Dysregulated Provision of Oxidisable Substrates to the Mitochondria in ME/CFS Lymphoblasts, 2021, Missailidis et al

[John Mac]

Abstract
Although understanding of the biomedical basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is growing, the underlying pathological mechanisms remain uncertain.

We recently reported a reduction in the proportion of basal oxygen consumption due to ATP synthesis by Complex V in ME/CFS patient-derived lymphoblast cell lines, suggesting mitochondrial respiratory inefficiency.

This was accompanied by elevated respiratory capacity, elevated mammalian target of rapamycin complex 1 (mTORC1) signaling activity and elevated expression of enzymes involved in the TCA cycle, fatty acid β-oxidation and mitochondrial transport.
These and other observations led us to hypothesise the dysregulation of pathways providing the mitochondria with oxidisable substrates.

In our current study, we aimed to revisit this hypothesis by applying a combination of whole-cell transcriptomics, proteomics and energy stress signaling activity measures using subsets of up to 34 ME/CFS and 31 healthy control lymphoblast cell lines from our growing library.

While levels of glycolytic enzymes were unchanged in accordance with our previous observations of unaltered glycolytic rates, the whole-cell proteomes of ME/CFS lymphoblasts contained elevated levels of enzymes involved in the
TCA cycle (p = 1.03 × 10−4),
the pentose phosphate pathway (p = 0.034, G6PD p = 5.5 × 10−4),
mitochondrial fatty acid β-oxidation (p = 9.2 × 10−3),
and degradation of amino acids including
glutamine/glutamate (GLS p = 0.034, GLUD1 p = 0.048, GOT2 p = 0.026),
branched-chain amino acids (BCKDHA p = 0.028, BCKDHB p = 0.031) and
essential amino acids (FAH p = 0.036, GCDH p = 0.006).

The activity of the major cellular energy stress sensor, AMPK, was elevated but the increase did not reach statistical significance. The results suggest that ME/CFS metabolism is dysregulated such that alternatives to glycolysis are more heavily utilised than in controls to provide the mitochondria with oxidisable substrates.

https://www.mdpi.com/1422-0067/22/4/2046

 

[borko2100]

The results suggest that ME/CFS metabolism is dysregulated such that alternatives to glycolysis are more heavily utilised than in controls to provide the mitochondria with oxidisable substrates.

That's how I experience it. When I do aerobic activities I feel lactate build up, this feeling comes up extremely quick, like my body immediately switches to some inefficient energy burning mode.

I think this is the second or third paper these researchers have published about Complex V problems, has anyone besides them found problems with Complex V? Seems to me like a good lead.

 

[Creekside]

has anyone besides them found problems with Complex V?

I was suspicious of Complex V long before I knew about ME. I thought I had some problem with kynurenine production, which involved superoxide and Complex V. There were some other factors, now forgotten, that kept me suspicious. Now I've become sensitive to proline, which reduces activity of cytochrome c oxidase (complex IV). Since proline gives me symptoms seemingly identical to PEM, this might indicate that PEM involves mitochondrial dysfunction.

 

[Trish]

Now I've become sensitive to proline,

I'm curious about how anyone would find out they were sensitive to proline, since it is an amino acid prevalent in a wide range of foods.

 

[Midnattsol]

I was suspicious of Complex V long before I knew about ME. I thought I had some problem with kynurenine production, which involved superoxide and Complex V. There were some other factors, now forgotten, that kept me suspicious. Now I've become sensitive to proline, which reduces activity of cytochrome c oxidase (complex IV). Since proline gives me symptoms seemingly identical to PEM, this might indicate that PEM involves mitochondrial dysfunction.

What do you mean by "sensitive to proline", sensitive to the amount we get from a regular diet or some supplement?

 

[Hoopoe]

The elevated expression of enzymes involved in mitochondrial glutamine degradation which we have observed here is consistent with the reductions in blood glutamine levels previously reported in ME/CFS patients [3,4,6]. This strongly suggests elevated usage of glutamine as a mitochondrial substrate by ME/CFS cells. Such dysregulation of glutamine metabolism would have far-reaching consequences given its importance in many cellular processes.

 

[Midnattsol]

Glutamine and glutamate can be drastically altered by dietary changes in otherwise healthy humans (I've seen around 1.5 fold increase in glutamine, with a similar reduction in glutamate, by changing diet alone), and reduced glutamine with increased glutamate is seen in many conditions.

 

[borko2100]

I get insomnia and some neuro symptoms from MSG, so glutamine / glutamate definately affects me in some way.

 

[Cheesus]

Glutamine and glutamate can be drastically altered by dietary changes in otherwise healthy humans (I've seen around 1.5 fold increase in glutamine, with a similar reduction in glutamate, by changing diet alone), and reduced glutamine with increased glutamate is seen in many conditions.

How do you measure that? What does the diet entail?

 

[Hutan]

@DMissa, nice to see another paper out. What is up next for you and the team? Do you have funding?

 

[Hutan]

A really nice background - it can be hard to get it right, but this one nails it, I think.
Background

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease characterized by debilitating fatigue and a worsening of symptoms following exertion referred to as post-exertional malaise (PEM). This can include pain, cognitive difficulty, flu-like symptoms and myriad other symptoms whose severity is disproportionate to the exertion-inducing activity. These properties of ME/CFS can significantly impair quality of life to the point of bed-bound disability in severe cases, rivalling the impact of other sim- ilarly devastating chronic diseases such as multiple sclerosis [1]. Much evidence for the biological basis of ME/CFS has been presented, but no underlying mechanism of disease has yet been identified. Insufficient cellular energy supply has been suspected, and in line
with this, we previously reported inefficient ATP synthesis by Complex V in ME/CFS lymphoblasts.

Page 3 typo

Much of this work has discussed potential dysregulation of glycolysis in ME/CFS of glycolysis which mediates the multi-step conversion of glucose to pyruvate, which can be converted by pyruvate dehydrogenase (PDH) to acetyl CoA, a major TCA cycle substrate.

 

[Milo]

I think the mistake many people make is translating what scientific experiments, at the cellular level, means or implies for their functioning as owners of a complex machine.

Remember that no diet and no supplements so far have worked for patients with ME including the one clinical trial for mito support from a couple years ago, and remember that the authors are not implying that changes in diets will change anything. Science is not there yet.

(That is when people add: but i feel better on x,y and z...)

 

[Midnattsol]

How do you measure that? What does the diet entail?

A bloodsample. In this case, it was a change from a "standard" diet to a diet following that country's specific dietary guidelines (these are pretty much the same all over the world: increase intake of whole foods, fiber-rich foods such as fruits/vegetables/legumes/grains, eat high quality protein and fats). But as I said, this was otherwise healthy humans, it might be different once someone has a disease. The reason for the change glutamine/glutamate values may also be different.

I think the mistake many people make is translating what scientific experiments, at the cellular level, means or implies for their functioning as owners of a complex machine.

Remember that no diet and no supplements so far have worked for patients with ME including the one clinical trial for mito support from a couple years ago, and remember that the authors are not implying that changes in diets will change anything. Science is not there yet.

(That is when people add: but i feel better on x,y and z...)

But I feel better...

Diet as a treatment in other disease is also for the most part in the "science is not there yet", except in the cases of nutrient deficiency diseases and celiac disease. There are cool results for inflammatory bowel disease, where certain dietary approaches can induce remission of inflammation in the gut, but I guess that's about it. Or, low-FODMAP or IBS might be included here, even if there are some questions about how it works.

There's impairment of the integrity of the intestinal wall ("leaky gut") and changes in gut microbiome composition in several diseases, and we know certain foods/dietary approaches (and other lifestyle approaches) that can improve things, but if that influences the state of the disease is more uncertain. In the case of the gut microbiome we don't know what the ideal microbiome should look like for anyone. That said, for any disease not meeting your nutrient requirements is an unnecessary strain on your body that has enough to deal with already.

Edit: Forgot inborn errors of metabolism.

 

[Hutan]

Since the Sweetman study examined non-immortalised PBMCs (from which lymphoblasts are derived), this confirms that the upregulated respiratory capacity and upregulation of substrate-provid- ing mitochondrial pathways exhibited by ME/CFS lymphoblasts is present independent of immortalisation.

So, if those differences are there between ME/CFS and controls, in both non-immortalised PBMCs and lymphoblasts, what does that tell us about what is causing the differences? The lymphoblasts will have been frozen, and presumably washed, and cultured, and treated with the EBV, umm, treatment that immortalises them. And presumably new cells are being made by the lymphoblasts, with the new cells also showing the differences. So, what is perpetuating the difference from controls, in the lymphocytes taken from the patients all the way through to new generations of lymphoblast cells?

Is it epigenetic changes? Could it be bits of virus?
(Sorry, I've run out of energy, so haven't read much of the last bit of the paper.)

4.1.1. Proteomics
This subset included 34 ME/CFS patients (88% female, median age 52.5, age range 26–71) and 31 controls (45% female, median age 30, age range 19–58). The difference in gender proportions (Fisher’s exact test p = 0.0004) and the distribution of ages (Fisher’s exact test, p = 0.000183 using 15 year bins) were statistically significant. However, there was no significant effect of either age (multiple regression) or gender (ANOVA) on ana- lysed experimental outcomes in either patients or controls (p > 0.05).

There are quite big differences between patient lymphoblasts and control lymphoblasts in gender (88% female vs 45% female) and age (53 years vs 30 years). No significant effect of age or gender was found, but it's less than ideal. I guess Covid may have made blood collection difficult in 2020 - but perhaps Emerge could help with control recruitment, to try to get better matched control samples going forward?

 

[Hoopoe]

The glutamine caught my attention because my mother says she needs to take glutamine before going to sleep or she will wake up from hypoglycemia or epilepsy (the two are connected). She has suffered from mystery illness for many years and also had postinfectious ME/CFS for a while and I suspect that there is a large overlap between our illnesses.

 

[butter.]

@Jonathan Edwards

would you line to comment?

 

[ME/CFS Skeptic]

Diet as a treatment in other disease is also for the most part in the "science is not there yet", except in the cases of nutrient deficiency diseases and celiac disease

Thanks for the interesting comments and background. What about diabetes, obesity, heart disease: is there reliable evidence that diet works there as a treatment?

 

[ME/CFS Skeptic]

The methods and various pathways are a bit too complex for me to understand but this seems to be the main conclusion/hypothesis behind it:

We previously found no changes in glycolytic rate, reserve and capacity in ME/CFS lymphoblasts [2] and here report unaltered expression of glycolytic enzymes at the protein level. Instead of an impaired capacity to undertake glycolysis, our results suggest that changes in ME/CFS lymphoblast metabolism might be driven by dysregulated energy stress signaling and elevated usage of alternatives such as the β-oxidation of fatty acids or increased shunting of glucose towards the PPP

 

[wigglethemouse]

Very happy to see @DMissa and team collaborating with other researchers. I also appreciate that Sweetman and co in NZ have been attempting to replicate some of their previous work with small cohorts.

Yes, we are very excited. These guys have been doing fantastic work as evidenced by this paper. I'm currently working with them to do the metabolomics portion of this project.

 

[Hoopoe]

The methods and various pathways are a bit too complex for me to understand but this seems to be the main conclusion/hypothesis behind it:

My interpretation is: in a previous study, their main finding was a mitochondrial complex V inefficiency, in cells that had been immortalized. Immortalization of cells is an invasive technique and they need to be sure that this complex V inefficiency is due to ME/CFS and not the immortalization. This study looks like an attempt to collect more data with different methods to show that this inefficiency is due to ME/CFS. The data appears to be consistent with this.