Figure 3. dmaKG treatment depletes aspartate levels in respiration-deficient cells via GOT1.

(A) Mass spectrometry analysis of UOK262 cell pairs indicating the relative abundances of TCA cycle intermediates (citrate, succinate, fumarate, malate) and aspartate in control or with 0.5 or 5 hr of dmaKG (6.6 mM) treatment. α-KG, alpha-ketoglutarate; OAA, oxaloacetate.

(B) The log₂ fold change in amino acids in UOK262 cell pairs after 5 hr of dmaKG (6.6 mM) treatment as compared to untreated control.

(C) Mass spectrometry analysis of 143B cell pairs in control or with 3 or 6 hr of dmaKG (9.9 mM) treatment.

(D) The log₂ fold change in amino acids in 143B cell pairs after 6 hr of dmaKG (9.9 mM) treatment as compared to untreated control.

(E) Depiction of the enzymatic function of GOT1/2.

(F) Mass spectrometry analysis of aspartate levels in UOK262 cell pairs. Cells were treated with or without 6.6 mM dmaKG (left panel) or 2 mM aminooxyacetic acid (AOA) (right panel) for 6 hr. AOA potently decreases aspartate levels in both UOK262-ev and UOK262-FH, whereas dmaKG causes a stronger reduction in aspartate in UOK262-ev than in UOK262-FH.

(G) Targeted metabolic flux analysis using 13C4,15N-aspartate in 143B-wt cells expressing SLC1A3. The 15N-glutamate/13C4,15N-aspartate ratio increased upon dmaKG treatment. There is an approximately 8-fold increase in the labelled glutamate/aspartate ratio upon treatment with dmaKG but a 40-fold increase with co-treatment of dmaKG and phenformin as compared to their respective controls without dmaKG.

(H) Cytotoxicity of dmaKG in UOK262-ev and 143B-CytB cells. These cells additionally carried pMXs empty vector (ev) or pMXs-GOT1 (GOT1). Cells were treated with dmaKG (4.4 mM for UOK262-ev and 6.6 mM for 143B-CytB) for 24 hr.

(I)The effects of various compounds on cell proliferation as determined by crystal violet staining. UOK262 cells carried pMXs empty vector (ev) or pMXs-GOT1 (GOT1) were treated with the vehicle (Control), 3.3 mM dmaKG, or 2 mM AOA for 72 hr.

Data are shown as mean and individual data points or mean ± SD. Statistical analysis was performed in (A, C, H-I) (n=4, two-way ANOVA with Tukey’s multiple comparisons) and in (F-G) (n=4, unpaired two-tailed t test with Welch’s correction, ns p>0.5, *p≤0.05, **p≤ 0.01, ***p≤0.001, ****p≤0.0001).