Department of Physiology and Lipid Research Unit, Laval University Hospital Research Center, Quebec, Canada G1V 4G2
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ABSTRACT |
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The aim of the present study was to investigate
the mechanism of adipose tissue inducible nitric oxide synthase (iNOS)
induction in endotoxemia. Systemic administration of the bacterial
endotoxin lipopolysaccharide (LPS) to rats for 8 h markedly increased
iNOS mRNA and protein levels in white and brown adipose tissues. This effect was comparable to or greater than the induction of iNOS in
liver, kidney, or skeletal muscle. iNOS activity was also found to be
greatly enhanced in both white and brown adipose tissues of LPS-treated
rats (an ~12- to 20-fold increase). Treatment of cultured 3T3-L1
adipocytes with LPS, tumor necrosis factor-
(TNF-
), or
interferon-
(IFN-
) alone failed to induce iNOS activity. However,
when used in combination, TNF-
, IFN-
, and LPS markedly and
synergistically increased iNOS activity in these cells. In conclusion,
these results suggest that adipose tissue is a major site of iNOS
expression in endotoxemia. Our data further indicate that iNOS
induction can be reproduced in vitro in cultured adipocytes and that a
concerted action of cytokines and endotoxin is needed for maximal
activation of the enzyme.
lipopolysaccharide; tumor necrosis factor-; interferon-
; adipocytes; skeletal muscle; liver; kidney
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INTRODUCTION |
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NITRIC OXIDE (NO) is an important messenger molecule that plays a critical role in a wide variety of physiological functions, including neuronal transmission, vascular relaxation, immune modulation, and cytotoxicity (25). There are at least three isotypes of NO synthase (NOS): the calcium-dependent endothelial cell NOS (eNOS), the neuronal type NOS (nNOS), and the so-called inducible, relatively calcium-independent NOS (iNOS). iNOS is implicated in host defense and is synthesized de novo in response to a variety of inflammatory stimuli. As opposed to eNOS and nNOS, iNOS can produce large amounts of NO over prolonged periods of time (26, 43).
Whereas NO produced by iNOS is beneficial or even critical for host survival in several infectious diseases, it is also known to be detrimental to the host in some inflammatory settings. For example, induction of iNOS expression in many tissues in endotoxic shock results in an enhanced formation of NO that contributes to hypotension, vascular hyporeactivity to vasoconstrictors, organ injury, and dysfunction (see Ref. 26 for a review). iNOS induction by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) has been reported in several tissues, including (but not limited to) blood vessels, lung, liver, kidney, cardiac, and skeletal muscle (22, 33, 35). More recently, one study reported that injection of LPS also increases iNOS activity and protein levels in epididymal adipose tissue of rats (30). This finding suggests that adipose tissue is a potential site of NO production in endotoxemia. However, the quantitative contribution of adipose tissue to systemic NO production in endotoxic shock remains unclear. Because important regional variations exist in adipose tissue metabolism (12, 23, 24), it is important to examine whether iNOS expression is widely induced in adipose tissues (white and brown) or in fact limited to certain fat depots. Furthermore, the mechanism of iNOS induction in adipose cells is still poorly understood. LPS is known to increase the release of a series of inflammatory cytokines from the peripheral immune system (1, 27, 32), and these molecules may act individually or in concert with LPS to increase iNOS expression at the tissue level.
The main goals of this study were, therefore, 1) to compare iNOS induction in different adipose depots and other tissues of LPS-injected rats, a well-established experimental model of endotoxemia, and 2) to test whether iNOS induction in adipocytes can be reproduced in adipose cells in vitro and to determine the role of cytokines and LPS in increasing iNOS expression in these cells. The results suggest that white and brown adipose tissues are major sites of iNOS expression in endotoxemia and that iNOS expression is induced by a direct and synergistic action of cytokines and LPS on the adipocyte.
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MATERIALS AND METHODS |
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Materials.
LPS and chromatographically purified LPS (Escherichia
coli serotype 055:B5), NADPH, FAD, riboflavin
monophosphate (FMN), calmodulin, EGTA, and
BH4 were obtained from Sigma (St.
Louis, MO). NG-nitro-L-arginine
methyl ester (L-NAME) was
obtained from Alexis (San Diego, CA). Cd filings were purchased from
Fluka Chemical (Ronkonkoma, NY).
L-[3H]arginine
was purchased from Amersham (Oakville, ON, Canada). Recombinant murine
tumor necrosis factor- (TNF-
) was purchased from R&D systems.
Murine interferon-
(IFN-
) was kindly supplied by Dr. Martin
Olivier (CHUL Research Center, QC, Canada). Anti-iNOS polyclonal
antibody was purchased from Cedarlane Laboratories (Hornby, ON,
Canada). Monoclonal and polyclonal antibodies against nNOS and eNOS
were obtained from Transduction Laboratories (Lexington, KY).
Animal treatment.
This study was approved by the Animal Care and Handling Committee of
Laval University. Male Sprague-Dawley rats (200-250 g) purchased
from Charles River (Montreal, QC, Canada) were used in these studies.
Rats were randomly assigned to LPS-treated (2, 4, or 8 h) or control
groups (n = 4) and were housed in
individual cages, maintained on a 12:12-h light-dark schedule, and fed
ad libitum with Purina rat chow. Rats were injected with LPS (15 mg/kg
ip) or saline (1 ml/kg ip) for endotoxin and control groups, respectively. Animals were euthanized after 2, 4 or 8 h,
and various organs (white and brown adipose tissues, kidney, liver, and
skeletal muscle) were removed and stored at 80°C until
further processing. In a subsequent study, rats were also injected with
a lower dose of LPS (0.6 mg/kg ip) or saline and euthanized 12 h
posttreatment. Blood was also collected to measure plasma levels of NO products.
Cell culture.
3T3-L1 adipocytes (kind gift of Dr. A. Klip, Hospital for Sick
Children, Toronto, ON, Canada) were grown and differentiated exactly as
previously described (40). Briefly, cells were grown and maintained in
monolayer culture in -DMEM containing 20% (vol/vol) calf serum and
1% (vol/vol) antibiotic-antimycotic solution (10,000 U/ml penicillin,
10,000 µg/ml streptomycin, and 25 µg/ml amphotericin B) in an
atmosphere of 10% CO2 at
37°C. Cells were plated in 12-well plates or 10-cm dishes at 10,000 cells/ml. One day postconfluence, differentiation was initiated by
incubating cells in
-DMEM containing 10% (vol/vol) fetal bovine
serum, IBMX (115 µg/ml), dexamethasone (390 ng/ml), and insulin (10 µg/ml) for 48 h, followed by a 72-h incubation period in the same
medium but without IBMX and dexamethasone. Differentiation was
completed by incubating cells in
-DMEM containing 10% (vol/vol)
fetal bovine serum for 8-12 days. Adipocytes were incubated with
or without cytokines (TNF-
and/or IFN-
) and/or LPS at the
concentrations indicated in figure legends for
48 h. In some
experiments, a chromatographically purified LPS was also used.
Nitrite/nitrate assay. The plasma nitrite and nitrate levels were measured after Cd-mediated reduction of nitrate to nitrite as described by Vodovotz (39). Plasma was first cleared of proteins by ZnSO4 precipitation. Plasma samples (50 µl) were brought up to 200 µl with water, and 10 µl of 30% (wt/vol) ZnSO4 solution were added. The samples were vortex mixed, incubated at room temperature for 15 min, and centrifuged for 5 min. The resulting supernatants were added to the Cd-containing microcentrifuge tubes (~0.5 g of Cd per sample) and incubated overnight at room temperature in a rotator. One hundred microliters of the supernatants were mixed with 100 µl of Greiss reagent (1% sulfanilamide-0.1% napthylethylenediamine dihydrochloride-2.5% H3PO4) in 96-well plates. The plates were read at 550 nm against standard curves of sodium nitrite. For 3T3-L1 adipocytes, the accumulation of nitrite was used as an index of iNOS activity (3). Nitrite was measured in 100-µl samples of the incubation medium by the Greiss method just described.
Measurements of NOS activity in tissue and cellular extracts. Rat tissues were homogenized in 20 vol of homogenization buffer containing 25 mM Tris · HCl (pH 7.4), 1 mM EDTA, and 100 µg/ml phenylmethylsulfonyl fluoride (PMSF) with a speed-controlled Brinkmann polytron (PT20 probe) at a setting of 10 (15 strokes). In experiments with 3T3-L1 adipocytes, cells were scraped off 10-cm dishes, collected by spinning at 800 g for 10 min, and resuspended in lysis buffer containing 20 mM Tris · HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol (DTT), and 100 µg/ml PMSF. Cells were left on ice for 15 min before sonication for 30 s at medium speed. The cell homogenate was centrifuged at 1,500 g for 15 min at 4°C, and the supernatant was used for the measurement of NOS activity. Protein concentrations of the homogenates were determined by the bicinchoninic acid method (Pierce) with BSA as the standard. NOS activity in tissue and cell extracts was quantified by the conversion of L-[3H]arginine to [3H]citrulline, as previously described (19). Briefly, 100-200 µg of proteins were incubated in 50 mM HEPES (pH 7.4) with 100 nM L-[3H]arginine (50 Ci/mmol), 120 µM NADPH, 60 mM L-valine, 12 mM L-citrulline, 1.2 mM MgCl2, 0.2 mM CaCl2, 10 µg/ml CaM, 3 µM BH4, 1 µM FAD, and 1 µM riboflavin monophosphate. The reaction was carried out for 1 h at 37°C with and without 2 mM L-NAME, an inhibitor of NOS, and 1 mM EGTA and was terminated by adding 2 ml of 20 mM HEPES (pH 5.5) containing 2 mM EDTA. Samples were applied to 1-ml columns of Dowex AG50W-X8 (Na+ form), which were eluted with 2 ml of water. [3H]citrulline was quantified by liquid scintillation spectroscopy of 4.0 ml flow through. The activity of Ca2+-independent NOS (or iNOS) was determined from the difference between samples containing EGTA and samples containing EGTA and L-NAME.
RNA extraction and RT-PCR. RNA extraction and RT-PCR analysis of iNOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were performed as recently described (3). Briefly, total cellular RNA was isolated using guanidium thiocyanate-phenol-chloroform extraction with the TRIzol reagent (Life Technologies, Burlington, ON, Canada). cDNA synthesis was performed with 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL) with 100-400 ng of total RNA in 20 µl of reverse transcriptase buffer (in mM: 50 Tris · HCl, pH 8.3, 75 KCl, 3 MgCl2, and 10 DTT) containing 1 mM each dNTP and 8 pmol of iNOS antisense primer or GAPDH antisense primer. The reaction was performed at 42°C during 1 h, and the enzyme was then denatured at 95°C for 10 min.
Samples were then supplemented with 3 µl of 10× PCR buffer (1× PCR buffer is 33.3 mM KCl and 3 mM MgCl2), 8 pmol of iNOS sense primer or GAPDH sense primer, and water to 30 µl. cDNAs were denatured for 5 min at 94°C, cooled to 72°C, and then 1 unit of Thermophylus aquaticus DNA polymerase (Boehringer Mannheim) was added to each sample. Amplification was performed by 30 cycles of temperature (94°C, 1 min; 60°C, 1 min; 72°C, 1 min with an extension of 3 s at each cycle) in a temperature cycler (DNA Thermal Cycler, Perkin-Elmer). Sequences of the antisense and sense oligonucleotides were 5'-TGGAACCACTCGTACTTGGGA-3' and 5'-CAAGAGTTTGACCAGAGGACC-3' for iNOS and 5'-AGATCCACAACGGATACATT-3' and 5'-TCCCTCAAGATTGTCAGCAA-3' for GAPDH. The expected sizes of amplification products were 653 base pairs for iNOS and 331 base pairs for GAPDH. Amplification products were run in 12% acrylamide gels and visualized by ethidium bromide staining.Western blot analysis. Protein samples (50-100 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5% polyacrylamide gels and were electrophoretically transferred (100 V, 2 h) to polyvinylidene difluoride (PVDF) filter membranes for 2 h. PVDF membranes were incubated for 1 h at room temperature with buffer I (50 mM Tris · HCl, pH 7.4, and 150 mM NaCl) containing 0.04% NP-40, 0.02% Tween 20, and 3% bovine serum albumin (fatty acid-free BSA), followed by overnight incubation at 4°C with the polyclonal anti-iNOS antibody (1:2,500 dilution). PVDF membranes were then washed for 30 min, followed by a 1-h incubation with anti-rabbit immunoglobulin G (1:10,000 dilution) conjugated to horseradish peroxidase (Amersham) in buffer I containing 1% BSA. The PVDF membranes were washed for 30 min in buffer I, and the immunoreactive bands were detected by the enhanced chemiluminescence method.
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RESULTS |
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The effect of LPS treatment on NO production was assessed by measuring
the concentrations of its stable metabolites nitrite and nitrate in the
plasma (Fig. 1). A significant increase in plasma nitrite/nitrate levels was already detectable 2 h after LPS
challenge, and those were still markedly elevated by 8 h posttreatment.
|
iNOS mRNA levels in adipose tissue, liver, kidney, and skeletal muscle
from control and LPS-treated rats were measured by RT-PCR (Fig.
2). iNOS mRNA was barely detectable in
tissues of control rats. LPS treatment induced iNOS expression in all
tissues, but more importantly in adipose tissues (Fig.
2A) and the liver (Fig.
2B). However, iNOS induction in
white adipose tissues was transient and peaked 4 h after LPS challenge.
At this time point, adipose iNOS induction was similar to that of the
liver and greater than that of the kidney and skeletal muscle. iNOS
mRNA levels in LPS-treated rats remained elevated for 8 h in brown
adipose tissue as well as in liver, kidney, and skeletal muscle. GAPDH mRNA levels were not different between samples from control and LPS-treated rats.
|
The effects of LPS treatment on iNOS protein levels and iNOS activity
in white and brown adipose tissues are illustrated in Fig.
3. iNOS protein and activity were barely detectable in
adipose tissues of control rats. LPS treatment significantly increased cellular iNOS protein concentrations and iNOS activity in all adipose
tissues investigated. However, iNOS protein levels and activity were
greater in brown adipose tissue, particularly 8 h post-LPS treatment.
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The increased iNOS expression in adipose tissue of LPS-treated rats has
been previously reported to be occurring mainly within adipocytes (30).
However, the mechanism of iNOS activation in these cells still remains
unknown. Systemic administration of the bacterial endotoxin LPS causes
the release of several cytokines (such as TNF-, interleukin-1 and
-6, and IFN-
) (1, 27, 32), and these inflammatory molecules may
activate iNOS expression in a concerted manner in adipose cells. We
have therefore used the 3T3-L1 adipocyte cell line to investigate the
individual and combined effects of two cytokines (TNF-
and IFN-
)
and LPS on iNOS activation in vitro. As shown in Fig.
4, incubation of the adipose
cells with TNF-
, LPS, or IFN-
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 cytokines were combined. A more robust response was obtained
when LPS was combined with IFN-
(but not TNF-
). However, maximal
activation of iNOS was observed when cells were incubated with all of
these immune molecules. Indeed, the combined effect of LPS, TNF-
,
and IFN-
was clearly synergistic compared with the effect of only
two of these factors. Similar results were obtained when a more
purified LPS (cell culture grade) was used, ruling out the possibility
that iNOS induction was potentiated by some contaminants in the LPS
preparation (data not shown).
|
The results shown in Fig. 5 further show
that iNOS protein levels (Fig. 5A)
and iNOS activity (Fig. 5B) were
markedly increased in homogenates prepared from adipose cells treated
with cytokines and LPS, and that this activation could be blocked by
the NOS inhibitor L-NAME (Fig.
5C). These results indicate that
iNOS protein expression and activity can be directly induced by
cytokines and LPS in 3T3-L1 adipocytes. The measured NOS activity must
be related to iNOS, because this activity was observed even in the
presence of EGTA (calcium-independent) and because neither eNOS nor
nNOS isoforms could be detected in control or cytokine/LPS-treated 3T3-L1 adipocytes (data not shown).
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DISCUSSION |
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The results of the present study indicate that white and brown adipose tissues are major sites of iNOS expression in LPS-treated rats, a widely used experimental model of endotoxemia. Ribière et al. (30) previously reported that iNOS is expressed in rat white adipose tissue and that endotoxin increases iNOS protein content in that tissue. Whether adipose tissue represents a major source of iNOS-mediated NO production at the whole body level could not be clearly established in this previous study, as only one adipose region (epididymal) was investigated. Our data indicate that iNOS is induced in adipose tissues from different anatomic regions and that interscapular brown adipose tissue is also a target for iNOS expression in endotoxemia.
After LPS challenge, adipose tissue iNOS expression increased markedly and to levels comparable to that of the liver and greater than that of muscle and kidney. Because we found iNOS expression and activity to be markedly increased in three distinct anatomic fat deposits, it is reasonable to assume that adipose tissue (white and brown) is a major source of iNOS-derived NO production in endotoxemia. Indeed, the total mass of adipose tissue represents as much as 5.6% of the total weight in the rat (18) compared with other organs that showed significant iNOS expression on LPS challenge, such as the liver (3.5%) and the kidney (0.9%). Skeletal muscle is quantitatively by far the major organ, representing ~41% of total body weight in the rat (18), and may also represent an important source of NO production in endotoxemia. However, iNOS induction in that tissue is comparably low (Fig. 2) and even failed to be detected in some muscles of LPS-challenged rats (5). The development of the microdialysis technique for the study of adipose tissue metabolism (2) may help to determine the actual NO production capacity of this tissue in vivo in future studies.
In contrast with brown adipose tissue, liver, kidney, and muscle, iNOS
mRNA expression in white adipose tissues is very transiently increased,
peaking 4 h after LPS challenge. Despite this drop in iNOS mRNA levels
in adipose tissue of LPS-treated rats, iNOS protein concentration and
enzymatic activity remained elevated for 8 h in that tissue, albeit
less that in brown fat. Whereas iNOS gene transcription may not have
been turned off long enough for detection of a drop in protein levels
after 8 h, we cannot rule out the possibility that endotoxin also
increased adipose tissue iNOS protein half-life in white adipocytes. In
any case, the lack of correlation between iNOS mRNA levels and iNOS
protein content and activity 8 h after LPS treatment indicates that the sole measurement of iNOS mRNA levels in white fat is not always a good
index of iNOS activation in that tissue.
The dose of LPS used in this study is commonly used to cause septic shock in rats. The hemodynamic changes and hypotension that occur in this condition may contribute to iNOS induction in adipose tissues. In additional experiments, we have used a much lower dose of LPS (0.6 mg/kg) and still found iNOS induction (mRNA and protein levels) in brown and white adipose tissues (data not shown). However, it is known that even very low doses of LPS can have regional hemodynamic effects (14), and thus it is virtually impossible to rule out that part of the in vivo effect of the endotoxin on iNOS induction is totally independent from vascular mechanisms. Nevertheless, the fact that iNOS induction could be reproduced in cultured adipocytes strongly supports the hypothesis that at least part of this effect can be observed in the absence of any hemodynamic changes.
iNOS expression is increased in monocytes and macrophages in tissues of
LPS-treated rats (6). A significant part of iNOS induction in adipose
tissue of LPS-treated rats could therefore occur in these cells and not
in adipocytes. However, iNOS induction in adipose tissue of
LPS-challenged rats could be mainly recovered in adipocytes isolated
from the tissue (30). On the other hand, LPS was administered in vivo
in the latter study, and thus a direct relationship between the
endotoxin and the adipocyte could not be established. LPS has a wide
spectrum of peripheral and central targets and stimulates the release
of numerous immunoregulatory factors that may be implicated in iNOS
induction in adipocytes. Furthermore, Finck et al. (10) recently
reported that in vivo LPS administration indirectly stimulates leptin
production from adipocytes by augmenting TNF- secretion. It is
therefore important to test whether LPS-induced iNOS induction could be
reproduced in vitro in a homogeneous population of adipose cells.
Because adipose cells have been shown to express receptors for
endotoxin (CD14) (9), TNF-
(both p55-60 and p75-80
receptors) (21, 29), and IFN-
(28), direct induction of adipocyte
iNOS expression by these immune factors appeared plausible.
As shown in Figs. 4 and 5, iNOS expression can be directly increased by
incubating 3T3-L1 adipocytes with combinations of TNF- and IFN-
or LPS and IFN-
. Moreover, a marked and synergistic iNOS induction
was observed when LPS and both cytokines were combined. Because neither
LPS alone nor individual cytokines could increase iNOS-mediated NO
production by the adipocytes, it appears that the induction of this
enzyme in vivo is mediated by a complex network of interactions between
inflammatory cytokines and endotoxin at the level of the adipocyte. The
requirement of multiple cytokines and LPS for a maximal activation of
iNOS has been observed in other cell types (3, 7, 15, 42). However, the
relative potency of these immune factors is cell specific. Indeed,
whereas macrophage iNOS can be maximally stimulated by a combination of IFN-
and LPS (7), hepatocyte iNOS is poorly activated by these two
factors and needs the combination of at least two cytokines (preferably
TNF-
and interleukin-1) and LPS to promote a full enzymatic response
(15). Our data indicate that, in adipocytes, IFN-
is the most potent
inducer of iNOS but that other cytokines and LPS must be present to
maximally induce iNOS. In future studies, it will be important to
define more precisely the transduction pathways involved in adipocyte
iNOS activation by cytokines and LPS. In particular, the potential
roles of interleukin-1 and interleukin-6 released from activated
adipose cells in contributing to iNOS activation remain to be
examined. In any case, the results of the present study clearly
establish that LPS and cytokines can induce iNOS expression by a
direct interaction with the adipocyte.
What is the physiological role of iNOS in adipose tissue? White and
brown fat are dispersed throughout the body and are widely distributed
along and even within major organs, such as the intestine, liver,
kidney, blood vessels, and skeletal muscle. It is therefore likely to
represent a major source of local NO generation in endotoxin shock.
This suggests that adipose cells may play a role in the local immune
defense during inflammatory processes. A link between the immune system
and adipose tissue has been previously suggested on the basis of the
observation that adipsin, the murine equivalent of human complement
Factor D, is mainly expressed in adipose tissue (37). Adipsin is the
initial and rate-limiting enzyme of the alternative pathway of
complement activation (31), and stimulation of this pathway leads to
the production of several peptide cleavage molecules with antimicrobial
activities (37). A possible role for adipose cells in immune function
is also supported by other recent studies showing that adipose tissue
is a target for LPS and inflammatory cytokines. Adipose tissue is known
as a major site of expression and synthesis of immunoregulatory
molecules, such as the cytokines TNF- and leptin, the latter being
the product of the ob gene (see Ref.
38 for a review). Administration of LPS or the recombinant cytokines
TNF-
and interleukin-1 has been reported to induce leptin expression
and secretion by adipose tissue (16, 34). Both TNF-
and
interleukin-1 appear to be essential for optimal leptin secretion by
the adipocytes (8, 10). Although leptin was first described for its
role in modulating food intake and energy expenditure, there is now
substantial evidence that leptin is also involved in immune function,
as evidenced by its effect to enhance cytokine production and
phagocytosis by macrophages (13). Thus adipose tissue seems to
represent an important target site for LPS, and the concerted induction of TNF-
, leptin, and iNOS expression strongly supports a role for
this tissue in immune function.
On the other hand, iNOS-derived NO is a double-edged sword in that it
can also be detrimental to the host in a variety of inflammatory
conditions. For example, the marked NO production that is consequent to
iNOS expression in many tissues in endotoxic shock is believed to cause
hypotension, organ injury, and dysfunction (recently reviewed in Ref.
26). Another deleterious effect of endotoxemia that appears to be
related to iNOS induction is some impairment of glucose metabolism.
Indeed, the endotoxic state is characterized by marked perturbations in
glucose metabolism in both animals and humans (11), and insulin
resistance is commonly observed in this condition (36, 41).
Furthermore, administration of endotoxin and cytokines has been found
to reduce insulin-stimulated glucose uptake in adipose tissue and
adipose cells (17, 20). We have recently demonstrated that cytokines
and LPS impair insulin-stimulated glucose transport by inducing iNOS
expression and NO production in skeletal muscle cells (3, 19).
Experiments to test whether NO is also an autocrine modulator of
glucose metabolism in adipose cells are currently underway in our
laboratory. Although the cytokine TNF- is also a potent inhibitor of
lipoprotein lipase in adipocytes, this pathway is unlikely to be
dependent on iNOS induction because the presence of LPS or other
cytokines is not required for this effect (4, 44). Whether
iNOS-mediated insulin resistance is an adaptive phenomenon to infection
rather than an uncontrolled secondary effect of high NO output on
energy metabolism is still unknown. However, the observations made in
this and other studies that LPS induces the expression of TNF-
,
leptin, and iNOS in adipose tissue support the emerging concept that
these immunoregulatory molecules are playing critical roles in
modulating energy metabolism in inflammatory conditions.
In summary, these studies show that iNOS expression and activity are markedly increased in white and brown adipose tissues upon endotoxin treatment. This effect could be reproduced in adipocytes in vitro, indicating that iNOS expression can be directly activated within adipose cells in endotoxin-treated rats. Our results further suggest that a concerted action of cytokines and endotoxin is needed for maximal induction of adipocyte iNOS expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Martin Olivier for the kind gift of interferon- and
Dr. Claude Côté for critical reading of the manuscript.
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FOOTNOTES |
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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 Santé du Québec to A. Marette. S. Kapur was supported by a fellowship from the Canadian Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: A. Marette, Dept. of Physiology and Lipid Research Unit, Laval Univ. Hospital Research Center, 2705 Laurier Boulevard, RC 9502, Ste-Foy, Québec, Canada G1V 4G2 (E-mail: andre.marette{at}crchul.ulaval.ca).
Received 20 July 1998; accepted in final form 2 December 1998.
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