Nitrate consumption preserves HFD-induced skeletal muscle mitochondrial ADP sensitivity and lysine acetylation: A potential role for SIRT1

Abstract

Dietary nitrate supplementation, and the subsequent serial reduction to nitric oxide, has been shown to improve glucose homeostasis in several pre-clinical models of obesity and insulin resistance. While the mechanisms remain poorly defined, the beneficial effects of nitrate appear to be partially dependent on AMPK-mediated signaling events, a central regulator of metabolism and mitochondrial bioenergetics. Since AMPK can activate SIRT1, we aimed to determine if nitrate supplementation (4 mM sodium nitrate via drinking water) improved skeletal muscle mitochondrial bioenergetics and acetylation status in mice fed a high-fat diet (HFD: 60% fat). Consumption of HFD induced whole-body glucose intolerance, and within muscle attenuated insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity (higher apparent K_m), submaximal ADP-supported respiration, mitochondrial hydrogen peroxide (mtH₂O₂) production in the presence of ADP and increased cellular protein carbonylation alongside mitochondrial-specific acetylation. Consumption of nitrate partially preserved glucose tolerance and, within skeletal muscle, normalized insulin-induced Akt phosphorylation, mitochondrial ADP sensitivity, mtH₂O₂, protein carbonylation and global mitochondrial acetylation status. Nitrate also prevented the HFD-mediated reduction in SIRT1 protein, and interestingly, the positive effects of nitrate ingestion on glucose homeostasis and mitochondrial acetylation levels were abolished in SIRT1 inducible knock-out mice, suggesting SIRT1 is required for the beneficial effects of dietary nitrate. Altogether, dietary nitrate preserves mitochondrial ADP sensitivity and global lysine acetylation in HFD-fed mice, while in the absence of SIRT1, the effects of nitrate on glucose tolerance and mitochondrial acetylation were abrogated.

Keywords: Insulin resistance; Mitochondrial dysfunction; Nitrate; Obesity; SIRT1.

Fig. 1

– The effects of dietary nitrate on body weight, whole-body glucose tolerance and substrate utilization, and skeletal muscle insulin signaling. Body weight gain during the 8 weeks of HFD and nitrate consumption (A, n = 9–10/group), body mass gain at the end of the experimental protocol (B, n = 9–10/group), intraperitoneal glucose tolerance test (C, n = 6–7/group) and area under the curve calculated from ipGTT (D, n = 6–7/group), lipid (E, n = 9–10/group) and carbohydrate (F, n = 9–10/group) whole-body utilization, and insulin-stimulated skeletal muscle Akt phosphorylation levels (G, n = 4–5/group). White bars are control-fed group, gray bars are HFD-fed group, and black bars are HFD + Nitrate group. Data are expressed with individual values and mean ± SD superimposed. *p < 0.05 compared to control-fed group; #p < 0.05 compared to HFD-fed group. CTL – control group; HFD – high-fat diet; Statistical analysis: One-way ANOVA with Fisher's LSD post hoc test.

Fig. 2

– Nitrate consumption does not alter maximal mitochondrial capacity or lipid utilization. Mitochondrial OXPHOS subunits and VDAC content quantification (A, 7–9/group), mitochondrial respiration using a modified SUIT protocol (B, n = 9–10/group), respiratory control ratio (C, n = 9–10/group), lipid-supported respiration (D, n = 9–10/group), and percentage of M-CoA inhibition on P-CoA supported respiration (E, n = 8–9/group). White bars are control-fed group, gray bars are HFD-fed group, and black bars are HFD + Nitrate group. Data are expressed with individual values and mean ± SD superimposed. *p < 0.05 compared to control-fed group. P – pyruvate; M − malate; d – adenosine diphosphate; G – glutamate; S – succinate; P-CoA – palmitoyl-CoA; M-CoA – malonyl-CoA; CI – complex I subunit NDUFB8; CII – complex II subunit SDHB; CIII – complex III subunit UQCRC2; CIV – complex IV subunit MTCO1; VDAC1 – voltage-dependent anion channel 1; CTL – control group; HFD – high-fat diet; Statistical analysis: One-way ANOVA with Fisher's LSD post hoc test.

Fig. 3

– Dietary nitrate preserves mitochondrial ADP sensitivity in association with lowerH₂O₂production and JNK phosphorylation. Mitochondrial kinetics ADP-supported respiration (A, n = 7–10/group), ADP apparent K_m (B, n = 7–10/group), mitochondrial respiration with 25 μM ADP (C, n = 7–9/group), mitochondrial hydrogen peroxide emission (D, n = 11–16/group), % of suppression by 100 μM ADP (E, n = 11–16/group), SOD2, catalase, and ANT1 protein content (F, n = 8–9/group), 4-HNE (G, n = 7–8/group), nitrotyrosine content (H, n = 7–8/group), and protein carbonylation (I, n = 8–9/group). White bars are control-fed group, gray bars are HFD-fed group, and black bars are HFD + Nitrate group. Data are expressed with individual values and mean ± SD superimposed. *p < 0.05 compared to control-fed group; #p < 0.05 compared to HFD-fed group. CTL – control group; HFD – high-fat diet; ADP – adenosine diphosphate; K_m – Michaelis-Menten constant; mtH₂O₂ – mitochondrial hydrogen peroxide; SOD-2 – superoxide dismutase 2; ANT-1 – adenosine transporter 1; 4-HNE – 4-hydroxynonenal. Statistical analysis: One-way ANOVA with Fisher's LSD post hoc test.

Fig. 4

– Nitrate supplementation preserves CaMK-II phosphorylation levels and rescue SIRT1 protein content. Quantification of western blotting for p-JNK1/total-JNK1 ratio (A, n = 8–9/group), p-JNK2/total-JNK2 ratio (B, n = 8–9/group), p-CaMK-II/total-CaMK-II ratio (C, n = 8–9/group), p-AMPK/total-AMPK ratio (D, n = 8–10/group), SIRT1 (E, n = 9–10/group), and SIRT3 content (F, n = 9–10/group). White bars are control-fed group, gray bars are HFD-fed group, and black bars are HFD + Nitrate group. Data are expressed with individual values and mean ± SD superimposed. *p < 0.05 compared to control-fed group; #p < 0.05 compared to HFD-fed group. CTL – control group; HFD – high-fat diet; CaMK-II – Ca²⁺/calmodulin-dependent protein kinase II; AMPK - AMP-activated protein kinase; SIRT1 – sirtuin 1; SIRT3 – sirtuin 3. JNK – c-Jun N-terminal. Statistical analysis: One-way ANOVA with Fisher's LSD post hoc test.

Fig. 5

– SIRT1 is required for positive metabolic effects and acetylation levels of nitrate. Quantification of acetylated lysine proteins in a whole-muscle lysate (A, n = 7–9/group), representative image of the mitochondrial isolation quality (B), quantification of acetylated lysine proteins in isolated mitochondria (C, n = 8–10/group), SIRT1 quantification in inducible SIRT1 knock out mice (D, n = 4–6/group), glucose tolerance test in HFD-fed SIRT1^−/− mice (E, n = 6/group), area under the curve from ipGTT in HFD-fed SIRT1^−/− mice (F, n = 6/group), quantification of acetylated lysine proteins in isolated mitochondria from gastrocnemius of HFD-fed SIRT1^−/− mice (G, n = 6/group). Data are expressed with individual values and mean ± SD superimposed. *p < 0.05 compared to control-fed group; #p < 0.05 compared to HFD-fed group. CTL – control group; HFD – high-fat diet; Ac-lysine – acetylated lysine; Cav3 – caveolin 3; GLUT4 – glucose transporter 4; SERCA2 – sarcoendoplasmic reticulum (SR) calcium transport ATPase; COXIV – complex IV subunit COX; CIV – complex IV subunit MTCO1; CII – complex II subunit SDHB; CI – Complex I subunit NDUFB8; SIRT1 – sirtuin 1; SIRT3 – sirtuin 3; WT – wild-type; LFD – low-fat diet; AUC – area under the curve. Statistical analysis: One-way ANOVA with Fisher's LSD post hoc test for A, C, and D; Unpaired student's t-test for F and G.