Figure 3: Glucose signals via mTORC1 and HIF1α to promote metabolic reprogramming of LPS-activated GM-DCs.

(a) GM-DCs were left unstimulated (Unstim) or stimulated with LPS (100 ng ml⁻¹) for 8 h or 24 h and analysed by qPCR for the expression of the glucose transporter Slc2a1 and key glycolytic enzymes: phosphofructose kinase (Pkf), pyruvate kinase 2 (Pkm2), and lactate dehydrogenase a (Ldha). (b) GM-DCs were treated+/−LPS (100 ng ml⁻¹) for 8 h, washed and then cultured in media containing 10 mM glucose (Glc) or 10 mM galactose (Gal) for 20 h and Slc2a1, Pkf and Pkm2 mRNA expression was measured. (c) GM-DCs generated from Hif1a^flox/flox (WT) or Hif1a^flox/flox VavCre (Hif1a^−/−) mice were treated+/−LPS (100 ng ml⁻¹) for 24 h, and then HIF1α protein and Phd3 mRNA levels were measured. (d) qPCR analysis of Phd3 mRNA levels in GM-DCs treated+/−LPS (100 ng ml⁻¹) for 8 h in normal media and then for 20 h in media with different glucose concentrations. (e) GM-DCs were treated+/−LPS (100 ng ml⁻¹) for 8 h, washed and then cultured in media containing different concentrations of glucose or galactose for 20 h before immunoblot analysis for HIF1α, phosphorylated and total acetyl-CoA carboxylase (pACC and ACC), phosphorylated p70 S6-kinase (p-S6K), phosphorylated S6 ribosomal protein (p-S6) and total protein kinase B (PKB, loading control). (f) GM-DCs were treated+/−LPS (100 ng ml⁻¹) for 8 h, washed and then cultured in media containing either 10 mM glucose (Glc) or 10 mM galactose (Gal) for 20 h and analysed by qPCR for the expression of Phd3 mRNA. (g) GM-DCs were treated+/−LPS (100 ng ml⁻¹)+/−rapamycin (20 nM) for 20 h and analysed by immunoblot analysis for HIF1α, p-S6K and total S6 and by qPCR for Phd3 mRNA. (h) GM-DCs were treated+/−LPS (100 ng ml⁻¹) for 8 h, washed and then cultured in media containing either 10 mM glucose or 10 mM galactose for 20 h and analysed for rates of OXPHOS coupled to ATP synthesis. Data are mean±s.e.m. of at least three separate experiments. Representative immunoblot of at least three separate experiments are shown. Data were analysed using a one-way analysis of variance with Tukey’s post test (**P<0.01, ***P<0.001). OCR, oxygen consumption rate.