Profiling the Metabolism of Human Cells by Deep 13C Labeling

Abstract

Summary Studying metabolic activities in living cells is crucial for understanding human metabolism, but facile methods for profiling metabolic activities in an unbiased, hypothesis-free manner are still lacking. To address this need, we here introduce the deep labeling method, which combines a custom 13C medium with high-resolution mass spectrometry. A proof-of-principle study on human cancer cells demonstrates that deep labeling can identify hundreds of endogenous metabolites as well as active and inactive pathways. For example, protein and nucleic acids were almost exclusively de novo synthesized, while lipids were partly derived from serum; synthesis of cysteine, carnitine and creatine was absent, suggesting metabolic dependencies; and branched-chain keto acids (BCKA) were formed and metabolized to short-chain acylcarnitines, but did not enter the TCA cycle. Remarkably, BCKA could substitute for essential amino acids to support growth. The deep labeling method may prove useful to map metabolic phenotypes across a range of cell types and conditions.

Gabapentin Can Suppress Cell Proliferation Independent of the Cytosolic Branched-Chain Amino Acid Transferase 1 (BCAT1)

“…In untreated cells, the 13 C 6 leucine, 13 C 6 isoleucine and 13 C 5 valine fractions were 90–95% (Figure 2A), showing that virtually all of these BCAA were obtained from the medium, while other sources (endogenous or serum proteins) were negligible. The corresponding BCKAs 4-methyloxopentanoate (4mop)/3-methyloxopentanoate (3mop) and 3-methyloxobutanoate (3mob) were similarly highly labeled (Figure 2B), indicating that the BCKAs are synthesized almost exclusively from BCAAs via the branched-chain aminotransferase, consistent with our previous study (15). Gabapentin treatment did not reduce BCKA 13 C fractions (Figure 2B), nor abundance of 13 C BCKA in cells (Figure 2C), indicating that synthesis of BCKAs from BCAA is not affected.…”

Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability

“…For example, PDAC cells consume large amounts of glucose and glutamine in culture (Son et al, 2013; Ying et al, 2012), and it has been assumed that these nutrients are depleted in the microenvironment (Commisso et al, 2013; Kamphorst et al, 2015; Lyssiotis and Kimmelman, 2017; Sherman et al, 2017; Sousa et al, 2016). Branched chain amino acids are also proposed to be limiting for proliferation of PDAC cells in tumors, requiring cancer cells to acquire those nutrients from alternative sources such as stromal cells or extracellular protein (Grankvist et al, 2018; Palm et al, 2015). We find that neither glutamine nor branched-chain amino acids are substantially depleted in PDAC tumors regardless of anatomical site.…”

Mapping metabolic oscillations during cell cycle progression

“…In addition, based on matching MS/MS fragments with Metlin database [35], some of the fragments (m/z 76.0223, 84.0451, 233.059) matched glutathione fragments. To further investigate methylglyoxal metabolism in cycling cells, we analyzed isotope tracing experiments in whole cell culture for 48 hours using 13 C 5glutamine [13], which should yield a 13 C 5 glutathione moiety, and with 13 C 1 -glucose [36], which is expected to label the lactoyl moiety as 50% 13 C 1 . Indeed, we observed these mass isotopomers in the S-Lactoylglutathione peak (Figure 3c), demonstrating that the compound was endogenously synthesized.…”