Oral NMN administration mitigates weight-gain during obesity in a SIRT1-dependent manner.

(A) Data showing body weight measurements over the course of 5 months in Control mice fed chow (n=25), high-fat diet (HFD) (n=26), or HFD concurrent with 400 mg/kg/day NMN (n=25). (B) Food and water intake values recorded over a 3-day period in the indicated experimental groups (Chow, n=6; HFD, n=7; HFD+NMN, n=7). (C) Data showing body weight measurements over the course of 5 months in SIRT1 iKO mice fed Chow (n=16), high-fat diet (HFD) (n=21), or HFD+NMN (n=18). (D) Food and water intake values recorded over a 3-day period in the indicated experimental groups (Chow, n=6; HFD, n=8; HFD+NMN, n=8). (E) Blood glucose data collected from the indicated experimental groups after a 6-hour fast (Control mice: Chow – n=13, HFD – n=9, HFD+NMN, n=8; SIRT1 iKO mice: Chow – n=10, HFD – n=12, HFD+NMN, n=10). (F, G) Body composition analysis of Control and SIRT1 iKO mice (Control mice: Chow – n=14, HFD – n=20, HFD+NMN, n=17; SIRT1 iKO mice: Chow – n=13, HFD – n=24, HFD+NMN, n=17). Fat mass, fluid mass, and lean mass were estimated, as indicated.

Oral NMN administration enhances energy expenditure in obese mice in a SIRT1-dependent manner.

(A) Energy expenditure (kcal/hr.) in Control mice fed HFD (n=8) or HFD+NMN (n=8). (B) Area under curve (AUC) values for energy expenditure (EE) recorded during 2 consecutive dark periods for the experiments exemplified in (A). (C) Energy expenditure (kcal/hr.) in SIRT1 iKO mice fed HFD (n=8) or HFD+NMN (n=8). (D) Area under curve (AUC) values for energy expenditure (EE) recorded during 2 consecutive dark periods for the experiments exemplified in (C). (E, F) Respiratory exchange ratio (RER) values recorded during 2 consecutive dark periods in Control mice fed HFD (n=8) or HFD+NMN (n=8). (G, H) (F) Respiratory exchange ratio (RER) values recorded during 2 consecutive dark periods in SIRT1 iKO mice fed HFD (n=8) or HFD+NMN (n=8).

Oral NMN administration improves dyslipidemia but not glucose-intolerance or insulin-sensitivity.

(A) Data from intraperitoneal glucose-tolerance tests (IPGTT) performed in Control and SIRT1 iKO mice fed Chow, HFD, or HFD with NMN. Mice were fasted overnight for 16 hours and injected with 2g/kg glucose (Control mice: Chow – n=13, HFD – n=9, HFD+NMN, n=8; SIRT1 iKO mice: Chow – n=10, HFD – n=12, HFD+NMN, n=10). (B) Data from intraperitoneal insulin-tolerance tests (ITT) performed in Control and SIRT1 iKO mice fed Chow, HFD, or HFD with NMN. Mice were fasted for 6 hours and injected with 0.75U/kg insulin (Control mice: Chow – n=13, HFD – n=9, HFD+NMN, n=8; SIRT1 iKO mice: Chow – n=10, HFD – n=12, HFD+NMN, n=10). (C, D) Lipid profile of Control and SIRT1 iKO mice fed Chow, HFD, or HFD+NMN (Control mice: Chow – n=6, HFD – n=6, HFD+NMN, n=6; SIRT1 iKO mice: Chow – n=6, HFD – n=6, HFD+NMN, n=6). (E) Epididymal fat (eWAT), mesenteric fat (mes. WAT), and liver weights of Control and SIRT1 iKO mice from the indicated experimental groups (Control mice: Chow – n=13, HFD – n=9, HFD+NMN, n=8; SIRT1 iKO mice: Chow – n=10, HFD – n=12, HFD+NMN, n=10).

Proteomics Design and Sample PCA.

A) Proteomics analysis flowchart depicting the main design and analysis steps B) PCA plots for Olink and Mass-Spec data. The first two principal components are shown. Ellipses are drawn to depict the shape of each group. The symbols and colors of each group are shown in the legend.

Diet effect in WT mice (Control HFD vs.Control Chow).

A) Volcano plot showing the top 50 significant DE proteins. B) String-db network analysis for all high confidence significant differentially expressed proteins (peptides>1, p-value <0.05, abs(FC) > 0.5) between High-fat diet and Chow in wild-type mice. The top 5 enriched Kegg pathways by false discovery rate are shown. C) Network diagram showing the interactions between proteins and enriched network membership. Proteins belonging to a specific network are colored in the interior based on the color shown in B. Proteins may have multiple interior colors. The fold-change value is shown as a colored halo. Edge width is proportional to the strength of the evidence for an interaction. D) Volcano plots labelling only the members of the top 5 enriched kegg pathways. The legend is the same as in A.

NMN effect in WT mice (Control HFD+NMN vs. Control HFD).

A) Volcano plot showing the top 50 significant DE proteins. B) String-db network analysis for all high confidence significant differentially expressed proteins (peptides>1, p-value <0.05, abs(FC) > 0.5) between High-fat diet and Chow in wild-type mice. The top 5 enriched Kegg pathways by false discovery rate are shown. C) Network diagram showing the interactions between proteins and which proteins belong in which network. Proteins belonging to a specific network are colored in the center based on the color shown in A and B. Proteins may have different center colors. The fold-change value is shown as a colored halo. Edges are colored based on the type of interaction as shown in the legend. D) Volcano plots labelling only the members of the top 5 enriched kegg pathways. The legend is the same as in A.

Comparison of SIRT1 KO effect.

A,B) Chow, C,D) high fat diet and E,F) NMN-treat high fat diet. A,C, E) Volcano plots show the differential expression. The top 50 significant protein groups are labelled. Point shapes refer to the assay used (triangle=DIA, circle=OLINK). Point colors refer to the significance thresholds (red: fdr<0.05, orange: pvalue<0.05 and grey: pvalue>=0.05). Vertical dashed lines are set at an abs(FC)>0.5 and horizontal dashed line is set at-log10( p-value) < 0.05. Only groups with more than one peptide are included in this figure. B,D,F) The top 5 enriched Kegg pathways in the string-db network analysis.

Grouped Analysis combining all significant proteins for the diet and NMN in control and chow.

A) MCL clustering diagram showing the different clusters. Each cluster is depicted in a different cluster. Nodes without color have no interactions with other nodes at the medium confidence level. Solid line width between nodes is proportional to the evidence. Dashed lines depict interactions between groups. B) The list of clusters with gene counts and genes belonging to each cluster. C) Pathway enrichment for each MCL Cluster. Where possible, KEGG pathways are shown. If no KEGG pathways are enriched, then enriched reactome pathways are shown. Note there are no enriched functional pathways for MCL Cluster 5.

SIRT1 Independent Diet and NMN effects.

Clustered heatmap of proteins with SIRT1 independent effects in showing the effect size of each comparison, the proteomics technology used (Tech), the MCL cluster and any enriched KEGG pathways (Blue text) and PFAM domains (Black text). The heatmap column names are shortened for clarity and are CC: Control Chow, CH: Control HFD, CH+N, Control HFD + NMN, SC: SIRT1 iKO Chow, SH: SIRT1 iKO HFD, SH+N: SIRT1 iKO + NMN. The asterisks inside each cell indicates significance: * = p-value < 0.05, ** = adj. p-value < 0.05.

SIRT1 Dependent Diet and NMN effects.

A) NMN-specific SIRT1 dependent clustered heatmap, B) Diet-specific SIRT1 dependent genes, C) Both diet and NMN SIRT1 dependent genes. Clustered heatmap shows the effect size of each comparison, the proteomics technology used (Tech), the MCL cluster and any enriched KEGG pathways (Blue text) and PFAM domains (Black text). The heatmap column names are shortened for clarity and are CC: Control Chow, CH: Control HFD, CH+N, Control HFD + NMN, SC: SIRT1 iKO Chow, SH: SIRT1 iKO HFD, SH+N: SIRT1 iKO + NMN. The asterisks inside each cell indicates significance: * = p-value < 0.05, ** = adj. p-value < 0.05.

Clustered Heatmap of all Master Regulators

with abs(z-score) ≥ 2 and the top 20 most significant by p-value in the NMN + HFD vs. HFD comparison. Different annotations are as indicated in the legend.

Inducible and specific deletion of SIRT1 in mice.

(a) SIRT1-deletion strategy using the tamoxifen-inducible Cre-ERT2 system. Cre-recombination leads to the deletion of exon 4, which encodes the catalytic site in SIRT1. (b) Genotyping to identify Cre-negative and Cre-positive mice. Male Cre-positive mice were bred with Cre-negative female mice to generate Cre-positive and – negative mice in the same litter. The data are shown for mice prior to tamoxifen administration. (c) Weight loss in tamoxifen-fed mice and recovery after tamoxifen withdrawal. (d) qPCR data showing the expression levels of Cre and SIRT1 in the indicated groups. (e) qPCR data showing a specific loss of SIRT1 mRNA in eWAT after tamoxifen administration.

Grouped Analysis Gene Expression Heatmap.

Clustergram shows the row and column clustered protein expression of all proteins in four differential expression comparisons. The row annotations are the Tech (Mass-spec or Olink) and the MCL cluster the protein belongs to.

FBXW7 Network in the NMN + HFD vs. HFD comparison.

Node colors represent expression with red increased and green decreased. Solid and dashed lines are direct and indirect interactions. FBXW7 is highlighted in orange.