RES E AR C H Open Access
Effect of nitric oxide synthase inhibition on the
exchange of glucose and fatty acids in human
, Bengt Saltin
, Jukka Kemppainen
, Pirjo Nuutila
, Juhani Knuuti
, Kari Kalliokoski
and Ylva Hellsten
Background: The role of nitric oxide in controlling substrate metabolism in humans is incompletely understood.
Methods: The present study examined the effect of nitric oxide blockade on glucose uptake, and free fatty acid
and lactate exchange in skeletal muscle of eight healthy young males. Exchange was determined by measurements
of muscle perfusion by positron emission tomography and analysis of arterial and femoral venous plasma
concentrations of glucose, fatty acids and lactate. The measurements were performed at rest and during exercise
without (control) and with blockade of nitric oxide synthase (NOS) with N
Results: Glucose uptake at rest was 0.40 ± 0.21 μmol/100 g/min and increased to 3.71 ± 2.53 μmol/100 g/min by
acute one leg low intensity exercise (p < 0.01). Prior inhibition of NOS by L-NMMA did not affect glucose uptake, at
rest or during exercise (0.40 ± 0.26 and 4.74 ± 2.69 μmol/100 g/min, respectively). In the control trial, there was a
small release of free fatty acids from the limb at rest (−0.05 ± 0.09 μmol/100 g/min), whereas during inhibition of
NOS, there was a small uptake of fatty acids (0.04 ± 0.05 μmol/100 g/min, p < 0.05). During exercise fatty acid
uptake was increased to (0.89 ± 1.07 μmol/100 g/min), and there was a non-significant trend (p = 0.10) for an
increased FFA uptake with NOS inhibition 1.23 ± 1.48 μmol/100 g/min) compared to the control condition. Arterial
concentrations of all substrates and exchange of lactate over the limb at rest and during exercise remained
unaltered during the two conditions.
Conclusion: In conclusion, inhibition of nitric oxide synthesis does not alter muscle glucose uptake during low
intensity exercise, but affects free fatty acid exchange especially at rest, and may thus be involved in the
modulation of energy metabolism in the human skeletal muscle.
Keywords: Nitric oxide, Metabolism, Energy substrates, Humans
The translocation of glucose transporter GLUT 4 to
muscle sarcolemma appears to be the key step in
mediating contraction induced glucose uptake, but the
mechanisms that trigger the process are still poorly char-
acterized [1-3]. One mediator that has been postulated
to trigger GLUT 4 translocation and the subsequent
increased glucose uptake is nitric oxide (NO), whose
formation is increased from rest to muscle contractions
[4,5]. An improved understanding of the regulation of
glucose uptake, including the role of NO, in human skel-
etal muscle is important, especially considering that
skeletal musculature accounts for ~70-80% of postpran-
dial glucose uptake in human body .
NO has, in some vitro and in vivo studies in animals
and humans been shown to play a role in the regulation
of glucose uptake in skeletal muscle [7-13]. Moreover,
exogenously applie d NO donors have also been shown
to enhance resting glucose uptake [10,14-16], although
not all stud ies support this finding . In terms of exer-
cise, there is also some evidence for an effect of NOS
* Correspondence: firstname.lastname@example.org
Turku PET Centre, PO Box 52, FI-20521, Turku, Finland
Research Centre of Applied and Preventive Cardiovascular Medicine,
University of Turku, Turku, Finland
Full list of author information is available at the end of the article
© 2013 Heinonen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Heinonen et al. Nutrition & Metabolism 2013, 10:43
inhibition on glucose uptake during muscle contractions
in animals [7,13] and during moderate intensity whole
body exercise in humans [8,11], wherea s other animal
studies have documented unchanged glucose uptake
during exercise or electrically-in duced contractions in
response to acute or chronic inhibition of NO synthesis
[9,10,18]. Moreover, in a human study where synthesis
of NO was inhibited locally by infusion of L-NMMA
into the exercisin g muscle via a microdialysis catheter,
only local blood flow but not glucose uptake was re-
duced . Also, in cardiac muscle, which displays a
similar energy substrate utilization as skeletal muscle
during low contraction intensity, it has been consistently
shown in animals that NO reduces glucose uptake
[12,20-23]. Hence, it is clear that the data in the litera-
ture show highly discrepant findings on the role of NO
on glucose metabolism and studies especially in humans
In a recent study with the use of positron emission
tomography (PET) methodology, we reported that the
inhibition of nitric oxide enhances oxygen consumption
in human skeletal muscle , a finding that potentially
could be related to an alteration in energy substrate
utilization, such as altered exchange of fatty acids. In this
regard, animal studies have indicated that the exchange
of free fatty acids may be enhanced in response to NOS
inhibition [25,26], leading also to enhanced free fatty
acid oxidation as recently reviewed , but this aspect
has not previously been investigated in humans. Thus,
the aim of the present study was to determine the role
for NO in glucose and fatty acid uptake in skeletal muscle
by measurements of muscle specific blood flow by PET
with radio-labelled water and arterial and venous concen-
trations of glucose and fatty acids. We hypothesised that
the inhibition of NOS will reduce the uptake of glucose
and enhance the exchange of free fatty acids at rest and
Eight healthy untrained young men (26 ± 2 yrs, 184 ± 4
cm, 82 ± 8 kg, 24.2 ± 1.9 kg/m
) volunteered to partici-
pate in this study. Central and local hemodynamic data,
but not any of the substrate metabolic findings have pre-
viously been published . The purpose, nature, and
potential risks of the study were explained to the sub-
jects before they gave their written informed consent to
participate. None of the subjects had chronic diseases,
were taking regular medication or were smokers. The
study was performed approximately at four hours after
the subjects had eaten their normal breakfast (approxi-
mately 450 Kcal, carbohydrates, proteins and fat contrib-
uting to 55%, 15% and 30% energy from total. The
subjects abstained from caffeine-containing beverages
for at least 24 h before the experiments. The subjects were
also requested to avoid strenuous exercise within 48 h
prior to the study. The study was performed according to
the Declaration of Helsinki and was approved by the Eth-
ical Committee of the Hospital District of South-Western
Finland and National Agency for Medicine.
Before the PET experiments, the antecubital vein was can-
nulated for tracer administration. For blood sampling, a
radial artery cannula was placed under local anesthesia in
the contralateral arm. Additionally, cannulas were placed
under local anesthesia into the femoral artery and vein for
local drug infusions and blood sampling, respectively.
Subjects were then moved to the PET scanner with the
femoral region in the gantry and the right leg was posi-
tioned in an in-house designed leg exercise dynamometer.
PET measurements were first performed at resting base-
line and thereafter during exercise without any drug infu-
sion, but only during control saline. Thirty minutes later,
resting and exercising measurements were performed
during NOS blockade with N
NMMA) (Clinalfa, Laufelfingen, Switzerland). L-NMMA
was infused intra-arterially with a concentration of 1.0 mg
kg leg mass
. The infusion of the drug started
ten minutes before the scanning (blood flow measure-
ment) and continued until the end of the experiments.
Additionally, radial artery and femoral vein blood samples
for energy substrate and blood gas parameters were drawn
for analysis (7 min after the onset of steady state exercise)
during each of the conditions described above. Systemic
mean arterial pressure (MAP) was measured (Omron,
M5-1, Omron Healthcare, Europe B.V. Hoofddorf, The
Netherlands) on every occasion studied.
Perfusion measurements and analysis
Radio water positron-emitting tracer [
produced as previously described  and the ECAT
EXACT HR + scanner (Siemens/CTI, Knoxville, TN,
USA) was used in 3D mode for image acquisition to
measure musc le blood flow. The oxygen-15 isotope was
produced with Cyclone 3 cyclotron (IBA Molecular,
Belgium). Photon attenuation was corrected by 5-min
transmission scans performed at the beginning of the
PET measurements performed at rest and during exer-
cise. All data were corrected for dead time, decay and
measured photon attenuation, and the images were
reconstructed into a 256 × 256 matrix, producing 2.57 ×
2.57 mm in-plane dimensions of voxels with 2.43 mm
plane thickness. For the measurement of perfusion at
rest, scanning began simultaneously with the inf usion,
and consisted of the following frames; 6 × 5 s, 12 × 10 s
and 7 × 30 s at rest and 6 × 5 s and 12 × 10 s during
exercise. During exercise, scanning was started five
Heinonen et al. Nutrition & Metabolism 2013, 10:43 Page 2 of 7
minutes after exercise onset to obtain a metabolic
steady-state situation and continued until the end of the
exercise bout, e.g. 2.5 min (7,5 min totally). Arterial
blood radioactivity was also sampled continuously with a
detector during imaging for perfusion quantification.
Exercise consisted of dynamic m. quadriceps femoris
(~2.5 kg muscle mass) one-legged exercise at 40 rpm
with an average work load of 4.5 kg and with a knee
angle range of motion of ~ 75–80 degrees . Local
muscle blood flow was measured from the m. quadriceps
femoris. The data analysis was performed using the
standard models  and methods [32,33].
Magnetic resonance imaging
Structural Magnetic Resonance Imaging (MRI) was
performed about one week before the PET study as de-
scribed earlier , when subjects were also accustomed
to the one-leg knee extension exercise model in a PET
scanner. MRI scanning was performed to obtain total leg
volume of the working leg since NOS inhibiting drug in-
fusions were based on effective concentrations per litre
leg volume . The mean total leg volume of the sub-
jects wa s 12.2 ± 1.5 l.
Blood samples for energy substrates (free fatty acids, glu-
cose and lactate) and blood gases were drawn from fem-
oral vein and radial artery in each study condition at the
mid time-point of PET measurement and analysed with
standardized hospital practises. Lactate and free fa tty
acids were analyzed with enzymatic methods (Roche
Modular P analyzer, Roche Diagnostics GmbH, Mannheim,
Germany) and glucose was determined in duplicates by the
Glucose hexokinase method (Roche Modular P analyzer,
Roche Diagnostics GmbH, Mannheim, Germany) and the
average was used for concentration and following calcula-
tions. Uptake or release of energy substrates were deter-
mined by the Fick principle, thus a-v differences were
multiplied with muscle blood flow.
Statistical analyses were performed with SAS 8.2 and
SAS Enterprise 4.2 programs (SAS Institute, Cary, NC).
Statistical analyses were performed using two-way
ANOVA for repeated measures (exercise and drug as
factors). If a significant main effect(s) was found, pair
wise differences were identified using the Tukey-Kramer
post hoc procedure. Results are expressed as mean ± SD.
A p value ≤ 0.05 was considered statistically significant.
The arterial concentrations of glucose and FFA were not
affected by inhibition either at rest (Figure 1) or during
exercise (Figure 2). Glucose uptake at rest was 0.40 ±
0.21 μmol/100 g/min and increased to 3.71 ± 2.53 μmol/
100 g/min by acute one leg exercise (p < 0.01) (Figure 1).
Inhibition of NOS did not affect glucose uptake at rest
(Figure 1) or during exercise (Figure 2), although it re-
duced (P < 0.05) resting muscle blood flow and increased
(P < 0.05) oxygen extraction and uptake substantially
(Table 1). Inhibition of NOS altered the release of free
fatty acids (FFA) at rest from a release of FFAs, to an up-
take (P < 0.05) during NOS blockade (Figure 1). During
exercise, FFA uptake wa s similar during the two condi-
tions , although there was a tendency for a higher uptake
(p = 0.10) during NOS inhibition. Arterial lactate con-
centrations and exchange of lactate over the muscle at
rest and during exercise remained unaltered during the
two conditions (Figures 1 and 2). During exercise muscle
blood flow and muscle oxyg en extraction and consump-
tion were similar during the control condition and NOS
inhibition (Table 2).
We report in the present study that inhibition of en-
dogenous NO formation does not alter glucose uptake
of human skeletal muscle at rest or during low intensity
exercise. However, by affecting the release and uptake of
free fatty acids, NO appears to contribute to the regula-
tion of muscle energy metabolism, at least when the
muscle is at rest.
The effect of nitric oxide on muscle glucose uptake at
rest and during exercise
In the present study we show that glucose uptake is un-
affected by prior NOS inhibition with L-NMMA both at
rest and during exercise. Previous human studies that
have addressed the role of nitric oxide for glucose up-
take in skeletal muscle have shown discrepant findings
where some have shown that glucose uptake during
exercise is reduced when NOS is inhibited [8,11],
whereas others have shown no effect . The discrep-
ancy between findings in the different studies is unclear,
however, differences in experimental conditions between
the studies could in part explain the findings. Firstly,
McConell and co-workers  began infusion of L-NMMA
ten minutes after steady state exercise, while we began in-
fusion ten minutes before exercise. Copp and colleagues
showed that the timing of NOS inhibition does have an ef-
fect on the blood flow response in relation to muscle fibre
type in rats , but it may also affect glucose uptake. An-
other experimental difference lies in the mode and inten-
sity of exercise. In our study single leg exercise at ~10
watts with a substantial isometric component, was used
whereas in the study by Bradley and co-authors the exer-
cise consisted of two leg cycling at the 60% of peak
, corresponding to ~142 watts . Consequently,
glucose uptake was increased 30-fold ~ in the study by
Heinonen et al. Nutrition & Metabolism 2013, 10:43 Page 3 of 7
Bradley et al. whereas we observed a ~10-fold increase in
our study. The approach and results of Kingwell et al. was
similar to Bradley et al. [8,11]. Thus, our present findings
combined with that of others  suggest that glucose up-
take at rest and during low-to-moderate exercise intensity
is not NO mediated, but NO affects glucose uptake during
higher exercise intensities [8,11]. Many previous animal
studies that have addressed the effect of NO on glucose
uptake in muscle have also resulted in contrasting conclu-
sions [7,9,10,13,18], whereas most studies on cardiac
muscle have all reported increased glucose uptake in par-
allel with increased carbohydrate metabolism during the
inhibition of NOS [12,20-22]. Thus, overall, the role of
NO for glucose uptake appears to be more important in
cardiac than skeletal muscle.
The effect of nitric oxide on free fatty acid exchange in
In line with the result s of Rottman et al. in mice ,
the present study demonstrates that NO inhibition alters
the exchange of FFAs over the limb. At rest in the
control condition, there was a release of FFA from the
limb, whereas during NOS inhibition an uptake of FFA
was detected (Figure 1). This finding, combined with the
unaltered glucose uptake during NOS blockade, is also
in line with the observation that oxygen consumption of
the muscle is increased during NOS inhibition, and sug-
gests that the overall metabolism, as indicated by the
change in oxygen consumption, of the muscle was en-
hanced during NOS inhibition. The finding suggests that
nitric oxide suppresses fatty acid metabolism in resting
human skeletal muscle, which is in accordance with
findings in vitro and animal studies showing that NOS
inhibition increases FFA oxidation .
Whether the observed increase in FFA utilization also
resulted in increased FFA oxidation is unclear as this
was not determined in the present study. Nevertheless,
the increa se in oxyg en consumption during the NOS
blockade could indicate that there was an increased FFA
oxidation. Many animal [36,37] and human  studies
indicate that increased rates of FFA oxidation lowers the
efficiency of the muscle. However, a switch to exclusive
Baseline NOS inhibition
a-v difference of glucose
Baseline NOS inhibition
Baseline NOS inhibition
Baseline NOS inhibition
a-v difference of FFA
a-v difference of lactate
Baseline NOS inhibition
Baseline NOS inhibition
Release of lactate
Baseline NOS inhibition
Figure 1 The effect of nitric oxide synthase (NOS) inhibition on arterial glucose, free fatty acids (FFA) and lactate and their arterial-
to-venous (a-v) differences and uptake/release at rest. * p <0.05 compared to baseline.
Heinonen et al. Nutrition & Metabolism 2013, 10:43 Page 4 of 7
use of fatty acids as an energy sourc e is calculated to im-
pair the efficiency of ATP production by only 10–15%
. Our results do not suggest a complete substrate
switch and, thus, the increased FFA uptake and utilization
cannot fully explain the observed increase in oxygen
consumption. Therefore, an additional explanation for the
increase in oxygen uptake during NOS blockade probably
was a reduced inhibitory influence of NO on mitochon-
drial respiration [40-42]. Alternatively, utilization of FFAs
may have actually been enhanced secondarily to this
phenomenon to fulfil increased cellular metabolism, but
Control NOS inhibition
a-v difference of glucose
Control NOS inhibition
a-v difference of FFA
a-v difference of lactate
Release of lactate
NOS inhibition Control NOS inhibition
Figure 2 The effect of nitric oxide synthase (NOS) inhibition on arterial glucose, free fatty acids (FFA) and lactate and their arterial-
to-venous (a-v) differences and uptake/release during exercise.
Table 1 Heart rate, blood pressure, blood flow and
oxygen uptake at rest
HR (bpm) 56 ± 6 53 ± 8
BPs (mmHg) 137 ± 15 139 ± 13
BPd (mmHg) 78 ± 10 85 ± 8*
MAP (mmHg) 98 ± 11 103 ± 9
Muscle blood flow (ml/100 g/min) 2.2 ± 0.8 1.3 ± 0.5**
Oxygen extraction (ml/L) 58 ± 18 108 ± 22***
Oxygen consumption (ml/100 g/min) 0.11 ± 0.03 0.13 ± 0.03*
HR heart rate, MAP mean arterial pressure, BPs systolic blood pressure,
BPd diastolic blood pressure, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared
Table 2 Heart rate, blood pressure, blood flow and
oxygen uptake during exercise
HR (bpm) 80 ± 11 73 ± 10
BPs (mmHg) 158 ± 23 160 ± 19
BPd (mmHg) 90 ± 12 95 ± 12
MAP (mmHg) 113 ± 15 117 ± 13
Muscle blood flow (ml/100 g/min) 36.2 ± 4.9 34.8 ± 7.9
Oxygen extraction (ml/L) 125 ± 13 132 ± 16
Oxygen consumption (ml/100 g/min) 4.50 ± 0.60 4.55 ± 0.99
HR heart rate, MAP mean arterial pressure, BPs systolic blood pressure,
BPd diastolic blood pressure.
Heinonen et al. Nutrition & Metabolism 2013, 10:43 Page 5 of 7
this possibility warrants further investigation. Finally, it
has been observed that the inhibition of NOS leads to
enhanced lipolysis in subcutaneous adipose tissue [43,44].
In our study arterial FFA levels appeared to be somewhat
increased both at rest and during exercise, but this did not
reach statistical significance, which points to the con-
clusion that indeed shift in the utilization rather than
increased availability accounted for the observed increase
in FFA uptake.
Muscle biopsies were not obtained in the current study
so direct measurement s of the degree of NOS inhibition
could not be obtained. The dose of L-NMMA used in
the present study was similar to that used in a number
of studies from our own as well as other laboratories.
These previous studies, e.g. Rådegran and Saltin 1999,
have shown that resting blood flow as well as the re-
sponses to acethylcoline infusions are approximately
halved with use of this L-NMMA dose, indicating an
effective inhibition . In the current study, resting
blood flow during L-NMM A infusion was reduced to a
similar extent as previously observed. Moreover, it has
been previously demonstrated that infusion of the NOS
blocker L-NAME that reduces limb blood flow to a simi-
lar extent as the L-NMMA dose used in the current
study, reduces NO synthase activity by approximately
70% . Hence, it appears likely that the extent of NOS
inhibition was similar in the present as in previous stud-
ies on humans [45,46]. Finally, as a vehicle control group
was not applied in the presen t study, it is not possible to
completely eliminate the possibility that there may have
been a carry over effect during the second bout of exer-
cise with NOS inhibition.
In conclusion, endogenous nitric oxide does not ap-
pear to change glucose uptake of human skeletal muscle
at rest or during low intensity exercise, but shifts a re-
lease of free fatty acids to uptake, thereby altering
muscle energy metabo lism, in particular at rest.
None of the authors had personal or financial conflict of interests.
All authors contributed to the conception and design of the experiments,
the collection, analysis and interpretation of data, and to drafting of the
article or revising it critically for important intellectual content. All authors
also approved the final version of the manuscript.
The study was conducted within the Finnish Centre of Excellence in
Molecular Imaging in Cardiovascular and Metabolic Research - supported by
the Academy of Finland, University of Turku, Turku University Hospital and
Abo Academy. Authors want to thank the contribution of the personnel of
the Turku PET Centre for their excellent assistance during the study. The
study was financially supported by The Ministry of Education of State of
Finland, Academy of Finland, The Finnish Cultural Foundation and its South-
Western Fund, The Finnish Sport Research Foundation, Turku University
Hospital (EVO funding), Novo Nordisk Foundation and The Danish Medical
Research Council .
Turku PET Centre, PO Box 52, FI-20521, Turku, Finland.
Research Centre of
Applied and Preventive Cardiovascular Medicine, University of Turku, Turku,
Department of Clinical Physiology and Nuclear Medicine, University
of Turku, Turku, Finland.
Department of Medicine, Turku University Hospital,
University of Turku, Turku, Finland.
Exercise and Sport Sciences, Section of
Human Physiology, University of Copenhagen, Copenhagen , Denmark.
Copenhagen Muscle Research Center, University of Copenhagen,
Received: 16 April 2013 Accepted: 10 June 2013
Published: 18 June 2013
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Cite this article as: Heinonen et al.: Effect of nitric oxide synthase
inhibition on the exchange of glucose and fatty acids in human skeletal
muscle. Nutrition & Metabolism 2013 10:43.
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