Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress - PubMed (original) (raw)
doi: 10.1186/2049-3002-1-23.
Xiaohu Tang, Julia Ling-Yu Chen, Jianli Wu, Chien-Kuang Cornelia Ding, Melissa M Keenan, Carolyn Sangokoya, Hsiu-Ni Kung, Olga Ilkayeva, László G Boros, Christopher B Newgard, Jen-Tsan Chi 1
Affiliations
- PMID: 24359630
- PMCID: PMC4178214
- DOI: 10.1186/2049-3002-1-23
Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress
Gregory Lamonte et al. Cancer Metab. 2013.
Abstract
Background: A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.
Results: Acidosis increased both glutaminolysis and fatty acid β-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.
Conclusions: Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.
Figures
Figure 1
The use of 13 C glucose isotope tracers to analyze the glucose metabolism under acidosis. (A) Schematic graph indicating various measured metabolites (and corresponding panels) in major metabolic pathways resulting from the 1,213C labeled glucose tracer (green). 13C labeled and unlabeled carbons are indicated in red and black, respectively. (B-G) Relative 13C enrichment under control or acidosis conditions for glucose (B), CO2**(C)**, lactate (D), glutamate (E), ribonucleic acids (F), and fatty acids (G). Lactate (D) is presented as the total 13C labeled lactate pool as well as 1 (C1) and 2 (C2) labeled carbon subpools. Glutamate (E) is presented as both the 2 (C2) and 4 (C4) labeled subpools. Ribonucleic acids (F) are presented as the 13C positions 1 to 4 and 13C positions 3 to 5 subpools. Fatty acids (G) are presented as 2-carbon 13C-labeled palmitate and oleate. Error bars are mean ± SD, significant P values are indicated (*P ≤0.05, **P ≤0.01, ***P ≤0.001).
Figure 2
The use of 13 C glutamine isotope tracer to analyze glutamine metabolism under acidosis. (A) Schematic graph indicating various measured metabolites (and corresponding panels) resulting from the uniformly 13C labeled glutamine tracer (green). The 13C labeled and unlabeled carbons are indicated in red and black, respectively. (B-G) Relative 13C enrichment in the glutamate (B,D), CO2**(C)**, lactate (E), ribonucleic acids (F) and fatty acids (G) under control or acidosis conditions. Glutamate (B,D) is presented as both the 2 (C2 (D)) and 4 (C2 (B)) labeled carbon subpools. Lactate (E) is presented as the uniformly labeled 13C3-labeled lactate. Ribonucleic acids (F) are presented as the 13C positions 1 to 4 and 13C positions 3 to 5 subpools. Fatty acids (G) are presented as 2-carbon 13C-labeled palmitate and oleate. Error bars are mean ± SD, significant P values are indicated (*P ≤0.05, **P ≤0.01, ***P ≤0.001).
Figure 3
Acidosis increases glutaminolysis and renders glutaminolysis essential. (A) Heatmap shows the changes in the intracellular amino acids under acidosis (HCl), lactosis (10 and 25 mM Lac), or lactic acidosis (10 and 25 mM LA) (n = 3) conditions. (B) Normalized levels of intracellular glutamine (Gln) and glutamate (Glu) in MCF-7 cells under control or lactic acidosis conditions (n = 4). (C) Relative cell numbers as determined by trypan blue exclusion, of MCF-7 and ZR-75-1 cells after 72 h under the indicated media conditions. (n = 4) (D) Relative ATP levels under acidosis (Acid) with (+) or without (-) of glutamine (Gln) in media. (E,F) Relative changes of indicated mRNAs (E) and proteins (F) under indicated conditions. (G) Relative cell numbers of MCF-7 cells when transfected with control or two GLS2 small interfering (si)RNAs under control and acidosis conditions. (H) Total glutathione (GSH) level of MCF-7 cells exposed to acidosis or lactic acidosis (n = 3). (I) Normalized glutathione disulfide (GSSG)/GSH ratios for MCF-7 and ZR-75-1 cells under control, acidosis or lactic acidosis conditions (n = 6). (J) Normalized cell numbers of MCF-7 cells under control, acidosis and lactic acidosis conditions when exposed to indicated level (uM) of H2O2 (n = 3). (K,L) Relative cell numbers (via trypan blue exclusion) (K) and cellular ATP level for MCF-7 (L) treated with vector or 0.2 mM amino-oxyacetate (AOA) for 72 h. The indicated samples were supplemented with 700 μm α-ketoglutarate (α-KG). Error bars are mean ± SEM, P values as indicated (*P ≤0.05, **P ≤0.001, ***P ≤0.0001).
Figure 4
Acidosis enhanced oxidative branch of pentose phosphate pathways (PPPs). (A) Percentage of glucose that enters the PPP under control or acidosis conditions. (B) NADP+/nicotinamide adenine dinucleotide phosphate (NADPH) ratio for MCF-7 and ZR-75-1 cells under control or acidosis conditions (n = 6). (C) Heatmap indicates the transcriptional changes of genes listed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) in the PPPs under hypoxia, lactic acidosis or glucose deprivation conditions. (D) Genes in PPP were induced (red) or repressed (green) by at least 1.7-fold under lactic acidosis. (E) The mRNA expression of glucosephosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PD), transketolase (TKT), and triose phosphate isomerase (TPI) under control or acidosis conditions with 0, 4, 10 and 25 mM lactate. (F) The protein levels of G6PD, TKT and β-tubulin in MCF-7 cells under acidosis, lactosis (25 mM), or lactic acidosis (10 mM and 25 mM pH 6.7) conditions with the relative changes (by densitometry) shown. (G) Relative cell numbers of MCF-7 cells that have been transfected with control or two small interfering (si)RNAs targeting G6PD under control or acidosis conditions. (H) Relative NADP+/NADPH ratios in MCF-7 cells, transfected with control or two G6PD-targeting siRNAs, under control or acidosis conditions (n = 4). (I) Relative ROS in MCF-7 cells transfected with control or two G6PD-targeting siRNAs, under control or acidosis conditions. (J) Relative cell numbers of MCF-7 cells transfected with control or _TKT_-targeting siRNA under control or acidosis conditions (A). Error bars are mean ± SEM, P values as indicated (*P ≤0.05, **P ≤0.001, ***P ≤0.0001).
Figure 5
Regulation of the metabolic response to acidosis by p53. (A) Protein levels of p53 under control, lactosis, acidosis (HCl) and lactic acidosis (LA). (B-D) Relative mRNA expression of p21, p53 and MDM2 genes in the shControl or shP53 MCF-7 cells under control or acidosis conditions. (C) Relative mRNA expression and protein levels of GLS2 in shControl or shP53 MCF-7 cells under control or acidosis conditions. (D) Relative ATP levels in shControl or shp53 cells under lactic acidosis (LA) with (+) or without (-) glutamine. (E) Relative mRNA expression and protein levels of glucose-6-phosphate dehydrogenase (G6PD) in shControl or shP53 MCF-7 cells under control or acidosis conditions. (F-H) Relative NADP+/nicotinamide adenine dinucleotide phosphate (NADPH) ratio (F), normalized ROS levels (G), and normalized cell numbers (H) in shControl or shP53 MCF-7 cells under control or acidosis conditions. Error bars are mean ± SEM, P values as indicated (*P ≤0.05, **P ≤0.001, ***P ≤0.0001).
Figure 6
Glucose-6-phosphate dehydrogenase (G6PD) and p53 status are correlated in vivo . (A) Relative mRNA expression of GLS2 in the indicated cell lines under control or acidosis conditions. (B) Relative mRNA expression of G6PD in the indicated cell lines under control or acidosis conditions. (C,D) Relative NADP+/nicotinamide adenine dinucleotide phosphate (NADPH) ratio (C) and normalized ROS levels (D) in the indicated cell lines under control or acidosis conditions. (E) Relative expression level of G6PD mRNA in among groups of breast tumors with wild-type or mutant p53.
Figure 7
Overview of the metabolic reprogramming mediated by p53 under acidosis conditions. Glucose-6-phosphate dehydrogenase (G6PD) is induced under acidosis by p53, which increases nicotinamide adenine dinucleotide phosphate (NADPH) from the oxidative PPP appears to help cells tolerate the increased ROS stresses and reduced novel GSH synthesis under acidosis conditions. Acidosis also dramatically reduces the novel RNA ribose synthesis via the PPP. Furthermore, the activation of p53 under acidosis contributes to increased glutaminolysis by inducing GLS2, leading to increased glutamate generation, which is then converted to α-ketoglutarate (α-KG) and enters the tricarboxylic acid cycle (TCA cycle). Enzymes are underlined and in bold.
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