5, 8, 24, and 48 hours after sustained moderate exercise.

Leukocyte mRNA of four metabolite-detecting receptors (acid-sensing ion channel 3, purinergic type 2X4 and 2X5 receptors, and transient receptor potential vanilloid type 1) and four adrenergic (?-2a, ?-1, and ?-2 receptors and catechol-O-methyltransferase) and five immune markers (CD14, toll-like receptor 4 [TLR4], interleukin [IL] 6, IL-10, and lymphotoxin ?) was examined using quantitative polymerase chain reaction.

Results: Patients with CFS had greater postexercise increases in fatigue and pain (1029 points above baseline, p < .001) and greater mRNA increases in purinergic type 2X4 receptor, transient receptor potential vanilloid type 1, CD14, and all adrenergic receptors than controls (mean standard error = 1.3 0.14- to 3.4 0.90-fold increase above baseline, p = .04.005).

Patients with CFS with comorbid fibromyalgia (n = 18) also showed greater increases in acid-sensing ion channel 3 and purinergic type 2X5 receptors (p < .05).

Patients with MS had greater postexercise increases than controls in ?-1 and ?-2 adrenergic receptor expressions (1.4 0.27- and 1.3 0.06-fold increases, respectively, p = .02 and p < .001) and greater decreases in TLR4 (p = .02). In MS, IL-10 and TLR4 decreases correlated with higher fatigue scores.

Conclusions: Postexercise mRNA increases in metabolite-detecting receptors were unique to patients with CFS, whereas both patients with MS and patients with CFS showed abnormal increases in adrenergic receptors.

Among patients with MS, greater fatigue was correlated with blunted immune marker expression.

Following Dolphin's example, I've given each sentence in the abstract its own paragraph for ease of reading.

 

[alex3619]

The thing I found most interesting is that MS also has adrenergic receptor issues, though of a different type. It might be a clue as to why MS and ME are so similar. Bye, Alex

 

[Snow Leopard]

This paper also overlaps with another paper by Kathleen Light on gene expression in FM:
http://forums.phoenixrising.me/showthread.php?15377-Kathleen-Light-New-Fibro-ME-CFS-Review

 

[Valentijn]

According to wikipedia, P2X4 causes "hypersensitivity to innocuous stimuli following upregulation of P2X4 in the spinal cord" according to wikipedia, and the function of TPRV-1 "is detection and regulation of body temperature. Additionally, TRPV1 provides sensation of scalding heat and pain (nociception)" as well as triggering a response that causes hypersensitivity to painful stimuli and the sensation of pain from non-painful stimuli.

Maybe topical capsaicin would work for some of the ME/CFS muscle pain, by desensitizing TPRV1.

 

[Vitalic]

The Light's work is incredible, they are really trying to get to the core of the issue. These are the people we really need to be supporting 100%.

p.s. did they mention whether the immune markers were upregulated postexercise? i know their other study detected this so i wondered if the results were consistent

 

[Camilla]

The Light's work is incredible, they are really trying to get to the core of the issue. These are the people we really need to be supporting 100%.

I agree! This is one of our most hopeful leads.

Some people have wondered about the name of the journal, Psychosomatic Medicine. Does anyone know why they would choose this journal for publication? Or is it rather a matter of taking what you get, i e not their first choice?

 

[alex3619]

Maybe topical capsaicin would work for some of the ME/CFS muscle pain, by desensitizing TPRV1.

Hi Valentijn, and may explain why I don't feel so well if I am not eating chili. Bye, Alex

 

[biophile]

It is very interesting, but I have concerns about the Lights' use of the "Reeves" CDC 2005 criteria in all their CFS exercise studies. I wrote a comment about it on the review thread that Snow Leopard posted above (http://forums.phoenixrising.me/show...-ME-CFS-Review&p=231367&viewfull=1#post231367).

 

[*GG*]

I agree! This is one of our most hopeful leads.

Some people have wondered about the name of the journal, Psychosomatic Medicine. Does anyone know why they would choose this journal for publication? Or is it rather a matter of taking what you get, i e not their first choice?

I am not really certain. But I think they probably tried to publish in other journals, but had to settle.

GG

 

[oceanblue]

It is very interesting, but I have concerns about the Lights' use of the "Reeves" CDC 2005 criteria in all their CFS exercise studies. I wrote a comment about it on the review thread that Snow Leopard posted above (http://forums.phoenixrising.me/show...-ME-CFS-Review&p=231367&viewfull=1#post231367).

That would be alarming, but in this paper, at least, it states that the 22 CFS patients were diagnosed using Fukuda/CDC '94. Also true for the 2011 paper, as Dolphin pointed out on another thread:

...
Information on cohort:

All CFS patients met the CDC criteria for CFS
[1], and 46 (96%) met the Canadian Criteria for ME/CFS as well [2]

1. Fukuda, K., S.E. Straus, I. Hickie, M.C. Sharpe, J.G. Dobbins and A. Komaroff. The chronic
fatigue syndrome: a comprehensive approach to its definition and study. International Chronic
Fatigue Syndrome Study Group. Ann.Intern.Med 1994; 121: 953-959.

So I'm a bit confused as to which studies the Empiric criteria were used in.

 

[Snow Leopard]

Who has read this? Any points of note would be helpful.

What is the reasoning for the adrenergic receptor expression? In my opinion, this increased expression might not so much be the cause of fatigue so much as a response to the fatigue and the cells way of trying to overcome fatigue by increasing its level of stimulation?

It is a mistake to assume that it is merely dysfunctional signalling causing the sensation of fatigue.

 

[biophile]

Thanks oceanblue for pointing out Dolphin's post on one of these studies (Gene expression alterations at baseline and following moderate exercise in patients with Chronic Fatigue Syndrome, and Fibromyalgia) mentioned in the Light et al 2011/2012 review, where 46/48 (~96%) of the CFS patients also met Canadian criteria in that particular study. That eases my concerns about the CFS cohort, but I would still like to see stress/anxiety controls too!

oceanblue wrote: So I'm a bit confused as to which studies the Empiric criteria were used in.

Probably most or all of them, but as the Reeves criteria apparently casts a very broad net, the use of other criteria like the Canadian one would discard a lot of fish.

Snow Leopard wrote: Who has read this? Any points of note would be helpful. What is the reasoning for the adrenergic receptor expression? In my opinion, this increased expression might not so much be the cause of fatigue so much as a response to the fatigue and the cells way of trying to overcome fatigue by increasing its level of stimulation? It is a mistake to assume that it is merely dysfunctional signalling causing the sensation of fatigue.

I haven't read the full text either but it seems consistent with all their other studies on CFS patients. Their recent chapter on the issue for a book (http://www.ncbi.nlm.nih.gov/books/NBK57253) is helpful and they do speculate about possible causes for the dysregulation. IIRC one of Cort's articles about the research states that the Light's also speculate about a chronic infection in the dorsal root ganglion?

I agree with your questioning, the body may attempt to overcome symptoms by increasing stimulation and the increases in adrenergic receptor expression would help to explain that, although wouldn't increased COMT activity also increase the breakdown of catecholamines and therefore partly counteract the stimulation at the expense of reduced dopamine? Also, blockade against adrenergic receptors may have negative effects for some of us, although Light et al have pointed out that the amount of beta blockers needed is only 1/5 to 1/10 of what is usually used for other conditions.

Some of the other receptors may be overexpressed for good reason too, to help prevent the body from further over-exertion.

 

[alex3619]

What is the reasoning for the adrenergic receptor expression? In my opinion, this increased expression might not so much be the cause of fatigue so much as a response to the fatigue and the cells way of trying to overcome fatigue by increasing its level of stimulation?

It is a mistake to assume that it is merely dysfunctional signalling causing the sensation of fatigue.

HI Snow Leapard, I don't know that you are right, but I do think its a valid hypothesis and I have had the same thought myself. Bye, Alex

 

[oceanblue]

Different reaction to exercise for CFS vs fatigued MS patients

I found this paper pretty heavy-going so I'll start with a basic finding, the self-reported reaction to 'moderate' exercise for pain, Physical Fatigue (PF) and Mental Fatigue (MF), all on a VAS scale. What I found really interesting was the different reactions between MS patients - who were all fatigued at baseline - and CFS patients.

Basically, MS patients showed no change in these measures post-exercise (using group-by-time repeated measure ANOVA), while CFS patients reported big increases in pain, PF and MF (p<0.001), though the gain in MF was not significant relative to MS patients:

patients with CFS had significantly higher postexercise PF and pain ratings than patients with MS ( p < .002)

This is the first direct comparison of exercise response in CFS vs fatigued MS patients, AFAIK.

This finding ties in with the different attitudes to graded exercise that CFS and MS patients have generally. CFS patients and their organisations often report problems, while the MS Society recommends exercise for fatiue, which was first trialled for MS patients following anecdotal reports that MS patients found it helpful. This could just be because we CFS types are a cussed lot, or it could be that we react quite differently to moderate exercise than MS patients.

 

[oceanblue]

Some general concerns with the study

Overall, I think the main findings are sound, but I do have a number of concerns which make me doubtful about many of the minor findings that actually just make the paper more confusing. So here are my concerns:

1. Relationship of mRNA changes to biologically significant changes

This concerns relates to all studies using gene expression in white blood cells as a proxy for biologically important change in different types of cells.

• mRNA changes in leucocytes vs mRNA changes in muscle nerves
  There was a large study done a few years back (which I can no longer find) which compared results between white blood cell gene expression and gene expression in various tissues. Overall, it found there was a correlation between changes in tissue gene expression and changes in the same genes in leucocytes. In some ways this is bizarre, as if the leucocytes are mimicing every other cell in the body, but there you go. However, the strength of correlation between leucocyte and tissue was very variable and I don't think it was ever extremely high (eg r>=0.8).
  
• mRNA changes vs changes in signaling from protein receptors
  From the wikipedia on gene expression study limitations
  

  Cells use many other mechanisms to regulate proteins in addition to altering the amount of mRNA, so these genes may stay consistently expressed even when protein concentrations are rising and falling. [and vice versa]. Other experiments, such as a Western blot of some of the protein products of differentially expressed genes, make conclusions based on the expression profile more persuasive, since the mRNA levels do not necessarily correlate to the amount of expressed protein.

  Additionally, there are many possible modifications of the protein receptors that influence activity levels regardless of the number to receptors.

Consequently, changes in mRNA in leucocytes won't necessarily correlate very accurately with changes in receptor signalling in muscle nerves - which is the underlying assumption of this experiment.

2. Use of mulitple comparsions
With 13 genes measured at 5 different time points for 3 different groups (MS, CFS and controls) there are a lot of potential comparisons, with the danger of false positives. The stats section says "To decrease the likelihood of obtaining false-positive results due to multiple comparisons, we pooled postexercise responses into a variable labeled area under the curve (AUC)" but many of the findings are looking at individual time points, not the pooled AUC and so do carry the risk of false positives. Note that their 2011 paper explicitly didn't consider individual timepoints for precisely this reason.

These 2 points make me suspicious of findings where p=0.05 or close to it; they could well just be noise.

 

[oceanblue]

Metabolite-Detecting Gene Expression

This covers 4 genes: Acid-sensing ASIC, ATP-sensing Purinergic receptors P2X4 and P2X5, and Heat/acid-sensing TRPV1. There were significant time effects for each gene, ie expression changed between baseline and post-exercise in every case.

Broadly speaking, MS patients and controls showed a similar pattern with most of the genes showed lower expression at 0.5 hours and 8 hours post-exercise:

These transitory decreases in expression of these receptors
after exercise may represent adaptive downregulation in
response to enhanced receptor activation. The full return to
preexercise levels for all four receptors that were evident in both
MS and control groups by 24 hours later is also consistent with
a well-regulated sensory pathway.

By comparison, most of these same genes appeared to increase expression substantially post-exercise. However, the standard error of the mean (SEM) was large and only P2X4 at 24 hours was significantly increased relative to baseline. The large SEM suggests a lot of variation in the data indicating, perhaps, some patients had large changes in expression while others had only minor ones (have I've got that right?)

Additionally, P2X4 (which is probably the most important gene in the experiment) showed different expression between CFS and both MS patients and controls, and, crucially, correlated with changes in pain too:

Greater P2X4 expression was also
directly associated with severity of postexertional fatigue and
pain only in the CFS group and may be important for sustaining
postexertional malaise and reduced pain threshold
observed in patients with CFS

This is perhaps overstating things: they reported that P2X4 expression correlated with Mental Fatigue only at 8 hours post-expression, and there was no mention of Physical Fatigue, suggesting that it didn't correlate with P2X4 expression at all. So the correlation appears to be more with pain than fatigue. No other genes in the whole experiment correlated with fatigue or pain for CFS, which is a little worrying.

 

[oceanblue]

Adrenergic Receptor gene expression

This covers 4 genes: (Adrenergic Receptors Alpha-2a, Beta-1 and Beta-2 that help regulate blood flow to working muscles, and catechol-O-methyltransferase, COMT [don't know the biological role of this]).

The control group response was broadly flat, with Beta 2 and COMT expression dipping significantly 30 minutes post-exercise before returing to baseline levels. This pattern is similar to that seen for control metabolite receptor genes.

Generally the adrenergic genes showed a boost in expression post-exercise in both MS and CFS patients.

The abstract says there were "Patients with CFS had.. greater mRNA increases in... all adrenergic receptors than controls"; however, figure 3 says that using repeated measures ANOVA there were only group differences for the Alpha-2a and Beta-2 receptors, ie the differences with Beta-1 and COMT between groups were not significant overall. So it looks like the authors have relied on significance at some individual time points to make the claims for Beta-1 and COMT, though these may well be false postives due to mulitple comparisons.

So what's the significance of MS & CFS patients showing greater expression of (some) adrenergic receptors than controls in response to exercise? 3 possibilities come to mind:

1. As the authors suggest: "adrenergic dysregulation may be linked to the pathological fatigue in both of these disorders". However, adrenergic receptor expression did not correlate with fatigue in CFS patients, though it did in MS patients
2. For CFS patients, the increase in adrenergic receptors may be a response to the higher level of metabolites indicated by higher metobolite-gene mRNA expression (ie trying to increase blood flow to clear the excess metabolites). I think this is what Snow Leopard suggested earlier
3. Both MS & CFS patients had higher exertion levels than controls and the higher expression of adrenergic receptors may be linked to this (though metabolite gene expression was different between MS & CFS groups).

Given the lack of response to my earlier posts I'll assume there's not much interest in this paper so will cut my losses and leave it here.

 

[Vitalic]

Appreciate the breakdowns oceanblue, I'm assuming we can infer from the increase in metabolite-detecting receptors that there is an over-abundance of metabolites being produced as a result of cell metabolism, the question is what could trigger those excesses, that is where my knowledge meets it's limit. The other possibility though is that the excess in metabolite-detecting receptors is a result of some other mechanism but is contributing to the disproportionate response to small amounts of exertion, i.e. the body misinterprets low levels of exertion as high intensity exertion due to the overabundance of detection receptors. Would be good to get some insight from Richvank etc. My initial thought was that it might be a consequence of "de-conditioning", or rather that as general fitness and tolerance to exertion decreases the body produces more lactic acid and other metabolites more quickly after crossing the anaerobic threshold, but given the fact that it was unique to the ME subjects makes that seem unlikely. The fact it was unique should make this big news, at least bigger than the reception it has got, it is another potential bio-marker.

 

[oceanblue]

Appreciate the breakdowns oceanblue, I'm assuming we can infer from the increase in metabolite-detecting receptors that there is an over-abundance of metabolites being produced as a result of cell metabolism, the question is what could trigger those excesses, that is where my knowledge meets it's limit. The other possibility though is that the excess in metabolite-detecting receptors is a result of some other mechanism but is contributing to the disproportionate response to small amounts of exertion, i.e. the body misinterprets low levels of exertion as high intensity exertion due to the overabundance of detection receptors. Would be good to get some insight from Richvank etc. My initial thought was that it might be a consequence of "de-conditioning", or rather that as general fitness and tolerance to exertion decreases the body produces more lactic acid and other metabolites more quickly after crossing the anaerobic threshold, but given the fact that it was unique to the ME subjects makes that seem unlikely. The fact it was unique should make this big news, at least bigger than the reception it has got, it is another potential bio-marker.

Thanks, Vitalic.

I agree, the increase in metabolite gene expression could be down to unusually high levels of metabolites (though I'm not sure if that would normally lead to greater gene expression of receptors), or could be part of an abnormality in sensing metabolites. A recent study by Julia Newton found excess build up of acid in some CFS patients, but I think that was based on a sub-sample of only 8 people.

As you say, the fact that MS patients don't have raised metabolite-receptor gene expression tends to argue against deconditioning. However, it's worth noting that the MS patients were only mildly-affected with "Expanded Disability Status Scale scores mean = 2.0, (range = 1.0-3.0)". A score of 2.0 on the EDSS scale equates to only 'minimal disability in one Functional System, no impairment in walking' while the maximum recorded score of 3.0 is still Moderate disability in one FS, or mild disability in three or four FS, with no impairment to walking. So it's not clear how well they were matched in physical condition to the CFS patients.

 

[Marco]

I appreciate your efforts Oceanblue and I do think the Lights may be onto something - although I also have some concerns that they assume the problem is just one of abnormal signalling.

The COMT gene is an interesting one. I've been looking at this recently and COMT val158met polymorphisms have been associated with schizophrenia, bipolar disorder, PTSD, anxiety disorders and fibromyalgia to name just a few. Of course the relationship is not straightforward as no single disease is directly linked with a particular polymorphism and its likely that a number of genes may interact to result in a genetic predisposition to develop a particular disease/disorder.

Nonetheless the val (high activity) or met (low activity) alleles do appear to contribute to a some general predispositions that may appear as symptoms in a range of diseases and it appears that have one or both met mutations results in an increased sensitivity to pain. It also confers some advantage in higher executive cognitive function but this advantage breaks down (i.e. higher executive cognitive functions are compromised) under conditions of stress.

One commentator rather crudely suggest that these polymorphisms may contribute to whether you are a warrior (val) or worrier (met).

I very much doubt whether these polymorphisms singly and directly cause ME/CFS, but in combination with other SNPs may constitute a risk factor and/or contribute to the constellation and severity of symptoms that an individual experiences.

PS If you ever wish to trace physiological pathways/links (in the interests of speculating about these things which I admit I like to do) I've found this a useful tool :


http://biograph.be/concept/graph/C0015674/C1135159