Mechanism of adipose tissue iNOS induction in endotoxemia

Sonia Kapur, Bruno Marcotte, and André Marette

Department of Physiology and Lipid Research Unit, Laval University Hospital Research Center, Quebec, Canada G1V 4G2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ), or interferon-gamma (IFN-gamma ) alone failed to induce iNOS activity. However, when used in combination, TNF-alpha , IFN-gamma , 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-alpha ; interferon-gamma ; adipocytes; skeletal muscle; liver; kidney


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (TNF-alpha ) was purchased from R&D systems. Murine interferon-gamma (IFN-gamma ) 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 alpha -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 alpha -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 alpha -DMEM containing 10% (vol/vol) fetal bovine serum for 8-12 days. Adipocytes were incubated with or without cytokines (TNF-alpha and/or IFN-gamma ) 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1.   Time course of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in plasma of control and LPS-treated rats. Rats were injected with saline (controls) or LPS and euthanized 2, 4, or 8 h later, and plasma was collected. NO production was assessed by the accumulation of nitrite and nitrate in the plasma, as described in MATERIALS AND METHODS. Bars represent means ± SE of 4 rats in each group. * P < 0.05, ** P<0.01, *** P < 0.001 vs. control values.

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.


View larger version (23K):
[in this window]
[in a new window]
 


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   LPS-induced expression of inducible nitric oxide synthase (iNOS) mRNA in white epididymal (EWAT), perirenal (PWAT), and interscapular brown adipose tissue (BAT) (A), as well as in liver, kidney, and skeletal (SK) muscle (B). Rats were treated for 2, 4, or 8 h with LPS or vehicle (C) as described in legend to Fig. 1. RNA was isolated, and iNOS mRNA levels were measured by RT-PCR as described in MATERIALS AND METHODS. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels were also determined as an internal control. Results are representative of 3 independent experiments with tissues from different animals.

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.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of LPS on iNOS protein levels and iNOS activity in EWAT, PWAT, and BAT adipose tissues. Rats were treated or not with LPS, as described in legend to Fig. 1, and tissue iNOS protein levels and iNOS activity were determined as described in MATERIALS AND METHODS. The immunoblot is representative of 3 independent experiments with tissues from different animals. Results for iNOS activity are represented as means ± SE of 2-3 individual experiments. ** P < 0.01 vs. control values.

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-alpha , interleukin-1 and -6, and IFN-gamma ) (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-alpha and IFN-gamma ) and LPS on iNOS activation in vitro. As shown in Fig. 4, incubation of the adipose cells with TNF-alpha , LPS, or IFN-gamma 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-gamma (but not TNF-alpha ). However, maximal activation of iNOS was observed when cells were incubated with all of these immune molecules. Indeed, the combined effect of LPS, TNF-alpha , and IFN-gamma 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).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   LPS and cytokines synergistically increase iNOS activity in adipocytes. 3T3-L1 adipocytes were treated or not with the cytokines tumor necrosis factor-alpha (TNF-alpha , 10 ng/ml) and/or interferon-gamma (IFN-gamma , 200 U/ml) and/or LPS (10 µg/ml). After incubation, nitrite accumulation in incubation medium was measured under identical conditions for all groups, as described in MATERIALS AND METHODS. Results are means ± SE of 3 independent experiments, each performed in triplicate with different batches of cells. ** P < 0.01 vs. control values, + P < 0.01 vs. TNF-alpha  + IFN-gamma values.

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of cytokines (TNF-alpha and IFN-gamma ) and LPS on iNOS activity and protein expression in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated or not with cytokines (TNF-alpha and/or IFN-gamma ) and/or LPS as described in the legend to Fig. 4. After incubation, iNOS activity was measured by conversion of L-[3H]arginine to L-[3H]citrulline under identical conditions for all groups, as described in MATERIALS AND METHODS. A: cellular iNOS protein levels in control vs. cytokines and/or LPS-treated (CYT-LPS) cells (duplicate samples) were measured by Western blotting with an iNOS-specific antibody (see MATERIALS AND METHODS). B: cytokines and LPS markedly increased iNOS activity in adipocytes. C: reversal of iNOS activation by co-incubation with NG-nitro-L-arginine methyl ester (L-NAME). Results are representative of 2 independent experiments, each performed in triplicate with different batches of cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha (both p55-60 and p75-80 receptors) (21, 29), and IFN-gamma (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-alpha and IFN-gamma or LPS and IFN-gamma . 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-gamma and LPS (7), hepatocyte iNOS is poorly activated by these two factors and needs the combination of at least two cytokines (preferably TNF-alpha and interleukin-1) and LPS to promote a full enzymatic response (15). Our data indicate that, in adipocytes, IFN-gamma 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-alpha 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-alpha and interleukin-1 has been reported to induce leptin expression and secretion by adipose tissue (16, 34). Both TNF-alpha 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-alpha , 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-alpha 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-alpha , 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.


    ACKNOWLEDGEMENTS

We thank Dr. Martin Olivier for the kind gift of interferon-gamma and Dr. Claude Côté for critical reading of the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersson, J., S. Nagy, L. Bjork, J. Abrams, S. Holm, and U. Andersson. Bacterial toxin-induced cytokine production studied at the single-cell level. Immunol. Rev. 127: 69-96, 1992[Medline].

2.   Arner, P., and J. Bolinder. Microdialysis of adipose tissue. J. Intern. Med. 230: 381-386, 1991[Medline].

3.   Bédard, S., B. Marcotte, and A. Marette. Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase. Biochem. J. 325: 487-493, 1997[Medline].

4.   Beutler, B., D. Greenwald, J. D. Hulmes, M. Chang, Y. C. Pan, J. Mathison, R. Ulevitch, and A. Cerami. Identity of tumour necrosis factor and the macrophage-secreted factor cachectin. Nature 316: 552-554, 1985[Medline].

5.   Boczkowski, J., S. Lanone, D. Ungureanu-Longrois, G. Danialou, T. Fournier, and M. Aubier. Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction. J. Clin. Invest. 98: 1550-1559, 1996[Abstract/Free Full Text].

6.   Cook, H. T., A. J. Bune, A. S. Jansen, G. M. Taylor, R. K. Loi, and V. Cattell. Cellular localization of inducible nitric oxide synthase in experimental endotoxic shock in the rat. Clin. Sci. (Colch.) 87: 179-186, 1994[Medline].

7.   Ding, A. H., C. F. Nathan, and D. J. Stuehr. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J. Immunol. 141: 2407-2412, 1988[Abstract/Free Full Text].

8.   Faggioni, R., G. Fantuzzi, J. Fuller, C. A. Dinarello, K. R. Feingold, and C. Grunfeld. IL-1beta mediates leptin induction during inflammation. Am. J. Physiol. 274 (Regulatory Integrative Comp. Physiol. 43): R204-R208, 1998[Abstract/Free Full Text].

9.   Fearns, C., V. V. Kravchenko, R. J. Ulevitch, and D. J. Loskutoff. Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide. J. Exp. Med. 181: 857-866, 1995[Abstract].

10.   Finck, B. N., K. W. Kelley, R. Dantzer, and R. W. Johnson. In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide. Endocrinology 139: 2278-2283, 1998[Abstract/Free Full Text].

11.   Frayn, K. N. Hormonal control of metabolism in trauma and sepsis. Clin. Endocrinol. 24: 577-599, 1986[Medline].

12.   Fried, S. K., M. Lavau, and F. X. Pi-Sunyer. Variations in glucose metabolism by fat cells from three different depots of the rat. Metabolism 31: 876-883, 1982[Medline].

13.   Gainsford, T., T. A. Willson, D. Metcalf, E. Handman, C. McFarlane, A. Ng, N. A. Nicola, W. S. Alexander, and D. J. Hilton. Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells. Proc. Natl. Acad. Sci. USA 93: 14564-14568, 1996[Abstract/Free Full Text].

14.   Gardiner, S. M., P. A. Kemp, J. E. March, and T. Bennett. Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity, and the effects of NOS inhibitors in conscious, endotoxaemic rats. Br. J. Pharmacol. 116: 2005-2016, 1995[Abstract].

15.   Geller, D. A., A. K. Nussler, M. Di Silvio, C. J. Lowenstein, R. A. Shapiro, S. C. Wang, R. L. Simmons, and T. R. Billiar. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl. Acad. Sci. USA 90: 522-526, 1993[Abstract].

16.   Grunfeld, C., C. Zhao, J. Fuller, A. Pollack, A. Moser, J. Friedman, and K. R. Feingold. Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters. J. Clin. Invest. 97: 2152-2157, 1996[Abstract/Free Full Text].

17.   Holley, D. C., and J. A. Spitzer. Insulin action and binding in adipocytes exposed to endotoxin in vitro and in vivo. Circ. Shock 7: 3-12, 1980[Medline].

18.   Hom, F. G., C. J. Goodner, and M. A. Berrie. A [3H]2-deoxyglucose method for comparing rates of glucose metabolism and insulin responses among rat tissues in vivo. Validation of the model and the absence of an insulin effect on brain. Diabetes 33: 141-152, 1984[Abstract].

19.   Kapur, S., S. Bedard, B. Marcotte, C. H. Cote, and A. Marette. Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action. Diabetes 46: 1691-1700, 1997[Abstract].

20.   Leach, G. J., and J. A. Spitzer. Endotoxin-induced alterations in glucose transport in isolated adipocytes. Biochim. Biophys. Acta 648: 71-79, 1981[Medline].

21.   Liu, L. S., M. Spelleken, K. Rohrig, H. Hauner, and J. Eckel. Tumor necrosis factor-alpha acutely inhibits insulin signaling in human adipocytes: implication of the p80 tumor necrosis factor receptor. Diabetes 47: 515-522, 1998[Abstract].

22.   Liu, S., I. M. Adcock, R. W. Old, P. J. Barnes, and T. W. Evans. Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA. Biochem. Biophys. Res. Commun. 196: 1208-1213, 1993[Medline].

23.   Marette, A., P. Mauriège, C. Atgié, C. Bouchard, G. Thériault, L. Bukowiecki, P. Marceau, S. Biron, A. Nadeau, and J.-P. Després. Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women. Diabetologia 40: 590-598, 1997[Medline].

24.   Mauriège, P., D. Prud'homme, S. Lemieux, A. Tremblay, and J. P. Després. Regional differences in adipose tissue lipolysis from lean and obese women: existence of postreceptor alterations. Am. J. Physiol. 269 (Endocrinol. Metab. 32): E341-E350, 1995[Abstract/Free Full Text].

25.   Moncada, S., R. M. J. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43: 109-142, 1991[Medline].

26.   Nathan, C. Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100: 2417-2423, 1997[Free Full Text].

27.   Nathan, C. F. Secretory products of macrophages. J. Clin. Invest. 79: 319-326, 1987[Medline].

28.   Patton, J. S., H. M. Shepard, H. Wilking, G. Lewis, B. B. Aggarwal, T. E. Eessalu, L. A. Gavin, and C. Grunfeld. Interferons and tumor necrosis factors have similar catabolic effects on 3T3 L1 cells. Proc. Natl. Acad. Sci. USA 83: 8313-8317, 1986[Abstract].

29.   Peraldi, P., G. S. Hotamisligil, W. A. Buurman, M. F. White, and B. M. Spiegelman. Tumor necrosis factor (TNF)-alpha inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase. J. Biol. Chem. 271: 13018-13022, 1996[Abstract/Free Full Text].

30.   Ribière, C., A. M. Jaubert, N. Gaudiot, D. Sabourault, M. L. Marcus, J. L. Boucher, D. Denis-Henriot, and Y. Giudicelli. White adipose tissue nitric oxide synthase: a potential source for NO production. Biochem. Biophys. Res. Commun. 222: 706-712, 1996[Medline].

31.   Rosen, B. S., K. S. Cook, J. Yaglom, D. L. Groves, J. E. Volanakis, D. Damm, T. White, and B. M. Spiegelman. Adipsin and complement factor D activity: an immune-related defect in obesity. Science 244: 1483-1487, 1989[Medline].

32.   Ruetten, H., C. Thiemermann, and J. R. Vane. Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaesthetized rat. Br. J. Pharmacol. 118: 198-204, 1996[Abstract].

33.   Salter, M., R. G. Knowles, and S. Moncada. Widespread tissue distribution, species distribution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide synthases. FEBS Lett. 291: 145-149, 1991[Medline].

34.   Sarraf, P., R. C. Frederich, E. M. Turner, G. Ma, N. T. Jaskowiak, D. J. Rivet, III, J. S. Flier, B. B. Lowell, D. L. Fraker, and H. R. Alexander. Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J. Exp. Med. 185: 171-175, 1997[Abstract/Free Full Text].

35.   Schulz, R., E. Nava, and S. Moncada. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br. J. Pharmacol. 105: 575-580, 1992[Abstract].

36.   Shangraw, R. E., F. Jahoor, H. Miyoshi, W. A. Neff, C. A. Stuart, D. N. Herndon, and R. R. Wolfe. Differentiation between septic and postburn insulin resistance. Metabolism 38: 983-989, 1989[Medline].

37.   Spiegelman, B. M., L. Choy, G. S. Hotamisligil, R. A. Graves, and P. Tontonoz. Regulation of adipocyte gene expression in differentiation and syndromes of obesity/diabetes. J. Biol. Chem. 268: 6823-6826, 1993[Free Full Text].

38.   Spiegelman, B. M., and J. S. Flier. Adipogenesis and obesity: rounding out the big picture. Cell 87: 377-389, 1996[Medline].

39.  Vodovotz, Y. Modified microassay for serum nitrite and nitrate. Biotechniques 20: 390-392, 394, 1996.

40.   Wang, Q., P. J. Bilan, T. Tsakiridis, A. Hinek, and A. Klip. Actin filaments participate in the relocalization of phosphatidylinositol 3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes. Biochem. J. 331: 917-928, 1998[Medline].

41.   Westfall, M. V., and M. M. Sayeed. Basal and insulin-stimulated skeletal muscle sugar transport in endotoxin and bacteremic rats. Am. J. Physiol. 254 (Regulatory Integrative Comp. Physiol. 23): R673-R679, 1988[Abstract/Free Full Text].

42.   Wileman, S. M., G. E. Mann, and A. R. Baydoun. Induction of L-arginine transport and nitric oxide synthase in vascular smooth muscle cells: synergistic actions of pro-inflammatory cytokines and bacterial lipopolysaccharide. Br. J. Pharmacol. 116: 3243-3250, 1995[Abstract].

43.   Xie, Q., and C. Nathan. The high-output nitric oxide pathway: role and regulation. J. Leukocyte Biol. 56: 576-582, 1994[Abstract].

44.   Zechner, R., T. C. Newman, B. Sherry, A. Cerami, and J. L. Breslow. Recombinant human cachectin/tumor necrosis factor but not interleukin-1 alpha downregulates lipoprotein lipase gene expression at the transcriptional level in mouse 3T3-L1 adipocytes. Mol. Cell. Biol. 8: 2394-2401, 1988[Medline].


Am J Physiol Endocrinol Metab 276(4):E635-E641
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society