Fig 1: Leucine protected against aging-induced vascular damage via Sirt1–Foxo1 pathway. Representative Western blot and quantification of vascular leucine transporters Slc3a2 and Slc7a5 from mice at different ages are in (A,B). Representative Western blot and quantification of Sirt1, Foxo1, and acetyl-Foxo1 from mice at different ages are shown in (C,D). Representative Western blot and quantification of vascular Slc3a2 and Slc7a5 from mice in different intervention groups are shown in (E,F). Representative Western blot and quantification of Sirt1, Foxo1, and acetyl-Foxo1 from mice in different intervention groups are shown in (G,H). Statistical analysis was performed using one-way ANOVA; data are presented as mean ± SEM, n = 6/group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ND: Normal diet.
Fig 2: Antiaging and antioxidant properties of the composition in human primary cell cultures: (A) Number of cumulative population doublings (CPDs) of human embryonic fibroblasts continuously treated with the indicated composition concentrations or the diluent DMSO 0.1% (control) as a function of time in culture. (B) (i) % CT-L activities, (ii) mRNA levels of β5, α7 and Rpt6 proteasome subunits, (iii) immunoblot analysis of the catalytic β5 proteasome subunit and of the structural core α7 subunit. (C) oxidized protein levels, (D) analysis of the indicative telomere length T/S ratio, (E) global DNA methylation levels, (F) FoxO1 transcriptional activity and (G) Sirtuin1 direct activation and in early passage (T0) and terminally senescent human fibroblasts grown in media continuously supplemented with the composition concentrations or the diluent DMSO 0.1% (control), throughout their lifespan. The signal in early-passage (T0) control cells was arbitrarily set to 100% in all described assays. GAPDH was used as a control for equal protein loading. ***: p < 0.001, **: p < 0.01, *: p < 0.05.
Fig 3: Inhibition of Foxo1 activity reversed the protective effects of leucine supplementation on VSMC phenotype transformation. Representative images and quantification of CD45 immunohistochemistry and DHE staining are shown in (A–D). Representative immunohistochemistry staining and quantification of contractile markers calponin, ɑ-SMA, vimentin, and synthetic marker osteopontin are shown in (E,F). Statistical analysis was performed using one-way ANOVA; data are presented as mean ± SEM, n = 6/group. * p < 0.05, *** p < 0.001, and **** p < 0.0001. ND: normal diet.
Fig 4: Inhibition of Foxo1 activity reversed the protective effects of leucine supplementation on vascular dysfunction and remodeling. Quantification of Foxo1 activation (A). Dose–response curves for acetylcholine-mediated endothelium-dependent relaxation (B) and SNP-mediated endothelium-independent relaxation (C). Representative images of H&E, Masson’s, and van Gieson’s in aortas from mice at 2M and 21M, and 21M mice with leucine supplementation from 15M, with or without Foxo1 inhibitor AS1842856 (D–F). Quantitative analysis is shown in (G–J). Statistical analysis of vascular relaxation curves was performed with two-way ANOVA, * p < 0.05 15M + Leu + AS1842856 vs. 2M group, † p < 0.05 21M vs. 2M group, †† p < 0.01 21M vs. 2M group. Histological statistical analysis was performed with one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. All data are presented as mean ± SEM, n = 6/group. ND: normal diet.
Fig 5: Mechanisms of leucine in the protection of aging-induced vascular remodeling. Transport of leucine depends on amino acid transporters Slc7a5 and Slc3a2. Intracellular leucine increases protein level of Sirt1, which subsequently induces Sirt1-mediated Foxo1 deacetylation. Foxo1 deacetylation enhances Foxo1 activity in nucleus, which represses VSMC transition to a synthetic phenotype, vascular inflammatory responses, and excessive ROS generation. Meanwhile, leucine or Sirt1 may also relieve vascular inflammation and ROS level via Foxo1-independent mechanisms. Dietary leucine supplementation at middle-age stage reversed aging-induced vascular remodeling and dysfunction.
Supplier Page from Abcam for FOXO1 Transcription Factor Assay Kit (Colorimetric)