Requirement of a High-Flux Metabolic State for Mouse Embryonic Stem Cell Self-Renewal

Unbiased profiling of global metabolite levels has revealed that cultured mouse embryonic stem (ES) cells exist in a unique metabolic state. Metabolites fluctuating dramatically in response to ES cell differentiation include purine nucleotides, acetyl-CoA, the amino acid threonine, and folic acid derivatives. These altered metabolic pathways, collectively known as the high-flux backbone (HFB) of metabolism, are surmised to be responsible for the rapid proliferation of this cell type. In particular, the amino acid threonine is shown here to be critical for mouse ES cell self-renewal. Gene and protein expression analysis has revealed that the enzyme threonine dehydrogenase (TDH) has the potential to play a major role in the establishment of HFB metabolism. TDH breaks down threonine into glycine and acetyl-CoA, molecules which are used to drive purine biosynthesis and ATP production, respectively. Using multiple approaches, we show here that TDH is strongly expressed both in ES cells and in the inner cell mass of the mouse blastocyst. Identification of potent and specific small molecule inhibitors has made possible the targeted elimination of the TDH enzyme in mouse ES cells. Using these compounds, we have determined that metabolic flux through this pathway is essential for ES cell selfrenewal. TDH inhibition is shown to cause an alteration in the cell’s metabolic state that results in increased autophagic activity and cell death. This study also reports on the generation of TDH conditional knockout mice, which will enable further elucidation of the role of HFB metabolism in adult and developing animals.