Nitrate Supplementation and Exercise Tolerance in Patients With Type 2 Diabetes
Phase: NA
Conditions: Type 2 Diabetes
Sponsor: Darren P Casey

 
 
 
Nitrate supplementation and exercise tolerance in patients with type 2 diabetes  
NCT02804932  
11/07/22 
 

 
Nitrate supplementation and exercise tolerance in patients with type 2 diabetes 
 
Principal Investigator: 
Darren Casey, Ph.D. 
 
Co-investigator(s):  
Kenichi Ueda, M.D. 
 
 
 
 
Vitor Lira, Ph.D. 
 
 
 
 
Richard Shields, Ph.D. 
 
 
 
 
Dale Abel, M.D., Ph.D. 
 
 
 
 
 
 
 

Abstract 
Patients with type 2 diabetes (T2D) demonstrate a reduced exercise capacity, a powerful predictor 
of cardiovascular mortality, which may be due to reductions in skeletal muscle perfusion and 
mitochondrial dysfunction. Nitric oxide (NO) is a key molecule involved in in the regulation of blood 
flow to contracting muscles, as well as a critical mediator in mitochondrial respiration.  However, 
there appears to be a decreased enzymatic synthesis of NO and an overall reduction of 
bioavailable NO in patients with T2D, which likely contributes to the reduced exercise capacity 
and tolerance. Accumulating evidence suggests that exogenous nitrate supplementation is an 
effective option for increasing NO bioavailability in vivo. The purpose of the proposal is to use a 
randomized, double-blind, placebo controlled study design to comprehensively investigate the 
impact of 8-weeks of dietary nitrate supplementation on skeletal muscle blood flow, mitochondrial 
function, and exercise capacity in patients with T2D.  The central hypothesis is that increasing 
NO bioavailability via dietary nitrate supplementation in patients with T2D will lead to improved 
oxygen delivery and utilization during exercise. To address this hypothesis a highly mechanistic 
and translational experimental strategy will be used to explore whether increased NO 
bioavailability via dietary nitrate supplementation improves skeletal muscle perfusion during 
exercise (Aim 1), enhances mitochondrial biogenesis and function (Aim 2), and improves 
exercise capacity and efficiency (Aim 3). Collectively, these studies will provide important 
mechanistic insight into the therapeutic potential of dietary nitrate supplementation for improving 
skeletal muscle blood flow, mitochondrial function and exercise capacity in patients with T2D. 
 
Research Plan 
I. Specific Aims: 
Aim 1: Determine if increased NO bioavailability via 8 weeks of dietary nitrate 
supplementation improves skeletal muscle perfusion during exercise in patients with T2D.  
The increase in muscle blood flow during exercise is substantially attenuated in T2D compared 
with relatively healthy subjects, which has been attributed to impaired endothelium-dependent 
(NO-mediated) 
vasodilation 
and/or 
potentially 
greater 
sympathetic 
restraint 
of 
the 
microvasculature.1-4 Our primary hypothesis is that increasing NO bioavailability through the 
nitrate-nitrite-NO pathway will improve blood flow to the contracting muscles of T2D patients.  Our 
secondary hypothesis is that the improved exercise blood flow will be due in part to an enhanced 
ability to blunt sympathetic vasoconstriction within the vascular beds of contracting skeletal 
muscle during exercise. 
 
Aim 2: Determine if dietary nitrate supplementation and subsequent increases in NO 
bioavailability lead to improved mitochondrial function in patients with T2D.  NO stimulates 
mitochondrial biogenesis and contributes to mitochondrial respiration. Individuals with T2D 
demonstrate a reduced content and impaired functional capacity of skeletal muscle mitochondria,5 
possibly due to reductions in bioavailable NO. Our hypothesis is that 8 weeks of dietary nitrate 
supplementation will improve various measures associated with skeletal muscle mitochondrial 
content and function (i.e. respiration) in patients with T2D. 
 

Aim 3: Determine if exercise capacity and efficiency are improved following 8 weeks of 
dietary nitrate supplementation in patients with T2D and examine whether these 
improvements are associated with changes in oxygen delivery and utilization (as assessed 
in Aims 1 and 2).  Relative to age- and weight-matched subjects, exercise capacity is reduced in 
T2D patients.6-9 Recent evidence suggests that nitrate supplementation improves exercise 
efficiency and tolerance in humans.10-14 Our primary hypothesis is that dietary nitrate 
supplementation will improve maximal exercise capacity, as measured by whole body maximal 
oxygen uptake (VO2), as well as improve exercise efficiency (reduce the oxygen cost for a given 
workload). 
 
II. Background 
Overview.  It is currently estimated that nearly 30 million Americans have diabetes mellitus, with 
the majority (up to 90%) of cases being Type 2 diabetes (T2D).15  Individuals with T2D are at a 
greater risk of developing CV disease;16, 17 with mortality rates from CV related disease being up 
to four times higher in this patient population.18 Compared to age-matched controls, patients with 
T2D often demonstrate a reduced exercise capacity, as measured by whole body maximal oxygen 
uptake (VO2).6-9 This is critical from a clinical standpoint, as VO2peak and measures of 
cardiorespiratory fitness are inversely related to mortality risk in healthy individuals and those with 
CV diseases.19-21  Moreover, among patients with T2D those with the lowest VO2peak have a 
greater tendency to develop future CV disease related events.22 Although the mechanism by 
which exercise capacity is reduced in persons with T2D is not completely understood, there is 
evidence to suggest that impairments in skeletal muscle perfusion or oxygen utilization play a 
significant role.23  Along these lines, patients with T2D often demonstrate an attenuated blood 
flow response to exercise compared to age- and weight-matched subjects.1-4 It has also been 
demonstrated that the ability to utilize available oxygen within skeletal muscle via mitochondrial 
respiration is also altered in T2D.5  Collectively, impairment in each of these parameters can 
contribute to the decreased exercise capacity associated with T2D. 
 
T2D and Nitric Oxide (NO) bioavailability. Since its initial discovery in the late 1980s,24 NO has 
been demonstrated to have multiple biological actions and can regulate physiology acutely or lead 
to long-term changes in cell function. Accumulating evidence suggests a decreased enzymatic 
synthesis of NO and an overall reduction of bioavailable NO in T2D.25, 26 From a vascular 
standpoint, patients with T2D demonstrate impairments in endothelial dependent vasodilation, 
which is largely a NO mediated response.1, 27-29 Important to this proposal, endothelial dysfunction 
has been proposed as a critical component involved with the reduced exercise tolerance in T2D.23 
Increasing evidence also demonstrates that endogenously produced NO plays an important role 
in regulating metabolism of energy substrates, various aspects of mitochondrial respiration, and 
in the activation of mitochondrial biogenesis.30-35 Taken together, a reduction in bioavailable NO 
in T2D could 1) limit vasodilation in active skeletal muscle and therefore attenuate the blood flow 
and oxygen delivery response to exercise and/or 2) alter skeletal muscle mitochondrial respiration 
and oxygen utilization, which in turn contributes to the exercise deficits observed in patients with 
T2D   
 
Source of NO and benefits of NO boosting compounds. In addition to the L-arginine-NOS 
system for endogenous synthesis of NO, evidence suggests that nitrite (NO2-) and nitrate (NO3-) 
metabolism occurs in blood and various tissues and contributes to the overall bioavailability of 
NO.36-38  Thus, NO2- reduction (i.e. nitrate-nitrite-NO pathway) represents an alternative and 
differentially regulated system for NO generation that operates in parallel to the classical L-
arginine-NOS pathway.39 A relatively easy method to increase plasma NO2- is through increasing 

dietary NO3- intake.  Recent evidence suggests that dietary nitrate supplementation can have 
physiological and therapeutic effects in both animal models and humans.40 Briefly, several of the 
reported physiological benefits of acute oral nitrate supplementation in young and older humans 
include but are not limited to improvements in exercise efficiency and tolerance,10-14 vascular 
function,41-43 and blood pressure.12, 14, 43-45 Considering the important link between NO 
deficiency and impairments in muscle blood flow and mitochondrial function, stimulation 
of the nitrate-nitrite-NO pathway may enhance NO bioavailability and thus have beneficial 
effects on exercise capacity in patients with T2D. Therefore, the findings of the present 
study may be rapidly translated to changes in therapy for patients with T2D. 
 
III. Research Design and methods 
APPROACH  
 
Figure 1 is an overview of the key concepts underpinning this application. The idea is that a 
decreased NO bioavailability contributes to I) a reduced blood flow in contracting muscle, II) 
impaired skeletal muscle mitochondrial function, and III) ultimately a reduced exercise capacity in 
patients with T2D.  We will use a novel therapeutic option (8 weeks of dietary nitrate) in an effort 
to boost bioavailable NO and subsequently help improve exercise tolerance.  The use of state-of-
the-art human physiology and pharmacology techniques will allow us to examine the potential 
beneficial effects of dietary nitrate supplementation in vivo, while our studies utilizing muscle 
tissue from patients with 
T2D will provide novel 
information 
at 
the 
molecular 
and 
cellular 
level regarding skeletal 
muscle 
mitochondrial 
function. 
  
 
 
 
 
 
 
 
 
 
 
 
Patients. 50 patients with documented Type 2 diabetes will be studied.  We will recruit patients 
(40-70 years of age) from the University of Iowa Hospitals and Clinics database.  The Institute 
for Clinical and Translational Science (ICTS) at the University of Iowa identifies potential 
research participants within the database based on specific inclusion and exclusion 
criteria (described below) provided by the investigator. Exclusion criteria will include 
diagnosis of type 2 diabetes < 3 years prior to enrollment, HbA1c <6.0% or >10.0%, body mass 
index > 40 kg/m2, incident cardiovascular events in the last year (heart attack, stroke), 
symptomatic coronary artery disease and/or heart failure, uncontrolled hypertension, hypotension 
(resting systolic BP < 90 mmHg), renal impairment with creatinine clearance (eGFR) of <50 
ml/min, insulin use, smoking or history of smoking within past one year, and the use of medication 
Figure 1. Conceptual overview of application and protocols.  Dashed line represents dietary 
nitrate supplementation as a potential therapy to improve bioavailable nitric oxide (NO). 

which contain nitrates.  We will also recruit 15 age- and weight-matched nondiabetic control 
subjects which will allow us to 1) quantify the degree of impairment in T2D prior to intervention 
and 2) assess the effectiveness of the nitrate therapy in restoring the primary outcome measures 
back to ‘normal’.    
 
Measurements: All studies required to accomplish the three specific aims of this application 
outlined below will employ state of-the-art techniques to assess blood flow, exercise capacity, and 
mitochondrial content and function in humans. Relevant publications from other groups and ours 
are listed next to the following primary measures/techniques to be used in the proposed studies: 
• Subject monitoring. Heart rate will be monitored via a three-lead electrocardiogram. Beat-by-
beat arterial blood pressure will be measured and monitored via artery catheter (described 
below).   
• Brachial Artery Catheterization. An indwelling 20-gauge Teflon catheter will be placed in the 
brachial artery using aseptic techniques after local anesthesia (1% lidocaine).46-51 The catheter 
will be connected to a 3-port connector as well as a pressure transducer, which will permit the 
simultaneous measurement of beat-by-beat brachial arterial pressure, blood draws and 
infusion of study drugs.  Placement of the arterial catheter will be performed by Dr. Kenichi 
Ueda (board-certified anesthesiologist) who has significant experience in this technique in 
research studies and clinical practice.  
• Intra-arterial Drug Administration. 
• Vasodilator responsiveness in the forearm using standard intra-arterial doses of 
acetylcholine (ACh) and sodium nitroprusside (NTP).46, 48, 50 Ach (a nonspecific muscarinic 
agonist) will be infused intra-arterially at 4.0, and 8.0 μg/dl forearm volume/min for 2 min 
each. Intra-arterial infusion of NTP will be infused at 1.0 and 2.0 μg/dl forearm volume/min 
for 2 min each. 
• Standard intra-arterial doses of phenylephrine (α₁-agonist) and dexmedetomidine (α₂-
agonist) will be administered to examine postjunctional alpha-adrenergic vasoconstrictor 
responses during exercise.52, 53 In male subjects, phenylephrine will be infused at 0.03125 
μg/deciliter of tissue/min for the final 3 min at rest, exercise and during the high flow trial. 
In male subjects dexmedetomidine will be infused at 6.25 ng/deciliter of tissue/min for the 
final 3 min at rest, exercise and during the passive vasodilation trial.  The doses of 
phenylephrine and dexmedetomidine will be doubled for young and older female 
participants because they typically have reduced vasoconstrictor responses to α-receptor 
stimulation compared with men.  In an effort to normalize the concentration of each drug in 
the blood perfusing the forearm, the infusions will be adjusted on the basis of forearm blood 
flow (measured via Doppler ultrasonography) and forearm volume (measured via water 
displacement). 
• Standard intra-arterial doses of adenosine will be administered as a control non-exercise 
vasodilator condition.47, 49 Adenosine will be infused at 6.25 μg/deciliter of tissue/min for a 
duration of 6 min.  
• Rhythmic forearm exercise will be performed using a handgrip device at 20% maximum 
voluntary contraction (MVC), as previously described.46-50 Briefly, subjects will be studied while 
in the supine position under standardized conditions.  Maximal voluntary contraction (MVC) of 
the nondominant hand will be measured by having subjects squeeze a handgrip device at 
maximal effort three separate times and then averaged. The MVC will be used to calculate a 
relative work rate of 20% MVC for the experimental protocol.  Based on data from our lab, this 
intensity is considered “moderate” and therefore, all subjects are expected to complete this 
intensity under each exercise condition. Rhythmic forearm exercise will be conducted with a 
handgrip dynamometer by squeezing and releasing two handles together by 4-5 cm which will 
then raise and lower a weight over a pulley connected by rope.  Forearm contractions will be 

at a rate of 20 times min-1 (7 minutes each exercise trial). In addition, this technique has been 
adapted to facilitate beat-to-beat measurements of brachial artery blood flow via Doppler 
ultrasound.   
• Forearm blood flow (FBF) will be determined using Doppler ultrasound. Briefly, brachial artery 
diameter and blood velocity will be measured with a probe insonation angle at 60 degrees. 
Velocity measurements will be recorded continuously; diastolic diameter measurements will be 
made under each condition. FBF will be calculated as the product of mean blood velocity and 
artery cross sectional area. Forearm vascular conductance (FVC) will be calculated using FBF 
and mean arterial pressure. For more details see references46-51. 
• Arterial and deep vein blood samples (retrograde catheter technique) will be obtained during 
the forearm exercise trials for determination of blood gases and calculation of oxygen delivery 
and extraction. 
• Exercise Testing. A 12-lead ECG, symptom limited cardiopulmonary exercise testing with gas 
exchange measurements (Parvo Medics, Sandy, UT) will be performed on a cycle ergometer 
(Lode Corival Bike Ergometer) using a ramp protocol to determine peak aerobic capacity 
(VO2peak). In addition to VO2peak, exercise efficiency will be analyzed at various submaximal 
workloads (i.e. VO2 for a given workload). 
• Venous blood samples will be obtained before and after the intervention for determination of 
blood glucose, HbA1C, plasma nitrate and nitrite, and cGMP concentration. Plasma nitrate and 
nitrite concentration will be measured using ozone chemiluminescence and quantified using a 
NO analyzer (NOA 280i, Sievers, Boulder, CO USA) as previously described.44 cGMP will be 
determined as an indicator for generation of bioactive NO using an enzyme immunoassay 
(cGMP EIA kit; Cayman Chemical, Ann Arbor, MI, USA). All subjects will follow a 3 day low 
nitrate diet prior to each study day. 
• Muscle biopsies will be obtained from the vastus lateralis using a modified Bergstrom needle 
with suction under local anesthesia, which will yield ~200-250mg of tissue.54, 55 
• Mitochondrial function. Fiber bundles will be chemically permeabilized with saponin and 
mitochondrial respiration will be analyzed by in situ high-resolution respirometry at 37°.56 
Briefly, state 3 (with ADP) and state 4 (without ADP) respiration will be measured using 
succinate as a substrate and the respiratory control index and P/O ratio will be calculated as 
previously described.5, 57 Additionally, mitochondrial ATP production will be measured.58-60 
• Mitochondrial biogenesis and content. Enzyme activity (citrate synthase), mitochondrial DNA 
and respiratory chain protein content (i.e. Complexes I - V), as well as peroxisome-proliferator-
activated receptor γ co-activator-1α (PGC-1α) mRNA and protein will be assessed as 
previously described. 35, 61  
• Oxidative Stress. Previous studies have demonstrated a link between increased mitochondrial 
oxidative stress and the development of insulin resistance.62  Whether this increased oxidative 
stress contributes to reduced exercise tolerance in T2D remains unknown. Because nitrate 
supplementation is expected to improve mitochondrial function, it may also reduce oxidative 
stress in skeletal muscle. Therefore, 4-Hydroxynonenal (4-HNE) adducts, indicative of lipid 
peroxidation, and protein carbonylation will be assessed in muscle homogenates via western 
blots as previously described.63 
• Autophagy. NO has been shown to improve mitochondrial function in different cell models. 
Because turnover of damaged/dysfunctional mitochondria can contribute to improved 
respiratory function we will assess autophagy levels in skeletal muscle. Autophagy flux will be 
assessed via western blot and quantification of LC3-II/LC3-I ratio and p62 levels from muscle 
homogenates, as previously shown.64, 65 In addition, we will assess levels of AMP-associated 
protein kinase (AMPK, and its phosphorylation at T172) and of its downstream target unc-51 
like autophagy activating kinase (ULK1, and its phosphorylation at S555). The rationale for 
these measurements is provided under Aim 2.      

 
Exploratory measures (Not necessary to address specific aims) 
• Oral glucose tolerance tests (OGTT). Recent evidence suggests that nitrite augments glucose 
uptake in adipocytes.66 Therefore OGTTs will be performed to assess if nitrate supplementation 
improves blood glucose clearance in T2D. Plasma glucose and insulin will be measured before 
and at 1 and 2 hours after consumption of 75 g dextrose drink as previously described.67 
 
Inorganic Nitrate Supplementation 
Patients will be randomized in a double-blind fashion to either a inorganic nitrate (beetroot juice 
with nitrate) or placebo (beetroot juice without 
nitrate) group for 8 weeks.  The daily dose of oral 
nitrate is similar to the doses previously used in 
human studies,11, 14, 45 which is effective at 
boosting plasma levels of nitrate and nitrite 
(Figure 2).  
 
 
 
 
 
 
 
 
Overview of Analysis Plan: For all analyses distributional assumptions will be assessed with 
data transformations or non-parametric methods used as appropriate.  Test statistics with an 
association probability of ≤ 0.05 will be considered statistically significant. When the stated 
hypothesis indicates a direction for the anticipated association a one-tailed test will be used; if a 
direction is not stated a two-tailed test will be used.  The primary outcome variables outlined for 
each aim will be analyzed as the change from baseline and compared between groups (inorganic 
nitrate vs. placebo) using repeated measures ANOVA. Pearson product-moment correlations will 
be used to assess whether improvements in exercise efficiency/capacity are associated with 
changes in skeletal muscle blood flow and/or mitochondrial function following the nitrate 
intervention. Additionally, unpaired t-tests will be used to compare group means between the 
patients with T2D at baseline (prior to 8 week intervention) and the age- and weight-matched 
nondiabetic control subjects.  This will allow us to 1) quantify the degree of impairment in T2D 
prior to intervention and 2) assess the effectiveness of the nitrate therapy in restoring the primary 
outcome measures back to ‘normal’.    
 
Sample-size/ Statistical Power: One of the primary aims of this investigation is to assess the 
potential efficacy of dietary nitrate supplementation for improving muscle blood flow during 
exercise in patients with T2D (Aim 1).  In a previous study of 12 older adults (see Figure 4) we 
found that the mean ± SD within subject change in forearm blood flow at 20% exercise following 
acute dietary nitrate supplementation was 32±30 ml/min corresponding to an effect size of 1.07 
standard deviation units. In general, for a repeated measures analysis (e.g. paired t-test) a 
sample-size in the treatment group of N=25 will provide statistical power (two-tailed, alpha=0.05) 
of 94% to detect a difference of 1 SD.  For Aims 2 and 3 we plan to use the same patient groups 
as proposed in Aim 1. Based on previously published data related to dietary nitrate and exercise 
efficiency,58 we assume the standard deviation of the change in whole body VO2 for a given 
workload from baseline to end of treatment will be approximately 0.09 l/min. Under the assumption 
that the standard deviation is 0.09 l/min; if dietary nitrate supplementation improves VO2 for a 
Figure 2. Preliminary data demonstrating the 
effectiveness 
of 
8 
weeks 
of 
NaNO3 
supplementation in boosting plasma levels of 
nitrate (NO3-) and nitrite (NO2-). (n=7).    *P < 
0.001 vs baseline (pre intervention) values.   

given workload by 0.06 l/min compared to placebo, a total sample size of N=50 (25 per group) 
will provide statistical power (two-tailed, alpha=0.05) of 85%. 
 
General Overview of Protocol 
Overall Strategy: Figure 3 on the following page is a schematic of our overall strategy. In 
keeping with ADA guidelines we focus on our overall approach and avoid excessive detail on 
well-established methods we have used previously. 
 
 
 
 
 
Strategy to Address our Specific Aims 
 
 
 
 
 
 
 
 
 
 
 
Rationale and hypothesis: Relative to age- and weight-matched subjects, blood flow to exercising 
muscles is reduced in patients with T2D,1-4 which has been attributed to impairments in NO-
mediated vasodilation.1  Recent evidence suggests that dietary nitrate supplementation can 
increase NO bioavailability and subsequently improve various measures of vascular function,41-43 
including muscle blood flow during exercise in animals.68, 69 Figure 4 is recent data from our lab 
demonstrating that acute dietary nitrate supplementation augments muscle blood flow during 
hypoxic exercise in older adults.70 It is also important to note that the magnitude of change in 
Figure 3. Proposed experimental approach. Patients with T2D will undergo pre intervention 
experimental measurements (3 separate study days) prior to being randomized to either 8 
weeks of 1) dietary nitrate supplementation or 2) placebo. All patients with T2D enrolled in the 
study will complete both pre and post intervention measurements. It is important to note that 
the age and weight matched nondiabetic subjects will only undergo pre intervention 
experimental measurements. The inclusion of the control subjects will allow us to 1) quantify 
the degree of impairment in T2D prior to intervention and 2) assess the effectiveness of the 
nitrate therapy in restoring the primary outcome measures back to ‘normal’.  
Aim 1 – Determine if increased NO bioavailability via 8 weeks of dietary nitrate 
supplementation improves skeletal muscle perfusion during exercise in patients with 
T2D 

vasodilation during hypoxic exercise was related to the rise in plasma nitrite post supplementation 
in aging humans (r=0.69; P<0.05).  Moreover, our preliminary data in a small sample of patients 
with peripheral artery disease (PAD), some of which had T2D, suggest that long term (8 weeks) 
dietary nitrate supplementation improves blood flow to the contracting muscles at multiple 
exercise intensities (Figure 5). Thus, our primary hypothesis is that 8-weeks of nitrate 
supplementation will effectively increase NO bioavailability via the nitrate-nitrite-NO pathway and 
improve blood flow to the contracting muscles of T2D patients. 
 
 
In healthy young adults, sympathetic vasoconstrictor responses are blunted in the vascular beds 
of contracting skeletal muscle,71-75 a phenomenon referred to as “functional sympatholysis.” The 
ability to blunt sympathetic vasoconstriction (i.e. functional sympatholysis) in vascular beds of 
contracting muscle is a key local regulatory mechanism to ensure adequate blood flow and 
oxygen delivery to the contracting muscle.  Along these lines, NO has been shown to inhibit 
sympathetic vasoconstriction in contracting skeletal muscle of experimental animals and 
humans.76-79 Therefore a decreased NO bioavailability1, 29 coupled with sympathetic overactivity80, 
81 commonly observed in patients with T2D might lead to a decreased ability to blunt sympathetic 
vasoconstriction in contracting skeletal muscle and consequently contribute to the attenuated 
blood flow responses to exercise.1-4 Thus, our secondary hypothesis is that the improved 
exercise blood flow following nitrate supplementation will be due in part to an enhanced ability to 
blunt sympathetic vasoconstriction within the vascular beds of contracting skeletal muscle during 
dynamic exercise. Our approach (described below) will allow us to determine whether these 
potential changes are selective for α1- or α2-adrenergic receptors. 
 
General Approach: All patients will be instrumented with a brachial artery catheter before and 
after the 8 week intervention. On each study day (before and after intervention) they will perform 
a total of two bouts of rhythmic handgrip exercise at 20% of maximum. The exercise bouts will be 
performed to assess 1) the effects of dietary nitrate supplementation on exercise blood flow 
(primary hypothesis), and 2) the vasoconstrictor responsiveness during exercise (i.e. functional 
sympatholysis) to direct α1- and α2-adrenergic receptor stimulation (via phenylephrine and 
dexmedetomidine, respectively – secondary hypothesis). Intra-arterial adenosine will be 
Figure 4. Our recent data showing 
blunted blood flow during hypoxic 
exercise 
with 
aging, 
which 
is 
improved following acute dietary 
nitrate intake. *P<0.05 vs. young; 
†P<0.05 vs. control.70  
Figure 5. Preliminary data showing an increase in 
blood flow during mild to moderate intensity 
forearm exercise following 8 weeks of NaNO3 in 
patients with PAD. (n=7 for NaNO3 and n=5 for 
placebo) *P<0.05 vs PRE  

administered to elevate blood flow to values similar to those observed during exercise and serve 
as ‘high flow-functional sympatholysis’ control trials. The order of adenosine vs. exercise, as well 
as phenylephrine vs. dexmedetomidine will be varied across subjects. ACh and NTP will be 
infused to assess endothelial-dependent and-independent vascular responsiveness. Non-
invasive measurements of arterial stiffness will also be performed using applanation tonometry.  
 
Potential Results, Interpretation & Follow-Up Studies: We expect that 8-weeks of dietary nitrate 
supplementation will increase plasma levels of NO3- and NO2- relative to the placebo group.  In 
the nitrate group we also anticipate increases in forearm blood flow (FBF) and vascular 
conductance (FVC) post treatment as a result of an enhanced ability to blunt sympathetic 
vasoconstriction (i.e. improved functional sympatholysis). The use of both intra-arterial 
phenylephrine (α₁-agonist) and dexmedetomidine (α₂-agonist) will permit us to determine if 
improvements in functional sympatholysis are selective to a specific α-adrenergic receptor 
subtype. Additionally, we have the experimental tools to pharmacodissect a number of potential 
dilator and constrictor pathways that might contribute to improvements in the FBF and FVC 
responses not explained by changes in sympathetic vasoconstriction or enzymatically produced 
NO. These studies will also set the stage for future mechanistic studies on how NO may modulate 
sympathetic vascular transduction in T2D. 
 
 
 
 
 
Rationale and hypothesis: Mitochondria are important dynamic organelles for cell survival and 
function, which often become dysfunctional and/or reduced in various disorders, including 
diabetes.5, 82 NO appears to play a critical role in mitochondrial function (i.e. respiration) as well 
as acts as a key messenger to activate mitochondrial biogenesis.30, 33-35 Interestingly, a recent 
study led by Dr. Vitor Lira (co-investigator in this application) demonstrated that in healthy insulin 
sensitive muscle mitochondrial biogenesis and autophagy, a catabolic process responsible for 
aggregate protein and organelle degradation, are regulated in parallel. This conclusion is based 
on a series of experiments that showed: a) oxidative muscles, which have higher mitochondrial 
content, also present higher autophagy machinery (i.e., expression of autophagy proteins) and 
basal autophagy flux when compared to glycolytic muscles; b) endurance exercise training leads 
to increased mitochondrial content in primarily glycolytic muscles, which also adopt basal 
autophagy levels that more closely resemble those of oxidative muscles; and c) muscle specific 
overexpression of peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α), a 
master regulator of mitochondrial biogenesis, results in increased autophagy flux primarily 
associated with mitochondrial autophagy (i.e., autophagy targeting mitochondria).65 The most 
logical interpretation of these findings is that a fine balance between mitochondrial biogenesis 
and removal, via autophagy, enables optimal organelle turnover, which seems instrumental for 
an ideal mitochondrial function in muscle. 
  
Aim 2 – Determine if dietary nitrate supplementation and subsequent increases in NO 
bioavailability lead to improved mitochondrial function in patients with T2D.   

Mechanistically, the processes of 
mitochondrial 
biogenesis 
and 
autophagy can be stimulated by 
AMP-activated 
protein 
kinase 
(AMPK).83, 
84 
This 
is 
further 
supported by our preliminary data 
shown in Figure 6. Dr. Lira has 
also demonstrated that NO donors 
induce mitochondrial biogenesis 
and 
Glut4 
expression 
in 
rat 
skeletal muscle cells via AMPK-
mediated activation of PGC-1α.35, 
85 Important to this proposal, short 
term (3 days) supplementation of 
NaNO3 
improves 
intrinsic 
mitochondrial efficiency in young 
humans.58 
However, 
whether 
NaNO3 improves mitochondrial 
function in T2D, and to what 
degree that is associated with 
increased 
mitochondrial 
biogenesis 
and 
autophagy 
remains unknown.  Thus we 
hypothesize that longer term (8 
weeks) nitrate supplementation 
will be effective in improving 
mitochondrial respiration as well 
as enhancing mitochondrial content in patients with T2D. We further postulate that the processes 
of mitochondrial biogenesis and autophagy will both be upregulated by the nitrate 
supplementation intervention. 
 
General Approach: We will obtain a biopsy of the vastus lateralis.  The sample will be obtained 
under local anesthesia by Dr. Richard Shields (co-investigator) with the assistance of Dr. Kenichi 
Ueda (licensed physician and co-investigator), using a modified Bergstrom needle with suction, 
which will yield ~200-250mg of tissue.  A portion (~50-100mg) of the biopsy will be dissected and 
rapidly frozen in liquid nitrogen and stored at -80°C for later analysis of enzymatic activity and 
protein concentration. The major part of the biopsy (~150mg) will be chemically permeabilized 
with saponin and mitochondrial function will be assessed through isolation of the electron 
transport chain complexes in situ.56 
 
Potential Results, Interpretation & Follow-Up Studies:  We expect that an increase in bioavailable 
NO following nitrate supplementation will lead to an enhanced maximal ADP-initiated 
mitochondrial respiration (state 3). We also expect that there will be an increase in citrate synthase 
activity, suggestive of mitochondrial biogenesis following dietary nitrate supplementation.  We 
further expect an increase in basal autophagy flux (i.e. increased LC3-I conversion into LC3-II, 
assessed via LC3-II/LC3-I ratio, and reduced p62 levels64 as shown in Figure 6) in response to 
nitrate supplementation. Increases in both mitochondrial content and autophagy flux will 
mechanistically point to improved mitochondrial turnover, which is dependent on both 
mitochondrial biogenesis and degradation, as a central molecular outcome of nitrate 
supplementation in improving mitochondrial function in T2D. Examination of various protein and 
mRNA levels within the biopsied muscle tissue will also provide us with insight how NO and 
Figure 6. Preliminary data showing the induction of 
AMPK-associated signals for autophagy activation in 
mouse skeletal muscle (A) and heart (B and C). In these 
studies an acute bout of treadmill exercise was used to 
induce autophagy in skeletal muscle and in the heart 
(n=3 in each group). Representative images denoting 
increased phosphorylation of AMPK (T172) and of its 
downstream target unc-51 like autophagy activating 
kinase 1 (ULK1, at S555) occurring in both skeletal 
muscle (A) and heart (B) in response to exercise. (A) 
Flexor digitorum brevis (FDB) muscle was removed from 
sedentary control mice (c), as well as from mice that 
exercised immediately and 45 min post-exercise (0 and 
45, respectively); (B) Left ventricle lysates from 
sedentary control mice (con) and from mice at the end of 
A 
B 
C 

associated metabolites may impact mitochondria function at different levels.  Lastly, we have the 
experimental tools to conduct follow-up microarray RNA studies to identify muscle transcriptional 
signatures associated with reduced baseline mitochondrial function, as well as improved function 
as a result of nitrate supplementation in T2D. This non-biased approach will allow us to develop 
future mechanistic proposals to identify novel therapeutic targets for diabetic patients. 
 
 
 
 
 
 
Rationale and hypothesis: Several studies suggest that patients with T2D have an impaired 
exercise tolerance,6-9 which is not associated with the degree of glycemic control.9  Of clinical 
significance, a lower VO2peak is associated with a higher rate of subsequent cardiovascular 
events in patients with T2D.22  Recent evidence suggests that dietary nitrate can improve exercise 
efficiency and tolerance in young and older adults.10-14 Therefore, we hypothesize that increasing 
bioavailable NO via nitrate supplementation will improve maximal exercise capacity and efficiency 
(e.g. reduce the oxygen cost for a given workload). In support of this hypothesis, our preliminary 
data suggests that 8 weeks of nitrate supplementation is effective in improving 6 minute walk test 
(6MWT) distance, a clinical measure of functional capacity, in patients with peripheral artery 
disease (Figure 7).  Ultimately, exercise tolerance is dependent on the delivery and utilization of 
oxygen to the working muscles, both of which involve NO-mediated mechanisms.30, 86  Therefore, 
we also hypothesize that the improvements in exercise efficiency will be associated with an 
increased blood flow response in contracting muscles (Aim 1) and/or mitochondrial function (Aim 
2). Along these lines, our preliminary data in older adults (Figure 8) support the idea that a greater 
blood flow response to muscle contractions, a NO-mediated response,51 is associated with 
enhanced exercise efficiency.  
 
 
 
 
 
Figure 8. Preliminary data highlighting the 
relationship between contraction-induced 
blood flow and exercise efficiency in 
humans.  
Figure 7. Preliminary data demonstrating 
a significant increase in walking distance 
during a 6-mintue walk test following 8 
weeks of NaNO3 in a clinical population 
with severe exercise intolerance. *P<0.05 
vs Placebo 
Aim 3 – Determine if exercise capacity and efficiency are improved following 8 weeks 
of dietary nitrate supplementation in patients with T2D and examine whether these 
improvements are associated with changes in oxygen delivery and utilization (as 
assessed in Aims 1 and 2).   

General Approach: Exercise efficiency and capacity will be assessed using a 12-lead ECG, 
symptom limited exercise test on a cycle ergometer with gas exchange measurements before 
and after 8 weeks of inorganic nitrate or placebo.  The main comparisons will be changes in 
VO2peak and exercise efficiency after 8 weeks of dietary nitrate supplementation.  The 
associations between improvements in exercise capacity/efficiency and muscle blood flow will 
also be explored. 
 
Potential Results, Interpretation & Follow-Up Studies: We expect that the dietary nitrate 
intervention will increase exercise capacity (VO2peak) as well as exercise efficiency.  Coupled 
with the protocols outlined in Aims 1 and 2, we will be able to determine if the potential 
improvements in exercise capacity and efficiency following nitrate supplementation are 
associated with changes in blood flow, mitochondrial function, or both. However, it is possible 
improvements in exercise capacity and efficiency may be observed without changes in local 
control of skeletal muscle blood flow or mitochondrial function.  If this is the case, follow-up studies 
could be aimed at looking at potential changes in myocardial function following dietary nitrate 
supplementation. 
 
Summary of Protocols: We have outlined a series of protocols to test our Aims and anticipate 
conducting ~40 sets of studies (either pre or post) per year to address our Aims. We have 
preliminary data that supports the questions we raise and demonstrates the technical ability 
needed to address them. Each protocol has clearly stated hypotheses with clear outcomes linked 
to each hypothesis and we have outlined key potential results, data interpretation and ideas about 
follow-up studies. 
 
 
Technical Expertise of Investigative Team. The table below shows the team’s experience with 
key elements of the proposal. 
 Technique/Approach 
Investigators 
# Performed 
Years of 
Experience 
Exercise blood flow studies 
Darren Casey, PhD 
>500 
10 
Arterial catheterization 
Kenichi Ueda, MD 
>500 
10 
Drugs to Study Human 
Physiology 
Darren Casey, PhD 
>500 
8 
Exercise Testing 
Darren Casey, PhD 
Kenichi Ueda, MD 
>500 
>50 
12 
2 
Muscle Biopsies 
Richard Shields, 
PhD 
>200 
25 
Mitochondrial content, enzyme 
activity and autophagy 
measures  
Vitor Lira, PhD 
~100 
8 
Mitochondrial function studies  
E. Dale Abel, MD 
PhD 
>500 
>10 
 
Potential Risks. 
Risks from participation in these studies are not considered minimal. 
 
1.  Brachial artery catheterization.  The brachial artery will be catheterized for ~4.5 hours in the 
proposed experiments.  A 2-inch standard 20-gauge Teflon catheter will be used, and 
continuously flushed with low dose saline (2 units/ml, 3 cc/hr).  Cannulation will be done using 

ultrasound guidance under aseptic technique after infiltration of the area with 1-2 ml of 
lidocaine.  Minor risk associated with this procedure would be the pain at the site of insertion, 
hematoma after withdrawal, and soreness over the site.  These should be transient and resolve 
after 2-10 days.  Major complications associated with brachial artery catheterization would include 
vasospasm, thromboembolism, or infection.  Two large reviews (N = 4,000 patients) of brachial 
artery catheterization for 24 or more hours in older patients with cardiovascular disease have 
been published.  These reviews report only two serious complications (one occlusive clot and one 
aneurysm) and indicate that brachial catheterization has less risk than radial artery 
catheterization.  Dr. Kenichi Ueda has extensive experience (10+ years) with these procedures 
as part of his routine clinical duties.  Dr. Casey has been involved with >300 arterial 
catheterization studies in the last 8 years without any major complications.  
 
2. Forearm drug infusions.  Because the doses used are very small and infused locally via the 
arterial catheter, there should be little or no effect on systemic hemodynamics (e.g., blood 
pressure and heart rate). Intra-arterially administration of adenosine and acetylcholine will cause 
an increase in blood flow (i.e. vasodilators) and thus carry minimal risk. Phenylephrine and 
dexmedetomidine cause a reduction in limb blood flow (i.e. vasoconstriction; < 25-50%) similar to 
that associated with the vasoconstriction seen when moving from the supine to upright posture. 
These compounds are well tolerated and we are unaware of any allergic or other untoward 
reactions when administered in this fashion. Dr. Casey and Dr. Ueda have extensive experience 
over the last several years with administration of vasoactive drugs via arterial catheter or IV.  
 
3. Lidocaine: 2% lidocaine will be used to numb the area on the arm for insertion of the brachial 
artery needle. Likely and expected effects include mild, temporary burning at the site of injection 
and a sensation of coolness or numbness at the site of the injection. Unlikely, but possible 
reactions include lightheadedness, dizziness, ringing in the ears, nervousness, and blurred vision. 
Individuals known to be allergic to anesthetics like lidocaine (e.g., novocaine- used for tooth 
fillings) should notify the investigator before participating and will not undergo the forearm blood 
flow test. 
 
4.  Venipuncture/IV catheters.  Risks of venipuncture or placing IV catheters in the arm include 
bruise or clot formation and infection. 
 
5.  Forearm exercise. There are no long term risks associated with rhythmic forearm handgrip 
contractions.  Rhythmic handgrip exercise may lead to minor fatigue in the hand and/or forearm 
and soreness for a day or two. 
  
6. Doppler ultrasound. There is no risk associated with Doppler ultrasound measurements. 
 
7. Applanation tonometry. There is no risk associated with arterial tonometry measurements. 
 
8. Maximal exercise testing.  This procedure will be used to determine each subjects exercise 
capacity. There is minor discomfort associated with maximal exercise testing including temporary 
fatigue, shortness of breath, and muscle soreness.  These sensations resolve within minutes after 
the test or exercise session is completed.  In healthy older subjects screened with a questionnaire 
who are normotensive, non-smokers, not obese, and non-diabetic the risk of a catastrophic event 
during graded exercise tests for screening purposes are thought to be less than 1 in 10,000 
(Squires, personal communication).  According to the American College of Sports Medicine the 
risk of a cardiovascular (CV) event during maximal exercise testing increases in individuals with 
CV disease and/or risk factors for CV disease.  However, safe testing with appropriate monitoring 
(blood pressure, ECG, and physician supervised) is still recommended in this patient population.  

The exercise tests performed in this study will be conducted by well-trained personnel and 
supervised by medical personnel (Dr. Ueda) who are familiar with the use of graded exercise 
testing. Moreover heart rate and rhythm will be monitored via 12-lead ECG and blood pressure 
measured throughout the test as well as during a recovery period following termination of 
exercise.  After maximal exercise, serious risks during submaximal exercise are extremely small, 
except for temporary fatigue and muscle soreness. 
 
9. Muscle biopsies: Biopsies will be performed under sterile conditions to minimize the risk of 
infection. However, there is a risk of bleeding, bruising, pain and/or swelling after the muscle 
sample is taken. To further minimize such risks subjects will be instructed to leave the bandage 
on the biopsy site for 24 hours and to not allow the area to become wet for 48 hours after the 
procedure. 
 
 
Summary of risks: The proposed study will include both invasive and non-invasive techniques.  
Regarding invasive approaches Drs. Casey and Ueda have extensive experience conducting 
brachial artery catheterization studies and Dr. Shields has extensive experience in performing 
muscle biopsies.  Along these lines, we are confident that at the very least, the standard care of 
practice will be met before, during and after artery catheterization, drug infusions, and muscle 
biopsies.  Additionally, with regard to the non-invasive approaches of the proposed study, the 
team has a long history of conducting these types of studies in conscious humans, and we have 
standard operating procedures in our laboratory to minimize the risks and to ensure that these 
studies can be done safely. 
 
Protection. 
Each subject will give written informed consent after all questions have been answered by an 
investigator or study coordinator.  Confidentiality of data will be assured by coding of subject 
identities, and that coding will be known only to the investigators.  No identified individual data will 
be presented or published, rather group mean data will serve as the basis for comparisons.  
Participation in these studies is safe, with minimal short-term (days) complications and minimal 
anticipated long-term risks.  Procedures used in this study are performed regularly in the 
laboratory or clinical setting by the investigators.   
 
Inclusion of Women and Minorities. 
The proposed studies will include older (40-70 yr) men and women of all races and ethnic 
backgrounds recruited from the University of Iowa and from persons in Iowa City, Iowa and 
surrounding communities.  We will strive to enroll an equal number of males and females and 
recruit minority participants at least to the extent representative of the participation of minority 
populations in Iowa City and Johnson County.   
 
Inclusion of Children 
The proposed studies will only include older (40-70 yr) men and women.  Therefore, children will 
be excluded from the proposed studies. 
 
 
 

References 
 
1. Kingwell BA, Formosa M, Muhlmann M, Bradley SJ, McConell GK. Type 2 diabetic 
individuals have impaired leg blood flow responses to exercise: Role of endothelium-
dependent vasodilation. Diabetes care. 2003;26:899-904 
2. Lalande S, Gusso S, Hofman PL, Baldi JC. Reduced leg blood flow during submaximal 
exercise in type 2 diabetes. Medicine and science in sports and exercise. 2008;40:612-617 
3. MacAnaney O, Reilly H, O'Shea D, Egana M, Green S. Effect of type 2 diabetes on the 
dynamic response characteristics of leg vascular conductance during exercise. Diabetes & 
vascular disease research : official journal of the International Society of Diabetes and 
Vascular Disease. 2011;8:12-21 
4. Zheng J, Hasting MK, Zhang X, Coggan A, An H, Snozek D, Curci J, Mueller MJ. A pilot 
study of regional perfusion and oxygenation in calf muscles of individuals with diabetes with 
a noninvasive measure. Journal of vascular surgery. 2014;59:419-426 
5. Mogensen M, Sahlin K, Fernstrom M, Glintborg D, Vind BF, Beck-Nielsen H, Hojlund K. 
Mitochondrial respiration is decreased in skeletal muscle of patients with type 2 diabetes. 
Diabetes. 2007;56:1592-1599 
6. Bauer TA, Reusch JE, Levi M, Regensteiner JG. Skeletal muscle deoxygenation after the 
onset of moderate exercise suggests slowed microvascular blood flow kinetics in type 2 
diabetes. Diabetes care. 2007;30:2880-2885 
7. Brandenburg SL, Reusch JE, Bauer TA, Jeffers BW, Hiatt WR, Regensteiner JG. Effects of 
exercise training on oxygen uptake kinetic responses in women with type 2 diabetes. 
Diabetes care. 1999;22:1640-1646 
8. Regensteiner JG, Bauer TA, Reusch JE, Brandenburg SL, Sippel JM, Vogelsong AM, Smith 
S, Wolfel EE, Eckel RH, Hiatt WR. Abnormal oxygen uptake kinetic responses in women 
with type ii diabetes mellitus. Journal of applied physiology. 1998;85:310-317 
9. Regensteiner JG, Sippel J, McFarling ET, Wolfel EE, Hiatt WR. Effects of non-insulin-
dependent diabetes on oxygen consumption during treadmill exercise. Medicine and 
science in sports and exercise. 1995;27:661-667 
10. Bailey SJ, Fulford J, Vanhatalo A, Winyard PG, Blackwell JR, DiMenna FJ, Wilkerson DP, 
Benjamin N, Jones AM. Dietary nitrate supplementation enhances muscle contractile 
efficiency during knee-extensor exercise in humans. Journal of applied physiology. 
2010;109:135-148 
11. Bailey SJ, Winyard P, Vanhatalo A, Blackwell JR, Dimenna FJ, Wilkerson DP, Tarr J, 
Benjamin N, Jones AM. Dietary nitrate supplementation reduces the o2 cost of low-intensity 
exercise and enhances tolerance to high-intensity exercise in humans. Journal of applied 
physiology. 2009;107:1144-1155 
12. Kelly J, Fulford J, Vanhatalo A, Blackwell JR, French O, Bailey SJ, Gilchrist M, Winyard PG, 
Jones AM. Effects of short-term dietary nitrate supplementation on blood pressure, o2 
uptake kinetics, and muscle and cognitive function in older adults. American journal of 
physiology. Regulatory, integrative and comparative physiology. 2013;304:R73-83 
13. Lansley KE, Winyard PG, Fulford J, Vanhatalo A, Bailey SJ, Blackwell JR, DiMenna FJ, 
Gilchrist M, Benjamin N, Jones AM. Dietary nitrate supplementation reduces the o2 cost of 
walking and running: A placebo-controlled study. Journal of applied physiology. 
2011;110:591-600 
14. Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate on oxygen cost 
during exercise. Acta physiologica. 2007;191:59-66 
15. Patti ME, Corvera S. The role of mitochondria in the pathogenesis of type 2 diabetes. 
Endocrine reviews. 2010;31:364-395 

16. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart 
disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior 
myocardial infarction. The New England journal of medicine. 1998;339:229-234 
17. Wannamethee SG, Perry IJ, Shaper AG. Nonfasting serum glucose and insulin 
concentrations and the risk of stroke. Stroke; a journal of cerebral circulation. 1999;30:1780-
1786 
18. Almdal T, Scharling H, Jensen JS, Vestergaard H. The independent effect of type 2 diabetes 
mellitus on ischemic heart disease, stroke, and death: A population-based study of 13,000 
men and women with 20 years of follow-up. Archives of internal medicine. 2004;164:1422-
1426 
19. Blair SN, Kampert JB, Kohl HW, 3rd, Barlow CE, Macera CA, Paffenbarger RS, Jr., Gibbons 
LW. Influences of cardiorespiratory fitness and other precursors on cardiovascular disease 
and all-cause mortality in men and women. JAMA : the journal of the American Medical 
Association. 1996;276:205-210 
20. Kokkinos P, Myers J, Kokkinos JP, Pittaras A, Narayan P, Manolis A, Karasik P, Greenberg 
M, Papademetriou V, Singh S. Exercise capacity and mortality in black and white men. 
Circulation. 2008;117:614-622 
21. Snader CE, Marwick TH, Pashkow FJ, Harvey SA, Thomas JD, Lauer MS. Importance of 
estimated functional capacity as a predictor of all-cause mortality among patients referred 
for exercise thallium single-photon emission computed tomography: Report of 3,400 patients 
from a single center. Journal of the American College of Cardiology. 1997;30:641-648 
22. Seyoum B, Estacio RO, Berhanu P, Schrier RW. Exercise capacity is a predictor of 
cardiovascular events in patients with type 2 diabetes mellitus. Diabetes & vascular disease 
research : official journal of the International Society of Diabetes and Vascular Disease. 
2006;3:197-201 
23. Reusch JE, Bridenstine M, Regensteiner JG. Type 2 diabetes mellitus and exercise 
impairment. Reviews in endocrine & metabolic disorders. 2013;14:77-86 
24. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing 
factor produced and released from artery and vein is nitric oxide. Proceedings of the 
National Academy of Sciences of the United States of America. 1987;84:9265-9269 
25. Tessari P, Cecchet D, Cosma A, Vettore M, Coracina A, Millioni R, Iori E, Puricelli L, 
Avogaro A, Vedovato M. Nitric oxide synthesis is reduced in subjects with type 2 diabetes 
and nephropathy. Diabetes. 2010;59:2152-2159 
26. Bender SB, Herrick EK, Lott ND, Klabunde RE. Diet-induced obesity and diabetes reduce 
coronary responses to nitric oxide due to reduced bioavailability in isolated mouse hearts. 
Diabetes, obesity & metabolism. 2007;9:688-696 
27. Preik M, Kelm M, Rosen P, Tschope D, Strauer BE. Additive effect of coexistent type 2 
diabetes and arterial hypertension on endothelial dysfunction in resistance arteries of human 
forearm vasculature. Angiology. 2000;51:545-554 
28. Sivitz WI, Wayson SM, Bayless ML, Sinkey CA, Haynes WG. Obesity impairs vascular 
relaxation in human subjects: Hyperglycemia exaggerates adrenergic vasoconstriction 
arterial dysfunction in obesity and diabetes. Journal of diabetes and its complications. 
2007;21:149-157 
29. Woodman RJ, Playford DA, Watts GF. Basal production of nitric oxide (no) and non-no 
vasodilators in the forearm microcirculation in type 2 diabetes: Associations with blood 
pressure and hdl cholesterol. Diabetes research and clinical practice. 2006;71:59-67 
30. Dai Z, Wu Z, Yang Y, Wang J, Satterfield MC, Meininger CJ, Bazer FW, Wu G. Nitric oxide 
and energy metabolism in mammals. BioFactors. 2013;39:383-391 
31. Kobzik L, Stringer B, Balligand JL, Reid MB, Stamler JS. Endothelial type nitric oxide 
synthase in skeletal muscle fibers: Mitochondrial relationships. Biochemical and biophysical 
research communications. 1995;211:375-381 

32. Sarti P, Arese M, Forte E, Giuffre A, Mastronicola D. Mitochondria and nitric oxide: 
Chemistry and pathophysiology. Advances in experimental medicine and biology. 
2012;942:75-92 
33. Nisoli E, Carruba MO. Nitric oxide and mitochondrial biogenesis. Journal of cell science. 
2006;119:2855-2862 
34. Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valerio A, 
Francolini M, Moncada S, Carruba MO. Mitochondrial biogenesis in mammals: The role of 
endogenous nitric oxide. Science. 2003;299:896-899 
35. Lira VA, Brown DL, Lira AK, Kavazis AN, Soltow QA, Zeanah EH, Criswell DS. Nitric oxide 
and ampk cooperatively regulate pgc-1 in skeletal muscle cells. The Journal of physiology. 
2010;588:3551-3566 
36. Bryan NS. Nitrite in nitric oxide biology: Cause or consequence? A systems-based review. 
Free radical biology & medicine. 2006;41:691-701 
37. Lundberg JO, Gladwin MT, Ahluwalia A, Benjamin N, Bryan NS, Butler A, Cabrales P, Fago 
A, Feelisch M, Ford PC, Freeman BA, Frenneaux M, Friedman J, Kelm M, Kevil CG, Kim-
Shapiro DB, Kozlov AV, Lancaster JR, Jr., Lefer DJ, McColl K, McCurry K, Patel RP, 
Petersson J, Rassaf T, Reutov VP, Richter-Addo GB, Schechter A, Shiva S, Tsuchiya K, 
van Faassen EE, Webb AJ, Zuckerbraun BS, Zweier JL, Weitzberg E. Nitrate and nitrite in 
biology, nutrition and therapeutics. Nature chemical biology. 2009;5:865-869 
38. Lundberg JO, Weitzberg E. No generation from nitrite and its role in vascular control. 
Arteriosclerosis, thrombosis, and vascular biology. 2005;25:915-922 
39. Lundberg JO, Carlstrom M, Larsen FJ, Weitzberg E. Roles of dietary inorganic nitrate in 
cardiovascular health and disease. Cardiovascular research. 2011;89:525-532 
40. Weitzberg E, Lundberg JO. Novel aspects of dietary nitrate and human health. Annual 
review of nutrition. 2013;33:129-159 
41. Heiss C, Meyer C, Totzeck M, Hendgen-Cotta UB, Heinen Y, Luedike P, Keymel S, Ayoub 
N, Lundberg JO, Weitzberg E, Kelm M, Rassaf T. Dietary inorganic nitrate mobilizes 
circulating angiogenic cells. Free radical biology & medicine. 2012;52:1767-1772 
42. Rammos C, Hendgen-Cotta UB, Sobierajski J, Bernard A, Kelm M, Rassaf T. Dietary nitrate 
reverses vascular dysfunction in older adults with moderately increased cardiovascular risk. 
Journal of the American College of Cardiology. 2014;63:1584-1585 
43. Webb AJ, Patel N, Loukogeorgakis S, Okorie M, Aboud Z, Misra S, Rashid R, Miall P, 
Deanfield J, Benjamin N, MacAllister R, Hobbs AJ, Ahluwalia A. Acute blood pressure 
lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to 
nitrite. Hypertension. 2008;51:784-790 
44. Kapil V, Milsom AB, Okorie M, Maleki-Toyserkani S, Akram F, Rehman F, Arghandawi S, 
Pearl V, Benjamin N, Loukogeorgakis S, Macallister R, Hobbs AJ, Webb AJ, Ahluwalia A. 
Inorganic nitrate supplementation lowers blood pressure in humans: Role for nitrite-derived 
no. Hypertension. 2010;56:274-281 
45. Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on 
blood pressure in healthy volunteers. The New England journal of medicine. 2006;355:2792-
2793 
46. Casey DP, Joyner MJ. Nos inhibition blunts and delays the compensatory dilation in 
hypoperfused contracting human muscles. Journal of applied physiology. 2009;107:1685-
1692 
47. Casey DP, Joyner MJ. Contribution of adenosine to compensatory dilation in hypoperfused 
contracting human muscles is independent of nitric oxide. Journal of applied physiology. 
2011;110:1181-1189 
48. Casey DP, Madery BD, Curry TB, Eisenach JH, Wilkins BW, Joyner MJ. Nitric oxide 
contributes to the augmented vasodilatation during hypoxic exercise. The Journal of 
physiology. 2010;588:373-385 

49. Casey DP, Madery BD, Pike TL, Eisenach JH, Dietz NM, Joyner MJ, Wilkins BW. Adenosine 
receptor antagonist and augmented vasodilation during hypoxic exercise. Journal of applied 
physiology. 2009;107:1128-1137 
50. Casey DP, Walker BG, Curry TB, Joyner MJ. Ageing reduces the compensatory 
vasodilatation during hypoxic exercise: The role of nitric oxide. The Journal of physiology. 
2011;589:1477-1488 
51. Casey DP, Walker BG, Ranadive SM, Taylor JL, Joyner MJ. Contribution of nitric oxide in 
the contraction-induced rapid vasodilation in young and older adults. Journal of applied 
physiology. 2013;115:446-455 
52. Kirby BS, Crecelius AR, Voyles WF, Dinenno FA. Modulation of postjunctional alpha-
adrenergic vasoconstriction during exercise and exogenous atp infusions in ageing humans. 
The Journal of physiology. 2011;589:2641-2653 
53. Kirby BS, Voyles WF, Carlson RE, Dinenno FA. Graded sympatholytic effect of exogenous 
atp on postjunctional alpha-adrenergic vasoconstriction in the human forearm: Implications 
for vascular control in contracting muscle. The Journal of physiology. 2008;586:4305-4316 
54. Adams CM, Suneja M, Dudley-Javoroski S, Shields RK. Altered mrna expression after long-
term soleus electrical stimulation training in humans with paralysis. Muscle & nerve. 
2011;43:65-75 
55. Kunkel SD, Suneja M, Ebert SM, Bongers KS, Fox DK, Malmberg SE, Alipour F, Shields 
RK, Adams CM. Mrna expression signatures of human skeletal muscle atrophy identify a 
natural compound that increases muscle mass. Cell metabolism. 2011;13:627-638 
56. Pesta D, Gnaiger E. High-resolution respirometry: Oxphos protocols for human cells and 
permeabilized fibers from small biopsies of human muscle. Methods in molecular biology. 
2012;810:25-58 
57. Mogensen M, Sahlin K. Mitochondrial efficiency in rat skeletal muscle: Influence of 
respiration rate, substrate and muscle type. Acta physiologica Scandinavica. 2005;185:229-
236 
58. Larsen FJ, Schiffer TA, Borniquel S, Sahlin K, Ekblom B, Lundberg JO, Weitzberg E. Dietary 
inorganic nitrate improves mitochondrial efficiency in humans. Cell metabolism. 
2011;13:149-159 
59. Boudina S, Bugger H, Sena S, O'Neill BT, Zaha VG, Ilkun O, Wright JJ, Mazumder PK, 
Palfreyman E, Tidwell TJ, Theobald H, Khalimonchuk O, Wayment B, Sheng X, Rodnick KJ, 
Centini R, Chen D, Litwin SE, Weimer BE, Abel ED. Contribution of impaired myocardial 
insulin signaling to mitochondrial dysfunction and oxidative stress in the heart. Circulation. 
2009;119:1272-1283 
60. Boudina S, Sena S, O'Neill BT, Tathireddy P, Young ME, Abel ED. Reduced mitochondrial 
oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in 
obesity. Circulation. 2005;112:2686-2695 
61. Bugger H, Boudina S, Hu XX, Tuinei J, Zaha VG, Theobald HA, Yun UJ, McQueen AP, 
Wayment B, Litwin SE, Abel ED. Type 1 diabetic akita mouse hearts are insulin sensitive but 
manifest structurally abnormal mitochondria that remain coupled despite increased 
uncoupling protein 3. Diabetes. 2008;57:2924-2932 
62. Anderson EJ, Lustig ME, Boyle KE, Woodlief TL, Kane DA, Lin CT, Price JW, 3rd, Kang L, 
Rabinovitch PS, Szeto HH, Houmard JA, Cortright RN, Wasserman DH, Neufer PD. 
Mitochondrial h2o2 emission and cellular redox state link excess fat intake to insulin 
resistance in both rodents and humans. The Journal of clinical investigation. 2009;119:573-
581 
63. Okutsu M, Call JA, Lira VA, Zhang M, Donet JA, French BA, Martin KS, Peirce-Cottler SM, 
Rembold CM, Annex BH, Yan Z. Extracellular superoxide dismutase ameliorates skeletal 
muscle abnormalities, cachexia, and exercise intolerance in mice with congestive heart 
failure. Circulation. Heart failure. 2014;7:519-530 

64. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, et al. Guidelines 
for the use and interpretation of assays for monitoring autophagy. Autophagy. 2012;8:445-
544 
65. Lira VA, Okutsu M, Zhang M, Greene NP, Laker RC, Breen DS, Hoehn KL, Yan Z. 
Autophagy is required for exercise training-induced skeletal muscle adaptation and 
improvement of physical performance. FASEB journal : official publication of the Federation 
of American Societies for Experimental Biology. 2013;27:4184-4193 
66. Khoo NK, Mo L, Zharikov S, Kamga-Pride C, Quesnelle K, Golin-Bisello F, Li L, Wang Y, 
Shiva S. Nitrite augments glucose uptake in adipocytes through the protein kinase a-
dependent stimulation of mitochondrial fusion. Free radical biology & medicine. 2014;70:45-
53 
67. Kanaley JA, Goulopoulou S, Franklin R, Baynard T, Carhart RL, Jr., Weinstock RS, Fernhall 
B. Exercise training improves hemodynamic recovery to isometric exercise in obese men 
with type 2 diabetes but not in obese women. Metabolism: clinical and experimental. 
2012;61:1739-1746 
68. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones AM, Musch TI, Poole 
DC. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle 
vascular control in rats. The Journal of physiology. 2013;591:547-557 
69. Ferguson SK, Hirai DM, Copp SW, Holdsworth CT, Allen JD, Jones AM, Musch TI, Poole 
DC. Dose dependent effects of nitrate supplementation on cardiovascular control and 
microvascular oxygenation dynamics in healthy rats. Nitric oxide : biology and chemistry / 
official journal of the Nitric Oxide Society. 2014;39:51-58 
70. Casey DP, Treichler DP, Ganger CTt, Schneider AC, Ueda K. Acute dietary nitrate 
supplementation enhances compensatory vasodilation during hypoxic exercise in older 
adults. Journal of applied physiology. 2015;118:178-186 
71. Casey DP, Joyner MJ, Claus PL, Curry TB. Vasoconstrictor responsiveness during 
hyperbaric hyperoxia in contracting human muscle. Journal of applied physiology. 
2013;114:217-224 
72. Dinenno FA, Joyner MJ. Blunted sympathetic vasoconstriction in contracting skeletal muscle 
of healthy humans: Is nitric oxide obligatory? The Journal of physiology. 2003;553:281-292 
73. Hansen J, Sayad D, Thomas GD, Clarke GD, Peshock RM, Victor RG. Exercise-induced 
attenuation of alpha-adrenoceptor mediated vasoconstriction in humans: Evidence from 
phase-contrast mri. Cardiovascular research. 1999;41:220-228 
74. Remensnyder JP, Mitchell JH, Sarnoff SJ. Functional sympatholysis during muscular 
activity. Observations on influence of carotid sinus on oxygen uptake. Circulation research. 
1962;11:370-380 
75. Tschakovsky ME, Sujirattanawimol K, Ruble SB, Valic Z, Joyner MJ. Is sympathetic neural 
vasoconstriction blunted in the vascular bed of exercising human muscle? The Journal of 
physiology. 2002;541:623-635 
76. Chavoshan B, Sander M, Sybert TE, Hansen J, Victor RG, Thomas GD. Nitric oxide-
dependent modulation of sympathetic neural control of oxygenation in exercising human 
skeletal muscle. The Journal of physiology. 2002;540:377-386 
77. Sander M, Chavoshan B, Harris SA, Iannaccone ST, Stull JT, Thomas GD, Victor RG. 
Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of 
children with duchenne muscular dystrophy. Proceedings of the National Academy of 
Sciences of the United States of America. 2000;97:13818-13823 
78. Thomas GD, Sander M, Lau KS, Huang PL, Stull JT, Victor RG. Impaired metabolic 
modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle. 
Proceedings of the National Academy of Sciences of the United States of America. 
1998;95:15090-15095 

79. Thomas GD, Victor RG. Nitric oxide mediates contraction-induced attenuation of 
sympathetic vasoconstriction in rat skeletal muscle. The Journal of physiology. 1998;506 ( 
Pt 3):817-826 
80. Huggett RJ, Scott EM, Gilbey SG, Stoker JB, Mackintosh AF, Mary DA. Impact of type 2 
diabetes mellitus on sympathetic neural mechanisms in hypertension. Circulation. 
2003;108:3097-3101 
81. Kobayashi D, Takamura M, Murai H, Usui S, Ikeda T, Inomata J, Takashima S, Kato T, 
Furusho H, Takeshita Y, Ota T, Takamura T, Kaneko S. Effect of pioglitazone on muscle 
sympathetic nerve activity in type 2 diabetes mellitus with alpha-glucosidase inhibitor. 
Autonomic neuroscience : basic & clinical. 2010;158:86-91 
82. Kelley DE, He J, Menshikova EV, Ritov VB. Dysfunction of mitochondria in human skeletal 
muscle in type 2 diabetes. Diabetes. 2002;51:2944-2950 
83. Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, Vasquez DS, Joshi 
A, Gwinn DM, Taylor R, Asara JM, Fitzpatrick J, Dillin A, Viollet B, Kundu M, Hansen M, 
Shaw RJ. Phosphorylation of ulk1 (hatg1) by amp-activated protein kinase connects energy 
sensing to mitophagy. Science. 2011;331:456-461 
84. Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, Shulman GI. Amp kinase is 
required for mitochondrial biogenesis in skeletal muscle in response to chronic energy 
deprivation. Proceedings of the National Academy of Sciences of the United States of 
America. 2002;99:15983-15987 
85. Lira VA, Soltow QA, Long JH, Betters JL, Sellman JE, Criswell DS. Nitric oxide increases 
glut4 expression and regulates ampk signaling in skeletal muscle. American journal of 
physiology. Endocrinology and metabolism. 2007;293:E1062-1068 
86. Joyner MJ, Casey DP. The catecholamines strike back. What no does not do. Circulation 
journal : official journal of the Japanese Circulation Society. 2009;73:1783-1792