International Journal of Obesity (2000) 24, Suppl 4, S36±S40
ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00
www.nature.com/ijo
Nitric oxide: a new player in the modulation of
energy metabolism
S Kapur1, F Picard1, M Perreault1, Y Deshaies1 and A Marette1*
Department of Physiology, Lipid Research Unit, Centre de Recherche sur le MeÂtabolisme EÂnergeÂtique (CREME), Laval University
Hospital Research Center, QueÂbec, Canada
1
Nitric oxide (NO) is a key messenger molecule in several cell types. NO formation is catalyzed by a family of NO
synthases (NOS) that use L-arginine as a substrate. Rat adipose tissue expresses the inducible, macrophage-type,
nitric oxide (NO) synthase isoform (iNOS). Systemic administration of the bacterial endotoxin lipopolysaccharide
(LPS) markedly increases the expression and activity of iNOS in both white and brown adipose tissues, as well as in
skeletal muscle. iNOS induction can be reproduced in vitro by treatment of cultured white or brown adipocytes or L6
myocytes with LPS and in¯ammatory cytokines (TNFaa, IFNgg). The physiological role of NO in adipose tissues and
skeletal muscle is still obscure. Recent evidence suggests that NO may be implicated in the regulation of energy
metabolism. Using both pharmacological and genetic models of iNOS invalidation, we have recently begun to uncover
a role for NO in the modulation of glucose transport and lipoprotein hydrolysis. These studies support the emerging
concept that NO may ful®ll the dual role of modulating energy metabolism in both physiological and pathological
conditions as well as contributing to local immune defense during in¯ammatory processes.
International Journal of Obesity (2000) 24, Suppl 4, S36±S40
Keywords: nitric oxide synthase isoform; lipopolysaccharide; tumor necrosis factor-a; adipocytes; skeletal muscle;
obesity; insulin resistance; in¯ammation; endotoxemia
Introduction
Skeletal muscle and adipose tissues are the principal
sites of glucose and lipid metabolism in the postabsorptive state. Both glucose and lipid metabolism
are greatly affected in conditions associated with
increased levels of in¯ammatory cytokines such as
endotoxemia, but also including metabolic diseases
such as obesity and diabetes. The mechanisms by
which these immune molecules modulate energy
metabolism are still unclear. In this paper, we will
review the increasing body of evidence supporting
the concept that NO generated by the inducible
NO synthase (NOS) iNOS is implicated in this
modulation.
NO synthases in skeletal muscle and adipose tissues
There are at least three isotypes of NOS: the endothelial cell NOS (eNOS), the neuronal type NOS (nNOS),
and the macrophage-type inducible NOS (iNOS).
When induced, this latter NOS isotype generates NO
at a much higher rate and for longer periods of time
than constitutive nNOS and eNOS enzymes.1 We have
*Correspondence: A Marette, Department of Physiology and
Lipid Research Unit, Laval University Hospital Research Center,
2705 Laurier Boulevard, RC 9502, Ste-Foy, QueÂbec, Canada
G1V 4G2.
E-mail: [email protected]
previously shown that, in the basal state, skeletal
muscle expresses both nNOS and eNOS isoforms.2
In fact, nNOS is mostly expressed in type IIb muscle
®bers, whereas eNOS detection in skeletal muscle is
in large part explained by the presence of vascular
endothelial cells in the tissue. In the rat, iNOS is not
detected to any signi®cant level in resting muscle but
it is induced following in vivo injection of lipopolysaccharide (LPS) (Figure 1). The endotoxin-mediated
iNOS induction results in marked NO production by
the isolated soleus and extensor digitorum longus
(EDL) muscles in vitro.
Unlike skeletal muscle, neither eNOS nor nNOS
can be detected to signi®cant levels in rat adipose
tissue (Figure 2). On the other hand, iNOS protein can
be detected in both white (Figure 2) and brown
adipose tissues (data not shown) of rats, even in the
basal state. Whether iNOS expression in adipose
tissues of normal rats is localized to adipocytes or
other cell types (eg resident macrophages) is still
unknown. As in muscle, endotoxin challenge markedly increased cellular iNOS protein concentrations
and iNOS activity in both white and brown adipose
tissues (Figure 3). The effect is marked (10 ± 20-fold
increase in iNOS activity) and can be detected within
2 h of LPS exposure in vivo.
Mechanism of iNOS induction in endotoxemia
We have recently investigated the mechanism of
iNOS activation in adipose tissue and skeletal
NOS in energy metabolism
S Kapur et al
S37
Figure 1 LPS-induced iNOS expression in skeletal muscle. Rats
were injected i.p. with LPS or saline and sacri®ced 8 h later.
Soleus and EDL muscles were removed and used for determination of iNOS protein contents or NO production. iNOS in soleus
was detected by immunoprecipitation followed by SDS ± PAGE
and Western blot analysis of iNOS immunoreactivity. Molecular
weight standards are shown on the left. NO production was
measured by the assessment of plasma concentrations of
NO2=NO3 by the Griess reaction. The results are representative
of three independent experiments. **P < 0.01 vs control values.
Figure 2 Expression of NOS isoforms in white adipose tissue.
Tissue extracts (50 mg) of epididymal white adipose tissue (WAT)
from a control rat were subjected to SDS ± PAGE and immunoblot analysis using isoform-speci®c antibodies against iNOS,
eNOS and nNOS. Extracts from L6 myocytes, endothelial cells
and skeletal muscle were used for positive controls of iNOS,
eNOS and nNOS, respectively. Only iNOS (mol wt 130 kDa) was
detected in control WAT.
muscle of LPS-treated rats. Systemic administration
of the bacterial endotoxin LPS causes the release of
several cytokines (such as tumor necrosis factor-a
(TNFa), interleukin-1 and -6, and interferon-g
(IFNg))3,4 and these in¯ammatory molecules may
activate iNOS expression in a concerted manner. We
have therefore used cell lines of skeletal muscle (L6),
and white adipose tissue (3T3-L1) to investigate the
individual and combined effects of two cytokines
(TNFa and IFNg) and LPS on iNOS activation in
vitro. As shown in Figure 4, incubation of muscle or
adipose cells with TNFa, LPS or IFNg alone did not
increase NO production, as measured by the accumulation of nitrite in the incubation medium. A small
increase in NO production was detected when the two
Figure 3 Effects of LPS on iNOS protein levels and iNOS activity
in epididymal (EWAT), perirenal (PWAT) and interscapular
brown (BAT) adipose tissues. Rats were treated or not with
LPS for 2, 4 or 8 h and tissue iNOS protein levels and iNOS
activity were determined. The immunoblot is representative
of three independent experiments with tissues from different
animals. Results for iNOS activity are represented as means
s.e. of two to three individual experiments. **P < 0.01 vs
control values. (Reproduced from Am J Physiol 1999; 276:
E635, with permission from the American Physiological Society.)
cytokines were combined. However, maximal activation of iNOS was observed when cells were incubated
with all these immune molecules. Indeed, the combined effect of LPS, TNFa and IFNg was clearly
synergistic as compared to the effect of only two of
these factors. Similar ®ndings were obtained with a
brown adipocyte cell line (T37i) (data not shown).
Since neither LPS alone nor individual cytokines
could increase iNOS-mediated NO production by the
muscle or adipose cells, it appears that the induction
of this enzyme in vivo is mediated by a complex
network of interactions between in¯ammatory cytokines and endotoxin at the cellular level. Our data
indicate that in myocytes and white adipocytes, IFNg
is the most potent inducer of iNOS but that other
cytokines and LPS must be present to maximally
induce the enzyme. Interestingly, we found that
TNFa is more potent than IFNg in increasing iNOS
activity in cultured brown adipocytes (Penfornis and
Marette, unpublished data). In future studies, it will be
important to de®ne more precisely the transduction
pathways involved in iNOS activation by cytokines
and LPS. In particular, the potential roles of interleukin-1 and -6 released from activated adipose cells
in contributing to iNOS activation remain to be
examined. In any case, our results clearly establish
that LPS and cytokines can induce iNOS expression
by a direct interaction with the adipocyte or myocyte.
Possible role of iNOS in coupling energy metabolism to
immune function
iNOS is implicated in host defense and it is synthesized de novo in response to a variety of in¯ammatory
stimuli (reviewed in Nathan5). Induction of iNOS in
International Journal of Obesity
NOS in energy metabolism
S Kapur et al
S38
Figure 4 LPS and cytokines synergistically increase iNOS activity in myocytes and adipocytes. L6 myocytes or 3T3-L1 adipocytes
were treated or not with the cytokines TNFa (10 ng=ml) and=or IFNg (200 U=ml) and=or LPS (10 mg=ml). Incubation time was 24 h for
muscle cells and 48 h for adipocytes. After the incubation, nitrite accumulation in the incubation medium was measured under
identical conditions by the Griess reaction. Results are mean s.e. of three independent experiments, each performed in triplicate with
different batches of cells. *P < 0.05 vs control values; ‡P < 0.05 vs TNFa ‡ IFNg values.
muscle and fat cells may therefore contribute to the
immune defence in in¯ammatory settings. It is also
noteworthy that white and brown adipose tissues are
dispersed throughout the body and are widely distributed along and even within major organs. It is therefore likely to represent a major source of local NO
generation in endotoxic shock. Another possible role
of iNOS in skeletal muscle and adipose tissues is to
mediate the profound changes in energy metabolism
that are known to occur during endotoxic shock.
Indeed, endotoxemia is characterized by marked perturbations in both glucose and lipid metabolism in
animals and humans,6,7 and insulin resistance is commonly observed in this condition (see Figure 5).8,9 We
have summarized below some of our recent ®ndings
that strongly support the hypothesis that iNOS-generated NO production is mediating the perturbations of
energy metabolism in endotoxemia and possibly other
in¯ammatory disorders.
Role of iNOS in endotoxin-mediated changes in glucose
metabolism
Figure 5 Proposed role of iNOS as a modulator of energy
metabolism in altered metabolic states such as obesity and
in¯ammatory diseases. Increased levels of in¯ammatory cytokines in these conditions lead to iNOS induction in skeletal
muscle and overexpression of iNOS in adipose tissues. iNOSgenerated NO overproduction in muscle causes insulin resistance for glucose uptake and inhibits LPL activity, leading to
insulin resistance and hypertriglyceridemia. On the other hand,
increased expression of iNOS in adipose tissues may increase
basal rates of lipolysis and inhibit insulin action on lipolysis,
leading to increase circulating FFA levels.
International Journal of Obesity
It is well documented that endotoxemia elicits profound changes in whole-body glucose homeostasis in
both animals and humans.7 Septic patients have an
accelerated rate of glucose clearance in the basal
state.10 On the other hand, sepsis is also associated
with a state of insulin resistance, as evidenced by
diminished glucose tolerance, hyperinsulinemia and
impaired insulin action on peripheral glucose
disposal.8 ± 10 We have previously demonstrated that
cytokines and LPS increase glucose uptake in muscle
NOS in energy metabolism
S Kapur et al
cells by inducing the expression of iNOS and NO.11
On the contrary, exaggerated NO production upon
iNOS induction also causes insulin resistance by
interfering with insulin action to promote glucose
transport into the muscle cells.2,11 This effect is
direct since cytokine-induced iNOS overexpression
and iNOS-mediated inhibition of insulin-stimulated
glucose uptake could be reproduced in L6 myocytes in
culture.11 The inhibitory effects of NO on insulin
action may be particularly relevant in in¯ammatory
conditions in which muscle insulin resistance is associated with elevated levels of cytokines such as TNFa,
IFNg and interleukins. Moreover, iNOS induction
may also be involved in obesity-linked metabolic
alterations since the obese state is also associated
with increased TNFa secretion by adipocytes and
muscle cells (see below).
Very recently, we have further con®rmed the role of
iNOS in LPS-induced muscle insulin resistance using
pharmacological and genetic models of iNOS invalidation. Thus we ®rst showed that LPS-induced muscle
insulin resistance in rats could be blocked by pretreatment with the speci®c iNOS inhibitor aminoguanidine (AGN) (Kapur and Marette, unpublished data).
Moreover, LPS-induced insulin resistance is signi®cantly blunted in mice de®cient for the iNOS gene
(Perreault and Marette, unpublished data). These
results strongly support our hypothesis that iNOS is
a key player in the development of insulin resistance
in endotoxemia and other in¯ammatory diseases.
Role of iNOS in endotoxin-mediated changes in lipid
metabolism
We have also recently explored the possible role of
iNOS in modulating lipid metabolism in skeletal
muscle and adipose tissues of endotoxin-challenged
rats. It is well known that endotoxemia increased
plasma triglycerides and that this is explained, at
least in part, through a clearance of lipoproteinbound triglycerides by peripheral tissues (see Hardardottir et al6). This led us to test whether iNOS may
be involved in this endotoxin-mediated alteration in
triglyceride hydrolysis. It was found that LPS administration increased triglyceride levels in rats but that
pre-treatment with the iNOS inhibitor AGN completely blocked this effect. Interestingly, LPS-mediated
inhibition of LPL activity in skeletal muscle was also
prevented by AGN pre-treatment, whereas the reduction in adipose tissue LPL activity was not affected by
the iNOS inhibitor (Picard, Kapur, Marette and
Deshaies, unpublished data). Very recently, we have
also found that LPS failed to affect plasma triglyceride levels and LPL activity in skeletal muscle of
iNOS-de®cient mice, further documenting the primary
role of iNOS in LPS-induced hypertriglyceridemia
(Picard, Perreault, Marette and Deshaies, unpublished
data). These results provide convincing evidence that
iNOS induction is responsible for the marked inhibition of LPL activity in skeletal muscle of endotoxic
rats and further indicate that muscle, and not adipose
tissue, is the primary site of decreased lipoprotein
hydrolysis in this condition.
Endotoxemia is also characterized by an increased
level of circulating free fatty acids (FFA), which is the
consequence of increased release by the adipose
cells.10,12 Whether iNOS-mediated NO production is
responsible for this increased fat cell lipolysis is not
known. There is some evidence that NO can regulate
adipocyte lipolysis. Indeed, Gaudiot et al 13 recently
reported that some NO-releasing compounds inhibit
catecholamine-stimulated lipolysis in rat fat cells. The
NO-dependent inhibition of b-adrenoceptor-stimulated lipolysis has been recently con®rmed in isolated
human fat cells.14 On the other hand, in the absence of
catecholamines, NO donors can also increase basal
lipolysis.13 Whether NO is mediating the known
stimulatory effects of cytokines (such as TNFa) on
lipolysis remains to be tested. Furthermore, NO may
also increase FFA release by interfering with insulin's
ability to blunt lipolysis since NO overproduction
inhibits insulin action in other cell types (myocytes).
More studies are needed to con®rm the role of NO in
the increased release of FFA into the circulation of
septic subjects.
S39
Is iNOS involved in the obesitylinked metabolic syndrome?
TNFa expression is increased in adipose tissue of
genetic and nutritional rodent models of obesity as
well as in obese individuals.15 ± 17 There is also evidence that TNFa secretion is increased in muscle cells
of type II diabetic subjects.18 This increased TNFa
secretion, especially in the presence of other in¯ammatory cytokines in vivo, may induce iNOS expression in muscle and fat cells. It is therefore possible
that an exaggerated production of NO through iNOS
induction may contribute to the development of insulin resistance and hypertriglyceridemia in obesitylinked diabetes (Figure 5). We have preliminary
evidence that iNOS expression is induced in skeletal
muscle of high-fat fed rodents (Perreault and Marette,
unpublished observations). Future studies will be
aimed at testing whether iNOS invalidation (pharmacologically or genetically) of obese animals could
reduce or even prevent the development of insulin
resistance, hypertriglyceridemia and associated cardiovascular complications.
Conclusions
The studies reviewed in the present paper strongly
support the tenet that iNOS is a key player in the
modulation of energy metabolism in altered metabolic
states such as in endotoxemia and other in¯ammatory
International Journal of Obesity
NOS in energy metabolism
S Kapur et al
S40
conditions. Changes in both glucose metabolism and
lipoprotein hydrolysis appear to be mostly explained
by iNOS induction in skeletal muscle. iNOS expression is also markedly increased in white and brown
adipose tissues of septic rats and the increased NO
production may be involved in the modulation of fat
cell lipolysis. What remains unclear is whether the
iNOS-mediated perturbations in energy metabolism
are part of an adaptative phenomenon to in¯ammatory
settings or simply an uncontrolled secondary effect of
high NO output. However, the concerted and tissuespeci®c effects of iNOS-produced NO on glucose and
lipid metabolisms strongly support the emerging concept that iNOS is playing a critical role in modulating
energy metabolism in in¯ammatory conditions. Future
studies may also reveal an important role for iNOS in
the pathogenesis of obesity-linked insulin resistance
and hyperlipidemia.
Acknowledgements
This work was supported by grants from the Medical
Research Council of Canada and by scholarships from
the Medical Research Council and the Fonds de la
Recherche en Sante du QueÂbec to A Marette. S Kapur
was supported by a fellowship from the Canadian
Diabetes Association. F Picard and M Perreault were
supported by studentships from the Medical Research
Council of Canada and by the `Association du diabeÁte
du QueÂbec'.
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