Influence of hypoxia and 3-hydroxybutyrate on the expression of ketone body metabolizing enzymes and the activity of HIF-1α signaling. (A) LNT-229 glioma cells were cultured in the absence or presence of 3-hydroxybutyrate (3OHB, 5 mM) at 21%, 1% or 0.1% oxygen for 24 h. mRNA levels of OXCT1, ACAT1, BDH1 and BDH2 were determined by real-time quantitative PCR. Data are presented as the fold change in gene expression normalized to the internal control 18S rRNA (mean and standard deviation, p < 0.05 compared with normoxic conditions, asterisks omitted for clarity). (B) Glioma cell lines were treated as in (A), and the expression of HIF-1α, BDH1, BDH2, OXCT1 and ACAT1 was analyzed by immunoblot. (C) HIF-specific transcriptional activity was examined by luciferase reporter assay (3HRE-pTK-luc construct) in the absence or presence of 3-hydroxybutyrate (24 h treatment, mean and standard deviation). (D) Glioma cells were incubated for 24 h at the indicated oxygen conditions in the absence or presence of 3-nitropropionic acid (3NPA, 10 mM), and the expression of HIF-1α was analyzed by immunoblot.