dietary sources of nitrate are found in a variety of foods including roots and green leafy vegetables , and more recently , some cured / processed meats . dietary sources of nitrate have become of interest for ergogenic and metabolic purposes because of the ability of dietary nitrate to increase nitric oxide ( no ) biosynthesis . nitrate consumption results in heighted plasma nitrite , which has been previously recognized as an in  vivo biomarker of no production.2 , 3 consumption of nitrate - rich foods prior to endurance events including cycling , rowing , and running results in improved performance,4 , 5 , 6 resulting in increasing popularity of nitrate - containing foods.7 , 8 , 9 , 10 , 11 , 12 , 13 as a result , commercially available dietary products high in nitrate content have emerged as ergogenic aids purported to increase athletic performance . no is a potent cellular stimulus capable of increasing intracellular ca and cyclic guanosine monophosphate ( cgmp ) leading to activation of ca / calmodulin - dependent protein kinase and 5-adenosine monophosphate - activated protein kinase ( ampk ) . dietary no precursors including arginine and arginine - like metabolites increase no , stimulating phosphorylation and activation of ampk , which later acts to increase the expression and phosphorylation of peroxisome proliferator - activated receptor gamma coactivator-1 alpha ( pgc-1).14 , 15 pgc-1 acts as a master metabolic regulator of energy metabolism and mitochondrial biogenesis by controlling the expression of nuclear respiratory factor ( nrf ) and its downstream target , mitochondrial transcription factor a ( tfam).16 , 17 , 18 , 19 in adipocytes , arginine was shown to increase mitochondrial content under ambient conditions , and intensify cold - induced adipocyte browning . despite the heightened interest in nitrate consumption , findings about the affects of beetroot (  tin ci ; br ) supplementation have been inconsistent.10 , 21 , 22 , 23 the primary aim of this work is to characterize the effects a commercially available br supplement beverage on metabolism and related metabolic gene expression in skeletal muscle . murine myocytes ( c2c12 ) were purchased from the american type culture collection ( manassas , va , usa ) and cultured in dulbecco 's modified eagle 's medium containing 4500  mg / l glucose and supplemented with 10% heat - inactivated fetal bovine serum and 100  u / ml penicillin / streptomycin , in a humidified 5% co2 atmosphere at 37  c ( standard conditions ) . following overnight seeding , cells were treated with various concentrations of the nitrate - containing commercially available beet it pro - elite shot from james white drinks ltd ( ipswich , uk ) for 24 hours ( ingredients are displayed in table  1 ) . beet it pro - elite 's nitrate / nitrite content was quantified using the nitrate / nitrite quantification assay kit from cayman chemical ( ann arbor , mi , usa ) . nitrate and nitrite were quantified according to the manufacturer 's protocol . briefly , nitrate was quantified by diluting samples in an assay buffer , followed by 1 hour of incubation at room temperature with nitrate reductase and vitamin cofactors . griess reagents were then added in equivocal volume to each well and incubated for 10  min at room temperature , and then sample absorbance was measured at 540  nm . all treatments were standardized to determined nitrate concentrations of 20 , 10 , and 5m , which correspond with 0.5% , 0.25% , and 0.125% br , respectively , by volume ( determined during preliminary cell viability and mitochondrial staining studies ) . cells were seeded overnight in 24-well culture plate from seahorse bioscience ( billerica , ma , usa ) at a density of 5    10 cells / well , and treated with either control medium or media containing br in dilutions corresponding with 0.5% , 0.25% , and 0.125% br by volume for 24 hours . following treatment , culture media was removed and replaced with xf assay media from seahorse bioscience containing 4500  mg / l glucose free of co2 and briefly incubated at 37  c . as per the manufacturer 's protocol , seahorse injection ports were loaded with oligomycin , an inhibitor of atp synthase that induces maximal glycolytic metabolism and reveals endogenous proton leak ( mitochondrial uncoupling ) at a final concentration of 1.0  m . oligomycin addition was followed by the addition of carbonyl cyanide p-[trifluoromethoxy]-phenyl - hydrazone , an uncoupler of electron transport that induces peak oxygen consumption ( an indirect indicator of peak oxidative metabolism ) at final concentration of 1.25m . rotenone was then added in 1.0m final concentration to reveal nonmitochondrial respiration and end the metabolic reactions.24 , 25 extracellular acidification , an indirect measure of glycolytic capacity , and oxygen consumption , a measure of oxidative metabolism , was measured using the seahorse xf24 extracellular analyzer from seahorse bioscience . seahorse xf24 extracellular analyzer was run using 8-minute cyclic protocol commands ( mix for 3 minutes , let stand for 2 minutes , and measure for 3 minutes ) in triplicate as previously described . following a 24-hour treatment with either the control medium or media containing br in dilutions corresponding with 0.5% , 0.25% , and 0.125% br by volume for various durations up to 24 hours , total rna was extracted using the rneasy kit from qiagen ( valencia , ca , usa ) and cdna was synthesized using the retroscript rt kit from ambion ( austin , tx , usa ) according to manufacturer 's instructions . polymerase chain reaction ( pcr ) primers were designed using the primer express software from invitrogen ( carlsbad , ca , usa ) and synthesized by integrated dna technologies ( coralville , ia , usa ) . amplifications of pgc-1 , nrf1 , tfam , glucose transporter 4 ( glut4 ) , and mitochondrial uncoupling protein 3 ( ucp3 ) were normalized to the housekeeping gene , tata binding protein ( tbp ) . quantitative reverse transcription- polymerase chain reactions were performed in triplicate using the lightcycler 480 real - time pcr system from roche applied science ( indianapolis , in , usa ) . sybr green based pcr was performed in triplicate with final primer concentrations at 10m in a total volume of 30  l . the following cycling parameters were used : 95c for 10 minutes followed by 45 cycles of 95c for 15 seconds , and 60c for 1 minute . relative expression levels were determined using the cp method and compared to the lowest expressing group as previously described . cells were seeded in six - well plates at a density of 1.0    10 cells / well and treated with either control medium or media containing br in dilutions corresponding with 0.5% , 0.25% , and 0.125% br by volume for 24 hours . following incubation , the medium was removed and the cells were resuspended in prewarmed media with 200 nm mitotracker green from life technologies ( carlsbad , ca , usa ) and incubated for 45 minutes in a humidified 5% co2 atmosphere at 37c . the cells were pelleted , the media with mitotracker was removed , and the cells were suspended in prewarmed media . group mean fluorescence was measured using facscalibur filtering 488  nm . to investigate the protein expression of metabolic proteins pgc-1 , cytochrome c ( cyt c ) , glut4 , and ucp3 , cells were seeded in chamber slides obtained from bd bioscience ( sparks , md , usa ) with 5000 cells / well and treated with the media containing 0.5% br by volume for 24 hours . cells were fixed using 3.7% formaldehyde in media , permeabilized with phosphate - buffered saline ( pbs ) with 0.1% triton 100x from sigma ( st . louis , mo , usa ) for 10 minutes , and blocked for 1 hour with pbs with 0.1% triton 100x and 3.0% bovine serum albumin ( bsa ) purchased from sigma . cells were stained with either an anti - pgc-1 , anti - cyt c from santa cruz biotechnologies ( santa cruz , ca , usa ) , or an anti - glut4 or anti - ucp3 primary polyclonal - antibody from abcam ( cambridge , ma , usa ) at 1:200 dilution in pbs with 0.1% bsa overnight . the cells were rinsed with pbs with 0.1% triton 100x and 3.0% bsa , and secondary alexfluor 488 or alexfluor 633 from invitrogen was applied at 1:200 dilution for 2 hours . slides were mounted with prolong gold with dapi from invitrogen , cured overnight , and imaged using the axiovert 25 microscope with axiocam mrc from zeiss ( thornwood , ny , usa ) . cells were treated with and without media containing 0.5% br by volume for 24 hours . whole - cell lysates were prepared by harvesting the cells on ice in high salt lysis buffer ( 25 mm tris base , 8 mm mgcl2 , 1 mm dithiothreitol , 15% glycerol , 0.1% triton 100x ) supplemented with protease inhibitor mix ( sigma ) , followed by incubation on ice for 60 minutes . the insoluble material was removed via centrifugation at 17,500    g for 3 minutes , and protein concentrations were determined using bradford assay ( protein assay dye reagent concentrate ; bio - rad laboratories , hercules , ca , usa ) . total protein ( 40  g per sample ) was size - separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes . after blocking in tbst5% nonfat milk powder for 1 hour , membranes were probed at 4c for 24  h with an anti - pgc-1 or anti - ampk primary polyclonal antibody from santa cruz biotechnologies , and an anti--actin primary monoclonal antibody from sigma in tbst1% nonfat milk powder overnight . bound antibodies were detected by horseradish peroxidase - conjugated secondary antibodies from sigma and by chemiluminescence using the ecl plus western blotting detection kit from ge healthcare life sciences ( little chalfont , buckinghamshire , uk ) . signal intensities were obtained by densitometry using the imagej software ( available from the nih at http://rsbweb.nih.gov/ij/ ) by quantifying lane intensities followed by normalizing intensity with corresponding -actin expression . cells were seeded in 96-well plates at a density of 5000 cells / well and grown overnight . cells were treated and incubated with either control medium or media with br ranging from 0.0025% to 25% by volume for 24  h hours . the medium and treatment were removed , and media containing 10% wst1 was added to each well and incubated . fluorescence was measured 1 hour following wst1 addition using wallac victor3v 1420 multilabel counter from perkinelmer ( waltham , ma , usa ) . metabolic measurements , flow cytometry , and cell viability data were analyzed using analysis of variance with dunnett 's pairwise comparisons . gene expression was quantified by relative expression using the cp method , and analyzed using analysis of variance with dunnett 's pairwise comparisons . confocal microscopy images ( performed using 3 randomly selected images ) , and western blots were assessed for cellular fluorescence determined by image j quantification and analyzed using student t tests . all data are presented as average    standard deviation normalized to the control mean ( control  =  100 ) with * , * * , and * * * indicating p  <  0.05 , p  <  0.01 , and p  <  0.001 statistical differences compared to control , respectively . to investigate the effects of br on myocyte metabolism , we first measured oxygen consumption rate ( ocr ) following treatment with and without br in dilutions corresponding with 0.5% , 0.25% , and 0.125% br by volume for 24 hours . basal ocr was significantly elevated above control levels in cells treated with br at all tested volumes , with 0.25% br yielding the greatest increase in basal oxidative metabolism ( fig . despite the significant elevations in basal oxygen consumption , br - treated cells exhibited no change in chemically induced peak oxidative metabolism ( fig . 1c ) . to further investigate the effects of br supplementation on myocyte metabolism , we measured the extracellular acidification rate ( ecar ) , an indirect measure of glycolytic metabolism . cells treated with 0.5% or 0.25% br exhibited significantly reduced basal and peak glycolytic metabolism compared with control cells ( fig . 1d and e ) . to determine the effect of br treatment on relative metabolic reliance , we compared the ocr / ecar ratio of br - treated cells to control cells . cells treated with br consistently displayed a significantly increased reliance on oxidative metabolism compared with control cells ( fig . , these data suggest that cells treated with br and grown in high - glucose media develop a greater propensity to completely oxidize glucose , leading to increased oxygen consumption with concurrent reduction in lactate export ( acidification ) . to investigate the effects of br on metabolic gene expression , we treated cells with various percentages of br for various durations and assessed the expression of pgc-1 , nrf1 , tfam , glut4 , and ucp3 , which were normalized to the housekeeping gene , tbp . treatment of myocytes with 0.5% br by volume induced pgc-1 and glut4 expression in a time - dependent manner compared with the corresponding control . tfam expression increased 8 hours after treatment , prior to returning to basal levels at 24 hours after treatment ( fig . additionally , treatment with 0.5% , 0.25% , and 0.125% br increased pgc-1 and glut4 expression following a 24-hour treatment ( fig . furthermore , treatment with 0.25% and 0.125% br increased tfam expression , whereas only 0.25% br increased nrf1 expression ( fig . mitochondrial ucp3 expression was not altered by br treatment at any of the tested doses or durations ( data not shown ) . similar to gene expression , pgc-1 protein expression was significantly elevated following br treatment , which led to increased cyt c content , an indirect indicator of increased mitochondrial content ( fig . 3a ) . moreover , while ucp3 rna expression was unchanged in br - treated cells , ucp3 protein expression was significantly reduced in cells treated with br ( fig . 3a ) . additionally pgc-1 and ampk protein expression were also significantly elevated in br - treated cells ( fig . br activation of metabolic gene expression also resulted in a dose - dependent increase in myocyte mitochondrial content ( fig . 3c ) . following a 24-hour treatment , only treatment with 0.5% br significantly increased mitochondrial content above control levels . in addition , cell viability was only significantly reduced in c2c12 myocytes following treatment with 2.5% br and greater ( fig . our observations are among the first to demonstrate that a br supplement beverage can stimulate myocyte metabolism in a favorable fashion by increasing mitochondrial biogenesis and oxidative metabolism in a pgc-1-specific fashion . because our experiments were conducted under high glucose conditions , increased oxygen consumption with concurrent reductions in glycolytic acidification suggest that br - treated myocytes undergo a more complete carbohydrate oxidation . additionally , our data also demonstrate that br can also promote glut4 expression , ( and although speculative ) nitrate - treated myocytes may have enhanced glucose transport and possibly insulin sensitivity in high glucose ( diabetic ) conditions ( observations that should be verified experimentally in  vivo ) . our observations also demonstrate that br containing nitrate increases myocyte mitochondrial efficiency in a dose - dependent manner , which is ultimately accompanied by an increase in total mitochondrial content ; specifically increased basal mitochondrial metabolism without increased mitochondrial density following supplementation with 0.25% and 0.125% br by volume . similarly , previous observations by larsen et  al showed that inorganic dietary nitrate consumption in male human volunteers led to enhanced mitochondrial efficiency with suppressed ucp3 content in biopsied muscle . despite reductions in ucp3 content , isolated mitochondria also displayed unaltered mitochondrial coupling indicated by oxygen consumption . additionally , larsen et  al reported no change in pgc-1 or tfam rna content or mitochondrial content . moreover and contrary to our findings , larsen et  al previously found that treatment of primary myotubes with sodium nitrate decreased oxygen consumption . perhaps most interesting , sodium nitrate consumption reduced resting energy expenditure in healthy individuals . this is a meaningful finding because it suggests that despite some potentially favorable metabolic adaptations induced by nitrate - containing foods , dietary nitrate consumption ( br ) may prove counterproductive to those with existing metabolic disease such as obesity . despite some similarities , larsen et  al 's investigation used isolated mitochondria from primary myocytes , which may account for some of the discrepancies between the two findings . moreover , larsen and colleagues used inorganic nitrate consumed orally , likely explaining some of the other discrepancies between our findings . previous in  vitro experiments with dietary no precursors demonstrate that no increases ampk activation , leading to heightened expression of pgc-1 , and potentially mitochondrial content.14 , 15 on a related note , mo et  al previously demonstrated that treatment of primary rat aortic smooth muscle cells with 50m nitrate mediates mitochondrial biogenesis via the activation of sirtuin 1 and pgc-1 by ampk phosphorylation . discrepancies between our observations and those made by mo and colleagues may be a function of different experimental models or differences in nitrate type / source . one possible explanation for this difference may be related to nitrate bioactivation and subsequent bioavailability ( considered biologically inert ) . given that our experimental approach lacked commensal bacteria ( microbes that express nitrate reductases catalyzing the conversation of the nitrate anion to nitrite ) , it is possible that an alternative mechanism exists that promotes nitrate - to - nitrite conversion of br extract . jansson et  al reported that in  vitro mammalian tissues ( gastrointestinal , liver , kidney , heart , and lung ) have nitrate reductase activities , notably higher in rodents . using allopurinol ( inhibitor of xanthine oxidoreductase ) , the group further documented reduced conversation of nitrate to nitrite , indicating a potential reduction mechanism in  vitro . another limitation of our experimental approach is that the br used in our study was a blend of numerous other dietary food chemicals or properties ( in addition to nitrate ) , any of which may have contributed to our findings . despite this limitation , our observations document that br reproducibly increases mitochondrial content and oxygen consumption in cultured myocytes . nitrate - containing supplements are rapidly becoming a topic of interest for many athletes , especially competitive endurance athletes . our data support the hypothesis that br ( containing nitrate ) consumption may promote a favorable metabolic phenotype in skeletal muscle cells , although the observations should be verified experimentally in  vivo .
beetroot (  tin ci ) juice consumption is of current interest for improving aerobic performance by acting as a vasodilator and possibly through alterations in skeletal muscle metabolism and physiology . 
 this work explored the effects of a commercially available beetroot supplement on metabolism , gene expression , and mitochondrial content in cultured myocytes . 
 c2c12 myocytes were treated with various concentrations of the beetroot supplement for various durations . 
 glycolytic metabolism and oxidative metabolism were quantified via measurement of extracellular acidification and oxygen consumption , respectively . 
 metabolic gene expression was measured using quantitative reverse transcription  polymerase chain reaction , and mitochondrial content was assessed with flow cytometry and confocal microscopy . 
 cells treated with beetroot exhibited significantly increased oxidative metabolism , concurrently with elevated metabolic gene expression including peroxisome proliferator - activated receptor gamma coactivator-1 alpha , nuclear respiratory factor 1 , mitochondrial transcription factor a , and glucose transporter 4 , leading to increased mitochondrial biogenesis . 
 our data show that treatment with a beetroot supplement increases basal oxidative metabolism . 
 our observations are also among the first to demonstrate that beetroot extract is an inducer of metabolic gene expression and mitochondrial biogenesis . these observations support the need for further investigation into the therapeutic and pharmacological effects of nitrate - containing supplements for health and athletic benefits .