Nicotinamide riboside supplementation confers marginal metabolic benefits in obese mice without remodeling the muscle acetyl-proteome

Abstract

Nicotinamide riboside supplements (NRS) have been touted as a nutraceutical that promotes cardiometabolic and musculoskeletal health by enhancing nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, mitochondrial function, and/or the activities of NAD-dependent sirtuin deacetylase enzymes. This investigation examined the impact of NRS on whole body energy homeostasis, skeletal muscle mitochondrial function, and corresponding shifts in the acetyl-lysine proteome, in the context of diet-induced obesity using C57BL/6NJ mice. The study also included a genetically modified mouse model that imposes greater demand on sirtuin flux and associated NAD⁺ consumption, specifically within muscle tissues. In general, whole body glucose control was marginally improved by NRS when administered at the midpoint of a chronic high-fat diet, but not when given as a preventative therapy upon initiation of the diet. Contrary to anticipated outcomes, the study produced little evidence that NRS increases tissue NAD⁺ levels, augments mitochondrial function, and/or mitigates diet-induced hyperacetylation of the skeletal muscle proteome.

Keywords: Nutrition; Physiology; Proteomics.

Graphical abstract

Figure 1

NR supplementation alters the plasma and muscle NAD metabolome in DIO CrAT-deficient and control mice (A) Prevailing view whereby oral NR administration impacts skeletal muscle metabolism. (B) Intervention cohort experimental design in CrAT^fl/fl (floxed control, FC) and CrAT^MCK (knock out, KO) mice. (C) Plasma NR. (D) Plasma NAM. (E) Skeletal muscle NAD. (F) Skeletal muscle NADH. (G) Skeletal muscle NAM. (H) Skeletal muscle NMN. Data are represented as mean ± SEM. (C–H) N = 5–8 per group. Data were analyzed by two-way ANOVA. ∗ represents a main effect of treatment, # represents a main effect of genotype, and ★ represents an interaction between treatment and genotype. ∗P≤0.05. N represents biological replicates. See also Figure S1 and Table S2.

Figure 2

Insulin tolerance is improved in NR-treated mice whereas other markers of glucose homeostasis and energy balance remain largely unaffected (A) Growth curve. (B) Respiratory Exchange Ratio (RER). (C) Oral glucose tolerance. (D) Insulin during the oral glucose tolerance test. (E) 5h fasting insulin. (F) Insulin tolerance. (G) ITT Area Above the Curve (AAC). (H) ITT-AAC vs. Plasma NAM. Data are represented as mean ± SEM. (A–H) N = 5–8 per group. Data in (A), (C), and (D) were analyzed by three-way ANOVA (treatment x genotype x time). In (A), § represents a time × treatment interaction, and Δ represents a time x genotype × treatment interaction. Data in (F) were analyzed by two-tailed Student's t-test and three-way ANOVA (treatment x genotype x time). In (F), ∗ represents a significant difference between HF- and HF + NR-fed mice by two-tailed Student's t-test, and ‡ represents a main effect of treatment by three-way ANOVA. Data in (B), (E), and (G) were analyzed by two-way ANOVA. In (E) and (G), ∗ represents a main effect of treatment. ∗P≤0.05. N represents biological replicates. See also Figures S2–S4.

Figure 3

NR slightly increases muscle mitochondrial respiratory function in HF-fed control but not CrAT-deficient mice (A–C) Maximal, ADP-supported mitochondrial oxygen consumption (JO₂) was assayed in isolated skeletal muscle mitochondria fueled by (A) octanoyl-carnitine/malate/ADP/cytochrome c, (B) palmitoyl-carnitine/malate/ADP, or (C) glutamate/malate/ADP/succinate. (D) Pyruvate titration conducted in the presence of ADP and malate. (E) Correlation matrix. Red lines indicate the line of best fit through the data points and the blue shaded region represents 95% confidence intervals. Data are represented as mean ± SEM. (A–E) N = 5–8 per group. (A–D) Data were analyzed by two-tailed Student's t-test. ∗ represents a significant difference between HF- and HF + NR-fed mice. ∗P≤0.05. N represents biological replicates. See also Figure S5.

Figure 4

NR decreased gene expression for several markers of the mitochondrial unfolded protein response (mtUPR) mRNA was extracted from tibialis anterior (TA) muscles and qPCR was performed for all experiments. (A) Gene expression for markers of the mtUPR in FC standard chow (SC) and HF-fed mice. Gene expression of (B) Chop, (C) ClpP, (D) Lonp1, (E) Hspd1, and (F) Hspe1 from FC and KO mice fed either an HF or HF + NR diet. Data are represented as mean ± SEM. (A–F) N = 5 per group. Data in (A) were analyzed by two-tailed Student's t-test and data in (B–F) were analyzed by two-way ANOVA. In (A), ∗ represents a significant difference between SC and HF mice analyzed by two-tailed Student's t-test; and in (C), (D), and (F), ∗ represents a main effect of treatment analyzed by two-way ANOVA. ∗P≤0.05. N represents biological replicates.

Figure 5

The muscle mitochondrial acetylproteome is largely unaffected by NR treatment (A) Proteomics workflow. Quadriceps muscle tissue was obtained from HF- and HF + NR-fed FC mice. Proteins were extracted, enzymatically digested, labeled with unique tandem mass tag (TMT) 10plex reagents, and pooled. After retaining a small portion of the input fraction, acetyl-peptides were enriched via immunoprecipitation, and the flow-through was subsequently used for phosphoenrichment via IMAC. After analyzing all fractions by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS), the quantitative data was analyzed using Proteome Discoverer 2.4 (PD 2.4) and in-house Python code. (B) Volcano plot of acetyl-peptide relative occupancy (protein-normalized acetylation) versus −log (p value), comparing FC mice with and without NR supplementation. Open and closed dots refer to non-mitochondrial and mitochondrial peptides, respectively, with size inversely correlated with quantitative FDR. N = 5 per group. N represents biological replicates. See also Figure S6 and Table S1.

Figure 6

NR modestly increases energy expenditure wild-type C57BL6/NJ mice without impacting glucose tolerance when NR is administered at the onset of a high-fat diet (A) Experimental design for the prevention cohort. (B) Growth curve. (C) Body composition at 8 and 18 weeks post HF or HF + NR diet. (D) Energy expenditure in the fed state monitored over 24h at 10 and 16 weeks post HF or HF + NR diet. (E) Oral glucose tolerance. (F) 5h fasting insulin at 15 weeks post HF or HF + NR diet. Data are represented as mean ± SEM. (A–F) N = 10 per group. Data were analyzed by two-tailed student's t-test. ∗ represents a significant difference between HF- and HF + NR-fed mice. ∗P≤0.05. N represents biological replicates. See also Figure S7.

Figure 7

NR does not alter muscle mitochondrial respiratory kinetics or H₂O₂ emissions in high-fat fed C57BL6/NJ mice under physiologically relevant energetic conditions Mitochondria were isolated from skeletal muscles of wild-type C57BL6/NJ mice fed an HF or HF + NR diet. (A–C) Relationship between (A) JO₂, (B) ΔΨ, and (C) NAD(P)H/NAD(P)⁺ redox state versus Gibb's Energy of ATP hydrolysis (ΔG_ATP) measured in mitochondria fueled by pyruvate + malate (Pyr/M), palmitoyl-carnitine + malate (Pc/M), or glutamate + malate (G/M). (D) Mitochondrial respiratory efficiency represented as JO₂ plotted against ΔΨ. (E) H₂O₂ emissions (JH₂O₂). Data are represented as mean ± SEM. (A–E) N = 5 per group. Data were analyzed by two-tailed Student's t-test. ∗ represents a significant difference between HF- and HF + NR-fed mice. ∗P≤ 0.05. N represents biological replicates.