Quantitative analysis of acetyl-CoA production in hypoxic cancer cells reveals substantial contribution from acetate

Abstract

Background: Cell growth requires fatty acids for membrane synthesis. Fatty acids are assembled from 2-carbon units in the form of acetyl-CoA (AcCoA). In nutrient and oxygen replete conditions, acetyl-CoA is predominantly derived from glucose. In hypoxia, however, flux from glucose to acetyl-CoA decreases, and the fractional contribution of glutamine to acetyl-CoA increases. The significance of other acetyl-CoA sources, however, has not been rigorously evaluated. Here we investigate quantitatively, using (13)C-tracers and mass spectrometry, the sources of acetyl-CoA in hypoxia.

Results: In normoxic conditions, cultured cells produced more than 90% of acetyl-CoA from glucose and glutamine-derived carbon. In hypoxic cells, this contribution dropped, ranging across cell lines from 50% to 80%. Thus, under hypoxia, one or more additional substrates significantly contribute to acetyl-CoA production. (13)C-tracer experiments revealed that neither amino acids nor fatty acids are the primary source of this acetyl-CoA. Instead, the main additional source is acetate. A large contribution from acetate occurs despite it being present in the medium at a low concentration (50-500 μM).

Conclusions: Acetate is an important source of acetyl-CoA in hypoxia. Inhibition of acetate metabolism may impair tumor growth.

Keywords: 13C-tracing; Acetate; Acetyl-CoA; Cancer metabolism; Fatty acids; Hypoxia; Lipogenesis; Mass spectrometry; Palmitate.

Figure 1

Percentage ¹³ C-labeling of cytosolic acetyl-CoA can be quantified from palmitate labeling. (A) Increasing ¹³C₂-acetyl-CoA labeling shifts palmitate labeling pattern to the right. ¹³C₂-acetyl-CoA labeling can be quantified by determining a best fit between observed palmitate labeling and computed binomial distributions (shown on right-hand side) from varying fractions of acetyl-CoA (AcCoA) labeling. (B) Steady-state palmitate labeling from U-¹³C-glucose and U-¹³C-glutamine in MDA-MB-468 cells. (C) Percentage acetyl-CoA production from glucose and glutamine. For (B) and (C), data are means ± SD of n = 3.

Figure 2

Acetyl-CoA labeling from ¹³ C-glucose and ¹³ C-glutamine decreases in hypoxia. (A) Steady-state palmitate labeling from U-¹³C-glucose and U-¹³C-glutamine in normoxic and hypoxic (1% O₂) conditions. (B) Percentage acetyl-CoA production from glucose and glutamine in hypoxia. (C) One or more additional carbon donors contribute substantially to acetyl-CoA production in hypoxia. Abbreviations: Gluc, glucose; Gln, glutamine. Data are means ± SD of n = 3.

Figure 3

Amino acids (other than glutamine) and fatty acids are not major sources of cytosolic acetyl-CoA in hypoxia. (A) Palmitate labeling in hypoxic (1% O₂) MDA-MB-468 cells, grown for 48 h in medium where branched chain amino acids plus lysine and threonine were substituted with their respective U-¹³C-labeled forms. (B) Same conditions, except that glucose and glutamine only or glucose and all amino acids, were substituted with the U-¹³C-labeled forms. (C) Palmitate labeling in hypoxic (1% O₂) MDA-MB-468 cells, grown in medium supplemented with 20 μM U-¹³C-palmitate for 48 h. Data are means ± SD of n = 3.

Figure 4

The main additional AcCoA source in hypoxia is acetate. (A) Percentage ¹³C₂-acetyl-CoA labeling quantified from palmitate labeling in hypoxic (1% O₂) and normoxic MDA-MB-468 cells grown in medium with U-¹³C-glucose and U-¹³C-glutamine and additionally supplemented with indicated concentrations of U-¹³C-acetate. (B) Acetate concentrations in fresh 10% DFBS, DMEM, and DMEM with 10% DFBS. (C) Percentage ¹³C₂-acetyl-CoA labeling for hypoxic (1% O₂) HeLa and A549 cells. For (A) and (C), data are means ± SD of n ≥ 2. For (B), data are means ± SEM of n = 3.