Eicosapentaenoic Acid Induces the Inhibition of Adipogenesis by Reducing the Effect of PPARγ Activator and Mediating PKA Activation and Increased COX-2 Expression in 3T3-L1 Cells at the Differentiation Stage.

Obesity has received increasing attention in recent years because it is a factor in the development of non-communicable diseases. The current study aimed to analyze how representative fatty acids (FAs) such as palmitic acid, stearic acid, oleic acid, α-linolenic acid (ALA), and eicosapentaenoic acid (EPA) affected adipogenesis when/if introduced at the differentiation stage of 3T3-L1 cell culture. These FAs are assumed to be potentially relevant to the progression or prevention of obesity. EPA added during the differentiation stage reduced intracellular triacylglycerol (TAG) accumulation, as well as the expression of the established adipocyte-specific marker genes, during the maturation stage. However, no other FAs inhibited intracellular TAG accumulation. Coexistence of Δ12-prostaglandin J2, a peroxisome proliferator-activated receptor γ activator, with EPA during the differentiation stage partially attenuated the inhibitory effect of EPA on intracellular TAG accumulation. EPA increased cyclooxygenase-2 (COX-2) expression and protein kinase A (PKA) activity at the differentiation stage, which could explain the inhibitory actions of EPA. Taken together, exposure of preadipocytes to EPA only during the differentiation stage may be sufficient to finally reduce the mass of white adipose tissue through increasing COX-2 expression and PKA activity.

Arachidonic Acid Added during the Differentiation Phase of 3T3-L1 Cells Exerts Anti-Adipogenic Effect by Reducing the Effects of Pro-Adipogenic Prostaglandins.

A linoleic acid (LA) metabolite arachidonic acid (AA) added to 3T3-L1 cells is reported to suppress adipogenesis. The purpose of the present study aimed to clarify the effects of AA added during the differentiation phase, including adipogenesis, the types of prostaglandins (PG)s produced, and the crosstalk between AA and the PGs produced. Adipogenesis was inhibited by AA added, while LA did not. When AA was added, increased PGE2 and PGF2α production, unchanged Δ12-PGJ2 production, and reduced PGI2 production were observed. Since the decreased PGI2 production was reflected in decreased CCAAT/enhancer-binding protein-ß (C/EBPß) and C/EBPδ expression, we expected that the coexistence of PGI2 with AA would suppress the anti-adipogenic effects of AA. However, the coexistence of PGI2 with AA did not attenuate the anti-adipogenic effects of AA. In addition, the results were similar when Δ12-PGJ2 coexisted with AA. Taken together, these results indicated that the metabolism of ingested LA to AA is necessary to inhibit adipogenesis and that exposure of AA to adipocytes during only the differentiation phase is sufficient. As further mechanisms for suppressing adipogenesis, AA was found not only to increase PGE2 and PGF2α and decrease PGI2 production but also to abrogate the pro-adipogenic effects of PGI2 and Δ12-PGJ2.

Prostaglandin D2 Added during the Differentiation of 3T3-L1 Cells Suppresses Adipogenesis via Dysfunction of D-Prostanoid Receptor P1 and P2.

We previously reported that the addition of prostaglandin, (PG)D2, and its chemically stable analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), during the maturation phase of 3T3-L1 cells promotes adipogenesis. In the present study, we aimed to elucidate the effects of the addition of PGD2 or 11d-11m-PGD2 to 3T3-L1 cells during the differentiation phase on adipogenesis. We found that both PGD2 and 11d-11m-PGD2 suppressed adipogenesis through the downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, the latter suppressed adipogenesis more potently than PGD2, most likely because of its higher resistance to spontaneous transformation into PGJ2 derivatives. In addition, this anti-adipogenic effect was attenuated by the coexistence of an IP receptor agonist, suggesting that the effect depends on the intensity of the signaling from the IP receptor. The D-prostanoid receptors 1 (DP1) and 2 (DP2, also known as a chemoattractant receptor-homologous molecule expressed on Th2 cells) are receptors for PGD2. The inhibitory effects of PGD2 and 11d-11m-PGD2 on adipogenesis were slightly attenuated by a DP2 agonist. Furthermore, the addition of PGD2 and 11d-11m-PGD2 during the differentiation phase reduced the DP1 and DP2 expression during the maturation phase. Overall, these results indicated that the addition of PGD2 or 11d-11m-PGD2 during the differentiation phase suppresses adipogenesis via the dysfunction of DP1 and DP2. Therefore, unidentified receptor(s) for both molecules may be involved in the suppression of adipogenesis.

Prostaglandin D2 and its analog, 11d-11m-PGD2, added during the differentiation phase contribute to adipogenic program inhibition in 3T3-L1 cells.

We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

Anti-inflammatory and bioavailability studies on dietary 3,5,4'-trihydroxy-6,7-methylenedioxyflavone-O-glycosides and their aglycone from indigo leaves in a murine model of inflammatory bowel disease.

Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities. We have recently demonstrated that indigo leaves contain high levels of flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) as an aglycone. In this study, we attempted to evaluate anti-inflammatory activities of TMF-O-glycosides and free TMF prepared from indigo leaves after extraction with hot water. Free TMF was found to appreciably down-regulate the gene expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, inducible nitric oxide, and tumor necrosis factor-α in cultured macrophage cells stimulated with lipopolysaccharide while up-regulating the expression of anti-inflammatory IL-10. However, no study has been conducted regarding in vivo anti-inflammatory activities of TMF-O-glycosides and free TMF until now. Here, we assessed in vivo anti-inflammatory effects of these dietary compounds on ulcerative colitis in a murine model of inflammatory bowel disease by the induction with dextran sulfate sodium (DSS). Histological evaluation revealed that both TMF-O-glycosides and free TMF effectively protected against DSS-induced ulcerative colitis. The analysis of digested products by liquid chromatography and mass spectrometry led us to detect free TMF as a predominant metabolite in the feces of mice fed with TMF-O-glycosides. Moreover, free TMF was later detected as glucuronyl conjugates of TMF in the liver of mice fed with both fractions. These results indicate the effective digestion of TMF-O-glycosides and the subsequent absorption of free TMF in the gut of mice for exerting anti-inflammatory effects. Taken together, our findings suggest that dietary TMF-O-glycosides could be promising natural sources for the utilization as herbal medicine and nutraceuticals to expect in vivo anti-inflammatory activities.

Constituents of flavonol O-glycosides and antioxidant activities of extracts from seeds, sprouts, and aerial parts of Polygonum tinctorium Lour.

Polygonum tinctorium Lour. (family Polygonaceae), known as indigo plant, has been useful as a medicinal or edible plant abundant in polyphenolic compounds. We have recently shown that flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) are predominant flavonoids in indigo leaves. However, no study has been performed regarding changes in the levels of flavonoid species during the germination and growth of indigo plant. Here, we attempted to determine the individual constituents of flavonol O-glycosides and the changes in their contents of the seeds, sprouts, and aerial parts. These results revealed that only the seeds predominantly contained flavonol O-(acetyl)-rhamnosides with quercetin or kaempferol as an aglycone. During the development of the sprouts and aerial parts, flavonol O-glycosides with TMF as an aglycone became mainly detectable and accounted for 79.4% and 74.9% of total flavonol O-glycosides from the extracts of aerial parts harvested in 2016 and 2017, respectively. Of the plant organs tested, the aerial parts exhibited the highest antioxidant activities concomitant with greatly increased levels of total polyphenols. Thus, we were able to conduct the identification and quantification of flavonol O-glycosides from the seeds, sprouts, and aerial parts of indigo plant and to evaluate antioxidant activities of their extracts. Taken together, our findings clearly provide the evidence that the aerial parts of indigo plant are a rich source of flavonol O-glycosides with TMF and exhibit much higher antioxidant activities, indicating the usefulness for the application to food and nutraceutical purposes.

Skatole regulates intestinal epithelial cellular functions through activating aryl hydrocarbon receptors and p38.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction. We found that skatole dose-dependently caused IEC death and time-dependently induced IEC apoptosis. Since skatole directly interacts with aryl hydrocarbon receptors (AhR), we investigated whether these receptors influence the skatole-induced death of IEC. In addition to increased AhR transcriptional activity induced by skatole, the AhR antagonist CH223191 partially suppressed of skatole-induced IEC death. Extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) are mitogen-activated protein kinases (MAPK) induced by skatole. None of them were repressed by CH223191, whereas the p38 inhibitor SB203580 promoted skatole-induced IEC death. These findings together indicated that skatole induces both AhR-dependent activation pathways and the AhR-independent activation of p38, consequently regulating the amount of IEC death. Accumulating evidence indicates that consuming large amounts of animal protein is associated with the pathogenesis and progression of inflammatory bowel diseases (IBD). Thus, intestinal skatole production induced by large amounts of dietary animal protein might be associated via IEC death with intestinal pathologies such as IBD.

Comparison of pro-adipogenic effects between prostaglandin (PG) D2 and its stable, isosteric analogue, 11-deoxy-11-methylene-PGD2, during the maturation phase of cultured adipocytes.

Prostaglandin (PG) D2 is relatively unstable and dehydrated non-enzymatically into PGJ2 derivatives, which are known to serve as pro-adipogenic factors by activating peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipogenesis. 11-Deoxy-11-methylene-PGD2 (11d-11m-PGD2) is a novel, chemically stable, isosteric analogue of PGD2 in which the 11-keto group is replaced by an exocyclic methylene. Here we attempted to investigate pro-adipogenic effects of PGD2 and 11d-11m-PGD2 and to compare the difference in their ways during the maturation phase of cultured adipocytes. The dose-dependent study showed that 11d-11m-PGD2 was significantly more potent than natural PGD2 to stimulate the storage of fats suppressed in the presence of indomethacin, a cyclooxygenase inhibitor. These pro-adipogenic effects were caused by the up-regulation of adipogenesis as evident with higher gene expression levels of adipogenesis markers. Analysis of transcript levels revealed the enhanced gene expression of two subtypes of cell-surface membrane receptors for PGD2, namely the prostanoid DP1 and DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)) receptors together with lipocalin-type PGD synthase during the maturation phase. Specific agonists for DP1, CRTH2, and PPARγ were appreciably effective to rescue adipogenesis attenuated by indomethacin. The action of PGD2 was attenuated by specific antagonists for DP1 and PPARγ. By contrast, the effect of 11d-11m-PGD2 was more potently interfered by a selective antagonist for CRTH2 than that for DP1 while PPARγ antagonist GW9662 had almost no inhibitory effects. These results suggest that PGD2 exerts its pro-adipogenic effect principally through the mediation of DP1 and PPARγ, whereas the stimulatory effect of 11d-11m-PGD2 on adipogenesis occurs preferentially by the interaction with CRTH2.

In Vitro Anti-inflammatory and Antioxidant Activities of 3,5,4'-Trihydroxy-6,7-Methylenedioxyflavone-O-Glycosides and Their Aglycone from Leaves of Polygonum tinctorium Lour.

Polygonum tinctorium Lour (indigo plant) has been regarded as a useful medicinal plant for traditional herbal medicine. The polyphenolic fraction of indigo leaves exhibited anti-inflammatory activities as determined by the suppressed synthesis of nitric oxide (NO) in cultured RAW264 macrophage cells. The acid hydrolysate of the fraction showed much more potent effect than the unhydrolyzed one. In sharp contrast, those fractions of indigo stems had almost no effect. 3,5,4'-Trihydroxy-6,7-methylenedioxyflavone (TMF)-O-glycosides and tryptanthrin were detected exclusively in the extracts of the leaves. The isolated flavonol species were furthermore tested for their anti-inflammatory activities against the synthesis of NO and prostaglandin E2 in the cultured macrophage cells. More potent anti-inflammatory effects were recognized with different aglycones of flavonols than their flavonol O-glycosides. Although the inhibitory effects of TMF were less effective than those of tryptanthrin, the levels of flavonol O-glycosides with TMF were much more abundant than those of tryptanthrin in the leaves. Oral administration of the fraction containing flavonol O-glycosides with TMF into mice revealed the detection of free TMF in the blood circulation, indicating that the aglycone moiety can be cleaved by digestive enzymes and absorbed in the gut. Alternatively, the assay of hydrophilic oxygen radical absorbance capacity revealed that the isolated species of flavonol O-glycosides with TMF and their aglycone had appreciable antioxidant activities. Taken together, our findings suggest that the predominant flavonol O-glycosides with TMF as an aglycone could be promising natural agents for the application to herbal medicine, nutraceuticals, and food additives.

Antioxidant activities and structural characterization of flavonol O-glycosides from seeds of Japanese horse chestnut (Aesculus turbinata BLUME).

We attempted to evaluate the contents and distribution of antioxidants in the whole seeds, seed shells, and peeled seeds of the Japanese horse chestnut. The seed shells exhibited the highest antioxidant activities due to the presence of highly polymeric proanthocyanidins as we have reported recently. On the other hand, the peeled seeds predominantly contained flavonols such as quercetin and kaempferol at a high level of 66.7% of total polyphenols, also contributing to the predominant antioxidant activities. The instrumental analysis of the extract from the whole seeds revealed the identification of eight flavonol O-glycosides, including six compounds with quercetin and two species with kaempferol as aglycones. The isolated species exhibited different antioxidant activities depending on the types of aglycones, glycosides, and acylated moieties. The results indicate that the peeled seeds are a good source of flavonol O-glycosides serving as antioxidants to be used for food additives and dietary supplements.

Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase.

Stimulation of fat storage by prostacyclin and selective agonists of prostanoid IP receptor during the maturation phase of cultured adipocytes.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from Arabidopsis thaliana, which is involved in the interaction between VSR4 and AP1M2, µ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4.

µ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.

New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth ß-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes.

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4-6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.

Cultured preadipocytes undergoing stable transfection with cyclooxygenase-1 in the antisense direction accelerate adipogenesis during the maturation phase of adipocytes.

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

A monoclonal antibody specific for Δ12-prostaglandin J2 and its utilization in the immunological assay in cell culture system of adipocytes.

Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ(12)-PGJ(2) and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ(12)-PGJ(2). The assay provided a sensitive calibration curve for Δ(12)-PGJ(2), allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ(2) and 15d-PGJ(2) were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ(12)-PGJ(2) in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ(12)-PGJ(2) in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ(12)-PGJ(2) became more pronounced after several hours of incubation of PGD(2) at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ(12)-PGJ(2) during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ(12)-PGJ(2), reflecting the biosynthesis of unstable PGD(2) in the culture system of adipocytes.

o-Quinone involvement in the prooxidant tendency of a mixture of quercetin and caffeic acid.

The oxidation products of a previously tested prooxidant mixture of quercetin (Q) and caffeic acid (CA) at 1:2 ratio were analysed by LC-MS. The UV-Vis and MS spectra of three chromatographic peaks eluting at t(R)=9.11min, t(R)=14.36min and t(R)=30.30min were studied further. The structures of the tentatively identified compounds indicate polymeric molecules. A pentamer formed by 3 units of quercetin and 2 units of caffeic acid was attributed in the case of peaks t(R)=9.11min and t(R)=14.36min. The quercetin quinone (QQ) - a polymerization intermediate - is a fragment of the compound identified in the MS spectrum with the m/z=323 coming from Q o-quinone m/z=300 plus 23 from Na(+). According to the UV-Vis spectrum, we suggest a different intermolecular arrangement which gives a more extended e-delocalisation. At t(R)=30.30min, the spectra helped us to tentatively identify this oxidation product as being a polymer of 4 CA units and 1 QQ.

Prostaglandin J2 series induces the gene expression of monocyte chemoattractant protein-1 during the maturation phase of cultured adipocytes.

Prostaglandin (PG) J(2) series including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is the dehydration products of PGD(2) that are biosynthesized through the cyclooxygenase (COX) pathway. These prostanoids are active ligands for peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis in adipocytes. Here we investigated whether PGJ(2) derivatives can modulate the gene expression of monocyte chemoattractant protein-1 (MCP-1), a pro-inflammatory chemokine, during the maturation phase of adipocytes. Each of selective or nonselective inhibitors for COX isoforms suppressed significantly the accumulation of fats by interfering the induced expression of the PPARγ gene. Immunological assays of PGJ(2) series revealed higher production of Δ(12)-PGJ(2) than 15d-PGJ(2) by cultured adipocytes, implicating the contribution of endogenous PGJ(2) series to the stimulated adipogenesis. In addition, the increased transcription of MCP-1 was detectable at later maturation phase of adipogenesis, which was prevented by co-incubation with aspirin. Although 15d-PGJ(2) was more potent than Δ(12)-PGJ(2), both PGJ(2) derivatives series had similar effects to rescue dose-dependently the expression of the MCP-1 gene attenuated by aspirin. These findings suggest that the expression of MCP-1 involved in adipocyte inflammation could be positively regulated by the PGJ(2) series during adipogenesis in adipose tissue.

Generation of monoclonal antibody for 15-deoxy-Δ¹²,¹4-prostaglandin J2 and development of enzyme-linked immunosorbent assay for its quantification in culture medium of adipocytes.

15-deoxy-Δ¹²,¹4-prostaglandin J2 (15d-PGJ2) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD2. To determine the endogenous synthesis of 15d-PGJ2, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ2. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ2 was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ2. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ2 from 0.5 pg to 9.7 ng with 71 pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ2 with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ2 in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ2 became more evident after several hours of incubation with authentic PGD2 at 37 °C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ2 and its roles in various cells and tissues.