ABSTRACT
Adenosine triphosphate (ATP) is the main energy provider for intracellular processes, through its cleavage into adenosine diphosphate (ADP) and hydrogen phosphate. The ATP/ADP ratio reflects the energy status of a cell. It is generally assumed that a high intracellular ATP/ADP ratio (i.e. a high energy potential) constitutes a signal for proliferation. Conversely, energy depletion leads to cell death. However, the consequences of cell cycle progression on cell energy consumption are unknown. Here, we quantified the ATP/ADP ratio in live cells using a single cell approach. We thus characterize a reproducible pattern of variation of the energy level throughout the cell cycle, notably with a post-mitotic decrease in the ATP/ADP ratio. In addition, the cellular energy status can be controlled by inhibiting or stimulating cell proliferation. Finally, we show that proliferating cells are more sensitive to an aggression than non-proliferating ones. Our results establish that the proliferation rate critically impacts the cellular energy state, with major implications for the pathophysiology of organ injury.
Adenosine triphosphate (ATP) is the main energy provider for intracellular processes, through its cleavage into adenosine diphosphate (ADP) and hydrogen phosphate 1,2. The ATP/ADP ratio reflects the energy status of a cell 3–5. It is generally assumed that a high intracellular ATP/ADP ratio (i.e. a high energy potential) constitutes a signal for proliferation 6–9. Conversely, energy depletion leads to cell death 10,11. However, the consequences of cell cycle progression on cell energy consumption are unknown. Here, we quantified the ATP/ADP ratio in live cells using a single cell approach. We thus characterize a reproducible pattern of variation of the energy level throughout the cell cycle, notably with a post-mitotic decrease in the ATP/ADP ratio. In addition, the cellular energy status can be controlled by inhibiting or stimulating cell proliferation. Finally, we show that proliferating cells are more sensitive to an aggression than non-proliferating ones. Our results establish that the proliferation rate critically impacts the cellular energy state, with major implications for the pathophysiology of organ injury.
Adenosine triphosphate (ATP) is the cell’s main cellular energy provider. In tumors, its levels are correlated with proliferation. Thus, increased cellular ATP is generally considered to be a signal for cell proliferation 12,13. Multiple molecules act as ATP sensors, either intracellular (adenosine monophosphate-activated protein kinase, APMPK) or extracellular (PY2 purinergic receptor), and foster proliferation in response to elevated ATP levels 6,14,15. Conversely, a decrease in the level of intracellular ATP is associated with cell death, both in tumor and non-tumor cells 11,16.
Thus, intracellular ATP is a major determinant of cell fate. In addition, however, several studies suggest that intracellular ATP levels can be affected by the rate of proliferation through increased energy consumption. Such an effect of proliferation on ATP could explain why an increase in cell proliferation aggravates the course of acute kidney injury caused by an ischemic episode 17. The kidney has a very high energy turnover and is thus very sensitive to injury (and especially ischemia), which causes an immediate energy depletion and epithelial cell death 18. After an injury, cell proliferation is necessary for epithelial repair 19. Surprisingly, an increase in cell proliferation caused by the inactivation of the anti-proliferative factors p53 and p21 was shown to worsen the lesions caused by an episode of ischemia-reperfusion in the kidney 20–22. Similarly, we found an association between increased proliferation, ATP depletion and increased cell death in renal epithelial cells in which Nupr1 (Nuclear Protein 1) was inactivated 23. Nupr1 is a downstream effector of ATF4, a master regulator of endoplasmic reticulum stress, the activation of which leads to excessive protein synthesis, causing energy depletion and cell death 24. Taken together, these studies suggest that an increase in cell proliferation in conditions of stress might cause critical energy depletion, and result in cell death.
In order to follow the cellular energy level during the course of a cell cycle, we measured the ATP/ADP ratio in proliferating human renal epithelial cells (HK2 cell line). Indeed, if the ATP concentration is positively correlated with the number of cells in vitro 25, the ATP/ADP ratio is a better indicator of the cellular energy status, with a low ATP/ADP indicating a decreased energy state 26. PercevalHR is a genetically encoded fluorescent marker enabling the live monitoring of the ATP/ADP ratio 26,27. To monitor the single cell-ATP/ADP ratio over time in proliferating epithelial cells, we generated human renal epithelial HK2 cells stably expressing PercevalHR (Figure 1a). We first validated the quantification of the ATP/ADP ratio by PercevalHR (ATP/ADP[Perceval]) by an independent technique based on the measurement of luciferin/luciferase bioluminescence (ATP/ADP[Luciferin]). The total ATP/ADP[Perceval] signal correlated well with the ATP/ADP[Luciferin] (r=0.96, p=0.0001). We also observed the expected decrease of ATP/ADP[Perceval] after chemical energy depletion with blockage of glycolysis and oxidative metabolism using 2-deoxyglucose (2 DG) and sodium azide (NaN3) (Figure 1b).
We then performed a single cell quantification of ATP/ADP[Perceval] in real-time. Although changes in mean ATP/ADP values over time with or without energy depletion were reproducible, this ratio was highly variable among proliferating cells in the same well and at the same time (Figure 1c). This observation suggests that the ATP/ADP ratio is a dynamic parameter, undergoing variations that are at least partly independent of the extracellular environment. We hypothesized that the ATP/ADP ratio could be correlated to the cell cycle stage. To test this hypothesis, we plated PercevalHR-expressing epithelial cells at various densities to achieve different levels of contact-inhibition of proliferation (Figure 2a). We found that the intracellular ATP/ADP ratio increased when the cell density was higher. To rule out a direct effect of the confluence, we cultured the cells at the same densities in the presence of rigosertib, a potent cell cycle inhibitor inducing G2/M arrest. Non-proliferating cells showed a strong increase in the ATP/ADP ratio, regardless of their degree of confluence. Conversely, performing a scratch assay in confluent cells induced the proliferation and migration of the cells, which then displayed a low ATP/ADP ratio. Again, rigosertib increased the cellular ATP/ADP ratio while inhibiting wound closure (Figure 2a).
A panel of pharmaceutical compounds was then used to study the changes in the ATP/ADP ratio over time when the cell cycle is perturbed (Figure 2b and 2c). Tenovin-1 (a p53 agonist) and Rigosertib (a PLK1 inhibitor) were used to inhibit the cell cycle during the G1/S or G2/M transition, respectively. Conversely, pifithrin-α (a p53 antagonist promoting G1/S and G2/M transition) and KU-55933 (an ATM inhibitor promoting G2/M transition) were used to stimulate cell proliferation. Single-cell monitoring demonstrated that cell cycle inhibition increased ATP/ADP progressively over time whereas cell proliferation decreased it (Figure 2c).
We observed that the single-cell ATP/ADP[Perceval] ratio was maximal around cytokinesis in non-synchronized cells (Figure 3a). Therefore, we quantified the single-cell ATP/ADP ratio over time of the cell cycle in cells undergoing division. We found that the variations of the ATP/ADP ratio before and after cytokinesis follow a systematic pattern (Figure 3a): after a gradual increase, the ratio peaks at mitosis before dropping dramatically. This pattern was highly reproducible, including in cells exposed to pharmaceutical inhibition or stimulation of the cell cycle (Figure 3b). We concluded that the intracellular energy level, evaluated by the ATP/ADP ratio, is directly influenced by the cell cycle.
In order to study potential variations in the single cell ATP/ADP ratio in successive cell cycles within the same cell, we then selected the ATP/ADP ratio trajectories of cells undergoing division twice in the same live-imaging experiment. After a first mitosis, cells treated with tenovin-1 increased their ATP/ADP ratio significantly more than cells treated with pifithrin-α (Figure 3b, upper right). The cells had the same pattern of ATP/ADP variations for each of the two successive mitoses. In cells incubated with the cell cycle inhibitor tenovin-1, we observed an increment in the mitotic maxima of the ATP/ADP ratio from one mitosis to the next, whereas we did not find such an increment with the cell cycle facilitator pifithrin-α (Figure 3b, bottom right). We conclude that cell cycle inhibition increases ATP/ADP ratio in cells independently of the cell cycle stage, in a time dependent manner (Figure 3b). This suggests that the slower the cell cycles, the more positive is the cellular energy balance.
We then tested if the variations of the intracellular energy level during the cell cycle could impact the cell survival after a toxin. HK2 cells were incubated with puromycin, in the presence of tenovin-1 or pifithrin-α (Figure 4a and 4b). We observed that the stimulation of cell proliferation by pifithrin-α promoted the death of the cells exposed to puromycin (a well characterized toxicant acting through inhibition of protein synthesis, i.e. not directly targeting energy metabolism), whereas inhibition of cell proliferation by tenovin-1 was protective. Pifithrin-α or tenovin-1 alone were not toxic by themselves as they didn’t cause cell death in the absence of puromycin (Figure 4a). The same result was obtained when cells were cultured under conditions of chemical hypoxia + no glucose versus control conditions (normoxia + glucose), showing that pifithrin-α caused cell death whereas tenovin-1 was protective under energy depleted conditions (Figure 4b). These data show that highly proliferating cells are more sensitive to an insult than non-proliferative ones.
Our observations provide important insight for the understanding of the consequences of a variation in the cell energy status in physiology and pathology (Figure 4c). We demonstrate that the ATP/ADP ratio oscillates throughout the cell cycle, a pattern reminiscent to cyclic variations of the TCA cycle flux described by Ahn and colleagues 28. We also show here that the intracellular energy level not only varies during the cell cycle but also depends on the proliferation rate. We provide experimental evidence that the association of high intracellular energetic levels with cell proliferation, although usually observed, is not absolute. This finding is supported by the work of Mendelsohn et al, who found in a high throughput CRISPRi screen that cell growth and ATP can follow dissociated patterns 29. In addition, the cell survival in adverse conditions depends on proliferation inhibition, as cells in which proliferation is stimulated are more stress-sensitive than non-proliferating ones, likely related to accelerated energy consumption due to the requirements for protein synthesis and other metabolic processes necessary for genetic amplification prior to cell division.
This hypothesis is supported by studies performed in various types of tumors. Indeed, less proliferative tumors are often more resistant to cell death whereas highly proliferative tumors frequently undergo spontaneous necrosis. In addition, it is known that cancer cells with a high intracellular ATP level are more resistant to cytotoxic chemotherapy 30. This could be due to the fact that these cells do not proliferate. It could also explain why these chemotherapies are more efficient in proliferating tumors 8,31,32.
Cell proliferation is also observed during organ repair, where restoring the balance between energy production and energy consumption could also increase cell survival after an injury. For example, recent studies have described the changes in energy metabolism occurring during acute kidney injury (AKI) 33–35. They showed that fatty acid oxidative phosphorylation is defective in proximal tubular cells after injury. Restoring oxidative metabolism not only reverses established ischemic AKI but also prevents AKI. On the other hand, stabilization of the Hypoxia Inducible Factor (HIF) shifts the energy metabolism to anaerobic glycolysis, inhibits cell proliferation36–38 and protects against ischemic acute kidney injury 39–41. Activation of AMPK signaling reduces the cellular energy consumption and protects the kidneys from ischemia-reperfusion injury 42, while suppressing cell proliferation 43,44. Our data shows that as a complement to metabolic reprogramming, inhibiting proliferation might be a useful strategy to prevent lethal energy deprivation in epithelial cells. This protective mechanism might be responsible for the protective effect of the extensively studied phenomenon of ischemic preconditioning 45–47, where a mild and transient ischemia triggers cytoprotective pathways concomitant with transient cell cycle arrest 48,49, offering an endpoint to select a time frame for the difficult clinical implementation of a promising preclinical therapeutic approach 50,51. With severe injury to an organ the increase in ATP/ADP ratio with cell cycle arrest may have adaptive effects to allow the cell more energy to repair damaged DNA. This may prevent malignant transformation and have a adaptive or maladaptive consequences with respect to long term sequelae of kidney injury depending upon whether the cell cycle arrest is at G1/S or G2/M due to a senescence associated secretory program resulting in production of pro-fibrotic cytokines52,53.
ONLINE METHODS
Cell cultures and pharmaceutical inhibitors
HK2 cells are immortalized human renal proximal tubular cells cultured in DMEM with 10% fetal bovine serum. The experiments were conducted in Leibovitz’s L-15 medium with no phenol red (Fisher, #21083027). L-15 was specifically designed for renal primary cell cultures. It provides a stable physiological pH without CO2 supplementation, as it is buffered with non- bicarbonate ions. Pharmaceutical inhibitors were purchased from Selleckchem: tenovin-1 (#S8000), pifithrin-α (#S2929), Rigosertib (#S1362) and KU-55933 (#S1092).
Viability experiments
Puromycin toxicity was tested on cells grown in DMEM with 10% fetal bovine serum with or without puromycin (2µg/mL). Energy depletion was achieved in cells grown in Leibovitz’s L-15 medium without glucose supplementation and hypoxia obtained by applying a 100% atmosphere (ref 26700 from Air products) in a airtight chamber (ref 27310 and 27311 from Stemcell) during 24h, according to the manufacturer’s instructions. The control cells were grown in Leibovitz’s L- 15 medium with 4,5 g/L glucose supplementation under ambient atmosphere. Flow cytometry analysis for assessment of dead cells was performed with the fixable viability dye Viobility 405/452 Fixable Dye (Miltenyi Biotec®). Viobility dyes react with amine groups of proteins and are non-toxic. In dead cells, intracellular proteins are stained, resulting in an increase in fluorescence.
ATP/ADP bioluminescence assay
ATP and ADP measurements were performed with the ApoSENSOR ADP/ATP ratio assay (Enzo Life Sciences), in accordance with the manufacturer’s instructions.
ATP/ADP ratio live imaging
PercevalHR was used to study the ATP/ADP ratio in live individual cells in real-time experiments, spanning multiple cell cycles. Using lentiviral transformation, we generated proximal tubular cells with stable expression of PercevalHR as well as pHRed, in order to adjust the PercevalHR signal on pH variations 27. ATP/ADP ratio and pH were assessed by dual signal acquisition for PercevalHR (ex 436/495; em 540) and pHRed (ex 436/550; em 640), followed by ratiometric normalization using the Ratio Plus plugin in Fiji 54. PercevalHR (ATP/ADP ratio) was further corrected for pH by normalization on pHRed 27, using the Ratio Plus plugin.
Bioinformatic analysis of ATP/ADP ratio
The Fiji Trackmate plugin was used to monitor single-cell ATP/ADP ratio over time, and to determine the time of cytokinesis for each cell 55. Briefly, every cell was assigned to a single trajectory at a specific timepoint, with a corresponding ATP/ADP value. The times of cytokinesis were noted for each cell trajectory. ATP/ADP values were represented on the y-axis as mean+/- standard error, and time was represented on the x-axis in hours from either the start of the experiment or from the time of cytokinesis.
Statistical analysis
Statistical analyses were conducted using JMP 11 (SAS). Log-transformation was performed to obtain normal distribution, when necessary. Correlations were evaluated using Pearson’s test. P- values were considered significant when <0.05. Comparison between groups were considered significant when the p-value was <0.05 using a Wilcoxon rank-test. Differences in ATP/ADP changes between treatment groups over time were assessed by analysis of variance using a standard least square model.
Supplemental material
Fiji Jython script for single cell ratiometric analysis Live PercevalHR videos
ACKOWLEGMENTS
PG received support from Monahan Foundation, Fondation pour la Recherche Médicale, Groupe Pasteur Mutualité, Société Francophone de Transplantation, Arthur Sachs fellowship, Philippe Foundation, Fulbright Scholarship, ATIP Avenir program.