A gene is required for the high activity of glycolysis and OXPHOS in naïve hESCs
Description
A(gene) depletion led to a marked reduction in the abundance of key glycolytic intermediates, including fructose-6-phosphate (F6P), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), phosphoenolpyruvate (PEP), pyruvate, and lactate, as well as tricarboxylic acid (TCA) cycle metabolites such as succinate and citric acid. Interestingly, aspartate and glutamine levels were elevated upon A(gene) depletion. To further evaluate the impact of A(gene) on energy metabolism in naïve human embryonic stem cells (hESCs), we measured the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), reflecting mitochondrial respiration and glycolytic activity, respectively. A(gene) depletion significantly impaired basal respiration, maximal respiration, and spare respiratory capacity, and also disrupted glycolytic function. Consistent with these metabolic alterations, cellular NADH levels were significantly decreased in A(gene) -depleted cells. Together, these results uncover a critical role of A(gene) as an essential regulator of the bivalent metabolic state in naïve hESCs, balancing both glycolysis and oxidative phosphorylation.
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Steps to reproduce
Naïve hESCs were seeded in 10 cm plates, and each group contained 4 plates. Three days later, cells were treated without or with DOX for additional 2 or 3 days. All samples were mechanically collected with 1 mL of dry ice-cold solution of 80% methanol: 20% water, sonicated to break up the cells to release metabolites, and then centrifuged at 13,500 g for 10 minutes, keeping the whole process at a low temperature. Supernatant was collected and further evaporated. Samples were diluted with 100 μL of mobile phase A (25% ACN 5 mM ammonium acetate) and then centrifuged at 20,000 g for 10 minutes to remove insoluble particles for LC-MS detection of metabolites. Metabolites were analyzed by LC-MS on an Ultimate 3000 UHPLC liquid chromatography system (Thermo Scientific) equipped with a C18 column (Phenomenex ,00B-4252-B0) and a TSQ Quantiva mass spectrometer (Thermo Scientific).
Institutions
- Shanghai Jiao Tong University School of Medicine