Hypoxia, anoxia, lactic acid fermentation, and naked mole rats…

Fermentation is the ancient and evolutionarily conserved metabolic process by which glycolytic pyruvate is reduced by the electron carrier NADH, thus regenerating NAD+ while producing any of a number organism-specific end products. For example in yeast, the fermentation end product is ethanol, while in mammals, the end product is lactate. In mammals, this process occurs during periods of little or no oxygen availability (e.g. when oxygen is rapidly depleted via electron transport in energy-demanding muscle cells, or when cells are otherwise hypoxic or anoxic).

If lack of oxygen persists in the intracellular environment, then the major ATP-generating process for mammalian cells, the electron transport chain, will eventually come to a standstill. In such conditions, the cell becomes reliant on substrate level phosphorylation to generate sufficient ATP to survive. Given a steady supply of glucose, this process of substrate level phosphorylation can only continue indefinitely if NAD+ is continuously regenerated (NAD+ is reduced to NADH during the oxidation and phosphorylation of glyceraldehyde-3-phosphate to produce 1,3-bisphosphoglycerate during the sixth step of glycolysis). Thus, fermentation and the production of lactic acid from pyruvate allow for continuous generation of ATP in anaerobic conditions.

Naked mole rats are unique among non-marine mammals in that they have been shown to survive prolonged periods of hypoxia and anoxia by utilization of fructose metabolism.1 Crucially, fructose appears to bypass phosphorylation by phosphofructokinase (which would otherwise be inhibited by metabolic products such as ATP, citrate, and low pH). Since the naked mole rat is totally dependent on substrate level phosphorylation during anoxia, this process generates considerable lactate, presumably leading to low extracellular pH as excess lactate is released into blood as lactic acid. Yet the naked mole rats don’t seem to suffer ill effects from acidosis during anoxia.

naked mole rat
You have to admit, they’re kinda cute.

A growing body of research has demonstrated that low levels of acidosis in the extracellular milieu actually enhance cell survival during hypoxia and anoxia, though the full mechanisms remain unclear. Khacho et al. (2014) found that acidosis during hypoxia induces morphological changes (i.e. elongation) in the mitochondria of mouse cortical neurons.2 Elongation of mitochondria was observed across a range of neuronal cell types in an in vitro hypoxic condition in which cells were incubated with 1% oxygen in media that allowed for acidification via lactic acid fermentation; mitochondrial elongation was also observed in hippocampal sections. However, proliferative cell types able to undergo mitosis (such as embryonic fibroblasts and several cancer cell lines) did not demonstrate mitochondrial elongation during acidosis and hypoxia.

These findings suggest that the mechanisms underlying mitochondrial elongation in hypoxic cells exposed to acidosis are in place throughout the central nervous system, but may be specific to post-mitotic cells. Interestingly, cells in the control condition (exposed to hypoxia but kept at neutral pH) failed to demonstrate mitochondrial elongation, instead showing severe fragmentation of mitochondria. Mitochondrial elongation was only observed within a pH range mirroring that of the immediate physiologic environment during lactic acidosis (pH 6.65-6.45). The authors used ratiometric fluorescence to confirm that intracellular pH decreased in accord with the decrease in extracellular pH, and that acidosis alone is sufficient to trigger mitochondrial elongation (independently of oxygen or glucose availability).

Hypoxia has previously been shown to induce mitochondrial fragmentation (also called “mitochondrial fission”) via activation and recruitment of the dynamin protein DRP1.3 Mild acidosis seems to inhibit activation of DRP1, effectively preventing mitochondrial fragmentation. Electron microscopy also seems to show that acidosis preserves mitochondrial ultrastructure, preventing disruption of cristae. The number of cristae actually seem to increase as a result of acidosis, possibly increasing surface area for cellular respiration. Along with the finding that an ATP synthase inhibitor caused a 90% reduction in ATP levels in hypoxia/acidosis exposed cells, this led the authors to conclude that oxidative phosphorylation is actually maintained during hypoxia (by lactic acidosis), and that mitochondrial capacity for oxidative phosphorylation is enhanced despite limitations in oxygen availability.

Lee et al., (2015) found that the protein NDRG3 is upregulated in hypoxic conditions, and that it is activated by binding to intracellular lactate.4 NDGR3 expression is increased during low oxygen availability through an unknown posttranslational mechanism (the authors used RT-PCR to confirm that NDGR3 mRNA levels were essentially unaltered by hypoxia). Using statistical analysis of transcriptome expression data, the authors determined that NDGR3 is correlated with increased activity in pathways associated with angiogenesis as well as the prevention of apoptosis (as well as other processes).

Furthermore, the authors were able to inhibit increases in the intracellular accumulation NDGR3 by suppressing lactate production (whereas oxygen availability alone was insufficient to alter NDGR3 levels). The authors obtained additional evidence for the dependence of NDGR3 on intracellular lactate by adding lactate to cell lines that lack the genes for its production; concomitant increases in NDGR3 were seen with lactate administration in such cells. Finally, the authors showed that NDGR3 bound to lactate mediates hypoxia-induced activation of the Raf-ERK pathway which has been implicated in improved cell survival in such conditions.

Taken together, these studies offer a glimpse into the myriad potential fates of the intracellular lactate produced by naked mole rats via fructolysis. Potentially lethal mitochondrial fission as a result of low oxygen availability is mediated by the dynamin protein DRP1, yet it’s been demonstrated that lactic acidosis as a result of excess lactate production during hypoxia inhibits DRP1 and, moreover, that it induces morphological changes in mitochondria that drastically enhance oxidative phosphorylation capacity despite the limited availability of oxygen; indeed, Khacho et al. claim to have discovered that “oxygen is not a limiting factor” for mitochondrial generation of ATP in such circumstances. Perhaps simultaneously, lactate binds to and activates NDGR3 (which accumulates during hypoxia), activating the Raf-ERK pathway and thereby improving cell survival.

[1] Park TJ, Reznick J, Peterson BL, et al. Fructose-driven glycolysis supports anoxia resistance in the naked mole-rat. Science. 2017;356(6335):307-311. doi:10.1126/science.aab3896. PubMed PMID: 28428423

[2] Khacho M, Tarabay M, Patten D, et al. Acidosis overrides oxygen deprivation to maintain mitochondrial function and cell survival. Nat Commun. 2014;5:3550. doi:10.1038/ncomms4550. PubMed PMID: 24686499; PubMed Central PMCID: PMC3988820

[3] Hu C, Huang Y, Li L. Drp1-Dependent Mitochondrial Fission Plays Critical Roles in Physiological and Pathological Progresses in Mammals. International Journal of Molecular Sciences. 2017;18(1):144. doi:10.3390/ijms18010144. PubMed PMID: 28098754; PubMed Central PMCID: PMC5297777

[4] Lee DC, Sohn HA, Park ZY, et al. A lactate-induced response to hypoxia. Cell. 2015;161(3):595-609. doi:10.1016/j.cell.2015.03.011. PubMed PMID: 25892225