Metabolic control of embryonic stem cell function by AMDHD2 in the hexosamine pathway

• Applicant: Dr. Ina Huppertz, since 12/2022
• Subject Area: Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 458980033

Energy metabolism of embryonic stem cells is characterized by a reliance on aerobic glycolysis, while the tricarboxylic acid (TCA) cycle provides substrates for biosynthesis and growth. Oxidative phosphorylation increases during differentiation and a shift to glycolysis, in turn, occurs during reprogramming to pluripotency. The causal links between metabolic state and embryonic stem cell fate decisions and function, however, are only partly understood.The metabolic hexosamine biosynthetic pathway (HBP) utilizes fructose-6-phosphate, glutamine, acetyl-CoA, and UTP to build up 5’-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a key precursor of various cellular glycosylation reactions. Through yet unpublished genetic screens we have uncovered a unique configuration of the HBP in mouse embryonic stem cells (mESCs). First, while the HBP is controlled by the rate-limiting glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) in most tissues, GFAT-2 controls HBP flux in mESCs. Second, through in vitro assays we found that GFAT-1 is under strong negative feedback regulation by UDP-GlcNAc while GFAT-2 shows much weaker inhibition. Third, the decarboxylase AMDHD2 is an essential component of the HBP, critically controlling the UDP-GlcNAc pool in mESCs. AMDH2 knockout leads to early embryonic lethality, suggesting a key role in differentiation decisions and development. Together, the HBP configuration in mESCs contains a previously unknown and largely unregulated futile metabolic cycle, likely consuming significant amounts of fructose-6-phosphate, glutamine, and acetyl-CoA. This observation plausibly positions the HBP at the cross roads of glycolysis and the TCA cycle. Further, mESCs show elevated cellular UDP-GlcNAc concentrations compared with more differentiated cells, likely affecting downstream glycosylation events. Here I propose to investigate the role of the HBP in mESC function. First, we will analyze the HBP in mESC maintenance and differentiation, as well as in pluripotency reprogramming. We will ask if this is dependent on specific downstream glycosylation events. Second, we will perform metabolic flux analyses to characterize the HBP futile cycle and its potential cross talk with the TCA cycle. We will functionally test a potential role of the HBP futile cycle in establishing and maintaining the metabolic state of mESCs. Finally, we will find out whether the switch in HBP configuration from GFAT-2 to GFAT-1 control is required for mESC differentiation and, vice versa, for pluripotency reprogramming. This hypothesis-driven research proposal aims to understand how elevated HBP flux affects mESC state. Further, it will delineate the role of the newly discovered HBP futile cycle. This cycle is a feature of mESCs, likely to enable sensitive and rapid regulation through the interactions with other key metabolic pathways. Together, this work will provide a deep understanding of the HBP in embryonic stem cell metabolism and function.

• DFG Programme: Research Grants
• Ehemalige Antragsteller: Dr. Martin Denzel, until 12/2021; Dr. Patrick Giavalisco, from 1/2022 until 11/2022