Stimulation of NGF expression and secretion in 3T3-L1 adipocytes by prostaglandins PGD2, PGJ2, and {Delta}12-PGJ2

Mònica Bulló, Muhammad R. Peeraully, and Paul Trayhurn

Neuroendocrine and Obesity Biology Unit, Liverpool Centre for Nutritional Genomics, School of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom

Submitted 10 January 2005 ; accepted in final form 14 February 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nerve growth factor (NGF) has recently been shown to be secreted from white adipocytes, its production being strongly stimulated by the proinflammatory cytokine tumor necrosis factor-{alpha}. In this study, we have examined whether a series of prostaglandins and other inflammation-related factors also stimulate NGF expression and secretion by adipocytes, using 3T3-L1 cells. Although interleukin (IL)-1{beta}, IL-10, and IL-18 each induced a small decrease in NGF mRNA level in 3T3-L1 adipocytes, there was no significant effect of these cytokines on NGF secretion. A small reduction in NGF expression and/or secretion was also observed with adiponectin and prostaglandins PGE2, PGF2{alpha}, and PGI2. In marked contrast, prostaglandin PGD2 induced a major, dose-dependent increase (up to 20- to 40-fold) in NGF expression and secretion. The PGD2 metabolites, PGJ2 and {Delta}12-PGJ2, also induced major increases (up to 30-fold) in NGF production. A further metabolite of PGJ2, 15-deoxy-{Delta}12,14-PGJ2, a peroxisome proliferator-activated receptor-{gamma} agonist, led paradoxically to a small increase in NGF mRNA level but a fall in NGF secretion. Both PGD2 and PGJ2 induced significant increases in NGF gene expression by 4 h after their addition. It is concluded that PGD2 and the J series prostaglandins, PGJ2 and {Delta}12-PGJ2, can play a significant role in the regulation of NGF production by white adipocytes. These results provide support for the view that NGF is an important inflammatory response protein, as well as a target-derived neurotrophin, in white adipose tissue.

adipokines; inflammation; neurotrophin; nerve growth factor; white adipose tissue


WHITE ADIPOSE TISSUE (WAT) is now recognized to be a major endocrine and secretory organ, in addition to its central role in the storage of energy as triacylglycerols (13, 23, 31, 32). The various protein signals and factors released by white adipocytes, termed adipokines, are functionally highly diverse (32). They include proteins involved in the regulation of energy balance, insulin sensitivity and glucose tolerance, vascular hemostasis, lipid metabolism, and angiogenesis (13, 23, 31, 32). An increasing number of adipokines are linked to inflammation and the acute-phase response [e.g., tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{beta}, IL-6, IL-8, haptoglobin, and plasminogen activator inhibitor-1], and the production of these factors is increased in obesity (31), the disorder being associated with a state of chronic low-grade inflammation (12, 23, 32, 38).

WAT is innervated by sympathetic nerves, and the sympathetic system is involved in the stimulation of lipolysis (2), the control of cell number (6), and in regulating the production of several adipokines, particularly leptin (24). We have recently shown that nerve growth factor (NGF), a target-derived neurotrophin that plays a key role in the growth and maintenance of sympathetic neurons within tissues, is secreted from white adipocytes (21). In addition to its neurotrophic function, NGF is also involved in immune and inflammatory responses (1, 17). It is present in the circulation of both rodents and humans, and the levels increase during stress and in certain autoimmune diseases and allergic inflammatory states, such as rheumatoid arthritis and asthma (4, 8). The proinflammatory cytokine TNF-{alpha} has a major stimulatory effect on NGF expression and secretion in 3T3-L1 cells and in human adipocytes, consistent with the concept that the neurotrophin is an inflammatory response protein in adipose tissue (21, 35). This view is also supported by the observation that dexamethasone and the peroxisome proliferator-activated receptor (PPAR) {gamma} agonist rosiglitazone, both of which have anti-inflammatory actions, inhibit NGF production and release by fat cells (21).

In the present study, we have examined the effect of a series of inflammation-related factors on NGF expression and secretion in adipocytes using 3T3-L1 cells, focusing particularly on prostaglandins. Prostaglandins (PGD2 and PGJ2 series) have recently been reported to be powerful inducers of NGF production in mouse astrocytes in culture (30), and prostaglandins are synthesized and released within WAT (3, 9, 16). We show here that PGD2, PGJ2, and 9-deoxy-{Delta}9,{Delta}12,13,14-dihydro-PGJ2 ({Delta}12-PGJ2) each strongly induce NGF expression and secretion in 3T3-L1 adipocytes; in contrast, PGI2 and 15-deoxy-{Delta}12,14-PGJ2 (15d-PGJ2), a PPAR{gamma} ligand, inhibit NGF production.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured at 37°C in a humidified atmosphere of 5% CO2-95% air, essentially as described previously (21). The cells were maintained in culture medium containing the following: DMEM (Invitrogen, Paisley, UK) with 25 mM glucose, 1 mM pyruvate, 4.02 mM L-alanyl-L-glutamine, and 10% vol/vol FCS (Sigma, Poole, UK). Differentiation was initiated 24 h after confluence by incubation for 2 days in culture medium containing 0.25 µM dexamethasone, 0.5 mM IBMX, and 5 µg/ml insulin (Sigma). This was followed by maintenance in feeding medium (renewed every 2/3 days) consisting of culture medium containing 5 µg/ml insulin.

After the induction of differentiation (11 days), the cells were preincubated for 24 h with FCS-free feeding medium. Cells were then treated on day 12 after the induction of differentiation and incubated for 24 h with FCS-free feeding medium containing each of the agents to be tested; the control cells had no agent added. The agents used were as follows: IL-1{beta} (low dose 5 ng/ml, high dose 20 ng/ml), IL-10 (5 ng/ml, 20 ng/ml), adiponectin (0.5 µg/ml, 2 µg/ml; PreProtech EC, London, UK), IL-18 (100 ng/ml, 500 ng/ml; R&D Systems, Minneapolis, MN), PGD2 (50 µM, 100 µM), PGF2{alpha} (50 µM, 100 µM; Sigma), PGE2 (50 µM, 100 µM; Calbiochem, Darmstadt, Germany), PGI2 (50 µM, 150 µM), PGJ2 (7.5 µM, 25 µM), {Delta}12-PGJ2 (7.5 µM, 25 µM), and 15d-PGJ2 (25 µM, 75 µM; Alexis Biochemicals, Nottingham, UK). The prostaglandins were employed at similar concentrations to those used with mouse astrocytes in culture (29). Between three and six individual wells in cell culture plates were treated with each agent, or used as controls; different batches of cells were used for each set of experiments. Cells were collected after 24 h in Tri-Reagent (Sigma). Medium was also collected and centrifuged at 1,000 rpm for 10 min, the supernatant being taken and stored together with the collected cells at –20°C until use.

The dose-dependent effect of PGD2 on NGF expression was studied by treating cells with different concentrations of PGD2 (0–150 µM) for 24 h. The time course of the effect of PGD2 (100 µM), PGJ2 (25 µM), and TNF-{alpha} (50 ng/ml; Sigma) on NGF gene expression was investigated by treating cells for incubation times ranging from 0 to 48 h.

RNA preparation. Total RNA was extracted from cells with Tri-Reagent. RNA samples were then treated using a DNA-free kit (Ambion, Huntingdon, UK), in accordance with the manufacturer's instructions, to remove any contamination with genomic DNA.

RT and real-time PCR. Mouse NGF mRNA levels were analyzed by relative quantitation with the 2 method (19) using real-time PCR with an ABI Prism 7700 instrument (Applied Biosystems, Foster City, CA). All samples were normalized to values of {beta}-actin, and results expressed as degree of changes of threshold cycle value relative to controls. Primer and Taqman probe sequences were as previously described (21); the primers and probes were synthesized commercially (Sigma-Genosys, Haverhill, UK, and Eurogentec, Romsey, UK, respectively).

Total RNA (1 µg) was reverse transcribed to cDNA in a 20-µl reaction volume with anchored oligo(dT) primer using the Reverse-iT first-strand synthesis kit (ABgene, Epsom, UK). Real-time PCR was performed in 96-well plates using a qPCR Core Kit (Eurogentec) according to the supplier's instructions, with 900 nM forward and reverse primers, 225 nM probe, and 1 µl of cDNA in a 26-µl final reaction volume. Each sample was run in triplicate with the NGF primers and probe and in duplicate with the {beta}-actin primers and probe. Amplifications were performed with a 2-min activation stage at 50°C and then a 10-min denaturation stage, followed by 40 cycles consisting of a denaturation step of 15 s at 95°C and a combined primer annealing and extension step for 60 s at 60°C. Data were collected and analyzed with Sequence Detector software (Applied Biosystems).

ELISA. NGF was measured in culture medium using the NGF Emax Immunoassay System (Promega, Southampton, UK), a specific and highly sensitive ELISA kit with a stated intra-assay coefficient of variation of 4.2%. ELISA assays were performed according to the manufacturer's instructions in Nunc MaxiSorp 96-well microplates (Fisher Scientific).

Statistical analysis. The statistical significance of differences between groups of treated cells was assessed by Student's unpaired t-test; differences were considered to be significant at P < 0.05.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the first set of experiments, the effect of several interleukins and prostaglandins, and of adiponectin, on NGF mRNA level and NGF secretion was examined. IL-1{beta}, IL-10, and IL-18 each induced a small, but statistically significant, reduction in NGF mRNA (by real-time PCR), the decrease being between 18 and 57% (Fig. 1A). Measurement of NGF protein by ELISA indicated, however, that these interleukins had no effect on the amount of the neurotrophin released in the medium (Fig. 1B). The hormone adiponectin induced a reduction in NGF in the medium; mRNA levels were, however, significantly reduced only with the lower dose (Fig. 1).



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Fig. 1. Effect of interleukin (IL)-1{beta}, IL-10, IL-18, adiponectin, and prostaglandins PGE2, PGF2{alpha}, PGI2, and PGD2 on nerve growth factor (NGF) mRNA levels (A) and protein secretion (B) in 3T3-L1 adipocytes. Cells were taken at day 12 after the induction of differentiation and incubated for 24 h in medium, to which different amounts of each agent were added. Control cells received no addition. IL-1{beta}, low dose (LD) 5 ng/ml, high dose (HD) 20 ng/ml; IL-10, LD 5 ng/ml, HD 20 ng/ml; IL-18, LD 100 ng/ml, HD 500 ng/ml; adiponectin, LD 0.5 µg/ml, HD 2 µg/ml; all PGs, LD 50 µM, HD 100 µM, except PGI2, which had an HD of 150 µM. Values for relative NGF mRNA levels, measured by real-time PCR, are expressed in relation to the controls. NGF protein (ELISA) values are actual concentrations in the medium. Results are given as means ± SE (bars) for groups of 4–6. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls.

 
Prostaglandins PGE2, PGF2{alpha}, and PGI2 had generally similar effects to those observed with the interleukins and with adiponectin. Thus there was a modest reduction in mRNA level with each of these prostaglandins of between 42 and 73% (Fig. 1A). In the case of PGF2{alpha}, there was also a dose-dependent reduction in NGF concentration in the medium; PGE2 and PGI2 led to a significant fall in NGF in the medium only with the lower dose (Fig. 1B). In marked contrast to PGE2, PGF2{alpha}, and PGI2, there was a substantial stimulatory effect of PGD2 on both NGF mRNA level and medium protein (Fig. 1). At the higher of the two doses of PGD2 used, the level of NGF mRNA in the adipocytes was >40-fold greater than in the controls, whereas the amount of NGF in the medium was increased 18-fold.

In view of the major effects of PGD2 on NGF expression and release, a dose-response study was performed with this prostaglandin. There was a significant increase in NGF in the medium with a 50 µM concentration of PGD2, whereas the mRNA level in the cells increased with a slightly higher concentration (Fig. 2). The peak in mRNA level occurred with 100 µM PGD2, whereas NGF release peaked at the lower concentration of 75 µM. In both cases, the peak was followed by a modest reduction with higher concentrations of PGD2. Nevertheless, at 150 µM PGD2, there was still a substantial increase in NGF mRNA and protein release relative to the controls. At the peak, NGF mRNA level was increased 30-fold, and the amount of the protein in the medium was increased by >20-fold.



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Fig. 2. Dose response of PGD2 on NGF mRNA levels (A) and protein secretion (B) in 3T3-L1 adipocytes. Cells were taken at day 12 after the induction of differentiation and incubated for 24 h in media to which different amounts of PGD2 were added. Values for NGF mRNA levels, measured by real-time PCR, are expressed relative to the controls. NGF protein (ELISA) values are actual concentrations in the medium. Results are given as means ± SE (bars) for groups of 5–6. **P < 0.01 and ***P < 0.001, compared with controls.

 
PGD2 spontaneously breaks down to PGJ2, and PGJ2 to {Delta}12-PGJ2 and 15d-PGJ2, depending on the conditions (29, 30). The effect of each of these metabolites was therefore investigated, given the powerful stimulatory effect of PGD2 on NGF production. Although there was little effect of PGJ2 at the low dose, the high dose induced a fourfold increase in NGF mRNA level, and the NGF concentration in the medium was increased ~35-fold (Fig. 3). A similar result was obtained with {Delta}12-PGJ2, the mRNA level increasing nearly ninefold with the high dose and the protein concentration approximately eightfold (Fig. 3). With 15d-PGJ2, there was paradoxically a small increase in NGF mRNA level that was statistically significant but a dose-dependent reduction in the amount of NGF (73% decrease at the high dose) in the medium (Fig. 3).



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Fig. 3. Effect of PGJ2, {Delta}12-PGJ2, and 15d-PGJ2 on NGF mRNA levels (A) and protein secretion (B) in 3T3-L1 adipocytes. Cells were taken at day 12 after the induction of differentiation and incubated for 24 h in medium to which different amounts of each agent were added. Control cells received no addition. PGJ2 LD 7.5 µM, HD 25 µM; {Delta}12-PGJ2 LD 7.5 µM, HD 25 µM; 15d-PGJ2 LD 25 µM, HD 75 µM. Values for NGF mRNA levels, measured by real-time PCR, are expressed relative to the controls. NGF protein (ELISA) values are actual concentrations in the medium. Results are given as means ± SE (bars) for groups of 5–6. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls.

 
In the final set of experiments, the time course of the response of NGF gene expression to PGD2 and PGJ2 was examined. The level of NGF mRNA was significantly elevated by 4 h after the addition of these prostaglandins (Fig. 4, A and B). By 8 h, NGF mRNA level was increased nearly eightfold in the presence of PGD2, and there were further increases up to 48 h. In the case of PGJ2, the level of NGF mRNA essentially plateaued by 8 h at five to six times that at time 0, but again there was a further increase between 24 and 48 h. The time course of the response to TNF-{alpha} was also studied for comparison. Although there was a small rise in NGF mRNA level by 1 h, by 8 h the level was only two times that of control cells (Fig. 4C); most of the response to TNF-{alpha} occurred after 8 h, and by 24 h the level was approximately eightfold higher.



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Fig. 4. Time course of effect of PGD2 (A), PGJ2 (B), and tumor necrosis factor (TNF)-{alpha} (C) on NGF mRNA levels in 3T3-L1 adipocytes. Cells were taken at day 12 after the induction of differentiation and incubated for different periods in media to which each agent was added. Control cells received no addition and were collected immediately. PGD2 100 µM; PGJ2 25 µM; TNF-{alpha} 50 ng/ml. Values for NGF mRNA levels, measured by real-time PCR, are expressed relative to the controls. Results are given as means ± SE (bars) for groups of 3–6. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with controls.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NGF plays an important role in the development and survival of sympathetic neurons within tissues and was the first of the neurotrophins to be discovered. We have recently reported that NGF is expressed in the major fat depots of mice and humans and is secreted both by 3T3-L1 cells and by human white adipocytes differentiated in culture (21, 35). Synthesis of NGF has also been reported in white adipocytes associated with cutaneous wound healing in rats, and in human subepicardial adipose tissue in association with atherosclerotic lesions (7, 14). In addition to its neurotrophic role, NGF is involved in immune and inflammatory responses (17, 34), and in both murine and human white adipocytes the inflammatory cytokine TNF-{alpha} strongly stimulates NGF synthesis and release (21, 35). The present study demonstrates that PGD2 and prostaglandins of the J2 series also have a major stimulatory effect on NGF expression and secretion by white adipocytes, consistent with a role for the protein in inflammation in adipose tissue.

The peak response to PGD2 resulted in a nearly 40-fold increase in the secretion of NGF from 3T3-L1 adipocytes over the 24-h incubation period employed. PGD2 can spontaneously break down to PGJ2 (29, 30), and it appears that these two prostaglandins are similarly potent in terms of NGF production. PGJ2 can in turn break down to {Delta}12-PGJ2 and 15d-PGJ2, depending on the conditions (29, 30). Of these two PGJ2 metabolites, {Delta}12-PGJ2 also had a stimulatory effect on NGF release by 3T3-L1 adipocytes.

In contrast, 15d-PGJ2 induced a dose-dependent reduction in NGF secretion, although paradoxically there was a small increase in mRNA level at the end of the 24-h incubation period. The mRNA measurements reflect, of course, the point at which the cells were taken, whereas the protein in the medium represents the sum of the total 24-h incubation period. It may be, therefore, that, although there is suppression of NGF secretion, the small increase in mRNA level at 24 h reflects a rebound from an earlier inhibition. An inhibitory effect of 15d-PGJ2 on NGF production would be predicted, since this prostaglandin is an endogenous ligand for the PPAR{gamma} nuclear receptor (29), and the PPAR{gamma} agonist rosiglitazone has been shown to inhibit NGF expression and secretion in 3T3-L1 adipocytes (21).

15d-PGJ2 can be considered a repressor of the inflammatory response because of its inhibition of NF-{kappa}B and IKK and downregulation of TNF-{alpha} and inducible nitric oxide synthase expression (29). It is also recognized that 15d-PGJ2 suppresses proinflammatory prostaglandin production via inhibition of COX-2 synthesis, this COX isoenzyme being under the transcriptional control of NF-{kappa}B (29). Inhibition of these autocrine stimulators of NGF expression would provide an additional mechanism for the observed inhibitory effect of 15d-PGJ2 on NGF production.

The powerful stimulatory effect of PGD2, PGJ2, and {Delta}12-PGJ2 on NGF synthesis and secretion by white adipocytes is similar to what has been recently reported with these prostaglandins in mouse astrocytes in culture (30). This suggests that PGD2 and J2 series prostaglandins may be general activators of NGF production. However, although 15d-PGJ2 inhibited NGF secretion from adipocytes, it stimulated release of the neurotrophin from astrocytes (30), a difference that may relate to the selective presence of the PPAR{gamma}2 receptor in fat cells. A further difference between astrocytes and adipocytes is that PGD2 was less potent than J2 series prostaglandins in the former. It should be noted that the prostaglandin concentrations employed were similar in the astrocyte and adipocyte studies, and micromolar concentrations of prostaglandins can occur in body fluids during inflammation and infection (18).

The time course in terms of NGF gene expression showed that most of the response to PGD2 and PGJ2 occurred within 16 h (although there were further increases up to 48 h), with the mRNA level increasing five- to eightfold in the initial 8 h. This contrasts with the time course of the response to TNF-{alpha}, where the level of NGF mRNA was only twofold higher at 8 h while the increase was over eightfold at 24 h. Thus the response to TNF-{alpha} is relatively delayed compared with the response to the two prostaglandins. This raises the possibility that TNF-{alpha} could stimulate NGF production in part through an activation of PGD2 synthesis. A stimulation of PGD2 production by TNF-{alpha} has been previously demonstrated in rat Kupffer cells (22), and TNF-{alpha} has been shown to upregulate expression of the PGD2 synthase gene in human chondrocytes (25), the encoded enzyme being necessary for the synthesis of PGD2, and therefore also of the PGJ2 series metabolites.

The strong stimulatory effect of PGD2, PGJ2, and {Delta}12-PGJ2 on NGF expression and secretion is in direct contrast not only with 15d-PGJ2, but also with the response to PGE2, PGF2{alpha}, and PGI2, each of which was inhibitory. The fall in NGF production induced by these prostaglandins was, however, small. Similarly, there was little effect of IL-1{beta}, IL-10, and IL-18 on NGF production; indeed, there was only a small decrease in mRNA level with these agents and no effect on NGF release in the medium. The adipocyte factor adiponectin led to a modest fall in NGF production (though not dose dependent), and this may reflect the anti-inflammatory action of the hormone (20, 36).

It has been recognized for some time that adipocytes can synthesize prostanoids (26). 3T3-L1 adipocytes are reported to secrete PGE2, 6-keto-PGF1{alpha} (the natural metabolite of PGI2), PGF2{alpha}, and 15d-PGJ2 (3). Similarly, isolated human adipocytes have been shown to release both PGI2 and PGE2, but at lower levels than the stromal vascular fraction of adipose tissue (9), and PGD2 synthase is expressed in both 3T3-L1 cells and in human adipose tissue (16). Thus WAT, whether through the fat cells themselves or from the other cell types within it (such as macrophages), can produce the major prostaglandins that markedly stimulate NGF expression and secretion by adipocytes.

The effects of different prostaglandins on adipose tissue function have not been extensively investigated. Nevertheless, PGE2, for example, has been shown to inhibit lipolysis and to stimulate leptin secretion in rodent adipose tissue and in adipocytes (10). Furthermore, lipolysis induced by TNF-{alpha} in adipocytes can be blocked by indomethacin, an inhibitor of prostaglandin synthesis (11) and also by both rosiglitazone and 15d-PGJ2 through the PPAR{gamma} receptor (28). 15d-PGJ2, which is a putative endogenous promoter of adipogenesis, has also been shown to inhibit the expression of the leptin gene through the PPAR{gamma} receptor (27). PGE2, on the other hand, appears to suppress the differentiation of 3T3-L1 adipocytes, this occurring through the prostaglandin EP4 receptor (33).

In addition to EP4, the expression of several other prostaglandin receptor genes has been identified in adipocytes, including FP, IP, EP1, and EP3, and these are selective for PGF2{alpha}, PGI2, and PGE2 (5). Each of these receptors is also expressed in preadipocytes, and the level of expression is higher than in mature fat cells; the exception is EP3, which is found only in the adipocytes themselves (5).

The demonstration here that PGD2, PGJ2, and {Delta}12-PGJ2 strongly stimulate NGF expression and secretion by 3T3-L1 adipocytes supports the view that the neurotrophin is an important inflammatory response protein in WAT. Adipose tissue secretes a wide range of inflammation-related adipokines, including acute-phase response proteins (e.g., haptoglobin, plasminogen activator inhibitor-1), cytokines (e.g., TNF-{alpha}, IL-1{beta}, and IL-6), and chemokines (e.g., monocyte chemoattractant protein-1; see Refs. 23 and 32). The production of several of these is increased in obesity, the obese being characterized by chronic low-grade inflammation (12, 23, 32, 38). There is a growing recognition of a link between the secretion of inflammation-related adipokines in obesity, particularly TNF-{alpha} and IL-6, and the development of associated diseases such as type 2 diabetes and the metabolic syndrome (15, 37). Whether adipocyte-derived NGF might also be implicated in these disorders is unknown; however, the circulating levels of the neurotrophin increase during stress and in autoimmune diseases and allergic inflammatory states, such as rheumatoid arthritis and asthma (4, 8). Finally, the present results raise the question of whether prostaglandins may be extensively involved in the regulation of the synthesis of inflammation-related adipokines.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
M. Peeraully is supported by a BBSRC Research Studentship. M. Bulló received funding from the Institut de Recerca en Ciències de la Salut Foundation and is supported by the Fondo de Investigación Sanitaria (FIS 02/0550), Red de Centros en Metabolismo y Nutrición (C03/08), and Rovira i Virgili University.

Present address for M. Bulló: Human Nutrition Unit, Facultat de Medicina i Ciències de la Salut de Reus, Universitat Rovira i Virgili, C/Sant Llorenç, 21–43201 Reus (Tarragona), Spain.


    ACKNOWLEDGMENTS
 
We are grateful for the help and advice of Leif Hunter and of Drs. Yi Bao, Sarah Dutton, Bohan Wang, and Stuart Wood.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Trayhurn, Neuroendocrine & Obesity Biology Unit, School of Clinical Sciences, Univ. of Liverpool, UCD Bldg., Liverpool L69 3GA, UK (e-mail: p.trayhurn{at}liverpool.ac.uk)

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. Section 1734 solely to indicate this fact.


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 DISCUSSION
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