Figure 3. Downregulation of visfatin by TNFα leads to decreases in NAD⁺ concentrations and Sirt1 deacetylating activity in 3T3-L1 adipocytes. Cells were incubated with or without TNFα (15 ng/mL) for 24 h. (A and B) Intracellular concentrations of visfatin and NAD⁺. After incubation, cells were collected and processed for visfatin and NAD⁺ quantification as described in Materials and Methods. Values were determined in ng visfatin/mg of cellular protein and in ng NAD⁺/mg of cellular protein, respectively. Values are presented as means ± SEM. *P < 0.05 (t test). (C) Sirt1 activity in 3T3-L1 cells. Total cell lysates (20 μg) were submitted to a Sirt1 activity assay as described in Materials and Methods. Values are presented as means ± SEM. *P < 0.05 (t test). (D) Quantification of Sirt1 mRNA levels by quantitative RT-PCR. Sirt1 data were normalized to 18S rRNA. Data are presented as means ± SEM. *P < 0.05 (t test).