Aerobic glycolysis is important for zebrafish larval wound closure and tail regeneration

Claire A. Scott, Tom J. Carney, Enrique Amaya

Preprint posted on 26 April 2021 https://www.biorxiv.org/content/10.1101/2021.04.23.441208v1

Article now published in Wound Repair and Regeneration at http://dx.doi.org/10.1111/wrr.13050

Turning up the power for healing; glycolysis is important for zebrafish wound closure

Selected by Jonny Coates

Updated 13 October 2022 with a postLight by Jonny Coates

This preprint has recently been published in Wound Repair and Regeneration. Overall, the published paper has minimal changes compared to the preprint, with some of the preprint supplemental figures being promoted to main text figures.

The biggest addition is the inclusion of two supplementary figures (S3 and S4) in which the authors tested a second inhibitor of LDH (GME-140) to further confirm the specificity of their results. This new data does strengthen the findings, but it does not impact the conclusions or substance of the paper which remain the same.

Reference:
https://onlinelibrary.wiley.com/doi/10.1111/wrr.13050

Background

ATP (Adenosine Triphosphate) is essential for cell survival and function. ATP is the primary energy source for molecular processes and is generated by glycolysis and oxidative phosphorylation. During these metabolic processes, glucose is first metabolised into pyruvate (glycolysis) which then enters the TCA cycle where it generates ATP. In total, one molecule of glucose yields 30-32 molecules of ATP with ~2 ATP molecules being generated by glycolysis and the remaining molecules by oxidative phosphorylation. Although glycolysis generates less ATP molecules per glucose, this process is much quicker than oxidative phosphorylation; thus cells will prioritise this pathway during times of stress, for example following injury (1).

Zebrafish are a well-established model for wound healing (2). Following wounding, an organism must close the wound, prevent infection and clear debris before tissue regeneration can occur. Wound closure is an essential process which restores the skin barrier and prevents infection. During the early stages of wound closure (within 10-15 minutes), actin and myosin are recruited to the wound edge. There, they assemble into contractile actomyosin rings which then contract “like a purse string” to close the wound (3,4). This process requires a large amount of energy forcing the muscle fibres to prioritise glycolysis to generate energy quickly.

In this preprint, the authors investigated glycolytic activity in a zebrafish wound model using a real-time reporter of lactate levels.

Key findings

Lactate increases post-wounding

The authors utilised a genetically encoded sensor of lactate (called Laconic) which can report lactate levels in real-time. With this, the authors demonstrated that following fin fold or tail amputation, the levels of lactate rapidly increased, forming a gradient from the wound border. In the bigger wound inflicted by the tail fin amputation, the lactate levels took longer to return to control levels.

During wound healing, muscle fibres contract to close the wound. This process involves actomyosin cables and demands a lot of energy. The authors suggest that the glycolysis activity is required for actomyosin cable contraction during wound closure.

Inhibition of glycolysis prevents wound contraction and tail regeneration

Following the observation that lactate levels increase post-wounding, the authors next investigated if blocking glycolysis could reduce or prevent wound healing. To do this, the authors treated zebrafish with oxamate, a drug that inhibits lactate dehydrogenase and prevents glycolysis.

Inhibition of glycolysis resulted in wider wounds following fin fold amputation. However, at later time points, the oxamate and control groups had similar regeneration suggesting that embryos are able to recover from temporary inhibition of glycolysis.

To assess the impact of inhibiting glycolysis over the course of regeneration, the authors used 2DG (an inhibitor a key enzyme in the glycolysis pathway). In these experiments, tails did not regenerate within 120 hours after amputation, while fin folds did regrow. This would suggest that glycolysis has essential roles at different stages of wound healing.

Figure 1. Fin fold and tail amputations following 2DG treatment. Figure shows that 2DG treatment limits tissue regeneration of both the tail and fin fold after amputation. Adapted from Fig 7 in the preprint under a CC-BY 4.0 license.

Why I selected this preprint

This preprint combines my previous experience in wound healing and my interests in metabolism. How organisms cope with wounding is an important question that directly translates into human health. The authors combine the powerful imaging capabilities of zebrafish with a novel lactate sensor which represents an exciting route for future work.

Open questions

Where does the lactate come from in wounds – is this just the muscle fibres?
You show that inhibition of glycolysis prevents rapid wound contraction which you attribute to actomyosin contraction. However, it’s also known that other cells responding to a wound utilise a glycolytic metabolism (for example neutrophils). Could this account for your findings of reduced regeneration when embryos were treated with 2DG? Is this something you ruled out?
Did you try genetically manipulating muscle cells / cells at the wound border to confirm that these cells were indeed utilising glycolytic metabolism to fuel the wound closure, perhaps by knocking out some of the genes you have targeted pharmacologically?
How relevant are your findings to human health and wound healing?
What’s the next big step for this work?

References

Kelly B, O’Neill LAJ. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. 2015 Jul;25(7):771–84.
Martin P, Feng Y. Wound healing in zebrafish. Nature. 2009 Jun;459(7249):921–3.
Abreu-Blanco MT, Watts JJ, Verboon JM, Parkhurst SM. Cytoskeleton Responses in Wound Repair. Cell Mol Life Sci. 2012 Aug;69(15):2469–83.
Nakamura M, Dominguez ANM, Decker JR, Hull AJ, Verboon JM, Parkhurst SM. Into the breach: how cells cope with wounds. Open Biol. 2018;8(10):180135.

Tags: cell biology, immunology, metabolism, microscopy, wound healing, zebrafish

Posted on: 13 May 2021 , updated on: 19 December 2022

doi: https://doi.org/10.1242/prelights.28916

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Author's response

Claire Scott shared

Thank you for highlighting our work!

In response to your questions:

We cannot say for certain which cell type(s) the lactate originates from as our imaging was not at a resolution allowing single cell analysis, but given the thin nature of the fin fold, it is likely from the epidermal cells as this is the tissue affected by the excision of amputation.
When imaging with Laconic during the period of regeneration, we did not witness elevated ratios in cells or areas of the image resembling neutrophils. This could be due to the widefield nature of imaging masking single cell effects rather than glycolytic metabolism being absent, so we cannot completely rule out this possibility. However, with this imaging we did find elevated Laconic ratio and lactate levels in the notochord bead, and therefore hypothesised this was the most likely element reliant on aerobic glycolysis. Furthermore, the inflammatory immune response occurs following both fin fold and tail amputations, but only tail regeneration is attenuated by inhibition of glycolysis.
We unfortunately did not attempt any genetic knockouts in addition to the pharmacological treatments – however, assuming it is the contracting epidermal cells that are producing the lactate, one would have to knock out the pharmacologically targeted enzymes in a temporally restricted manner, as the epidermis is a tissue and such inhibition would have significant effects throughout development.
If glycolysis is indeed important for formation of blastema-like structures, it may be a potential target for inducing blastema formation in non-regenerative organisms like humans. Though there is a long way to go!
We would say that the next interesting avenue for investigation would be examining the effects of glycolysis/inhibition of glycolysis on proliferation. Proliferation is clearly an important element of regeneration to replace lost tissues, and a glycolytic switch required for the dedifferentiation and proliferative capacity of induced pluripotent stem cells (Folmes et al., 2011; Prigione et al., 2014). The possibility of achieving an induced pluripotent stem cell population within a blastema-like structure in mammals by metabolic reprogramming would be an exciting finding!

Thanks again for your interest!

References:

Folmes, C. D. L., Nelson, T. J., Martinez-Fernandez, A., Arrell, D. K., Lindor, J. Z., Dzeja, P. P., Ikeda, Y., Perez-Terzic, C., & Terzic, A. (2011). Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming. Cell Metabolism, 14(2), 264–271. https://doi.org/10.1016/j.cmet.2011.06.011

Prigione, A., Rohwer, N., Hoffmann, S., Mlody, B., Drews, K., Bukowiecki, R., Blümlein, K., Wanker, E. E., Ralser, M., Cramer, T., & Adjaye, J. (2014). HIF1α modulates cell fate reprogramming through early glycolytic shift and upregulation of PDK1-3 and PKM2. Stem Cells (Dayton, Ohio), 32(2), 364–376. https://doi.org/10.1002/stem.1552

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Aerobic glycolysis is important for zebrafish larval wound closure and tail regeneration

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