Frustrated for 10 years in his desire to study ME/CFS, Paul Fisher of LaTrobe University in Australia made the most of it when he got the chance He and his lead researcher on these studies, Daniel Missailidis, have rolled off four studies and two reviews in the last three years. They include the most sophisticated analyses of mitochondrial functioning done yet in ME/CFS and have provided a new slant on what’s possibly going on.
First, though, a quickie overview of mitochondrial energy production.
Mitochondrial Energy Production Simplified
Producing ATP involves two processes that work side by side. The TCA, or Krebs, cycle provides the fuel the electron transport chain (ETC) needs to function. The ETC then uses electrons to convert ADP to the usable form of energy for the cells – ATP.
As the electrons go through Complexes I-IV in the electron transport chain, they cause protons to get pumped out into the spaces between the mitochondrial membranes. When the protons reach a certain level, Complex V is able to transform ADP to ATP. With that, the electron transport chain’s job is done: it’s produced the energy the cell needs to run on.
It’s critical, then, that the electron transport chain has the fuel – NADH, and FADH2 – it needs to run on. These compounds are produced by another process entirely, which runs alongside the electron transport chain and provides it with the fuel it needs. It’s known by a couple of names: the Krebs, TCA (tricarboxylic acid cycle) or citric acid cycle.
(Hans Kreb was a German Jew who was dismissed by the Nazis in 1933 and ended up in Oxford, England on his way to a Nobel Prize and enormous acclaim. His letter describing the cycle was rejected by Nature, but his manuscript was later published elsewhere. He won the Nobel Prize for the discovery in 1953. He also discovered a couple of other cycles. :))
First, two compounds, NADH and FADH2 need to be “reduced”; i.e. electrons need to be added to them so that they can give them up later in the first four complexes of the electron transport chain. (Adding negatively charged electrons to a compound results in that compound being “reduced“).
Glucose, fatty acids, and amino acids can all be used by the TCA, or Krebs, cycle to produce NADH and FADH2. First, though, these substances have to be metabolized into a compound that the TCA cycle can use. For glucose and fatty acids – the two main sources of fuel – that compound is acetyl CoA.
- Glucose – glycolysis provides the mitochondria with pyruvate, which is then converted by pyruvate dehydrogenase (PDH) into acetyl CoA.
- Fatty acid – the beta-oxidation of fatty acids provides acetyl CoA.
Amino acids are different. The relevant amino acids are broken down by a variety of methods. For instance, glutamine is converted to glutamate, which is then converted to alpha-ketoglutarate – which can be used by the TCA cycle.
The Glycolysis Hypothesis
Since three plasma studies pegged a deficient glycolytic process as the main potential culprit in ME/CFS glycolysis – which uses glucose to produce acetyl CoA – it has been the main focus. (Plasma is a clear liquid that doesn’t contain red blood or other cells).
Studies using serum, however, were unable to replicate their findings. (Serum doesn’t contain cells either but it also doesn’t contain any clotting factors.) Seahorse studies have also failed to find evidence that glycolysis is inhibited in ME/CFS. Some studies have also suggested that amino acids – rather dirty fuels – are being preferentially utilized to produce energy in ME/CFS.
With virtually every study finding something wrong, but different studies pointing at different things, the quest to pin down the energy problem in ME/CFS patients’ cells has become something of a “dog’s breakfast” at this point; i.e. it’s kind of a mess.
Special Cell Line Plays Significant Role
Enter the Fisher group with a new method of studying the mitochondria they hoped would bring clarity.
In the 2020 review, “Lymphoblastoid Cell Lines as Models to Study Mitochondrial Function in Neurological Disorder“, Annesley and Fisher explained why they’ve turned to cell lines to study mitochondrial functioning. In the review, they explain that while system-wide mitochondrial dysfunction would be most like to show up first in tissues with higher energetic needs like the muscles and the neurons, it should still be evident in all types of cells – including cells in the blood that are easier to get and work with.
Fisher believes the cells he’s using – continuously proliferating lymphoblastoid cell lines (LCLs) to which Epstein–Barr virus (EBV) DNA has been added – are perfect for assessing mitochondria. When used in his matter, EBV will make very small changes to the cell but will prompt them to become activated. It’s the ability to analyze the activated cell – and the burst of mitochondrial activity that comes with it – that Fisher was apparently after.
In the review article, Annesley and Fisher showed how LCLs have been valuable in understanding the mitochondrial issues in neurological diseases like Parkinson’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis (ALS), Fragile X-Associated Tremor Ataxia Syndrome (FXTAS), and ME/CFS.
They noted that the direct measures of mitochondrial energy production done in non-proliferating, quiescent cells, such as PBMCs and muscle cells, have, thus far, revealed no clear, consistent differences in ME/CFS. It was the use of LCLs, Fisher believes, that enabled him to find a “clear and specific defect” in the mitochondria of ME/CFS patients.
“A Clear and Specific Defect”
Their first major study, “An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients“, indicated that oxygen wasn’t being well utilized by the ME/CFS patients’ mitochondria; that relative to the amount of oxygen they were getting, the amount of ATP they were producing was low.
At the same time that was happening, several aspects of mitochondrial functioning were elevated. The first complex in the electron chain (Complex I) was working overtime, maximum oxygen consumption and maximum nonmitochondrial oxygen consumption rate were increased. Plus, the upregulation of the mTORC1 stress signaling complex associated with Complex I, the overactive fatty acid transporters, and the enzymes that break down that fuel so the mitochondrial can use it and power the Krebs cycle, suggested that the ME/CFS patients’ mitochondria were working awfully, awfully, hard.
The authors proposed that all that work was an attempt to compensate for damage to the last Complex (Complex V) in the electron transport chain where ADP gets turned into ATP. The compensation worked – but only up to a point. The ME/CFS patients’ cells were probably doing fairly well at rest, but once stressed – they were running out of gas.
Altered Diet for ME/CFS Patients’ Mitochondria?
That hypothesis in hand, they tested it. In their 2021 paper, “Dysregulated Provision of Oxidisable Substrates to the Mitochondria in ME/CFS Lymphoblasts“, Missailidis and Fisher looked to see if the mitochondria were getting the fuel they need to thrive. Once again, they compared lymphoblasts from people with ME/CFS (n=34) to healthy controls (n=31).
Glycolysis Hypothesis Down – Fatty Acid and Mitochondrial Dysregulation Hypotheses Up
“The potential for abnormal utilization of fatty acid β-oxidation has arisen clearly in our own work.” Missailidis et al.
Normal levels of glycolytic enzymes, plus former findings suggesting that the normal glycolytic rates and reserves are present, suggest that the glycolytic pathways are operating normally; i.e. glucose was being metabolized correctly and providing normal amounts of “food” to the Krebs or TCA cycle.
Instead of the cells turning to other sources of energy because glycolysis – the glucose pathway – is damaged, Fisher now believes they’re doing that for two reasons: in order to compensate for damage to the Complex V in the electron transport chain, and in response to increased levels of cellular stress; i.e. a “dysregulated energy stress signaling” process.
This study’s results concurred with those of past studies which suggested that fuels other than glucose – which provides an easy and clean source of energy – such as fatty acids and amino acids, are indeed being preferentially used to produce energy in ME/CFS patients’ cells.
Activated Fatty Acid Oxidation Pathway Stands Out
One energy source, in particular, stood out – fatty acids. B-oxidation refers to the process by which enzymes break down long-chain fatty acids in the mitochondria to get them ready to be used. for fuel; It was remarkable to see that almost half (9/20) of the most significantly upregulated pathways “pertained directly to fatty acid β-oxidation”.
Overall, the mitochondrial issues were highlighted to a remarkable degree. Besides the innate/adaptive immune regulation, the significantly upregulated pathways all involved the mitochondria. They included the TCA cycle, respiratory electron transport, other mitochondrial proteins, and the pentose-phosphate pathway.
Enzymes involved in peroxisomal B-oxidation were also upregulated in ME/CFS. The peroxisomal fatty acid-b-oxidation finding was intriguing given Ian Lipkin’s latest study, which suggested that peroxisomal dysregulation plays a key role in the mitochondrial problems in ME/CFS.
Interestingly, the mRNA in the ME/CFS patients’ cells did appear to be telling the glycolytic enzymes to pick up the pace a bit, but the protein analysis indicated they didn’t do that. This is what happens, though, when cells begin favoring fatty-acid B-oxidation instead of glycolysis.
All of this strongly “suggested that an upregulation of fatty acid β-oxidation was occurring in ME/CFS patients’ cells.”
Still, there were questions. While this study overwhelmingly supported the idea that fatty acids are increasingly being used for fuel in ME/CFS, some study results suggest that fatty acid oxidation is actually being hindered. One study which specifically looked for increased fatty acid utilization in muscle cells did not find it. The authors had a possible answer for that but asserted that more direct measures of fatty acid β-oxidation utilization in lymphoblasts, as well as protein expression in muscle cells, need to be done.
Branched-Chain-Amino-Acids (BCAAs) Getting Chewed up
The mitochondria’s need for more and more fuel (electrons) appears to be causing them to break down the BCAAs as well. As the mTORC1 complex stimulates BCAA breakdown, and the mTORC1 pathway was chronically activated, this was expected. The authors also asserted there was “compelling evidence” that proteolysis – the breakdown of proteins – in order to release amino acids for fuel was occurring in ME/CFS.
Complex V Problems Are Key?
“More importantly, our observations here support our previous proposal that upregulated fatty acid β-oxidation in ME/CFS cells provides acetyl CoA to the TCA cycle more rapidly, provisioning the upregulated respiratory complexes with reducing equivalents to accelerate respiration and compensate for inefficient ATP synthesis by Complex V.” The authors
Why, though, were ME/CFS patients’ cells choosing to rely on fatty acids or amino acids for fuel when glycolysis provided such an easy pathway to energy?
If I have it right, the authors believe this is all an attempt to make up for problems in the final and determinate step of the electron transport chain in Complex V, where ADP is converted to ATP. The reason fatty acid metabolism is being emphasized is that it operates more quickly than the other pathways – and Complex V is screaming for more fuel.
Far-Reaching Consequences for Glutamine-mTORCI-Rapamycin Connection?
Another possibly critical finding concerned the elevated activation of enzymes involved in breaking down mitochondrial glutamine. This finding could explain the reduced levels of glutamine found in several studies and suggested that the mitochondria in ME/CFS patients’ cells were also preferentially breaking down the glutamine amino acid for fuel. The authors believed that this metabolic dysregulation could have “far-reaching consequences given its importance in many cellular processes”.
Glutamine degradation activates mTORC1 (mechanistic target of rapamycin complex 1) signaling and, as noted, chronic mTORCI activation was also found. The mTOR complex is a nutrient/energy/redox sensor and controller of protein synthesis.
- It took Paul Fisher of LaTrobe University in Australia ten years to get to ME/CFS, but when he did he made the most of it.
- Using a novel technique using immortalized immune cells, Fisher and his lead researcher, Daniel Missailidis, have unveiled a series of significant abnormalities in the mitochondria of people with ME/CFS.
- Problems with the penultimate complex in the electron transport chain (Complex V) appear to be causing ATP production to decline dramatically when the mitochondria are put under stress.
- Despite a series of compensatory reactions, the ATP production of the mitochondria appears to decline by 25%.
- While problems with glycolysis – long thought to be present in ME/CFS – have not shown up – glucose is being shunted to a different pathway than usual. More importantly, Fisher found that the mitochondria in ME/CFS are preferentially turning – perhaps in an attempt to feed a broken Complex V – to amino acids, and particularly fatty acids.
- The authors pointed to evidence proteins were being broken down for fuel in ME/CFS. Glutamine degradation, in particular, was highlighted. Glutamine degradation triggers the mTORC1 pathway.
- Inhibiting the mTORC1 pathway has been associated with longevity in several animals. The authors did not recommend Rapamycin (or any other treatment option), but it has been used to inhibit the mTORC1 pathway.
- “Compelling evidence” also suggests that higher than normal numbers of cells are being broken down as well.
- The authors cited numerous studies they believe should be done to further understand the mitochondrial problems found in ME/CFS.
The search has been on to find ways to tamp down mTORC1 activity. Some dietary compounds (resveratrol, curcumin, caffeine, and alcohol) appear to be able to, and rapamycin (Soriolimus, Rapamune) – a drug that has elicited some interest in ME/CFS – has as well.
The authors asserted more work on mTORC1 activation in ME/CFS should be done. Plus, the amount of glutamine possibly being degraded indicated that studies focused on glutamine metabolism are needed as well.
The authors also asserted “compelling evidence” that the ubiquitin-proteasome system that attacks and breaks down cells is upregulated in ME/CFS patients’ cells as well.
Fisher has put together a set of novel studies that suggest that problems with glycolysis are not to blame but that something in the last complex of the electron transport chain has gone haywire in people with ME/CFS. (Fisher did find that glucose was being shunted through the pentose-phosphate pathway, though).
Fisher’s studies are different in that instead of assessing serum or plasma, they’re using immortalized lymphoblasts in the laboratory. This means the mitochondria are not exposed to factors in the blood which could be impacting them. On the other hand, this method appears to be providing a purer test of mitochondrial functioning and has been used to good effect in neurological diseases.
Fisher is the first that I know of to identify a deficiency in a specific complex in the electron transport chain. His prior study found a 25% drop in ATP production by Complex V when it was put under stress. That drop apparently triggered a wide range of compensatory reactions: extra copies of mitochondrial complexes were created, Complex 1 activity skyrocketed, proton pump activity increased, as did enzymes that consumed oxygen. All that compensatory activity was able to maintain the mitochondrial production at rest – but not when the mitochondria were put under stress.
This study is the last of several which have suggested that the fuel utilization by the mitochondria has shifted dramatically in ME/CFS. Instead of glucose, fatty acids, in particular, but also amino acids (BCAAs), are being preferentially broken down to provide fuel.
Fisher believes ME/CFS patients’ cells may be seeking out other fuels in order to compensate for the striking deficiency he found in Complex V. A chronic elevation of the mTORC1, and perhaps AMPK, may be adding to the mess. Except for the possible use of Rapamycin or other mTORC1 drugs or food sources, the consequences of this study for treatments are unclear. (The authors made no treatment recommendations and noted that more study is needed.)
It’s not clear, at least to me, how the problems utilizing long-chain fatty acids that have been found in some fibromyalgia patients relate to this study. This study’s findings, on the other hand, seem to fold in well with those from Ian Lipkin’s latest study, which suggested the peroxisomes may play a key role in the energy production problems in ME/CFS. Fisher’s study suggests, but does not prove, that the mitochondrial problems found in ME/CFS may be present system-wide; i.e. they could be present in every cell of the body.
With Fisher, we got a new approach to studying the mitochondria – which thus far has unveiled new and dramatic possible abnormalities – and may be clearing up some issues in the mitochondrial field as well. Fisher’s studies have also opened up many new opportunities for study as he and Missailidis, again and again, pointed out areas that need investigation.
- Coming up – A Mitochondrially Based Diagnostic Tool for ME/CFS?
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