Fig. 2

Regulation of NAD salvage pathway by palmitate and oleate. HepG2 cells and primary human hepatocytes were stimulated with palmitate (0.5 mM), oleate (1 mM) and a combination of both (0.5 mM/1 mM) for the indicated time points. a Intracellular NAD concentration was measured with EnzyChrom™ NAD⁺/NADH Assay Kit (E2ND-100) or by HPLC (48 h). b NAMPT mRNA expression was quantified by qPCR and normalised to β-actin in HepG2 cells (4 h, 24 h, 48 h) and primary human hepatocytes (48 h). c NAMPT protein abundance was measured by Western blot analysis (48 h). GAPDH was used as loading control. One representative Western blot is shown. d After 48 h exposure to the indicated free fatty acids NAMPT enzyme activity assay was performed. e NAMPT protein in the supernatant of HepG2 cells (4 h, 24 h, 48 h) and primary human hepatocytes (48 h) was determined by Western blot analysis. One representative Western blot is shown. Data were normalised to control (serum free medium) which was set 1. Data represent three independent experiments performed in triplicates shown as means ± SEM. *p < 0.05, **p < 0.01 compared to control. ^# p < 0.05, ^## p < 0.01 compared to palmitate. ^§§§ p < 0.001 palmitate in a time dependent manner