Propionate and Mitochondria

Propionic acid, in this post propionzte, from the gut microbiome is generally considered a bad player in the the autism spectrum of disorders. [1] Part of this stems from the injection of propionate into the ventricals of the brains of rats rats resulting in anti-social behavior. [1] The Roginski study started with the aim of understanding a rare and lethal mutation of the gene that codes for propionyl CoA decarboxylase.  [2] This study utilized embryonic rat cardiac mitochondria.  TCA cycle enzymes analyzed:   α-ketoglutarate dehydrogenase (α-KGDH), citrate synthase (CS) and malate dehydrogenase (MDH) using commercial microtiter plate assays.  Mitochondrial membrane potential and ATP production were also parameters of analysis. The Roginski study took into account that propionate and hydroxy propionate are metabolized in the liver to maleate [3] that was found to be more toxic. [2,3] This post is a figure by figure exploration of the Roginski publication [2] with adding cartoons to help me, and perhaps other readers, understand what is going on.

Oxygen consumption was measured in three states of respiration

1. State 3 stimulated with 1 mM ADP. Most mitochondria cartoons only show ADP binding to complex V, which technically is not part of the electron transport chain. Kelath Murali Manoj & Abhinav have Parashar have written a chaptor on ADP binding to electron transport complexes. The Rice University page on State II respiration discussed it being created after the establishment of State 4 but didn’t really discuss the mechanism.
2. State 4 non phosphorylating produced by adding μg·mL-1 oligomycin A, a blocker of the H⁺-coupled ATP synthase. Rice University has a nice piece on State IV respiration that informs us that the rate of respiration depends on the carbon source and how quickly NADH is generated.
3. Uncoupled by adding 1.0 μM CCCP (two pulses of 0.5 μM) was added to induce the uncoupled respiration.  This compound destroys the H⁺ gradient.

Tricarboxylic acids and propionate metabolites were added to isolated mitochondria simply to test the hypothesis that propionate metabolites inhibit generation of NADH by the TCA cycle. Oxygen consumption is simply a way to measure generation of NADH in a meaningful way. The trick to reading these graphs is realizing that there is a pattern:

• the control
• a concentration gradient of maleic acid
• propionic acid and likely less important metabolites

1. TCA cycle NADH from malate

Malate is the source of NADH, electrons and H⁺, to fuel the electron transport of the mitochondria. The first thing to note is that malate is not the same compound as maleic acid.  Both are four carbon dicarboxylic acids.  The OH group on the 2^nd carbon in malate is removed and replaced with a double bond between the 2^nd and 3^rd carbon.  This could put a block in the TCA cycle between malate and oxaloacetate

My first thought was that maleate was a competitive inhibitor at this NADH generating site of the TCA cycle and inhibited oxygen consumption this way.

2. TCA cycle NADH from α–ketoglutarate

Is maleate simply acting as a competitive inhibitor log jam in the NADH producing TCA cycle such that O₂ consumption requiring a H⁺ gradient is abolished?  This group fed the mitochondria with α-ketoglutarate.  Adipic acid is structurally similar being a six-carbon dicarboxylic acid.  The difference is that ketoglutarate has a ketone group on the 2^nd carbon. 

There really was no such inhibition, but it is worth noting that propionic acid, and potentially 3-hydroxy propionic acid feed into the TCA cycle right after α-ketoglutarate.  Both PA and 3-OH-PA slightly inhibit state 3 respirations the source of tricarboxylic acids, state 4 O2 consumption maleate is not as inhibitory to α-ketoglutarate is quite a bit upstream of malate, a putative site of maleate inhibition.  When succinate, somewhat less upstream of malate, maleate is not that inhibitory to state 4 respiration. .  It should be remembered that succinate feeds into the electron transport chain via Complex II (Cx II).

3. TCA cycle NADH and FADH₂ from succinate

4. Maleate appears to compete with succinate for the dicarboxylate carrier

Continuing on this theme of succinate feeding into the ETC, allamethacin is a peptide ionophore that destroys the H+ gradient uncoupling the H+ gradient from ATP synthesis. This image of alamethacin in a membrane was taken from slide 18 of 28 of a University of Alberta slideshare. The dicarboxylate carrier is an inner mitochondrial membrane that exchanges malate and succinate for inorganic phosphate and sulfate.

Roginski and coauthors claim that these data suggest that maleate and succinate compete for the same dicarboxylate carrier in inner membrane. Alamethacin seems to relieve some of this inhibition. The authors did not really discuss how this happens.

5. Does maleate sequester CoA?

A 1981from the Roginski publication stated that maleate-CoA can undergo an isomerization that renders CoA-SH metabolically inactive. Does spiking the sytem with extra CoA relieve this inhibition?

The answer appears to be “maybe a little bit’ but not enough to explain the full inhibition going in at either of the CoA entry points of the TCA cycle.

6. TCA cycle source of the propionate/maleate log jam

This particular figure documents experiments to determine which enzymes in the TCA cycle were inhibited by maleate and propionate. α-Ketoglutarate dehydrogenase was markedly inhibited by maleate but not propionate. Citric acid synthase (C) and malate dehydrogenase (B) were unaffected.

CoA was added to test the hypothesis that the maleate induced inhibition was due to CoA depletion.

7. Enter glutamate, adding more targets of inhibition

Roginski followed up on another report demonstrating that maleate inhibits glutamate dehydrogenase. Just as a reminder

• State 3 stimulated with 1 mM ADP
• Uncoupled respiration is produced by adding 1.0 μM CCCP to destroy the H⁺ gradient.

Propionate has no effect. 3-hydroxy propioanate and maleate only inhibit when malate, pyruvate, and glutamate are the source of carboxylate acids. Down stream succinate makes no difference.

8. In permeabilized cardiac myocytes

Cardiac cell membranes were permeabilized with the detergent digitonin so that the mitchondria could be loaded with the same carboxylic acids in a more cellular environment.

These preparations are thought more closely resemble what is seen in vivo.

9. The affect on ATP production

Up until now, Roginski et al were using CCCP to uncouple the H⁺ gradient that drives ATP production and uncouples respiration, i.e. the reduction of O₂ to H₂O from ATP production. In this figure, they actually examined ATP production. The Complex V inhibitor oligomycin was used as a control.

Note the excess of H⁺ on the intermembrane space. This gradient forms the mitochondrial membrane potential, ΔΨm. The authors measured the affect of maleate on actual ATP production. This next step was to measure the ΔΨm.

10 The mitochondrial membrane potential.

Cyclosporin A (CsA, 1 μM) was used to keep the mitochondrial permeability transition pore from opening. The MPTP is a pore in the membrane of the mitochondria that can open in response to excess intracellular Ca²⁺, reactive oxygen species, and loss of membrane poteintial, Δψ. In this particular set of experiments, a Δψ sensitive fluorescent indicator was used.

• A. Propinonate had no influence on the Δψ but the higher concentration of Maleate did
• B. Adding the MPTP inhibitor cyclosporin A prevented this maleate induced decrease in Δψ
• E. The Ca²⁺ chelator EDTA did nothing.
• C Propionate had a small effect on Δψ when α-ketoglutarate was the carboxylic acid source. Maleate was more effective in decreasing Δψ.
• D Adding the MPTP inhibitor cyclosporin A partially inhibited this decrease in Δψ.

Apoptosis related events

The mitochondrial Ca²⁺ uniporter (MCU) is a means for Ca²⁺ to enter the mitochondria matrix. It is of note that MPTP opening provokes non-selective permeabilization that may lead to ΔΨm collapse and mitochondrial swelling, loss of matrix components (Ca²⁺, Mg²⁺, glutathione, NADH, NADPH, and release of mitochondrial proapoptotic factors. This image is presented as a reminder of NADH consumed by the mitochondria.

11. Not to forget NADH

In these experiments, the mitochondria do not have an active complex V. NADH, but not NAD⁺, has intrinsic fluorescence.

Propionate is indistinguishable from the control. What is not clear is if NADH is being consumed faster or simply escaping through the MPTP pore. Does removal of the H⁺ gradient favor faster oxidation of NADH to NAD⁺ + H⁺?

12 Mitochondrial swelling

Ca²⁺ is being added to open the MPTP. The mechanism for swelling is less clear.

13 Ca²⁺ retention

It helps to consult the BMG Labtech website to truly understand what the final figure of the Roginsky paper [2] is showing.

Panel A, Calcium Green is in the bulk solution. Until it binds Ca^2+, it is non fluorescent. All the available Ca²⁺ is in the matrix of the mitochondria because MPTP is closed. Panel B MPTP opens releasing Ca²⁺ from the matrix of the mitochondria. Calcium Green becomes fluorescent.

Roginski et al added external Ca²⁺ causing the Green-5N to spike. The mitochondria took up the Ca²⁺ causing the slow decline in fluorescence. Then they spiked in more Ca²⁺. In the presence of maleate they never saw Ca²⁺ uptake suggesting that MPTP was open.

Conclusions

The featured image of this post is the conclusion of Roginski and coauthors. [2] We may need to rethink the concept that propionate is directly the cause of the neurological symptoms of ASD. This is not to say that its metabolite maleate [3] is not a key player. There are still many unanswered questions.

1. Do other cells have the same capacity as hepatocyte [3] to metabolize propionate to maleate?
2. Can maleate get past the plasma membrane of most cells in the circulation?
3. Can plasma levels of maleate ever reach a high enough concentration to cuase mitochondrial dysfunction?

A 2014 review on the maleic acid, found in food packaging and modified starch, found literature supporting an association with mental and nervous system diseases, cardiovascular diseases, and cancer. [4]

References

1. Frye RE, Rose S, Slattery J, MacFabe DF. Gastrointestinal dysfunction in autism spectrum disorder: the role of the mitochondria and the enteric microbiome. Microb Ecol Health Dis. 2015 May 7;26:27458. PMC free article
2. Roginski AC, Wajner A, Cecatto C, Wajner SM, Castilho RF, Wajner M, Amaral AU. Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats. Biochim Biophys Acta Mol Basis Dis. 2020 May 1;1866(5):165682. free paper
3. Wilson KA, Han Y, Zhang M, Hess JP, Chapman KA, Cline GW, Tochtrop GP, Brunengraber H, Zhang GF. Inter-relations between 3-hydroxypropionate and propionate metabolism in rat liver: relevance to disorders of propionyl-CoA metabolism. Am J Physiol Endocrinol Metab. 2017 Oct 1;313(4):E413-E428. PMC free paper
4. Lin YC, Wang CC, Tung CW. An in silico toxicogenomics approach for inferring potential diseases associated with maleic acid. Chem Biol Interact. 2014 Nov 5;223:38-44.