D 1 14c glucose

Carbon flux from glucose through the PPP was measured in cells treated for 120 min, as previously described [8 (link)], with minor modifications. Briefly, suspensions of known amounts of cells (3–5 × 10⁵ cells) obtained by smooth detaching from the cultures were incubated in sealed vials containing a central well with KOH (4 mol), which was used for ¹⁴CO₂ trapping, in the presence of 0.5 μCi of D-[1-¹⁴C]glucose (activity 56 mCi/mmol/L; Perkin Elmer) or D-[6-¹⁴C]glucose (activity 60 mCI/mmol/L; Perkin Elmer) in a Krebs–Ringer phosphate buffer (5.7 mmol/L Na₂HPO₄, 145 mmol/L NaCl, 4.86 mmol/L KCl, 1.22 mmol/L MgSO₄, 0.54 mmol/L CaCl₂; pH 7.4) containing 5.5 or 22 mmol/L glucose at 37 °C. In order to ensure an adequate O₂ supply for oxidative metabolism by the cells throughout the 120 min incubation period, the gas phase in the vials containing the cells was supplied with extra O₂ before the vials were sealed. The PPP flux was measured by assessing the difference between ¹⁴CO₂ production from [1-¹⁴C]glucose—which decarboxylates via the 6-phosphogluconate dehydrogenase-catalyzed reaction and the tricarboxylic acid cycle—and that of [6-¹⁴C]glucose—which decarboxylates only via the tricarboxylic acid cycle [8 (link), 9 (link)].

Glucose oxidation via the PPP was measured as previously described based on the difference in ¹⁴CO₂ production from [1-¹⁴C] glucose (decarboxylated in the 6-phosphogluconate dehydrogenase-catalyzed reaction and in the Krebs cycle) and [6-¹⁴C] glucose (only decarboxylated in the Krebs cycle) (48 (link)). Hippocampal slices were washed with ice-cold PBS and collected, then slices were kept in O₂-saturated Krebs Henseleit buffer (11 mM Na₂HPO₄, 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH₂PO₄, 1.2 mM MgSO₄, and 1.3 mM CaCl₂, pH 7.4), and this suspension was placed in Erlenmeyer flasks with another 0.5 mL of the Krebs Henseleit solution containing 0.5 μCi D-[1-¹⁴C] glucose or 2 μCi D-[6-¹⁴C] glucose and 5.5 mM D-glucose (final concentration). The Erlenmeyer flasks were equipped with a central well containing an Eppendorf tube with 500 μL of benzethonium hydroxide. The flasks were flushed with O₂ for 20 s, sealed with rubber caps, and incubated for 60 min in a 37°C water bath with shaking. The incubations were stopped by the injection of 0.2 mL of 1.75 M HClO₄ into the main well, although shaking was continued for another 20 min to facilitate the trapping of ¹⁴CO₂ by benzethonium hydroxide. Radioactivity was assayed by liquid scintillation spectrometry. Both D-[1-¹⁴C]glucose (#NEC043) and D-[1-¹⁶C]glucose (#NEC045) were purchased from Perkin-Elmer.

Glucose oxidation via the PPP was measured as previously described based on the difference in ¹⁴CO₂ production from [1-¹⁴C] glucose (decarboxylated in the 6-phosphogluconate dehydrogenase-catalyzed reaction and in the Krebs cycle) and [6-¹⁴C] glucose (decarboxylated only in the Krebs cycle) [92 (link)]. Hippocampal neurons were treated with lithium (10 mM) for 30 min, washed with ice-cold PBS, and collected by trypsinization. The lysates were then resuspended in O₂-saturated Krebs–Henseleit buffer and 500 μL of this suspension (~10⁶ cells) were placed in Erlenmeyer flasks with another 0.5 mL of Krebs–Henseleit solution containing 0.5 μCi D-[1-¹⁴C] glucose or 2 μCi D-[6-¹⁴C] glucose and 5.5 mM D-glucose (final concentration). The Erlenmeyer flasks were equipped with a central well containing an Eppendorf tube with 500 μL of benzethonium hydroxide. The flasks were flushed with O₂ for 20 s, sealed with rubber caps, and incubated for 60 min in a 37 °C water bath with shaking. The incubations were stopped by the addition of 0.2 mL of 1.75 M HClO₄ into the main well, and shaking was continued for another 20 min to facilitate the trapping of ¹⁴CO₂ by benzethonium hydroxide. Radioactivity was quantified as previously described by liquid scintillation spectrometry [93 (link),101 (link)]. Both D-[1-¹⁴C] glucose (#NEC043) and D-[1-¹⁶C] glucose (#NEC045) were purchased from Perkin-Elmer.