Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.     

Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously

The capacity of cultured mesothelial cells to produce prostaglandins from both exogenous an endogenous arachidonic acid has been investigated. Incubations with labelled [1-14C]arachidonic acid and [1-14C]prostaglandin endoperoxide H2 indicated the formation of prostacyclin and prostaglandin E2. Evaluation of the transformation of endogenously released arachidonic acid, however, could only confirm the production of prostacyclin.     

Prostaglandin E(2) regulates wound closure in airway epithelium

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E(2) (PGE(2)) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE(2) stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.     

Regulation of endothelial cell prostaglandin synthesis by glutathione

Prostaglandin synthesis in in vitro systems is dependent on glutathione and peroxide concentrations. We tested the effects of glutathione depletion and H2O2 exposure on prostaglandin synthesis in cultured porcine aortic endothelial cells. Depletion of glutathione using buthionine sulfoximine (BSO), diethylmaleate, and 2,4-chlorodinitrobenzene increased prostaglandin synthetic capacity. Production of prostacyclin, but not prostaglandin E2, from exogenous arachidonic acid was significantly greater than in controls. Glutathione depletion also resulted in enhanced production of prostacyclin from exogenous prostaglandin H2. These responses were not due to direct effects of glutathione-depleting agents on prostaglandin synthetic enzymes. Exposure to H2O2 also altered prostaglandin synthetic capacity in endothelial cells. While 5 microM H2O2 stimulated prostaglandin production from exogenous arachidonate, 25 and 50 microM were found to be inhibitory. Prostaglandin synthetic capacity was greater in BSO-treated cells which were exposed to 5 and 10 microM H2O2 than in cells exposed to H2O2 alone. However, prostaglandin synthetic capacity was greatly reduced in BSO-treated cells exposed to 50 microM H2O2. Thus, normal levels of cellular glutathione exert an inhibitory influence on prostaglandin synthesis. However, glutathione depletion increases the sensitivity of prostaglandin synthesis to inhibition by 50 microM H2O2.     

Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase

Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.     

Radioimmunologic identification of prostaglandins produced by serum-stimulated mouse embryo palate mesenchyme cells

High-performance liquid chromatography and radioimmunoassay were used to identify the prostaglandins synthesized by mouse embryo palate mesenchyme cells. Serum stimulated the release of several different metabolites of arachidonic acid including 6-ketoprostaglandin F1 alpha (the stable product of prostacyclin, prostaglandin I2), prostaglandin E2 and prostaglandin F2 alpha. Compared to control cells, the serum-stimulated cells produce elevated levels of prostaglandin E2 (36-fold), 6-ketoprostaglandin F1 alpha (15-fold) and prostaglandin F2 alpha (7-fold). The acetylenic analogue of arachidonic acid, 5,8,11,14-eicosatetraynoic acid prevented this accelerated synthesis.     

17beta-estradiol decreases vascular tone in cerebral arteries by shifting COX-dependent vasoconstriction to vasodilation

We have previously shown that estrogen treatment increases cerebrovascular cyclooxygenase-1, prostacyclin synthase, and production of prostacyclin. Therefore, vascular tone and prostanoid production were measured to investigate functional consequences of estrogen exposure. Middle cerebral arteries were isolated from ovariectomized female Fischer-344 rats with or without chronic in vivo 17beta-estradiol treatment. In vivo 17beta-estradiol treatment increased cerebral artery diameter; functional endothelium was required for expression of these differences. The nonspecific cyclooxygenase inhibitor indomethacin constricted, whereas arachidonic acid dilated, cerebral arteries from estrogen-treated animals. Estrogen exposure increased production of prostacyclin by cerebral arteries. Conversely, in estrogen-deficient animals, indomethacin dilated and arachidonic acid constricted cerebral blood vessels. This correlated with vasorelaxation following inhibition of the thromboxane-endoperoxide receptor with SQ-29548 but not after selective blockade of thromboxane synthase with furegrelate, suggesting prostaglandin endoperoxide (i.e., PGH2) activity. Removal of the endothelium or selective blockade of cyclooxygenase-1 with SC-560 abolished estrogen-mediated differences in the effects of arachidonate on vessel diameter and on prostacyclin production by cerebral arteries. These data suggest 17beta-estradiol decreases cerebrovascular tone by shifting the primary end product of the endothelial cyclooxygenase-1 pathway from the constrictor prostaglandin PGH2 to the vasodilator prostacyclin. These effects of estrogen may contribute to the heightened thromboresistance and enhanced cerebral blood flow documented in pre-versus postmenopausal women.     

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.     

Arachidonic acid metabolites in pregnant rat uterus

Production of prostaglandin E2 (PGE2), F2 alpha (PGF2 alpha) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) by pregnant rat uterus were measured in vitro. At mid-pregnancy, myometrium incubated with decidua attached released more prostanoids into the culture medium than when incubated without. As pregnancy progressed to 21 days more prostanoids were detected in the culture medium. However, no significantly increased conversion of exogenous arachidonic acid (AA) by myometrium was found.     

Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 production by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) stimulated prostaglandin E₂ (PGE₂) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE₂ production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE₂ by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE₂ production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE₂ production from endogenous AA than that from exogenous AA. These data suggest that PGE₂ production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs.     

Induction of prostacyclin formation by sodium n-butyrate in a cloned epithelial liver cell line

The effect of sodium n-butyrate on prostaglandin synthesis in cultured cells was examined. Exposure of BC-90 cells, a clone of an epithelial rat liver cell line, to 1 mM sodium n-butyrate for 40 h induced prostacyclin production. Prostacyclin synthesis was proved by demonstrating: (1) production of labeled 6-ketoprostaglandin F1 alpha by treating [14C]arachidonic acid pre-labeled cells with calcium ionophore A23187, (2) production of unstable substance that inhibited adenosine diphosphate-induced platelet aggregation, and (3) conversion of [14C]arachidonic acid to 6-ketoprostaglandin F1 alpha in homogenates of n-butyrate-treated cells. Untreated control cells showed negligible prostaglandin synthesis. Untreated cell homogenates did not convert [14C]arachidonic acid to any prostaglandins, but they converted [14C]prostaglandin H2 to prostacyclin. Induction of prostacyclin production by n-butyrate was also demonstrated with cells that had been treated with acetylsalicylic acid before n-butyrate treatment in acetylsalicylic acid-free medium. Incorporation of [3H]acetylsalicylic acid by sodium n-butyrate-treated cells increased in accordance with treatment time, while that of untreated cells did not change during culture. There was no difference in the phospholipase A2 activities of n-butyrate-treated and -untreated cells. From these findings, the possibility that n-butyrate induced prostacyclin in BC-90 cells through induction of fatty acid cyclooxygenase activity is discussed.     

Prostacyclin as a potent effector of adipose-cell differentiation.

The terminal differentiation of Ob1771 pre-adipose cells induced by arachidonic acid in serum-free hormone-supplemented medium containing insulin, transferrin, growth hormone, tri-iodothyronine and fetuin (5F medium) was strongly diminished in the presence of inhibitors of prostaglandin synthesis, namely aspirin or indomethacin. Carbaprostacyclin, a stable analogue of prostacyclin (prostaglandin I2) known to be synthesized by pre-adipocytes and adipocytes, behaved as an efficient activator of cyclic AMP production and was able, when added to 5F medium, to mimic the adipogenic effect of arachidonic acid. Prostaglandins E2, F2 alpha and D2, unable to affect the cyclic AMP production, failed to substitute for carbaprostacyclin. However, prostaglandin F2 alpha, which is another metabolite of arachidonic acid in pre-adipose and adipose cells, able to promote inositol phospholipid breakdown and protein kinase C activation, potentiated the adipogenic effect of carbaprostacyclin. In addition, carbaprostacyclin enhanced both a limited proliferation and terminal differentiation of adipose precursor cells isolated from rodent and human adipose tissues maintained in primary culture. These results demonstrate the critical role of prostacyclin and prostaglandin F2 alpha on adipose conversion in vitro and suggest a paracrine/autocrine role of both prostanoids in the development of adipose tissue in vivo.     

Prostanoid synthesis in peripheral nerve

The transformation of [1-14C]arachidonic acid into radiolabeled prostanoids was studied with homogenates and desheathed sciatic nerves of rats and frogs. All of the preparations studied were shown to synthesize prostaglandins; the specific prostanoids made were characterized by their migration on thin-layer chromatograms in three separate solvent systems. Both desheathed rat nerve and homogenates synthesize prostaglandin E2, prostaglandin F2 alpha, prostaglandin D2, 6-ketoprostaglandin F1 alpha and thromboxane B2. With preparations from frog nerve, prostaglandin E2 was the major prostanoid product formed. Several conditions were able to modulate the production of prostaglandin E2 with desheathed frog nerve. Electrical stimulation at high frequency (100 Hz) for 30 min increased the formation of labeled prostaglandin E2. Inclusion of glutathione also affected prostaglandin E2 formation. A lower concentration (0.1 mM) stimulated prostaglandin synthesis, while 1 mM glutathione was partially inhibitory. In both the rat and frog system, prostanoid synthesis was suppressed by indomethacin and aspirin.     

The effect of endotoxin on the lipoxygenase-mediated conversion of exogenous and endogenous arachidonic acid in mouse peritoneal macrophages

The influence of lipopolysaccharide (LPS, endotoxin) or its lipid A component (bacterial and synthetic) on the synthesis of zymosan induced leukotriene C4, prostaglandin E2 and prostacyclin and on the conversion of exogenous arachidonic acid was studied in mouse peritoneal macrophages. It was found that following preincubation with LPS the amount of leukotriene C4 released during phagocytosis of zymosan was substantially decreased. The levels of prostaglandin E2 and prostacyclin, however, were the same in LPS-treated cells and controls. Likewise, pretreatment with LPS impaired the capacity to convert exogenously added arachidonic acid to mono- and di-HETE's. Lipid A (bacterial and synthetic) exhibited the same activity as LPS. LPS had no effect on macrophages of the endotoxin low responder mouse strain (C3H/HeJ). Several explanations could be possible for the observed LPS effect. The finding that low doses of alpha-tocopheryl acetate prevented the LPS-induced decrease of LTC4 synthesis indicates a protective role of this agent. We would, therefore, favour the idea that lipoxygenases undergo oxidative selfinactivation during LPS action.     

Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase.

We report here that lipopolysaccharide (LPS) priming of rabbit alveolar macrophages leads to amplified synthesis of prostanoids, at least in part, by induction of a novel prostaglandin H synthase (PGH synthase). Rabbit alveolar macrophages were cultured with or without added LPS derived from Escherichia coli 0111:B4 for 4 h and then stimulated with opsonized zymosan (OPZ). LPS priming of alveolar macrophages resulted in enhanced release of thromboxane (TX) upon stimulation with OPZ, when compared to stimulated non-LPS controls. Addition of exogenous arachidonic acid to LPS-primed alveolar macrophages also resulted in increased production of TX. The LPS-induced increase in TX formation, in response to OPZ or arachidonic acid, was abolished by the addition of actinomycin D or cycloheximide during the priming period. Gas chromatography/mass spectrometry analysis indicated that levels of prostaglandins D2, E2, and F2 alpha, along with TX, were augmented in stimulated LPS-primed alveolar macrophages, implicating PGH synthase in the priming process. PGH synthase enzymatic activity, as determined by addition of arachidonic acid to macrophage sonicates, was markedly enhanced in LPS-primed alveolar macrophages. This correlated with increased PGH synthase levels detected by immunoprecipitation of 35S-labeled proteins and by Western blot analysis. Finally, Northern blot analysis using a cDNA probe to the recently described mitogen-inducible mouse PGH synthase revealed strong induction of approximately 4.3-kilobase mRNA in LPS-primed alveolar macrophages. Taken together, these results reveal that induction of a novel PGH synthase, probably the rabbit homologue of PGH synthase-2, plays a role in the enhanced synthesis of prostanoids by LPS-primed alveolar macrophages.     

Isoprostanes and PGE2 production in human isolated pulmonary artery smooth muscle cells: concomitant and differential release.

The isoprostanes are a group of biologically active arachidonic acid metabolites initially thought to be formed under conditions of oxidative stress and independently of cyclooxygenase. However, recent studies have demonstrated isoprostane production under conditions in which cyclooxygenase is intentionally activated/induced. Here we describe for the first time formation of isoprostanes by human vascular cells via independent pathways of oxidative stress and cyclooxygenase induction. We compared the release of the isoprostane with that of the traditional prostaglandin, prostaglandin E2. Cyclooxygenase-2 induction was confirmed by Western blot. When cells were stimulated with cytokines, the release of isoprostanes was inhibited by the cyclooxygenase-1 and -2 inhibitor indomethacin as well by as the cyclooxygenase-2 selective inhibitor L-745,337. However, treatment of cells with the superoxide-producing enzyme xanthine oxidase also resulted in isoprostane release, which was not affected by cyclooxygenase inhibition, unlike PGE2 release under the same condition. Thus, two independent pathways relating to oxidative stress and cyclooxygenase-2 induction form isoprostanes. These findings may have particular importance in diseases such as sepsis and ARDS in which oxidant stress occurs and cyclooxygenase is induced.     

Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced approximately 30 ng/mg wet tissue of 6-oxo-prostaglandin F1 alpha (the stable hydrolysis product from prostacyclin) and approximately 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures or aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2 to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediately after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are greatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local changes in their environment.     

Effects of ozone on lung mechanics and cyclooxygenase metabolites in dogs.

To determine if acute exposure to ozone can cause changes in the production of cyclooxygenase metabolites of arachidonic acid (AA) in the lung which are associated with changes in lung mechanics, we exposed mongrel dogs to 0.5 ppm ozone for two hours. We measured pulmonary resistance (RL) and dynamic compliance (Cdyn) and obtained methacholine dose response curves and bronchoalveolar lavagate (BAL) before and after the exposures. We calculated the provocative dose of methacholine necessary to increase RL 50% (PD50) and analyzed the BAL for four cyclooxygenase metabolites of AA: a stable hydrolysis product of prostacyclin, 6-keto-prostaglandin F1 alpha (6-keto-PgF1 alpha); prostaglandin E2 (PgE2); a stable hydrolysis product of thromboxane A2, thromboxane B2 (TxB2); and prostaglandin F2 alpha (PgF2 alpha). Following ozone exposure, RL increased from 4.75 +/- 1.06 to 6.08 +/- 1.3 cm H2O/L/sec (SEM) (p less than 0.05), Cdyn decreased from 0.0348 +/- 0.0109 TO .0217 +/- .0101 L/cm H2O (p less than 0.05), and PD50 decreased from 4.32 +/- 2.41 to 0.81 +/- 0.49 mg/cc (p less than 0.05). The baseline metabolite levels were as follows: 6-keto PgF1 alpha: 96.1 +/- 28.8 pg/ml; PgE2: 395.8 +/- 67.1 pg/ml; TxB2: 48.5 +/- 11.1 pg/ml; PgF2 alpha: 101.5 +/- 22.6 pg/ml. Ozone had no effect on any of these prostanoids. These studies quantify the magnitude of cyclooxygenase products of AA metabolism in BAL from dog lungs and demonstrate that changes in their levels are not prerequisites for ozone-induced changes in lung mechanics or airway reactivity.     

Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha.

The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6-keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio-immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion.