Fig. 4: mTOR inhibition restricts NAD⁺ synthesis in LPS-stimulated primary human monocytes.

a–c Donor monocytes (n = 7) were pretreated with an mTORi (AZD2014 at 5 μM, 6 h) or vehicle (DMSO) and stimulated with LPS (1 ng, 18 h) prior to metabolome extraction and analysis via LC-MS. a Partial least squares discriminant analysis (PLS-DA) and b hierarchical clustering analysis of “top 25” metabolites identified via one-way ANOVA performed using MetaboAnalyst⁸³. c Shows relative peak area of the individual metabolite NAD⁺. Significance was assessed with a Friedman test and Dunn’s multiple comparisons. d Monocytes from four independent donors were pretreated and stimulated ex vivo as in a and intracellular NAD⁺ content was quantified differently using an enzymatic assay. Significance was evaluated by one-way ANOVA and Tukey’s multiple comparisons. e Schematic outlining pathways supporting NAD⁺ biosynthesis. f Donor monocytes (n = 3 unique donors) were pretreated as in a and stimulated with LPS (1 ng) for the specified duration (3 or 6 h) prior to lysis and western analysis. Representative blot series from one donor of three shown. G-H Donor monocytes (n = 4 unique donors) were pretreated as indicated with inhibitors of either mTOR (AZD2014) (LM, light gray), IDO1 (epacadostat) (LI, red), NAMPT (FK866) (LN, blue), or G6PD (G6PDi-1) (LG, yellow), cultured in media supplemented with ¹³C-tryptophan for 6 h and then stimulated with LPS (1 ng, 18 h). Note that key to abbreviations of conditions is listed in panel h for conditions used in g and h. g The metabolome was extracted and targeted analysis was obtained via LC-MS. The abundance of the unlabeled (M + 0, gray columns) and labeled (maroon columns) species of interest are shown as a percentage of the total abundance of each metabolite. Representations of labeled species with each ¹³C marked in maroon are under graphs in g. h Shows relative peak area of three metabolites of interest: tryptophan, kynurenine, and NAD⁺. For g and h, significance was determined by two-tailed t-tests, comparing LPS-induced response after each inhibitor pretreatment to the DMSO pretreatment, LPS-stimulated condition. For g, relative abundances of only the labeled species of kynurenine (in maroon) were compared, evaluating LPS-induced response after each inhibitor pretreatment compared to the DMSO pretreatment, LPS-stimulated condition. Statistical evaluation was not performed for tryptophan or NAD⁺ in g. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent mean ± SD in any panel showing them.