Supporting Information

Supporting Information Carlström et al. 10.1073/pnas.1008872107 SI Materials and Methods Animals and Treatments. Female endothelial nitric oxide synthase (eNOS)-deficient (strain: B6.129P2-Nos3tm1Unc/J; stock no.: 002684), wild-type mice (strain: C57BL/6J; stock no.: 000664), or neuronal nitric oxide synthase (nNOS)-deficient (strain: B6.129S4Nos1tm1Plh/J; stock no.: 002986) were obtained from Jackson Laboratories and were randomly assigned to treatment groups to ensure that each group had the same average age. Sodium nitrate (NaNO3) was added to the drinking water during 8 to 10 wk at a concentration of 85 mg·L−1 (1 mM). To ensure a low overall nitrate intake in the control group, we first measured nitrate content in several standard rodent diets and found that it varied considerably (0.14–1.5 mM). The chow containing 0.14 mM nitrate was given to all animals (R34, Lactamine). The measured nitrate content of regular drinking water was 18 to 22 μM and did not vary throughout the study period. The additional sodium load from the NaNO3 supplementation was less than 2% of that provided by the regular chow (0.7% sodium content). Body Weight Development and Metabolism Studies. Mice were weighed weekly during a 7-wk observation period. At the end of the period, animals were housed in individual metabolism cages for 48 h, with food and water given ad libitum. Water and food consumption were determined gravimetrically. Intraperitoneal Glucose Tolerance Test. For the glucose tolerance test, mice were fasted overnight (14 h). Before starting the experiments, the animals were weighed to determine the amount of glucose to inject. The glucose tolerance test was performed in a quiet room and handling was kept down to a minimum to reduce stress during the procedure. A bolus of glucose (2 g·kg−1) was injected into the intraperitoneal cavity (30% D-glucose:H2O solution) and blood was sampled from the tail tip at 0, 15, 30, 60, and 120 min; blood-glucose levels were determined with a portable glucose meter (Glucocard X-SENSOR; OneMed). Termination and Sample Preparation. Animals were weighed and then fasted overnight. On the day of the terminal experiments, anesthesia was induced by spontaneous inhalation of isoflurane (Forene; Abbot Scandinavia AB) and continued by inhalation of 2.2% isoflurane in air. The body temperature was measured and kept stable at 37.5 °C with a servo-regulated heating pad. The abdomen was opened sterile through a midline incision, the visceral fat was inspected. Blood samples from vena cava inferior were collected in tubes containing EDTA (final concentration 2 mM) and N-ethylmaleimide (final concentration 5 mM), immediately centrifuged at +4 °C (4,700 × g, 5 min) and frozen at −80 °C. Tissues were rapidly excised, weighed, rinsed, snap-frozen in liquid nitrogen, and stored at −80 °C. Analysis of stored plasma and tissue samples were always performed within 3 wk. Frozen tissue samples were cut in small pieces, placed in PBS buffer (1:4 wt/vol, pH 7.4) containing EDTA (2 mM) and N-ethylmaleimide (5 mM), and then immediately homogenized (BBX24 Bullet Blender, Next Advance Inc.). Supernatants were collected after centrifugation (15 min, 17,000 × g, 4 °C) for determination of nitrate, nitrite, and nitros(yl)ation products (see below). Blood Pressure Measurements in Rats. A telemetric device (PA-C40; Data Sciences International) was implanted in the abdominal aorta of male Sprague-Dawley rats (8 wk old, weight 250–300 g; B&K) for continuous blood pressure measurements. After surgery, all animals were allowed to recover for at least 10 d before Carlström et al. www.pnas.org/cgi/content/short/1008872107

any measurements commenced. Blood pressure was recorded during a control period for 72 h and then continuously (72 h) with L-NAME supplementation in the drinking water (1 g·L−1). Data were collected every second minute throughout the measurement periods. Measurements of Nitrate, Nitrite, and Nitros(yl)ation Products. Chemiluminescence assay for nitrogen oxides. Nitrite, nitrate, and

nitroso products were determined by chemiluminescence after reductive cleavage and subsequent determination of the NO released into the gas phase (1, 2). The samples were directly introduced via a gas-tight syringe into the reduction solution of a microreaction purge vessel coupled with a condenser and heating jackets unit (Sievers). The condenser jacket temperature was controlled by a continuous flow of cold water and the temperature of the heating jacket was controlled by a continuous flow of warm water regulated by a constant-temperature circulating bath (MGW Lauda M3). Nitrogen, at a rate of 192 mL/min, was used as the carrier gas of NO. The flow could be adjusted with a needle valve integrated with the purge vessel and the outlet of the gas stream was passed through a scrubbing bottle containing sodium hydroxide (1 M; 0 °C) to trap traces of acid before transfer into the NO analyzer. A rapid-response chemiluminescence NO system (ECO Physics) was used to detect the NO signals. Nitrite and nitros(yl)ation products. The reducing mixture, consisting of 45 mmol/L potassium iodide (KI) and 10 mmol/L iodine (I2) in glacial acetic acid, was kept at a constant temperature of 56 °C and continuously bubbled with nitrogen. Nitrite measurements were performed by direct sample injection (100 μL) into the reducing solution, and the amount of nitrite was quantified by simple subtraction of the peak areas of sample aliquots pretreated with sulfanilamide from that of untreated aliquots [10% (vol/vol) of a 5% solution of sulfanilamide in 1 M HCl is added to the biological sample (final concentration 29 mmol/L) and incubated for 15 min at room temperature]. Under these conditions, nitrite reacts with sulfanilamide to form a stable diazonium ion that is not converted to any appreciable extent to NO by the reducing mixture. The calibration curve was obtained with freshly prepared nitrite standard solutions in ultrapure water. Nitros(yl)ation products (RXNO) represent the sum of all S-nitrosation, N-nitrosation and iron nitrosyl products. These species are represented by the NO signal that remains from the original sample after the addition of sulfanilamide. S-nitrosothiols represent a subpopulation of the RXNOs. These measurements were performed by direct sample injection (300 μL) into the reducing solution and quantified by simple subtraction of the peak areas of sample aliquots pretreated with sulfanilamide plus mercuric chloride (0.2% HgCl2 in 1 N HCl) from that of sample aliquots treated with only sulfanilamide. HgCl2 selectively cleaves the S-NO bond without affecting peak shape or recovery of other detectable NO species. Nitrate. Nitrate was reduced to NO with a solution of Vanadium (III)chloride in hydrochloric acid 1 N (saturated solution) at 95 °C. Because Vanadium(III)/HCl will also convert nitrite to NO, the amount of nitrate was quantified by subtraction of the nitrite concentration. The calibration curve was obtained with freshly prepared nitrate standard solutions in ultrapure water. Glycosylated Hemoglobin. Glycosylated hemoglobin (HbA1c) was measured in whole blood collected from the tail tip at the end of the 10-wk nitrate treatment period using an immunoassay analyzer (DCA Vantage analyzer; Siemens). 1 of 5

Insulin and Proinsulin. Proinsulin and insulin were measured in plasma collected at the termination of the experiment, using commercial ELISA kits (Mercodia). Animals had been fasting for 14 h. Mitochondrial Parameters. Transmission electron microscopy. In each muscle sample, the total mitochondrial area was determined from 10 randomly selected muscle fibers by a blinded pathologist (3). The soleus muscle was dissected and small pieces were fixed in 2% glutaraldehyde + 0.5% paraformaldehyde in 0.1 M sodiumcacodylate buffer containing 0.1 M sucrose and 3 mM CaCl2, pH 7.4 at room temperature for 30 min, followed by 24 h at 4 °C. Specimens were rinsed in 0.1 M phosphate buffer, pH 7.4, and postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4 at 4 °C for 2 h, dehydrated in ethanol followed by acetone and embedded in LX-112 (Ladd). Semi-thin sections were cut and stained with toluidine blue and used for light microscopic analysis. Ultrathin sections (≈40–50 nm) were cut and contrasted with uranyl acetate followed by lead citrate and examined in a Leo 906 transmission electron microscope at 80 kV (Oberkochen). Digital images were taken by using a Morada digital camera (Soft Imaging System, GmbH). Citrate synthase activity. Muscle samples from Rectus abdominis were trimmed free of connective tissue and homogenized in a glass homogenizer. The activity of citrate synthase (CS) was measured spectrophotometrically as described elsewhere (4). CS activity was determined as moles per gram muscle protein per minute (BCA Protein Assay Kit; Pierce). Gene expression studies. Gene expression of PGC-1α, a master regulator of mitochondrial biogenesis, was measured in liver and muscle samples using quantitative RT-PCR. Total RNA was extracted from frozen tissues using a power homogenizer (KEBO-lab) and TRIzol reagent. One microgram of total RNA was reverse transcribed with M-MLV reverse transcriptase. The relative expression levels of PGC-1α were determined by real1. Lundberg JO, Govoni M (2004) Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radic Biol Med 37:395–400. 2. Feelisch M, et al. (2002) Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: Implications for the fate of NO in vivo. FASEB J 16:1775–1785.

time PCR in a 7900 sequence-detection system (Applied Biosystems) and amplification was carried out with SYBR green master mix. Sequences of the primers used were mPGC-1α forward; 5′-ggc agt aga tcc tct tca aga tc-3′ and reverse 5′-tca cac ggc gct ctt caa ttg-3′; and β-actin forward 5′-gct cct cct gag cgc aag t-3′ and reverse 5′-gtg gac agt gag gcc agg at-3′. Thermal conditions were 2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C. Western blots. Protein was extracted from snap-frozen tissues (liver and heart) using a power homogenizer (KEBO-lab) and lysis buffer (20 mM hepes pH 7.9, 125 mM NaCl, 1% Nonidet P-40, 1 mM EDTA), supplemented with 0.5% protease inhibitor mixture (Sigma). Protein samples were resolved on SDS/PAGE, transferred onto Hybond-C extra membrane (Amersham), and analyzed by Western blot with the SuperSignal West Pico chemiluminescence detection system (Thermo Scientific). The analysis of the Western blot images was done with Image J 1.43. Antibodies: rabbit polyclonal anti-PGC-1α (H-300), mouse monoclonal anti-β-actin (C4), horseradish peroxidase-conjugated secondary antibodies antirabbit and anti-mouse, from Santa Cruz. cGMP. Liver and aortic tissue samples from eNOS-deficient mice,

treated for 10 wk with regular drinking water (n = 14, controls) or water supplemented with sodium nitrate (0.1 mmol·kg−1·d−1, n = 16), were quickly removed, snap frozen on dry ice and stored at −80 C. Frozen tissue samples were homogenized (BBX24 Bullet Blender) in the presence of IBMX (10 μM) and then analyzed for cGMP content using a commercial ELISA method (Biotrak EIA System; Amersham). Triglycerides. Blood was drawn from vena cava inferior in iso-

flurane-anesthetized mice. Samples were immediately centrifuged and plasma was stored at −80 C until analysis, which was done with a commercial kit (Cayman Chemical). 3. Park CB, et al. (2007) MTERF3 is a negative regulator of mammalian mtDNA transcription. Cell 130:273–285. 4. Reisch AS, Elpeleg O (2007) Biochemical assays for mitochondrial activity: Assays of TCA cycle enzymes and PDHc. Methods Cell Biol 80:199–222.

Fig. S1. Dietary nitrate does not significantly affect mitochondrial numbers in skeletal muscle. Total mitochondrial cross-sectional area (presented as percent of total reference area) was measured in the soleus muscle of eNOS-deficient mice using transmission electron microscopy. The mice (n = 4–6) had been treated with regular water or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1) for 10 wk. ns, nonsignificant.

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Fig. S2. Dietary nitrate does not significantly affect citrate synthase activity. Enzyme activity (CS) was measured in skeletal muscle samples from eNOSdeficient mice treated with regular water or sodium nitrate supplemented drinking water (0.1 mmol·kg−1·d−1) for 10 wk. Horizontal lines represent mean; ns, nonsignificant.

Fig. S3. Dietary nitrate does not significantly affect gene expression of PGC1-α. Skeletal muscle (A) and liver samples (B) from eNOS-deficient mice (n = 4–6), treated with regular water or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1) for 10 wk, were analyzed for mRNA of PGC1-α. ns, nonsignificant.

Fig. S4. Dietary nitrate does not affect protein levels of PGC1-α. Liver and heart samples from eNOS-deficient mice, treated with regular water (controls) or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1) for 10 wk, were analyzed for protein expression (Western blot) of PGC1-α. The results displayed are representative of blots from 16 nitrate-treated and 14 control animals. Relative values (PGC1-α/β-actin) from densitometric analysis are displayed below the blots where the first control is given a value of 1.0. Each relative value represents the mean from three separate gels.


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Fig. S5. Dietary nitrate does not affect tissue levels of cGMP. Liver and aortic tissues were harvested from eNOS-deficient mice and analyzed for cGMP content. The mice had been treated with regular water (control) or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1) for 10 wk. (Scale bars, mean ± SEM.)

Fig. S6. Dietary nitrate has no effect on glucose tolerance in young wild-type mice. (A) Effects on glucose tolerance in young wild-type mice (age 3–4 mo). Glucose tolerance tests were performed after 10 wk of treatment with regular water or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1), with lines indicating the time course of glucose excursion following i.p. injection of glucose (2 g·kg−1) in control mice (n = 11) and nitrate treated (n = 12) mice. (B) Changes in plasma glucose levels during the glucose tolerance test (expressed as area under the curve) in eNOS-deficient and wild-type mice. *P < 0.05.


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Fig. S7. Dietary nitrate has no effect on glucose tolerance in nNOS-deficient mice. Glucose tolerance tests were performed after 10 wk of treatment with regular water (n = 8) or sodium nitrate-supplemented drinking water (0.1 mmol·kg−1·d−1, n = 9), with lines indicating the time-course of glucose excursion following i.p. injection of glucose (2 g·kg−1).


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