Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells

Abstract

Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

Figure 1. Cytochrome B Mutant 143B Cybrid Cells are Auxotrophic for Electron Acceptors that can Regenerate NAD+

(A) Proliferation rate of 143B CytB cells was determined in the presence or absence of pyruvate. (left) Cell counts, normalized to cell number at t=0 when media conditions were applied, were assessed over time and used to calculate proliferation rate (right). (B) Pyruvate is a substrate of lactate dehydrogenase (LDH), accepting electrons from NADH to produce NAD+ and lactate. (C) Alpha-ketobutyrate (AKB) can also act as an electron acceptor from NADH and yield NAD+ and alpha-hydroxybutyrate (AHB). (D) Proliferation rate for 143B CytB cells in the presence or absence of AKB was determined as in (A). (E) Intracellular ratio of NAD+/NADH was determined in 143B CytB cells in untreated media or in the presence of pyruvate (Pyr) or AKB. (F) The concentration of AKB and AHB in the media of 143B CytB cells cultured in the presence of AKB was determined over time by GCMS analysis. Values in all figure panels denote mean ± standard error of the mean (SEM), n=3. See also Figure S1.

Figure 2. Proliferation of Respiration-Inhibited Cells is Restored by Exogenous Electron Acceptors

(A) The proliferation rate of 143B cells was determined in media with or without AKB supplementation in the absence (Untr) or in the presence of the mitochondrial respiration inhibitors rotenone (Rot), phenformin (Phen), antimycin (Ant), myxothiazol (Myxo), azide (N₃), or oligomycin (Oligo). (B) The ratio of NAD+/NADH was determined in 143B cells with or without AKB supplementation in the absence or presence of respiration inhibitors as in (A). (C) The proliferation rate of A172, H1299, HeLa, U87, FL5.12, and MEF cell lines was determined with or without AKB supplementation in the absence or presence of rotenone, antimycin, or oligomycin. Values in all figure panels denote mean ± SEM, n=3. See also Figure S2.

Figure 3. Oxygen Utilization in the Absence of Mitochondrial ATP Production is Sufficient for Cell Proliferation

(A) Schematic illustrating the effects of oligomycin and FCCP on mitochondrial membrane potential and ATP synthesis. Oligomycin treatment inhibits ATP synthase, resulting in a hyperpolarized mitochondrial membrane. This hyperpolarization inhibits proton pumping and thereby inhibits ETC activity resulting in decreased NADH oxidation and O₂ consumption (left). Treatment with FCCP in addition to oligomycin relieves the hyperpolarization of the mitochondrial membrane allowing restoration of NADH oxidation and mitochondrial O₂ consumption without restoring ATP production (right). (B) Proliferation rate of 143B cells treated with oligomycin in the presence or absence of FCCP treatment. (C) Mitochondrial oxygen consumption rate of 143B cells treated with oligomycin with or without FCCP. (D) Intracellular NAD+/NADH ratio in 143B cells treated with oligomycin in the presence or absence of FCCP. Values in all figure panels denote mean ± SEM, n=3 (B, D), n=5 (C).

Figure 4. Electron Acceptor Insufficiency Affects Purine Nucleotide Levels

(A) Analysis of DNA content by propidium iodide staining and flow cytometry of 143B CytB cells cultured with our without AKB supplementation. (B) LCMS quantification of purine nucleotide levels in 143B CytB cells cultured with or without AKB supplementation. (C) Cell doublings were measured over time of 143B CytB cells cultured in unsupplemented media or media supplemented with AKB, adenine, hypoxanthine, or guanine. (D) Schematic illustrating the use of IMP for GMP and AMP synthesis. Synthesis of GMP uses NAD+, whereas synthesis of AMP requires aspartate. Values in (B) and (C) denote mean ± SEM, n=3. See also Figure S3.

Figure 5. Electron Acceptor Insufficiency Suppresses TCA Metabolite and Aspartate Levels

(A) Schematic detailing the TCA cycle reaction routes for the biosynthesis of aspartate from glutamine. (B) Isotopomer distribution of aspartate from 143B CytB cells supplemented with AKB and 143B cells cultured in the presence of U-¹³C Glutamine for 8 hours. (C) GCMS quantification of TCA cycle metabolites, glutamate, and aspartate from 143B CytB cells cultured with or without AKB. (D) GCMS quantification of TCA metabolites, glutamate, and aspartate from 143B cells cultured with or without AKB in the presence or absence of the indicated mitochondrial inhibitors. Ion counts are relative to untreated 143B cells, which is denoted by the dashed grey line in each panel. Values denote mean ± SEM, n=3. See also Figure S4.

Figure 6. Aspartate is the Key Biosynthetic Precursor Provided by Respiration

(A) Proliferation rate of 143B CytB cells determined in the presence or absence of aspartate. (B) The proliferation rate of 143B cells cultured with or without aspartate in the presence of the mitochondrial respiration inhibitors rotenone (Rot), phenformin (Phen), antimycin (Ant), myxothiazol (Myxo), azide (N₃), or oligomycin (Oligo). (C) The proliferation rate of A172, H1299, HeLa, U87, FL5.12, and MEF cells cultured with or without aspartate in the presence of rotenone, antimycin, or oligomycin. (D) Schematic detailing the role of respiration and exogenous electron acceptors in aspartate biosynthesis. The conversion of nutrients into aspartate requires the removal of electrons and therefore requires access to electron acceptors, which can be supplied by respiration (O₂) or exogenous electron acceptors such as AKB. Maintenance of aspartate pools supports nucleotide and protein biosynthesis. Values in all figure panels denote mean ± SEM, n=3. See also Figure S5.