All posts tagged glycolysis

ME/CFS Seahorse Energy Production Study Shows Surprises

Dr. Maureen Hanson leads one of the three NIH funded ME/CFS research centers, but her ME/CFS research doesn’t stop there.  Using samples from Dr. Daniel Peterson provided by the Simmaron Research Foundation, she’s also been assessing the metabolism of one of the most important cells in our immune systems: our T-cells.

T-cells affect a large part of our adaptive immune response that clears out infections. They do this by regulating our immune response (CD-4 or Helper T-cells) and/or by killing off pathogens that have infected other cells (CD-8 or cytotoxic T-cells).

Hanson and Mandarano and Seahorse machine

Maureen Hanson, Alexandra Mandarano and the Seahorse machine

Prior to getting activated, T-cells are primarily on sentry duty.  Once activated by dendritic cells presenting little bits of pathogens to them things change dramatically, however. The T-cells rev up their cellular engines to order to start pumping out cytokines or clones en masse in order to stop the infection. Both parts of energy production – glycolysis and oxidative phosphorylation –  have to jump into action.

In short, assessing the energy production of activated T-cells is a perfect way to determine if their energy metabolism has been affected in ME/CFS – and that’s just what Maureen Hanson’s group did.

Alexandra Mandarano, a graduate student in Hanson’s lab, took T-cells from 53 healthy controls and 45 pretty long duration (avg. a@ 12.7 years) ME/CFS patients and healthy controls and tricked them into going into high alert with antibodies plus IL-2. Then, using the Seahorse Flux Analyzer, she examined how well the two parts of their cellular energy production system did in both unactivated and activated T-cells:  glycolysis – the anaerobic part which takes place outside the mitochondria, and oxidative phosphorylation – the aerobic part which takes place inside the mitochondria (and produces far more ATP) .

Dr. Hanson presented on her results at the recent Open Medicine Foundation sponsored Harvard Symposium

Results

Whether they were activated or not, mitochondrial energy production; i.e. oxidative phosphorylation (the main ATP producer) was normal for both the CD4 and CD8 cells in the ME/CFS group. When pushed, the mitochondria in the ME/CFS patients’ cells quickly got energy production up to speed. That was a surprise. Usually when you push a cell or system in ME/CFS it fails- but, in this case, the T-cells responded normally.

Then came the real surprise.  Systems in ME/CFS often test out fine or at least not strongly abnormal at baseline or rest, but in this case Hanson found low glycolysis activity in both the T-helper cells (CD4) and the CD8 cells at baseline.  Simply prowling around the body, they had considerably lower levels of glycolytic activity.  When pushed, though, their glycolytic activity was normal.  The pattern was opposite to what we usually see.

That wasn’t all. It’s possible with the Seahorse to turn off different energy production pathways in order to assess how effectively the other pathways are at compensating.  When the oxidative phosphorylation pathways were turned off experimentally, the ME/CFS patients’ glycolytic pathways failed to compensate as effectively as did those of healthy controls.

Mandarano did not find problems with mitochondrial ATP production but did find issues with glycolysis

Thus no problems with mitochondrial energy production were found but three potential issues with glycolysis popped up: low glycolytic activity in both forms of unactivated T-cells, and poor glycolytic compensation with the oxidative phosphorylation pathways were turned off.

Hanson’s group next examined a critical part of energy production called the mitochondrial membrane potential. Our mitochondria need to maintain a certain membrane potential to keep up the flow of positively charged ions into the mitochondria. It does this by keeping more positively charged ions outside of the mitochondria and more negatively charged ions inside the mitochondria. Her group used a flow cytometer to assess the levels of mitochondria present and to determine how strong the membrane potential was.

The mass and membrane potential of the ME/CFS patients’ CD4 T-cells and the mitochondrial mass of the CD8 cells was normal, but the membrane potential of the CD8 T-cells – whether activated or not – were significantly impaired in the ME/CFS patients.

Four potential problems, then, were found:

  • low glycolytic activity in unactivated CD4 and CD8 T-cells
  • poor glycolytic compensation when the oxidative phosphorylation pathways were turned off
  • The mitochondrial membrane potential was impaired in the CD8 T-cells

Dr. Hanson left her presentation with the  encouraging statement that we are starting to put the pieces of the puzzle together in ME/CFS and the tantalizing suggestion that ME/CFS might be something different than what we think it is right now; i.e. keep an open mind, don’t put all your eggs in one basket, and be prepared for surprises.

Overview

Hanson and her co-authors have submitted a paper and we will get more details when their paper is published but, with these preliminary results, we have a few more data points on cellular energy production in ME/CFS. While noting that several study results are pending, maybe it’s time for a look at what we have.

It should be noted that measuring energy production is very complex. Different researchers are doing it in different ways, and I am no judge of any of them.  Researchers are using different instruments, different criteria, different kinds and numbers of patients, and they are reporting things differently. Solving those problems is one of the reasons for the NIH funded ME/CFS research centers where larger studies can use proven technologies and rigorously defined patient populations.

Check out some of the different protocols which have assessed mitochondrial functioning in isolation from the blood in ME/CFS:

Study protocols

  • Hanson’s group activated her T-cells using antibodies and IL-2 and then tested activated and unactivated cells in the Seahorse Machine
  • Tomas took PBMC’s and stressed them in the Seahorse machine
  • Stanford took PBMC’s and then used laboratory assays to test each of the complexes and flow cytometry to assess mitochondrial membrane potential
  • Fisher (unpublished) appears to have taken PBMC’s and stressed them in the Seahorse machine
  • Vermeulen measured ATP PBMC’s etc. in the lab
  • Smits measured ATP production rate in muscle biopsies

The Land of Mixed Signals

We seem to find ourselves in a familiar place – the land of mixed signals! One encouraging unmixed signal is that everyone seems to be finding something wrong – just often different things.

MITOCHONDRIA

Mitochondria Mass – Normal

  • Hanson – CD4 and CD8 (T-helper cells)
  • Fisher

Mitochondria ATP production – Normal

  • Hanson (T-cells)
  • Stanford study (not a Ron Davis study) (PBMC’s)
  • Fisher (PBMC’s)
  • Vermoulen (PBMC’s)
  • Smits (muscle biopsy)

Increased ATP Production Overall

  • Stanford
  • Preliminary results from NIH Intramural study

Reduced ATP production

  • Tomas – under both low and high glucose conditions

Functioning of Complexes – Normal

  • Stanford (I-IV)
  • Vermeulen (I-II)

Functioning of Complex V – reduced

  • Fisher
GLYCOLYSIS

Increased Glycolysis at Baseline (PBMC’s)

  • Stanford

Reduced Glycolysis at Baseline (T-cells)

  • Hanson

Reduced Glycolysis (low glucose conditions)

  • Tomas

Reduced Compensatory Glycolysis

  • Hanson
  • Tomas (?)

Glycolysis Stress Test (Glycolysis, glycolytic reserve, glycolytic capacity)- normal

  • Tomas
  • Fisher

It’s quite a muddle.  Surprisingly, though, the most consistent finding thus far is normal (or in two cases) increased mitochondrial production (!) Not many studies have directly measured glycolysis, but in these early days the results are mixed.

Isolation

cells in the blood

The most consistent result so far is normal (or increased) mitochondrial function but none of the above studies tested cells in the blood – where an inhibiting factor may lurk. (Seahorse machine cannot test cells in the blood.)

Note that all these studies are assessing the energy production of the mitochondria in isolation. None tested cells in the blood where Davis, Fluge and Mella and Prushty have found evidence that some sort of inhibiting factor may be present. The metabolomic findings which suggest problems with glycolysis are present have been assessing factors in the blood and urine as well.

Adding an exercise stress test would, of course, add another important factor. At the NIH ME/CFS Conference, Brian Walitt reported that the NIH is finding that exercise causes mitochondrial oxygen consumption (ATP production) to increase in the healthy controls but to decrease in about half of the ME/CFS patients. Several recent studies have validated that exercise impairs energy production in ME/CFS (blog coming up). Where and how the energy depletions are occurring is unclear. (Note that most of these studies examined immune cells not muscle cells.)

We obviously have long way to go to fit all the different pieces of the energy production puzzle in ME/CFS together but the good news is that an increasing amount of research is now being aimed at deciphering what’s inhibiting energy production in this disease.

The Simmaron Research Foundation’s collaboration with Maureen Hanson – which paired rigorously diagnosed patients with a respected researcher –  is just one way the Foundation is contributing to solving that puzzle.

Immune Study Adds to Evidence Of Body-Wide Problems With Energy Production in Chronic Fatigue Syndrome (ME/CFS)

Increased expression of CD24 …could thus reflect abnormalities in maintaining appropriate ATP generation (in ME/CFS). The authors

Numerous studies suggest problems with energy production exist in chronic fatigue syndrome (ME/CFS). The huge energy needs exertion places on the muscles and brain suggest they’re an obvious place for energy production problems to show up. Ramping up to fight off pathogens also places extreme demands on energy production in immune cells. Now comes a study which suggests that energy production problems in a subset of B-cells could be setting people with ME/CFS up for problems with inflammation and autoimmunity.

B-cells and ME/CFS

The first couple of successful Rituximab trials brought renewed interest in the immune cells most effected by the drug – the B-cells. The last, large Rituximab trial unfortunately failed – the drug does not work in ME/CFS – but the Rituximab effort succeeded in other ways.

First off, it brought two creative and dedicated researchers, Oystein Fluge and Olav Mella to the field and energized ME/CFS research in Norway.

B-cell

A B-cell producing antibodies to fight pathogens

Secondly, the B-cells – which play a huge role in immunity (and autoimmunity) – finally got some study in ME/CFS.  Keeping with ME/CFS’s time-honored tradition of falling between the cracks in medicine, several studies found no indication of altered levels of “classical B-cell markers”.

Something unusual did, however, pop up, in an extended analysis, which went well beyond the classical markers usually explored. In 2015 a study found that a molecule called CD24 was highly expressed in a group of B-cells.

CD24 is an adhesion molecule which turns on various signaling networks – it basically tells cells what to do. It is most highly expressed on early stage or transitional B-cells as they emerge from the bone marrow.

During the normal transition from immature to mature metabolically active B-cells, early B-cells are tested again and again for evidence that they may be turning into autoantibody producing cells and many are removed.  Over time, as these cells transform themselves into mature B-cells, the CD24 molecule gradually disappears from their surfaces. High levels of these molecules in people with ME/CFS suggested that a problem with B-cell maturation might be present.

Since antibody producing B-cells play a major role in fighting off infections, having a bunch of immature B-cells hanging around the immune systems of ME/CFS patients could constitute a problem.

Plus there’s evidence that the CD24 molecule plays a role in several diseases.  CD24 polymorphisms (genetic variants of CD24) have been associated with increased risk for and accelerated progression of autoimmune diseases including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematous. CD24 can also be over‐expressed in many cancers, including B cell lymphomas.

In short, it’s not a molecule you want to ignore.

Energy Production Problems in the Immune System

Front Immunol. 2018 Oct 22;9:2421. doi: 10.3389/fimmu.2018.02421. eCollection 2018. CD24 Expression and B Cell Maturation Shows a Novel Link With Energy Metabolism: Potential Implications for Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Mensah FFK1, Armstrong CW2, Reddy V1, Bansal AS3, Berkovitz S4, Leandro MJ1, Cambridge G1.

In a 2018 study, a UK and Australian group took B-cells from ME/CFS patients and healthy controls, stimulated them and then monitored what happened.  As before, they found an increased frequency of CD24+ B-cells in ME/CFS patients.

What made the increased frequency of these naïve CD24 packed B cells in ME/CFS so interesting, though, was their mode of energy production.

Examining the metabolism of the ME/CFS patients’ cells, the researchers found a “strong(ly) positive” association between the amount of glycolysis and lactate produced and the expression of CD24 molecules on ME/CFS patients’ B-cells. Put another way, the more glycolysis was used to produce energy and the greater the lactate production (a by-product of glycolysis) – the more the CD24 molecule showed up on ME/CFS patient’s B-cells.

energy production me-CFS

Findings suggesting that a body-wide problem with energy production are present in ME/CFS are piling up.

Lower levels of mitochondria in these cells suggested one reason ME/CFS patients’ B-cells may be stuck in this mode.  Not surprisingly, the authors found this more prominent B-cell subset in ME/CFS was also largely unresponsive to stimulation.

That suggested that not only do the energy problems in ME/CFS extend to the immune cells but that they could be impacting immune functioning – in this case by keeping ME/CFS B-cells in a naïve state – that may be associated with disease.

Plus increased levels of the CD24 molecule have also been associated with a damaging state called “senescence”.  Instead of undergoing a process called autophagy during which a cell’s contents are safely recycled, during senescence – which is often associated with aging –  damaged mitochondria cause cells to slowly deteriorate while producing scads of pro-inflammatory factors.

An exercise physiologist, Graham Salmun, recently reported that his exercise study results suggest senescence is indeed occurring in ME/CFS.  He believes problems with aerobic energy production are a) impairing ME/CFS patients ability to produce energy and b) creating a senescent state that is causing chronic inflammation.

Anaerobic Thresholds, Fatty Acid Problems and Autophagy: Dr. Klimas’s Exercise Study

Conclusion

This study provided an intriguing metabolic snapshot of the immune system. The fact that increased expression of the CD24 molecule has been associated with autoimmune disorders and cancer makes the CD24 finding in ME/CFS interesting, but the metabolic connection the researchers found may be more important.

Their findings suggest that the same problems producing energy found elsewhere in ME/CFS may also be occurring in their immune cells.  Plus the findings suggest that a state of senescence, chronic inflammation and cellular unresponsiveness may be present as well. The possible penalties of having an immune system with a broken aerobic energy production system could go beyond fatigue and pain and extend to problems with autoimmunity, and perhaps in rare cases, cancer.

Studies finding an increased reliance on anaerobically produced energy in the muscles, the brain, in neutrophils and now in some of the B-cells all suggest that a body-wide disruption in energy production may be present in ME/CFS.

 

Major Research Group Highlights Inflammation Energy Production Connection in ME/CFS

We propose that chronic low-grade inflammation induces and/or maintains persistent fatigue by inducing an imbalance between cellular-energy availability and cellular- and behavioral energy expenditure.  Lacourt et al. 2018

Neurosci. 2018 Apr 26;12:78. doi: 10.3389/fnbeh.2018.00078. eCollection 2018. The High Costs of Low-Grade Inflammation: Persistent Fatigue as a Consequence of Reduced Cellular-Energy Availability and Non-adaptive Energy Expenditure. Lacourt TE1, Vichaya EG1, Chiu GS1, Dantzer R1, Heijnen CJ1.

Inflammation, the brain and energy metabolism – it’s like the trifecta in chronic fatigue syndrome (ME/CFS) research. It seems like virtually everyone in the ME/CFS field believes that all three are involved but that belief only carries so much weight in a small field. What this field really needs is buy-in from outside researchers who can help move it forward.

That appears to have happened recently when a major research group lead by Robert Dantzer penned a review paper proposing that low-grade inflammation is causing energy production problems in chronic fatigue syndrome (ME/CFS) and probably many other diseases. The authors didn’t shy away from the chronic fatigue syndrome (ME/CFS) connection. In fact, they lead their review paper off with it, placing the fatigue in ME/CFS in the same context as the fatigue in cancer, MS, rheumatoid arthritis and others.

Major Stanford Study Indicates Chronic Fatigue Syndrome (ME/CFS) is Inflammatory Disorder

The study was published in the Frontiers in Neuroscience journal series which is touted as the 1st most cited series in the Neurosciences journal field.

The Dantzer group’s involvement in the intersection between inflammation and energy production is welcome but not entirely surprising; it’s a logical outcome of their past work. Dantzer spearheaded the now accepted idea that the immune system produces the symptoms of “sickness behavior” (fatigue, headache, muscle aches, sore throat, etc.) that occur during an infection which serve to reduce our energy usage and to keep us isolated from others (they posit to prevent pathogen spread).

What’s new is his group’s focus on the energy production process itself – a focus, interestingly, made possible largely by the work of ME/CFS researchers. The piece, with lead author Tamara LaCourt, shows how low-grade inflammation can cause the same energy problems we’re seeing in ME/CFS: a metabolic switch from energy-efficient, oxygen-based energy production process to a fast-acting, inefficient glycolysis-based approach.

Immune cells aren’t like other cells; jumping into action causes them to rev their motors up tremendously, placing enormous stress on their energy production systems. As they do this, they switch from a focus on aerobic energy metabolism to what the authors call “aerobic glycolysis” in order to churn out energy more quickly. That process results in less mitochondrial energy production and the increased production of toxic by-products like lactate.  Plus, over time this process results in reduced nutrient availability and less energy for the rest of the body.

low energy chronic fatigue

The authors believe that inflammation and metabolic and energy problems come together to produce a final common endpoint: fatigue.

Several studies from the Solve ME/CFS Initiative are examining whether the energy production of immune cells in ME/CFS is up to the task.

Prolonged inflammation also tends to result in two other energy production problems: increased insulin resistance and reduced glucose tolerance. Reduced glucose tolerance smacks glucose uptake by immune cells at the very time that they’re clamoring for it, causing the body to break down fats and proteins, thus removing resources it would ordinarily use elsewhere.  In yet another whack at the energy production, inflammation increases reactive oxygen species production which can hammer mitochondrial energy production.

The authors believe that neurons – which rely on glycolytic processes in astrocytes to get their energy – may be hit hardest by chronic inflammation.  This is because insulin resistance – a common outcome of chronic inflammation – destroys the glycolytic process in astrocytes, causing neurons to get their energy from fats – a slower and less efficient process.

Miller’s work on ME/CFS suggests that problems with the basal ganglia – the dopamine-producing center of the brain – may be causing problems with movement, reward and fatigue in ME/CFS. That’s a particularly interesting finding given that dopaminergic neurons in the brain are particularly vulnerable to inflammation. Shungu’s studies, which have consistently found high lactate and low gluthathione levels in the ventricles of ME/CFS patients brains, suggest that high levels of oxidative stress could be causing inflammation in the brain itself.

Plus, even low-level inflammation can disrupt a key element in ME/CFS and FM – sleep – which, in turn, increases fatigue. Simply altering one’s circadian rhythm (i.e. one’s sleep times) can have significant metabolic effects, leading to increased glucose levels and decreased insulin sensitivity.  The effects don’t end with sleep; sleep deprivation results in the need for increased energy expenditures the next day.

Is Poor Sleep Pummeling the Immune System in ME/CFS and Fibromyalgia? A Vicious Circle Examined

Then add in the extra ten percent in extra energy needs that chronic low-level inflammation imposes on the body – and the potential for a dramatic drop in energy production rises.   (We’ll find out more about total energy production in ME/CFS during the metabolic chamber tests in the NIH’s intramural study).

The authors believe that impaired energy production represents a “final common pathway” in persistent fatigue.

Leader in the Field

“In sum, most evidence for an association between fatigue and mitochondrial functioning comes from CFS, indicating lower levels of antioxidants and possible reductions in mitochondrial ATP production.” The authors.

We understandably don’t think of researchers in the small ME/CFS research field as being pioneers in the medical research field at large, but some have ploughed brand new ground. Suzanne Vernon’s computational biology work at the CDC was so novel that an entire issue of the Pharmacogenomics journal was devoted to it. Gordon Broderick and Travis Craddock’s expansion of that work at Dr. Klimas’s Institute of NeuroImmune Medicine has taken computational biology further – much further – in ME/CFS than in any other field. Ron Davis and Mark Davis at Stanford are using new HLA gene typing and T-cell technologies to try and nail down what is activating ME/CFS patients’ immune systems.

ME/CFS researchers’ attempts to understand the intersection between mitochondrial problems and fatigue are clearly breaking new ground as well.  According to the authors of this review article, 21 of the 25 studies examining the intersection between mitochondrial problems and fatigue have been produced by ME/CFS researchers. Researchers we all know ( e.g. Naviaux, Montoya, Hornig and Lipkin, Fluge and Mella) were cited again and again in the overview.

The authors even cited Workwell’s groundbreaking 2013 study which indicated that a shift to glycolytic energy production occurred during the second day of a two day exercise test in ME/CFS. They also singled out the 2017 Tomas study which found that under conditions of cellular stress, the mitochondria in ME/CFS patients’ cells were unable to rise to the occasion.

Turning to the metabolomics studies, the authors cited three ME/CFS studies which have pointed to “reduced metabolic activity”.  They believe the metabolic changes seen in ME/CFS reflect a chronic over-reliance and eventual depletion and abandonment of lipid metabolism, which results in a greater use of carbohydrate stores; hence the greater reliance on glycolysis and impaired aerobic energy production. In short, the authors believe the metabolomic studies in ME/CFS are demonstrating the same metabolic shift that the authors propose occur in states of chronic low-grade inflammation.

Interestingly, the authors proposed that many ME/CFS patients are probably exceeding their daily energy stores.  That, of course, makes perfect sense given Staci Stevens’s and Workwell’s findings that, for some patients, simply sitting upright puts them into an aerobic energy deficit.

For all its possible connections, the idea that fatigue in ME/CFS is simply the result of “low-grade inflammation” seems untenable given the disability present – unless that inflammation is found in the brain. The Simmaron Research Foundation is bringing the brain, the immune system and metabolism together in a way that’s never been seen before in ME/CFS.

The Simmaron Research Foundation’s first ME/CFS cerebral spinal fluid study suggested that an immune dysregulation, the likes of which approached that found in multiple sclerosis, may be present in the ME/CFS patients’ central nervous systems. Their second outlined an atypical ME/CFS subset. Their current CSF (cerebrospinal fluid) study – an expanded version of the first study which includes a metabolomic component – will be the first to potentially merge immune and metabolic findings in the most energetically active part of the body – the brain.

Peterson’s Atypical Subset Opens New View of ME/CFS in Columbia/Simmaron Publication

Plus, stay tuned for a report suggesting that inflammation is not just present, but pervasive, in ME/CFS patients’ brains.