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Am J Physiol Heart Circ Physiol 307: H840H847, 2014.

First published July 18, 2014; doi:10.1152/ajpheart.00312.2014.

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Cardiovascular Consequences of Obesity and Type 2 Diabetes

Exercise-mediated vasodilation in human obesity and metabolic syndrome:


effect of acute ascorbic acid infusion
Jacqueline K. Limberg,1 J. Mikhail Kellawan,1 John W. Harrell,1 Rebecca E. Johansson,1
Marlowe W. Eldridge,2 Lester T. Proctor,3 Joshua J. Sebranek,3 and William G. Schrage1
1

Department of Kinesiology, School of Education, University of Wisconsin, and Departments of 2Pediatrics


and 3Anesthesiology, School of Medicine and Public Health, University of Wisconsin Hospital and Clinics,
Madison, Wisconsin
Submitted 9 May 2014; accepted in final form 17 July 2014

Limberg JK, Kellawan JM, Harrell JW, Johansson RE,


Eldridge MW, Proctor LT, Sebranek JJ, Schrage WG. Exercisemediated vasodilation in human obesity and metabolic syndrome:
effect of acute ascorbic acid infusion. Am J Physiol Heart Circ
Physiol 307: H840 H847, 2014. First published July 18, 2014;
doi:10.1152/ajpheart.00312.2014.We tested the hypothesis that infusion of ascorbic acid (AA), a potent antioxidant, would alter
vasodilator responses to exercise in human obesity and metabolic
syndrome (MetSyn). Forearm blood flow (FBF, Doppler ultrasound)
was measured in lean, obese, and MetSyn adults (n 39, 32 2 yr).
A brachial artery catheter was inserted for blood pressure monitoring
and local infusion of AA. FBF was measured during dynamic handgrip exercise (15% maximal effort) with and without AA infusion. To
account for group differences in blood pressure and forearm size, and
to assess vasodilation, forearm vascular conductance (FVC FBF/
mean arterial blood pressure/lean forearm mass) was calculated. We
examined the time to achieve steady-state FVC (mean response time,
MRT) and the rise in FVC from rest to steady-state exercise (,
exercise rest) before and during acute AA infusion. The MRT (P
0.26) and steady-state vasodilator responses to exercise (FVC, P
0.31) were not different between groups. Intra-arterial infusion of AA
resulted in a significant increase in plasma total antioxidant capacity
(174 37%). AA infusion did not alter MRT or steady-state FVC in
any group (P 0.90 and P 0.85, respectively). Interestingly, higher
levels of C-reactive protein predicted longer MRT (r 0.52, P
0.01) and a greater reduction in MRT with AA infusion (r 0.43,
P 0.02). We concluded that AA infusion during moderate-intensity,
rhythmic forearm exercise does not alter the time course or magnitude
of exercise-mediated vasodilation in groups of young lean, obese, or
MetSyn adults. However, systemic inflammation may limit the MRT
to exercise, which can be improved with AA.
blood flow; reactive oxygen species; antioxidant
OVER 25% OF YOUNGER ADULTS (ages 20 40 yr) are obese, and
greater than one-third of the United States population meets the
criteria for metabolic syndrome (MetSyn; obesity, hypertension, dyslipidemia, hyperglycemia) (8). Exercise is an effective
way to prevent and/or combat obesity and obesity-related
disorders, such as MetSyn. Unfortunately, adults with MetSyn
exhibit exaggerated blood pressure responses to whole body
exercise (30, 38), which may be the result of decreased skeletal
muscle vasodilation and/or increased vasoconstriction. In sup-

Address for reprint requests and other correspondence: W. G. Schrage;


2000 Observatory Dr., Rm 1149A, Madison, WI 53706 (e-mail wschrage
@education.wisc.edu).
H840

port of this idea, animal models of obesity and MetSyn exhibit


blunted vasodilation, increased vasoconstriction, and impaired
blood flow distribution within the skeletal muscle microcirculation in response to simulated exercise (2, 10, 13). Such
impairments have been linked to increased reactive oxygen
species (ROS), such that scavenging of ROS via antioxidant
therapies can improve exercise-mediated vasodilation in these
animal models (9, 12).
We have shown previously that rapid-onset vasodilation in
response to forearm muscle contraction is blunted in human
obesity (1). On the other hand, steady-state vasodilator responses to dynamic forearm exercise are preserved, or even
increased, in young obese humans and those with MetSyn (25,
26). The potential reasons behind this discrepancy are not
completely understood, although mechanisms responsible for
vascular responses to exercise onset are unlikely to mirror
those important in maintaining blood flow and vascular tone
during steady-state exercise (24). Furthermore, data from our
laboratory are consistent with the concept that blood flow
distribution may be altered in human MetSyn during steadystate exercise (28).
How ROS might influence these vascular responses to exercise
in obese and MetSyn humans is largely unknown. As noted
above, excessive ROS may impair exercise-mediated vasodilation
(11, 21), and recent research suggests that treatment with antioxidants may improve blood flow distribution in animal models (2,
10, 13). Conversely, increased ROS may serve as an important,
alternative vasodilator mechanism that compensates for loss of
other vascular control mechanisms. Consistent with this concept,
ROS signaling is important to the maintenance of blood flow and
vascular tone in the coronary circulation of humans with cardiovascular disease (16, 23), such that increasing antioxidant capacity
might limit vasodilation in diseased patients.
With these ideas in mind, we aimed to examine the effect of
increasing total antioxidant capacity on vasodilator responses
to exercise onset and steady-state exercise in human obesity
and MetSyn. We hypothesized that acute antioxidant (ascorbic
acid, AA) administration would increase vasodilation at exercise
onset yet may blunt responses during steady-state, moderateintensity exercise in obese adults and adults with MetSyn. Furthermore, given that levels of oxidative stress increase with
obesity and further rise in MetSyn (14), we hypothesized that
acute AA infusion would have a graded effect on exercisemediated vasodilation, with adults with MetSyn seeing the greatest changes.

0363-6135/14 Copyright 2014 the American Physiological Society

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EXERCISE HYPEREMIA IN OBESITY AND METSYN


MATERIALS AND METHODS

Procedures completed as part of the present study were part of a


larger protocol testing resting microvascular endothelial and smooth
muscle function (27).
Ethical approval. Procedures were submitted to and approved by
the Institutional Review Board at the University of Wisconsin Madison and conformed to the standards set by the Declaration of
Helsinki. Written informed consent was obtained before study participation.
Subjects. Subjects were between the ages of 18 40 yr to limit the
confounding vascular effects of aging (21). Subjects were nonsmokers, generally healthy, and were not taking any cardiovascular medications, as determined by self-report. Two subjects reported consuming supplemental antioxidants [obese n 1 (acai berry), MetSyn n
1 (American ginseng, coenzyme Q10)]; however, when excluded
from analysis, conclusions were maintained. Therefore, both subjects
were included in the present study. Adults were characterized as
MetSyn if they met three of the following National Cholesterol
Education Program Adult Treatment Panel III criteria as modified by
the American Diabetes Association: central obesity (waist circumference 88 cm for women/102 cm for men), prehypertension (resting
blood pressure 130 mmHg/85 mmHg), hypertriglyceridemia (triglycerides 150 mg/dl), hyperglycemia (fasting glucose 100 mg/dl),
and/or dyslipidemia [high-density lipoprotein (HDL) 50 mg/dl
women/40 mg/dl men] (15). Obese subjects had a body mass index
(BMI) 30 kg/m2 but were otherwise healthy (not meeting MetSyn
criteria). Female subjects were not pregnant and were studied during
the early follicular phase (days 15) of the menstrual cycle. Hormonal
contraception was allowed, and women on contraception (Lean n 2,
Obese n 1, MetSyn n 1) were studied during the placebo phase.
Subjects were instructed to refrain from exercise, nonsteroidal antiinflammatory drugs, alcohol, and caffeine for 24 h before the study
day.
Measurements. Weight and height were measured, and body composition was determined by waist circumference, BMI (kg/m), and
dual-energy X-ray absorptometry (GE Lunar Prodigy; GE Healthcare,
Milwaukee, WI). Lean forearm mass was analyzed from whole body
DEXA scans using anatomical landmarks (33). Maximal voluntary
contraction (MVC, kg) of the nondominant arm was determined as the
average of the two highest measurements from five trials using a hand
dynamometer. Arterial blood was collected after a 10-h fast, and
levels of triglycerides, HDL, low-density lipoprotein (LDL), and
glucose were measured immediately (CardioChek; PTS Panels, Indianapolis, IN). Additional plasma samples were collected before study
interventions, frozen at 80C and analyzed at a later date for insulin
(Millipore, Billerica, MA), C-reactive protein (CRP; R&D Systems,
Minneapolis, MN), thiobarbituric acid-reactive substances (TBARS;
Cayman Chemical, Ann Arbor, MI), and total antioxidant capacity
(TAC; Ref. 34). A second plasma sample was collected for measures
of TAC at the conclusion of study interventions, just before removal
of the arterial catheter. A change in TAC is reported relative to
baseline [(Intervention Baseline)/Baseline 100]. To examine
potential insulin resistance, the homeostatic model assessment
[HOMA-IR (Fasting Glucose Fasting Insulin)/405] and quantitative insulin sensitivity check index [QUICKI 1/log(Fasting Insulin) log(Fasting Glucose)] were assessed. To examine physical
activity levels, subjects completed the Paffenbarger questionnaire
(31), a validated tool for assessing physical activity over the course of
a year based on blocks walked, flights of stairs taken, and participation
in other sports and recreational activities.
Brachial artery catheterization. A 20-gauge, 5-cm catheter was
placed in the brachial artery of the nondominant forearm under aseptic
conditions and after local anesthesia (2% lidocaine). The catheter was
used for continuous blood pressure measurement, blood sampling, and
local administration of AA (8 mgdl forearm volume1min1 fol-

H841

lowed by a 2.5 mg/min maintenance infusion). AA (Bioniche Pharma


USA, Lake Forest, IL) was used as a potent antioxidant and was
mixed specifically for each study visit. The dose of AA was greater
than or equal to that shown previously to increase endothelialdependent vasodilation (20, 21, 27, 32) and blood flow responses to
exercise (7, 21) in humans.
Blood flow. Forearm blood flow (FBF; artery diameter, blood
velocity) was measured using Doppler ultrasound (Vivid 7; General
Electric, Milwaukee, WI). The ultrasound probe operator placed the
12-MHz linear array probe medial to the biceps brachii muscle, with
position adjustments to maintain a fixed insonation angle of 60
degrees, and the sample volume was adjusted to cover the width of the
brachial artery (27). A mark was made on the skin over the brachial
artery to ensure measurements were taken in the same anatomical
position for each trial.
Forearm exercise. Each subject was supine with the nondominant
arm extended to the side, at heart level. Dynamic and rhythmic,
nondominant forearm exercise required subjects to squeeze and release two handles together 4 5 cm to raise and lower a weight over a
pulley. Exercise was completed at a rate of 20 times per minute (at
a duty cycle of 1-s contraction:2-s relaxation) to the rhythm of a
metronome. A mild (15% MVC) workload was used to minimize
systemic effects of exercise (i.e., increases in sympathetic nervous
system activity, baroreflex activation, etc.). This forearm exercise
model is similar to that used in several laboratories (3, 21).
Study protocols. Procedures completed as part of the present study
were part of a larger protocol (27). Room temperature was controlled
at 2122C. Relevant to the present investigation, a total of two study
conditions were conducted: 1) 15 min of dynamic forearm exercise,
with AA administered during the last 10 min, 2) 5 min of dynamic
forearm exercise at 15% MVC during a continuous maintenance dose
infusion of AA. In this way, the immediate effect of acute AA
administration could be examined during steady-state exercise (Trial
1), in addition to the effect of AA infusion on exercise onset (Trial 2)
(21). Two minutes of resting data were acquired at the start of each
trial. Trials were separated by a minimum of 30 min of quiet rest, and
both exercise trials were preceded by identical intra-arterial infusions
of endothelial-dependent and -independent agonists, followed by
appropriate wash-out periods, as part of the larger protocol (27).
Importantly, we have previously shown vascular responses to steadystate exercise at 15% MVC to be repeatable across exercise trials
spanning 120 min in both lean subjects and adults with MetSyn
(main effect of trial, P 0.96; unpublished observations in n 24).
Data acquisition and analysis. Heart rate (ECG; Datex-Ohmeda,
Helsinki, Finland), blood pressure, and brachial artery blood velocity
measurements were obtained beat to beat throughout each trial. FBF
was determined as the product of mean blood velocity (MBV, cm/s,
during the last 30 s of steady-state rest and exercise) and vessel cross
sectional area (CSA, radius in cm2) and was reported in ml/min
[FBF (MBV)(CSA)(60 s/min)]. The angle-corrected, intensityweighted Doppler audio information from the ultrasound system was
processed by a commercial interface unit (Multigon Industries, Yonkers, NY) into a blood velocity signal that was sampled in real time
using an analog-to-digital data collection system (PowerLab; ADInstruments, Colorado Springs, CO). All hemodynamic data were stored
on a computer at 400 Hz. Postprocessing using PowerLab Chart5
application package yielded MBV, blood pressures, and heart rates.
To determine vessel CSA, digital video of the brachial artery was
recorded, and brachial artery diameters were taken manually as the
median of five measurements in late diastole. Arterial diameter was
measured on B-mode images in the part of the artery running perpendicular to the ultrasound beam and was identified by strong wall
signals in the longitudinal section of the artery in each image by a
trained investigator. All measurements were assessed offline.
The primary analysis was to test whether exercise-mediated vasodilation was altered after AA infusion and whether vascular responses

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EXERCISE HYPEREMIA IN OBESITY AND METSYN

Fig. 1. Effect of acute ascorbic acid (AA) infusion on mean response time (MRT). A: change in forearm vascular conductance (FVC) averaged in 3-s bins during
5 min of dynamic forearm exercise. Data from a representative healthy control before AA infusion (Trial 1). B: Lean (n 13), Obese (n 10), metabolic
syndrome (MetSyn) (n 9). MRT was not different between groups (main effect of group: P 0.26). MRT was not altered when AA was infused (Trial 2),
compared with exercise alone (Trial 1) (main effect of AA: P 0.50). C: change in MRT with AA infusion () was not different between groups (interaction
between group and AA: P 0.90) although individual responses were variable (a negative value indicates MRT decreased with AA infusion).

to AA infusion were different between groups. To account for group


differences in resting blood pressure and forearm size and to assess
vasodilation, the main dependent variable was a change in relative
forearm vascular conductance (FVC) [FBF measurements (ml/min)
normalized for blood pressure and lean forearm mass (mlmin1100
mmHg1100 g1)]. In addition to steady-state vasodilation, we
examined time to steady-state responses (Fig. 1A). For this analysis,
beat-by-beat data were averaged into 3-s time bins (length of the
exercise duty cycle) and fit using a single component model [FVC
(t) G0 G1(1 e(t TD)/)], with G1 representing the amplitude,
TD representing time delay, and representing the time constant of
the response. Thus, FVC (t) is indicative of the time-dependent
change in FVC from baseline (G0) relative to lean forearm mass (29).
The mean response time (MRT, time taken to reach 63% of the
increase to steady state) was then calculated as MRT TD (29).
Statistical analysis was done using SigmaPlot Version 12.0 (Systat
Software, Evanston, IL). Subject characteristics were analyzed using
a one-way ANOVA. Hemodynamic variables during 15 min of
exercise (Trial 1) were analyzed using a two-way ANOVA approach
to determine the significance of the fixed effect of group (Lean,
Obese, MetSyn) and/or exercise (Rest, Exercise, Exercise AA) on
parameters of interest. Hemodynamic variables during the first 5 min
of exercise (Trial 1 vs. Trial 2) were analyzed using a three-way
ANOVA to determine the significance of the fixed effect of group
(Lean, Obese, MetSyn), exercise (Rest, Exercise), and/or AA (PreAA, Post-AA) on parameters of interest. MRT and changes (,
Exercise Rest) in main outcome variables were assessed using a
two-way ANOVA to determine the significance of the fixed effect of
group (Lean, Obese, MetSyn) and/or AA (Pre-AA, Post-AA). Distributional assumptions were assessed, and log transformations were
used as appropriate. Bonferroni or Dunns post hoc comparisons were
performed when significant effects were observed. Pearsons productmoment correlations were conducted post hoc to determine the association between main outcome variables ( FVC and MRT) and
measures of cardiovascular disease risk (age, weight, waist circumference, body fat percentage, plasma glucose, HDL cholesterol, triglycerides, TBARS, CRP, TAC, physical activity, insulin, HOMA-IR,
and QUICKI). The number of subjects was determined a priori based
on previous research from older adults (21), which showed that n
15 per group would provide 0.92, and n 10 per group would provide
0.75 power, to detect a 32 7% increase in steady-state FVC with
infusion of AA. All data are presented as means SE, and significance was determined a priori at P 0.05.

RESULTS

Subject characteristics. Subject characteristics are summarized in Table 1. Fourteen adults with MetSyn, 10 obese, and
15 lean healthy adults completed the present study (74% White
non-Hispanic, 5% White Hispanic, 13% Asian American, 3%
Table 1. Subject demographics
Sex, M/F
Age, yr
Height, cm
Weight, kg
BMI, kg/m2
Waist, cm
Body fat, %
Total forearm mass, g
Lean forearm mass, g
MVC, kg
MABP, mmHg
Glucose, mg/dl
Insulin, U/ml
HOMA-IR, AU
QUICKI, AU
Total cholesterol, mg/dl
Triglyceride, mg/dl
HDL, mg/dl
LDL, mg/dl
CRP, mg/l
TBARS, MDA units
TAC, Trolox, M
TAC increase with AA, %
Physical activity, kcal/wk

Lean

Obese

MetSyn

12/3
30 3
174 2
70 3
23 1
79 3
22 2
1028 63
970 59
39 2
90 2
81 3
12 1
31
0.34 0.01
151 8
76 7
46 4
91 6
0.4 0.1
1.5 0.2
3.9 1.5
224 97
2354 523

8/2
33 3
174 3
110 7*
36 2*
114 4*
40 2*
1436 93*
1063 68
46 3
98 4
75 3
31 9*
62
0.31 0.01
162 14
98 12
32 4
115 8
1.4 0.4
1.5 0.2
2.3 0.1
137 34
1528 233

10/4
33 3
176 3
122 8*
39 2*
119 5*
41 2*
1457 175
1040 114
41 3
105 2*
92 5
43 9*
10 2*
0.28 0.01*
173 12
189 28*
37 4
99 10
2.1 0.5*
1.9 0.3
2.2 0.2
166 57
1889 461

Values are means SE. Lean (n 15), Obese (n 10), metabolic


syndrome (MetSyn) (n 14), unless otherwise noted: Waist (Lean n 14),
Body Fat (Obese n 9), Insulin/homeostatic model assessment of insulin
resistance (HOMA-IR)/quantitative insulin sensitivity check index (QUICKI)
(Lean n 11, Obese n 8, MetSyn n 11), low-density lipoprotein (LDL)
(Lean n 13, Obese n 6), high-density lipoprotein (HDL) (Obese n 9),
C-reactive protein (CRP) (Lean n 14), thiobarbituric acid reactive substances (TBARS) (Lean n 14), total antioxidant capacity (TAC) (Lean n
8, Obese n 8, MetSyn n 12), physical activity (MetSyn n 12). Effect
of group: *P 0.05 vs. Lean, P 0.05 vs. Obese. BMI, body mass index;
MVC, maximal voluntary contraction; MABP, mean arterial blood pressure;
MDA, malondialdehyde; AA, ascorbic acid.

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EXERCISE HYPEREMIA IN OBESITY AND METSYN

Table 2. Steady-state responses to 15-min forearm exercise


with intra-arterial infusion of AA (Trial 1)
Lean

Obese

Mean arterial blood pressure, mmHg


Rest
97 3
101 3
Exercise
100 3
101 3
Exercise AA
105 4
104 4
Brachial artery diameter, cm
Rest
0.40 0.02
0.44 0.02
Exercise
0.42 0.01
0.46 0.02
Exercise AA
0.43 0.01
0.47 0.02
Forearm blood flow, ml/min 100 g
Rest
71
92
Exercise
26 2
33 5
Exercise AA
31 2
37 5
Forearm vascular conductance, ml/min 100 mmHg 100 g
Rest
71
92
Exercise
27 2
33 5
Exercise AA
30 3
37 5

MetSyn

105 2*
106 3*
109 3*
0.43 0.02
0.45 0.02
0.46 0.02
14 2*
43 7*
46 7*
13 3*
41 6*
43 6*

Values are means SE. Lean (n 14), Obese (n 10), MetSyn (n 13)
(2 subjects did not complete all 15 min of exercise). Main effect of group:
*P 0.05 vs. Lean. Main effect of exercise: P 0.05 vs. Rest.

African American, 5% not disclosed). Five additional subjects


(4 obese, 1 MetSyn) participated in the present investigation
but were excluded because of inability to obtain measures of
lean forearm mass as a result of limitations of the DXA
scanner. There were no significant differences between groups
in regard to age, height, lean forearm mass, MVC, total
cholesterol, LDL cholesterol, HDL cholesterol, TBARS, TAC,
or physical activity (P 0.05). Subjects in the obese and
MetSyn groups were clinically obese, displaying significantly
higher weight, BMI, waist circumference, and percentage of
body fat (P 0.05) compared with lean adults. Glucose,
HOMA-IR, QUICKI, triglycerides, resting mean blood pressure, and CRP were not significantly different between lean
and obese adults (P 0.05). Adults with MetSyn exhibited
higher fasting glucose, insulin, triglycerides, HOMA-IR,
QUICKI, resting mean blood pressure, and CRP compared
with lean and/or obese adults (P 0.05). Intra-arterial infusion
of AA resulted in a significant increase in plasma TAC (174
37%, n 28 attributable to sample loss), and the increase was
not different between groups (P 0.05).

Systemic hemodynamic responses to steady-state exercise.


See Tables 2 and 3. Brachial artery diameter was larger in
obese and MetSyn adults when compared with controls (main
effect of group, P 0.05) and did not change with exercise or
AA infusion (main effect of exercise/AA, P 0.05). Mean
arterial blood pressure was greatest in adults with MetSyn
(main effect of group, P 0.05) and did not change with
exercise (main effect of exercise, P 0.05). Mean arterial
blood pressure increased from Trial 1 to Trial 2 (5 mmHg;
main effect of AA, P 0.01; Table 2) although responses were
not different between groups (interaction between group and
AA, P 0.05).
AA infusion and time to achieve steady-state exercise-mediated vasodilation. Data are summarized in Fig. 1. Time course
data were analyzed from a subset of participants (Lean n 13,
Obese n 10, MetSyn n 9) because of incomplete blood
velocity data during the transition from rest to steady state
(defined as 90% of data points available for analysis, as a
result of repositioning the ultrasound probe). MRT was not
different between groups (main effect of group, P 0.26). AA
infusion did not alter the time to achieve steady state in any
group (MRT: main effect of AA, P 0.50; interaction between
group and AA, P 0.90).
AA infusion and steady-state exercise-mediated vasodilation.
Data are summarized in Table 3. FBF and FVC were greatest
in adults with MetSyn (main effect of group, P 0.05). FBF
and FVC increased with exercise (main effect of exercise, P
0.01), and the rise in blood flow and conductance with exercise
() was not different between groups (Fig. 2, A and C). There
appeared to be no effect of AA infusion on steady-state FBF or
FVC (main effect of AA, P 0.05) regardless of whether AA
was infused during steady-state exercise (Table 2, Fig. 2, A and
B) or before exercise onset (Table 3, Fig. 2, C and D).
Relationship between cardiovascular disease risk factors
and exercise hyperemia. Relationships between main outcome
variables and measures of cardiovascular disease risk were
examined using data pooled from all groups. A significant
positive relationship was observed between MRT and CRP
(r 0.52, P 0.01; n 31), such that subjects with the
highest CRP levels exhibited the longest MRT before infusion
of AA. Additionally, CRP was negatively correlated with

Table 3. Steady-state responses to 5-min forearm exercise before and after intra-arterial infusion of AA, Trial 1 (Pre-AA)
vs. Trial 2 (Post-AA)
Lean
Pre-AA

Obese
Post-AA

Mean arterial blood pressure, mmHg


Rest
96 3
100 3
Exercise
99 3
104 3
Brachial artery diameter, cm
Rest
0.40 0.02
0.40 0.01
Exercise
0.42 0.01
0.42 0.01
Forearm blood flow, ml/min 100 g
Rest
71
81
Exercise
27 2
30 2
Forearm vascular conductance, ml/min 100 mmHg 100 g
Rest
71
81
Exercise
27 2
30 3

Pre-AA

MetSyn
Post-AA

Pre-AA

Post-AA

101 3
101 3

104 3
105 3

105 2*
106 2*

113 3*
112 3*

0.44 0.02*
0.46 0.02*

0.44 0.02*
0.47 0.02*

0.44 0.02*
0.46 0.02*

0.44 0.02*
0.46 0.02*

92
33 5

12 2
37 4

16 3*
45 6*

15 2*
43 5*

92
33 5

11 2
36 4

15 3*
42 6*

14 2*
39 5*

Values are means SE. Lean (n 15), Obese (n 10), MetSyn (n 14). Main effect of group: *P 0.05 vs. Lean, P 0.05 vs. Obese. Main effect
of AA: P 0.05 versus Pre AA.
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EXERCISE HYPEREMIA IN OBESITY AND METSYN


40

Pre AA (5 min exercise)


Post AA (15 min exercise)

30

20

20

10

10

0
MetSyn

Lean

25
20

30

10

10

5
0

-10

-5

-20

-10

-30
Obese

Lean

MetSyn

Obese

MetSyn

DISCUSSION

This study directly examined exercise-mediated vasodilation


before and during acute AA infusion in young (32 yr old)
obese adults and adults with MetSyn. Results from the present
study confirm previous findings that steady-state vascular responses to dynamic forearm exercise are not impaired in obese
adults and adults with MetSyn and further suggest that the time
to achieve steady-state is also preserved. Contrary to our
hypothesis, increasing total antioxidant capacity did not alter
the time course or steady-state vascular responses during dynamic, moderate-intensity exercise in groups of lean or obese

60

10

50

MRT with AA (s)

Mean Response Time (s)

MetSyn

20

MRT after AA infusion (r 0.43, P 0.02, n 31),


indicating that subjects with the highest CRP experienced
the greatest reduction in MRT during acute AA infusion.
Consistent with clinical guidelines, those subjects with CRP
levels 3 mg/l (n 6) tended to exhibit longer baseline
MRT (P 0.09) and a greater reduction in MRT with AA
infusion (P 0.07) (Fig. 3). A trend was also observed
between the change in steady-state FVC with AA infusion
( FVC) and physical activity (r 0.31, P 0.07; n
36). In this way, those subjects with the lowest level of
physical activity exhibited the greatest increase in FVC
with infusion of AA.

Fig. 3. Effect of inflammation on MRT and


change with acute ascorbic acid infusion.
A: C-reactive protein (CRP) 3 mg/l (n
25), CRP 3 mg/l (n 6). Consistent with
clinical guidelines, those subjects with CRP
levels 3 mg/l tended to exhibit longer baseline MRT (P 0.09). B: those subjects with
CRP levels 3 mg/l tended to exhibit a
greater reduction in MRT () with ascorbic
acid infusion (P 0.07) compared with
adults with CRP levels 3 mg/l. P 0.10
vs. CRP 3 mg/l.

Obese

15

Lean

70

Pre AA (5 min exercise)


Post AA (5 min exercise)

0
Obese

-15

40

30

Lean

Change in FVC with AA


(mL/min*100mmHg*100g)

Fig. 2. Effect of acute ascorbic acid infusion on


the exercise-mediated increase in FVC. A: Lean
(n 14), Obese (n 10), MetSyn (n 13).
The exercise-mediated increase FVC from rest
(, Exercise Rest) was not altered when AA
was infused (Trial 1, Min 15), compared with
exercise alone (Trial 1, Min 5) (main effect of
AA: P 0.34), and responses were not different between groups (main effect of group: P
0.16). B: change in the exercise-mediated increase in FVC with AA infusion was not different between groups (interaction between
group and AA: P 0.94) although individual
responses were variable (a positive value indicates FVC increased after AA infusion).
C: Lean (n 15), Obese (n 10), MetSyn
(n 14). The exercise-mediated increase in
FVC from rest () was not different between
groups (main effect of group, P 0.31), and
responses did not increase when AA was infused (Trial 2, Min 5), compared with exercise
alone (Trial 1, Min 5) (main effect of AA: P
0.93). D: change in the exercise-mediated increase in FVC with AA infusion was not different between groups (interaction between
group and AA: P 0.85) although individual
responses were variable (a positive value indicates FVC increased after AA infusion).

FVC (mL/min*100mmHg*100g)

40
30
20

-10

-20

-30

-40

10

-50

C-Reactive Protein
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C-Reactive Protein

EXERCISE HYPEREMIA IN OBESITY AND METSYN

humans or those with MetSyn. Despite the lack of group effect,


individual analyses suggest that the impact of increasing total
antioxidant capacity on exercise-mediated vasodilator responses differs considerably across subjects in all groups
studied, with greatest effect in adults with high levels of
systemic inflammation.
Time course of vasodilator responses to exercise and impact
of acute AA infusion. Rodent models of MetSyn exhibit impaired vasodilation within 30 s of the onset of contraction
during simulated exercise (9). Thus we proposed that vasodilator responses to exercise onset and the time to achieve
steady-state vasodilation would be impaired in obese adults
and adults with MetSyn. In contrast, the MRT to dynamic
forearm exercise was not altered in obese adults and adults
with MetSyn (Fig. 1B); rather, MRT across all groups was
similar to those published previously in young, healthy men
using a similar exercise model (18). Such findings were surprising, given that contraction-induced rapid (6 s) vasodilation is blunted in human obesity (1). Furthermore, research
from healthy humans suggests that rapid vasodilation is primarily potassium channel and nitric oxide mediated (3, 6), and
both mechanisms are altered in animal models of obesity and
MetSyn (12, 17). In this context, differences in key vascular
control mechanisms between rapid-onset vasodilation and the
transition to and maintenance of steady-state exercise hyperemia may explain the discrepancy between findings. However,
mechanisms mediating the transition from rest to steady state
were previously unexamined in human obesity and MetSyn.
On the basis of rationale from animal studies in which acute
antioxidant administration altered contraction-induced vasodilation (9), we examined the effect of acute AA infusion on
MRT in obese adults and adults with MetSyn. Interestingly,
there appeared to be no significant effect of an increase in total
antioxidant capacity on hemodynamic responses in any group
(Fig. 1B). Despite these initial findings, with closer inspection,
our data suggest that chronic inflammation may play a modest
role in determining the time course of the exercise vasodilator
response. Specifically, when data from all groups were combined, adults with the highest CRP levels took the longest time
to achieve steady-state exercise-mediated vasodilation, suggesting that inflammation may account for 27% (r 0.52,
r2 0.27, P 0.01) of the variance in MRT. Along these
lines, adults with MetSyn exhibited significantly higher CRP
and an 20% greater MRT to exercise before AA infusion
compared with lean and obese individuals (Fig. 1; Pre-AA,
Lean 30 6; Obese 31 8; MetSyn 36 8 s).
Interestingly, acute AA infusion was able to improve MRT in
those individuals with high CRP, exposing a potential relationship between inflammation, ROS, and the time course of the
exercise vasodilator response independent of obesity and/or MetSyn (Fig. 3). Given that a slower vasodilator response to exercise
may result in earlier onset of fatigue and poor exercise tolerance
(18), such findings have important clinical implications.
Steady-state exercise-mediated vasodilation and impact of
acute AA infusion. Confirming our previous observations,
steady-state vasodilator responses to moderate-intensity, dynamic forearm exercise are not impaired in human obesity (25)
and MetSyn (26) and, if anything, are increased in obese and
MetSyn adults (Tables 23, Fig. 2, A and C). Although not
directly examined, previous research from our laboratory is
consistent with impaired blood flow distribution in human

H845

MetSyn (28), and such ideas are supported by recent research


from animal models (2, 10, 13). In this way, impaired flow
distribution may result in ventilation-perfusion mismatch, thus
requiring a greater whole limb exercise blood flow response to
meet the metabolic demand of working tissues. Increased ROS
has been shown to contribute to the observed increase in flow
heterogeneity in response to simulated exercise in animal models
of MetSyn (2, 10, 13). Thus we hypothesized that infusion of AA
may contribute to a reduction in exercise blood flow in MetSyn,
possibly as a result of improved flow distribution.
Alternatively, increased levels of ROS commonly observed
in obesity and MetSyn may act as compensatory mechanisms
important in preserving exercise hyperemia in response to a
loss of primary vascular control mechanisms. In support of this
idea, ROS signaling is known to play a role in the maintenance
of blood flow in the coronary circulation of humans with
cardiovascular disease (16, 23). Along these lines, oral antioxidant administration may actually reduce exercise-mediated
vasodilation even in young healthy adults (35), supporting a
potential contribution of ROS to the normal vasodilator response to exercise in humans.
Despite the above rationale, increasing total antioxidant
capacity via intra-arterial infusion of AA neither increases nor
significantly limits steady-state exercise hyperemia in younger
lean, obese, or MetSyn adults during steady-state exercise
(Trial 1) or before exercise onset (Trial 2). Consistent with our
findings, antioxidant supplementation does not affect total limb
blood flow or flow distribution in older rats during treadmill
exercise (5), nor does it impact the oxygen delivery/utilization
balance in skeletal muscle of young healthy rats (4). It is
important to note that, when mechanisms important to vascular
control (e.g., ROS) are blunted, redundant signals may compensate to produce a normal hyperemic response (36). Thus,
by examining acute responses to AA infusion during exercise,
in addition to the effect of AA infusion before exercise onset,
we can rule out the possibility of the presence of a compensatory mechanism masking the normal contribution of ROS to
exercise-mediated vasodilation in human obesity and/or MetSyn in the present study.
Interestingly, post hoc analysis revealed a possible relationship between physical activity and FVC, such that adults with
lower levels of physical activity tend to exhibit increased
steady-state exercise-mediated vasodilation with infusion of
AA (r 0.31, P 0.07). Although speculative, these results
may suggest that, rather than obesity-related disease per se,
ROS-mediated impairments in vascular responses to exercise
might be the result of relatively sedentary behaviors observed
in the current research cohort. Along these lines, physical
activity has been shown to increase production of endogenous
antioxidant enzymes, which may be important to counteract the
presence of chronic and/or exercise-mediated ROS production
(19). Consistent with this idea, low levels of physical activity
are associated with increased oxidative stress, inflammation,
impaired endothelial function, and increased cardiovascular
disease risk (37). It is important to note that all subjects were
relatively inactive (3 h of organized activity each week),
typical physical activity consisted of low-intensity walking,
and subjects refrained from exercise for a minimum of 24 h
before participation (a standard protocol for these types of
studies). It may be possible, although unlikely, that physical
activity completed more than 24 h before study participation

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H846

EXERCISE HYPEREMIA IN OBESITY AND METSYN

modified the observed relationship between low physical activity and increased steady-state vasodilation with AA. Future
studies are necessary to explore this idea, in addition to
examining the effect of initiation of an exercise training program in sedentary individuals.
Experimental considerations. As summarized above, group
data indicate that increasing total antioxidant capacity with AA
infusion does not alter time course (MRT) or magnitude (
FVC) of vasodilator responses to moderate-intensity, dynamic
forearm exercise. This observation may be the result of the
relatively young age and likely short disease duration in the
present study population, resulting in only small group differences in baseline measures of systemic oxidative stress, antioxidant capacity, and inflammation (Table 1). However, the
range of individual responses to exercise and AA infusion
within the present data, independent of group, is consistent
with the concept of unique physiological phenotypes. Specifically, when the change in vascular responses to acute AA
infusion was examined, MRT and FVC to exercise increased,
decreased, or did not change (Figs. 1C, and 2, B and D).
Furthermore, our data suggest that systemic measures of oxidative stress may not be representative of what is occurring
locally within the skeletal muscle vasculature, and thus large
differences in systemic oxidative stress may not be necessary
to observe impairments in vascular control and/or improvements with infusion of AA. Thus physiological changes likely
occur in some subjects very early in the disease process, before
large impairments in standard clinical measures. Considering
that ROS scavenging improves exercise-mediated vasodilation
in a group of healthy older adults (21) and endothelial-dependent vasodilation in older obese adults (32), our data indicate a
complex transition between early disease processes and advancing age, with the potential for age-by-disease and/or lifestyle interactions. These speculations require more rigorous
testing both acutely and longitudinally.
It is important to note that CRP is a marker of systemic
inflammation and CRP elevations likely occur in response to
disturbances in IL-1, TNF-, and IL-6 signaling. Thus CRP may
not be mediating the observed impairments in MRT to dynamic
handgrip exercise and may not be an appropriate direct target for
future therapies (22); however, our results are suggestive of the
contribution of an inflammatory pathway in observed responses.
Furthermore, although we observed significant increases in TAC,
we did not directly measure changes in ROS during study interventions. Taken together, future studies will be necessary to better
understand the specific mechanisms behind inflammation-mediated impairments in MRT and the direct effect of ROS scavenging
(both acute and chronic therapies) on measures of exercisemediated vasodilation.
Conclusion. In summary, our findings add to the growing
body of knowledge indicating that the ability to achieve steadystate vasodilation in response to moderate-intensity, dynamic
forearm exercise is not impaired by obesity or MetSyn in
younger (32 yr) humans. Despite preserved exercise responses, factors linked to cardiovascular disease risk (e.g.,
inflammation) might alter control mechanisms important to
exercise-mediated vasodilation. Furthermore, the variety of
both time-course and steady-state vasodilator responses to
exercise, as well as the range of individual responses to acute
AA infusion, suggest that a normal exercise response can be
achieved by numerous physiological mechanisms, some of which

may be ROS mediated. Interestingly, averaged responses across


groups suggest that the collective effects of obesity and/or MetSyn
per se are not sufficient to impart negative ROS-mediated vasodilator responses similar to those reported in other human conditions, such as aging. In this way, future work will be necessary to
systematically test alternative mechanisms, in addition to disease
duration and/or disease-by-age interactions and their influence on
exercise-mediated vasodilation.
ACKNOWLEDGMENTS
We thank all our participants. In addition, we thank Trent Evans, Molly
Dixon, Caitlin Zillner, Jessica Danielson, and Meghan Crain for technical
assistance.
GRANTS
This study was supported by the American Heart Association predoctoral
awards 10PRE3870000 (J. Limberg) and 11PRE7390038 (J. Harrell), and the
National Institutes of Health (NIH) HL091397 (W. Schrage) and HL105820
(W. Schrage). In addition, we acknowledge the involvement of WNPRC Assay
Services and the partial support of NIH grant RR000167. This study was also
supported by the Clinical and Translational Science Award program through
the NIH National Center for Advancing Translational Sciences Grant
UL1TR000427. The content is solely the responsibility of the authors and does
not necessarily represent the official views of the NIH.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: J.K.L., J.W.H., R.J., M.W.E., L.T.P., J.J.S., and
W.G.S. performed experiments; J.K.L., J.M.K., J.W.H., and R.J. analyzed
data; J.K.L., J.M.K., J.W.H., R.J., M.W.E., L.T.P., J.J.S., and W.G.S. interpreted results of experiments; J.K.L. and J.M.K. prepared figures; J.K.L. and
W.G.S. drafted manuscript; J.K.L., J.M.K., J.W.H., R.J., and W.G.S. edited
and revised manuscript; J.K.L., J.M.K., J.W.H., R.J., M.W.E., L.T.P., J.J.S.,
and W.G.S. approved final version of manuscript; M.W.E., L.T.P., J.J.S., and
W.G.S. conception and design of research.
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