RESUMO
Obesity is a complex disease highly related to diet and lifestyle and is associated with low amount of thermogenic adipocytes. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to fight overweight and associated comorbidities. Recent studies suggest a role for several fatty acids and their metabolites, called lipokines, in the control of thermogenesis. The purpose of this work was to analyze the role of several lipokines in the control of brown/brite adipocyte formation. We used a validated human adipocyte model, human multipotent adipose-derived stem cell model (hMADS). In the absence of rosiglitazone, hMADS cells differentiate into white adipocytes, but convert into brite adipocytes upon rosiglitazone or prostacyclin 2 (PGI2) treatment. Gene expression was quantified using RT-qPCR and protein levels were assessed by Western blotting. We show here that lipokines such as 12,13-diHOME, 12-HEPE, 15dPGJ2 and 15dPGJ3 were not able to induce browning of white hMADS adipocytes. However, both fatty acid esters of hydroxy fatty acids (FAHFAs), 9-PAHPA and 9-PAHSA potentiated brown key marker UCP1 mRNA levels. Interestingly, CTA2, the stable analog of thromboxane A2 (TXA2), but not its inactive metabolite TXB2, inhibited the rosiglitazone and PGI2-induced browning of hMADS adipocytes. These results pinpoint TXA2 as a lipokine inhibiting brown adipocyte formation that is antagonized by PGI2. Our data open new horizons in the development of potential therapies based on the control of thromboxane A2/prostacyclin balance to combat obesity and associated metabolic disorders.
Assuntos
Ácidos Graxos , Tromboxano A2 , Humanos , Tromboxano A2/metabolismo , Rosiglitazona/farmacologia , Ácidos Graxos/metabolismo , Adipócitos Marrons/metabolismo , Obesidade/metabolismo , Prostaglandinas I/metabolismoRESUMO
Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.
Assuntos
PPAR beta , Dourada , Animais , Ciclo-Oxigenase 2/metabolismo , Ciclopentanos , DNA Bacteriano , Granulócitos/metabolismo , Inflamação , Mamíferos , PPAR alfa , PPAR gama/genética , PPAR gama/metabolismo , Prostaglandina D2/química , Prostaglandina D2/farmacologia , Prostaglandinas , Espécies Reativas de Oxigênio , Dourada/metabolismoRESUMO
Background: Prematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs). Methods: Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1ß as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph. Results: 15dPGJ2 inhibited IL-1ß-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1ß-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs. Conclusion: We conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.
Assuntos
NF-kappa B , Nascimento Prematuro , Âmnio , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/farmacologia , Citocinas/metabolismo , Dinoprostona/metabolismo , Dinoprostona/farmacologia , Dinoprostona/uso terapêutico , Células Epiteliais/metabolismo , Feminino , Humanos , Recém-Nascido , Inflamação/metabolismo , Interleucina-6 , Interleucina-8/metabolismo , Interleucina-8/farmacologia , Interleucina-8/uso terapêutico , Lipopolissacarídeos , Camundongos , Células Musculares/metabolismo , NF-kappa B/metabolismo , Prostaglandina D2/análogos & derivados , RNA Mensageiro/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/farmacologia , Fator de Transcrição AP-1/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recent studies have shown that prostaglandin D2 (PGD2) and its nonenzymatic metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2 (15-dPGJ2), inhibit in vitro growth of explanted human hair follicles and inhibit hair growth in mice through the GPR44 (DP2). However, the underlying mechanism is still unclear. In this study, we first investigated the expression of DP2 in human hair follicles and in cultured follicular cells. We found that DP2 is strongly expressed in the outer root sheath (ORS) cells and weakly expressed in the dermal papilla (DP) cells. We observed slight growth stimulation when ORS and DP cells were treated with PGD2. We also observed slight growth stimulation when DP and ORS cells were treated with low concentrations (0.5 and 1 µM) of 15-dPGJ2. However, 5 µM 15-dPGJ2 inhibited the viability and caused apoptosis of both cell types. Exposure of cultured human hair follicles to 15-dPGJ2 resulted in significant apoptosis in follicular keratinocytes. Altogether, our data provide an evidence that 15-dPGJ2 promotes apoptosis in follicular keratinocytes and provide rationale for developing remedies for the prevention and treatment of hair loss based on DP2 antagonism.
Assuntos
Apoptose/efeitos dos fármacos , Folículo Piloso/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Prostaglandina D2/análogos & derivados , Alopecia/metabolismo , Alopecia/patologia , Alopecia/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Folículo Piloso/patologia , Humanos , Queratinócitos/patologia , Prostaglandina D2/toxicidade , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismoRESUMO
Peroxisome proliferator activated receptors (PPARs) are ligand activated transcription factors with crucial functions in lipid homeostasis, anti-inflammatory processes and placental development. Maternal diabetes induces a pro-inflammatory environment and alters placental development. We investigated whether PPARs regulate lipid metabolism and nitric oxide (NO) production in placental explants from healthy and type 2 diabetic (DM2) patients. We found decreased PPARα and PPARγ concentrations, no changes in PPARδ concentrations, and increased lipids, lipoperoxides and NO production in placentas from DM2 patients. PPARα agonists reduced placental concentrations of triglycerides and both PPARα and PPARδ agonists reduced concentrations of phospholipids, cholesteryl esters and cholesterol. PPARγ agonists increased lipid concentrations in placentas from DM2 patients and more markedly in placentas from healthy patients. Endogenous ligands for the three PPAR isotypes reduced NO production and lipoperoxidation in placentas from DM2 patients. We conclude that PPARs play a role in placental NO and lipid homeostasis and can regulate NO production, lipid concentrations and lipoperoxidation in placentas from DM2 patients.