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Sphingolipid metabolism is dysregulated in many cancers, allowing cells to evade apoptosis through increased sphingosine-1-phosphate (S1P) and decreased ceramides. Ceramidases hydrolyze ceramides to sphingosine, which is phosphorylated by sphingosine kinases to generate S1P. The S1P allows cells to evade apoptosis by shifting the equilibrium away from ceramides, which favor cell death. One tumor type that exhibits a shift in the sphingolipid balance towards S1P is glioblastoma (GBM), a highly aggressive brain tumor. GBMs almost always recur despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Understanding sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target dysregulation of sphingolipid metabolism in GBM. Carmofur, a derivative of 5-fluorouracil, inhibits acid ceramidase (ASAH1), a key enzyme in the production of S1P, and is in use outside the USA to treat colorectal cancer. We find that the mRNA for ASAH1, but not other ceramidases, is elevated in recurrent GBM. When TMZ-resistant GBM cells were treated with carmofur, decreased cell growth and increased apoptosis were observed along with cell cycle perturbations. RNA-sequencing identified decreases in cell cycle control pathways that were specific to TMZ-resistant cells. Furthermore, the transcription factor and G1 to S phase regulator, E2F8, was upregulated in TMZ-resistant versus parental GBM cells and inhibited by carmofur treatment in TMZ-resistant GBM cells, specifically. These data suggest a possible role for E2F8 as a mediator of carmofur effects in the context of TMZ resistance. These data suggest the potential utility of normalizing the sphingolipid balance in the context of recurrent GBM.
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Extracellular vesicles play major roles in cell-to-cell communication and are excellent biomarker candidates. However, studying plasma extracellular vesicles is challenging due to contaminants. Here, we performed a proteomics meta-analysis of public data to refine the plasma EV composition by separating EV proteins and contaminants into different clusters. We obtained two clusters with a total of 1717 proteins that were depleted of known contaminants and enriched in EV markers with independently validated 71% true-positive. These clusters had 133 clusters of differentiation (CD) antigens and were enriched with proteins from cell-to-cell communication and signaling. We compared our data with the proteins deposited in PeptideAtlas, making our refined EV protein list a resource for mechanistic and biomarker studies. As a use case example for this resource, we validated the type 1 diabetes biomarker proplatelet basic protein in EVs and showed that it regulates apoptosis of ß cells and macrophages, two key players in the disease development. Our approach provides a refinement of the EV composition and a resource for the scientific community.
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Vesículas Extracelulares , Proteómica , Antígenos CD/metabolismo , Biomarcadores , Vesículas Extracelulares/metabolismo , Proteínas , Transducción de Señal , Conjuntos de Datos como Asunto , Humanos , AnimalesRESUMEN
INTRODUCTION: Type 1 diabetes (T1D) is an autoimmune disease in which pro-inflammatory and cytotoxic signaling drive the death of the insulin-producing ß cells. This complex signaling is regulated in part by fatty acids and their bioproducts, making them excellent therapeutic targets. AREAS COVERED: We provide an overview of the fatty acid actions on ß cells by discussing how they can cause lipotoxicity or regulate inflammatory response during insulitis. We also discuss how diet can affect the availability of fatty acids and disease development. Finally, we discuss development avenues that need further exploration. EXPERT OPINION: Fatty acids, such as hydroxyl fatty acids, ω-3 fatty acids, and their downstream products, are druggable candidates that promote protective signaling. Inhibitors and antagonists of enzymes and receptors of arachidonic acid and free fatty acids, along with their derived metabolites, which cause pro-inflammatory and cytotoxic responses, have the potential to be developed as therapeutic targets also. Further, because diet is the main source of fatty acid intake in humans, balancing protective and pro-inflammatory/cytotoxic fatty acid levels through dietary therapy may have beneficial effects, delaying T1D progression. Therefore, therapeutic interventions targeting fatty acid signaling hold potential as avenues to treat T1D.
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Enfermedades Autoinmunes , Diabetes Mellitus Tipo 1 , Ácidos Grasos Omega-3 , Humanos , Ácidos Grasos/metabolismo , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Transducción de Señal , Dieta , Ácidos Grasos Omega-3/uso terapéuticoRESUMEN
Impaired glucose tolerance (IGT) and ß-cell dysfunction in insulin resistance associated with obesity lead to type 2 diabetes (T2D). Glucose-stimulated insulin secretion (GSIS) from ß-cells occurs via a canonical pathway that involves glucose metabolism, ATP generation, inactivation of K ATP channels, plasma membrane depolarization, and increases in cytosolic concentrations of [Ca 2+ ] c . However, optimal insulin secretion requires amplification of GSIS by increases in cyclic adenosine monophosphate (cAMP) signaling. The cAMP effectors protein kinase A (PKA) and exchange factor activated by cyclic-AMP (Epac) regulate membrane depolarization, gene expression, and trafficking and fusion of insulin granules to the plasma membrane for amplifying GSIS. The widely recognized lipid signaling generated within ß-cells by the ß-isoform of Ca 2+ -independent phospholipase A 2 enzyme (iPLA 2 ß) participates in cAMP-stimulated insulin secretion (cSIS). Recent work has identified the role of a G-protein coupled receptor (GPCR) activated signaling by the complement 1q like-3 (C1ql3) secreted protein in inhibiting cSIS. In the IGT state, cSIS is attenuated, and the ß-cell function is reduced. Interestingly, while ß-cell-specific deletion of iPLA 2 ß reduces cAMP-mediated amplification of GSIS, the loss of iPLA 2 ß in macrophages (MØ) confers protection against the development of glucose intolerance associated with diet-induced obesity (DIO). In this article, we discuss canonical (glucose and cAMP) and novel noncanonical (iPLA 2 ß and C1ql3) pathways and how they may affect ß-cell (dys)function in the context of impaired glucose intolerance associated with obesity and T2D. In conclusion, we provide a perspective that in IGT states, targeting noncanonical pathways along with canonical pathways could be a more comprehensive approach for restoring ß-cell function in T2D. © 2023 American Physiological Society. Compr Physiol 13:5023-5049, 2023.
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Diabetes Mellitus Tipo 2 , Intolerancia a la Glucosa , Humanos , Secreción de Insulina , Insulina/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Glucosa/metabolismo , Obesidad , Adenosina Trifosfato/metabolismoRESUMEN
Effective energy expenditure is critical for maintaining body weight (BW). However, underlying mechanisms contributing to increased BW remain unknown. We characterized the role of brain angiogenesis inhibitor-3 (BAI3/ADGRB3), an adhesion G-protein coupled receptor (aGPCR), in regulating BW. A CRISPR/Cas9 gene editing approach was utilized to generate a whole-body deletion of the BAI3 gene (BAI3-/-). In both BAI3-/- male and female mice, a significant reduction in BW was observed compared to BAI3+/+ control mice. Quantitative magnetic imaging analysis showed that lean and fat masses were reduced in male and female mice with BAI3 deficiency. Total activity, food intake, energy expenditure (EE), and respiratory exchange ratio (RER) were assessed in mice housed at room temperature using a Comprehensive Lab Animal Monitoring System (CLAMS). While no differences were observed in the activity between the two genotypes in male or female mice, energy expenditure was increased in both sexes with BAI3 deficiency. However, at thermoneutrality (30 °C), no differences in energy expenditure were observed between the two genotypes for either sex, suggesting a role for BAI3 in adaptive thermogenesis. Notably, in male BAI3-/- mice, food intake was reduced, and RER was increased, but these attributes remained unchanged in the female mice upon BAI3 loss. Gene expression analysis showed increased mRNA abundance of thermogenic genes Ucp1, Pgc1α, Prdm16, and Elov3 in brown adipose tissue (BAT). These outcomes suggest that adaptive thermogenesis due to enhanced BAT activity contributes to increased energy expenditure and reduced BW with BAI3 deficiency. Additionally, sex-dependent differences were observed in food intake and RER. These studies identify BAI3 as a novel regulator of BW that can be potentially targeted to improve whole-body energy expenditure.
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BACKGROUND: Type 1 diabetes (T1D) is a complex autoimmune disorder whose pathogenesis involves an intricate interplay between ß cells of the pancreatic islet, other islet cells, and cells of the immune system. Direct intercellular communication within the islet occurs via cell surface proteins and indirect intercellular communication has traditionally been seen as occurring via secreted proteins (e.g., endocrine hormones and cytokines). However, recent literature suggests that extracellular vesicles (EVs) secreted by ß cells constitute an additional and biologically important mechanism for transmitting signals to within the islet. SCOPE OF REVIEW: This review summarizes the general mechanisms of EV formation, with a particular focus on how lipids and lipid signaling pathways influence their formation and cargo. We review the implications of EV release from ß cells for T1D pathogenesis, how EVs and their cargo might be leveraged as biomarkers of this process, and how EVs might be engineered as a therapeutic candidate to counter T1D outcomes. MAJOR CONCLUSIONS: Islet ß cells have been viewed as initiators and propagators of the cellular circuit giving rise to autoimmunity in T1D. In this context, emerging literature suggests that EVs may represent a conduit for communication that holds more comprehensive messaging about the ß cells from which they arise. As the field of EV biology advances, it opens the possibility that intervening with EV formation and cargo loading could be a novel disease-modifying approach in T1D.
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Diabetes Mellitus Tipo 1 , Vesículas Extracelulares , Comunicación Celular , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , LípidosRESUMEN
Glioblastoma (GBM) remains one of the most aggressive cancers, partially due to its ability to migrate into the surrounding brain. The sphingolipid balance, or the balance between ceramides and sphingosine-1-phosphate, contributes to the ability of GBM cells to migrate or invade. Of the ceramidases which hydrolyze ceramides, acid ceramidase (ASAH1) is highly expressed in GBM samples compared to non-tumor brain. ASAH1 expression also correlates with genes associated with migration and focal adhesion. To understand the role of ASAH1 in GBM migration, we utilized shRNA knockdown and observed decreased migration that did not depend upon changes in growth. Next, we inhibited ASAH1 using carmofur, a clinically utilized small molecule inhibitor. Inhibition of ASAH1 by carmofur blocks in vitro migration of U251 (GBM cell line) and GBM cells derived from patient-derived xenografts (PDXs). RNA-sequencing suggested roles for carmofur in MAPK and AKT signaling. We found that carmofur treatment decreases phosphorylation of AKT, but not of MAPK. The decrease in AKT phosphorylation was confirmed by shRNA knockdown of ASAH1. Our findings substantiate ASAH1 inhibition using carmofur as a potential clinically relevant treatment to advance GBM therapeutics, particularly due to its impact on migration.
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Ceramidasa Ácida , Glioblastoma , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Línea Celular Tumoral , Movimiento Celular , Ceramidas/metabolismo , Fluorouracilo , Glioblastoma/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt , ARN Interferente PequeñoRESUMEN
Endoplasmic reticulum (ER) stress contributes to pancreatic beta-cell apoptosis in diabetes, but the factors involved are still not fully elucidated. Growth differentiation factor 15 (GDF15) is a stress response gene and has been reported to be increased and play an important role in various diseases. However, the role of GDF15 in beta cells in the context of ER stress and diabetes is still unclear. In this study, we have discovered that GDF15 promotes ER stress-induced beta-cell apoptosis and that downregulation of GDF15 has beneficial effects on beta-cell survival in diabetes. Specifically, we found that GDF15 is induced by ER stress in beta cells and human islets, and that the transcription factor C/EBPß is involved in this process. Interestingly, ER stress-induced apoptosis was significantly reduced in INS-1 cells with Gdf15 knockdown and in isolated Gdf15 knockout mouse islets. In vivo, we found that Gdf15 deletion attenuates streptozotocin-induced diabetes by preserving beta cells and insulin levels. Moreover, deletion of Gdf15 significantly delayed diabetes development in spontaneous ER stress-prone Akita mice. Thus, our findings suggest that GDF15 contributes to ER stress-induced beta-cell apoptosis and that inhibition of GDF15 may represent a novel strategy to promote beta-cell survival and treat diabetes.
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Diabetes Mellitus Experimental , Células Secretoras de Insulina , Animales , Apoptosis , Diabetes Mellitus Experimental/genética , Estrés del Retículo Endoplásmico , Factor 15 de Diferenciación de Crecimiento/genética , Factor 15 de Diferenciación de Crecimiento/farmacología , RatonesRESUMEN
Bone modeling can be modulated by lipid signals such as arachidonic acid (AA) and its cyclooxygenase 2 (COX2) metabolite, prostaglandin E2 (PGE2), which are recognized mediators of optimal bone formation. Hydrolysis of AA from membrane glycerophospholipids is catalyzed by phospholipases A2 (PLA2s). We reported that mice deficient in the Ca2+- independent PLA2beta (iPLA2ß), encoded by Pla2g6, exhibit a low bone phenotype, but the cause for this remains to be identified. Here, we examined the mechanistic and molecular roles of iPLA2ß in bone formation using bone marrow stromal cells and calvarial osteoblasts from WT and iPLA2ß-deficient mice, and the MC3T3-E1 osteoblast precursor cell line. Our data reveal that transcription of osteogenic factors (Bmp2, Alpl, and Runx2) and osteogenesis are decreased with iPLA2ß-deficiency. These outcomes are corroborated and recapitulated in WT cells treated with a selective inhibitor of iPLA2 ß (10 µM S-BEL), and rescued in iPLA2ß-deficient cells by additions of 10 µM PGE2. Further, under osteogenic conditions we find that PGE2 production is through iPLA2ß activity and that this leads to induction of Runx2 and iPLA2ß transcription. These findings reveal a strong link between osteogenesis and iPLA2ß-derived lipids and raise the intriguing possibility that iPLA2ß-derived PGE2 participates in osteogenesis and in the regulation of Runx2 and also iPLA2ß.
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Células Secretoras de Insulina , Osteogénesis , Animales , Huesos , Dinoprostona/farmacología , Fosfolipasas A2 Grupo VI/genética , Ratones , Fosfolipasas A2RESUMEN
Type 1 diabetes (T1D) development, in part, is due to ER stress-induced ß-cell apoptosis. Activation of the Ca2+-independent phospholipase A2 beta (iPLA2ß) leads to the generation of pro-inflammatory eicosanoids, which contribute to ß-cell death and T1D. ER stress induces iPLA2ß-mediated generation of pro-apoptotic ceramides via neutral sphingomyelinase (NSMase). To gain a better understanding of the impact of iPLA2ß on sphingolipids (SLs), we characterized their profile in ß-cells undergoing ER stress. ESI/MS/MS analyses followed by ANOVA/Student's t-test were used to assess differences in sphingolipids molecular species in Vector (V) control and iPLA2ß-overexpressing (OE) INS-1 and Akita (AK, spontaneous model of ER stress) and WT-littermate (AK-WT) ß-cells. As expected, iPLA2ß induction was greater in the OE and AK cells in comparison with V and WT cells. We report here that ER stress led to elevations in pro-apoptotic and decreases in pro-survival sphingolipids and that the inactivation of iPLA2ß restores the sphingolipid species toward those that promote cell survival. In view of our recent finding that the SL profile in macrophages-the initiators of autoimmune responses leading to T1D-is not significantly altered during T1D development, we posit that the iPLA2ß-mediated shift in the ß-cell sphingolipid profile is an important contributor to ß-cell death associated with T1D.
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Apoptosis , Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/metabolismo , Lipasa/metabolismo , Proteínas de la Membrana/metabolismo , Esfingolípidos/metabolismo , Apoptosis/genética , Línea Celular , Cromatografía Liquida , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/genética , Expresión Génica , Humanos , Lipasa/genética , Lipidómica/métodos , Proteínas de la Membrana/genética , Modelos Biológicos , Espectrometría de Masas en TándemRESUMEN
The Ca2+-independent phospholipase A2ß (iPLA2ß) is a member of the PLA2 family that has been proposed to have roles in multiple biological processes including membrane remodeling, cell proliferation, bone formation, male fertility, cell death, and signaling. Such involvement has led to the identification of iPLA2ß activation in several diseases such as cancer, cardiovascular abnormalities, glaucoma, periodontitis, neurological disorders, diabetes, and other metabolic disorders. More recently, there has been heightened interest in the role that iPLA2ß plays in promoting inflammation. Recognizing the potential contribution of iPLA2ß in the development of autoimmune diseases, we review this issue in the context of an iPLA2ß link with macrophages and T-cells.
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Fosfolipasas A2 Grupo VI/metabolismo , Inmunidad Innata , Animales , Humanos , Inflamación , Linfocitos/inmunología , Macrófagos/inmunologíaRESUMEN
The field of bioactive lipids is ever expanding with discoveries of novel lipid molecules that promote human health. Adopting a lipidomic-assisted approach, two new families of previously unrecognized saturated hydroxy fatty acids (SHFAs), namely, hydroxystearic and hydroxypalmitic acids, consisting of isomers with the hydroxyl group at different positions, were identified in milk. Among the various regio-isomers synthesized, those carrying the hydroxyl at the 7- and 9-positions presented growth inhibitory activities against various human cancer cell lines, including A549, Caco-2, and SF268 cells. In addition, 7- and 9-hydroxystearic acids were able to suppress ß-cell apoptosis induced by proinflammatory cytokines, increasing the possibility that they can be beneficial in countering autoimmune diseases, such as type 1 diabetes. 7-(R)-Hydroxystearic acid exhibited the highest potency both in cell growth inhibition and in suppressing ß-cell death. We propose that such naturally occurring SHFAs may play a role in the promotion and protection of human health.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ácidos Grasos/farmacología , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Citocinas/farmacología , Ácidos Grasos/síntesis química , Ácidos Grasos/química , Expresión Génica/efectos de los fármacos , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Leche/química , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Ácidos Esteáricos/farmacología , Estereoisomerismo , Espectrometría de Masas en TándemRESUMEN
To examine the role of group VIA phospholipase A2 (iPLA2ß) in specific cell lineages in insulin secretion and insulin action, we prepared mice with a selective iPLA2ß deficiency in cells of myelomonocytic lineage, including macrophages (MØ-iPLA2ß-KO), or in insulin-secreting ß-cells (ß-Cell-iPLA2ß-KO), respectively. MØ-iPLA2ß-KO mice exhibited normal glucose tolerance when fed standard chow and better glucose tolerance than floxed-iPLA2ß control mice after consuming a high-fat diet (HFD). MØ-iPLA2ß-KO mice exhibited normal glucose-stimulated insulin secretion (GSIS) in vivo and from isolated islets ex vivo compared to controls. Male MØ-iPLA2ß-KO mice exhibited enhanced insulin responsivity vs. controls after a prolonged HFD. In contrast, ß-cell-iPLA2ß-KO mice exhibited impaired glucose tolerance when fed standard chow, and glucose tolerance deteriorated further when introduced to a HFD. ß-Cell-iPLA2ß-KO mice exhibited impaired GSIS in vivo and from isolated islets ex vivo vs. controls. ß-Cell-iPLA2ß-KO mice also exhibited an enhanced insulin responsivity compared to controls. These findings suggest that MØ iPLA2ß participates in HFD-induced deterioration in glucose tolerance and that this mainly reflects an effect on insulin responsivity rather than on insulin secretion. In contrast, ß-cell iPLA2ß plays a role in GSIS and also appears to confer some protection against deterioration in ß-cell functions induced by a HFD.
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Fosfolipasas A2 Grupo VI/genética , Células Secretoras de Insulina/metabolismo , Fosfolipasas A2/genética , Animales , Glucemia/genética , Dieta Alta en Grasa/efectos adversos , Glucosa/genética , Intolerancia a la Glucosa/tratamiento farmacológico , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/genética , Insulina/metabolismo , Secreción de Insulina/genética , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Fosfolipasas A2/deficienciaRESUMEN
Glioblastoma (GBM) is a primary malignant brain tumor with a dismal prognosis, partially due to our inability to completely remove and kill all GBM cells. Rapid tumor recurrence contributes to a median survival of only 15 months with the current standard of care which includes maximal surgical resection, radiation, and temozolomide (TMZ), a blood-brain barrier (BBB) penetrant chemotherapy. Radiation and TMZ cause sphingomyelinases (SMase) to hydrolyze sphingomyelins to generate ceramides, which induce apoptosis. However, cells can evade apoptosis by converting ceramides to sphingosine-1-phosphate (S1P). S1P has been implicated in a wide range of cancers including GBM. Upregulation of S1P has been linked to the proliferation and invasion of GBM and other cancers that display a propensity for brain metastasis. To mediate their biological effects, SMases and S1P modulate signaling via phospholipase C (PLC) and phospholipase D (PLD). In addition, both SMase and S1P may alter the integrity of the BBB leading to infiltration of tumor-promoting immune populations. SMase activity has been associated with tumor evasion of the immune system, while S1P creates a gradient for trafficking of innate and adaptive immune cells. This review will explore the role of sphingolipid metabolism and pharmacological interventions in GBM and metastatic brain tumors with a focus on SMase and S1P.
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Neoplasias Encefálicas/genética , Glioblastoma/genética , Esfingolípidos/metabolismo , Esfingomielina Fosfodiesterasa/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Proliferación Celular/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Lisofosfolípidos/genética , Lisofosfolípidos/metabolismo , Metástasis de la Neoplasia , Fosfolipasa D/genética , Esfingolípidos/genética , Esfingomielina Fosfodiesterasa/metabolismo , Esfingosina/análogos & derivados , Esfingosina/genética , Esfingosina/metabolismo , Fosfolipasas de Tipo C/genéticaRESUMEN
Type 1 diabetes (T1D) is a consequence of autoimmune ß cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2ß (iPLA2ß) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2ß can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering ß cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2ß-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2ß reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.
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Biomarcadores/metabolismo , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/etiología , Metabolismo de los Lípidos , Macrófagos Peritoneales/metabolismo , Adolescente , Animales , Niño , Diabetes Mellitus Tipo 1/terapia , Eicosanoides/metabolismo , Ácidos Grasos/metabolismo , Femenino , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/metabolismo , Humanos , Cetonas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/trasplante , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Naftalenos/farmacologíaRESUMEN
Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)ß (iPLA2ß). Here, we assessed the link between iPLA2ß-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2ß-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2ß.Tg mice with selective iPLA2ß overexpression in ß-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2ß-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2ß.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2ß-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that ß-cell iPLA2ß-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.
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Calcio/metabolismo , Eicosanoides/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Macrófagos/metabolismo , Transducción de Señal , Animales , Fosfolipasas A2 Grupo IV/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
The integrity of the Golgi and trans-Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A-inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF's targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.
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Factores de Ribosilacion-ADP/metabolismo , Dominio Catalítico , Aparato de Golgi/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Homeostasis , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Humanos , Red trans-Golgi/metabolismoRESUMEN
Ca2+-independent phospholipase A2 (GVIA iPLA2) has gained increasing interest recently as it has been recognized as a participant in biological processes underlying diabetes development and autoimmune-based neurological disorders. The development of potent GVIA iPLA2 inhibitors is of great importance because only a few have been reported so far. We present a novel class of GVIA iPLA2 inhibitors based on the ß-lactone ring. This functionality in combination with a four-carbon chain carrying a phenyl group at position-3 and a linear propyl group at position-4 of the lactone ring confers excellent potency. trans-3-(4-Phenylbutyl)-4-propyloxetan-2-one (GK563) was identified as being the most potent GVIA iPLA2 inhibitor ever reported ( XI(50) 0.0000021, IC50 1 nM) and also one that is 22 000 times more active against GVIA iPLA2 than GIVA cPLA2. It was found to reduce ß-cell apoptosis induced by proinflammatory cytokines, raising the possibility that it can be beneficial in countering autoimmune diseases, such as type 1 diabetes.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Lactonas/farmacología , Inhibidores de Fosfolipasa A2/farmacología , Fosfolipasas A2 Calcio-Independiente/antagonistas & inhibidores , Animales , Apoptosis/fisiología , Citocinas/fisiología , Diseño de Fármacos , Humanos , Mediadores de Inflamación/fisiología , Lactonas/química , Inhibidores de Fosfolipasa A2/química , Inhibidores de Fosfolipasa A2/metabolismo , Fosfolipasas A2 Calcio-Independiente/metabolismo , Relación Estructura-ActividadRESUMEN
The Ca2+-independent phospholipases, designated as group VI iPLA2s, also referred to as PNPLAs due to their shared homology with patatin, include the ß, γ, δ, ε, ζ, and η forms of the enzyme. The iPLA2s are ubiquitously expressed, share a consensus GXSXG catalytic motif, and exhibit organelle/cell-specific localization. Among the iPLA2s, iPLA2ß has received wide attention as it is recognized to be involved in membrane remodeling, cell proliferation, cell death, and signal transduction. Ongoing studies implicate participation of iPLA2ß in a variety of disease processes including cancer, cardiovascular abnormalities, glaucoma, and peridonditis. This review will focus on iPLA2ß and its links to male fertility, neurological disorders, metabolic disorders, and inflammation.
Asunto(s)
Fertilidad/fisiología , Inflamación/metabolismo , Enfermedades Metabólicas/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Animales , Humanos , Masculino , Transducción de Señal/fisiologíaRESUMEN
Macrophages are important in innate and adaptive immunity. Macrophage participation in inflammation or tissue repair is directed by various extracellular signals and mediated by multiple intracellular pathways. Activation of group VIA phospholipase A2 (iPLA2ß) causes accumulation of arachidonic acid, lysophospholipids, and eicosanoids that can promote inflammation and pathologic states. We examined the role of iPLA2ß in peritoneal macrophage immune function by comparing wild type (WT) and iPLA2ß-/- mouse macrophages. Compared with WT, iPLA2ß-/- macrophages exhibited reduced proinflammatory M1 markers when classically activated. In contrast, anti-inflammatory M2 markers were elevated under naïve conditions and induced to higher levels by alternative activation in iPLA2ß-/- macrophages compared with WT. Induction of eicosanoid (12-lipoxygenase (12-LO) and cyclooxygenase 2 (COX2))- and reactive oxygen species (NADPH oxidase 4 (NOX4))-generating enzymes by classical activation pathways was also blunted in iPLA2ß-/- macrophages compared with WT. The effects of inhibitors of iPLA2ß, COX2, or 12-LO to reduce M1 polarization were greater than those to enhance M2 polarization. Certain lipids (lysophosphatidylcholine, lysophosphatidic acid, and prostaglandin E2) recapitulated M1 phenotype in iPLA2ß-/- macrophages, but none tested promoted M2 phenotype. These findings suggest that (a) lipids generated by iPLA2ß and subsequently oxidized by cyclooxygenase and 12-LO favor macrophage inflammatory M1 polarization, and (b) the absence of iPLA2ß promotes macrophage M2 polarization. Reducing macrophage iPLA2ß activity and thereby attenuating macrophage M1 polarization might cause a shift from an inflammatory to a recovery/repair milieu.