Glycolysis-respiration relationships in a neuroblastoma cell line

Abstract

Background: Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods: We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results: Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions: These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance: Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches.

Figure 1. Adding glucose to glucose-deprived SH-SY5Y cells induces a Crabtree effect

The change in the ECAR is shown as a percent change from baseline (A), and the absolute ECAR values under the different glucose conditions are shown in (B). The change in the OCR is shown as a percent change from baseline (C), and the absolute OCR values under the different glucose conditions are shown in (D). Rot=rotenone, myx=myxothiazol.

Figure 2. In addition to reducing the ECAR, adding 2-DG to SH-SY5Y cells induces an OCR increase

The change in the ECAR is shown as a percent change from baseline (A), and the absolute ECAR values before and after 2-DG are shown in (B). The change in the OCR is shown as a percent change from baseline (C), and the absolute, true mitochondrial OCR values after the different injections are shown in (D). To demonstrate system stability, for the first injection half the wells received 25 mM 2-DG, while the other half of the wells received a control injection (25 mM glucose, same medium as the medium in the wells). After the 2-DG injection, the OCR was significantly increased relative to the pre-injection baseline of those wells, and also to the wells that had received the control injection. Blue line=2-DG treated cells, red line=control-treated cells.

Figure 3. In addition to reducing the ECAR, adding iodoacetate to SH-SY5Y cells induces an OCR increase

The change in the ECAR is shown as a percent change from baseline (A), and the absolute ECAR values before and after iodoacetate are shown in (B). The change in the OCR is shown as a percent change from baseline (C), and the absolute, true mitochondrial OCR values after the different injections are shown in (D). To demonstrate system stability, for the first injection half the wells received 25 uM iodoacetate, while the other half of the wells received a control injection (2.5 mM glucose, same medium as the medium in the wells). After the iodoacetate injection, the OCR was significantly increased relative to the pre-injection baseline of those wells, and also to the wells that had received the control injection. Blue line=iodoacetate-treated cells, red line=control-treated cells. IA=iodoacetate.

Figure 4. In addition to reducing the ECAR, glucose starvation of undifferentiated SH-SY5Y cells induces an OCR increase

The difference in the absolute ECAR values from cells in 25 mM and 0 mM glucose is shown in (A). The difference in the absolute total and mitochondrial OCR values are shown in (B) and (C).

Figure 5. In addition to reducing the ECAR, glucose starvation of differentiated SH-SY5Y cells induces an OCR increase

The difference in the absolute ECAR values from cells in 25 mM and 0 mM glucose is shown in (A). The difference in the total mitochondrial OCR values is shown in (B).

Figure 6. Galactose effects on SH-SY5Y cell OCR and ECAR

Cells in galactose medium had a higher OCR than cells in a comparable amount of glucose (A), as well as a lower ECAR (B). The OCR/ECAR ratio, accordingly, was higher for the cells in the galactose medium (C).

Figure 7. Effect of glucose deprivation on proteins regulated by cell bioenergetic fluxes

After four hours, PCG1a levels were unchanged (A), ERK phosphorylation, HIF1a, and SIRT1 decreased (B–D), and AKT phosphorylation, p38, and AMPK increased (E–H). These experiments were conducted in serum-free medium. NS=not significant.

Figure 8. Relative ATP levels from SH-SY5Y cells maintained in 25 mM glucose and 0 mM glucose

ATP levels are reduced in glucose-starved cells.