Fluge and Mella seem to be working at almost lightning speed. Besides managing their huge Rituximab study (and all the sub-

Dr. Fluge

Dr. Fluge – Fort Lauderdale IACFS/ME Conference – 2016

studies within it) and the cyclophosphamide trial, they’re also carrying out large research studies.

For years, of course, some researchers and doctors championed the idea that problems with mitochondrial energy production were at the heart of ME/CFS. For many, though, the idea seemed almost too simple…too easy in a way. The body throws too many curves at us for something so obvious to be the cause. But it may be.

The work of Bob Naviaux of UCSD, Ron Davis of the Open Medicine Foundation, McGregor and Armstrong et. al. in Australia, Maureen Hanson at Cornell, and Fluge and Mella in Norway suggest that problems producing energy could, in fact, be causing the physical and mental fatigue in (ME/CFS).

Of course, it’s going to be complex. Exercise studies and other studies have suggested that the aerobic energy production pathways are severely blunted in a significant number of ME/CFS patients. Thus far, though, the metabolomics data suggests that the breakdown comes not in the aerobic energy production pathway but just before it.

Some key facts – such as I understand them.

  • Key Factor in Glycolysis – Pyruvate –  Pyruvate is produced by glycolysis and then gets broken down into acetyl-CoA for use in the mitochondria. When oxygen levels are low, the same process is used to produce ATP anaerobically. Anaerobic energy gets its bad rep because it produces toxins like lactate which build up and cause pain and fatigue. All this occurs in the cell’s cytoplasm.
  • Key Factor in Aerobic Energy Production – Acetyl-CoA – The first goal in aerobic energy production is to produce acetyl-CoA. This occurs in three ways: preferentially by converting pyruvate and/or by converting fatty acids or amino acids. The acetyl-CoA is then broken down further to produce ATP by a process called oxidative phosphorylation. All aerobic energy production occurs in the cell’s mitochondria.

The Study

At 302 patients (200 ME/CFS patients and 102 healthy controls) this was a nice big study. A different type of study than the recent metabolomic studies, it used a mass spectrometer to measure the levels of 20 amino acids involved in energy metabolism in the blood.

QT-RCT PCR was used to examine gene expression. Cells were also cultured, dropped in ME/CFS or healthy control’s serum (blood), tweaked with metabolic factors, and their lactate production and cellular respiration was measured.

Results

Amino Acid Results

Fluge and Mella did a simple but telling thing with the 20 amino acids by dividing them into one of three energy production pathways:

  • Glycolytic Amino Acids – Amino acids used in the glycolysis pyruvate producing pathway which require PDH to be metabolized (alanine (Ala), cysteine (Cys), glycine (Gly), serine (Ser), and threonine (Thr))
  • Aerobic Amino Acids – Amino acids that fuel aerobic energy production but which do not require PDH to be broken down((isoleucine (Ile), leucine (Leu), lysine (Lys), phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr)) – mostly ketogenic amino acids
  • Other Amino Acids – not found in the first category but which are essential to aerobic energy production (anaplerotic – methionine (Met), valine (Val), histidine (His), glutamine (Gln), glutamate (Glu), and proline (Pro), aspartate (Asp), (Asn + Asp = Asx))

Chronic Fatigue Syndrome (ME/CFS) – A Pyruvate Dehydrogenase Disease?

Fluge and Mella found no difference in the levels of the amino acids used in glycolysis – pyruvate production is fine – but reductions in the levels of the ketogenic amino acids used to power aerobic energy production.

glucose

Our cells much prefer using glucose to produce energy but the results and the Aussie study suggest that our cells are turning elsewhere.

Remember that our cells – use three different substrates (pyruvate, fatty acids and amino acids) to produce acetyl-CoA but they much prefer glucose.  If pyruvate isn’t being broken down into acetyl-CoA, however, then our cells will turn to another energy source; in this case amino acids. The fact that virtually all the amino acids used to produce acetyl-CoA were depleted in the female ME/CFS patients suggested that their cells, starved of metabolized pyruvate, were turning to and using up amino acids to to produce energy.

Amino acids, unfortunately, are kind of like the body’s last straw for energy production. Our cells would much prefer to use glucose, but even fatty acids are a better source of energy than amino acids. In order for body to use amino acids it has remove an amino group (producing ammonia) and turn them into a sugar.  (The fact ME/CFS patients appear to be turning to amino acids might suggest that they’re having problems with fatty acid metabolism as well.)

There’s another problem, though. If all this unused pyruvate is hanging around, it has to go somewhere. Unused pyruvate gets converted into lactate – a toxin responsible for much of the fatigue and pain associated with exercise. Lactate ultimately gets dumped into the blood stream. Fluge and Mella believe lactate accumulations are probably a key problem in ME/CFS as well. High lactate levels in ME/CFS patients’ brains have been found in several studies but reports on lactate accumulations in the blood are mixed.

Since pyruvate production doesn’t appear to be the problem, the problem must lie in the pyruvate dehydrogenase enzyme complex (PDH) which breaks down pyruvate to produce acetyl-CoA.

Or a Pyruvate Dehydrogenase Kinase Disease?

Fluge and Mella then asked why pyruvate dehydrogenase is not working in ME/CFS and may have found an answer in the increased gene expression (or activity) of the enzymes (PDH kinases (PDK)) that inhibit PDH. Rather encouragingly, they found that increased expression of one form of the PDK enzyme (PDK1) was associated with increased severity and longer duration patients.

pyruvate dehydrogenase

Fluge and Mella uncovered numerous irregularities that could be affecting the pyruvate dehydrogenase enzyme in ME/CFS

Digging deeper still they found increased gene expression of the transcription factor (PPAR) which upregulates PDH kinase in ME/CFS patients as well.  Then they discovered that the gene expression levels of another enzyme (SIRT4) that limits pyruvate dehydrogenase production were increased as well.

Things were really humming along; Fluge and Mella’s findings suggested that every step in the chain needed to limit pyruvate dehydrogenase levels were activated in ME/CFS. Their consistently positive findings suggested that an inhibited PDH enzyme really may be a problem in ME/CFS.

Different Sexes – Different Compensatory Responses

The same problem (low acetyl-CoA levels associated with low PDH and PDK levels) was found in both sexes but men and women appeared to try to compensate for those reductions differently. While women demonstrated across the board depletions in the amino acids used to produce Acetyl-CoA, the depletions seen in men, on the other hand, were minor.

Fluge and Mella noted the small sample size of men  (n=38) could have limited their ability to uncover significant differences but they also found increased levels of a substance in men called 3-Mhis which is indicative of protein breakdown or muscle atrophy in the men.  (If the guys are wondering if their muscle atrophy might be due to more than lack of exercise, the increased levels of this substance could explain why. I’ve long felt that exercise results in a puffy look; could this be due to inflammation associated with muscle breakdown?)

One cautionary finding was that amino acid levels were not associated with disease severity; i.e. the more severely ill patients did not have lower amino acid levels than less severely ill patients. That was something of a surprise.

Cell Cultures

In one of the more fascinating aspects of this study, Fluge and Mella incubated (grew) muscle cells, put them in the serum from severely ill ME/CFS patients or healthy controls, and then analyzed their rates of mitochondrial respiration and lactate production (glycolysis).

They assessed the energy production of these cells under these different scenarios:

  • Resting – cells in culture.
  • Resting II – cells with glucose (ATP enhancer) added to the culture but with not extra energetic demands being placed on them.
  • Under Anaerobic Strain – oligomycin is used to block ATP production causing the cells to rely strictly on anaerobic energy production.

Under Aerobic Strain – oligomycin plus CCCP – is used to produce a more severe blockade of energy production causing both anaerobic and aerobic energy production systems to work at maximum capacity.

  • Rotenone and antimycin A – were administered to inhibit respiratory complex I and III, respectively, in order to assess nonmitochondrial oxygen consumption (subtracted as OCR background).

When the cells were not placed under any energetic demands they behaved the same whether they were in ME/CFS patients’ or controls’ blood.

When they forced the cells to exist solely upon anaerobic production, and when they put the cells under a severe energy strain, however, they found slightly higher rates of glycolysis in the cells exposed to ME/CFS patients’ serum. This suggested that something in ME/CFS patients’ blood was causing the cells to turn more to glycolysis for energy. Note, though, that the rate of glycolysis was only slightly higher.

fatigued

The increased lactate production in cells put in ME/CFS patients serum suggested that minimal exercise could end up causing pain and fatigue

A further analysis indicated that when the cells in the ME/CFS serum were put under a severe energetic strain lactate production increased significantly. This time it was not a slight increase; the graph indicated huge increases in lactate production had occurred.  That is what Fluge and Mella predicted what would happen when pyruvate dehydrogenase stopped working; the unused pyruvate would end up as lactate.

The surprise (at least to me) came when Fluge and Mella found that the ME/CFS patients’ serum did not cause the cells to reduce their oxygen consumption; instead, the cell in the ME/CFS patients serum actually increased their oxygen consumption both at baseline and when put under stress compared to the cells placed in the healthy controls’ serum.

Fluge and Mella suggested that the high oxygen consumption was a compensatory response to the problems with PDH they found. It may be that the mitochondria – missing their normal levels of acetyl-CoA – are sending out the message to get that glycolytic pathway moving to produce more pyruvate and hence more acetyl-CoA. No matter how much pyruvate it’s producing, though, it’s not enough for the mitochondria, because the pyruvate dehydrogenase enzyme has been turned off.

  • The pattern of amino acid depletion seen suggests that women with ME/CFS are turning to amino acids to fuel energy production rather than the bodies preferred substrate, glucose.
  • Possibly because of their lower sample size, men didn’t show the amino acid depletions the women did. They did, however, exhibit a significant increase in a substance which suggests they are breaking down muscle to fuel energy production
  • Problems with an enzyme complex (PDH) which breaks down pryruvate so that it can be used to fuel the aerobic energy production process in the mitochondria are present
  • Increased activity of the genes responsible for inhibiting the PDH enzyme complex is found in ME/CFS.
  • Putting cells into the serum of severely ill ME/CFS patients caused the cells lactate levels to climb dramatically when the cells were put under energetic stress.
  • Cells put into severe ME/CFS patients serum showed increased, not decreased oxygen consumption. Fluge and Mella believe the increase reflects an attempt to compensate for energy reductions associated with the PDH enzyme issues.
  • A Stanford study, suggested, that the increased cellular activity appeared tied more to glycolysis than to mitochondrial energy production.
  • Similar to what’s found in another severely fatiguing disease called biliary cirrhosis, Fluge and Mella believe that autoantibodies are attacking the metabolic pathways that regulate energy production in the body.
It seemed strange to see the cells in ME/CFS patients’ blood using more oxygen; i.e. being more active rather than less active, but Fluge and Mella’s finding seems to agree with a recent Stanford study that found increased energy production (ATP production) in ME/CFS patients’ cells. The catch was that the increased energy production came mostly from glycolysis rather than from the mitochondria. That appears to be exactly what Fluge and Mella’s study predicts.

So far two attempts to compensate for the lack of mitochondrial energy production are possibly being made. The cell is upping its glycolytic activity (but notice that it was only slightly raised) and it’s breaking down amino acids for fuel. Fluge and Mella believe other attempts to compensate that we don’t know about may be being made as well.

The fascinating Stanford study will be covered more in a future blog. In any case, despite the twists and turns, the evidence thus far consistently points an arrow at the early part of the energy production process – glycolysis. Since glycolysis, and ultimately aerobic energy production mostly relies upon carbohydrates, the main metabolic problem in ME/CFS may involve the inability to properly metabolize carbohydrates. That, of course, would seem to make some sense given the problems many ME/CFS patients have dealing with carbs. Similarly, the depletion of ketogenic amino acids in the ME/CFS women suggests that a more ketogenic (i.e. fat and protein-rich, low carb) diet might be helpful.

We’re not at all done with the possible metabolic problems seen in ME/CFS. Fluge and Mella believe and Bob Naviaux’s recent study suggest that problems in fat metabolism in women – the third substrate used by the mitochondria – to produce energy also probably exist, and Chris Armstrong in an email stated that he thought that problems with both glycolysis and the Krebs cycle in the mitochondria exist.

PEM at the Cellular Lever

It’s kind of comforting to see the exertional problems show up even at the cellular level.  As in the exercise studies, the cells in the ME/CFS patients’ blood needed to be put under strain to show significant differences.  It turns out there’s an enormous difference (70-100 fold) between a cells at rest or being involved in strenuous exercise. That kind of leap in action, of course, leaves plenty of room for problems to show up if the energy production process is not working smoothly.

Next Up

Next up for Fluge and Mella: trying to determine: (a) what in the blood is affecting energy production; and (b) what is turning the pyruvate dehydrogenase enzyme off.

They believe their Rituximab success probably indicates that autoantibodies are attacking metabolic signaling pathways that are turning off the pyruvate dehydrogenase enzyme. They believe the same autoantibodies are also involved in the blood vessel issues found in ME/CFS.

They’re not grasping at straws. Dr. Julia Newton has found that an autoimmune disease called primary biliary cirrhosis (PBC) bears some startling similarities to ME/CFS. PBC is characterized by multiple autoantibodies that impair mitochondrial functioning and target the PDH enzyme complex believed to play a role in ME/CFS. Besides the enormous fatigue they experience, PBC patients also display increased sympathetic nervous system activity, and problems with orthostatic intolerance and cognition are found.

In fact, last year Julia Newton, stating that she hopes the results will be similar to those in ME/CFS, began a Rituximab trial in PBC patients.

Coming up next on HR – more studies are flooding in. Coming up shortly on HR, a look at the Aussie and Stanford studies.

 Health Rising’s Big (Little) Donation Drive is Successful! – Thanks to over 214 people who donated we have met (and exceeded) our target :). ( I, of course, am much relieved :)) You’ve ensured that Health Rising will continue to bring you the latest news on ME/CFS and FM research and treatment into what appears to be a very exciting 2017.

Thanks to all who have supported HR this year including Dennis Sylvain for providing research papers, Marilyn for her rapid editing help :), an anonymous friend for her support in many areas, to Stavya for his impeccable work supporting the technical side of HR, to Glori for her artwork, to the Simmaron Research Foundation and Prohealth for their financial support, and to everyone who has provided helpful comments.

 

 

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