Fig 1: Cascade of Sirt1 activation. SIRT 1, Sirtuin 1; Nam, nicotinamide; NMN, nicotinamide mononucleotide; NAD, nicotinamide adenine dinucleotide; Nnmt, nicotinamide N-methyltransferase; MNAM, N1-methylnicotinamide.
Fig 2: SIRT1 expression in the cochlea: the age-related SIRT1 downregulation of expression observed in OHFD mice is absent in OHFD-MNAM mice. (A) SIRT1 staining (red) of the whole cochlea at 3 and 6 months after commencement of the experiment. SIRT1 is prominently localized in the OCs, SGCs, and type II and IV SLi cells in all turns within the cochlea. The arrowheads indicate a decrease in SIRT1 expression in the cochlea of the OHFD mice. (B) SIRT1 staining (red) of the middle turn of the cochlea at 3 and 6 months after commencement of the experiment. The arrowhead indicates a decrease in SIRT1 expression in the SGCs in the cochlea of the YHFD mice after experiment initiation. The asterisks indicate a decrease in SIRT1 expression in the SGCs in the cochlea of the OHFD mice after experiment initiation. The double asterisk indicates a decrease in SIRT1 expression in the OCs in the cochlea of the OHFD mice after experiment initiation. The dagger symbol indicates a decrease in SIRT1 expression in the SLi in the cochlea of the OHFD mice after experiment initiation. (C) The SIRT1 protein expression in the cochlea quantitatively analyzed using ELISA at 3 and 6 months after commencement of the experiment. The SIRT1 protein expression analyzed via ELISA decreased with aging (p < 0.001 in each group). The YHFD and OHFD groups exhibited a decrease in the SIRT1 protein expression compared with the YLFD and OLFD groups (p = 0.002 and p = 0.04). In the YHFD-MNAM and OHFD-MNAM, decrease in the SIRT1 protein expression was lower than that in the YHFD and OHFD groups (p = 0.004 and p = 0.01; All groups, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001). (D) The SIRT1 protein expression in the cochlea quantitatively analyzed using WB analysis at 3 and 6 months after commencement of the experiment. β-actin expression was used as a reference. The OHFD mice exhibited a greater decrease in the SIRT1 protein expression compared to the OLFD mice (p = 0.001). In the OHFD-MNAM group, decrease in the SIRT1 protein expression was less than that in the OHFD group (p < 0.001; All groups, n = 5; **p < 0.01, ***p < 0.001). (E) Sirt1 mRNA expression in the cochlea quantitatively analyzed using qRT-PCR at 3 and 6 months after commencement of the experiment. GAPDH expression was used as a reference. Sirt1 mRNA expression analyzed via ELISA decreased with aging (p = 0.03 in the YLFD and OLFD, p = 0.02 in the YHFD and OHFD, p = 0.04 in the YHFD-MNAM and OHFD-MNAM). The YHFD and OHFD mice did not exhibit a decrease in Sirt1 mRNA expression compared with the YLFD and OLFD groups (p = 0.14 and p = 0.49). In the YHFD-MNAM and OHFD-MNAM groups, there were no decreases in Sirt1 mRNA expression compared with those in the YHFD and OHFD groups (p = 0.11 and p = 0.45; Bars: 100 μm; Sirt1, Sirtuin 1; YLFD, low-fat diet-fed 3-month-old mice; OLFD, low-fat diet-fed 6-month-old mice; YHFD, high-fat diet-fed 3-month-old mice; OHFD, high-fat diet-fed 6-month-old mice; YHFD-MNAM, high-fat diet plus 1% N1-methylnicotinamide-fed 3-month-old mice; OHFD-MNAM, high-fat diet plus 1% N1-methylnicotinamide-fed 6-month-old mice; OCs, the organ of Corti; SGCs, spiral ganglion cells; SLi, spiral ligament; SV, stria vascularis; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WB, western blotting; mRNA, messenger ribose nucleic acid; qRT-PCR, quantitative reverse transcription-polymerase chain reaction).
Fig 3: Cardiac lipidomic signature in MCM is modulated by rSirt1 treatment. A Heat map showing levels of different lipid species across the three experimental groups (n = 6 for each group). B-D Radar plots describing Medium and long-chain triacylglycerols, long-chain triacylglycerols, and very long-chain triacylglycerols in the three experimental groups. Red dots: statistically significant difference between db/db mice treated with rSirt1 and db/db mice. Black dots: statistically significant difference between db/db mice treated with rSirt1 and db/ + mice; E–G Total triacylglycerols content, total 22:6- containing triacylglycerols content, total triacylglycerols containing only saturated fatty acids content in the three experimental groups. Data are presented as median and internal normalised within each species to provide a standardised measurement A-D; in radar plots, data are expressed in log10 changes and each grey line represent a 0.2 fold change) or as box plots E–G showing median [IQR] and compared by the Kruskal–Wallis test (upper bold bar) and corrected for multiple testing by the two stage step-up Benjamini, Krieger and Yekutieli false discovery rate method at an alpha level of 0.05. *p < 0.05, **p < 0.01. Full data are reported in Supplementary Table 1. LCT Long-chain triacylglycerols MLCT medium and long-chain triacylglycerols, rSirt1 recombinant Sirt1, SFA saturated fatty acid, TAG triacylglycerols VLCT very long-chain triacylglycerols
Fig 4: Mechanisms underpinning rSirt1-induced preservation of cardiac function in MCM. A Heat map showing differential mean relative expression levels of genes involved in lipid metabolism, trafficking and inflammation in the three experimental groups (n = 4 for each group). B–C In vitro assays showing levels of oxidative stress and apoptosis in H9c2 cardiomyocytes exposed to normal glucose (black dots and plots; n = 6), high glucose (red dots and plots; n = 6), high glucose and rSirt1 (green dots and plots; n = 6), high glucose and vehicle (pink dots and plots; n = 6). Data are presented as box plots showing median [IQR] and compared by the Kruskal–Wallis test (upper bold bar) with Dunn post hoc test. *p < 0.05, **p < 0.01. D Heat map showing differential mean relative expression levels of genes involved in lipid metabolism, trafficking and inflammation in the four experimental groups (n = 5 for each group). HG high glucose MDA malondialdehyde; NG normal glucose; rSirt1 recombinant Sirt1
Fig 5: rSirt1 treatment rescues cardiac function in a mouse model of MCM. A Myocardial levels of rSirt1 in the three experimental groups. B Indices of systolic function (ejection fraction, fractional shortening, aortic ejection time and isovolumic contraction time) across the three experimental groups. C Diastolic function assessed as isovolumic relaxation time and E/A in the three experimental groups. D Myocardial performance index in the three experimental groups. Data are presented as box plots showing median [IQR] and compared by the Kruskal–Wallis test (upper bold bar) with Dunn post hoc test. *p < 0.05, **p < 0.01. AET aortic ejection time, EF ejection fraction, FS fractional shortening IVCT isovolumic contraction time, IVRT isovolumic relaxation time MPI Myocardial performance index rSirt1 recombinant Sirt1
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