The NAD+/NADH ratio is proportional to serine synthesis rate.

(A) α-ketobutyrate (AKB) is an exogenous electron acceptor that promotes the oxidation of NADH into NAD+ through its conversion to α-hydroxybutyrate (AHB). Rotenone inhibits activity of complex I of the mitochondrial electron transport chain (ETC) to block NADH oxidation into NAD+. (B) NAD+/NADH ratio measured in A549 cells cultured without serine and indicated concentrations of AKB (left) and rotenone (right) for 24 hours. NAD+/NADH ratios are normalized to untreated cells cultured without serine, n=3. (C) Schematic depicting NAD+ requirement to produce serine labeled (M+3) from U-13C-glucose. Under culture conditions without serine, intracellular serine rapidly effluxes from cells into culture media. Thus, cells and media were jointly analyzed at each time point of kinetic tracing to better capture serine levels. (D) Serine labeled (M+3) from U-13C-glucose over time in A549 cells cultured without serine and with indicated concentrations of AKB and rotenone for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (E) Relative NAD+/NADH ratios from A549 cells cultured without serine after exposure to 250 μΜ AKB, 1000 μΜ AKB, 20 nM rotenone, or 40 nM rotenone for 24 hours are plotted against the serine synthesis rates from corresponding conditions as shown in D. Serine synthesis rates are calculated from labeled serine (M+3) after 1 and 15 minutes of U-13C-glucose exposure and normalized to untreated and serine-free culture conditions, n=3. (F) Proliferation rate (doublings per day) of A549 cells cultured with and without serine and the indicated concentration of AKB for 72 hours, n=3. (G) Proliferation rate (doublings per day) of A549 cells cultured with and without serine and the indicated concentration of rotenone for 72 hours, n=3. Data shown represent means ± SD.

The NAD+/NADH ratio differs between cancer cells upon serine withdrawal and correlates with proliferation in serine-depleted conditions.

(A) Proliferation rate (doublings per day) of indicated cancer cells cultured without serine for 72 hours normalized to proliferation rate of corresponding cancer cells cultured with serine, n=3. (B) NAD+/NADH ratios of indicated cells cultured with and without serine for 24 hours. Yellow indicates cells with unaltered NAD+/NADH ratios and blue indicates cells with elevated NAD+/NADH ratios when cultured without serine. Values are normalized to the NAD+/NADH ratios of cells cultured with serine, n=3. (C) Relative NAD+/NADH ratios of cells cultured without serine as shown in B are plotted against proliferation rates of corresponding cells cultured without serine. Data points denote the different cell lines (Calu6, A549, 8988T, MIA PaCa-2, H1299, and HCT116). (D) Serine labeled (M+3) from U-13C-glucose over time in A549 and H1299 cells cultured with and without serine for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (E) Absolute fold change of serine labeled (M+3) from U-13C-glucose in A549 and H1299 cells cultured without serine relative to serine labeled (M+3) from U-13C-glucose in A549 and H1299 cells cultured with serine for 24 hours after twelve minutes of U-13C-glucose exposure, n=3. Data shown are means ± SD. P-values were calculated by unpaired Student’s t-test, **p<0.01, ***p<0.001

Elevated mitochondrial respiration is associated with increased NAD+/NADH ratios and serine synthesis rates.

(A) Oxygen consumption rate (OCR) of indicated cells cultured with and without serine for 24 hours. Values are averages of three repeat measurements, n=14-20. (B) OCR of cells (H1299 top, A549 bottom) cultured with indicated concentrations of serine for 24 hours. Values are averages of three repeat measurements, n=3-9. (C) NAD+/NADH ratios of cells (H1299 top, A549 bottom) cultured with indicated concentrations of serine for 24 hours. Values are normalized to the NAD+/NADH ratio of cells cultured in 400μM serine (serine concentration in DMEM), n=3. (D) Relative OCR plotted against NAD+/NADH ratios in H1299 and A549 cells cultured in 0, 25, 50, and 100 μM serine for 24 hours. OCR and NAD+/NADH ratios are normalized to measurements from the 100 μΜ serine condition, n=3. (E) Serine labeled (M+3) from U-13C-glucose over time in cells (H1299 top, A549 bottom) cultured with indicated serine concentrations for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (F) Relative NAD+/NADH ratios of cells (H1299 top, A549 bottom) cultured with 0, 25, 50, and 100 μM serine for 24 hours plotted against relative serine synthesis rates of cells cultured in corresponding conditions. Serine synthesis rates are calculated from labeled serine (M+3) after 1 and 15 minutes of U-13C-glucose exposure and normalized to culture conditions with serine, n=3. Data shown are means ± SD. P-values were calculated by unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001, “ns” denotes not significant.

Mitochondrial respiration governs the cell NAD+/NADH ratio and influences serine synthesis.

(A) Schematic depicting the mitochondrial electron transport chain (ETC) and the protonophore activity of FCCP that uncouples the ETC from ATP synthase activity. IMS, intermembrane space; CoQ(H2) indicates the oxidized and reduced (H2) forms of coenzyme Q; cytc, cytochrome c. (B) Proliferation rate (doublings per day) of A549 cells cultured with and without serine with indicated concentrations of FCCP for 72 hours, n=3. (C) Proliferation rate (doublings per day) of H1299 cells cultured with and without serine with indicated concentrations of FCCP for 72 hours, n=3. (D) Serine labeled (M+3) from U-13C-glucose over time in A549 cells cultured without serine, and with and without 2 μM FCCP treatment for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (E) Serine labeled (M+3) from U-13C-glucose over time in H1299 cells cultured without serine, and with and without 2 μM FCCP treatment for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (F) Oxygen consumption rate (OCR) of A549 cells cultured with and without serine with indicated FCCP treatment for 24 hours. Values are averages of three repeat measurements, n=5-9. (G) Relative NAD+/NADH ratio of A549 cells cultured with and without serine with indicated FCCP treatment for 24 hours. NAD+/NADH ratios are normalized to the NAD+/NADH ratio in A549 cells culture with serine, n=3. (H) Proliferation rate (doublings per day) of A549 cells cultured with and without serine, 2 μM FCCP, and indicated concentrations of rotenone for 72 hours, n=3. (I) Serine labeled (M+3) from U-13C-glucose over time in A549 cells cultured without serine, and with and without 2 μM FCCP and 20 nM rotenone as indicated, for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (J) Relative NAD+/NADH ratio of A549 cells cultured with and without serine, 2 μM FCCP, and 20 nM rotenone as indicated. NAD+/NADH ratios are normalized to the NAD+/NADH ratio in untreated A549 cells cultured with serine, n=3. Data shown are means ± SD. P-values were calculated by unpaired Student’s t-test, *p<0.05, **p<0.01, ***p<0.005, ****p<0.001, “ns” denotes not significant.

Lipid depletion leads to a cell-specific elevation in mitochondrial respiration and the NAD+/NADH ratio, influencing oxidative citrate and serine synthesis.

(A) Schematic depicting glucose and glutamine oxidative routes for synthesizing citrate, highlighting NAD+ requiring oxidation reactions. (B) Citrate labeled (M+2) from U-13C-glucose over time in H1299 and A549 cells cultured with and without serine for 24 hours prior to U-13C-glucose exposure. Citrate levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (C) Citrate labeled (M+2) from U-13C-glucose over time in A549 and H1299 cells cultured without serine, and with and without 2 μM FCCP for 24 hours prior to U-13C-glucose exposure. Citrate levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (D) Citrate labeled (M+2) from U-13C-glucose over time in A549 cells cultured with serine, and with indicated concentrations of AKB or FCCP for 24 hours prior to U-13C-glucose exposure. Citrate levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (E) Oxygen consumption rate (OCR) in A549 and H1299 cells cultured with and without lipids for 24 hours. Values are averages of three repeat measurements, n=10. (F) Relative NAD+/NADH ratios of A549 and H1299 cells cultured with and without lipids for 24 hours. NAD+/NADH ratios are normalized to the NAD+/NADH ratio of the corresponding cell type cultured with lipids, n=3. (G) Citrate labeled (M+2) from U-13C-glucose over time in A549 and H1299 cells cultured with and without lipids for 24 hours prior to U-13C-glucose exposure. Citrate levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (H) Serine labeled (M+3) from U-13C-glucose over time in A549 and H1299 cells cultured with and without lipids for 24 hours prior to U-13C-glucose exposure. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. Data shown are means ± SD. P values were calculated by unpaired Student’s t-test, *p<0.05.

Lipid depletion increases mitochondrial respiration and the NAD+/NADH ratio in a cell specific manner to influence serine synthesis and proliferation in serine-depleted conditions.

(A) Oxygen consumption rate (OCR) of A549 and H1299 cells cultured with and without serine or lipids for 24 hours. Values are the average of three repeat measurements, n=14-15. (B) Relative NAD+/NADH ratios of A549 and H1299 cells culture with and without serine or lipids for 24 hours. NAD+/NADH ratio is normalized to the NAD+/NADH ratio in corresponding cell type cultured with serine and lipids, n=3. (C, D) Left: Serine labeled (M+3) from U-13C-glucose over time in A549 cells (C) and H1299 cells (D) cultured without serine, and with or without lipids for 24 hours prior to U-13C-glucose exposure. Right: Serine synthesis rates of A549 cells (C) and H1299 cells (D) cultured in conditions shown on left. Serine synthesis rates are calculated from labeled serine (M+3) after 1 and 15 minutes of U-13C-glucose exposure and normalized to the serine synthesis rates of the corresponding cell type cultured without serine and with lipids. Serine levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. Single data point (H1299, -serine +lipid, t=15 min) removed due to error in sample processing. (E,F) Left: Citrate labeled (M+2) from U-13C-glucose over time in A549 cells (E) and H1299 cells (F) cultured without lipids, and with or without serine for 24 hours prior to U-13C-glucose exposure. Right: Citrate synthesis rates of A549 cells (E) and H1299 cells (F) cultured in conditions shown on left. Citrate synthesis rates are calculated from labeled citrate (M+2) after 1 and 15 minutes of U-13C-glucose exposure and normalized to the citrate synthesis rates of the corresponding cell type cultured without lipids and with serine. Citrate levels are normalized to internal norvaline standard and cell number measured in indicated conditions, n=3. (G) Proliferation rate (doublings per day) of A549 cells cultured with and without serine or lipids for 72 hours, n=3. (H) Proliferation rate (doublings per day) of H1299 cells cultured with and without serine or lipids for 72 hours, n=3. Data shown are means ± SD. P-values were calculated by unpaired Student’s t-test, *p<0.05 ***p<0.005. ****p<0.001.