Extended Data Fig. 2: Trigonelline and Preiss-Handler pathway metabolomics and effects on NAD+ levels and mitochondrial homeostasis.
a, LC-HRMS measurements of trigonelline levels in liver, gastrocnemius muscles, kidney, whole blood and urine of C57BL/6J mice, collected at 2 hours (2 h) or overnight (o/n) after labelled trigonelline gavage (unpaired two-tailed Student’s t-test compared to control group, mean ± s.e.m, n = 5 biological replicates per group, except in urine samples where only 2 samples were collected in controls and 4 in treated group); LOQ, level of quantification. b, NAD+ levels relative to untreated control mice measured in liver, gastrocnemius and kidney tissues from the same groups as in (a) (unpaired two-tailed Student’s t-test, mean ± s.e.m, n = 5 biological replicates per group). c, LC-HRMS-based relative trigonelline incorporation into NAD+ in the same tissues as in (a), related to the NAD+ measured in Fig. 2b (unpaired two-tailed Student’s t-test compared to control group, mean ± s.e.m, n = 5 biological replicates per group). d, Naprt mRNA levels by qPCR in HSMM cells following 48 h incubation with an adenovirus carrying either a scrambled or NAPRT shRNA construct (unpaired two-tailed Student’s t-test, mean ± s.e.m, n = 3 biological replicates per group). e, NAD+ levels relative to untreated control measured in HSMM myotubes following 24 h incubation with NA in presence or absence of the NAPRT shRNA construct (unpaired two-tailed Student’s t-test, mean ± s.e.m, n = 6 biological replicates per group). f, NAD+ levels relative to untreated control measured in HSMM myotubes following 24 h trigonelline or NR treatment, in the presence or absence of FK866 (one-way ANOVA; mean ± s.e.m, n = 6 biological replicates per group). g, NAD+ levels relative to untreated control measured in HSMM myotubes following 24 h trigonelline or NR treatment, in the presence of FK866 and with or without co-treatment with 2-OHNA (one-way ANOVA; mean ± s.e.m, n = 10 biological replicates per group). h-i, LC-HRMS based measurements of trigonelline and NAM in HSMM myotubes treasted as in Fig. 2g–j (One-way ANOVA, mean ± s.e.m, n = 3 biological replicates per group; in red ****, unpaired two-tailed Student’s t-test for comparisons in conditions without trigonelline); AU, abitrary units. j, Naprt mRNA expression by qPCR in gastrocnemius muscle of wildtype (WT) and Naprt knockout (KO) C57BL/6N mice (One-way ANOVA, mean ± s.e.m, n = 4–6 animals per group). k, LC-MS measurements of trigonelline levels in liver, plasma and gastrocnemius muscles harvested 2 hours post trigonelline gavage in WT or Naprt KO mice (unpaired two-tailed Student’s t-test, mean ± s.e.m, n = 4–6 animals per group); AU, abitrary units. l-n, LC-MS measurements of NAD+ metabolites in (l) liver, (m) blood, (n) gastrocnemius of the same groups as in (k) (One-way ANOVA, mean ± s.e.m, n = 4–6 animals per group); AU, abitrary units. o, Nampt and Nrk mRNA levels by qPCR in liver and muscle of the same groups as in (j) (One-way ANOVA, mean ± s.e.m, n = 4–6 animals per group). p, Apoptosis detection via annexing staining time course in HSMM myotubes following treatments of trigonelline, FK866 or 2-OHNA and their combinations. Staurosporin (2.5 µM) is used as a positive control for apoptosis induction (two-way ANOVA; mean ± s.e.m, n = 6 biological replicates per group). q, Seahorse-based substrate-driven OCR measurement in permeabilized HSMM myotubes following 24 h trigonelline treatment, in the presence or absence of 2-OHNA, and with a sequence of substrate/inhibitors to extract contribution of different complexes (one-way ANOVA; mean ± s.e.m, n = 11 biological replicates per group). r, GPR109A agonist assay in cells incubated with increasing doses of NA or trigonelline (mean ± S.D., n = 4). P: <0.05 (*); < 0.01 (**); < 0.001 (***); < 0.0001 (****); n.s., non-significant. For all the individual p values, see the Extended Data Fig. 2 Source file.
