Figure 2.

NRH utilization in mammalian cells. (A) Scheme of the proposed hypothesis for NHR cellular utilization as a NAD⁺ precursor. (B) AML12 cells were treated for 5 min with either PBS (as vehicle), NR (0.5 mM), or NRH (0.5 mM). The cells were immediately frozen and used for NAD⁺ metabolomic analyses by mass spectrometry. All the results are expressed as intensity of the signal corrected by internal standard (IS) and protein (C) AML12 cells were treated with nitrobenzylthioinosine (NBTI) (10 μM) for 1 h prior to NRH treatment at the doses indicated. Then, 1 h later, acidic extracts were obtained to measure NAD⁺ levels. (D) AML12 cells were treated with FK866 (2 μM) for 1 h prior to NRH treatment at the doses indicated. Then, 1 h later, acidic extracts were obtained to measure NAD⁺ levels. (E) Primary hepatocytes from either wild-type or NRK1 KO mice were obtained and then treated for 1 h with NRH at the doses indicated. Then, 1 h later, acidic extracts were obtained to measure NAD⁺ levels. (F) AML12 cells were treated with an adenosine kinase inhibitor (5-IT; 1 μM) for 1 h prior to NRH treatment at the doses indicated. Then, 1 h later, acidic extracts were obtained to measure NAD⁺ levels. (G) AML12 cells were treated with 5-IT for 1 h prior to NRH treatment (0.05 mM). Then, 5 min later, the cells were snap frozen and processed for NAD⁺ metabolomics analyses. All the results are expressed as intensity of the signal, corrected by internal standard (IS) and protein. (H) AML12 cells were treated with gallotannin (100 μM) for 1 h prior to NRH treatment at the doses indicated. Then, 1 h later, acidic extracts were obtained to measure NAD⁺ levels. All the values in the figure are expressed as mean+/-SEM of 3 independent experiments. * indicates statistical difference at p < 0.05 vs the respective vehicle-treated group.