Octanol/water partition coefficients estimated using retention times in reverse-phase liquid chromatography and calculated in silico as one of the determinant factors for pharmacokinetic parameter estimations of general chemical substances.

The octanol/water partition coefficient P (logP) is a hydrophobicity index and is one of the determining factors for the pharmacokinetics of orally administered substances because it influences membrane permeability. To illustrate the wide-ranging variety of compounds in the chemical space, a two-dimensional data plot consisting of 25 blocks was previously proposed based on a substance's in silico chemical descriptors. The logP values of approximately 200 diverse chemicals (test plus reference compounds covering all 25 blocks of the chemical space) were estimated experimentally using retention times in reverse-phase liquid chromatography; these values were compared with those of authentic reference compounds with established logP values (available for 17 of 60 reference substances in the Organization for Economic Co-operation and Development Test Guideline 117). The logP values of 140 of 165 chemicals successfully estimated using four different mobile phase conditions (pH 2, 4, 7, and 10 for molecular forms) correlated significantly with those calculated using the in silico packages ChemDraw and ACD/Percepta (r > 0.72). Although substances that neighbored authentic compounds in the chemical space had precisely correlated logP values estimated experimentally and in silico, some compounds that were more distant from authentic substances showed lower logP values than those estimated in silico. These results indicate that additional authentic reference materials with wider ranging chemical diversity and their logP values from reverse-phase liquid chromatography should be included in the international test guidance to promote simple and reliable estimation of octanol/water partition coefficients, which are important determinant factors for the pharmacokinetics of general chemicals.

An Updated In Silico Prediction Method for Volumes of Systemic Circulation of 323 Disparate Chemicals for Use in Physiologically Based Pharmacokinetic Models to Estimate Plasma and Tissue Concentrations after Oral Doses in Rats.

Updated algorithms for predicting the volumes of systemic circulation (V1), along with absorption rate constants and hepatic intrinsic clearances, as input parameters for physiologically based pharmacokinetic (PBPK) models were established to improve the accuracy of estimated plasma and tissue concentrations of 323 chemicals after virtual oral administrations in rats. Using ridge regression with an enlarged set of chemical descriptors (up to 99), the estimated input V1 values resulted in an improved correlation coefficient (from 246 compounds) with the traditionally determined values. The PBPK model input parameters for rats of diverse compounds can be precisely estimated by increasing the number of descriptors.

Prediction of permeability across intestinal cell monolayers for 219 disparate chemicals using in vitro experimental coefficients in a pH gradient system and in silico analyses by trivariate linear regressions and machine learning.

For medicines, the apparent membrane permeability coefficients (Papp) across human colorectal carcinoma cell line (Caco-2) monolayers under a pH gradient generally correlate with the fraction absorbed after oral intake. Furthermore, the in vitro Papp values of 29 industrial chemicals were found to have an inverse association with their reported no-observed effect levels for hepatotoxicity in rats. In the current study, we expanded our influx permeability predictions for the 90 previously investigated chemicals to both influx and efflux permeability predictions for 207 diverse primary compounds, along with those for 23 secondary compounds. Trivariate linear regression analysis found that the observed influx and efflux logPapp values determined by in vitro experiments significantly correlated with molecular weights and the octanol-water distribution coefficients at apical and basal pH levels (pH 6.0 and 7.4, respectively) (apical to basal, r = 0.76, n = 198; and basal to apical, r = 0.77, n = 202); the distribution coefficients were estimated in silico. Further, prediction accuracy was enhanced by applying a light gradient boosting machine learning system (LightGBM) to estimate influx and efflux logPapp values that incorporated 17 and 19 in silico chemical descriptors (r = 0.83-0.84, p < 0.001). The determination in vitro and/or prediction in silico of permeability coefficients across intestinal cell monolayers of a diverse range of industrial chemicals/food components/medicines could contribute to the safety evaluations of oral intakes of general chemicals in humans. Such new alternative methods could also reduce the need for animal testing during toxicity assessment.

In Silico Prediction of Input Parameters for Simplified Physiologically Based Pharmacokinetic Models for Estimating Plasma, Liver, and Kidney Exposures in Rats after Oral Doses of 246 Disparate Chemicals.

Recently developed computational models can estimate plasma, hepatic, and renal concentrations of industrial chemicals in rats. Typically, the input parameter values (i.e., the absorption rate constant, volume of systemic circulation, and hepatic intrinsic clearance) for simplified physiologically based pharmacokinetic (PBPK) model systems are calculated to give the best fit to measured or reported in vivo blood substance concentration values in animals. The purpose of the present study was to estimate in silico these three input pharmacokinetic parameters using a machine learning algorithm applied to a broad range of chemical properties obtained from several cheminformatics software tools. These in silico estimated parameters were then incorporated into PBPK models for predicting internal exposures in rats. Following this approach, simplified PBPK models were set up for 246 drugs, food components, and industrial chemicals with a broad range of chemical structures. We had previously generated PBPK models for 158 of these substances, whereas 88 for which concentration series data were available in the literature were newly modeled. The values for the absorption rate constant, volume of systemic circulation, and hepatic intrinsic clearance could be generated in silico by equations containing between 14 and 26 physicochemical properties. After virtual oral dosing, the output concentration values of the 246 compounds in plasma, liver, and kidney from rat PBPK models using traditionally determined and in silico estimated input parameters were well correlated (r ≥ 0.83). In summary, by using PBPK models consisting of chemical receptor (gut), metabolizing (liver), excreting (kidney), and central (main) compartments with in silico-derived input parameters, the forward dosimetry of new chemicals could provide the plasma/tissue concentrations of drugs and chemicals after oral dosing, thereby facilitating estimates of hematotoxic, hepatotoxic, or nephrotoxic potential as a part of risk assessment.

Physiologically Based Pharmacokinetic Models Predicting Renal and Hepatic Concentrations of Industrial Chemicals after Virtual Oral Doses in Rats.

Recently developed high-throughput in vitro assays in combination with computational models could provide alternatives to animal testing. The purpose of the present study was to model the plasma, hepatic, and renal pharmacokinetics of approximately 150 structurally varied types of drugs, food components, and industrial chemicals after virtual external oral dosing in rats and to determine the relationship between the simulated internal concentrations in tissue/plasma and their lowest-observed-effect levels. The model parameters were based on rat plasma data from the literature and empirically determined pharmacokinetics measured after oral administrations to rats carried out to evaluate hepatotoxic or nephrotic potentials. To ensure that the analyzed substances exhibited a broad diversity of chemical structures, their structure-based location in the chemical space underwent projection onto a two-dimensional plane, as reported previously, using generative topographic mapping. A high-throughput in silico one-compartment model and a physiologically based pharmacokinetic (PBPK) model consisting of chemical receptor (gut), metabolizing (liver), central (main), and excreting (kidney) compartments were developed in parallel. For 159 disparate chemicals, the maximum plasma concentrations and the areas under the concentration-time curves obtained by one-compartment models and modified simple PBPK models were closely correlated. However, there were differences between the PBPK modeled and empirically obtained hepatic/renal concentrations and plasma maximal concentrations/areas under the concentration-time curves of the 159 chemicals. For a few compounds, the lowest-observed-effect levels were available for hepatotoxicity and nephrotoxicity in the Hazard Evaluation Support System Integrated Platform in Japan. The areas under the renal or hepatic concentration-time curves estimated using PBPK modeling were inversely associated with these lowest-observed-effect levels. Using PBPK forward dosimetry could provide the plasma/tissue concentrations of drugs and chemicals after oral dosing, thereby facilitating estimates of nephrotoxic or hepatotoxic potential as a part of the risk assessment.

Determination and prediction of permeability across intestinal epithelial cell monolayer of a diverse range of industrial chemicals/drugs for estimation of oral absorption as a putative marker of hepatotoxicity.

Apparent permeability coefficients (P app) across a human intestinal epithelial Caco-2 cell monolayer were measured for a range of industrial/drug chemicals. A predictive equation for determining in vitro P app values of fifty-six substances was set up using multivariate regression analysis based on in silico-estimated physicochemical properties (molecular weights and water distribution coefficients for apical and basal pH environments) (r = 0.77, p <  0.01). Predicted logP app values of a secondary set of 34 compounds were correlated with the measured values. Under the medicinal logP app values associated with their reported fraction absorbed, a significant inverse non-linear correlation was found between the logarithmic transformed values of observed P app values and reported hepatic no-observed-effect levels of industrial chemicals (r = -0.55, p <  0.01, n = 29). In vitro determination and/or in silico prediction of permeability across intestinal cells could be effective for estimating oral absorption as a putative indicator for hepatotoxicity.

Plasma and Hepatic Concentrations of Chemicals after Virtual Oral Administrations Extrapolated Using Rat Plasma Data and Simple Physiologically Based Pharmacokinetic Models.

Only a small fraction of chemicals possesses adequate in vivo toxicokinetic data for assessing potential hazards. The aim of the present study was to model the plasma and hepatic pharmacokinetics of more than 50 disparate types of chemicals and drugs after virtual oral administrations in rats. The models were based on reported pharmacokinetics determined after oral administration to rats. An inverse relationship was observed between no-observed-effect levels after oral administration and chemical absorbance rates evaluated for cell permeability ( r = -0.98, p < 0.001, n = 17). For a varied selection of more than 30 chemicals, the plasma concentration curves and the maximum concentrations obtained using a simple one-compartment model (recently recommended as a high-throughput toxicokinetic model) and a simple physiologically based pharmacokinetic (PBPK) model (consisting of chemical receptor, metabolizing, and central compartments) were highly consistent. The hepatic and plasma concentrations and the hepatic and plasma areas under the concentration-time curves of more than 50 chemicals were roughly correlated; however, differences were evident between the PBPK-modeled values in livers and empirically obtained values in plasma. Of the compounds selected for analysis, only seven had the lowest observed effect level (LOEL) values for hepatoxicity listed in the Hazard Evaluation Support System Integrated Platform in Japan. For these seven compounds, the LOEL values and the areas under the hepatic concentration-time curves estimated using PBPK modeling were inversely correlated ( r = -0.78, p < 0.05, n = 7). This study provides important information to help simulate the high hepatic levels of potent hepatotoxic compounds. Using suitable PBPK parameters, the present models could estimate the plasma/hepatic concentrations of chemicals and drugs after oral doses using both PBPK forward and reverse dosimetry, thereby indicating the potential value of this modeling approach in predicting hepatic toxicity as a part of risk assessments of chemicals absorbed in the human body.

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.

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.

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.

[Trends of activities in international organization and authorities in each country regarding risk management of nanomaterials].

The Organisation for Economic Co-operation and Development (OECD) estimates that in 2015 the worldwide nanotechnology-related market will reach about US$1 trillion and will create about two million additional jobs. The field began with the general technical industry, including many areas of the pharmaceutical, medical, agricultural, and environmental/energy industries, and recently has expanded to the formulation of nanotechnology. In particular, elements of nanometer-sized research and development (R & D) in the areas of drug delivery systems and nanomedicine are attracting widespread interest. On the other hand, the potential negative effects on human health and the environment of nanomaterials manufactured on the industrial scale have not been investigated in detail. Therefore, although the risks have not been fully clarified, research institutions and international organizations such as the OECD are working in earnest to elucidate them. This paper outlines the status of initiatives in industry and regulatory conditions and trends in individual countries and institutions to determine the safety of nanomaterials from the perspective of international relations.

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.

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.

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids.

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD(2) and its non-enzymatic dehydration products, PGJ(2) series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD(2) and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD(2) from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE(2) remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize Δ(12)-PGJ(2) increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor γ (PPARγ) agonists including troglitazone and Δ(12)-PGJ(2). Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPARγ signaling pathway without the contribution of endogenous pro-adipogenic prostanoids.

Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes.

Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD(2) and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD(2) synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE(2) was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE(2) and PGF(2α), while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ(12,14)-prostaglandin J(2). These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ(2) series.

15-Deoxy-Δ(12,14)-prostaglandin J(2) interferes inducible synthesis of prostaglandins E(2) and F(2α) that suppress subsequent adipogenesis program in cultured preadipocytes.

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E(2) and PGF(2α) involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ(2) interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ(12)-PGJ(2) and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ(2). Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ(2) or PDTC although 15d-PGJ(2) alone has no stimulatory effect. Moreover, 15d-PGJ(2) did not block the inhibitory effects of exogenous PGE(2) and PGF(2α) on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ(2) can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.