## 3 Results
### Nicotinamide Riboside Treatment Reduces Terminal Adipocyte Differentiation of hASCs  
Previous studies have presented conflicting findings regarding the role of nicotinamide mononucleotide in regulating adipogenesis in mesenchymal stem cells derived from various tissue sources [31; 32]. In this study, we focused on human adipose-derived stem cells (hASCs) and investigated the effects of nicotinamide riboside (NR), a readily cell permeable NAD\({}^{+}\) precursor on adipogenesis. To assess the impact of NR on adipogenesis, we cultured hASCs for 7 days with NR and examined the expression of key transcriptional factors involved in adipocyte differentiation, namely CEBP\(\alpha\), CEBP\(\beta\), and PPAR\(\gamma\). These factors play a crucial role in regulating the process of adipogenesis. Remarkably, our analysis revealed a significant upregulation of all three transcriptional factors following NR treatment (Figure 2A-C). These findings suggest a potential positive role of NR in supporting adipogenesis. To further evaluate the effect of NR on terminal adipogenesis and lipid accumulation, we treated hASCs with varying concentrations (0.5, 1, and 2 mM) of NR for 7 days before initiating adipogenesis. Subsequently, we continued NR treatment during the 14-day differentiation period. Surprisingly, when we stained the differentiated cells with Oil-Red-O, a marker for lipid accumulation, we observed a dose-dependent decrease in the number of Oil-Red-O positive cells (Figure 2D,E). Figure 1: (**A**) Mean fluorescence intensity (MFI) of differentiated and undifferentiated hASCs stained with Bodipy stain was measured by flow cytometry at day 14 post-adipogenesis (n = 3). (**B**) Mean fluorescence intensity of differentiated and undifferentiated hASCs stained with TMRM stain was measured by flow cytometry at day 14 post-adipogenesis (n = 3). (**C**) Differentiated and undifferentiated ASCs were stained with DCFDA dye and mean fluorescence intensity (MFI) was measured by employing flow cytometry (n = 3). * \(p\) < 0.05, *** \(p\) < 0.00005. To validate this observation, we performed Bodipy staining, another lipid marker, on differentiated ASCs, which consistently demonstrated a similar trend (Figure 3A). Figure 2: (**A–C**) hASCs were treated with control media or 2 mM NR containing media for 7 days. Cells were lysed for RNA extraction and reverse-transcribed for real-time PCR quantification of CEBP\(\alpha\) (**A**), CEBP\(\beta\) (**B**), and PPAR\(\gamma\) (**C**) genes (n = 3). (**D**) hASCs were grown to confluence in 6-well plates and differentiated in the absence or presence of different concentrations of NR. On day 14 post-differentiation, cells were fixed and stained with Oil-Red-O stain and imaged at different magnifications, as indicated. (**E**) Percentage Oil-Red-O positive cells were counted and plotted. *_p_ value < 0.05, **_p_ < 0.005, ***_p_ < 0.0005, ***_p_ < 0.00005. Figure 3: **(A)** hASCs were grown to confluence in 6-well plates and were differentiated in the absence or presence of different concentrations of NR. On day 14 post-differentiation induced cells were fixed and stained with Bodipy (Green) and Hoechst (Blue) and imaged using fluorescence microscope. **(B,C)** hASCs were differentiated in the presence or absence of different concentrations of NR. On day 14 post-differentiation induction cells were lysed for RNA extraction and reverse-transcribed for real-time PCR quantification of Perilipin (**B**), and AdipQ**(**C**) genes. **(D)** hASCs were differentiated in the presence or absence of different concentrations of NR. On day 14 post-differentiation induction cells were lysed for protein extraction and analyzed for Perilipin protein by western blotting. Perilipin protein western blot band intensity was analyzed by Image J and normalized to \(\beta\) actin housekeeping control. **(E)** hASCs were treated with 2 mM NR. NR was washed from the pre-treatment group and differentiation was induced in the absence of NR.