Structures of human prostaglandin F2α receptor reveal the mechanism of ligand and G protein selectivity.

Prostaglandins and their receptors regulate various physiological processes. Carboprost, an analog of prostaglandin F2α and an agonist for the prostaglandin F2-alpha receptor (FP receptor), is clinically used to treat postpartum hemorrhage (PPH). However, off-target activation of closely related receptors such as the prostaglandin E receptor subtype EP3 (EP3 receptor) by carboprost results in side effects and limits the clinical application. Meanwhile, the FP receptor selective agonist latanoprost is not suitable to treat PPH due to its poor solubility and fast clearance. Here, we present two cryo-EM structures of the FP receptor bound to carboprost and latanoprost-FA (the free acid form of latanoprost) at 2.7 Å and 3.2 Å resolution, respectively. The structures reveal the molecular mechanism of FP receptor selectivity for both endogenous prostaglandins and clinical drugs, as well as the molecular mechanism of G protein coupling preference by the prostaglandin receptors. The structural information may guide the development of better prostaglandin drugs.

The Roles Played by FP/EP3 Receptors During Pressure-lowering in Mouse Eyes Mediated by a Dual FP/EP3 Receptor Agonist.

Purpose: We investigated the intraocular pressure (IOP)-lowering effect of topical sepetaprost (SPT), a dual agonist of the FP and EP3 receptors. We explored whether certain receptors mediated the hypotensive effect of SPT and outflow facility changes in C57BL/6 mice (wild-type [WT]) and FP and EP3 receptor-deficient mice (FPKO and EP3KO mice, respectively). Methods: IOP was measured using a microneedle. Outflow facility was measured using a two-level, constant-pressure perfusion method. Results: SPT significantly reduced IOP for 8 hours after administration to WT mice. The 2-hour IOP reductions afforded by latanoprost were 15.3 ± 2.5, 1.8 ± 2.0, and 12.3 ± 2.4% in WT, FPKO, and EP3KO mice, respectively; the SPT figures were 13.6 ± 2.1, 5.9 ± 2.7, and 6.6 ± 2.6%, respectively. Latanoprost-mediated IOP reduction was significantly decreased in FPKO mice, and SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. At 6 hours after administration, latanoprost did not significantly reduce the IOP in any tested mouse strain. SPT-mediated IOP reduction was reduced in both FPKO and EP3KO mice. IOP reduction at 6 hours was significantly higher after simultaneous administration of selective FP and EP3 receptor agonists, but IOP did not fall on administration of (only) a selective EP3 receptor agonist. SPT significantly increased outflow facility in WT mice, but less so in FPKO and EP3KO mice. Conclusions: The IOP-lowering effect of SPT lasted longer than that of latanoprost. Our data imply that this may be attributable to augmented outflow facility mediated by the FP and EP3 receptors.

Sexual dimorphism in prostacyclin-mimetic responses within rat mesenteric arteries: A novel role for KV 7.1 in shaping IP receptor-mediated relaxation.

BACKGROUND AND PURPOSE: Prostacyclin mimetics express potent vasoactive effects via prostanoid receptors that are not unequivocally defined, as to date no study has considered sex as a factor. The aim of this study was to determine the contribution of IP and EP3 prostanoid receptors to prostacyclin mimetic iloprost-mediated responses, whether KV 7.1-5 channels represent downstream targets of selective prostacyclin-IP-receptor agonist MRE-269 and the impact of the oestrus cycle on vascular reactivity. EXPERIMENTAL APPROACH: Within second-order mesenteric arteries from male and female Wistar rats, we determined (1) relative mRNA transcripts for EP1-4 (Ptger1-4 ), IP (Ptgi) and TXA2 (Tbxa) prostanoid receptors via RT-qPCR; (2) the effect of iloprost, MRE-269, isoprenaline and ML277 on precontracted arterial tone in the presence of inhibitors of prostanoid receptors, potassium channels and the molecular interference of KV 7.1 via wire-myograph; (3) oestrus cycle stage via histological changes in cervical cell preparations. KEY RESULTS: Iloprost evoked a biphasic response in male mesenteric arteries, at concentrations ≤100 nmol·L-1 relaxing, then contracting the vessel at concentration ≥300 nmol·L-1 , a process attributed to IP and EP3 receptors respectively. Secondary contraction was absent in the females, which was associated with a reduction in Ptger3. Pharmacological inhibition and molecular interference of KV 7.1 significantly attenuated relaxations produced by the selective IP receptor agonist MRE-269 in male and female Wistar in dioestrus/metoestrus, but not pro-oestrus/oestrus. CONCLUSIONS AND IMPLICATIONS: Stark sexual dimorphisms in iloprost-mediated vasoactive responses are present within mesenteric arteries. KV 7.1 is implicated in IP receptor-mediated vasorelaxation and is impaired by the oestrus cycle.

Prostaglandin D2 metabolites activate asthmatic patient-derived type 2 innate lymphoid cells and eosinophils via the DP2 receptor.

BACKGROUND: Prostaglandin D2 (PGD2) signaling via prostaglandin D2 receptor 2 (DP2) contributes to atopic and non-atopic asthma. Inhibiting DP2 has shown therapeutic benefit in certain subsets of asthma patients, improving eosinophilic airway inflammation. PGD2 metabolites prolong the inflammatory response in asthmatic patients via DP2 signaling. The role of PGD2 metabolites on eosinophil and ILC2 activity is not fully understood. METHODS: Eosinophils and ILC2s were isolated from peripheral blood of atopic asthmatic patients. Eosinophil shape change, ILC2 migration and IL-5/IL-13 cytokine secretion were measured after stimulation with seven PGD2 metabolites in presence or absence of the selective DP2 antagonist fevipiprant. RESULTS: Selected metabolites induced eosinophil shape change with similar nanomolar potencies except for 9α,11ß-PGF2. Maximal values in forward scatter of eosinophils were comparable between metabolites. ILC2s migrated dose-dependently in the presence of selected metabolites except for 9α,11ß-PGF2 with EC50 values ranging from 17.4 to 91.7 nM. Compared to PGD2, the absolute cell migration was enhanced in the presence of Δ12-PGD2, 15-deoxy-Δ12,14-PGD2, PGJ2, Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2. ILC2 cytokine production was dose dependent as well but with an average sixfold reduced potency compared to cell migration (IL-5 range 108.1 to 526.9 nM, IL-13 range: 125.2 to 788.3 nM). Compared to PGD2, the absolute cytokine secretion was reduced in the presence of most metabolites. Fevipiprant dose-dependently inhibited eosinophil shape change, ILC2 migration and ILC2 cytokine secretion with (sub)-nanomolar potencies. CONCLUSION: Prostaglandin D2 metabolites initiate ILC2 migration and IL-5 and IL-13 cytokine secretion in a DP2 dependent manner. Our data indicate that metabolites may be important for in vivo eosinophil activation and ILC2 migration and to a lesser extent for ILC2 cytokine secretion.

Addition of EP2 agonists to an FP agonist additively and synergistically modulates adipogenesis and the physical properties of 3D 3T3-L1 sphenoids.

The additive effects of prostaglandin (PG)-EP2 agonists on a PG-FP agonist toward adipogenesis in two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells was examined by lipid staining, the mRNA expression of adipogenesis related genes, and extracellular matrixes (ECMs) including collagen molecules (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D sphenoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced 1) an enlargement in the sizes of 3D sphenoids, 2) a substantial enhancement in lipid staining, the expression of the PParγ, Ap2 and Leptin genes, and 3) a significant decrease in the stiffness of the 3D sphenoids. These effects were inhibited by bimatoprost acid (BIM-A), but 4) adipogenesis induced significant down-regulation of Col1 and Fn, and the significant up-regulation of the Col4 and Col6 genes were unchanged by BIM-A. On the addition of an EP2 agonist, such as omidenepag (OMD) or butaprost (Buta), to BIM-A, 1) the sizes of the 3D sphenoids were further decreased, 2) lipid staining was decreased (2D; OMD, 3D; Buta) 3) the stiffness of the 3D sphenoids was increased by Buta, 4) the expression of PParγ was up-regulated (2D; Buta) or unchanged (3D), the expression of Ap2 was down-regulated (2D; OMD) or up-regulated (3D; Buta), and the expression of Leptin was increased (2D), 5) the expression of all four (OMD) or all except Col4 (buta) in 2D, and Col1and Col4 (OMD) in 3D were up-regulated. These collective findings indicate that the addition of an EP2 agonist, OMD or Buta significantly modulated the BIM-A induced suppression of adipogenesis as well as physical properties of 2D and 3D cultured 3T3-L1 cells in different manners.

DP1 prostanoid receptor activation increases the severity of an acute lower respiratory viral infection in mice via TNF-α-induced immunopathology.

Respiratory syncytial virus (RSV) bronchiolitis is a leading cause of infant hospitalization and mortality. We previously identified that prostaglandin D2 (PGD2), released following RSV infection of primary human airway epithelial cells or pneumonia virus of mice (PVM) infection of neonatal mice, elicits pro- or antiviral innate immune responses as a consequence of D-type prostanoid receptor 2 (DP2) or DP1 activation, respectively. Here, we sought to determine whether treatment with the DP1 agonist BW245c decreases the severity of bronchiolitis in PVM-infected neonatal mice. Consistent with previous findings, BW245c treatment increased IFN-λ production and decreased viral load in week 1 of the infection. However, unexpectedly, BW245c treatment increased mortality in week 2 of the infection. This increased morbidity was associated with viral spread to the parenchyma, an increased cellular infiltrate of TNF-α-producing cells (neutrophils, monocytes, and CD4+ T cells), and the heightened production of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß. These phenotypes, as well as the increased mortality, were significantly attenuated following the administration of anti-TNF-α to PVM-infected, BW245c-treated mice. In summary, pharmacological activation of the DP1 receptor in PVM-infected neonatal mice boosts antiviral innate and adaptive immunity, however, this is ultimately detrimental, as a consequence of increased TNF-α-induced morbidity and mortality.

Patient and disease characteristics of the first 500 patients with pulmonary arterial hypertension treated with selexipag in real-world settings from SPHERE.

BACKGROUND: Selexipag is a selective oral prostacyclin receptor agonist indicated for pulmonary arterial hypertension (PAH) treatment. SelexiPag: tHe usErs dRug rEgistry (SPHERE) (NCT03278002) is collecting data from selexipag-treated patients in real-world clinical practice to elucidate and describe the clinical characteristics, outcomes, and dosing/titration regimens of patients treated with selexipag in routine clinical practice. METHODS: SPHERE is a United States (US)-based, ongoing, multicenter, prospective observational study (target N = 800). This study enrolls patients who are either newly initiated on selexipag (≤60 days before enrollment) or were previously receiving selexipag with documentation of dose titration at study enrollment. Data collection for the study occurs at routine clinic visits. In this paper, we report on the first 500 patients enrolled. RESULTS: Median follow-up was 17.8 months; 77.6% of patients completed the planned 18 months follow-up, and 22.4% discontinued early from the study. At diagnosis, 94.8% of patients had PAH (World Health Organization [WHO] Group 1), most commonly idiopathic (49.2%) and connective tissue disease associated (26.4%). Most patients (72.4%) initiated selexipag more than 60 days before enrollment. At initiation, 31.0% of patients had WHO functional class (FC) II disease, and 49.6% had WHO FC II or III disease. In addition, 55.0% of patients were receiving double therapy (most commonly an endothelin receptor antagonist plus phosphodiesterase type 5 inhibitor [42.3%]), whereas 30.6% were receiving monotherapy. Despite most patients already receiving PAH-specific therapy, at selexipag initiation, 67.2% (336 of 500) were at intermediate risk, and 9.6% (48 of 500) were at high risk of 1-year mortality. Risk scores remained stable in ∼55% of patients and improved in ∼20% at the end of the study. In total, 72.2% of patients had at least 1 adverse event (AE), and 37.6% reported a serious AE. The median selexipag maintenance dose was 1,200 µg twice daily (interquartile range: 800-1,600 µg twice daily). CONCLUSIONS: Real-world, US-based patients with PAH initiating selexipag typically have WHO FC II/III disease and are at intermediate risk, despite receiving PAH-specific treatment. Selexipag was prescribed as part of a combination regimen in most patients. The study identified no unexpected adverse effects.

Contribution of FP receptors in M1 macrophage polarization via IL-10-regulated nuclear translocation of NF-κB p65.

Macrophages are the effector immune cells with plasticity to differentiate as M1 (classically activated) and M2 (alternatively activated) phenotypes. Prostaglandins (PGs) have various important roles and are involved in the regulation of macrophage activation. However, the role of PGF2α in macrophage activation remains unclear. We investigated the role of PGF2α receptor (FP)-mediated signaling in the M1 macrophage polarization using murine macrophage RAW264.7 cells. Stimulation with lipopolysaccharide (LPS) + interferon (IFN)-γ increased the mRNA expression of the M1 macrophage markers such as inducible nitric oxide synthase, tumor necrosis factor-α, and CD11c. Pre-treatment with AL8810, an FP receptor antagonist, further enhanced the expression of these genes. In contrast, treatment with fluprostenol, an FP receptor agonist, decreased the LPS + IFN-γ-induced expression of M1 markers. LPS-induced M1 macrophage polarization was dependent on the activation of NF-κB p65. Treatment with IκB kinase ß inhibitor reduced AL8810-induced mRNA expression of the M1 markers. Stimulation with LPS + IFN-γ increased the expression of IL-10. Pre-treatment with AL8810 lowered LPS + IFN-γ-induced IL-10 expression, and further enhanced LPS + IFN-γ-stimulated nuclear translocation of NF-κB p65. In contrast, co-treatment with IL-10 reversed AL8810-induced nuclear translocation of NF-κB p65. These results indicate that the FP receptor signaling was involved in the control of M1 polarization of macrophages via IL-10-regulated nuclear translocation of NF-κB p65.

DP1 Activation Reverses Age-Related Hypertension Via NEDD4L-Mediated T-Bet Degradation in T Cells.

BACKGROUND: Blood pressure often rises with aging, but exact mechanisms are still not completely understood. With aging, the level of proinflammatory cytokines increases in T lymphocytes. Prostaglandin D2, a proresolution mediator, suppresses Type 1 T helper (Th1) cytokines through D-prostanoid receptor 1 (DP1). In this study, we aimed to investigate the role of the prostaglandin D2/DP1 axis in T cells on age-related hypertension. METHODS: To clarify the physiological and pathophysiological roles of DP1 in T cells with aging, peripheral blood samples were collected from young and older male participants, and CD4+ T cells were sorted for gene expression, prostaglandin production, and Western blot assays. Mice blood pressure was quantified by invasive telemetric monitor. RESULTS: The prostaglandin D2/DP1 axis was downregulated in CD4+ T cells from older humans and aged mice. DP1 deletion in CD4+ T cells augmented age-related hypertension in aged male mice by enhancing Th1 cytokine secretion, vascular remodeling, CD4+ T cells infiltration, and superoxide production in vasculature and kidneys. Conversely, forced expression of exogenous DP1 in T cells retarded age-associated hypertension in mice by reducing Th1 cytokine secretion. Tumor necrosis factor α neutralization or interferon γ deletion ameliorated the age-related hypertension in DP1 deletion in CD4+ T cells mice. Mechanistically, DP1 inhibited Th1 activity via the PKA (protein kinase A)/p-Sp1 (phosphorylated specificity protein 1)/neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) pathway-mediated T-box-expressed-in-T-cells (T-bet) ubiquitination. T-bet deletion or forced NEDD4L expression in CD4+ T cells attenuated age-related hypertension in CD4+ T cell-specific DP1-deficient mice. DP1 receptor activation by BW245C prevented age-associated blood pressure elevation and reduced vascular/renal superoxide production in male mice. CONCLUSIONS: The prostaglandin D2/DP1 axis suppresses age-related Th1 activation and subsequent hypertensive response in male mice through increase of NEDD4L-mediated T-bet degradation by ubiquitination. Therefore, the T cell DP1 receptor may be an attractive therapeutic target for age-related hypertension.

Prostaglandin receptor agonists as antiglaucoma agents (a patent review 2013 - 2018).

Introduction: Elevated intraocular pressure (IOP) is the most prevalent risk factor for glaucoma. Prostaglandin analogs are a first-line therapy for glaucoma due to their ability to reduce IOP, once-daily dosing, efficacy, and minimal side-effect profile. Many compounds targeting different PG receptors have been developed in the last years, some of them being in clinical use. Latanoprost, Bimatoprost, Travoprost, and Tafluprost are clinically used as antiglaucoma drugs and act as agonists of the PGF2α receptor. The inability to fully understand the mechanism of action of clinically used PGF2α analogs is thus a strong driver for additional research into the mechanism of action of ocular hypotensive drugs belonging to this class of pharmacological agents. Areas covered: This review explores the last 5 years (2013-2018), where many patents describing new compounds acting on different prostaglandin receptors, and mainly targeting EP1-4 and FP receptors, were released. Expert opinion: To date, there has been a growing awareness over recent years of the therapeutic use of novel derivatives as new antiglaucoma pharmaceutical products. Patents involved in discovering new approaches and new molecules for the treatment of glaucoma diseases encouraged the scientific community to increase the variety of drugs available for the treatment of ocular diseases.

Prostaglandin FP receptor antagonists: discovery, pharmacological characterization and therapeutic utility.

In contrast to the availability of potent and selective antagonists of several prostaglandin receptor types (including DP1 , DP2 , EP and TP receptors), there has been a paucity of well-characterized, selective FP receptor antagonists. The earliest ones included dimethyl amide and dimethyl amine derivatives of PGF2α , but these have failed to gain prominence. The fluorinated PGF2α analogues, AL-8810 and AL-3138, were subsequently discovered as competitive and non-competitive FP receptor antagonists respectively. Non-prostanoid structures, such as the thiazolidinone AS604872, the D-amino acid-based oligopeptide PDC31 and its peptidomimic analogue PDC113.824 came next, but the latter two are allosteric inhibitors of FP receptor signalling. AL-8810 has a sub-micromolar in vitro potency and ≥2 log unit selectivity against most other PG receptors when tested in several cell- and tissue-based functional assays. Additionally, AL-8810 has demonstrated therapeutic efficacy as an FP receptor antagonist in animal models of stroke, traumatic brain injury, multiple sclerosis, allodynia and endometriosis. Consequently, it appears that AL-8810 has become the FP receptor antagonist of choice. LINKED ARTICLES: This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc.

Prostaglandin analogues and nitric oxide contribution in the treatment of ocular hypertension and glaucoma.

In patients with ocular hypertension or glaucoma, all treatments aim to lower intraocular pressure (IOP) by modulating aqueous humour (AH) production and/or uveoscleral and trabecular meshwork/Schlemm's canal AH drainage. PG analogues are considered to be the 'gold standard' treatment and are the most frequently used IOP-lowering agents. Recent data support an important role for NO in regulating IOP. Thus, novel PG analogues carrying a NO-donating moiety were recently advanced. Latanoprostene bunod (LBN) and NCX 470, NO-donating derivatives of latanoprost and bimatoprost, respectively, are examples of such compounds. LBN ophthalmic solution, 0.024% (Vyzulta™), showed greater IOP-lowering efficacy compared with that of Xalatan® (latanoprost ophthalmic solution, 0.005%) or 0.5% timolol maleate in clinical settings. NCX 470 was found to be more effective than bimatoprost in animal models of ocular hypertension and glaucoma. Selective EP2 receptor agonists (i.e. taprenepag isopropyl, omidenepag isopropyl and aganepag isopropyl) and non-selective prostanoid receptor agonists (i.e. ONO-9054, sepetaprost isopropyl) that concomitantly stimulate FP and EP3 receptors have also been shown to hold promise as effective IOP-lowering agents. LINKED ARTICLES: This article is part of a themed section on Eicosanoids 35 years from the 1982 Nobel: where are we now? To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.8/issuetoc.

PTGFR activation promotes the expression of PTGS-2 and growth factors via activation of the PKC signaling pathway in bovine endometrial epithelial cells.

The endometrium of domestic animals has a remarkable capacity to self-repair. Prostaglandin F2α (PGF2α) is one of the major prostaglandins secreted from the endometrium. The role of PGF2α in endometrial repair, however, is still unknown. In the present study, it was investigated whether prostaglandin F2α receptor (PTGFR) activation could induce expression of prostaglandin-endoperoxide synthase 2 (PTGS-2) and growth factors associated with endometrial repair via activation of protein kinase C (PKC) signaling in endometrial epithelial cells (bEECs) of cattle. Results of the present study indicated that the treatment with the PTGFR agonist, fluprostenol, resulted in an increase in abundance of proteins for PTGS-2, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGF-ß1), and interleukin-8 (IL-8). The increased abundances of these proteins were suppressed by the treatment with the PTGFR antagonist, AL8810.Furthermore, fluprostenol treatment also induced PKC phosphorylation. Subsequently, treatment with AL8810 inhibited the fluprostenol-induced PKC phosphorylation. Additionally, treatment with the PKC inhibitor, chelerythrine, reduced the fluprostenol-induced increase in the relative abundance of VEGF, CTGF, TGF-ß1, and IL-8 mRNA in bEECs. Taken together, these results suggest that PTGFR activation may induce endometrial repair by upregulating PTGS-2 gene expression and stimulating VEGF, CTGF, TGF-ß1, and IL-8 gene expression via activation of the PKC signaling pathway.

Activation of Th2 cells downregulates CRTh2 through an NFAT1 mediated mechanism.

CRTh2 (encoded by PTGDR2) is a G-protein coupled receptor expressed by Th2 cells as well as eosinophils, basophils and innate lymphoid cells (ILC)2s. Activation of CRTh2, by its ligand prostaglandin (PG)D2, mediates production of type 2 cytokines (IL-4, IL-5 and IL-13), chemotaxis and inhibition of apoptosis. As such, the PGD2-CRTh2 pathway is considered important to the development and maintenance of allergic inflammation. Expression of CRTh2 is mediated by the transcription factor GATA3 during Th2 cell differentiation and within ILC2s. Other than this, relatively little is known regarding the cellular and molecular mechanisms regulating expression of CRTh2. Here, we show using primary human Th2 cells that activation (24hrs) through TCR crosslinking (αCD3/αCD28) reduced expression of both mRNA and surface levels of CRTh2 assessed by flow cytometry and qRT-PCR. This effect took more than 4 hours and expression was recovered following removal of activation. EMSA analysis revealed that GATA3 and NFAT1 can bind independently to overlapping sites within a CRTh2 promoter probe. NFAT1 over-expression resulted in loss of GATA3-mediated CRTh2 promoter activity, while inhibition of NFAT using a peptide inhibitor (VIVIT) coincided with recovery of CRTh2 expression. Collectively these data indicate that expression of CRTh2 is regulated through the competitive action of GATA3 and NFAT1. Though prolonged activation led to NFAT1-mediated downregulation, CRTh2 was re-expressed when stimulus was removed suggesting this is a dynamic mechanism and may play a role in PGD2-CRTh2 mediated allergic inflammation.

Prostaglandin F2α Facilitates Hepatic Glucose Production Through CaMKIIγ/p38/FOXO1 Signaling Pathway in Fasting and Obesity.

Gluconeogenesis is drastically increased in patients with type 2 diabetes and accounts for increased fasting plasma glucose concentrations. Circulating levels of prostaglandin (PG) F2α are also markedly elevated in diabetes; however, whether and how PGF2α regulates hepatic glucose metabolism remain unknown. Here, we demonstrated that PGF2α receptor (F-prostanoid receptor [FP]) was upregulated in the livers of mice upon fasting- and diabetic stress. Hepatic deletion of the FP receptor suppressed fasting-induced hepatic gluconeogenesis, whereas FP overexpression enhanced hepatic gluconeogenesis in mice. FP activation promoted the expression of gluconeogenic enzymes (PEPCK and glucose-6-phosphatase) in hepatocytes in a FOXO1-dependent manner. Additionally, FP coupled with Gq in hepatocytes to elicit Ca2+ release, which activated Ca2+/calmodulin-activated protein kinase IIγ (CaMKIIγ) to increase FOXO1 phosphorylation and subsequently accelerate its nuclear translocation. Blockage of p38 disrupted CaMKIIγ-induced FOXO1 nuclear translocation and abrogated FP-mediated hepatic gluconeogenesis in mice. Moreover, knockdown of hepatic FP receptor improved insulin sensitivity and glucose homeostasis in ob/ob mice. FP-mediated hepatic gluconeogenesis via the CaMKIIγ/p38/FOXO1 signaling pathway, indicating that the FP receptor might be a promising therapeutic target for type 2 diabetes.

Impact of dietary ω3 polyunsaturated fatty acid supplementation on brown and brite adipocyte function.

The recent characterization of functional brown adipose tissue in adult humans has opened new perspectives for regulation of energy expenditure with respect to obesity and diabetes. Furthermore, dietary recommendations have taken into account the insufficient dietary intake of ω3 PUFAs and the concomitant excessive intake of ω6 PUFA associated with the occurrence of overweight/obesity. We aimed to study whether ω3 PUFAs could play a role in the recruitment and function of energy-dissipating brown/brite adipocytes. We show that ω3 PUFA supplementation has a beneficial effect on the thermogenic function of adipocytes. In vivo, a low dietary ω6:ω3 ratio improved the thermogenic response of brown and white adipose tissues to ß3-adrenergic stimulation. This effect was recapitulated in vitro by PUFA treatment of hMADS adipocytes. We pinpointed the ω6-derived eicosanoid prostaglandin (PG)F2α as the molecular origin because the effects were mimicked with a specific PGF2α receptor agonist. PGF2α level in hMADS adipocytes was reduced in response to ω3 PUFA supplementation. The recruitment of thermogenic adipocytes is influenced by the local quantity of individual oxylipins, which is controlled by the ω6:ω3 ratio of available lipids. In human nutrition, energy homeostasis may thus benefit from the implementation of a more balanced dietary ω6:ω3 ratio.

The prostaglandin E2 receptor PTGER2 and prostaglandin F2α receptor PTGFR mediate oviductal glycoprotein 1 expression in bovine oviductal epithelial cells.

Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2α (PGF2α) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2α (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 µM H-89 (a PKA inhibitor) and 3 µM U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 µM chelerythrine chloride (a PKC inhibitor) and 3 µM U0126 negated OVGP1 downregulation by PGF2α. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2α through the PTGFR-Ca2+-PKC pathway.

DP1 receptor agonist, BW245C inhibits diet-induced obesity in ApoE-/- mice.

BACKGROUND/OBJECTIVE: Lipocalin Prostaglandin D2 synthase (LPGDS) contributes to the production of PGD2, which has been associated with adipogenesis. In this study, we aimed to investigate the role of PGD2 on obesity through its DP1 and DP2 receptor signaling using intraperitoneal injection of their respective agonists and antagonists. METHODS: ApoE-/- mice were divided into five groups: vehicle control (n=5), DP1 receptor agonist (n=5), DP1 receptor antagonist (n=5), DP2 receptor agonist (n=5), and DP2 receptor antagonist (n=5), and the study was carried out for 10 weeks. RESULTS: Despite being on high fat diet, mice receiving DP1 receptor agonist sustained a significant inhibition of weight gain throughout the study gaining only 11.4% body weight compared to the controls gaining 61% body weight. Interestingly, parallel to the body weight, the DP1 receptor agonist group showed a significant reduction in food intake throughout the study. Consistently, fasting leptin, insulin and bile acids levels were elevated in the DP1 receptor agonist group compared to controls. As expected, there was a significant reduction in fasting glucose level in DP1 receptor agonist group. At last, as a result of weight gain inhibition, DP1 receptor agonist also imparted cardiovascular benefits showing significant reduction in aortic wall thickness, intima, adventia and lumen size. CONCLUSION: Based on the obtained results, we believe DP1 receptor agonism inhibited diet induced weight gain possibly through controlling appetite which consequently imparted beneficial cardiometabolic effects. DP1 receptor agonism may represent a novel therapeutic target for the management of obesity.

Sex differences in the role of phospholipase A2 -dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.

KEY POINTS: The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. ABSTRACT: Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.