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Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes
Published on: January 7, 2013
Metabolic reprogramming contributes to radioprotection by protein kinase Cδ
Angela M Ohm1, Trisiani Affandi1, Julie A Reisz2
1Department of Craniofacial Biology, School of Dental Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.
Abstract:
Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition.
Insights
Loss of protein kinase Cδ (PKCδ) activity confers radioresistance by reprogramming cell metabolism. Depleted PKCδ enhances the pentose phosphate pathway and glutamine utilization for nucleotide synthesis, aiding cell survival against DNA damage.
Area of Science:
- Cellular Biology
- Metabolic Pathways
- Radiation Biology
Background:
- Protein kinase Cδ (PKCδ) loss confers resistance to DNA damaging agents like irradiation.
- The underlying mechanisms of this radioresistance are not fully understood.
- Metabolic reprogramming is a potential contributor to cellular resistance.
Purpose of the Study:
- To investigate if metabolic reprogramming by PKCδ contributes to radioprotection.
- To elucidate the specific metabolic pathways involved in PKCδ-mediated radioresistance.
Main Methods:
- Global metabolomics analysis to assess metabolic pathway alterations.
- Stable isotope tracing (using U-[13C6] glucose and [13C5, 15N2] glutamine) to track metabolic flux.
- Assays for pentose phosphate pathway (PPP) activation, nucleotide and glutathione biosynthesis, glycolysis, and oxidative phosphorylation.
- Inhibition studies targeting the PPP and glutaminase to assess their role in radioprotection.
Main Results:
- PKCδ depletion altered metabolic pathways, impacting energy production, antioxidant, nucleotide, and amino acid biosynthesis.
- Increased NADPH and nucleotide production in PKCδ-depleted cells correlated with PPP upregulation (evidenced by G6PD activation and increased 5-phosphoribosyl-1-pyrophosphate).
- Isotope tracing revealed reduced glucose utilization for glycolysis but increased glutamine utilization for nucleotide, glutathione, and TCA intermediate synthesis in PKCδ-depleted cells.
- PKCδ-depleted cells showed increased anaerobic glycolysis, reduced oxidative phosphorylation, and acquired a dependency on the PPP and glutamine for survival post-irradiation.
Conclusions:
- Metabolic reprogramming, specifically increased glutamine utilization and nucleotide synthesis, contributes to radioprotection when PKCδ is inhibited.
- The pentose phosphate pathway and glutamine metabolism are critical for the survival of PKCδ-depleted cells exposed to irradiation.
- Targeting the PPP or glutaminase can reverse the radioresistance observed in PKCδ-depleted cells.
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