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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Free Radic Biol Med. 2012 Aug 4;53(7):1440–1450. doi: 10.1016/j.freeradbiomed.2012.07.080

Nitrite Activates AMP Kinase to Stimulate Mitochondrial Biogenesis Independent of soluble Guanylate Cyclase

Li Mo 1, Yinna Wang 1, Lisa Geary 1, Catherine Corey 1, Matthew J Alef 2, Donna Beer-Stolz 3, Brian S Zuckerbraun 2, Sruti Shiva 1,4,*
PMCID: PMC3477807  NIHMSID: NIHMS405371  PMID: 22892143

Abstract

Nitrite, a dietary constituent and endogenous signaling molecule, mediates a number of physiological responses including modulation of ischemia/reperfusion injury, glucose tolerance and vascular remodeling. While the exact molecular mechanisms underlying nitrite’s actions are unknown, current paradigm suggests that these effects depend on the hypoxic reduction of nitrite to nitric oxide (NO). Mitochondrial biogenesis is a fundamental mechanism of cellular adaptation and repair. However, the effect of nitrite on mitochondrial number has not been explored. Herein, we report that nitrite stimulates mitochondrial biogenesis through a mechanism distinct from NO. We demonstrate that nitrite significantly increases cellular mitochondrial number by augmenting the activity of adenylate kinase, resulting in AMP kinase phosphorylation, downstream activation of sirtuin-1, and de-acetylation of PGC1, the master regulator of mitochondrial biogenesis. Unlike NO, nitrite-mediated biogenesis does not require the activation of soluble guanylate cyclase and results in the synthesis of more functionally efficient mitochondria. Further, we provide evidence that nitrite mediates biogenesis in vivo. In a rat model of carotid injury, two weeks of continuous oral nitrite treatment post-injury prevents the hyperproliferative response of smooth muscle cells. This protection is accompanied by a nitrite-dependent upregulation of PGC1 and increased mitochondrial number in the injured artery. These data are the first to demonstrate that nitrite mediates differential signaling than NO. They show that nitrite is a versatile regulator of mitochondrial function and number both in vivo and in vitro, and suggest that nitrite-mediated biogenesis may play a protective role in the setting of vascular injury.

Keywords: mitochondria, nitrite, nitric oxide, hypoxia, AMP kinase, biogenesis

INTRODUCTION

Mitochondria, once thought solely to play a role in energy production, are now recognized as dynamic signaling entities within the cell, which not only generate ATP, but also modulate redox signaling and initiate apoptosis. Thus, mitochondrial number and function are tightly regulated and change rapidly based on cellular metabolic and signaling demand. Mitochondrial biogenesis, the genetic program by which new mitochondria are synthesized is integral in maintaining sufficient numbers of mitochondria and underlies the adaptive processes initiated by stimuli such as exercise training [1-3], hypoxia [4-6] and caloric restriction [7-9]. Central to the biogenesis signaling cascade is the activation of peroxisome proliferator-activated receptor-coactivator 1 (PGC1 ), whose activity is regulated by sirtuin-1 (SIRT1) and AMP Kinase (AMPK), two major metabolic sensors [10, 11]. Deficiencies in PGC1 lead to failure to adapt to cardiac pressure overload [12], decreased contractile function [13], and increased susceptibility to oxidative stress [14], highlighting the essential role of mitochondrial biogenesis in cellular adaptation and repair.

Nitrite (NO2−), a dietary constituent present in the blood (0.1-0.3 μmol/L)[15] and tissues (1-20μmol/L)[16], is now established as an endocrine reservoir of nitric oxide (NO) and a mediator of hypoxic cell signaling [17]. Once thought to be physiologically inert, it is now recognized that NO2− is bioactivated along a decreasing physiological oxygen and pH gradient through its reaction with deoxygenated heme proteins, particularly myoglobin in the heart and smooth muscle [17-20]. Mechanistically, the reduction of NO2− to NO is thought to be the initial step in a number of vascular responses that NO2− mediates in vivo, including stimulation of angiogenesis [21], protection from ischemia/reperfusion injury [22-25] and reversal of intimal hyperplasia after vascular injury [26]. However, little is known about the events downstream of NO2− bioactivation that lead to tissue protection in these models. Furthermore, recent data suggesting that the reaction of NO2− with ferric heme can yield dinitrogen trioxide (N2O3) presents the possibility that NO2− may mediate cellular signaling through mechanisms distinct from those mediated by NO [27, 28].

NO is a well characterized regulator of mitochondrial function that stimulates mitochondrial biogenesis through the activation of soluble guanylate cyclase (sGC) and the production of cyclic guanosine monophosphate (cGMP)[9, 29]. While we and others have shown that NO2− modulates mitochondrial function through its modification of specific respiratory chain proteins, the role of NO2− in regulating mitochondrial number is not known [30, 31]. Herein, we demonstrate that NO2− increases the cellular number of functional mitochondria by stimulating mitochondrial biogenesis and show that the mechanism of nitrite’s action occur through the activation of AMPK but are not dependent on sGC. We demonstrate that NO − 2mediated biogenesis occurs both in vitro and in vivo and discuss the potential role of this pathway as a mechanism underlying physiological adaptation as well as the know therapeutic effects of NO2−.

MATERIALS AND METHODS

Materials

Reagents were obtained from Sigma-Aldrich unless otherwise noted.

Cell culture and staining

Primary rat aortic smooth muscle cells (RASMC) were treated every other day with either nitrite or NONOate. Adherent cells were treated with MitoTracker Green FM (Invitrogen; 100nmol/L; 37°C; 30 min). Fluorescence was quantified at Ex490nm/Em516nm. Nuclei were stained with crystal violet (0.25%; MP Biomedicals, Solon, OH) and absorption was read at 550nm.

Isolation of primary RASMC

Sprague-Dawley rats (6-8 weeks old) were anesthetized with an intraperitoneal injection of pentobarbital. The aorta was isolated from the aortic arch to the abdominal aorta, and immersed in 20% FBS-DMEM containing 1,000 U/ml of heparin. Fat and connective tissue was rapidly removed and the aorta was incubated in serum-free DMEM with collagenase type II (2mg/ml) for 45 min at 37 °C. After removal of the endothelium, the vessel was cut lengthwise, and the smooth muscle cells removed mechanically in gelatin as described in [32].

Transmission electron microscopy

Cells were fixed in 2.5% gluteraldehyde and post-fixed in 1% OsO4/1% Fe6CN3 (1 h). They were subsequently dehydrated and embedded in Polybed 812 resin (Polysciences, Warrington, PA). Cross sections (60 nm) were obtained on a Riechart Ultracut E microtome, post-stained in 4% uranyl acetate and viewed on a JEOL JEM 1011 transmission electron microscope (JEOL, Peobody MA) at 80 KV. Images were taken using an AMT 2k digital Danvers, MA).

PCR

RNA was extracted with TRIzol (Invitrogen). Real time PCR using SYBR Green was performed on the ABI 7300 real time PCR system. Results were normalized against the gene GAPDH. The main primer pairs included: (1) PGC1 (5′-a t g a g a a g c g g g a g t c t g a a-3′, reverse: 5′-g c g g t c t c t c a g t t c t g t c c-3′); (2) SIRT 1 (5′- c c a g a t c c t c a a g c c a t g t t-3′, reverse: 5′- t g c t g a g t t g c t g g a t t t t g-3′); (3) Tfam (5′- g g a a g a g c a a a t g g c t g a a g-3′, reverse: 5′- c c c a a t c c c a a t g a c a a c t c-3′); (4) NRF-2 (5′-a t g c c a g a a c c a a a g t g g a c-3′, reverse: 5′-t t t g c a t t a a c a t c a g c a c c a-3′); (5) GAPDH (5′-a g a c a g c c g c a t c t t c t t g t-3′, reverse: 5′-t g a t g g c a a c a a t g t c c ac t-3′).

Complex activities

Activities of mitochondrial complexes I-IV and citrate synthase were measured spectrophotometrically in cell lysates as previously described [31].

Measurement of cGMP

RASMC treated with nitrite or NONOate (3 h, 1% O2) were scraped and cGMP was measured according to the directions outlined in the cGMP kit (Cayman Chemicals; Ann Arbor, MI).

Measurement of NO, RSNO and Fe-NO by chemiluminescence

For the measurement of RSNO, nitrite treated RASMC were scraped in Triton X-100 (0.01%) and subjected to tri-iodide based reductive chemiluminescence in the presence and absence of acidified sulfanilamide (15% in 2M HCl) and mercuric chloride as described in [33]. Fe-NO was measured by injecting the treated cells into a solution of potassium ferricyanide (0.1M) to oxidize the heme iron and release the bound NO. The NO was detected by a Sievers Nitric Oxide Analyzer. Nitrite reduction to NO was measured by adding nitrite to cells in suspension in serum free media at 1% O2 in a vessel connected in line to the Nitric Oxide Analyzer as described in [33].

Oxygen consumption rate (OCR)

RASMC (30×103 cells/well) were treated in XF24 microplates. Media was changed to un-buffered DMEM after treatment and the plate incubated in a non-CO2 incubator (37°C; 60 min) before running on the XF24 Analyzer (Seahorse Bioscience). Basal OCR was measured after which pre-warmed pharmacological modulators (see text) were injected into each well. Viability was assessed by crystal violet staining and OCR normalized to cell number.

ATP and ROS generation

ATP and H2O2 generation were assessed using the ATP determination kit and Amplex Red Hydrogen Peroxide assay kit respectively (Invitrogen). NAD+/NADH ratio: NAD+/NADH ratio was measured in cell extracts using the NAD+/NADH quantification kit (Biovision).

Adenylate kinase activity

Adenylate kinase activity was assessed by measuring luciferin-luciferase luminescence using the AK determination kit (PromoKine).

PGC1 acetylation

PGC1 was immunoprecipitated from RASMC using anti-PGC1 antibody (Santa Cruz). Immunoprecipitated PGC1 was electrophoresed and immunoblotted with antibody for acetylated lysine (Santa Cruz) and for PGC1.

Transfection of RASMC

RASMC were seeded in antibiotic free medium and transfected with siRNA to AMPK 2, PGC1 or sGC 1 (100nmol/L; Santa Cruz) using the siRNA reagent system (Santa Cruz). After 5 hrs, fresh antibiotics were added to the medium and fetal bovine serum increased to 10% serum. Expression of proteins were assayed by Western blot 2 d later.

Carotid Artery Balloon Injury

All procedures were performed in accordance with the Institutional Animal Care and Use Committee of the University of Pittsburgh. Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN; 350-450 g) were anesthetized with intraperitoneal sodium pentobarbital (Nembutal; 45mg/kg) and supplemental inhalational isoflurane (Forane). The left carotid artery was injured as previously described [26]. Oral sodium nitrite supplementation (10.5mg sodium nitrite/L water) was delivered daily for 2 weeks post-injury. Carotid intima:media ratios, RT-PCR and microscopy were measured at 2 weeks.

Statistical Analysis

All results are expressed as means ± SEM; n represents the number of individual experiments. Single comparisons were tested for significance using a two-tailed Student’s t-test. ANOVA followed by the Bonferroni post hoc test was used for multiple comparisons.

RESULTS

Nitrite increases mitochondrial number

To determine whether chronic NO2− treatment regulates mitochondrial number, rat aortic smooth muscle cells (RASMC) were treated every other day with or without NO − 2(50 mol/L) in normoxia (21% O2) or hypoxia (1% O2), after which the cells were stained with crystal violet and Mitotracker green to quantify nuclear number and mitochondrial content respectively. After 6 days, Mitotracker fluorescence was increased in hypoxic NO2−-treated cells compared to untreated cells, while no significant difference was observed in nuclear staining between these groups (Figure 1A-D), suggesting that NO2− increased cellular mitochondrial content. Calculation of the ratio of mitochondrial to nuclear staining demonstrated that hypoxic NO2− treatment mediated a concentration-dependent biphasic increase in cellular mitochondrial mass, with a significant increase at 5 μM, a maximal effect occurring between 25 and 50 M and an EC50 of 7.5 μM (Figure 1E). Notably, no change in mitochondrial content was observed with normoxic NO2− treatment, consistent with the hypoxic bioactivation of NO2−. This increase in mitochondrial number was further confirmed by electron microscopy (Figure 1F-I). To ensure that NO2− induced the synthesis of new mitochondrial proteins, the expression of respiratory complex IV and cytochrome c were measured and shown to increase 1.58±0.06 and 1.80±0.09-fold respectively after hypoxic NO2− treatment compared to controls (Figure 1J). Collectively, these data corroborate that NO2− increased cellular mitochondrial content in hypoxia.

Figure 1. Nitrite increases mitochondrial number.

Figure 1

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Representative mitotracker (green) and nuclear (blue) staining of (A) Untreated 21% O2, (B) NO2− (50 M) treated 21% O2, (C) Untreated 1% O2, and (D) NO2− (50 M) treated 1% O2 cells. (E) Ratio of the intensity of mitotracker: nuclear staining in RASMC treated with NO − 2(0-100 M) at 21% or 1% O2 for 6 d. (F-I) Representative electron micrographs (25,000X) of (F) untreated 21% O2, (G) NO2− (50 M; 21% O2) treated, (H) Untreated 1% O2 and (I) NO2− (50 M; 1% O2) treated cells. Arrows denote mitochondria. (J) Relative density of protein expression of complex IV, cytochrome c and - actin after 6 d treatment (1% O2) with or without NO2− (normalized to -actin). Data are means ± SEM * p<0.01 vs untreated. n>5.

Nitrite mediates biogenesis through activation of PGC1, SIRT1, and AMPK

Activation of PGC1 leads to the downstream activation of nuclear respiratory factor 2 (NRF-2) and transcription factor A of the mitochondrion (Tfam), ultimately initiating the coordinated expression of nuclear and mitochondrial genes and the synthesis of mitochondrial proteins [34]. To determine whether NO2− activates PGC1, the mRNA and protein expression of PGC1 was measured in RASMC. After 1 day of hypoxic NO2− (50μM) treatment, PGC1 mRNA was upregulated 1.79±0.08 fold (Figure 2A), while its protein expression increased by approximately 2.5 fold after 6 d (data not shown). The mRNA expression of Tfam and NRF-2 also increased by 26.8±0.6% and 21.0±1.5% respectively (Figure 2A). Silencing of PGC1 using siRNA abolished the NO2−-mediated increase in mitochondrial number (Figure 2B), demonstrating that NO2− stimulates the classical PGC1 biogenesis pathway.

Figure 2. Nitrite activates AMPK, SIRT1 and PGC1.

Figure 2

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RASMC were treated with or without NO2− (50 M) at 1% O2. (A) mRNA expression of PGC1, SIRT1, NRF2, and Tfam after 1 d. (B) Mitochondrial: nuclear ratio 4 d after nitrite treatment of cells transfected with an empty vector (open bars) or siRNA to PGC1 (filled bars). (C) Representative immunoblot and quantitation of the ratio of phosphorylated AMPK to total AMPK 3 h after treatment. (D) SIRT1 deacetylase activity 24 h after treatment. (E) Representative immunoblot of acetylated and total PGC1 protein with quantification 4 h after treatment. (F) Mitochondrial: nuclear ratio 6 d after treatment in the presence (filled bars) or absence (open bars) of splitomycin (100 M) *p<0.01, #p<0.05 vs. control. n>3

The activity of PGC1 is regulated by metabolic sensors including AMPK and the NAD+-dependent deacetylase SIRT1, which de-acetylates PGC1 to increase its activity [10]. We next determined whether nitrite activates AMPK and SIRT1. Hypoxic NO2− treatment of RASMC induced a 4-fold increase in the phosphorylation of Thr172 on the catalytic subunit of AMPK as early as 3h after treatment (Figure 2C). Additionally, NO2− increased the expression of SIRT1 mRNA (Figure 2A) as well as its de-acetylase activity (Figure 2D). To determine whether NO2−-mediated biogenesis was dependent on SIRT1 activity, the acetylation state of PGC1 was measured after NO2− treatment. Consistent with a role for SIRT1-dependent de-acetylation, hypoxic NO2− treatment significantly decreased the acetylation of PGC1 (Figure 2E). Additionally, the NO2−-induced upregulation of PGC1 mRNA (data not shown) and increase in mitochondrial number (Figure 2F) was inhibited by splitomycin (100 μM), a pharmacological inhibitor of SIRT1, demonstrating that NO2−-mediated biogenesis was dependent on SIRT1 activity.

We next determined the mechanism by which SIRT1 was activated. Phosphorylation of AMPK has been shown to activate SIRT1 by increasing cellular NAD+/NADH ratios [10]. To determine whether NO2−-dependent AMPK phosphorylation regulates SIRT1 activation, the NAD+/NADH ratio was measured in NO2− treated RASMC transfected with siRNA to the catalytic 2 subunit of AMPK. In the presence of fully functional AMPK, NO2− increased the NAD+/NADH ratio 1.41±0.16-fold compared to controls. However, this NO2−-induced increase was significantly attenuated in cells lacking AMPKα2 (Figure 3A). Further, in the absence of AMPKα2 NO2 was unable to increase PGC1 mRNA expression (Figure 3B). Consistent with these results, the NO2−-induced increase in mitochondrial number was inhibited in cells treated with Compound C, a pharmacological inhibitor of AMPK (Figure 3C). These data demonstrate that NO2−-mediated activation of SIRT1 and subsequent stimulation of PGC1 is dependent on AMPK phosphorylation.

Figure 3. Nitrite-induced biogenesis is dependent on AMPK.

Figure 3

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RASMC were treated with or without NO2− (50 M) at 1% O. (A) NAD+/NADH measured 6 h after treatment in cells transfected with an empty vector (control) or siRNA to AMPK 2. (B) PGC1 mRNA levels measured 1 d after treatment in cells transfected with an empty vector (control) or siRNA to AMPK 2. (C) The ratio of mitochondrial to nuclear staining measured 6 d after treatment in cells co-treated with or without Compound C (5μM). *p<0.01, #p<0.05. n>3.

Nitrite and NO-mediated activation of PGC1 are mechanistically distinct

NO is a known stimulator of mitochondrial biogenesis and activator of AMPK [9, 35]. While the exact mechanisms of NO-mediated stimulation of PGC1 is unclear, it is known that NO-mediated biogenesis is dependent on the activation of sGC and the production of cGMP[9, 29]. Thus, we next determined whether NO2−-mediated biogenesis occurs through the reduction of NO2− to NO and subsequent sGC activation. The extent of biogenesis was first compared in RASMC treated with either NO2− or the NO donor diethylaminetriamine NONOate (NONOate). After 6 days of treatment, NONOate (12.5-25μM) and NO2− (25-50μM) both induced significant hypoxic mitochondrial biogenesis (Figure 4A). It was next investigated whether cGMP was required for NO2−-dependent biogenesis. Measurement of cGMP levels 3 h after hypoxic NO2− or NONOate treatment showed that both species resulted in a concentration dependent increase in cGMP, although NONOate was a much more potent inducer of cGMP production than NO2− (1114 ±0.90 fmol/mg versus 102 ± 10.12 fmol/mg at 25 μM) (Figure 4B). Comparison of the relationship between cGMP produced and the extent of biogenesis stimulated at each concentration of NO/NO2− was found to differ significantly between the species (Figure 4C). Figure 4C demonstrates that NO2− stimulated greater biogenesis at significantly lower levels of cGMP production (20-110fmol/mg) in comparison to NO, while NO-mediated biogenesis did not occur until significantly higher concentrations of cGMP (~130 fmol/mg) accumulated. These data suggested that NO2−-induced biogenesis occurs through a mechanism at least partially independent of cGMP. To more definitively test the role of sGC in NO2−-mediated biogenesis, RASMC were transfected with siRNA to the -subunit of sGC. This treatment inhibited cGMP production in response to NO/NO2−. While the lack of sGC activity significantly inhibited the NO (25μM)-mediated increase in PGC1 mRNA expression, it had no significant effect on the NO2− induced increase in PGC1 mRNA (Figure 4D). Further, the inhibition of sGC activity by siRNA or by the pharmacological inhibitor ODQ (1 M) had no significant effect on the NO2−-induced increase in the phosphorylation of AMPK (Figure 4E) or in the increase of the cellular NAD+/NADH ratio (Figure 4F).

Figure 4. Nitrite induced biogenesis is not dependent on sGC.

Figure 4

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(A) Mitochondria:Nuclear content measured 6 d and (B) cGMP levels measured 3 h after treatment with NO − 2(0-200 μM) or NONOate (0-100 μM) at 1% O2 (C) Mitochondria:nuclear plotted as a function of cGMP produced at each concentration of NO − 2or NONOate. (D-F) RASMC were treated with or without NO2− (50 M) at 1% O2 after transfection with an empty vector, siRNA to sGC 1 or ODQ (1 M). (D) PGC1 mRNA levels in sGC 1 +/+ (open bars) and sGC 1 -/- (filled bars) cells, (E) phospho-AMPK levels in the presence (filled bars) or absence (open bars) of ODQ and (F) NAD+/NADH measured 1 d, 3 h and 6 h after treatment respectively. *p<0.01, #p<0.05 vs control; n>3.

Nitrite mediated biogenesis is not dependent on the reduction of nitrite to NO

Since NO potentially stimulates mitochondrial biogenesis through mechanisms independent of sGC activation, we next sought to determine whether nitrite-mediated biogenesis was dependent on the reduction of nitrite to NO. The fate of nitrite in hypoxic RASMC was first assessed. Six days of hypoxic nitrite (50μM) treatment produced significant levels of S-nitrosated (RSNO) and iron-nitrosyl (Fe-NO) proteins (Figure 5A), indicative of nitrite-dependent NO formation. To directly determine whether nitrite was reduced to NO in these conditions, a suspension of RASMC were treated with nitrite (1mM) at 1%O2 and NO generation measured by chemiluminescence. A significant rate of NO formation was measured that persisted for at least 30 min (Figure 5B). To determine the enzyme responsible for nitrite reduction, the cells were pre-treated with inhibitors of known nitrite reductase enzymes, including potassium ferricyanide, cyanide and Allopurinol to inhibit heme globins, mitochondrial complex IV and xanthine oxidoreductase respectively. Consistent with prior studies [26], treatment with Allopurinol (100μM) inhibited over 97% of nitrite reduction to NO (Figure 5B-C). To test whether nitrite mediated biogenesis was dependent on the reduction of nitrite to NO, RASMC were treated with Allopurinol (100μM) for 6 days (replenished daily) in the presence and absence of nitrite (50μM). Consistent with the inhibition of nitrite reduction to NO, Allopurinol treatment abolished nitrite-dependent Fe-NO formation and significantly attenuated RSNO levels in the cells (Figure 5A). However, measurement of mitochondrial number in these cells showed that nitrite-mediated biogenesis was only inhibited by approximately 10% (Figure 5D). These data suggest that nitrite potentially activates signaling pathways independent of nitrite reduction to NO.

Figure 5. Inhibition of nitrite reduction to NO does not abolish nitrite-mediated biogenesis.

Figure 5

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(A) Concentration of RSNO and Fe-NO generated in RASMC treated with nitrite (50μM) in the presence (open bars) or absence (filled bars) of Allopurinol (100μM) for 6 days. (B) Representative chemiluminescence trace of NO production by RASMC treated with nitrite (1mM) in the absence (black) or presence (gray) of Allopurinol (100μM). (C) Quantitation of NO production by RASMC treated with nitrite (1mM) and pre-treated with nothing (control), Allopurinol (100μM), Potassium ferricyanide (100 μM), or Potassium Cyanide (100 μM). (D) Mitochondrial to Nuclear ratio of RASMC treated with (open bars) or without (filled bars) nitrite (50 μM) in the presence of absence of Allopurinol (100μM). *p<0.01, #p<0.05. n>3.

Nitrite increases adenylate kinase activity

Given that NO2−-mediated biogenesis was independent of sGC, but dependent on AMPK activation, we next determined the mechanism by which NO2− activates AMPK. Increases in AMP:ATP ratios are known to activate AMPK [36]. Cellular AMP levels are regulated predominantly by adenylate kinase 1 (AK1) and AMP deaminase, which catalyze the formation and metabolism of AMP respectively. While no change in AMP deaminase activity was observed 1 h after hypoxic NO2− treatment, AK1 activity increased by 34.1±6.0% compared to untreated hypoxic cells (Figure 6A). Further, pre-treatment with Di(adenosine-5’) Pentaphosphate pentaammonium (Ap5A; 50 M), a pharmacological inhibitor of AK1, abolished the NO2−-induced upregulation of SIRT1 mRNA, consistent with a regulatory role for AK1 in NO2−-mediated biogenesis (Figure 6B).

Figure 6. Nitrite increases AK1 activity.

Figure 6

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(A) AK1 activity in RASMC treated with or without NO2− (50 M) at 1% O2. (B) SIRT1 mRNA expression after co-treatment with or without Ap5A (50 M). #p<0.05 vs control; n>3.

Nitrite-mediated biogenesis generates functional mitochondria

To determine whether the new mitochondria generated after NO2− treatment were functional, the specific activity of respiratory complexes I-IV in RASMC was measured after 6 days of hypoxic NO − 2treatment. Consistent with the synthesis of mitochondrial proteins, the activities of all the respiratory complexes and the matrix enzyme citrate synthase were significantly increased (Figure 7A). To determine whether this increased activity resulted in increased function, oxygen consumption was measured in the intact cells (Figure 7B). After measurement of basal respiratory rate, RASMC were treated with the ATPase inhibitor oligomycin (1μM) to measure the respiratory rate not linked to ATP generation (proton leak), followed by treatment with the respiratory uncoupler FCCP (2.5 μM) to measure maximal rate of uncoupled respiration, and rotenone (10 μM) to inhibit mitochondrial oxygen consumption. While normoxic NO2− treatment did not change bioenergetic function, cells treated with NO2− (25 μM) in hypoxia showed increased basal and maximal respiratory rate (234.0±7.5 pmoles/min/cell, 396.2±8.9 pmoles/min/cell) compared to hypoxic controls (181.4±11.8pmoles/min/cell, 241.7±18.3pmoles/min/cell), but no change in non-ATP linked respiration (Figure 7C). Consistent with necessity of AMPK phosphorylation for nitrite-mediated biogenesis, pharmacological inhibition of AMPK with Compound C (5μM) inhibited the nitrite-mediated increase in oxygen consumption (Figure 7D). The lack of increase in non-ATP linked oxygen consumption by NO2− treated cells suggests that NO2−-mediated biogenesis generates not only greater numbers of mitochondria, but also more efficient mitochondria that have more coupling of oxygen consumption to ATP production (ie less proton leak). In addition to increasing respiratory rate, hypoxic NO2− treatment increased basal ATP generation rate, but did not increase H2O2 generation (Figure 7E, F). These data demonstrate that NO2− stimulates the production of new more functionally efficient mitochondria.

Figure 7. Nitrite generates functional mitochondria.

Figure 7

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(A) Activity of complexes I, II, IV and citrate synthase 6 d after NO2− (50 M) treatment at 1% O2. (B) Representative oxygen consumption (OCR) traces for RASMC treated with or without NO2− (50 M) at 1% O2 for 6 d. Arrows denote addition of oligomycin (1 μM), FCCP (2.5 μM) and rotenone (10 μM). (C) Quantitation of several traces such as those shown in (B) as well as similar traces with NO2− treatment at 21% O2. (D) Basal and uncoupled OCR of RASMC treated with nothing (open bars), Compound C (5μM; hatched bars), NO2− (50 M; black bars), or NO2− and compound C (grey bars). (E-F) Rates of (E) ATP and (F) H2O2 generation in cells treated with NO2− (50 μM) for 6 d at 1% O2. * p<0.01, #p<0.05 vs control. n>3.

Nitrite stimulates biogenesis in vivo

Nitrite has recently been shown to protect against neointimal smooth muscle proliferation after balloon injury of the carotid artery [26]. To determine whether NO2−-dependent mitochondrial biogenesis occurs in vivo, rats were subjected to balloon injury in the left carotid artery and then treated with oral NO2− (10.5 mg/L supplemented in the drinking water daily). After 2 weeks neointimal proliferation was increased in the injured artery compared to the corresponding uninjured artery in the same animal. However, this smooth muscle proliferation was significantly inhibited by NO2− treatment (Figure 8A-C). Indicative of NO2−-dependent stimulation of the biogenesis pathway, expression of PGC1 mRNA was significantly increased in the NO2− treated injured vessel compared to the uninjured vessel or the non-NO2− treated vessel (Figure 8D). Additionally, quantification of mitochondrial volume in these vessels showed that the ratio of mitochondrial to tissue area in the NO2− treated vessel was significantly greater (0.061±0.006) than in the untreated injured vessel (0.032±0.008) (Figure 8E-G). These data demonstrate that NO2− stimulates mitochondrial biogenesis in vivo and is associated with the protective mechanism mediated by NO2− after balloon injury.

Figure 8. Nitrite mediates biogenesis in vivo.

Figure 8

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Rats were treated with or without daily oral sodium NO2− (10.5 mg/L water) for 2 wks after balloon injury to the left carotid artery. Controls are uninjured artery of the same animal. (A & B) Representative H&E stain of a carotid artery 14 d after injury in the (A) absence or (B) presence of NO2− treatment. (C) Quantification of the ratio of intimal to media thickness in several arteries such as those in A/B. (D) PGC1 mRNA expression in injured or non-injured arteries from rats supplemented with NO2− or not. (E&F) Representative electron micrographs of a section of injured carotid artery from (E) untreated and (F) NO2− treated rats. (G) Quantification of the mitochondrial volume per tissue area for arteries such as those shown in E/F. *p<0.01, #p<0.05 vs control. n>3.

DISCUSSION

The primary observation of this study is that NO2− activates the AMPK-SIRT1-PGC1 pathway to stimulate mitochondrial biogenesis in vitro and in vivo, leading to the production of new mitochondria with improved function. Further, the current study demonstrates that the actions of NO2− are distinct from those of NO, as NO2−-does not rely on sGC activation to mediate biogenesis. It is unclear why separate pathways exist for NO and NO2− mediated biogenesis and which pathway dominates in vivo. It is likely that they work in parallel and that considerable overlap exists since NO is oxidized to NO2− rapidly in cells and at least a small portion of NO2− is reduced to NO in hypoxia. This is reflected in our data showing that inhibition of sGC does not completely abrogate NO mediated biogenesis, and likewise, inhibition of sGC also has a small inhibitory effect on NO2−-mediated biogenesis. Physiologically, endothelial nitric oxide synthase (eNOS) activity has been shown to be essential for biogenesis mediated by physiological stimuli such as exercise and caloric restriction [9]. Given that eNOS generates not only NO, but also NO2− (through NO oxidation), it is possible that these redundant pathways exist in the cell to ensure the ability to modulate mitochondrial number even if one or the other pathway is damaged.

Interestingly, our data show that while NO2− increases basal and uncoupled respiration, non-ATP linked respiration is unchanged. This suggests that NO2− not only increases mitochondrial number, but also modulates function to generate more coupled mitochondria (ie a larger percentage of basal respiration is linked to ATP generation). This is consistent with previous studies demonstrating that NO2− increases mitochondrial efficiency in healthy human subjects [30]. Larsen and colleagues showed that increased efficiency was associated with a decrease in the expression of the adenine nucleotide translocase and uncoupling protein-3 [30]. While we did not explicitly investigate the cause of increased coupling, further study is needed to determine whether NO2−-dependent increased coupling is linked to or independent of stimulation of the biogenesis pathway. Further, these data suggest that NO − 2may provide added benefit over other inducers of biogenesis, which do not increase efficiency. This could be particularly beneficial in the treatment of genetic mitochondrial diseases, for which recent studies have shown that induction of mitochondrial biogenesis alleviates symptoms in patients and corrects biochemical deficiencies in animal models [37-39]. Many of these diseases are propagated by the inability to produce sufficient ATP. Thus, NO2− may be beneficial by not only increasing mitochondrial number but also ATP generating capacity.

Though our study demonstrates that NO2−-mediated biogenesis is distinct from NO and does not rely on the reduction of nitrite to free NO, the ability of NO − 2to increase mitochondrial number only in hypoxia suggests that NO2− is bioactivated to another species. Nitrite can mediate protein nitration through its heme-catalyzed reaction with H2O2 [40, 41] or be converted to electrophilic nitrated lipids, which signal through PPAR as well as adduction to critical cysteine residues [42]. Additionally, recent reports show that through its reaction with ferric heme proteins in hypoxia, NO2− can be reduced to N2O3 [27]. Evidence of significant S-nitrosothiol formation, even in the presence of allopurinol, in our model suggests that this may be a plausible mechanism by which nitrite mediates biogenesis. Though we did not specifically investigate the target of S-nitrosation or the mechanism by which NO2− increases AK1 activity, we have previously shown that NO2− inhibits complex I activity by S-nitrosation [31]. Inhibition of oxidative phosphorylation in this manner could decrease ATP production, potentially resulting in increased AK1 activity.

While the benefits of increasing mitochondrial number and hence oxygen consumption in hypoxic conditions appears counter-intuitive, our findings are consistent with other studies demonstrating hypoxia-induced biogenesis [43-45]. Mitochondrial ROS production has been shown to be essential in the stabilization of hypoxia inducible factor-1, leading to hypoxic adaptation [46, 47]. Further, brief episodes of hypoxia induce preconditioning responses, including upregulation of mechanisms to generate ATP and increased antioxidant capacity, to protect against future hypoxic/ischemic episodes [5, 48]. It is possible that the benefits of these type adaptive responses outweigh the cost of increased oxygen consumption in our hypoxic model. Further, while hypoxia is used in the in vitro model presented here, the role of nitrite as a physiological inducer of biogenesis is likely more relevant in models of inflammation or disease, as shown in the in vivo model of vessel injury, in which hypoxia is a component.

While we demonstrate that NO2− mediates biogenesis in vivo, prior studies have reported a lack of NO2−-mediated biogenesis in other animal models [31, 49]. This discrepancy is likely due to the fact that previous studies have not measured biogenesis in an area of injury. Here we show that NO2− mediates biogenesis only in the injured vessel, while no change in mitochondrial number is observed in the uninjured vessel of the same animal, despite increases in systemic NO2− levels. This suggests that the injured tissue provides an optimal environment for NO2− bioactivation. This ability for NO2− to mediate biogenesis in an injured area suggests a potential therapeutic advantage over NO, which cannot be targeted to a specific site of injury.

Prior studies have demonstrated that the activation of AMPK inhibits smooth muscle proliferation through modulation of mTOR as well as cyclin dependent kinases [50]. Similarly, PGC1 expression and mitochondrial biogenesis have been associated with decreased neointimal formation after carotid injury [51]. While this study links the protective effects of NO2− to these signaling pathways, our data have implications beyond the reversal of intimal hyperplasia. Nitrite mediates a number of other therapeutic responses including increasing glucose tolerance [49], protection against myocardial infarction [23, 24] and reversal of pulmonary hypertension [52]. While the mechanisms underlying these phenomena remain unclear, the NO2−-dependent AMPK-SIRT1-PGC1 pathway described here may play a role in some of these protective effects of NO2−. For example, activators of AMPK increase insulin sensitivity as well as provide protection from pulmonary hypertension [53] and myocardial infarction [54]. PGC1 also mediates effects beyond biogenesis including muscle fiber type switching from glycolytic to oxidative fibers, which may play a role in NO2−-induced increased exercise tolerance [55].

In summary, these data expand the function of NO2− as a modulator of metabolism through the activation of AMPK and SIRT1. Further, this study extends the role of NO2− in regulating mitochondrial function from acute post-translational modulation of activity to more chronic changes in mitochondrial number and bioenergetics. Additionally, these data suggest a role for NO2− in mitochondrial regulation distinct from that of NO. Future studies will determine how these pathways and species harmonize to meet the dynamic bioenergetic and signaling demands of the cell.

HIGHLIGHTS.

  • Nitrite activates AMP kinase, sirtuin-1 and PGC1 to mediate biogenesis

  • Nitrite stimulates the synthesis of mitochondria with increased respiratory coupling

  • Unlike NO, nitrite-mediated biogenesis does not require sGC activation

  • Biogenesis associates with nitrite-mediated protection in a model of carotid injury

ACKNOWLEDGEMENTS

This work was supported by the Institute of Transfusion Medicine and the Hemophilia Center of Western Pennsylvania, the National Institutes of Health (1R01HL096973) and by the American Heart Association (09SDG2150066).

Footnotes

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