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

Abstract

Prostaglandin (PG) D2 is inherently unstable and undergoes non-enzymatic dehydration, transforming into PGJ2 derivatives. These derivatives are recognized as pro-adipogenic factors, primarily through their activation of peroxisome proliferator-activated receptor (PPAR) γ, a key regulator of adipogenesis. 11-Deoxy-11-methylene-PGD2 (11d-11m-PGD2) is a novel, chemically stable, isosteric analog of PGD2, where the 11-keto group is substituted with an exocyclic methylene.

This study aimed to investigate the pro-adipogenic effects of both PGD2 and 11d-11m-PGD2, and to compare their mechanisms of action during the maturation phase of cultured adipocytes. Dose-dependent analysis revealed that 11d-11m-PGD2 was significantly more potent than natural PGD2 in stimulating fat storage, particularly when fat storage was suppressed by indomethacin, a cyclooxygenase inhibitor. These pro-adipogenic effects were attributed to the upregulation of adipogenesis, evidenced by increased gene expression levels of adipogenesis markers.

Transcript level analysis showed enhanced gene expression of two PGD2 cell-surface membrane receptor subtypes: prostanoid DP1 and DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)), along with lipocalin-type PGD synthase, during the maturation phase. Specific agonists for DP1, CRTH2, and PPARγ effectively restored adipogenesis that had been attenuated by indomethacin.

The pro-adipogenic action of PGD2 was diminished by specific antagonists for DP1 and PPARγ. In contrast, the effect of 11d-11m-PGD2 was more strongly inhibited by a selective CRTH2 antagonist than by a DP1 antagonist, while the PPARγ antagonist GW9662 had minimal inhibitory effects.

These results suggest that PGD2 primarily promotes adipogenesis through the DP1 and PPARγ pathways. Conversely, 11d-11m-PGD2 preferentially stimulates adipogenesis through its interaction with CRTH2.

Introduction

The differentiation of preadipocytes into mature adipocytes is a process orchestrated by a cascade of transcriptional factors, including the CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs). Among these nuclear factors, PPARγ, a member of the nuclear hormone superfamily, is abundantly expressed in adipocytes and is considered a master regulator of adipogenesis.

The activation of PPARγ requires active ligands, as this nuclear hormone receptor functions as a ligand-dependent transcription factor. PPARγ can be activated by various lipophilic ligands, including polyunsaturated fatty acids and their metabolites, which serve as natural ligands. While the precise endogenous ligands for this receptor in vivo remain to be definitively established, 15-deoxy-Δ12,14-prostaglandin (PG) J2 (15d-PGJ2) is recognized as a potent natural activator of PPARγ. 15d-PGJ2 is formed through the non-enzymatic dehydration of unstable PGD2. Consequently, PGD2 and related PGJ2 derivatives, such as 15d-PGJ2 and Δ12-PGJ2, act as pro-adipogenic factors in cultured adipocytes expressing PPARγ.

Alternatively, prostaglandin I2 (PGI2) is also known to promote adipogenesis, but its action is mediated through the cell-surface membrane prostanoid IP receptor. In contrast, other prostanoids, such as prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), exhibit anti-adipogenic effects through their respective cell-surface membrane EP4 and FP receptors. Thus, different classes of prostaglandins exert opposing effects on adipogenesis through their specific nuclear hormone receptors or cell-surface membrane receptors.

Similar to some prostanoids, PGD2 can mediate its biological activities in certain immune cells through two types of G protein-coupled membrane receptors: the DP1 receptor and the DP2 receptor. DP2 is also known as the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). However, the role of these cell-surface membrane receptors for PGD2 in regulating adipogenesis in adipose tissues remains largely unexplored.

Mouse preadipogenic 3T3-L1 cells are a valuable model for studying adipogenesis, encompassing the growth, differentiation, and maturation phases. Cultured preadipocytes are grown in differentiation medium until they reach confluence. The growth-arrested cells are then typically exposed to a hormonal mixture of insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX) within the differentiation medium.

This mixture initiates the adipogenesis program by inducing the expression of specific CCAAT enhancer-binding proteins (C/EBPs). Following the induction of the differentiation phase, the treated cells begin to accumulate lipids during the maturation phase, a process dependent on PPARγ expression.

The arachidonate cyclooxygenase (COX) pathway is responsible for the biosynthesis of prostaglandin D2 (PGD2), which readily undergoes non-enzymatic dehydration to form prostaglandins of the J series. Using cultured 3T3-L1 cells, previous studies have demonstrated the enhanced expression of lipocalin-type PGD synthase (L-PGDS) during the maturation phase of adipocytes.

Furthermore, we have previously reported that cultured adipocytes have the capacity to increasingly synthesize endogenous PGJ2 derivatives, including 15d-PGJ2 and Δ12-PGJ2, known activators of PPARγ. Additionally, exogenous PGD2 and PGJ2 derivatives effectively restore lipid accumulation that has been suppressed by COX inhibitors during the adipocyte maturation phase.

However, the biological activity of PGD2 is complicated by its instability in biological fluids and its ability to act through various nuclear and cell-surface receptors, such as PPARγ, DP1, and CRTH2.

Previously, we successfully generated a monoclonal antibody specific for PGD2 using 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), a chemically stable, isosteric analog of PGD2, as a hapten mimic to develop a highly sensitive and specific immunological assay for PGD2.

Therefore, we hypothesized that 11d-11m-PGD2 could serve as a useful analog to mimic the biological action of PGD2 in certain biological systems. However, the biological activity of this stable PGD2 analog remained to be fully evaluated. An earlier study indicated that 11d-11m-PGD2 did not exhibit significant DP1 agonist activity on human platelets.

In this study, we aimed to evaluate the pro-adipogenic effects of 11d-11m-PGD2 during the maturation phase of cultured adipocytes and compare them with those of natural PGD2 and related PGJ2 derivatives. We paid particular attention to the potential involvement of nuclear and cell-surface membrane receptors in mediating these effects.

Materials and methods

Materials

Dulbecco’s modified Eagle medium with 25 mM HEPES (DMEM-HEPES), penicillin G potassium salt, streptomycin sulfate, dexamethasone, fatty acid-free bovine serum albumin, recombinant human insulin, and Oil Red O were obtained from Sigma (St. Louis, MO, USA). L-Ascorbic acid phosphate magnesium salt n-hydrate, IBMX, and Triglyceride E-Test Kit were supplied by Wako (Osaka, Japan). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Solon, OH, USA).

PGD2, 11d-11m-PGD2, PGE2, PGF2α, 15d-PGJ2, Δ12-PGJ2, indomethacin, aspirin, troglitazone, GW9662, BW245C, 15R-15-methyl-PGD2 (15R-15m-PGD2), BWA868C, and CAY10471 were products of Cayman Chemical (Ann Arbor, MI, USA). M-MLV reverse transcriptase (RT) (Ribonuclease H minus, point mutant) and polymerase chain reaction (PCR) MasterMix were obtained from Promega (Madison, WI, USA). Oligonucleotides used for PCR amplification were provided by Sigma Genosys Japan (Ishikari, Japan). Petri dishes of Iwaki brand for tissue culture were supplied by Asahi Glass (Tokyo, Japan). All other chemicals used were of reagent or tissue culture grade.

Cell culture of 3T3-L1 cells and storage of fats during the maturation phase

The mouse 3T3-L1 preadipogenic cell line (JCRB9014) was obtained from JCRB Cell Bank (Osaka, Japan). The cells were seeded at a density of 5 × 10^4 cells/mL in growth medium (GM), which consisted of DMEM-HEPES, 10% fetal bovine serum (FBS), 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, and 200 μM ascorbic acid. Cells were grown to confluence at 37°C in a 7% CO2 atmosphere.

Under standard culture conditions, the confluent monolayer cells were exposed to differentiation medium (DM). This DM consisted of GM supplemented with 1 μM dexamethasone, 0.5 mM IBMX, and 10 μg/mL insulin for 45 hours to induce the differentiation phase, as previously described. To promote lipid storage during the maturation phase, the treated cells were further cultured in maturation medium (MM). MM consisted of GM supplemented with 5 μg/mL insulin and was refreshed every two days for a total culture period of 12 days.

To examine the effects of various compounds, including exogenous prostaglandins (PGs), inhibitors, agonists, and antagonists, on lipid storage during the maturation phase, cells that had completed the differentiation phase were cultured in MM supplemented with the test compounds. The MM was refreshed every two days with fresh medium containing the same compounds, and the culture was continued until the indicated time points. The test compounds were dissolved in ethanol as a vehicle, and added to MM such that the final ethanol concentration was 0.2%.

Microscopic and macroscopic observation of cultured adipocytes

The morphology and lipid accumulation of cultured adipocytes were meticulously examined using a combination of microscopy techniques. Phase-contrast microscopy, performed with a Nikon Eclipse TE300 inverted microscope system, was used to observe the overall cellular status. Images were captured using a Nikon D-5200 digital camera attached to the microscope.

To visualize intracellular lipid droplets, cells were stained with Oil Red O. This allowed for observation via differential-interference microscopy, providing detailed images of lipid accumulation within individual cells. Additionally, macroscopic views of the cultured cells in 35-mm Petri dishes were recorded, offering a broader perspective of adipocyte differentiation.

To quantitatively assess adipogenic efficiency, phase-contrast micrographs were analyzed using ImageJ 1.52a. This software was used to determine the number of oil droplets per cell, the proportion of the cell occupied by oil droplets, and the relative cell size. These quantitative parameters provided a robust measure of adipocyte differentiation and lipid accumulation.

Results

Stimulation of fat storage by PGD2 and 11d-11m-PGD2, a chemically stable, isosteric analogue of PGD2, during the maturation phase of cultured adipocytes

To assess the impact of PGD2 and its stable analog, 11d-11m-PGD2, on adipogenesis, 3T3-L1 cells were treated with increasing concentrations of each compound during the maturation phase, in the presence of 1 μM indomethacin, a COX inhibitor.

Both compounds effectively stimulated triacylglycerol accumulation in a dose-dependent manner. Notably, 11d-11m-PGD2, at concentrations from 10 nM to 1 μM, exhibited significantly greater potency than PGD2. Microscopic observation revealed an increased number of intracellular oil droplets with increasing concentrations of both compounds, with 11d-11m-PGD2 demonstrating a higher capacity for lipid deposition.

The efficacy of PGD2 and 11d-11m-PGD2 was then compared to other prostanoids in rescuing lipid storage during the maturation phase. In the presence of indomethacin, lipid accumulation was significantly suppressed, likely due to reduced biosynthesis of endogenous pro-adipogenic prostanoids, such as PGJ2 derivatives and PGI2.

The indomethacin-induced attenuation of lipid storage was significantly reversed by treatment with exogenous PGD2, 11d-11m-PGD2, 15d-PGJ2, and Δ12-PGJ2, all known pro-adipogenic factors. In contrast, PGE2 and PGF2α, anti-adipogenic prostanoids, did not enhance lipid storage.

Microscopic observation of adipocytes treated with PGD2 or 11d-11m-PGD2, alongside indomethacin, confirmed increases in oil droplet number, oil droplet area, and cell size, compared to indomethacin alone. These findings were evident in both phase-contrast and Oil Red O stained images. The rescuing effects of PGD2 and 11d-11m-PGD2 were also confirmed in the presence of aspirin, another COX inhibitor.

Action of PGD2 and 11d-11m-PGD2 on fat storage suppressed by a specific PPARγ antagonist

In this study, we investigated the role of activated PPARγ in the pro-adipogenic effects of PGD2 and 11d-11m-PGD2 during the adipocyte maturation phase. Treatment of cultured adipocytes with GW9662, a potent PPARγ antagonist, at 0.1 and 1 μM significantly suppressed lipid storage.

The inhibitory effect of 0.1 μM GW9662 was effectively reversed to the level of vehicle-treated cells by co-incubation with PPARγ activators, including troglitazone, 15d-PGJ2, and Δ12-PGJ2, at 1 μM. Notably, both PGD2 and 11d-11m-PGD2 also exhibited potent rescuing effects.

However, the PPARγ activators at 1 μM failed to fully reverse the lipid accumulation suppression caused by 1 μM GW9662. Despite this, PGD2 and 11d-11m-PGD2 were more effective in rescuing lipid storage suppressed by 1 μM GW9662 than the PPARγ activators.

These results suggest that receptors other than PPARγ contribute significantly to the pro-adipogenic effects of PGD2 and 11d-11m-PGD2. Specifically, under our culture conditions, the contribution of activated PPARγ to 11d-11m-PGD2-mediated adipogenesis appears to be minimal.

Discussion

Previous research has indicated that cultured adipocytes can synthesize endogenous PGD2 and related PGJ2 derivatives, which function as pro-adipogenic factors during the adipocyte maturation phase. PGJ2 derivatives, including 15d-PGJ2 and Δ12-PGJ2, are well-established activators of the nuclear hormone receptor PPARγ, a key regulator of adipogenesis.

Given that PGD2 undergoes non-enzymatic dehydration to form J2 series prostaglandins in biological fluids, it might also stimulate adipogenesis through PPARγ. However, the chemical instability of PGD2 complicates its analysis and understanding of its in vivo actions.

Therefore, this study aimed to investigate the biological activity of 11d-11m-PGD2, a chemically stable analog of PGD2, despite its biological activity being previously unclear. Interestingly, 11d-11m-PGD2, at concentrations ranging from 10 nM to 1 μM, was found to be more potent than natural PGD2 in stimulating adipogenesis in the presence of indomethacin or aspirin during the adipocyte maturation phase. The stimulatory effects of 1 μM PGD2 and 11d-11m-PGD2 were comparable to those of PGJ2 derivatives. Similar results were observed in the absence of COX inhibitors.

These findings raised questions about the cellular mechanisms by which this isosteric analog exerts its pro-adipogenic effect. A plausible explanation was that 11d-11m-PGD2 acts through PPARγ, similar to PGD2 and PGJ2 derivatives. However, the chemical structure of 11d-11m-PGD2, with an exocyclic methylene replacing the 11-keto group, makes it unlikely to activate PPARγ.

This study, using varying concentrations of GW9662, a selective PPARγ antagonist, provided evidence that the pro-adipogenic effect of 11d-11m-PGD2 was largely independent of PPARγ activation. Conversely, the stimulatory effect of 1 μM PGD2 on adipogenesis was partially mediated by PPARγ. However, other mechanisms are likely involved in PGD2-mediated adipogenesis beyond PPARγ activation.

These results highlight a clear difference in the pro-adipogenic mechanisms of PGD2 and 11d-11m-PGD2.

PGD2 is known to mediate its inflammatory effects through specific interactions with two cell-surface membrane receptors: the G protein-coupled receptors DP1 and CRTH2 (also known as DP2). However, the involvement of DP1 and CRTH2 in the pro-adipogenic actions of PGD2 and its stable analog, 11d-11m-PGD2, is poorly understood.

Our current study revealed the gene expression of both DP1 and CRTH2 in cultured 3T3-L1 adipocytes. Their transcript levels peaked around day 6 of the maturation phase, mirroring the expression pattern of the nuclear hormone receptor PPARγ. In contrast, the expression of L-PGDS, responsible for PGD2 biosynthesis, gradually increased up to day 10. These findings suggest that the DP receptors may contribute to the upregulation of adipogenesis during the terminal differentiation phase.

We then investigated the effects of BW245C, a specific DP1 agonist, and 15R-15m-PGD2, a selective CRTH2 agonist, on the gene expression of adipocyte-specific markers during the maturation phase. Both compounds effectively promoted the gene expression of adipogenesis markers, such as PPARγ, GLUT4, LPL, and adiponectin, which were attenuated by indomethacin treatment. This suggests that DP1 and CRTH2 receptors significantly contribute to the pro-adipogenic effects of PGD2 and 11d-11m-PGD2 by promoting the adipogenesis program during adipocyte maturation. Consistent with the gene expression data, both DP1 and CRTH2 agonists stimulated triacylglycerol accumulation, which was suppressed by indomethacin.

In this study, we compared the cellular mechanisms of the pro-adipogenic effects of PGD2 and 11d-11m-PGD2 during adipocyte maturation. The following evidence suggests that PGD2 primarily exerts its pro-adipogenic effect through DP1 and PPARγ, while 11d-11m-PGD2 preferentially stimulates adipogenesis through CRTH2. First, the promoting effect of PGD2, in the presence of indomethacin, was significantly attenuated by the DP1 antagonist BWA868C.

Furthermore, the pro-adipogenic effect of PGD2 was partially sensitive to the PPARγ antagonist GW9662. However, the stimulatory effect of PGD2 was not significantly sensitive to the selective CRTH2 antagonist CAY10471 at either 0.1 or 1 μM in our culture system. It is important to acknowledge that other metabolites derived from PGD2 may also play a role, as various prostanoids of the D2 and J2 series have been reported to interact with CRTH2 in other systems.

Conversely, the pro-adipogenic effect of 11d-11m-PGD2, in the presence of indomethacin, was much more effectively suppressed by CAY10471 than by BWA868C. This highlights the predominant role of CRTH2 in 11d-11m-PGD2-mediated adipogenesis.

Additionally, GW9662 failed to block the stimulatory effect of 11d-11m-PGD2 on lipid storage. These findings reveal a clear distinction between natural PGD2 and its steric analog 11d-11m-PGD2 in their pro-adipogenic mechanisms. Our study provides evidence for the efficacy and potency of 11d-11m-PGD2 in stimulating adipogenesis in cultured adipocytes.

To our knowledge, the actions of 11d-11m-PGD2 through the DP1 and CRTH2 receptors have not been previously described in other biological systems. However, we have previously demonstrated the utility of 11d-11m-PGD2 as a hapten mimic for generating a highly sensitive and specific monoclonal antibody for PGD2, which was used to develop an immunological assay.

Our study showed that PGD2 can stimulate adipogenesis, at least in part, through the DP1 receptor. The DP1 receptor is coupled to Gs proteins and increases intracellular cAMP levels in immune cells. These elevated cAMP levels could contribute to the gene expression of adipogenic factors in cultured adipocytes, potentially through a transcriptional cascade initiated by C/EBPβ. Recently, we reported that cAMP analogs and forskolin, at specific concentrations, can rescue lipid storage suppressed by aspirin during the maturation phase of 3T3-L1 adipocytes.

The rescuing effects of those compounds are partial, and higher concentrations tend to attenuate the positive effects on adipogenesis. It is possible that other signaling pathways specific for the DP1 receptor by PGD2 could act in this system. On the other hand, the CRTH2 receptor is known to decrease intracellular cAMP levels through Gi proteins and to enhance Ca2+ in immune cells, which mediate proinflammatory effects.

Previous studies have implicated phospholipase Cβ and various kinases, including phosphatidylinositol 3 kinase and Akt, in the signal propagation of CRTH2. Therefore, these signaling pathways might be involved in the pro adipogenic action of 11d 11m PGD2 through CRTH2 in our system. More recently, a study has described that PGD2 and the CRTH2 agonist 15R 15m PGD2 stimulates the expression level of adipogenic and lipogenic enzymes in adipocytes.

This report also suggests that PGD2 enhances lipid accumulation through the suppression of lipolysis through CRTH2. Thus, the role of CRTH2 in adipogenesis and lipolysis has become an interesting subject. However, the action of PGD2 is complex and different results can be obtained depending on the experimental culture conditions and cell lines of adipocytes. More detailed studies are required to unravel the precise understanding of the cellular mechanism for the pro adipogenic action of PGD2 and the related derivatives through the DP receptors.

Adipose tissue is a major metabolic organ controlling systemic energy homeostasis in vivo through the storage of fats by lipogenesis and the mobilization of fatty acids by lipolysis. Our results suggest the potential usefulness of 11d 11m PGD2 and 15R 15m PGD2 as chemically stable synthetic analogues to stimulate adipogenesis of white pre adipocytes through the preferential activation of the PGD2 receptor CRTH2.

The efficacy of these compounds remains unknown since the metabolic stability of them has not been studied extensively. As the activation of CRTH2 is known to promote allergic responses in immune cells, the use of the synthetic analogues of PGD2 to activate CRTH2 might raise the possibility of side effects in vivo. Further studies are required for the clarification of their control of adipogenesis in the body.

In conclusion, our study was undertaken to study the pro adipogenic effects of PGD2 and its isosteric analogue, 11d 11m PGD2, in the presence of indomethacin during the maturation phase of adipocytes. The dose dependent study revealed that 11d 11m PGD2 was more potent than PGD2 in stimulating the storage of fats.

Their promoting effects of both compounds were due to upregulation of adipogenesis. On the basis of the results with selective agonists and antagonists, we found that the action of 11d 11m PGD2 was mediated mainly through the CRTH2 receptor while PGD2 exerted its effect through the DP1 and PPARγ receptors.

Our findings suggest a useful application of 11d 11m PGD2 to the adipogenesis study through CRTH2. GW9662