Alanyl-glutamine-free William's medium E (PAN Biotech GmbH) that was supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), and Gibco Insulin-Transferrin-Selenium (100X) supplement (Invitrogen, Karlsruhe, Germany).

Extra- and intracellular samples were collected in triplicate directly before and after the stimulus, as well as 1, 2, 5, 10, 30, 60, 120, and 180 min after glucose deprivation. The sampling approach and the processing of the samples were done as previously described [1]. ... r />The concentrations of Lanine, serine, glucose, lactate, pyruvate, fumarate, malate, cis-aconitate, isocitrate, and citrate were determined by GC-MS as described before [1,2]. After glucose deprivation, extracellular glucose concentrations were determined in 10 μl of diluted (1:9 v/v) medium samples, the intracellular glucose concentrations before and after perturbation were determined in 5 and 50 μl of cell extract, respectively. Phosphoenolpyruvate, 3-phosphoglycerate, dihydroxyacetonphosphate, fructose-1,6-bisphosphate, glucose-6-phosphate, 6-phosphogluconate, sedoheptulose-7-phosphate, ribose-5-phosphate, and ribulose-5-phosphate were determined by LC-MS-MS as described by Hofmann [1] with the following modifications: HPLC separation was performed at 20 °C on a Synergi Hydro-RP column (150 × 2 mm, 4 μm; Phenomenex, Aschaffenburg, Germany) at a flow rate of 0.2 ml/min. Nucleotide analysis was performed by reversed phase ion pair high performance liquid chromatography. The HPLC system (Agilent Technologies, Waldbronn, Germany) consisted of an Agilent 1200 series autosampler, an Agilent 1200 series Binary Pump SL, an Agilent 1200 series thermostatted column compartment, and an Agilent 1200 series diode array detector set at 260 and 340 nm. The nucleotides were separated and quantified on an RP-C-18 column that was combined with a guard column (Supelcosil LC-18-T; 15 cm × 4.6 mm, 3 μm packing and Supelguard LC-18-T replacement cartridges, 2 cm; Supelco, Bellefonte, USA) at a flow rate of 1 ml/min.