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

Abstract

The underlying mechanisms of appendage regeneration remain largely unknown, and uncovering these mechanisms in capable organisms have far-reaching implications for potential treatments in humans. Recent studies implicate a requirement for metabolic reprogramming reminiscent of the Warburg effect during successful appendage and organ regeneration. Changes are thus predicted to be highly dynamic, methods permitting direct visualization of metabolites at the tissue and organismal level, in real time, would offer a significant advance in defining the influence of metabolism on regeneration and healing. We sought to examine whether glycolytic activity was altered during larval fin regeneration, utilising the genetically encoded biosensor, Laconic, enabling the spatiotemporal assessment of lactate levels in living zebrafish. We present evidence for a rapid increase in lactate levels within minutes following injury, with a role of aerobic glycolysis in actomyosin contraction and wound closure. We also find a second wave of lactate production, associated with overall larval tail regeneration. Chemical inhibition of glycolysis attenuates both contraction of the wound and regrowth of tissue following tail amputation, suggesting aerobic glycolysis is necessary at two distinct stages of regeneration.SUMMARY STATEMENTBy combining a genetically encoded lactate FRET sensor with chemical inhibitors, we demonstrate a critical role for the Warburg effect and metabolic reprogramming during zebrafish wound closure and tail regeneration.

The Warburg effect is necessary to promote glycosylation in the blastema during zebrafish tail regeneration

“…3e). These results are consistent with the observation that there is increased lactate production in the notochord bead during regeneration, indicative of the Warburg effect 34 . Taken together, these data demonstrate that a metabolic shift to aerobic glycolysis takes place in the regenerating tail prior to blastema development.…”

“…2e). Moreover, 2-DG has been shown to suppress lactate production in uninjured zebrafish embryos, and in support of our results no gross morphological defects were observed 34 . Next, we amputated tails of embryos treated with MB-6.…”

“…Post amputation there is an increase of uptake of the glucose analog 2-NBDG, indicating a requirement for additional glucose during regeneration. Additionally, work from another group has shown increased lactate production during tail regeneration, indicating that glucose metabolism is uncoupled from OxPhos during this process 34 . Addition of 2-DG to embryo medium completely blocks regeneration without affecting early development, further indicating the shift to glucose metabolism is specific to regeneration.…”

Gill and Liver Transcript Expression Changes Associated With Gill Damage in Atlantic Salmon (Salmo salar)

“…Wound closure (e.g., through actomyosin contraction) was concurrent with elevated glycolysis during zebrafish larval wound healing and tail regeneration ( 77 ). In the current study, glucose-metabolism related genes, phosphoglycerate mutase 2 ( pgam2 ) and aldolase a, fructose-bisphosphate 1 ( aldoa ), were microarray-detected as responsive to moderate gill damage.…”

Tissues and Cell Types of Appendage Regeneration: A Detailed Look at the Wound Epidermis and Its Specialized Forms

“…Likewise, amputations have been indicated to promote epithelial proliferation and involve forming an actomyosin cable during zebrafish fin regeneration ( Mateus et al, 2012 ). Moreover, in zebrafish caudal fin and tail regeneration, a transient metabolic shift to glycolysis has been shown to influence actomyosin networks in epithelial cells for successful epithelial closure, presumably forming the WE ( Scott et al, 2021 ). How such molecular processes and metabolic changes mediate rapid wound covering during limb regeneration remains unknown.…”