Fig. 6.

RetSat depletion prevents the glucose-induced nuclear accumulation of ChREBP independent of 13,14-dihydroretinol generation. a Primary hepatocytes were seeded on cover slips in 2.5 mM glucose. 24 h later, hepatocytes were treated with Control or RetSat siRNA overnight. The next day hepatocytes were exposed to 2.5 or 25 mM glucose and insulin as indicated for 24 h. After fixation, endogenous ChREBP was stained by immunocytochemistry and its localization determined by confocal microscopy. Nuclei were stained using DAPI, scale bars = 20 µm. Representative images of three independent experiments are shown. b Quantification of nuclear staining. Data are shown as mean ± s.d., n = 6 random optical fields of averaged nuclei intensities (total of >15 nuclei for each condition) from hepatocyte cultures from the same mouse; two-way ANOVA with Bonferroni post test revealed significances between low and high glucose (^# P < 0.05) and between siControl and siRetSat (*P < 0.05). c Hepatocytes were treated with Control or RetSat siRNA overnight. The next morning, cells were incubated with vehicle (DMSO) or 1 µM 13,14-dhretinol for 24 h at the indicated glucose concentrations and mRNA expression of Txnip determined by qPCR. Data are shown as mean ± s.d., n = 4. Two-way ANOVA with Bonferroni post test revealed significances between low and high glucose (^# P < 0.05) and between siControl and siRetSat (*P < 0.05), treatment with 13,14-dhretinol had no effect. d Incorporation of ¹⁴C-acetate into extractable lipids was assessed in hepatocytes depleted of RetSat for 48 h and supplemented with 13,14-dhretinol for the final 24 h. Data are shown as mean ± s.d., n = 4 independent transfections of hepatocyte cultures from the same mouse; *P < 0.05 between siControl und siRetSat by one-way ANOVA with Bonferroni post test. e, f Primary hepatocytes were treated with Control or RetSat siRNA and adenoviruses expressing GFP or a GFP-ChREBP fusion protein. Forty-eight hours after transfection/infection, e mRNA expression of RetSat and ChREBP and f ChREBP target genes were analyzed by qPCR. Data are shown as mean ± s.d., n = 6 independent transfections/infections of hepatocyte cultures from two mice; two-way ANOVA with Bonferroni post test showed significances between GFP and ChREBP (^# P < 0.05) and between siControl and siRetSat (*P < 0.05). n.s., not significant