SIRT3-AMP-Activated Protein Kinase Activation by Nitrite and Metformin Improves Hyperglycemia and Normalizes Pulmonary Hypertension Associated With Heart Failure With Preserved Ejection Fraction

Abstract

Background: Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is an increasingly recognized clinical complication of metabolic syndrome. No adequate animal model of PH-HFpEF is available, and no effective therapies have been identified to date. A recent study suggested that dietary nitrate improves insulin resistance in endothelial nitric oxide synthase null mice, and multiple studies have reported that both nitrate and its active metabolite, nitrite, have therapeutic activity in preclinical models of pulmonary hypertension.

Methods and results: To evaluate the efficacy and mechanism of nitrite in metabolic syndrome associated with PH-HFpEF, we developed a 2-hit PH-HFpEF model in rats with multiple features of metabolic syndrome attributable to double-leptin receptor defect (obese ZSF1) with the combined treatment of vascular endothelial growth factor receptor blocker SU5416. Chronic oral nitrite treatment improved hyperglycemia in obese ZSF1 rats by a process that requires skeletal muscle SIRT3-AMPK-GLUT4 signaling. The glucose-lowering effect of nitrite was abolished in SIRT3-deficient human skeletal muscle cells, and in SIRT3 knockout mice fed a high-fat diet, as well. Skeletal muscle biopsies from humans with metabolic syndrome after 12 weeks of oral sodium nitrite and nitrate treatment (IND#115926) displayed increased activation of SIRT3 and AMP-activated protein kinase. Finally, early treatments with nitrite and metformin at the time of SU5416 injection reduced pulmonary pressures and vascular remodeling in the PH-HFpEF model with robust activation of skeletal muscle SIRT3 and AMP-activated protein kinase.

Conclusions: These studies validate a rodent model of metabolic syndrome and PH-HFpEF, suggesting a potential role of nitrite and metformin as a preventative treatment for this disease.

Keywords: AMP-activated protein kinases; SIRT3 protein; heart failure; hypertension, pulmonary; metabolic syndrome.

Figure 1

Development of a novel rat model of PH-HFpEF. A, A single subcutaneous injection of SU5416 (Sugen, 100 mg/kg) was administrated to eight-week old obese ZSF1 rats. Fourteen weeks after SU5416 administration, B–G, right ventricular systolic pressure (RVSP, B), left ventricular end diastolic pressure (LVEDP, C), left ventricular ejection fraction (LV EF, D), mean right atrial pressure (mRAP, E), mean arterial blood pressure (MABP, F), and pulmonary vascular resistance (PVR, G) were measured. H, Medial index (%) were calculated (n = 5). I–K, RV (I) and LV (J) mass normalized to tibial length and Fulton index (K) were measured. Data are mean ± SEM. Mann-Whitney U test was used for two-group comparison.

Figure 2

Chronic oral nitrite treatment improves hyperglycemia and glucose intolerance in obese ZSF1 rats. A, Two doses of nitrite (50 and 100 mg/L) were given in drinking water chronically for 14 weeks to obese ZSF1 rats (Ob). B, Body weights were measured during a 14-week period, n = 3–8 rats per group. C, Fasting blood glucose levels were measured in whole blood samples collected at week 0, 7 and 14 weeks. Both Ob N50 and Ob N100 groups are associated with significant lower blood glucose levels compared to Ob alone (mixed effect model with bootstrapping: ^*P = 0.048 and 0.016, respectively). D, HbA1c levels were measured in whole blood samples collected at week 0, 7 and 14 weeks. Both Ob N50 and Ob N100 groups are associated with significant lower HbA1c levels compared to Ob alone (mixed effect model with bootstrapping: ^**P = 0.005 and < 0.0001, respectively). E, At week 14, rats were challenged with oral glucose (2 g/kg) and tail-vein blood was sampled for glucose at the indicated times. Both Ob N50 and Ob N100 groups are associated with significant lower blood glucose levels compared to Ob alone (mixed effect model with bootstrapping: both at ^***P < 0.0001). All data are presented as mean ± SEM.

Figure 3

Chronic oral nitrite treatment improves glucose metabolism via AMPK phosphorylation and GLUT4 membrane translocation. At week 14, plasma samples and skeletal muscle were collected from lean and obese ZSF1 rats, treated or untreated with nitrite (50 and 100 mg/L). A, Plasma insulin levels were measured. B–C, Effects of nitrite on phosphorylation of insulin-dependent Akt signaling (B) and insulin-independent AMPK signaling (C) were detected by Western blot analyses in skeletal muscle. Each lane represents the skeletal muscle sample from an individual rat. The dot plots show pAkt/tAkt and pAMPK/tAMPK ratio, accounting for Akt and AMPK activation, respectively. D, Representative Western blots for GLUT4 expression in membrane protein extracts from skeletal muscle. Equal membrane protein loading was ensured by examination of Na⁺-K⁺-ATPase. Global significance among four groups was determined by Kruskal-Wallis test, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure. All data are presented as mean ± SEM.

Figure 4

Nitrite increases activation of SIRT3 and AMPK in human skeletal muscles cells (HSKMCs). HSKMCs cultured from vastus lateralis muscles obtained from lean and obese volunteers were chronically treated with nitrite (10 µM) and metformin (1 mM) throughout the differentiation period. These cells were then stimulated with 0.2 mM palmitic acid, 25 mM glucose and 120 nM insulin (PGI) for 24 hours, and a further short-term insulin stimulation (120 nM, 40 min) to induce insulin resistance. A, Representative Western blots for AMPK phosphorylation in HSKMCs obtained from lean and obese volunteers. Four to five different biopsies were used for density analyses of Western blots for AMPK activation. Mann-Whitney U test was used to compare the effect of nitrite on AMPK activation in HSKMCs. B, Net glucose uptake was determined relative to basal uptake in HSKMCs. Mann-Whitney U test was used to compare the effect of nitrite on glucose uptake in HSKMCs. C, Phosphorylation and protein expression levels of upstream activators of AMPK (LKB1 and CaMKII) and downstream substrate of AMPK (ACC). D, Representative Western blot for activation levels of SIRT3. E, Western blot of protein acetylation in mitochondria isolated from HSKMCs. Red arrows show decreased acetylation of several mitochondrial proteins due to nitrite and metformin supplementations. F, HSKMCs were incubated with ROS scavengers, peg-catalase and peg-SOD (CAT/SOD, 50 U/ml each), in the presence or absence of nitrite and/or PGI for 1 and 4 days. Effect of ROS on SIRT3 activation was measured by Western blot. Dot plots show the fold change of activated SIRT3 relative to PGI alone. Decrease in SIRT3 activation levels correlates to the duration of CAT/SOD treatment in either nitrite or metformin group was determined by Kruskal-Wallis test, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure. All data are presented as mean ± SEM.

Figure 5

SIRT3 is required for the glucose lowering effect of nitrite in HSKMCs and in mice fed a high-fat diet (HFD). A, HSKMCs were chronically treated with nitrite (10 µM) and metformin (1 mM) throughout the differentiation period. After cells were differentiated to 70% confluence, cells were transiently transfected with siRNA targeting SIRT3 or scrambled control 48 hours before stimulation with PGI. Effect of SIRT3 knockdown (KD) on nitrite-mediated AMPK activation was measured by Western blots. Four different biopsies were used for density analyses of Western blots for SIRT3 and AMPK activation. B, Net glucose uptake was determined relative to basal uptake in HSKMCs (n = 4). Mann-Whitney U test was used to compare two groups. C, WT and SIRT3 KO mice were fed with a HFD in the presence or absence of nitrite (50 mg/L, in drinking water) for 20 weeks. D, At week 20, mice were fasted for 6 hours and challenged with glucose (1.8 mg/g) and tail-vein blood was sampled for glucose at the indicated times. Kruskal-Wallis test was performed, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure at each time point. ^*P < 0.05 nitrite-treated WT mice compared to WT mice fed with HFD alone; ^#P < 0.05 and ^##P < 0.01 nitrite-treated SIRT3 KO mice compared to nitrite-treated WT mice fed with a HFD. All data are presented as mean ± SEM.

Figure 6

Increased SIRT3 activation levels in skeletal muscle obtained from obese ZSF1 rats and human patients with metabolic syndrome. A, Representative Western blot for SIRT3 activation levels in skeletal muscle obtained from lean and obese ZSF1 rats treated or untreated with nitrite (50 and 100 mg/L, in drinking water). Data are mean ± SEM. Global significance among four groups was determined by Kruskal-Wallis test, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure. For comparison, AMPK activation levels are shown in Figure 3C. B, Representative Western blots and individual changes of SIRT3 and AMPK activation levels from pre-to-post (12 weeks) of combined nitrite/nitrate treatment in vastus lateralis muscle obtained from human patients with metabolic syndrome. Statistical differences were P = 0.028 by paired t-test and P = 0.11 by Wilcoxon signed-rank test for SIRT3 activation. P = 0.078 by paired t-test and P = 0.11 by Wilcoxon signed-rank test for AMPK activation.

Figure 7

Early preventative treatment with oral nitrite and oral metformin treatments reduce pulmonary pressures and vascular remodeling in SU5416/ZSF1 rats. A, Nitrite (50 and 100 mg/L) and metformin (300 mg/kg) were given in drinking water chronically for 14 weeks to eight-week old SU5416/ZSF1 rats (Ob-Su). B, Right ventricular systolic pressures (RVSP) were measured. C, Representative images of lung sections stained with α-smooth muscle actin (α-SMA) and quantification of medial index from the mean of 5 vessels per lung section from 3–6 rats per group. Data are mean ± SEM. Global significance among five groups was determined by Kruskal-Wallis test, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure.

Figure 8

Nitrite and metformin normalize PH-HFpEF associated with metabolic syndrome via activation of SIRT3 and AMPK. Nitrite (50 and 100 mg/L) and metformin (300 mg/kg) were given in drinking water chronically for 14 weeks to eight-week old SU5416/ZSF1 rats (Ob-Su). A, Representative images of lung sections stained with pAMPK, α-smooth muscle actin (α-SMA), and DAPI. B–C, Activation levels of SIRT3 (B) and AMPK (C) were analyzed by Western blot in skeletal muscles obtained from SU5416/ZSF1 rats. Data are mean ± SEM. Global significance among five groups was determined by Kruskal-Wallis test, followed by post-hoc pairwise comparisons with the Dunn-Bonferroni procedure.