Role of Exogenous Pyruvate in Maintaining Adenosine Triphosphate Production under High-Glucose Conditions through PARP-Dependent Glycolysis and PARP-Independent Tricarboxylic Acid Cycle

Abstract

Pyruvate serves as a key metabolite in energy production and as an anti-oxidant. In our previous study, exogenous pyruvate starvation under high-glucose conditions induced IMS32 Schwann cell death because of the reduced glycolysis-tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production. Thus, this study focused on poly-(ADP-ribose) polymerase (PARP) to investigate the detailed molecular mechanism of cell death. Rucaparib, a PARP inhibitor, protected Schwann cells against cell death and decreased glycolysis but not against an impaired TCA cycle under high-glucose conditions in the absence of pyruvate. Under such conditions, reduced pyruvate dehydrogenase (PDH) activity and glycolytic and mitochondrial ATP production were observed but not oxidative phosphorylation or the electric transfer chain. In addition, rucaparib supplementation restored glycolytic ATP production but not PDH activity and mitochondrial ATP production. No differences in the increased activity of caspase 3/7 and the localization of apoptosis-inducing factor were found among the experimental conditions. These results indicate that Schwann cells undergo necrosis rather than apoptosis or parthanatos under the aforementioned conditions. Exogenous pyruvate plays a pivotal role in maintaining the flux in PARP-dependent glycolysis and the PARP-independent TCA cycle in Schwann cells under high-glucose conditions.

Keywords: PARP; Schwann cells; adenosine triphosphate depletion; cell death; exogenous pyruvate; glycolysis; high-glucose; tricarboxylic acid cycle.

Figure 1

Rucaparib ameliorated glycolytic, but not mitochondrial, adenosine triphosphate (ATP) production under high-glucose and pyruvate-starved conditions. Glycolytic (A) and mitochondrial (B) ATP production of IMS32 cells under exposure to 5 mM glucose in the presence (blue) and absence (yellow) of pyruvate conditions, 100 mM glucose in the presence (brown) and absence (green) of pyruvate conditions, and 100 mM glucose in the absence of pyruvate conditions supplemented with rucaparib (red) were determined. Values represent mean + SD from nine to ten experiments (individual values in glycolytic and mitochondrial ATP production are depicted as circles and triangles, respectively). ** p < 0.01. ND: not detected.

Figure 2

Pyruvate starvation and rucaparib had no significant effects on oxidative phosphorylation under high-glucose conditions. Representative image of blue native PAGE and Western blotting of the supercomplex of IMS32 cells under exposure to 5 mM glucose in the presence and absence of pyruvate, 15 mM glucose in the presence and absence of pyruvate, and 15 mM glucose in the absence of pyruvate conditions supplemented with rucaparib (A). The bands of the complex in the electric transporter chain were estimated as referred to in the manuscript [26]. Relative complex I (CI; NDUFB8), CII (SDHB), CIII (MTCO1), CIV (UQCR2), and CV (ATP5A) expression (B) and CI activities (C) were measured. The representative blots for these complexes and β actin (B) are shown in Figure S1. (B,C) These bars exhibit the conditions of 5 mM glucose in the presence (blue) and absence (yellow) of pyruvate conditions, 15 mM glucose in the presence (brown) and absence (green) of pyruvate conditions, and 15 mM glucose in the absence of pyruvate conditions supplemented with rucaparib (red). The values represent the mean + SD from three (B) and four (C) experiments (individual values are depicted as circles).

Figure 3

Pyruvate starvation decreased pyruvate dehydrogenase (PDH) activity without PDHE1α phosphorylation under high-glucose conditions. PDH activity (A) and PDHE1α (Ser293) phosphorylation (B) of IMS32 cells under exposure to 5 mM glucose in the presence (blue) and absence (yellow) of pyruvate conditions, 15 mM glucose in the presence (brown) and absence (green) of pyruvate conditions, and 15 mM glucose in the absence of pyruvate conditions containing rucaparib (red) were determined. The representative blots for PDHα1 (Ser293) and PDH (B) are shown in Figure S2. The values represent the mean + SD from three experiments (individual values are depicted as circles). ** p < 0.01.

Figure 4

GAPDH was not poly-ADP-ribosylated under high-glucose pyruvate-starved conditions. (A) The degrees of poly-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) under high-glucose conditions in the presence and absence of the pyruvate condition were assessed by the immunoprecipitation of poly-(ADP-ribose) polymer or isotype control and Western blotting of GAPDH. Blots for GAPDH (A) and the image of the Ponceau stain are shown in Figure S4. (B) Images of the immunostaining of GAPDH (green) and nuclear staining of DAPI (blue) of IMS32 cells under 5 or 15 mM glucose in the presence and absence of pyruvate and 15 mM glucose in the absence of pyruvate containing rucaparib. Enlarged images of the white box in merged images are shown. Scale bar: 10 μm.

Figure 5

Pyruvate-starvation-induced necrosis-like cell death under high-glucose conditions. The cell viability, toxicity, and caspase 3/7 activity of IMS32 cells at 3 (A) and 6 (B) h under exposure to 5 mM glucose in the presence (blue) and absence (yellow) of pyruvate conditions, 15 mM glucose in the presence (brown) and absence (green) of pyruvate conditions, and 15 mM glucose in the absence of pyruvate conditions supplemented with rucaparib (red) were determined. (C) Images of immunostaining for apoptosis-inducing factor (AIF; green) and TOMM40 (red) and nuclear staining of DAPI (blue) of IMS32 cells under 5 or 15 mM glucose in the presence and absence of pyruvate. Enlarged images of the white box in merged images are shown. Scale bar: 10 μm. The values represent the mean + SD from six experiments (individual values are depicted as circles). * p < 0.05, ** p < 0.01.

Figure 5

Pyruvate-starvation-induced necrosis-like cell death under high-glucose conditions. The cell viability, toxicity, and caspase 3/7 activity of IMS32 cells at 3 (A) and 6 (B) h under exposure to 5 mM glucose in the presence (blue) and absence (yellow) of pyruvate conditions, 15 mM glucose in the presence (brown) and absence (green) of pyruvate conditions, and 15 mM glucose in the absence of pyruvate conditions supplemented with rucaparib (red) were determined. (C) Images of immunostaining for apoptosis-inducing factor (AIF; green) and TOMM40 (red) and nuclear staining of DAPI (blue) of IMS32 cells under 5 or 15 mM glucose in the presence and absence of pyruvate. Enlarged images of the white box in merged images are shown. Scale bar: 10 μm. The values represent the mean + SD from six experiments (individual values are depicted as circles). * p < 0.05, ** p < 0.01.

Figure 6

The schematic representation of metabolic changes in IMS32 cells under high-glucose pyruvate-starved conditions. Pyruvate starvation under high-glucose conditions resulted in impaired glycolysis–tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production due to the impaired glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate dehydrogenase (PDH) activities (A). Rucaparib treatment under these conditions led to the recovery of NAD, NADH, GAPDH activity, and glycolytic flux and ATP production. However, PDH activity, TCA cycle flux, and mitochondrial ATP production remained unaltered (B). PDH activity was found to be independent of PARP and PDH kinase (PDK). These metabolic changes resulted in necrosis-like IMS32 cell death. Blue allows: decrease, Red allows: increase, ×: no regulation, G3P: glyceraldehyde-3-phosphate, PEP: phosphor enol pyruvate.