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Effect of nitric oxide synthase inhibition on the exchange of glucose and fatty acids in human skeletal muscle

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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 NG-monomethyl-l-arginine (L-NMMA). 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.
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RES E AR C H Open Access
Effect of nitric oxide synthase inhibition on the
exchange of glucose and fatty acids in human
skeletal muscle
Ilkka Heinonen
1,2*
, Bengt Saltin
6
, Jukka Kemppainen
1,3
, Pirjo Nuutila
1,4
, Juhani Knuuti
1
, Kari Kalliokoski
1
and Ylva Hellsten
5
Abstract
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
G
-monomethyl-l-arginine (L-NMMA).
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
Introduction
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 [6].
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 [17]. In terms of exer-
cise, there is also some evidence for an effect of NOS
* Correspondence: ilkka.heinonen@utu.fi
1
Turku PET Centre, PO Box 52, FI-20521, Turku, Finland
2
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
http://www.nutritionandmetabolism.com/content/10/1/43
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 [19]. 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
are warranted.
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 [24], 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 [27], 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
during exercise.
Methods
Subjects
Eight healthy untrained young men (26 ± 2 yrs, 184 ± 4
cm, 82 ± 8 kg, 24.2 ± 1.9 kg/m
2
) 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 [24]. 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.
Study design
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
G
-monomethyl-l-arginine (L-
NMMA) (Clinalfa, Laufelfingen, Switzerland). L-NMMA
was infused intra-arterially with a concentration of 1.0 mg
min
1
kg leg mass
1
[28]. 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 [
15
O]-H
2
Owas
produced as previously described [29] 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
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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 ~ 7580 degrees [30]. Local
muscle blood flow was measured from the m. quadriceps
femoris. The data analysis was performed using the
standard models [31] 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 [34], 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 [28]. The mean total leg volume of the sub-
jects wa s 12.2 ± 1.5 l.
Biochemical analysis
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 analysis
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.
Results
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).
Discussion
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 [19]. 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 [8] 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 [35], 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
VO
2max
, corresponding to ~142 watts [8]. Consequently,
glucose uptake was increased 30-fold ~ in the study by
Heinonen et al. Nutrition & Metabolism 2013, 10:43 Page 3 of 7
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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 [19] 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
muscle
In line with the result s of Rottman et al. in mice [26],
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 [27].
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 [38] studies
indicate that increased rates of FFA oxidation lowers the
efficiency of the muscle. However, a switch to exclusive
Arterial glucose
Baseline NOS inhibition
4.5
5.0
5.5
6.0
Concentration (mmol/L)
a-v difference of glucose
Baseline NOS inhibition
0.0
0.2
0.4
0.6
0.8
Difference (mmol/L)
Glucose uptake
Baseline NOS inhibition
0.0
0.5
1.0
1.5
Uptake (µmol/100g/min)
Arterial FFA
Baseline NOS inhibition
0.0
0.2
0.4
0.6
0.8
Concentration (mmol/L)
a-v difference of FFA
-0.3
-0.2
-0.1
0.0
0.1
Difference (mmol/L)
FFA release/uptake
-0.3
-0.2
-0.1
0.0
0.1
Release/uptake (µmol/100g/min)
a-v difference of lactate
Baseline NOS inhibition
-0.4
-0.3
-0.2
-0.1
0.0
Difference (mmol/L)
Arterial lactate
Baseline NOS inhibition
0.0
0.5
1.0
1.5
Concentration (mmol/L)
Release of lactate
Baseline NOS inhibition
-0.5
-0.4
-0.3
-0.2
-0.1
-0.0
Release (µmol/100g/min)
Baseline
enilesaBnoitibihni
SO
N
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
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use of fatty acids as an energy sourc e is calculated to im-
pair the efficiency of ATP production by only 1015%
[39]. 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
Arterial glucose
Control NOS inhibition
4.5
5.0
5.5
6.0
Concentration (mmol/L)
a-v difference of glucose
Control
NOS inhibition
0.0
0.1
0.2
0.3
Difference (mmol/L)
Glucose uptake
Control
NOS inhibition
0
2
4
6
8
10
Uptake (µmol/100g/min)
Arterial FFA
Control NOS inhibition
0.0
0.2
0.4
0.6
0.8
Concentration (mmol/L)
a-v difference of FFA
-0.10
-0.05
0.00
0.05
0.10
0.15
Difference (mmol/L)
FFA uptake
-2
0
2
4
Uptake (µmol/100g/min)
Arterial lactate
Control
NOS inhibition
0.0
0.5
1.0
1.5
2.0
Concentration (mmol/L)
a-v difference of lactate
-1.5
-1.0
-0.5
0.0
0.5
Difference (mmol/L)
Release of lactate
-60
-40
-20
0
20
Release (µmol/100g/min)
Contro
l
NOS inhibition Control NOS inhibition
Control
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
BASELINE L-NMMA
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
to BASELINE.
Table 2 Heart rate, blood pressure, blood flow and
oxygen uptake during exercise
CONTROL L-NMMA
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
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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.
Methodological considerations
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 [45]. 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% [46]. 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.
Competing interests
None of the authors had personal or financial conflict of interests.
Authors contributions
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.
Acknowledgements
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 .
Author details
1
Turku PET Centre, PO Box 52, FI-20521, Turku, Finland.
2
Research Centre of
Applied and Preventive Cardiovascular Medicine, University of Turku, Turku,
Finland.
3
Department of Clinical Physiology and Nuclear Medicine, University
of Turku, Turku, Finland.
4
Department of Medicine, Turku University Hospital,
University of Turku, Turku, Finland.
5
Exercise and Sport Sciences, Section of
Human Physiology, University of Copenhagen, Copenhagen , Denmark.
6
Copenhagen Muscle Research Center, University of Copenhagen,
Copenhagen, Denmark.
Received: 16 April 2013 Accepted: 10 June 2013
Published: 18 June 2013
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doi:10.1186/1743-7075-10-43
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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... In addition, it has been reported to have an inhibitory effect on platelet aggregation and prevents the activation, adhesion and migration of leukocytes (Hegyi and Rakonczay, Jr. 2011). Studies have also documented the involvement of NO in glucose metabolism, especially as it affects skeletal muscle glucose uptake, with conflicting results (Bradley et al. 1999, Durham et al. 2003, Heinonen et al. 2013. Previous studies demonstrated that the administration of exogenous NO donors enhanced resting glucose uptake (Higaki et al. 2001, Durham et al. 2003. ...
... In agreement, the blockage of nitric oxide synthase (NOS) was demonstrated to decrease muscle glucose uptake during exercise in animals and humans (Balon and Nadler 1997, Bradley et al. 1999, Kingwell et al. 2002. However, some studies in animals and humans reported that the inhibition of NOS did not alter skeletal muscle glucose uptake during exercise (Inyard et al. 2007, Heinonen et al. 2013. ...
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The inhibition of renin angiotensin system pathway has been largely documented to be effective in the control of cardiovascular events. The present study investigated the effect of angiotensin converting enzyme (ACE) inhibitor on fasting blood glucose level in hypertension induced by the inhibition of nitric oxide synthase (NOS) in male Wistar rats. Hypertension was induced by the inhibition of NOS using a non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). The blockade of NOS resulted in an increase in blood pressure, ACE, angiotensin II and endothelin-1 levels, and a decrease in fasting blood glucose and nitric oxide (NO) levels. The hypertensive rats treated with ACE inhibitor (ramipril) recorded a decrease in blood pressure, ACE, angiotensin II, endothelin-1, NO and fasting blood glucose levels, and an increase in prostacyclin level. In conclusion, ACE inhibitor potentiated the hypoglycaemic effect of NOS inhibitor and this effect is independent of NO and pancreatic insulin release.
... Hypoxic state associated with increased breathing intensity at the moment of transition to physical activity in tissue cells is one of the factors in formation of activated mitochondrion state [12]. Hypoxia initiates the formation of reactive oxygen forms with subsequent deployment of free-radical and peroxide reactions through moderate mobilization of endogenous fatty acids and stimulation of sympathoadrenal system [13]. The accumulation of endogenous oxygen in the process of free-radical reactions provides maintenance of intensive energy exchange and attraction of free-radical oxidation products to metabolic processes [14]. ...
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Hemato-ophthalmic barrier is one of the mechanisms of body resistance. One of the complications of mechanical trauma of the eye and violation of the hemato-ophthalmic barrier is the emergence of oxidative stress on the background of the general inflammatory process. Normally, oxidative stress in the skeletal muscle tissue is not a damaging agent, but when intensified by other factors, it promotes pathological changes in the body. Objective: to study the dynamics of superoxiddismutase (SOD) activity in rat skeletal muscle tissue under oxidative stress caused by mechanical action on the hemato-ophthalmic barrier. Materials and methods: The study was carried out on pedigree matured male rats in the amount of 150 pieces. The activity of SOD in skeletal muscle tissue was studied before the experiment, as well as on the 1st, 3rd, 5th, 7th and 14th day of the experiment using the standard technique of V.S. Gurevich. The obtained digital material was subjected to statistical processing by means of non-parametric statistical analysis. Conclusion: SOD activity in rat skeletal muscle tissue under oxidative stress caused by mechanical action on hemato-ophthalmic barrier is most effectively stabilized in standard therapy of mechanical eye injury with the addition of quercetin in the form of injections.
... nNOSμ is the main isoform expressed in skeletal muscle (210), is constitutively active, and muscle contraction causes a twofold increase in NO production (233). However, there is some disagreement in the field as to whether NOS inhibition decreases exercise-stimulated glucose uptake (27,143), as this is not universally observed (94,138). ...
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The skeletal muscle is the largest organ in the body, by mass. It is also the regulator of glucose homeostasis, responsible for 80% of postprandial glucose uptake from the circulation. Skeletal muscle is essential for metabolism, both for its role in glucose uptake and its importance in exercise and metabolic disease. In this article, we give an overview of the importance of skeletal muscle in metabolism, describing its role in glucose uptake and the diseases that are associated with skeletal muscle metabolic dysregulation. We focus on the role of skeletal muscle in peripheral insulin resistance and the potential for skeletal muscle-targeted therapeutics to combat insulin resistance and diabetes, as well as other metabolic diseases like aging and obesity. In particular, we outline the possibilities and pitfalls of the quest for exercise mimetics, which are intended to target the molecular mechanisms underlying the beneficial effects of exercise on metabolic disease. We also provide a description of the molecular mechanisms that regulate skeletal muscle glucose uptake, including a focus on the SNARE proteins, which are essential regulators of glucose transport into the skeletal muscle. © 2020 American Physiological Society. Compr Physiol 10:785-809, 2020.
... [21][22][23] Interestingly, this technique also controversially showed that nitric oxide synthase (NOS) inhibition increases the permeability of the capillaries to insulin, increasing the delivery of insulin to the skeletal muscle, and accelerating glucose lowering. 29 Local NOS inhibition had no effect on capillary blood flow during contraction, [30][31][32] while systemic 33 and local 34 NOS inhibition can block insulin-induced microvascular recruitment, suggesting a direct local effect on the microvasculature. NOS inhibition also suppressed muscle glucose uptake due to the reduction in perfusion during insulin exposure, 33 however, in exercise, impairments of glucose uptake by NOS inhibition occurred without changes in microvascular recruitment. ...
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The endocrine system relies on the vasculature for delivery of hormones throughout the body, and the capillary microvasculature is the site where the hormones cross from the blood into the target tissue. Once considered an inert wall, various studies have now highlighted the functions of the capillary endothelium to regulate transport and therefore affect or maintain the interstitial environment. The role of the capillary may be clear in areas where there is a continuous endothelium, yet there also appears to be a role of endothelial cells in tissues with a sinusoidal structure. Here we focused on the most common endocrine disorder, diabetes, and several of the target organs associated with the disease, including skeletal muscle, liver and pancreas. However, it is important to note that the ability of hormones to cross the endothelium to reach their target tissue is a component of all endocrine functions. It is also a consideration in organs throughout the body and may have greater impact for larger hormones with target tissues containing a continuous endothelium. We noted that the blood levels do not always equal interstitial levels, which is what the cells are exposed to, and discussed how this may change in diseases such as obesity and insulin resistance. The capillary endothelium is, therefore, an essential and understudied aspect of endocrinology and metabolism that can be altered in disease, which may be an appropriate target for treatment.
... NO exerts other effects: it inhibits platelet aggregation (interestingly, activated-plateletderived substances increase the activity of eNOS, thus producing more NO) and the adhesion of leucocytes to the vascular wall by decreasing the expression of adhesion molecules on endothelial surface [90]. Moreover, it interferes with cellular metabolism [125] by modulating mitochondrial function, and oxygen metabolism [106,126]. As stated, NO forms ROS (ONOO − ) with increased levels of O 2− which, among others, impairs the mitochondrial respiratory chain [127]. ...
Chapter
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... Nitric oxide synthase (NOS) activity and nitric oxide (NO) production are increased during electrical stimulations in muscle cells (34,42), muscle contractions, or exercise in rodents (14,15,31,32,36,38) and exercise in humans (28). Several studies have demonstrated that pharmacological inhibition of NOS attenuates the increase in skeletal muscle glucose uptake during contractile activity (1,3,14,24,31,32,37,38), although this is not a universal finding (7,10,12,13,39). Neuronal NOS (nNOS) is considered the predominant source of NO in contracting skeletal muscle (14,26) and is largely targeted to the mechanosensing dystrophin-glycoprotein complex at the sarcolemma (4). ...
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... In addition to its direct positive effects on cardiac function, apelin has been shown to increase the release of nitric oxide, which has many important functions in peripheral vasculature (Heinonen et al., 2011(Heinonen et al., , 2013 and in the heart (Seddon et al., 2007;Simon et al., 2014). Consequently, apelin may be one of the key mediators regulating myocardial function and circulation, which are positively affected by regular physical activity (Heinonen et al., 2008(Heinonen et al., , 2014a. ...
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... There are at least three isoenzymes of NOS: constitutive neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and constitutive endothelial NOS (eNOS or NOS-3), located on different chromosomes and expressed in different cell lines [218]. eNOS has been described as a major regulator of adipose tissue metabolism and energy balance by affecting lipolysis [219]. The adipose tissue and skeletal muscle of obese humans and rodents have decreased eNOS [220][221][222]. ...
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The control of vascular tone during exercise is highly complex and integrated. Specifically, in regards to the contribution of nitric oxide (NO), the observed magnitude and muscle fiber-type dependency of the NO contribution to exercise hyperemia may differ depending on the timing of NO synthase (NOS) inhibition with respect to the exercise bout (i.e., administration prior to vs. during exercise). We tested the hypothesis that, in the presence of prior cyclooxygenase inhibition (indomethacin, 5 mg/kg(-1)), NOS inhibition (N(G)-nitro-L-arginine methyl ester, L-NAME; 10 mg/kg) administered during submaximal treadmill exercise would blunt blood flow and vascular conductance (VC) in the hindlimb muscle(s) of the rat with the greatest reductions in blood flow and VC occurring in the predominantly oxidative muscles. Adult female Wistar rats (n = 10, age: 3-4 mo) ran on a motor-driven treadmill (20 m/min, 10% grade). Total and regional hindlimb muscle blood flow and VC were determined via radiolabeled microspheres before (control) and after L-NAME administration during exercise. L-NAME reduced (P < 0.05) total hindlimb muscle blood flow (control: 123 + or - 10, L-NAME: 103 + or - 7 ml x min(-1) x 100g(-1)) and VC (control: 0.95 + or - 0.09, L-NAME: 0.63 + or - 0.05 ml x min(-1) x 100g(-1) x mmHg(-1)). There was a significant correlation (r = 0.51, P < 0.05) between the absolute reductions in VC after L-NAME and the percent sum of type I and IIa fibers in the individual muscles and muscle parts; however, there was no correlation (P = 0.62) when expressed as blood flow. Surprisingly, the highly oxidative muscles demonstrated a marked ability to maintain oxygen delivery, which differs substantially from previous reports of L-NAME infusion prior to exercise in these muscles. The demonstration that NO is an important regulator of blood flow and VC in the rat hindlimb during treadmill exercise, but that the fiber-type dependency of NO is altered markedly when NOS inhibition is performed during, vs. prior to, exercise, lends important insights into the integrated nature of vascular control during exercise.
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Adenosine is a widely used pharmacological agent to induce a "high-flow" control condition to study the mechanisms of exercise hyperemia, but it is not known how well an adenosine infusion depicts exercise-induced hyperemia, especially in terms of blood flow distribution at the capillary level in human muscle. Additionally, it remains to be determined what proportion of the adenosine-induced flow elevation is specifically directed to muscle only. In the present study, we measured thigh muscle capillary nutritive blood flow in nine healthy young men using PET at rest and during the femoral artery infusion of adenosine (1 mgmin-1l thigh volume -1), which has previously been shown to induce a maximal whole thigh blood flow of ∼8 1/min. This response was compared with the blood flow induced by moderate- to high-intensity one-leg dynamic knee extension exercise. Adenosine increased muscle blood flow on average to 40 ± 7 ml,min -1,100 g muscle-1 with an aggregate value of 2.3 ± 0.61/min for the whole thigh musculature. Adenosine also induced a substantial change in blood flow distribution within individuals. Muscle blood flow during the adenosine infusion was comparable with blood flow in moderate- to high-intensity exercise (36 ± 9 ml.min-1.100 g muscle -1), but flow heterogeneity was significantly higher during the adenosine infusion than during voluntary exercise. In conclusion, a substantial part of the flow increase in the whole limb blood flow induced by a high-dose adenosine infusion is conducted through the physiological non-nutritive shunt in muscle and/or also through tissues of the limb other than muscle. Additionally, an intra-arterial adenosine infusion does not mimic exercise hyperemia, especially in terms of muscle capillary flow heterogeneity, while the often-observed exercise-induced changes in capillary blood flow heterogeneity likely reflect true changes in nutritive flow linked to muscle fiber and vascular unit recruitment.
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Possible nitric oxide (NO)-mediated effects on lipolysis were investigated in vivo in human subcutaneous adipose tissue using microdialysis, as well as in vitro on isolated fat cells of non-obese, healthy volunteers. NO donors were added to the ingoing dialysate solvents. Changes in lipolysis and local blood flow were investigated by measuring glycerol levels and ethanol ratios, respectively, in the microdialysates. It was shown that the NO synthase inhibitor, NG-monomethyl L-arginine (L-NMMA), but not the biologically inactive enantiomer NG-monomethyl D-arginine (D-NMMA), increased glycerol levels in the microdialysates without causing a change of local blood flow. In addition, L-NMMA increased glycerol levels in the microdialysate when local blood flow was stimulated with hydralazine. Nitric oxide gas as well as the NO donor, nitroglycerine, reduced glycerol release from isolated adipocytes in vitro. Expression of inducible nitric oxide synthase (iNOS) in human adipose tissue was shown by Western blot analysis. Biologically active NOS was demonstrated by measuring total enzymatic activity. In conclusion, the data demonstrate that inhibition of NO release in subcutaneous adipose tissue results in an increased lipolysis in vivo. These effects, which were also observed in vitro, are independent of local blood flow changes. Furthermore, the demonstration of enzymatic NOS activity and the expression of inducible nitric oxide synthase (iNOS) in adipose tissue indicate that locally synthesized NO may play a role in the physiological control of lipolysis in human adipose tissue. British Journal of Pharmacology (1999) 126, 1639–1645; doi:10.1038/sj.bjp.0702430
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Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 +/-2 years, BMI 31.7 +/-1.2 kg/m(2), fasting plasma glucose 9.52+/-0.80 mmol/L) and eight healthy individuals (aged 46 +/-2 years, BMI 27.1 +/- 1.5 kg/m(2), fasting plasma glucose 4.69+/-0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 microM) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was also examined at a concentration of 50 microM. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P=0.03) but not the therapeutic SNP doses (P=0.60) or SNAP (P=0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.
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The effect of myocardial uptake of free fatty acids (FFAu) on myocardial oxygen consumption (MVO2) in relation to increased heart rate and inotropic stimulation was determined in patients with coronary artery disease. Submaximal atrial pacing and isoproterenol stimulation increased MVO2 by 66% and 142%, respectively, at similar heart rates. Inhibition of lipolysis with beta-pyridyl carbinol almost abolished FFAu and reduced MVO2 significantly. Increased heart rate contributed 47% and FFAu 50% of the raised MVO2 attributed to inotropic stimulation was 30%. Augmentation of FFAu by triglyceride/heparin infusion increased MVO2 significantly above control levels, both during pacing and isoproterenol infusion. We conclude that MVO2 is closely correlated to FFAu, catecholamines sensitize the heart to FFA, and increased FFAu account for a major part of the increased MVO2 during catecholamine stimulation. The importance of reducing heart rate and lipolysis to reduce myocardial oxygen requirements is emphasized.