Figure 7
From: Regulation of glycolysis in brown adipocytes by HIF-1α
The importance of HIF-1α for β-adrenergically stimulated glycolysis and thermogenesis. Mature brown adipocytes (WT-1) were reverse transfected with scramble siRNA or two different siRNAs against Hif-1α. Four days later, the adipocytes were stimulated with vehicle or 1 μM ISO for 6 h, after which glucose (A) and lactate (B) measurements were performed on the medium. (C) Representative results from Seahorse XF Glycolysis Stress Tests performed on mature adipocytes (WT-1), which were reverse transfected with scramble siRNA or an siRNA against Hif-1α (siRNA 1) (n = 3). (D–G) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured four days after transfection (n = 5). (D) Representative time-course of ECAR during basal conditions and after injection of 1 μM ISO. (E) Fold change in ECAR between the basal level and the measurement 30 min after ISO stimulation. (F) Representative time-course of OCR during basal conditions and after injection of 1 μM ISO. (G) Fold change in OCR between the basal level and the measurement 30 min after ISO stimulation. (H–K) ECAR and OCR were measured at day 8 on a Seahorse XF96 Flux Analyzer after treatment with 5 μM MitoQ or vehicle (n = 4). (H) Representative time-course of ECAR during basal conditions and after injection of 1 μM ISO. (I) Fold change in ECAR between the basal level and the measurement 30 min after ISO stimulation. (J) Representative time-course of OCR during basal conditions and after injection of 1 μM ISO. (K) Fold change in OCR between the basal level and the measurement 30 min after ISO stimulation. (L) Total RNA was isolated from mature adipocytes (WT-1) transfected with scramble siRNA or two different siRNAs against Hif-1α. Relative gene expression was analyzed by RT-qPCR and presented as fold change relative to the scramble siRNA-transfected cells. The genes measured were Cyc1, CoxII, Cs, Pgc-1α and Tfam (n = 3). mRNA expression levels were normalized to Tbp. Data represent mean + SEM (panel C-K) or mean of means + SEM (panel A, B and L). *p < 0.05 versus vehicle-treated cells. #p < 0.05 versus scramble siRNA transfected cells.
