Fig. 4: NTS regulates OXPHOS through PGC1α inhibition.

A Effect of SR48692 (NTSR1 antagonist, 10 μM) on NTS-induced PGC1α expression in hepatocytes (C57/BL6); normalized to actin. N = 4 independent experiments. B Promoter activity of PGC1α (2 kb luciferase promoter) with NTS and PD98059 (MEK inhibitor, 1 µM) treatment in transfected HepG2 cells (Dual Glo Luciferase assay). N = 3. C Mito stress test in control (Hep-pcDNA) and PGC1α-overexpressing (Hep-PGC1α) clone 2 treated with NTS; N = 16 datapoints from two independent experiments. D, E Effect of NTS (10 nM) and PA (100 μM) stimulation (16 h) on protein expression (western blot) in hepatocytes (C57/BL6). PGC1α was normalized to actin and pAMPK was normalized to AMPK. N = 3 independent experiments. F Gene expression analyses in Nts^+/+ hepatocytes treated with NTS and PA treated as in (D). Dashed line represents expression levels in control group; N = 4–5 mice; ns = not significant. G Lipid utilization assay. Nts^+/+ hepatocytes were incubated with 100 µM PA for 24 h in presence/absence of NTS (10 nM), washed extensively and then incubated in lipid-free media for another 24 h. Confocal images show amount of unmetabolized lipids (BODIPY) at 48 h after lipid addition. Hoechst = nuclear stain. Scale bar = 50 μm. Quantified data from 30 cells/group are shown on the right. H Measurement of unmetabolized lipids by Ntsr1^+/+ and Ntsr1^−/− hepatocytes at 48 h in 96-well plates (BODIPY staining was normalized to Hoechst). Representative data from two independent experiment is shown. Data are expressed as mean ± SD, and p ≤ 0.05 is considered significant.