Figure 5. LSD1 inhibition enhances mitochondrial metabolism in adipocytes.
Mitochondrial energy metabolism was assessed in differentiating 3T3-L1 cells under LSD1-KD (a) and TC treatment (b). siRNA-introduced or drug-treated cells were subjected to adipogenic induction for 24 h, and were stained with JC-1 followed by flow cytometric analyses. Red fluorescent JC-1 aggregates (FL2) and green fluorescent monomers (FL1) were measured. Hatched histograms indicate the unstained control samples. Each histogram shows the representative result of triplicate samples. (c) The effect of LSD1-KD on the OXPHOS capacity of differentiating 3T3-L1 cells. OXPHOS activity was determined by measuring the OCR using the XF24 Analyzer. During the real-time measurement, respiratory chain inhibitors were added to the culture at the indicated time points. Values are means±s.d. of five assay wells at each time point. (d) LSD1-KD reduces lipid accumulation in insulin-stimulated adipocytes. Differentiated 3T3-L1 cells were cultured in the absence or presence of insulin, followed by adenovirus-mediated introduction of LSD1 shRNA (Ad-shLSD1) or control (Ad-sh-ctrl). Cellular lipid was analysed at 4 days after infection. Values are means±s.d. of five mice. Scale bars in the whole-well and magnified images indicate 5 mm and 50 μm, respectively. (e,f) The effects of LSD1-KD and TC treatment on the OCR in mature 3T3-L1 adipocytes. Insulin-stimulated adipocytes were treated with shRNA-containing adenoviruses (control or LSD1, e) or 10−4 M TC (f), and cultured for 4 days. OCR was determined using the Oxygen Biosensor System, and was calculated as the fold increase during 30 min-measurement. (g,h) Enhanced lipolysis in LSD1-inhibited adipocytes. Insulin-stimulated mature 3T3-L1 adipocytes were infected with shRNA-containing adenovirus (g) or were treated with TC (h), and cultured for 4 days. All values (except for those in c) are means±s.d. of three independent samples. *P<0.05, **P<0.01 versus control siRNA, control shRNA or vehicle by Student's t-test